Proceedings of the

International Food Research

Conference 2017

Date: 25-27 July 2017

Venue: Complex of the Deputy Vice Chancellor
(Research and Innovation),
Universiti Putra Malaysia

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© Universiti Putra Malaysia 2017

All rights reserved. No part of this publication may be produced, stored in a retrieval
system or transmitted, in any form or any means, electronic, mechanical, photocopying,
recording or otherwise without the prior permission of the copyright owner.

Published by:
Faculty of Food Science and Technology
Universiti Putra Malaysia
43400 UPM Serdang
Selangor Darul Ehsan
MALAYSIA

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Table of Content
Page
Foreword by the Vice Chancellor, Universiti Putra Malaysia xiv

Foreword by the Dean, Faculty of Food Science and Technology xv

Foreword by the IFRC 2017 Chairman xvi

IFRC 2017 Proceedings

Plenary 2 1
Supercritical Fluid Technology for Food Processing
Masaki Ota and Hiroshi Inomata

Food Safety and Quality

1. IFRC 2017: 035-049 5

Toxigenic Campylobacter jejuni in Vegetables Farms and Retail Outlets in Terengganu
Tang, J.Y.H., Khalid, M.I. and Radu, S.

2. IFRC 2017: 037-024 9

Molecular Typing of Bacillus cereus Isolated from Sago Processing Mills in Sarawak.
Jaraee J., Bilung M. L., Nolasco C. H. and Vincent M.

3. IFRC 2017: 071-051 13

Variation of Microbial and Chemical Quality of Two Major Food Fishes in Sri Lanka with Gamma
Irradiation.
Surendra, I.H.W., Edirisinghe, E.M.R.K.B. and Rathnayake, R.M.N.P.

4. IFRC 2017: 138-096 17

Shiga Toxin Escherichia coli Survival in Different Blending Ratio of Fresh Pineapple-Mango Juice
Blends.
Kamarul, Zaman, A.A., Shamsudin, R., Mohd Adzahan, N. and Sulaiman, A.

5. IFRC 2017: 139-163 21

Proper Hand Washing Practices in School Canteen: A Qualitative Study on Food Handlers’ Belief.
Ahmad, I. A., Abidin, U.F.U.Z., Mahyudin, N.A. and Ab-Rashid, N.K.

6. IFRC 2017: 141-098 25

Effect of Poster and Video Intervention on The Knowledge, Attitude and Practice (KAP) Level of
Personal Hygiene Among Food Handlers in 24 Hours Mamak Restaurants in Sungai Petani, Kedah.
Masyita, M. and Nur Amalina, M.J.

7. IFRC 2017: 148-107 33

Trace Level Determination of Organophosphorus Pesticides in Fruit Samples Using
Tetramethylguanidine-Silica Nanoparticles as Solid Phase Extraction Sorbent.
Veloo, K.V., Adam, F. and Batagarawa, M. S.

8. IFRC 2017: 149-149 37

Effect of Thickness of Antimicrobial Film-Coated Paper for Food Packaging on Antimicrobial Agent
Migration Rate and Biodegradability.
Mustapha, F.A., Jai, J., Sharif, Z.I.M., and Yusof, N.M.

iii
9. IFRC 2017: 152-168 41
Antioxidant Activity and Estragole Content Of Ethanolic and Methanolic Extract of Fennel (Foeniculum
Vulgare Mill.) and Nutmeg (Myristica Fragrans Houtt) and its Risk Assessment Using Margin of
Exposure (MOE).
Martati, E., and Akmalina, M.A.

10. IFRC 2017: 159-116 45

Selection of Lactic Acid Bacteria Can Reduce the Cyanide Compound on The Processing Yam Flour
(Dioscorea Hispida Dennst.)
Winarti, S., Murtinngsih and Amalia, S.R.

11. IFRC 2017: 163-148 49

Antimicrobial and Mechanical Properties of Gelatin Film Plasticized With Nigella Sativa (Black Seed Oil)
Han low, Ademola M Hammed, and Munirat A Idris

12. IFRC 2017: 168-124 53

Application of Hydroxyl Radical Aerosolization on E. coli/Coliform Reduction for Rapid Surface
Disinfection in Food Processing
Boonchan, W., Chayasitthisophon, A., Foster, K.W., Weeranoppanant, N. and Thipayarat, A.

13. IFRC 2017: 179-144 61

The Lipolytic Activity of Pseudomonas fluorescens BIOTECH 1123 in Commercially Available Salted
Butter under Refrigerated Conditions and Its Relation to Product Quality Deterioration
Babaran, G.M.O., and Mopera, L.E.

14. IFRC 2017: 182-147 65

Quality Evaluation of Microwave and Conventional Pasteurised Pineapple Juice
Abd Aziz, N. A., Mohd Jusoh, Y. M., Nik Mahmood, N. A., Yunus, N. A. and Endut, A.

15. IFRC 2017: 184-152 69

Development of Selective Esculin Hydrolysis Broth for Rapid Screening of Vibrio parahaemolyticus
Sangadkit, W., Deepatana, A. and Thipayarat, A.

16. IFRC 2017: 186-156 73

Evaluation of the Marketability, Microbial Quality, and Safety of Fresh-Cut Vegetable Mixes from
Selected Wet Markets and Supermarkets in Los Baños, Laguna
Sotiangco, I.D.G., Piamonte, S.B.H. and Castillo-Israel, K.A.T.

17. IFRC 2017: 187-153 77

Combined Ozonation and UV-C Treatment to Inactivate E. coli Contaminants in Model Fish Sauce.
Sangadkit, W., Kunpanya, P., Deepatana, A., Foster, K.W. and Thipayarat, A.

18. IFRC 2017: 195-174 81

Case Study of Profiling of Adulterant in Tongkat Ali Herbal Product using Real Time Coupled with High
Resolution Melting Analysis
N.F. Fadzil, A.Wagiran, F. Mohd Salleh and S. Abdullah
19. IFRC 2107: 214-200 85
Microflora Identification of Enteral Feeding Tubes in Neonatal Intensive Care Unit Setting
Mahirah Mohamad, Shareena Ishak, Rohana Jaafar and Norrakiah Abdullah Sani

20. IFRC 2017: 216-202 89

Effects of Hydrocolloids on Physicochemical and Sensory Qualities of Noodles
Chiew, C.S. and Thed, S.T.

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21. IFRC 2017: 223-209 93
Perceptions of Plastic Packaging Usage to Pack Hot Foods: The Perspectives of Food Hawkers In
Kuala Selangor, Malaysia.
Mat Issa, Z. and Ab Rahim, N.F.

22. IFRC 2017: 225-213 97

Atmospheric Cold Plasma Treatment Effect on Microbiological Evaluation and Moisture Effect of
Mango-Fortified Noodles
Zabidi N.Z.A., Zaaba S.K., Abidin N.S.A. and Rukunudin IH

23. IFRC 2017: 242-225 101

Microbiological Quality of Municipal Tap Water and Filtered Drinking Water in Serdang, Selangor
Nor-Khaizura M.A.R., Ahmad, N.A., Ahmad Zainal,A.S. and Mahyudin, N. A.

24. IFRC 2017: 309-268 105

Antimicrobial Activity of Plant Extracts against Foodborne Pathogens
Mat Issa, Z., Othman, N., Mustakim, M. and Jipiu, L. B.

Food Processing and Post-Harvest Technology

25. IFRC 2017: 014-112 109

Antifungal Activity of Aloe vera gel Towards the Pathogenic Fungus of Papaya Fruit
Mendy, T.K., Misran, A., Mahmud, T.M.M and Ismail, S.I

26. IFRC 2017: 027-142 117

Supercritical Fluid Extraction of Date Seed Oil
Jaih, A.A.M., Rahman, R.A., Razis, A.F.A., Ariffin, A.A., Al-Awaadh, A. and Selamat, J.

27. IFRC 2017: 036-023 121

Effect of Heat Adaptation and Spray Drying Outlet Temperature on the Survival of Lactobacillus sp.
Strain 3C2-10
Kunnathep, J. and Oonsivilai, R.

28. IFRC 2017: 044-029 125

Influence of Maturity Stage of Rhizomes on the Physicochemical and Sensory Properties of Ginger
(Zingiber officinale Roscoe) powder
Rabang, J.C.T. and Castillo-Israel, K.A.T.

29. IFRC 2017: 047-047 129

Development of an Artificially-Carbonated Fruit Wine Blend from Mango (Mangifera indica L.),
Pineapple (Ananas comosus) and Passion Fruit (Passiflora edulis Sims)
Zubia C.S., Hurtada, W.A. and Dizon, E.I.

30. IFRC 2017: 051-034 133

Development of Vegan Patties for Young Adults as a Source of Calcium using Tofu and Tempeh
Neo, Y.P., Au, J.E. and Tan, K.L.

31. IFRC 2017: 054-054 137

Production Process Technology and Its Characteristics of Probiotic Instant Chocolate Drink
Heny Herawati, Sri Yuliani, Widaningrum, Tatang Hidayat

32. IFRC 2017: 056-041 141

Effects of Virgin Coconut Oil on Qualities of Low Fat Pork Meatball
Oonmetta-aree, J.

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33. IFRC 2017: 057-042 145
Rambutan (Nephelium lappaceum Linn) Fruit Processing: Development of Preserve and Ice Cream
Rivadeneira, J., Juanico, C., Damaso, C., Espiritu, R., Jolejole, T.K., Lanorio, M.C., Parani, M.S., San
Juan, H.O., Sunico, D.J. and Sumague, M.J.

34. IFRC 2017: 058-043 149

Storage and Processing Stability of Natural Food Colorant from Philippine Wild Raspberry (Rubus
rosifolius Linn.)
Palomeno,Jr., A.M. and Lizardo, R.C.M.

35. IFRC 2017: 058-095 153

Resistant Starch Content and Physico-Chemical Properties of Flour from ‘Saba’ and ‘Latundan’ Banana
(Musa sp.) Varieties
Mendoza, C.A.J.G. and Lizardo, R.C.M.

36. IFRC 2017: 063-048 157

Effect of Squid Ink Addition on the Physicochemical Properties and Acceptability of Noodle
Aishah, B., Maisarah, K. and Fadhilah, J.

37. IFRC 2017: 066-084 161

Effect of Extraction Temperature, pH, and Time on Pectin Yield of Katmon (Dillenia philippinensis Rolfe)
Belan, D.L. and Israel, K.A.C.

38. IFRC 2017: 080-053 165

Physicochemical Properties of Two Varieties of Rambutan (Nephelium lappaceum L.) Fruit
Chai, K.F., Karim, R., Adzahan, N.M., Rukayadi, Y. and Ghazali, H.M.

39. IFRC 2017: 085-058 169

Comparative Study on the Quality and Storage Stability of Instant Mashed Sweet Potato [Ipomea
batatas (L) Lam] Prepared Using Three Different Varieties
Castillo-Israel, K.A.T., Perez, P.R.G. and Reginio, F.C., Jr.

40. IFRC 2017: 116-075 177

Optimization of Oil, Whey Protein Concentrate and Carboxymethyl Cellulose Levels on Rheological
Properties and Stability of Sky Fruit (Swietenia Macrophylla) Seed Oil-In-Water Emulsion
Nor Hayati, I., Chong, P.Y, and Yusof, H.M.

41. IFRC 2017: 120-078 181

Physical and Nutritional Properties of Malaysian Avocado (Persea americana Mill) Fruit
Tan, C.X., Chong, G.H., Hazilawati, H. and Ghazali, H.M.

42. IFRC 2017: 131-089 185

Determination of the Potential of Kamuning (Murraya paniculata) Flowers for Tea Development
Navarro, B.R.R. and Iñigo, H.B.R.

43. IFRC 2017: 134-092 189

Characteristic of Edible Film from Pectin of Citrus (Citrus Aurantifolia), Papaya (Carica papaya L.) and
Latundan Bananas (Musa acuminata × M. balbisiana) Peel Wastes: A Comparative Study
Hapsari N., Rosida, D.F.,Ramadhani, P.V., Sudaryati., 5Dewati, R

44. IFRC 2017: 142-099 193

Effect of Maltodextrin, Tricalcium Phosphate and Glycerol Monostearate on Moisture Sorption
Characteristics of Jamun (Syzygium cumini L.) Pulp Powder
Dey Paul, I., and Das, M.

vi
45. IFRC 2017: 158-132 201
The Effect of Pectinase, Glucoamylase and Cellulase Enzymes on the Extraction Yield of Roselle
Petals.
Mardiah,, Noli Novidahlia, Ma’rifat Khoirunnisa, and Hanafi

46. IFRC 2017: 166-150 205

Effect of Emulsifier at Different Concentrations on the Properties and Characteristics of Biodegradable
Films Based on Gelatin with Palm Oil for Food Packaging Application
Zazalli, S.A., Nabilah, B., de la Caba, K., Guerrero, P. and Nur Hanani, Z.A.

47. IFRC 2017: 170-126 209

Low Fat Coconut Flour as a Coconut Milk Powder Supplement for Improving Health and Reducing Cost
of Product
Dharmasena, D. A. N., Herath, H.M.T.K. and Madujith, T.

48. IFRC 2017: 196-175 213

Optimization of Sweet Cassava (Manihot esculents crantz.) Crude Extract with High Maltodextrin Level
Using Response Surface Methodogy.
Posridee, K. and Oonsivilai, R.

49. IFRC 2017: 207-188 217

Effect of Gamma Irradiation and Different Packagings on the Shelf Life of Mushrooms )Agaricus
bisporus(
Fartash, E., Khoshtaghaza, M. H., Abbasi, S

50. IFRC 2017: 211-194 221

Effect of Ultrasound Treatment on the Functional Properties of Jackfruit Seed Starch
Mohamad Yazid, N.S., Abdullah, N., Muhammad, N.

51. IFRC 2017: 212-196 225

Influence of Drying Methods on the Bioactive Compound and Antioxidant Activity of Pomelo Residue
Abd Rahman, N.F., Shamsudin, R., Ismail, A. Shah, N.N.A.K. and Varith, J.

52. IFRC 2017: 247-226 229

Development of Fish Gelatin Coatings Incorporated with Lemon Peel Extracts as Antimicrobial
Packaging to Extend the Shelf Life of Flammulina velutipes.
L. Naphawan, Z.A Maryam Adilah, Z.A. Nur Hanani

53. IFRC 2017: 248-230 233

Non-Evaporative Method to Remove High Boiling Point Solvent (Ethyl Lactate) from Palm Oil Extract at
Atmospheric Conditions
Kua, Y.L., Gan, S., Morris, A. and Ng, H.K.

54. IFRC 2017: 255-241 237

Effect of Aloe Vera Powder as Fat and Corn Flour Replacers in the Production of Reduced Fat Beef
Meatballs
Nurfazwin, Z., Nur Izzah Arifah, Z.A., Mohamad Afifi, I., Mat Yusoff, M. and Ismail-Fitry, M.R.

55. IFRC 2017: 259-272 241

Optimization of Natural Red Colorant Production from Roselle Using Ultrasound-Assisted Extraction
Mokhtar, N., Pak-Dek, M.S., Hamid, A.A., Mohd-Johar, A.H.H., and Jaafar, A.H.

56. IFRC 2017: 277-254 245

Rheological Properties, Emulsion and Oxidative Stability of Cocoa Butter Based Salad Dressing During
Storage
ishak, I., Hussain, N. and Mohd Hariri, N.A.
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57. IFRC 2017: 279-255 249
Effect of Extraction Methods with Different Matrix for Gelatin Recovery and Properties: A Review
Ee, S. C. and Jamilah Bakar

58. IFRC 2017: 310-269 253

Comparison of Sensory Quality and Preference Between Fermented Barleys, Glutinous Rice and the
Combination of Barley and Glutinous Rice
Ibrahim, N., Ezani, N. A. B., Ahmad Kamal, N., Mazlan, N., Abu Kassim, N. A., Jipiu, L. B., Abdul Aziz,
S. A. and Mat Issa, Z

Functional Food

59. IFRC 2017: 018-113 261

‘Ceri’Terengganu, Lepisanthes fruticosa the Rare Fruits of Malaysia, With New Potential.
Sukirah Abdul Rahman, Muhammad Anas Othaman, Nur Yuhasliza Abdul Rashid, Nur Diyana Alyas,
Hazniza Adnan, Nor Hazniza Aziz and Musaalbakri Abdul Manan.

60. IFRC 2017: 029-018 265

Bioactive Compounds and Nutritional Properties of Khao-Mao
Singthong, J., Oonsivilai, R., Oonmetta-aree, J. and Onsaard, E.

61. IFRC 2017: 043-030 269

Comparative Analysis of The Nutrient Content and Antioxidant Activity Of Lagikway (Abelmoschus
manihot L), Alugbati (Basella alba L), Camote (Ipomoea batatas L), and Saluyot (Corchorus capsularis
L) Leaves
Algar, A.F.C. and Sediño, D.J.I.

62. IFRC 2017: 070-055 273

Effect of Solvents on Extraction and Bioactive Properties of Commercial Grape Cultivars in Taiwan
Sridhar, K. and Charles, A.L.

63. IFRC 2017: 079-086 277

Quality of Dried Rice Noodles Incorporated with Differently Encapsulated Carrot Powders
Ismail, H. Karim, R. and Muhammad, K.

64. IFRC 2017: 081-087 281

Effect of Sago and Tapioca Starches on The Physicochemical Properties of Expanded Rice Products
Coloured with Red Beetroot (Beta vulgaris) Powder
Abdul Alam, N.A., Karim, R. and Muhammad, K

65. IFRC 2017: 092-088 285

Anti-browning and Antioxidant Properties of Clinacanthus nutans (Burm. F.) Lindau on “Granny Smith”
Apple Juice.
Husain N. F., Rahman R.A., and Suleiman N.

66. IFRC 2017: 098-066 289

Prebiotic Potential of Oligosaccharides Derived From Kappaphycus alvarezii Using Microwave-Assisted
Hydrolysis
Chan, S.T., Chye, F.Y. and Siew, C.K.

67. IFRC 2017: 102-067 293

Antioxidant and Metabolite Identification of Different Varieties of Dates (Phoenix dactylifera L.)
Hana Kadum, Azizah Abdul Hamid, Faridah Abasa, Abdul Karim Sabo Mohammed, Nurul Shazini
Ramli

viii
68. IFRC 2017: 106-069 297
Effectivity of Ethanol Extract of Purple Sweet Potato Var. Ayamurasaki as Natural Antihypertension in
Doca-Salt Hypertensive Rats
Irma Sarita Rahmawati, Soetjipto, Annis Catur Adi, Aulanni’am

69. IFRC 2017: 110-070 301

Chemical Composition And Physicochemical Properties Of Red Seaweeds (Kappaphycus alvarezii,
Eucheuma spinosum and Eucheuma striatum) from Sabah, Malaysia
Mohd Subakir, F.N., Wan Ishak, W.M.F., Mohd Azman, N.A., and Ibrahim, A.I.

70. IFRC 2017: 118-077 305

Quality Attributes of Malaysian Coconut Water (MATAG and MAWA)
Halim, H.H., Williams-Dee, E., Pak Dek, M.S., Hamid, A., Ahmad, N. and Jaafar, A.H.

71. IFRC 2017: 119-235 309

Proximate Composition and Vitamins of Mangifera odarata from Fruit pulp and Peel
Nur Diyana Alyas, Muhammad Anas Othaman, Hazniza Adnan, Sukirah Abdul Rahman, and Nur
Yuhasliza Abd Rashid and Norhazniza Aziz

72. IFRC 2017: 121-079 313

Response Surface Optimization on the Total Phenolic Content and Antioxidant Activities of Sabah
Snake Grass (Clinacanthus nutans) Leaves Peleg Kinetic Modelling Extract
Fazil, F.N.M., Azzimi, N.S.M. and Zubairi, S.I.

73. IFRC 2017: 123-083 317

Insect Powder: A New Protein Source
Valenzuela, K.M. and Duque, S.M.

74. IFRC 2017: 124-166 321

Extracted Water Soluble Polysaccharide from Gum Arabic as Potential Prebiotic
Ahallil Hammad., Aminah Abdullah., Shahrul R. Sarbini and Mohamad Yusof Maskat

75. IFRC 2017: 129-212 325

Effect of Taste Genetic Determinants on Oral Fatty Taste Sensitivity and Perception among Obese and
Non-Obese Subjects
Ahmad Riduan Bahauddin, Roselina Karim, Nazamid Shaari and Zalilah Mohd Shariff

76. IFRC 2017: 133-091 329

Antioxidant Activity of Tea Formulation of Leaves of Leucaena Leucocephala (Lam) de Wit, Soursop
(Annona muricata L.) and Bay Leaf (Syzygium polyanthum) With Black Tea
Rosida, D.F., Putri, C.A., Murtiningsih

77. IFRC 2017: 135-094 333

Sabah Snake Grass (SSG) Pearls for Food Application
Kong,H. S., Mohd-Kasim, Z. and Abdullah Sani, N.

78. IFRC 2017: 137-122 337

Development of Convenient Fruit Bars as Sources of Dietary Fiber and Potassium from Thai Fruits
Racha Saiprasongsin, Visith Chavasit and Aurawan Kettawan

79. IFRC 2017: 160-117 341

Profile of Antioxidant in Dark Chocolate Product that Enriched with Herbs
Suprayatmi,M., Hutami, R., Tiastadia, I.P., Purnamasari, D

80. IFRC 2017: 162-119 345

Effect of Thermal Treatment on Total Phenolic Content and Antioxidant Activity of Garcinia atroviridis
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 and Fenugreek Seed
Ummi Kalthum Ibrahim, Umirah Rashidah Dalip, Suzihaque, M.U.H., Syafiza Abd Hashib and Siti
Fatma Abd Karim

81. IFRC 2017: 176-140 349

Inhibitory effects of mungbean soup on the enzymes and regulator related to type 2 diabetes
Saeting, O. and Sae-tan, S.

82. IFRC 2017: 188-157 353

Ovarian Histomorphological Changes in Rats Supplemented with Edible Bird’s Nest
Albishtue, A. A., Yimer, N., Zakaria, M.A.,Haron ,A.W.,Rosnina, Y

83. IFRC 2017: 189-158 355

Effects of pH and Storage Temperature on the Stability of Encapsulated Anthocyanins from Red
Dragon Fruit (Hylocereus polyrhizus (Weber) Britton & Rose)
Zaidel, D.N.A., Makhtar, N.A., Mohammad, N.A., Mohd Jusoh, Y.M. and Muhamad, I.I.

84. IFRC 2017: 191-162 359

Total Phenolics, Flavonoids and Antioxidant Activity of Sudanese Baobab (Adansonia
digitata) Fruit Pulp
Idris, Y.M. A., Ibraheem, S. A., Mustafa, S. E.and Kabeir, B. M

85. IFRC 2017: 223-210 363

Comparison of Sensory Quality and Preference between Fermented Barleys, Glutinous Rice and the
Combination of Barley and Glutinous Rice
Ibrahim, N., Ezani, N. A. B., Ahmad Kamal, N., Mazlan, N., Abu Kassim, N. A., Jipiu, L. B., Abdul Aziz,
S. A. and Mat Issa, Z.

86. IFRC 2017: 231-216 367

Glycemic Index of Chocolate Fortified with Pumpkin (Cucurbita moshata) and Taro (Colocasia
esculenta) Powder and its Effect on Mood and Cognitive Functions of Female students
Shahidan, N., Salleh, N.Z., Rois Anwar, N.Z., Zakaria, Z.

87. IFRC 2017: 240-220 371

Chemical Composition of Mesocarp and Exocarp from Borassus flabellifer
Rodiah M. H., Jamilah B., Russly A. R., and Sharifah Kharidah S. M.
88. IFRC 2017: 244-224 375
Physico-chemical Properties of Spray Dried Powders from Two Varieties of Amaranth (Amaranthus
viridis)
Siti Faridah, M.A., Tan, L.Y. and Muhammad, K.
89. IFRC 2017: 246-228 379
Investigation of Nutritional and Bio-active Properties of Selected Sri Lankan Marine Macro-algae
Warnasooriya, S.G.V.B., Jayawardana, B.C., Liyanage, N.L.B.R. and Nirooparaj, B.
90. IFRC 2017: 253-240 383
Comparative Evaluation of Total Phenolics, Total Flavonoids and Antioxidant Capacity of Dried Shrimp
and Fermented Shrimp Products
Kaida, S.T., Rahmat, A. and Ramli, N.S.
91. IFRC 2017: 281-257 387
Effect of Germination Treatment in Amino Acids and Proteins Content of Jackfruit Seeds
I.Zuwariah, H. Hadijah, I.Aida Hamimi and R. Rodhiah
92. IFRC 2017: 284-261 391
Hypocholesterolemic Effect Of Dietary Fibre Powder From Pink Guava By-Product
Ibrahim A. H,. Hassan H., Ismail A., Samad A.N., Nordin N.
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Halal Food
93. IFRC 2017: 140-097 395
Perception of Food Sellers towards Halal Labelled Fish Ball in Kelantan
Zul Ariff Abdul Latiff, Mohamad Izwani Halim and Mohamad Amizi Ayob

94. IFRC 2017: 153-110 399

Halal Malaysia Brand Equity Mishap: False Recognition of Brand Mere Recognition using Implicit
Association Test.
Wan Rusni Wan Ismail, Mohhidin Othman, Russly Abdul Rahman, Nitty Hirawaty Kamarulzaman and
Suhaimi Ab. Rahman

95. IFRC 2017: 210-195 403

Do SMEs Halal Food Products Measure Up to Customer Expectation? : An Empirical Investigation
Abdul Salam, S. S., Othman, M., Ungku Zainal Abidin, U. F. and Kamarulzaman, N. A.

96. IFRC 2017: 224-217 407

Revisiting the Theory of Planned Behaviour (TPB) In Halal Food Purchasing: After the Case of Cadbury
Mohd Helmi Ali, Azman Ismail, Syed Shah Alam and Zafir Mohd Makhbul

97. IFRC 2017: 229-232 411

Muslim Consumers’ Awareness and Perception of Halal Food Fraud
Ruslan, A.A.A., Kamarulzaman, N.H and Sanny, M.

98. IFRC 2017: 230-227 416

Muslim Consumer’s Awareness and Acceptance on Halal Genetically Modified Food Labelling
Md Rapi, N.R., Kamarulzaman, N.H. and Ismail, N.W.

99. IFRC 2017: 258-244 420

Halal Assurance System (HAS) Cost Analysis Using Descriptive Quantitative Methods and Prevention,
Appraisal, Failure (PAF) (Case Study at the Chicken Slaughterhouse Mitra Karya Unggas Batu East
Java Indonesia)
Sucipto Sucipto, Riska A. Novita, Danang T. Setiyawan, Mas’ud Effendi and Retno Astuti

100. IFRC 2017: 273-252 424

Comparative Study Of Acid And Alkaline Pre-Treatment Process Prior To Gelatin Extraction From Rohu
(Labeo Rohita) Scales
Khairulnizam, A.B., Jamilah, B., Nur Hanani, Z.A., Russly, A.R. and Kharidah, M.

101. IFRC 2017: 205-192 428

Halal practices integrity and halal supply chain trust in Malaysian halal food supply chain
Kamisah, S., Mokhtar, A., and Hafsah, A.

Food Bioprosessing

102. IFRC 2017: 034-031 432

Survival, physicochemical properties and digestive stability of microencapsulated Lactobacillus spp.
strains 21C2-10 in probiotic ice cream.
Sengsaengthong, S., Oonsivilai, R.

103. IFRC 2017: 045-039 436

Effects of different processing methods on hydroxycitric acid content of “batuan” [Garcinia binucao
(Blanco) Choisy] fruits
Bainto, L.C., Dizon, E.I., Israel, K.A.C. and Laurena, A.C.

xi
104. IFRC 2017: 125-085 440
Physical properties of heat treated purple potato (Solanum tuberosum cv. Shadow-Queen) flour
Santiago, D.M.O., Yamauchi H., Koaze H.

105. IFRC 2017: 104-093 444

Comparative study on the phytochemical and antioxidant properties of fermented jackfruit leaves
(Artocarpus heterophyllus L.) leaves using single and mixed starter cultures
Norhazniza, A., Koh, S.P., Rosmawati A.,Nur Syazwani, A.H.,and Razali, M.
106. IFRC 2017:107-167 448
Changes in Phenolic Content and Antioxidant Activity of Rice Bran by Aspergillus oryzae as Influenced
by Different Initial Moisture Content
Jamaluddin, A., Abd. Rashid, N., Abd. Razak, D.L., Abd. Ghani, A., Mansor, A., Abdul Manan, M., Md.
Saad, A.Z., Sani, N. and Jonit, M.J.
107. IFRC 2017: 221-206 452
Selection of Acetobacter Species Isolated from Fermented Cocoa Beans in Dong Nai Province for Their
Potential Use as Starter Cultures
Vu T.L.A., Nguyen M.H., Phan T.H.
108. IFRC 2017: 114-215 456
Fermentation Characteristic of Kuini (Mangifera odorata) and Its Potential as Substrate to Acetic Acid
Bacteria
Adnan, H., Othaman, M.A. and Alyas, N.D.

109. IFRC 2017: 271-250 460

Development of GABA Malted Milk Drink from Germinated Brown Rice
I.Zuwariah, I.Aida Hamimi, R. Rodhiah, and H. Hassan
Food Service and Management
110. IFRC 2017: 157-115 464
Pilot interviews of job satisfaction with offshore catering employee
Majid, M. A. A., Othman, M., Mohamad, S. F., and Lim, S. A. H.
111. IFRC 2017: 222-207 468
Identifying Possible Factors of Job Stress and Employees Intention to Leave a Job: A Case of Casual
Typed Restaurant Employees in Johor Bahru.
Majid A., N., Ghazali, H. and Farahwahida, A.
112. IFRC 2017: 222-242 472
Initial Findings of Possible Factors Contribute to Job Stress among Casual Dining Restaurant
Employees in Klang Valley, Malaysia.
Farahwahida, A. and Ghazali, H.
113. IFRC 2017: 256-264 475
Internship satisfaction factors and instruments: A review and research directions for the undergraduate
hospitality programs
Ruslan, S., Mohamad, S.F., and Othman, M.
114. IFRC 2017: 276-251 479
A qualitative study on factors influencing older consumer dining out behaviour
Ganesan, L., Abu Bakar, A.Z., and Othman, M.

xii
Others
115. IFRC 2017: 275-253 483
Hemicellulose Extraction and Characterization of Oil Palm Empty Fruit Bunches
Nor Nadiha, M.Z., Russly, A.B. and Jamilah, B.
116. IFRC 2017: 112-071 (Halal Food)
Effect of Heating time and temperature to the dielectric properties of pork and chicken in the range of
0.5 – 50 GHz
Zurina Zainal Abidin, Fatin Nordalila Omar, Dayang Radiah Awang Biak

Sponsors
Organizing committee

xiii
Foreword by the Vice Chancellor, Universiti Putra
Malaysia
Assalamualaikum W.B.T. and greetings.

On behalf of Universiti Putra Malaysia, it gives me great pleasure to

welcome all of you to the International Food Research Conference
(IFRC 2017).

To mankind, food is the primary source of energy and therefore, life. Since time immemorial,
mankind has exemplified ardent interest and creativity towards food creation and
consumption which sprang solely from basic instinct and natural curiosity. This curiosity
consequently gave birth to what the modernist would now call food research.

In Malaysia at present, the Government has made UPM as the centre of excellence for
agricultural education and research to develop skilled manpower for the related industries.
The dynamic food sector is thus considered the heartbeat of these industries. Therefore, to
be able to face the emerging challenges in the food sector, the Faculty of Food Science and
Technology (FSTM) is tasked to spearhead the development of human capital as well as the
direction in food research and innovation. Among the approaches taken by FSTM is through
the organisation of research conferences such as IFRC 2017. By aiming to disseminate the
latest advancement and information in food researches, and also to elevate the industry-
university collaboration to new heights, IFRC 2017 will certainly serve as the best platform
by which the scientific and academic communities as well as the stakeholders could benefit
from.

Congratulations to FSTM for their continuous effort in championing food-related disciplines

both locally and internationally as reflected by the organisation of IFRC 2017. The great leap
in ranking from 270th (2015) to 229th (2016) as published by the Quacquarelli Symonds’s
(QS) World University Ranking is indeed the just outcome of the collective efforts taken by
all faculties and entities in UPM.

To all IFRC 2017 participants, I would like to urge you to actively participate and engage in
the three-day conference by contributing your ideas and insight. I sincerely hope that you will
also have an enjoyable stay in Kuala Lumpur and get to experience the warmth of Malaysian
hospitality.

‘WITH KNOWLEDGE WE SERVE’

Agriculture • Innovation • Life

PROF. DATIN PADUKA DR. AINI IDERIS, FASc

Vice Chancellor
Universiti Putra Malaysia

xiv
Foreword by the Dean, Faculty of Food Science and Technology
Assalamualaikum W.B.T and very good day.

On behalf of the Faculty of Food Science and Technology, I am truly

delighted to welcome all of you to Universiti Putra Malaysia for the
International Food Research Conference (IFRC 2017).

Food research is an important and well-established avenue to

spearhead the food science and technology niche area. The food
we consume on a daily basis is the result of extensive food research
which is a systematic investigation into a variety of foods’ properties
and compositions. The investigation starts from the food
components (macro– and microcomponents of foods, food biochemistry, nutrient changes in
foods), to the preparation and technology involved (food processing, food engineering, food
packaging, culinary), to the end products (sensory analyses, food safety and quality,
functional food, new food development, food service) and ultimately to the dining table for
our consumption. As the consumers’ knowledge, perception and preference expanded, so
too has the food niche which now also includes food marketing, heritage food, halal food and
so on. The organisation of the IFRC 2017 is hoped to achieve yet another milestone in
bringing food researches to greater heights.

The theme appropriately selected for the IFRC 2017 is “Emerging Challenges in Food
Research”. To that end, we are very fortunate to have several of the world’s leading
researchers as our keynote and plenary speakers. I am positive that the IFRC 2017 will offer
you a sound basis for academic discussions and the ensuing exchange of ideas. I therefore
look forward to facilitating the invaluable dialogue among academics, researchers and
market professionals in the spirit that such debate will only pave way to new and exciting
approaches and technologies for the food industry.

I would also like to acknowledge the contribution of our co-organisers, supporters, and
sponsors. Congratulations are also conveyed to the organising committee without whom the
conference would have not been possible.

Lastly, I wish you all a fruitful and wonderful time at the IFRC 2017.

Best wishes,

PROF. DR. NAZAMID SAARI

Dean
Faculty of Food Science and Technology

xv
Foreword by the IFRC 2017 Chairman
Assalamualaikum W.B.T. and good day.

After months of careful planning and preparation, finally the

International Food Research Conference (IFRC 2017) is upon us.
With nearly 300 participants from around 20 countries, the IFRC
2017 is now on full swing. Having been tailored to cater to wider
audience from the food sector, the IFRC 2017, which encompasses
the major food areas such as food processing and post-harvest
technology, food bioprocessing, food safety and quality, functional
food, food service and management, heritage food and halal food, is
aimed to provide a platform on which we could face and respond to the emerging challenges
in food research.
To our co-organiser, the Institute of Food Science and Technology, College of Agriculture
and Food Science, University of the Philippines Los Baños; and our supporters, the
Malaysian Institute of Food Technology (MIFT) and the Associated Chinese Chambers of
Commerce and Industry of Malaysia (ACCCIM), we extend our sincere gratitude and
appreciation for the collaboration that makes the organisation of the IFRC 2017 possible.
Credit also goes to all the invited speakers; Prof. Dr. Da-Wen Sun (University College
Dublin, Ireland), Prof. Dr. Hiroshi Inomata (Tohoku University, Japan), and Prof. Dr. Farooq
Anwar (University of Sargodha, Pakistan), for graciously sharing their vast knowledge and
wisdom. Their years of experience in their respective fields are certainly an asset that will
enrich the IFRC 2017.
To fellow academics, researchers, entrepreneurs, industry practitioners, and policy makers
who participate in the IFRC 2017, I am entirely certain that you will all benefit from the
arranged keynote and plenary speeches as well as the oral and poster presentations.
To the organising committee, you have my endless admiration for your months of effort and
energy poured into materialising the IFRC 2017. A job well done!

To our guests from abroad, have a pleasant stay in Malaysia!

Warm regards,
PROF. DR. RUSSLY ABDUL RAHMAN
Chairman
International Food Research Conference 2017

xvi
 IFRC 2017
Proceedings
 Plenary 2 Food Processing and Post-Harvest Technology

Supercritical Fluid Technology for Food Processing

Masaki Ota and *Hiroshi Inomata

Tohoku University
Research Center of Supercritical Fluid Technology
Sendai, Japan

Abstract

The lecture covers fundamental principles of supercritical carbon dioxide for use in food research and processing.
Specific topics include flavonoid solubility and extraction and the separation of hops with staged-processes. Chemical
engineering approach includes consideration of partition coefficients (K-values) and the use of unit operations for
obtaining the desired separations. There are many opportunities for using supercritical carbon dioxide to isolate new
compounds and to develop new products.

Corresponding author’s email: inomata@scf.che.tohoku.ac.jp

Introduction-Features of using supercritical CO2

CO2 is thermodynamically stable and very low toxicity/environmental impact. Its supercritical state, sc-CO2, has
become an important commercial and industrial solvent especially for food fields.

One merit of supercritical carbon dioxide (scCO2) is that the solvent power can be tuned by changing the extraction
temperature or pressure allowing control of compound isolation selectivity. Another merit of scCO 2 is that separation
into solid or liquid states is simple through the use of depressurization. Relatively low temperature operation The
relatively low temperature of the process and the stability of CO2 also allows most compounds to be extracted with
little damage or denaturing.

For designing the sc-CO2 extraction/separation process, phase equilibria, solubility and distribution coefficient of
target compounds under sc-CO2 conditions.

Phase equilibrium, solubility and distribution coefficient(K-values)

Most of functional food compounds are high melting and exist as solid phase at ambient temperature. In this case,
phase equilibrium information for target compound+CO2 is solid-fluid equilibrium, meaning solubility of the compound
into sc-CO2. As mentioned later, vapor-liquid equilibria are necessary for the rectification from induced phase
separation by temperature gradient.

A typical experimental setup in our group is shown in Fig.1 which is a flow type apparatus designed originally with
back pressure control values.

For solubility measurements, semi-batch apparatus is widely used with various composition determining methods.

1
 flavanone tangeritin
Ethanol aqueous solution CO2(1)+Ethanol(2)+Water(3)
Back pressure
V3
P regulator Dry gas
Entrainer Pump flow meter
Solutes
Chiller Preheat
coil
Mixer
T Trap Graduated
Filter Pump V1 cylinder nobiletin 6-hydroxy
V2
Separation Reservoir
flavanone
cell
CO2 Safety Oven
valve

CO2
313.2 K (□)

7-hydroxy 323.2 K(▲)

Vapor-Liquid equilibria flavanone
333.2 K (○)

Fig.1 Phase equilibrium measuring apparatus Fig. 2. Pressure dependence of solubility of Flavonoids in
supercritical CO2.

Isothermal solubility data measured in our group are plotted against the pressure in Fig.2 It can be seen that
temperature dependence for each flavonoid was not so large, while the solubility increased with increasing pressures
at isothermal conditions. It would be noted that the logarithm of the solubility of each flavonoid increased linearly with
CO2 density calculated from the temperature and pressure conditions.

Distribution coefficients can be measured by using VLE apparatus such as Fig.1 by adding a target compound into
liquid feed (as ethanol solution) and additional composition analyses are required to determine the target compound
compositions in both liquid and vapor phases. In presentation, we will show our K-value data of hop extract
components.
EXT_Cell Consist.Parts
Int Volume 85 cm3 ：Heating part
・Feeding ・Extraction
Development of supercritical fluid extraction Tower
I.D. 9.0 mm ・Tower ・Liq. Receiver
and rectification processes Height 45 mm
Int Volume 28 cm3
P BPR Flow meter ・sampling
Dixon Packing T5 T Temp-Press Control
Supercritical fluid extraction with a (D1.5 mm)
Rectification ・Max T 373 K
rectification process was developed towards
Liq. Receiver Trap
Int.Volume 23 cm3 Tower T4 Independent Control
separation of natural components. Trial Chiller V4
Temp Profile Designable
experiments for separation of 7- Pre-heater T3

hydroxyflavone from anthracene were carried CO2 Pump

V5 ・Press
T1 Max-P30 MPa
out by using a developed apparatus for V1 T2
BPR control
Liq. Receiver
verification of methodology. When Pump V2 Ext Cell V6
supercritical CO2 + ethanol extraction and P V3
T
rectifier were respectively controlled at 333 K Entrainer
Fig.3 Supercritical Extraction/Rectification System Trap
and 353 K with a feed ethanol concentration

2
of 12 mol% and a CO2 flow rate of 1.4 L/min, an only anthracene was recovered from the top of the rectifier. The
optimization of rectification condition should be achieved by selecting low feed ethanol concentration condition with
taking into consideration of phase separation of CO2-ethanol systems.

i) Nobiletin separation from citrus peels

The rectification process was applied to the separating nobiletin from citrus peels. With CO 2-ethanol extraction-
rectification systems, nobiletin with carotenoids were recovered from top of the rectification tower, while chlorophylls
were recovered from the bottom of the tower. With CO2-ethanol-water systems, nobiletin was recovered from top of
the rectification tower, while carotenoids and chlorophylls were recovered from bottom and separated from each
other. Rectification conditions for separation of nobiletin from extracts were optimized by taking into consideration
the phase separation of CO2-ethanol-water systems.

0.025 0.8

[g_comp./g_feed comp.]
Top
[g_comp./g_feed comp.]

Bottom

Total bottom recovery

0.7
0.02 10 MPa 10 MPa
Total top recovery

0.6
313 - 333 K 313 - 333 K
0.015 0.5
0.4
0.01 0.3
0.2
0.005
0.1
0 0
0 0.0005 0.001 0.0015 0 0.2 0.4 0.6
Total top yield [g/g_feed] Total bottom yield [g/g_feed]
Fig.4 Fraction behavior for CO2+ethanol rectification

ii) Hop extracts separation

Among the components in hop extracts, 8 compounds were selected to follow the fractionation behavior by the
supercritical rectification process.
The table shows the 8 compounds where no.1 – no.4 are flavors and no.5-no.8 are bitters. It is well known that flavors
are more volatile than bitters whose melting temperatures are relatively high.
The results clearly showed that flavors effluent only from
top and no bitter compounds were not found in the CO2 ① 1,5-heptadiene, (fHep)
stream from top. While bitter compounds were involved in
the bottom stream together with small amount of flavors. ② β-myrcene (fMyr)
flavor

However, it can be pointed out that we should improve the ③ caryophyllene (fCar)
operation condition for increasing the yield of top effluent.
The modelling of rectification process based on the K- ④ humulene (fHum)
values will be powerful tool for discussing the optimization
of operation conditions in terms of extraction/rectification ⑤ cohumulone (bCoh)
purposes.
⑥ humulone,adhumulone (bHum)
bitter

⑦ colupulone (bCol)

⑧ lupulone, adlupulone (bLup)

3
Summary
Supercritical CO2 extraction and/or fractionation are promising technology for food treatment processes because its
various merits in this field. For the practical process developments, we have to know its fundamental features, phase
equilibria, solubility, etc. for mixtures together with the features of solvents. The balanced research activities on
fundamental and engineering approaches are required for proper evaluation of obtained results in terms of
economical, environmental and scientific viewpoints.

4
 IFRC 2017: 035-049 Food Safety and Quality

Toxigenic Campylobacter jejuni in Vegetables Farms and Retail Outlets in Terengganu

1*Tang, J.Y.H., 1Khalid, M.I. and 2Radu, S.

1Faculty of Agriculture, Biotechnology and Food Sciences, Universiti Sultan Zainal Abidin, 22200 Besut, Terengganu,
Malaysia
2Food Safety Research Centre, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor, Malaysia

This study was conducted to determine the prevalence and antibiotic resistance among toxigenic Campylobacter
jejuni in ulam at farms and retail outlets located in Kuala Terengganu, Malaysia. A total of 526 samples (ulam, soil,
and fertilizer) were tested for the presence of C. jejuni and the gene for cytolethal distending toxin (cdt) by using a
multiplex PCR method. Antibiotic susceptibility to 10 types of antibiotics was determined using the disc diffusion
method for 33 C. jejuni isolates. The average prevalence of contaminated samples from farms, wet markets, and
supermarkets was 35.29, 52.66, and 69.88%, respectively. The cdt gene was not detected in 24 of the 33 C. jejuni
isolates, but 9 isolates harbored cdtC. Antibiotic resistance in C. jejuni isolates was highest to penicillin G (96.97% of
isolates) followed by vancomycin (87.88%), ampicillin (75.76%), erythromycin (60.61%), tetracycline (9.09%),
amikacin (6.06%), and norfloxacin (3.03%); none of the isolates were resistant to ciprofloxacin, enrofloxacin, and
gentamicin. In this study, C. jejuni was present in ulam, and some isolates were highly resistant to some antibiotics
except quinolones. Thus, appropriate attention and measures are required to prevent C. jejuni contamination on
farms and at retail outlets.

Keywords: Toxigenic Campylobacter jejuni, vegetables farms, retail outlets, antibiotics resistance
*Corresponding author’s email: Dr. John Yew Huat Tang, jyhtang@unisza.edu.my

Introduction
Campylobacter has been recognized as a major cause of human bacterial gastroenteritis that cause millions of cases
worldwide every year (Friedman, et al., 2000). Campylobacter jejuni is Gram negative, spiral shape, motile, non-spore
forming and grow in microaerophilic environment. Cytolethal distending toxin (CDT) has been identified as one of the
potential virulence factors in C. jejuni (Findik et al., 2011) which produced by the cdt gene, which comprises cdtA,
cdtB, and cdtC (Bae et al., 2005). CDT targets epithelial cell layer which cause progressive distension and death of
the epithelial cell (Pickett et al., 1996). Campylobacter strains with cdt genes were found in humans, animals and
fresh produce (Rozynek et al., 2005). Low infectious dose at 500 to 800 cells of highly virulent campylobacter strains
are capable of causing foodborne illness in humans (Black et al., 1988).

The objective of this study was to determine the presence of toxigenic C. jejuni in vegetables in small scale farms and
retail outlets found in Terengganu. Antibiotic resistance profile among C. jejuni isolates from vegetables were also
determined.

Materials And Methods

Sampling.
Samples were taken from five farms, two wet markets, and two supermarkets in Kuala Terengganu were selected as
sampling locations. A total of 526 samples were collected from farms and retail outlets in Terengganu which include
vegetables (386 samples), soils (80 samples), and fertilizers (60 samples).

Detection of C. jejuni.
Samples were enriched with Bolton selective enrichment broth (BB) without blood as described by Williams et al.
(2009). DNA was extracted from the enriched samples with the boiled-cell method with minor modifications as
5
described by Tang et al. (2010). PCR reactions were performed on a Veriti 96-well FastThermal Cycler (Applied
Biosystems, Foster City, CA). PCR products were visualised under UV transilluminator (AlphaImager HP, CA).

Isolation of C. jejuni.
C. jejuni was isolated as described by Tang et al. (2010) using modified charcoal cefoperazone deoxycholate agar
(mCCDA; CM0739B, Oxoid) supplemented with CCDA selective supplement (SR0155, Oxoid). Confirmed C. jejuni
isolates were stored in glycerol at -20ºC.

Antimicrobial susceptibility testing.

Thirty three of C. jejuni isolates were recovered from raw salad vegetables and soil samples from farms and retail
outlets in Terengganu: 9 isolates from supermarkets, 9 isolates from farms, and 15 isolates from wet markets.
Antibiotic resistance profile were determined with the disk diffusion method according to the guidelines of the Clinical
and Laboratory Standard Institute (CLSI).

Results and Discussion

A total of 526 samples were examined for theprevalence of Campylobacter spp. and the presence of cdtgenes using
a multiplex PCR method. Table 1 summarized the prevalence of C. jejuni in the samples.

Most C. jejuni isolates (95.70%) wereresistant to penicillin G, followed by vancomycin (82.6% of isolates), ampicillin
(65.20%), and erythromycin (43.50%). All C. jejuni isolates were susceptible to ciprofloxacin,enrofloxacin, and
gentamicin. C. jejuni resistance to otherantibiotics used in this study was low: 4.30% for norfloxacin and 8.70% for
both of amikacin and tetracycline. Antimicrobial agents are not likely to be used in vegetable farming, which might
explain the low resistance to quinolones found in the present study.

Conclusion
C. jejuni and cdt genes detected in this study indicates that salad vegetables were contaminated and pose significant
health risks to consumers.

Acknowledgement
The authors would like to thank for the grant provider International Foundation of Sciences, Sweden (E/5237-2F) and
Universiti Sultan Zainal Abidin.

6
Table 1: Number and percent fraction of samples and isolates from supermarkets and wet markets that were positive
for C. jejuni and cdt.
PCR
Location Vegetables N
C. jejuni cdtA cdtB cdtC
n (%) n (%) n (%) n (%)
Supermarket I A 10 5(50.00) ND ND 6(60.00)
B 10 7(70.00) ND ND 1(10.00)
C 10 6(60.00) ND ND 1(10.00)
D 3 ND ND ND ND
F 10 9(90.00) ND ND 2(20.00)
G 10 8(80.00) ND ND 8(80.00)
Total 53 35(66.04) ND ND 18(33.96)

Supermarket II A 10 8(80.00) ND ND ND
B 10 6(60.00) ND ND 7(70.00)
D 10 9(90.00) ND ND ND
Total 30 23(76.67) ND ND 7(23.33)

Wet Market I A 24 8(33.33) ND 1(4.17) 6(25.00)

B 20 12(60.00) 1(5.00) 1(5.00) 10(50.00)
D 20 1(5.00) ND ND 1(5.00)
F 20 11(55.00) ND ND ND
H 20 17(85.00) ND ND 17(85.00)
J 24 14(58.33) 3(12.5) 1(4.17) 11(55.00)
Total 128 63(49.20) 4(3.13) 3(2.34) 45(35.16)

Wet Market II A 2 ND ND ND ND
B 10 10(100.00) ND ND 7(70.00)
C 10 5(50.00) ND ND ND
D 12 7(58.33) ND ND 10(83.30)
E 3 ND ND ND ND
H 10 8(80.00) ND ND 9(90.00)
J 13 6(46.15) ND 1(7.69) 1(7.69)
Total 60 36(60.00) ND 1(1.67) 27(45.00)

Farm I Aa 15 1(6.67) ND ND ND
Ba 10 5(50.00) ND ND 6(60.00)
K 20 ND ND ND ND
L 20 4(20.00) ND ND 10(50.00)
Total 65 10(15.38) ND ND 16(24.62)

Farm II Ha 10 7(70.00) ND ND ND
K 10 ND ND ND ND
L 20 3(15.00) ND ND ND
Total 40 10(25.00) ND ND ND

Farm III Ha 10 3(30.00) ND ND ND

Ja 10 ND ND ND ND
K 10 1(10.00) ND ND ND
L 20 14(70.00) ND ND 1(5.00)
Total 50 18(36.00) ND ND 1(2.00)

Farm IV Ea 10 5(50.00) ND ND 1(10.00)

Ha 10 9(90.00) ND ND 3(30.00)
Ia 10 7(70.00) ND ND ND
K 10 4(40.00) ND ND ND
L 10 5(50.00) ND ND ND
Total 50 30(60.00) ND ND 4(8.00)

Farm V Da 10 2(20.00) ND ND 3(30.00

Ea 10 7(70.00) ND ND 4(40.00)
Ha 10 6(60.00) ND ND ND
K 10 4(40.00) ND ND ND
L 10 3(30.00) ND ND ND
Total 50 22(44.00) ND ND 7(14.00)
ND- Not detected. n-number of samples
A, winged bean; B, long yard bean; C, indian pennywort; D, water spinach; E, cucumber; F, cabbage; G, mung bean sprout; H, wild cosmos; I,
vietnamese coriander; J, japanese parsley; K, fertilizer; L, soil.

7
References

Black, R. E., Levine, M. M., Clements, M. L., Hughes, T. P., and Blaser, M. J. 1988. Experimental Campylobacter
jejuni infection in humans. The Journal of Infectious Diseases 157:472–479.
Findik, A., Ica, T., Onuk, E. E., Percin, D., Kevenk, T. O., and Ciftci, A. 2011. Molecular typing and cdt genes
prevalence of Campylobacter jejuni isolates from various sources. Tropical Animal Health and Production
43:711–719.
Friedman, C. R., Neimann, J., Wegener, H. C., and Tauxe, R. V. 2000. Epidemiology of Campylobacter jejuni
infection in the United States and other industrialized nations, p. 121–138. In I. Nachamkin and M. J. Blaser
(ed.), Campylobacter, 2nd ed. ASM Press, Washington, DC.
Pickett, C., Pesci, E. C., Cottle, D. L., Russell, G., Erdem, A. N., and Zeytin, H. 1996. Prevalence of cytolethal
distending toxin production in Campylobacter jejuni and relatedness of Campylobacter spp. cdtB genes.
Infection and Immunity 64:2070–2078.
Rozynek, E., Dzierzanowska-Fangrat, K., Jozwiak, P., Popowski, J., Korsak, D., and Dzierzanowska, D. 2005.
Prevalence of potential virulence markers in Polish Campylobacter jejuni and Campylobacter coli isolates
obtained from hospitalized children and from chicken carcasses. Journal of Medical Microbiology 54:615–619.
Tang, J. Y. H., Saleha, A. A., Jalila, A., Farinazleen, M. G., Tuan Zainazor, T. C., Noorlis, A., Afriani, S., Nishibuchi,
M., and Radu, S. 2010. Thermophilic Campylobacter spp. occurrence on chickens at farm, slaughter house and
retail. International Journal of Poultry Science 9:134–138.
Williams, L. K., Jørgensen, F., Grogono-Thomas, R., and Humphrey, T. J.. 2009. Enrichment culture for the isolation
of Campylobacter spp: effects of incubation conditions and the inclusion of blood in selective broths.
International Journal of Food Microbiology 130:131–134.

8
 IFRC 2017: 037-024 Food Safety and Quality

Molecular typing of Bacillus cereus isolated from sago processing mills in Sarawak.

Jaraee J.1, *Bilung M. L.1, Nolasco C. H.2 & Vincent M.1

1Molecular Microbiology Laboratory, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak,
94300 Kota Samarahan, Sarawak
2Department of Chemical Engineering and Energy Sustainability, Faculty of Engineering, Universiti Malaysia
Sarawak, 94300 Kota Samarahan, Sarawak

Abstract

Bacillus cereus is an ubiquitous bacteria which is commonly found in soil and plant-based food. In this study, thirty-
nine Bacillus cereus isolates were detected with the presence of hly gene by using specific PCR. These isolates were
further characterized and differentiated by using the Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-
PCR) to determine their genomic fingerprints. The ERIC-PCR generated several genetic profiles consisting of 1 to 7
bands with sizes in the range of 120 bp to 990 bp. Based on the dendrogram generated from the DNA fingerprinting
profiles (ERIC-PCR), all of the isolates can be divided into 2 main clusters that is further divided into few sub-clusters.
The heterogeneity of the isolates indicated the cross-contamination of Bacillus cereus occurred in sago processing
mills in Sarawak.

Keywords : Bacillus cereus, sago processing, ERIC-PCR, genetic diversity, cross-contamination

Corresponding author’s email: mblesley@unimas.my

Introduction

Sago processing industry is a highly potential industry in Sarawak whereby the export of sago product from Sarawak
procuring income approximately RM 81 million per year (Department of Agriculture, 2013). However in food
processing industries, some of the preparation, processing and storage procedures were exposed to the risk of
contamination of bacterial contaminants. Bacillus cereus is one of the known causes of foodborne illness. Bacillus
cereus is a Gram-positive and rod-shaped foodborne pathogen that can cause gastrointestinal diseases such as food
poisoning (Sandra et al., 2012; Kotiranta et al., 2000). Bacillus cereus is an ubiquitous bacterium commonly found in
soil and is able to live in it because it does not have complex nutrient requirements (Kotiranta et al., 2000).
Pasteurized food such as dairy foods of cooked chill foods, starchy food such as rice and starch, and dry foods such
as spices, ready-to-eat cereals are among of food products under risk of Bacillus cereus contamination (Lesley et al.,
2013; Sandra et al., 2012; Greenhill, 2010; Kontiranta, 2000). To the best of our knowledge, this is the first study
providing genotypic epidemiologic data regarding the Bacillus cereus isolated from the processing steps of sago
especially in Sarawak. However, the study on genetic diversity of Bacillus cereus had been reported in other food
processing plant such as fresh-cut vegetable salad processing in Korea (Kim et al., 2016), red pepper powder
processing plant (Oh et al., 2012), dairy plant (Svensson et al., 2006) and chilled zucchini puree processing plant
(Guinebrietre et al., 2003). In the present study, the presence of Bacillus cereus was monitored in sago flour and sago
samples from each stage of sago processing which consist of debarking, pulping, starch extraction, drying, packaging
and discharging point. Molecular typing of Bacillus cereus was also investigated using ERIC-PCR method to identify
genetic diversity of Bacillus cereus in sago processing in Sarawak.

Material and Methods

Sample collection and preparation

9
A total of 120 samples were collected from sago processing mill A and sago processing mill B in January 2016 and
May 2016 respectively. The samples were obtained from each stage of sago processing which consists of debarking
(n=20), pulping (n=20), starch extraction (n=20), drying (n=20), packaging (n=20) and discharging (n=20). Ten gram
or 10 ml of samples collected were enriched with Tryptone Soy Broth in stomacher bag for 12 hours at 37oC.

Enterobacterial Repetitive Intergenic Consensus Polymerase reaction (ERIC-PCR)

Out of 120 samples, a total of 42 Bacillus cereus strain collected from debarking (n=15), pulping (n=9), starch
extraction (n=4), drying (n=2) and discharging (n=12) were detected with hly gene of Bacillus cereus were further
characterized using Enterobacterial Repetitive Intergenic Consensus Polymerase reaction (ERIC-PCR). Prior to
ERIC-PCR, the DNA were extracted using Bacterial Genomic DNA Isolation Kit by Norgen Biotek Corp. ERIC-PCR of
Bacillus cereus was conducted according to Merzougui et al. (2013). The primer pairs used were 5′-
ATGTAAGCTCCTGGGGATTCAC-3′ and 5′-AAGTAAGTGACTGGGGTGAGCG-3.To prepare 25 μl of PCR mixture,
1.0 M of forward primer, 1.0 M of reverse primer, 3.0 l of DNA template, 5.0 μl of 5×Taq PCR buffer, 0.2 mM dNTP,
2.0 mM MgCl2, and 1.0 unit of Taq DNA polymerase were mixed together. The PCR products were then separated on
1% agarose gel with 100 bp DNA ladder for 90 min. After that, the gel was stained with ethidium bromide and viewed
under a UV transilluminator (Maestrogen). The DNA band patterns were analyzed and a dendrogram was generated
for the Bacillus cereus isolates by using BioNumerics 7.6.2 software program (Applied Maths, Sint-Martens-Latem,
Belgium) using Dice coefficient and the unweighted pair group method (UPGMA).

Result and Discussion

The electrophoretic profile of DNA fragments obtained after ERIC-PCR amplification for both sago processing mills
yielded 1 - 7 bands with size approximately 100 bp to 990 bp. A total of 32 DNA fingerprint profiles were produced in
this analysis whereby 12 DNA fingerprint profiles from sago processing A and 26 DNA fingerprint profiles from sago
processing B. The dendrogram shown in Figure 1 were constructed by using Bionumerics 7.6.2 software. All the
isolates were divided in two main clusters and each cluster was divided in several sub-clusters. Isolates in the same
lineage are usually found to be genetically related and/or indicate possible cross-contamination (Shi et al., 2015). In
this study, ERIC-PCR yielded 1 - 7 bands with size approximately 100 bp to 990 bp. From both sago processing mills,
32 DNA fingerprint profile were shown. The findings in the present study showed that the Bacillus cereus isolates
were not group together based on the types of samples and the source of isolation. It is in agreement with a study by
Merzougui et al. (2013), they isolated Bacillus cereus from different food samples such as rice salads, dairy products,
milk and spices and they were not classified into specific cluster neither by sampling area nor the type of samples.
The present study is also comparable to a study conducted by Kim et al. (2014), whereby Bacillus cereus isolated
from fermented soy bean products showed high genetic diversity and the banding pattern of rep-PCR were not
associated with specific product types. In the present study, the Bacillus cereus isolated from each sago processing
mills were genetically diverse and heterogeneous. The heterogeneity was expected as the isolates were collected
from different types samples. It may also due to the ubiquitous nature of Bacillus cereus where there are easily found
in different types of environment (Swiecicka, 2008). Hence, the contamination can occur from different sources such
as soil, water, tree bark and sago processing mills equipment. Some of the cluster showed that they were classified
into specific cluster by stages of samples were collected. It revealed that high prevalence of similar strain of Bacillus
cereus and it had become dominant and better adapted in those specific stages. The findings of present study was
meaningful and should be considered because it suggest that cross-contamination occurred between the processing
steps in sago processing mills. Cross-contamination could lead to serious foodborne illness which is a widespread
health problem that create social and economic burden as well as human suffering. Therefore, strict hygiene and
management practices of sago processing must be adopted to avoid unwanted illness due to consumption of
contaminated sago and sago products.

10
 Figure 1:
Dendrogram
constructed for
Bacillus cereus
isolated from sago
processing mills in
Sarawak.

Conclusion

Contamination of Bacillus cereus in raw materials and samples of processing steps in sago processing in Sarawak
were analyzed and the genetic diversity compared among isolates from different processing steps including raw
materials to describe the transmission of Bacillus cereus along the processing step and how Bacillus cereus cross-
contamination could occur. These results the heterogeneity suggest that further intervention study is needed to
prevent pathogen contamination during sago processing to lower the risk of contamination. Our molecular typing
result based on ERIC-PCR analysis will be able to provide useful information for elucidating the route of
contamination of Bacillus cereus in sago processing. The results were also useful in developing appropriate
intervention strategies in sago processing in Sarawak thus contribute in lowering the disease burden and assist in
providing safer food to the society.

Acknowledgement

This study was supported by UNIMAS Tun Openg Chair Grant F07(ORC)/1223/2015(04).

References
Department of Agriculture Sarawak. 2013. Sarawak Agriculture Statistics 2013. Malaysia. Retrieved January 2, 2017,
from http://www.doa.sarawak.gov.my/modules/web/pages.php?mod=webpage&sub=page&id=712
Greenhill, A. R., Shipton, W. A., Blaney, B. J., Amoa, B., Kopel, E., Pelowa, D. & Warner, J. M. 2010. Hazards and
critical control points for traditional sago starch production in Papua New Guinea: Implications for food safety
education. Food Control, 21(5), 657–662.
Guinebretiere, M., Y. Broussolle, & C. Nguyen-The. 2002. Enterotoxigenic profiles and food-
Kim, H. J., Koo, M., Hwang, D., Choi, J. H., Kim, S. M., & Oh, S. W. 2016. Contamination patterns and molecular
typing of Bacillus cereus in fresh-cut vegetable salad processing. Applied Biological Chemistry, 59(4), 573–
577.
Kim, Y.R. & Carl A. B. 2014. Riboprint and Virulence Gene Patterns for Bacillus cereus and Related Species. Journal
of Microbiology and Biotechnology. 18(6), 1146–1155.

11
Kotiranta, A., Lounatmaa, K., & Haapasalo, M. 2000. Epidemiology and pathogenesis of Bacillus cereus infections.
Microbes and Infection2(2), 189–198.
Lesley, M. B., Velnetti, L., Yousr, A. N., Kasing, A., & Samuel, L. 2013. Presence of Bacillus cereus s.l. From ready-
to-eat cereals (RTE) products in Sarawak. International Food Research Journal, 20(2), 1031–1034.
Merzougui, S., Lkhider, M., Grosset, N., Gautier, M., & Cohen, N. 2013. Differentiation by Molecular Typing of Bacillus
Cereus Isolates from Food in Morocco : PFGE-Eric PCR, 3(4), 223–227.
Oh, S. W., Koo, M., & Kim, H. J. 2012. Contamination patterns and molecular typing of Bacillus cereus in red pepper
powder processing. Journal of the Korean Society for Applied Biological Chemistry, 55(1), 127–131.
poisoning and foodbome Bacillus cereus strains. Journal of Clinical Microbiology. 40(2), 3053-3056.
Sandra, A., Afsah-Hejri, L., Tunung, R., Tuan Zainazor, T. T. C., Tang, J. Y. H., Ghazali, F. M., Son, R. 2012. Bacillus
cereus and Bacillus thuringiensis in ready-to-eat cooked rice in Malaysia. International Food Research Journal,
19(3), 829–836.
Shi, W., Qingping, W., Jumei, Z., Moutong, C. & Zean, Y. 2015. Prevalence, antibiotic resistance and genetic diversity
of Listeria monocytogenes isolated from retail ready-to-eat foods in China. Food Control, 47, 340-347.
Svensson, B., Ekelund, K., Ogura, H., & Christiansson, A. 2004. Characterisation of Bacillus cereus isolated from milk
silo tanks at eight different dairy plants. International Dairy Journal, 14(1), 17–27.
Swiecicka I. 2008. Natural occurrence of Bacillus thuringiensis and Bacillus cereus in eukaryotic organisms: a case
for symbiosis. Biocontrol Science and Technology. in press

12
 IFRC 2017: 071-051Food Safety and Quality

Variation of Microbial and Chemical Quality of Two Major Food Fishes in Sri Lanka with Gamma Irradiation.

1*Surendra, I. H. W., 1Edirisinghe, E.M.R.K.B. and 2Rathnayake, R.M.N.P.

1Department of Physical Sciences, Faculty of Applied Sciences, Rajarata University of Sri Lanka, Mihintale, Sri Lanka.
2Sri Lanka Gamma Center, Atomic Energy Authority of Sri Lanka, Biyagama

Abstract
The present study was carried out to evaluate the effect of different gamma irradiation doses with bacterial load, fatty
acid composition and free fatty acids (FFA) of two major food fishes in Sri Lanka. The fillets of freshwater fish tilapia
and marine water fish yellowfin tuna were gamma irradiated at 3, 5, 7 and 10 kGy doses and results were examined
with non-irradiated samples. After gamma irradiation, total bacterial counts were reduced to 2.39, 2.61, 0.33 and 0.57
log units for tilapia samples irradiated with the dose of 3, 5 7 and 10 kGy respectively with non-irradiated control of
5.27 log 10cfu/g. Tuna also showed reduction in bacterial load with dose and at 7 kGy it reduce significantly with the
irradiation. Among all fatty acids, polyunsaturated, omega-3 and total omega fatty acids showed significant reduction
at 3 kGy irradiated tilapia fillets respect to control and other irradiated fillets. In tuna, at 3 kGy irradiation showed
significant increment in total saturated fatty acids while total unsaturated, omega-3 and total polyunsaturated fatty
acids showed significant reduction at 3 kGy irradiation. FFA of each fish variety, showed a fluctuation with the
irradiation. In tilapia, lowest FFA was found in 10 kGy irradiated sample (1.41 ± 0.0) and highest value was found in 3
kGy irradiated fillets (29.04 ±16.7). In tuna fillets highest value was found in 3 kGy irradiated fillets 71.06 ± 9.8) and
lowest value was found in 5 kGy irradiated fillets (15.6735 ± 3.3). Although bacterial load showed significant reduction
with the irradiation, fatty acids and FFA showed inconsistence variation.

Keywords: gamma irradiation, total bacterial counts, free fatty acid, polyunsaturated fatty acid, total omega fatty
acids
*Corresponding author’s email: isiri.hiranya@gmail.com

Introduction
The interest on food quality has contributed to guide consumers' choice toward fishery products which are considered
to be of high nutritional value and capable of influencing human health in a positive way (Mercogliano et al., 2012). In
recent years, consumers are eating more raw fish products such as sushi and surimi. But the major problem of
distribution of fishery products is their spoilage. Gamma irradiation reported to have potential for decontamination of
foods and thus having a ability to extend shelf life (Ozden and Erkan, 2009).
Therefore, the present study was carried out to investigate the effect of gamma irradiation on major food fishes in Sri
Lanka.

Methodology
Freshwater fish tilapia (Oreochromis sp) collected from local fishermen in Mahakanadarawa tank, Mihintale and
marine fish tuna (Thunnus albacares) were collected from landing sites of Kalpitiya coast. Both were degutted,
deheaded and filleted and packed in labeled sealed polythene bags and kept on ice box at a temperature below 4˚C.
Then the samples were divided into five lots: non-irradiated (control) and irradiated (3, 5, 7 and 10 kGy). Each group
consisted of 100 ± 10 g fish fillet and was packed in polystyrene boxes with ice for irradiation. Samples were
irradiated using cobalt 60 source supplied by Sri Lanka Gamma Center. The Irradiated doses were examined by
using amber 3042 perspex. Then the microbiological and chemical analysis were carried out to each set with the non-
irradiated control sample set.

To determine the microbial changes Total Plate Count (TPC) was determined by decimal dilution technique followed
by pour plate method according to the ISO 4833:2003 standard.
13
 Free fatty acid values (FFA) were determined according to the method describe in AOAC 1992 method. Fatty acid
compositions of each were determined by capillary column gas chromatography (Agilent 7890 B) with DB WAX
Column (30 m x 250 µm x 0.25 µm film thickness) and flame ionization detector. .

Results and Discussion

The initial bacterial load of control was 9.61 x 104 cfu/g and 4.55 x 104cfu/g in tilapia and tuna respectively. The
bacterial load was reduce with the irradiation (Figure 01).

The bacterial load of tilapia was 2.50 x 102 cfu/g at 3 kGy irradiation followed by 5 kGy irradiated tilapia with 1.23 x 103
cfu/g. In tuna bacterial load was 7.77 x 102 cfu/g at 3 kGy irradiation and at 5 kGy irradiation it was reduced to 2.48 x
102 cfu/g. At 7 and 10 kGy irradiation both samples showed sterilized conditions resulting significant reduction (< 1.0 x
101cfu/g) in bacterial growth. In Chakraborty et al., (2012) also showed the same results with climbing perch and
recorded that, at 6 kGy dose samples were sterilized resulting very low bacterial growth. Silva et al., (1994)
investigated the microbial quality of irradiated Bluejack Mackerel and found that the extension of shelf-life of the
product two three times than non-irradiated samples. It revealed that irradiation produces a reduction in the initial
number of microorganisms.

Table 01. FFA (%) of gamma irradiated fish fillets with respect to control samples

Fish variety Control 3 kGy 5 kGy 7 kGy 10 kGy

Tilapia 8.59a±0.54 29.04a±16.76 4.31a±0.73 11.86a±0.14 1.41a±0.0
Tuna 61.23ab±13.21 71.06a±9.76 15.67b±3.29 16.26b±4.95 68.32ab±23.80
Values are expressed as the mean ± SD. a-c Means followed by different letters in one row are significantly different
(P<0.001)

Hydrolysis of ester bonds in lipids by enzymatic action or heating in the presence of water liberates free fatty acids
(FFA). The accumulation of FFA during the processing or storage of foods influences the quality of the final product
and the period of shelf life. FFA of each fish variety in the study showed a fluctuation with the irradiation. In tilapia
lowest FFA was found in 10 kGy irradiated sample and highest value was found in 3 kGy irradiated fillets. In tuna
fillets highest value was found in 3 kGy irradiated fillets and lowest value was found in 5 kGy irradiated fillets. In both
species, irradiation dose does not showed any relation with the FFA values. Each values were fluctuating and
comprehensible.
Total saturated fatty acids (SFAs) in the non-irradiated tilapia fish muscle was 41.174% (Table 02). The highest and
lowest SFAs content between each dose treatments were in samples irradiated with 3 kGy (47.452%) and 5 kGy
(39.365%) respectively.

14
 Table 02: Fatty acid composition of tilapia fish fillets with gamma irradiation

Fatty acids Control 3 kGy 5 kGy 7 kGy 10 kGy

Total Saturated fatty acids (SFAs) (%) 41.17a ± 0.98 47.45b ± 0.27 39.36a ± 1.25 41.61a ± 0.00 41.21a ± 2.10
Total monounsaturated fatty acids (MUFAs) (%) 20.18 ± 0.06 21.10 ± 3.14 17.36 ± 3.34 23.41 ± 0.00 20.54 ± 0.64
Total Polyunsaturated fatty acids (PUFAs) (%) 34.65a ± 1.09 22.08b ± 0.97 38.90a ± 2.38 36.06a ± 0.00 35.12a ± 1.13
Total unsaturated fatty acids (%) 54.82a ±1.15 44.07b ± 4.11 56.27a ± 0.96 59.46a ± 0.00 55.66a ± 1.78
Omega 3 fatty acids (%) 23.83ab ± 0.56 13.93c ± 0.72 26.40a ± 2.16 21.52b ± 0.00 24.05ab ± 0.25
Omega 6 fatty acids (%) 10.82ab ± 0.53 8.15b ± 0.25 12.50bc ± 0.22 14.54c ± 0.00 11.07b ± 1.39
Total Omega fatty acids (%) 34.65a ± 1.09 22.08b ± 0.967 38.90a ± 2.38 36.06a ± 0.00 35.12a ± 1.13
unidentified 4.00± 0.17 12.78 ± 4.84 4.77 ± 0.29 5.18 ± 0.00 4.26± 0.32
Values are expressed as the mean ± SD. a-c Means followed by different letters in one row are significantly different
(P<0.001)

Table 03: Fatty acid composition of tuna fish fillets with gamma irradiation
Fatty acids Control 3 kGy 5 kGy 7 kGy 10 kGy
Total Saturated fatty acids (SFAs) (%) 32.21a ± 0.11 45.48b ± 0.00 37.59c ± 1.60 37.59c ± 0.41 33.14a ± 0.29
Total monounsaturated fatty acids (MUFAs) (%) 15.79a ± 1.90 17.87a ± 0.00 16.42a ± 2.99 20.03a ± 1.97 15.96a ± 0.19
Total Polyunsaturated fatty acids (PUFAs) (%) 51.08a ± 1.93 27.76b ± 0.00 46.17ac ± 4.43 40.24c ± 1.55 45.75ac ± 1.55
Total unsaturated fatty acids (%) 66.87a ± 0.03 45.24b ± 0.00 62.59c ± 1.44 59.60d ± 0.52 61.71cd ± 1.35
Omega 3 fatty acids (%) 39.35a ± 2.10 14.07b ± 0.00 34.85ac ± 5.08 31.30c ± 0.21 34.67ac ± 1.15
Omega 6 fatty acids (%) 11.73ab ± 0.17 12.81a ± 0.00 11.32ab ± 0.65 8.41c ± 1.33 10.63b ± 0.40
Total Omega fatty acids (%) 51.08a ± 1.93 26.88b ± 0.00 46.17 a ± 4.43 39.71c ± 1.55 45.30ac ± 1.55

unidentified 1.33 ± 0.09 8.54± 0.00 0.65 ± 0.16 2.69 ± 0.48 5.89± 1.25

Values are expressed as the mean ± SD. a-c Means followed by different letters in one row are significantly different
(P<0.001)

At 3 kGy dose treatment, tuna muscle samples has showed highest SFAs and lowest value was shown by non-
irradiated samples (45.479 % and 32.208% respectively). The content of MUFAs in the muscle of non-irradiated, 3,
5,7 and 10 kGy irradiated tilapia were 20.176% , 21.996% , 17.364% , 23.406% and 20.539% respectively. In tuna
fish muscles MUFAs does not showed significant increment with irradiation but it showed slight increase with the
irradiation (Table 03). The highest MUFAs content was found with 7 kGy irradiated tuna muscle (20.030 %) and
lowest was found with non-irradiated sample (15.790%).

At 3 kGy irradiation tilapia muscle showed significantly lower (P<0.001) PUFAs (22.077%) than all other treated
samples. In tuna at 3 kGy and 7 kGy irradiated samples showed significant reduction in PUFAs respect to other
irradiated samples and non-irradiated samples. In tilapia total omega 3 fatty acids were significantly lower at 3 kGy
irradiation dose (13.927%) than all other irradiated and non-irradiated samples. The omega 6 fatty acids showed
steadily increment with irradiation in tilapia. In tuna, omega 6 fatty acids were not markedly changed during the
irradiation process except 7 kGy irradiated sample which showed significant reduction (P<0.001) in omega 6 fatty
acids than non-irradiated and irradiated samples. Also Grcgel (2013) reported that,saturated fatty acids increased
with the irradiation, monounsaturated and polyunsaturated fatty acids decreased with the irradiation in meatballs.

Conclusion
The results obtained in this study suggest that bacterial load showed significant reduction with the irradiation, but fatty
acids and FFA showed inconclusive variation, thus required to study further.

Acknowledgement
This work was supported by National Research Council grant no. 15-020 from National Research council (NRC) of Sri
Lanka, 120/7, Ground Floor, Institute of Architects, Colombo 7, Sri Lanka.

15
References

AOAC 1992, Official methods and recommended practices of the American Oil Chemists Society, 4th edition.,
Champaingn American Oil Society, USA, 1992.
Chakraborty, S., MD. Mustafa, G., MD. Alam, Z. and Jannat, M. (2012). Effect of gamma irradiation on the sensory,
chemical and microbiological changes in two strains of climbing perch (Anabas testudineus, Bloch 1792).
J.Asiat. Soc. Bangladesh, Sci. 38 (2):183-188.
Gecgel, U., 2013. Changes in some physicochemical properties and fatty acid composition of irradiated meatballs
during storage. Journal of Food Science and Technology, 50(3):505–513.
Mercogliano, R., De Felice, A., Cortesi, M. L., Murrru, N., Marrone, R. and Anastasio, A. (2012), Biogenic amines
profile in processed bluefin tuna (Thunnus thunnus) products, CyTA-Journal of food, 11(2):101-107.
Ozden, O. and Erkan, N. (2009). Impacts of gamma irradiation on nutritional components of minimal processed
cultured sea bass (Dicentrarchus labrax). Iranian Journal of Fisheries Sciences. 9 (2):265-278.
Silva, H. A. and Nunes, M. L. (1994), Sensory and Microbiological Assessment of Irradiated Bluejack Mackerel
(Trachurus picturatus), Journal of the Science of Food and Agriculture, 66 (1) :175-180.

16
 IFRC 2017: 138-096 Food Safety and Quality

Shiga Toxin Escherichia coli survival in different blending ratio of fresh pineapple-mango juice blends

1Kamarul, Zaman, A. A., 1, 3, *Shamsudin, R., ²Mohd Adzahan, N. and 1Sulaiman, A.

1Department of Process and Food Engineering, Faculty of Engineering, University Putra Malaysia, 43300 UPM
Serdang, Selangor
2Department of Food Technology, Faculty of Food Science and Technology, University Putra Malaysia, 43300 UPM
Serdang, Selangor
3Institute of Advance Technology (ITMA), University Putra Malaysia, 43300 UPM Serdang, Selangor

Abstract
Shiga toxin producing E. coli (STEC) of serotype O157:H7 widely known to cause hemorrhagic colitis (HC) and
hemolytic uremic syndrome (HUS) which may lead to fatality. Mixed fruit juice were among the reported outbreak of
E. coli O157:H7 as the pathogen able to adapt to acidic environment of fruit juice. Different blending ratio of pineapple
and mango juice blend results with acidic range pH of 3.96±0.010 to 4.86±0.006 with blending ratio of 70%
pineapple:30% mango; 50% pineapple:50% mango and 30% pineapple: 70% mango. The initial microbiological
analysis on pineapple and mango juice blends however, results with no significant detection of STEC on all blending
ratios as the colonies were too few to count. Inoculation of pineapple and mango juice blends with STEC of serotype
O157:H7 shows blending ratio of 50% pineapple: 50% mango have highest STEC count (2.81 log cfu/ ml) and yeast
and mould counts (YM) (8.65±0.64 log cfu/ml) compared to other ratios. Total plate count (TPC) however, shows
deviate results as blending ratio of 70% pineapple: 30% mango results with highest TPC counts (8.29±0.25 log cfu/
ml).

Keywords: shiga toxin E. coli; pineapple; mango; juice blends; blending ratio
*Corresponding author’s email:: rosnahs@upm.edu.my, Fax/tel: +603- 89466366

Introduction
Fruit juice extracted directly from fruits or reconstituted of fruits which generally have a low pH value as the organic
acid content is high (Dewanti-Hariyadi, 2013). Codex (2005) defines juice as liquid extract from the edible part of
mature fresh fruit which can be either fermented or unfermented. Single fruit juice obtained from one fruit while mixed
fruit juice produced from blending two or more different type of fruit (Codex, 2005) to improve the juice nutritional and
organoleptic quality (Akusu et al., 2016). Tropical mixed fruit juice are the popular fruit juice blend product. Tropical
fruits widely known with their exotic taste with pineapple (Ananas comosus) and mangoes (Mangifera indica) among
commonly processed tropical fruits into juices, concentrates and pulp alongside bananas and passion fruits (Olesen,
1996).

pH, oxidation reduction potential, water activity, nutrients availability, antimicrobial compound presence and
competing micro flora are crucial factors impacting on juices spoilage with pH and water activity are the most
influential factors (Aneja et al., 2014).

Shiga toxin producing E. coli (STEC) with serotype E. coli O157:H7 able to cause hemorrhagic colitis (HC) and
hemolytic uremic syndrome (HUS) (Perera et al., 2015). Vojdani et al., (2008) revised the outbreak associated from
Shiga toxin producing E. coli O157:H7 with 5 numbers of outbreaks with pineapple and mixed fruit juice among the
reported juice outbreak. Acid tolerant bacteria may cause spoilage and produce undesirable organoleptic changes in
juice product. Food and Drug Administration (2000) foods with natural pH of 4.6 and below are classified as acidic
foods. The effect of blending ratio on the survival of STEC with E. coli O157:H7 serotype in pineapple mango juice
blends was not yet explored. Therefore, the objective of this study is to investigate the pH changes at a different

17
blending ratio of fresh pineapple and mango juice blends on survival of Shiga toxin Escherichia coli of serotype
O157:H7.

Material and methods

Juice preparation
Pineapple and mango fruits were obtained from retailer in Selangor. Josapine variety of pineapple and Chock Anan
mango were used in this study. Pineapple and mango fruits were cleaned, peeled and cut into smaller pieces. Both
pineapple and mango juice were extract separately using juice extractor (PENSONIC, PJ-37, Malaysia) and blend
according to the blending ratio as stated by Kamarul Zaman et al., (2016) which were 70% pineapple:30% mango,
50% pineapple:50% mango and 30% pineapple:70% mango.

pH and titratable acidity

pH of pineapple-mango juice blends were measured using pH meter, (pH 200, HM Digital, Korea). Titratable acidity
was measured using AOAC, 2012 method. The measurement were done in triplicate. Titratable acidity of pineapple-
mango juice blend were expressed as % of malic acid.
% malic acid = ((ml NaOH used × 0.1 N NaOH × 0.067)/grams of sample) × 100

Microbiological analysis
Shiga toxin Escherichia coli (STEC) culture media were obtained from Bacteriological Food Safety Laboratory, Food
Science and Technology Faculty, University Putra Malaysia. The STEC media were growth in Tryptic Soy Broth, TSB
(BactoTM, Becton, Dickinson and Company, USA) and incubated for 24 hours at 37°C. The culture then centrifuge at
5000 rpm for 5 minutes (Benchtop Centrifuge, Universal 320/320 R, Hettich Zentrifugen, Germany) to obtained the
cell pellet. The pellet then washed with 0.85% NaCl (Tosun & Gönül, 2006) and inoculated in juice sample at different
blending ratio for 2 hours. The juice then incubated for 2 hours at 37°C. Pineapple-mango juice then serially diluted in
0.1 % peptone water (10-1 until 10-5) and spread plating on Tryptic soy agar, TSA (Difco TM, Dickinson and Company,
USA), and McConkey agar (Difco TM, Dickinson and Company, USA). The dilution was done in triplicate and
incubated at 37°C for 24 hours.

Aerobic plate count or total plate count (TPC) (Maturin & Peeler, 2001) and yeast and mould count (YM) (AOAC,
2002) analysis were also conducted. Plate count agar, PCA (Merk, Germany) and Dichloron rose Bengal
Chloramphenicol, DRBC agar (Condalab, Spain) were used for TPC and YM respectively. Similarly, triplicate serial
dilution of juice inoculate with STEC were spread onto the solidified agar and incubated at 25±2°C for 5 days and
35±2°C for 2 days for PCA and YM respectively.

All the obtained data were analyzed using IBM SPSS Statistic 22 tools for significant level.

Results and discussion

The initial microbiological counts of E. coli O157:H7 on McConkey plate agar were null indicated the pineapple and
mango juice at all different blending ratios were not contaminated with E. coli O157:H7 as the colonies were too few
to be counted as significant as shown in Table 1. However, on tryptic soy agar (TSA) juice with higher mango ratios
resulted with higher counts of 4.43 log cfu/ml (30 P: 70 M) and pH (4.68). Total plate counts (TPC) measured the
aerobic mesophilic microbial counts in which single mango juice (100 M; 7.03 cfu/ml) was higher compare to single
pineapple juice (100 P; 5.22 log cfu/ml). The results in agreement with Leul and Kibret (2012) as the pH of mango
juice was higher compare to pineapple juice which inhibit the growth of bacteria. Whereas, yeast and mould counts
(YM) were not significantly differ (p>0.05) in log counts for all different blending ratios of pineapple-mango juice
blends.

Increment of titratable acidity of pineapple-mango juice blends at higher mango ratio results with lower pH. According
to Chia et al. (2012) and Akusu et al., (2016) as the pH increase the titratable acidity decrease. Pineapple (100 P)
have slightly lower pH value (3.96±0.010) compare to mango (100 M) juice. Similarly, studies by Kamarul Zaman et
al. (2016) and Shamsudin et al. (2007) on pineapple juice and Guan et al. (2016) for mango juice results with pH
range of 3.81 to 3.96 and 4.22 to 4.86 respectively.
18
 Table 1 Initial colonies count in pineapple-mango juice blends at different blending ratio.
Blending pH Titratable acidity TPC YM McConkey TSA
ratio (%malic acid) (log cfu/ml) (log cfu/ml) (log cfu/ml) (log cfu/ml)
100 P 3.96±0.010a 0.70±0.002a 5.22±0.23a 8.96±0.19a 0a 2.23±0.09a
70 P:30 M 4.32±0.006a 0.66±0.004a 7.50±0.14a 6.03±0.95b 0a 2.10±0.11a
50 P:50 M 4.62±0.010 a 0.52±0.005 a 5.01±0.42 a 8.55±0.64 a 0 a 3.67±0.17a
30 P:70 M 4.68±0.011 a 0.46±0.003 a,b 4.76±0.15 a 7.93±0.40 a,b 0 a 4.43±0.21a
100 M 4.86±0.006a 0.29±0.005b 7.03±0.38a 8.92±0.54a 0a 4.40±0.25a
P and M were pineapple and mango respectively. TPC, YM, TSA were total plate count, yeast and mould count and
tryptic soy agar count respectively. Same subscript indicate results were not significantly different (p>0.05) from each
other.

Table 2 Colonies count in different plate agar for pineapple-mango juice blends at different blending ratio after
inoculation.
Blending ratio TPC YM McConkey TSA
(log cfu/ml) (log cfu/ml) (log cfu/ml) (log cfu/ml)
100 P 8.50±0.25a 8.93±0.19a 3.17±0.20a 5.37±0.09a
70 P:30 M 8.29±0.10a 5.27±0.09b 1.02±0.87a 5.90±0.12a
50 P:50 M 4.67±0.42 b 8.65±0.64 a 2.81±0.27 a 8.38±0.92b
30 P:70 M 4.96±0.15 b 8.46±0.40 a 1.00±1.0 a 7.80±0.54b
100 M 8.43±0.38 a 8.82±0.54 a 2.36±1.17 a 8.26±1.37b
P and M were pineapple and mango respectively. TPC, YM, TSA were total plate count, yeast and mould count and
tryptic soy agar count respectively. Same subscript indicate results were not significantly different (p>0.05) from each
other.

Table 2 shows the colonies count at different blending ratios of pineapple-mango juice blends after inoculated with
STEC. Blending ratio of 50 P: 50 M results with highest STEC counts on both McConkey (2.81 log cfu/ml) and TSA
agar (8.38 log cfu/ml) with highest YM counts of 8.65 log cfu/ml indicate the juice at the respective blending ratios
were favorable for the pertinent pathogen to grow. According to Mohd Hanif et al. (2016) colonies on TSA detect the
possible background microflora and unretrieved STEC counts on McConkey plate agar. However, the TPC of juice
blends at 50 P: 50 M were the lowest (4.67 log cfu/ml).

pH of pineapple-mango juice blends at all blending ratios were not significantly different and in the range of acidic
juice as stated by Food and Drugs Administration (2000) which explained the growth of STEC in pineapple-mango
juice blends. Leul and Kibret (2012) added that total acidity of juice were due to presence of organic acids mixture in
which bacteria were more sensitive towards citric acids. According to Hsin-Yi and Chou (2001), acidic foods usually
able to control bacteria growth, but acid adaptation may occur as microorganism shows an increased resistance
towards environmental stress after exposure to moderate acidic condition.

Conclusion
The study revealed that at different blending ratio of fresh pineapple-mango juice blends pathogenic bacteria may
survive due to the adaptation of STEC on the acidic environments which may contribute to food borne outbreak if
consumed. Pineapple-mango juice blends at balance pineapple and mango ratio (50 P: 50 M) was more susceptible
towards pathogenic bacteria as STEC growth counted more
in both McConkey and TSA agar.

Acknowledgement
This research studies were funded by University Putra Malaysia Postgraduate grant (VOT 9473900).

19
References

Akusu, O. M., Kiin-Kabari, D. B. and Ebere, C. O. (2016) Quality characteristics of orange/pineapple fruit juice blends.
American Journal of Food Science and Technology 4 (2), 43-47.
Aneja, K. R., Dhiman, R., Aggarwal, N. M. and Aneja, A. (2014). Emerging preservation techniques for controlling
spoilage and pathogenic microorganisms in fruit juices. International Journal of Microbiology, 1-14.
AOAC International (2002). Official Method of Analysis, Official Method 997.02: Yeast and Mould counts in foods.
AOAC International, Gaithersburg, MD.
AOAC (2012). Official Methods of Analysis of the Association of Official Analytical Chemists, 20th ed.
Chia, S. L., Shamsudin, R., Mohd Adzahan, N., and Wan Ramli, W. D. (2012). The effect of storage on the quality
attributes of ultraviolet-irradiated and thermally pasteurized pineapple juices. International Food Research
Journal 19 (3), 1001-1010.
Codex Stan 247. (2005). Codex General Standard for fruit juices and nectars, 1-19.
Dewanti-Hariyadi, R. (2013). Microbiological quality and safety of fruit juices. Food Review International 1 (1), 54-57.
Food and Drug Administration (2000) Code of Federal Regulations Title 21, Food and Drugs. Chap 1. Part 114.
Acidified foods.
Guan, Y., Zhou, L., Bi, J., Yi, J., Liu, X., Chen, Q., Wu, X. and Zhou, M. (2016). Change in microbial and quality
attributes of mango juice treated by high pressure homogenization combined with moderate inlet temperature
during storage. Innovative Food Science and Emerging Technologies 36, 320-329.
Perera, A., Clarke, C. M., Dykes, G. A. and Fegan, N. (2015) Characterization of shiga toxigenic Escherichia coli
O157 and non-O157 isolates from ruminant feces in Malaysia. BioMed Research International, 1-8.
Hsin-Yi, C. and Chou, C. C. (2001). Acid adaptation and temperature effect on the survival of E. coli O157:H7 in
acidic fruit juice and lactic fermented milk product. International Journal of Food Microbiology 70, 189-195.
Kamarul Zaman, A. A., Shamsudin, R. and Mohd Adzahan, N., (2016). Effect of blending ratio on quality of fresh
pineapple (Ananas comosus L.) and mango (Mangifera indica L.) juice blends. International Food Research
Journal 23 (Suppl.), S101-S106.
Leul, A. and Kibret, M. (2012). Bacteriological safety of freshly squeezed mango and pineapple juices served in juice
houses of Bahir Dar Town, Northwest Ethiopia. International Journal of Sciences: Basic and Applied Research
6 (1), 24-35.
Mohd Hanif, H. A., Shamsudin, R. and Mohd Adzahan, N. (2016). Effects of UVC irradiation and thermal treatment on
the physic-chemical properties and microbial reduction of clear and turbid tamarind juice. International Food
Research Journal 23 (Suppl.), S107-S112.
Olesen, R. K. (1996). Tropical fruit products: A well establish market. International Trade Forum 3, 10-17.
Shamsudin, R., Wan Daud, W. R., Takriff, M. S. and Hassan, O. (2007). Physicochemical properties of the Josapine
variety of pineapple fruit. International Journal of Food Engineering 3 (5), 1-12.
Vojdani, J. D., Beuchat, L. R. and Tauxe, R.V. (2008). Juice associated outbreaks of human illness in the United
States, 1995 through 2005. Journal of Food Protection 71 (2), 356-364.

20
 IFRC 2017: 139-163 Food Safety and Quality

Proper Hand Washing Practices in School Canteen: A Qualitative Study on Food Handlers’ Belief

1Ahmad, I. A., Abidin, 1*U. F. U. Z., 1Mahyudin, N. A., and 2Ab-Rashid, N. K.

1Department of Food Service and Management, Faculty of Food Science and Technology, Universiti Putra Malaysia,4
3400 UPM, Serdang, Selangor, Malaysia
2Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM,
Serdang, Selangor, Malaysia

Abstract
Improving hand washing practice among food handlers is one of the ways to reduce food borne illness. This study
aimed to assess behavioural belief, normative and control beliefs underpinning food handlers’ decision to perform
hand washing practices in school canteen in Malaysia. A qualitative study comprising eight focus group discussions
with 71 food handlers were conducted to elicit the underlying belief specific to hand washing practices. Respondents
from school canteen within Klang Valley were recruited using purposive sampling strategy. All focus group audio
recordings were transcribed verbatim. A thematic content analysis was conducted using Atlas.ti software to code,
manage and analyze data. Results obtained from the focus group discussions were coded according to key concepts
in the Theory of Planned Behaviour (TPB) framework which explain behavioural belief (i.e. advantages and
disadvantages of hand washing), normative belief (i.e. an individual’s perception of social normative pressure) and
control belief (i.e. barriers and facilitators) to perform hand washing. Themes emerge for the advantages of proper
hand washing are cleanliness, prevent bacteria/virus infection, reduce food poisoning risk, while the disadvantages
are time pressure, hassle, and work load pressure. The barriers for proper hand washing identified were time
pressure, work load pressure, the frequency of hand washing required, steps in proper hand washing, and availability
of hand washing soap. The facilitators for hand washing practices include poster reminder, reminder from
manager/owner and rewards/punishment from manager/owner. Themes found under normative belief are the
influence from school administration, manager, co-worker and health inspector. In summary, all the beliefs elicited
may provide valuable information for the development and evaluation of intervention strategies to improve food
handlers’ proper hand washing practices.

Keywords: Hand washing practices, food handlers, school canteen, focus group, Theory of Planned Behaviour
*Corresponding author’s email:: ungkufatimah@upm.edu.my

Introduction
Foodborne illness is one of the serious public health threats in the world. According to WHO (2015), 1 in 10 people
fall ill every year because of contaminated food. Previous studies have shown that many reported food borne illness
occurred in foodservice establishment such as school food service, cafeterias and restaurant (Green et al., 2007;
Pilling, Brannon, Shanklin, Howells, & Roberts, 2008). In Malaysia, most of the outbreaks incidents are coming from
school and academic institutions (Soon, Singh, & Baines, 2011). A number of studies indicated that food handlers’
food safety practices need to be improved in order to reduce the rate of foodborne illness outbreak. Soon et al.,
(2011) further stated that epidemiological selection, lack of quality assurance in foodservice and food handlers’ poor
safe food handling practices are the reason why foodservice is considered as the main setting for food borne illness
outbreaks. On the other hand, good hand washing practice is very important to prevent the spread of microbial
infection (Fendler, Dolan, & Williams, 1998; Pragle, Harding, & Mack, 2007). Current intervention in food service
establishment use enforcement to ensure food safety and legal requirements in food service establishment are
maintained. Other typical intervention is by imparting knowledge to food handlers via training (Clayton, Griffith, Price,
& Peters, 2002). However, according to Mitchell, Fraser, & Bearon, (2007) these interventions neglected other factors
that may contribute to failure in practicing food safety behaviours among food handlers. Recent studies showed that
identification of belief using TPB could be used to gain information to develop interventions and improve food
21
handlers’ practices (Nik Husain, Wan Muda, Noor Jamil, Nik Hanafi, & Abdul Rahman, 2016; White et al., 2015;
Zoellner et al., 2012). Therefore, this study aimed to assess behavioural belief, normative and control beliefs of food
handlers’ decision to perform hand washing practices.

Method
Study Participants
Eight focus group (FG) discussions with 71 food handlers from school canteens were conducted around Klang Valley
area during Mac and April 2016. FG participants were recruited using purposive sampling technique.

Data collection
Each FG generally ran for 90 minutes and consisted of 6 to 12 participants for each session. FG discussion were
facilitated by one moderator and one assistant moderator. First, the moderator briefed the participants the purpose of
study and explained that the FG will be audio-recorded & transcribed. To guide the FG, a semi structured FG
questions were developed within a TPB framework and designed to elicit the underlying belief specific to hand
washing practices among the food handlers. The FG questions were constructed based on previous studies and TPB
guidelines (White et al., 2015; York, Brannon, Roberts, Shanklin, & Howells, 2009). A video of 7-step hand washing
practice was played before the key questions were asked to ensure participants shared the same understanding
about the proper hand washing practice. Data collection stopped when data was saturated after eight FGs were
conducted. The key questions for the FG are developed based on three TPB components. The first component is
behavioral belief (i.e. advantages & disadvantages), and the example of question is “What do you believe/think would
be the advantages/disadvantages of washing your hands properly whenever you should at work?”. Second
component in the key question is control belief (advantages/disadvantages) and the example of question is “What
factors/circumstances make it difficult/easier for you to wash your hands properly whenever you should at work?”.
Third component is normative belief (i.e. social normative approval/disapproval) and the sample of question is “Are
there any individuals or groups important to you that would approve/disapprove you in performing hand washing?”

Data analysis
Each FG was audio-recorded and transcribed verbatim. A thematic content analysis was conducted using Atlas.ti
software to code, manage and analyze data.

Result and Discussion

Demographic
In total, 71 food handlers (Female=48; Male=23) participated in eight focus group discussions. The majority of the
participants were Malaysian (77.46%) and the rest were foreign workers (22.54%). The participants’ age ranged from
18 to 64 years old. Their food industry experience ranged from one month to 28 years. The majority of participants
had attended food handlers training (69.01%) and the rest are not (26.76%). Most of them had vaccinated for typhoid
(N=62, 87.32%), and only nine (12.68%) participants were not vaccinated. Majority of the participants (73.24%) had
graduated from high school, nine (12.68%) graduated from primary school, six (8.45%) graduated from diploma and
only two (2.817%) do not received any formal education.

Behavioural beliefs: advantages and disadvantages

Many food handlers perceived cleanliness as the biggest advantage in performing proper hand washing. When asked
about the advantages of performing proper hand washing, food handlers agreed that proper hand washing make
themselves and food preparation cleaner. Besides, preventing bacteria/virus infection and potentially reduce food
poisoning has been identified as the other major advantages of proper hand washing practices. Many participants
perceived both factors as the advantages of proper hand washing especially when related to their customer safety
and protection for the food handlers themselves. Some food handlers explained:

“…. Ah ...can prevent children from getting (food) poisoning, we also become healthy, right? Cleaner. Then we
can bring that practice back home, teaching our children, our parents.” Focus group 1.

“.…. [When] the hands are always clean, [we] protected from bacteria/virus infection” Focus group 2.
22
This finding is consistent with the study conducted by White et al., (2015). White et al., (2015) studied beliefs of hand
hygiene among nurses in hospital using TPB component. Nurses have identified that patient protection, self-
protection and infection control as main advantages of performing proper hand hygiene. Additionally, food handlers
had also recognized time, work pressure and hassle as the major disadvantages of proper hand washing in school
canteen.

Control beliefs: barriers and facilitators

Some food handlers agreed that reminder poster and reward/punishment from manager are important facilitators to
help them performing proper hand washing practice. But the most frequently reported facilitator for proper hand
washing practice is having enough resources (e.g. soap, dispenser and sanitizer) in school canteen. The food
handlers mentioned:
“.…. [If] We have soap dispenser, so it is very convenient.” Focus group 3.

“... Soap must be available first [in school canteen].” Focus group 4.

Food handlers were asked about the barriers for proper hand washing practices. Most of them mentioned that time
pressure as the main barrier, followed by work load pressure, the frequency of hand washing required, the steps of
proper hand washing and availability of hand washing soap. Some food handler described:

“... because in school time we have a brief period [for break]. We are time constraint, at 10 [am] for example
students are having their break. So, if we are busy washing hand [laughing], when are we going to cook, when
are we going to work, right?” Focus group 5.

Most of the barriers identified in this study were supported by previous research (York, Brannon, Shanklin, et al.,
2009). For example, York et al., (2009) identified a few barriers such as not having enough time, not having reminder
poster, and not having reminder from manager as factors that hindered food workers from performing the behavior.
Other studies also reported similar result, where lack of time, work load pressure, the availability of hand washing
soap have been identified as barriers to improve food safety practices (Arendt, Strohbehn, & Jun, 2015; Green et al.,
2007; Pragle et al., 2007).

Normative belief
Food handlers described school administration (teacher, principal) as the most important individual/group who can
give an impact to their hand washing practice. Most of them considered school administration as supportive referent.
One of the food handler explained:
“They (canteen teacher) helped a lot, without the canteen teacher, our cafeteria will…. we just don’t care.”

Other important referents for food handlers for performing hand washing practices are co-workers, supervisor/owner,
family members and health officer (government officer). This finding is similar to the study conducted by White et al.,
(2015), where the researcher identified co-workers, supervisor and patient as important referents for nurses in
performing hand hygiene in hospital. Additionally, the results described co-workers as the main group that support
hand hygiene performance.

Conclusion
Overall, the identified themes provided critical insight in understanding beliefs associated with hand washing
practices. The findings may assist the development and evaluation of the future intervention to improve food handlers’
proper hand washing practices in school canteens in Malaysia.

References

Arendt, S., Strohbehn, C., & Jun, J. (2015). Motivators and barriers to safe food practices: Observation and interview.
Food Protection Trends, 35(5), 365–376.
Clayton, D. A., Griffith, C. J., Price, P., & Peters, A. C. (2002). Food handlers â€TM beliefs and self- reported
practices. International Journal of Environmental Health Research, 12(1), 25–39.

23
Green, L. R., Radke, V., Mason, R., Bushnell, L., Reimann, D. W., Mack, J. C., … Selman, C. A. (2007). Factors
related to food worker hand hygiene practices. Journal of Food Protection, 70(3), 661–666.
Mitchell, R. E., Fraser, A. M., & Bearon, L. B. (2007). Preventing food-borne illness in food service establishments:
Broadening the framework for intervention and research on safe food handling behaviors. International Journal
of Environmental Health Research, 17, 9–24. http://doi.org/10.1080/09603120601124371
Nik Husain, N. R., Wan Muda, W. M., Noor Jamil, N. I., Nik Hanafi, N. N., & Abdul Rahman, R. (2016). Effect of food
safety training on food handlers’ knowledge and practices. British Food Journal, 118(4), 795–808.
http://doi.org/10.1108/BFJ-08-2015-0294
Pilling, V. K., Brannon, L. A., Shanklin, C. W., Howells, A. D., & Roberts, K. R. (2008). Identifying specific beliefs to
target to improve restaurant employees’ intentions for performing three important food safety behaviors.
Journal of the American Dietetic Association, 108(6), 991–7. http://doi.org/10.1016/j.jada.2008.03.014
Pragle, A. S., Harding, A. K., & Mack, J. C. (2007). Food workers’ perspectives on handwashing behaviors and
barriers in the restaurant environment. Journal of Environmental Health, 69(June), 27–32.
Soon, J. M., Singh, H., & Baines, R. (2011). Foodborne diseases in Malaysia: A review. Food Control, 22(6), 823–
830. http://doi.org/10.1016/j.foodcont.2010.12.011
White, K. M., Jimmieson, N. L., Obst, P. L., Graves, N., Barnett, A., Cockshaw, W. and Paterson, D. (2015). Using a
theory of planned behaviour framework to explore hand hygiene beliefs at the “5 critical moments” among
Australian hospital-based nurses. BMC Health Services Research, 15(1), 59. http://doi.org/10.1186/s12913-
015-0718-2
WHO (World Health Organization). October 2016. 10 facts of food safety. Retrieved on June 1, 2017 from WHO
website: http://www.who.int/features/factfiles/food_safety/en/
York, V. K., Brannon, L. A., Roberts, K. R., Shanklin, C. W., & Howells, A. D. (2009). Using the Theory of Planned
Behavior to Elicit Restaurant Employee Beliefs about Food Safety: Using Surveys Versus Focus Groups.
Journal of Foodservice Business Research, 12(2), 180–197. http://doi.org/10.1080/15378020902910777
York, V. K., Brannon, L. a., Shanklin, C. W., Roberts, K. R., Barrett, B. B., & Howells, A. D. (2009). Intervention
improves restaurant employees’ food safety compliance rates. International Journal of Contemporary
Hospitality Management, 21(4), 459–478. http://doi.org/10.1108/09596110910955703
Zoellner, J., Krzeski, E., Harden, S., Cook, E., Allen, K., & Estabrooks, P. A. (2012). Qualitative Application of the
Theory of Planned Behavior to Understand Beverage Consumption Behaviors among Adults. Journal of the
Academy of Nutrition and Dietetics, 112(11), 1774–1784. http://doi.org/10.1016/j.jand.2012.06.368

24
 IFRC 2017: 141-098 Food Safety and Quality

Effect of Poster and Video Intervention on The Knowledge, Attitude and Practice (KAP) Level of Personal
Hygiene Among Food Handlers in 24 Hours Mamak Restaurants in Sungai Petani, Kedah.

*Masyita, M. and Nur Amalina, M.J.

Environmental Health Section, Universiti Kuala Lumpur, Institute of Medical Science Technology, 43000 Kajang

Abstract
Frequenting Mamak restaurants has been a culture in Malaysia and it signifies the importance of food safety since it
runs on 24 hour operation. This present study is to assess the knowledge, attitude and practice on personal hygiene
among food handlers at 24 hours Mamak operation restaurants before and after poster & video intervention. A survey
and observation checklist was conducted among 100 food handlers selected from Mamak restaurants. A poster was
placed at the restaurants for 4 weeks and educational video on personal hygiene was showed to the food handlers.
There was an increase of good personal hygiene knowledge and practice after the intervention (51% to 80% and 42% to
73% respectively) and slight increase was observed on the awareness level (77% to 100%).There was a significant
difference within the median score of knowledge, attitude and practice before and after intervention, indicating the
efficiency of poster & video intervention. It was observed that there was no significant correlation between knowledge
and practice (r= -0.082, p=0.415) similarly there was no significant correlation observed between knowledge and
attitude (r= -0.009, p=0.930). The finding adds to the growing body of evidence on the effectiveness of poster and video
intervention in educating food safety.

Keywords: KAP, poster & video intervention, personal hygiene, food handlers, 24 hours Mamak restaurants
*Corresponding author’s email:: masyita@unikl.edu.my

Introduction
Mamak culture or “frequenting a Mamak restaurant” has evolved and become a phenomenon in Malaysia. It is also
believed that the Mamak culture inculcates values of national unity with providing a place where Malaysians and
foreigners can come to relax while enjoying great food and great conversation in a laid back environment.
Consequently, this phenomenon has led to the increase operational hours of Mamak restaurants (Zarina et al.,
2012).The culture of Mamak frequenting restaurants in Malaysia however, has raised several issues on the hygiene
of food premise and food handling. Recently, one Mamak restaurant in Klang was ordered to close for failing to
comply with hygiene requirements (Bernama, 2016). In addition, three Mamak restaurants in Selayang were ordered
to close due to three major errors committed: the unused apron and hat, raw food on the floor and the food wastes
stream down the drain (Sinar online, 2015). The incidence raised a public concern on the mishandling of food during
food preparation and serving that may cause food borne illness (MOH, 2016). As part of the initiative, Ministry of
Health, Malaysia has enforced all food handlers to undergo Food Handler trainings that are recognized by the Ministry
of Health (MOH, 2016). Despite the enforcement done, the incidence rate of foodborne illness has increased 3% from
the year 2012 to 2012 (44.79% and 47.79% respectively) (Abdul-Mutalib et al., 2015). The increase of foodborne
illness indicates the food safety negligence among the food handlers (Abdul-Mutalib et al., 2015). In addition, this
issue also highlight the comprehension of food safety concept among those food service workers or managers with
little to no knowledge of national language or English (Li, 2015). Therefore, the usage of visual-based tools is
considered as useful in addressing language and cultural differences (Li, 2015). This study aim to assess the food
handlers’ knowledge, attitude and practices on personal hygiene in Mamak restaurants before and after poster and
video intervention.

Material and Methods

Study design and sample

25
This study employed quasi-experimental design of one group pre test-post test. 100 food handlers were conveniently
selected from six 24- hour operated Mamak restaurants in Sungai Petani, Kedah. Respondents must at least had 6
months of working experience in handling food (which may include food and beverage preparation and serving,
cooking, washing and cleaning dishes) in the respective restaurants. Written informed consent was obtained from the
respondents.

3.2 Assessment and intervention tools

A set of bi language questionnaire (Bahasa Malaysia and English) on socio demographic and personal hygiene
knowledge and attitude was constructed based on the modified questions from previous studies (Tokuç et al., 2009)
and pre-tested on 10 food handlers in Mamak restaurants in Kajang in order to ensure the wording consistency and
reliability. A standard checklist on personal hygiene constructed by Ministry of Urban Wellbeing, Housing and Local
Government (2014) was utilised in order to observe the personal hygiene practice of the food handlers.

A poster was designed for intervention purpose, illustrating simple picture of a person wearing proper attire for food
handling and vice versa. The illustration was accompanied by written and illustration instruction of dos and don’ts
during food handling. In addition to the poster intervention, an educational video on personal hygiene developed by
Ministry of Health was projected to the respondents in order to increase educational experience and level of personal
hygiene practices (MOH, 1998). The video duration was five minutes.

3.3 Study administration

The study was in collaboration with Sungai Petani Municipal Council. At least four respondents from a premise were
selected to answer the questionnaire, either by self-completion or interview. Prior to assess the personal hygiene
practices, an observation was conducted. The respondents later were briefed on the personal hygiene, assisted by
the educational video viewing. Additionally, posters on personal hygiene were placed in the restaurants at designated
areas such as food or beverage preparation area, washroom, kitchen and front counter. A post- evaluation was
conducted on the same respondents after four weeks using the similar questionnaire and observation checklist.

3.5 Data analysis

Data were expressed as n (percentage) for descriptive analysis. Wilcoxon signed ranked was employed in order to
compare the Knowledge, Attitude and Practice (KAP) scores at baseline and post intervention. This analysis was
expressed as median (interquartile range). A Spearman correlation test was assigned to determine the association
within knowledge, attitude and practice on personal hygiene. p< 0.05 was considered as statistically significant. All
data were analysed using SPSS version 20 (SPSS Inc., Chicago, Illionis, USA).

Results and Discussion

Table 1 exhibit the characteristic of the respondents. Most of the respondents were males with age range 20 – 40
year old and working experience more than a year. 84% of the respondents reported have attended food safety
training. With regards to level of personal hygiene during baseline and post intervention, the scores obtained for
knowledge, attitude and practice (KAP) were categorised into poor and excellent according to the midpoint score
calculated. Those who scored above midpoint score were defined as ‘Excellent’ whilst below midpoint score indicated
as ‘Poor’. There was an increase of percentage on knowledge and practice during the baseline and post intervention.
A slight increase on attitude percentage was observed between pre and post intervention (77% to 100%) (Table 2).
The result was consistent with the percentage reported where there were significant increases in knowledge and
practice scores between baseline and post intervention (See Table 2). The finding is concordant with previous studies
(Ansari-lari et al., 2010; Nurul Huda 2008) that suggest such initiatives are necessary in order to promote appropriate
knowledge and practice on food handling (Nurul Huda, 2008). Moreover, the visual-based intervention utilised in this
study might be helpful in improving food safety knowledge among the non-natives food handlers (Li, 2015). It is also
proposed that by incorporating the importance of food safety with science-based information may assist the food
handlers to understand better and help the health inspectors from being perceived as the ‘trouble makers’ (Li, 2015).

26
The correlation between knowledge on food safety with attitude and practice showed insignificant negative weak
correlation within the variables (p> 0.05) (Table 2). This was Ansari- Lari et al. (2010) who observed positive
correlation between knowledge, attitude and practice. However, the finding demonstrated that knowledge alone does
not always result in safe food handling behaviour and this is agreed by Mullan et al. (2013).

Table 1: Respondent socio demographic characteristic (N=100)

Variables n(%)
Gender
Male 89 (89%)
Female 11(11%)
Age
20 -30 years old 61(61%)
> 30 years old 39(39%)
Years of working experience
6 months 9(9%)
1-3 years 51(51%)
> 3 years 40 (40%)
Food handling training
Yes 16 (16%)
No 84(84%)

Data was presented as n (%) for categorical variables.

Table 2: Personal hygiene during pre and post intervention (N= 100)
Personal Hygiene
Variable Pre Intervention Post Intervention p- value
Knowledge Poor : 49 (49%) Poor : 20 (20%)
Excellent : 51 (51%) Excellent : 80 (80%) < 0.001*
Score : 7.00(7.00) Score : 8.00(7.50)
Attitude Poor : 23 (23%) Poor : 0 (0%)
Good : 77 (77%) Good :100 (100%) 0.965
Score : 7.00 (7.00) Score : 7.00 (7.00)
Practice Poor : 58 (58%) Poor : 27 (27%)
Good : 42 (42%) Good : 73 (73%) <0.001*
Score : 4.0 (4.50) Score : 6.00 (5.50)
**Knowledge Attitude r= - 0.009 0.930
Practice r = - 0.082 0.415
Data was presented as n (%) for categorical variables. Wilcoxon ranked is used to compare continuous variable. Data
is presented as median (IQR). *indicates significant (p< 0.05). IQR: Interquartile range. **Spearman rho is used to
correlate continuous data. Data is presented as r coefficient.

Conclusion
Finding from this study indicated that the usage of visual-based intervention was helpful in educating the food safety
component of personal hygiene among the non-native speaker food handlers, which in this study were the Mamak
food handlers. Nevertheless, the incorporation of knowledge, attitude and element in this study predict limited
effectiveness of food hygiene intervention. An intervention that imparts health-related behaviours framework such as
theory-planned behaviour (TPB) should be warranted for future study. In addition, the employment of one group quasi
27
experimental design was noted to be lacked in internal validity due to the respondent’s maturity and selection-history
threats. Therefore, a randomised controlled trial designed is proposed in order for the population of participating
individuals are clearly identified.

Acknowledgement
The authors would like to express their gratitude to all participants for their contribution and participation throughout
the research.

References

Abdul-Mutalib, N.A., Syafinaz A.N., Sakai, K. & Shirai, Y. (2015) An overview of food borne illness and food safety in
Malaysia, International Food Research Journal, 22(3): 896-901
Soon, J.M., Singh, H.& Baines, R. (2011) Food borne diseases in Malaysia: a review, Food Control, 22(6): 823-830
Ansari-Lari, M., Soodbakhsh, S., & Lakzadeh, L. (2010). Knowledge, attitudes and practices of workers on food
hygienic practices in meat processing plants in Fars, Iran. Food Control, 21, 260e263.
Bernama (March 29, 2016) 16 Premis Makanan Di KL Abaikan Kebersihan Diarah Tutup.myputarajayanews.
Retrieved from: http://myputrajayanews.com/v2/16-premis-makanan-di-kl-abaikan-kebersihan-diarah-tutup/
Dawei, Li (2015) Development and assessment of visual-based training on Chinese-speaking foodservice workers in
independently owned Chinese restaurants. Iowa, United States: Iowa State University, MSc thesis
Ministry of Health Malaysia (MOH),Food Safety and Quality Division. December 29, 2016: Guidelines for
Accreditation of Food Handlers Training Program. Retrieved from: http://fsq.moh.gov.my/v5/ms/garis-panduan-
skim-akreditasi-program-latihan-pengendali-makanan-lpm-3/
Ministry of Urban Wellbeing, Housing and Local Government (2014) Food premise inspection and grading form.
Retrieved from: http://www.mdbg.gov.my/sites/default/files/perniagaan/pelesenan/garis_panduan_sistem_
penggredan_ premis_makanan_di_kawasan_pihak_berkuasa_tempatan.pdf
Mullan, B.B., Wong, C. & Kothe, E.J. (2013) Predicting adolescents’ safe food handling using an extended theory of
planned behavior, Food Control, 31(2): 454-460
Nurul Huda, H. 2008. Tahap pengetahuan, sikap dan amalan kebersihan dan keselamatan makanan di kalangan
pengendali makanan di hospital. MSc Thesis. Universiti Kebangsaan Malaysia.
Sinar [online] (13 March, 2015) Tiga restoran di arah tutup. Sinar Online. Retrieved from
http://www.sinarharian.com.my/edisi/selangor-kl/tiga-restoran-diarah-tutup-1.368227
Tokuc, B., Ekuklu, G., Berberoglu, U., Bilge, E. & Dedeler, H. (2009), “Knowledge, attitude and self-reported practices
of food services staff regarding food hygiene in Edirne, Turkey”, Food Control, 20(6), 565-568.
Zarina Z, Faisal I.(2012). Jom mamak! Examining the role of sociocultural and technological determinants in a local
pop-culture phenomenon: School of Communication Studies, SEGi University, Selangor, Malaysia, Vol.5,
No.2.

28
 This page is intentionally left blank

Detection of Irradiated Cucurma longa and Cariandrum sativum Using Photo-stimulated Luminescence (PSL)
Technique

1*Ros Anita Ahmad Ramli, 2Ainul Hafiza Abdul Hair & 1Zainon Othman

1Agrotechnology & Biosciences Division, Malaysian Nuclear Agency, 43000 Kajang, Selangor, Malaysia
2 Department of Biology, Pusat Asasi, Universiti Teknologi Mara Selangor Cawangan Dengkil, 43800, Dengkil,
Selangor, Malaysia

Abstract
Photo-stimulated luminescence (PSL) measurement was conducted to detect irradiated Cucurma longa and
Cariandrum sativum after dark storage for 1 day, 6 and 12 months. Using screening PSL, all the samples were
correctly distinguished between non-irradiated and irradiated samples at doses (1, 5 and 10 kGy) based on photon
count values. The PCs of irradiated samples showed a general trend of increase with increasing doses. However, the
signal intensity response to irradiation dose varied with samples and this is possibly attributed to the varying quantity
and quality of silicate minerals present in each samples. Among all samples, irradiated Cucurma longa (10 kGy)
showed the highest intensity with the value of 485178. All irradiated samples showed sensitivity index ratio of less
than 10 while non-irradiated samples showed higher value than 10. The results of this study provide a useful
database on the applicability of PSL technique for the detection of Malaysian irradiated food.

Keywords: Photo-stimulated luminescence, irradiated spices, photon count values

*Corresponding author’s email: anita@nuclearmalaysia.gov.my

Introduction
The use of food irradiation is increasing throughout the world as a suitable and safe process to reduce the number of
microorganisms and disinfestations various for products, such as spices, herbs, fish, meat, legumes and fruits. Spice
irradiation is the treatment with ionizing energy of the shelf life by the inactivation spoilage organisms and
improvement of the safety of spices by inactivating food-borne pathogens (Khan et al., 2008). According to
Rodríguez-Lazcano et al., 2013, irradiated spices in the range 1-10 kGy (medium doses) can eliminate
microorganism and pathogenic organisms. Studies on detection methods for irradiated food have been carried out on
the basic of the physical, biological and chemical in foods expose to ionizing during the last few decades (Delincée,
2002).

The PSL detection method can be used for rapid screening of many irradiated foods including herbs, seasoning and
shell fish (Sanderson et al., 1996). Therefore, the objective of this study was to investigate whether PSL
measurement can be employed to identify the status of irradiation of these two Malaysia spices. Then, it is an aim to
establish baseline data on PSL measurement of locally available spices that not examined before, for identification of
γ-ray irradiation treatment. Principal characteristics of sensitivity, dose response and signal stability during storage (6
and 12 months) were also studied.

Materials and Methods

Preparation of samples
Turmeric (Curcuma longa) and coriander (Cariandrum sativa) were purchased from Nasuha Herb and Spice Farm,
Johor Darul Takzim, Malaysia. The samples were exposed to γ-radiation at doses ranging 1 to 10 kGy using a cobalt-
60 gamma source (dose rate 37.4 Gray/min). All samples (non-irradiated and irradiated) were stored under dark
condition for 1 day, 6 and 12 months to confirm the stability of detection parameters.

29
 Measurement of photo-stimulated luminescence (PSL) This page is intentionally left blank

The PSL of the control and irradiated samples were measured using a PSL Irradiated Food Screening System at
Malaysian Nuclear Agency. The PSL measurements were performed according to EN 13751 (2009). About 3g of
samples were placed in 50 mm diameter disposable petri dish. The PSL signals (photon counts, PCs) of the samples
were recorded in the measuring mode at the rate of counts/60s and were presented at PCs/ 60s. All measurements
were done in triplicates. Samples producing signals below the lower threshold value of 700 counts/6s (T 1) are
categorized as non-irradiated (negative) while above the upper threshold of 5000 counts/60 s (T2) suggests irradiated
samples (positive). Samples with signal levels between the two thresholds (700-5000 counts/60s) were classified as
intermediate (M) and was further investigated using calibrated PSL to determine the PSL sensitivity of the sample.
Calibrated PSL was performed by subjecting all the spices to 1 kGy radiation dose after the initial PSL measurement,
and then re-measured.

Results and Discussion

The effect of radiation dose on signal intensity shows a general trend of increasing PCs with increasing dose up to 10
kGy. The PSL photon counts (PCs) for all samples, measured as a function of irradiation dose and storage periods,
are presented in Figure 1. The PCs of the 1 kGy irradiated turmeric and coriander measured were higher than the
upper threshold value (T2 =5000 counts/60 s) indicating there was an irradiation treatment. From Figure 1, it can be
seen that the PCs of all irradiated spices were higher than 5000 (T2) even after 12 months of storage, making it
possible to discriminate them from non-irradiated ones. The result also showed a trend of increasing PCs with
increasing dose in all the spices. The results of this study agree with the finding of 19 different irradiated herbs
reported by Sukdeb et al., 2010.

The other hand, the PCs of store in spices at dose 10 kGy, for 6 and 12 months storage were decrease 485178 to
88782 PCs (turmeric) and 241483 to 163524 PCs (coriander). Respectively, which were upper threshold values 5000
counts/60s indicated the samples irradiated (EN 13751: 2009). According to Yi (2000), if the PSL responses of the
materials isolated from irradiated food are significantly greater than those of non irradiated ones or if the fading of the
PSL response is low during the long-term storage, the PSL measurement may be suitable for the detection of food
irradiation.

Figure 1(a)

Figure 1(b)

Figure 1. Effect of irradiation dose on the signal intensity of turmeric (a) and coriander (b) after 6 and 12 months
storage.

Although PSL signals below the lower threshold (T1) are generally associated with non-irradiated materials, it can also
derive from low sensitivity irradiated materials. Calibrated PSL can help to distinguish these since irradiated samples
show only a small increase in PSL after re-irradiation whereas non-irradiated samples usually show a substantial
increase in PSL signal after irradiation (EN 13751, 2009). The index sensitivity of the spices after storage is shown in
Table 1. All non-irradiated spices produced index sensitivity above 10, with values ranging from 401 for turmeric to
375 for coriander. Index sensitivity value below 10 indicates irradiated whilst values above 10 showed non irradiated
samples (Liwen., 2013).

30
Differences in the index sensitivity values determined for the non-irradiated two spices were likely due to the variation
in the types and quantity of silicate materials such as feldspar and quartz contaminating the spices, as reported by
Ijaz et al. 2008. In contrast, the index sensitivity values of all irradiated spices This page is intentionally left blank

were below 10, confirming the treatment even after 12 months storage. Based on the results obtained, it was clear
that index sensitivity value is a better indicator than using the PSL response to discriminate between non-irradiated
and irradiated samples.

Table 1. Index sensitivity of irradiated spices after 6 months and 12 months storage.
Radiation dose Storage time
Spice
(kGy) 1 day 6 months 12 months
Turmeric Control 356 401 134
1 kGy 2 3 1
5 kGy 1 1 1
10 kGy 1 1 1
Coriander Control 198 189 375
1 kGy 1 1 1
5 kGy 1 1 1
10 kGy 1 1 1
* Calibrated PSL/initial PSL

Conclusion
Photo-luminescence detection method (PSL) has been shown to be can be used to detec the irradiation treatment of
the spices. The PSL signal intensity remained high even after 12 months of storage for irradiated turmeric and
coriander, indicating its suitability for detecting spicess and dry food ingredients which have long shelf-life. All kinds of
irradiated samples were successfully distinguished from the non-irradiated control samples analyzed in this study.
The results of this study provide a useful database on the applicability of PSL technique in detection of irradiated
spices. The availability of the method to detect irradiated food in Malaysia will be useful to the Ministry of Health for
enforcing labeling control in Malaysia.

Acknowledgment
The authors wish to express their gratitude to the Malaysian Nuclear Agency (MOSTI) for their support in this project.

References:

Delincée, H. 2002. Analytical methods to identify irradiated food - A review. Radiat. Phys. Chem. (63):455-458.
European Standard EN 13751. 2009 Foodstuff-detection of irradiated food using photostimulated luminescence.
Brussels, Belgium: European Committee for Standardization.
Ijaz, B., Byeong-Keun, K., Mi-Yeung, K., Jeongeun, L., Hyun-Ku, K. and Joong-Ho, K.. 2008. The screening and/or
identification of different types of irradiated eggs by analyzing photo-stimulated luminescence and
thermoluminescence. Food Control (19): 587–591.
Khan, H. M. and Bhatti. I. A. 2008. Thermoluminescen Methods for Detection Irradiated Black Pepper. J. Chem. Soc.
Pak. Vol, 30 (4).
Liwen, Z. L. and Tong, Yingqiao, J. and Fujun, B. 2013. A new criterion of Photostimulated Luminescence (PSL)
method to detect irradiated traditional Chinese medicinal herbs, Radiation Physics and Chemistry: 1-21.
Rodríguez-Lazcano, Y. Correcher, V. J. Garcia-Guinea and Cruz-Zaragoza, E. 2013. Gamma radiation-induced
thermoluminescence emission of minerals adhered to Mexico sesame seeds. Radiat. Phys. Chem. (83) 15-18.
Sanderson, D. C. W., Carmichael, L. A. Spencer, J. Q. and Naylor, J. D. 1996. Luminescence detection of shell fish.
In C. H. McMurray, E. M. Stewart, R. Gray, and J. Pearce (Eds.), Detection methods of irradiated foods, 139–
148. Cambridge: The Royal Society of Chemistry.
Sukdeb, P. B., Kim, K., Kim, W.Y., Kim, M.J., Ki, H.A., Kang, W.S., Kang I. H., Kan

31
 This page is intentionally left blank

g, S. J. and Song, J. M. 2010. Pulsed photostimulate and thermo-luminescence investigations of γ-ray irradiated
herbs. Food chemistry 122: 1290-1297.
Yi, S. D and Yang, J.S. 2000. The application of pulsed photostimulated luminescence (PPSL) methods for the
detection of irradiated foodstuffs. J. Food Sci. Nutri. (5): 136-141.

32
 IFRC 2017: 148-107 Food Safety and Quality

Trace Level Determination of Organophosphorus Pesticides in Fruit Samples Using Tetramethylguanidine-

Silica Nanoparticles as Solid Phase Extraction Sorbent

1*Veloo, K.V., 2Adam, F. and 2Batagarawa, M. S.

1Faculty of Agro-Based Industry, Universiti Malaysia Kelantan, 17600 Jeli, Kelantan, Malaysia.
2School of Chemical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia.

Abstract
A highly sensitive solid phase extraction method for determination of selected organophosphorus pesticides (OPPs)
in fruits were successfully developed using tetramethylguanidine-silica nanoparticles as a sorbent. Several effective
SPE parameters such as sample loading volume, type of elution solvent and elution solvent volume were optimised.
Under the optimum SPE design, the developed method exhibits good linearity (10-1000 pg mL-1), excellent LODs (1-
5 pg mL-1), and excellent precision (repeatability and reproducibility) of 1.2-2.9%. The LODs achieved were excellent
compared to LODs achieved with commercial SPE C18 (1-5 µg mL-1). The feasibility of the SPE-
tetramethylguanididine silica nanoparticles in the determination of the four targeted OPPs from real samples was
tested using spiked fruits samples (grape, green apple, red apple, orange). Recoveries ranging from 99-104% with
RSD 1.5-2.8% (n=5) were achieved for all real samples employed.

Keywords: Solid phase extraction, organophosphorus pesticides, tetramethylguanidine-silica nanoparticles, gas

chromatography-mass spectrometry.
*Corresponding author’s email: veni.v@umk.my

Introduction
Organophosphorus pesticides (OPPs) are widely used in the world especially in agricultural field as it is inexpensive
and their broad biological activity to pest. However, OPPs are also known among most dangerous environmental
pollutant, because of their stability, mobility and long term effects on living organisms. Their presence in water is
particularly danger as it can undergo transformations that can lead bioaccumulation to production of substances of
even greater toxicity (Bjorseth and Ramdahl, 1985). It can take part in physical, chemical and biological process and
act as cholinesterase inhibitors (Santodonato, 1997). The maximum admissible concentration of a single compound
of all pesticides in the environmental and drinking waters set by European Union (EU) are 0.1 µg L -1 and 0.5 µg L-1,
respectively (EPA, 1984; WHO, 2003). Therefore, sensitive sample preparation techniques are required for trace
analysis of OPPs in environmental sample. Solid phase extraction (SPE) is a well-known sample preparation
technique for trace analysis of complex samples. The common sorbent for SPE is C 8 and C18 (Frontanals et al., 2007;
Kloskowski et al., 2010). Nanotechnology has been one of the emerging technologies in material science. Due to the
ultra-small size, nanomaterials as per reported in literatures poses excellent physical and chemical properties (Tian et
al., 2013). Nanomaterial has been considered as the promising sorbent material for sample preparation and pre-
concentration technique. Siliceous nanoparticles, have large specific surface area and high adsorption capacity which
can help to increase the analytes concentration and reduce the matrix effect and thus, improve the detection
sensitivity of analytes. The silica material also can be modified at low temperature whereby, most of the materials can
be chemically modified by grafting with the desired functional groups when used as sorbent in sample preparation
techniques such as SPE (Yuan et al., 2011; Deng et al., 2012) and SPME (Li et al., 2009; Bagheri and Roostaie,
2012) to enhance the selectivity of extraction of the targeted analyte in various samples. Therefore, in this work,
tetramethylguanidine-silica nanoparticles which was successfully proven as an efficient and recyclable catalyst for the
synthesis of propylene carbonate (PC) from propylene oxide (PO) and carbon dioxide (Adam and Batagarawa, 2013),
has been further extended as a sorbent for solid phase extraction.. Tetramethylguanidine-silica nanoparticles with
specific surface area of 527 m2 g−1 were used for analysis of four targeted OPPs in water samples using GC-MS.
Although there are many reported developments of new sorbents for SPE, this is the first report on the application of
silica based nanomaterial with tetramethylguanidine as functional monomer.
33
Materials and Methods
Chemicals and reagents
Pesticides standards (purity 99.0%) (methidathion, chlorpyrifos, profenofos and diazinon and were from Dr.
Ehrenstorfer (Ausburg, Germany). Acetonitrile, methanol, dichloromethane, acetone, ethyl acetate, ethanol and
hexane were from QReC (99.0%). Distilled water was distilled using water stills (Favorit W4L, Malaysia).

Instrumentation
Separations of OPPs were carried out using a Elite-5 MS fused capillary GC column (30 m × 0.32 mm (ID) and × 0.25
µm film thickness) with the Clarus 600 GC system which comprises of Clarus 600 MS. Helium was used as carrier
gas at a flow rate of 1.0 mL min-1. The injection port and detector temperature was set at 250ºC and 300ºC,
respectively. Gas chromatography temperature profile was set as follows: initially temperature at 80ºC (held 1 min)
ramped at 15ºC min-1 and increased to 280ºC (held 5 min). Sample (1 µL) was injected manually into the injection
port under split-less mode (with the solvent delay of 3.00 min).

Solid phase extraction procedure

SPE was carried out using SPE manifold from Supelco (Bellefonte, PA, USA). 100 mg of tetramethylguanidine-silica
nanoparticles was placed into an empty 3-mL polypropylene SPE tube (Supelco, Shanghai, China) and manually
packed. PTFE frits (Supelco, Shanghai, China) were placed above and below the sorbent bed. Before the samples
were processed, the cartridge was preconditioned with 1 x 5 mL of MeOH followed by 1 x 10 mL double deionised
water. 10 mL of spiked water sample with mixture of OPPs spiked at known concentration was passed through.
Retained analytes in the cartridge were eluted with 3 mL methanol. Eluted fraction was dried under a stream of
gentle nitrogen gas until all the solvent evaporated. Finally, the residue was reconstituted in 100 µL acetonitrile prior
to GC-MS analysis.

Sample preparation
Stock solution of each OPPs (200 mg L-1) were prepared in and methanol and stored in the dark at 4ºC. Working
solutions containing the mixture of OPPs were prepared at different range of concentrations by spiking them in
methanol. All standard solutions were stored in dark at 4◦C when not in use. Real samples (green apple, red apple,
strawberry, and grapes) were were purchased from local market and were used without washing. Analytical portion of
real sample (12.0 g) were weighed into a 50 mL centrifuge tubes and 20 mL of acetonitrile was added respectively
into each tubes. Then the samples were left to be extracted for 30 min in an ultrasonic bath. After ultrasonification, the
aliquot was filtered throughout a Whatman filter paper (0.45 µm).

Results and discussion

SPE optimization
Selected SPE parameters namely sample loading volume, type and elution solvent volume were carefully studied in
order to attain an optimum SPE design. Sample loading volume is the maximum volume of samples which can be
loaded in a SPE system to ensure that the breakthrough volumes of analytes are not exceeded. Sample volumes
ranging between 1-100 mL were examined. Based on the results, the peak area of OPPs increases as the sample
loading volume increases. This shows the remarkable enrichment capacity of sorbent. However 10 mL sample
loading volume was selected as the best parameter. This is to save the analytical time and furthermore 10 mL of
sample loading volume was observed to be sufficient to obtain high enrichment with the sensitivity of GC-MS.
Selecting appropriate solvents for elution is crucial as elution is an important factor in recovery and enrichment of
analyte. Since the selected OPPs were of different polarities, desorption were carried out with different solvent
ranging from non-polar to polar. Methanol (MeOH) was chosen as the best eluting solvent as it gave the highest peak
area for all of the targeted OPPs. The effect of eluting solvent volume was studied with 1-10 mL of MeOH and 3 mL of
MeOH was selected as the best eluting volume, as it gave the highest peak area for all the analyte; meaning that the
volume is sufficient to recover all of the OPPs studied. Therefore, 10 mL of mixture of OPPs as sample loading
volume and 3 mL of MeOH as eluting solvent were found to be the optimum condition of SPE along with 100 mg of
tetramethylguanidine-silica nanoparticles which were kept constant throughout the SPE optimization studies.

34
Method validation
Table 1 shows the analytical performances of the proposed SPE-tetramethylguanidine-silica nanoparticles method for
the determination of the selected OPPs. Good linearity range, (10-1000 pg mL-1) was obtained for the SPE-
tetramethylguanidine-silica nanoparticles method with correlation of determination, r2> 0.9998 for methidhation,
diazinon, profenofos and quinalphos. Good linearity was also obtained with the use of SPE-C18 cartridge for all of
targeted OPPs with correlation of determination, r2> 0.9996. The LODs were calculated by equating the S/N of the
lowest detectable concentration to a value of 3. Low LODs down to trace level (part per trillion) (1-5 pg mL-1) were
achieved using SPE-tetramethylguanidine-silica nanoparticles compared to 1-5 µg mL-1 which was obtained with the
usage of commercial SPE C18. This showed that the developed method is highly sensitive as the maximum residue
levels (MRL) as established by World Health Organization (WHO) International Standards for drinking water is less
than 0.0002 mg L-1 (WHO, 2003). The inter- and intra-day precision studies were carried out for five repeated
extractions per day and consequently for five days using water samples fortified at 50 pg mL -1 of each OPPs for SPE-
tetramethylguanidine-silica nanoparticles and 50 µg mL-1 of each OPPs for SPE-C18 since their linearity range
obtained were different. Excellent RSDs values (based on the peak areas of the five replicate extractions) were
obtained from the intra- and inter-day precision studies were 1.4-2.1% and 2.1-2.9%, respectively using SPE-
tetramethylguanidine-silica nanoparticles compared to the intra- and inter-day precision 6.4-8.1% and 7.1-9.9%
respectively using SPE-C18. The SPE-tetramethylguanidine-silica nanoparticles cartridge was found to be reusable for
all the 25 repeated extractions (inter- and intra-day precision studies).

Table 1. Analytical performances of the SPE-tetramethylguanidine-silica nanoparticles method for the determination
of OPPs compared to SPE-C18.

OPPs SPE-tetramethylguanidine- SPE-C18

Corr. of silica nanoparticles
*RSD *RSD aLOD b Corr.of **RSD **RSD aLOD bLOQ
det. (%, (%, det. (%, (%,
(pg mL-1) L (µg mL- (µg mL-1)
(r2) n=5) n=25) O (r )
2 n=5) n=25 1)
Diazinon 0.9999 6 Q 0.9996 10 41
Quinalphos 0.9998 2.5 3.2 5 2 ( 0.9995 6.7 8.7 11 19
Chlopyrifos 0.9999 2.1 3.3 3 20 p0.9994 6.8 8.3 13 11
Methidathio 0.9997 2.4 3.5 8 2 g0.9994 7.5 8.3 12 14
n 2.7 3.5 31 7.6 8.9
Application to real sample 3 m
Since, no targeted OPPs could be detected under the optimal L SPE-tetramethylguanidine-silica nanoparticles method
-
developed in this work, thus to assess matrix effect, the tap grape, green apple, red apple, orange were spiked at 50
1
pg mL-1 (SPE-tetramethylguanidine-silica nanoparticles) and at 50 µg mL-1 for SPE-C18 to evaluate the matrix effect
and applicability of the developed analytical method towards ) real samples. Excellent recoveries ranging from 98-
104% were achieved for all the employed real samples using SPE-tetramethylguanidine-silica nanoparticles
compared to 75-78% using SPE-C18, making this method highly feasible for trace analysis of real aqueous matrices of
environmental origin without much interference of matrix effect.

Conclusion
A simple, precise, accurate and highly sensitive SPE method has been successfully developed. Under the optimized
conditions, the proposed method showed good linearity, repeatability, and reproducibility. Low LODs down to pg mL-1
and excellent recoveries in fruits samples were achieved, making this method suitable for trace level analysis of
OPPs without much matrix effect. The simple preparation, cost effective, porous nature and large surface area
exhibited by tetramethylguanidine-silica nanoparticles is a promising new sorbent for sample preparation and clean-
up device.

35
Acknowledgement
KVV would like to thank Universiti Sains Malaysia for the Post-Doctoral Fellowship and Fundamental Research Grant
Scheme (FRGS) for funding of the research.

References

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Ali, W.H., Derrien, D., Alix, F., Pérollier, C., Lépine, O., Bayoudh, S., Chapuis Hugon, F. and Pichon, V. 2010. Journal
of Chromatography A 1217: 6668.
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Bjorseth, A. and Ramdahl, T. 1985. Handbook of Polycyclic Aromatic Hydrocarbon in Sources and Emission of PAH.
Vol. 2, p. 1-20. Marcel Dekker, New York, Basel.
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Advances 2: 956.
Frontanals, N., Marcé, R. M. and Borrull, F. 2007. Journal of Chromatography A 1152: 14.
Li, T., Xu, J., Wu, J. H. and Feng, Y. Q. 2009. Journal of Chromatography A 1216: 2989.
Santodonato, J. 1997. Chemosphere. 34: 835.
United States Environmental Protection Agency. 1984. Guidelines Establishing Test Procedures for the Analysis of
Pollutants. Proposed Regulations, (49) 209. Federal Register, EPA, Washington, DC.
World Health Organization. 2003. Background Document for Preparation of WHO Guidelines for Drinking-water
Quality (WHO/SDE/WSH/03.04/59), World Health Organization, Geneva.
Yuan, L. Y., Liu, Y. L., Shi, W. Q., Lv, Y. L., Lan, J. H., Zhao, Y. L. and Chai, Z. F. 2011. Dalton Trans. 40: 7446

36
 IFRC 2017: 149-149 Food and Safety and Quality

Effect of Thickness of Antimicrobial Film-Coated Paper for Food Packaging on Antimicrobial Agent Migration
Rate and Biodegradability
*Mustapha, F.A., Jai, J., Sharif, Z.I.M., Yusof, N.M.

Department of Chemical Engineering, Faculty of Chemical Engineering, Universiti Teknologi MARA, 40450, Shah
Alam, Selangor, Malaysia.
Abstract
Even though paper and paperboard are the common materials used in food packaging industries because of its
recyclability, however papers are commonly laminated with petroleum-based plastic such as polyethylene to enhance
its barrier and mechanical properties. Problem arising in usage of plastic from petroleum sources are environmental
pollution during manufacturing process and accumulation of solid waste in landfill. In this study, polyethylene-coated
paper packaging was replaced with biodegradable antimicrobial film-coated paper packaging. The biopolymer
sources of the film based were cassava starch and carboxymethylcellulose (CMC) enriched with turmeric oil as
natural preservatives. Response Surface Methodology (RSM) was used to evaluate two independent variables (A:
coating thickness and B: turmeric oil concentrations) on the antimicrobial agent migration rate and biodegradability of
the coated-films. The films thickness was controlled by varying the gap distance of blade during casting process in
range of 0.5 to 2.0 mm and the concentration of TO in range of 0.5 to 30 ppm of film-forming solutions. Effects of film
thickness on migration rate of antimicrobial agent from turmeric oil (TO) at different concentration were evaluated on
antimicrobial activity of bread wrapped with film-coated paper and solubility test of the film in conjunction with
ultraviolet-visible spectroscopy (UV-VIS). Biodegradability of different thickness coated-paper was studied by
conducting biodegradation in soil test. The result showed that the antimicrobial film can extend the bread shelf life
longer compared to commercial packaging. Meanwhile, coating thickness significantly affects the biodegradation rate
as value of P>F of A was less than 0.05 Thus, the biodegradable antimicrobial film could extend the shelf life of bread
and used as an alternative technology for biodegradable polymer in food packaging.
Keywords: Paper Food Packaging, Biodegradable, Antimicrobial, Turmeric Oil
*Corresponding author’s email: alia534492@gmail.com

Introduction
Microbial development in foods commonly started on foods surface and thus most of the research has been done with
the goal of joining antimicrobial substances into the food packaging to keep up quality and delay the time span of
usability of these foods (Appendini & Hotchkiss, 2002). The system of packaging can inhibit the microbial
development in non-sterilized as well as pasteurized foods and avoid contamination of post processing. It is essential
to control the migration rate of antimicrobials from the package to the food products. The discharged antimicrobials
must have the capacity to control microbial development and to keep up the antimicrobial level over the base
inhibitory concentration (Seiler, 2000).
Objective of the study is to study the effect of antimicrobial film thickness that been coated on Kraft paper used as
food packaging on migration rate of antimicrobial agent and their biodegradability.
Materials and Methods
3.1. Materials used in film-forming solution formulations was Malaysia originated cassava starch, CS (Cap Kapal
ABC.Co) which was applied as based polymer for coating film, carboxymethylcellulose, CMC (Waris NOVE,
Malaysia) from oil palm empty fruit bunch which was used as composite biopolymer together with CS as filler and
thickener, glycerol (Merck, Germany) as plasticizer and turmeric oil, TO (Soul, pure essential oil) as antimicrobial
agent sources. Unbleached Kraft papers (brown paper) were used as paper food packaging material.
3.2. Film-forming preparation was done by cassava starch gelatinization (5% w/v of distilled water) at temperature of
75°C ± 5°C for 20 mins under continuous stirring at 500 rpm before plasticized with glycerol (30% w/w of starch).
0.5g of CMC was dissolved in 100mL distilled water at room temperature. Both of the solutions were mixed and

37
slowly stirred at 250rpm for 20 minutes at 40°C to remove air bubble and to mix well the mixtures. In order to obtain
uniform thickness of the film-coated paper, bar coating (casting) technique was applied. The film-forming solutions
were casted at volumes of 3mL on flat Kraft paper (20cm length × 9cm width) using automatic film casting machine at
speed of 1cm/s. After that, the wet-coated paper was dried in oven at temperature 50°C for 2 and half hours. The
samples were stored for 14days in controlled conditions.
3.3. Shelf life of bread was determined by observing the growth of mold on the food surface. 5 cm length × 5 cm width
in dimensions of bread were wrapped inside the coated paper and commercial packaging. The growth of mold on the
bread was observed daily.
3.4. Migration rate of antimicrobial agents from film coated on Kraft paper into water as a moisture-food simulant was
quantified by using UV-Vis spectrometer (PerkinElmer, Lambda 750). The film-coated Kraft paper samples of 2.5 cm
length × 2.5 cm width in dimensions were immersed in 30mL distilled water in a closed container for 2 hours. The
concentration of turmeric oil antimicrobial agent that was extracted from the film into 30mL of distilled water was
quantified by standard curve concentration of turmeric oil from UV-Vis spectroscopy. The migration rate was
measured at 297 nm corresponding to the maximum wavelength absorption of turmeric oil. The test was conducted at
room temperature and the intensity of the peak was tabulated at 120 minutes.
3.5. Biodegradation in soil test was conducted by cut the samples into 2.5 cm height × 2.5 cm width, weighed and
buried under the soil with depth of 13cm. The test was done by exposed the buried samples to rain and sun for one
weeks. After one week, the samples were removed and dried at 100°C for 15 minutes as the wet soil was attached to
the samples. The dry soil was removed from the samples and weighed. The changes in samples weight were
recorded. The average temperature of the weeks was 29°C and the rainfall occurred after 5 days of the test was
started with average precipitation of 3.05mm (“Shah Alam, Malaysia Forecast | Weather Underground,” 2017)
3.6. Response Surface Methodolgy (RSM) was employed to study the data of 16 experimental runs using central
composite design (CCD) method with two independent factors which were coating thickness (mm, A) and
concentration of TO (ppm, B) and two responses; antimicrobial agent migration rate (ppm/min) and biodegradation
rate (mg/day) by using Design Expert software (version 6.0). All listed experiments were conducted randomly to
minimize the effects of uncontrolled factors that may introduce bias.
Results and Discussions
Aspergillus sp was one of the microorganism that cause bread spoilage (Saranraj & Geetha, 2012). Study by Singh et
al. (2012) on essential oil of C.longa rhizome (turmeric) identify that it can inhibit growth of Aspergillus niger with
minimum inhibitory concentration of 6.7 μL/L (Singh et al., 2012). The shelf life of bread was studied by wrapped the
bread in both the commercial packaging and the antimicrobial film coated on Kraft paper for food packaging as shown
in Figure 1. Similar behaviour was found in this study where all the film that been incorporated with different
concentration of turmeric oil extends shelf life of bread by 7 days after the expiration date compared to commercial
packaging as there was no growth of mold and spores observed.

a) b)

Figure 1 Comparison study on shelf life of bread after 7 days of the expired date a) Commercial packaging b)
Antimicrobial film-coated on Kraft paper food packaging (5mm thickness, 0.5ppm of TO)

Analysis of variance (ANOVA) was studied to analyse the significance and fitness of the two independent factors for
the antimicrobial migration rate and biodegradability rate as shown in Table 1 and Table 2, respectively. The model F-
value of 7.50 (migration rate) and 9.51 (biodegradability rate) as shown in Table 1 and Table 2, respectively indicates
a significant model. There was only 0.68% and 0.15% chance that a “Model F-Value” this large could occur due to
38
noise. Value of P>F less than 0.05 indicated that the model was significant and both studies showed that A
(thickness) were significant model terms which means that the responds was significantly affected by coating
thickness instead of concentration of TO. Besides, both of the model was fit to be used in determination of optimum
condition as the p-value for lack of fit was higher than 0.05 as confirmed by Jamil et al. (2016) studies (Jamil, Halim, &
Sarbon, 2016).
Table 1 Analysis of variance (ANOVA) after choosing linear model for antimicrobial agent migration rate at 120 mins
Source Sum of Squares DF Mean square F Value P>F
Model 0.066 2 0.033 7.50 0.0068 significant
A 0.060 1 0.060 13.66 0.0027
B 5.906E-003 1 5.906E-003 1.33 0.2688
Residual 0.058 13 4.426E-003
not
Lack of Fit 0.039 2 7.734 E-003 3.28 0.0662
significant
Pure Error 0.019 8 2.358E-003
Cor Total 0.12 15
R2= 0.5356, A= Thickness (mm), B= TO concentration (ppm)
Table 2 Analysis of variance (ANOVA) after choosing quadratic model for biodegradability rate
Source Sum of Squares DF Mean square F Value P>F
Model 62.58 5 12.52 9.51 0.0015 significant
A 51.54 1 51.54 39.15 <0.0001
B 3.95 1 3.95 3.00 0.1138
A2 0.00 1 0.00 0.00 1.0000
B2 1.74 1 1.74 1.32 0.2766
AB 5.26 1 5.26 4.00 0.0735
Residual 13.17 10 1.32
not
Lack of Fit 5.83 2 2.92 3.18 0.0962
significant
Pure Error 7.33 8 0.92
Cor Total 75.75 15
R2= 0.8262, A= Thickness (mm), B= TO concentration (μL/L of solutions)
A 3-dimensional (3D) response graph of the regression coefficient illustrated the effect of A (thickness, mm) and B
(concentration of TO, ppm) on the migration rate of antimicrobial agent and biodegradability test was shown in Figure
2. Referring to the Figure 2(a), migration rate increased as the thickness and TO concentration value was increased.
This may attribute to the presence of higher amount of TO as the film thickness increased.

a) b)

Figure 2 Response surface plots for a) Migration rate of antimicrobial agent (ppm/min and b) Biodegradability rate
(mg/days) as function of coating thickness and concentration of TO.
39
Meanwhile, referring to Figure 2(b), the biodegradation rate was directly proportional with coating thickness and
increased as TO concentration increased but decreased when TO concentration exceeded 15ppm. This is because,
presence of TO concentration higher than 15ppm may change the hydrophilic properties surface of the film which
reduce water uptake and thus reduced the biodegradation rate. From the optimization analysis done using Design
Expert software found that the minimum migration rate of antimicrobial agent and maximum biodegradation rate can
be achieved with coating thickness at 0.89mm and 12.46 ppm concentration of TO. The estimated migration rate was
0.10ppm/min and biodegradation rate was 11mg/days.
Conclusions
From this study, it can be concluded that antimicrobial film based cassava starch and carboxymethylcellulose
incorporated with turmeric oil can be used as alternative green technology for food packaging. Shelf life of bread can
be extended longer by minimize the migration rate of antimicrobial agent and in term of waste disposal in soil, it
degraded faster compared to the commercial packaging (8mg/day). However, further analysis on the functional
properties of the film such as on its mechanical properties and barrier properties should be studied in future to satisfy
the requirements of food packaging system.
Acknowledgement
Support from Universiti Teknologi MARA, Shah Alam, Selangor, Malaysia and financial aid from 600-IRMI/DANA
5/3/LESTARI (30/2015) are gratefully acknowledged.
References
Appendini, P., & Hotchkiss, J. H. (2002). Review of antimicrobial food packaging. Innovative Food Science and
Emerging Technologies, 3(2), 113–126.
Jamil, N. H., Halim, N. R. A., & Sarbon, N. M. (2016). Optimization of enzymatic hydrolysis condition and functional
properties of eel (Monopterus sp.) protein using response surface methodology (RSM). International Food
Research Journal, 23(1), 1–9.
Saranraj, P., & Geetha, M. (2012). Microbial Spoilage of Bakery Products and Its Control by Preservatives, 3(1), 38–
48.
Seiler, D. A. . (2000). Modified atmosphere packaging of bakery products. In A. L. Brody (Ed.), Controlled/ Modified
Atmosphere/Vaccum Packaging of Foods (pp. 119–133). Trumball: Food and Nutrition Press.
Shah Alam, Malaysia Forecast | Weather Underground. (2017). Retrieved June 14, 2017, from
https://www.wunderground.com/my/shah-alam
Singh, K., Sankar, B., Rajesh, S., Sahoo, K., Subudhi, E., & Nayak, S. (2012). Chemical Composition of turmeric oil
(Curcuma longa L. cv. Roma) and its antimicrobial activity against eye infecting pathogens. Journal of
Essential Oil Research, 23(6), 11–18.

40
 IFRC 2017: 152-168 Food Safety and Quality

Antioxidant Activity and Estragole Content Of Ethanolic and Methanolic Extract of Fennel (Foeniculum
Vulgare Mill.) and Nutmeg (Myristica Fragrans Houtt) and its Risk Assessment Using Margin of Exposure
(MOE).

* Martati, E., and Akmalina, M.A.

Study Program of Food Science and Technology, Department of Agricultural Product Processing, Universitas
Brawijaya, Jalan Veteran 64145 Malang, Indonesia

Abstract
Botanicals and its derived products should be considered with care since some botanicals are known containing
genotoxic and carcinogenic compounds such as alkenylbenzenes. The aims of this study were to know the total
phenolic content (TPC), total flavonoid (TFC) and the concentration of alkenylbenzene of estragole in ethanolic and
methanolic extract of fennel (Foeniculum vulgare Mill.) and nutmeg (Myristica fragrans Houtt). Furthemore,
concentration of estragole in the extract and simulation of intake of the extract are used to do risk assessment using
Margin of Exposure approach (MOE). The results showed that TPC, TFC and capacity of antioxidant of methanolic
extract of fennel is higher than that of ethanolic extract. For nutmeg, TFC of ethanolic extract is higher than that of
methanolic extract, whereas, TPC and antioxidant capacity are not significant different. Simulation consumption of
methanolic extracts of fennel > 36.2 mg/60 kg bw per day and ethanolic extracts > 8.4 mg/60 kg bw per day; gives an
MOE < 10,000, so it was considered as a high priority for risk management actions and would be of high concern
from a public health point of view.

Keywords: fennel, nutmeg, estragole, risk assessment, margin of exposure

*Corresponding author’s email: erryana_m@ub.ac.id

Introduction
Spices either raw material or its isolated have been used as traditional medicine, flavoring agents, food supplement
and food preservation for many years. Some spices or botanicals are consumed to maintain and promote health or
reduce risk of diseases. On the other side, some spices or herbs contain genotoxic and carcinogenic compounds
(Berg et al., 2011; Rietjens et al., 2008). In some countries, the using of alkenylbenzenes estragole, methyleugenol,
safrole and β-asarone as flavorings in food products is regulated and prohibited, while, some plant food supplements
containing these ingredients are not regulated (Berg et al., 2011). Fennel (Foeniculum vulgare Mill.) contains
estragole (Berg et al., 2011; Leela, 2008). Methanolic basil extract, a herb containing estragole, inhibit
sulfotransferase catalyzed bioactivation and DNA adduct formation in HepG2 cells exposed of estragole (Jeurissen et
al., 2008). Nutmeg (Myristica fragrans Houtt) is commonly used as important flavour and fragrance ingredients,
having immunomodulatory effects, used as antimicrobial activity and antimutagenic activity (Gunasekar et al., 2012;
Akinboro et al., 2011; Checker et al., 2008; Chung et al., 2006). Fennel and nutmeg contain alkenylbenzenes
compounds which high intake may pose a potential risk for human.
The aims of this study were to know the total phenolic content (TPC), total flavonoid (TFC), antioxidant capacity and
risk assessment of estragole using Margin of Exposure (MOE) in ethanolic and methanolic extract of fennel and
nutmeg.

Material and Methods

3.1 Extract preparation
Fennel and nutmeg were obtained from Griya Wisata Herbal, Batu, East Java Indonesia. Samples were milled, sieved
using 30 mesh, packed in a plastic bag and stored at a refrigerator. Sample of 10 g was dissolved in either 100 ml of
ethanol 96% or methanol. The mixture were agitated in a shaker water bath for 24 h. Then, It was filtered through
paper filter. The extraction was repeated two times with the same procedure. The filtrate was evaporated in a rotary
vapor at 40°C. The extract was stored at -20°C until analysis.
41
3.2. Moisture content of the raw material was measured according AOAC.

3.3.Total phenolic content was determined using Folin-Ciocalteu reagents and the absorbance was measured at 745
nm (Singleton and Rossi, 1965). The calibration curve was made using galic acid.

3.4.Total flavonoid content was measured according to Chang et al. (2002)

3.5. The antioxidant activity of the extract was measured using DPPH radical scavenging activity method (Brand-
Williams et al., 1995).

3.6. HPLC analysis

Estragole was analyzed on a HPLC (Shimadzu) using column of Ultima C1
ultrapure water and acetonitrile (Martati et al., 2011).

3.7. Risk assessment of estragole

Calculated MOE is BMDL10 divided by intake. Simulation was performed to know how much consumption of the
extract of nutmeg and fennel to have MOE of <10,000 (a high priority of risk management) or >10,000 (a low priority
of risk management). In this simulation, MOE of 11,000 is used to calculate the intake of the extract which result a low
priority of risk management and MOE of 1000 which result a high priority of risk management. The BMDL10 for
estragole was calculated using BMD software version 2.4 based on the data Miller et al. (1983).

3.8. Data analysis

The data obtained was expressed as mean ± standard deviation (except the estragole content). The differences of
the parameters measured between methanolic and ethanolic extract was tested with T test using Microsoft Excel
2016.

Result and Discussion

Table 1 shows that TPC and TFC of methanolic fennel extract are higher than that of ethanolic extract. Methanol may
be used as an solvent in food, provided that the residue of methanol in the food does not exceed 10 ppm (European
Union).The phenolics determined the antioxidant activity (Do et al., 2014). Methanolic extract of fennel has antioxidant
capacity higher (lower IC50) than that of ethanolic extract.

Table 1. Results analysis of methanolic and ethanolic fennel and nutmeg extract.
Fennel Nutmeg
Raw Methanolic Ethanolic Raw Methanolic Ethanolic
extract Extract extract Extract
Moisture 6.64 ± 0.10 n.a n.a 4.81 ± 0.05 n.a n.a
content
(%)
TPC (mg 3.13 ± 0.04 13.95 ± 1.34a 5.80 ± 0.13b 22.06 ± 1.74 47.81 ± 3.49 a 51.77 ± 3.06 a
GAE/g)
TFC (mg 12.71 ± 0.43 30.07 ± 2.39a 16.80 ± 1.65b 62.67 ± 3.04 32.32 ± 2.41a 48.58 ± 2.43 b
QE/g)
IC50 (ppm) 6033.14 1587.01 ± 204.76a 2409.66 ± 133.14b 466.58 228.79 ± 21.96 a 253.46 ± 22.68 a
Estragole n.a 0.51 2.22 n.a n.d n.d

Note: n.a: not available

n.d: not detected
Estragole analysis was measured one repeat

42
In case of fennel, there is a correlation between TPC and TFC and antioxidant activity. The presence of water in
ethanol 96% decreases TPC and TFC in the extract. This result agrees with Do et al. (2014) on extraction of
Limnophila aromatica using aqueous methanol and ethanol. Ethanolic nutmeg extract has higher TFC than that of
methanolic extract. TFC and antioxidant activity of methanolic nutmeg extract are not statistically different with those
of ethanolic extract. Gupta et al. (2013) showed that TFC of ethanolic nutmeg extract was higher than that of
methanolic extract. Nutmeg extract has higher antioxidant capacity than fennel extract.

Table 2. Results from BMD analysis based on the data of induction of hepatoceluler in mice administered 0, 2300 or
4600 mg/kg bw diet containing estragole (Miller et al., 1983)
Model No. of Log p-value Accepted BMD10 (mg/kg BMDL10
parameters Likelihood bw/ day) (mg/kg bw/
day)
Null 1 -100.10
Full 3 -62.21
Gamma 1 -62.74 0.59 Yes 7.62 6.15
Logistic 2 -70.99 0 No 22.43 17.82
LogLogistic 1 -62.21 0.10 Yes 4.46 3.11
LogProbit 2 -62.21 1 Yes 0.95 4.44
Multistage 1 -62.74 0.59 Yes 7.62 6.15
Multistage 1 -62.74 0.59 Yes 7.62 6.15
cancer
Probit 2 -70.70 0 No 21.71 17.40
Weibull 1 -62.74 0.59 Yes 7.62 6.15
Quantal-linear 1 -62.74 0.59 Yes 7.62 6.15

The presence of estragole or other compounds which are carcinogenic and genotoxic in spices cannot be avoided
resulting in a certain level daily exposure. The MOE approach needs reference point of BMDL10. The BMDL10 is a
lower confidence limit of the benchmark dose level causing 10% extra tumor incidence above background level
(BMD10) (Berg et al., 2011). It is necessary to predict the cancer risk at low dose level representing realistic human
dietary intake because estragole is genotoxic and carcinogenic. Table 2 shows the calculated BMDL10 value of
estragole is 3.11-6.15 mg/kg bw. Simulation of MOE of 11,000 and 1,000 resulted an intake of methanolic fennel
extract of 3.15 and 36.2 mg/60 kg bw day, respectively. Simulation of MOE of 11,000 and 1,000 resulted an intake of
ethanolic fennel extract of 0.7 and 8.4 mg/60 kg bw day, respectively.

Conclusion
The present study showed that type of solvent affects the amount of bioactive compounds, therefore, it is determined
the antioxidant capacity. Consumption of spices or herbs should consider both the health benefit and the risk potential
to the health.

References

Akinboro, A., Mohamed, K., Asmawi, M., Sulaiman, S. and Sofiman, O. 2011. Antioxidants in aqueous extract of
Myristica fragrans (Houtt.) suppress mitosis and cyclophosphamide-induced chromosomal aberrations in
Allium cepa L cell. Journal of Zhejiang University - Science B 12(11), 915-922.
Berg, S.J.P.L.v.d., Restani, P., Boersma, M.G., Delmulle, L.,and Rietjens, I.M.C.M., 2011. Levels of genotoxic and
carcinogenic compounds in plant food supplements and associated risk assessment. Food and Nutrition
Sciences 2, 989-1010.
Brand-Williams, W., Cuvelier, M.E., and Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity.
Lebensm. Wiss. Technol. 28, 25–30.
43
Chang C.C, Yang M.H, and Wen H.M. Estimation of total flavonoid content in propolis by two complementary
colorimetric methods. J Food Drug Anal 2002;10:178-82.
Checker, R., Chatterjee, S., Sharma, D., Gupta, S., Variyar, P., Sharma, A. and Poduval, T. B. 2008.
Immunomodulatory and radioprotective effects of lignans derived from fresh nutmeg mace (Myristica fragrans)
in mammalian splenocytes. Int. Immunopharmacol. 8(5), 661-669.
Chung, J. Y., Choo, J. H., Lee, M. H. and Hwang, J. K. 2006. Anticariogenic activity of macelignan isolated from
Myristica fragrans (nutmeg) against Streptococcus mutans. Phytomedicine 13(4), 261-266.
Do, Q.D., Angkawijaya, A.E., Tran-Nguyen, P.L., Huynh, H.L., Soetaredjo, F.E., Ismadji, S. and Ju, Y.H. 2014. Effect
of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of Limnophila
aromatica. Journal of Food and Drug Analysis (22): 296-302.
Gunasekar, Geemon, K. and Mariwala, S. J. 2012. Health benefits of bioactive molecules from spices and aromatic
plants.
Gupta,S.D, Bansal, V.K., Babu,V., Maithil, N. 2013. Chemistry, antioxidant and antimicrobial potential of nutmeg
(Myristica fragrans Houtt). Journal of Genetic Engineering and Biotechnology 11, 25–31.
Jeurissen, S.M.F., Punt, A., Delatour, T.,and Rietjens, I.M.C.M., 2008. Basil extract inhibits the sulfotransferase
mediated formation of DNA adducts of the procarcinogen 1'-hydroxyestragole by rat and human liver S9
homogenates and in HepG2 human hepatoma cells. Food Chem. Toxicol. 46, 2296-2302.
Leela, N.K., 2008. Nutmeg and mace, in: V.A. Parthasarathy, B. Chempakam, T.J. Zachariah (Eds.), Chemistry of
spices. CABI, Wallingford, pp. 165-189.
Miller,E,C. Swanson, A. B., Phillips, D.H., Fletcher, T.L., Liem, A. and Miller,J.A., 1983. Structure-activity studies of
the carcinogenicities in the mouse and rat of some naturally occurring and synthetic alkenylbenzene
derivatives related to safrole and estragole,Cancer Research, Vol. 43, No. 3, 1983, pp. 1124-1134.
Rietjens, I.M.C.M., Slob, W., Galli, C., and Silano, V., 2008. Risk assessment of botanicals and botanical preparations
intended for use in food and food supplements: Emerging issues. Toxicol. Lett. 180, 131-136.
Singleton, V. L.; Rossi, J. A. 1965.Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid
reagents. Am. J. Enol. Vitic.16, 144-158.

44
 IFRC 2017: 159-116 Food Safety and Quality

Selection of Lactic Acid Bacteria Can Reduce the Cyanide Compound on The Processing Yam Flour
(Dioscorea Hispida Dennst.)
Winarti, S., Murtinngsih and Amalia, S.R.
Food Technology Program, Enginering Faculty, University of Pembangunan Nasional “Veteran” Jawa Timur, Jl.
Raya Rungkut Madya, Surabaya, Indonesia, 60294

Abstract
The study was carried out to select lactic acid bacteria can reduce effectively the cyanide compounds in the
processing yam flour. Lactic acid bacteria used in the study were Lactobacillus plantarum FNCC-0027; Lactobacillus
casei FNCC-90; Lactobacillus acidophillus FNCC-0051; Bifidobacterium bifidum BRL-130; Bifidobacterium breve
BRL-131. Data were analyzed using One Way ANOVA and continued with Tukey's test (HSD). Lactobacillus
plantarum FNCC-0027 as lactic acid bacteria the most effectively reduced cyanide compound in the yam flour was of
41.98% for 24 hours. At 72 hours fermentation can reduce cyanide compound from 411.65 ppm to 23.917 ppm. This
level of cyanide is safe. Reducing sugar of yam flour was 0.06%.

Keywords: cyanide, intoxicating yam, Dioscorea, Lactobacillus plantarum

*Corresponding author’s email: swin_tpupn@yahoo.co

Introduction
The yam plant produce the tubers contain edible nutrients that are good enough, but its beneficial is limited. This
is because in the yam tubers contain poisonous compounds, namely the Glycoside cyanogenic. Its compound is
poisonous in the form of free acid cyanide (HCN). Removal of cyanide poison in yam tubers with traditionally
method takes a long time so it is less efficient. These constraints can be overcome by fermentation using
mushrooms or bacteria. Research conducted by Sasongko (2009), cyanide decrease from yam tuber 425,44 ppm
into yam flour 21,74 ppm through fermentation with 15% concentration of fungus and 72 hours fermentation time.
The effectiveness of cyanide reduction was 94.9%. Lactic acid bacteria play a role in the process of cyanide
reduction such as Lactobacillus acidophilus L10, Lactobacillus casei L26 (Donkor and Shah, 2007); Lactobacillus
plantarum pentosus FNCC 235 (Sumarna, 2010); Bifidobacterium longum 536 (Otieno, 2007).
The objective of the study is selective the most effective lactic acid bacteria and fermentation time can reduce
cyanide in the processing of yam flour.

Material and Methods

3.1. Sample preparation

The sample used are the yam tubers from Mojokerto District, aquades, MRS broth, and lactic acid bacteria
(Lactobacillus plantarum FNCC-0027, Lactobacillus casei FNCC-90, Lactobacillus acidophillus FNCC-0051,
Bifidobacterium bifidum BRL-130, Bifidobacterium breve BRL-131) from Food and Nutrition Center, Gadjah Mada
University, Yogyakarta.

Inoculum preparation: taken 100 μl of bacterium from ependoff, inoculated into MRS Broth medium 5 ml, incubated
at 37°C for 24 hours. This study was conducted in 2 stages; stage I selection bacteria the most effective in reducing
cyanide levels on the yam tubers, stage II determines the most optimal time to reduce cyanide levels at safe limits.
3.2. Selection of bacteria
Bacteria were selection for the most effective in reducing cyanide levels on the yam tubers, were done: yam tubers
washed, peeling, size reduction as “sawut”, then fermentation for 24 hours at room temperature, laundering for
running water, drying with cabinet dryer at 60°C at 17 hours, milling and sifting using an 80 mesh sieve.

45
3.3. Optimation of Fermentation Time
The “sawut” of yam tubers fermentation with the selected bacteria from the stage I at the time 24, 48 and 72 hours
3.4. Data were analyzed using one-way ANOVA using SPSS version 16 (SPSS Inc., Chicago, Illinois, USA).
Results and Discussion
The results showed that total of Lactobacillus plantarum FNCC-0027 during the 24-hour fermentation was highest
11,071 log cfu/ml, whereas the lowest Bifidobacterium bifidum BRL-130 bacteria was 10.811 log cfu/ml (Figure 1).
Each lactic acid bacteria has different capabilities in utilizing nutrients in the medium of yam tubers. Increase the
number of bacteria because in the raw material there is a source of nutrients needed by microorganisms for
metabolism. Increasing the total number of bacteria, because LAB can be use the medium or hydrolyze sugar into
simpler components to lactic acid, organic acids, CO2, H2O and energy (Retnaningtyas, et al. 2014).
The average of acidity degree (pH) on fermented tubers ranged from 3.95 to 4.1, whereas the degree of acidity (pH)
in tubers without fermentation was 5.85 (Figure 2). Decrease of pH is due lactic acid bacteria able to break down
starch and sugar in yam tuber into lactic acid during fermentation. Lactobacillus plantarum FNCC-0027 has the lowest
pH lowering ability due to the highest total colonies (11.07 log cfu/ml) so that the ability to form lactic acid is greater.
Lactic acid bacteria oxidize glucose to pyruvate and energy, wherein energy is used to reduce pyruvate to lactic acid
(Sumarna, 2007).
Lactic acid bacteria had the ability to reduce cyanide on the yam tuber the ability of each type of bacteria was different
(Figure 3). Cyanide content in control tuber (without fermentation) was 411,6465 ppm (db). Decreased cyanide levels
are suspected because lactic acid bacteria have the ability to produce β-glucosidase enzymes capable of hydrolyzing
cyanogenic glucoside compounds into water-soluble cyanide acid. According to Kobawila, et al. (2005) that lactic acid
bacteria produce β-glucosidase enzymes that can eliminate cyanogenic glucosides. Lactobacillus plantarum FNCC-
0027 is the most effective lactic acid bacteria in reducing cyanide in the yam tuber by fermentation. The effectiveness
of cyanide reduction using Lactobacillus plantarum FNCC-0027 was 41.98% higher than Bifidobacterium bifidum
BRL-130 of 24.67%. According to Meryandini, et al. (2011), Lactobacillus plantarum may produce β-glucosidase
enzymes that can hydrolyze cyanogenic glucosides. The activity of β-glucosidase enzyme by Lactobacillus plantarum
bacteria was 3.08 nM / mL/min (Kobawila, et al., 2005).
The results from stage 2, showed that the fermentation time had significant effect on the cyanide content on the yam
flour. Fermentation 72 hours can reduce cyanide levels from 411.65 ppm (0 hours) to 23.92 ppm (Figure 4).
Decreased cyanide during fermentation due Lactobacillus plantarum FNCC-0027 produce β-glucosidase enzyme
capable to hydrolyzed cyanogenic glucoside into water-soluble cyanide acid. Sasongko (2009), that CN will be
hydrolyzed by enzyme at acid condition. Meryandini, et al. (2011), Lactobacillus plantarum may produce β-
glucosidase enzymes that can hydrolyze cyanogenic glucosides. Alsuhendra and Ridawati (2013) found, cyanogenic
glucosides are hydrolyzed by β-glucosidase enzymes into sugars and cyanohydrin acetone and the cyanohydrin
acetone is broken down by the hydroxynitrile liase enzyme into acetone and cyanide acid. This is showed that
fermentation time had significant effect on the reducing sugar content of the yam flour. At the fermentation time 24
hours reduces the reducing sugar content, then rises again at 48 hours and 72 hours (Figure 5).
Total LAB (log cfu/ml)

11.5 11,07a
10,86a 10,90a 10,91a
10,81a
11

10.5
Lactic Acid Bacteria

B. Bifidum B. Breve L. acidophillus L. casei L. plantarum

Figure 1: Total lactic acid bacteria in yam tubers after fermentation 24 hours. Values are the mean ± SD (n=3); mean value
not significantly different (p<0.05) as measured by Duncan test

46
 5,85a
6
4,1a 4,1a 4,05a 4a 3,95a

Score pH
4

umbi gadung B. Bifidum B. Breve

L. acidophillus L. casei L. plantarum

Figure 2: pH score in yam tubers after fermentation 24 hours. Values are the mean ± SD (n=3); mean value not significantly
different (p<0.05) as measured by Duncan test

500 411,65
310,08a 301,72b 298,67b
Cyanide (ppm) (bk)

400 283,21c
(24,67%) (26,70%) (27,44%) (31,20%) 238,83d
300 (41,98%)

200
100
0

Tepung gadung B. bifidum B. breve

L. acidophillus L. casei L. plantarum

Figure 3: Cyanide content in yam flour after fermentation 24 hours. Values are the mean ± SD (n=3); mean value significantly
different (p<0.05) as measured by Duncan test

500
Conten of HCN (ppm)

411,65a
400
300 238,83b
(41,98%)
200
32,73c 23,92d
100
(92,05%) (94,19%)
0
0 24 48 72
Fermentation time (hours)

Figure 4: Cyanide in yam flour after fermentation 24, 48 and 72 hours. Values are the mean ± SD (n=3); mean value with
different letter as significantly different (p<0.05) as measured by Duncan test

47
 0.6

Reduction sugar (%)

0,53a
0.5
0.4
0.3
0.2
0,06b
0.1 0,03b
0,00c
0
0 24 48 72
Fermentation time (hours)

Figure 5: Reduction sugar in yam flour after fermentation 24, 48 and 72 hours. Values are the mean ± SD (n=3);
mean value with different letter as significantly different (p<0.05) as measured by Duncan test

Conclusion
Lactobacillus plantarum FNCC-0027 is the most effective bacteria can reduce the highest level of cyanide, so as
selected lactic acid bacteria. Decrease of cyanide compound in the yam tuber 41.98% during 24 hours of
fermentation. The level of cyanide in the yam flour at fermentation time 72 hours was 23.92 ppm (lower than 50
ppm). This content is safe level.

References

Alsuhendra and Ridawati. 2013. Toxic Compound in the Foods. PT Remaja Rosdakarya. Bandung.
Donkor, O. N., Shah, N. P. (2007). Production of β-glucosidase and hydrolysis of isoflavone phytoestrogens by
Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus casei in soymilk. Journal of Food Science.
177-186.
Kobawila SK, Louembe, Keleke, Hounhouigan J, and Gamba C. 2005. Reduction of the cyanide content during
fermentation of cassava roots and leaves to produce bikedi and ntoba mbodi, two food products from Congo.
Academic Journals. 4(7): 689-696.
Meryandini, A., Melani, Vitria and Sunarti, T.C. 2011. Addition of Cellulolitic Bacteria to Improve the Quality of
Fermented Cassava Flour. Journal Bogor Agricultural University. Bogor.
Otieno, D.O., Ashton, J.F., Shah, N.P., 2007. Stability of β-glucosidase activity produced by Bifidobacterium and
Lactobacillus spp. in fermented soymilk during processing and storage. Journal of Food Science. 70(4) : 236 –
241.
Retnaningtyas, Dyah A., W.D.R. Putri. 2014. Characterization of Physicochemical properties of Sweet Potato Starch
Modify with STPP. Journal of Food and Agroindustry Vol 2 (4): 68-77
Sasongko, P. 2009. Detoxification Yam Tubers (Dioscorea hispida Dennst.) by Fermentated Process Using Mucor
sp.. Journal of Agricultural Tehnology. 10 (3): 205-215.
Sumarna. 2010. Hydrolysis of bioactive isoflavone in soymilk fermented with β–glucosidase producing lactic acid
bacteria from local fermented foods of Indonesian. Malaysian Journal of Microbiology. 6(1): 30-40.

48
 IFRC 2017: 163-148 Food Safety and Quality

Antimicrobial and Mechanical Properties of Gelatin Film Plasticized With Nigella Sativa (Black Seed Oil)

Han low1, Ademola M Hammed2, Munirat A Idris2*

1Department of Chemistry, University of Malaya, 50603, Kuala Lumpur, Malaysia

2International Institute for Halal Research and Training, International Islamic University, Malaysia

Abstract
Natural polymers are beginning to gain attention as eco-friendly packaging materials to eliminate environmental and
ecological pollution caused by synthetic packaging materials and packaging of food helps to protect the food quality,
extend the shelf lives of products, prevent food oxidation and microbial degradation. Natural polymers such as lipids,
protein and polysaccharides are currently being harnessed for packaging in food industries. Gelatin, is an excellent
biomaterial commonly used in packaging of food products and it is obtained from hydrolysis of collagen in the
insoluble state, available in bones of animals and in fish processing. It usage in the food industry reveals an excellent
film forming ability utilized as coating to minimize contamination, prevent oxidation and dehydration and maintain food
quality during storage. The properties such as colour, light absorption, water absorption, antioxidant and antimicrobial
ability of gelatin film can be enhanced by adding active ingredients such as oils. Nigella sativa commonly known as
black seed is an emerging as a miracle herb and the seeds of N. sativa and their oil have been widely studied for the
treatment of various ailments throughout the world. It has been extensively studied for its biological activities and
therapeutic potentials such as diuretic, antihypertensive, antidiabetic, anticancer and immunomodulatory, analgesic,
antimicrobial, anti-inflammatory, and antioxidant properties etc. Incorporating Nigella sativa seed oil into gelatin films
provides the possibilities of improving the physicochemical and antimicrobial properties. Hence, the aim of this study
was to determine the physical, mechanical and antimicrobial properties of gelatin film plasticized with Nigella sativa.

Keywords: Gelatin, Nigella sativa, antimicrobial

*Corresponding author’s email: osenimunirat@gmail.com

Introduction
The use of natural polymers is increasingly high due to their eco-friendly usage as packaging materials which
enhances and protect food quality, eliminate microbial disintegration and food oxidation(Ramos, Valdés, Beltrán, &
Garrigós, 2016). Gelatin is a common biopolymer used as food packaging material that is obtained from hydrolysis of
collagen available either from animal bones or from fish. Gelatin can be transformed into films that can be used as
food packaging materials. Gelatin is characterized with high molecular weight polypeptide consisting of amino acids
about glycine (27%), hydroxyproline and proline (25%)(da Silva & Pinto, 2012). The polar groups present in gelatin
are bounded with hydrogen bonds which enhances the moisture absorption causing brittleness. The properties of
gelatin films can be greatly improved by adding other ingredients such as essential oils, seed extracts etc. Nigella
sativa oil is very known in the middle eastern countries and the oil is shown have antimicrobial, anti-inflammatory,
anticancer, antifungi properties etc(Forouzanfar, Bazzaz, & Hosseinzadeh, 2014). Based on its numerous properties,
it is envisaged that enhancing gelatin film with black seed oil will improve the physical properties as well as the
antimicrobial properties. This research aims to investigate the properties both physical and antimicrobial properties of
gelatin film fortified with black seed oil.

2. Materials and methodology

2.1 Gelatin and Nigella sativa (Black seed oil)
The main materials used in this study are the black seed oil (Nigella sativa) which was bought locally from
supermarket in Malaysia and bovine hide gelatin obtained from Sigma Chemicals Co. USA.

49
2.2 Preparation of Gelatin-Nigella sativa films emulsions
Gelatin was dissolved in distilled water at 3%(w/v) which was heated, stirred at about 60 0C for about 30 minutes.
Glycerol of about 0.75ml was added to the solution. Black seed oil solution was prepared by mixing Tween 20 the oil
with in the ratio 1:4. The oil mixture was added to the gelatin solution at different concentrations. The solution was
homogenised using homogenizer 2000 series for about 30 minutes. the films were casted in petric dishes and dried at
470C for 24h and the films were peeled from the dish for characterization.

2.3 Bacterial strain and preparation

Bacteria strain (E.coli) were locally obtained from stock present in the Microbiology laboratory of Biotechnology
Department, International Islamic University Malaysia and maintained on LB (Merck Sdn Bhd) slants. The strains
were sub-cultured very two weeks and stored at 40C. Bacterial cells obtain from laboratory stock solution will be
inoculated into 10mL of LB broth. The inoculum density of the bacterial cells was done using UV spectrophotometer
to determine its absorbance.

2.4 Antimicrobial determination

The antibacterial activity of gelatin-black seeds oil was done against E.coli bacteria strains using the agar well
diffusion method. The LB agar plates were seeded with suspension (106cfu/ml) of the bacterial strains. Wells of 9mm
in size were dug inside the seeded agar plate and 9mm gelatin-black seed films was put inside the wells. The plates
were incubated at 370C for 24 hours. The antibacterial activity was evaluated by measuring the diameter of inhibition
zones in mm (Ahmad, Benjakul, Prodpran, & Agustini, 2012).

2.5 Water absorption capacity

The geletin-black seed oil films were suspended in glass bottles containing 50mL of phosphate buffered saline (PBS)
of pH 7.4 at room temperature. At an appropriate time intervals, the films were taken out and the excess water was
removed carefully with filter paper then weighed immediately. Measurements were taken thrice and the average was
determined. The percentage water absorption capacity was calculated as
wt − w0
%Water absorption capacity = × 100
w0
where w0 and wt are weights of film at initial and after immersion respectively.

2.6 Light transparency and opacity

The light barrier properties of the gelatin-black seed oil films were measured according to a procedure by exposing
the films to light wavelengths ranging from 200 to 700nm using a UV-1601 spectrophotometer (Model CPS-240,
Shimadzu, Kyoto Japan). The transparency of the films was calculated using the equation
A
Film opacity ( ) = −logT/x
mm
where A is the absorbance at each wavelength, T is the transmittance (%) at each wavelength, x is the film thickness
(mm).

2.7 FTIR analysis

FTIR spectra was determined using ATR system model FTIR-Spectrum 400 Perkin Elmer brand. Absorbance mode
the spectra is from 4000-500 cm-1. The characteristic bands were determined for the pure gelatin films and gelatin film
incorporated with black seed oil. The spectra were collected in 32 scans, resolutions of about 4cm -1 for each of the
sample, three replicates were made.

2.8 Statistical analysis

All the results obtained from this study except where otherwise stated, were carried out in triplicates hence, their
mean values were determined with Microsoft Excel or SPSS software version 20. The significance levels of the
generated data in this study were also determined with SPSS (v.20) obtained.

3. Results and discussion

3.1 Water absorption capacity behaviour
50
The water absorption capacity of the film was significantly increased with a slight concentration of the black seed oil,
as compared with pure gelatin shown in figure 1. This property is greatly enhanced as a result of hydrophilicity and
swelling properties of gelatin. However, addition of black seed oil improved the water absorption as compared with
pure gelatin.

1200

1000

800

600

400

200

0
gel-black seed oil gel-black seed oil gel-black seed oil gel
0.0625 0.125 0.25

Percentage water absorption

Figure 1 Percentage water absorption at varying black seed oil concentration

3.2 Film transparency and opacity

One important properties of packaging materials are their ability to transmit light which have a direct effect on the
appearance of the product. As a result, transparency of films is crucial criteria that judge the compatibility of
components present in the films. When components in the film are incompatible, transmittance will be low which
results in poor light reflection. Table 1 shows that film opacity was high with addition of black seed oil at the low dose
as compared with the original gelatin indicating that the film was less transparent which was similar to previous
research(Ma et al., 2012). From the table it is observed that as the concentration of the black seed oil increases the
opacity decreased.
Table 1 Film transparency and opacity
Film opacity Thickness Transmittance Transmittance Film opacity at
@280nm @600nm 600nm
gel-black seed 0.157 51 96.1 2.79
oil 0.0625

gel-black seed 0.171 47.4 81.5 2.68

oil 0.125
gel-black seed 0.29 45.7 72.5 2.40
oil 0.25
gel 0.283 71.6 99.1 2.54

3.3 FTIR analysis

The spectrum of the film with the highest concentration of black seed oil showed more peaks than the rest with lesser
concentration of black seed oil. When compared with gelatin, significant peaks were not observed. Similar trend was
observed for gelatin-black seed oil at the lowest concentration as shown in figure 2 below. The main absorption peaks
appeared at 2347 cm-1 to 2449 cm-1 indicating the presence of strong triple bonds stretching (o=c=o). Absorption
peaks at 3367 cm-1 indicates the presence of hydroxyl groups, N-H stretching amide group. Peaks at 1021 cm-1
shows the strong presence of primary alcohol and peaks at 715 cm-1 shows C-H bending, presence of alkene.

51
 919

1837

2755
1
103
205
307
409
511
613
715
817

1021
1123
1225
1327
1429
1531
1633
1735

1939
2041
2143
2245
2347
2449
2551
2653

2857
2959
3061
3163
3265
3367
3469
Gelatin Gel-black seed 0.0625 Gel-black seed 0.125 Gel-black seed 0.25

Figure 2 FTIR analysis

3.4 Antimicrobial analysis

The inhibitory effect of gelatin-black seed oil film against E.coli and S. aureus reveals that the film did not exhibit any
antimicrobial property against the tested bacteria strains. There was no clear zone diameter which indicated that no
inhibition zone and the diameter was valued as zero. Although previous research reveals that the black seed oil has
antimicrobial property, the incorporation into gelatin film might have reduced its antimicrobial property. Non
antimicrobial property of the film indicates that the protein content of gelatin cannot inhibit the growth or it may
promote the growth of the microbes (Nguyen, Nguyen, & Hsieh, 2013).

Conclusion
The results of this research have shown incorporating black seed oil into gelatin film was successfully characterized
and its water absorption capacity was greatly improved. The addition of the seed oil also impacts the film
transparency and opacity. The FTIR analysis shows the presence of functional groups that are present in the film and
lastly the enhanced films did not exhibit antimicrobial properties. Further analysis is recommended to determine its
tensile strength, elongation, colour and thermal properties.

Acknowledgement
The authors would like to thank the assistance of IIUM in utilizing the research initiative grant (RIGS16-075-0239).

References

Ahmad, M., Benjakul, S., Prodpran, T., & Agustini, T. W. (2012). Physico-mechanical and antimicrobial properties of
gelatin film from the skin of unicorn leatherjacket incorporated with essential oils. Food Hydrocolloids, 28(1),
189-199.
da Silva, R. S., & Pinto, L. A. (2012). Physical cross-linkers: alternatives to improve the mechanical properties of fish
gelatin. Food Engineering Reviews, 4(3), 165-170.
Forouzanfar, F., Bazzaz, B. S. F., & Hosseinzadeh, H. (2014). Black cumin (Nigella sativa) and its constituent
(thymoquinone): A review on antimicrobial effects. Iranian journal of basic medical sciences, 17(12), 929.
Ma, W., Tang, C.-H., Yin, S.-W., Yang, X.-Q., Wang, Q., Liu, F., & Wei, Z.-H. (2012). Characterization of gelatin-
based edible films incorporated with olive oil. Food Research International, 49(1), 572-579.
Nguyen, V. C., Nguyen, V. B., & Hsieh, M.-F. (2013). Curcumin-loaded chitosan/gelatin composite sponge for wound
healing application. International Journal of Polymer Science, 2013.
Ramos, M., Valdés, A., Beltrán, A., & Garrigós, M. C. (2016). Gelatin-Based Films and Coatings for Food Packaging
Applications. Coatings, 6(4), 41.

52
 IFRC 2017: 168-124 Food Safety and Quality

Application of Hydroxyl Radical Aerosolization on E. Coli/Coliform Reduction for Rapid Surface Disinfection
in Food Processing

1Boonchan, W., 2Chayasitthisophon, A., 3Foster, K.W., 4Weeranoppanant, N. and 2,5*Thipayarat, A.

1Department of Environmental Engineering, Faculty of Engineering, Burapha University, 20131, Muang, Chon Buri,
Thailand.
2Department of Food Engineering, Faculty of Engineering, King Mongkut’s University of Technology Thonburi,10140,
Tungkru, Bangkok, Thailand.
3Department of Physics, Syracuse University, 13244-1130, Syracuse, NY, USA.
4Department of Chemical Engineering, Faculty of Engineering, Burapha University, 20131, Muang, Chon Buri,
Thailand.
5Office of Education, Faculty of Engineering, Burapha University, 20131, Muang, Chon Buri, Thailand.

Abstract
Hydroxyl radical fumigation was generated by an ultrasonic fogging system to disinfect food-processing surfaces and
difficult-to-reach areas. . The hydroxyl radicals were produced via an advance oxidation processes (AOPs) of H 2O2
solutions (1-5%), which were also exposed to O3 and UV-C light. The surface disinfection performance was tested on
E.coli/coliform. Duplicates of agar plates spiked with the E. coli/coliform inoculum at 7-8 log CFU/mL were installed
inside the testing chamber. Estimates of colony numbers after the treatment indicated the performance of different
fumigation schemes. The higher concentration of the H2O2 solution (e.g., 5% H2O2) resulted in a faster rate of the E.
coli/coliform destruction than that at the lower concentration. The combination of O3 and UV-C effects was found to
increase the rate of E. coli/coliform destruction significantly. In addition, the experimental data were fitted reasonably
to a modified Chick-Watson model to reflect the effects of different treatments.

Keywords: Aerosolization, Advanced oxidation process, Disinfection, Hydrogen peroxide, E. coli/coliform.

*Corresponding author’s email: aluck@eng.buu.ac.th

Introduction
Inadequate sanitary facilities in food industry cause a significant number of foodborne outbreaks, illnesses and death
every year worldwide. As reported in the bulletin of U.S. Department of Agriculture, Hoffmann et al. (2015) estimated
a financial loss as a result of food-related illnesses to be $15.5 billion annually. The presence of pathogenic
microorganisms on equipment surfaces increases contamination in both intermediate and final products and causes
illnesses in the food supply chain (Choi et al., 2012).

Bacteria are able to multiply and attach to both engineered plastic and metal surfaces such as polystyrene,
polypropylene, and stainless steel (Barnes et al., 1999). Therefore, eliminating or reducing bacterial contamination is
of vital importance in achieving basic food safety standards. There is a persistent need to develop less toxic and fast
sanitizing means or protocols (Nitschke et al., 2009).

Alternative aqueous sanitizers (e.g., organic acids, chlorine dioxide, hydrogen peroxide, and ozonated water) have
highly effective antimicrobial activity but minimal toxicity of the residual chemical (Kingsley et al., 2014). Hydrogen
peroxide (H2O2) solution has been proven effective in controlling and decontaminating the spread of pathogens in
hospital settings (Ali et al., 2016). As a highly active biocide, H2O2 exhibits antimicrobial activity through generation of
hydroxyl free radicals that penetrate cell walls to attack lipids, proteins and DNA (McDonnell and Russell, 1999).
Owing to its non-selective biocidal property, H2O2 is active against viruses, spores and fungi and bacteria, but it does
not react with organic substances to form toxic residues (Herdt and Feng, 2009).

53
In this work, the oxidation of H2O2 was used along with an ultrasonic fogging system to create and disperse a
hydroxyl radical aerosol to disinfect contact surfaces, especially those that are difficult to reach such as overhead
surfaces, cracks and crevasses of food equipment. O3 and UV-C generators were installed to enhance the formation
of hydroxyl radicals (Kommineni et al., 2000). While the system with O3 and UV-C has been widely demonstrated, the
study of this system upon fumigation is limited. We built a fumigating setup to investigate the effects of H 2O2 strength
as well as the enhancement via O3 and UV-C. The system was further validated with an empirical kinetic model.
Highly effective disinfecting protocol, which allowed the lower concentration of H 2O2 to be used, was constructed by
incorporating O3 and UV-C.

Materials and Methods / Methodology

3.1 Equipment and instruments
The system which consists of an ozone generator, UV-C lamp, and fumigator was constructed (Figure 1). An oxygen
tank, capable of adjusting flowrates from 0 to 2 L/min, was connected to the ozone generator. The ozone gas was
subsequently forced into a venturi in which the ozone gas was mixed with circulating water before transferring into a
10 L reservoir. This reservoir contained three ultrasonic atomizers with water evaporation at a rate of 133 mL/h,
producing aerosols. The aerosols were further dispersed by a fan at the top of the reservoir. The system also has four
UV-C lamps (15W each unit) installed in the circulation line to activate more hydroxyl radicals. The fumes, containing
the radicals, were then sent to a testing chamber (34 × 34 × 34 cm3).

Figure 1: The schematic diagram of AOPs aerosolization consisting of (1) oxygen tank, (2) ozone generator, (3)
venturi, (4) 4-lamp UV-C sterilizer, (5) flow meter, (6) valve, (7) water pump, (8) 10-L liquid tank, (9) ultrasonic mist
maker, (10) conduit, (11) testing chamber

3.2 Media and chemical reagents

Trypticase soy broth (TSB) was purchased from Himedia (USA). Plate Count Agar (PCA) was purchased from Difco
(USA). Chromogenic coliform agar (CCA) was purchased from Merck (USA). Hydrogen peroxide solution was
purchased from Merck (Germany).

3.3 Microorganism and agar media preparation

Escherichia coli stock cultures were kept in TSB containing 20% glycerol and stored at –18 °C. Prior to use, the
bacterial stocks were grown in TSB for 18–24 h at 37 °C. The TSB and PCA solution were prepared by mixing 3 g of
TSB and 7.8 g of PCA into 100 mL and 200 mL of distilled water, respectively. Both solutions were sterilized at 121 ᵒC
for 15 min in an autoclave. The strain of E. coli was confirmed and enumerated in Chromocult® Coliform Agar (CCA)
using the spread plate technique (Supanivatin et al., 2011). 2.65 g of CCA mixture was dissolved in 100 mL of
distilled water. The CCA solution was heated and stirred for approximately 35 min to ensure homogeneity.

3.4 Empirical model for disinfection kinetics

The bacterial inactivation was fitted to a modified Chick-Watson model (Cho et al., 2013), which assumed the pseudo-
first order kinetics and accounted for the existence of a tail at the end:

54
 C
log  k1 1  exp  k2t  
Co
Where C/C0 is the reduction in the bacterial concentration at time t, k1 and k2 are kinetic constants that were obtained
by nonlinear least-square solver in MATLAB® (tolerance = 10-6).

Results and Discussion

Estimates of colony numbers after treatment were used to evaluate effectiveness of different hydroxyl radical
formation treatments. The degree of E. coli/coliform destruction was estimated upon the fume saturation at the top
side of the chamber. A higher strength of H2O2 (e.g., 5% H2O2) enabled faster destruction of E. coli/coliform on the
agar surface than that at a lower strength, as depictedin Figure 2(a). The H2O2/O3/UV treatment demonstrated
significantly faster E. coli/coliform destruction at 7-8 log CFU/mL than other treatments, even with 1% H2O2 The
experimental data were fitted to the modified Chick-Watson model, which also accounted for the deceleration of the
microbial inactivation at the end when the concentration of bacteria was very low. The fitted kinetic parameters were
tabulated in Fig 1. The fitted curves validated the previous discussion that the combined H 2O2/O3/UV resulted a high
rate of inactivation compared to other treatments as shown in Figure 2(b). A parity plot, as shown in Figure 2(c),
indicated the model captured the experimental data reasonably well, especially during the initial and middle
inactivation regions (i.e. high log CFU/mL), although their deviation became larger towards the end of the inactivation
(i.e. low log CFU/mL). The H2O2/O3/UV enhanced the rate of radical formation through the complex mechanism
including many steps such as the UV-activated cleavage of the H2O2 into radicals, the photolytic reaction between
ozone and hydrogen peroxide to form radicals and oxygen gas.
(a) (b)
8 8
1% H2O2 1% H2O2 (model)
E. coli/Coliform (log CFU/mL)

7
E. coli/coliform (log CFU/mL)

3% H2O2 7 3% H2O2 (model)

6 5% H2O2 5% H2O2 (model)
6
1% H2O2 + UV + O3 1% H2O2 + UV + O3 (model)
5 5
4 4
3 3
2 2
1 1
0 0
0 10 20 30 40 50 0 10 20 30 40 50
Time (min) Time (min)
(c)
8
7
6
Model (log CFU/mL)

5 Experiment k1 k2 (min-1)
4 1% H2O2 19.78 0.0118
3 3% H2O2 7.84 0.0573
5% H2O2 7.80 0.0740
2
1% H2O2 + UV + O3 7.00 1.0524
1
0
0 1 2 3 4 5 6 7 8
Experimental (log CFU/mL)
Figure 2: Concentrations of E. coli/coliform (in log scale) at different time points (a) E. coli/coliform inactivation using
different treatments (b) fitting of the modified Chick-Watson kinetic model to experimental data (c) parity plot

55
 comparing experimental data against model’s predicted data. The table shows two rate constants obtained from the
non-linear fitting to the model. In all experiments, the initial bacterial concentration was 107 CFU/mL. (Error bars were
standard deviations calculated from repeated experiment)

Conclusion
This work presents a study of E. coli/coliform disinfection using hydroxyl radicals from H2O2 in the form of aerosols.
The kinetics of the bacterial inactivation increased as the concentration of H2O2 increased. The inactivation was
significantly enhanced through the exposure of the O3 and UV-C light. The experimental data were validated with the
modified Chick-Watson model to further demonstrate the effects of different treatments. The O3 and UV light tended
to activate several reactions that formed more hydroxyl radicals.

Acknowledgement
This work was financially supported by the Research Grant of Burapha University through National Research Council
of Thailand (Grant No. 115/2560). I grateful to have strong industrial partnership with Rano Tech Co.,Ltd. for their
funding to support equipment and chemical agents in my laboratory. Special thanks are extended to the Thailand
Research Fund, namely research and researcher for industry: MAG master research Grants (Grant No. MSD58I0110)
and the National Science and Technology Development Agency for master degree student (Grant No. SCA-CO-2560-
3504-TH).

References

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Vapour Aerial Decontamination Systems for Enhanced Disinfection of Meticillin-resistant Staphylococcus
aureus, Klebsiella pneumoniae and Clostridium difficile in Single Isolation Rooms. Journal of Hospital Infection
93(1): 70-77.
Barnes, L. M., Lo, M. F., Adams, M. R., and Chamberlain, A. H. L. 1999. Effect of milk proteins on adhesion of
bacteria to stainless steel surfaces. Applied and environmental microbiology 65(10): 4543-4548.
Cho, M., Chung, H., and Yoon, J. 2013. Disinfection of water containing natural organic matter by using ozone-
initiated radical reactions. Applied and environmental microbiology 69(4): 2284-2291.
Choi, N. Y., Baek, S. Y., Yoon, J. H., Choi, M. R., Kang, D. H., and Lee, S. Y. 2012. Efficacy of aerosolized hydrogen
peroxide-based sanitizer on the reduction of pathogenic bacteria on a stainless steel surface. Food control
27(1): 57-63.
Herdt, J. and Feng, H. 2009. Aqueous Antimicrobial Treatments to Improve Fresh and Fresh-Cut Produce Safety. In
Fan, X., Niemira, B. A., Doona, C. J., Feeherry, F. E. and Gravani, R. B., (Eds.) Microbial Safety of Fresh
Produce, p. 169-190. Oxford: Wiley-Blackwell.
Hoffmann, S., Maculloch, B., and Batz, M. 2015. Economic burden of major foodborne illnesses acquired in the
United States. Current Politics and Economics of the United States, Canada and Mexico, 17(4): 543.
Kingsley, D. H., Vincent, E. M., Meade, G. K., Watson, C. L., and Fan, X. 2014. Inactivation of human norovirus using
chemical sanitizers. International journal of food microbiology 171: 94-99.
Kommineni, S., Zoeckler, J., Stocking, A., Liang, P. S., Flores, A., Rodriguez, R., and Brown, A. 2000. 3.0 Advanced
Oxidation Processes. Treatment Technologies for removal of Methyl Tertiary Butyl Ether (MTBE) from drinking
water: air stripping, Granular Actived carbon, Sinthetic resin sorbents. Melin, G. and National Water Research
Institute, (2nd ed.) Advanced Oxidation Process, p. 109-208. California: The California MTBE Research
Partnership.
McDonnell, G. and Russell, A. D. 1999. Antiseptics and disinfectants: activity, action, and resistance. Clinical
microbiology reviews 12(1): 147-179.
Nitschke, M., Araújo, L. V., Costa, S. G. V. A. O., Pires, R. C., Zeraik, A. E., Fernandes, A. C. L. B., and Contiero, J.
2009. Surfactin reduces the adhesion of food‐borne pathogenic bacteria to solid surfaces. Letters in applied
microbiology, 49(2): 241-247.
Supanivatin, P., Khueankhancharoen, J., Saeung, W., and Thipayarat, A. 2011. Fast and less thermal degradation
protocol for Chromocult® Coliform Agar (CCA) preparation to detect E. coli. Thai Journal of Agricultural
Science 44(5): 459-465.
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Residual Heavy Metal and Bio-Accumulated Pesticide Estimation in Commonly Used Fish Food of Pakistan

Muhammad Danish1*, Yad-e-baiza1, Maida Fatima1, Muhammad Waseem Mumtaz1

1Department of Chemistry, University of Gujrat, Punjab, Pakistan

Abstract
A scientific study was conducted to evaluate the level of heavy metals and pesticides in fish namely, Morakhi
(Cirrihinus mrigala), Sanghara (Aorichthys seenghala), Rohu (Labeo rohita), and Mahseer (Tor tor) using atomic
absorption spectroscopy and high performance liquid chromatography (HPLC). The orders of heavy and toxic metal
concentrations were projected to be Cd > Fe > Pb > Zn > Co > Ni > Cu > Cr > Hg in Cirrihinus mrigala; Fe > Cd > Zn
> Pb > Ni > Co > Cr > Cu > Hg in Aorichthys seenghala; Fe >Cd >Zn > Pb >Ni > Cr > Cu > Co > Hg in Labeo rohita
and Fe > Zn > Cd > Pb > Ni > Co > Cu > Cr > Hg in Tor tor. Majority of heavy metals were found above the
permissible limits set by World Health Organization (WHO) and Environment Protection Agencies (EPA). Levels of
accumulation of Imedacloprid, Furadan, Fenitrothion and Dimethoate pesticides were tested in fish samples and
found within the permissible limits set by said organizations. It was established that regular monitoring and remedial
measures were required on urgent basis to avoid further contamination of food chain and to minimize the hazardous
health impacts.

Keyword: Fish, Heavy Metals, Pesticides, Bioaccumulation, Pakistan

*Corresponding author’s email: muhammad.danish@uog.edu.pk

Introduction
Food security for human consumption is a critical issue, as developments in its production and processing technology
has expanded the threat of contamination of food stuffs with various poisonous substances. These contagions have
ability to accumulate in the organs of the animals that ingest them [Järup, 2003].Fish is an important human diet as it
contains I2, Se, Zn, Fe, Ca, P, K, vitamins A, vitamin B and vitamin D [Dhasarathan et al., 2000]. Fish consumption
reduces risk of heart infirmities and diabetes, keeps up the neural system of body and development of the brain
tissues [Halwart, 2013]. Human activities are affecting the environment by adding various toxic substances like
phenols, pesticides, heavy metals and distinctive chemicals that change the nature of the water bodies too which has
lethal consequence on the marine life [Pazhanisamy & Indra, 2007; Murthy et al., 2013]. Fish have the ability to
gather hazardous substances; for example, harmful metals and pesticides through diet and polluted water [Dirilgen,
2001]. These toxins get transferred to human body and bring the metabolic system at risk [Raikwar et al., 2008].
Rapid industrialization and natural sources both are responsible for the entry of heavy metals and other toxins
continuously into the water bodies [Parvathi et al., 2012]. The abnormal levels of heavy metals result in different
diseases [APHA, 2005], [FAO, 1983].

Similar is the effect of pesticides, insect repellants, manufacturing, processing, storing and transportation of food and
animal feedstuffs [Allsop et al., 1993]. Pesticides being non-biodegradable substances have high potential to
accumulate in biological systems especially aquatic and marine animals like fish [Jaradat, 2009] and cause spectrum
of diseases when enters food chain [Fürst et al., 1190]. These substances change phagocytic capacity, respiratory
burst and immune system, due to which death rate is increased [Helfrich et al., 2009]. The purpose of current
investigation is to evaluate the status of heavy metals concentration and selective pesticide accumulation in four
famous fish varieties commonly used as food in Punjab province of Pakistan.

57
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Materials and Methods

Fish Collection
Four freshwater fish; Singhara (Aorichthys seenghala), Morakhi (Cirrihinus mrigala), Rohu (Labeo rohita) and
Mahseer (Tor tor) used in this study were collected from local market of Jhelum, Pakistan in November 2015. Fish
were properly washed with double distilled water and dissected. A weighed portion was separated with the stain less
steel knife and placed in sterilized polyethylene bags. Bags were sealed and stored at -25°C for further analysis.

Determination of Heavy Metals

Heavy Metal Extraction
20 g each of stored fish sample was taken in a china dish and placed in a furnace for 2 hours at 350 °C till ash. 3 g
each of the ash was digested in conc. HNO3, cooled, added 10ml HCl and heated followed by filtration.

Heavy Metal Analysis

The heavy and toxic metals including Cd, Pb, Cr, Hg, Cu, Zn, Co, Ni and Fe were estimated in fish samples C.
mrigala, L. rohita, T. tor and A. seenghala using AAS (Solaar 969). Triplicate measurements were taken for all
digested samples. Heavy metal concentration was determined from standard absorption curve.

Preparation of Fish Samples for Pesticide Residues

Fish samples (10 g each) were dried at 105 °C to remove water contents and ground to fine powder. These ground
samples were transferred to a conical flask containing 50 ml of acetonitrile followed by 48 hours shaking. Flasks were
removed from the shaker and the extract of each fish sample was filtered, the filtrate was treated with anhydrous
Na2SO4 and left it for 24 hours. The extract was later on passed through a cleanup step using Florisil and refiltered.

Pesticide Analysis
The samples were estimated by HPLC for bioaccumulation of said pesticides in these fish using the C18 reverse
phase (150 × 4.6 mm, 5 µm) column at 30 °C. A mixture of 70% acetonitrile and 30% water was used as a mobile
phase at a flow rate 1.2ml/min. The results obtained were subjected to statistical evaluation. Parameters evaluated
were grand mean and standard deviation (SD) using ANOVA and SPSS- 16 software.

Results and Discussions

Heavy metals
Data based on heavy metals in these fish was analyzed from the descriptive analysis of mean and standard deviation.
Concentration of various metals in these fish is given the Table 1.

Table 1. Concentration of toxic metals in fish samples

Fish Name Cd Pb Cr Hg Cu Zn Co Ni Fe
Morakhi 7.82 + 6.18+0.2 0.734 0.01+0.0 0.80+0.0 4.99 1.12+0 6.76
0.98
(Cirrihinus mrigala) 0.14 1 +0.05 0 5 +0.18 .09 +0.22
+0.32
Singhara 5.27 + 3.56+0. 0.69 0.01+0. 0.42+0. 3.66+0. 1.96+0 7.64
2.28
(Aorichthys 0.32 06 +0.01 00 02 14 .08 +0.19
+0.08
seenghala)
Rohu (Labeo 6.82 5.13+0. 0.94 0.01+0. 0.69+0. 5.98+0. 0.68 8.07
2.94
rohita) +0.21 14 +0.02 01 01 21 +0.05 +0.15
+0.08
Mahseer (Tor tor) 3.81 2.04+0. 0.37 0.01+0. 0.71+0. 4.76 1.64 5.39
1.67
+0.31 07 +0.03 00 01 +0.18 +0.09 +0.17
+0.19

58
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By above mentioned data, it was concluded that understudy fish samples Cirrihinus mrigala, Labeo rohita, Tor tor and
Aorichthys seenghala showed bioaccumulation of major heavy metals beyond the fish food safety standards set by
WHO, 1985 and FEPA 2003. It is established fact that fish bioaccumulate heavy metals to great extent but which
metal is be accumulated truly depends upon the topography of the sampling site or habitat. Types of industry,
agricultural intensity and population exposure may be the key factors for distribution of heavy metals in environment
especially water bodies. So the comparison of bioaccumulation of substances in fish and other aquatic life of one
specific habitat to the life of other habitat seems very immature.

Pesticides
Data related to various pesticides found in these fish have been presented in Fig.1 (A-D).

Fig 1. Status of (A) Furadan (B) Imedacloprid (C) Dimethoate (D) Fenitrothion concentrations in fish samples
under investigation

Levels of all the understudy pesticides in Aorichthys seenghala and Cirrihinus mrigala, Labeo rohita & Tor tor were
found to have a significant impact with p values, i.e., 0.000 lesser than 0.05 for Imedacloprid, Furadan, Dimethoate
and Fenitrothion, respectively. Furthermore, the levels of understudy pesticides, i.e. Imedacloprid, Furadan,
Dimethoate and Fenitrothion were found to be significantly different in all the understudy feed stocks but remained in
permissible limits.

Conclusion
The above mentioned analytical investigation emphasized on the fact that all the fish species involved in study
accumulated certain levels of heavy metals and toxic pesticides. So it is need of hour to design the strategy keeping
in view the national and international scenario for stopping the entrance of these toxic entities in water bodies. This
check may fruitful to prevent the contamination of food chain hence limiting the detrimental health impacts associated
with these substances.

References

Allsop, P. J., Chisti, Y., Moo‐Young, M., and Sullivan, G. R. 1993. Dynamics of phenol degradation by Pseudomonas
putida. Biotechnology and bioengineering 41(5): 572-580.
American Public Health Association (APHA). 2005.Standard Methods for the Examination of Water and Wastewater
Analysis, 21st Edition, American Water Works Association/Water Environment Federation, Washington D.C.,
pp. 289.
Awofolu, O. R., Mbolekwa, Z., Mtshemla, V., and Fatoki, O. S. 2005. Levels of trace metals in water and sediment
from Tyume River and its effects on an irrigated farmland. Water Sa 31(1), 87-94.
Dhasarathan, P., Palaniappan, R., and Singh, A. J. A. R. 2000. Effect of Endosulfan and Butachlor on the digestive
enzyme and proximate composition of the fish Cyprinus carpio. Indian Journal of Environment &
Ecoplanning 3(3): 611-614.
Dirilgen, N. 2001. Accumulation of heavy metals in freshwater organisms: Assessment of toxic interactions. Turkish
Journal of Chemistry 25(2): 173-180
FAO- Food and Agricultural Organization. 1983. Compilation of legal limits for h
59
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ardous substances in fish and fishery products. FAO Fishery Circular, No. 46, 5-100.
FAO. 1983. Compilation of legal limits for hazardous substances in fish and fishery products. FAO Fishery Circular,
464, 5-100.
FEPA. 2003. Guideline and Standards for Environmental Pollution and Control in Nigeria. Nigeria, Federal
Environmental Protection Agency.
Fürst, P., Fürst, C., and Groebel, W. 1990. Levels of PCDDs and PCDFs in food-stuffs from the Federal Republic of
Germany. Chemosphere 20(7): 787-792.
Halwart, M. 2013. Valuing aquatic biodiversity in agricultural landscapes. Diversifying Food and Diets: Using
Agricultural Biodiversity to Improve Nutrition and Food Security. Routledge/Earthscan, 88-108.
Helfrich, L. A., Weigmann, D. L., Hipkins, P., and Stinson, E. R. 2009. Pesticides and aquatic animals: a guide to
reducing impacts on aquatic systems.
Jaradat, K. A. 2009. Adsorption and Desorption Charecteristics of Endosulfan Pesticide in Three soils in
Palestine. An-Najah National University .
Järup, L. 2003. Hazards of heavy metal contamination. British Medical Bulletin 68(1): 167-182.
Murthy, K. S., Kiran, B. R., and Venkateshwarlu, M. 2013. A review on toxicity of pesticides in fish. International
Journl of Open Science Research 1, 15-36.
Parvathi, K., Mathan, S., and Sarasu, R. 2011. Sublethal effects of chromium on some biochemical profiles of the
freshwater teleost, Cyprinus carpio. Int J App Biol Pharma Techno 2: 295-300.
Pazhanisamy, K., and Indra, N. 2007. Toxic effects of arsenic on protein content in the fish, Labeo
rohita(Hamilton). Nature, Environment and Pollution Technology 6(1): 113-116.
Raikwar, M. K., Kumar, P., Singh, M., and Singh, A. 2008. Toxic effect of heavy metals in livestock health. Veterinary
world 1(1): 28-30.
Sarikaya, R., Selvı, M., and Erkoç, F. 2004. Investigation of acute toxicity of fenitrothion on peppered corydoras
(Corydoras paleatus) (Jenyns, 1842).Chemosphere 56(7): 697-700.
World Health Organization WHO. 1985 Chromium, zinc, lead, in drinking-water. Background document for
preparation of WHO Guidelines for drinking-water quality, Geneva, (WHO/SDE/WSH/03.04/4).

60
 IFRC 2017: 179-144 Food Safety and Quality

The Lipolytic Activity of Pseudomonas fluorescens BIOTECH 1123 in Commercially Available Salted Butter
under Refrigerated Conditions and Its Relation to Product Quality Deterioration

Babaran, G.M.O., and *Mopera, L.E.

Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines Los
Baños, Los Baños, 4031 College, Laguna, Philippines

Abstract
The normal storage temperature for butter is usually less than 4°C for the preservation of its important properties.
Butter’s susceptibility to oxidation and lipolysis, which leads to the production of off odors and flavors upon prolonged
storage, is attributed to its very high-fat content. This study aimed to identify the extent of lipolytic spoilage of
Pseudomonas fluorescens BIOTECH 1123 in commercially available salted butter. The microbiological properties
(lipolytic, psychrophilic, proteolytic, coliform, and yeasts and mold counts) of salted butter were evaluated after 2, 7,
12, and 15 days. The chemical attributes (peroxide value, and free fatty acid and acid value) were also monitored to
account for the extent of rancidity. The results indicate that there is no significant difference between the chemical
attributes of the samples taken randomly in the four incubation periods. The correlation between the chemical and
microbial attributes was examined using the Spearman’s correlation test and the results range from very weakly
negative to moderately positive association among the variables compared. Sensory evaluation of the sample’s odor
after microbial inoculation and incubation was not significantly different. The study showed that the lipolytic
capabilities of Pseudomonas fluorescens BIOTECH 1123 within a period of 15 days is weak. However, the presence
of these microorganisms must not be neglected as it should be noted that the activity of lipases varies widely based
on the microorganism itself, even within the same species the enzyme activity and its effect on the product quality
deterioration will not be the same.

Keywords: Pseudomonas fluorescens BIOTECH 1123, salted butter spoilage, lipolytic activity, microbial spoilage,
rancidity
*Corresponding author’s email: lemopera@up.edu.ph, Tel: +63 927-702-1975

Introduction
Salted butter typically is composed of 80-81% milk fat, 16-17% moisture, 1% carbohydrates and proteins and 1.2-
1.5% sodium chloride (Ghodduci and Özer, 2014). Butter’s susceptibility to oxidation and lipolysis is attributed to its
high−fat content.

Pseudomonas fluorescens is a psychrotrophic to psychrophilic bacterium that is one of the microorganisms

responsible for spoilage of dairy products. Suryavanshi and Gosh (2010) carried out a study to determine the
optimum pH and temperature which Pseudomonas aeruginosa NCIM 2036 can produce lipase in unsalted white
butter. The results showed that Pseudomonas aeruginosa produced lipase with significant activity at 5°C. Such
activity at 25°C leading to fat hydrolysis of butter by the microbial enzymes was also observed. Generally, the heat
stable lipases secreted by Pseudomonas are able to hydrolyze tributyrin and milk fat at both 6°C and 20°C (Batt and
Tortorello, 2014). Some Pseudomonas strains secrete two to three types of proteases, and there is a large diversity of
lipases within different species. Lipolytic activity has been less studied compared with proteolytic activities, both can
occur during spoilage and they are both associated with one another (Batt and Tortorello, 2014). A study conducted
by Hussong (1932) showed the relationship of Pseudomonas fragi to rancidity in butter. This study revealed that at
21°C the organism increased rapidly in unsalted butter and brought about a rancid condition in as short as 4 days.
The development of the condition was accompanied by a rapid increase in the amount of free fatty acid in the butter.
Batt and Tortorello (2014) on the other hand, reported that all Pseudomonas groups found in butter are psychrophiles
traced in the wash water. These organisms grow well at refrigerated temperatures and produce putrid or lipolytic

61
flavors in 5-10 days. The aim of the current work was therefore to identify the extent of lipolytic spoilage of
Pseudomonas fluorescens BIOTECH 1123 in commercially available salted butter.

Methodology
Commercially available salted butter was randomly obtained in a local supermarket in Los Baños Laguna, Philippines.
Its initial microbial (coliform, lipolytic, psychrophilic, proteolytic, yeasts and mold count) and chemical quality (iodine
value, saponification number, peroxide value, free fatty acid (FFA) and acid value) were evaluated. The samples were
inoculated with approximately 2x106 cfu/ml and incubated in a sterile glass jar covered with aluminum foil under
refrigerated conditions for a period of 2, 7, 12 and 15 days. The incubation periods were based on the study
conducted by Hussong (1932) and the Encyclopedia of Food Microbiology by Batt and Tortorello (2014). Three trials
were made for each incubation period. An uninoculated sample served as the control.
The microbial and chemical quality of the samples after incubation was evaluated using the same techniques used for
the preliminary analysis. The iodine value and saponification number were not evaluated after incubation. The
procedure for the microbial analysis is based on the 17th edition of the Standard Methods for the Examination of
Dairy Products and the procedures for the chemical analysis is based on the laboratory manual in Food Analysis used
by the Institute of Food Science and Technology, University of the Philippines Los Baños, in the course entitled Food
Analysis (FST 102). The data analysis was done using the F-test ANOVA and the Spearman’s correlation test. The
lipase of P. fluorescens BIOTECH 1123 was qualitatively detected by using butterfat agar and egg yolk agar. Sensory
evaluation of the sample’s odor was performed using the difference from the control test, using fresh butter as the
control. The data was analyzed using analysis of variance (ANOVA).

Results Discussion
Preliminary microbial and chemical analysis of the sample was done to determine its quality.
The initial microbial count for all treatments was <20 cfu/g (1.3 log cfu/ml). The initial chemical analysis revealed that
the sample’s iodine value and saponification number was 33.46 + 8.33 and 215.21 + 6.84, respectively. Its peroxide
value and FFA and acid value was 1.29 + 1.12, 0.28 and 0.55 + 9.17 x 10-3, respectively. According to guidelines set
by the Philippines Food and Drug Administration (FDA) Circular No. 2013-010 (Revised Guidelines for the
Assessment of Microbiological Quality of Processed Foods) and Davis (1963) the values obtained from the initial
microbial analysis of the sample did not surpass the level which when exceeded would cause the product to be
rejected.

The results of the iodine value and saponification number during the initial chemical analysis coincide with the range
of the typical values for bovine butterfat in Nielsen (2003). The most abundant fatty acid present in butter is oleic acid
thus, the FFA and Acid value was expressed with respect to oleic acid throughout the experiment. The initial peroxide
value and the acid value of the samples are also within the acceptable conditions according to the Food and
Agriculture Organization (FAO) (1999) and Pike (2003).Thus, the sample is microbiologically and chemically of good
quality to be used in the study.

The lipolytic activity of Pseudomonas fluorescens BIOTECH 1123 was qualitatively detected by using Butter Fat Agar
(BFA) and Egg Yolk Agar (EYA). The positive lipolytic colonies in Butter Fat Agar were indicated by the Turquoise
Blue zone beneath or around the colony (Harringan and McCane, 1976). On the other hand, the positive lipolytic
colonies in Egg Yolk Agar were indicated by dirty white to cream colonies with opalescent precipitate around them
with no change in the media color (Zamora et al., 2015).
The values obtained from the chemical and microbial analysis after incubation is summarized in Table 1. The average
values for the chemical analysis were analyzed by using the F- test and its correlation with the microbial counts were
evaluated using Spearman’s Correlation test (Table 2). The assumptions for both statistical tests used were satisfied.
There is no significant difference in the mean chemical values among the four incubation periods at 5% level of
significance. This indicates that there are no drastic changes that are observed in the sample within the incubation
periods. Furthermore, the peroxide, FFA, and acid values obtained shows that the samples are still within the
acceptable standards. There is a very minimal correlation between the chemical and microbial data obtained which
indicates a lower likelihood of a relationship between the two.

62
 The results of the sensory analysis showed that there is no significant difference between the odor of the inoculated
samples incubated at different incubation periods and the control, which is fresh butter. This study revealed that FFA,
mostly volatiles, in salted butter caused by the presence of Pseudomonas fluorescens BIOTECH 1123 lipase for a
period of 15 days is not enough to be detected by consumers in terms of odor.
Table 1. Chemical and microbial analysis of commercially available salted butter during the four incubation periods
Yeast and
Proteolytic Lipolytic Psychrophi Coliform
Incubation

Peroxide FFA Value Acid Value Molds

Period,

Count Count lic Count Count

days

Value (x̄) (x̄) (x̄) Count

Log CFU/ml
C T C T C T C T C T C T C T C T
2 5.85 5.41 0.36 0.27 0.71 0.53
+ + + + + + 1.30 2.73 1.70 2.78 1.30 2.77 1.48 1.30 1.30 1.30
3.86 3.57 0.12 0.01 0.23 0.02
7 3.24 3.02 0.24 0.42 0.47 0.54
+ + + + + + 1.30 2.92 1.30 3.39 1.30 3.42 1.30 4.23 1.30 3.29
1.12 1.38 0.03 0.22 0.05 0.11
12 3.25 2.33 0.26 0.30 0.51 0.55
+ + + + + + 1.30 3.12 1.30 3.27 1.30 3.28 1.30 1.30 1.30 3.04
0.12 0.79 0.03 0.04 0.05 0.02
15 0.00 3.91 0.25 0.27 0.49 0.53
+0 + + + + + 4.23 3.40 1.30 3.40 1.30 3.40 1.70 1.48 1.30 1.70
0.67 0.04 0.02 0.07 0.04
C = Control; T = Treated

Table 2. The Correlation between the chemical and microbial analysis of commercially available salted butter during
the four incubation periods
POV :Proteolytic Count 0.2232 Weak (+)
Lipolytic Count 0.1056 Very weak (+)
Psychrophilic Count 0.3422 Weak (+)
FFA: Proteolytic Count 0.2598 Weak (+)
Lipolytic Count 0.4718 Moderately (+)
Psychrophilic Count 0.4234 Moderately (+)
Acid Value: Proteolytic Count 0.03348 Very weak (+)
Lipolytic Count -0.04225 Very weak (-)
Psychrophilic Count -0.1363 Very weak (-)

Conclusion
The lipase of Pseudomonas fluorescens BIOTECH 1123 in salted butter is very weak based on the results of the
study. It is unable to cause significant changes in a span of 15 days. In future studies, it is recommended that an
incubation period longer than 15 days be used to evaluate the lipolytic activity of Pseudomonas fluorescens
BIOTECH 1123. Furthermore, gas chromatography can be used in order to monitor the volatiles produced.

Acknowledgement
The authors would like to thank the Institute of Food Science and Technology for conducting the experiment.

References
Batt, C.A. and Tortorello, M.L. (Eds.). 2014. Encyclopedia of Food Microbiology (2nd ed., Vol. 2). London, UK:
Academic Press: an imprint of Elsevier Ltd.
Davis, J.G. 1963. Microbiological Standards for Dairy Products. Journal of the Society of Dairy Technology.16 (4) p.
224

63
Duncan, S.E., Yaun, B.R. and Summer, S.S., 2004. Microbial Methods for Dairy Products. In Wehr, H.M. and Frank,
J.F. (Eds.). Standard Methods for the Examination of Dairy Products. (17th ed.). Washington, DC: American
Public Health Association.
Food and Agriculture Organization (FAO). 1999. Section 1. Codex General Standard for Fats and Oils. Retrieved
June 13, 2017 from FAO Website: http://www.fao.org/docrep/004/y2774e/y2774e03.htm
Food and Drug Administration Philippines. 2013. Revised Guidelines for the Assessment of Microbiological Quality of
Processed Foods. Retrieved on April 30, 2016 from FDA Website: http://www.fda.gov.ph/issuances-2/food-
laws-and-regulations-pertaining-to-all-regulated-food-products-and-supplements/food-fda-circular/17218-fda-
circular-no-2013-010
Ghoducci, H. and Özer, B.H. 2014. Microbiology of Cream, Butter, Ice cream and related products. Özer, B.H. and
Akdemir-Evrendilek, G. (Eds.). Dairy Microbiology and Biochemistry: Recent developments. Boca Raton FL:
CRC Press
Harringan, W.F. and McCane, M.E. 1976. Laboratory Methods in Food and Dairy Microbiology. Acads Press, London
New York, San Francisco.
Hussong, R.V.1932. The relationships of a lipolytic organism to rancidity of butter. Ames: Iowa State College, Thesis
Nielsen, S. (ed.). 2003. Food Analysis. (3rd ed.).New York: Kluwer Academic/ Plenum Publishers
Pike, O.A. 2003. Fat Characterization. In Nielsen, S. (ed.). Food Analysis. (3rd ed.).New York: Kluwer Academic/
Plenum Publishers
Santiago, D.M.O., Dia, V.P. and Del Rosario, O.M. 2014. Food Analysis Laboratory Manual. Philippines:
Suryanvanshi, M.V. and Ghose, J.S. 2010. Spoilage of White Unsalted Butter by Psychrophillic lipolysis of
Pseudomonas aeruginosa NCIM 2036. British journal of dairy science 1(1):26-29
Zamora, A.F., Dalmacio, I.F. and Sabino, N.G. 2015. Dairy Microbiology Laboratory Manual. Philippines: Microbiology
Division Institute of Biological Sciences, College of Arts and Sciences, UPLB

64
 IFRC 2017: 182-147 Food Safety and Quality

Quality Evaluation of Microwave and Conventional Pasteurised Pineapple Juice

1Abd Aziz, N. A., 2*Mohd Jusoh, Y. M., 2Nik Mahmood, N. A., 1Yunus, N. A., 3Endut, A.

1Department of Chemical Engineering, Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia,
81310 Skudai, Johor, Malaysia.
2Department of Bioprocess Engineering, Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia,
81310 Skudai, Johor, Malaysia.
3Faculty of Innovative Design and Technology, Universiti Sultan Zainal Abidin, 21300 Kuala Terengganu,
Terengganu, Malaysia.

Abstract
This study evaluated the effect of microwave treatment and conventional thermal treatment on bromelain and vitamin
C content as well as the shelf life of pineapple juice during storage period of 30 days. The juices were heated until
60°C and held for 5 minutes. The results showed that pineapple juice pasteurised by microwave had significantly
retained higher content of bromelain and vitamin C compared to conventional thermal treatment with the values of
87.36 ± 3.15% and 90.87 ± 1.13%, respectively. Meanwhile for conventional treated sample, the percentage of
residual bromelain and vitamin C were only 70.83 ± 7.01% and 74.72 ± 0.37%, respectively. Approximately 50% of
these nutrients were retained after 30 days in microwave treated juice. In contrast, a rapid degradation of bromelain
and vitamin C was observed after storage. In addition, the shelf life of microwave pasteurised juice was longer
compared to conventional pasteurised juice. From these promising results, microwave technology has a potential as
an alternative for fruit juice pasteurisation, a method dedicated for small and medium scale industries.

Keywords: microwave pasteurisation, food safety, pineapple juice, bromelain, vitamin C.

*Corresponding author’s email: yanti@cheme.utm.my

Introduction
Pineapple (Ananas comosus) is one of the most important tropical fruits in Malaysia and it contains an abundant
amount of nutritional values such as bromelain and vitamin C. Bromelain and vitamin C attract a lot of interest from
the consumers and food processing industries due to their health benefits. Bromelain can be used for therapeutic
purposes such as reducing inflammation, treating indigestion and anti-tumour agent (Abdul Majid et al., 2008).
Meanwhile, vitamin C is crucial for formation of antibodies, synthesis of collagen and bones and absorption of iron
(Uckiah et al., 2009).

Pineapple is usually consumed fresh and as processed products. Fresh pineapple juice is prone to spoilage due to
high water content which makes the juice susceptible to microorganisms. Usually, thermal pasteurisation was applied
to ensure a safe consumption of fruit juice (Goh et al., 2012). However, conventional pasteurisation method causes
adverse effect towards nutritional value in fruit juices, therefore microwave technology is considered an alternative for
juice pasteurisation. This study aimed to evaluate the effect of microwave and conventional thermal treatment on the
bromelain, vitamin C and microbial contents of pineapple juice.

Materials and Methods

Sample preparation
Pineapple fruits of the Morris variety at a commercial maturity were purchased from a local supermarket in Johor
Bahru. The fruits were rinsed with tap water, the shell and core were removed using a stainless steel knife. The flesh
was cut into small pieces approximately 3 cm3 and the juice was extracted using a juice extractor (Philips HR1833).

65
Treatments
100 mL of pineapple juice was heated in a beaker at 900 W until the temperature reached 60° and was held for 5 min
using a microwave oven (Sineo MAS-II Plus) with a stirrer. For conventional thermal method, 100 mL of fresh
pineapple juice was heated to 60°C in a beaker using hotplate with stirrer and was held for 5 min. The temperature of
the both samples were measured with a thermocouple with an accuracy of ±1°C. The treated samples were
immediately cooled until the temperature reached 25°C and kept in the dark at 4°C for 30 days.

Determination of bromelain and vitamin C

Bromelain activity measurement was carried out according to the method described by Sew et al. (2014). Method for
determination of vitamin C was conducted as described by Shamsudin et al. (2014).

Microbiological analysis
A plate count agar (PCA) was used to determine the total plate counts (TPC). 0.1 mL of juice samples from each
serial dilution (10-1 to 10−5) was spread onto the solidified agar. Then, the PCA plate was incubated for 2 days at
37°C. The results were expressed as log CFU/ml.

Statistical analysis
The treatments were conducted in triplicate and the results were expressed as means ± standard deviation. The
statistical evaluation was performed using Minitab 17 Statistical Software and the data was analysed using 2 sample
t-test.

Results and Discussions

The changes of bromelain, vitamin C, and microbial content of pineapple juices after microwave and conventional
thermal treatment were tabulated in Table 1. It was found that microwave heating effectively retained a higher amount
of bromelain compared to conventional heating. Statistical analysis showed a significant reduction (p<0.05) of
bromelain content in conventional pasteurised pineapple juice. The reason of this reduction could be due to the longer
exposure time during conventional heating to reach the target temperature resulted in a higher inactivation rate of
proteolytic activity. The degradation of bromelain contents in both pasteurised pineapple juices during storage period
of 30 days are shown in Figure 1(a). Storage had significantly (p<0.05) affected the contents of bromelain in both
pasteurised pineapple juices. However, a greater loss was observed when the juice was conventionally heated
compared to microwaved samples. After 30 days, the loss of bromelain in microwaved and conventional treated
samples were 46% and 71.94%, respectively. The reduction of bromelain in pasteurised pineapple juices was best
fitted with a first-order kinetic model with k values of 0.0175 ± 0.0002 day -1 (microwave) and 0.0322 ± 0.0027 day-1
(conventional). A lower k value for microwave treated sample proved that retention of bromelain is higher in
microwaved juice.

Table 1: Comparison of different parameters for microwaved and conventional treated juice
Parameters Microwave treatment Conventional treatment
Residual bromelain activity (%) 87.36 ± 3.15a 70.83 ± 7.01b
Residual vitamin C (%) 90.87 ± 1.13 a 74.72 ± 0.37b
Microbial content
1.30 ± 0.06a 1.00 ± 0.09a
(log CFU/ml)
Values are mean ± standard deviation of three replicates and values with different superscript letter within the same
row are statistically different from each other (p < 0.05).

Heating processes had a significant effect on vitamin C as this nutrient is sensitive to heat. Although microwave
processing did not show a significant loss in vitamin C, but it was significantly reduced (p<0.05) by conventional heat
treatment. During 30 days of storage period, it can be seen that the degradation was rapid in conventional heated
sample as shown in Figure 1(b). The residual percentage of vitamin C in microwaved and conventional treated
samples after 30 days were 49.88 ± 1.60% and 32.40 ± 1.21, respectively. The reduction of vitamin C over time can
be attributed to the oxidation process resulting from the exposure of oxygen, light as well as the activities of ascorbate

66
oxidase and peroxidase enzymes (Davey et al., 2000). Degradation of vitamin C followed a first-order kinetic model
for microwaved and conventional treated samples. The k values are 0.0182 ± 0.001 day -1 and 0.0257 ± 0.001 day-1
for microwaved and conventional heated pineapple juice, respectively. The lower k value of microwaved sample
further confirmed that pineapple juice was better preserved when it was processed using microwave heating
technology.

The present study showed that both microwaved and conventional treated samples showed a significant reduction
(p<0.05) in microbial contents of pineapple juice. Based on the fact that heat can cause a cytolytic effect towards
microorganism (George et al., 2015), this clearly shows that both treatments were effective in the inactivation of
microbes. Statistical analysis showed no significant differences in the microbial reduction for both methods. The
destruction of microbial was mainly because of the free radical production, disruptive of cell membranes and localized
heating (Lagnika et al., 2017). The shelf life of both samples were determined based on the acceptable limit under EU
legislation. The microbial limit for total aerobic and mesophilic bacteria is ≤3.10 log10 CFU/ml (EU, 2005). On this
basis, the shelf life of microwaved and conventional pasteurised pineapple juice were 22 days and 14 days,
respectively as depicted in Figure 2.
0 0
0 10 20 30 -0.1 0 10 20 30
-0.2
R² = 0.9615 -0.2
-0.4 -0.3 R² = 0.96
ln C/C0

ln C/C0

-0.4
-0.6
-0.5
-0.8 -0.6
R² = 0.9907 -0.7 R² = 0.9736
-1
-0.8
-1.2 -0.9
Storage period (Day) Storage period (Day)

Microwave Conventional Microwave Conventional

(a) (b)
Figure 1: First-order kinetic model for degradation of (a) bromelain and (b) vitamin C.
8
7
6
5
log CFU/ml

4
3
2
1
0
-1 0 5 10 15 20 25 30
Storage period (Day)

Microwave Conventional

Figure 2: Changes in microbial content of microwave-treated and conventional treated pineapple juice during storage
at 4°C. Dashed line (---) indicates the limit of microbial shelf life for juices.

67
Conclusion
From the findings on this study, it is proven that microwave heating treatment can preserve nutritional values of
pineapple juice better than using conventional thermal treatment. Additionally, the inactivation of microorganisms in
pineapple juice were successfully achieved in this study. Therefore, this technology can potentially be employed as
pasteurisation method in medium scale processing industry of pineapple juice. Further investigations that involves
scaled-up processes, process design, and optimization should be conducted to maximize the potential of microwave
technology in fruit-juice processing and at the same time maintaining a high quality of fruit juice.

Acknowledgements
The authors would like to thank Universiti Teknologi Malaysia for the financial support and facilities provided
throughout this study.

References

Abdul Majid, F. A., Abdul Gani, M., Talib, S. Z., Hasyim, K. K. (2008). Stability of bromelain-polyphenol complex in
pineapple juice. Jurnal Teknologi, 49(F), 27-38.
Davey, M. W., Van Montagu, M., Inze, D., Sanmartin, M., Kanellis, A., Smimoff, N., Benzie, L. J. J., Strain, J.
J.,Favell, D. & Fletcher, J. (2000). Plant L-ascorbic: chemistry, function, metabolism, bioavailable and effects
od processing. Journal of the Science of Food and Agriculture, 80(7), 825-860.
EU. (2005). Commisison regulation (EC) No 2073/2005 of 15 November 2005 on the microbiological criteria of
foodstuffs. Official Jornal of the Europian Union, L338, 1-26.
George, D. S., Razali, Z., Santhirasegaram, V., & Somasundram, C. (2015). Effects of Ultraviolet Light (UV-C) and
Heat Treatment on the Quality of Fresh-Cut Chokanan Mango and Josephine Pineapple. Journal of Food
Science, 80(2), S426-S434.
Goh, S. G. (2012). Effect of thermal and ultraviolet treatments on the stability of antioxidant compounds in single
strength pineapple juice throughout refrigerated storage. International Food Research Journal, 19(3), 1131-
1136.
Lagnika, C., Adjovi, Y. C. S., Lagnika, L., & Gogohounga, F. O., Do-Sacramento, O., Koulony, R. K. & Sanni, A.
(2017). Effect of Combining Ultrasound and Mild Heat Treatment on Physicochemical , Nutritional Quality and
Microbiological Properties of Pineapple Juice. Food and Nutrition Sciences, 8(2), 227-241.
Sew, C.C., Mohd Ghazali, H., Martin-Belloso, O., Noranizan, M. A. (2014). Effects of combining ultraviolet and mild
heat treatments on enzymatic activities and total phenolic contents in pineapple juice. Innovative Food Science
and Emerging Technologies, 26, 511-516.
Shamsudin, R., Mohd Adzahan, N., Pui Yee, Y., & Mansor, A. (2014). Effect of repetitive ultraviolet irradiation on the
physico-chemical properties and microbial stability of pineapple juice. Innovative Food Science and Emerging
Technologies, 23, 114-120.
Uckiah, A., Goburdhun, D., Ruggoo, A. (2009). Vitamin C content during processing and storage of pineapple.
Nutrition & Food Science, 39(4), 398-412.

68
 IFRC 2017: 184-152 Food Safety and Quality

Development of selective esculin hydrolysis broth for rapid screening of Vibrio parahaemolyticus

1Sangadkit, W., 2Deepatana, A. and 1,3*Thipayarat, A.

1Department of Food Engineering, Faculty of Engineering, King Mongkut’s University of Technology Thonburi, 126
Pracha-u-Tid Road, Bangmod, Tungkru, Bangkok 10140, Thailand.
2Department of Chemical Engineering, Faculty of Engineering, Burapha University, 169 Long-Hard Bangsaen Road,
Saensook, Muang, Chon Buri 20131, Thailand.
3Office of Education, Faculty of Engineering, Burapha University, 169 Long-Hard Bangsaen Road, Saensook, Muang,
Chon Buri 20131, Thailand.
Abstract
This research explored alternative rapid screening strategies of V. parahaemolyticus detection by the occurrence of
blackened broth as a result of the esculin-ferric ammonium citrate reaction. V. parahaemolyticus as well as other
Gram-positive competitors and Gram-negative non- V. parahaemolyticus bacteria were cultured in these substrates
and incubated at 37C for 24 h. Broth color was monitored using the micro scale cultivation technique and
spectrophotometer at discrete wavelengths from 340 to 650 nm, respectively. The results showed that the optimal
signal in detecting esculin activity was at 450 nm and detected as early as 18-24 h after incubation. The esculin
activity itself was able to differentiate Gram-negative from – positive due to the lack of -glucosidase (no dark brown
broth for S. aureus). In this developed esculin based broth, the concentrations of sodium chloride (3, 6, 8 and 10%
w/v) and pH values (7, 8, 9 and 10) were varied to determine the most optimal selectivity towards V.
parahaemolyticus selection. The optimized esculin-ferric ammonium citrate based broth, named EFA (esculin, ferric
ammonium citrate, and arabinose), effectively increased black color and OD450 of V. parahaemolyticus and displayed
good selectivity. The modified esculin broth was able to presumptively screen the presence of V. parahaemolyticus in
industrial food samples within 1 day in contrast to the conventional method.

Keywords: selective esculin hydrolysis broth, rapid screening, Vibrio parahaemolyticus.

*Corresponding author’s email: aluck@eng.buu.ac.th

Introduction
Vibrio parahaemolyticus, a kind of Gram-negative motile bacteria inhabiting marine and estuarine environments
throughout the world. It is a major foodborne pathogen that causes diarrhea primarily after the consumption of raw or
undercooked seafood. Current BAM method for the detection of V. parahaemolyticus in foods requires minimum 3
days to obtain presumptive positive or negative results. This conventional method is time-consuming, labour-intensive
for reliable identification and high operating cost (Kaysner and DePaola, 2004). The requirement of food industry is to
determine the presumptive results as early as possible to make a decisive production judgment for the food industry.
It can provide financial saving in time and operating cost for routine monitoring of food products. The analytical time
can be decreased by applying specific biochemical properties enabling differentiation of V. parahaemolyticus from
other competing microbes.

In this paper, the optical density change due to esculin hydrolysis by V. parahaemolyticus and other competitive
bacteria in modified esculin broth was studied to show its application for primary screening of V. parahaemolyticus.
The effect of selective agents (combined sodium chloride and adjusted pH) was evaluated on the reaction of esculin
hydrolysis by false positive bacteria to differentiate these bacteria from V. parahaemolyticus.

Material and Methods

3.1 Bacterial strains and culture preparation

69
V. parahaemolyticus DMST 17802 and other competitive bacteria were prepared. One loopful of the stock culture
were recovered in tryptic soy broth at 37C for 24 h and then diluted to obtain the bacterial culture at specified
concentrations.

3.2 Medium broths

Proposed esculin indicative broth based on contained (g/l): peptone 10; beef extract 5; ferric ammonium citrate 0.5
and esculin 1 g. The esculin broths were varied with different sodium chloride concentrations at 3, 6, 8, and 10% (w/v)
and pH adjusting to initial level at pH 7, 8, 9, and 10 by HCl 1 N and NaOH 1 N and then sterilized by filtration through
a sterile nylon syringe filter membrane (13 mm diameter, 0.45 μm pore size) before using.

3.3 Microscale enrichment of V. parahaemolyticus and non- V. parahaemolyticus

The 3-4 log CFU/ml inoculum of V. parahaemolyticus and other negative and positive esculin bacteria were prepared.
Each inoculum of 20 μl was transferred into modified esculin (180 μl) contained in a 96-well flat bottom microplate.
The esculin without inoculation 200 μl was the control sample. Visible wavelength at 340, 405, 450, 490, 550, 600 and
650 nm absorption spectra of the modified broth at sodium chloride 3% and pH 7 was obtained by the
spectrophotometer during the incubation at 37°C for 24 h (Fig. 1a). The absorbance difference between the peak
absorption of the positive media at different wavelength and peak absorption of the control media was proposed (Fig.
1b). The highest absorbance difference was determined for detection of V. parahaemolyticus.

3.4 Effect of carbohydrate on the esculin-ferric ammonium citrate reaction for V. parahaemolyticus
The esculin was a base broth for making 19 test broths (i.e., dextrose, maltose, arabinose) by adding 3.5 g/l of
fermentable carbohydrate. The broths were prepared as shown in 3.2

Results and Discussion

Optical spectrum scans
When V. parahaemolyticus was inoculated in esculin broth based, the natural color broth noticeably transformed to a
darker with 18 h and measured the absorbance at different wavelengths as shown in Figure 1a. The differences of the
absorbance reading from the positive and control broth indicated that there was a few optimal wavelength at 450 nm
to differentiate the positive- and negative-samples (Fig. 1b). The esculin broth was used to evaluate esculin
metabolism by 8 food-borne microorganism models. The selected positive esculin hydrolysis groups (V.
parahaemolyticus, L. monocytogenes, K. pneumonia, E. faecalis, S. marcescens, and B. cereus) was able to convert
esculin to esculatin which then reacted with ferric ions to darken the esculin broth (Netten et al., 1989). For the
negative models (V. cholerae and S. aureus), the color of broth remained unchanged and the color broth stayed
yellowish throughout the course of incubation (Fig. 1c), which supported the use of spectrophotometry at 450 nm as
described above.

Practically, proper selective inhibitors must be incorporated to increase selectivity towards V. parahaemolyticus
species. The most desirable inhibitory system for modified esculin should facilitate the growth of V. parahaemolyticus
and promote the chromatic change of the broth as a result of esculin-ferric ammonium citrate activity. Table 1 Effect
of different concentration of sodium chloride and pH levels on V. parahaemolyticus and non-V. parahaemolyticus. The
control treatment without any inhibitor showed no selective preference towards any Gram-positive or -negative
microorganisms whereas the esculin broth at 6% sodium chloride concentration and adjusted pH level at 10 inhibited
all positive esculin competitors. It was hypothesized that high concentration of sodium chloride combined with alkaline
pH helped regulate the tonicity of the enrichment medium (Glass, 1992). This composition provided good inhibitory
effect against Gram-positive bacteria while minimally affected the recovery and growth of V. parahaemolyticus. In
industrial application, this formula is potentially useful for safety V. parahaemolyticus screening. This broth has a
sensitive color indicator and minimal inhibitory effect, consequently no false-negative result.

To improve the ability on esculin ferric-ammonium citrate leading to increase blacken production, carbohydrate
fermentable groups were evaluated. V. parahaemoyticus was tested in prepared media at 6% sodium chloride
concentration and pH 10 to study effects of carbohydrate fermentation on the increase of blacken broth. Among 19

70
 carbohydrate substances, arabinose showed the highest absorbance signal value. Thus the rapid from arabinose
supported and accelerated esculatin production.

2.5 2.5 3.5

control sample positive - control sample

V. parahaemolyticus (positive) 3.0
2.0 2.0

Absorbance difference
2.5
Absorbance

1.5 1.5
2.0

A450
1.5
1.0 1.0

1.0

0.5 0.5
0.5

0.0 0.0 0.0

300 350 400 450 500 550 600 650 700 300 0 350 5
400 45010 500 15 550 20
600 65025 700 30

Time (h) TimeTime

(h) (h)

(a) absorption spectra (b) absorbance difference (c) absorbance profiles

Figure 1. Absorbance spectral scans (a); absorbance difference between positive and control sample (b) and time
course of absorbance reflecting esculin activity in positive and negative esculin bacteria.

Table 1: Esculin activity of V. parahaemolyticus and competitive bacteria enriched in esculin broth supplemented with
different sodium chloride concentrations and pH levels. The results were based on absorbance reading at 450 nm
and detected by microplate reader along 24 h.
3% NaCl 6% NaCl 8% NaCl 10% NaCl
Test strains control pH levels
7 8 9 10 7 8 9 10 7 8 9 10 7 8 9 10
V. parahaemolyticus + + + + + + + + + + + + - - - - -
E. aerogenes + + + + + + + + - + - - - - - - -
K. pneumoniae + + + + + + + + - + - - - - - - -
S. marcescens + + + + + + + + - - - - - - - - -
L. monocytogenes + + + + - + + - - + + - - + - - -
E. faecalis + + + + + + + + - - - - - - - - -
E. avium + + + + + + + + - - - - - - - - -
B. cereus + + + + - - - - - - - - - - - - -

1.4

1.2

1.0

0.8
A450

0.6

0.4

0.2

0.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Figure 2. Effect of carbohydrate fermentation on esculatin production in V. parahaemolyticus detection by optical

method. (1:no sugar; 2:salicin; 3:arabinose; 4:xylose; 5:melibiose; 6:mannitol; 7:galactose; 8:sorbitol; 9:rhamnose;
10:dextrose; 11:lactose; 12:sucrose; 13:inositol; 14:mannose; 15:adonitol; 16:trehalose; 17:fructose; 18:maltose;
19:cellobiose; 20:ducitol)

71
Conclusion
The use of esculin activity provided a good indication for V. parahaemolyticus screening. The reaction produced
reliable A450 reading to differentiate the esculin-positive apart from esculin-negative results. The absorbance
measurement of esculin broth corresponded well with visual detection of broth color but provided fast numerical
evaluation using a spectrophotometer. For inhibitory agents, modified esculin with 6% sodium chloride and adjusted
pH level at 10 was chosen for good recovery and high selectivity in pure culture. The optimized esculin-based broth
combined with arabinose effectively increased blacken broth. The formula is potentially a new effective indicative
broth for rapid precautionary V. parahaemolyticus screening.

Acknowledgements
This work was financially supported by a Research Grant from Burapha University through National Research Council
of Thailand (Grant No. 156/2559) and a graduate scholarship from the Thailand Research Fund through the Royal
Golden Jubilee Ph.D. Program (Grant No. PHD/0216/2552) to Wipavadee Sangadkit and Asst. Prof. Dr. Aluck
Thipayarat.

References
Glass, K.A., Jodi, M., Loeffelholz, J., Ford, P. and Doyle, M.P. 1992. Fate of Escherichia coli 0157:H7 as affected by
pH or sodium chloride and in fermented, dry sausage. Applied and Environmental Microbiology 58(8): 2513-
2516.
Kaysner, C.A., DePaola Jr., A., 2004. Chapter 9, Vibrio. U.S. Food and Drug Administration Bacteriological Analytical
Manual. Retrieved on June 2, 2017 from BAM Website: www.fda.gov/Food/ScienceResearch/
LaboratoryMethods/BacteriologicalAnalyticalManualBAM/UCM070830.
Netten, P. van., Perales, A., van der Moosdijk, A., Curtis, G.D.W. and Mossel, D.A.A. 1989. Liquid and solid selective
differential media for the detection and the enumeration of Listeria monocytogenes and other Listeria spp.
International Journal of Food Microbiology 8(4): 299-316.

72
 IFRC 2017: 186-156 Food Safety and Quality

Evaluation of the Marketability, Microbial Quality, and Safety of Fresh-Cut Vegetable Mixes From Selected
Wet Markets and Supermarkets in Los Baños, Laguna
*1Sotiangco, I.D.G., 2Piamonte, S.B.H., 1Castillo-Israel, K.A.T.
1Food Science Cluster, College of Agriculture and Food Science, University of the Philippines Los Baños, Laguna,
Philippines
2Department of Social Sciences, College of Arts and Sciences, University of the Philippines Los Baños, Laguna,
Philippines
Abstract
With the consumers’ increased awareness in healthy lifestyle, consumption of fruits and vegetables has increased by
about 30% for the past decades. The study aimed to evaluate the marketability, processing practices, microbial
quality, and safety of fresh-cut vegetable mixes sold in selected wet markets and supermarkets in Los Baños,
Laguna. From the 500 survey respondents in Los Baños, results showed that fresh-cut vegetable mixes have
moderate marketability with quality as the most important factor considered when buying. The compliance of food
processors to food safety practices in processing fresh-cut vegetable mixes was evaluated through interviews and
checklists. Based on the evaluations, poor compliance was observed among processors in wet markets. For the
analysis of microbial quality, pour plating technique was used. All had high counts for APC (7.01-7.49 log(10) cfu g-1),
total coliforms (4.60-7.35 log(10) cfu g-1), and yeasts and molds (5.26-8.52 log(10) cfu g-1. Salmonella was not
detected in the fresh-cut samples; however, E. coli were present in all of the samples. The high persistence and
proliferations of these microorganisms reflect poor hygienic and sanitation practices of food handlers resulting to poor
microbial quality and food safety issues.
Keywords: Fresh-Cut Vegetable Mixes, Microbial Quality, Marketability, Food Safety
*Corresponding author’s email: isotiangco@gmail.com
Introduction
The fresh-cut produce is now a fast growing segment of the agricultural sector. The increase in consumption of fresh
produce could be attributed to the increased awareness in healthy eating habits (Barth et al., n.d.). In Asia, fresh-cut
produce are sold in open-air markets, food stands and supermarkets. Fresh produce being sold without refrigeration
may encourage the growth of microorganisms which could cause foodborne illness when consumed (FAO, 2011).
Furthermore, practicing proper sanitation procedures and proper hygiene greatly reduce the possibility of microbial
infiltration and cross contamination. Hence, promoting consciousness and awareness of the possible health risks
caused by spoilage and contamination to consumers and food handlers is important. The study aimed to determine
the marketability of fresh-cut vegetable mixes based on consumer buying behavior, evaluate the processing practices
done by food handlers in the selected wet markets and supermarkets, and assess the microbial quality and safety of
samples in terms of aerobic plate count, yeasts and molds, and total coliforms, and the presence of Escherichia coli
and Salmonella.
Methodology
Market Research.
500 survey respondents buying vegetables in Los Baños, Laguna were selected using non-probability sampling from
the total population of Los Baños of 101,884 with a confidence level of 97%, margin error of 4.84%, and a response
distribution of 50%. Respondents were limited to ages 18 to 60 year-olds purchasing at supermarkets and wet
markets.
Process Evaluation.

73
Using a checklist in reference to FDA’s Guidance for Industry, different parameters related to processing of fresh-cut
vegetable mixes were observed and evaluated in two selected supermarkets and wet markets, in Los Baños, Laguna.
Parameters included personnel, buildings/area and equipment, sanitation operations, production and process control,
and distribution and marketing. Interviews with the food handlers were conducted for further assessment of their
processing practices.
Microbial Analysis.
Sixteen fresh-cut vegetable mixes (pinakbet mix, pancit mix, and chopsuey mix) and whole vegetables sold in wet
markets and supermarkets were collected and analysed at the Food Science Cluster, UPLB, for yeasts and molds,
total scoliforms, aerobic plate count (APC), and the presence for Escherichia coli. The method for microbial analysis
was adapted from the Compendium of Methods for the Microbiological Examination of Foods. For the detection for
the presence of Salmonella, samples were delivered to Biotech UPLB for analysis using PCR-based Salmonella
DAS™ Kit.
Results and Discussion
Fifty four percent of the market-goers in Los Baños, Laguna were buying fresh-cut vegetable mixes; 59% of which
buy in wet markets and the remaining 41% buy in supermarkets. The top three reasons for purchasing these fresh-cut
vegetable mixes were: cheap, saves time, and less waste. When respondents were asked which among the three
factors enumerated was the most important in buying fresh-cut vegetable mixes, 53% answered quality, 29% for
price, and 18% answered safety. On the other hand, the top two reasons why market-goers do not purchase fresh-cut
vegetable mixes were: poor quality and look dirty/unsafe.
With the fast-paced lifestyle today, convenience is one of the main factors consumers look at in purchasing food
products. In interviews, consumers who buy fresh-cut vegetable mixes were those in need to save money and/or
those on a tight budget. It is cheaper to buy fresh produce in wet markets. Hence, the preference of buying fresh-cut
vegetable mixes in wet markets. Despite being cheap, consumers still considered quality as the most important factor
when buying fresh-cut vegetable mixes. However, the perception of consumers on quality is limited to what they see
and is therefore, subjective. Overall, fresh-cut vegetable mixes have moderate marketability with the odds of males
buying fresh-cut vegetables mixes is 10% higher as compared to females; 15% higher for those who are not single as
compared to those who are single; 24% higher for the old age group (36 years old and above) as compared to the
young ones (18 to 35 years old); and 54% higher for persons belonging to a small household (1 to 6 persons) as
compared to those belonging to a large household (7 members and above). These show that demographic
characteristics affect the preference of consumers in choosing between buying fresh-cut vegetables mixes and whole
vegetables.
There was poor compliance of hygiene and sanitation practices by processors in wet markets. Processors can be
carriers for possible contamination of the produce through microorganisms present on the skin, hair, hands, digestive
systems or respiratory tracts. It is also important to consider infrastructures, facilities, and equipment during
processing for they may also be possible sources of direct contamination and cross-contamination. Since
microorganisms are naturally occurring in the environment, it is necessary to emphasize the importance of sanitation
practices and personal hygiene especially those into the fresh produce sector (FDA, 2008).
Table 1. Summary of microbial analysis of fresh-cut vegetable Table 2. Summary of microbial analysis of fresh-cut vegetable
mixes bought in selected wet markets in Los Baños, Laguna. mixes bought in selected supermarkets in Los Baños, Laguna.
Market 1 Market 2 Market 1 Market 2
Fresh-Cut Whole Fresh-Cut Whole Fresh-Cut Whole Fresh-Cut Whole
Aerobic Plate Count Aerobic Plate
7.49 5.40 7.42 5.70 Count (log cfu g-1)
(log cfu g-1) 7.01 5.29 6.61 5.34
Total Coliforms Total Coliforms
7.05 5.40 7.35 5.17 5.57 5.14 6.81 4.60
(log cfu g-1) (log cfu g-1)
Yeasts and Molds Yeasts and Molds
8.52 5.84 7.60 5.28 7.86 5.26 5.48 4.98
(log cfu g-1) (log cfu g-1)
Presence of E.coli Presence of E.coli
Positive Positive Positive Positive Positive Negative Positive Negative
colonies colonies
Presence of Presence of
Negative Negative Negative Negative Negative Negative Negative Positive
Salmonella Salmonella

74
As seen in Tables 1 and 2, the APC values of fresh-cut vegetables fall between the expected range of raw, ready-to-
eat salad and vegetables which is 6 log(10) to 8 log(10) cfu g-1. Total coliforms, on the other hand, had values from
5.57 to as high as 7.35 log(10) cfu g-1 for fresh-cut vegetable mixes and 4.60 to 5.40 log(10) cfu g-1 for whole
vegetables which exceeded the limit for fecal coliforms for minimally processed salads, 2 log(10) cfu g -1, set by the
Brazilian standard (Health Protection Agency, 2009). These results indicate the possibility of improper handling and
overall poor hygiene and sanitation practices by the farmers, processors, distributors, and vendors. For yeasts and
molds counts, samples had values from 5.26 to 8.52 log(10) cfu g-1. Significant numbers of yeasts and molds are
predominant in fresh, raw vegetables. Their presence in high numbers decreases the shelf-life of the fresh-cut
vegetables as well as its quality. However, yeasts may cause spoilage at slightly lower levels, 106 to 107 cfu g-1, due
to acid and gas production (Health Protection Agency, 2009). Furthermore, spoilage caused by molds does not pose
a major problem to the quality of fresh vegetables. Possible health problems associated with molds are their ability to
produce toxins and cause allergic reactions when enough conidia are produced (Abadias et al., 2008).

Whole vegetables were observed to have lower microbial counts than fresh-cut vegetable mixes showing that
processing can affect the microbial load. Bigger surface area exposed to the atmosphere meant easier access to
oxygen, which is important to microbial growth. Since more flesh is exposed for fresh-cut vegetable mixes, more
available nutrients are available for the microorganisms. Unlike with the whole vegetables, the skin is intact
preventing the entry of microorganisms to the flesh.

All samples from both wet markets and supermarkets were positive for the presence of E. coli and only one sample,
from whole vegetables, was positive for the presence of Salmonella. Most E. coli are harmless however certain
strains are pathogenic. Salmonella spp. on the other hand, is one of the pathogenic microorganisms of greatest
health concerns. It causes two types of illnesses which are gastrointestinal and typhoidal, the latter being a very
serious condition wherein 10% of the people infected who doesn’t get treatment die (FDA, 2012). Presence of these
microorganisms could pose serious health risks to consumers.

Conclusion
Demographic characteristics are contributing factors on consumers’ preference in buying fresh produce and
consumers’ assessment on quality of these fresh produce is only based on appearance, which is very subjective. One
may see it of good quality based from appearance but could actually contain high microbial loads, including
pathogenic microorganisms. Fresh-cut vegetable mixes have moderate marketability which means a lot of consumers
could still be at risk if these fresh-cut mixes are contaminated. Moreover, microbial analysis, if used correctly, can
provide useful information about the general quality, safety and shelf-life of fresh-cut vegetable mixes, thus
highlighting the potential problems of storage and handling (Health Protection Agency, 2009). The high persistence
and proliferations of these microorganisms reflects the hygienic and sanitation practices of food handlers and
processors. Strict compliance, implementation and surveillance system of good manufacturing practices and existing
food laws should be improved. Currently, there are no standard limits set in the Philippines for fresh-cut vegetables or
produce. It is recommended to start developing guidelines and establish standard limits for microbial quality and
safety.

Acknowledgements
The authors would like thank the Emerging Interdisciplinary Research (EIDR) Grant of UP-OVPAA for the funding of
the research.

References

Abadias, M., Usall, J., Anguera, M., Solsona, C., & Viñas, I. (2008). Microbiological quality of fresh, minimally-
processed fruit and vegetables, and sprouts from retail establishments. International Journal of Food
Microbiology, 123(1-2), 121-129. doi:10.1016/j.ijfoodmicro.2007.12.013

Barth, M., Hankinson, T. R., Zhuang, H., and Breidt, F. (n.d). Microbiological Spoilage of Fruits and Vegetables.
Retrieved September 5, 2015 from fbns.ncsu.edu/USDAARS/Acrobatpubs/P351-375/p363.pdf.
75
Food and Agriculture Organization of the United Nations (FAO). 2011. Processing of Fresh-Cut Tropical Fruits and
Vegetables: A Technical Guide. Retrieved on September 5, 2015 from
www.fao.org/docrep/014/i1909e/i1909e00.htm
Food and Drug Administration (FDA). 2008. Guide to minimize microbial food safety hazards of fresh-cut fruits and
vegetables guidance for industry. College Park, MD: U.S. Dept. of Health and Human Services, Food and
Drug Administration, Center for Food Safety and Applied Nutrition. Retrieved on May 23, 2016, from
http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/ProducePlantProdu
cts/ucm064458.htm#ch5
Food and Drug Administration (FDA). 2012. Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural
Toxins. Second Edition. Salmonella spp., 9–13; Escherichia coli, 75–77.
Health Protection Agency. (2009, November). Guidelines for Assessing the Microbiological Safety of Ready-To-Eat
Foods Placed on the Market. London: Health Protection Agency.

76
 IFRC 2017: 187-153 Food Safety and Quality

Combined Ozonation and UV-C Treatment to Inactivate E. Coli Contaminants in Model Fish Sauce.

1Sangadkit, W., 1Kunpanya, P., 2Deepatana, A., 3Foster, K.W. and 1,4*Thipayarat, A.

1Department of Food Engineering, Faculty of Engineering, King Mongkut’s University of Technology Thonburi, 126
Pracha-u-Tid Road, Bangmod, Tungkru, Bangkok 10140, Thailand.
2Department of Chemical Engineering, Faculty of Engineering, Burapha University, 169 Long-Hard Bangsaen Road,
Saensook, Muang, Chon Buri 20131, Thailand.
3Department of Physics, Syracuse University, Physics Building, Syracuse, NY 13244-1130, USA
4Office of Education, Faculty of Engineering, Burapha University, 169 Long-Hard Bangsaen Road, Saensook, Muang,
Chon Buri 20131, Thailand.

Abstract
The application of ozonation and UV-C applied together, as well as individually, to eradicate Escherichia coli
contamination was tested on a model fish sauce, a liquid with caramel color and salt. A batch-type prototype of the
O3-UV treatment was constructed with a circulation flow of 5 l/min, an O 3 gas generator with a flow rate of 2.0 l/min,
and four UV-C (254 nm) lamp pasteurizers adjustable for 1.44 or 2.88 kJ/m2. Salt concentrations were 1, 10 or 20%
(w/v) and caramel was 0.03, 0.06 or 0.13% (w/w) adjusting the absorbance to 0.25, 0.50, or 1.00 OD 405nm,
respectively. Salt in the model solution increased the level of soluble solids and affected the efficacy of UV-C and O3.
The effectiveness of UV-C depended on the transmittance of UV-C in the solution whereas that of ozonation
depended on the total soluble solid (or the salt concentration) in the solution. Ionized salt critically altered the solubility
of ozone and largely affected the ozonation efficacy of microbial inactivation. The effect of caramel color was two-fold.
In addition to reducing the transmission of UV-C light due to increased optical density of the solution, the caramel also
changed the concentration of total soluble solids. The effectiveness of UV-C treatment was significantly compromised
and additional soluble solids also decreased the O3 solubility making ozonation less effective.

Keywords: Combined ozonation and UV-C treatment, inactivate, Escherichia coli, caramel and brine solutions.
*Corresponding author’s email: aluck@eng.buu.ac.th

Introduction
Fish sauce industry together with other traditional food products suffers from poor hygienic production practices and
microbial contamination of finished and intermediate products. Advance oxidation processes (AOP) have emerged as
a competent technology to control and disinfect microbial contamination with minimal effect on the overall quality of
food products. Many AOP applications have been demonstrated on food materials; for example, development of non-
thermal disinfection technology by combining ozone and ultraviolet-C (UV-C) was developed and shown effective in
wash water for the fresh-cut vegetable industry. The use of UV-C treatment alone has also demonstrated high
efficacy in inactivating various foodborne pathogens (e.g., Salmonella spp., Escherichia coli O157:H7, Listeria
monocytogenes, etc.) in water and various food matrices (Mukhopadhyay et al., 2014). In this paper, a water mixture
of different salinity and/or optical density levels was utilized to represent a fish sauce model. This rather complex
liquid food was pasteurized by the combination of ozonation and UV-C sterilization to reduce microbial contamination.
In the UV-C/O3 application, the effectiveness of combined AOP treatment was evaluated on a model aqueous solution
containing two main interferences (different caramel concentrations and salt contents). The efficacy of UV-C/ozone
treatment on E. coli inactivation was assessed. The use of UV-C/ozone was proposed as an alternative nonthermal
sterilization of food liquids with complex interfering nature.

77
Material and Methods
3.1. Bacterial strain and media
The E. coli DMST 4609 culture was prepared at 107 CFU/mL and spiked onto the caramel and brine solution. The
abundance of E. coli cells in the UV-C/O3 treated solution were evaluated intermittently every 2.5, 5, 10, 15, 20, 25
and 30 min. Colony count (CFU/ml) of E. coli counts were quantified using the micro inoculation culture (MIC) method
(Khueankhancharoen et al., 2011).

3.2 Preparation of fish sauce model

Caramel solutions were prepared at 5 different optical densities (i.e., 0.25, 0.50 1, 2 and 3 OD405 nm) using 2, 4, 8, 18,
32 g of caramel liquid in 6 l of DI water, respectively. The OD of caramel solution was measured using a microplate
reader (M965, Metertech, Taiwan). The brine solutions were prepared by adjusting salt concentrations at 1, 10 and
20% (w/v) using NaCl.

3.3 O3, UV, and O3-UV treatment

An UV-C/O3 reactor was constructed by connecting 10 l storage tank with a O3 venturi mixer and four UV-C lamp sets
(254 nm wavelength at 16 W fluency each). The maximum fluence dose of the four UV-C set combined is 2.88 kJ/m2.
Pure DI water was used as a control and compared the microbial destruction efficacy against caramel and/or brine
solutions. The flow rate of O3 gas was fixed at 2 l/min of O2 gas. The dissolved O3 concentration was monitored and
maintained at 450-550 mV using an oxidation reduction potential (ORP) sensor. For each run of UV-C/O3 treatment, 6
l of the prepared solution was filled in the storage tank and the solution was circulated in the system with different UV-
C and O3 settings for 30 min.

Results and Discussion

Comparison of the effect of UV-C, O3 and UV-C/O3 on caramel water
Simulating a variety of common liquid foods (e.g., apple juice, soy sauce, fish sauce, etc.), the absorbance and color
of the DI water was adjusted from clear to dark brown by adding the caramel food additive. The interference of liquid
color did affect the E. coli inactivation profiles in all treatments (Fig. 1). Increasing the optical densities reduced the
effectiveness of the UV-C and/or O3 treatment significantly. At the 1 OD405nm treatments, the ozonation did not show
any E. coli reduction. The profiles of E. coli survival ratio for both the ozonation and control experiments were
practically identical (Fig. 1c). Perhaps as the OD increased, the O 3 solubility in the solution was restricted and less
residual dissolved O3 remained to disrupt cells. Also the efficacy of the UV-C radiation was substantially limited as the
absorbance increased. At 1 OD405nm, both 1.44 and 2.88 kJ/m2 UV-C treatments were unable to produce zero cell
count within 30 min of treatment. Only 5 log reduction of E. coli cells were achieved. The effect of OD on UV-C
inactivation efficacy can be observed from the time increase to obtain complete E. coli inactivation from 2.5 to more
than 15 min in the clear DI water to 0.25 OD405nm caramel water, respectively. In Fig. 1a, the time to achieve 7 log
reduction of E. coli density was shifted from less than 2.5 min in the DI water experiment to 20 and 15 min for 1.44
and 2.88 kJ/m2 UV-C radiation, respectively. As the absorbance was increased from 0.03 to 0.13% caramel color,
both UV-C intensities were able to inactive (Fig. 1a and c). From the observation of the E. coli survival profiles, it can
be construed that the addition of caramel color affects the efficacy of ozonation more than the UV-C sterilization. The
profiles of the O3-1.44 kJ/m2 UV and -2.88 kJ/m2 UV treatments did resemble to those of 1.44 and 2.88 kJ/m2 UV-C
treatments than the ozonated treatment, especially at higher absorbance experiments. In the presence of color
compounds, organic solutes and suspended matter, lower UV-C light was able to penetrate turbid and colored liquid
foods and degraded the performance efficacy of the UV-C pasteurization in reducing the levels of microbial
contamination (Choudhary and Bandla, 2012).

UV-C, O3 and UV-C/O3 treatments in brine solutions

Salt contents produced subtle effects on the E. coli inactivation in this UV-C/O3 treatment. At relatively low salt
content approximately 1% similar to standard saline, this level of salt deteriorated the effectiveness of the O 3 and UV-
C/O3 treatment (Fig. 2a). Also the improvement of UV-C sterilization in the UV-C/O3 treatment was affected. For
instance, the O3-2.88 kJ/m2 UV treatment in the 1% NaCl salt did not longer produce absolute sterility and only 5.20
log reduction was observed for 30 min of treatment time. As the salt content in DI water was increased from 1 to 10%,
higher salt concentration began to impact the UV-C transmission in the sterilizing chambers (Fig. 2b). At 10% NaCl,
78
 only high UV-C dose treatment (2.88 kJ/m2) was able to sterilize the tested spiked water within 2.5 min. The
sterilization time of the lower UV-C dose (1.44 kJ/m2) required twice as long extended to 5 min. Higher salt did
negatively affect the effectiveness of ozonation as seen in the 1% NaCl treatment. However, high salt solution had
some profound impacts on the E. coli survival ratio. High salt concentration produced higher E. coli reduction in both
O3-2.88 kJ/m2 UV-C and O3-1.44 kJ/m2 UV treatments showing steeper slopes of E. coli inactivation profile and lower
E. coli counts. The salt concentration was double and the level of NaCl was intensified. The growth condition at 20%
NaCl was not suitable for E. coli. In Figure 2c, the cell counts of E. coli with no UV-C and/or O3 treatments showed the
decline of growth over 30 min. At this level of NaCl, the effect of ozonation was minimal. The only effective treatment
was the UV-C radiation as noticed by the E. coli inactivation profiles of the UV-C and UV-C/O3 treatments. The O3-
2.88 kJ/m2 UV treatment was almost identical to the 2.88 kJ/m2 UV treatment. The same result was applied to the O3-
1.44 kJ/m2 UV treatment. It is noticeable that the high concentration of salt synergize with the treatment with UV to
inactivate E. coli cells.

Even though in this transparent solution of NaCl, the UV-C pasteurization was relatively effective to curve E. coli
growth, the combined ozone and UV-C system was able to produce 2.7 to 7-log reduction where ozonation alone
rendered poor performance. With ozone acting as a protoplasmic oxidant and UV-C fluence causing the disrupt
transcription and DNA replication, high NaCl concentration exerted addition hurdles to the sequential use of these
advanced oxidation processes for antimicrobial treatments (Glass et al., 1992).

no UV
UV 1.44 kJ/m2

O3-UV 1.44 kJ/m2

O3 0.13% caramel
UV 2.88 kJ/m2 O3-UV 2.88 kJ/m2
0.03% caramel 0.06% caramel

a) 0.03% caramel b) 0.06% caramel c) 0.13% caramel

Figure. 1 Reduction kinetics of E. coli by UV illumination, O3 and their combination in caramel solution.

no UV

O3

O3-UV 1.44 kJ/m2

O3-UV 2.88 kJ/m2

UV 1.44 kJ/m2 UV 2.88 kJ/m2

a) 1% NaCl b) 10% NaCl c) 20% NaCl

Figure. 2 Inactivation curves of E. coli inoculated in brine solution with UV, O3 and UV-C/O3

79
Conclusion
A model fish sauce, a liquid with caramel color and salt was tested to study the ability of O3 and UV-C applied
together, as well as individually in activating Escherichia coli contamination. Salt in the solution increased the level of
soluble solids and affected the efficacy of UV-C and O3. The effect of caramel color was two-fold when increasing
optical density of the solution. It was affected by reducing the transmission of UV-C light. The effectiveness of UV-C
treatment was significantly compromised and additional soluble solids also decreased the O3 solubility making
ozonation less effective. The combined treatment outperformed the individual treatments when the ozonation was the
least effective and the UV-C treatment offered mediocre microbial reduction. The combined treatment takes
advantage of the beneficial characteristics of the two technologies. E. coli inactivation by the combined treatment
improved with increased contact time.

Acknowledgements
This work was financially supported by a Research Grant from Burapha University through National Research Council
of Thailand (Grant No. 114/2560) and a graduate scholarship from the Thailand Research Fund through the Royal
Golden Jubilee Ph.D. Program (Grant No. PHD/0216/2552) to Wipavadee Sangadkit and Asst. Prof. Dr. Aluck
Thipayarat.

References
Bandla, S., Choudhary, R., Watson, G.D. and Haddock, J. 2012. UV-C treatment of soymilk in coiled tube UV
reactors for inactivation of Escherichia coli W1485 Bacillus cereus endospores. LWT-Food Science and
Technology 46(1): 71 – 76.
Glass, K.A., Jodi, M., Loeffelholz, J., Ford, P. and Doyle, M.P. 1992. Fate of Escherichia coli 0157:H7 as affected by
pH or sodium chloride and in fermented dry sausage. Applied and Environmental Microbiology 58(8): 2513-
2516.
Khueankhancharoen, J. and Thipayarat, A. 2011. Application of modified drop plate technique (MDPT) and logistic
model to optimize non-selective substrates for Samonella typhi resuscitation. Asian Journal of Food and Agro-
industry 4: 349-358.
Mukhopadhyay, S., Ukuky, D.O., Juneja, V. and Fan, X. 2014. Effect of UV-C treatment on inactivation of Salmonella
enteric and Escherichia coli O157:H7 on grape tomato surface and stem scars, microbial loads, and quality.
Food Control 44: 110-117.

80
 IFRC 2017: 195-174 Food Safety and Quality

Case Study of Profiling of Adulterant in Tongkat Ali Herbal Product using Real Time Coupled with High
Resolution Melting Analysis

N.F. Fadzil1, A.Wagiran1,*, F. Mohd Salleh1 and S. Abdullah2

1Department of Biotechnology & Medical Engineering, Faculty of Biosciences and Medical Engineering, UTM Skudai,
81310, Johor, Malaysia.
2 Faculty of Plantation and Agrotechnology Universiti Teknologi MARA (UiTM), Selangor,Malaysia.

Abstract
Eurycoma longifolia or known as Tongkat Ali belongs to the family of Simaroubaceae is a prominent herbal plant in
Malaysia. Regarding to the aphrodisiac and therapeutic properties it offers, E. longifolia is vastly harvested as a
source of raw ingredient in herbal products. However, the increasing demand of this herbal remedies had raised
concern in food safety due to possible adulteration, substitution and contamination problems intentionally or
unintentionally. Therefore, in this study, we have endeavored to assess the authenticity of E.longifolia in herbal
product by using real time PCR combined with a new sensitive method of High Resolution Melting analysis (HRM).
The DNA barcode, rbcLa chloroplastid marker was chosen as the plant DNA marker. To determine the profiling
melting temperature, this barcode was amplified from fresh root and three selected herbal products in market
designated as T1, T2 and T3. Melting temperature data obtained was then analyzed and compared with fresh root
samples as the reference to verify whether the tested product contained the indicated species. In our study, the
melting profile of rbcLa showed clear distinction of melting temperature (Tm) between fresh root and commercial
herbal product. Two products (T2 and T3) showed a deviate Tm and normalized curve from the positive fresh plant
control (rbcLa- 81.52°C, 85.66°C) while one product (T1) showed a close Tm with E.longifolia root. For the
conclusion, from this study, real time coupled with HRM can serve as a reliable tool to detect adulteration in herbal
products with more sensitive and effective way.

Keywords: Eurycoma longifolia, rbcLa, High Resolution Melting Analysis.

*Corresponding author’s email: alina@utm.my

Introduction
The tremendous market in herbal medicine and food based products had caught the attention of many consumers to
choose herbal remedies because of safe perception. However, with the increasing demand of herbal products
nowadays, there are some problems regarding the quality and purity of those herbal remedies marketed worldwide.
The issues of misidentification of herbal plants, adulteration of herbal product’s content and contamination during
processing stage has raised concern in food safety (Newmaster et al., 2013).

Eurycoma longifolia is widely acclaimed for its energy boosting and aphrodisiac properties. Tongkat Ali was
established as health supplement with high market value with 20-25 US dollar/kg for the roots (Bhat and Karim,
2010), while water extract sold in 60 capsules form for 26 US dollar (Kaur et al., 2003). This research focused on
Tongkat Ali herbal product marketed in Malaysia including tea bag and capsule. In HRM concept, melt curve is used
as an indicator to differentiate DNA samples between one another according to the melting behavior at specific range
of temperature. HRM was developed to monitor the dissociation of double stranded DNA with small temperature
increment (0.01°C-0.2°C) and analyzed using special instrument software. The objective of this study is to profile the
differences of adulterants in herbal products by comparing the melting temperature with fresh E.longifolia root using
real time PCR coupled with a new sensitive method of High Resolution Melting analysis.

81
Materials and Methods
3.1 Plant Materials, Herbal Product and DNA Isolation
Fresh Eurycoma longifolia plant was purchased from Forest Research Institute Malaysia (FRIM) and three
commercial herbal products chosen randomly from market. Approximately 100 mg of E.longifolia root and 200mg of
products were grind in liquid nitrogen separately and extracted using NucleoSpin Plant II Kit (Macherey-Nagel,
Germany) following manufacturer’s instruction. Yield and purity was assess by using spectrophotometric Nanodrop
1000 Spectrophotometer (Thermo Scientific) and visualize in 1% (w/v) agarose gel electrophoresis, viewed by Gel
Documentation (Fisher Scientific).

3.2 Real-Time PCR and HRM Analysis

Real time PCR was conducted in 25µL total reaction volume containing 2x 10µL Type-it HRM PCR master mix
(QIAGEN), 10ng DNA for fresh E.longifolia root or 30ng for commercial herbal products, 0.2µM of each primer set
and top up with Nano-pure water. Primer used in this experiment is rbcLa-Forward; 5’-
ATGTCACCACAAACAGAGACTAAAGC-3’ (Levin et al., 2003) and rbcLa-Reverse; 5’-
GTAAAATCAAGTCCACCRCG-3’ (Kress & Erickson, 2007). The amplification was then performed by using QIAGEN
Rotor Gene Q real time cycler. HRM with pre-amplification file was chosen to set up the run. Thermocycling reaction
was conducted in 36 well plates starting with 95 °C step for 5 minutes, followed by 40 cycles of 95°C for 30 seconds,
51°C for 40 seconds and 72°C for 30 seconds. Subsequently, HRM was performed by increasing the temperature at
0.1°C for each step from 75°C to 90°C. Fluorescence signals captured was then analyzed by using Rotor-Gene Q
Series Software version 2.3.1.

Results and Discussion

Table 1: Type of products, Ct value and melting temperature (Tm) with standard deviation obtained from real time
coupled with high resolution melting analysis using rbcLa primer.
Sample Source Ct ± SD Peak 1 Peak 2 Peak 3
Tm ± SD Tm ± SD Tm ± SD
E. longifolia Fresh root 16.83 ± 0.35 81.52 ± 0.05 85.66 ± 0.06 -
Product 1(T1) Tea Bag 22.83 ± 0.70 81.54 ± 0.08 85.67 ± 0.07 -
Product 2(T2) Tea Bag 20.53 ± 0.59 81.88 ± 0.11 84.54 ± 0.08 85.68 ± 0.14
Product 3(T3) Capsule 26.30 ± 0.09 81.89 ± 0.01 84.24 ± 0.02 85.48 ± 0.04

In the present study, the universal primer used yielded amplicon of ~550 bp long. This explained the multiple peaks
present. It is because long amplicon or amplicon with high GC content tend to create two melt domains although gel
electrophoresis shows one clear band. According to an article by (Robinson et al., 2006) melting of DNA was a multi-
state process which means sequence with high GC content does not melt simultaneously until it reach certain
temperature to melt completely create multiple peaks phenomenon. From the results, there were two products (T2
and T3) that were observed to have shifted melting temperature from the root. The presence of these adulterants can
be observed from melting point of rbcLa amplicon in product T2 (81.88°C, 84.54°C) and 3 (81.89 °C, 84.24 °C) which
is highly deviates from the root (81.52°C, 85.66 °C). Furthermore additional peak (Peak 3) on product T2 and T3 also
strengthen the result that it was highly contradict from E.longifolia root peak. Additional peak was also present which
in agreement with other finding which can be used as an extra indicator for species discrimination (Decat et al., 2012;
Kalivas et al., 2014).

With the aid of difference curve from HRM analysis, any samples with close or similar melt temperature can be
differentiated by analyzing both melting temperature and shape of normalized curve (Osthanunkul et al., 2015; Witwer
et al., 2003). As can be observed from the melting peak (Table 1) and normalized graph (Figure 1), product T1 seems
to have a very similar melting point (81.54 °C, 85.67 °C) and shape of normalized fluorescence curve with
E.longifolia. As shown in both figures, rbcLa marker was very useful and specific to diverse herbal species as the
melting temperature and shape of normalized curve can be differentiated clearly compared to E.longifolia root. In
order to further analyze the similarities between those sequences, difference curve was used to clearly distinguish

82
subtle differences between all of the samples by subtracting fluorescence of each sample with E.longifolia root as the
reference. This analysis evidently showed that product T2 and T3 do not have the indicated species as claimed on
the product’s information/packaging.

Figure 1: Normalized fluorescence graph compared between samples with the same starting and final fluorescence
between samples. Red line: E.longifolia; blue line: product T1; purple line product T2; green line: product T3.

Figure 2: Difference graph comparing all of the samples with E.longifolia as reference. Red line: E.longifolia root; blue
line: product T1; purple line: product T2; green line: product T3

Conclusion
For the conclusion, this work has asses the dependability of High Resolution Melting Analysis to profile adulterants in
Tongkat Ali commercial herbal products. Out of three products tested two shows a different melting temperature
compared to the positive control while one product shows a high similarity of melting temperature and melting curve
shape to E.longifolia. Hence, this study can be used as a promising method to detect adulterants in processed food
with simple procedures, high sensitivity and time saving.

Acknowledgement
This project was supported by Ministry of Higher Education under Fundamental Research Grant Scheme; FRGS
Q.J130000.2545.4F893.Thank you to all members and staff of Plant Biotechnology Lab, T02 Cluster Building, Faculty
of Biosciences and Medical Engineering Universiti Teknologi Malaysia.

References
Bhat R. and Karim A.A. (2010). Tongkat Ali (Eurycoma longifolia Jack): A Review on its Ethnobotany and
Pharmacological Importance. Fitoterapia (81)669–679.
Decat E., Mechelen E.V., Saerens B., Vermeulen S. J.T., Boekhout T., De Blaiser S., Vaneechoutte M. and
Deschaght P. (2013). Rapid and Accurate Identification of Isolates of Candida species by Melting Peak and
Melting Curve Analysis of the Internally Transcribed Spacer Region 2 Fragment (ITS2-MCA). Research in
Microbiology. (164) 110-117.
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Kalivas A., Ganopoulos I., Xanthopoulou A., Chatzopoulou P., Tsaftaris A. and Madesis P. (2014). DNA Barcode
ITS2 Coupled with High Resolution Melting (HRM) Analysis for Taxonomic Identfification of Sideritis Species
Growing in Greece. Molecular Biology Reports. (41) 5147-5155.
Kaur I, Kumaresan S, Sarmidi MR. A study into the Effect of Laboratory Scale Processing Parameters and Scale up
on Eurycoma longifolia Water Extract Yield. Proceedings of the 17th Symposium of Malaysian Chemical
Engineers, 29–30th December 2003, Penang; 2003. p. 294–9.
Kress W.J. & Erickson D.L. A Two-Locus Global DNA Barcode for Land Plants: The Coding rbcL Gene Complements
the Non-Coding trnHpsbA Spacer Region. PLoS ONE. 2(6): e508.
Levin R.A., Wagner W.L., Hoch P.C., Nepokroeff M., Pires J.C., Zimmer E.A., and Sytsma K.J. (2003). Family-Level
Relationships of Onagraceae Based on Chloroplastrbcl and Ndhf Data. American Journal of Botany. 90(1).
107-115.
Newmaster S.G., Grguric M., Shanmughanandhan D., Ramalingam S. and Raguphaty S. (2013). DNA barcoding
detects contamination and substitution in North American herbal products. BMC Medicine, (11) 222-235.
Osathanunkul, M., Suwannapoom, C., Ounjai, S., Rora, J. A., Madesis, P., and de Boer, H. (2015). Refining DNA
Barcoding Coupled High Resolution Melting for Discrimination of 12 Closely Related Croton Species. PLoS
ONE. 10(9):e0138888.
Robinson B.S., Monis P.T. and Dobson P.J. (2006). Rapid, Sensitive and Discriminating Identification of Naegleria
spp. By Real-time PCR and Melting Curve Analysis. Applied and Environmental Microbiology.72 (9) 5857-
5863.
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Amplicon Melting Analysis using LCGreen. Clinical Chemistry. (49) 85

84
 IFRC 2107: 214-200 Food Safety and Quality

Microflora Identification of Enteral Feeding Tubes in Neonatal Intensive Care Unit Setting

1Mahirah Mohamad, 2Shareena Ishak, 2Rohana Jaafar and 1*Norrakiah Abdullah Sani

1School of Chemical Sciences and Food Technology,Faculty of Science and Technology, Universiti
Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia.
2Department of Paediatric, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, Bandar
Tun Razak, 56000 Cheras, Kuala Lumpur, Malaysia.

Abstract
Neonates with low-level immune system in the Neonatal Intensive Care Unit (NICU) are highly exposed to the risk of
infection from the contamination of enteral feeding tubes during feedings. This study aims to identify the microflora
and biofilm formations in the enteral feeding tubes which were in place for 24, 48 and 96 hours in the NICU of the
Universiti Kebangsaan Malaysia Medical Centre (UKMMC). A total of 30 tubes from ten neonates (A-J) with different
feeding regimes and tube sizes were evaluated. Enumeration of aerobic colony count, Staphylococcus aureus,
Enterobacteriaceae spp., Enterococcus spp. and detection for the presence of Cronobacter sakazakii and Salmonella
spp. in the enteral feeding tubes were tested. It was indicated that the highest level of aerobic colony count and S.
aureus were enumerated from the tubes with similar tube (size 10) of a neonate (D) used for up to 48 hours and
received mixed feeding regime of ready to feed formula (RTF) and pasteurised expressed breast milk (PEBM) with
fortifiers, with mean 7.15 log cfu/g (1.4 x 107 cfu/g) and 4.08 log cfu/g (1.2 x 104 cfu/g) respectively. The most
enumerated Enterobacteriaceae was from the tube (size 8) of neonate (H) fed with powdered infant formula, myotein,
PEBM and MCT oil (supplement) placed for 96 hours, with mean 2.92 log cfu/g (8.4x 102 cfu/g). The enteral feeding
tube (size 8) of a neonate (E) fed with mixed feeding regime of (RTF) and (PEBM) used for 48 hours showed the
highest level of Enterococcus spp., with mean 4.41 log cfu/g (2.6 x 104 cfu/g). None of C. sakazakii and Salmonella
spp. was detected in all tubes. Cocci and rod-shaped biofilm of bacteria were also detected in 14 tubes through a
Scanning Electron Microscope (SEM). Poor flushing step could have contributed to the bacterial contamination in
these tubes Therefore, the cleaning step prior and after feeding and its placement period should be strictly taken into
consideration in order to reduce the number of microbial load in them.

Keywords: Enteral feeding tube, microflora, biofilm, neonatal intensive care unit
*Corresponding author’s email: norrasani@ukm.edu.my

Introduction
Enteral feeding tube can act as loci for bacterial colonization with prolonged placement and feeding regime
influences. Biofilm attached on the enteral feeding tubes may detach and passes into neonate intestines,
consequently exposed them to the neonatal risk of infections (Kim et al., 2006). The enteral feeding tube provides
ideal condition for bacterial multiplication as it was in-place for long period with regular additional of nutrient from
neonates feeding (Hurell et al., 2009).

The objective of the study is to identify the microflora and biofilm formations in the enteral feeding tubes which were in
place for 24, 48 and 96 hours in the NICU UKMMC.

Material and Methods

3.1. Sample preparation
Enteral feeding tubes were collected from Neonatal Intensive Care Unit Universiti Kebangsaan Malaysia Medical
Centre upon informed consent from parents.

3.2. Microbiological analysis for enumeration aerobic colony count and Enterobactericeae Staphylococcus aureus,
Enterococcus, Cronobacter sakazakii and Salmonella according to AOAC, BS and ISO Standard Methods.
85
3.3 Formation of biofilm was identified through Scanning Electron Microscope (SEM) based on the method
recommended by Hurell et. al (2009).

3.4. All data were analysed using SPSS version 23.

Results and Discussion

Microbiological analysis for several bacteria enumeration and detection for enteral feeding tubes was tested
according to AOAC, BS and ISO Standard Methods. Table 1 presents the microbiological analysis of aerobic colony
count, Staphylococcus aureus, Enterobacteriaceae spp., Enterococcus spp. and detection for the presence of
Cronobacter sakazakii and Salmonella spp on thirty enteral feeding tubes from ten neonates. The biofilm formation in
enteral feeding tubes was examined through Scanning Electron Microscope based on the method recommended by
Hurell et. al (2009) as shown in Figure 1 and 2.

Result of the study showed that the enteral feeding tube (size 10) of neonate (D) placed for 48 hours and received
mixed feeding regime of ready to feed formula (RTF) and pasteurized expressed breast milk (PEBM) with fortifiers,
was the highest aerobic colony count and S. aureus with mean 7.15 log cfu/g (1.4 x 107 cfu/g) and 4.08 log cfu/g (1.2
x 104 cfu/g) respectively. The most enumerated Enterobacteriaceae was from the tube (size 8) of neonate (H) fed with
powdered infant formula, myotein, PEBM and MCT oil (supplement) and placed for 96 hours with mean 2.92 log cfu/g
(8.4x 102 cfu/g). The enteral feeding tube (size 8) of a neonate (E) fed with mixed feeding regime of (RTF) and
(PEBM) used for 48 hours showed the highest level of Enterococcus spp., mean 4.41 log cfu/g (2.6 x 104 cfu/g). None
of C. sakazakii and Salmonella spp. was detected in all tubes.

Cocci and rod-shaped biofilm of bacteria were also detected in 14 tubes through a Scanning Electron Microscope
(SEM). Enteral feeding tube neonate (F) fed with PEBM mixed with HMF for 96 hours contained cocci shape biofilm
as shown in Figure 1 while both rod and cocci-shaped biofilm bacteria were found on enteral feeding tube neonate (B)
fed with PEBM and HMF for 48 hours (Figure 2). Hurell et al. (2009) reported Enterobacteriaceae biofilm formation
achieved 105–106 cfu/cm after 24 hours and highlighted the enteral feeding tubes with any feeding regime can be a
site for bacteria attachment and multiplication. Proper cleaning the feeding tube prior and after feeding is important to
prevent the contamination. Flushing the tube with small volume of sterile water is suggested for proper cleaning
besides flushing with air (RCH, 2016).

86
 Table 1: Microbiological analysis of enteral feeding tubes for 24, 48 and 96 hours

Aerobi C.
Enterobacteri sakazakii
Placeme Tub c Staph. Enterococc
Neonat cea &
nt period e Feeding regime colony aureus us
e count Salmonell
(hour) size
a
(cfu/g)
Mean log cfu/g
96 8 PEBM +HMF 2.85 0.00 1.18 0.00 ND
A
48 8 PEBM 3.68 0.00 1.88 0.00 ND
24 5 PEBM+HMF 2.85 0.00 0.00 0.00 ND
96 8 PEBM+HMF 4.84 0.00 0.00 2.26 ND
B 48 8 PEBM+HMF 3.38 0.00 0.00 0.00 ND
24 8 PEBM+HMF 5.18 2.82 1.30 0.00 ND
96 8 PEBM+HMF 1.60 0.00 0.00 0.00 ND
C 48 8 PEBM+HMF 1.66 0.00 0.00 0.00 ND
24 8 PEBM+HMF 0.00 0.00 0.00 0.00 ND
96 10 PEBM+HMF 7.14 2.00 0.00 3.93 ND
D 48 10 RTF,PEBM+HMF 7.15 4.08 0.00 0.00 ND
24 10 PEBM+HMF 6.57 4.07 1.30 0.00 ND
FEBM+HMF+
96 8 4.70 0.00 1.30 4.40 ND
CARBONIC
E
48 8 RTF+PEBM 6.11 0.00 0.00 4.41 ND
24 8 RTF+PEBM 0.00 0.00 2.26 0.00 ND
96 8 PEBM+HMF 5.95 0.00 0.00 2.78 ND
F 96 8 PEBM+HMF 5.43 0.00 2.04 0.00 ND
48 8 PEBM+HMF 4.4 0.00 0.00 0.00 ND
24 8 PEBM_HMF 3.79 0.00 1.00 ND
G 96 8 RTF 3.00 2.49 0.00 1.18 ND
48 8 RTF 1.70 0.00 0.00 1.40 ND
24 8 RTF 4.67 2.62 0.00 0.00 ND
PIF,MYOTEIN,PE
96 8 2.15 0.00 2.92 2.92 ND
H BM,MCT OIL
PIF, MYOTEIN,
48 8 5.04 2.46 0.00 3.11 ND
PEBM, MCT OIL
24 8 PEBM 4.15 2.18 0.00 3.18 ND
I 96 8 PEBM 5.18 3.00 1.00 3.15 ND
48 8 PEBM 4.76 1.81 2.18 3.15 ND
PEBM+HMF+MCT
24 8 4.04 2.40 0.00 0.00 ND
OIL
PEBM+HMF+MCT
J 96 8 4.26 2.56 0.00 0.00 ND
OIL
PEBM+HMF+MCT
48 8 3.76 3.63 0.00 0.00 ND
OIL

*PEBM= Pasteurised expressed breast milk

HMF=Human Milk Fortifier
RTF=Ready to feed milk

87
Figure 1. Electron microscopy of enteral feeding tube inner wall from neonate (F) fed pasteurized expressed breast
milk and human milk fortifier for 96 hours.

Figure 2. Electron microscopy of enteral feeding tube inner wall from neonate (B) fed pasteurized expressed breast
milk and human milk fortifier for 48 hours.
Conclusion
This study demonstrates that aerobic colony count, S. aureus, Enterobactericeae spp., Enterococcus spp. and also
rod and cocci shaped biofilm are found in enteral feeding tubes of neonates fed with mixed feeding regimes. Further
analysis of the isolate Enterobacteriaceae spp. and Enterococcus spp on enteral feeding tubes should be carried out
in future. Therefore, the cleaning step prior and after feeding and its placement period should be strictly taken into
consideration in order to reduce the number of microbial load in them.
Acknowledgements
The authors would like to thank the financial support for this research via Dana Impak Perdana DIP-2014-007 and
Electron Microscope Unit UKM for Scanning Electron Microscope service. The cooperation from NICU staffs is highly
appreciated.
References
Hurrell, E., Kucerova, E., Loughlin, M., Caubilla-Barron, J., Hilton, A., Armstrong, R., Smith, C., Grant, J., Shoo, S.,
Forsythe, S. 2009. in press. Neonatal enteral feeding tubes as loci for colonisation by members of the
Enterobacteriaceae. BMC Infectious Diseases.
Kim, H., Ryu, J.-H., Beuchat, L.R., 2006. Attachment and biofilm formation by Entero- bacter sakazakii on stainless
steel and enteral feeding tubes. Applied Environmental Microbiology 73: 5846–5856.
Royal Children Hospital. Enteral Feeding Medication and Administration Retrieved on June 14, 2017 from The Royal
Children’s Hospital Melbourne Website:
http://www.rch.org.au/rchcpg/hospital_clinical_guideline_index/Enteral_feeding_and_medication_administratio
n/

88
 IFRC 2017: 216-202 Food Safety and Quality

Effects of Hydrocolloids on Physicochemical and Sensory Qualities of Noodles

Chiew, C.S. and *Thed, S.T.

Faculty of Applied Sciences, Tunku Abdul Rahman University College, Jalan Genting Kelang,
53300 Setapak, Kuala Lumpur.

Abstract
Hydrocolloids were incorporated into noodles made from all-purposed wheat flour and their effects on the cooking
quality, physicochemical properties and sensory characteristics of the noodles were investigated. Hydrocolloid blends
(set at 5%) were prepared by combining konjac glucomannan (KGM) and kappa-carrageenan (KC) at different ratios,
namely KGM:KC at 0:10, 2:8, 4:6, 6:4, 8:2, and 10:0. The noodles contained KGM:KC (8:2) significantly increased the
cooking yield by 29.12% as compared to the control without hydrocolloids. Noodles contained KGM:KC (8:2) showed
the highest swelling index (72.17%) and the lowest cooking loss in terms of solid particles (10.63%). Addition of
KGM:KC (8:2) into the noodles significantly improved the firmness (2.769 ± 0.155 N) and chewiness (1.970 ± 0.134
N) of the samples. Noodle contained KGM:KC (8:2) had an overall acceptability score of 4 out of 5 on a hedonic
scale. In conclusion, noodles contained KGM:KC (8:2) had the most desirable quality among all the samples. The
addition of small quantity of hydrocolloids could potentially reduce or replace other functional ingredients such as
eggs, fats and high protein flour, thus providing a more economical way in noodle making.

Keywords: Hydrocolloid noodles, physicochemical properties, sensory quality

*Corresponding author’s email: thedst@acd.tarc.edu.my

Introduction
Noodle is one of the most consumed staple foods around the world. The quality of the noodles is affected by the flour
composition, moisture content, cooking yield, and other ingredients like fats, boric acid, sodium polyphosphate,
sodium silicate and food hydrocolloids. The addition of hydrocolloids in starch-based products can improve
myofibrillar protein gel in terms of adhesiveness and strength (Xiong et al., 2009). Konjac glucomannan (KGM) and
kappa-carrageenan (KC) are natural plant-based hydrocolloids. The outstanding characteristics of KGM are its
indigestible dietary fiber, water-absorbing ability, and high viscosity. KGM has demonstrated potential health benefits
such as treatment of constipation (Passaretti et al., 1991), weight management (Keithley et al., 2013), lowering of
cholesterol and improvement in Type 2 diabetes (Chen et al., 2003). KC is widely used as food additives for its
gelling, thickening, and stabilizing properties. Their main application is in dairy and meat products due to their strong
binding to food proteins. KGM can interact synergistically with other hydrocolloids such as carrageenan, furcellaran,
xanthan to form a stronger gel network structure (Jimenez-Colmenero et al., 2013). The aim of this study is to
examine the effects of KGM:KC blends on the physicochemical and sensory qualities of noodles; and to obtain the
optimum KGM:KC ratio for producing the most desirable noodle quality.

Materials and Methods

Noodles preparation
Noodles were prepared using all-purposed wheat flour, distilled water (55%), corn oil (5.6%), fine salt (2%), KMC:KC
blends (5%), based on the flour weight.

Cooking properties
Cooking yield, swelling index, cooking losses in term of solid particles were determined according to the methods
used by Zhou et al. (2013). Optimum cooking time was determined by observing the disappearance of white core
center and powdery particles of noodles.

89
Texture profile analysis (TPA) and Colorimetry
TPA on the noodle samples was done by using the TA:XTplus texture analyzer (Stable Micro Systems) and the
values were computed according to the method used by Zhou et al. (2013). Cohesiveness= area 2/area 1;
springiness=time 2/time 2; chewiness=firmness x cohesiveness x springiness. The lightness (L*), greenness (a*) and
yellowness (b*) of the noodles samples were measured using a calorimeter.

Sensory evaluation
The color, appearance, springiness, smoothness and the overall acceptability of the cooked noodle samples
contained KGM:KC (8:2) were evaluated by 50 untrained panelists using a 5-point hedonic scale. Ranking test was
conducted on three cooked noodle samples contained different KGM:KC ratios (0:0, 6:4 and 8:2).

Statistical analysis
Data collected were analyzed using SPSS version 19 and Graphpad Prism 6.

Results and Discussion

The cooking yield and swelling index of the noodles samples with different ratios of KGM:KC were displayed in Table
1. The sample contains KGM:KC (8:2) had the highest percentage of cooking yield (216.50%) and swelling index
(72.17%). The cooking yield and swelling index of sample contained KGM:KC (8:2) were higher than that of samples
contained KGM or KC alone, suggesting a synergistic interaction between KGM and KC in the gelatinization process.
Majzoobi et al. (2011) stated that the cooking yield is strongly attributed by the swelling of gluten network and
gelatinization of starch. The larger the swollen gluten network, the more water can be held within it. Hydrocolloid has
strong water imbibing property, its presence would enhance the gelatinization of starch and the reinforcement of the
swollen gluten network (Zhou et al., 2013) during cooking.

Higher cooking yield and low cooking loss is demanded from point of view of industries and consumers. Noodles
contained KGM:KC (8:2) showed the lowest cooking losses in term of solid particles (Table 1). Hydrocolloids could
prevent the amylose from leaching out the granule during starch gelatinization (Zhou et al., 2013). For control
sample, the weak and discontinuous gluten protein network permits large amount of amylose and water-soluble
components to be leached out into the boiling water (Skrabanja and Kreft, 1998). Zhou et a (2013) explained that the
colloidal matrix formed by gluten is further compacted by KGM, thus delaying amylose leakage from starch granule.
Both control and sample contained KGM:KC (8:2) had the desirable short optimum cooking time. The optimum
cooking time was the time taken for complete disappearance of white and powdery center in cooked noodles.

The results of TPA were presented in Table 2. The addition of KGM:KC (8:2) into the noodles significantly improved
the firmness (2.77± 0.15 N), springiness (0.95 ± 0.01) and chewiness (1.97± 0.13 N) of the cooked noodle samples.
Color perception is one of the factors used to gauge the desirability and acceptability of food products. The lightness
(L*) of noodles gradually decreased (from 69.70 to 62.17) with increasing ratio of KGM levels (from KGM:KC of 0:10
to 10:0). This is due to the color of KGM is light brown while KC is white in color.

Table 1. Cooking yield, swelling index and cooking loss of noodle samples with different ratios of KGM:KC
Sample Cooking yield (%) Swelling index (%) Cooking loss (%)
Control 167.67 ± 0.50 a - 22.48 ± 1.09 c
KGM:KC (0:10) 174.30± 4.39 ab 9.80 ± 6.48 a 15.41 ± 1.18 ab
KGM:KC (2:8) 182.23± 4.87 b 21.53 ± 7.19 a 13.65 ± 1.66 ab
KGM:KC (4:6) 199.47± 4.82 c 47.04 ± 7.13 b 13.94 ± 0.02 ab
KGM:KC (6:4) 205.37± 0.45 c 55.71 ± 0.67 b 13.78 ± 0.41 ab
KGM:KC (8:2) 216.50± 2.48 d 72.17 ± 3.66 c 10.63 ±0.37 a
KGM:KC (10:0) 207.40± 0.60 c 58.72 ± 0.89 bc 10.95 ±2.01 a
Each value is the mean of three replicates ± standard deviation. Means within each column with the same letters are
not significantly different (p > 0.05).

90
 Table 2. Texture profile analysis of noodles samples with different ratios of KGM:KC

Sample Textural attributes

Firmness, N Cohesiveness Springiness, mm Chewiness, N mm
Control 1.81 ± 0.01 a 0.76 ±0.02 b 0.78 ±0.01 a 1.03 ±0.20 a
KGM:KC (0:10) 1.87 ±0.03 a 0.76 ±0.03 b 0.82 ±0.01 a 1.17 ±0.05 ab
KGM:KC (2:8) 2.01 ±0.11 bc 0.70 ±0.02 ab 0.83 ±0.01 a 1.30 ±0.20 ab
KGM:KC (4:6) 2.00 ±0.18 ac 0.69 ±0.04 ab 0.83 ±0.02 a 1.14 ±0.06 ab
KGM:KC (6:4) 2.37 ±0.03 d 0.74 ±0.03 ab 0.82 ±0.02 a 1.44 ±0.08 ab
KGM:KC (8:2) 2.77 ± 0.15 e 0.75 ±0.00 ab 0.95 ±0.01 b 1.97 ±0.13 c
KGM:KC (10:0) 2.24 ±0.09 bcd 0.67 ±0.04 a 0.84 ±0.02 a 1.24 ±0.04 ab
Each value is the mean of three replicates ± standard deviation. Means within each column with the same letters are
not significantly different (p > 0.05).

70
60
Percentage, %

50
40
30 66
50 52
20 46 42
10 24 26 22 24 24 24
20 20
0 6 12 18 10 0 12
0
Colour Appearance Springiness Smoothness Overall
Acceptability
Sensorial Attributes

Dislike moderately Neither like nor dislike Like moderately Like very much

N=50 panelists
Figure 1: Percentage distribution against sensorial attributes of KGM:KC (8:2) noodles.
.

The various sensory attributes (color, appearance, springiness, smoothness and the overall acceptability) of the
noodles contained KGM:KC (8:2) were evaluated and the results were presented in Figure 1. The mean scores for
color=4.0, appearance=3.9, springiness=3.8, smoothness=3.8 and the overall acceptability=4.0, where 1:dislike very
much to 5:like very much. Ranking test results showed that noodle sample with KGM:KC (8:2) was the most
preferred, followed by sample with KGM:KC (6:4) and control was the least preferred.

Conclusion
The incorporation of KGM:KC (8:2) into noodles prepared from all-purposed wheat flour significantly improved the
cooking properties, textural and sensory qualities. The health promoting hydrocolloids could potentially reduce or
replace other functional ingredients such as eggs, fats and high protein flour, thus providing a more economical way
in noodle making.

91
Acknowledgements
The authors would like to thank Tunku Abdul Rahman University College for financial support.

References
Chen, H.L., Sheu, W.H., Tai, T.S., Liaw, Y.P. and Chen, Y.C. 2003. Konjac supplement alleviated
hypercholesterolemia and hyperglycemia in type 2 diabetic subjects--a randomized double-blind trial. Journal
of the American College of Nutrition 22 (1): 36-42.
Jimenez-Colmenero, F., Cofrades, S.; Herrero, A.M.; Solas, M.T. and Ruiz-Capillas, C. 2013. Konjac gel for use as
potential fat analogue for healthier meat product development: Effect of chilled and frozen storage. Food
Hydrocolloids 30, 351-357.
Keithley,J., Swanson, B., Mikolaitis, M., DeMeo, J., Zeller, L., and Fogg, A.J. 2013. Safety and efficacy of
glucomannan for weight loss in overweight and moderately obese adults. Journal of Obesity 2013: 610908.
Majzoobi, M., Ostovan, R. and Farahnaky, A. 2011. Effect of hydroxypropyl cellulose on the quality of wheat flour
spaghetti. Journal of Texture Studies 42: 20-30.
Passaretti, S., Franzoni, M., and Comin, U. 1991. Action of glucomannans on complaints in patients affected with
chronic constipation: a multicentric clinical evaluation. The Italian Journal of Gastroenterology 23 (7): 421-5.
Skrabanja, V. and Kreft, I. 1998. Resistant starch formation following autoclaving of buckwheat (Fagopyrum
esculentum Moench) groats. An in vitro study. Journal of Agriculture and Food Chemistry 46:2020-2023.
Xiong, G., Cheng, W., Ye, L., Du, X., Zhou, M., Lin, L., Geng, S., Chen, M., Corke, H. and Cai, Y.Z. 2009. Effects of
konjac glucomannan on physicochemical properties of myofibrillar protein and surimi gels from grass carp
(Ctenopharyngodon idella). Food Chemistry 116:413-418.
Zhou, Y., Cao, H., Hou, M., Nirasawa, S., Tatsumi, E., Foster, T.J. and Cheng, Y.Q. 2013. Effect of konjac
glucomannan on physical and sensory properties of noodles made from low-protein wheat flour. Food
Research Journal 51:879-885.

92
 IFRC 2017: 223-209 Food Safety and Quality

Perceptions of Plastic Packaging Usage to Pack Hot Foods: The Perspectives of Food Hawkers In Kuala
Selangor, Malaysia.

*Mat Issa, Z. and Ab Rahim, N.F.

Faculty of Hotel and Tourism Management, Universiti Teknologi MARA, Kampus Puncak Alam, 42300 Bandar
Puncak Alam, Selangor, Malaysia.

Abstract
The plastic packaging has widely been used by hawkers to pack food items within the foodservice industry. Despite of
the various benefits brought about by plastic in the foodservice industry, two particular concerns have been raised
i.e., food safety and solid waste disposal issue. It was on such grounds that the present study was carried out. It
involved 131 food hawkers in Kuala Selangor, in the state of Selangor to assess the perceptions among them in
relation to plastic packaging usage to pack hot food items. The present study also aims to investigate the relationship
identified between dependent and independent variables and the most relevant predictor in relation to the plastic
usage practices. A cross-sectional survey study involving a structured questionnaire was employed. The data of the
study were analysed using SPSS version 21. Findings of the study revealed that the sampled food hawkers appeared
to have good perceptions related to environmental hazard and regulation, but poor perceptions were found in relation
to awareness and health hazard. However, positive moderate significant correlations were observed between health
hazard, environmental hazard, and regulation, and on plastic packaging practices. The study also revealed that health
hazard was the main predictor for the practices. It is recommended that future studies involving larger respondents at
various locations in Malaysia may have to be carried out since this study is only able to provide a basis for
understanding the food hawkers’ view on plastic packaging to pack hot food items.

Keywords: plastic packaging, hot foods, safety, perceptions.

*Corresponding author’s email: zurainim@salam.uitm.edu.my

Introduction
Malaysia is reportedly one of the largest plastic producers in Asia besides being one of the leading suppliers of the
plastic bags. The plastic products are exported to countries such as the EU, China, Hong Kong, Singapore, Japan
and Thailand (Market Watch, 2012). Malaysia is also one of the developing countries, which consumes relatively a
larger number of plastic bags on a daily basis (The Sun Daily, 2015; Rahman & Rahman, 2010) especially for food
packaging purposes (Maidin and Latiff, 2015). Some of the characteristics associated with the materials such as
inexpensive, lightweight, strong, durable, corrosion-resistant, with high thermal and electrical insulation properties,
make plastics become the best choice among the consumers and business owners (Thompson et al., 2009; Hopewell
et al, 2009). However, two particular concerns have thus far been raised in relation to the usage of this packaging,
which are solid waste disposal and food safety issues (Jayaraman et al, 2011).

It is a widely established fact that plastic materials cannot be degraded naturally owing to the composition
characteristics associated with it. In addition, it has also been highlighted in some studies that plastics and their
associated degradation products may potentially cause threats to organisms at all tropic levels (Seltenrich, 2015;
Lambert, Sinclair, and Boxall, 2014), hence cause hazards to food web (Reisser et al., 2013). Increased costs and
decreasing space of landfills have also been associated with disposing plastic materials (Zia et al., 2007). Besides,

93
packing food items with plastics has also been reported to cause health hazards due to the migration of chemicals
from the plastics to foods (Farhoodi et al., 2014; Moreira, André and de Lourdes Cardeal, 2015; Chang et al., 2016). It
was on such grounds that the present study was carried out. Specifically, it aims to look into the perceptions among
the food hawkers in relation to this issue. It is hoped that the findings of the present study would help Malaysian
authorities to draw regulations on the usage of plastic packaging, especially the ones used to pack hot food items.

Material and Methods

Study design and sample
A descriptive cross-sectional survey study was employed for the present study. The survey was administered among
131 food hawkers at three different night market areas in Kuala Selangor, Selangor, Malaysia between April 1, 2016
and May 30, 2016. It is worthy of note that only respondents who sold hot edible foods were approached to participate
in this study. Enrolment and completion of the study took place on-site.

Questionnaire
The bilingual questionnaire in the present study was adapted from related previous studies (Jayaraman et al., 2011;
Gooi, 2010, Maibach et al., 2008; Thompson et al., 2009; D’Souza et al., 2006; Ayalon et al., 2009; van Birgelen et
al., 2009; Troschinetz and Meilcic, 2009). Prior to the use of it, the content of the questionnaire was validated by a
number of competent and credible experts within the field. The reliability of the survey instrument had been tested
among the food handlers in Puncak Alam area with a Cronbach’s alpha value, which was within an acceptable level
of 0.55 and 0.89. The questionnaire consisted of demographic profiles, perceptions related to awareness (10 items),
health hazards (10 items), environmental hazard (8 items), and regulation (8 items), and also on practices in using
plastic packaging (10 items). For perceptions and plastic packaging practices, a 5-point Likert scale with strongly
agree, agree, neutral, disagree and strongly disagree responses was designed. Prior to data collection, the
questionnaire was pre-tested among conveniently selected food hawkers at an area other than the area in which the
main data were collected.

Statistical Analysis
The data of the study were analysed using SPSS (SPSS Inc., version 21) for descriptive statistics and multiple linear
regression analyses.

Results and Discussion

Of the total 131 respondents, majority are male (55.7%), aged between 40 and 59 years old (37.4%) and Malay
(88.5%). Almost half of the respondents (49%) completed their secondary education level and had three to five years
of experience in selling hot food items such as fried food, soup or porridge and other food items. Most of the
respondents (33%) used plastic bags and polystyrene to pack their food, and 21.4% of them used plastic bag and
plastic film due to cost effectiveness, convenience and for easy storage. In addition, fried food was the most frequent
type of food being sold, followed by soup and porridge, and other food combinations. It is worthy of note that this
scenario has been worrisome due to the fact that fatty foods, storage time and food temperature can accelerate the
migration of chemicals from plastic to the foods (Farhoodi et al., 2014; Moreira, André and de Lourdes Cardeal, 2015;
Chang et. al., 2016).

As presented in Table 1, among the perception variables, environmental hazard (3.52 ± 0.479) was perceived as the
most significant by the food hawkers, followed by regulation (3.51 ± 0.38), awareness (2.73 ± 0.656), and health
hazard (2.55 ± 0.636), and it has to be noted that similar findings were also reported by Eltayeb, Zailani and Filho
(2010). Remarkably, food hawkers perceived health hazard as the least to be considered even though the plastic
usage implications to health were considered lethal. However, the correlation analysis between the practice of plastic
packaging to pack food and independent variables showed that health hazards (r = 0.453, p < 0.05), environmental
health (r = 0.434, p < 0.05) and regulation (r = 0.415, p < 0.05) exhibited a moderate significant relationship.
Moreover, it was also revealed that with a total variance explained by the model of 27.9%, F (4, 126) = 12.187, P <
0.001, health hazard was the main predictor in determining the practices of using the plastic packaging to pack hot
food items (β= 0.315, p < 0.05). One possible reason that leads to the findings was moderate to good awareness
94
related to cancer and its possible causing factors among Malaysians (Suan, Mohammed and Abu, 2015; Al-Naggar et
al., 2015). Apart from that, similar to the findings of Jayaraman (2011), the results of the present study indicated that
regulation and awareness have little or no influence on the practice of using plastic packaging among consumers.
Despite the various campaigns carried out to reduce the usage of plastic to pack foods, without strict regulations,
consumers have the tendency to ignore it.

Table 1: Standard multiple regression on perception variables and practices in using the plastic packaging to pack hot
edible food items
Variables Practice Aware- Health Environmental Regulation B β
(DV) ness hazard hazard
Awareness 0.204* -0.06 -0.10
Health hazard 0.45** 0.68** 0.18 0.32*
Environmen-tal 0.43** 0.43** 0.62** 0.16 0.20***
hazard
Regulation 0.42** 0.04 0.48** 0.48** 0.17 0.17***
Means 4.03 2.73 2.55 3.52 3.51
Standard 0.37 0.66 0.64 0.48 0.38
deviation R2 = 0.279
Adjusted R2 = 0.256
R = 0.528**
Note: *p < 0.05, **p < 0.001, p < 0.01

Conclusion
It was revealed in the present study that health hazard was the main predictor and significantly correlated to the
plastic usage practices among the sampled food hawkers. Moreover, the findings also indicated that awareness and
regulations did not play a significant role in the usage of plastic as packaging material. Hence, the government of
Malaysia has to increase the effort to create awareness and improvements in the enforcement related to plastic
usage to pack hot foods is of utmost importance. As highlighted in previous related studies, plastic packaging can
create environmental problems and cause health problems at all trophic levels.

References
Ali, N. and Abdullah, M.A., 2012. The food consumption and eating behaviour of Malaysian urbanites: Issues and
concerns. Geografia: Malaysian Journal of Society and Space, 8(6), pp.157-165.
Al-Naggar, R.A., Jillson, I.A., Abu-Hamad, S., Mumford, W. and Bobryshev, Y.V., 2015. Knowledge and beliefs of
Malaysian adolescents regarding cancer. Asian Pacific Journal of Cancer Prevention, 16(3), pp.1097-1103.
Ayalon, O., Goldrath, T., Rosenthal, G., and Grossman, M. 2009. Reduction of Plastic carrier bag use: an analysis of
alternative in Israel. Waste Management, 29, 2025-2032.
Chang, N., Zhang, C.H., Zheng, F.E., Huang, Y.L., Zhu, J.Y., Zhou, Q., Zhou, X. and Ji, S.J., 2016. Migration of
toluene through different plastic laminated films into food simulants. Food Control, 59, pp.164-171.
D'Souza, C., Taghian, M., Lamb, P., and Peretiatkos, R. 2006. Green products and corporate strategy: an empirical
investigation. Society and Business Review, 1(2), 144-157.
Eltayeb, T., Zailani, S., and Filho, W. L. 2010. Green Business among Certified companies in Malaysia towards
environmental sustainability: benchmarking on the drivers, initiatives, and outcomes. International Journal
Environmental Technology and Management,12(1), 2010.
Farhoodi, M., Mousavi, S.M., Sotudeh‐Gharebagh, R., Emam‐Djomeh, Z. and Oromiehie, A., 2014. Migration of
aluminum and silicon from PET/clay nanocomposite bottles into acidic food simulant. Packaging Technology
and Science, 27(2), pp.161-168.

95
Gooi, B. S. (2010). Ban on plastic bag usage: is it the right move? An empirical study on consumer perception and
practice (Master's thesis, Universiti Sains Malaysia).
Hopewell, J., Dvorak, R., and Kosior, E. 2009. Plastics recycling: challenges and opportunities. Philosophical
Transactions of the Royal Society B: Biological Sciences, 364(1526), 2115-2126.
Jayaraman, K., Haron, H., Gooi, B. S., and Soh, K. L. 2011. Consumer reflections on the usage of plastic bags to
parcel hot edible items: an empirical study in Malaysia. Journal of Cleaner Production, 19, 1527-1535.
Lambert, S., Sinclair, C. and Boxall, A., 2014. Occurrence, degradation, and effect of polymer-based materials in the
environment. In Reviews of Environmental Contamination and Toxicology, Volume 227 (pp. 1-53). Springer
International Publishing.
Maibach, E. W., Renouf, C. R., and Leiserowitz, A. 2008. Communication and marketing as climate change-
intervention assets. American Journal of Preventive Medicine, 35(5).
Maidin, S. and Latiff, A.N., 2015. Nasi lemak packaging: a case study of food freshness and design flexibility. Journal
of Advanced Manufacturing Technology (JAMT), 9(1), pp.13-19.
Market Watch 2012” The Malaysian Plastic Industry. 2012. Retrieved from
“www.malaysia.ahk.de/.../ahk_malaysia/Market.../The_Malaysian_Plastic_Industry.pdf
Moreira, M.A., André, L.C. and de Lourdes Cardeal, Z., 2015. Analysis of plasticiser migration to meat roasted in
plastic bags by SPME–GC/MS. Food chemistry, 178, pp.195-200.
Rahman, H. Y., and Rahman, M. M. 2010. Solid waste generation at Bandar Baru Bangi: a statistical analysis on the
community awareness. Journal of Energy and Environment, 2(1), 31-34.
Reisser, J., Shaw, J., Wilcox, C., Hardesty, B.D., Proietti, M., Thums, M. and Pattiaratchi, C., 2013. Marine plastic
pollution in waters around Australia: characteristics, concentrations, and pathways. PloS one, 8(11), p.e80466.
Seltenrich, N., 2015. New link in the food chain? Marine plastic pollution and seafood safety. Environmental health
perspectives, 123(2), pp.A34-A41.
Suan, M.A.M., Mohammed, N.S. and Abu, M.R., 2015. Colorectal Cancer Awareness and Screening Preference: A
Survey during the Malaysian World Digestive Day Campaign. Asian Pacific Journal of Cancer
Prevention, 16(18), pp.8345-8349.
The Sun Daily. 2015. Letters- Plastic packaging pollution poses profound peril. The Sun Daily.
Thompson, R. C., Swan, S. H., Moore, C. J., and Vom Saal, F. S. (2009). Our plastic age. Philosophical Transactions
of the Royal Society B: Biological Sciences, 364(1526), 1973-1976.
Troschinetz, A. M., and Mihelcic, J. R. 2009. Sustainable recycling of municipal solid waste in developing
countries. Waste Management, 29, 915-923.
van Birgelen, M., Semeijin, J., and Keicher, M. 2009. Packaging and pro-environmental consumption
behavior. Environment and Behavior, 41(1), 125-146.
Zia, K.M., Bhatti, H.N. and Bhatti, I.A., 2007. Methods for polyurethane and polyurethane composites, recycling and
recovery: a review. Reactive and functional polymers, 67(8), pp.675-692.

96
 IFRC 2017: 225-213 Food Safety and Quality

Atmospheric Cold Plasma Treatment Effect on Microbiological Evaluation and Moisture Effect of Mango-
Fortified Noodles

1Zabidi NZA, 2*Zaaba SK, 3Abidin NSA, 3Rukunudin IH

1Department of Mathematics, Science and Computer, Politeknik Tuanku Syed Sirajuddin, Pauh Putra, 02600 Arau,
Perlis, Malaysia
2School of Mechatronic Engineering, Universiti Malaysia Perlis, Kampus Pauh Putra, 02600 Arau, Perlis, Malaysia
3School of Bioprocess Engineering, Universiti Malaysia Perlis, Kompleks Pengajian Jejawi 3, 02600 Arau, Perlis,
Malaysia

Abstract
Atmospheric Cold Plasma (ACP) can offer advantage for food quality maintenance by decontamination thus
extending food shelf life. The aim of this paper is to evaluate the effects of microbiological and moisture content of
mango fortified noodle (MFN) after treated with ACP. The addition of mango in noodle enriched the nutrition,
antioxidant and phytochemical properties. MFN was prepared according to 3%, 5%, and 10% of mango powder
concentration. Samples of MFNs were exposed to ACP for 2,4,6,8 and 10 minutes. They were kept under room
temperature and chilled temperature. Samples were observed for 10 days for bacteria growth. Results showed that
decontamination decreased as plasma exposure time increases. Four minutes of ACP exposure is the optimum time
to inactivate bacteria growth. Results from moisture content experiment showed that water content were retained
about 98% in 4% MFN for both temperatures. Results also demonstrated that treated MFN have better antimicrobial
properties under room temperature and also retained moisture content.

Keywords: Cold atmospheric plasma, non-thermal plasma, food safety, food technology, microbial decontamination,
mango noodle
*Corresponding author’s email: skzaaba@unimap.edu.my; Tel: +60195589220, Fax: +6049885167

Introduction
Noodle is one of the favorite meal for some east country such China, Japan, Korea, Thailand, Malaysia and others
(Bin Xiao Fu, 2008). Because of high consumers demand in healthy life style, and updated existing technologies in
stimulating and development of alternative preferable on non-thermal approaches, most of the food producers are
seeking method on how to extend noodles shelf life and minimize or to avoid conventional chemical preservatives.
This also to ensure the optimum microbiological safety and quality of minimally processed foods. One of the novel
technologies now a days is by modifying starch preparation, altering surface and chemical properties is plasma
technology. (Mir, S.A. et al., 2016). Atmospheric pressure cold plasma (ACP) offers distinct advantages for
decontamination of foods.

Methodology
Atmospheric cold plasma setup
The setup of atmospheric cold plasma in this study is shown in Figure 1.0. It consist of glass quartz tube with inner
diameter (di) is 1.5 mm, the outer diameter (do) is 2.5mm, gas regulator, gas tube and two electrodes. The voltage
that was applied is approximately 16.6 V for voltage supply and 0.8 A for currents. Two electrodes were connected to
the glass tube. The gap between the glass tube and sample is 0.5 cm. Helium gas (99.9%) with flow rate 1 L/min was
used to generate atmospheric cold plasma. The gas will flow through the quartz glass tube through the gas regulator,
into the gas tube, flow meter, and then to the quartz glass tube, where the excitation of gas occurs (Nor Aimi Saad et
al, 2015). Samples were exposed to atmospheric cold plasma for 2, 4, 6, and 8 mins.

97
 Figure 1.0 Atmospheric cold plasma treatment

Noodles Preparation
The noodles were prepared by conventional method with some modification with additional of some mangoes
powder. The noodle formulation consisted of 100 g of high protein flour, 2 g of salt, 3.2 g of lye water (potassium
carbonate) and 45 g of water (N. S. Aizee, 2016). The high protein flour dough without mango powder served as
normal noodles (CN). Three kinds of Mango Fortified Noodles (MFN) that prepared known as MFN1 (3% MFN),
MFN2 (5% MFN) and MFN3 (10% MFN). All the ingredients are mixed together until the noodles sheets are properly
formed (Bin Xiao Fu, 2008, Gary G. Hou, 2010). The ingredients will be mixed at manual mode for 7-10 mins, mixing
will make all the ingredients added together uniformly. Then followed by mode auto for 5-10 mins. This was produced
by using the low speed (5-8 rpm) for 10-20 mins. After mixing process the dough would be rested for a while for the
crumbly mixture and flour particle to accelerate the hydration and to redistribute water in the dough. Then the dough
will become noodles according to the mold. The ready noodles were pre-boiled in 800 ml hot water for 45 s (Bin Xiao
Fu, 2008). The noodles were drained, cut to 4 cm and arranged on petri dish for evaluation.

Microbiological Evaluation
The microbiological evaluation experiment was conducted to study the effect of plasma treatment. The activity of
bacteria growth of samples were observed. The samples were divided into two processing conditions; treated plasma
and untreated plasma. Each condition were divided into two more conditions; room temperature and chiller. Samples
were arranged in a petri dish about 2 cm apart separated by partitions. Microbiological evaluation were conducted
daily for 10 days.

Moisture Content
The measurement of moisture content is useful to determine yield and to predict food safety and quality. Samples are
prepared as mentioned in the above section. A drying oven was utilized to dry the samples. Metal dishes and lid were
pre-dried at 100°C for 3 hours to remove the moisture. Pre-dried metal dishes were cooled and weighted (A1). Then,
dishes, lids and samples before drying were weighted (A2). Lastly the dishes, lids and samples after oven dry at
100°C for 3 hours and cooled at room temperature were weighted (A3). The result of moisture content were
calculated using equation 1.0.

A2−(A3−A1)
Moisture = A2
x100

Results and Discussions

Microbiological Evaluation
Experiments were conducted in triplicates for 10 days and repeated twice (60 samples). In general, all noodles that
was chilled showed no growth of microbes until day 10. Calculations of percentage were made based on the number
of appearance of microbe observed for 10 days. Table 1.0 shows results of survival percentage of microorganism on
noodles after plasma treatment time for 2, 4, 6, 8 and 10 mins for 10 days. From table 1.0 we can see that the

98
survival rates for treated noodles is approximately 13-58% less than untreated noodles. Comparing between treated
and untreated noodles, treated noodles have lower microorganism survival percentage. Comparing between
treatment time and microorganism survival percentage, 10 minutes treatment time showed the highest reduction of
survival percentage.

Table 1.0 Survival percentage of microorganism on noodles after plasma treatment stored at room temperature
Plasma treatment time (mins)
Sample 2 4 6 8 10
Control normal noodles untreated (CNUT) 0.00 40.00 40.00 68.33 11.67
Control normal noodles treated
0.00 26.67 26.67 13.33 0.00
(CNT)
3% mango fortified noodles untreated
34.44 40.00 40.00 63.33 58.33
(MFN1UT)
3% mango fortified noodles treated (MFN1T) 20.00 13.33 26.67 25.00 0.00
5% mango fortified noodles untreated
30.00 40.00 50.00 40.00 0.00
(MFN2UT)
5% mango fortified noodles treated (MFN2T) 8.89 13.33 40.00 13.33 0.00
10% mango fortified noodles untreated
64.44 40.00 50.00 65.00 43.33
(MFN3UT)
10% mango fortified noodles treated (MFN3T) 26.67 0.00 13.33 28.33 0.00

Moisture content
Moisture content test were conducted on samples that were treated and untreated by plasma for 2 and 4 mins stored
at room temperature and in chiller. This is because plasma treatment after 4 mins The results of moisture content
between these samples are shown in Table 3.1. Comparing the moisture content between untreated noodles, noodles
stored in a chiller has higher moisture content. There was no significant difference between CN and MFN. At room
temperature, noodles treated with 4 mins of plasma showed a almost 100% increase in moisture content. Other
samples such as 2 mins treated noodles and chilled noodles does not show significant increase.

Table 2.0: Moisture Content for 2 and 4 Mins Plasma Treatment Stored at Room Temperature and in Chiller.

Room Temperature Chiller

2 minutes 4 minutes 2 minutes 4minutes

Sample
Untreated

Untreated

Untreated

Untreated
Treated

Treated

Treated

Treated
(%)

(%)

(%)

(%)

(%)

(%)

(%)

(%)

CN 48.95 49.02 65.33 97.98 97.95 98.22 97.97 98.20

MFN1 48.88 48.98 48.95 98.16 97.91 98.05 97.67 98.26

MFN2 48.86 32.57 48.96 98.32 97.56 98.41 97.73 98.48

MFN3 48.84 49.22 48.82 98.35 97.81 98.35 97.65 98.25

99
Conclusion
Our experiment showed that noodles exposed to plasma at room have shown a decrement in microbial activity. The
longer the treatment time, survival rate of microorganism could deplete to zero. Cold plasma treatment at 10 mins
showed a complete decontamination and was achieved. This was also proven by our group through experiments on
bacteria on agar (Nor Aimi Saad et al, 2015). We also note that the texture became dry after 10 mins of plasma
treatment. The addition of mango into noodles showed and increment in microorganism survival rate. This is because
the mango flour in MFN has been enriched in carbohydrate, fiber, fat and protein (S. A. A. Noor et al., 2015).

For moisture content experiments, results showed that the samples exposed for 4 mins at room temperature have
higher moisture content. This is because plasma itself contains active species such as OH- that binds and could
increase the moisture content during exposure.

Acknowledgement
This work was supported by Fundamental Research Grant Scheme by Ministry of Higher Education Malaysia (FRGS
9003-00526).

References

Bin Xiao Fu 2008. Asian noodles : History, classification, raw materials, and processing. Food Research International
41(9):888–902.
Gary G. Hou 2010. Asian Noodles: Science, Technology and Processing. John Wiley & Sons, New Jersey.
Mir, S.A., Shah, M.A. & Mir, M.M. 2016. Understanding the Role of Plasma Technology in Food Industry. Food
Bioprocess Technol. 9(5):734-750.
Nor Aimi Saad, SK Zaaba, M.T. Mustaffa, A. Zakaria, Roslan Md Nor, AB Shahriman, Tetsuya Akitsu 2015. Optical
Emission Spectroscopy Analysis Of Atmospheric Plasma Jet Plume On Bacteria Inactivation. Jurnal Teknologi,
77 (6).
S. A. A. Noor, N. M. Siti, N. J. Mahmad. 2015, Chemical Composition, Antioxidant Activity and Functional Properties
of Mango (Mangifera indica L. var Perlis Sunshine) Peel Flour (MPF). Applied Mechanics and Materials, 754-
755:1065-1070.

100
 IFRC 2017: 242-225 Food Safety and Quality

Microbiological Quality of Municipal Tap Water and Filtered Drinking Water in Serdang, Selangor

1*Nor-Khaizura M. A. R., 1Ahmad, N. A., 1Ahmad Zainal, A. S., 2Mahyudin, N. A.

1Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor Darul Ehsan, Malaysia
2Department of Food Service & Management, Faculty of Food Science and Technology, Universiti Putra Malaysia,
43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

Abstract
Microbiological quality is one of the primary indicators of the safety of drinking water supply. Contaminated municipal
and drinking water may result in waterborne diseases like gastrointestinal illness or diarrhea. The microbiological
quality of tap water from municipal water supplies and drinking water from water cooler were comparatively studied.
Heterotrophic plate count (HPC) and total coliform (TC) were enumerated from both samples that collected from
Serdang area. The method used in this study for water analysis was membrane filtration (MF) method with using
acetate cellulose membrane filter (0.45 mm). The heterotrophic plate count was found not significantly different (p >
0.05) in municipal tap water (MTW) and filtered drinking water (FDW), which was 1.60 log10 cfu ml-1 and 1.45 log10 cfu
ml-1, respectively. For total coliform, MTW (1.44 log10 cfu ml-1) were found significantly (P ≤ 0.05) higher compared to
FDW (1.00 log10 cfu ml-1). Heterotrophic plate count in MTW and FDW among the samples were not significantly
different (p > 0.05) with the range value is 1.51 to 1.71 log10 cfu ml-1 and 1.01 to 1.65 log10 cfu ml-1 respectively. In
conclusion, FDW possesses lower heterotrophic plate count and total coliform count than MTW, and microbiological
quality of the drinking water system depends on regular cleaning of filters in the water cooler to ensure the safety of
the water cooler users.

Keywords: Microbiological quality, total coliform, membrane filter, municipal tap water, filtered drinking water
*Corresponding author’s email: norkhaizura@upm.edu.my

Introduction
Safe and hygienic water supply is essential for healthy living to decrease the chances of contamination of harmful
bacteria, viruses, cysts and worms that caused by unclean water (WHO, 2008). Water-borne diseases are prone in
the developing countries and still pose major threats to those who cannot afford clean water. Waterborne diseases
like cholera, typhoid fever, and bacillary dysentery are reported more frequently during any drinking water associated
outbreaks than it was reported before (Chan, 2009). In many countries, microbiologically safe drinking water is
considered a fundamental human right and about 80% of communicable diseases in the world are waterborne. Water
quality is often related to the degree of bacterial contamination, and drinking water distribution systems are colonized
by heterotrophic saprophytic microorganisms that grow on the biodegradable organic matter (Servais et al., 1992). It
is well known that municipal water is characterized by its bacterial flora, chemical and physical composition
(Zamberlan da Silva et al., 2008). The aim of this study is to investigate the bacteriological quality of filtered drinking
water in the water cooler in Serdang area.

Materials and Methods

3.1 Water Sampling
There were two types of water samples collected for this experiment that is tap water represent as municipal water
and water cooler represent as drinking water. The water samples were collected from 6 areas around Serdang,
Selangor. The technique used in this water sampling was simple convenience sampling. The water samples were
collected for three consecutive weeks in a month. The container used for this microbiological analysis is sterilized
Schott’s glass bottle. To make sure that the samples were representative of the water consumed, no flush before
sampling, and there was no attempt to sterilize the outer surfaces of the faucets (Levesque et al., 1994). Five drops of
aqueous sodium thiosulphate solution (100g/L) was added to each clean bottle before sterilizing (121°C at 20
101
minutes) all the bottles. This addition would neutralize any residual disinfectant that may be present in the water
sample. A total of 36 water samples were analyzed, including 18 samples of municipal tap water and 18 samples of
drinking water from the water cooler.

3.2 Media
The medium used in this study were standard agar which is Plate Count Agar and MacConkey Agar. Plate Count
Agar was supplied by Oxoid Ltd (Basingstoke, Hampshire, England) while MacConkey Agar was supplied by Criterion
Ltd (England).

3.3 Bacteriological Analysis

The microbiological parameters determined were total coliforms (TC) and heterotrophic plate count (HPC). Each
sample was analyzed using membrane filtration method and pour plate method. In membrane filtration method, a
volume of 100mL of the samples was filtered through cellulose membrane filters with 0.45 µm pores size with a
diameter of 47mm. The cellulose membrane filters were then placed on solid media employed for each bacteria
(Zamberlan da Silva et al., 2008). Total coliform: incubation at 37°C for 48hr using MacConkey agar and
Heterotrophic plate count: incubation at 35°C for 24hr using Plate Count Agar
3.4 Statistical analysis
T-test and One-way ANOVA with Tukey were used to determine a family error rate, at (p≤ 0.05) of confidence level
by using Minitab 16.

Results and Discussion

The bacteriological analysis using membrane filtration method to compare municipal tap water (MTW) and filtered
drinking water (FDW) were presented in Figure 1. MTW showed higher heterotrophic plate count than FDW in all
samples except for sample D. A significant reduction (P ≤ 0.05) of heterotrophic plate count after a filtration process
was observed for sample A, B, E and F. The total coliform for MTW and FDW were presented in Figure 2.
From the results, almost all water sample collected had exceeded acceptable value limit in Malaysian Water Quality
Standard which was 5000 MPN/100ml for municipal water and no trace at all in 100ml in drinking water for the total
coliform test. MTW was more contaminated than FDW. Insufficient cleaning and service of the water cooler cause
bacterial contamination in the filtered drinking water. The presence of total coliform in water supply especially in
drinking water cannot be taken lightly, and their presence in drinking water must at least be considered as a possible
threat or indicative microbiological water quality deterioration. All MTP and FDW water samples show positive total
coliform. This may indicate treatment ineffectiveness, loss of disinfectant, breakthrough, and intrusion of
contaminated water into the potable water supply or regrowth problems in the distribution system or water from
dispenser (Traistaru, 2011.)

102
 MTP
FDW
2
1.8
1.6
1.4
1.2
Heterotrophic
Plate Count 1
log cfu/100ml 0.8

0.6
0.4
0.2
0
A B C D E F
Samples

Figure 3: Heterotrophic Plate Count (HTC) for municipal tap water (MTW) and filters drinking water (FDW)

MTW

2 FDW

1.8

1.6

1.4

1.2
Total Coliform 1
log cfu/100ml
0.8

0.6

0.4

0.2

0
A B C D E F

Samples
Figure 4: Total Coliform for municipal tap water (MTW) and filters drinking water (FDW)

103
Conclusion
As the conclusion, the filtration system could reduce the heterotrophic plate count, in the range of 0.1 to 0.8 log cfu
/100 ml and total coliform, in the range of 0.1 to 1.0 log cfu /100 ml that present in municipal tap water (MTW).
However, the filters drinking water (FDW) was still exceeded the limitation of Malaysia drinking water regulation.

References
Chan, N. W. .2009. Issues and Challenges in Water Governance in Malaysia. Iranian Journal of Environmental Health
Science and Engineering 6(3): 143–152.
Levesque, B., Simard, I. P., Gauvin, D., Dewailly, E., and Letarte, R. 1994. Comparison of the Microbiological Quality
of Water Coolers and That of Municipal Water Systems 60(4): 1174–1178.
Servais, P., Billen, G., Laurent, P., Levi, Y., & Randon, G. 1992. Studies Of BDOC And Bacterial Dynamics in The
Drinking Water Distribution System of The Northern Parisian Suburbs. Revue Des Science De L'eau 5, 69–89.
Traistaru, E. .2011. Comparative Study on The Microbiological Quality of Water Cooler Acta Universitatis Cibiniensis
Series E: Food Technology Vol. XV: 3–10.
World Health Organisation, (WHO) .2008. Safe water, Better Health: Costs, benefits and sustainability of interventions
to protect and promote health.
Zamberlan da Silva, M. E., Santana, R. G., Guilhermetti, M., Filho, I. C., Endo, E. H., Ueda-Nakamura, T., Dias Filho,
B. P. 2008. Comparison of The Bacteriological Quality of Tap Water and Bottled Mineral Water. International
Journal of Hygiene and Environmental Health 211(5-6), 504–9.

104
 IFRC 2017: 309-268 Food Safety and Quality

Antimicrobial activity of plant extracts against foodborne pathogens

*1Mat Issa, Z., 2Othman, N., 2Mustakim, M. and 1Jipiu, L. B.

1Faculty of Hotel and Tourism Management, Universiti Teknologi MARA, Kampus Puncak Alam, 42300 Bandar
Puncak Alam, Selangor, Malaysia.
2Faculty of Health Sciences, Universiti Teknologi MARA, Kampus Puncak Alam, 42300 Bandar Puncak Alam,
Selangor, Malaysia.

Abstract
Centella asiatica, Moringae oleifera and Clitoria ternatea are three widely used plants in Malaysian dishes, which are
either eaten raw or as part of cooking ingredients. It is noteworthy that their potentials in preventing foodborne
pathogens activity are not much explored. Therefore, this study seeks to investigate the antimicrobial activities of C.
asiatica, M. oleifera and C. ternatea extracts, and their synergistic effect against foodborne pathogens. In doing so,
the solvent extracts of the plants were tested against Listeria monocytogenes and Escherichia coli. Several tests were
carried out using disc diffusion method for antimicrobial-sensitive testing (AST), broth micro-dilution method for
minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) by means of subculturing
the MIC sample in the Mueller-Hinton agar. The results indicated that all the plant extracts exhibited an inhibitory
activity against the pathogens. Moreover, the combination of the plant extracts exhibited almost similar measurement
of the inhibition zone to single plant extract. The MIC and MBC showed that the combination of the three extracts
needed 25% concentration, while single extract needed 50% concentration in order to inhibit the pathogen growth. To
conclude C. asiatica, M. oleifera and C. ternatea extracts were proven to have antimicrobial activity against the
foodborne pathogens and the combination of all the three types of extract did show some synergistic effect when
tested against L. monocytogenes and E. coli. However, it has to be noted that the effectiveness of antimicrobial
activity may vary in natural host since this study was performed in vitro entirely.

Keywords: Centella asiatica, Moringae oleifera, Clitoria ternatea, antimicrobial activity, Listeria monocytogenes,
Escherichia coli
*Corresponding author’s email: zurainim@salam.uitm.edu.my

Introduction
Centella asiatica, Moringae oleifera and Clitoria ternatea are cultivated among Malaysian population and are also
commonly used in food preparation. Apart from being used in food, these plants were also believed to have some
natural medicinal values since they were commonly used to cure various ailments in traditional treatment (Farush
Khan, 2014; Ripen and Noweg, 2016; Yatim, 2013). Many studies were carried out with the aim to identify the
medicinal plant extracts that can be used as conventional natural alternatives in combating the growth of foodborne
pathogens due to the fact that they are environmentally friendly and safe to human (Zhang, 2011; Pitinidhipat and
Yasurin, 2012; Al-Daihan et. al., 2013; Tibolla, Pelissari, and Menegalli, 2014; Zhang, Khan, Bakht, and Nair, 2016).
Unlike the synthetic drugs that act directly upon bacteria, the herbal plant extracts work by creating a detrimental
effect on the environment and thus, minimising the survival chance of bacteria. (Paper, Banerjee, and Sciences,
2015; Yasurin, Sriariyanun, and Phusantisampan, 2015). Additionally, these plants were also reported to have
bioactive components that can result in pharmacological or toxicological effects in man and animals (Kamilla, Mnsor,
Ramanathan, and Sasidharan, 2009). Different parts of the plants possessed numerous phytochemical constituents,
which can exhibit antitumor in vitro (Gopalakrishnan, 2016). Therefore, the present study aims at investigating the
antimicrobial activities of C. asiatica, M. oleifera and C. ternatea extracts and their synergistic effect against
foodborne pathogens.

105
Material and Methods
Plant extract preparation
In March 2017, the plants used in the present study were obtained from local growers and were later sent to the
Forest Research Institute Malaysia (FRIM) for identification. The leaves of the plants were washed with distilled water
and dried at 60°C and were ground using a dry blender to make fine powder. The ratio of the ground powder with the
solvent used was 1: 6. Next, 50 g of the fine powder was weighted with the analytical balance before it was soaked in
300 ml diethyl ether for 72 h and placed onto an orbital shaker for constant agitation. The soluble compounds were
then filtered using Whatman No. 1 filter paper into a Schott bottle. The filtered product was concentrated by a rotary
evaporator for an hour at 550 mbar. Then, the crude extract obtained was sealed in a sterile container. Upon its
usage, the crude extract was put into 10% dimethyl sulfoxide (DMSO) (1g crude extract + 1000µl DMSO) and
dissolved with the aid of vortex. Finally, the extraction products and their combinations (1:1 ratio for combination of
two extracts, and 1:1:1 ratio for three combinations) were subjected to several tests.

Preparation of plant extract disc

The sterile disc approximately 6 mm in diameter was injected with 30 µl of the plant extract. The disc was allowed to
dry at room temperature for 24 h.

Bacteria strains and inoculum preparation

Antibacterial activity of the plant extracts was investigated against two gram negative bacterial isolates (L.
monocytogenes (ATCC 14038) and E. coli (ATCC 25925)). The L. monocytogenes was cultured on Blood Agar
(Merck) while the E.coli was cultured on Nutrient Agar (Merck). The inocula were prepared according to Islam et al.
(2008) and were incubated at 37°C for 24 hours.

Antimicrobial susceptibility testing (AST) by disc diffusion method

Antimicrobial activity assay by disk diffusion method was carried out based on Matuscke, Brown and Khalmeter
(2014). As for negative control, 30 µl of DMSO was injected into the sterilised disc and 30 µg of commercial
gentamycin was used as positive control.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
A 96-micro titer plate was used to perform MIC. The preparation of the plate was carried out according to the
manufacturer’s instructions (Greine Bio-One, Germany). Next, the lowest inhibitory concentration was determined by
selecting the clear well, in which no viability of microorganism was observed. In order to obtain a valid MIC result, the
well no 1, 12, and 13 must show the expected result as control panel. Later, the MBC was performed by means of
sampling the clear MIC, which showed no viability of the organism. All the cleared well, including the control well,
were streaked on MH agar and were incubated at 37°C for 24 hours. Notably, this procedure was carried out to
determine which extract concentration inhibit the bacterial growth. The lowest concentration in the medium, which had
less than five colonies or no growth were reported as the MBC.

Results and Discussion

Three plant species were studied to evaluate their potential antimicrobial activities against two types of food-borne
bacteria i.e., L. monocytogenes (ATCC 14038) and E. coli (ATCC 25925). The extracts were divided into three
groups, which are comprised of single extract for each plant, combination of two and three plant extracts. Table 1 and
2 demonstrated the qualitative evaluation of antimicrobial activities from the extracts by means of using disc diffusion
method. All the three plant extracts exhibited an inhibitory activity in confining growth of the studied microorganisms.
A mean diameter in the range of 26 – 28 mm was observed at the zone of inhibition from a single plant extract
measured from MH agar. Interestingly, the mixed plant extracts (i.e., two and three extracts) yielded measurement of
the zone of inhibitions in the range of 26 – 29 mm. Apart from that, qualitative assessment of MIC as the lowest
concentration of antimicrobial agent required to inhibit the growth of bacteria after 24 hours of incubation showed 25
% for the combination of the three plant extracts and 50 % for single and mixture of two extracts.

106
 TABLE 1: Mean diameter of inhibition zones of diethyl ether of plants (single extracts) against tested gram positive
and gram negative bacteria
Zone of Inhibition (Diameter in mm)
L. monocytogenes (ATCC Positive E. coli Positive Control
14038) Control (ATCC 25925) Gentamycin
Plant extracts Gentamycin (30 (30 µg)
(1g/mL) µg)
Centella asiatica 28.90 27.80 25.50 28.50
Moringa oleifera 27.42 27.60 21.70 26.30
Clitoria ternatea 26.67 27.50 24.50 26.00

TABLE 2: Mean diameter of inhibition zones of diethyl ether of plants (combination of two and three extracts) against
tested gram positive and gram negative bacteria
Zone of Inhibition (Diameter in mm)
L. monocytogenes Positive E. coli Positive Control
(ATCC 14038) Control (ATCC 25925) Gentamycin
Plant extracts Gentamycin (30 (30 µg)
(1g/mL) µg)
C.asiatica and 26.07 27.63 26.17 27.53
M. oleifera (1:1)
C.asiatica and 27.67 26.83 22.67 26.00
C. ternatea (1:1)
M.oleifera and 26.67 27.37 22.70 27.5
C.ternatea (1:1)
C.asiatica, M.oleifera 29.20 28.56 28.23 26.83
and C. ternatea
(1:1:1)

Discussions and Conclusions

Plant sources have traditionally been used as cooking ingredients, treating ailments and cleansing agent in many
parts of the world. The promising potential use of biological active compounds isolated from plants as an antimicrobial
agent to combat resistant pathogen has been reported by many studies (Alvin, Miller and Neilan, 2014; Cushie,
Cushie and Lamb, 2014; Nabavi et. al., 2015). As regards the present study, the antibacterial activity of diethyl ether
leaves extract of C.asiatica, M.oleifera and C. ternatea were screened against the tested gram positive and gram
negative bacteria. Moreover, it has to be noted that this is the very first attempt of using simple, rapid and cost-
effective methods of antibacterial screening through disc diffusion assay to assessing the combination of plant
extracts. Although further insights with regards to the mechanism associated with this susceptibility still needed, the
presence of secondary metabolites soluble in diethyl ether may contribute to the plant's characteristics as an
antibacterial agent (Syed et al., 2015; Marmouzi et al., 2016). In conclusion, the antibacterial activities demonstrated
by a combination of the three plant extracts indicated their potential use as antimicrobial therapy. However, further
evaluations on the clinical, pharmaceutical and therapeutic values of the studied plants are deemed necessary.

Acknowledgements
The authors would like to thank Universiti Teknologi MARA for the facilities and financial support (600-IRMI/ MyRA
5/3/LESTARI (0154/2016).

107
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Thymus as antibacterial agents: From farm to pharmacy. Food chemistry, 173, pp.339-347.
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 IFRC 2017: 014-112 Food Processing and Post-Harvest Technology
Antifungal Activity of Aloe Vera Gel Towards the Pathogenic Fungus of Papaya Fruit
1Mendy, T.K., 1*Misran, A., 1Mahmud, T.M.M and 2Ismail, S.I

1Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
2Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
Abstract
The aim of this research is to evaluate the inhibitory effect of aloe vera on the mycelia growth of five pathogenic fungi
that caused spoilage in papaya fruits. In this experiment, two types of aloe vera extract; fresh aloe vera and food
grade were used. Both of the extracts with different concentrations (0, 15 %, 25 %, and 50 % (v/v)) were tested
against mycelium growth of five pathogenic fungi of papaya fruits (Fusarium sp., Aspergillus niger, Colletotrichum
gloeosporioides, Lasiodiplodia theobromae and Rhizopus sp) that were isolated from papaya fruits.At 15 %
concentration, fresh aloe vera effectively inhibited all the fungi as compared to food grade aloe vera. Analysis on the
composition of the aloe vera demonstrated phenolic, flavonoid content and DPPH radical scavenging activity were
found to be concentration dependent, with the fresh aloe exhibiting higher phenolic content, flavonoid and DPPH
activity than the food grade aloe vera gel. The finding of present study concluded that both fresh and food grade aloe
vera controlled the mycelia growth of major postharvest fungus of papaya fruit in vitro. Fresh aloe vera extract has a
significant inhibitory effect on the fungus that caused spoilage in papaya and can be potentially used as edible coating
to extend the shelf life of the fruits and vegetables.
Keywords: Aloe Vera, inhibition, fungi, papaya, postharvest
*Corresponding author’s email: azizahm@upm.edu.my
Introduction
Carica papaya is sub-tropical and tropical plant belonging to the small Caricaceae family. Papaya has a luscious taste
and are a rich source of antioxidant nutrient such as carotenoids, vitamin C and minerals. Postharvest losses account
for great reduction in the availability of quality and quantity in the marketing of papaya because of its short post-
harvest life and also the invasion of postharvest pathogens (de Oliveira and Vitória, 2011). To control the postharvest
damages cause by these fungi on papaya, several approaches are used including the non-chemical approach such
as the use of biological materials with less or no effects on the consumer’s health (Misir et al., 2014). Several studies
have shown that aloe vera gel was effective in controlling the growth of fungi on fruits and vegetable, thereby
prolonging their post-harvest life (Valverde et al., 2005; Serrano et al., 2006; Castillo et al., 2010). Present
investigation is carried out to assess the antifungal properties of fresh aloe vera gel and food grade aloe vera gel in
suppressing fungal growth in papaya fruits.
Materials and method
3.1 Materials
Aloe vera: Fresh Aloe vera was purchased from PIJ Holding Sdn Bhd., Johor Bahru, Johor, Malaysia and immediately
shipped to the laboratory under cold conditions. While commercial aloe vera gel (food grade) was obtained from PPA
BIO Sdn Bhd Johor Bahru, Johor, Malaysia. Both products of Aloe vera were obtained from the same farm.

3.2 Extraction of fresh aloe vera gel.

Gel extraction was done using the method of Brishti et al., (2013). To increase the efficacy of fresh aloe vera gel, the
resulting mixture had to be filtered twice. First, the gel had to be filtered using muslin cloth to remove fibre and
secondly it was further filtered using 0.45 uM nylon filter.

109
3.3 Antifungal activity of Aloe vera
In vitro screening of antifungal activity of Aloe Vera was determined according to (Awoite et al., 2013).

Determination of phenolic, flavonoid content and DPPH scavenging activity of aloe vera
3.4 Extraction of the sample
Aloe vera extraction was done by the method of (Addai et al., 2013).

3.4.1 Determination of phenolic content

Total phenolic content (TPC) was determined spectrophotometrically using Folin–Ciocalteu reagent by the method of
Singleton and Rossi (1965)

3.4.2 Determination of flavonoid content

Total flavonoids was determined using spectrophotometer by the methods of (Nor Qhairul Izzreen and Mohd
Fadzelly, 2013)

3.4.3 Determination of DPPH scavenging activity.

The determination of antioxidant activity through 1,1-diphenyl-2-picrylhydrazyl radical- scavenging was done by
the method of Nor Qhairul Izzreen and Mohd Fadzelly (2013)

3.4 Experimental design and statistical analysis

The study involved a three factorial experiment and treatments were laid-out in a Completely Random Design (CRD)
with two types of Aloe vera (fresh and food grade) for the treatment at five different concentration (0, 15, 25, and 50
%, v/v) on five different fungi with 3 replications. Data obtained was subjected to analysis of variance (ANOVA) and
Least Significance Difference (LSD) at p≤0.05 level was performed for mean comparisons. Data was analyzed using
Statistical Analysis System (SAS version 9.4 Cary, NC, USA).

Results and discussion

Fungal decay is a major contributor to the losses in the postharvest quality of papaya. In this study, we investigated
the antifungal effects of aloe vera on the major fungi pathogen of papaya and determined their mycelial growth
inhibition. Our results showed that fresh aloe vera has higher antifungal activity than food grade aloe vera on most
fungi studied at lower concentration (15 %) as summarized in Table 1. As compared to food grade Aloe vera, higher
concentration of aloe vera was needed to inhibit the fungal growth. Among fungi studied, both fresh and food grade
aloe vera gel inhibited Colletotrichum gloeosporioides growth better than any other fungi. Antifungal activity of aloe
vera in this study are consistent with several reports of this against various plant pathogenic fungi (Rodríguez et al.,
2005). These activities can be attributed to the presence of bioactive constituents in aloe vera gel. Analysis on
phenolic, flavonoid content and DPPH radical scavenging activity were found to be concentration dependent, with the
fresh aloe vera exhibited higher phenolic and flavonoids content and higher DPPH radical scavenging activity than
food grade aloe vera (Table 2).

110
 120 Pooled LSD=14.9

100

80
% Inhibitioin

60

40

20

0
0% 15% 25% 50% 0% 15% 25% 50%
Fresh filtered aloe vera Food grade aloe vera
Fusarium sp L. theobromae Aspergillus niger
Rhizopus sp C. gloeosporioides

Figure 1. Effect of aloe vera application (fresh and food grade) at different concentrations on the growth inhibition of postharvest fungi in papaya
fruit. Evaluation was done when the control plates have reached maximum growth. Data shown are the means of three replicates. Vertical
bar=pooled LSD at the 5% level.

Table 2. Effect of different concentration on the antioxidant activity of fresh filtered aloe and food grade aloe vera gel.

Phenolic content (mg Flavonoids DPPH

GAE/100g of FW ) (mg QE/100g (% inhibition)
of FW)
Treatment
Fresh filtered aloe vera 0% 1.72 ± 0.2d 10.97 ± 9.3d 13.48 ± 0.7
15% 5.92 ± 0.3c 62.70 ± 9.6c 22.18 ± 1.7
25% 7.38 ±0.3b 93.09 ± 3.3c 43.67 ± 15.1
50% 8.70 ± 0.4a 113.39 ±10.9 a 47.70 ± 11.7

Food grade aloe vera 0% 1.12 ±0.4c 8.55 ± 1.6d 12.97 ± 2.1
15% 1.55 ± 0.1c 51.88 ± 13.5c 23.23 ± 3.2
25% 2.42 ± 0.2b 76.12 ± 3.7b 38.71 ± 10.9
50% 3.40 ±0.4a 108.85 ± 19.8a 43.60 ± 10.0

Aloe vera*Concentration ** ns ns

Mean (n=3) with different letter within the same column are significantly different. Significant different at p ≤ 0.05 using LSD. Significant (**).Not
Significant (ns). Fresh weight (FW).Garlic acid equivalent (GAE). Quercetin equivalent (QE)

111
Conclusion
The finding of the present study concluded that fresh aloe vera at 15 % effectively controlled the mycelia growth of the
fungus. Each aloe vera extract contained different chemical composition that may contribute to the different mode of
inhibition. In summary, the study paves the way of Aloe vera as potential edible coating for controlling the postharvest
fungus of papaya fruits.
Acknowledgements
The authors would like to thank to Government of Gambia and UPM for the financial support,

Reference
Addai, Z. R., Abdullah, A., Mutalib, S. A., and Musa, K. H. (2013). Effect of gum arabic on quality and antioxidant
properties of papaya fruit during cold storage. International Journal of ChemTech Research, 5(6): 2854–2862
Awoite, T.M, Olorunfemi, M.F., Ajani, A.O and Oyelakin, M.O (2013). Studies on Fungi Associated With Post Harvest
Spoilage of Pawpaw Carica PAPAYA Fruit, IOSR Journal of Pharmacy and Biological Sciences, 4(6): 1–4
Brishti, F.H., Misir, J. and Sarker, A. (2013). Effect of biopreservatives on storage life of papaya fruit (Carica Papaya
L.). International of Journal Food Studies, 2(1): 126-136.
Castillo, S., Navarro, D., Zapata, P. J., Guillén, F., Valero, D., Serrano, M., & Martínez-Romero, D. (2010). Antifungal
efficacy of Aloe vera in vitro and its use as a preharvest treatment to maintain postharvest table grape quality.
Postharvest Biology and Technology, 57(3): 183–188
de Oliveira, J. G., and Vitória, A. P. (2011). Papaya: Nutritional and pharmacological characterization, and quality loss
due to physiological disorders. An overview. Food Research International, 44(5): 1306–1313
Misir, J., H. Brishti, F., and Hoque, M. (2014). Aloe vera gel as a Novel Edible Coating for Fresh Fruits: A Review.
American Journal of Food Science and Technology, 2(3): 93–97.
Nor Qhairul Izzreen, M. N., and Mohd Fadzelly, A. B. (2013). Phytochemicals and antioxidant properties of different
parts of Camellia sinensis leaves from Sabah Tea plantation in Sabah, Malaysia. International Food Research
Journal, 20(1): 307–312.
Rodríguez, D.J., Hernández-Castillo, D., Rodríguez-Garcia, R. and Angulo-Sánchez, J.L. (2005). Antifungal activity in
vitro of Aloe vera pulp and liquid fraction against plant pathogenic fungi. Industrial Crops and Products, 21: 81–
87
Serrano, M., Miguel., Guillen, F., Castillo, S., Martinez-Romero, D & Valero, D (2006). Use of Aloe Vera gel coating
preserves the functional properties of table grapes. Journal ofAgricultural and Food Chemistry, 54,3882-3886.
Singleton, V. L. and Rossi, J. A. 1965. Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid
reagents. American Journal of Enology and Viticulture 16(1): 144-158
Valverde, M. J., Valero, D., Romero, M. D., Guillen, F., Castillo, S., and Serrano, M. (2005). Novel edible coating
based on Aloe vera gel to maintain table grape quality and safety. Journal of Agricultural and Food Chemistry,
53: 7807–7813

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Effect of Different Drying Methods on the Quality of Pink and Grey Oyster Mushrooms

Raseetha, S.* and Siti-Nuramira, J.

Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia
Abstract
The aim of this study was to determine the impact on the quality of different oyster mushroom (grey and pink) after
drying. Cabinet drying, microwave oven and vacuum oven were used to dry mushrooms in this study. Pleurotus
djamor (pink) using cabinet dryer has higher protein (29.94%), fat (0.79%), vitamin B3 (1.96 mg/100 g) and most of
the mineral content while Pleurotus ostreatus (grey) has higher carbohydrate (68.35%), has highest value for element
K, Mg, Zn, Fe, vitamin B3 and B6. Meanwhile, vacuum drying cause severe damage on cell structure of the
mushroom. On the other hand, microwave drying treatment mushroom has the highest value for browning index
(75.11). Based from the study, cabinet drying is more likely to be chosen as post harvest treatment as it causes least
degradation.
Keywords: Oyster mushroom, drying, Pleurotus ostreatus, Pleurotus djamor
*Corresponding author’s email: raseetha@salam.uitm.edu.my

Introduction

Oyster mushroom has a short shelf life due to its high water content compared to most vegetable at ambient
temperature. Therefore, it is necessary to apply preservation technologies to extend the shelf life of oyster
mushrooms. Drying is one of the most important preservation methods employed for its storage (Taghian et al.,
2014). Dehydrated mushrooms can be stored in airtight packages for more than one year (Jaworska et al., 2014).
Pleurotus djamor (pink) was studied to increase the awareness among consumer on the health benefits and
nutritional value. Thus, the objectives of this study were to determine the effect of different drying method (cabinet
drying, microwave drying and vacuum drying) on the nutrient content of pink and grey oyster mushroom and the cell
structure of the mushroom subjected to different drying method which was cabinet drying, microwave drying and
vacuum drying method.

Materials and methods

Preparation of samples
Fresh cultivated Pleurotus ostreatus and Pleurotus djamor were purchased from Nas Agro Farm in Jenderam Hulu,
Sepang, Selangor. Mushroom samples were transported to the laboratory and stored at a temperature at 4°C before
use. Raw oyster mushrooms were washed, cleaned and placed on absorbent paper to remove excess surface water.
Mushrooms (1500 ± 5 g) were subjected to the following treatments; cabinet drying treatment (CD), vacuum drying
treatment (VC) and microwave drying treatment (MW).

Drying method
Drying method was adapted from Tian et al. (2016). For hot air drying, the samples were hot air dried (Vision
Scientific, Malaysia) using an electric cabinet dryer at 60°C overnight. Vacuum drying was conducted with pump
(Valucell 22, Germany) and dried at 60°C for 15 hours. Microwave drying was carried using microwave oven
(Samsung, Model: MW71E, Malaysia) with a maximum power output of 1150 W and 2450 MHz were used and dried
at 539 W for 6 minutes.
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 Proximate analysis This page is intentionally left blank

Moisture content, protein, fat, ash and carbohydrate content were determined by the standard AOAC (1995).

Analysis of elements
Mineral content were analysed (Zn, Fe, Ca, Mg, Na and K) using Inductively Coupled Argon Plasma Optical Emission
Spectrometer (ICP-OES) (Drewnowska et al., 2015).

Vitamin B analysis
Quantification of vitamin B3 and B6 content was accomplished using HPLC by comparison to vitamin B standards
based on previous method (Aslam et al., 2008).

Browning index determination

The colour measurement was carried out using chroma meter (CR 400, Konica Minolta Optics, Inc. Osaka, Japan)
according to Jiang (2013).

Microstructure analysis
Scanning Electron Microscopy (SEM) was used to visualize the microstructure of dried mushroom using Scanning
Electron Microscope (Quanta 200 FEI (2004)) with magnification of (x500), (x1500) and (x2000) (Tian et al., 2016).

Statistical analysis
All data on dried mushroom were presented as means ± standard deviation. The statistical analysis was carried out
using analysis of variance (ANOVA) by duncan’s multiple test at (p<0.05) and independent t-test procedure of SPSS
software.

Result and discussion

Proximate, mineral and vitamin B analyses for pink and grey oyster mushrooms
Table 1 shows proximate composition of the mushroom samples based on different drying method. Dried Pleurotus
djamor has lower moisture content (11.54%) compared to Pleurotus ostreatus (13.14%). Protein content of Pleurotus
djamor was significantly higher (p<0.05) which was 29.94% compared to Pleurotus ostreatus, 15.26%, for cabinet
drying treatment. According to Zhang et al., (2006) hot air drying technique leads to high energy demand and
prolonged drying time which cause severe shrinkage and reduced bulk density. Pleurotus djamor has higher protein
content (26.6%) compared to Pleurotus ostreatus (20.82%) (Chinarang & Intarapichet, 2009; Rodrigues et al., 2015).

Table 1 Proximate composition of dried oyster mushrooms

Grey oyster mushroom Pink oyster mushroom
Composition (%)
Cabinet dryer oven Microwave oven Vacuum oven Cabinet dryer oven Microwave oven Vacuum oven
Carbohydrate 64.75 ± 1.05 a,A 59.85 ± 0.26 b,A 59.22 ± 1.25 b,A 50.75 ± 0.70 b,B 48.36 ± 0.82 c,B 52.37 ± 0.70 a,B
Protein 15.26 ± 0.78 a,B 13.50 ± 0.37 b,B 12.89 ± 0.86 b,B 29.94 ± 0.60 a,A 24.05 ± 0.73 b,A 18.90 ± 0.19 c,A
Moisture 13.14 ± 0.30 c,A 20.61 ± 0.12 b,A 23.41± 0.19 a,A 11.54 ± 0.36 c,B 21.06 ± 0.42 b,A 22.97 ± 0.44 a,A
Ash 6.27 ± 0.24 a,B 5.58 ± 0.42 b,A 4.27 ± 0.28 c,B 6.98 ± 0.22 a,A 5.92 ± 0.15 b,A 5.24 ± 0.41 c,A
Fat 0.58 ± 0.05 a,B 0.46 ± 0.08 a,A 0.20 ± 0.03 b,B 0.79 ± 0.07 a,A 0.61 ± 0.02 b,A 0.52 ± 0.05 b,A
Note: Analysis data were obtained from three triplicate samples. Values are expressed as mean ± standard deviation. Means with different
small letter indicate a significant difference using ANOVA (p<0.05) for different drying treatment and means with different capital letter indicate a
significant difference using independent t-test (p<0.05) for different type of oyster mushroom respectively.

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Table 2 Mineral composition of dried oyster mushrooms

SAMPLE K Mg Na Ca Zn Fe
GCD 550.60 ± 2.12 a,A 90.24 ± 0.37 a,B 9.07 ± 0.27 b,B 8.14 ± 0.07 b,B 7.72 ± 0.11 b,B 4.81 ± 0.006 a,B
GMW 427.45 ± 1.91 b,B 63.26 ± 0.29 c,B 7.23 ± 0.21 c,B 3.35 ± 0.20 c,B 0.72 ± 0.10 c,B 1.65 ± 0.01 b,B
GVC 398.80 ± 0.42 c,A 88.49 ± 0.27 b,B 14.33 ± 0.11 a,B 36.24 ± 2.16 a,A 13.33 ± 0.006 a,B 2.28 ± 0.02 c,B
PCD 558.25 ± 1.77 a,A 134.27 ± 0.06 a,A 10.10 ± 0.34 c,A 9.04 ± 0.18 b,A 45.08 ± 0.55 a,A 5.04 ± 0.04 a,A
PMW 241.25 ± 2.90 c,B 124.83 ± 0.38 b,A 12.08 ± 0.11 b,A 9.87 ± 0.34 b,A 38.67 ± 0.34 c,A 4.82 ± 0.03 b,A
PVC 439.35 ± 1.35 b,A 118.73 ± 1.06 c,A 16.38 ± 0.17 a,A 18.26 ± 0.75 a,B 41.56 ± 0.55 b,A 4.73 ± 0.05 c,A
Note: Concentration in mg/100 g sample, mean ± standard deviation)
Means with different small letter indicate a significant difference using ANOVA (p<0.05) for different drying treatment and means with different
capital letter indicate a significant difference using independent t-test (p<0.05) for different type of mushroom.
(GCD; Grey oyster mushroom cabinet dried, GMW; Grey oyster mushroom microwave dried, GVC; Grey oyster mushroom vacuum dried, PCD;
Pink oyster mushroom cabinet dried, PMW; Pink oyster mushroom microwave dried, PVC; Pink oyster mushroom vacuum dried)

The mineral content analysed in different type of oyster mushroom of different drying treatment was shown in Table
4.2. Element potassium, magnesium, zinc and iron, cabinet dried samples have retained most of their mineral
compared to other treatment. The data showed that, Pleurotus djamor has higher content of all the elements
determined. Pleurotus species contain high potassium to sodium ratio, which makes mushrooms an ideal food for
patients suffering from hypertension & heart diseases (Subramanian et al., 2014). Pleurotus djamor was indicated to
have high amount of potassium and magnesium (Guo et al., 2007; Rodrigues et al., 2015).

Pleurotus djamor has higher (0.2521 mg/100 g and 0.1136 mg/100 g vitamin B3 and B6, respectively, compared to
Pleurotus ostreatus (0.2196 mg/100 g and 0.0251 mg/100 g). P. ostreatus contains more folacine, vitamin B1, vitamin
B3 but less vitamin B12 than other mushroom species (Deepalakshmi, & Mirunalini, 2014).

Cabinet drying showed higher browning index in gill of the pink oyster mushroom (84.35) compared to grey oyster
mushroom (51.21). Variation in colour from observation may be caused by cultivation of the mushroom, which
affected by the environment factors, such as climate, substrate used, time of harvest and storage temperature (Islam
et al., 2014).

Scanning electron microscopy of the microstructure indicated for gill surface, cabinet drying shows a flaky cracked
surface and severe shrinkage. According to Garcia-Segovia et al., (2011), hot air drying treatment cause hyphae to
lose their structure and flattened. This explained the hard texture of cabinet dried mushroom. While, for both vacuum
and microwave dried mushroom, the surface of their gills are uneven and gritty. However, vacuum dried mushroom
showed more shrinkage surface compared to microwave dried mushroom.

Conclusion
In conclusion, pink oyster mushroom has high protein (29.94%) and fat content (0.79%), vitamin B3 (1.96 mg/100g)
value and most of the mineral content while grey oyster mushroom has significant value of carbohydrate (68.35%).
Based from the study, cabinet drying is more likely to be chosen as post harvest treatment as it causes least
degradation and it is low cost and easily maintain drying treatment.

References
AOAC, 1995. Official Methods of Analysis, AOAC International, Washington, p. 7.
Aslam, J., Mohajir, M. S., Khan, S. A. and Khan, A. Q. 2008. HPLC analysis of water-soluble vitamins (B1, B2, B3, B5,
B6) in vitro and ex vitro germinated chickpea (Cicer arietinum L.). African Journal of Biotechnology 7(14): 2310-
2314.
Deepalakshmi, K. and Sankaran, M. 2014. Pleurotus ostreatus: an oyster mushro
115
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nal and medicinal properties. Journal of Biochemical Technology 5(2): 718-726.

Drewnowska, M. and Falandysz, J. (2015). Investigation on mineral composition and accumulation by popular edible
mushroom common chanterelle (Cantharellus cibarius). Ecotoxicology and Environmental Safety 113: 9-17.
Garcia-Segovia, P., Andres-Bello, A. and Martinez-Monzo, J. 2011. Rehydration of air-dried shiitake mushroom
(Lentinus edodes) caps comparison of conventional and vacuum water immersion processes. Food Science
and Technology 44(2): 480–488.
Guo, L. Q., Lin, J. Y. and Lin, J. F. 2007. Non-volatile components of several novel species of edible fungi in
China. Food Chemistry 100(2): 643-649.
Islam, M. N., Zhang, M., Adhikari, B., Xinfeng, C. and Xu, B. G. 2014. The effect of ultrasound-assisted immersion
freezing on selected physicochemical properties of mushrooms. International Journal of Refrigeration 42: 121-
133.
Jaworska, G., Pogon´, K., Bernas´, E. and Skrzypczak, A. 2014. Effect of different drying methods and 24-month
storage on water activity, rehydration capacity, and antioxidants in boletus edulis mushrooms. Drying
Technology 32(3): 291–300.
Jiang, T. (2013). Effect of alginate coating on physicochemical and sensory qualities of button mushrooms (Agaricus
bisporus) under a high oxygen modified atmosphere. Postharvest Biology and Technology 76, 91-97.
Rodrigues, D. M., Freitas, A. C., Rocha-Santos, T. A., Vasconcelos, M. W., Roriz, M., Rodríguez-Alcalá, L. M. and
Duarte, A. C. 2015. Chemical composition and nutritive value of Pleurotus citrinopileatus var cornucopiae, P.
eryngii, P. salmoneo stramineus, Pholiota nameko and Hericium erinaceus. Journal of Food Science and
Technology 52(11): 6927-6939.
Subramanian, K., Shanmugasundaram, K. and Muthu, N. 2014. Spawn production and cultivation strategies for
Pleurotus eous (pink oyster mushroom). World Journal of Pharmacy and Pharmaceutical Sciences 3(10): 915-
924.
Taghian Dinani, S., Hamdami, N., Shahedi, M. and Keramat, J. 2014. Optimization of carboxymethyl cellulose and
calcium chloride dip-coating on mushroom slices prior to hot air drying using Response Surface Methodology.
Journal of Food Processing and Preservation 38(3): 1269–1278.
Tian, Y., Zhao Y., Huang, J., Zeng, H. and Zheng B. 2016. Effects of different drying methods on the product quality
and volatile compounds of whole shiitake mushrooms. Food Chemistry 197(1): 714–722.
Zhang, M., Tang, J., Mujumdar, A. S. and Wang, S. 2006. Trends in microwave-related drying of fruits and
vegetables. Trends in Food Science & Technology 17(10): 524–534.

116
 IFRC 2017: 027-142 Food Processing and Post-Harvest Technology

Supercritical Fluid Extraction of Date Seed Oil

1Jaih, A.A.M., 1,2,4*Rahman, R.A., 3Razis, A.F.A., 2Ariffin, A.A., 5Al-Awaadh, A. and 3Selamat, J.

1Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
2Faculty of Food Science and Technology, Department of Food Technology, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor, Malaysia
3Faculty of Food Science and Technology, Department of Food Science, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor, Malaysia
4Faculty of Engineering, Department of Process and Food Engineering, Universiti Puta Malaysia, 43400 UPM
Serdang, Selangor, Malaysia
5Department of Agriculture Engineering, King Saud University, P.O. Box 2460, Riyadh, 11451, Saudi Arabia

Abstract
Date seed is a by-product of date fruit (Phoenix dactylifera) industry. It is normally being discarded as waste or
traditionally processed into animal feed or caffeine-free coffee in small scale. In this study, seed from Mabroum date
variety was cleaned and grinded, and oil was extracted using supercritical fluid extraction method at pressure range
from 20 to 40 MPa and temperature range from 40 to 70 ºC. The extraction was last for 120 mins and flowrate of
carbon dioxide, the supercritical fluid was made constant at 5 ml/min. Supercritical fluid extraction that was performed
at combination of 20 MPa and 40 ºC resulted in the highest oil yield and recovery. Oil recovery ranged from 4.3 to 8.1
%, while oil yield ranged from 52.4 to 99.2 %. Oleic acid was found as the major fatty acid present in the date seed
oil. However, different combination of temperature and pressure in supercritical fluid extraction resulted in different
fatty acid composition of the oil.

Keywords: Date seed oil, supercritical fluid extraction, fatty acid composition.
*Corresponding author’s email: russly@upm.edu.my

Introduction
Supercritical fluid extraction (SFE) is a solvent-free extraction method, developed as alternative to the conventional
solvent extraction for oil extraction which involves the use of chemical-solvent. Carbon dioxide (CO2) is widely used
as extraction fluid in SFE method due to its characteristics which is non-flammable, harmless, noncorrosive,
inexpensive, nontoxic, classified as “GRAS”, as well as low critical temperature which help to prevent thermal
degradation of solute being extracted (Ashkin et al., 2009). In addition, the application of CO2 seems to benefit the
users which demand for more natural and chemical-solvent-free products, compared to the products obtained from
solvent extraction method. Parameters of the extraction method in SFE play a very important role in determining the
characteristics of the products that will be obtained from the extraction. There are lot of studies have been conducted
on date seed oil (DSO), however, all the studies uses chemical-solvent to extract the oil such as chloroform-methanol
(Abou-Zeid and Baeshin, 1992), hexane (Nehdi et al., 2010) and petroleum ether (Besbes et al., 2004; 2005;
Dammak et al., 2007; Ben Abdallah et al., 2009). This study focuses on the effect of two parameters of SFE method
namely pressure and temperature to the oil extract in terms of yield and fatty acid composition (FAC). From our
review (Abdul Afiq et al., 2013), the application of SFE method for extraction of DSO has never been reported at the
present time, hence this will be as preliminary study of using SFE for extraction of date seed oil.

117
Materials and Methods
2.1 Preparation of date seed
Mabroum variety date fruits were obtained at final maturity stage (“tamar” stage). The seeds were cleaned and dried
at 50 ºC until moisture content was below 5 %. A high speed grinder (Hamilton Beach Commercial, Hamilton Beach
Brands Inc., USA, Canada) was used to grind the date seeds into powder and sieved using a strainer with 0.5 mm
pore size.

2.2 Oil extraction from date seed

In each extraction process, 25 g of date seed powder was placed into a 50 ml extraction vessel (Model EV-3; Jasco
Inc., Easton, United States). The pressure and temperature setting used for extraction was developed based on Chan
and Ismail (2009), with modifications. Modified pressure range from 20 to 40 MPa and modified temperature range
from 40 to 70 ºC. The extraction last for 120 mins and CO2 flowrate was 5 ml/min throughout the time. A back
pressure regulator (Model BP-1580-81; Jasco Inc., Easton, United States) was connected to the extractor as pressure
controller. Oil was collected in glass vessel and stored at 4 ºC until further used. For Soxhlet method, the extraction
by using petroleum ether was performed according to Ariffin et al. (2009).

2.3 The total oil recovery from the date seed by using SFE and Soxhlet extraction method was determined by dividing
the weight of oil obtained to the weight of sample and the result is expressed in percentage. Whilst, the total oil yield
of date seed oil was determined by dividing the percentage of DSO from SFE method to the percentage of DSO from
Soxhlet extraction method and expressed in percentage.

2.4 Fatty acid composition of oils were analysed by using Gas Chromatography equipped with Fluoresce Ionisation
Detector (GC-FID) (Agilent 6890 N Network GC System, Agilent Technologies, Singapore) and a polar capillary
column DB-WAX (Agilent Technologies, USA) having 0.25 mm internal diameter, 30 m length and 0.25 µm film
thickness. Fatty acid methyl ester (FAME) was prepared according to PORIM Test Method (1990).

2.5 The tests were performed in duplicate and the results presented as mean ± standard deviation value. Data were
statistically analysed by One-way Analysis of Variance (ANOVA) using MINITAB Ver. 14 (Minitab Inc., State College,
PA, USA) at 0.05 probability level.

Results and Discussion

The total oil recovery and total oil yield from SFE and Soxhlet extraction methods are shown in Table 1. Three
different pressures (20, 30 and 40 MPa) and temperatures (40, 55, 70 ºC) were used to extract DSO by using SFE.
The highest oil recovery (8.1 %) was extracted at combination of 20 MPa and 40 ºC, while the lowest (4.3 %) at 30
MPa and 55 ºC. Oil recovery from Soxhlet extraction is 8.2 ± 0.01 %. Overall, oil recoveries from SFE at all
parameters are lower than the Soxhlet extraction. However, extraction of DSO at 20 MPa and 40 ºC was the only
extraction from SFE that is not significantly different to oil recovery from Soxhlet extraction, which has 99.2 % of oil
yield. The lowest oil yield was obtained at 52.4 % (30 MPa and 55 ºC).

The results shows that, at a constant low pressure condition 20 MPa, the increase of temperature had decreased the
amount of oil being extracted. The increase of temperature has decreased CO2 density, which then reduces its
solubility, and as a result decrease in extraction yield. Whilst, at a constant high pressure condition 40 MPa, the
increase of temperature had increased the extraction yield, due to the oil vapour pressure that has overcome the
effect of CO2 density (Zaidul et al., 2007).

Analysis of fatty acid profile of DSO by FAME determination using Gas Chromatography is shown in Table 2. Oleic
acid (C18:1) was found as the major fatty acid in DSO. Other fatty acids that were obtained in DSO from this study
are lauric acid (C12:0). myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), and linoleic acid (C18:2).
From SFE method, the highest oleic acid (94.3 %) was obtained by the extraction at 40 MPa, and 40 and 55 ºC.
Previous studies conducted by Zaidul et al. (2006) and Markom et al. (2001) found that the selectivity of fatty acid
constituents at certain temperature and pressure has affect its elution from the seed. This has explained the findings
of this study which found that different parameters of SFE resulted in different fatty acid profile of the oils, as obtained
118
by the extraction at combination of 20 MPa and 70 ºC, 30 MPa and 40 ºC, 20 MPa and 55 ºC, and 20 MPa and 40 ºC.
Generally, FAC of DSOs from SFE are significantly different compared to FAC of DSO from Soxhlet extraction,
though the difference only present at small amount, except for oil from SFE at certain parameters.

Table 1: Total oil recovery and oil yield extracted from date seed
Temp. Total oil recovery (%) Total oil yield (%)
(oC) Pressure (MPa) Pressure (MPa)
20 30 40 20 30 40
40 8.10 ± 6.00 ± 6.29 ± 99.21 ± 73.53 ± 77.02 ±
0.01aA 0.23abB 0.78aAB 0.95aA 2.77abB 9.62aAB
55 6.63 ± 4.28 ± 6.38 ± 81.25 ± 52.39 ± 78.13 ±
1.00aA 0.77bA 0.02aA 13.35aA 9.45bA 0.26aA
70 6.22 ± 6.87 ± 6.86 ± 76.17 ± 84.13 ± 84.07 ±
0.97aA 0.33aA 0.21aA 11.87aA 4.07aA 2.60aA
Values are means ± standard deviation of duplicate measurements. Means with different lower letter and upper letter in the same columns and
rows, respectively, in separate column of total oil recovery and total oil yield, are significantly different at p<0.05.

Table 2: Fatty acid composition of date seed oil from SFE and Soxhlet extraction method
SFE Fatty acid composition (%)
Myristic Palmitic Stearic Linoleic
Press. Temp. Lauric acid acid acid acid Oleic acid acid
(MPa) (oC) (C12:0) (C14:0) (C16:0) (C18:0) (C18:1) (C18:2)
20 40 4.29 ± 3.21 ± 3.09 ± 2.46 ± 83.83 ± 3.13 ±
0.04d 0.03d 0.03d 0.03d 0.16f 0.03c
20 55 .6.22 ± 4.48 ± 4.10 ± 3.30 ± 77.61 ± 4.29 ±
0.04c 0.04c 0.03c 0.03c 0.18g 0.05b
20 70 10.18 ± 6.02 ± 4.99 ± 4.50 ± 69.21 ± 5.11 ±
0.13a 0.08a 0.02b 0.06a 0.38i 0.09a
30 40 8.53 ± 5.66 ± 5.08 ± 4.33 ± 71.25 ± 5.14 ±
0.07b 0.05b 0.03a 0.03b 0.17h 0.01a
30 55 2.93 ± 1.81 ± 1.60 ± 1.38 ± 90.77 ± 1.51 ±
0.09e 0.05e 0.03e 0.05e 0.28e 0.07d
30 70 1.51 ± 1.26 ± 0.85 ± 92.58 ± 1.05 ±
2.77 ± 0.04f 0.02f 0.01 f 0.01 j 0.06c 0.02e
40 40 1.64 ± 1.14 ± 1.08 ± 1.14 ± 94.32 ± 0.68 ±
0.07g 0.02g 0.10g 0.04g 0.37b 0.13f
40 55 1.65 ± 1.04 ± 0.96 ± 1.26 ± 94.37 ± 0.72 ±
0.02g 0.01h 0.01h 0.01f 0.16b 0.15f
40 70 2.79 ± 1.53 ± 1.31 ± 0.95 ± 92.44 ± 0.98 ±
0.11ef 0.03f 0.04 f 0.01 h 0.05d 0.12d
Soxhlet 1.00 ± 0.73 ± 0.68 ± 0.65 ± 96.51 ± 0.43 ±
extraction 0.13h 0.11i 0.07i 0.11k 0.32a 0.09g
Values are means ± standard deviation of duplicate measurements. Means with same letter in the same columns are not significantly different
at p<0.05.

Conclusion
In SFE method, low density of CO2 at low pressure (20 MPa) condition influenced the oil yield, while oil vapour
pressure has greater effect to the oil yield at high pressure condition (40 MPa). Solubility of certain fatty acid at certain
pressure and temperature resulted in the variation of fatty acid profile of DSO. Date seed oil from Mabroum variety
can be categorised as unsaturated oil due to the high content of oleic acid. Consumption of oleic acid in our diet could
increase the high density lipoprotein (HDL) and decrease the low density lipoprotein (LDL) level in blood, thus reduce
the risk of cardiovascular diseases.
119
Acknowledgement
Thank you to the Malaysian Ministry of Higher Education for financial support.

References

Abdul Afiq, M.J., Abdul Rahman, R., Che Man, Y.B., AL-Kahtani, H.A. and Mansor, T.S.T. 2013. Date seed and date
seed oil. International Food Research Journal 20(5): 2035-2043.
Abou-Zeid, A. A-Z. and Baeshin, N. A. 1992. Utilization of date-seed lipid and hydrolysate in the fermentative
formation of oxytetracycline by Steptomyces rimosus. Bioresource Technology 41: 41-43.
Ashkin, R., Goto, M. and Sasaki, M. 2009. Supercritical fluid extraction in food analysis, in Semih Otles (Ed).
Handbook of Food Analysis Instrument, p 25-53. United States: CRC Press.
Ariffin, A.A., Bakar, J., Tan, C.P., Rahman, R.A., Karim, R. and Loi, C.C. 2009. Essential fatty acids of pitaya (dragon
fruit) seed oil. Food Chemistry 114: 561-564.
Ben Abdallah, F., Dammak, I., Mallek, H., Hentati, B. and Keskes, L.A. 2009. Effects of date seed oil on testicular
antioxidant enzymes and epididymal sperm characteristics in male mice. First International Journal of
Andrology 41: 229-234.
Besbes, S., Blecker, C., Deroanne, C., Drira, N.E and Attia, H. 2004. Date seeds: chemical composition and
characteristic profiles of the lipid fraction. Food Chemistry 84: 577-584.
Besbes, S., Blecker, C., Deroanne, C., Lognay, G., Drira, N.E. and Attia, H. 2005. Heating effects on some quality
characteristics of date seed oil. Food Chemistry 91: 469-476.
Dammak, I., Abdallah, F.B., Boudaya, S., Keskes, L., Besbes, S., El Gaied, A., Attia, H., Turki, H. and Hentati, B.
2007. Effects of date seed oil on normal human skin in vitro. European Journal of Dermatology 17: 516-519.
Markom, M., Singh, H. and Hasan, M. 2001. Supercritical CO2 fractionation of crude palm oil. Journal of Supercritical
Fluids 20: 45-53.
Nehdi, I., Omri, S., Khalil, M.I. and Al-Resayes, S.I. 2010. Characteristics and chemical composition of date palm
(Pheonix canariensis) seeds and seeds oil. Industrial Crops and Products 32: 360-365.
PORIM Test Methods. 1995. Methods of Test for Palm Oil and palm Oil Products, p 72-91. Malaysia: Palm Oil
Research Institute of Malaysia.
Zaidul, I.S.M., Nik Norulaini, N.A., Mohd Omar, A.K. and Smith Jr., R.L. 2006. Supercritical carbon dioxide (SC-CO2)
extraction and fractionation of palm kernel oil from palm kernel as cocoa butter replacers blend. Journal of
Food Engineering 73: 210-216.
Zaidul, I.S.M., Nik Norulaini, N.A., Mohd Omar, A.K. and Smith Jr., R.L. 2007. Supercritical carbon dioxide (SC-CO2)
extraction of palm kernel oil from palm kernel. Journal of Food Engineering 79: 1007-1014.

120
 IFRC 2017: 036-023 Food Processing and Post-Harvest Technology
Effect of Heat Adaptation and Spray Drying Outlet Temperature on the Survival of Lactobacillus sp. Strain
3C2-10
1, 2 Kunnathep, J. and 1, 2,*Oonsivilai, R.
1School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon
Ratchasima, 30000, Thailand
2Postharvest Technology Innovation Center, Commission on Higher Education,
Bangkok 10400, Thailand
Abstract
The effect of heat adaption, core-to-wall ratio and spray drying outlet temperature on Lactobacillus sp. strain 3C2-10
survival during storage at the temperature of 4, 25 and 40°C were studied. The microencapsulation of Lactobacillus
sp. strain 3C2-10 was performed with wall materials such as fructooligosaccharides (FOS), resistance maltodextrin
and the combination of FOS and resistance maltodextrin at two different core-to-wall ratios of 1:1 and 1:2. The spray
drying outlet temperatures were varied from 70-90°C, and the heat adaptation was carried out by heating cell
suspensions at 47°C for 15 min before spray drying. The Fourier Transform Infrared spectrophotometer (FTIR) was
applied for chemical composition of encapsulated bacterial cells. The results showed that FTIR results confirmed the
presence of FOS and resistance maltodextrin in the microencapsulated Lactobacillus sp. strain 3C2-10 after passed
through spray drying process. In addition, the survival rate of Lactobacillus sp. strain 3C2-10 in all media with and
without heat adaption condition was decreased. Heat adapted Lactobacillus sp. strain 3C2-10 cells showed higher
survival rate than non-adapted Lactobacillus sp. strain 3C2-10 cells. In conclusion, the heat adaption helped prevent
microencapsulated Lactobacillus sp. strain 3C2-10 cells at microencapsulation with all media of core-to-wall ratio at
1:2 had better storage and simulated gastrointestinal digestion stability. The higher spray drying outlet temperature
would resulted in a lower water activity of encapsulated Lactobacillus sp. strain 3C2-10 cells and improved
survivability storage at temperature of 4 °C.
Keywords: Microencapsulation, heat adaption, Spray drying, FOS, Resistance Maltodextrin
*Corresponding author’s email: ratchadaporn.oonsivilai@gmail.com
Introduction
Lactobacilli strains are among the lactic acid bacteria (LAB) that are the most widely used as oral probiotics to
improve gut health and immunity (Bourdichon et al., 2012). The genus Lactobacillus has a long history of safety use
including dairy and non-dairy foods application. Delivery foods or nutritive supplements should contain probiotic
populations of at least 106 – 107 live microorganisms per gram or milliliter at the time of consumption in order to
provide health benefits to the host (Kent & Doherty, 2014). Prebiotics are defined as non-digestible foods that are
necessary for stimulating the growth of probiotic bacteria in the colon and improve host health. The major prebiotics
used are carbohydrate and fibers. In manufacturing probiotics and other fermented products, freeze drying and spray
drying are frequently applied techniques. While spray drying is a promising low-cost alternative because it is allows
the continuous production of high yields dehydration within short time periods. However, in the spray drying process
loss of viability of the cells. When bacterial cells sense a high temperature that change membrane fluidity, alter cell
protein structure or disrupt ribosomes or affect nucleic acids. It is possible to induce a thermal sub-lethal effect on
cells which adapts them to adverse conditions during drying and storage (Anekella & Orsat, 2013). Maltodextrin are
used for microencapsulation by spray drying but only few have reported the use of fructooligosaccharides (FOS) as
wall material. Stickiness behavior limits the use of FOS. Resistance maltodextrin (RMD) is resistant to digestion in the
human digestive system. The research work on the nutritional benefits such as increase probiotics populations in
colon and increase in fecal weight (Lefranc-Millot et al., 2012).
The purpose of this study was to investigate the influence of heat stress and wall materials FOS, RMD and the
combination of both at different core-to-wall ratios for microencapsulation of Lactobacillus sp. 3C2-10. The effect of
121
wall material on physical properties of microcapsules and viability of cells in simulated gastric and intestinal conditions
were evaluated.
Materials and Methods
Bacterial strains and culture conditions
Lactobacillus sp. strain 3C2-10 isolated from cassava pulp (Nawong, 2015) was used in this study. The cells were
sub-cultured in 9 mL MRS broth at 37°C for 24 h. under anaerobic conditions. The cells were harvested until reach to
the early stationary phase (24 h.) and centrifuged (5804R, eppendorf, Bangkok, Thailand) at 3,234g for 10 min at
25°C.
Heat adaptation
In order to study the effects of heat adaption on active cells of Lactobacillus sp. 3C2-10, the bacterial cells were
cultured at 37 °C until early stationary phase (24 h.). Each cell suspensions were incubated for 15 min at 37, 42, 47,
50 and 52 °C in water bath to induce heat adaption. The adapted and non-adapted cells were treated at 60 °C for 15
min (Anekella & Orsat, 2013). The viable cell counts were determined on MRS agar plate.
Spray drying
Microencapsulation of Lactobacillus sp. strain 3C2-10 with wall materials at different ratios was performed in
laboratory-scale spray dryer (BÜCHI B-290, Bangkok, Thailand). The pellets (30 g) were mixed with each wall
material solutions (20%w/w FOS, RMD, and combination of both) to obtain a required core-to-wall ratio of 1:1 and 1:2
before spray drying. The air flow rate at 40 m3/h and aspiration was maintained at 100 % at constant air inlet
temperature of 150 ± 5 °C. After spray drying, spray dried powder were collected from bottom under the cyclone,
stored in aluminium foil at 4, 25 and 40 °C.
Gastro-intestinal tract simulation
Simulated gastric juice (SGJ) and Simulated intestinal juice (SIJ) was prepared according to (Rajam &
Anandharamakrishnan, 2015)
Results and Discussion
The D-value of heat adapted cell at the temperature of 37, 42, 47, 50 and 52 °C after exposed to 60 °C for 15 min
were 3.21, 4.48, 10.14, 2.24 and 2.13 min, respectively (Fig. 1). So the temperature of 47 °C was the optimal for heat
adaptation. These results are approval with those of (Corcoran, Ross, Fitzgerald, & Stanton, 2004; Kang, Jeon, Shin,
Kwon, & So, 2015) who showed that L.lactis HE-1 was treated at the temperature of 42 °C for 15 min in MRS broth
before exposed to 60 °C for 10 min, the D-values were the highest as 7.85 min. And according to the theory that the
temperature increase 10 °C from optimal the temperature of bacterial growth leads to thermal chock (Teixeira, Castro,
& Kirby, 1994).

122
 12

Viable cell count (Log cfu/ml)

10

8 37 °C

42 °C
6
47 °C
4 50 °C

2 52 °C

0
0 5 7 10 15
Time (min)

Figure 1. The viability of Lactobacillus sp. strain 3C2-10 in MRS broth were treated at the temperature of 37, 42, 47, 50 and 52
°C before exposed 60 °C 15 min.
Table 1. Effect of wall material and core-to-wall ratio on encapsulation efficiency

Wall material Core-to-wall ratio Encapsulation efficiency

FOS 1:1 76.76 ± 0.65

1:2 80.62 ± 0.16
RMD 1:1 88.46 ± 0.50
1:2 89.37 ± 1.00
FOS+RMD 1:1 91.30 ± 0.27
1:2 91.57 ± 0.92

The effect of spray drying outlet temperature on cell viability in RMD with and without heat adaptation (47 °C for 15
min.) data not showed. The adaptive cell show more heat adaption higher than non-adaptive cell at 84-90 and 91-97
°C of out let temperature While the cell viability at the outlet temperature of 70-76 °C and 77-83 °C showed not
significantly different (p<0.05) because at these the temperature may be good survival of non-adapted cells.
(Corcoran, Ross, Fitzgerald, & Stanton, 2004) And the outlet temperature of 84-90 °C was the optimal for
encapsulation.
The viability of Lactobacillus sp. strain 3C2-10 encapsulated with three different wall materials with core-to-wall ratio
of 1:1 and 1:2 showed encapsulation efficiency in Table 1. The result showed that the combination of FOS and RMD
of both ratio had almost similar encapsulation efficiency while RMD microcapsules of both ratio showed significant
(p<0.05) higher than FOS microcapsules. However, FOS microcapsules core-to-wall ratio of 1:2 showed significant
(p<0.05) higher than core-to-wall ratio of 1:1. While the combination of FOS and RMD showed significant (p<0.05) the
highest of encapsulation efficiency. The effect of hydrocolloid properties of FOS protective mechanism of the cells
when the cells were exposed to high temperature during spray drying. The molecules of hydrophilic carbohydrate may
be accumulated into the cells cause prevent proteins of the cells denature. The hydrogen bonds formation ensured
stable tertiary structure of the proteins in the lack of water (Leslie, Israeli, Lighthart, Crowe, & Crowe, 1995)
Encapsulation of Lactobacillus sp. 3C2-10 significant (p<0.05) higher survival than non-encapsulated free cells after
exposure to SGJ (pH2) 60 min and follow SIJ at 37°C for up to 240 min (data not showed). The survival of
encapsulated ratio 1:2 significant (p<0.05) higher survival than ratio 1:1 for all wall materials. The viability of
encapsulated ratio 1:2 after exposure to SGJ at 37°C for 30 min was decreased by approximately 1.5-1.7 log cfu/g
and the viability of encapsulated ratio 1:1 was decreased by approximately 2.0-2.30 log cfu/g. However, the viability of
each encapsulated by FOS, RMD and the combination of FOS with RMD at the same ratio showed non- significant
different (p<0.05). Viability was observed for RMD, FOS and the combination encapsulated cells, indicating the

123
moderate protection. This may be due to the dissolution effect of wall material (Crittenden & Playne, 1996) causes
some released the cells when suspended in gastric and intestinal juice.
The spray dried Lactobacillus sp. 3C2-10 microcapsules were analyzed by FTIR to obtain spectral
information for each wall material data not showed. The spectra of all wall material had fingerprint region of 1,200 to
800 cm-1 region the general of carbohydrates. The result showed for all wall materials had strong absorptions at 1,026
cm-1 represent glucose and 990 cm-1 represent fructose. The Lactobacillus sp. 3C2-10 after spray drying (storage 0
day) showed higher spectral absorption than storage at 4 °C for 60 days, storage at 25 °C for 60 days and storage at
40 °C for 60 days is minimal, respectively. While the spectral bands of amides I and II (1,650 to 1540 cm -1) increases
relative to reduced carbohydrates. This may be fatty acids such as acetic acid, propionic acid and butyric acid which
are produced by Lactobacillus sp. strain 3C2-10.
The cell viability during storage depend on moisture content and storage temperature. The effect of wall
materials on stability of encapsulated Lactobacillus sp. strain 3C2-10 not showed. The survival rate of encapsulated
cells accorded first-order kinetics. The results at the temperature of 4°C showed lower rate constant than the
temperature of 25°C. Considering that core-to-wall ratio of 1:2 showed higher rate constant than ratio of 1:1.
Conclusion
The effect of heat adaption on cell viability of Lactobacillus sp. strain 3C2-10 in MRS broth, at the temperature of 47°
C for 15 min was the optimal for heat adaptation. The combination of FOS and RMD after spray drying showed the
highest encapsulation efficiency about 91%. Finally the best storage temperature at 4 °C with the wall material,
combination of both at ratio of 1:2.
Acknowledgement
This study was supported by Suranaree University of Technology, Thailand.
References
Anekella, K., and Orsat, V. 2013. Optimization of microencapsulation of probiotics in raspberry juice by spray drying.
Food Science and Technology 50(1) 17-24.
Bourdichon, F., Casaregola, and Farrokh, C. 2012. Food fermentations: microorganisms with technological beneficial
use. International journal of food microbiology 154(3) 87-97.
Corcoran, B. and Ross, R. 2004. Comparative survival of probiotic lactobacilli spray‐dried in the presence of prebiotic
substances. Journal of applied microbiology 96(5) 1024-1039.
Ibrahim, M., Alaam, M., El-Haes, H., Jalbout, A. F., & Leon, A. d. 2006. Analysis of the structure and vibrational
spectra of glucose and fructose. Ecletica quimica, 31(3), 15-21.
Kang, C.-H., Jeon, H., Shin, Y., Kwon, Y.-J., & So, J.-S. 2015. Heat adaptation improves viability of L. lactis HE-1
after heat stress. Food Science and Biotechnology 24(5) 1823-1827.
Kent, R. M., & Doherty, S. B. 2014. Microencapsulation using milk and pea proteins to improve microbiological
quality. Food research international 64 567-576.
Nawong, S. 2015. Isolation and selection of potential probiotic lactic acid bacteria from cassava pulp for cholesterol
lowering property.
Rajam, R., & Anandharamakrishnan, C. 2015. Microencapsulation of Lactobacillus plantarum (MTCC 5422) with
fructooligosaccharide as wall material by spray drying. Food Science and Technology 60(2) 773-780.
Teixeira, P., Castro, H., & Kirby, R. 1994. Inducible thermotolerance in Lactobacillus bulgaricus. Letters in Applied
Microbiology 18(4) 218-221.

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 IFRC 2017: 044-029 Food Processing and Post-Harvest Technology

Influence of Maturity Stage of Rhizomes on the Physicochemical and Sensory Properties of Ginger (Zingiber
officinale Roscoe) Powder

Rabang, J.C.T. and *Castillo-Israel, K.A.T.

Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines Los
Baños, Laguna, 4031

Abstract

Ginger (Zingiber officinale Roscoe) powder was produced using different maturities of the rhizomes and evaluated for
quality in order to determine the maturity stage of rhizomes which will yield the best quality powder for application as
a food ingredient.The maturity stages used were: mature (10-12 months), half-mature (6-9 months), and immature (3-
5 months) ginger rhizomes. The study was conducted using several tests classified as production yield comparison,
physicochemical analysis, solubility tests, and sensory evaluation. The production yield of rhizomes were determined
prior to and after sieving. Results showed that the more mature the rhizome utilized, the higher was the production
yield prior to sieving, and the lower was the production yield after sieving. Results of physico-chemical analyses
showed the increasing moisture content, crude fat, and crude fiber of ginger powders as maturity of rhizomes
increased. Solubility tests were performed in terms of Water Solubility Index (WSI), Water Absorption Index (WAI),
Water Holding Capacity (WHC), and Solubility in terms of time. Results showed that the more mature the rhizome
utilized in the production process, the higher WAI and WHC, while WSI and solubility were lower. The different ginger
powders were applied as an ingredient in ginger cookie, ginger tea, and ginger soup. Results of the sensory
evaluation of the food products showed that the half-mature rhizome was the most acceptable among the ginger
powders. Taking all parameters and tests into account, it was determined that the best maturity for ginger powder
production was at half-mature stage (6-9 months).

Keywords: ginger powder, ginger rhizomes, water absorption index, rhizome maturity
*Corresponding author’s email: kcisrael@up.edu.ph

Introduction

Ginger (Zingiber officinale Roscoe) is a popular crop in Southeast Asia, well known not only for its flavor but also for
various therapeutic properties. Ginger contains about 82.6% moisture and phytochemical compounds such as
oleoresin and essential oils that give ginger its pungent and characteristic odor. These essential oils (n-gingerol, n-
shogaol, zingerone, etc) have beneficial effects (stimulation of the digestion, high antioxidant properties and
alleviation of chronic inflammatory diseases) that are now exploited in the food and pharmaceutical industry. In the
food industry, ginger is used to produce functional foods: foods that have a specific health function. Ginger is
traditionally consumed fresh as a spice, however, certain process are now applied to ginger to give more variety to
the diet and to improve its shelf-life. Products such as ginger syrup, ginger chutney, ginger bar, ginger paste, and
ginger powder are also available. Dried or dehydrated ginger products are also some of the shelf-stable forms which
commonly have a moisture content of 6-10% (Yeh et al. 2014; Nampoothiri et al., 2012; Hasan et al., 2012). In this
study, various powders were prepared from rhizomes at different maturity stages, and compared for their physico-
chemical and sensory properties to determine the best maturity stage of ginger rhizome for ginger powder production.

Material and Methods

1.1. Experimental Set-up
The best maturity stage of rhizome was determined using mature (10-12 months), half-mature (6-9 months), and
immature (3-5 months) ginger rhizomes. The different batches of ginger powder produced were tested for %yield,
125
physicochemical characteristics, and solubility characteristics. The ginger powders were applied to ginger cookie,
ginger tea, and ginger soup, and were tested for sensory properties.

1.2. Proximate analysis was done according to the AOAC Standard Methods including moisture content, fat, fiber and
water activity. WSI, WAI and solubility were measured using the method used by Phoungchandang and Sertwasana
(2009). WHC was determined by placing 2.5g of sample and 30mL distilled water at 30oC in a centrifuge tube, and
then allowing the mixture to stand for 30 minutes. The mixture was then centrifuged for 10 minutes. The supernatant
of the mixture was decanted and discarded. The remaining wet powder was then weighed in an analytical balance
with 0.0001 capacity.

1.3. Sensory evaluation for acceptability, quality, and the flavor intensity attributes of the ginger powder as applied to
ginger cookies, ginger tea, and ginger soup were measured Quality Scoring, wherein the panelists rated the intensity
of the properties of the samples on a 6-point hedonic scale.

1.4. All analyses were done in triplicates and the data were reported as mean ± SD and were analyzed using one-
way ANOVA using DNMRT.

Results and Discussion

In the determination of the best maturity stage for ginger powder production, as seen in Table 2, the moisture content
of the ginger powders from the different stages of rhizome maturity increases as the rhizome matures. Yield after
drying is highest for the mature rhizomes, however, after powdering, lowest yield was obtained due to its high fiber
content. This was confirmed by the crude fiber analyses as shown in Table 2. Highest crude fat was also true for the
mature rhizomes. This is due to the increasing essential oil and oleoresin content of the ginger rhizomes as they
mature(Phoungchandang and Sertwasana, 2010), which dictates the extent of their pungency or their gingery aroma.
Since ginger powders with high level of pungency is a characteristic of a good quality powder, it is therefore assumed
that ginger powders from mature rhizomes and half-mature rhizomes will give a more pungent aroma and flavor.
Table 4 shows the decreasing water solubility as rhizomes mature as evidenced by WSI and solubility values. WAI
on the other hand, increases with maturity. WAI and WSI are valuable parameters in the quality testing of powdered
products in terms of their instant capacity to be dissolved in water during rehydration. WAI will determine the amount
of powder that will be saturated quickly and would settle in the bottom, whereas WSI will determine the amount of
powder that will be solubilized and dissolved without forming any lumps (Phoungchandang and Sertwasana, 2010).
This inverse relationship of WAI and WSI of the ginger powders are expected due to the higher fiber content of the
more mature powders. The higher the fiber content of the ginger powders, the less soluble they are, however more
fiber particles are present to bind the water molecules, thereby decreasing WSI and increasing WAI at the same time.
A good quality ginger powder has a higher WSI value than WAI value that would mean a high solubility and low
amount of suspended particulates. WHC is also another quality indicator, good quality ginger powders should have a
relatively low water holding capacity to signify that the powder does not only absorb water, but are soluble. It is also a
relative measure of the product’s emulsification capacity, viscosity, and organoleptic properties (Kneifel, et. al., 1991).
Half-mature and Immature ginger rhizomes have low WHC.

The ginger powders were applied to cookies, tea and soup and sensory evaluations were conducted to determine the
best maturity stage for product applications. In Tables 5-7, generally, for all the products significant differences in
organoleptic properties were observed for the 3 maturity stages of ginger powder. Aroma, color, flavor and
astringency were observed to become stronger with maturity. For all three products, the half-mature rhizomes
obtained significantly higher acceptability scores. Since the general acceptability attribute is the index of the choosing
the appropriate ginger powder in making ginger cookies, ginger tea and ginger soup during product development, it is
therefore concluded that the best ginger powder that can be utilized is the half-mature ginger powders.

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 Table 1. Moisture content and %Yield of ginger powders produced from rhizomes at different stages of maturity of rhizomes.
MOISTURE CONTENT, frb %YIELD(after
MATURITY OF RHIZOMES %YIELD(after drying)
(%) sieving)
Mature (10-12 mos) 10.60±0.06a 27.17 3.18
Half-Mature (6-9 mos) 8.87± 0.00b 23.43 6.50
Immature (3-5 mos) 8.35±0.00c 22.98 8.30

Table 2. Moisture content, crude fat, and crude fiber of ginger powders from rhizomes at different stages of maturity.
MATURITY OF RHIZOMES Crude Fat, frb (%) Crude Fiber, (%)
Mature (10-12 mos) 3.52±0.98a 5.32±0.10a
Half-Mature (6-9 mos) 3.03±0.32a 3.33±0.29b
Immature (3-5 mos) 2.78±0.64a 2.42±0.62c

Table 3. Evaluation of solubility of ginger powders from different stages of maturity of rhizomes.
Water Water Holding
Water Solubility Solubility (s),
Maturity of Rhizomes Absorption Index Capacity (WHC),
Index (WSI) seconds
(WAI) g/g
Mature (10-12 months) 14.02±0.53a 85.15±0.45a 0.37±0.00a 117.82±8.09a
Half-Mature (6-9 months) 16.23±0.29b 83.22±0.19b 0.33±0.00b 52.62±1.29b
Immature (3-5 months) 19.79±0.74c 79.85±0.35c 0.26±0.01c 31.36±0.98c

Table 4. Quality Scoring Analysis of Ginger Cookies

MATURITY
PARAMETERS LEGEND Mature (10-12 Half-Mature (6-9 Immature (3-5
Months) Months) Months)
AROMA 6- Strong ,1- Weak 3.47a 2.07b 1.93b
FLAVOR 6- Strong, 1- Weak 4.20a 3.40b 2.07c
ASTRIGENCY 6- Strong, 1- Weak 4.07a 3.33b 2.07c
AFTERTASTE 6- Strong, 1- Weak 3.47a 2.87b 1.47c
6- Very Acceptable
ACCEPTABILITY 3.07a 3.80b 1.67c
1- Unacceptable

Table 5. Quality Scoring Analysis of Ginger Tea

MATURITY
PARAMETERS LEGEND Mature (10-12 Half-Mature (6-9 Immature (3-5
Months) Months) Months)
COLOR 6- Intense ,1- Light 4.00a 1.40b 1.13b
AROMA 6- Strong , 1- Weak 4.20a 3.13b 1.60c
FLAVOR 6- Strong , 1- Weak 4.73a 3.73b 2.07c
ASTRIGENCY 6- Strong, 1- Weak 4.60a 3.93b 2.40c
CLARITY 6- Clear, 1- Turbid 1.73a 3.47b 4.20c
6- Very Acceptable
ACCEPTABILITY 1.67a 3.73b 2.60c
1- Unacceptable

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 Table 6. Quality Scoring Analysis of Ginger Soup
MATURITY
PARAMETERS LEGEND Mature (10-12 Half-Mature (6-9 Immature (3-5
Months) Months) Months)
COLOR 6- Intense , 1- Light 4.27a 2.87b 1.67c
AROMA 6- Strong , 1- Weak 3.47a 2.13b 1.40c
FLAVOR 6- Strong , 1- Weak 3.87a 2.13b 1.73b
ASTRIGENCY 6- Strong, 1- Weak 3.73a 2.47b 1.47c
CLARITY 6- Clear, 1- Turbid 1.53a 2.40b 3.80c
6- Very Acceptable
ACCEPTABILITY 3.33a 3.80a 1.93b
1- Unacceptable

Conclusion
The best maturity of rhizomes for ginger powder production was determined to be the half-mature stage (6-9 months).
At this stage, the ginger powder produced contains a relatively high amount of essential oil, as expressed by %crude
fat that contributes to the relatively high pungency of the product. The ginger powder produced at this stage of
maturity also has a relatively low fiber content that signifies the relatively high solubility of the powder. In addition, the
production yield of half-mature ginger powders after sieving are also higher compared to the mature ginger powders.
Sensory evaluation of various food products, namely cookies and tea, applied with the ginger powder made with half-
mature rhizomes also showed the significantly highest acceptability among the panelists.

Acknowledgment
We would like to acknowledge CHED-PHERNet for funding this research.

References
Hasan, H. A., Rasheed Raauf, A. M., Abd Razik, B. M., and Rasool Hassan, B. A. 2012. Chemical Composition and
Antimicrobial Activity of the Crude Extracts Isolated from Zingiber Officinale by Different Solvents. Pharmaceut
Anal Acta 3:184.
Kneifel,W., Paquln, P., Abert, T., and Richard, J.P. 1991. Water-Holding Capacity of Proteins with Special Regard to
Milk Proteins and Methodological Aspects-A Review. Journal of Dairy Science Vol. 74, No.7.
Nampoothiri, S. V., Venugopalan, B. J., Sreekumar, A. N. M. 2012. Comparison of Essential Oil Composition of Three
Ginger Cultivars from Sub Himalayan Region. Asian Pacific Journal of Tropical Biomedicine.
Phoungchandang, S., Sertwasana, A., Sanchai, P., Pasuwan, P. 2009. Development of a Small Scale Processing
System for Concentrated Ginger Powders. World Applied Sciences Journal. Vol 6. pp 488-493.
Phoungchandang, S., and Sertwasana, A. 2010. Spray-drying of ginger juice and physicochemical properties of
ginger powders. ScienceAsia. Vol 36. pp 40-45.

128
 IFRC 2017: 047-047 Food Processing and Post-Harvest Technology
Development of an Artificially-Carbonated Fruit Wine Blend from Mango (Mangifera indica L.), pineapple
(Ananas comosus) and Passion Fruit (Passiflora edulis Sims)
1*Zubia C.S., 2Hurtada, W.A. and 1Dizon, E.I.
1Food Science Cluster, University of the Philippines Los Baños, College, Laguna 4031, Philippines
2Institute of Human Nutrition and Food, University of the Philippines Los Baños, College, Laguna 4031, Philippines

Abstract
With the aim of developing an artificially-carbonated fruit wine blend, three locally available fruits from the Philippines
particularly mango (Mangifera indica L.), pineapple (Ananas comosus) and passion fruit (Passiflora edulis Sims) were
fermented individually and the resulting wine product of each was blended at different proportions. Four formulations
were created which were evaluated for physico-chemical, antioxidant, and sensory properties. Physico-chemical
properties evaluated were pH, titratable acidity (TA) total soluble solids (TSS), total sugar, and alcohol content. Total
phenolic content (TPC) was measured by Folin-Ciocalteau Assay and antioxidant activity (free-radical scavenging
activity) was evaluated by DPPH assay. Based on overall acceptability, best wine blend formulation for carbonation
consisted of 50% mango wine, 25% pineapple wine, and 25% passion fruit wine. This formulation exhibited highest
TSS (6.5 oBrix), total sugar content (5.16 mg/mL), total phenolic content (275 mg/L GAE) and antioxidant activity
(45.26% inhibition). It was perceived as the sweetest, least bitter and least sour compared to other treatments
evaluated.
Keywords: fruit wine, antioxidant, carbonation, blending
*Corresponding author’s email: cszubia@up.edu.ph

Introduction
Wine is a highly flavored alcoholic beverage resulting from the interaction of fermentative yeasts and fruit must. In
making wine, grape is most commonly used because it produces high quality wine from its high levels of fermentable
sugars. But non-viniferous fruits and berries can also be used. Though they are considered less noble than grape
wine, their quality can be very high (Tarko et al., 2008). Several studies already investigated the potential of different
fruits in winemaking but no study had been conducted yet on blending different fruit wines. Blending and carbonation
of fruit wines would not only offer new variants to wine but can also improve wine’s quality and consumer
acceptability. Blending is usually done to balance out acids, sugars and color making a better or more complex wine
(Rivard, 2009).
This study aimed to develop an artificially-carbonated wine blend from a combination of locally available fruits that will
serve as an alternative to imported sparkling wines.
Materials and Methods
Fruit wine blend formulation and carbonation
Mango, pineapple and passion fruit were individually fermented and aged following the procedure of Dizon (2010).
They were then blended at different proportions coming up with four formulations. Blends were carbonated using a 1-
liter soda siphon and a soda charger containing 8 grams of CO2.

129
Physico-chemical analyses
The pH and total soluble solids (TSS) were measured using a pH meter (Eutech- pH 510) and hand-held
refractometer (Atago), respectively. Estimation of titratable acidity (TA) (as citric acid) was done by titration. Alcohol
content was determined using an ebulliometer while total sugars were analyzed by phenol-sulfuric acid method
(DuBois et al., 1956). The total phenolic content of each wine sample was estimated by Folin-Ciocalteu method
(Singleton and Rossi, 1965). All tests were done in triplicate.
Antioxidant properties
The total antioxidant activity of the wine was determined by 2,2’ diphenylpicrylhydrazyl (DPPH) assay where 1000 µL
of wine sample was added to 4 mL of 0.004% (w/v) of DPPH in methanol. The solution was then allowed to stand at
room temperature for 60 minutes and absorbance was read at 517nm against a reagent blank. DPPH radical-
scavenging activity was calculated as % Inhibition = [(A0-A1/A0)x100] where A0 is the absorbance without sample
and A1 is the absorbance with samples.
Sensory evaluation
The fruit wine samples were evaluated by 15 panelists familiar with drinking wines using quality scoring on a 7-point
Hedonic scale.
Data analysis
Analysis of Variance (ANOVA) using Microsoft Excel, version 2007 was used to statistically analyze the data at
p<0.05 level of significance. Differences among means where differences in treatments were found significant were
located using Duncan’s New Multiple Range Test (DNMRT).
Results and Discussion
Physico-chemical and antioxidant properties of fruit wine blends
Physico-chemical and antioxidant activities of the different formulations were analyzed (Table 1). These formulations
yielded pH ranging from 3.10 to 3.43 which were all within the recommended pH of 2.8 to 4.0 (Ribéreau-Gayon et al.,
2006). Lowest pH was observed in formulation 2M which mainly consisted of mango wine. The %TA ranged from
0.446% to 0.577% with 2PF exhibiting the highest. In wine, the organic acids present can enhance flavor and add to
palatability of the wine when in acceptable levels. They also aid in the precipitation of pectins and proteins which is
essential to achieve a clear wine.
TSS content varied from 6.1 oBrix to 6.5 oBrix. Highest level of TSS was observed in 2M formulation which also had
the highest total sugar content. The total sugar content in the wine blends ranged from 3.75 mg mL-1 to 5.16 mg mL-1.
Lowest total sugar content was recorded in 2PF. Resulting alcohol content of the formulations were from 12.33% to
13.33%. Highest ethanol content was observed in formulation MPAPF which contained equal portions of the
component wines while 2M had the lowest among the four samples.
In terms of the total phenolic content of the formulations, 2M achieved highest level with 275 mg L -1 gallic acid
equivalent (GAE). Lowest amount was observed in 2PA. Phenolic compounds are a large class of plant secondary
metabolites that are structurally diverse (Cheynier, 2012). They contribute to the sensory qualities of wine mainly on
the color, flavor (odor and taste), astringency and bitterness (Macheix and Fleuriet, 1990). Other than the impact of
phenolics on the sensory properties of food, they are also associated with antioxidant properties.
Antioxidants are compounds that have the ability to scavenge free radicals. Based on the assay conducted, an
increasing trend in the free radical scavenging activity was observed as the portion of mango wine concentration in
the blend increases. 2M blend had the highest free radical scavenging activity with 45.26% inhibition followed by

130
MPAPF formulation. 2PA and 2PF had free radical scavenging activities of 42.67% and 41.27%, respectively which
are not significantly different from each other.
Sensory properties of fruit wine blend formulations
The carbonated fruit wine blends were evaluated in terms of their sensory properties. The mean scores for the
sensory attributes of the blended wines are summarized in Table 2. It was found out that the formulations differed
significantly in terms of color, aroma and overall acceptability. Formulation 2M which was perceived to have the
highest overall acceptability followed by MPAPF which both contained high portions of mango wine. Formulation 2M
also had the lightest color and was evaluated to be the sweetest. It obtained the lowest score for bitterness as well as
for sourness. On the other hand, 2PF had the highest yellow hue and was also found to have highest clarity score. It
had the strongest aroma but was perceived to be the least sweet and most sour formulation. 2PA formulation, which
consisted mostly of pineapple wine exhibited the weakest aroma.
Table 1. Physico-chemical and antioxidant properties of blended fruit wines.
BLEND FORMULATION
PARAMETER
MPAPF 2M 2PA 2PF
pH 3.20b 3.10c 3.43a 3.13bc
TTA, % (citric acid) 0.540b 0.566a 0.446c 0.577a
TSS, oBrix 6.3b 6.5a 6.1b 6.1b
Total Sugar, mg mL-1 4.50b 5.16a 4.10c 3.75c
Alcohol Content (Ethanol), % (v/v) 13.33c 12.53a 12.93b 12.99b
Total Phenolic Content, 256bc 275b 230a 239ac
mg L-1 GAE
Antioxidant Activity, 44.47bc 45.26c 42.67ab 41.27a
% inhibition
1Means within rows followed by the same letter(s) are not significantly different from each other at p<0.05.

Blend Formulation MPAPF=33.33% mango:33.33% pineapple:33.33% passion fruit; 2M= 50% mango:25 % pineapple:25% passion fruit; 2PA=
25% mango:50% pineapple:25% passion fruit; 2PF= 25% mango:25 pineapple:50%passion fruit

Table 2. Mean sensory scores of the different attributes of the different formulations of blended fruit wines.
BLEND FORMULATION
ATTRIBUTE
MPAPF 2M 2PA 2PF
Color 2.00a 1.60a 1.87a 2.80b
Clarity 5.53a 5.00a 5.20a 5.73a
Aroma 4.87b 4.87b 2.87a 5.4b
Sweetness 3.07a 3.2a 2.80a 2.13a
Bitterness 4.13a 3.53a 3.60a 3.60a
Sourness 3.87a 3.67a 3.87a 4.06a
Overall Acceptability 4.73a 5.20ac 4.26c 3.40b
1Means within rows followed by the same letter(s) are not significantly different from each other at P<0.05.

Sensory Scores:
Color = 1- pale yellow, 7-dark yellow
Clarity = 1-very turbid, 7- very clear
Aroma = 1 – weak, 7-very strong
Sweetness = 1- dry, 4-just right, 7- very sweet
Bitterness = 1-bland, 4- just right,7-very bitter
Sourness = 1-bland, 4- just right, 7-very sour
General Acceptability =1- not acceptable, 4-acceptable,7- very acceptable
131
Conclusion
A carbonated fruit wine blend from locally available fruits of acceptable quality was developed. The best formulation
consisting of 50% mango, 25% pineapple and 25% passion fruit wines was found to be of better quality in terms of
antioxidant and sensory properties as compared to other formulations. The right sourness, bitterness and sweetness
of the said formulation created a balance resulting in a high overall acceptability. The sensory quality of wine depends
on the harmony of the different tastes present in wine. The basic flavor of a wine is formed from the balance of the
sweet, sour and bitter taste. The harmony of the different tastes in wine determines its overall sensory quality. One of
these attributes should not mask another excessively.
References
Cheynier V. 2012. Phenolic compounds: from plants to foods. Phytochemistry Reviews. 11(2):153–177.
Dizon E. I. 2010. The art of tropical fruit wine processing. Institute of Food Science and Technology. College of
Agriculture, University of the Philippines Los Baños, College, Laguna. Philippines.
DuBois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. 1956. Colorimetric method for determination of
sugars and related substances. Analytical Chemistry. 28(3):350–356. doi: 10.1021/ac60111a017.
Macheix, J. J., and Fleuriet, A. 1990. Fruit phenolics. CRC Press.
Ribéreau-Gayon, P., Glories, Y., Maujean, A., and Dubourdieu, D. 2006. Handbook of enology Vol. 2: The chemistry
of wine: stabilization and treatments. 2nd ed. John Wiley & Sons Inc. U.S.A.
Rivard, D. 2009. The ultimate fruit winemaker’s guide. 2nd ed. Bacchus Enterprises Ltd.
Singleton, V.L.and Rossi, J.A. 1965. Colorimetry of total phenolics with phosphomolybdic-phospohotungstic acid
reagents. American Journal of Enology and Viticulture 16(1):144-158
Tarko, T., Duda-Chodak, A., Sroka, P. Satora. P. and Jurasz, E. 2008. Physicochemical and antioxidant properties of
selected Polish grape and fruit wines. Acta Scientiarum Polonorum Technologia Alimentaria 7(3):35-45.

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 IFRC 2017: 051-034 Food Processing and Post-Harvest Technology
Development of Vegan Patties for Young Adults as a Source of Calcium Using Tofu and Tempeh

*Neo, Y.P., Au, J.E. and Tan, K.L.

School of Biosciences, Taylor’s University, No 1, Jalan Taylor’s, 47500 Subang Jaya, Selangor, Malaysia

Abstract

Osteoporosis is a global public health burden affecting 200 million people worldwide and causing more than nine
million fragility fracture every year. The current median calcium intake in Malaysian diet was 357 mg per day, which is
only 43% of the recommended nutrient intake (RNI) of calcium and the calcium intake among young adults having to
be lower than other age groups. This study aims to develop a vegan patty that serves as a non-dairy source of
calcium with young adults as the targeted consumer using traditional soyfoods such as tofu and tempeh. A total of 5
vegan patties with different formulations and a meat control were developed that subjected to acceptance tests,
physical and proximate analyses. It was found that calcium content was the highest in vegan patties formulated with
the highest proportion of tofu and lowest proportion of tempeh. Consumer panelists chose vegan patties with 70:30
(tofu:tempeh) formulation as the most well received formulation, and the calcium content for this formulation was
found to be approximately 132.5 mg of calcium. There were no significant differences (P > 0.05) in water activity,
color and chewiness among the patties determined in this study. Diameter shrinkage was inconsistent between
different formulations, whereas cooking loss was found to be generally similar between the patties. Both meat control
and vegan patties with 70:30 (tofu:tempeh) formulation exhibited significantly different (P < 0.05) proximate
compositions from one another. Overall, the acceptance of young adults towards these vegan patties is expected to
bring more exposure on the traditional local cuisine, in addition to improve their status of calcium intake.

Keywords: vegan patties, calcium, tofu, tempeh, product development, young adults.
*Corresponding author’s email: yunping.neo@taylors.edu.my

Introduction
A balanced and healthy diet is one of the keys to good health as it provides sufficient energy and nutrients needed for
survival. Despite the importance of a balanced diet, young adults aged 18 to 30 years old generally do not prioritize
healthy eating due to busy lifestyles (Sharkawi et al., 2014). It was found that many young adults did not meet their
daily recommended intakes of calcium and calcium deficiency in the long run would cause low bone mass,
osteoporosis and bone fractures. Interestingly, daily calcium intake by Malaysian remains low despite increasing
purchase of dairy products. This has implied a need of other platforms to deliver calcium to improve the calcium
status among Malaysian. In this study, tofu (a soy product that is known to be high in calcium) and tempeh (a
fermented soy product that commonly used as meat substitutes) were incorporated into the production of vegan
patties as a source of calcium for young adults.
The aim of this study is to develop a vegan patty that serves as a source of calcium by using traditional soyfoods such
as tofu and tempeh. The objectives of this study are to: a) evaluate the acceptance of the vegan patties among young
adults and b) determine the calcium content and texture of the vegan patties, which were prepared using different
formulations.

Materials and Methods

3.1 Formulations of vegan patties
All food ingredients were obtained from the local market in the Klang valley, Malaysia. Six types of patties were
prepared depending on the addition of varied tofu to tempeh ratio, including a control patty prepared using skinless,
deboned chicken breast. For the production of vegan patties, the chicken breast meat was replaced with tofu and
tempeh at a tofu to tempeh ratio of 100:0, 70:30, 50:50, 30:70, and 0:100. The patties were prepared by mixing 38.2 g
133
of prepared meat or tofu-tempeh blends with 9.5 g of roasted sesame paste, 9.5 g of king oyster mushroom, 9 g of
breadcrumbs, 7.1 g of lemon juice, 6.2 g of honey, 5.5 g of onion and garlic, 4.5 g of soy sauce, 3.5 g of ground
flaxseed, 3 g of balsamic vinegar, 1.8 g of white rice vinegar, 1.2 g of garlic paste and 1 g of brown sugar. Patties
(approximately 90 g/patty) were manually formed using hands to give average dimensions of 8.5 cm (diameter) × 1.7
cm (thickness), wrapped and kept at -22 °C. The patties were then individually cooked by heating approximately 3g of
olive oil in a small frying pan, adding the patties and frying for 5 minutes with turning until thoroughly cooked.

3.2 Diameter shrinkage and cooking loss of each patty were measured using a weighing balance and ruler before and
immediately after cooking. Texture profile analyses of the cooked patties were determined using a computer-
controlled texture analyzer (CT3, Brookfield Engineering Lab, Inc., USA) equipped with a 10 kg load cell. The cooked
patties were cut into 2 cm x 2 cm pieces and texture profile analysis was applied to the patties according to the
method of Hayes et al. (2005). Surface colour measurements of the patties were performed using a colorimeter
(ColorFlex EZ®, Hunter Lab, USA) and water activity was determined using a AquaLab water activity meter. Sensory
evaluation for consumer acceptance testing was done with 138 young adults (50 males, 88 females) using a 9-point
hedonic scale (dislike extremely to like extremely). The acceptance test was approved by the Taylor’s Human Ethics
Committee with the ethics reference number of HEC/2016/SBS/006.

3.3 Proximate analysis (fat, protein, moisture and ash) and calcium content determination were done in triplicate
according to AOAC Standard methods.

3.4 All data were expressed as mean standard deviation and were done in triplicate independent analyses. Data were
analyzed using one-way ANOVA using SPSS version 21 (SPSS Inc., Chicago, USA)

Results and Discussion

The physical properties and sensory attributes scores of the patties are shown in Table 1. The average diameter
shrinkage was inconsistent and varied between 1.22-8.50%. Vegan patties with 100:0 formulations had the highest
diameter shrinkage followed by vegan patties with 50:50,
control, 0:100, 70:30 and 30:70 tofu to tempeh formulations. The inconsistent diameter shrinkage may be due to the
lack of firmness of the patties. Nevertheless, the percentage of cooking loss by weight was more consistent where the
values were found to range from 2.22-3.31%. According to Vittadini et al. (2005), the losses were based on the mass
transfer process during thermal treatment which was highly influenced by cooking procedures, characteristics and
compositions of the patties. Apparently, protein, fat and fiber content were determinant factors for reducing cooking
loss and higher protein content may have increased the fat retention during cooking, hence reduced the cooking loss.
Water activity of the samples ranged from 0.87- 0.93 and were not significantly different (P > 0.05) among all the six
samples. Similarly, there were no significant differences (P > 0.05) for the L*, a* and b* values between all the patties
and the range of the values was found to be 31.50 - 43.63, 8.30 - 9.20 and 16.06 - 20.56 for L*, a* and b*,
respectively.

The sensory analysis showed that vegan patties obtained a lower score in most attributes (appearance, aroma, taste,
texture, aftertaste and overall acceptance) than meat control. The mean score for color was not significantly different
(P > 0.05) for meat control and the vegan patties, but other attributes were significantly different (P < 0.05). However,
the sensory attribute scores of the vegan patties were generally within the range of 5.0 – 6.0, which could be
translated to an overall opinion of between “neither like nor dislike” and “like slightly”.

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 Table 1: Physical properties and sensory attribute score of the patties
Vegan patties (tofu to tempeh ratio)
Physical
properties Control 100:0 70:30 50:50 30:70 0:100
Diameter 4.26±1.04 8.50±3.70 2.50±3.54 6.47±0.83 1.22±1.72 4.22±0.85
shrinkage (%)
Cooking loss 2.27 2.32 3.31 2.76 2.22 3.07
(%)
Aw 0.93±0.00 a 0.91±0.02 a 0.90±0.01 a 0.88±0.02 a 0.88±0.01 a 0.87±0.04 a
Colour:
L* 38.28±2.96a 43.63±6.84a 39.78±3.96a 37.46±2.60a 31.50±5.43a 32.99±5.70a
a* 9.02±0.88a 8.57±0.44a 9.20±0.54a 8.30±0.35a 8.30±0.64a 9.10±1.12a
b* 19.00±0.98a 20.56±2.93a 20.33±1.72a 18.83±2.13a 16.06±3.91a 17.18±5.35a
Sensory:
Colour 6.0±0.1a 5.9±0.1a 5.8±0.1a 5.7±0.1a 5.6±0.1a 5.5±0.1a
Appearance 6.3±0.1a 5.8±0.1b 5.7±0.1b 5.6±0.1b 5.4±0.1b 5.5±0.1b
Aroma 6.7±0.1a 5.6±0.1b 5.8±0.1b 5.6±0.1b 5.6±0.1b 5.7±0.1b
Taste 6.7±0.1a 5.3±0.1bc 5.5±0.2b 5.0±0.1b 4.9±0.1bc 4.5±0.1c
Texture 6.0±0.2a 5.2±0.1bc 5.3±0.1b 4.9±0.1bc 4.8±0.1bc 4.7±0.1c
Aftertaste 6.2±0.1a 5.2±0.1bc 5.5±0.1b 5.1±0.1bcd 4.9±0.2cd 4.6±0.1d
Overall
acceptance 6.6±0.1a 5.4±0.1bc 5.6±0.1b 5.0±0.1bcd 5.0±0.1cd 4.7±0.1d
Acceptance score: 1 = dislike extremely, 9 = like extremely. Different superscript letter in the same row indicate significant differences among samples (P <
0.05).Data are means ± standard deviation.

Table 2 shows the texture profile analysis of the patties. Cohesive and springiness values were found to be ranged
from 0.184 – 0.243 and 0.236 – 0.319, respectively. There was a significant (P < 0.05) difference in cohesiveness
values between 100:0 (0.243 ± 0.026), 50:50 (0.184 ± 0.030) and 30:70 (0.194 ± 0.013) formulations. During the
sensory evaluation, 20 out of 138 sensory panelists commented the texture of the vegan patties were too mushy,
sticky and/or soft. This was supported by the overall lower hardness and chewiness values of all vegan patties as
compared to control despite the increasing content of tempeh in the formulations. This may be the reason which
attributed to the significantly lower (P < 0.05) degree of likeness of the vegan patties than the meat control. Higher
content of calcium was observed in the vegan patties formulations when higher proportion of tofu was used (Table 2).
This might be due to calcium sulphate that is generally found in tofu, which is responsible for soy milk coagulation.

The most preferred vegan patty formulation was chosen to be 70:30, and the proximate composition of the most
preferred formulation was compared with the meat control. Moisture and protein content were found to be higher in
control (48.83% and 10.83%, respectively) as compared to 70:30 formulation (45.81% and 9.42%, respectively),
whereas fat, ash and carbohydrates were lower in control (10.95%, 2.19% and 27.20%, respectively) than 70:30
formulation (12.99%, 2.65% and 29.13%, respectively).

135
 Table 2: Texture profile analysis and calcium content of the patties
Sample Hardness (g) Cohesiveness Springiness Chewiness (mJ) Calcium (mg
(mm) Ca/100g)
Control 2952.7±642.7a 0.201±0.022ab 0.319±0.031a 0.019±0.004a 23.3 ± 1.2e
100:0 841.4±237.3b 0.243±0.026a 0.303±0.027ab 0.007±0.005b 179.0 ± 5.0a
70:30 929.4±301.3b 0.209±0.015ab 0.273±0.016abc 0.004±0.005b 132.5 ± 0.3b
50:50 1117.3±146.9ab 0.184±0.030b 0.236±0.022c 0.005±0.005b 102.9 ± 1.3c
30:70 1097.4±281.0ab 0.194±0.013b 0.260±0.027bc 0.007±0.005b 87.8 ± 1.5d
0:100 948.7±360.9b 0.201±0.042ab 0.257±0.048bc 0.003±0.007b 83.7 ± 0.8d

Different superscript letter in the same column indicate significant differences among samples (P < 0.05).Data are means ± standard deviation.

Conclusion
The formulation of vegan patties was found to influence the acceptance of young adults, where 70:30 tofu to tempeh
ratio was the most liked formulation. The calcium content in vegan patties with 100:0 (tofu:tempeh) formulation
exhibited the highest calcium content followed by vegan patties with 70:30 (tofu:tempeh) formulation. All the vegan
patties were significantly different (P < 0.05) with the meat control patties on several attributes such as proximate
compositions and textural properties. Majority of the vegan patties had several aspects that were similar to the control
patties such as color, and water activity. Sensory evaluation showed that panelists preferred the color, appearance
and aroma of the vegan patties as compared to its taste, texture and aftertaste. The results obtained in this study will
be useful for discovery and generation of new ideas in the development of alternative calcium source using traditional
soyfoods.

Acknowledgements
The authors would like to thank Taylor’s University for financial support.

References
Hayes, J. E., Desmond, E. M., Troy, D. J., Buckley, D. J., and Mehra, R. 2005. The effect of whey protein-enriched
fractions on the physical and sensory properties of frankfurters. Meat Science, 71(2): 238-243.
Sharkawi, I., Mohamed, Z. and Rezai, G. 2014. Healthy eating: the preventive factors among Malaysians. Journal of
Economics, Business and Management, 2(4): 257-261.
Vittadini, E., Rinaldi, M., Chiavaro, E., Barbanti, D. and Massini, R. 2005. The effect of different convection cooking
methods on the instrumental quality and yield of pork Longissimus dorsi. Meat Science, 69(4): 749-756.

136
 IFRC 2017: 054-054 Food Processing and Post-Harvest Technology
Production Process Technology and Its Characteristics of Probiotic Instant Chocolate Drink

*Heny Herawati, Sri Yuliani, Widaningrum, Tatang Hidayat

Indonesia Center for Agricultural Postharvest Research and Development, Jl. Tentara Pelajar No 12, Cimanggu-
Bogor-Indonesia
+62-251-8321762/ +628122025272
Abstract
Indonesia is one of the renowned manufacturers of chocolate in the world. In 2010, Indonesia is the third largest
exporter of cocoa beans world. Indonesia is generally sold in the form of cocoa beans. Based on this potential, the
market opportunity is still very open to increase the added value of products resulting from the processing of cocoa
beans. One of the technologies that can be developed is a technology processing of probiotic instant chocolate drink.
Functional food products that are circulating widely on the market is fermented food products containing probiotics.
Probiotics are live microbes that can affect health by balancing the microbes in the intestine and inhibits the growth of
microbial pathogens. The purpose of this research is to produce a product, characterization as well as the shelf life of
instant chocolate drink probiotic products. The mixture of four types of Lactic acid bacteria (L. Plantarum, L. Casei, B.
Longum and L. Acidopilus) shows the number of colonies that are still quite high at 2.1 x 1013. The number of
bacteria is still showing a high enough value that is equal to 1.4 x 1013, after the spray drying process. This shows
that, the process of heating during the spray drying does not significantly decrease the number of lactic acid bacteria.
Based on the research results, obtained the best formula is the result of instant chocolate drink by the addition of
powdered sugar, invert sugar, full cream milk, cmc, pasta and vanilla as well as the addition of probiotics is the best
treatment. Results mikrosturtur SEM for instant chocolate drink probiotic shows spherical granules stick together.
Treatment of storage at cold temperatures provide stability lactic acid bacteria growth is higher than the normal
temperature storage and high temperatures.

Keywords: technology, production, probiotic, instant, cocoa drink

*Corresponding author’s emaill: herawati_heny@yahoo.com

Introduction
In 2010, Indonesia is the third largest exporter of cocoa beans in the world. Indonesia is generally sold in the form of
cocoa beans. Based on this potential, the market opportunity is still open to increase the added value of products
resulting from the processing of cocoa beans. One of the technologies that can be developed is a technology
processing of probiotic instant chocolate drink. Lactic acid bacteria used as a probiotics bacteria in chocolate
products. Probiotics are dietary supplements in the form of living microbes that benefit humans especially in the
balance of intestinal microflora (Shortt 1999; Fuller 1999). Probiotics are very beneficial to the body because they
show an important physiological role in maintaining the balance of the gastrointestinal microflora to form a unique
ecosystem, where complex interactions work synergistically and antagonally depending on the strains involved, their
amount and metabolic activity (Matilla-Sandholm et Al. 1999). Lactic acid bacteria that are as probiotic in human
digestion are normal intestinal microflora, which consists of Bifidobacteria and Lactobacillus acidophilus (Gomes and
Malcata 1999; Shortt 1999).The instant drink means final product reduce dust, improve liquidity, add rapid solubility,
and reduce hygroscopicity (Kowalska et al. 2011). Reliable measurements method are required for determination of
water state in food. Optimal control to produce the characterized of probiotic instant cocoa drink. The purpose of this
research is to produce a product, characterization as well as the shelf life of instant chocolate drink probiotic products.

Methodology
The mixture of four types of Lactic acid bacteria (L. Plantarum, L. Casei, B. Longum and L. Acidopilus) commercially
from Gajah Mada University, Maltodextrin, Skim Milk, Cocoa, Sugar, Lecitin, CMC, Glucose, Peanut Oil, MRSA,
MRSB, Hexan, NaOH, H3BO3, H2SO4, K2SO4, HgO, HCl. The tools that used included Pan frying, Hotplate,
Magnetic strirer, Ultraturax, Spray Drier, SEM (Scanning Electrone Microscope), glass ware, soxhlet, oven,
refrigerator . Spray Drying of the mixture lactic acid bacteria to get the highest Total Plate Count of Lactic Acid
137
Bacteria. Skim milk is added water and stirred until dissolved. Then in the ultra torax with a speed of 11,000 rpm for 2
minutes and slowly added a culture that has been dissolved in the broth. After homogeneous then in spray drier with
inlet temperature 150 ° C with speed 25 rpm.The process of making instant brown probiotics included: 150 gram
chocolate powder formulation, 350 gram powdered sugar, 14 gram inversion sugar, 0-3,5 gram peanut oil, 1.05 gram
lecithin, 100 grams of skim milk, CMC 0 to 0, 5 grams, 1 gram of salt and vanilla 0.5 gram. The granulation process is
carried out at a temperature of 40-45 ° C with a stirring speed of 15-30 rpm. The instant chocolate beverage powder
obtained, then mixed with the addition of probiotics by 10% which is a combination of Lactic Acid Bacteria. Optimized
the instant cocoa drink to insert the mixture of the dried of Lactic Acid Bacteria. Characterizing the physic-chemical
properties and shelf life of instant chocolate drinks.

Results and discussion

The Characteristics of Lactic Acid Bacteria
The bacteria used were a mixture of bacteria: Lactobacillus plantarum, L. Casei, B. Longum and L.
Acidopilus.The total plate count of lactic acid bacteria with differences treatment, L. Acidopilus 1,1 x 1013, L. Casei
2,3 x 1014, L. Platarum 1,9 x 1012, B. Longum 5,9 x 1010, Mixture of forth culture 2,1 x 1013, spray drying result 2,1 x
1013. Lian et al. (2002) stated that in all treatment of encapsulation materials, spray drying resulted in the reduction of
Bifidobacteria by a population reduction of 1.0-2.0 log / g dry weight. Mosilhey (2003) also reported that spray drying
with various encapsulation materials caused a decrease of L. acidophilus bacteria from 1.0-2.0 log / g dry weight.
Harmayani et al. (2001) conducted spray drying method for preservation of Lactobacillus sp culture obtained via cell
viability from 1011 cfu / ml to 108 cfu / g.To know the characteristic of encapsulation result by using skim milk matrix
with spray drying method, microstructure analysis was done by using SEM (Scanning Electrone Microscope). The
result of SEM as shown in Figure 1.

Figure 1. Results of SEM (Scanning Electrone Microscope) Analysis of Lactic Acid Bacteria Encapsulation (L.
Acidopilus, L. Casei, L. Platarum, B. Longum)

The result of microstructural analysis from mixed lactic acid bacteria encapsulation as the result of Rizqiati
(2006) research, which used the skim milk skeletal ingredient using L. Plantarum bacteria. L. Plantarum
microcapsules are rounded with cracked, uneven surface or deep folds on the surface. The size of the microcapsules
varies, ie about 5-12 μm. As observations made by Charpentier et al. (1998), gum arabic microcapsules, gelatin and
starch-shaped solvents that have been dehydrated. According to Lian et al. (2002), the crack may facilitate the
release of heat from within the particle after drying, causing less heat injury to the microorganism trapped in it.
Threatment of Lactic Acid Bacteria in Instant Chocolate Powder and Drink
The results obtained then carried out the implementation of instant chocolate powder drink. The treatment
used is the manufacture of instant chocolate beverage powder mixed with chocolate powder blend which is
processed by dry mixing process. Treatments include: S21 (2.5 grams of probiotics and 7.5 grams of brown mixed
powder); S22 (5 grams of probiotic and 5 grams of brown mixed powder); S23 (7.5 grams probiotics and 2.5 grams of
brown mixed powder). Based on the results observations obtained results as listed in the following Table.

Table 1. The Influences of Microbial by Lactic Acid Bacteria Concentration Addition Treatments
Sample Treatments
Microbial (cfu/ml)
Cocoa Powder Cocoa Drink
S21 7,9 x 109 2,1 x 108
S22 4,4 x 1010 3,7 x 108
S23 4,1 x 1010 6,0 108
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Chemical Analysis of Control and Probiotic Instant Chocolate Powder
Based on the best formula, then added a mixture of lactic acid bacteria that has been in spray drying by
10%. Based on the results of chemical analysis obtained results as listed in the table below.
Table 2. The Chemical Analysis Result of Control Compare with Probiotic Instant Artificial Chocolate Powder
Content CONTROL PROBIOTIC
Moisture (%) 1,61 1,665
Ash (%) 3,085 2,53
Fat (%) 9,49 8,86
Protein (%) 4,685 5,325
Carbohydrat e(%) 81,13 81,62
Energy(ccal) 428,67 427,52
Moisture content of instant chocolate powder drink is still in accordance with 01-4320-1996 Indonesia
standard traditional beverage powder, where maximum moisture content of 3%. Water content of instant chocolate
powder drink 1.61% and instant probiotic chocolate powder drink of 1.665%. Fat content in the instant powder control
drink has a higher content of 9.49% compared with instant powder chocolate probiotic 8.86%.

Organoleptic Analysis
The potimal results were then used as control and compared with the addition of lactic acid bacteria by 10%.
Based on the results was organoleptic analysis resulted as shown in the table below. Based on the results of
organoleptic analysis showed that there was no significant difference between control and treatment of 10% probiotic
addition. This thus indicates that instant powder and instant chocolate drink is acceptable to panelists.
Table 3. Organoleptic Analysis of Cocoa Powder and Drink
Cocoa Powder Cocoa Drink
Sample Colour Flavour Taste Texture Acceptance Colour Flavour Taste Viscosity Acceptance
Control 3,90 4,13 4,17 3,87 4,00 3,83 4,17 3,90 3,40 3,93
Probiotic 4,13 4,13 4,17 3,90 4,10 4,07 3,83 3,90 3,63 4,00

Shelflife Analysis Probiotic Instant Powder Chocolate

Based on the results of the water content analysis during the storage process showed an increase as shown
in Figure below. A substantial increase occurred in the second week. A sharp increase also occurs in storage
conditions at 45 ° C. Based on SNI 01-4320-1996 standard, maximum water content for traditional beverage powder
is 3%. Thus, based on the results of analysis of water content, probiotic chocolate drinks are still feasible to be
consumed until the eighth week. Increased moisture content is increased in storage of probiotic chocolate drink
products with a storage temperature of 4 ° C. The moisture content of the instant chocolate powder beverage
probiotic has a water content of 2.21% under storage conditions of 4 ° C at 8 weeks. The extent of the damage can
be known through the analysis of free fatty acid (FFA) and tio barbituric acid (TBA) (Herawati 2008). The main
parameters used are the parameters considered to be the most affecting the deterioration of product quality, ie
moisture content, total microbial, and free fatty acid (FFA) content (Utami 2015).
2.5 0.4
Suhu 4°C
Moisture Content (%)

FFA Content (%)

2 0.3 Suhu 25°C

1.5 Suhu 4° Suhu 35°C
Suhu 25°C 0.2
1 Suhu 35°C Suhu 45°

0.5 Suhu 45°C 0.1

0 0
0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9
Shelf Life (Weeks) Shelf Life (Weeks)

Figure 3. Moisture Content and FFA Result Analysis as Shelflife Parameter

139
Based on the results of shelflife analysis based on BAL total parameters obtained results total lactic acid bacteria
dropped dramatically after the second week. The decrease in the total amount of lactic acid bacteria decreased for all
three storage temperature treatments. At 25 ° C storage conditions decreased by about 1.6 x 10 3, while under
storage conditions 35 and 45 ° C, the total lactic acid bacteria decreased to below 10. Meanwhile, at storage
temperature of 4 C indicates the number of lactic acid bacteria more stable.

Conclusion
The number of bacteria is still showing a high Enough value that is equal to 1.4 x 10 13, after the spray drying process.
This shows that, the process of heating during the spray drying does not decrease the amount of lactic acid bacteria.
Based on the research results, obtained the best formula is the result of instant chocolate drink by the addition of
powdered sugar, invert sugar, full cream milk, cmc, pasta and vanilla as well as the addition of probiotics is the best
treatment. Treatment of storage at cold temperatures provide stability of lactic acid bacteria growth.

Aknowledment
This research work is financially supported by ICAPRD (Indonesia Center For Agricultural Postharvest Research and
Development), under Indonesia Agency For Agricultural Research and Development, Indonesia Ministry of
Agriculture.

Refferences
Charpentier, C.A, Gadille, P., Digat, B., Benoit, J.B. 1998. Microencapsulation of Rhizobacteria by spray drying :
formulation and survival studies. J Microencapsulation 15:639–659.
Fuller, R. 1999. Probiotics from animals. Didalam Probiotics: A Critical Review. Editor : G.W. Tannock. Horizon
Scientific Press.
Gomes, A.M.P, Malcata, G.A. 1999. Bifidobacterium ssp. and L. Acidophilus: Biological , technological and
therapeutical properties relevant for use as probiotics. Review. Trend in Food Sci Tech 10:139-157.
Harmayani, E., Ngatirah, Rahayu, E.S., Utami, T. 2001. Ketahanan dan viabilitas probiotik bakteri asam laktat selama
proses pembuatan kultur kering dengan metode freeze dan spray drying. J Tek dan Ind Pangan 12:126-132.
Herawati, H. 2008. Penentuan umur simpan pada produk pangan. Jurnal Litbang Pertanian, 27(4): 124-130.
Lian, W.C., Hsio, H.C., Chou, C.C. 2002. Survival of Bifidobacterium longum after spray drying. Int J Food Microbiol
74:79– 86.
Mattila-Sandholm, T. 1999. Probiotics: towards demonstrating efficacy. Trends in Food Sci and Tech 10:393-399.
Mosilhey, S.H. 2003. Influence of different capsule materials on the physiological properties of microencapsulated
Lactobacillus acidophilus. Institute of Food Technology, Faculty of Agriculture University of Bonn. 153 pages.
Rizqiati, H. 2006. Ketahanan dan viabilitas Lactobacillus p-lantarum yang dienkapsulasi dengan susu skim dan gum
arab setelah pengeringan dan penyimpanan. Tesis Magister Ilmu Pangan, IPB-Bogor.
Shortt, C. 1999. The probiotic century: Historical and current perspectives. Review. Trend Food Sci and Tech 10:
411-417.
Utami, N.M. 2015. Penentuan masa kadaluarsa produk bubur bekatul instan dengan metode accelerated shelf life
test (ASLT) berdasarkan pendekatan arhenius. BIMGI Volume 3 No.2: 1-7. Juli-Desember 2015.

140
 IFRC 2017: 056-041 Food Processing and Post-Harvest Technology
Effects of virgin coconut oil on qualities of low fat pork meatball
*Oonmetta-aree, J.
Food Science and Technology Program, Faculty of Science and Technology,Nakhon Ratchasima Rajabhat
University, Muang, Nakhon Ratchasima, 30000, Thailand
Abstract
Pork meatball is ground pork with back fat and seasoning, which shaped into a small emulsifier ball. It has good
emulsion stability and firmness but it has high fat and calories. Therefore, this research was to evaluate the emulsion
stability, cooking yields, fatty acid composition, texture profile and the shelf life of the low fat meatball (LFM) which
was supplemented with 4%, 6% and 8% virgin coconut oil (VCO) to replacing back fat in developing LFM compare to
the control with 20% pork back fat. LFM with 4% VCO showed the best of emulsion stability: 7.52% total expressible
fluid and 5.36% fat (p≤0.05). It had 34.2502% unsaturated fatty acid and 65.7500% saturated fatty acid, which
contained mainly 26.5718% lauric acid. The control had 60.0406% unsaturated fatty acid and 39.9595% saturated
fatty acid. There were no significant difference in the texture profile and overall acceptability between LFM with
4%VCO and control (p>0.05). At 4ºC, LFM with 4%VCO could stored in vacuum packaging for 4 days, which could
kept longer than the control (2 days).
Keywords: low fat pork meatball, emulsion meatball, virgin coconut oil
*Corresponding author’s email: ojirawan@gmail.com

Introduction
Emulsified pork meatball, namely ‘Luk chin’ in Thailand, is ground pork with back fat and seasoning, which shaped
into a small ball. Nowadays, consumers are interested in healthy foods and high nutrition. Virgin coconut oil (VCO)
reduces total cholesterol, triglycerides and phospholipids (Marina et al., 2009). Low fat meatball is regarded as
healthy products that contain low amounts of fat, additive agents, salt content, which the low fat meatball with VCO is
one of the interesting aspects.
The objective of the study were to evaluate the emulsion stability, cooking yields, fatty acid composition, texture
profile and the shelf life of low fat meatball (LFM) which was supplemented with 4%, 6% and 8% VCO to replacing
back fat in developing LFM compare to the control with 20% pork back fat.
Materials and methods
3.1 Low fat pork meatball (LFM) preparation
Pork meatball ingredients were main mixtures, which composed of pork loin (75.93%), ice (18.98%), salt (1.52%),
tapioca starch (2.28%) and spice mix (1.39%), and back fat pork meatball. Then, the virgin coconut oils (VCO) were
added 4%, 6% and 8% (of pork loin) for replacing 20% pork back fat (Control) in making LFM. Batters were
thoroughly mixed by chopping in the bowl chopper and manually shaped into meatballs and boiled in 60±2ºC, 10 min
and then in 80±2ºC, 15 min. Final products were packed in laminated (Nylon/PE) bags.
3.2 The emulsion stability of batters were determined according to the method of Crehan et al. (2000). The volumes
of total expressible fluid (TEF) and the percentage fat were calculated as follows:
TEF = (weight of centrifuge tube and batter) – (weight of centrifuge tube and pellet)
% TEF = TEF/batter weight x 100
% fat = (weight of crucible + dried supernatant) – (weight of empty crucible)/TEF x 100
3.3 Cooking yields were determined by measuring the difference in the sample weight before and after cooking and
was calculated.
Cooking yields (%) = Weight of meatball after cooking x 100
Weight of meatball before cooking

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3.4 The texture profile analyses (TPA) of pork meatballs were determined using a texture analyzer (Model TA-XT2i,
England). Values for hardness (N), springiness, cohesiveness, and chewiness (N) were determined according to the
method of Bourne (1973).
3.5 Shelf life storage of Pork meatball
LFM with VCO that showed the best results, was selected for studying various physical, microbiological properties,
proximate composition, fatty acid composition and compared to the control. The control and the selected LFM were
packed in vacuum laminated (Nylon/PE) bags, were studied the shelf life storage at refrigerated temperature (4°C).
3.6 Fatty acid composition was determined by using gas liquid chromatography (Agilent technologies 7890 A, United
States). The lipid fraction of pork meatballs were extracted according to Folch et al. (1957).
3.7 Compositional properties (moisture content, fat and protein) of pork meatballs were performed using AOAC
(1995).
3.8 Sensory evaluation of pork meatball (control) and the selected LFM were tested for acceptance by 100 panelists.
A nine-point hedonic scale, in which a score of 1 = dislike extremely, 5 = neither like nor dislike and 9 = like extremely,
was used for evaluation.
3.9 Microorganisms which contaminated in the selected LFM were determined and compare to control. Total viable
counts, E. coli, Staphylococcus aureus, Salmonella sp. and Clostridium perfringens were performed according to
FDA’s Bacteriological Analytical Manual (BAM).
3.10 All experiments were conducted in at least triplications. One-way ANOVA was used and mean comparison was
performed by Duncan’s multiple range test.
Results and Discussion
Total expressible fluid and %fat exhibited similar trends to cooking yield. Increasing the oil content of the LFM caused
the emulsion stability lowering (p≤0.05) (Table 1). The results showed that emulsion stability of the different batters
was affected by the oil contents. High oil pork meatballs had the cooking yield lowering, due to high loss of %fat and
moisture during cooking. Choi et al. (2010) reported that the cooking yield of meat products relies on the cooking time
and temperature, condition of meat emulsion, added level of fat and dietary fiber. Although, the pork meatballs had
different oil contents but texture attribute (hardness, springiness, chewiness and cohesiveness) were not significant
difference (p>0.05) (Table 2). From these results, LFM with 4%VCO was selected to determine the shelf life,
proximate composition and fatty acid composition compare to the control.
Table 3 showed the fatty acid profile of emulsion meatballs. The highest saturated fatty acid was observed in LFM
with 4%VCO, which mainly consist of high lauric acid (26.5718%) and palmitic acid (15.5476%). Lauric acid is a
common acid in coconut oil, which was known as medium chain fatty acid (Handayani et al., 2009). It caused
increasing in high density lipoprotein (HDL).
Proximate analysis (Table 4) indicated that %fat of LFM and control were 11.46 and 24.42, respectively, which fat
level of LFM was 50% of control since Hsu and Yu (2002) reported that 10% plant oils was used to replace 25% pork
back fat in making low-fat Taiwanese meatball. LFM with 4% VCO had more moisture content (62.73%) than control
(56.29%) because its lipid and water could be embedded in the meat matrices to form completing solid emulsion.
However, these differences did not affect their sensory scores on color, odor, taste, texture and overall liking to
statistically significant level (Table 5) (p>0.05).
At day 4, pork meatball (control) was detected total viable count more than LFM with 4%VCO and safety regulation
standard (Table 6). Therefore, the control was able to keep at 4ºC for 2 days. For shelf life of LFM with 4%VCO could
store at 4ºC for 4 days because total plate count were more than safety standard at day 6. Both samples were not
detected Staphylococcus aureus, Clostridium perfringens and Enterbacteriaceae.

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 Table 1 Effects of virgin coconut oil on cooking yield (%) and emulsion stability (%) of meat batters formulated with various virgin coconut oil
contents.
Treatments Cooking yield (%) Emulsion stability (%)
TEF (%) Fat (%)
Control 94.89ab±0.40 9.24b±0.71 7.98b±0.61
T1 95.66a±1.24 7.52c±0.20 5.36c±0.31
T2 94.40b±0.06 10.32a±0.64 9.36a±0.45
T3 94.60ab±1.10 9.88ab±0.45 11.29a±1.34
ab different superscripts in the same column indicate significant differences (p ≤0.05)
Control: Original pork meatball with 20% pork back fat, T1: pork meatball with 4% VCO, T2: pork meatball with 6% VCO, T3: pork meatball with
8% VCO, %TEF: Total expressible fluid separation, %Fat: Fat separation
Table 2 Effects of VCO on the textural attributes of pork meatballs formulated with various VCO contents.
Treatments Hardnessns Springinessns Cohesivenessns Chewinessns
(g/force) (g/force)
Control 219.65±5.09 0.67±0.02 0.53±0.01 44.39±3.11
T1 217.12±4.73 0.65±0.02 0.53±0.01 42.25±1.25
T2 216.67±3.93 0.65±0.01 0.51±0.01 42.06±1.15
T3 215.32±4.18 0.65±0.02 0.52±0.01 42.73±2.15
ns in the same column indicate no significant differences (p >0.05)
Table 3 Fatty acid composition of pork meatballs with 20% back fat (control) and 4% VCO

Fatty acid composition (%norm) Control pork meatball with 4% VCO

Saturatad Fatty acid 39.9595 65.7500
Caproic acid (c6:0) - 0.0501
Capric acid (c10:0) 0.0654 3.1343
Undecanoic acid (c11:0) - 0.0160
Lauric acid (c12:0) 0.2067 26.5718
Tridecanoic acid (c13:0) - 0.0196
Myristic acid (c14:0) 1.5613 10.8051
Behenic acid (c22:0) 0.0192 -
Unsaturatad Fatty acid 60.0406 34.2502
Myristoleic acid (c14:1) 0.0135 -
Elaidic acid (c18:1n9t) - 20.9178
Linoleic acid (c18:2n6c) 18.6977 8.6571

Table 4 Proximate composition of pork meatballs with 20% back fat (control) and 4% VCO
Treatments % Moisture* %Fat* %Protein*
Control 56.29±0.19 24.42±0.44 12.69±0.28
pork meatball with 4% VCO 62.73±0.03 11.46±0.33 17.54±0.08
* in the same row indicate significant differences (p ≤0.05)
Table 5 Sensory evaluation of the pork meatball with 20% pork back fat (control) and 4% VCO.
Treatments Colorns Odorns Tastens Texture ns Overall likingns
Control 7.37±0.85 7.15±0.89 7.12±0.99 6.71±0.97 7.23±0.74
pork meatball 7.75±0.78 7.42±0.90 7.32±1.04 7.11±1.04 7.70±0.82
with 4%VCO
ns in the same column indicate no significant differences (p >0.05)

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 Table 6 Microbiological analysis of the pork meatball (control) and 4% VCO during storage at 4ºC.

Products Day Total plate count S. aureus Enterobacteriaceae C. perfringens

(CFU/g) (0.1/g) (CFU/g) (0.1/g)
Pork 0 <10 ND ND ND
meatball 2 200 ND ND ND
(control) 4 3.5×104 ND 960 ND
pork 0 <10 ND ND ND
meatball 2 <10 ND ND ND
with 4% 4 2×103 ND ND ND
VCO 6 2.8×104 ND 110 ND
ND: Not detect microorganisms in the sample.

Conclusion
Four percent of VCO content was suitable for adding to LFM production that showed the best emulsion stability. It
contained 26.5718% of lauric acid, which was the useful saturated fatty acid, and low fat and high protein. Its shelf life
storage was 4 days at 4ºC, which was longer than the control (2 days). Therefore, LFM with VCO is a choice for
healthy and nutritious food.
Acknowledgement
This research was financially supported by NRCT and NRRU.
References
Bourne, M.C. 1973. Use of the penetrometer for deformation testing of foods. Journal of food science. 38 : 720-721.
Choi, Y. S., Choi, J. H., Han, D. J., Kim, H. Y., Lee, M. A., Jeong, J. H., Chung, H. J., and Kim, C. J. 2010. Effects of
replacing pork back fat with vegetable oil and rice bran fiber on the quality of reduced-fat frankfurters. Meat
Science 84, 557-563.
Handayani, R., Sulistyo, J., and Rahaya, R.D. 2009. Extraction of coconut oil (Cocos nucifera L.) through
fermentation system. Biodiversitas 10(3):151-157.
Hsu, S.Y. and Yu, S.H. 2002. Comparisons on 11 plant oil fat substitutes for low-fat Kung-wans. Journal of food
engineering 51: 215-220.
Marina, A.M., Che Man, Y.B., Nazimah, S.A.H. and Amin, I. 2009. Chemical properties of virgin coconut oil: Journal of
the American Oil Chemists’ Society. 86: 301-307.
Crehan CM, Hughes E, Troy DJ, and Buckley DJ. 2000. Effects of fat level and maltodextrin on the functional
properties of frankfurters formulated with 5%, 12% and 30% fat. Meat Science. 55:463–469.

144
 IFRC 2017: 057-042 Food Processing and Post-Harvest Technology

Rambutan (Nephelium lappaceum Linn) Fruit Processing: Development of Preserve and Ice Cream

*Rivadeneira, J., Juanico, C., Damaso, C., Espiritu, R., Jolejole, T.K., Lanorio, M.C., Parani, M.S., San Juan, H.O.,
Sunico, D.J. and Sumague, M.J.
Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines Los
Baños, College 4031, Laguna, Philippines

Abstract
Value-added products suitable for rambutan (Nephelium lappaceum Linn), a tropical fruit in the Philippines, were
developed and evaluated. Rambutan from local growers was processed into preserve and ice cream. Treatments
were the types of anti-browning agent (sodium metabisulfite and Vitamin E) for the rambutan preserve and the
presence and absence of rambutan nut brittle for the ice cream. Products were evaluated for their sensory properties.
Treatments with higher sensory scores were subjected to physicochemical characterization and microbial analysis.
Results showed that for both preserve and ice cream, at p=0.05, there is no significant difference on the over-all
acceptability of the two treatments. Nutritional analysis showed that rambutan preserve has low minerals, fats, and
protein content but is a good source of carbohydrates. Analysis of total coliform count for both preserve and ice cream
gave negative result.

Keywords: Nephelium lappaceum Linn, Rambutan Processing, Preserve

*Corresponding author’s email: jprivadeneira@up.edu.ph

Introduction
Rambutan (Nephelium lappaceum Linn.) is a tropical fruit tree that belongs to Sapindaceae and is widely distributed
throughout the tropics and warm regions, such as in the Philippines, Malaysia, and Thailand. In the Philippines, this
fruit is represented by about 33 genera and 124 species (Galindo and Loquias, 2016). The fruits are produced in
terminal, loose cluster of 10-13 fruits. They are globose or ovoid, about 4-5 cm long and 2.5-3.7 cm wide and turns
from green to various shades of yellow or red as they ripen. The fruit is surrounded by short to long, soft spines. The
pearly white translucent aril or the edible portion of the fruit varies in flavor from very sweet to distinctly acid, maybe
thin or thick, and maybe dry to very juicy. It adheres to the seed coat which may or may not be easily separated from
the seed. The single seed is oblong or ovoid, flattened, 2.5-3.5 cm long and 1.0-1.5 cm wide (Galindo and Loquias
2016.).

Peeled rambutan fruits are occasionally stewed as dessert. These are canned in syrup on a limited scale. In some
parts of Asia, a preserve is made by first boiling the peeled fruit to separate the flesh from the seeds. After cooling,
the testa is discarded and the seeds are boiled alone until soft. They are combined with the flesh and plenty of sugar
for about 20 minutes, and 3 cloves maybe added before sealing in jars (Morton, 1987). In the Philippines, these can
be consumed similarly as fresh or in excess, peeled and placed in a refrigerator to prolong shelf life. The seeds are
sometimes roasted and eaten although they are reputedly poisonous when raw. There are traces of an alkaloid in the
seed, and the testa contains sapponin and tannin that are known to be bitter and narcotic (Galindo and Loquias,
2016.).

When the rambutan season arrives, its price drops dramatically. Processing of fresh rambutan into new products is
one way to take advantage of increased supply and low prices to have a form of livelihood. Processing can make
rambutan available for household consumption longer.

The objective of this study is to utilize rambutan to innovate or develop food products, thus, increasing the value of
the said commodity.

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Materials and Methods
1. Preparation of materials
Fruits were washed in sodium hypochlorite solution and drained. Peel was removed while flesh and seeds were kept.
Some of the flesh was converted into puree.

2. Preparation of preserve
Three sets of 75 g sugar solutions were prepared. Each set was added with 1.5 g citric acid and dissolved in 75 mL
water. The first set served as the control while the second and third sets were added with 0.5 g sodium metabisulfite
and 60 mg Vitamin E, respectively. All mixtures were heated to boiling for 5 minutes. Rambutan was then arranged in
pre-sterilized glass jars, and prepared syrups were hot-filled to designated treatments. Each set-up underwent
exhaustion for 10 minutes and then sealed, immediately. Upon sealing, processing was done for 10 minutes.

3. Preparation of ice cream

Rambutan seeds were roasted in oven at 320°F until it turns crunchy and light brown in color. Seeds were chopped in
blender. Half cup sugar was dissolved in ¼ cup water, added to the chopped seeds, and then heated to caramelize.
One-eight teaspoon of baking soda was added and then stirred immediately.

Separately, two sets of seven hundred grams rambutan was converted into puree. Three hundred milliliters of
condensed milk was added to each set. Resulting mixture was then added to pre-beaten 250 mL cream and then
blended. The first set served as control while the second set was added with ½ cup rambutan brittle. Ice creams were
packaged in 2.5 oz cups and kept in freezer for at least six hours.

4. Sensory, physico-chemical, and microbiological analyses

Sensory evaluation by quality scoring (maximum value of 9) was carried out for both products using 20 panelists.
Parameters evaluated for the preserve were color, taste, mouthfeel, gumminess, off-flavor, and overall acceptability;
while for the ice cream, parameters were appearance, taste, flavor, sweetness, mouthfeel, and overall acceptability.
Moreover, proximate analysis of preserve was done to calculate for its nutritional properties. Lastly, both products
underwent microbiological testing for total coliform bacteria using serial dilution test (Blodgett, 2010).

Results and Discussion

Sensory analysis using quality scoring was carried out for both products. Table 1 and Table 2 show the sensory
analysis for rambutan preserve and ice cream, respectively. Results showed that at 95% confidence level, there is no
significant difference on the taste, mouthfeel, off-flavor, and general acceptability of the three treatments for rambutan
preserve. Thus, the use of sodium metabisulfite and vitamin E has no negative effect on the products, and may be
used as anti-browning agent. For ice cream, no significant difference was found in the overall acceptability of the two
rambutan ice creams at 95% confidence level. However, samples were found to be significantly different in terms of
appearance and taste. Rambutan ice cream without nut brittle had a higher appearance and taste preference score
than the sample with nut brittle.

Proximate analysis for rambutan preserve was carried out according to AOAC Methods. Table 3 presents nutrition
label calculated from the proximate composition of moisture content, ash, protein, total fat, crude fiber and
carbohydrate in the said product. It was found out the final product was low in minerals but is high in carbohydrate
content, equivalent to 11% of the daily requirement of a Filipino based on a 2,000 caloric diet, making it a good
source of calories. High carbohydrate content of the preserve is due to the amount of sugar added during the
processing.

For the microbiological analysis of rambutan preserve and ice cream, Table 4 shoes that both products were found to
have <3 MPN/g total coliform count.

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 Table 1. Sensory evaluation for rambutan preserve.
Attributes* Samples
Citric acid only (control) With Sodium metabisulfite With Vitamin E
Color 4.70
c
6.10
a
5.30
b

Taste 5.50
a
5.00
a
5.40
a

Mouthfeel 5.50
a
5.10
a
5.70
a

Gumminess 5.50
b
5.50
b
6.30
a

Off-flavor a a a
1.70 2.70 2.40
Overall acceptability 5.60
a
5.20
a
5.30
a

Table 2. Sensory evaluation for rambutan ice cream.

Attribute Sample
With nut brittle Without nut brittle
Appearance b a
5.1 5.3
Taste b a
5.7 6.4
Rambutan flavor a a
5.7 6.1
Sweetness a a
6.2 6.5
Mouthfeel a a
5.3 6.3
Overall Acceptability a a
5.3 6.0

Table 3. Nutrition label for rambutan ice cream.

NUTRITION FACTS
Servings Per Container:
Amount per Serving: 100 g
Calories 130 Calories from Fat 0
%Daily Value*
Total Fat 0g 0
Total Carbohydrates 33g 11
Dietary Fiber <1g 3
Protein 0g 0
*Percent Daily Values are based on a 2,000 calorie diet
Calories per gram: Fat=9, Carbohydrate=4, Protein=4

147
 Table 4. Enumeration of total coliform bacteria in rambutan preserve.
Dilution (Amount of # of (+) tubes (tubes showing gas & growth) / total # of tubes used per dilution
Inoculum per tube in LSB BGLB
ml) 24 hrs 48 hrs 24 hrs 48 hrs
-1
10 0/3 0/3 - -
-2
10 0/3 0/3 - -
-3
10 0/3 0/3 - -
Control*(-) – Sterility NG NG - -
Combination: 0-0-0 = < 3 MPN/g Total Coliform Count

Table 5. Enumeration of total coliform bacteria in rambutan ice cream.

Dilution (Amount of # of (+) tubes (tubes showing gas & growth) / total # of tubes used per dilution
Inoculum per tube in LSB BGLB
ml) 24 hrs 48 hrs 24 hrs 48 hrs
-1
10 0/3 0/3 - -
-2
10 0/3 0/3 - -
-3
10 0/3 0/3 - -
Control*(-) – Sterility NG NG - -
Combination: 0-0-0 = < 3 MPN/g Total Coliform Count

Conclusion
The use of sodium metabisulfite and vitamin E may be used as antibrowning agent for rambutan preserve. Moreover,
preserve is a good source of calories due to its sugar content. For ice cream, the presence of rambutan brittle has the
same acceptability with that of plain rambutan ice cream. Lastly, microbiological analysis of both products showed
very low total coliform count. Good manufacturing practices may further be improved to ensure safety consumption of
the food products.

References
Blodgett, R. 2010. Most probable number for serial dilutions. Bacteriological Analytical Manual. Retrieved on April 3,
2017 from US Food and Drug Administration Website:
https://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm109656.htm
Galindo, R., Loquias, M. 2016. Rambutan production guide. Retrieved on June 9, 2017 from Bureau of Plant Industry
website: http://www.bpi.da.gov.ph/index.php/production-guide/58-mangosteen-2
Morton, J. 1987. Rambutan. In: Fruits of warm climates. Retrieved on June 9, 2017 from Purdue Agriculture:
https://hort.purdue.edu/newcrop/morton/rambutan.html

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 IFRC 2017: 058-043 Food Processing and Post-Harvest Technology
Storage and processing stability of natural food colorant from Philippine wild raspberry (Rubus rosifolius
Linn.)

*Palomeno,Jr., A.M. and Lizardo, R.C.M.

Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines Los
Baños, College, Laguna 4031

Abstract

It has become an interest in many researches to replace synthetic colorants with natural colorants derived from
plants. In the Philippines, one potential source of anthocyanin is the Philippine wild raspberry or ‘Sapinit’ (Rubus
rosifolius Linn.). In this study, the stability of natural food colorant as affected by storage and processing conditions
has been evaluated. The extracted crude pigment was subjected to varying storage conditions: refrigerated (4°C),
ambient (25°C), and incubated (37°C); and processing temperatures: 60°C and 80°C; in combination with pH: 3.0,
5.0, and 7.0. The anthocyanin concentration of the sample was analyzed using pH differential method and measured
spectrophotometrically. Results showed that refrigerating the samples at 4°C was the most effective storage condition
with the highest mean total monomeric concentration of anthocyanin observed after 4 weeks. An 85% retention of
anthocyanin pigments was obtained after 7 days of storage at pH 3 and temperature of 4°C. The highest mean
pigment concentration after processing for 30 minutes was observed at pH 3 for both 60°C and 80°C with 983.56µg/g
and 961.30µg/g, respectively. Maintaining the samples in an acidic condition (pH 3) also helped in stabilizing the
pigments. Aqueous and ethanolic extracts of powdered sapinit also had significant percent scavenging activity with
60.95% and 71.16%. A linear relationship was also observed between the anthocyanin content and antioxidant
activity. 87.10% of the variation in the % scavenging activity was explained by the total monomeric anthocyanin
content of the extracts.

Keywords: Rubus rosifolius Linn., Philippine wild raspberry, natural food colorant, pH differential method, antioxidant
activity.
*Corresponding author’s email: ampalomeno@up.edu.ph

Introduction

The appearance of the food, especially its color, is an important factor that could influence its perceived taste and
acceptability. Synthetic colorants have been widely used in the food industry however, according to Jenie, et al.
(1994), it can be toxic and carcinogenic. Consequently, it has become an interest in many researches to replace
synthetic colorants with natural colorants derived from plants. Philippine Wild Raspberry or ‘Sapinit’ (Rubus rosifolius
Linn.), a high-value crop rich in anthocyanin that is found at Mt. Banahaw of Quezon Province, is a potential source of
natural food colorant because the extract is not only visually appealing but is also beneficial to health.

The objectives of the study are to evaluate the stability of pigments from sapinit when subjected to various storage
and processing conditions and to evaluate the antioxidant activity of the extracts.

Materials and Methods

3.1 Sapinit was purchased at Mt. Banahaw, Lucban, Quezon Province. The washed fruit pieces were dried in a
cabinet dryer at 40°C for 24 hours and pulverized using Shimono® blender.

3.2 The extraction of anthocyanin pigments was done by conventional solvent extraction employing the method of
Amelia et al. (2013).
149
3.3 Total monomeric anthocyanin content was determined spectrophotometrically using pH-differential method
according to AOAC Official Method 2005.02.

3.4 The storage and processing stability of the pigments was analyzed by subjecting the filtered extracts at different
temperatures of storage: refrigerated (4°C), ambient (25°C) and incubated (37°C); pH of 3, 5, and 7; and at
processing temperatures 60 and 80°C up to a period of 30 mins.

3.5 The percent scavenging activity was analyzed using 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) free radical assay.

3.6 Statistical Analysis

All data were statistically analyzed using ANOVA (p < 0.05) and Tukey’s Honest Significance Difference test using the
Statistical Analysis System 9.1.3.

Results and Discussion

For this study, three storage temperatures along with three pH levels were employed in a factorial experiment.
Analysis of variance showed that there is no interaction among the storage time (week), storage temperature and pH.
Meanwhile, only the interaction between storage time (week) and pH was significant. Hence, the main effect of
storage temperature was investigated.
The effect of storage temperature of 4°C (refrigerated condition) is significantly different from storage temperatures of
25°C (ambient condition) and 37°C (incubated condition) as illustrated in Figure 1. At higher storage temperature, a
greater degree of destruction in anthocyanins is observed.
Brouillard, et al. (1989) reported that thermodynamic measurements show that the formation of the chalcone species
from anthocyanin hemiacetal is endothermic (ΔH = 3.7 Kcal/mol); therefore, any rise in temperature favors the
increase of the chalcone form (colorless) present at equilibrium, which was confirmed by each of the samples, since
at 4°C they were more stable than at 25°C and 37°C, given the same pH. Thus, the lower the temperature, the more
stable the extracted pigment will be. Aqueous extracts of sapinit will present greater stability and, in consequence, will
behave best for natural consumption if stored in low temperature condition.
Furthermore, results showed that when stored at 4°C and pH 3, it is possible to achieve 31% pigment retention
throughout the 4 weeks of storage. The extracts kept at the same temperature but with pH 5 retained 21% of the
anthocyanins; whereas, at high temperature of incubation (37°C) at pH 3, pigment loss is significantly higher,
retaining about 10% of the original residual anthocyanin. Incubating the extracts with pH 5 and 7 after four weeks
incurred a negative percent of retention. Retentions of 85% of anthocyanin pigments have been reported after 7 days
of storage at 4°C and with pH 3, while at pH 5, retention was 72% in the same storage temperature.
Many foods which contain anthocyanins are thermally processed prior to consumption. Thermal processing of foods
generally involves heating at temperatures of 50-150°C, depending upon pH of the product and desired shelf life.
Results showed that there was no interaction among the processing time (minutes), processing temperature and pH
and only the interaction between processing temperature and pH was significant. The main effect of processing time
was also examined. As illustrated in Figure 2, the anthocyanin concentration of the sapinit extract after 30 minutes of
heating significantly decreased, regardless of the processing temperature employed.
Results also showed that at the end of the 30-minute heating at 60°C, the highest amount of anthocyanin was
retained, 69%, at pH 3 compared to the other processing temperature and pH conditions. According to Delgado-
Vargas et al. (2000), acidic pH favors the appearance of the colored forms of anthocyanin. In an acidic medium, the
predominant form is that of the flavylium ion, which gives a red color when it is subjected to basic or alkaline pH. The
flavylium ion is susceptible to nucleophilic attack by water, producing, at pH 4.5, the carbinol pseudobase, followed by
chalcone; the two forms are colorless (Hutchings,1999).

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Moreover, analysis of the antioxidant activity of extracts from sapinit fruit using DPPH assay showed that both the
aqueous and ethanolic extracts of powdered sapinit contained significant amounts of antioxidants with 60.95% and
71.16% scavenging activity, respectively.
Table 1 shows the mean total monomeric anthocyanin content and mean % scavenging activity of the different
extracts. Correlation analysis showed 87.10% of the variation in the % scavenging activity can be attributed to the
total monomeric anthocyanin content of the extracts.

Table 1. Anthocyanin content and antioxidant activity of the sapinit extracts.

Sapinit Extract Total anthocyanin content, µg/g Scavenging activity, %

Ethanolic extract of the fresh sapinit 1196.21a >100 a
Aqueous extract of the powdered sapinit 247.53c 60.95 c
Ethanolic extract of the powdered sapinit 450.95b 71.16 b
Means (n=3) followed by the same letter within each column are not significantly different (p<0.05) using Tukey’s HSD test.

180
157.69a
160
concentration, µg/g

140
Anthocyanin

120
100 84.69b
76.67b
80
60
40
20
Refrigerated (4°C) Ambient (25°C) Incubated (37°C)

Figure 1. Anthocyanin concentration of sapinit extract at different storage temperature. Means (n=75) with the same letter are not
significantly different using Tukey’s HSD test at p ≤ 0.05.

1300

1183.5a 1192.7a
1200
Anthocyanin concentration, µg/g

1120.9a
1100

1000 1052.1a 1056.1a

900

800
806.7b
700
5 10 15 20 25 30
processing time, mins

Figure 2. Anthocyanin concentration of sapinit extract at varying processing time. Means (n=36) with the same letter are not
significantly different using Tukey’s HSD test at p ≤ 05.

151
Conclusion
The study had shown that pigments present in sapinit extract maintained a desirable red color at around refrigerated
and ambient storage conditions. The maintenance of this visual physical state, aside from the anthocyanin
concentration, indicates that this crude extract has the potential to be used as a food colorant. Meanwhile, processing
stability test showed that there shorter time periods should be practiced when processing anthocyanin enriched food.
Maintaining the extracts in an acidic condition also helped in stabilizing the pigments. Extracts of powdered sapinit
have significant antioxidant activity. Further analysis of the purification and identification of the pigments present in
the sapinit extract should be carried out in the future.

Acknowledgements
The authors would like to thank the Institute of Food Science and Technology of the University of the Philippines Los
Baños for providing the necessary reagents and laboratory equipment used in the analyses.

References
Amelia, F., Afnani, G.N., Musfiroh, A., Fikriyani, A.N., Ucche, S., Murrukmihadi, M. 2013. Extraction and Stability Test
of Anthocyanin from Buni Fruits (Antidesma Bunius Linn) as an alternative Natural and Safe Food Colorants.
Journal of Food and Pharmaceutical Sciences 1:49-53
Brouillard, R., Mazza, G., Saad, Z., Albrecht-Gray, A. M. and Cheminat, A. 1989. The Copigmentation Reaction of
Anthocyanins: A Microprobe for the Structural Study of Aqueous Solutions. Journal of the American Chemical
Society 111(7): 2604-2610.
Delgado-Vargas, F., Paredes-Lopez, O., Jimenez, A.R. 2000. Natural pigments: carotenoids, anthocyanins, and
betalains — characteristics, biosynthesis, processing, and stability. Critical Reviews in Food Science and
Nutrition 40(3):173–289.
Hutchings, J. 1999. Food Color and Appearance. 1st ed. USA: Springer US, Aspen Publishers Inc.
http://dx.doi.org/10.1007/978-1-4615-2373-4
Jenie, B.S.L., Helianti and Fardiaz, S. 1994. Pemanfaatan Ampas Tahu, Onggokdan Dedakuntuk Produksi Pigmen
Meraholeh Monascus purperus Buletin Teknologidan Industri Pangan 2:22-29

152
 IFRC 2017: 058-095 Food Processing and Post-Harvest Technology
Resistant Starch Content and Physico-Chemical Properties of Flour from ‘Saba’ and ‘Latundan’ Banana
(Musa sp.) Varieties
1 Mendoza, C.A.J.G. and 2*Lizardo, R.C.M.
1Food and Nutrition Research Institute, Department of Science and Technology, Bicutan, Taguig, Philippines
2Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines
Los Baños, College, Laguna 4031 Philippines
Abstract

This study aimed to determine the resistant starch content and physico-chemical properties of Saba and Latundan
flour and to utilize this as a gluten-free alternative for wheat flour-based biscuits. Results showed that Latundan and
Saba flours contain 52-77% of resistant starch which can increase the dietary fiber of baked products. Moreover, the
physical properties of Saba and Latundan flours are significantly different from wheat flour. The values obtained for
the physical properties of banana flour were as follows: swelling power, 7.2-7.8g/g; water solubility index, 1.2-1.8%;
water holding capacity, 2.1-2.g/g; and oil holding capacity, 2.0-2.4%. In addition, the physico-chemical properties of
Saba and Latundan are as follows: pH, 5.5-5.7; total soluble solids, 0.80-0.87 °Brix, and bulk density of 0.74-
0.76g/mL. Chemical compositions of both Saba and Latundan flours are comparable with each other: moisture
content, 8.1-8.5%; crude protein, 3.1-3.4%; crude fat, 0.7-0.8%, crude fiber, 2.9-3.0%; nitrogen free extract, 82.6-
81.0%; ash content, 1.9-3.7%. The high resistant starch observed was reflected in the higher crude fiber content of
the Saba and Latundan flour (2.9- 3.0%) compared to wheat flour (0.30%). Data also showed that there is no
significant difference in the crude fiber content of flour and biscuits. Overall, though there are significant differences in
some sensory attributes, the general acceptability of using 100% Saba and Latundan flour as a gluten-free
alternative raw material to wheat flour is not reduced.

Keywords: Enzyme-resistant starch, Saba banana flour, Latundan banana flour, gluten-free flour
*Corresponding author’s email: rmlizardo@up.edu.ph
Introduction
Dietary fiber, which includes enzyme-resistant starch, plays a major role in determination of health-related diseases
such as obesity, diabetes and gastrointestinal disorders. Green bananas are some of the tropical fruits which contain
resistant starch (RS), specifically Type II, also known as a functional fiber. Moreover, flour from green bananas is
considered gluten-free and may be utilized as a functional ingredient in starch- or flour-based products. Because of
these characteristics, green banana is regarded as one of the promising substitutes to wheat flour (Mota, 2000) and
with the high content of these functional components, regular consumption of green banana flour-based products can
be beneficial to human health (Rodriguez-Ambriz, et al. 2008).
The objectives of this study are to determine the resistant starch content and physico-chemical properties of ‘Saba’
and ‘Latundan’ banana flour and to utilize this as a gluten-free alternative for wheat flour-based biscuits.
Materials and Methods
3.1. Sample preparation
Banana varieties, specifically Musa balbisiana (BBB group), locally called Saba, and Musa paradisiaca (AAB group),
Latundan, which are at the stage 2 of maturity, were purchased from the local markets of Los Baños and San Pablo,

153
respectively. Banana fruits were processed into flour - peeled and immediately soaked in citric acid solution (1g L-1),
cut into 4 mm slices, dried at 50C using cabinet dryer for 6 h and pulverized using commercial grinder to pass a US
80-mesh sieve (0.3 mm) - and stored at 25C in sealed containers for further analysis.
3.2. Physico-chemical, proximate and resistant starch content analysis
Swelling power and solubility index were measured using the method of Leach, et. al.(1959). Water and oil
absorption were determined using the methods of Sathe and Salunke (1981). Bulk density was measured using the
method employed by Aina and Oladele, 2007. pH, TSS and proximate composition were analyzed using AOAC
standard methods. Resistant starch analysis was performed based on the AOAC standard methods with some
modifications.
3.3. Processing of biscuits from banana flour and sensory evaluation
Biscuits were processed using ‘Saba’ and ‘Latundan’ banana flour and wheat flour (control) as the base ingredients.
Sensory characteristics of the biscuits namely color, aroma, hardness, fracturability, crispness, sweetness, off-flavor
and general acceptability were evaluated by 20 laboratory panelists using quality scoring.
3.4. Statistical analysis
All experiments were done in triplicates. The data gathered from the experiments were subjected to the Analysis of
Variance (ANOVA) and results were expressed as mean ± standard deviation.
Results and Discussion
The physico-chemical properties of the banana flour from ‘Saba’ and ‘Latundan’ varieties are shown in Table 1. Saba
and Latundan flour have higher swelling capacity which can be associated with low levels of fat (Table 2) that leads
to formation of amylose-lipid complexes and restricts swelling.
Bainbridge, et.al. (1996), stated that good quality starch with high starch content have low solubility index and high
swelling volume and power. The low values of water solubility index of both Saba and Latundan flours are associated
with the high resistant starch content of the Saba and Latundan flour (Figure 1).
Water and oil absorption capacity are important functional properties of baked products such as doughs and
custards. Water binding capacity indicates the convenience of using flour in foods such as bakery products in which
they require hydration to improve handling characteristics. Compared to the control, wheat flour, green banana flour
has higher water holding capacity (2.1-2.9 g/g). Giami and Alu (1993) stated that water binding capacity values of
1.25 g/g and above is an indication of good bakery material. In addition, oil absorption capacity is useful in flavor
retention, improvement of palatability and extension of shelf-life in bakery products (Adebowal and Lawal, 2004). The
amount of oil absorbed by the products also depends on the physical characteristics, i.e. porosity, which is highly
correlated with the fiber content (Table 2); thus, high fiber content means high oil-binding capacity. Table 1 shows
that the flour from ‘Saba’ and ‘Latundan’ has higher oil absorption, 2.4 and 2.1 g/g, respectively, compared to wheat
flour, which also indicates higher fiber content (Table 2).
The bulk density of ‘Saba’ and ‘Latundan’ flour, 0.74g/mL and 0.76g/mL, are higher than that of wheat flour,
0.68g/mL. This property is influenced by the structure of the starch polymer; a loose structure within the starch
polymer could result in low bulk density (Plaami, 1997).
As shown in Figure 1, Saba and Latundan flour contain significant amount of resistant starch (RS). RS provide better
appearance, texture and mouthfeel than conventional fibers to food products (Martinez-Flores, et.al., 1999). Table 3
shows the results of sensory evaluation wherein the biscuits with ‘Saba’ banana flour have comparable hardness,
fracturability, crispiness and general acceptability with that of wheat flour.

154
 Table 1. Physico-chemical properties of ‘Saba’ and ‘Latundan’ banana flour compared to wheat flour.
Physico-chemical Property ‘Saba’ flour ‘Latundan’ flour Wheat flour
Swelling power, g/g 7.29 ± 0.17a 7.81 ± 0.40a 6.37 ± 0.35a
Water solubility index, % 1.87 ± 0.01 b 1.83 ± 0.01 b 5.03 ± 0.01a
Water holding capacity, g/g 2.19 ± 0.05 b 2.85 ± 0.14 a 1.58 ± 0.13c
Oil holding capacity, g/g 2.42 ± 0.07a 2.08 ± 0.08b 1.89 ± 0.10b
Bulk density, g/ml 0.74 ± 0.00a 0.76 ± 0.01a 0.67 ± 0.02b
pH 5.50 ± 0.10b 5.70 ± 0.00b 6.47 ± 0.15a
Total soluble solids, °Brix 0.80 ± 0.10a 0.87 ± 0.06a 0.50 ± 0.00b
Means (n=3) followed by the same letter within each row are not significantly different (p ≤ 0.05) using Duncan test.

Table 2. Proximate composition of ‘Saba’ and ‘Latundan’ banana flour compared to wheat flour.
Component ‘Saba’ flour ‘Latundan’ flour Wheat flour
Moisture Content, % 8.55 ± 0.08b 8.16 ± 0.06c 10.47 ± 0.16a
Ash, % 1.92 ± 0.17 b 3.73 ± 0.08 c 0.37 ± 0.12a
Crude Protein, % 3.10 ± 0.03 b 3.16 ± 0.63 b 9.52 ± 1.00a
Crude Fiber, % 2.97 ± 0.24 a 3.00 ± 0.07 a 0.31 ± 0.02b
Crude Fat, % 0.84 ± 0.04 b 0.72 ± 0.10 b 1.57 ± 0.48a
Nitrogen-free extract, % 82.62 81.23 77.76
Means (n=3) followed by the same letter within each row are not significantly different (p ≤ 0.05) using Duncan test.

Figure 1. Resistant starch content of ‘Saba’ and ‘Latundan’ banana flour.

Table 3. Sensory properties of wheat-flour based and banana-flour based biscuits.

Mean sensory scores1
Sensory Attributes
Wheat-flour based biscuit Saba-flour based biscuit Latundan-flour based biscuit
Color 1.23 c 5.97 b 7.93 a
Aroma 3.42 c 7.33 a 4.92 b
Hardness 6.30 a 6.18 a 2.75 b
Fracturability 5.64 a 5.46 a 3.16 b
Crispness 7.21 a 7.03 a 3.33 b
Sweetness 5.80 a 5.25a 3.56 b
Off flavor 2.07 b 3.54 a 3.42 a
General Acceptability 7.12 a 6.76 a 4.97 a
1Means (N=20) followed by the same letter within each row are not significantly different (p≤0.05) using Duncan test.

Range of sensory scores: Color: 1- light brown; 10- dark brown; Aroma: 1- weak; 10- strong; Hardness: 1- very soft; 10- very hard;
Fracturability: 1- crumbles; 10- fractures; Crispness: 1- not crisp; 10- crispy; Sweetness: 1- weak; 10- strong; Off-Flavor: 1- weak; 10- strong;
General Acceptability: 1- not acceptable; 10- highly acceptable

155
Conclusion
The physicochemical properties of the banana flour– low water solubility index, high swelling power, high water
holding and oil absorption capacity – are indicative that the flour from ‘Saba’ and ‘Latundan’ has desirable functional
characteristics that can be useful in many food preparations involving both hydration and oil mixing such as in bakery
products. The high fiber and resistant starch contents of these flours from green banana can help improve nutritional
properties as well as the sensory quality, specifically texture, of selected baked products like biscuits.
Acknowledgements
The authors would like to thank UPLB and CHED-PHERNet for the financial support.
References
Adebowale, K.O. and Lawal, O.S. 2004. Comparative study of the functional properties of Bambara groundnut
(Voandzeia subterranean), jack bean (Canavalia ensiformins) and mucuna bean (Mucuna pruriens) flours.
Food Research International 37(4): 355-365.
Aina, J.O., and Oladele, A.K. 2007. Chemical composition and functional properties of flour produced from two
varieties of tigernut (Cyperus esculentus). African Journal of Biotechnology 6 (21): 2473-2476.
AOAC.1980. Official methods of analysis of the Association of Official Analytical Chemists. 13th ed. W. Horwitz
(ed.). Association of Official Analytical Chemists, Washington, D.C.
Bainbridge, Z., Tomlins, K., Wellings, K. and Wesby, A. 1996.(1st ed). Methods of assessing quality
characteristics of non-grain starch staples (Part 3-Laboratory Methods). p 1-70. Clatham, UK: Natural
Resource Institute.
Giami S.Y. and Alu, D.A. 1993. Changes in the composition and certain functional properties of ripening plantain
(Musa spp. AAB group) pulp. Food Chemistry 50: 137-140
Leach, H.W., Cowell, L.D. and Schoch, T.J. 1959. Structure of the starch granule: Swelling and solubility patterns of
various starches. Cereal Chemistry. 1959 (3)6:534–544.
Mota, R.L. 2000. Composition and functional properties of banana flour from different varieties. Journal of Starch/
Staerke 52:63-68.
Plaami, S.P. 1997. Content of dietary fibre in foods and its physiological effects. Food Reviews International 13: 27-
76.
Rodriguez-Ambriz, S.L., Islas-Hernandez, J.J., Agama-Acevedo, F., Tovar, J. and Bello-Perez, L.A. 2008.
Characterization of a fiber-rich powder prepared by liquefaction of unripe banana flour. Food Chemistry 107
(2008): 1515-1521.
Sathe, S.K. and Salunke, D.K. 1981. Analysis of Starch. New Delhi: Mc

156
 IFRC 2017: 063-048 Food Processing and Post-Harvest Technology
Effect of Squid Ink Addition on the Physicochemical Properties and Acceptability of Noodle

1,2Aishah, B., 2Maisarah, K. and 2,3*Fadhilah, J.

1Malaysia Institute of Transport (MITRANS), Universiti Teknologi MARA, Shah Alam, 40450 Selangor, Malaysia
2Faculty of Applied Sciences, Universiti Teknologi MARA, Shah Alam, 40450 Selangor, Malaysia
3Functional Food Research Interest Group, Universiti Teknologi MARA, Shah Alam, 40450 Selangor, Malaysia

Abstract
Noodles are a type of staple food mainly consumed in Asian countries and also internationally recognised. The
ingredients used to make noodles depend on the type of noodles which differs in size, shape, texture and taste. The
main aim of this study is to develop a new variety of noodle with addition of squid ink. Effect on the physicochemical
properties and acceptability of noodle added with 1, 2 and 3 ml of pasteurised squid ink were evaluated and
compared with control (without squid ink added). The results showed that addition of squid ink significantly change
the colour of noodle. The highest +a* (redness) and lowest L* (lightness) and +b* (yellowness) were observed with 3
ml squid ink added noodle. In term of texture, increase in squid ink amount reduces hardness, adhesiveness,
gumminess, chewiness and resilience of the noodle, while no changes were observed for springiness and
cohesiveness attributes. Significant increase in mineral content of Na, Ca and Mg were obtained with the increase
addition of squid ink. Based on sensory evaluation, noodle added with 3 ml squid ink is most preferred with highest
preference score for texture, after taste and overall acceptability. Therefore, utilisation of squid ink in food products
has great potential either as biocolourant and functional ingredient.

Keywords: squid ink, noodle, natural colourant, texture profile

*Corresponding author’s email: fadhi478@salam.uitm.edu.my

Introduction
Cephalopods such as squid, cuttlefish and octopus are one of the major marine food resources for human
consumption. The defining features of cephalopods is the ejection of dark ink for defense which constituted of a
suspension of melanin granules (71.55%) in a viscous colourless medium (28.45%) or melanin-free ink (Neifar et al.,
2009). The composition of crude ink from cuttlefish Sepia officinalis includes 65.54% water, 0.18% lipids, 15.75%
protein, 5.78% ash and 13.75% carbohydrate content with pH of 7.30 (Neifar et al., 2009). The ink also contains
other chemicals such as tyrosinase, dopamine and L-DOPA, and small amounts of free amino acids, including
taurine, aspartic acid, glutamic acid, alanine and lysine (Wang et al., 2014; Nair et al., 2011).

The medicinal value of squid ink has been reported by many researchers. It been revealed that the bioactive
polysaccharides in squid ink could increase antioxidant ability of some tissues including liver, heart, lung and kidney
(Liu & Simon, 2012). Squid ink helped reduce iron deficiency anemia in rats (Naraoka et al., 2000). Some studies
have reported that squid ink possesses a wide range of biological roles as antitumour, anticancer, antioxidant,
antiradiation antiretrovirus and antibacterial properties (Sasaki et al., 2014; Fahmy & Soliman, 2013; Liu et al., 2009;
Lei et al., 2007; Guan et al., 2007; Rajaganapathy et al., 2000, Naraoka et al., 2000). An angiotensin-converting
enzyme purified from squid ink causes dilation of blood vessels, resulting in lower blood pressure which is a potential
treatment for hypertension (Kim et al., 2003). Cephalopod ink has antimicrobial properties against diverse organisms
including human pathogens (Chacko & Patterson, 2005) and antibacterial activity of squid ink has been reported
against biofilm bacteria by Santhana & Murugan (2005).

Commercially available squid ink is derived from cuttlefish which is larger and thick-fleshed relative to the squid and
has been use traditionally in the cuisine of Japan, Italy and Spain. The ink is naturally high in glutamic acid, which
produces the deep, rich flavour associated with umami taste (Marquinet & Inaki, 2001) therefore, suitable for

157
application as natural food flavouring. Currently, processed ink is used as natural food colouring to enhance the
colour of food products (Marquinet & Inaki, 2001). The ink is also a healthy natural ingredients with dietary mineral for
humans (Wang et al., 2014). However, unfortunately there are still very little study done on the application of squid
ink in food products. The objective of this study is to evaluate the effect of squid ink addition on physicochemical
properties and sensory acceptability of noodle.

Materials and Methods

All of the food grade ingredients such as wheat flour, eggs (grade C) and salt for preparation of noodle were
purchased from Giant Hypermarket, Section 7, Shah Alam, Selangor.

Preparation of squid ink

Squid of Loligo spp. was purchased from wet market at Section 6, Shah Alam. The sac was removed and the ink
collected, homogenised to get a better representation of ink sample and then pasteurised at 90 oC for 20 mins to kill
pathogenic microorganisms.

Preparation of noodle
For control noodle, the formulation used were wheat flour (450 g), whole egg (50 g), salt (5 g) and distilled water (50
ml). For squid ink added noodle, similar amount of water was substituted with 1, 2 and 3 ml of pasteurised squid ink
for NS1, NS2 and NS3 noodle samples respectively. Noodle samples were produced by mixing all ingredients in
mixing bowl and kneaded for 15 mins. The dough was rolled to required thickness using pasta making machine
(Deluxe 150mm, Italy) and then slit at 3 mm width. Raw noodles were cooked in boiling water added with salt for 4
mins. The cooked noodles were transferred into cold water and left to drained-off excess water on a metal strainer for
30 mins prior to analysis.

Determination of physicochemical properties and sensory evaluation

Texture profile analysis was conducted using texture analyser (TA.XT2i plus, Stable Micro Systems, UK) attached
with probe (P/36R) with 50 kg load cell and 50% compression. The force-deformation curve obtained was analysed
by the instrument software for textural attributes. Noodle colour was measured using chroma meter (CR400, Konica
Minolta, Japan) based on CIE L*a*b* colour system with standard illuminator of D65, observation angle 2o visual field
and 1 cm pathlengh cuvette. Content of selected minerals were determined by wet digestion and measured using
ICP-OES (Optima 5300 DV, Perkin Elmer Inc., USA). For sensory evaluation, noodle samples (5 g) were boiled
separately in 100 ml of plain chicken flavoured soup as carrier and served at 40±5 ºC. Total of 30 untrained panelists
consisted of staff and students of UiTM Shah Alam were used. Panelist preference was assessed using 9- points
hedonic scale with scale 1 corresponding to ‘extremely dislike’ and 9 ‘extremely like’.

Statistical analysis
All data were expressed as mean ± standard deviation and analysed using Statistical Package for the Social Science
(SPSS Inc., Chicago, Illinois, USA) version 19.0. Significant difference at p≤ 0.05 was evaluated by one-way ANOVA
and Duncan’s new multiple range test.

Results and Discussion

Table 1 shows the physicochemical properties and acceptability score by sensory panelists of noodle added with
squid ink compared to control noodle.

158
 Table 1: Physicochemical properties of noodle added with squid ink and control noodle
Chroma Control NS1 NS2 NS3
L* 58.49 ± 0.03a 55.25 ± 0.01b 53.24 ± 0.04c 50.50 ± 0.23d
+a* 0.14 ± 0.01c 0.18 ± 0.01b 0.17 ± 0.01b 0.19 ± 0.01a
+b* 8.07 ± 0.08a 5.24 ± 0.10b 4.67 ± 0.10c 3.91 ± 0.04d
Texture attribute
Hardness (g) 453.83 ± 14.27a 415.37 ± 6.12b 372.94 ± 15.51c 309.56 ± 6.09d
Adhesiveness (g/s) -0.49 ± 0.08a -0.51± 0.09a -0.86 ± 0.12b -1.39 ± 0.05c
Springiness 0.78 ± 0.02b 0.87 ± 0.03a 0.84 ± 0.02a 0.85 ± 0.02a
Cohesiveness 0.87 ± 0.02a 0.75 ± 0.05b 0.73 ±0.03b 0.71 ± 0.04b
Gumminess (g) 413.57 ± 12.57a 378.03 ± 8.16b 347.07 ± 13.08c 310.02 ± 13.04d
Chewiness (g) 569.69 ± 18.96a 494.06 ± 4.13b 435.97 ± 10.57c 329.01 ± 13.97d
Resilience 0.85 ± 0.05a 0.52 ± 0.02bc 0.47 ± 0.02c 0.55 ± 0.04d
Mineral content (%)
Na 0.97 ± 0.02d 1.05 ± 0.03c 1.52 ± 0.02b 1.77 ± 0.05a
Ca 0.76 ± 0.02d 0.85 ± 0.05c 1.01 ± 0.03b 1.23 ± 0.04a
Mg 0.13 ± 0.02d 0.35 ± 0.04c 0.75 ± 0.05b 0.99 ± 0.04a
Sensory Attribute
Appearance 6.60 ± 1.16a 6.37 ± 1.15a 6.78 ± 1.25a 6.20 ± 1.78a
Colour 6.87 ± 1.22a 6.20 ± 0.92b 7.26 ± 0.91a 6.88 ± 1.18a
After taste 6.77 ± 0.77b 6.80 ± 0.83b 6.80 ± 0.84b 7.40 ± 1.07a
Texture 6.30 ± 1.31b 6.67 ± 0.92b 6.63 ± 0.96b 7.70 ± 0.87a
Overall accceptability 6.30 ± 1.32b 6.67 ± 0.92b 6.63 ± 0.96b 7.50 ± 1.04a
Values are means ± standard deviation (n=5)
Means within same row having different letters showed significant difference (p<0.05).

Colour is one of the major quality attributes of noodle along with flavour and texture. An increase in darker colour of
noodle samples can be directly observed with increase addition of the black squid ink. The values of L* and b*
decreases while a* increases with increase amount of squid ink added. Texture plays an important role in
determining the quality of noodle. Significant decrease in hardness, gumminess, chewiness and resilience were
observed with the increase amount of squid ink added. A decrease of 32% in hardness, 25% in gumminess, 42% in
chewiness and 35% in resilience attributes were obtained between control and NS3 noodle. These indicates that
squid ink addition affects the texture in which it becomes much softer and easier to chew. No significant difference
was observed for adhesiveness between control and NS1 noodle but further addition showed significant increase for
NS2 and NS3. The more negative the value of adhesiveness, the stickier the sample becomes. For cohesiveness
and springiness, significant difference was observed between control and NS1 however no significant difference was
observed among the squid ink added noodles. This shows that the increase amount of squid ink does not affect
cohesiveness and springiness of the noodle and therefore indicates no further extent of deformation and destruction
on noodle texture. It is known that squid ink has considerable amount of minerals similar to other marine animals. As
expected, addition of squid ink contributes to the increase of mineral contents in noodles. Addition of only 1 ml of
squid ink compared to control showed an increase of 8.2% in Na, 11.8% in Ca and 169.2% in Mg content. For a
newly developed product, knowledge on the consumer acceptability is an essential part in ensuring the marketability
of the product prior to launching. The results showed no significant difference in preference for appearance and
colour of noodles added with squid ink compared to control. However, better acceptability was shown by panelists for
after taste, texture and overall acceptability between control and NS3 but no significant increase was observed for
NS1 and NS2. The increase amount of squid ink of 3 ml is probably the detectable level that influence preference
score by panelists due to the savoury umami taste imparted by glutamate present in squid ink.

159
Conclusion
This study has shown that addition of squid ink in noodle significantly affects the colour, texture and nutritional value
in terms of mineral content. Although the improved noodle has noticeable darker colour but it showed better
acceptability score. This indicates a good commercialisation potential for squid ink as functional food ingredient.
However, further investigation are still needed to look into the stability of squid ink towards different processing and
storage conditions practice by the food industry.

Acknowledgements
The authors would like to thank laboratory staffs of Food Technology Programme, UiTM Shah Alam for their
assistance and Faculty of Applied Sciences for financial support and facilities provided.

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Marquinet, A. and Inaki, J. (2001). Process for producing a food colorant, colorant thus obtained and uses Thereof.
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Nair, J.R., Pillai, D., Joseph, S.M., Gomathi, P., Senan, P.V. and Sherief, P.M. (2011). Cephalopod research and
bioactive substances. Indian Journal Marine Science, 40, 13–27.
Naraoka, T., Chung, H.S., Uchisawa, H., Sasaki, J. and Matsue, H. (2000). Tyrosinase activity in antitumor
compounds of squid ink. Food Science and Technology Research, 6, 171–175.
Neifar, A., Ben Rebah, F., Gargouri, A., & Abdelmouleh, A. (2009) Physicochemical characterization of Sepia
officinalis ink and the effects of storage conditions on the coagulation process. Journal of the Marine
Biological Association of the United Kingdom, 89(4), 803-807.
Rajaganapathy, J., Thyagarajam, S.P. and Edward, J.K. (2000). Study on cephalopod's ink for anti-retroviral activity.
Indian Journal Experiment Biology, 38, 519– 520.
Santhana, R.M. and Murugan, A. (2005). Potential antimicrobial activity of marine molluscs from southeast coast of
India against 40 biofilm bacteria. Journal Shellfish Research, 24, 243-251.
Sasaki, J., Ishita, K.,Takaya, Y., Uchiswa, H. and Matsue, H. (2014). Antitumor on activity of squid ink. Journal of
Nutrional Science and Vitaminology, 43, 455– 461.
Wang, F.R., Xie, Z.G., Ye, X.Q., Deng, S.G., Hu,Y.Q., Guo, X. and Chen, S.G.(2014) Effectiveness of treatment of
iron deficiency anemia in rats with squid ink melanin. Food and Function, 5, 123–128.

160
 IFRC 2017: 066-084 Food Processing and Post-Harvest Technology
Effect of Extraction Temperature, pH, and Time on Pectin Yield of Katmon
(Dillenia philippinensis Rolfe)

*Belan, D.L. and Israel, K.A.C.

1Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines
Los Baños, College, Laguna, 4031, Philippines

Abstract

Utilization of indigenous neglected crops may play an important role in achieving food security and reduction of food
waste. This research reports on the acid extraction of pectin from whole katmon (Dillenia philippinensis Rolfe) fruit for
possible food applications. This aimed to determine the effect of temperature, pH and time on the pectin yield of
katmon. Acid extraction of pectin revealed that the yield is dependent on the extraction temperature, pH and
extraction time. A decreasing pectin yield was observed with increasing pH where the maximum yield
(2.5132+0.2049% fresh weight) was obtained at pH 1.5 at 80°C for 2 hours. Same trend was observed with
increasing extraction time where a maximum yield of 2.9502+0.6339% was obtained after 1 hour at 80°C and pH 1.5.
The yield was also found to be highest at this temperature and is low at very low and very high extraction
temperatures. The pectin content of katmon was found to be comparable to the pectin content of orange (2.34-
2.38%) and lemon (2.80-2.99%) expressed against the fresh weight of the edible portion.

Keywords: Dillenia philippinensis Rolfe, pectin extraction, time, temperature, pH

*Corresponding author’s email: dlbelan@up.edu.ph

Introduction

Katmon (Dillenia philippinensis Rolfe) is a tree endemic to the Philippines. It bears considerable masses of fruit every
year. The fruits have a characteristic sweet-sour and astringent flavor and are traditionally made into jams, jellies and
other sugar concentrates (Sabandar et al., 2017). D. philippinensis species are also used as cough and fever
(Macahig et al., 2011) and hair loss and dandruff (Quattrocchi, 2012) treatment. However, katmon can still be
considered as one of the underutilized fruits in the country. The large volume of the fruit is accounted for its peels
which largely contribute to waste. The goal of this study is to determine other possible uses of katmon fruit such as
pectin extraction.

Pectins are water-soluble plant polymers found in soft plant tissues especially in fruits (Imeson, 1992). They are
employed in the food industry as gelling agents and stabilizers (Rolin, 1993). They are usually used in the
manufacture of sugar concentrates like jams and jellies.

The objective of this study is to determine the effect of extraction pH, time, and temperature, on the pectin yield of
katmon fruit.

Materials and Methods

Raw Materials and Sample Preparation

Katmon fruits were obtained from Camarines Sur, Philippines. The fruits were washed, sliced and homogenized
using a blender. Homogenized samples were stored in a clean container at 4°C until use.

161
Pectin Extraction

Extraction of pectin was conducted by boiling the homogenized sample in acidified water (1:1 w/v) under different
conditions of pH (1.5, 2.5, 3.5), extraction time (1, 2, 3 hr), and extraction temperature (70, 80, 90°C) with continuous
agitation. The temperature was controlled using a hotplate stirrer (Corning Hot Plate PC-101). The pH of the solution
was adjusted using 0.25N HCl and measured using a pH meter (pH600, Milwaukee, USA). The heated solution was
cooled and filtered through a cheesecloth double-layer. The residue was washed with minimal deionized water. The
filtrate was collected and precipitated with 95% analytical grade ethanol (1:2 v/v). The mixture was left to stand
overnight and filtered through a pre-weighed filter paper. The residue was washed with minimal 95% ethanol, air
dried and then weighed.

Experimental Design

The optimal extraction condition was determined using one-factor-at-a-time. The optimum pH was determined first at
80±5°C for a period of 1.5 hours. The optimum extraction time was determined at the optimum pH at 80±5°C. Lastly,
the optimum extraction temperature was determined at the optimum pH and extraction time. Due to limited sample
resources, all treatments were done in duplicates. Analysis of Variance was conducted to determine significant
differences among treatments at 5% significance level. A significant ANOVA was followed by Tukey’s test.

Results and Discussion

Pectin yield of katmon at different conditions of pH, time and temperature is summarized in Table 1. The yield only
differed significantly at different values of pH while it showed no significant difference at different time and
temperature conditions.

Effect of pH on Pectin Yield

Highest pectin yield was obtained pH 1.5 (2.51%) while pH 3.5 gave the lowest (1.57%). A significant increase in
pectin yield was observed when pH was decreased (Figure 1). This increase at lower pH values was due to the
softening of the plant cell walls in acidic conditions making pectin molecules more easily extracted. Also, at low pH
values, pectin recovery is increased when the insoluble pectin constituents are hydrolyzed to soluble pectin (Maran,
2015). The decrease in pectin yield with the increase in pH is because less acidic environments cause the
aggregation of pectin molecules, reducing pectin recovery. This result is in agreement with the results obtained by
Raji et al. (2017) for melon peel pectin and Chaharbaghi et al. (2017) for pistachio green hull pectin.

Table 1. Pectin yield of katmon at different extraction conditions.

pH Time, hr Temperature, °C
1.5 2.5 3.5 1 2 3 70 80 90
%pectin

2.5132 1.6650 1.5740 2.9503 2.4023 2.3155 1.5279 2.9503 2.3616

±0.2049a ±0.1131b ±0.0219b ±0.6339a ±0.4847a ±0.0216a ±0.2839a ±0.6339a ±0.3702a

*means with different superscripts are significantly different (α=0.05)

*yields are expressed in fresh basis
*ANOVA was performed separately for each parameter

162
 Effect of Extraction Time on Pectin Yield
pH vs yield
Maximum pectin yield was achieved after 1hr (2.95%).
3.0000
Yield values varied only slightly and are not significantly
2.5000 different across all levels of extraction time. A general
2.0000 decrease in pectin yield was observed as the extraction
% pectin

1.5000 time was increased (Figure 2). Generally, the time of

1.0000 extraction usually range from 20 to 60 minutes. Also,
0.5000 generally within this range, pectin yield increases with
0.0000 increasing extraction time (Abid et al., 2009). Extraction
0 1 2 3 4 times greater than this range may either increase or
pH
decrease pectin yield depending on the pectin source.
Pectin yield of melon peels (Raji et al., 2017) and
pistachio green hull (Chaharbaghi et al., 2017) increased
Figure 1. Effect of pH on the pectin yield of further up to 200 and 150 minutes of extraction,
katmon. respectively. However, generally, longer heating time will
cause a reduction in pectin yield. The decrease in pectin
yield as extraction time was increased was due to
time vs yield prolonged heating which promoted the degradation of
3.5000 pectin molecules (Chang et al., 1994). This result is in
3.0000 agreement with the results of obtained by Maran (2015)
2.5000 for durian rind.
% pectin

2.0000
1.5000 Effect of Extraction Temperature on Pectin Yield
1.0000
0.5000 Maximum pectin yield was obtained at 80°C (2.95%).
0.0000 Generally, extraction temperatures range from 80 to
0 1 2 3 4 100°C. Temperatures lower than this range will give
time, hr lower pectin yields since heat aids in the extraction of
pectin from the plant cell wall. On the other hand, higher
temperatures will promote degradation of pectin
Figure 2. Effect of extraction time on the
molecules, also causing lower pectin yields (Graham,
pectin yield of katmon. 1977; Imeson, 1992; Chang et al., 1994). This was the
reason for the observed increase in pectin yield as the
temperature was increased from 70 to 80°C and
temperature vs yield decrease as the temperature was increased from 80 to
3.5000 90°C (Figure 3). This result is in agreement with the
3.0000 results for orange peels obtained by Abid et al. (2009)
2.5000 and Hosseini et al. (2016) and for durian rind obtained by
% pectin

2.0000 Maran (2015).

1.5000
1.0000
0.5000
0.0000
0 20 40 60 80 100
temperature, °C

Figure 3. Effect of extraction temperature

on the pectin yield of katmon.

163
Summary and Conclusion

When the effect of pH, extraction time and temperature were studied, it was found out that highest yield of pectin was
obtained at pH 1.5 at 80°C for 1 hour. These results indicated that pectin extraction from katmon should be done at
lower pH values and intermediate values of time and temperature to prevent pectin degradation.

In order to optimize the extraction conditions, it is recommended that the type of acid, particle size, solute to solvent
ratio be also optimized using response surface methodology and obtain second order models.

References

Abid, H., Hussain, A., Ali, S. and Ali, J. 2009. Technique for optimum extraction of pectin from sour orange peels and
its chemical evaluation. Journal of the Chemical Society of Pakistan Vol.31, No.3: 459-461.
Chaharbaghi, E., Khodaiyan, F. and Hosseini, S. S. 2017. Optimization of pectin extraction from pistachio green hull
as a new source. Carbohydrate Polymers 173: 107-113.
Chang, K.C., Dhurandhar, N., You, X. and Miyamoto, A. 1994. Sunflower head residue pectin extraction as affected
by physical conditions. Journal of Food Science 59:1207-1210.
Graham, H. D. 1977. Food Colloids. The AVI Publishing Company, Inc.: Westport Connecticut, pp. 418-435.
Hosseini, S. S., Khodaiyan, F., Yarmand, M. S. 2016. Aqueous extraction of pectin from sour orange peel and its
preliminary physicochemical properties. International Journal of Biological Macromolecules 82: 920-926.
Imeson, A. 1992. Thickening and Gelling Agents for Food. Chapman & Hall, UK, pp. 124-152.
Macahig, R. A. S., Matsunami, K., Otsuka, H. 2011. Chemical studies on an endemic Philipppine plant, sulfated
glucoside and seco-A-ring triterpenoids from Dillenia philippinensis. Chemical and Pharmaceutical Bulletin 59:
397-401.
Maran, J. P. 2015. Statistical optimization of aqueous extraction of pectin from waste durian rinds. International
Journal of Biological Macromolecules 73: 92-98.
Quattrocchi, U. F. L. S. 2012. CRC World Dictionary of Medicinal and Poisonous Plants. CRC Press, New York, pp.
1407-1408.
Raji, Z., Khodaiyan, F., Rezaei, K., Kiani, H. and Hosseini, S. S. 2017. Extraction optimization and physicochemical
properties of pectin from melon peel. International Journal of Biological Macromolecules 98: 709-716.
Rolin, C. 1993. Pectin. In Whistler, R. L. and BeMiller, J. N. (Eds). Industrial Gums, 3rd ed. New York: Academic
Press.
Sabandar, C. W., Jalil, J., Ahmat, N. and Aladdin, N. 2017. Medicinal uses, chemistry and pharmacology of Dillenia
species (Dilleniaceae). P

164
 IFRC 2017: 080-053 Food Processing and Post-Harvest Technology
Physicochemical Properties of Two Varieties of Rambutan (Nephelium lappaceum L.) Fruit

1Chai, K.F., 2Karim, R., 2Adzahan, N.M., 1Rukayadi, Y. and 1*Ghazali, H.M.

1Department of Food Science and Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia,
43400 UPM, Serdang, Selangor, Malaysia.
²Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM,
Serdang, Selangor, Malaysia.

Abstract
Rambutan (Nephelium lappaceum L.) is an exotic fruit which is native of Southeast Asia. It is normally consumed
fresh, canned or processed. Although many rambutan varieties or clones have been registered, the physicochemical
properties of their fruit pulp, especially the underutilized wild types remained unknown. It is important to study the
characteristics of the fruit prior transforming it into various products. Hence, this study was carried out to determine
the physicochemical properties of fruit pulp from a registered clone (R7) of rambutan and a wild type rambutan.
Results showed that Clone R7 comprised 58.4±2.4 % peel, 22.5±2.1 % pulp and 19.1±3.2 % seed while the wild
type was made up of 55.4±3.4 % peel, 27.5±1.3 % pulp and 17.1±3.1 % seed. The pulp-to-seed ratio of the wild type
(5:3) variety was significantly (p<0.05) higher than that of Clone R7 (5:4). The pH value, titratable acidity and total
soluble solids of Clone R7 and wild type were pH 5.15±0.04 and 4.55±0.17, 0.11±0.01 and 0.27±0.02 % expressed
as citric acid, and 14.17±0.41 and 16.33±0.82 °Brix, respectively. HPLC analysis showed that Clone R7 and wild
type rambutan contained high amounts of sugars which were mainly contributed by sucrose (5.39±1.24 % for Clone
R7 and 7.74±1.17 % for wild type), fructose (2.34±0.34 % for Clone R7 and 2.48±0.21 % for wild type) and glucose
(2.05±0.35 % for Clone R7 and 2.25±0.20 % for wild type). The major organic acid found in the rambutan pulp was
citric acid (0.29±0.06 g/100 g for Clone R7 and 0.53±0.06 g/100 g for wild type), followed by lactic, ascorbic, acetic,
malic and tartaric acids. The present study suggested that the high organic acid contents in the wild type could mask
its sweet flavor and give a more sour taste compared to the Clone R7.

Keywords: Rambutan, Nephelium lappaceum, physicochemical properties, sugars, organic acids.

*Corresponding author’s email: hasanah@upm.edu.my

Introduction
Rambutan (Nephelium lappaceum L.) is a popular tropical fruit from the family Sapindaceae (Wall, 2006). It is closely
related to lychee (Litchi chinesis), pulasan (Nephelium mutabile Blume) and longan (Dimocarpus longan) (Zee,
1993). It is an important commercial crop in Asia and in Malaysia and Thailand, rambutan is industrially processed
into juice, jam, jelly, marmalade and spread. In addition, the fruit is also processed as rambutan stuffed with a chunk
of pineapple and canned in syrup (Sirisompong et al., 2011). However, during rambutan harvest season, the fruit,
especially the wild type, is often wasted as the demand of the fruit is always lower than supply, more so for the wild
type. Excessive supply leads to a severe problem in the community as they gradually ferment and release off-odors.
Hence, in order to reduce wastage, rambutan fruit should be fully utilized by transforming it into various products.
However, prior to the transformation process, it is crucial to have information on the properties of the fruit. Previous
studies were mainly focused on popular clones which have been registered such as Jitlee (Wall, 2006), Rongrien
(Sirichote et al., 2008) and Seechompoo (Thitilertdecha & Rakariyatham, 2011) but not the underutilized wild types.
Hence, the objective of this study was to compare the physicochemical properties of fruit from a registered clone (R7)
of rambutan and a wild type rambutan.

Materials and Methods

3.1. Sample collection and sampling procedures
Fruits from Rambutan Clone R7 and one wild type were collected from the University Agricultural Park, Universiti
Putra Malaysia. Fruits selected were free from blemishes and were of uniform color (red) and size (20.6 g per fruit).

165
3.2. The proportion of different parts of the fruit was determined by weighing the peel, pulp and seed from one kg of
fruit (done in triplicate). Results were expressed as percent and pulp-to-seed ratio.

3.3. pH, titratable acidity and total soluble solids of the fruit were determined according to the method described by
Ranganna (1977).

3.4. The sugar profile and composition of the fruit was determined by a HPLC (Waters Corp., Milford, MA, USA)
using the method described by Hunt et al. (1977) with slight modifications. Once the sugars were extracted from 10 g
of fruit by using 100 mL of 85 % methanol for 30 minutes at 80 °C in a water bath, it was then concentrated by
removal of methanol and making up to 10 mL with deionized water. Then, 20 μL of the extract were injected into the
HPLC connected to a Waters 2414 refractive index (RI) detector. The chromatographic column used was a
Purospher® Star NH2 column (259×4.6 mm, particle size of 5 µm from Merck, Germany) connected to a Purospher®
NH2-18e guard column (4×4 mm I.D from Merck, Germany), thermostated at 35 °C. The eluent used in this analysis
was 80 % degassed HPLC-grade acetonitrile: water and the flow rate was at 1.5 mL/min.

3.5. The organic acid profile and composition of the fruit pulp was determined according to the method described by
Sturm et al. (2003) with slight modifications. The HPLC system was similar to that used for sugar analysis but with a
different detector (Waters 2478 two-channel UV detector). A Purovspher® Star RP18 end-capped column (250×4.6
mm I.D, 5 μm particle size, from Merck, Germany) connected to a Purovspher® RP-18e guard column (4×4 mm I.D
from Merck, Germany) thermostated at 30 °C was used. Diluted 4 mmol H2SO4 was used to elute 15 μL of the
extract at the flow rate of 0.5 mL/min.

3.6. The analytical data were analyzed by one-way analysis of variance followed by Tukey’s test using Minitab v. 16
statistical software (Minitab Inc., Coventry, UK). The results were expressed as mean value ± standard deviation.
Statistical significance differences were considered at the level of p<0.05.

Results and Discussion

The proportion and pulp-to-seed ratio of rambutan Clone R7 and wild type are shown in Table 1. Regardless of
variety, rambutan was made up of 55.4-58.4 % peel, 22.5-27.5 % pulp and 17.1-19.1 % seed. Ng & Thamboo (1967)
found that the rambutan fruit they studied was composed of 47.5 % peel, 46.1 % pulp and 6.3 % seed. There is a
significant difference (p<0.05) in pulp and pulp-to-seed ratio between these two varieties. The pulp-to-seed ratio of
the wild type (5:3) variety was significantly (p<0.05) higher than that of Clone R7 (5:4). Thus, the wild type rambutan
has a higher pulp content compared to Clone R7, and indicates that it could be used in food products requiring a
higher quantity of pulp such as jam.

As shown in Table 1, the pH, titratable acidity and total soluble solids of rambutan Clone R7 were significantly
different (p<0.05) from those of wild type variety. The wild type rambutan has a lower pH (4.55) and higher total
soluble solids (16.33 °Brix) compared to Clone R7. Besides, the titratable acidity of the wild type rambutan was at
least two times higher than that of Clone R7. Lower pH and higher titratable acidity in wild type rambutan show that it
is more acid than Clone R7.

Figure 1 shows the sugar contents of rambutan fruit. The high amounts of sugars in rambutan were mainly
contributed by sucrose (5.39-7.74 %), fructose (2.34-2.48 %) and glucose (2.05-2.25 %). Similar observations were
made by Lee et al. (2013) and Tindall (1994). Wild type rambutan contained a significantly (p<0.05) higher sucrose
content compared to Clone R7 making the wild type rambutan having a slightly sweeter taste.

As depicted in Figure 2, the major organic acid in the rambutan was citric acid which comprised 36.16 % and 56.68
% of the total organic acid in Clone R7 and wild type, respectively. Thus, the citric acid content in wild type was
almost two times higher than that of Clone R7. This corresponds well to their titratable acidity values as shown in
Table 1, indicating that the wild type rambutan is more acidic. Other organic acids (lactic, ascorbic, acetic, malic and

166
tartaric) were present in the rambutan as well. These findings are comparable to those reported by Lee et al. (2013)
and Tindall (1994).

Table 1: Proportion, pulp to seed ratio pH, titratable acidity and total soluble solids of rambutan fruit
Relative percent (%) Pulp-
Titratable
to- Total soluble
Variety pH acidity (as %
Peel Pulp Seed seed solids (°Brix)
citric acid)
ratio
R7 58.4±2.4a 22.5±2.1b 19.1±3.2a 5:4b 5.15±0.04a 0.11±0.01b 14.17±0.41b
Wild
55.4±3.4a 27.5±1.3a 17.1±3.1a 5:3a 4.55±0.17b 0.27±0.02a 16.33±0.82a
type
Mean ± SD values with similar letters within the same column are not significantly different (p>0.05).

Figure 1. Sugar contents of rambutan fruit. Means that do not share a letter in a respective sugar are
significantly different (p<0.05).

Figure 2. Organic acid contents of rambutan fruit. Means that do not share a letter in a respective organic acid
are significantly different (p<0.05).

Conclusion
Rambutan is high in sugar and organic acid contents which allow it to have a sweet and sour taste. However, wild
type rambutan contained higher concentrations of sugars and organic acids compared to Clone R7 which made the
wild type to be sourer and slightly sweeter. Due to the wild type is sour in nature, the food industry could process the
fruit into products such as jam and subsequently, wastage can be reduced.

167
Acknowledgements
The authors would like to thank UPM for the financial support (grant 02-02-12-2049RU) awarded to H.M. Ghazali and
GRF awarded to K.F. Chai.

References
Hunt, D. C., Jackson, P. A., Mortlock, R. E., & Kirk, R. S. 1977. Quantitative determination of sugars in foodstuffs by
High-performance liquid chromatography. Analyst 102: 917–920.
Lee, P., Tan, R., Yu, B., Curran, P., & Liu, S. 2013. Sugars, organic acids, and phenolic acids of exotic seasonable
tropical fruits. Nutrition & Food Science 43(3): 267–276.
Ng, S.K. and Thamboo, S. 1967. Nutrient removal studies on Malaysian fruits – durian and rambutan. Malaysian
Agricultural Journal 46:164-183.
Ranganna, S. 1977. Manual of analysis of fruit and vegetable products. Nova Delhi: Tata McGraw-Hill.
Sirichote, A., Jongpanyalert, B., Srisuwan, L., Chanthachum, S., Pisuchpen, S., & Ooraikul, B. 2008. Effects of
minimal processing on the respiration rate and quality of rambutan cv. “Rong-Rien.” Songklanakarin Journal of
Science and Technology 30(SUPPL. 1): 57–63.
Sirisompong, W., Jirapakkul, W., & Klinkesorn, U. 2011. Response surface optimization and characteristics of
rambutan (Nephelium lappaceum L.) kernel fat by hexane extraction. LWT - Food Science and Technology
44(9): 1946–1951.
Sturm, K., Koron, D., & Stampar, F. 2003. The composition of fruit of different strawberry varieties depending on
maturity stage. Food Chemistry 83(3): 417–422.
Thitilertdecha, N., & Rakariyatham, N. 2011. Phenolic content and free radical scavenging activities in rambutan
during fruit maturation. Scientia Horticulturae 129(2): 247–252.
Tindall, H. D. 1994. Sapindaceous fruits: Botany and horticulture. In J. Janick (Ed), Horticultural Reviews, Vol. 16 p.
143–196. London: John Wiley & Sons.
Wall, M. M. 2006. Ascorbic acid and mineral composition of longan (Dimocarpus longan), lychee (Litchi chinensis)
and rambutan (Nephelium lappaceum) cultivars grown in Hawaii. Journal of Food Composition and Analysis
19(6-7): 655–663.
Zee, F.T. 1993. Rambutan and pilinuts: potential crops of Hawaii. In Janick, J. and Simon, J.E. (Eds). New Crops, p.
461-465. New York: John Wiley and Sons

168
 IFRC 2017: 085-058 Food Processing and Post-Harvest Technology

Comparative study on the quality and storage stability of instant mashed sweet potato [Ipomea batatas (L)
Lam] prepared using three different varieties

Castillo-Israel, K.A.T., Perez, P.R.G. and *Reginio, F.C., Jr.

Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines Los
Baños, College, Laguna, 4031, Philippines
Abstract

Instant mashed sweet potato was prepared using three varieties (purple, white, orange) and analyzed to determine
the influence of variety on the physico-chemical characteristics, sensory properties, and storage stability. Each
variety was made into instant mashed sweet potato, formulated with water and salt (250 mL, 275 mL, 300 mL:0.6 g,
0.7 g, 0.8 g per 100 g sample), and evaluated for sensorial characteristics. The most acceptable formulation from
each variety was analyzed for proximate composition, physical properties, and storage stability at 30°C, which was
conducted at intervals of every 2 weeks for 2 months, using moisture content, pH, total solids, and microbial count as
quality parameters. Results showed that treatments within the three varieties had no significant difference on the
sensory attributes, except for consistency; therefore, the treatments with the lowest amount of water (250 mL) and
salt (0.6 g) per 100 g instant mashed sweet potato, which established acceptable scores, were subjected to quality
and storage stability evaluation. Physical properties, which included bulk density (0.84 g/cm 3), swelling capacity
(27.03%), and rehydration ratio (9.26%), were best in purple variety. On the other hand, white and orange varieties
showed significant effect on the proximate composition. The former was highest in moisture (8.36%), ash (3.37%),
and crude fat (1.95) while the latter was highest in nitrogen free extract (83.13%). In terms of storage stability, all
samples were stable up to 2 months of storage at room temperature with no observed significant difference. The
study has shown that, in general, the variety of sweet potato does not impart significant differences in physico-
chemical and sensory properties as well as storage stability of its instant mashed counterpart.

Keywords: instant mashed sweet potato, sweet potato variety, Ipomea batatas (L) Lam, proximate analysis, storage
stability
*Corresponding author’s email: fcreginio@up.edu.ph

Introduction
Sweet potato [Ipomea batatas (L) Lam], a tuberous rooted perennial plant, is one of the most important crops
worldwide aside from wheat, rice, maize, potato and cassava (Truong et al., 2011). Considering its composition and
agricultural potential, sweet potatoes are used as raw material for several industrialized products. One of the
potential products from sweet potato is instant mashed sweet potato. In this study, instant mashed sweet potato was
made from three diverse varieties, and the physico-chemical characteristics, sensory properties, and storage stability
were investigated to compare the varietal effects on the quality properties of the end products.

Materials and Methods

3.1. Instant mashed sweet potato preparation
The sound sweet potato tubers were washed, steamed in a pressure cooker, peeled, grated, dried in a cabinet drier
at 60-65⁰C for 6 hours, and ground using a hammer mill. After milling, sweet potatoes were passed through an 80
mesh sieve. The resulting instant mashed sweet potato from three varieties, purple, white and orange, were
formulated with varying amounts of water and salt (250ml, 275ml, 300ml water: 0.6g, 0.7g, 0.8g salt) per 100 g. The
samples were subjected to sensory analysis and the most acceptable formulation in each variety was subjected to
physical, proximate, and storage stability test.

169
3.2. Sensory evaluation
Preliminary analysis was conducted to determine the most acceptable formulation in each variety. The acceptability
of different varieties of sweet potato as a primary component of instant mashed sweet potato was compared using
quality scoring. The attributes, evaluated by 15 laboratory panelists, were color, aroma, texture, flavor, consistency,
appearance, and general acceptability.

3.3. Physical properties

The physical properties such as bulk density (Okezie and Bello, 1998), water absorption capacity (Sathe and
Salunkhe, 1981), swelling capacity (Okaka and Potter, 1977), and rehydration ratio (Gupta et al., 2012) of the three
varieties of sweet potato were evaluated following the previous standard methods with some modifications.

3.4. Proximate analysis

The proximate compositions of instant mashed sweet potato from three different varieties were determined following
the AOAC standard methods (AOAC, 2005).

3.5. Storage stability test

Storage stability of instant mashed sweet potato, produced from the most acceptable formulation upon sensory
evaluation, was determined by monitoring the changes in moisture content, pH, TSS, and microbiological count of the
sample at intervals of every 2 weeks for 2 months.

3.6. Data analysis

One-way ANOVA with Scheffe’s test and LSD were performed to statistically analyze the variations of means of
multiple groups at p≤0.05 using STAR software version 2.0.1. All data were done in triplicates.

Results and Discussion

Among the three varieties of sweet potato, orange variety yielded the highest amount of instant mashed counterpart
(29.38%), followed by purple and white varieties which produced 26.94% and 20.83%, respectively. From each sweet
potato variety, 9 treatments of instant mashed sweet potato were formulated with varying amounts of salt and water.
Sensory evaluation was conducted to determine the most acceptable formulation among the treatments. In general,
no significant difference was observed among the treatments in each variety, except for consistency. Therefore, the
treatments with the lowest water:salt ratio were used for the succeeding analyses. Table 1 shows the comparison of
mean sensory scores of three sweet potato varieties formulated using the lowest water:salt ratio. No significant
difference was found among the samples in color, aroma, texture, and flavor attributes. However, orange and white
varieties showed comparable results in consistency, appearance, and general acceptability. In addition, both samples
were more preferred by the panelists than purple variety.

The results of physical properties of instant mashed sweet potatoes prepared from three varieties are shown in Table
2. Purple variety was found to have the highest swelling capacity among the sweet potato varieties. According to
Ruales et al. (1993), the variation in the swelling capacity indicates the degree of exposure of the internal structure of
the starch present in the flour to the action of water. In terms of bulk density and rehydration ratio, both purple and
white varieties showed comparable values. High bulk density of instant mashed sweet potato indicates heaviness
and offers greater packaging advantage, as a greater quantity may be packed within a constant volume (Fagbemi,
1999). Likewise, all varieties showed high percentage values of water absorption capacity but the difference was not
significant. This could be attributed to the presence of higher amount of carbohydrates (starch) and fiber in sweet
potato.

The high value of water absorption capacity of white variety was reflected in its high moisture content (Table 3).
Moreover, it also had the highest ash and fat contents among the sweet potato varieties. Crude fiber and protein
contents were not significantly different among the samples. For a 100-g serving amount, the instant mashed sweet
potato from three different varieties could contribute a caloric value of approximately 350 kcal.

170
The results of storage stability test on instant mashed sweet potato prepared using three varieties are summarized in
Table 4. The pH of three varieties did not differ from its initial after 6 weeks of storage at room temperature. Likewise,
total soluble solids and moisture contents did not vary through time but decreasing and increasing trends were
observed, respectively. For the microbiological analysis, microbial loads of the three varieties were still below the
allowable limit of 104 cfu/g for yeasts and molds (BAFPS, 2010). Thus, the products were stable up to two months of
storage at room temperature (30°C).

Table 1. Mean scores of instant mashed sweet potato prepared using three varieties.
VARIETY* SENSORY ATTRIBUTE
Color Aroma Texture Flavor Consistency Appearance GA
Purple 3.04a 2.22a 2.61a 2.79a 2.79b 2.60b 2.25b
Orange 2.98a 1.99a 2.34a 2.53 a 3.58ab 3.65a 3.66a
White 2.93a 2.72a 2.79a 2.92a 4.07a 3.37ab 3.50a
N= 45; mean scores within the same column followed by the same superscripts are not significantly different at p≤0.05; GA: general
acceptability.
*Instant mashed sweet potato from three varieties prepared with 250 mL water and 0.6 g salt (per 100 g).
Range of scores: color: 0- light, 6- dark; aroma: 0- very weak, 6- very strong; texture: 0- very fine, 6- very coarse; flavor: 0- very weak, 6- very
strong; consistency: 0- thin, 6- thick; appearance: 0- very unacceptable, 6- very acceptable; general acceptability: 0- very unacceptable, 6- very
acceptable.

Table 2. Mean (±SD) values of physical properties of instant mashed sweet potato prepared using three varieties.
VARIETY* PHYSICAL PROPERTIES
Bulk Density Water Absorption Swelling Capacity Rehydration
(g/cm3) Capacity (%) (%) Ratio (%)
Purple 0.84±0.01a 346.69±7.75a 27.03±1.03a 9.26±0.58a
Orange 0.80±0.01b 355.95±8.75a 26.32±1.00b 6.63±0.75b
White 0.82±0.01ab 396.11±23.21 a 25.00±1.02c 8.29±0.47a
N=3; mean scores within the same column followed by the same superscripts are not significantly different at p≤0.05.
*Instant mashed sweet potato from three varieties prepared with 0.6 g salt per 100 g flour.

Table 3. Mean (±SD) values of proximate composition (%) of instant mashed sweet potato prepared using three varieties.
VARIETY* PROXIMATE COMPOSITION (%)
Moisture Ash Crude Fat Crude Fiber Crude Carbohydrate
Protein
Purple 7.72±0.03b 2.46±0.04b 0.97±0.10b 3.70±1.82a 3.25±1.35a 82.15±0.65ab
Orange 7.45±0.06b 2.30±0.11b 1.80±0.33ab 3.73±0.17a 1.72±0.03a 83.13±0.28a
White 8.36±0.30a 3.37±0.07a 1.95±0.42a 3.67±0.72a 2.36±1.36a 80.46±1.43b
N=3; mean scores within the same column followed by the same superscripts are not significantly different at p≤0.05.
*Instant mashed sweet potato from three varieties prepared with 0.6 g salt (per 100 g).
Table 4. Mean values of pH, TSS (°Brix), and moisture (%) of instant mashed sweet potato prepared using three varieties stored at different
time periods.
VARIETY* pH TSS (°Brix) MC (%)
W Wee Wee Wee Wee Wee Wee Wee Wee Wee Wee Wee
ee k2 k4 k6 k0 k2 k4 k6 k0 k2 k4 k6
k0
Purple 5.9 5.9 5.8 5.8 7.0 7.0 6.0 6.0 7.46 7.45 7.44 7.43
Orange 5.8 5.8 5.8 5.8 10.5 9.0 9.0 9.0 5.33 5.40 5.48 5.50
White 5.7 5.7 5.8 5.8 6.0 5.0 5.0 5.0 8.00 8.10 8.18 8.21
N=12; *instant mashed sweet potato from three varieties prepared with 0.6 g salt (per 100 g).

171
Conclusion
The variations in the data obtained among the instant mashed sweet potatoes prepared from three varieties were
possibly due to maturity of sweet potatoes at harvest, elapsed time between harvesting and processing period,
conditions during processing, and probable environmental factors in areas of cultivation under which the sweet
potatoes were grown. In general, the study has shown that the variety of sweet potato from which instant mashed
sweet potato was prepared does not impart significant differences in physico-chemical composition, sensory
properties, and storage stability of the end products.

Acknowledgements
The authors would like to thank the Institute of Food Science and Technology, College of Agriculture and Food
Science, UPLB for providing the necessary equipment and utensils.

References
[AOAC] Association of Official Analytical Chemist. Official Method of Analysis. 2000. Washington, D.C.
[BAFPS] Bureau of Agriculture and Fisheries Product Standards. 2010. Philippine National Standard: Sweet potato –
Classification and grading. Department of Agriculture.
Fagbemi, T. N. 1999. Effect of blanching and ripening on functional properties of plantain (Musa aab) flour. Plants
Food for Human Nutrition 54: 261-269.
Gupta, M. K., Sehgal, V. K., Singh, A. K. and Singh, R. K. 2012. Effect of process parameters and storage length on
quality of dried cauliflower during storage. Indian Journal of Traditional Knowledge 11(1): 177-184.
Okaka, J. C. and Potter, N. N. 1977. Functional and storage properties of cow pea-wheat flour blends in bread
making. Journal of Food Science 42: 828-833.
Okezie, B. O. and Bello, A. B. 1988. Physicochemical and functional properties of winged bean flour and isolate
compared with soy isolate. Journal of Food Science. 53: 450-454.
Ruales, J., Valencia, S. and Nair, B. 1993. Effect of processing on the physiochemical characteristics of guinea flour
(Chenopodium guinea Wild). Starch 46(1): 13-19.
Sathe, S. K. and Salunkhe, D. K. 1981. Functional properties of great northern bean (Phaseolus vulgaris) proteins:
emulsion, foaming, viscosity and gelation properties. Journal of Food Science. 46: 71-75.
Truong, V. D., Avula, R. Y., Pecota, K. and Yencho, C. G. 2011. Sweet potatoes. In handbook of vegetables and vegetable
processing. N. K. Sinha ed., p. 717-737. USA: Bla

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Potential of Canned and Pouched Adlai (Coix lacryma-jobi L.)- Duck (Anas platyrhynchos L.) Meat Congee as
Emergency Relief Food
*Tapia, M.S.R.C., Horiondo E.J.T., Maghirang, M.C., Peñaflor, L.M., and Reginio, F.C., Jr.
Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines Los
Baños, College, Laguna, 4031, Philippines

Abstract
In response to the challenge of food security during and after natural disasters, the potential of pouched and canned
adlai-duck meat congee to be a suitable emergency relief food was studied through evaluation of its microbial quality
by commercial sterility test, chemical composition through proximate analysis, shelf life using accelerated
temperature method, and sensory analysis through consumer test. Results revealed that the thermally processed
products were commercially sterile indicating that efficient processing was achieved. Based on Q 10 approach, the
estimated shelf life of canned and pouched adlai-duck meat congee were 169 and 287 days, respectively. Proximate
composition of the product showed that it could provide 130 kcal (per 200 g) and contained large proportion of
moisture (83%) and carbohydrates (14%). In addition, a liked moderately consumer rating was obtained in both
samples with retort pouch as the most preferred packaging material. The study has shown that canned and pouched
adlai-duck meat congee are safe for consumption, shelf-stable, and can serve as relief food products at the onset of
emergency.

Keywords: adlai, emergency relief food, commercial sterility, shelf life, proximate analysis, consumer test,
*Corresponding author’s email mctapia@up.edu.ph

Introduction
Philippines is considered as the third most disaster-prone country in the world (Santos, 2016). The negative impact of
the disaster is on the reduction of viable land and water as well as shortage of food supply in affected areas. Food
rations are given to affected areas as part of the immediate disaster relief assistance program. The food ration must
not need further cooking and preparation and it should be eaten directly. Most of the products given to disaster
victims lack variety and nutritional value (Israel and Briones, 2012). Therefore, it is important to find other suitable
food products that can serve as an immediate response at the onset of the emergency, like the formulated adlai-duck
meat congee. The formulation and heat penetration data of adlai-duck meat congee were already done by Genoveso
(2016). However, data regarding its quality properties to be regarded as disaster food were not yet studied.
Therefore, this study evaluated the commercial sterility, proximate composition, shelf life, and consumer acceptability
of pouched and canned adlai-duck meat congee to determine their potential as emergency relief food products.

Materials and Methods

3.1. Preparation of Adlai-Duck Meat Congee
Ingredients were purchased at Los Baños Market, Brgy. Batong Malake, Laguna, Philippines while the utensils and
equipment were obtained from the Institute of Food Science and Technology, UPLB. The water retort used for
thermal processing of the products was acquired from the High Impact Technology Solutions (HITS) Project of the
Department of Science and Technology (DOST).
Formulation and time-temperature of the processing was based on the study of Genoveso (2016).

3.2. Commercial sterility test and proximate analysis were done in accordance to AOAC Standard Methods (2000).

3.3. Shelf life test method was based on combined methodology of Labuza and Schmidl (1985) and Office of
Technology Assessment (2007).

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 3.4. Consumer accept This page is intentionally left blank

ability was assessed using 9-point hedonic scale with 125 panelists, 81 females and 44 males, taken as respondents.

3.5. All data were expressed as mean + standard deviation and were done in triplicates. T-test was done to
determine differences between data.

Results and Discussion

The summary of observations and findings during the commercial sterility test is presented in Table 1. Since the
packaging condition remained the same even after incubation, product appearance and odor were normal, pH was
within the range of low-acid food product, cultural evaluation showed negative growth in aerobic and anaerobic tubes,
and microscopic examination of adlai-duck meat congee showed similar number of occasional cells before and after
incubation (Figure 1), it can therefore be concluded that the product was commercially sterile and efficient processing
was achieved.

Table 2 shows the proximate composition of adlai-duck meat congee packaged in can and pouch. It was observed
that both products had high water content of about 83%. This means that the ready-to-eat food can be consumed
even without any water accompaniment. The computed calorie contents were 63.18 kcal/100g for canned and 64.96
kcal/100g for pouched congee. For a 200-g serving amount, the product could contribute a caloric value of
approximately 130 kcal. This means that the product can be used as an immediate response at the onset of the
emergency when general food distribution systems are not adequate and no other foods are available (WHO, WFP,
UNICEF and UNHCR, 2002).

Using Q10 approach, the estimated shelf life of canned and pouched adlai-duck meat congee were 169 and 287 days,
respectively. pH, water activity and flavor had the highest impact on the acceptability of congee as these quality
parameters exhibited a consistent pattern of change throughout the shelf life monitoring (Table 3). pH decreased
through time due to protein deterioration which releases free ions, such as glutamic acid, aspartic acid, lysine and
methionine (Aronal et al., 2012). Water activity increased during storage due to the the change from amorphous to
crystalline state of food components (Barbosa-Canovas et al., 2008). Whereas, flavor of the duck meat decreased
over time because of the onset of lipid oxidation and accumulation of its by-products that contributed to the
production of off-flavors and unpleasant odor (Jayasena et al., 2013).

Based on the sensory analysis (Table 4), canned and pouched samples were perceived as not significantly different
in all attributes by consumers at different age groups (p≤0.05). The R values of the products’ general acceptability
were found out to be strongly dependent on almost all attributes, especially aftertaste. In addition, most of the age
groups (94 out of 125) had a unanimous preference for retort pouches over tin cans as packaging material for adlai-
duck meat congee.
Table 1. Summarize results of commercial sterility test of canned (A) and pouched (B) adlai-duck meat congee (n=6).
SAMPLE CONDITION OF PRODUCT NUMBER OF POSITIVE NUMBER OF POSITIVE pH RANGE
PACKAGING APPEARANCE AND TUBES IN TRYPTONE TUBES IN MODIFIED PE-2
ODOR BROTH MEDIUM
A Normal Normal 0/2 0/2 6.13 –
6.23
B Normal Normal 0/2 0/2 6.15 –
6.25

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Figure 1. Microscopic examination of smears of canned (A) and pouched (B) adlai-duck meat congee before1 and after2
incubation.

Table 2. Mean (±SD) values of proximate composition (%) of canned (A) and pouched (B) adlai-duck meat congee.
SAMPLE PROXIMATE COMPOSITION (%)
Moisture Ash Crude Fiber Crude Protein Crude Fat Carbohydrates
A 83.33±0.20a 0.93±0.19a 0.56±0.39a 0.38±0.13a 0.51±0.06b 14.28±0.44a
B 84.15±0.02a 0.89±0.18a 0.27±0.24a 0.25±0.00a 1.00±0.06a 13.70±0.14a
N= 6; mean scores within the same column followed by the same superscripts are not significantly different at p≤0.05.

Table 3. Quality properties of canned (A) and pouched (B) adlai-duck meat congee before and after storage at 35, 45, and 55˚C
(n=144).
PARAMETERS BEFORE AFTER STORAGE
STORAGE 35˚C 45˚C 55˚C
A B A B A B A B
pH 5.94 5.92 4.50 4.30 4.49 4.48 4.20 4.17
±0.03 ±0.06 ±0.24 ±0.30 ±0.23 ±0.36 ±0.09 ±0.14
Water Activity 0.92 0.92 0.94 0.96 0.94 0.94 0.95 0.94
±0.00 ±0.00 ±0.01 ±0.02 ±0.01 ±0.01 ±0.01 ±0.01
Flavor 7.00 7.00 6.10 5.40 4.30 4.00 4.00 4.00
±0.00 ±0.00 ±0.74 ±0.52 ±0.82 ±0.67 ±0.67 ±0.67

Table 4. Mean (±SD) scores of sensory attributes of canned (A) and pouched (B) adlai-duck meat congee.
SAMPLE SENSORY ATTRIBUTES
Color Aroma Flavor Texture Aftertaste General
Acceptability
A 6.82±1.32 6.61±1.68 6.71±1.65 6.39±1.78 6.43±1.75 6.76±1.62
B 6.92±1.21 6.69±1.51 6.76±1.80 6.53±1.72 6.46±1.75 6.75±1.59
N= 250; Range of scores: 1 = extremely unacceptable, 9 = extremely acceptable

Conclusion
This study showed that the thermally-processed canned and pouched adlai-duck meat congee were commercially
sterile and safe for consumption. Based on the shelf life study, the canned and pouched samples could be stored for
169 and 287 days, respectively at ambient temperature. The proximate compositions proved that products can be
used as an immediate response at the onset of the emergency due its high water content and caloric value.
Moreover, both canned and pouched samples were liked moderately by consumers. Further analysis on the behavior
of the adlai-duck meat congee using different deteriorating attributes at elevated temperatures is recommended. It is
also suggested to validate the results of the accelerated shelf life test using real time determination.

Acknowledgement

175
 This work was part of the research project supported by the Office of the Vic This page is intentionally left blank

e Chancellor for Research and Extension through University of the Philippines Los Baños Basic Research.

References
[AOAC] Association of Official Analytical Chemist. Official Method of Analysis. 2000. Vol I and II. Washington, D.C.
Aronal, A.P., Huda N. and Ahmad R. 2012. Amino Acid and Fatty Acid Profiles of Peking and Muscovy Duck Meat.
International Journal of Poultry Science. 11(3): 229-236.
Barbosa-Canovas, G., Fontana A., Schmidt S. and Labuza T. 2008. Water Activity in Foods: Fundamentals and
Applications. New York: Wiley-Blackwell. pp. 32-35.
Labuza, T. P. and Schmidl, M. K. 1985. Accelerated shelf Life testing of foods. Food Technology. 39:57-62.
Genoveso, J. 2016. Heat Penetration Test of Canned and Pouched Adlai (Coix lacryma-jobi L.) Duck (Anai
Plaibrbynchos L.) Meat Congee. Laguna Philippines: University of the Philippines Los Banos, BS Thesis.
Israel, D. and Briones, R. 2012. Impacts of Natural Disasters on Agriculture, Food Security, Natural Resources and
Environment in the Philippines. In: Y. Sawada and S. Oum (Eds.). Economic and Welfare Impacts of Disaster
in East Asia and Policy Responses. ERIA Research Project Report 2011-8, Jakarta: ERIA. pp. 553-599.
Jayasena D. D., Ahn D. U., Nam, K. C. and Jo, C. 2013. Flavour Chemistry of Chicken Meat: A Review. Asian-
Australas Journal of Animal Science 26: 732-742.
[OTA] Office of Technology Assessment. 1979. Open shelf life dating of food. US Government Printing Office,
Washington, D.C.
World Health Organization (WHO), World Food Programme (WFP), United Nations Children’s Fund (UNICEF), and
United Nations High Commissioner For Refugees (UNHCR). 2002. Food and Nutrition Needs in Emergencies.
http://www.who.int/nutrition/publications/ emergencies/a83743/en. Retrieved on November 29, 2016.

176
 IFRC 2017: 116-075 Food Processing and Post-Harvest Technology
Optimization of Oil, Whey Protein Concentrate and Carboxymethyl Cellulose Levels on Rheological
Properties and Stability of Sky Fruit (Swietenia macrophylla) seed oil-in-water emulsion

*Nor Hayati, I., Chong, P.Y. and Yusof, H.M.

School of Food Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu,
Malaysia
Abstract

Sky fruit (Swietenia macrophylla) seed oil (SFSO) is known to have various therapeutic values yet scientific studies
that explore its potential as a functional food are hardly found. This study utilized a response surface methodology to
investigate effects of SFSO (10 – 30%), whey protein concentrate (WPC) (4 – 8%) and carboxymethyl cellulose
(CMC) (0.5 – 1.5%) on rheological properties and stability of oil-in-water emulsions and ultimately to optimize their
levels in the system. For the rheological properties, apparent viscosity, yield stress and storage modulus (G’) ranges
were observed to be 1.06 – 287.29 Pa.s, 0.21 – 64.70 Pa, 1.38 – 3265.94 Pa. respectively. The emulsion stability
parameters were demonstrated by turbidity loss rate (1.15 x 10-3 – 8.36 x 10-3), creaming index (CI) (3 – 100%) and
peroxide value (PV) (2.0 – 6.5 mEqO2/kg). It seemed that CI, yield stress and PV were influenced significantly (p <
0.05) by WPC, SFSO and CMC levels. The most predominant interaction effect was SFSO–CMC showing significant
(p < 0.05) synergisms on apparent viscosity, yield stress, G’ and PV of the emulsions. On the other hand, turbidity
loss rate and CI were significantly (p < 0.05) influenced by the interaction of WPC–CMC. The experimental data were
satisfactorily fitted to regression models with high coefficients of determination (R2 > 80%). The optimized levels of
SFSO (29.6%), WPC (5.9%) and CMC (1.1%) were successfully validated to provide a desired emulsion with
acceptable rheological properties (i.e. apparent viscosity = 60.09 Pa.s, yield stress = 27.37 Pa and G’ = 285.15 Pa)
and stability (i.e. turbidity loss rate = 1.63x10-3, CI = 22% and PV = 2.67 mEqO2/kg of PV.

Keywords: Swietenia macrophylla seed oil, emulsion, whey protein concentrate, carboxymethyl cellulose,
interaction, optimization.
*Corresponding author’s email: yati@umt.edu.my

Introduction
Sky fruit (Swietenia macropylla) has been used widely as a traditional medicine and the fruit seed contains 19.42%
with linoleic and linolenic acids being predominant polyunsaturated fatty acids. To promote its consumption as a
nutraceutical, an attempt has been made on converting the sky fruit seed oil (SFSO) into a functional food emulsion
(Nor Hayati et al., 2014). However, the stability and rheology of the emulsion depends on the interaction between oil
droplets on the interfacial composition. Polysaccharide like carboxymethyl cellulose (CMC) and whey protein isolate
(WPC) are commonly used ingredients in the system that significantly influence the interfacial interaction. However,
more research should be made in order to better understand the interaction for formulation optimization. The
interaction effect of the ingredients will be better elucidated by using a response surface methodology (RSM) (Nor
Hayati et al., 2016) and this approach also could reduce experimental cost efficiently. Previous study of Gharibzahedi
et al. (2012), have successfully used RSM in optimizing formulation variables for beverage emulsions of walnut oil,
which predicted that 9.62% of Arabic gum and 3% of walnut oil shall provide the emulsion with optimum properties
and stability. Thus, the objective of the study is to determine optimized levels of SFSO, WPC and CMC in the
emulsions based on the rheological properties and stability response.

177
Material and Methods
Emulsion preparation
Oil-in-water emulsions were prepared by using different combinations of SFSO, CMC and WPC following a face
centred composite design (CCD) with 20 runs. The coarse emulsion was firstly prepared by using a high speed
homogenizer and finally homogenized using an ultrasonic processor at 20 kHz and 500 W at 30 % amplitude.

Rheology and stability analyses

Rheological properties were measured using a rheometer (Discovery Hybrid Rheometer) at 25°C. Flow curves with
apparent viscosity (ɳ) and yield stress (τo) were obtained at shear rate of 0.1 – 50 s-1 while storage modulus (G’) was
recorded from frequency sweep experiments at 1 – 100 rad/s and strain of 0.5%. Emulsion turbidity was measured
at 500 nm as a function of time by means of a UV–visible spectrophotometer and turbidity loss rate was calculated.
The emulsions were centrifuged at 5000 rpm to induce phase separation. The height of the aqueous layer was
measured and converted to creaming index (CI) as follows: % layers = (height of the aqueous layer/the total height of
the emulsion) x 100%. Peroxide value (PV) of the emulsion was measured after storage at 60°C for 24 hours,
following the standard procedure.

Experimental design and statistical analysis

A CCD of RSM was employed in this study using a Minitab 14 statistical software at α = 0.05, involving 20
experimental units with 6 replications at the center point. Independent variables of SFSO (X1) (10 – 30 %), WPC (X2)
(4 – 8 %) and CMC (X3) (0.5 – 1.5 %) and their relationship with dependent variables (Yi) was expressed by a
multiple linear regression equation as follow: Yi = β0 + β1X1 + β2X2 + β3X3 + β11X12 + β22X22 + β33X32 + β12X1X2 +
β13X1X3 + β23X2X3. Optimum levels of SFSO, WPC and CMC was determined by using a response optimizer and the
experimental data obtained were verified against the predicted data provided by the fitted regression equations.

Results and Discussion

For the rheological properties, on ɳ, τo and G’ ranges were observed to be 1.06 – 287.29 Pa.s, 0.21 – 64.70 Pa, 1.38
– 3265.94 Pa, respectively. The emulsion stability parameters were demonstrated by turbidity loss rate (1.15 x 10-3 –
8.36 x 10-3), CI (3 – 100%) and PV (2.0 – 6.5 mEqO2/kg). Table 1 summarizes ANOVA results from a multiple
regression analysis of RSM for each response. It seemed that CI, τo and PV were influenced significantly (p < 0.05)
by WPC, SFSO and CMC levels. The interaction effect of SFSO and CMC shows significant synergism (p < 0.05) on
ɳ, τo and G’ of the emulsions as expressed by positive coefficient of estimation (8.60, 2.10 and 88.71, respectively) of
the interaction term (X1X3). It is suggested that CMC has decreased the oil droplet size, which indirectly caused
flocculation to give more resistance to flow, leading to increase in the emulsion viscosity (Nor Hayati et al., 2016).
This flocculation phenomenon also has resulted in more stress needs to be given to cause an initial flow, as
indicated by the high τo value. Moreover, flocs which trapped some of the continuous phase were formed. Hence, the
effective volume fraction of the disperse phase was increased which then increased the apparent shear viscosity and
yield stress in a synergistic manner (Wu et al., 2013). It also promoted a gel-like behaviour giving rise to high G’ of
the emulsions and hence, formed a more stable gel at constant conditions. Differently, significant antagonism of
SFSO and CMC with a negative coefficient (0.14) of the interaction term (X1X3) was observed. In other words,
increasing level of both ingredients in a small magnitude is desirable in reducing peroxide value for better stability of
the emulsion towards lipid oxidation. For turbidity loss rate, antagonism between WPC and CMC gave the strongest
effect (p < 0.05) with a negative coefficient (0.60) of the X2X3 interaction term. This reflects that increase in both
ingredients will successfully maintain the emulsion turbidity as a function of time. Otherwise, synergism between both
ingredients seemed to pronouncedly increase CI of the emulsion, as shown by a positive coefficient (13.96) of the
X2X3 interaction term. At high amount of WPC, the pH of the emulsion seemed to rise near to neutral pH. At neutral
pH, the aggregated oil droplet network is weak and depletion occurred (Damianou & Kiosseoglou, 2006). At the
meantime, increased CMC level caused un-adsorbed CMC exceed a certain critical value, which led to depletion
flocculation and hence increased the CI, indicating emulsion destabilization.

178

Responses
Apparent viscosity at
0.53 s-1
Yield stress (Pa)
Storage modulus at 10
rad/s
Peroxide value (meqO2
/kg
Turbidity loss rate
Creaming index (%)
*Significant term at p > 0.05.
Table 1: Summary of ANOVA results for the fitted regression models
Fitted regression models R2
487.26 – 20.74X1 – 75.22X2 – 321.49X3 + 0.20X12 + 2.90X22 + 36.12X32 + 86.3
1.35X1X2 + 8.60X1X3* + 24.49X2X3
101.57 – 4.88X1 – 15.29X2 – 62.68X3 + 0.06X12 + 0.68X22 + 6.19X32 + 0.25X1X2 89.3
+ 2.10X1X3* + 4.22X2X3
5976.31 – 228.88X1* – 955.45X2 – 4047.06X3 + 1.79X12 + 32.85X22 + 454.57X32 82.5
+ 18.09X1X2 + 88.71X1X3* + 345.91X2X3
4.88 + 0.22X1 – 0.06X2 – 10.20X3 + 0.01X12 + 0.02X22 + 8.31X32 – 0.02X1X2 – 96.6
0.14X1X3* + 0.06X2X3
6.39 + 0.38X1 – 2.79X2 – 4.10X3 – 0.005X12 + 0.25X22* + 1.42X32 – 0.02X1X2 – 81.7
0.06X1X3 + 0.60X2X3*
– 204.12 – 2.99X1 – 55.86X2* – 99.32X3 – 0.01X12 + 2.81X22 + 5.03X32 + 84.4
0.51X1X2* – 0.54X1X3 + 13.96X2X3*

Figure 1 shows representative contour plots, visualizing the interaction between two independent variables. By using
a RSM response optimizer function with superimposed plots, the optimum levels for SFSO, WPC and CMC predicted
to be at 29.6 %, 5.9 % and 1.1 %, respectively.

(a) (b)
Figure 1. Contour plots showing interaction effect between two independent variables on (a) storage
modulus (Pa) and (b) creaming index (%) of the emulsion.

This optimization achieved composite desirability value of 0.928 which indicated that all responses fell within the
acceptable limits. Table 2 reveals that all the experimental data fell within the predicted range at 95% confidence
interval generated by the RSM. Therefore, the optimum ingredient combination in the emulsion achieved by RSM
was practical and all fitted models were verified as valid.

179
 Table 2: Predicted range of response variables under optimum condition
Response variables Fit Predicted range at 95% CI Experimental data (Mean ±
SD)
Turbidity loss rate 1.17x10-3 0.17x10-3 to 2.17x10-3 1.63 x 10-3 ± 0.3 x 10-3
Creaming index (%) 32 16 to 47 22 ± 3
Apparent viscosity at 0.53 74.11 22.71 to 125.50 60.09 ± 7.55
s-1
Yield stress (Pa) 20.25 9.67 to 30.82 27.37 ± 3.50
Storage modulus at 10 666.21 11.89 to 1320.50 285.15 ± 76.54
rad/s
Peroxide value (meqO2 2.6 1.9 to 3.3 2.6 ± 0.3
/kg)

Conclusion
This study demonstrated that rheology and stability of the emulsions were mainly affected by the concentration of
CMC. Moreover, desirable interaction between SFSO and CMC or WPC and CMC seemed to produce more
desirable emulsions with better rheological properties and stability. Optimum levels of SFSO (29.6%), WPC (5.9%)
and CMC (1.1%) achieved by RSM was proven to be practical and all fitted models were verified as valid.

Acknowledgements
The authors acknowledged financial supports from UMT and FRGS fund (Vote. No.59284).

References
Damianou, K. and Kiosseoglou, V. 2006. Stability of emulsions containing a whey protein concentrate obtained from
milk serum through carboxymethyl cellulose complexation. Food Hydrocolloids 20 (6): 793-799.
Nor Hayati, I., Cheong, W. C., Rozaini, M. Z. H. 2016. Flow properties of o/w emulsions as affected by xanthum gum,
guar gum and carboxymethyl cellulose interactions studied by a mixture regression modelling. Food
Hydrocolloids 53: 199-208.
Nor Hayati, I. Yap, Y. H. and Nurul Farhanah, M. A. 2014. Physical properties and stability of oil-in-water emulsions
as affected by sky fruit (Swietenia macrophylla) seed oil and emulsifier concentrations. In Masni, M. A.,
Shahrizad, Y., Ramlan, O., Mahanem, M. N., Abdullah, S. and Norrakiah, A. S. (Eds). Proceedings of the 13th
Symposium of the Malaysian Society of Applied Biology, p. 270. Cherating: Malaysian Society of Applied
Biology.
Wu, B., Degner, B., McClements, D. J. 2013. Microstructure and rheology of mixed colloidal dispersions: Influence of
pH induced droplet aggregation on starch granule-fat droplet mixtures. Journal of Food Engineering 116 (2):
462-471.

180
 IFRC 2017: 120-078 Food Processing and Post-Harvest Technology

Physical and Nutritional Properties of Malaysian Avocado (Persea americana Mill) Fruit
1Tan, C.X., 2Chong, G.H., 3Hazilawati, H. and *1Ghazali, H.M.
1Department of Food Science, 2Department of Food Technology, Faculty of Food Science and Technology,
3Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia
(UPM), 43400 Serdang, Selangor, Malaysia

Abstract
The tropical climate, along with abundant of rainfall throughout the years, produces a wide diversity of succulent and
edible fruits in the Malaysia. In Malaysia, avocado (Persea americana Mill) is an underutilized fruit grown in semi-wild
conditions of the rural areas or in the small orchards along with other commercial fruit trees. As the global market for
avocado fruit is increasing, the underutilized avocado has the potential to be developed as a new commercial fruit
crop in Malaysia. Since data on the physical and nutritional properties of Malaysian avocado fruit are necessary for
the food industry to determine which part of the fruit can be used for commercialization, a study was conducted with
this aim in mind. Results indicated the Malaysian avocado is a medium sized fruit with an average mass, length and
diameter of 216.92 g, 9.50 cm and 7.20 cm, respectively. The major part of Malaysia avocado fruit is the pulp
(56.01%), followed by the seed (33.04%) and the peel (10.94%). Each of these parts was further investigated for their
nutrient composition. All the fruit parts were found to be low in protein (< 1%), but high with moisture content (> 60%).
Compared to the peel and seed, the pulp has the highest lipid content (20.79 ± 0.27%), but the lowest ash (1.47 ±
0.13%) and carbohydrate (3.39 ± 0.76%) contents. Malaysian avocado fruit, particularly the pulp, contains a
significant amount of lipid content, suggesting the potential for extraction of an edible oil.
Keywords: avocado, Persea americana Mill, underutilized fruit, proximate composition
*Corresponding author’s email: hasanah@upm.edu.my

Introduction

The tropical climate, along with abundant of rainfall throughout the years, produces a wide diversity of succulent and
edible fruits in the Malaysia. Durian (Durio zibethinus L.), rambutan (Nephelium lappaceum L.) and papaya (Carica
papaya L.) are some examples of popular tropical fruits planted in large quantities in Malaysia. These fruits are
commercial fruits, selling at good prices in the local and export markets. On the other hands, avocado (Persea
americana Mill), dabai (Canarium odontophyllum Miq) and kuini (Mangifera odorata) are some examples of less
popular fruits, often grown in the semi-wild conditions of the rural areas or in the small orchards along with other
commercial fruit trees (Halim et al., 2016). Due to lack of popularity, these underutilized fruits are sometimes viewed
as nutritionally inferior by Malaysians. Among the underutilized fruits mentioned, avocado has the potential to be
developed as the new commercial fruit crop in Malaysia. This is because the demand for avocado fruit is increasing.
Between 1993 to 2013, the average global per capita consumption of avocado fruit increased from 0.36 kg in 1993 to
0.66 per kg in 2013, with a growth rate of 87% (Paz-Vega, 2015). Fresh avocado fruit contributes a large market
worldwide, alongside with its use in the oil, cosmetic and food processing industries (Mostert, 2007).

Avocado is an evergreen plant from the Lauraceae family. The avocado tree can reach up to 20 meters in height
when fully grown and the leaves are 15 to 25 cm long with well-developed petioles that are spirally arranged near the
branch ends (Afahkan, 2012). More than 500 varieties of avocado have been identified worldwide and the fruiting
seasons vary according to the variety (Mostert, 2007). As the global market for avocado fruit is increasing, the

181
underutilized Malaysian avocado fruit has the potential for commercial development. The information on the physical
properties of Malaysian avocado fruit is necessary to design equipment for harvesting, handling and storing the fruit.
Previous studies reported the nutritional composition of avocado fruit is affected by variety and geographical
locations (Mostert, 2007; Tango et al., 2004), but the data on the nutritional profile of Malaysia grown avocado fruit
are scarce. Therefore, this study was carried out to determine the physical properties and nutrient contents of
Malaysian avocado fruit.

Materials and methods

3.1. Sample preparation
Mature avocado fruits were collected from UPM campus, Malaysia in February 2016. Fruits without physical
damages were washed under running tap water and wiped dry before storing at room temperature to promote
ripening. The peel, pulp and seeds were manually separated and then dried in a tray-type dryer (Memmert UFB 400,
Germany) at 35ºC for three consecutive days. The dried fruit parts (moisture content: 2-3%) were ground into powder
using a home blender and sieved to obtain a uniform particle size (360 µm).

3.2. Methods
The mass of the whole fruits and fruit parts (peel, pulp and seeds) were measured using a digital balance (Shimadzu,
Japan) while the length and diameter were measured using a vernier calliper (Mitutoyo, Japan). The moisture
content, crude protein, crude lipid, crude fiber and ash content were determined using the AOAC methods (AOAC,
2007). Total carbohydrate was calculated by difference (Coimbra et al., 2011). The data were analyzed using one-
way ANOVA accompanied with Turkey’s post hoc using SPSS software.

Results and discussion

4.1. Physical properties
Malaysian avocado fruit is similar in appearance to a pear fruit. The unripe fruit is green in color, and turns dark red
when fully ripe. The fruit has a glossy smooth and waxy skin texture. Information on the physical properties of
Malaysian avocado fruit is important for designing and fabricating postharvest equipment for processing, packing,
grading and storing the fruit. Furthermore, these data will also be useful to the food industry to determine which part
of the fruit can be used for commercialization. Table 1 shows the results for the physical properties of Malaysian
avocado fruit. The mass (216.92 ± 4.29 g), length (9.50 ± 0.26 cm) and diameter (7.20 ± 0.28 cm) of Malaysian
avocado fruit were slightly lower compared with the values reported by Galvao et al. (2014) on Brazilian avocado
fruit. Young et al. (1978) classify avocado sizes into three categories, namely small (156-186 g), medium (187-218 g)
and large (219-346 g). From this classification, Malaysian avocado fruit is categorized as a medium sized fruit. An
avocado fruit can be divided into three anatomical regions, which are the peel, pulp and seed. The major portion of
Malaysian avocado fruit was found to be the pulp (56.01 ± 1.23%), followed by seed (33.04 ± 1.03%) and peel
(10.94 ± 0.28%). A similar observation was made by Galvao et al. (2014) with respect to the proportion of the peel,
pulp and seed in the avocado fruit.

Table 1: Physical properties of Malaysian avocado fruit

Parameters Obtained values Galvao et al. (2014)1
Mass (g) 216.92 ± 4.29 336.22
Length (cm) 9.50 ± 0.26 10.74
Diameter (cm) 7.20 ± 0.28 8.09
Distribution of fruit parts (%)
Peel 10.94 ± 0.28 10.09
Pulp 56.01 ± 1.23 72.91
Seed 33.04 ± 1.03 16.98
1Average values from three geographical areas

182
4.2. Proximate analysis of Malaysian avocado fruit
Proximate analysis provides an overview of the nutritional composition of a food. The proximate composition of
various parts of the Malaysian avocado fruit is presented in Table 2. Moisture content measures the perishability and
solid content of food (Vinha et al., 2013). High moisture contents (> 60%) were observed in various parts of
Malaysian avocado fruit, in which the pulp (73.67 ± 0.49%) contained significantly higher (p<0.05) amount of
moisture content than the peel (68.44 ± 1.91%) and seed (60.51 ± 0.80%). These findings suggest Malaysian
avocado fruit is not advantageous in terms of the shelf life and improper storage conditions could easily deteriorate
the fruit quality. The data in the current study was in agreement with Vinha et al. (2013), who reported the peel, pulp
and seed of avocado contained high moisture contents (> 50%). Protein is an essential nutrient for maintaining the
function of body tissues and growth. Low protein contents (< 1%) were exhibited by different parts of Malaysian
avocado fruit, whereby the seed (0.20 ± 0.09%) contained a significantly lower (p<0.05) of protein content than the
peel (0.52 ± 0.15%) and pulp (0.73 ± 0.07%). Generally, fruits are not good sources of protein, and animal-derived
foods such as meat and cheese are excellent sources of protein. All the fruit parts of Malaysian avocado displayed
lower protein contents than the flesh of durian (2.60%) and banana (1.30%) (Voon & Kueh, 1999).

Commercialized oils are mostly extracted either from the seeds or pulp of plants. Seeds with high lipid contents such
as soybean, canola, peanut and sunflower are the main sources of plant oils in the market whereas fruit pulp with
high lipid contents such as avocado, olive and palm fruits are sources for the latter. (Gunstone, 2011). The results of
the present study indicated the crude lipid content in the pulp (20.79 ± 0.27%) of Malaysian avocado was significantly
greater (p<0.05) than the peel (8.62 ± 0.20%) and seed (2.09 ± 0.07%). These findings are in accordance with
Gunstone (2005), who reported that avocado seed has very little lipid (2.0%). The lipid content in Malaysian avocado
pulp was higher than the pulp of Guariroba (13.60%) and Jeriva (7.48%) palm fruits, but lower than the seeds of
rambutan (38.0%) and papaya (27.0%) (Coimbra et al., 2011; Yanty et al., 2013). High fiber intake promotes health
benefits such as reduction of postprandial blood sugar level and improvement of gastrointestinal function. The
amount of crude fiber in the peel (12.74 ± 0.67%) was significantly higher (p<0.05) than the pulp (3.89 ± 0.04%) and
seed (3.08 ± 0.13%). These are in good agreement with Anhwange (2008), who stated that fruit peels are the
excellent source of fiber. Malaysian avocado pulp showed a higher fiber content than mandarin orange flesh
(0.40%), but lower than the guava flesh (6.80%) (Voon & Kueh, 1999).

Ash content measures the total amount of minerals in food (Borchani et al., 2010). The ash content in the peel (2.89
± 0.17%) of Malaysian avocado was significantly greater (p<0.05) than the pulp (1.47 ± 0.13%) and seed (1.93 ±
0.12%). This indicates the mineral content of the avocado peel is the highest. The ash contents of different fruit parts
of Malaysian avocado were comparable to the date flesh (1.58-2.59%) (Borchani et al., 2010). The main energy
source of the human diet is contributed by carbohydrate. Carbohydrate is almost exclusively derived from plant-
originated food (Lunn & Buttriss, 2007). The amount of carbohydrate in the pulp (3.34%) of Malaysian avocado was
lower than the peel (19.53%) and seed (35.27%). Malaysian avocado pulp exhibited lower carbohydrate content than
the flesh of papaya (7.10%), pineapple (10.60%) and orange (10.40%) (Voon & Kueh, 1999).

Table 2: Proximate composition of Malaysian avocado fruit

Parameters (%) Peel Pulp Seed
Moisture content 68.44 ± 1.91a 73.67 ± 0.49b 60.51 ± 0.80c
Crude protein 0.52 ± 0.15a 0.73 ± 0.07a 0.20 ± 0.09b
Crude lipid 8.62 ± 0.20a 20.79 ± 0.27b 2.09 ± 0.07c
Crude fiber 12.74 ± 0.67a 3.89 ± 0.04b 3.08 ± 0.13b
Ash content 2.89 ± 0.17a 1.47 ± 0.13b 1.93 ± 0.12c
Total carbohydrate1 19.53 3.34 35.27
Different letters in the same row are significantly different at p<0.05. 1Obtained by difference.

183
Conclusion
The Malaysian avocado is a medium sized fruit with waxy and glossy smooth surface. All the fruit parts of Malaysian
avocado were characterized by high moisture content, but low in protein content. Fruits with high moisture content
are highly perishable, one way to counter this shortcoming is by processing them into food products such as oil and
jam. Malaysian avocado fruit, particularly the pulp, contains a substantial amount of lipid. This suggests the potential
of Malaysian avocado pulp as raw material for edible oil extraction. However, further studies are needed to identify
the fatty acid and triacylglycerol compositions of the oil extracted from the pulp of Malaysian avocado.
Acknowledgments

The authors would like to thank UPM for financial support

References:

Afahkan, M. E. 2012. Effects of Petroleum Ether and Methanolic Fractions of Avocado Pear Seeds on Wistar Rats
Fed a High Fat-High Cholesterol Diet. Nigeria: Ahmadu Bello University.
Anhwange, B. 2008. Chemical composition of Musa sapientum (banana) peels. Journal of Food Technology, 6(6),
263–266.
AOAC. 2007. Official methods of analysis of AOAC international. 18th ed. Washington: Association of Official
Analytical Chemists.
Borchani, C., Besbes, S., Blecker, C., Masmoudi, M., Baati, R. and Attia, H. 2010. Chemical properties of 11 date
cultivars and their corresponding fiber extracts. African Journal of Biotechnology, 9(12), 4096–4105.
Coimbra, M.C. and Jorge, N. 2011. Proximate composition of guariroba (Syagrus oleracea), jeriva (Syagrus
romanzoffiana) and macauba (Acrocomia aculeata) palm fruits. Food Research International, 44(7), 2139–
2142.
Dorantes, L., Parada, L. and Ortiz, A. 2004. Avocado Post-harvest Operations. Italy: Food and agriculture
Organization of the United Nations.
Galvao, M.D., Narain, N. and Nigam, N. 2014. Influence of different cultivars on oil quality and chemical
characteristics of avocado fruit. Food Science and Technology, 34(3), 539–546.
Gunstone, F.D. 2011. Vegetable oils in food technology: composition, properties and uses. New Jersey: John Wiley
and Sons.
Halim, N.A., Rozana, N., and Mohd, N. 2016. Underutilized Fruit Species Conservation in Malaysia. Mardi, 1–4.
Lunn, J. and Buttriss, J. 2007. Carbohydrates and dietary fibre. Nutrition Bulletin, 32(1),21–64.
Mostert, M. 2007. Characterization of micro-components of avocado oil extracted with supercritical carbon dioxide
and its oxidative stability. South Africa: University of Pretoria.
Tango, J.,Carvalho, C. and Soares, N. 2004. Physical and chemical characterization of avocado fruits aiming for oil
extraction. Revista Brasileira de Fruticultura, 26(1), 17–23.
Vinha, A., Moreira, J., Jorge, R. and Ferreira, V. 2013. Physicochemical Parameters, Phytochemical Composition
and Antioxidant Activity of the Algarvian Avocado (Persea americana Mill.). Journal of Agricultural Science,
5(12), 100–109.
Voon, B. and Kueh, H. 1999. The nutritional value of indigenous fruits and vegetables in Sarawak. Asia Pacific
Journal of Clinical Nutrition, 8(1), 24–31.
Yanty, N., Marikkar, J.M.N., Long, K. and Ghazali, H.M. 2013. Physico-chemical characterisation of the fat from red-
skin rambutan (Nephellium lappaceum L.) seed. Journal of Oleo Science, 62(6), 335–343.
Young, R.E. and Lee, S. 1978. Avocado Fruit Maturity. California Avocado Society Yearbook, 62, 51–57

184
 IFRC 2017: 131-089 Food Processing and Post-Harvest Technology
Determination of the Potential of Kamuning (Murraya paniculata) Flowers
for Tea Development
*Navarro, B.R.R. and Iñigo, H.B.R.
Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines Los
Baños, College, Laguna, 4031 Philippines

Abstract
In the Philippines, there is a high incidence of noncommunicable diseases, making healthy interventions crucial in
improving the health of the Filipinos. One such intervention is encouraging tea consumption among the population
owing to the long-established therapeutic and health benefits of tea. To this purpose and to develop food use for
Philippine indigenous flowers, the potential of kamuning (Murraya paniculata) flowers for tea development was
evaluated. Tea samples were produced by drying fresh kamuning flowers using cabinet, sun, and freeze-drying. They
were then subjected to physicochemical tests [i.e., pH, total soluble solids, vitamin C content, total phenol content
(TPC), and antioxidant activity determinations] and sensory evaluation. The most sensory-acceptable sample was
then compared with commercially available acacia flower tea by quality scoring. From the results, the three kamuning
flower tea samples did not differ in all physicochemical properties evaluated, except total phenolic content, regardless
of the type of drying. However, they significantly differed in TPC. The cabinet-dried flower tea had the highest TPC of
6.15×10-3 GAE/mg followed by the freeze-dried (4.66×10-3 GAE/mg) and sun-dried (1.891×0-3 GAE/mg) samples.
The three samples also showed no significant differences in general acceptability. However, the cabinet- and freeze-
dried flower tea samples were significantly darker than the sun-dried flower tea. Moreover, the freeze-dried flower tea
had a significantly stronger aroma than the other two samples. In comparison with acacia flower tea, the KFT showed
no significant differences in all evaluated attributes. It is thus concluded that kamuning flowers have high potential for
tea development.
Keywords: Murraya paniculata, kamuning, flower tea, noncommunicable diseases, drying
Corresponding author’s email: rrnavarro1970@yahoo.com
Introduction
Recent statistics by the World Health Organization (2015) shows that Filipinos have a 28% probability of dying from
noncommunicable diseases (NCDs), which is highly preventable, as NCDs develop only through poor lifestyle and
eating habits. NCDs occupy a large percentage of the top ten causes of death in the Philippines (Department of
Health, 2013). Therefore, addressing the major predisposing risk factors of NCDs can greatly improve the health of
the Filipinos. One potentially viable means is encouraging tea concumption among the Filipinos.
Tea, which originated from China 5000-6000 years ago (Chen, 1994), has nutritional and therapeutic properties
(Zhen et al., 2002) and is effective in preventing some important diseases (Chen & Lin, 2015) and lowering the risk of
heart diseases (Nutraceutical Science and Technology, 2009). Originally, tea refers to only beverage prepared by
steeping young dried leaves of Camellia sinensis; nowadays, it is also used to pertain even to decoctions prepared
from dried parts of various plants, including flowers.
Here, we aimed to determine whether flowers of kamuning, which is indigenous to the Philippines and is included in
the list of underutilized horticultural crops, can be developed into tea. Kamuning has various medicinal benefits, since
extract of its leaves, bark, and roots has analgesic, antioxidant, antimicrobial, and antidiabetic activities (Gill et al.,
2014). Our specific objectives were to determine the physicochemical properties of kamuning flower tea (KFT), to
determine the functional properties [vitamin C, antioxidant activity, and total phenol content (TPC)] of KFT, to test the
general acceptability of KFT by sensory evaluation, to determine which drying method will yield the most acceptable
KFT, and to determine the preference of general consumers between KFT and a conventional flower tea, i.e., acacia
flower tea, from Korea.

185
Materials and Methods
Procurement and processing of raw materials
Kamuning flowers were harvested in Los Baños, Laguna by handpicking and then dried by three techniques: sun,
cabinet, and freeze-drying. In sun drying, the flowers were placed top-side down in a winnowing basket and exposed
to the sun from 8:00 am-4:00 pm. In cabinet drying, they were evenly distributed on a mesh tray, which was then
placed in a cabinet drier kept at 42° C for 20 h. Lastly, in freeze-drying, overnight-prefroze flowers were placed in a
freeze dryer for 16 h operated under standard settings. All the dried flowers were then sealed in tea bags in 500 mg
portions and stored in polypropylene containers at room temperature until analysis.
Chemical analyses
Total soluble solids (TSS) measurement. The TSS of the samples was directly measured using a hand refractometer.
pH determination. pH was determined using a pH meter.
Vitamin C analysis. Vitamin C content was determined by the 2,6-dichloroindophenol titrimetric method (AOAC
Method 967.21) (Suzanne Nielsen, 2009).
Total phenolic analysis. TPC (gallic acid equivalent) was determined using the Folin-Ciocalteu reagent method
(Saeed et al., 2012). It was calculated using
total phenolic content (GAE) = (conc. from std. curve)(vol. of sple. soln.) .
mass of dry sample
Antioxidant activity analysis. Free radical scavenging activity was examined in vitro using DPPH radical, as described
by Shimada et al. (1992). %DPPH scavenging activity was calculated using
%DPPH scavenging activity = (1 - sample absorbance ) x 100 .
blank absorbance
Sensory evaluation
The sensory evaluation of the KFT was performed using quality scoring test. The panel comprised 25 Japanese,
Koreans and tea-drinking Filipinos.
Statistical analyses
Analysis of variance (ANOVA) was used to determine if there are significant differences among the samples
produced by the three drying methods. For populations of data that do not satisfy the assumptions of ANOVA,
Kruskal-Wallis test was used. In cases where significant differences exist, Dunn’s procedure was used to determine
which treatments are significantly different. To analyze the data obtained from the sensory analysis by quality
scoring, Friedman’s Test and Wilcoxon Signed Rank Test were employed.
Results and Discussion
Physicochemical properties of KFT
From Table 1, the samples have a pH range of 8.03-8.10 and 0°Brix reading, indicating the lack of dissolved solutes.
Regardless of the drying method, they have a vitamin C content of <1. Among the samples, the cabinet-dried sample
had the highest TPC and antioxidant activity. But, by Kruskal-Wallis test, the pH showed no significant differences
between samples. The pHs were slightly basic and in line with the vitamin C content results. Vitamin C is a direct
source of acidity and directly contributes to the pH of the sample.
Table 1. Physicochemical and functional properties of KFT among different drying methods
Physicochemical property Sun-dried Freeze-dried Cabinet-dried
pH 8.03 a* 8.10 a 8.10 a
TSS, °Brix 0 a 0 a 0a
Vitamin C, mg/mL <1 a <1 a < 1a
TPC, GAE/mg 1.89×10 -3 a 4.66×10 -3 ab 6. 15×10-3 b
%DPPH scavenging activity 83.33 a 68.67 a 90.67 a
* Means followed by the same superscript are not significantly different.
In terms of TPC, the freeze-dried sample was not significantly different from the sun- or cabinet-dried sample.
Phenols include several substances that differ in heat sensitivity but are commonly destroyed by heat. However, this
is based on studies of food products that use cooking and processing temperatures to measure the extent of

186
degradation of phenolic compounds (Chipurura et al., 2010). Here, note that cabinet drying used a temperature of
only 42°C, much different from the common food processing temperature range of 75-160°C.
In terms of antioxidant activity, all KFT samples showed no significant differences, despite the wide %DPPH
scavenging activity range of 68.67-90.67. The low scavenging activity of the freeze-dried sample is explained by the
fact that, during freeze-drying, plant cells are disrupted, causing the decompartmentalization of enzymes, substrates,
and activators (Chang et al., 2006).

Table 2. Average ratings of KFT samples among three drying methods

Average Rating Friedman's Test
Attribute
Freeze-Dried Sun-Dried Cabinet-Dried (p-value)
Color 3 2 3 0.0015*
Aroma 5 4 3 0.0013*
Flavor 4 3 3 0.3451ns
Aftertaste 4 3 5 0.5004ns
General Acceptability 4 4 4 0.1339ns
*Significant at α=5%

Effect of the three drying methods on sensory properties

From Table 2, the freeze-dried sample had the highest average aroma and flavor ratings, while the sun-dried sample
had the highest aftertaste rating. All the samples had equal general acceptability ratings. Statistically, there were no
significant differences in flavor, aftertaste, or general acceptability, only in color and aroma. Since there were no
significant differences in general acceptability among the KFT samples produced using the three drying methods, the
ratings for the other attributes of KFT were used as reference to determine the most acceptable flower tea. Table 4
shows that the freeze- and cabinet-dried samples had the highest color rating, while the freeze-dried sample had a
higher aroma rating than the other two. Thus, the freeze-dried flower tea was compared with commercial acacia
flower tea.

Fig. 1. Distribution of the 25 panelists based on their preference between acacia and KFT among sensory attributes.
Figure 1 shows that more panelists rated the KFT higher in general acceptability than the acacia flower tea, and that
fewer panelists rated the KFT in the 1- to 2-point general acceptability. Also, KFT and acacia flower tea showed no
significant differences in general acceptability(p=0.1371). This points to the high potential of KFT for
commercialization and consumer acceptance.

Summary and Conclusion

Kamuning flowers were dried by cabinet, sun, and freeze-drying to produce flower tea and then evaluated of their
physicochemical properties and sensory attributes. All three flower tea samples significantly differed in TPC, but not
in physicochemical properties; they also exhibited high scavenging activities. Sensory evaluation results showed that
the three samples did not significantly differ in general acceptability, but that the freeze-dries samples showed high
color and aroma ratings and thus was used for comparison with acacia flower tea. In the comparison between
kamuning and acacia flower tea, no significant differences were noted in the attributes tested. Thus, it is concluded
that KFT is at par with acacia flower tea, and thus has high potential for tea development. For further study, we
suggest that the same physicochemical analyses be carried out on both fresh and dried kamuning flowers to

187
accurately evaluate the effect of drying on the physicochemical properties of KFT. Also, tests on other
phytoconstituents not examined in this study should be done, and the effect of the steeping time of the flowers on the
physicochemical and sensory properties should be evaluated. It was observed that varying the steeping time resulted
in different sensory properties, which was very evident in terms of color.

References
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hot-air-dried tomatoes. Journal of Food Engineering, 77(3): 478-485.
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Chen, Z-M. and Lin, Z. 2015. Tea and human health: biomedical functions of tea active components and current
issues Journal of Zhejiang University Science B. 16(2): 87-102.
Chipurura, B., Muchuweti, M. and Manditseraa, F. 2010. Effects of thermal treatment on the phenolic and antioxidant
activity of some vegetables. Asian Journal of Clinical Nutrition, 2: 93-97.
Department of Health. 2013. Leading Causes of Mortality. Retrieved on July 7, 2015, from the Department of Health
Website: http://www.doh.gov.ph/node/198.html
Gill, N., Kaur, N., and Arora, R. 2014. An Overview on: Murraya paniculata Linn. International Journal of Institutional
Pharmacy and Life Sciences, 4(4): 3-9.
Nutraceutical Science and Technology. 2009. Tea and Tea Products: Chemistry and Health Promoting Properties.
C.-T. Ho, J.-K. Lin, and F. Shahidi (Eds.). New York: CRC Press.
Saeed, N., M.R. Khan, and M. Shabbir. 2012. Antioxidant activity, total phenolic and total flavonoid contents of whole
plant extracts Torilis leptophylla L. BMC Complementary and Alternative Medicine. 12: 221.
Shimada, K., K., Fujikawa, K., Yahara, and T. Nakamura. 1992. Antioxidative properties of xanthan on the
autoxidation of soybean oil in cyclodextrin emulsion. Journal of Agriculture and Food Chemistry 40: 945-948.
Suzanne Nielsen, S. 2009. Vitamin C determination by indophenol method. In Food Analysis Laboratory Manual,
p. 55-60, New York: Springer US.
World Health Organization. 2015. Philippines: WHO Statistical Profile. World Health Organization. Retrieved in April
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Zhen, Y., Chen, Z., Cheng, S., and Chen, M. (Eds.). 2002. Tea: Bioactivity and Therapeutic Potential. New York:
Taylor & Francis.

188
 IFRC 2017: 134-092 Food Processing and Post-Harvest Technology

Characteristic of Edible Film from Pectin of Citrus (Citrus Aurantifolia), Papaya (Carica papaya L.) and
Latundan Bananas (Musa acuminata × M. balbisiana) Peel Wastes: A Comparative Study

1Hapsari N., *2Rosida, D.F.,3Ramadhani, P.V., 4Sudaryati., 5Dewati, R

1,5Departement of chemical engineering, Faculty of Engineering University of Pembangunan Nasional “Veteran” Jawa
Timur, Surabaya
2,3,4 Departement of Food Technology, Faculty of Engineering University of Pembangunan Nasional “Veteran” Jawa
Timur, Surabaya Jalan Raya Rungkut Madya Gunung Anyar Surabaya 60294 Indonesia

Abstract
Citrus (Citrus Aurantifolia) peel wastes found in many soto stalls, while the banana peel was found in fried snack
sellers on the roadside and papaya peel wastes was found in the seller of fresh fruit ready to eat. Fruit peel waste is
very much in the community and has not been widely utilized. The peel of these fruits still contain a lot of pectin.
Pectin is a polymer of D-galactaconic acid linked by a 1.4 glycosidic bond. Pectin is an additive compound that
functions as a gelling agent. Pectin compounds are commonly found in fruits, but more abundantly in the peel of the
fruit. One of the uses of pectin is by applying it to an environmentally friendly product, such as an edible film. Edible
film made from carbohydrates has a good ability to protect products from oxygen, carbon dioxide, has the desired
mechanical properties and can improve the unity of structural products. Edible film serves as a food packer, a mass
transfer inhibitor such as moisture, oxygen and fat. The purpose of this study was to determine the characteristics of
edible film from three types of raw materials. This study used a complete randomized design of factorial pattern. The
first factor was the Origin of raw materials pectin (peel wastes of citrus, papaya and banana ), while the second
factor was glycerol concentration (15%, 20% and 25%). The best result was edible film from pectin of citrus peel with
15% glycerol concentration. The characteristics of Edible film had a thickness of 0.51 mm, tensile strength 12.56 kgf /
cm2, elasticity 32.57% and the rate of water vapor transmission of 124.315 gr / m2.hr.

Keywords: edible film, pectin, peel, citrus, papaya, banana

*Corresponding author’s email: rosy.upnsby@gmail.com

Introduction
The main components of edible film compilers are grouped into hydrocolloids (proteins and carbohydrates), lipids
(fatty acids, acyl glycerol, and waxes) and composites. Edible films made from carbohydrates have a good ability to
protect products from oxygen, carbon dioxide, have the desired mechanical properties and can improve the structural
unity of the product, while the weakness of the film of this carbohydrate is less good used to regulate the migration of
water vapor so it needs to be added plasticizer ( Bourtoom, 2008).
One of the raw materials of edible film is pectin. Pectin is a polymer of D-galactaconic acid linked by a 1.4 glycosidic
bond (Maulidiyah et al., 2014). Pectin compounds are commonly found in fruit, but are more commonly found in the
peel of the fruit because their function is a structural element in tissue growth and the main component of middle
lamella and also acts as adhesive and maintains tissue and cell stability (Darmawan et al., 2014) .

Currently the fruit peel has not been optimally utilized. Generally just just thrown away causing increasing waste. The
waste one of them is to be used as raw materials pectin making which then produced further into edible film. The
study of edible film from pectin derived from fruits waste (peel of lime, papaya skin and banana peel) is intended to
utilize waste that has not been widely utilized to be processed into one of the environmentally friendly. Differences
pectin of these three materials one of them lies in the content of metoksil. The content of methoxyl found in banana
peel according to the research of is 4.43% (Hanum et al. 2012), while the content of metoxyl in papaya skin is 8.87%
(Widodo et al. 2011) and the methoxyl content of lime peel according to the results of was 7.02% (Perina et al.

189
2007). This metoxyl content will affect the ability of gel formation when applied to the making of edible film
(Prasetyowati et al., 2009). The process of making edible films also added plasticizer to overcome the fragile of the
film, so it will get a strong film, flexible, and not easily broken. Commonly used plasticizers are glycerol, sorbitol, and
poly ethylene glycol (PEG) (Khotimah et al. (2006).

Material and Methods

Sample preparation
fruit peel waste were collected from region of Engineering Faculty, University of Pembangunan
Nasional Veteran Jawa Timur, Indoensia. This research uses Completely Randomized Design, which was arranged
with factorial pattern, consist of two factors. The first factor was a pectin type consisting of three levels (pectin of lime,
papaya and banana peel). The second factor was glycerol concentration consisting of three levels (15%, 20% and
25%). The data obtained from the analysis is processed using Analysis of Variance.
Production of Pectin
The fruit peel waste was extracted at 70 ° C. for 2 hours using HCl 0.05 N. The suspension was filtered to added
ethanol 96% with a ratio 1: 1, then precipitated. The resulting precipitate was dried at 60 ° C. The dried pectin was
mashed. The test was yield, moisture, ash content (AOAC), Metoxyl and Equivalent Weight (Prasetyowati et al.,
2009), Galacturonic Acid and Esterification Degree (Sulihono et al., 2012)

Production of Edible Film

The 1.6% tapioca solution was heated using a temperature of 70 ° C for 25 minutes. Pectin1.4% solution was poured
into tapioca solution, then heated using magnetic strirrer at 70 ° C for 10 minutes. Concentrated glycerol 15% (v / w)
of pectin weight was added and heated at 70 ° C. for 10 min. The pectin and glycerol mixture was poured into a 10
cm x 10 cm x 0.2 mm mold and dried in an oven at 80 ° C. for 8 hours. Edible dry film taken from the mold and tested

Results and Discussion

The highest metoxyl levels were found on pectin of lime peel (3.53%) (Table 1) and the lowest methoxyl content was
found in banana peel pectin (2.72%). Methoxyl content is directly proportional to galacticonic acid. The highest
equivalent weight was in banana peel pectin (4168.88 mg) and the lowest equivalent weight was in lime peel pectin
(2174.77 mg).

Table 1. Results of Pectin Analysis

Pectin
Parameter
Lime peel Papaya peel Banana peel
yield (%) 18.6 ± 0.14 4.93 ± 0.05 3.15 ± 0.03
moisture (%) 6.35 ± 0.16 4.85 ± 0.16 4.42 ± 0.31
ash (%) 3.38 ± 0.29 5.41 ± 0.11 5.42 ± 0.40
methoxyl (%) 3.53 ± 0.00 2.76 ± 0.03 2.72 ± 0.00
equivalent weight (mg) 2174,77 ± 0.75 3333.99 ± 0.66 4168.88 ± 0.47
Galacturonic acid (%) 112.56 ± 0.03 83.96 ± 0.81 78.83 ± 0.01
Derajat esterifikasi (%) 17.81 ± 0.00 18.71 ± 006 19.64 ± 0.00

The highest yield of galacturonic acid was found in pectin of lime peel (112.56%) and the lowest was in banana peel
pectin (78.83%). The highest of esterification degree was found in banana peel pectin (19.64%) and the lowest was
in pectin of lime peel (17.81%) (Table 1). The degree of esterification is obtained from a comparison between the
methoxyl content and the degree of galacturonic acid.
The thickness of edible film ranged from 0.25 to 0.55 mm. The strength of edible film ranged from 10.15 to 12.56 kgf /
cm2. Measurement of tensile strength is useful to know the magnitude of force achieved to achieve maximum
attraction in every unit of film area to stretch or lengthen (Gontard et al., 1993). The result of elasticity of edible film
ranged from 29.52 to 32.71% (Table 1). In Figure 1 showed that the higher the concentration of glycerol, the value of
tensile strength decreases. The amount of residue present in the pectin is smaller and the bond formed between

190
galacturonic acid is blocked by its glycerol molecule. The entry of glycerol into the galacturonic acid polymer chain
will result in intermolecular interaction reduction so that the film matrix formed will decrease.(Gontard et al., 1993).

tensile strength
12,565 12,325 12,260
11,330 11,320 11,295
10,525 10,350 10,155

(kgf/cm2)
Pektin type
banana peel
papaya peel
lime peel

Gliserol concentration (%)

Figure 1. The relationship between pectin of fruit peel type and glycerol concentration to the tensile strength of edible
film.

Water vapor permeability rate (WVTR) should be as low as possible since an edible film or coating should retard
moisture transfer between the food and the environment, or between two components of a heterogeneous food
product. as compared to the films prepared from gluten extracted from wheat cultivars Raj 3765 using higher
concentration of glycerol, had WVTR (85.74 g/m2 d) lower than this research (Sharma et al 2017).

256,955
vapor transmission

242,175
232,750

167,295
(gr/m2.d)

142,080 147,675 Pektin type

124,315 127,215 130,825
banana
peel

Gliserol concentration (%)

Figure 2. The relationship between pectin of fruit peel type and glycerol concentration to the water vapor
transmission rate.

The velocity value of vapor transmission is inversely proportional to the thickness value, where the thickness of a film
the lower transmission rate value. The thickness value is influenced by the metoxyl content, the equivalent weight,
the galacturonic acid and the degree of esterification present in pectin. (Figure 2).

Conclusion
The best edible film obtained from pectin of lime peel with 15% glycerol concentration, where the treatment yielded
0.51 mm thickness, tensile strength 12,56 kgf / cm2, elasticity 32,57% and vapor transmission rate equal to 124,315
gr / m2 hr .

Acknowledgment
The authors would like to express their sincere thanks to the Directorate General of Higher Education, Ministry of
Education and Culture in Indonesia through the superior research college grant (PUPT 2016-2018)

191
References
Bourtoom, T. 2008. Edible Films and Coatings: Characteristics and Properties. International Food Research Journal.
15 (3): 1-2
Darmawan, K., Nainggolan, R.J. and Limbong, L.N. 2014. Method of Washing and Screening on Pectin Extraction
from Durian Skin. Journal of Food and Agriculture Engineering. Food Science and Technology. University of
Northern Sumatra. 2 (2): 105-110
Gontard, N., Guilbert S., Cuq, J.L. 1993. Water and Glycerol as Plasticizer Affect Mechanical
and Water Vapor Barrier Properties of an Edible Wheat Gluten Film. J. Food Sci. 58 (1) 206 – 211
Hanum, F., Kaban, I.M.D., Tarigan, M.A. 2012. Pectin Extraction from Banana Leather (Musa sapientum). Journal of
Chemical Engineering. University of Northern Sumatra. 1 (2): 21-26
Khotimah, K., Diana, P.S., Febrianing, D.K. 2006. Characterization of Edible Film from Pati cassava (Manihot
utilissima Pohl). Department of Biology Education. FMIPA. Yogyakarta State University
Maulidiyah., Halimatussadiyah, Susanti, F., Nurdin, M., Ansharullah. 2014. Pectin Isolation from Cocoa Fruit Leather
(Theobroma cacao L.) and Absorption Power Test against Copper (Cu) and Zinc Metals (Zn). Agroteknos
Journal. IV (2): 112-118
Perina, I., Satiruiani., Soetaredjo, F.E., Hindarso, H. 2007. Pectin Extraction from Various Types of Citrus Peels.
Widya Technique. 6 (1): 1-10
Prasetyowati, K.P., Sari, H., Pesantri. 2009. Pectin Extraction from Mango Leather. Journal of Chemical Engineering.
Faculty of Engineering. Sriwijaya University. 16 (4): 42-49
Sharma, N., 1 Khatkar, B. S., 1*Kaushik, R., 2 Sharma, P. and 3 Sharma, R.2017. Isolation and development of
wheat based gluten edible film and its physicochemical properties. International Food Research Journal 24(1):
94-101
Sulihono, A., Tarihoran, B., Agustina, T.E. 2012. Effect of Time, Temperature, and Type of Solvent Against Pectin
Extraction from Citrus Skin of Bali (Citrus maxima). Journal of Chemical Engineering. Sriwijaya University. 18
(4): 1-8
Widodo, L.U., Karaman, N., Candra K. 2011. Pectin from Papaya Fruit Leather. Journal of Chemical Engineering. 6
(1): 783-786

192
 FRC 2017: 142-099 Food Processing and Post-Harvest Technology

Effect of Maltodextrin, Tricalcium Phosphate and Glycerol Monostearate on Moisture Sorption

Characteristics of Jamun (Syzygium cumini L.) Pulp Powder

*Dey Paul, I., and Das, M.

Agricultural and Food Engineering Department, Indian Institute of Technology Kharagpur, Pincode-721302, West
Midnapore, West Bengal, India

Abstract
Syzygium cumini L. (jamun) is a seasonal and highly perishable medicinal fruit rich in antidiabetic and antioxidant
properties. Drying of seasonal fruits into powders is an effective way to develop a shelf-stable marketable product
with good handling properties. However, the primary obstacle in safe handling and storage of fruit powder is its
stickiness caused by the presence of low molecular weight sugars and organic acids. Food additives viz.,
maltodextrin (MD), tricalcium phosphate (TCP), and glycerol monostearate (GMS) are usually added to the pulp prior
to drying to induce desired flow properties in fruit powder. The objective of this work was to study the effect of these
additives on the moisture sorption property and color of jamun pulp powder. Fresh jamun pulp was mixed with MD,
TCP, and GMS at levels ranging from 1-15%, 0-2% and 1-3% of the pulp on dry basis, respectively. The mixture was
dried using microwave-convective hot air dryer at 70 ºC and 0.5 m/s air velocity using power density of 1 Watt/g.
Rotatable central composite design was used to make the formulations. The powder samples were analysed for
moisture sorption characteristics and overall color difference (∆E*). MD, TCP and GMS improved moisture sorption
characteristics and destroyed the color of jamun pulp after drying, but did not follow any specific trend. At humid
atmosphere (~80% RH), the rate of moisture sorption changed exponentially with time. The formulation containing
MD, TCP and GMS as 12.2%, 0.4%, and 1.4%, respectively, of the pulp (dry basis) produced minimum rate of
1.105x10-3 g water/min at 60 min, and was selected as the best suitable. Original color of jamun was moderately
deteriorated (∆E*= 6.98) in this formulation. The selected formulation could help in improved handling and storage of
the pulp powder.

Keywords: Jamun, dehydration, food additives, moisture sorption.

*Corresponding author’s email: indiradeypaul27@agfe.iitkgp.ernet.in

Introduction
Syzygium cumini L. (jamun) is a well-known medicinal fruit having antidiabetic and antioxidant properties. Due to
short seasonal availability and highly perishable nature, jamun is considered as underutilized. Drying of fruits to make
powder is a popular preservation technique as the handling, marketing and storage are more industrially feasible
compared to that of fruit juices. Hence, development of shelf-stable jamun pulp powder is necessary to widen the
scope of its application. However, preparation and storage of fruit powder is difficult because of the presence of low
molecular weight sugars and acids, which enhance its moisture sorption characteristics leading to poor flowability.
Drying aids, e.g., isolated protein and maltodextrin (MD) with different dextrose equivalents (DE) can help in
producing non-sticky free flowing powder. MD generally increases the glass transition temperature (T g) of the
product, thus, reducing stickiness vis-à-vis improving product stability. Anti-caking agents (tricalcium phosphate
(TCP), silicon dioxide, silicates, phosphates, etc.) are often added along with drying aids to improve flowability and
reduce caking tendency. Glycerol monostearate (GMS), a mostly used surfactant in food industry, could also be
added for attaining a puffed structure in fruit powder. However, concentration of these additives for any particular fruit
pulp to produce non-sticky fruit powder is dependent upon its chemical composition. The aim of the present study
was to find the suitable amount of MD, TCP and GMS to be added to jamun pulp to render the least moisture
sorption characteristics and color deterioration in the produced powder.

193
Material and Methods
Raw material and sample preparation
The fully matured jamun fruits were procured from the Technology market of IIT Kharagpur, West Bengal, India. The
pulp along with the skin was manually collected, made into paste using domestic grinder and stored in sterilized
containers at -30 ºC.
Prior to drying, the jamun pulp-skin paste was thawed and mixed with MD (DE < 20), TCP and GMS at different
concentrations obtained through rotatable central composite design (RCCD) (Design Expert Version 7.0.3., Stat-
Ease Inc., Minneapolis, USA) that provides as much information as a full factorial design but requiring much fewer
tests. The range of MD, TCP and GMS was kept as 1-15%, 0-2% and 1-3% of the pulp on dry basis, respectively
(Table 1), based on preliminary trials and available literature.
The blends were dried using microwave-convective hot air dryer (Enerzi Microwave Systems Pvt. Ltd., Karnataka,
India) at 70 ºC, 1 watt/g (W/g), 0.5 m/s air velocity. The dried products were obtained as flakes and ground into fine
powder using grinder. The samples were analyzed for moisture sorption both by static and dynamic methods, and
overall color difference (∆E*) with respect to (w.r.t.) control (C) that contained no additive.
Moisture sorption by static method
Hygroscopicity (HG) is the ability of food powder to absorb moisture from high RH environment. It was measured (3
replicates) using the isopiestic vapour transfer technique at 40 ºC under two RHs 75% and 100%, after the time
period of 2 and 5 days. The HG (%, on the basis of equilibrated mass of the sample), was calculated using equation
1.

(WI%+FW%)x 100
HG (%) = (1)
100 +WI%
(Final weight of the sample−Initial weight of the sample) x 100
Where, WI% =
Initial weight of the sample
FW% = Initial moisture content of the sample taken (wet basis).
Moisture sorption by dynamic method
Air at ~80% relative humidity (RH) was passed over the sample and the weight (w, g) was recorded at 30 min interval
till attainment of constant weight. The rate of moisture sorption, i.e., time t (min) was plotted against change in weight
divided by the taken time interval (∆w/∆t, g/min) following graphical differential method. Dynamic method provides
the kinetics of moisture sorption over the full exposure tenure. The regressed equation followed exponential nature
(discussed later) and, therefrom the rate of moisture sorption at 60 min (arbitrarily selected), RMS60, was evaluated to
judge the promptness in moisture sorption.
Overall color difference
The color parameters L*, a* and b* were measured (3 replicates) using Hunter colorimeter, and ∆E* w.r.t. C was
calculated by equation 2.

∆E* = [(∆L*)2 + ( ∆a*)2 + (∆b*)2]1/2 (2)

Where, ∆L*, ∆a* and ∆b* represent the difference of L*, a* and b* values between any sample (from R1 to R20) and
that of the control.

194
Results and Discussion

0.006

0.005 R1
∆w/∆t (g/min)

0.004 Control (C)

0.003

0.002

0.001

0
0 20 40 60 80
Time (t, min)

Ο Control: ∆w/∆t=0.006e-0.02t; R2=0.961

∆ R1: ∆w/∆t=0.008e-0.033t; R2=0.9872

Figure 1: Exponential relationship between time (t, min) and ∆w/∆t (g/min) of RCCD sample R1 and control (C).

Table 1: The HG, RMS60 and ∆E* of twenty RCCD samples and control (C)
Sample Composition (% on dry HG$ by static method (%) RMS60 (x103 ∆E*$
basis) g water/ min) (w.r.t C)
MD TCP GMS 75% RH, 40 ºC 100% RH, 40 ºC
2 days 5 days 2 days 5 days
R1 12.2 0.4 1.4 17.90 21.64 25.43 35.88 1.105 6.98
R2 8 1 3 19.52 22.84 27.06 37.21 1.531 7.63
R3 8 1 1 19.50 23.02 28.33 38.68 1.667 5.44
R4 3.8 0.4 1.4 20.37 24.27 30.25 40.39 1.897 5.83
R5 12.2 1.6 2.6 18.16 22.13 26.38 36.01 1.381 5.55
R6 3.8 1.6 2.6 18.90 23.11 27.01 37.03 1.352 7.43
R7 12.2 0.4 2.6 18.81 23.09 26.83 36.89 1.872 9.41
R8 8 1 2 18.75 23.09 26.25 36.42 1.765 7.97
R9 8 1 2 18.32 22.53 26.09 36.41 1.658 9.22
R10 8 1 2 18.91 22.89 27.18 37.48 1.730 8.36
R11 8 1 2 17.81 21.73 25.97 35.53 1.738 10.08
R12 15 1 2 17.86 21.42 25.82 35.84 1.370 12.01
R13 8 1 2 19.80 23.66 27.90 37.81 1.527 9.64
R14 8 1 2 20.14 24.12 29.21 38.98 1.727 11.28
R15 8 0 2 18.66 22.94 27.97 38.12 1.597 7.93
R16 1 1 2 18.29 22.71 26.85 37.34 1.790 9.22
R17 8 2 2 18.69 22.85 27.64 37.89 1.504 8.65
R18 12.2 1.6 1.4 19.09 23.09 27.27 37.52 1.706 7.43
R19 3.8 1.6 1.4 19.78 23.81 28.88 39.05 1.432 7.55
R20 3.8 0.4 2.6 18.75 23.03 27.84 37.92 1.740 8.69
C No additives 21.10 24.61 31.06 41.96 1.807 0
$mean of 3 replications, effect of composition on HG and ∆E* (F test) is positive (p<0.05)

195
The HG (shown in Table 1) of all the samples containing additives was lower compared to that of C. The samples,
R1, R11 and R12 showed maximum reduction, i.e., approximately 19% and 12%, and 19% and 14% decrease after 2
and 5 days exposure at 75% RH and 100% RH, respectively. Thus, higher concentration of MD (at and above 8%)
along with moderate to low combined concentration of TCP and GMS in the pulp powder helped in decreasing %HG.
MD possesses low HG (Tonon et al., 2008), and hence, increasing MD concentration in the powdered sample
decreases its moisture content, as opined by Abadio et al. (2004). Additionally, TCP and GMS In the present study
might have also increased the total solids to reduce HG.
In addition to HG over storage, the information on instant moisture sorption is vital in food processing, as it may
interfere in taking the measured amount of powder. The t vs. ∆w/∆t plot for all the samples including C followed
negative exponential relation, where for initial ≈ 80 min the rate was faster followed by a sluggish decrease. Figure 1
displays the representative profile along with the equation for R1 and C up to 75 min. The RMS60 calculated from the
equations for all the samples is shown in Table 1, wherein no specific trend with concentration of MD is exhibited.
Tze et al. (2012) and Patil et al. (2014), while working on spray-dried pitaya and guava fruit powder, respectively,
also observed that the moisture content showed no specific relation with the concentration of maltodextrin. Except R4
and R7, all other samples showed lower RMS60 values compared to that of C. Sample R1 showed slowest RMS60 of
1.105x10-3 g water/min, i.e., 38.8% decrease w.r.t. C with RMS60 of 1.807x10-3 g water/min.
Apparently, color of all the twenty samples containing additives were paler or lighter compared to that of C. Negative
influence of additives on the product color has also been reported by Kalil and Sial (1974). Table 1 shows that ∆E* of
R12 (containing 15% MD, 1% TCP and 2% GMS) was maximum (12.01) while R3 (containing 8% MD, 1% TCP and
1% GMS) exerted minimum deviation (5.44), indicating maximum and minimum deterioration respectively. Sample
R1 was observed to have moderate ∆E* value of 6.98.
Based on the lowest RMS60 (g water/min) and least %HG, sample R1 containing 12.2% MD, 0.4% TCP and 1.4%
GMS was selected to be the best formulation. The moderate alteration in product color was compromised to fulfill the
target of modifying moisture sorption characteristics of the product needed for desirable flow properties.

Conclusion
It can be concluded that MD, TCP and GMS influence moisture sorption characteristics and deteriorate the color of
jamun pulp after drying, but follow no specific trend perhaps because of the concomitant contribution by the additives.
Sample R1 containing 12.2% MD, 0.4% TCP and 1.4% GMS showed least rate of moisture sorption and %HG, with
moderate change in color. The low moisture sorption of the selected formulation will help in improving the handling
and storage of the product.

References
Abadio, F. D. B., Domingues, A. M., Borges, S. V. and Oliveira, V. M. 2004. Physical properties of powdered
pineapple (Ananas comosus) juice––effect of maltodextrin concentration and atomization speed. Journal of
Food Engineering 64(3): 285-287.
Kalil, M. A. and Sial, M. B. 1974. Spray drying of mango juice powder. Mesopotamia Journal of Agriculture 9(1/2): 47
56.
Patil, V, Chauhan, A. K. and Singh, S. P. 2014. Influence of spray drying technology on the physical and nutritional
properties of guava powder. International Journal of Current Microbiology and Applied Sciences 3(9): 1224-
1237.
Tonon, R. V., Brabet, C. and Hubinger, M. D. 2008. Influence of process conditions on the physicochemical
properties of açai (Euterpe oleraceae Mart.) powder produced by spray drying. Journal of Food
Engineering 88(3): 411-418.
Tze, N. L., Han, C. P., Yusof, Y. A., Ling, C. N., Talib, R. A., Taip, F. S. and Aziz, M. G. 2012. Physicochemical and
nutritional properties of spray-dried pitaya fruit powder as natural colorant. Food Science and
Biotechnology 21(3): 675-682.

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Optimization of Pretreatment Conditions and Drying Temperature in White Taro

[Colocasia esculenta (L.) Schott] Flour Production

1*Lirio, A.L.C., 2Reginio, F.C., Jr., 1Ignacio, M.C.C. and 1Dantes, P.T.

1Agricultural and Bio-Process Division, Institute of Agricultural Engineering, College of Engineering and Agro-
Industrial Technology, University of the Philippines Los Baños, College, Laguna, 4031, Philippines
2Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines
Los Baños, College, Laguna, 4031, Philippines

Abstract
Nowadays, flour plays an essential role in the Philippines since flour products are used as substitute staple for rice;
however, wheat flour is not readily available in the country. The starchy characteristic of taro makes it a potential
alternative source of flour. Hence, this study optimized the drying temperature, concentration of sodium metabisulfite,
and cutting thickness for the production of taro flour using response surface methodology. The values for the
independent variables were 50°C, 60°C and 70°C for the drying temperature, 0.01%, 0.05% and 0.1% for the
concentration of sodium metabisulfite, and 3 mm, 4 mm and 5 mm for the cutting thickness. The Box and Behnken
design with three levels was used as the experimental design with 15 test runs. Drying characteristics,
physicochemical properties, and sensory attributes were the dependent variables to determine the optimized pre-
treatment conditions and drying temperature. Analysis of variance showed that independent variables had no
significant difference among each other (p<0.05). As a result, the optimized values of drying temperature,
concentration of sodium metabisulfite, and cutting thickness were 50°C, 0.076%, and 3.665 mm, respectively. The
optimized taro flour was compared to commercially available cassava flour in terms of its physicochemical properties,
sensory attributes, and proximate composition. Using paired t-test of two samples for means, among the
physicochemical properties, pH and lightness index of both flours have significant differences (p<0.05). Sensory
analysis showed that cassava flour was found to be more acceptable than taro flour. However, the proximate
composition of optimized taro flour was more comparable to wheat flour than to cassava flour. The study has shown
that pretreatment conditions and drying temperature of taro flour production can be optimized to improve the quality
characteristics of the product.

Keywords: physicochemical properties, proximate analysis, sensory attributes.

*Corresponding author’s email: analuisalirio@yahoo.com

Introduction
In the Philippines, pasta, noodles, white bread, and biscuits are used as alternatives for rice due to its increasing
price (Moya & Casiwan, 2006). Though wheat flour is the most common flour in the Philippines, it is not readily
available in the country. Wheat flour in the Philippines is imported from Turkey, and the wheat used by flour
manufacturers are from US, Canada, and Australia (Miller Degirmerci, 2015). Since the supply of wheat flour is
lacking, food manufacturers have sought alternative solutions to this problem. One promising substitute to wheat is
taro. Based on the United States Department of Agriculture [USDA] National Nutrient Database, taro has high protein
content and dietary fiber. Furthermore, if the flour being used to produce these products has vitamins and minerals,
the victory against hunger and malnutrition will be attainable (Miller Degirmenci, 2015).

The main objective of this study was to optimize the pretreatment conditions and drying temperature in white taro
flour production and to compare the physicochemical properties and sensory attributes of optimized taro flour and
commercially available cassava flour.

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Materials and Methods
3.1. Sample Preparation This page is intentionally left blank

Taro tubers were bought from a supplier in Los Banos, Laguna dry market. In each trial, approximately 500grams
were used.

3.2. Experimental Design

Box and Behnken Design was used with three independent variables, namely: the percent solution of sodium
metabisulfite in pre-treatment, the thickness of the taro strip in pre-treatment, and the drying temperature of taro.
Dependent variables were drying rate, final moisture content, bulk density, lightness, color, aroma, dryness in texture,
particle interaction in texture and general acceptability.

Table 3-1. Coded values for the independent variables.

X Solution of Sodium Thickness of Cut, t (mm) Air Temperature, T (°C)
Metabisulfite, S (%)
-1 0.01 3 50
0 0.05 4 60
1 0.1 5 70

3.3. Taro Flour Production

Initially, taro tubers were peeled, and cut into specific dimension that differs in thickness. In pretreatment procedure,
taro strips with different thickness were soaked for 5 minutes in different sodium metabisulfite concentration. After
such, drying of taro strips was done in different drying temperatures depending on the set-up. Dried taro was then
subjected to grinder in order for it to be pulverized. Lastly, to ensure fineness of the taro flour, it was sieved through a
300 micromesh screen.

3.4. Determination of Physicochemical Properties of Taro Flour

Lightness determination was obtained using Minolta Chromameter. On the other hand, in pH determination, pH meter
was used. For the bulk density determination, one (1) gram of taro flour was transferred to a graduated cylinder with
ten (10) mL of distilled water at ambient temperature.

3.5. Sensory Evaluation of Taro Flour

It was done using quality scoring method. 30 untrained panelists evaluated the color, aroma, dryness, particle
interaction and overall acceptability of the samples in a 15 cm line scale. Each received 6 subsets of samples in
incomplete block design order. Zero scores corresponded to white, imperceptible, moist, loose particles and
unacceptable, and fifteen were light brown, perceptible, dry, clumpy and extremely acceptable, for color, aroma,
dryness, particle interaction and overall acceptability, respectively.

3.6. Statistical Analysis of Taro Flour

The study made use of analysis of variance (ANOVA), regression analysis and regression coefficient for the
calculations of the optimum drying condition. Response surface methodology (Design Expert 10) was used to
establish the levels of process variables to optimize the taro flour production conditions. Analysis of variance and
Duncan’s New Multiple Range Test (DMRT) was used to determine the effects of the independent variables.
Regression analysis was used to determine the relationship of the independent variables to the dependent variables,
and regression coefficients from the 15 experimental runs that were obtained to get the optimum drying parameters.

3.7. Verification of Optimum Pretreatment Conditions and Drying Temperature

The optimum drying conditions were verified to assess its reliability and accuracy.

3.8. Comparison of Taro and Cassava Flour

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3.8.1 Physicochemical analysis of Taro and Cassava Flour
The physicochemical properties, such as pH, bulk density and light This page is intentionally left blank

ness, of taro and cassava flour were determined.

3.8.2. Sensory Evaluation of Taro and Cassava Flour
Fifteen (15) untrained panelists evaluated the sensory attributes of the optimized taro flour and commercially
available cassava flour using the same method and evaluation sheet as described in the previous section.
3.8.3. Proximate Analysis of Taro and Cassava Flour
Proximate composition included the moisture, ash, crude protein, crude lipid, crude fiber, and digestible carbohydrate
content of the taro and cassava flour.
3.8.4 Statistical Analysis for Comparison of Taro and Cassava Flour
Paired t-test of two samples for means was used to determine the comparison of the taro and cassava flour based on
its physicochemical properties, proximate composition and sensory attributes.

Results
The lightness, pH and bulk density was on the range of accepted values for the wheat flour’s physicochemical
properties. Based on ANOVA, all of the dependent variables had no significant (p<0.05) differences. Therefore, the
values of the dependent variables were not significantly (p<0.05) different from the means. For the optimization of
independent variables, RSM (Design Expert 10®) was used. The generated values of optimized independent
variables were 50ᵒC, 3.67 mm and 0.076% for drying temperature, cutting thickness and concentration of sodium
metabisulfite, respectively.

After optimizing the independent variables in taro flour production, the taro flour was then compared to commercially
available cassava flour. Based on ANOVA, the lightness index, and pH had significant (p<0.05) difference from each
other, while bulk density had no significant (p<0.05) difference. Sensory attributes of taro and cassava flour were also
compared. All of the sensory attributes were significantly (p<0.05) different from the means. Based on the results of
sensory evaluation, panelists perceived cassava flour as more acceptable than taro flour. Meanwhile, of all the
proximate compositions, only crude fat was not significantly (p<0.05) different from the means.

Discussion
Previous studies for drying rate showed that temperature and cutting thickness had an effect to the drying rate, which
in this study had the same results. But for sodium metabisulfite concentration, it was different. Difference may be due
to difference in span of time it was soaked. In addition, sodium metabisulfite may restrict the mass transfer of
moisture.

Lightness was affected inversely by temperature, while percent sodium metabisulfite and cutting thickness had no
specific trend. Moreira et al. (2014) cited that increase in drying temperature would yield low value in lightness. This
is due to partial non-enzymic browning that had occurred during oven drying. Meanwhile, sulfites could increase in
value of lightness if the percent sulfite was increased. An increase in cutting thickness showed an increase in
lightness index. Non-enzymic browning would occur less with an increase in thickness since it would only occur on
the outer surface that is exposed to drying (Omidiran et. al, 2015).

In previous studies, drying temperature, cutting thickness and percent of sodium metabisulfite have a significant
effect on the bulk density. The variation in the moisture contents of the taro flour may have caused the difference in
the effect of the independent variables since bulk density is affected by the moisture content of the produce.

Increase in cutting thickness and percent sodium metabisulfite would increase the pH value. While in drying
temperature, no specific trend was showed. Based on Buckman et al. (2014), pH should decrease with increasing
value of sulfite since sodium metabisulfite was likely to be a little acidic with a value of 3.9 to 4.4. But in contrary, the
pH of the taro flour increased with the sodium metabisulfite in which pH of taro might cause the difference.

199
 Cutting thickness, drying temperature, and percent sodium metabisulfi This page is intentionally left blank

te had no specific effect on the overall acceptability of taro flour. Related research studies used sensory evaluation
when the flour was used as an ingredient to make products such as biscuits, breads,etc., thus overall acceptability
has limited or no information when it comes to sensory evaluation of flour alone.

Summary and Conclusion

Based on the results of proximate analysis, the composition of optimized taro flour was comparable to wheat flour but
further treatments can be done to substitute the product to wheat flour.

For future studies, it is recommended to use different drier since an error had occurred on the sensing of the
temperature inside the drier and this might affect further studies. If possible, there should be one working area to
ensure continuity of the study. Equipment should also be updated to ascertain highest efficiency of the machine.
Mechanized equipment in slicing and peeling should be used to guarantee accuracy of the experiment. The effects of
the environmental conditions, such as humidity and temperature should be accounted since it could increase or
decrease the moisture in the samples if subjected to ambient conditions.

Acknowledgments
I would like to thank my family (Manuel Luis, Angelo, Andrei and Maricar), friends and mentors.

References
Buckman, E.S., Plahar, W.A., Oduro, I.N., & Carey, E.E. 2014. Effects of sodium metabisulphite and blanching
pretreatments on the quality characteristics of yam bean flour. British Journal of Applied Science and
Technology Article no.BJAST.2015.074
Miller Degirmenci. 2015. Grain and flour market in the Philippines. Retrieved on October 20, 2015 from
http://en.millermagazine.com/grain-and-flour-market-in-philippines/
Moreira, R., Chenlo, F., Torres, M.D., Rama, B., & Afure, S. 2015. Air drying of chopped chestnuts at several
conditions: effect on colour and chemical characteristics of chestnut flour. International Food Research
Journal 22(1). pp. 407-413
Moya, P., Casiwan, C.B. 2006. Why does the Philippines import rice?: Meeting the challenge of trade liberalization.
International Rice Research Institute. p.43
Omidiran, A.T., Sobukola, O.P., Sanni, A. 2012. Effect of some processing variables on the quality of attributes of
yam flour. Journal of Agricultural Science and Technology. pp. 621-626

200
 IFRC 2017: 158-132 Food Processing and Post-Harvest Technology

The Effect of Pectinase, Glucoamylase and Cellulase Enzymes on the Extraction Yield of Roselle Petals.

Mardiah1,*, Noli Novidahlia1, Ma’rifat Khoirunnisa1, and Hanafi2

1Department of Food Techology and Nutrition, Faculty of Halal Food Science, Djuanda University, Bogor - Indonesia
2Polytechnic of Bogor Chemical Institute, Bogor - Indonesia

Abstract
Rosella contains anthocyanin pigments that can act as antioxidants and natural dye substances in food that were
beneficial and safe to the health. The objective of this research was to study the effect of three enzymes, pectinase,
cellulase, and glucoamylase, in increasing the extraction yield. The research used fresh and dried purple roselle
petals, then they were extracted by aquadest solvent with ratio of 1:4 and citric acid 1%, maltodextrin 10%, and
added pectinase (P) of 1000 ppm, pectinase and cellulase (P:C) of 500:500 ppm, pectinase and glucoamylase (P:G)
of 500:500 ppm, and pectinase, cellulase and glucoamylase (P:C:G) of 333:333:333 ppm to the extract.
Determination of best treatment based on residue extract, anthocyanin analysis and pH value. The results showed
that fresh rosella extract with P enzyme has yield value of 7.60% and was not significantly different to the extract with
PCG enzymes which has yield of 7.37%. Dried rosella extract with PCG enzymes has the highest yield which was
22.10% compared to the control of 12.96%. The anthocyanin content of fresh roselle extract and its control were
141.05 ppm and 131.89 ppm, respectively, while that of dried roselle extract and its control were 23.51 ppm and
23.25 ppm. These data showed that the PCG enzymes combination can increase the extraction yield of fresh and
dried roselle petals.

Keywords: rosella, extraction yield, enzyme, anthocyanin.

*Corresponding author’s email: mardiah@unida.ac.id, mardiahrohman@yahoo.com

Introduction
Roselle (Hibiscus sabdariffa L.) is a tropical plant that grows in Indonesia which has red or purple flower petals that
contain an important compound that is anthocyanin pigments that form the flavonoid that acts as an antioxidant.
Roselle petals contain high levels of anthocyanin, so roselle petals have the potential to be utilized as a source of
natural dyes on healthful food ingredients.
One study of roselle petals extraction has been conducted by Subagyo (2009) which using mixture of 95% water and
alcohol with the addition of citric acid 1% and macerated at 60oC for 3.5 hours and resulted the highest anthocyanin
level of 318.6708 mg/L. Another studies are also conducted by Mardiah (2015) and Setiawan (2012), which using
mixture of roselle petals and water as solvent with ratio of 1: 4, and then drying with spary dryer after adding 10%
maltodextrin as a filler. However, it is also mentioned that roselle petals extraction with spray drying method
resulting in low yields. This is due to their residues (pectins) which are attached to the wall of spray dryer that lead to
the decreasing in the extraction yield. Based on these, researchers start to use several enzymes to increase the
extraction yield of roselle petals.
Utilization of enzymes especially pectinase has been done by some researchers. Iriani, et al (2005) reported that
addition of pectinase, which degrades the pectin compounds, can increase the yield of mango juice. The addition of
pectinase at incubation time of 60 minutes, concentration of 1000 ppm and incubation temperature of 55 oC resulted
94% yield. The utilization of enzymes was also reported by Mintarti (2006), which used extraction method from fresh
vanillin fruit using 3 commercial enzymes namely pectinase, β-glucosidase, and cellulase and resulted in increasing
the extraction yield. The objective of this research was to study the effect of three enzymes, pectinase, cellulase, and
glucoamylase, in increasing the extraction yield of fresh and dried roselle petals.

201
Research Method
Fresh and dried roselle petals were used in this research which was obtained from West Palimanan, Cirebon,
Indonesia. Pectinase, cellulase and glucoamylase enzymes were taken from Sinobios Bioscience Company China,
aquadest, citric acid, maltodextrin and othres chemicals for analysis. This research consists of two stages of
extraction and analysis methods.
Extraction method
The anthocyanin extraction process from fresh and dried roselle petals were conducted using aquadest solvent with
ratio of 1:4. Then, added several treatments, namely pectinase enzyme (P) of 1000 ppm, mixture of pectinase and
cellulase enzymes (PC) of 500:500 ppm, mixture of pectinase and glucoamylase enzymes (PG) of 500:500 ppm,
mixture of pectinase, cellulase and glucoamylase enzymes (PCG) of 333:333:333 ppm, and without enzyme as
control. The extraction was conducted at temperature of 50°C for 60 minutes. The resulted extract then was filtered
and dried with a spray dryer, where the inlet and the outlet temperatures were 150 °C and 80 °C, respectively.
Analysis method
Chemical analysis of pH value using pH meter and total determination of anthocyanin with differential pH method
using UV-VIS spectrophotometry were measured in this research.
Result and Discussion
Extraction Yield
Note:
Extraction yield of fresh roselle petals P = Pectinase enzyme (1000 ppm)
7.60a P:C = Pectinase enzyme : Cellulase
8.00 7.37ab
6.56bc enzyme (500:500 ppm)
5.84c 5.80c
Yield value (%)

P:G =Pectinase enzyme :

6.00 Glucoamylase enzyme
4.00 (500:500 ppm)
P:C:G = Pectinase enzyme:Cellulase
2.00 enzyme : Glucoamylase
(333:333:333 ppm)
0.00 T.E = Control (0 ppm)
P P:S P:G P:S:G T.E
Enzyme treatment

Figure 1. Extraction Yield of Fresh and Dried Roselle Petals

According to Iriani et al (2005), the addition of pectinase will degrade pectin compounds so that in the pressing
process will more easily come out. Chaovanalikit et al (2012) and Landbo et al (2006), explained that the use of
pectinase enzymes can increase the extraction yields and decrease the turbidity. Pectinase enzymes may break
pectin bonds in tissue cells or degrade cell walls to increase procyanidin extract.
Extraction yield of dried roselle petals
25.00
Note:
22.10a P = Pectinase enzyme (1000 ppm)
18.54b P:C = Pectinase enzyme : Cellulase
20.00 17.30c enzyme (500:500 ppm)
Yield value (%)

14.79d P:G =Pectinase enzyme :

15.00 12.96e Glucoamylase enzyme
(500:500 ppm)
10.00 P:C:G = Pectinase enzyme:Cellulase
enzyme : Glucoamylase
5.00 (333:333:333 ppm)
T.E = Control (0 ppm)
0.00
P P:S P:G P:S:G T.E
enzyme treatment
Figure 1. Extraction Yield of Dried Roselle Petals

202
The results are also supported by Mintarti (2006) and Hanafi (2009), which stated that extraction by using more than
one enzyme can produce higher yields. The using of the three enzymes will cause a combination to degrade the cell
wall of plants to be better than just using single enzyme, so the yield value of the extract becomes higher. The cell
wall of plants is a very complex structure, which contains cellulose, xylan, pectin and others. Degradation of the cell
wall would be better done through a combination of several enzyme activities such as cellulase, xylanase and
pectinase than single enzyme.

Anthocyanin content
Table 1. Total anthocyanin content of fresh roselle petals
Fresh roselle petals Dried roselle petals
Enzyme
Anthocyanin content (mg/L) Anthocyanin content (mg/L
P 101.29±0.53e 26.40±0.46c
P:C 184.02±0.15 a 35.09±0.04a
P:G 156.64±1.30b 33.20±0.80b
P:C:G 141.05±0.56c 23.51±0.53d
T.E 131.89±0.76d 23.25±0.08d
Different superscripts in a column means different significantly (P<0.05)
Note:
P = Pectinase enzyme (1000 ppm)
P:C = Pectinase enzyme : Cellulase enzyme (500:500 ppm)
P:G = Pectinase enzyme : Glucoamylase enzyme (500:500 ppm)
P:C:G = Pectinase enzyme:Cellulase enzyme : Glucoamylase (333:333:333ppm)
T.E = Control (0 ppm)

Table 1 shows fresh roselle petals with addition of enzymes. The addition of enzyme combination (P:C, P:G, and
P:C:G) will increase the anthocyanin content of fresh roselle extract, while fresh roselle petals with addition of P:C
enzymes has the highest anthocyanin content (184.02±0.15 mg/L) than the others. However, fresh roselle with P
enzyme produces the lowest total anthocyanin content, even compared to the control. This is due to the use of
pectinase may decrease the anthocyanin content in roselle extract which has been reported by Chaovanalikit et al
(2012) that mentioned the pectinase can hydrolyze β1-2 cyanidin-3-sophoroside and cyanidin-3-glucosylrutinoside
glucoside groups to cyaniding-3-glucoside and cyaniding-3-sikosida which leads to the decreasing in stability of
anthocyanin.

Conclusion
The use of pectinase enzyme with 1000 ppm concentration can increase the extraction yield of fresh roselle petals as
much as 7.60%. This result was not significantly different with mixture of pectinase, cellulase and glucoamylase
which has yield of 7.37%. However, the levels of anthocyanin which was obtained by treatment of pectinase,
cellulase and glucoamylase enzymes produce higher anthocyanin value of 141.05 ppm compared to the treatment of
pectinase enzyme of 101.49 ppm. While, on the extract of dried roselle petals, the mixture of pectinase, cellulase and
glucoamylase enzymes has the highest extraction yield of 22.1045% and anthocyanin level of 23.51 ppm.

Acknowledgement
This research was supported by Grant of PUPT Ministry of Research, Technology, and Higher Education, Republic
of Indonesia

203
References

Chaovanalikit., A, Mingmuang, T., Kitbunluewit, N., Choldumrongkool, J., Sondee, and S, Chupratum. 2012.
Anthocyanin and Total Phenolics Content of Mangosteen and Effect of Processing on The Quality of
Mangosteen Products. International Food Research Journal 19 (3): 1047-1053
Hanafi. 2009. Sifat Aktivitas Enzim Sellulase dan Pektinase dalam Mendegradasi Dinding Sel Tanaman Untuk
Tujuan Ekstraksi Pigmen. Akademi Kimia Analis Bogor. Bogor
Iriani, SE., Gumbira, E., Suryani, A., Setyadjit. 2005. Pengaruh Konsentrasi Penambahan Pektinase dan Kondisi
Inkubasi terhadap Rendemen dan Mutu Jus Mangga Kuini (Mangifera odorata Griff). J Pascapanen 2(1):11-
17.
Landbo, AK., Pinelo, M., Vikbjerg, AF., Let, M.B., and Meyerm A.S. 2006. Protease-assisted Claification of Black
Currant Juice: Synergy With Other Clarifying Agents and Effects on The Phenol Content. Journal of
Agricultural and Food Chemistry 54: 6554-6563.
Mardiah. Zakaria, RF., Prangdimurti, E., Damanik, R. 2015. Perubahan Kandungan Kimia Sari Rosela Merah dan
Ungu (Hibiscus sabdariffa.) Hasil Pengeringan Menggunakan Cabinet Dryer dan Fluidzed Bed Dryer. J
Teknologi Industri Pertanian 25(1): 1-7
Mintarti, SI. 2006. Ekstraksi Vanili Secara Enzimatik dari Buah Vanili (Vanilla planifolia ANDREWS) Segar. Skripsi.
Institut Pertanian Bogor. Bogor
Setiawan, B. 2012. Pembuatan Tablet Efervesen dari Ekstrak Kelopak Bunga Rosela (Hibiscus sabdariffa L.)
sebagai Food Suplement. Skripsi. Jurusan Teknologi Pangan dan Gizi. Fakultas Ilmu Pangan Halal.
Universitas Djuanda Bogor.

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 IFRC 2017: 166-150 Food Processing and Post-Harvest Technology

Effect of Emulsifier at Different Concentrations on the Properties and Characteristics of Biodegradable Films
Based on Gelatin with Palm Oil for Food Packaging Application

1Zazalli, S.A., 1Nabilah, B., 2de la Caba, K., 2Guerrero, P. and 1,3*Nur Hanani, Z.A.

1Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43000 UPM
Serdang, Selangor, Malaysia.
2BIOMAT research group, Department of Chemical and Environmental Engineering, University of the Basque
Country (UPV/EHU), Polytechnic School, Plaza Europa 1, 20018 Donostia-San Sebastián, Spain.
3Halal Products Research Institute, Universiti Putra Malaysia, 43000 UPM Serdang
Selangor, Malaysia.
Abstract
Biodegradable materials derived from polysaccharides, proteins and lipids are edible and have gained high concern
nowadays due to the abilities and potential that can replace conventional plastics, thus it also can turn as food
contact edible coatings and/or films. This work focused on the preparation and characterization of biodegradable
films based on fish gelatin with enhanced flexibility by the incorporation of palm oil and different contents of an
emulsifier. Emulsifiers act as surface active agents that capable in reducing the surface tension of the water-lipid
interface or the water-air surface. The effect of emulsifier contents on physical, optical and mechanical properties of
fish gelatin films was analyzed. The emulsifier addition caused a significant increase in film flexibility, without
affecting other functional properties, such as water vapor permeability, solubility or color. Morphology analysis
showed a better distribution of palm oil by using the emulsifier, indicating the beneficial effect of the combination of
palm oil and emulsifier to manufacture flexible films.

Keywords: Gelatin films, palm oil, emulsifier, flexibility, biodegradable films.

*Corresponding author’s email: hanani@upm.edu.my

Introduction
Gelatin has been extensively studied on account of its film-forming ability and its renewable, biocompatible and
biodegradable nature. The addition of non-polar or hydrophobic substances (fats, fatty acids and oils) is widely used
in gelatin films to increase the water vapour barrier property (Tongnuanchan et al., 2016). Palm oil is a cheap and
abundant hydrophobic substance in the Southeast Asia and considered as potential non-polar candidate to
incorporate with biodegaradable films.
However, gelatin films manufactured with the incorporation of non-polar substances required emulsifiers to reduce
the surface tension of the water-lipid interface (Xu et al., 2014). The purpose of emulsifier is to produce a colloidal
suspension (emulsion) from the mixture of two immiscible liquids phases (Carpiné et al., 2016). The aim of this study
was to analyze the effect of emulsifier on the physical and mechanical properties of fish gelatin films with palm oil.

Materials and methods

Materials
Gelatin derived from fish skin was obtained from Scienfield Expertise Plt. (Sabah, Malaysia) Glycerol was obtained
from Sigma Aldrich (St. Louis, MO, USA). Palm oil and Tween 20 were purchased from Spectrum Chemical
Manufacturing Corp. and R & M (Essex, UK), respectively.

Film preparation
Gelatin powder was mixed with distilled water to form film solutions of 6% (w/v) protein concentration. The mixture
was heated at 80°C followed by adding 30% (w/w) of glycerol, 25% of palm oil (w/w) based on protein content and

205
Tween 20 from 5 to 30% (w/w, based on oil content). Films were prepared by casting the emulsions onto a plates
and were dried.

Optical properties
The instrumental measurement of film color was carried out with a Colorimeter Minolta Chroma Meter CR-300.

Mechanical properties
Tensile strength (TS), and elongation at break (EAB) of films were measured five replicates according to the standard
method of ASTM D638-77 (ASTM, 1977).

Water vapour permeability (WVP)

WVP was measured according to the ASTM E-96 (ASTM, 2000) standard method.

Water solubility
Solubility of film was determined according to method of Nur Hanani et al., (2012).

Morphological properties
Microstructure of the film surface was evaluated by using scanning electron microscopy (SEM).

Statistical analysis
Data obtained from the experiment were analyses using Minitab 16 software by conducting one-way analysis of
variance (ANOVA).

Results and discussion

Film color
Results from Table 1 showed that the film color was not affected by the emulsifier used, since a and b values did not
changed significantly. However, as emulsifier concentration increased, L values slightly decreased (P≤0.05).

Mechanical properties
Increasing emulsifier contents caused a slight reduction (P≤0.05) in TS values; however, a significant (P≤0.05)
increase of EAB was observed. The good compatibility promoted by the emulsifier increased oil-gelatin interactions,
decreasing interactions among protein chains and increasing chain mobility and thus, flexibility (Xu et al., 2014).

Water vapor permeability

The increase of free volume among protein chains also facilitated water vapor diffusion through the films and WVP
values increased (P≤0.05) as indicated in Figure 1. Glycerol can be injected between protein chains because of its
relatively small hydrophilic molecule which can decrease the intermolecular attractions. While Tween 20 is a nonionic
surfactant with a hydrophilic-lipophilic balance (HLB) of 16.7, it was the most hydrophilic surfactant, which gave
higher permeability values (Rodríguez et al., 2006).

Water solubility
Results in Figure 2 showed that the solubility of the film increased as the emulsifier concentrations increased.
However, films maintained their integrity after being immersed in water and solubility values were lower than 30%.

Film morphology
From SEM micrographs (Figure 3), observations indicated that particle size and lipid globule distribution were seen to
be reduced as the emulsifier concentration increased. It was observed that the surface of the film with 0% emulsifier
had a rough matrix and exhibited lipid granules. Meanwhile, films 30% emulsifier showed a descending trend as the
emulsifier contributed to a more homogeneous distribution of smaller lipid globules in the film matrix, probably due to
a better distribution or dissolution of the oil droplet in the gelatin matrix.

206
 Table 1. Thickness, optical and mechanical properties of gelatin-palm oil films.
Emulsifier Thickness
L* a* b* TS (MPa) EAB (%)
(%) (mm)
0 0.41 ± 0.03a 90.59 ± 0.21a 1.67 ± 0.25a - 3.59 ± 0.30a 32.73 ± 1.00a 196.47 ± 60.48c
5 0.40 ± 0.02a 90.38 ± 0.09ab 1.67 ± 0.43a - 3.55 ± 0.27a 29.82 ± 3.04ab 318.53 ± 70.07b
10 0.40 ± 0.03a 90.34 ± 0.08 ab 1.67 ± 0.19 a - 3.41 ± 0.20 a 27.25 ± 3.25ab 331.87 ± 29.14ab
15 0.41 ± 0.01a 90.32 ± 0.05 ab 1.85 ± 0.16 a - 3.44 ± 0.30 a 26.93 ± 3.03ab 341.30 ± 25.25ab
20 0.41 ± 0.02a 90.04 ± 0.14 bc 1.55 ± 0.58 a - 3.18 ± 0.59 a 26.06 ± 3.98ab 366.33 ± 21.05ab
25 0.41 ± 0.03a 89.82 ± 0.34 c 1.78 ± 0.44 a - 3.25 ± 0.34 a 25.52 ± 5.18ab 378.87 ± 12.45ab
30 0.43 ± 0.03 a 88.69 ± 0.21 d 1.30 ± 0.39 a - 2.79 ± 0.65 a 20.94 ± 2.16b 434.67 ± 10.62a
Different letters in the same column indicate significant differences (P≤0.05).

a
WVP 10-7 (g mm s-1 m-2

20
ab
15 bc
c c c
c
Pa-1)

10

0
0 5 10 15 20 25 30
Emulsifier content (%)

Figure 1. WVP values of gelatin films as a function of emulsifier content.

40
Water solubility (%)

ab ab ab a a
30 bc
c
20

10

0
0 5 10 15 20 25 30
Emulsifier content (%)

Figure 2. Solubility values of gelatin films as a function of emulsifier content.

0% Tween 20 15% Tween 20 30% Tween 20

Figure 3. SEM images of gelatin films as a function of emulsifier content.

207
Conclusion
Different contents of Tween 20 were used in gelatin formulations in order to improve the compatibility between the
components and reduce the brittleness of gelatin films. Tween 20 provided good compatibility among the hydrophilic
components, gelatin and glycerol, and the lipophilic component, palm oil. This good compatibility resulted in the
increase of elongation at break up to values higher than 400%, maintaining a tensile resistance higher than 20 MPa.
This behavior highlights the potential use of these materials for their application as flexible films.

Acknowledgments
The authors would like to thank Universiti Putra Malaysia (UPM) for financial support.

References
ASTM. 1977. Standard Test Method for Tensile Properties of Plastics (Metric). Annual Book of ASTM Standards D
638M-77a.
ASTM. 2000. E 96-00. Standard test method for water vapour transmission of materials. Annual Book of ASTM
Standards, Philadelphia, USA, pp. 1–8.
Carpiné, D., Dagostin, J. L. A., de Andrade, E. F., Bertan, L. C. And Mafra, M. R. 2016. Effect of the natural
surfactant Yucca schidigera extract on the propertiesof biodegradable emulsified films produced from soy
protein isolate and coconut oil. Industrial Crops and Products 83:364-371.
Nur Hanani, Z. A., Roos, Y. H. and Kerry, J.P. 2012. Use of beef, pork and fish gelatin sources in the manufacture of
films and assessment of their composition and mechanical properties. Food Hydrocolloids 29: 144-51.
Rodríguez, M., Osés, J., Ziani, K. and Maté, J. I. 2006. Combined effect of plasticizers and surfactants on the
physical properties of starch based edible films. Food Research International 39(8): 840–846.
Tongnuanchan, P., Benjakul, S., Prodpran, T., Pisuchpen, S. and Osako, K. 2016. Mechanical, thermal, and heat
sealing properties of fish skin gelatin film containing palm oil and basil essential oil with different surfactants.
Food Hydrocolloids 56: 93-107.
Xu, J., Xia, Y., Qiao, C. D., Zhu, W., Wang, Y. and Li T. D. 2014. Solid-state structure of gelatin-mono epoxy
terminated polydimethylsiloxane polymer: Effect of electrostatic and hydrophobic interactions. Colloid Surface
B 123: 945-50.

208
 IFRC 2017: 170-126 Food Processing and Post-Harvest Technology

Low Fat Coconut Flour as a Coconut Milk Powder Supplement for Improving Health and Reducing Cost of
Product

1*Dharmasena, D. A. N., 1Herath, H.M.T.K. and 2Madujith, T.

1Department of Agricultural Engineering, Faculty of Agriculture, University of Peradeniya, 20400 Sri Lanka.
2 Department of Food Science & Technology, Faculty of Agriculture, University of Peradeniya, 20400 Sri Lanka

Abstract
About 40% of the kernel is converted to low fat press cake in commercial coconut milk extraction and it is mainly
used for animal feed. The commercial coconut milk powder contains 65% fat, 25% CHO, 2.3% fibre and 7% protein
and not very healthy. The press cake is a rich source of high quality dietary fibre and also considered as a functional
food due to its ability to prevent cardiovascular disease, colon cancer and chronic diseases. Therefore, the objective
of this study is to formulate a new coconut milk powder mixture by blending press cake flour with coconut milk
powder without sacrificing the physical and organoleptic properties in cooking. The new product will be healthy and
less expensive. Initially, the suitable particle sizes were determined based on their physical properties. Then sensory
evaluation tests were conducted to determine the best mixing ratio of coconut milk powder to coconut flour for
different particle sizes. The particle sizes considered were <75 µm, 75-150 µm, 150-250 µm, 250-425 µm and 425-
850 µm. Based on the physical properties, lowest three were selected and the consumer preference for milk curry
was assessed for the particle size in making the mixture. The highest preference was obtained for particles less than
150 µm. Then, the coconut flour to coconut milk powder mixing ratios of 15:85, 25:75, 35:65 were investigated taking
commercial coconut milk powder as the control. There is no significant different (P <0.05) between the control with
three ratios, but the preference for 15:85 is significantly higher than the control.

Keywords: Coconut milk powder, coconut press cake flour, particle size, coconut milk powder mixture
*Corresponding author’s email: nimal.dharmasena@gmail.com

Introduction
In coconut milk extraction, about 40% of the kernel is converted to press cake which is mainly used as animal feed.
The commercial coconut milk powder contains 65% fat, 25% CHO, 2.3% fiber and 7% protein (Trinidad et al., 2006)
and therefore, the healthiness is questionable as it is rich in fat but low in fiber. The by product, press cake is a rich
source of high quality dietary fiber; 56% insoluble and 4% soluble (Trinidad et al., 2006; Raghavendra et al., 2006),
that can also be used as an ingredient in preparation of foods (Jena & Das, 2012). It is also a functional food, it
increases feces mass and shortens passage time, helps proper control and prevention of cardiovascular disease,
colon cancer and chronic diseases as well (Trinidad et al., 2006). Therefore the objective of this research was to
formulate a new coconut milk and coconut press cake powder mixture by adding low fat white coconut flour without
sacrificing the physical and organoleptic properties in cooking for improving health benefits and reducing the cost of
coconut milk powder.

Materials and Methods

Coconut milk press cake was obtained from a virgin oil processing plant. The by product, coconut press cake was
used for coconut flour preparation. The initial moisture and fat content of the press cake was determined according to
the AOAC 934.01 (AOAC, 2005) and the crude fat content was determined by the Soxhlet extractor method.
Coconut Flour Sample Preparation
Coconut milk press cake sample was further dried in a vacuum oven at 70 °C and at an absolute pressure of
62 ± 5 mmHg for 24 hours. Then the sample was ground using a kitchen grinder (Jaipan kitchen master) for nearly
10 minutes at 18,000 rpm to obtain coconut flour. The dry coconut flour was sieved to obtain five coconut flour

209
samples with different particle sizes. The sieve sizes used are; 0.075 mm, 0.15 mm, 0.25 mm, 0.425 mm and 0.850.
(IMPACT Laboratory test sieve BS 410 – 1:2000).

Determination of the Best Particle Size for Mixing With Coconut Milk Powder
Best particle size was determined from those five particle sizes based on their physical properties. Viscosity, turbidity,
Total Dissolve Solids (TDS), sedimentation velocity, whiteness (L*) and the shape of the particle were analyzed.
Whiteness is a key parameter of coconut milk powder and therefore, the L* value was determined using a colorimeter
(HNspec model CS10) based on the CIELAB color space.
Viscosity of five different particle sizes of their 100 °C hot water preparations (20 gl-1) were determined using a rotary
viscometer (TOKIMEC INC, Model BL) viscosity after cooling down to room temperature (27 °C). The turbidity of
each sample was measured using a portable turbidity meter (HACH 2100Q). TDS is another similar measurement to
turbidity that could be related to the solubility of different particle sizes in hot water. The TDS values of five milk
samples were determined using a conductivity meter (Thermo Orion, model 145).

Determination of Sedimentation Velocity of Five Different Particle Sizes

The settling velocity of five particle sizes was calculated using the Stokes law (Eq. 01) to investigate the stability of
colloidal particles in a suspension. Sedimentation is not preferred in a coconut milk curry and the colloidal suspension
should be stable. The shape of the particle was assumed as spherical and the flow condition was assumed as
laminar at the room temperature (Reynolds Number <1). True density of coconut flour was measured by the
pycnometer method using Toluene as the liquid (ISO 1183-1:2004).
𝑑 2 (𝜌𝑠− 𝜌° )𝑔
𝑉𝑠𝑒𝑑 = ………………………………………………… (Eq. 01)
18𝑛°
Where, Vsed= sedimentation velocity (ms-1), d = diameter of particle (m), ρs = density of disperse phase (kgm-
3), ρ = density of disperse media (kgm-3), g = acceleration due to gravity (ms-2), h = viscosity of disperse medium
o o
(Pa.s).

Determination of the Best Mixing Ratio of Coconut Flour to Coconut Milk Powder
Best mixing ratio of commercially available coconut milk powder to coconut flour was determined using a sensory test
using a 7 point hedonic score system with 30 untrained panelists.

Selection of the Best Particle Size Based on Sensory Test

Three particle sizes were selected based on the physical properties of coconut flour. Therefore, sensory tests were
conducted to further analyze the best particle size by using a predetermined mixing ratio of 1:3 (coconut flour:
coconut milk powder). Three coconut milk based plain curries were cooked under uniform conditions and evaluated
by the untrained panel of 30. The best particle size was selected based on the results.

Optimization of the Best Mixing Ratio

Three different mixtures of flour to milk powder (15: 85, 25: 75 and 35: 65) formulations and commercial coconut milk
powder (0: 100 -control) were evaluated by an untrained panel. Non-parametric Friedman test was conducted using
MINITAB statistical package (MINITAB 17) to analyze the variance (P< 0.05) of sensory scores of the sensory
evaluation.

Results and Discussion

The initial moisture and fat content of the coconut press cake was 4.89% and 12.4%, respectively. Generally,
moisture content below 5% (db) maintains a water activity below 0.6 (Jena & Das, 2012). Rancidity also affects loss
of freshness and unfavorable odors. Therefore, low fat content is a good indication for stable shelf life for further
processing.

210
Physical Properties of Particles
The viscosity of the smallest hydrocolloid suspension was observed as Newtonian while the other suspensions
demonstrated non-Newtonian behavior. Fluid foods those contain significant amount of compounds and higher than
25% Total Solids exhibit non-Newtonian behavior (Noraziah, 2002). Viscosity of these five solutions cannot be
compared with two shear rates because of the Newtonian and non-Newtonian fluid behaviors. Therefore, comparison
of viscosity was done at 60 rpm shear rate. According to the results, particles lower than 75 µm has the lowest
viscosity (2.67±0.29 mPas) than all other particles. Larger particle cause high shear force due to particle friction with
suspended particles. Therefore, lower viscosity indicates good soluble and dispersion property of the particle in the
suspension which is needed for cooking.

Total Dissolved Solids (TDS) are the amount of organic and inorganic dissolved substances contained in a liquid that
are smaller than 2 micron in size. TDS values increased with decreasing particle size, 75-150 and 75> had similar
values of 722 and 727, respectively. Similarly, less than 75 µm (856 NTU) and 75-150 µm (940 NTU) had high
turbidity than other particles. According to the results of sedimentation velocities, the less than 75 µm (4.5 * 10-5) and
75-150 µm particle (9.6 * 10-5) have similar velocities. The larger particles had higher values ie. 3.1*10-3 ms-1 for
425-850 µm. Low sedimentation velocity causes the increase of settling time of the particle making a suspension
stable for some time. Therefore, lower than 150µm particle was selected based on low settling velocity for this
experiment.

Whiteness of the Particle

Whiteness is an important parameter for coconut milk. The color analysis of different sizes of particles indicated a
significantly higher value of L* for lower than 75 µm particle size due to smooth surface packing. Although, all five
samples were significantly different from each other (P < 0.05) for whiteness (Table 1), they are also acceptable.

Table 3: Whiteness (L* values) for different size of particles

Particle Size (µm) Sample L*

425-850 1 70.66±0.57a
250-425 2 73.88±0.03b
150-250 3 76.75±0.18c
75-150 4 78.21±0.19d
75> 5 87.43±0.08e

a – c Values in rows followed by the same superscript letters are not significantly different (p ≤ 0.05).

According to the viscosity, sedimentation rate, whiteness and shape of the five samples, below 250 µm particle size
samples were selected for further evaluations. The larger the particle size, it is easy and energy efficient to produce.

Selection of the Best Particle Size Based on Sensory Test

According to the result, coconut milk curry prepared using three particle sizes<75, 75-150 and 150-250 µm are not
significantly different (P < 0.05) with respect to the sensory attributes. However, 150-250 µm particle included milk
curry had the lowest rank for all attributes in comparison to other two samples. Therefore, below 150µm particle size
is selected for further evaluations of mixing ratios.

Optimum Mixing Ratio of Coconut Flour to Coconut Milk Powder

Result of the sensory evaluation is illustrated in Figure 1. The results reveal that overall acceptability is not
significantly different (P> 0.05) among three mixing formulations. However, the ratio of 15:85 would increase the
eating quality even better than the commercial product, while mixing up to 35: 65 ratio does not influence the

211
consumer satisfaction. Therefore, the new coconut milk formulations with coconut flour have a very good market
potential for healthy cooking.

7
Median Quality score

6
5
Formulation 4 (Control)
4
Formulation 5 (15:85)
3
Formulation 6 (25:75)
2
Formulation 7 (35:65)
1
0
Aroma Taste Texture Gravy Overall
Sensory Attribute

Figure1: Mean quality score for three different formulations of coconut milk curry (Kiri hodi)

Conclusion
Coconut press cake flour with the particle size lower than 150 µm could be mixed with commercially available
coconut milk up to a ratio of 35: 65. The mixing ratio of 15:85, consumer acceptability of taste is greater than the
commercial coconut milk powder.

References

AOAC, 2005. Official method of analysis. Washington: DC.

Jena, S. & Das, H., 2012. Moisture sorption studies on vacuum dried coconut presscake. Journal of Food Science
and Technology, 49(5):638-42.
Noraziah, M.O., 2002. Rheological behaviour of coconut milk: Effects of concentration and temperature. University of
Putra, Malaysia, Masters Thesis.
Raghavendra, S.N., Swamy, S. R. R., Rastogi, N. K. and Tharanathan, R. N. 2006. Grinding Characteristics and
Hydration properties of coconut residue: A source of dietary fiber. Journal of Food Engineering, 72(3):281-286
Trinidad, T.P., Mallilin, A.C., Valdez, D.H., Loyola, A.S., Askali, M.F., Castillo, J.C., Encabo, R.R., Masa, D.B.,
Maglaya, A.S., Chua, M.T., 2006. Dietaryfiber from coconut flour: a functional food, Innovative Food Science
and Emerging Technologies, (7): 309-317

212
 IFRC 2017: 196-175 Food Processing and Post-Harvest Technology
Optimization of Sweet Cassava (Manihot esculents crantz.) Crude Extract with High Maltodextrin Level Using
Response Surface Methodogy.

1,2 Posridee, K. and 1,2* Oonsivilai, R.

1School of Food Technology, Institute of Agricultural Technology, Suranaree University of

Technology,NakhonRatchasima, 30000, Thailand
2Postharvest Technology Innovation Center, Commission on Higher Education, Bangkok 10400, Thailand

Abstract
The purpose of this study was to obtain optimize condition for extracting sweet cassava crude extract by using
enzymatic hydrolysis extraction including the chemical composition of the cassava crude extract were investigated.
The optimal extraction condition to prepare sweet cassava crude extract with highest maltodextrin was determined
using Box-Behnken design experimental and the Response Surface Method was applied to get the optimization
condition. Three factors with three level used in this experiment are the enzyme concentration, temperature, and
extraction time. In addition, the proximate composition, mineral content and cyanogenic potential were determined
using standard methods .The result showed that the optimum extraction conditions were as following: enzyme
concentration at 0.3 %w/v, extraction temperature at 95oC extraction time at 45 minutes that gave the highest
maltodextrin of sweet cassava crude extract. From the experiment, the result showed that the affected to the high
percentage yield of sweet cassava crude extract (P<0.05) more than enzyme concentration and extraction time.
Furthermore, The cassava crude extract contain moisture content 9.425±0.05 , (% protein) 3.25±0.34, (% ash)
2.02±0.09, (% crude fiber) 2.97±0.41, (% fat) 1.15±0.08 ,(% carbohydrate) 85.75±0.53 and cyanogenic potential
0.09±0.011 mg HCN/kg.

Keywords: sweet cassava, maltodextrin, optimization, response surface method

*Corresponding author’s email: posridee.ka@gmail.com

Introduction
Maltodextrin is a mixture of saccharides with a molecular weight between polysaccharides and oligosaccharides with
DE lower than 20 (not sweet), which is available as white powders mostly or concentrated solution. Maltodextrin is
more soluble in water than native starches, also cheaper in comparison with other major edible hydrocolloid
(Alexander, 1992). The maximum activities of the α-amylase are usually in the pH range between 4.8 and 6.5, but the
activity-pH profile and location of the pH optima differ depending on the enzyme source. For production of low DE
maltodextrin, BAN 480L (a type of α-amylase) may be used. The major steps in the enzyme conversion of starch are
liquefaction and saccharification. In liquefaction, the enzyme hydrolyses the α-1,4-glycosidic bonds in starch (Bravo,
2006).

The purpose of study was determining the possibility of maltodextrin production by α-amylase from sweet cassava
and evaluation of different parameters in the process (enzyme concentrations, temperatures and hydrolysis time).

Materials and Methods

3.1 Sweet cassava was obtained from Nakhon Ratchasima, Thailand. They were dried at 60 C in tray dryer and
finely ground to powder, kept in vacuum package at 4 C until used.

3.2 Sweet cassava flour were analysed for moisture, ash, fat, crude fiber and protein by AOAC (2000) as the
following; moisture (AOAC 925.10), ash (AOAC 900.02A), protein (AOAC 928.08), fat (AOAC 963.15), crude fiber
(AOAC 978.10).

213
3.3 Dextrose equivalent determination the modified Lane and Eynon titration (Corn Refiner Association- Method E-
26). Suluble solids (°Brix) determination has been done by refractometer method.

3.4 A suspension containing 30% dry matter was liquefied to make the starch susceptible to further enzymatic
breakdown by α-amylase. The method used modified Kachenpakdee et al. (2016) method and pH for starch
hydrolysis by α-amylase adjusted to pH 6.

3.5 Response surface method was applied to identify optimum levels of three variables of the extraction enzyme –
amylase concentration (w/v), extraction temperature (C) and extraction time (min) regarding of responses extract
yields. The design independent and dependent variables are enzyme concentration (X1) (0.1, 0.2 and 0.3% w/v),
extraction temperature (X2) (90, 95 and 100oC) and extraction time (X3)(15, 30 and 45 minutes). Experiments were
designed according to the Box-behnken design. Order of experiments has been fully randomized. Data were
analyzed by One-Way ANOVA.

Results and Discussion

Sweet cassava crude extract contain moisture content 9.425±0.05, (% protein) 3.25±0.34, (% ash) 2.02±0.09, (%
crude fiber) 2.97±0.41, (% fat) 1.15±0.08, (% carbohydrate) 85.75±0.53 and cyanogenic potential 0.09±0.011 mg
HCN/kg.

Maltodextrin production (Y1) of sweet cassava obtained from all the experiments are listed in Table 1. According to
Myer (1995), R2 should be at least 0.80 for a good fit of a model. The high value of the coefficient of multiple
determination (R2 = 0.8946) exhibited that the model adequately represents the experimental results.

Results were statistically analyzed by Analysis of Variance. Mean analysis was performed using Duncan’s procedure
at p ≤ 0.05. RSM was focused on determining regression coefficients and statistical significance of model terms.
RSM helps in fitting regression models to experimental data to achieve an overall optimal region for all response
variables studied. Experimental design, data analysis and optimization procedure were carried out using Design-
Expert® software (Version 8.0.7.1, Stat-Ease, Inc., Minneapolis, MN). The variables of extraction was analyzed by
One-Way ANOVA to determine correlation between independent variable including get optimal extraction condition
for highest percentage yield as shown in Table 1.

From Table 1, highest percentage maltodextrin production had shown at the condition of the enzyme concentration at
0.3 %w/v, extraction temperature at 95C and extraction time at 45 minutes that gave the highest maltodextrin
production at the level of 17.337%w/w. While, replace value of optimum condition into regression equation obtained
(Y1= 82.83758+ 10.29375X1 -0.88265X2 +0.28016X3; linear) showed predicted yield value was 14.68%w/w. From the
experiment, the result showed that the extraction temperature affected to the maltodextrin percentage yield (P<0.05)
more than enzyme concentration and extraction time. In addition, data was analyzed for correlation between
independent variable and maltodextrin percentage, statically analysis showed significant at p value less than 0.01.

Response surfaces could be illustrated with three-dimensional plots by representing the response in function of two
factors and keeping the other constant. The highest maltodextrin level showed in relation to α-amylase concentration,
extraction temperature, and extraction time as Figures 1-3.

214
 Table 1. Experiment design and response of independent variables to the extract parameters.
Independent variables
Maltodrextrin(%w/w)
Exp. No.a α-amylase (%w/v) Temperature (C) Time (min)
Y1
X1 X2 X3
1 0.3 90 30 14.059±0.29c
2 0.1 100 30 2.755±0.22g
3 0.1 95 45 13.900±0.37c
4 0.2 95 30 11.950±0.05d
5 0.2 100 15 3.302±0.05fg
6 0.1 90 30 13.249±1.15c
7 0.3 100 30 3.426±0.35fg
8 0.2 100 45 6.831±0.02f
9 0.2 95 30 11.950±0.11d
10 0.1 95 15 1.701±0.20g
11 0.2 90 15 9.370±1.26e
12 0.2 90 45 14.942±0.21b
13 0.2 95 30 11.960±0.06d
14 0.3 95 45 17.337±0.03a
15 0.3 95 15 5.018±0.11f

a Experiments were conducted in a random order.

Figure 1. Response surface plots indicating the interaction effect of α-amylase concentrations
and extraction temperature on maltodextrin production at extraction time 45 minutes.

Figure 2. Response surface plots indicating the interaction effect of α-amylase concentrations and extraction time on
maltodextrin production at extraction temperature 95C.

215
 Figure 3. Response surface plots indicating the interaction effect of extraction temperature and extraction time on
maltodextrin production at α-amylase concentrations 0.3%w/v.

Conclusion
The optimum condition to prepare highest maltodextrin from sweet cassava was determined using randomized Box-
Behken design and data was used for statistically analysis by ANOVA. The condition that showed the enzyme
concentration at 0.3 %w/v, extraction temperature at 95C and extraction time at 45 minutes that gave the highest
maltodextrin of sweet cassava crude extract. For future research, study the functional properties for application in
food product for endurance athletes.

Acknowledgment
This research was supported by research and development institute funds, Suranaree University of Technology,
Nakhon Ratchasima, Thailand, and Postharvest Technology Innovation Center, Commission on Higher Education,
Bangkok Thailand.

References

O. A. C. 2000. Official Method of Analysis. (15th ed). The Association of Official Chemists,
Arlington, Verginia.
Alexander, J.R. 1992. Maltodextrins; production, properties and applications in starch hydrolysis products. Schenck
EW, Hebeda RE, Eds., VCH, New York.
Bravo, R.V. 2006. Modification of the activity of an -amylase from Bacillus licheniformis by several surfactants.
Electronic Journal of Biotechnology 9: 1-6.
Kachenpakdee, N., Santarre, C.R., Ferruzzi, M., and Oonsivilai, R. 2016. Enzymatic digestion optimization of dietary
fiber from cassava pulp and their effect on mercury bioaccessibility and intestinal uptake from fish using and in
vitro digestion/Caco-2 model. International Food research Journal 23(2): 660-666.

216
 FRC 2017: 207-188 Food Processing and Post-Harvest Technology
Effect of Gamma Irradiation and Different Packagings on the Shelf Life of Mushrooms )Agaricus bisporus(
1Fartash, E.,1 *Khoshtaghaza, M. H., 2Abbasi, S.
1Department of Biosystems Engineering, Tarbiat Modares University, Tehran, Iran
2Department of Food Science and Technology, Tarbiat Modares University, Tehran, Iran

Abstract

This study was conducted to evaluate the influence of packaging materials and gamma irradiation doses on the shelf
life of edible button mushroom )Agaricus bisporus). To do so, the fresh mushrooms were packed in two different
packages: (polyethylene or nanocomposite based on nanoemulsion silicone) and immediately irradiated (0, 1, 2 and
3 kGy) then stored (2–3°C). The effect of these parameters on some qualitative characteristics of mushrooms
(weight loss, cap diameter, pH, total soluble solids, firmness, and whiteness) during storage (1, 5, 10, 15 and 20 days
after irradiation) was evaluated. Our findings showed that the mutual interaction of irradiation dose and packaging
material were significant (p<0.01) in all measured properties except firmness. Moreover, the effect of storage time
was significant (p<0.01) in all measured properties and decreased quality of mushrooms over time. The maximum
amount of weight, cap diameter, pH, total soluble solids, firmness, and whiteness (L*) were observed in the samples
which packed in nanocomposite packaging and irradiated. It can be concluded that using appropriate packaging
material (nanocomposite) and proper doses of gamma irradiation (1-2 kGy) could significantly increase the shelf life
of edible button mushrooms.

Keywords: Button mushroom, Gamma irradiation, Nanocomposite, Polyethylene, Packaging

*Corresponding author’s email: khoshtag@modares.ac.ir

Introduction
The cultivation of edible mushrooms is importance in the world and have significant progress. Mushroom is a
perishable product and its shelf life is 1-3 days at the room temperature (Akram and Kwon, 2010). High respiration
rate and the lack of physical protection to prevent moisture loss and changes due to the attack of microbial often lead
to losing mushroom quality that occurs through browning, softening, increased cap diameter and weight loss (Singh
et al., 2010). Gamma irradiation is a food processing methods that has more energy compared with other irradiation
(Arvanitoyannis, 2010). In the range of energy used in this method, foods do not radiolysis and the only increases
stability in storage period (Arvanitoyannis, 2010). Irradiation method, beside other methods can be used as a co-
factor in protecting agricultural products.
Packaging is one of the important topics in the environment and the one of problems of industrial countries is waste
related to packaging materials. However, packaging films which are produced from recycled material, have an
important role in reducing losses. The Production of nanocomposite improve the quality of packaging materials and
thus ensure the safety of food (Kumar et al., 2010).

Irradiation and various packages have been used to investigate the chemical properties and antioxidant (Fernandes
et al., 2017) and physicochemical properties (Jiang et al., 2010) of mushrooms. In this study, the combined effects of
gamma irradiation and type of packing material on qualitative properties of mushrooms have been studied to
determine shelf life of product.

Materials and Methods

Packaging and irradiation of the samples

Button edible mushroom (Agaricus bisporus) immediately after harvesting was packed in the two types of packaging
including polyethylene (P, zip kip with dimensions of 35×25 cm) and nanocomposite based on nanoemulsion silicone

217
(N, zip kip with dimensions of 35×25 cm). The packed samples irradiated by gamma cell (Nordion, GC-220, Canada)
at doses of 0 (control), 1 (d1), 2 (d2) and 3 (d3) kGy with rate of 1.98 G/s in the Atomic Energy Organization of
Tehran-Iran, then were kept at a temperature of 2-3℃. The qualitative changes of edible mushroom during storage
investigate in five stages (the first day, 5, 10, 15 and 20 after irradiation treatment).

Measuring of quality characteristics

Percentage of weight loss at different days was calculated by the following equation:
𝑊0 −𝑊𝑓
100 ×WL=
𝑊0

where, WL is the percentage of weight loss, W0 and Wf are the samples weight in the first day (g) and other days of
storage (g), respectively. The Mushrooms cap diameter was measured by a digital caliper. Brix were measured by
handy refractometer (Erma-optical, works, LTD, Japan). The pH of the extracted mushroom juice was measured by
digital pH meter (Metrohm 827, USA) (Aday, 2016). The firmness of the samples was measured by material testing
machine (H50 K-S, Hunsfield, England). Loading performed by penetrating (5 mm) cylindrical probe with 2.3 mm
diameter to the sample. The whiteness (L*) of the mushroom samples was calculated by image processing method.
Imaging was performed by 13 megapixel camera (Samsung, J500F). Also the image processing was performed by
MATLAB 2015.

Statistical analyses

The experimental data was analyzed based on completely randomized design with three replications. Its analysis of
variance (ANOVA) was performed by SPSS 16.0 software.

Results and Discussion

Analysis of variance (ANOVA) showed that the effect of irradiation dose (ID), packaging type

(PT) and storage time (ST) on all quality parameters was significant (p<0.01). But interaction between ID and PT, ID
and ST and the interaction of each three factors on the firmness was insignificant (Table 1).

Percentage of weight loss and cap diameter

Figure 1 shows that by increasing storage time, the weight of the samples decreased (percentage of weight loss
increased). The amount of weight loss in the control samples (no irradiation) within polyethylene packaging (d0-P)
was higher than samples irradiated at doses of 1 and 2 kGy within packaging nanocomposite. Gamma irradiation can
delay the physiological processes, increase shelf life of mushroom through control respiration and reducing moisture
loss. Migration of moisture to the outside and high permeability in polyethylene packages were significant factor in
severe reducing the weight of mushrooms (Montanez et al., 2010).

The results obtained from investigating cap diameter of the samples showed that with the appropriate irradiation
doses (1-2 kGy) and the usage of nanocomposite packaging can prevent the opening of cap diameter and its
deterioration.

218
 Table 1. Mean square from STAT/ANOVA on the main effects and interaction

Source of df Weight Cap pH TSS Firmnes L*

Variables loss diameter s index
ID 3 45.33* 0.43* 0.017* 1* 0.38* 503.48*
PT 1 315.60* 0.18* 0.084* 3.10* 16.77* 4320*
ST 4 987.09* 6.69* 0.599* 17.74* 9.23* 2492.4*
ID×PT 3 0.55* 0.07* 0.005* 0.116* 0.07ns 7.62*
PT×ST 4 64.43* 0.030* 0.007* 1.06* 1.39* 322.10*
ID×ST 12 8.25* 1.17* 0.084 0.17* 0.11ns 40.71*
ID×PT×ST 12 0.29* 0.013* 0.002* 0.089* 0.087ns 4.67*
ns and *, nonsignificant and significant difference at 1% level, respectively.

pH and total soluble solids

The pH value of control samples and irradiated with doses of 3 kGy increased from the first to the tenth day and
decreased after that day. The increase of irradiation doses more than 2 kGy caused to damage the texture, the
reduction of sugars and the increase of respiration rate and organic acid that reduced the pH value. Also, in the
control samples, the spoilage of the product began since the tenth day was the main reason for a drop in pH value.
Irradiation doses 1-2 kGy were effective in delay ripeness. The pH value of the samples packed in nanocomposite
based on nanoemulsion silicone was more than of polyethylene package.

For a given dose of irradiation in the last day of storage, nanocomposite packaging had fundamental role in
preservation the total soluble solids, so that the maximum brix was related to doses 1 and 2 kGy in this type of
packaging. Appropriate dose of irradiation delay the ripeness of mushroom by removing microorganisms, so brix
reduction in doses of 1 and 2 was less.

Firmness
The firmness of samples pretreated with gamma irradiation were more than control samples during storage. In the
first day, the control samples had firmness more than the irradiated samples. But after the fifth day, the firmness of
irradiated samples increased. Also, the firmness of samples treated with doses of 3 kGy was less than 1 and 2 kGy.
The power of gamma rays at doses higher than 2 kGy caused the destruction of the cell wall and the reduced the
firmness of texture. Also the firmness of samples in nanocomposite packaging was more than polyethylene.

Whiteness (L* index)

With increase in storage time, the L* index in all cases decreased and the amount of reduction for the control
samples were more than irradiated samples. The appropriate dose of irradiation increases the shelf life of product
through remove microorganisms, so this index was higher in irradiated samples. The value of L* index of samples
packed in nanocomposite were more than polyethylene. The preservation of moisture and reducing input oxygen and
output dioxide carbon in nanocomposite packaging caused the protection of appearance characteristic such as color
in period of storage.

219
 ۲۵ d0-N

۲۰ d1-N

Weight loss (%)

d2-N
۱۵
d3-N
۱۰ d0-P

d1-P
۵
d2-P
۰
d3-P
۱ ۵ ۱۰ ۱۵ ۲۰
Storage time (day)

Figure 1. Effect of dose irradiation and the type of packaging on weight loss

Conclusion
The results of this study showed that the appropriate irradiation doses increased shelf life of mushrooms by removing
microorganisms and delay in the physiological action. Also the use of nanocomposite packaging addition to
biodegradable, caused to retain moisture and reduce input oxygen and output carbon dioxide in the product.
Therefore, it is recommended that new technologies such as irradiation at doses of 1-2 kGy and nanocomposite
packaging can increase the shelf life of edible mushrooms before entering these products to market.

References

Aday, M.S. 2016. Application of electrolyzed water for improving postharvest quality of mushroom. LWT-Food
Science and Technology 68: 44-51.
Akram, K. and Kwon, J. H. 2010. Food irradiation for mushrooms: a review. Journal of the Korean Society for Applied
Biological Chemistry 53(3): 257-265.
Arvanitoyannis, I.S. 2010. Irradiation of food commodities: techniques, applications, detection, legislation, safety and
consumer opinion: Academic Press.
Fernandes, A., Barreira, J. C., Gunaydi, T., Alkan, H., Antonio, A. L., Oliveira, M. B. P. and Ferreira, I. C. 2017. Effect
of gamma irradiation and extended storage on selected chemical constituents and antioxidant activities of
sliced mushroom. Food Control 72: 328-337.‫‏‬
Jiang, T., Luo, S., Chen, Q., Shen, L. and Ying, T. 2010. Effect of integrated application of gamma irradiation and
modified atmosphere packaging on physicochemical and microbiological properties of shiitake mushroom
(Lentinus edodes). Food Chemistry 122(3): 761-767.
Kumar, P., Sandeep, K., Alavi, S., Truong, V. D. and Gorga, R. 2010. Preparation and characterization of bio-
nanocomposite films based on soy protein isolate and montmorillonite using melt extrusion. Journal of Food
Engineering 100(3): 480-489.
Montanez, J.C., Rodriguez, F.A., Mahajan, P.V. and Frias, J.M. 2010. Modelling the gas exchange rate in
perforation-mediated modified atmosphere packaging: Effect of the external air movement and tube
dimensions. Journal of Food Engineering 97(1): 79-86.
Singh, P., Langowski, H.C., Wani, A.A. and Saengerlaub, S. 2010. Recent advances in extending the shelf life of
fresh Agaricus mushrooms: a review. Journal of the Science of Food and Agriculture 90(9): 1393-1402.

220
 IFRC 2017: 211-194 Food Processing and Post-Harvest Technology
Effect of Ultrasound Treatment on the Functional Properties of Jackfruit Seed Starch

Mohamad Yazid, N.S., *Abdullah, N., Muhammad, N.

Department of Technology and Heritage, Faculty of Science, Technology and Human Development, Universiti Tun
Hussein Onn Malaysia (UTHM), 86400 Parit Raja, Batu Pahat, Johor, Malaysia.

Abstract

The aim of this study was to investigate the effect of ultrasound treatment on the functional properties of jackfruit
seed starch (JSS). Starch was extracted from jackfruit seeds and treated with different times (15, 30 and 45 minutes)
and temperatures (30, 40, 50, 60, 70 and 80°C). Paste viscosity, solubility, swelling power, and gelatinisation
properties were determined for untreated and ultrasonically treated jackfruit starch suspensions. The functional
properties of ultrasonically treated starch were changed statistically significant (p<0.05). Paste viscosity, solubility
and swelling power of ultrasonically treated JSS are lower compared to the untreated JSS. Ultrasound is able to
reduce and increase the gelatinisation enthalpy of JSS. In conclusion, the native jackfruit starch is able to be modified
effectively using ultrasound-assisted treatment to improve its performance in different applications in food industry.

Keywords: Jackfruit, modified starch, native starch, seed, ultrasound, food waste.
*Corresponding author’s email: norazlinh@uthm.edu.my; Tel: +607-4537942 Fax: +607-4536051

Introduction
Jackfruit (Artocarpus heterophyllus Lam.) is one of the six high-value non-seasonal fruits listed in Malaysian National
Key Economic Areas (NKEA) under Malaysian Economic Transformation Programme. Leong et al. (2016) reported
that jackfruit in Malaysia has been extensively processed into numerous luxuries food products such as dried chips
and battered fried snacks. Pahang State Farmers Association (PASFA) is currently producing jackfruit ice-cream and
paste using J33 and Mastura varieties (Rafi, 2016). Jackfruit rinds and cores which holds the edible jackfruit pods
contribute to about 60% of the whole fruits and have been discarded as waste from jackfruit-processing industry
(Begum et al., 2017). Jackfruit seed contains high amount of starch (Madruga et al., 2014). Mukprasirt and
Sajjaanantakul (2004) proposed that native starch from seed of jackfruit can be used in food industry requiring high
thermal properties and can act as a substitute to commercialised modified starch such as Purity 4 starch. The ability
of starch in offering desired texture and stability in formulations of food making it one of the most important polymers
in food manufacturing (Silva et al., 2017). Food properties can be improved by applying ultrasound to starch based
products (Zhu, 2015). Thus, aim of this study is to investigate the effect of ultrasound treatment on paste viscosity,
swelling, solubility and gelatinisation properties of jackfruit seed starch.

Materials and Method

Fresh jackfruit was obtained from Malaysia’s Federal Agricultural Marketing Authority, (FAMA) in Tangkak, Johor
(2.2555° N, 102.5444° E). The fruits were cleaned and separated into pulp, seed and rind. Starch isolation were
performed according to the method of (Andrabi et al., 2015, Majeed et al., 2017) with slight modifications. Jackfruit
seeds (500 g) was soaked in 2 L of distilled water and kept at 4°C for 12 h. Seed coats were removed by hand
scraping and were reduced to fine particles using a laboratory blender (8010EG, Waring® Commercial, USA) for 5
min. Slurry obtained was diluted 10 times (v/v) by using distilled water and pH obtained were adjusted to 10 using 0.5
M NaOH. The slurry was continuously shaken in an incubator shaker (New Brunswick TM INNOVA® 42, Eppendorf,
Germany) for 1.5 h, and then was filtered through a 4-mm mesh sieve to separate the fibre portion. The slurry was
then centrifuged at 3000 g for 15 min at 4°C using a centrifuge (MPW-351R, MPW Med. Instruments, Poland). The
aqueous phase obtained on centrifugation was discarded. The sediment obtained was scrapped off from the surface
and the lower white portion was washed three times with distilled water and was recovered as starch by
sedimentation at 4°C. The starch was dried at 50°C in a hot air oven (G512.1433, Memmert, Germany) for 24 h.

221
Dried starch was then grounded at 22 000 rpm or at high speed for 5 minutes into powdered form using the
laboratory blender.

Starch granules of known moisture content (14%) were then suspended in distilled water to give a concentration of
1% (w/v) (Luo et al., 2008). Moistened samples were placed in a glass beaker and gently stirred for 30 min. An
ultrasound processor (FB15055, FisherbrandTM, Germany) was used to treat 100 mL of jackfruit seed starch (JSS) in
a 250-mL glass beaker. Samples were treated with different times (15, 30 and 45 min) and temperatures (30, 40, 50,
60, 70 and 80°C) at 37 kHz with an input power of 550 W. The experiment was designed using full factorial design
with three replications for a total of 54 runs (Table 1). Samples were then dried at 50°C in a hot air oven (G512.1433,
Memmert, Germany) until the final moisture content achieved 30%.

Apparent viscosity of 1% starch was measured with a viscometer (DV+ II, Brookfield, USA) using spindle no. 64 at
28°C and at shear rate speed of 50 rpm for 2 min (Sujka and Jamroz, 2013). Starch solubility and swelling power
were determined according to Leach et al. (1959) and Madruga et al. (2014). Thermal properties was determined by
using a Differential Scanning Calorimeter (DSC) (Perkin Elmer model DSC7; CT, USA) (Mukprasirt and
Sajjaanantakul, 2004). The DSC analysis was done at a temperature between 25 and 120°C in a 30% starch
concentration (dry basis). All data were analysed by generating analysis of variance (ANOVA) with significance level
of 5% using a commercial software (Minitab® 17.3.0, Minitab Inc. US).

Results and Discussion

Table 1 presents the effect of ultrasound treatment on paste viscosity, solubility, swelling power, gelatinisation
transition temperatures and gelatinisation enthalpy of JSS.

Ultrasound treatment causes the paste viscosity of JSS to be reduced significantly (R2 = 0.98, p<0.05). Starch treated
at 30°C for 30 min showed the most decreasing value at 380±2.8 cP as compared to untreated sample at 839±27.0
cP. This result is complied with Sujka and Jamroz (2013), who found that potato, wheat, corn and rice starch treated
with ultrasound gave same trend in decreasing paste viscosity values as compared to untreated sample. This is due
to the reduction of molecular weight of starch molecules after ultrasonic treatment (Huang et al., 2007).

Swelling power and solubility of JSS are temperature dependent. As the temperature increases, both parameters
increase. This result is consistent with Madruga et al. (2014), who found that Brazilian JSS gave huge rise in both
parameters after 75°C. The ultrasound causes the starch intermolecular bond to be broken and in turns making the
arrangement to be less close to each other (Luo et al., 2008). Ultrasound treatment also statistically proved to cause
a significant effect on swelling power and solubility of starch sample by having a strong positive correlation (R2 =
0.97, p<0.05) at all temperatures treated.

For the onset temperatures (To) of ultrasound treated starch at 40°C for 30 min showed a slight increase in value as
compared to untreated sample. Phrukwiwattanakul et al. (2014) claimed that less enthalpy will be needed to
solubilised starch granules with lower gelatinisation temperatures (To, Tc and Tp). However, in this study samples with
lower gelatinisation temperatures showed higher enthalpy values. This was supported by Zhang et al. (2016) study
which declared that larger starch granules are more stable due to its greater amount of amylopectin crystallites. In
conjunction with decreasing in difference of onset (To) and peak values (Tp), the ultrasound causes samples with
different stabilities to undergo crystallites breakage.

Conclusion
Jackfruit seed starch has potential to be used in food industry in addition to current commercialised starch source.
Moreover, ultrasound treatment applied in this study showed the ability in modifying starch functional properties
physically. Thus, JSS will be functionally utilised as a raw material in food processing rather than discarded the seeds
as waste.

Acknowledgement
This research is partly funded by the Universiti Tun Hussein Onn Malaysia Centre for Graduate Studies, and
Postgraduate Research Grant (UTHM-U538-2016).
222
 Table 1. Functional properties of untreated and ultrasonically treated JSS.
Solubility (%) Swelling Power (g/g) DSC
Viscosity
(cP) To Tp Tc Tc-To
min °C 55°C 65°C 75°C 85°C 95°C 55°C 65°C 75°C 85°C 95°C ΔH (J/g)
(°C) (°C) (°C) (°C)

Ultrasound- treated

15 30 494±15.8 0.5±0.1 3.1±0.1 6.5±0.1 13.9±0.1 18.1±0.1 2.5±0.3 4.5±0.1 10.3±0.4 23.5±0.4 27.5±0.4 125.9 143.6 152.4 26.5 302.900

40 478±18.5 0.5±0.1 1.5±0.1 3.2±0.1 19.1±0.1 25.4±0.3 4.7±0.1 11.5±0.4 19.7±0.2 40.4±0.4 51.4±0.4 123.6 147.5 172.3 48.7 1500.000

50 474±4.90 0.5±0.1 6.3±0.3 8.8±0.1 19.1±0.1 25.3±0.2 2.5±0.3 4.4±0.4 12.3±0.3 23.5±0.4 33.5±0.4 115.5 132.2 155.9 40.4 1031.000

60 745±5.70 0.6±0.1 4.3±0.2 11.1±0.1 20.1±0.1 22.8±0.6 3.2±0.3 3.4±0.3 7.5±0.4 22.4±0.4 34.4±0.3 108.4 116.2 141.5 33.1 1565.000

70 522±4.90 0.7±0.1 1.5±0.1 7.8±0.1 13.9±0.1 18.4±0.3 4.3±0.2 11.3±0.4 15.7±0.2 33.5±0.4 44.5±0.3 139.6 145.3 165.1 25.5 1398.000

80 692±41.1 1.0±0.1 6.6±0.1 15.5±0.1 19.7±0.2 22.2±0.1 3.2±0.1 18.4±0.3 23.4±0.4 31.5±0.4 44.4±0.4 143.7 148.8 164.4 20.7 0.028

30 30 380±2.80 0.6±0.1 6.2±0.2 8.1±0.1 19.1±0.1 19.6±0.1 2.2±0.1 9.4±0.4 18.5±0.4 31.4±0.4 37.4±0.3 94.4 98.8 104.5 10.1 280.400

40 514±15.7 0.5±0.1 3.1±0.1 5.1±0.1 10.7±0.1 22.1±0.1 3.4±0.4 9.3±0.4 21.3±0.2 34.4±0.3 55.2±0.2 152.8 158.1 184.8 32.0 1522.000

50 456±4.90 0.7±0.1 4.2±0.2 6.5±0.1 20.0±0.1 25.3±0.2 3.4±0.3 13.3±0.2 19.6±0.4 31.3±0.3 44.5±0.4 108.1 127.3 118.6 10.5 0.207

60 734±24.2 0.6±0.1 6.5±0.1 10.1±0.1 13.9±0.1 19.6±0.4 4.3±0.2 5.6±0.4 11.1±0.1 45.3±0.3 55.4±0.3 138.4 152.4 168.2 29.8 368.200

70 580±7.50 0.7±0.1 4.4±0.1 6.7±0.2 22.1±0.1 25.2±0.2 4.4±0.2 21.2±0.2 35.4±0.3 44.4±0.4 50.3±0.4 143.1 150.1 175.9 32.8 1644.000

80 450±13.0 1.0±0.1 8.1±0.1 13.3±0.1 13.7±0.1 18.6±0.5 3.3±0.4 9.3±0.3 14.5±0.4 22.6±0.4 33.6±0.4 143.5 146.0 172.3 28.8 1229.000

45 30 392±2.80 0.5±0.1 2.0±0.1 4.4±0.1 13.9±0.1 15.4±0.3 2.5±0.3 5.2±0.4 12.4±0.4 23.5±0.4 40.3±0.4 118.8 136.2 148.1 29.3 0.591

40 402±4.90 0.5±0.1 6.1±0.1 8.8±0.1 16.5±0.1 18.3±0.2 5.7±0.2 18.5±0.4 34.4±0.4 41.4±0.4 56.5±0.4 102.0 104.9 129.5 27.5 1980.000

50 486±4.90 0.6±0.1 4.4±0.1 6.5±0.1 16.1±0.1 25.3±0.2 2.1±0.1 10.2±0.2 21.7±0.5 45.3±0.4 51.4±0.3 132.5 136.1 160.9 28.4 1628.000

60 384±4.90 0.5±0.1 2.9±0.1 7.8±0.1 13.9±0.1 25.4±0.3 2.4±0.4 12.3±0.4 30.5±0.4 44.3±0.2 49.6±0.4 136.5 136.9 153.7 17.2 43.660

70 575±12.7 0.6±0.1 8.1±0.1 13.0±0.1 20.4±0.4 23.9±0.6 2.2±0.3 19.5±0.3 27.6±0.1 41.6±0.3 55.3±0.3 102.4 127.2 156.0 53.6 0.014

80 400±7.50 1.0±0.1 8.1±0.1 15.5±0.1 18.8±0.1 22.6±0.3 3.3±0.2 8.4±0.3 11.5±0.4 18.5±0.4 26.5±0.4 143.5 146.5 170.8 27.3 0.056

Untreated

0 839±27.0 0.1±0.1 1.5±0.1 3.1±0.1 10.7±0.1 16.6±0.1 1.4±0.2 2.4±0.4 5.2±0.2 11.5±0.4 19.6±0.2 151.9 157.9 183.5 31.6 1245.000

To, onset temperature of gelatinisation; Tp, peak temperature of gelatinisation; Tc, conclusion temperature of gelatinisation; ΔH, enthalpy of gelatinisation.

223
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Andrabi, S. N., Wani, I. A., Gani, A., Hamdani, A. M. and Masoodi. F. A. 2016. Comparative study of physico‐
chemical and functional properties of starch extracted from two kidney bean (Phaseolus vulgaris L.) and green
gram cultivars (Vigna radiata L.) grown in India. Starch‐Stärke 68: 416–426.
Begum. R., Yusof, Y. A., Aziz, M. G. and Uddin, M. B. 2017. Structural and functional properties of pectin extracted
from jackfruit (Artocarpus heterophyllus) waste: effects of drying. International Journal of Food Properties,
DOI:10.1080/10942912.2017.1295054 (just-accepted).
Huang, Q., Li, L. and Fu. X. 2007. Ultrasound effects on the structure and chemical reactivity of cornstarch granules.
Starch‐Stärke 59(8): 371-378.
Leach, H.W., McCowen, L.D. and Schoch, T.J. 1959. Structure of starch granule. I. Swelling and solubility patterns of
various starches. Cereal Chem 36(6): 534-544.
Leong, C. M., Adzahan. M., Muhammad, S., Noranizan., Kharidah, S., Choo. and Sim. W. 2016. Physicochemical
properties of pectin extracted from jackfruit and chempedak fruit rinds using various acids. International Food
Research Journal 23(3): 973-978.
Luo, Z., Fu, X., He, X., Luo, F., Gao, Q. and Yu, S. 2008. Effect of ultrasonic treatment on the physicochemical
properties of maize starches differing in amylose content. Starch‐Stärke 60(11): 646-653.
Madruga, M. S., de Albuquerque, F. S. M., Silva, I. R. A., do Amaral, D. S., Magnani, M. and Neto, V. Q. 2014.
Chemical, morphological and functional properties of Brazilian jackfruit (Artocarpus heterophyllus L.) seeds
starch. Food chemistry 143: 440-445.
Majeed, T., Wani, I. A. and Hussain, P. R. 2017. Effect of dual modification of sonication and - irradiation on
physicochemical and functional properties of lentil (Lens culinaris L.) starch. International Journal of Biological
Macromolecules, DOI: 10.1016/j.ijbiomac.2017.03.110.
Mamat, M. R. Dec 2016. Aiskrim Nangka. Retrived on June 13, 2017 from The Star Website:
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Mukprasirt, A. and Sajjaanantakul, K. 2004. Physico‐chemical properties of flour and starch from jackfruit seeds
(Artocarpus heterophyllus Lam.) compared with modified starches. International Journal of Food Science and
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properties of starches from jackfruit seed and mung bean. International Journal of Food Properties 17(9):
1965-1976.
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 IFRC 2017: 212-196 Food Processing and Post-Harvest Technology
Influence of Drying Methods on the Bioactive Compound and Antioxidant Activity of Pomelo Residue
1 Abd Rahman, N.F., 1,2,* Shamsudin, R., 2,3 Ismail, A. 1 Shah, N.N.A.K. and 4 Varith, J.
1Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor, Malaysia
2Halal Science Research Laboratory, Halal Products Research Institutes, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor, Malaysia
3Department of Nutrition and Dietician, Faculty of Medicine and Health Science, University Putra Malaysia, 43400
UPM Serdang, Selangor, Malaysia
4Division of Food Engineering, Faculty of Engineering and Agro-Industry, Maejo University, Chiang Mai, Thailand
Abstract

Pomelo (Citrus grandis L. Osbeck) fruit is a rich source of bioactive compound incorporating outstanding antioxidant
properties. However, the pomelo residue known as pulp waste is usually discarded after the juice has been
extracted. Pomelo residue is extremely perishable which simultaneously leads to difficulty of handling, processing
and transporting due to its high moisture content. Thus, pomelo residue needs dehydration process to remove the
moisture and preserve the bioactive compounds. Therefore, the effect of the method used for drying on the bioactive
compound and antioxidant activities of pomelo residue was investigated by three different methods of drying: freeze
(control), conventional and vacuum drying. Antioxidant includes total phenolic content (TPC) whereas its activities
were indicated by using ferric reducing antioxidant potential (FRAP) and 2, 2- diphenyl-1-picrylhydrazyl (DPPH)
scavenging activity. Bioactive compound known as total phenolic content (TPC) showed significant increased
(p<0.05) in the overall temperature of conventional drying method whereas vacuum drying showed no significant
difference. In addition, reduction of DPPH was observed in both drying methods (conventional and vacuum dried) in
contrast with FRAP, no significant difference (p>0.05) was observed in conventional drying. In conclusion, this study
concludes supportive proof for the superiority oven dried pulp waste of Citrus grandis L. Osbeck showed a potentially
excellent source of the bioactive compound and could be exploited as functional ingredients in the nutraceutical field.

Keywords: Pomelo, antioxidant capacities, conventional drying, vacuum drying, pulp waste.
*Corresponding author’s email: rosnahs@upm.edu.my

Introduction

Pomelo (Citrus grandis (L.) osbeck) is commonly known as the biggest citrus fruit in its family Rutaceae. Antioxidant
from the juice could be retained in pomelo residue and it will be a great loss to dispose of the potential bioactive
compound that benefits the human health. After extraction, high moisture content of pomelo residue is not a shelf-
stable product and it can easily be spoiled. Therefore, the drying process is vital for evaporation of moisture from the
material and eventually minimizes the occurrences of spoilage (Earl and Earle, 2004). However, drying methods
highly influenced the quality of bioactive compound and antioxidant activity (AOA) of the product. Therefore, it is
significant to investigate the effect of different drying methods on thebioactive compound and its antioxidant activity
(AOA) of pomelo residues.

Material and Methods

3.1. Plant material.

Tambun White (Citrus grandis) PO52 is the most cultivated and popular in Malaysia. The pulp and the peels were
manually separated and the pulp was extracted using a juice extractor (CL-003AP, Interglobal International, LTD,

225
Taiwan). During post extraction, the fresh pomelo residue was stored in the freezer at -20°C until further used for the
drying process.

3.2. Freeze, conventional and vacuum drying

The pulp waste was freeze at -80 °C overnight in an ultra-low temperature freezer (MDF-U2086S; Sanyo, Japan).
According to Rahman et al., (2016), 100 g fresh pulp residue was lyophilized for 96 hr by using a freeze dryer (VirTis
Benchtop K, PA, USA). Freeze drying process was used as a control. The pomelo residue was then dried at 50, 60,
70, 80 and 90 °C in an oven (DO6836, Memmert, Germany) and vacuum oven (VD 23, Binder GmbH, Germany) with
the pressure adjusted in the range of 90-110 mbar for 24 hr. The pomelo pulp residue was spread evenly with the
thickness of 0.25 - 0.30 cm on the aluminum foil (21 cm x 21 cm).

3.3. Bioactive compound

Total phenolic content (TPC) was determined using Folin-Ciocalteu reagent spectrophotometrically at 725nm
(Singleton and Rossi, 1965).

3.4. Antioxidant activity (AOA)

DPPH radical scavenging activity of the pomelo residues was measured using 2, 2- diphenyl-1-picrylhydrazyl (DPPH)
scavenging activity. The ferric-reducing antioxidant potential (FRAP) was determined according to a modified method
by Benzie and Strain (1996).

3.5. The data collected were analyzed using SPPS Statistics 21.0 edition whereby Duncan’s test was tested to
evaluate the significant difference between mean values.

Results and Discussion

A bioactive compounds also known as secondary metabolites from plants are significant to scavenge a free radical
and possess multiple medicinal and physiological functions (Sultana, et al., 2012). Bioactive compound such as total
phenolic content affected by drying methods was shown in Figure 1. For the conventional oven, total phenolic content
(TPC) increased (762.36 - 1387.42 mg GAE/100g DW) significantly (p<0.05) compared to vacuum oven (254.20-
569.66 mg GAE/100g DW) and freeze (554.65 mg GAE/100g DW) drying. High bioactive compound (TPC) might be
because of exposure to the oxygen in conventional drying methods formed Maillard reaction products and
development of new phenolic compounds from their precursor (Sultana et al., 2012) compared to vacuum drying
process. Meanwhile, vacuum dried pomelo residue at a lower temperature (50 and 60°C) resulted in similar TPC
value with freeze drying.

Meanwhile, Table 1 presents the antiradical activity and ferric-reducing antioxidant power of pomelo residue affected
by the oven and vacuum dryer. Freeze dried pomelo residue showed higher radical scavenging activity (90.47%)
compared to conventional (85.14-67.79%) and vacuum dried (81.48-66.46%). As temperature increase in oven dryer,
the value of DPPH reduces significantly (p<0.05) based on some compounds are liberated during the drying process
(Jeong et al., 2004). In contrast, increased in radical scavenging activity of vacuum dried pomelo residue have been
attributed to the release of bound phenolic compounds which due to the breakdown of cellular constituents, and the
formation of new compounds with enhanced DPPH value (Chan et al., 2009).Briefly conventional drying at 60°C
(85.14%) is considered higher than vacuum dried pomelo residue and is selected as the most equivalent scavenging
radical activity with freeze dried pomelo residue (90.47%).

Furthermore, the values of FRAP was found to increase in conventional (2.84 –18.81 mM Fe(II)/g DW) and vacuum
drying (1.31-4.03 mM Fe(II)/g DW) in elevated temperature compared to freeze drying (3.07mM Fe(II)/g DW) (Table
1). This result is in agreement with Jeong et al. (2004) which showed similar trend on citrus unshiu fruits peels after
heat treatment. The results suggested that the increment of FRAP is attributed to the action of a hydroxyl group in

226
phenolic compounds which acts as an electron donor (Prasad et al., 2010). Therefore, the results proposed that both
drying methods could successfully enhance FRAP in pomelo residue.

1600

Total phenolic content(mg GAE/100g

a
1400

1200
b bc
1000 c
d
DW)

800 FD
e e CD
600 e e
f VD
400 g
200

0
FD 50 60 70 80 90
Drying temperature(°C)

Figure 1. Total phenolic content of pulp waste affected by different drying methods
Note: FD: Freeze drying; CD: Conventional drying; VD: Vacuum drying

Table 1. Antiradical activity and ferric reducing antioxidant power of dried pulp waste affected by different drying
methods.

Treatment/
Temperature DPPH (%) FRAP (mM Fe(II)/ g DW)
FD (control) 90.47 ± 0.84a 3.07 ± 0.10d
CD 50 80.28 ± 2.31c 2.84 ± 0.05de
60 85.14 ± 0.40b 5.22 ± 0.09b
70 81.42 ± 0.18c 2.90 ± 0.05de
80 81.09 ± 0.22c 3.11 ± 0.13d
90 67.79 ± 0.10e 18.81 ± 0.58a
VD 50 66.46 ± 1.24e 1.31 ± 0.03g
60 71.86 ± 1.77d 1.74 ± 0.06f
70 80.95 ± 0.81c 2.68 ± 0.20e
80 81.30 ± 0.22c 2.88 ± 0.03de
90 81.48 ± 0.51c 4.03 ± 0.19c
The similar consonant in the same column showed no significant different (p >0.05) value. Note: FD: Freeze drying;
CD: Conventional drying; VD: Vacuum drying

Conclusion

This work has shown that the temperature and methods used in the drying process greatly affected the total phenolic
content and antioxidant activity of dried pomelo pulp waste. Conventional drying afforded superior results in terms of
bioactive compound (TPC) and antioxidant activity compared to vacuum oven drying. This article provides the
guidance for dehydration process of pomelo residue that potentially act as a natural source of bioactive compound
and nutraceutical product.

227
Acknowledgement

The authors would like to thank the Ministry of Education of Malaysia, for providing financial support under the
fundamental research grants scheme (FRGS) 03-01-14-1412FR and also to University Putra Malaysia for the
financial support to conduct this research work.

References
Abd Ghafar, M. F., Prasad, K. N., Weng, K. K., and Ismail, A. 2010. Flavonoid, hesperidine, total phenolic contents
and antioxidant activities from Citrus species. African Journal of Biotechnology, 9(3), 326–330.
Benzie, I. F. F., and Strain, J. 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant
power”: The FRAP assay. Analytical Biochemistry, 239, 70–76.
Chan, E.W.C., Lim, Y.Y., Wong, S.K., Lim, K.K., Tan, S.P., Lianto, F.S., Yong, M.Y.2009. Effects of different drying
methods on the antioxidant properties of leaves and tea of ginger species. Food Chemistry,113(1), 166–172
Earl, R. L., and Earle, M. D. 2004. Unit operations in Food Processing, Web Edition, The New Zealand Institute of
Food Science and Technology (Inc). Copyright.1983, Earle. R. L. Published by NZIFST (Inc.).
Jeong, S.-M., Kim, S.-Y., Kim, D.-R. et al. 2004. Effect of heat treatment on the antioxidant activity of extracts from
citrus peels. Journal of Agricultural and Food Chemistry, 52, 3389–3393
Prasad, N.K., Xie, H., Hao, J., Yang, B., Qiu, S., Wei, X., Jiang, Y. 2010. Antioxidant and anticancer activities of
8-hydroxypsoralen isolated from wampee (Clausena lansium (lour.) skeels) peel. Food Chemistry,
18(1), 62-66
Rahman, N. F. A., Shamsudin, R., Ismail, A., and Karim Shah, N. N. A. 2016. Effects of post-drying methods on
pomelo fruit peels. Food Science and Biotechnology, 25(S1), 85–90.
Singleton, V. L., and Rossi, Jr., J. A. 1965. Colorimetry of total phenolics with phosphomolybdic–phosphotungstic
acid reagents. American Journal of Enology and Viticulture, 16, 144–158.
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antioxidant activity of selected fruits. Journal of Medicinal Plants Research, 6(1), 161–167.

228
 IFRC 2017: 247-226 Food Processing and Post-Harvest Technology
Development of Fish Gelatin Coatings Incorporated with Lemon Peel Extracts as Antimicrobial Packaging to
Extend the Shelf Life of Flammulina velutipes.

L. Naphawan1, Z.A Maryam Adilah1, Z.A. Nur Hanani1,2*

1Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43000 UPM
Serdang, Selangor, Malaysia.
2Halal Products Research Institute, Universiti Putra Malaysia, 43000 UPM Serdang Selangor, Malaysia.

Abstract

Antimicrobial coatings based on fish gelatin with lemon peel extract (LPE) were developed to extend the shelf life of
mushroom (Flammulina velutipes). Fish gelatin coatings incorporated with LPE successfully reduced up to 20%
microbial growth on the mushroom on the 12th day of storage as compared with control. Lower temperature (4ºC)
maintained the quality of mushrooms better than the higher temperature (25ºC) for all analyses. The sensory
evaluation revealed that browning became prominent only on 8th day onwards at both temperatures. The overall
acceptability showed that the coated mushroom sample is still acceptable even after the 8th day.

Keywords: Flammulina velutipes, lemon, gelatin, active packaging

*Corresponding author’s email: hanani@upm.edu.my

Introduction

Flammulina velutipes (F.velutipes) is also well known as golden needle mushroom, winter mushroom and enokitake
(Liu, Feng, Li, Yan, & Yang, 2016). Mushrooms are rich in nutrition such as protein, amino acid, fiber and low
amount of fat thus it is not surprising that this species ranked fourth as the most produced mushroom (Yang et al.,
2016). F.velutipes is particularly special as it contains numerous bioactive compounds such as flavonoid, glycosides,
proteins, polysaccharides, and phenols (Hu et al., 2016). Although mushroom has many benefits, it has one
detrimental weakness. Right after it is harvested, it can only last up to 3 days at room temperature (Han et al., 2015).
This is mainly due to microbial spoilage and evaporation of water due to the absence of protective layer on
mushroom (Donglu et al., 2016). This will lead to browning and thus decreases the organoleptic properties of
mushroom (Qin et al., 2015).

The objective of this study is to evaluate the potential of lemon peel extract (LPE) as a source of natural antimicrobial
incorporated into fish gelatin coatings to extend the shelf life of golden needle mushroom at different storage
temperatures.

Materials and Methods

3.1 Mushroom sample

Mushroom samples were obtained fresh from the market at Sri Serdang. The mushrooms were selected according to
their color, size and harvest time.
3.2 Lemon Peel Extraction
The extraction was performed using ethanol as according to Otang and Afolayan (2016).
3.3 Coating Preparation
The coating solution was prepared by using the wet method according to Ojagh et al. (2010).
3.4 Microbiological Analysis
The antimicrobial effect of LPE on mushroom was analysed using the method by Donglu et al. (2016).

229
3.5 Sensory Evaluation
The colour and overall acceptability of mushroom samples were determined in the sensory test. The panellists were
asked to rank the attributes from 0 to 10.
3.6 Statistical Analysis
One-way analysis of variance (ANOVA) and Tukey’s multiple tests using Minitab 17 software was used for statistical
analysis. The significant level was set for p≤0.05.
Results and Discussion
4.1 Microbiological Analysis
Microbiological analysis is very important in the food industry to ensure the safety of food. Figure 1 and Figure 2
show the Total Plate Count (TPC) of the mushroom sample as affected by the temperatures, days and different
concentrations of LPE. At 25˚C, the TPC values of mushroom were higher than 4˚C as lower temperature decreases
the microbial activity. An increase in microbial growth was seen from day 1 to 12 regardless of temperature and
concentration. This was ascribed to the transpiration rate of mushroom which in turn increases the relative humidity
in the packaging. This is illustrated by the formation of water droplets inside the packaging. This condition is very
favourable for microbial growth as described by Srinivasa et al. (2002). However, at 0.10% LPE concentration, 18%
to 20% microbial growth was inhibited as compared to control at both temperatures. This is probably due to the
antimicrobial effect of LPE. Research on the antimicrobial potential of LPE by Casquete et al. (2015) proved that
lemon peel extract can inhibit the growth of a wide range of gram-positive and gram-negative bacteria.

4.2 Sensory Evaluation

The enzymatic browning is catalyzed by polyphenol oxidase (Aguiló-Aguayo et al., 2014). The browning started to be
visible after day 4 as shown Table 1. The browning started to be very prominent on day 8 and day 12 at 25°C and
4°C, respectively.

Browning is a very undesirable characteristic perceived by the consumer. This is supported by the overall
acceptability result in which the acceptability was the lowest on day 12 which had the highest browning. Yet, up until
day 8, the overall acceptability was still quite high for both temperatures as shown in Table 2. Normally, the shelf-life
of mushroom were only 1 to 3 days (Han et al., 2015). However, based on this physical sensory analysis, the
mushrooms coated with fish gelatin incorporated with LPE were still acceptable up to 8th day of storage. This showed
that the fish gelatin coatings incorporated with LPE have the potential to prolong the acceptability of mushrooms up
to 8 days.

Table 1: Colour of mushroom packaged in fish gelatin coatings incorporated with 0.05 and 0.10% LPE

25°C Day 1 Day 4 Day 8 Day 12

Control 0.00±0.00 Ac 1.80±0.83 Ac 6.00±0.83 Ab 8.80±1.09Aa
0.05% 0.00±0.00 Ad 1.80±0.83 Ac 5.80±0.83 Ab 8.40±1.14Aa
0.10% 0.00±0.00 Ad 1.80±0.83 Ac 5.60±0.54 Ab 8.60±1.14Aa
4°C Day 1 Day 4 Day 8 Day 12
Control 0.00±0.00Ac 0.40±0.55Ac 4.40±1.14Ab 6.40±1.52Aa
0.05% 0.00±0.00 Ac 5.60±0.54 Ac 3.80±1.30 Ab 5.60±0.55Aa
0.10% 0.00±0.00 Ab 0.40±0.54 Ab 4.20±1.64 Aa 5.60±0.54Aa
Values are given as mean ± standard deviation. Different superscript lowercase letters in the same column indicate
significant differences (p≤0.05). Different superscript uppercase letters in the same row indicate significant
differences (p≤0.05).

230
Table 2: Overall acceptability of mushroom packaged in fish gelatin coatings incorporated with 0.05 and 0.10% LPE

25°C Day 1 Day 4 Day 8 Day 12

Control 10.00±0.00Aa 7.6±0.54Ab 4.60±1.34Ac 0.20±0.44Ad
0.05% 10.00±0.00Aa 8.20±0.44Ab 5.00±1.00Ac 0.20±0.44Ad
0.10% 10.00±0.00Aa 8.20±0.44Ab 5.20±0.83Ac 0.20±0.44Ad
4°C Day 1 Day 4 Day 8 Day 12
Control 10.00±0.00Aa 8.40±0.54Aab 6.60±0.54Ab 4.40±1.51Ac
0.05% 10.00±0.00 Aa 8.80±0.83 Aab 7.40±1.34 Abc 6.40±1.34Ac
0.10% 10.00±0.00Aa 8.60±0.89Aab 7.40±0.89Abc 6.40±1.34Ac
Values are given as mean ± standard deviation. Different superscript lowercase letters in the same column indicate
significant differences (p≤0.05). Different superscript uppercase letters in the same row indicate significant
differences (p≤0.05).

Total Plate Count at 25˚C

10
TPC (Log10 CFU/ g)

6
Control
4
0.05%
2 0.10%

0
Day 1 Day 4 Day 8 Day 12
Storage time (days)

Figure 1. TPC of mushroom packaged in fish gelatin coatings incorporated with 0.05 and 0.10% LPE (Log10 CFU/ml)
at 25˚C.

Total Plate Count at 4˚C

8
TPC (Log10 CFU/g)

4 Control
0.05%
2
0.10%
0
Day 1 Day 4 Day 8 Day 12
Storage time (days)

Figure 2. TPC of mushroom packaged in fish gelatin coatings incorporated with 0.05 and 0.10% LPE (Log10 CFU/ml)
at 4˚C.

231
Conclusion

This research has proven that fish gelatin coatings incorporated with lemon peel extracts are able to help in
minimizing the microbial spoilage in mushroom due to its antimicrobial properties thus extending the shelf life. Up to
20% of microbial count reduction on the mushroom sample is seen at 4°C. The present study has demonstrated that
combination of fish gelatin coatings incorporated with lemon peel extracts and low temperature are able to prolong
the acceptability of mushrooms up to 8 days.

References

Aguiló-Aguayo, I., Oms-Oliu, G., Martín-Belloso, O., & Soliva-Fortuny, R. (2014). Impact of pulsed light treatments on
quality characteristics and oxidative stability of fresh-cut avocado. LWT - Food Science and Technology,
59(1), 320–326.
Casquete, R., Castro, S. M., Martín, A., Ruiz-Moyano, S., Saraiva, J. A., Córdoba, M. G., & Teixeira, P. (2015).
Evaluation of the effect of high pressure on total phenolic content, antioxidant and antimicrobial activity of
citrus peels. Innovative Food Science and Emerging Technologies, 31, 37–44.
Donglu, F., Wenjian, Y., Kimatu, B. M., Mariga, A. M., Liyan, Z., Xinxin, A., & Qiuhui, H. (2016). Effect of
nanocomposite-based packaging on storage stability of mushrooms (Flammulina velutipes). Innovative Food
Science and Emerging Technologies, 33, 489–497.
Han, L., Qin, Y., Liu, D., Chen, H., Li, H., & Yuan, M. (2015). Evaluation of biodegradable film packaging to improve
the shelf-life of Boletus edulis wild edible mushrooms. Innovative Food Science and Emerging Technologies,
29, 288–294.
Hu, Q., Yu, J., Yang, W., Kimatu, B. M., Fang, Y., Ma, N., & Pei, F. (2016). Identification of flavonoids from
Flammulina velutipes and its neuroprotective effect on pheochromocytoma-12 cells. Food Chemistry, 204,
274–282.
Liu, X. Bin, Feng, B., Li, J., Yan, C., & Yang, Z. L. (2016). Genetic diversity and breeding history of Winter Mushroom
(Flammulina velutipes) in China uncovered by genomic SSR markers. Gene, 591(1), 227–235.
Ojagh, S. M., Rezaei, M., Razavi, S. H., & Hosseini, S. M. H. (2010). Effect of chitosan coatings enriched with
cinnamon oil on the quality of refrigerated rainbow trout. Food Chemistry, 120(1), 193–198.
Otang, W. M., & Afolayan, A. J. (2016). Antimicrobial and antioxidant efficacy of Citrus limon L. peel extracts used
for skin diseases by Xhosa tribe of Amathole District, Eastern Cape, South Africa. South African Journal of
Botany, 102, 46–49.
Qin, Y., Liu, D., Wu, Y., Yuan, M., Li, L., & Yang, J. (2015). Effect of PLA/PCL/cinnamaldehyde antimicrobial
packaging on physicochemical and microbial quality of button mushroom (Agaricus bisporus). Postharvest
Biology and Technology, 99, 73–79.
Srinivasa, P., Baskaran, R., Ramesh, M., Prashanth, K. H., & Tharanathan, R. (2002). Storage studies of mango
packed using biodegradable chitosan film. European Food Research and Technology, 215(6), 504–508
Yang, W., Yu, J., Pei, F., Mariga, A. M., Ma, N., Fang, Y., & Hu, Q. (2016). Effect of hot air drying on volatile
compounds of Flammulina velutipes detected by HS-SPME-GC-MS and electronic nose. Food Chemistry,
196, 860–866.

232
 IFRC 2017: 248-230 Food Processing and Post-Harvest Technology
Non-Evaporative Method to Remove High Boiling Point Solvent (Ethyl Lactate) from Palm Oil Extract at
Atmospheric Conditions
1*Kua, Y.L., 1Gan, S., 2Morris, A. and 3Ng, H.K.
1Department of Chemical and Environmental Engineering, University of Nottingham Malaysia Campus, 43500
Semenyih, Selangor, Malaysia.
2Faculty of Science, University of Nottingham Malaysia Campus, 43500 Semenyih, Selangor, Malaysia.
3Department of Mechanical, Materials and Manufacturing Engineering, University of Nottingham Malaysia Campus,
43500 Semenyih, Selangor, Malaysia.
Abstract
Ethyl lactate was demonstrated as a green and efficient agrochemical solvent to concentrate phytonutrients such as
carotenes and tocols in palm oil. However, the removal of this solvent from the extract is difficult and expensive due
to its very high boiling point and low volatility. Heating at high temperature in an effort to evaporate the solvent is
undesirable even under reduced pressure as the extracted phytonutrients are heat-sensitive compounds. In this
paper, a non-evaporative method using only water was proposed for the first time to remove the polar solvent at
atmospheric conditions based on its solubility difference instead of the vapour pressure difference. The proposed
method was proved to be more effective, faster and cheaper as compared to conventional approaches such as rotary
evaporation, freeze drying and vacuum drying.
Keywords: Carotene, ethyl lactate, palm oil, tocol.
*Corresponding author’s email: kebx4kye@nottingham.edu.my
Introduction
Ethyl lactate is a food grade solvent derived from carbohydrate feedstock from the corn and soybean industries. In
2005, the US Food and Drug Administration (USFDA) approved the direct use of ethyl lactate in food and
pharmaceutical products. It is also environmental friendly as the US Environmental Protection Agency (USEPA)
recognized ethyl lactate as a Significant New Alternatives Policy Program (SNAP) solvent. Ethyl lactate has great
solvency power because it can dissolve in both aqueous and hydrocarbon environments and is capable of extracting
compounds of a wide range of polarity (Strati and Oreopoulou, 2011). It has been discussed in Pereira et al. (2011)
to promote the replacement of existing solvents in manufacturing processes with greener alternatives. As published
in Scientists Solve Solvent Production Puzzle (1998), ethyl lactate has the potential to replace up to 80% of the
current industrial applications. Several papers have been published to demonstrate the usefulness of ethyl lactate as
a greener alternative. In our previous paper, the potential of ethyl lactate has been discussed to extract both polar
and non-polar phytonutrients from various fruit and vegetable wastes (Kua et al., 2016b).
Although ethyl lactate is green, safe and efficient, its removal from the extract is a challenge. The solvent has high
boiling point up to 154oC and low volatility. The evaporation rate of ethyl lactate is 0.29 as compared to butyl acetate
at 1 (Smith, 1998). Prolonged heating at high temperature is highly undesirable for current system as it involves heat-
sensitive phytonutrients such as carotenes and tocols. As the conventional evaporative methods were found
unsuitable, time- and energy-intensive for a system that composed of ethyl lactate, an alternative approach that
depends on solubility difference instead of vapour pressure difference was proposed in order to achieve the
separation.

In this paper, a simple, green and efficient non-evaporative approach was demonstrated for the first time to remove
the high boiling point ethyl lactate from palm oil extract under atmospheric temperature and pressure using only
water as the extractive agent. The potential of this process to simultaneously deacidify the oil was also reported.

233
Material and Methods
A total volume of 250 mL of solution composed of palm oil extract and distilled water were loaded into a temperature-
controlled mixer-settler system. The palm oil extract contained approximately 88.7 mass% of solvents (ethyl lactate
and ethanol) and 11.3 mass% of palm oil containing 540 mg/L of carotenes and 1500 mg/L of tocols. The solution
was mixed at 25oC for 3 min followed by settling for 1 h. Then, the upper (oil-rich) phase and lower (solvent-rich)
phase were collected for analysis.
Carotenes (α- and β-carotene) and tocols (α-T, α-, γ- and δ-T3) were quantified by high-performance liquid-
chromatography (HPLC) using methods as published in Kua et al. (2016a). As described in MPOB Test Method
p2.5:2004, free fatty acids (FFA) was quantified via titration.
Results and Discussion
Enrichment factor is used to compare the mass (mg) of carotenes and tocols over the volume (mL) of oil in the oil-
rich phase and the solvent-rich phase. It is a measure of system selectivity towards the distribution of carotenes and
tocols in the two phases. Percentage recovery is defined as the percentage mass recovery of carotenes, tocols and
oil into the oil-rich phase over the feed. It accounts for the amount recovered regardless of the selectivity. Figure 1a
shows the enrichment factor of carotenes and tocols with respect to the amount of water added to remove the
solvents. As compared to tocols, carotenes preferably diffused into the oil-rich phase because carotenes are more
hydrophobic due to the presence of unsaturated chain and hydroxyl group in tocols. Thus, relatively more tocols
diffused into the polar solvent-rich phase. Nonetheless, the enrichment factors were close to unity and hence, the
partition of carotenes and tocols during this process was not apparent.
Figure 1b shows the percentage recovery of carotenes, tocols and palm oil into the oil-rich phase. The average
recovery of carotenes was the highest, followed by tocols and oil. The values were 94.6%, 92.4% and 91.2%,
respectively. The recovery of carotenes was the highest because they are the most hydrophobic (non-polar) followed
by tocols and oil. This method of solvents removal was promising because at least 90% of the desired compounds
(carotenes, tocols and palm oil) were recovered into the oil-rich phase with minimal loss in the solvent-rich phase.
In Figure 2a, the solvent content in the oil-rich phase decreased with increasing vol% of water. As more water was
added, more extractive force was introduced to remove the polar solvents from the oil-rich phase. The solvent
content in the palm oil extract was initially 88.7 mass% and it had reduced to 8.6 mass% to 13.0 mass% as 30 to 60
vol% of water was added. As the solvent content reduced by approximately 78%, the energy required to remove the
remaining solvents would be much reduced.

(a) (b)

Figure 1. The enrichment factor of carotenes and tocols (a) and the percentage recovery of carotenes, tocols and oil
(b) with respect to the amount of water.

234
Figure 2a also shows the change of FFA content after the solvent removal process. The FFA content in the palm oil
extract was measured at 17.3%. As the vol% of water increased from 30% to 50%, the FFA content increased from
11.6% to 17.3%. As 30 vol% of water was added, the FFA reduced by 33% while there was no separation as 50 vol%
water was added. Beyond 50 vol% of water, there was no more observable reduction in FFA contained in the
phytonutrients-enriched palm oil. Similar results were obtained by Ansolin et al. (2013) as the addition of water into
ethanol was found to reduce the distribution coefficient of FFA in oil/solvent systems. As water enhanced the
system’s immiscibility, less FFA was removed. Even though the FFA content was much higher than the Palm Oil
Refiners Association of Malaysia (PORAM) standard specification at 0.1%, the findings showed the possibility of
achieving a certain degree of deacidification while removing the solvents in the palm oil extract. At lower FFA
content, the cost required in subsequent oil-deacidification step would be reduced.

Vacuum drying was tested to remove the solvents from the palm oil extract as well as the remaining solvents in the
recovered palm oil. The temperature to evaporate the solvents must be controlled at the lowest temperature possible
to conserve the sensitive phytonutrients. A temperature of less than 70oC is recommended to preserve carotenes. A
preliminary test was simulated using a mixture of 10 vol% palm oil in ethyl lactate. It was found that ethyl lactate
could be progressively removed in vacuum oven at conditions of 20 mbar and 40oC. Rotary evaporator and freeze
dryer were also tested within the allowable conditions with no success. The conditions were 50 mbar/80 oC using
rotary evaporator and 0.1 mbar/-42oC using freeze dryer.

Figure 2b shows the reduction of solvents in mass from palm oil extract and the recovered palm oil. After 3 h, the
solvent content in palm oil extract reduced by half (from 88.7 mass% to 42.9%). The solvent content in the recovered
palm oil reduced from 10.9 mass% to 7.7 mass%. As the graph for palm oil extract was extrapolated, it was found
that approximately 6 h of vacuum drying was required to reduce the solvent content from 88.7 mass% to 10.9
mass%. This step could be easily achieved in 1 h by liquid-liquid extraction using only water with no heat application.
Even though no obvious degradation of carotenes and tocols were detected after 3 h of vacuum drying at 40 oC, the
drying curves indicated the difficulty of solvents removal under conventional approach of evaporation via heating.

(b)
(a)

Figure 2. The solvent and FFA content in the oil-rich phase with respect to the amount of water (a). The reduction of
the solvents (%) in palm oil extract and recovered palm oil (b).

235
Conclusion

In summary, the presented method to remove ethyl lactate from palm oil extract was fast, efficient and inexpensive.
More than 90% of palm oil, carotenes and tocols were recovered while the solvent content reduced by approximately
78% within an hour of operation at atmospheric conditions. Additionally, the FFA could be removed simultaneously
which reduced the subsequent oil refining effort to deacidify the oil. The application of this method is not limited to
current system, but can be extended to remove other polar solvents easily from oil samples.

References

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fatty systems with emphasis on the distribution of tocopherols and tocotrienols. Fluid Phase Equilibria 338: 78-
86.
Kua, Y.L., Gan, S., Morris, A. and Ng, H.K. 2016a. A validated, rapid, simple and economical high-performance
liquid-chromatorgaphy method to quantify palm tocopherol and tocotrienols. Journal of Food Composition and
Analysis 53: 22-29.
Kua, Y.L., Gan, S., Morris, A. and Ng, H.K. 2016b. Ethyl lactate as a potential green solvent to extract hydrophilic
(polar) and lipophilic (non-polar) phytonutrients simultaneously from fruit and vegetable by-products.
Sustainable Chemistry and Pharmacy 4: 21-31.
Pereira, C.S.M., Silva, V.M.T.M. and Rodrigues, A.E. 2011. Ethyl lactate as a solvent: properties, applications and
production processes-a review. Green Chemistry 13: 2658-2671.
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Strati, I.F. and Oreopoulou, V. 2011. Effect of extraction parameters on the carotenoid recovery from tomato waste.
International Journal of Food Science and Technology 46: 23-29.

236
 IFRC 2017: 255-241 Food Processing and Post-Harvest Technology

Effect of Aloe Vera Powder as Fat and Corn Flour Replacers in the Production of Reduced Fat Beef Meatballs

Nurfazwin, Z., Nur Izzah Arifah, Z.A., Mohamad Afifi, I., Mat Yusoff, M. and *Ismail-Fitry, M.R.

Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia 43400 UPM
Serdang, Selangor, Malaysia

Abstract
Meatballs could contain high amount of fat that can cause many health problems. This study aimed to produce and
analyse the reduced fat meatballs by replacing the fat and corn flour (CF) with aloe vera powder (AP). The
formulations were formula with 0% AP (6% fat + 3% CF + 0% AP), formula with 3% AP (4% fat + 2% CF + 3% AP),
formula with 6% AP (2% fat + 1% CF + 6% AP) and formula with 9% AP (0% fat + 0% CF + 9% AP). The production
of AP was carried out by using spray dryer. Proximate analysis, cooking yield, fat retention, juiciness, colour, texture
profile analysis and sensory test were carried out to analyse the new formulated meatballs. The fat contents were
decreased as the AP in the meatballs increased with 0% AP (2.43% fat), 3% AP (2.38% fat), 6% AP (2.24% fat) and
9% AP (2.15% fat). The moisture content in meatballs was decreased with the increased of AP substitution,
respectively. The protein and ash contents were increased as the AP percentage in meatballs increased. Cooking
yield showed some decrement with the cooked meatballs compared to the uncooked meatballs, while the juiciness
showed higher value as the amount of AP increased in each meatballs formulation. The colour analysis showed that
L*(lightness) was reduced (p>0.05) when the AP percentage increased, while no significant differences (P>0.05)
were observed for the a*(redness) and b*(yellowness). Hardness, cohesiveness, gumminess, and chewiness have
no significant differences (p>0.05) for all the samples, except for springiness. Meatballs with 3% AP could be an ideal
formulation for reduced fat meatballs as it has similar sensory preference compared to the original meatballs.

Keywords: Aloe vera powder (AP), fat replacer, reduced fat meatballs
*Corresponding author’s email: ismailfitry@upm.edu.my

Introduction

Based on the current trends, consumers are demanding healthy and nutritive meat based products such as low-fat
meat products. Meatballs is common meat based products that compose of meats, fat and starches as the main
ingredients. Fat content influences the flavour, texture, mouthfeel and overall sensation to the meat based products.
Thus, the reduction of fat can significantly alters all those behaviours. Combination strategies on reducing fat in meat
products involves the lowering the overall animal fat level and replacing it with other part or functional ingredient such
as hydrocolloid gums, starches, and fibers. The functional ingredients addition increase the fibre content which lead
to production of healthier meat based products. Aloe vera are widely used as a functional food ingredient in food
industry and the leaves consists of 99.5% water and 0.5% of solid materials. Aloe vera available commercially in form
of concentrated powder and safe to be use in food products thus it had been incorporated in burgers patties
(Soltanizadeh & Ghiasi-Esfahani, 2015).

The objective of the study was to determine physicochemical and sensory properties of meatballs with aloe vera
powder as the fat replacer.

Materials and Methods

Aloe vera powder (AP).

Fresh aloe vera leaves were collected from Taman Pertanian Universiti (TPU), Universiti Putra Malaysia, Selangor,
Malaysia. The outer part were washed, cut and stored at -4 ºC. The mucilage gel of the aloe vera was homogenized

237
by blender to produce juice. The juice then added with maltodextrin that acts as the carrier. Then, the solution was
spray dried by using spray dryer (NIRO) under specific condition (inlet temperature = 150 °C – 170 °C; feed rate =
1.5 – 1.7 L/h and atomization speed = 23 000 – 27 500 rpm).

Meatballs processing.
Fresh beef and beef fat were purchased from Selangor Wholesale Market. Other ingredients such as onion powder,
corn starch, sugar, sodium tripolyphosphate (STPP), monosodium glutamate, and salt were purchase from Giant,
Taman Equine, Selangor. The ground meat and fat were mixed together with iced water, salt, STPP, corn starch,
monosodium glutamate, sugar, and onion powder. The mixture was kneaded for 15 min to obtained homogenous
meatballs dough. Four formulas prepared were formula with 0% AP (6% fat + 3% CF + 0% AP), formula with 3% AP
(4% fat + 2% CF + 3% AP), formula with 6% AP (2% fat + 1% CF + 6% AP) and formula with 9% AP (0% fat + 0%
CF + 9% AP). Each formulation was kneaded for additional 15 min to obtain homogenous dough. Meatball dough
then formed into meatballs of approximately 10 g. The meatballs were deep in hot water at 45 °C for 20 min and then
cooked at 90 °C for 20 min. The meatballs were cool down and packed for storage at -20 °C.

Cooking yield and juiciness.

Cooking yield was measured by calculating weight differences for samples before and after cooking. Juiciness was
measured by calculating the weight of filter paper before and after pressing the meatballs.

Proximate analysis.
Moisture, protein, fat and ash contents measurements were done in triplicate according to the methods described by
AOAC Standard Methods (1996).

Texture Profile Analysis.

The texture of the meatballs was measured by using a TA-XT plus texture analyser (Stable Micro system Ltd.,
Loughborough, UK). Hardness, adhesiveness, springiness, cohesiveness, gumminess, and chewiness were
calculated from the forces deformation curves.

Colour Determination.
The colour of the meatballs (L* (lightness), a* (redness) and b* (yellowness)) were measured by using a Mini Scan
XE Plus colourimeter (Hunter Associates Laboratory Inc., USA).

Sensory analysis.
Sensory analysis of meatballs was performed according to the guidelines of American Meat Science Association
(AMSA) for number of sensory characteristics (i.e. texture, juiciness, tenderness, colour, flavour, overall appearance
and overall acceptability). The characteristics were evaluated using 9-point hedonic scale (9 = like extremely; 5 =
neither like nor dislike; 1 = dislike extremely) with 30 untrained panellists.

Statistical Analysis.
The difference between means of values of the cooking yield, fat retention, juiciness, proximate composition, texture,
colour and sensory attributes among different formulations were analysed by using one-way analysis of variance
(ANOVA) of Minitab 16 (Minitab Statistical Software, USA).

Results and discussion

Table 1 presents the proximate composition for the new formulated meatballs with aloe vera powder. The fat
contents were decreased as the AP in the meatballs increased with 0% AP (2.43% fat), 3% AP (2.38% fat), 6% AP
(2.24% fat) and 9% AP (2.15% fat). The result obtained shows that AP can act as replacer of fat and binder to
produce low fat meat products. The low-fat content in formulated meatballs also might be due to the low fat content in
the aloe vera pulp. The moisture content in meatballs were decreased with 72.97%, 70.77%, 66.80%, and 64.20%,
for meatballs with 0%, 3%, 6% and 9% AP substitution, respectively. It shows that high amount of AP incorporated
resulted the meatballs to contain less moisture content rather that aloe vera pulp substitutions. According to

238
Serdaroǧlu (2006), the moisture content of meatballs might be lower when higher amount of dry ingredients included
in the formulations. The protein and ash contents were increased as the AP percentage in meatballs increased. This
indicates that AP provide source of protein and minerals to the meatball formulations (Soltanizadeh & Ghiasi-
Esfahani, 2014).
Results for cooking yield, fat retention, juiciness, colour, and texture profile analysis are shown in Table 2. Cooking
yield of the meatball showed some decrement with the cooked meatballs compared to the uncooked meatballs. The
addition of AP in the formulated meatballs has increased the cooking loss, which may be attributed to the low water
holding capacity and moisture retention of aloe vera during cooking the meatballs (El-Magoli, Laroia, & Hansen,
1996). The juiciness of the meatballs showed higher value as the amount of AP increased in each formulation. The
percentage of the juiciness increased from 0.7% to 1.91% which shows positive result when using AP substitution in
the meatballs.
The colour analysis showed that L*(lightness) was reduced (p>0.05) when the AP percentage increased, while no
significant differences (P>0.05) were observed for the a*(redness) and b*(yellowness). This was affected by the
amount of fat and corn flour substitute in the formulations being reduced (Serdaroğlu & Değirmencioğlu, 2004).
Hardness, cohesiveness, gumminess, and chewiness have no significant differences (p>0.05) for all the samples,
except for springiness. When percentage of AP is increased, the meatballs become harder than the control.
Table 3 shows the results for sensory evaluation. The more preferable meatballs were the one with 0% and 3% of AP
substituted. These two formulations were slightly similar which makes the panellist can accept the presence of fat
and AP substitution. Fat and corn flour level were reduced as the AP substituted increased in the new formulated
meatballs. According to Soltanizadeh & Ghiasi-Esfahani (2014) the reason is because the bitter taste from the aloin
in the AP make the panellist reject the taste.

Table 1: Proximate analysis of meatballs with aloe vera powder (AP) as fat replacer
Proximate Analysis
Aloe vera Moisture Ash Fat Protein Carbohydrate
powder(AP)
0% 72.97±0.23a 1.25±0.09a 2.43±0.08a 0.38±0.17ab 23.02±0.32d
3% 70.77±0.36 b 1.00±0.73 a 2.38±0.03 a 0.25±0.03 b 25.60±1.13c
6% 66.80±0.05 c 1.67±0.08 a 2.24±0.05 b 0.27±0.02 b 29.02±0.02b
9% 64.20±0.13 d 1.68±0.12 a 2.15±0.03 b 0.56±0.03 a 31.42±0.03a
a-d Means with similar letter are significantly different (p<0.05) within the same column.

Table 2: Cooking yield, juiciness, fat retention, colour and texture profile analysis of meatballs with aloe vera powder
(AP) as fat replacer
Parameters Aloe vera powder (AP)
0% 3% 6% 9%
Cooking Yield 93.38±0.448a 78.44±0.477b 53.33±0.42d 55.14±0.255c
Juiciness 0.69±0.76 a 1.12±0.52 a 1.13±0.79 a 1.91±1.0a
Colour
L* 49.86±2.62a 46.52±3.29ab 41.83±5.56b 44.10±4.00ab
a* 2.15±0.73 a 2.07±0.86 a 2.58±0.81 a 2.85±0.79a
b* 11.72±0.53 a 11.10±0.36 a 10.96±0.56 a 11.62±0.97a
Texture
Hardness 4574.50±480.03a 4112. 46±63.60a 4598.40±804.92a 4832.69±587.95a
Springiness 0.93±0.01 a 0.91±0.009 ab 0.91±0.008 ab 0.91±0.004b
Cohesiveness 0.74±0.03 a 0.66±0.05 a 0.67±0.04 a 0.69±0.06a
Gumminess 3379.55±247.29 a 2719.55±178.76 a 3099.74±711.51 a 3359.06±693.22a
Chewiness 3150.05±195.20 a 2480.05±163.43 a 2829.07±666.58 a 3056.15±636.38a
a-d Means with similar letter are significantly different (p<0.05) within the same row.

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 Table 3: Sensory evaluation of meatballs with aloe vera powder (AP) as fat replacer
Aloe vera powder (AP)
Attributes
0% 3% 6% 9%
Colour 5.63±1.65a 5.30±1.71a 5.53±1.22a 5.60±1.65a
Aroma 5.93±1.55 a 5.67±1.54 a 5.40±1.22 a 5.63±1.19a
Texture 6.50±1.64a 5.83±1.53a 3.87±1.89b 3.60±2.06b
Taste 6.30±1.66 a 6.10±1.71 a 4.27±1.84 b 4.13±1.69b
Juiciness 6.60±1.35 a 5.57±1.94 a 3.37±1.79 b 3.37±1.83b
Overall 6.47±1.78 a 5.87±1.61 a 3.93±1.78 b 3.83±1.82b
acceptability
a-b Means with similar letter are significantly different (p<0.05) within the same column.

Conclusion
Meatballs with fat replaced with AP were successfully produced. The fat contents were reduced with several
physicochemical properties and textural qualities retained. Meatballs with 3% AP could be an ideal formulation for
reduced fat meatballs as it has similar preference compared to the original meatballs.

References

El-Magoli, S. B., Laroia, S. & Hansen, P. T. M. 1996. Flavour and texture characteristics of low fat ground beef
patties formulated with whey protein concentrate. Meat Science, 42(2), 179–193.
Serdaroğlu, M. & Değirmencioglu, Ő. 2004. Effects of fat level (5%, 10%, 20%) and corn flour (0%, 2%, 4%) on some
properties of Turkish type meatballs (koefte). Meat Science, 68(2), 291–296.
Soltanizadeh, N. & Ghiasi-Esfahani, H. 2014. Qualitative improvement of low meat beef burger using Aloe vera. Meat
Science, 99, 75–80.

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 IFRC 2017: 259-272 Food Processing and Post-Harvest Technology

Optimization of Natural Red Colorant Production from Roselle Using Ultrasound-Assisted Extraction

Mokhtar, N., *Pak-Dek, M.S., Hamid, A.A., Mohd-Johar, A.H.H., and Jaafar, A.H.

Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang,
Selangor, Malaysia
Abstract:
Roselle (Hibiscus sabdariffa L.) contains high level of anthocyanin and it gains a great consideration as natural red
food colorant. Previous study showed several extraction methods have been used to extract natural colorant from
plant but no study on the extraction of colorant from Roselle using ultrasound-assisted extraction (UAE) has been
reported. Similarly, the information on the stability of Roselle red colorant at different temperature and storage
materials is not available in the literature. Therefore, a study aims to optimize UAE conditions for producing red
colorant from Roselle. The stability of Roselle red colorant at different temperature and packaging materials was
evaluated. UAE optimization started with single factor experiments followed by response surface methodology
(RSM). The studied UAE variables for RSM were percentage of sample weight (10-20%), sonication time (10-30
minutes) and pre-incubation time (0-30 minutes). Results of the study showed an optimum UEA condition to extract
highest red colorant from Roselle was at 15% sample weight, 10 minutes sonication time and 20 minutes pre-
incubation time. The experimental values for these responses were not significantly different with predicted values.
Further analysis on the stability of red Roselle colorant at chilled and room temperature using different storage
materials namely opaque bottle and clear bottle showed the Roselle red colorant was stable at chilled temperature
for both packaging materials. In conclusion, the study showed that UAE can be used to produce red colorant from
Roselle and the storage at low temperature is recommended for longer shelf life. This information provides another
natural red colorant that can be incorporated in food.
Keywords: Roselle; red colorant; ultrasonic assisted extraction; storage temperature; packaging material
*Corresponding author’s email: mhdsabri@upm.edu.my

Introduction
Colorant is important in food processing. There are two types of colorants known as natural and synthetic. The food
colorants are also known as food additive (Dafallah et al., 2015). Food additive is a manufactured or natural
substance which can be added into the food to restore the loss of colors during processing. However, there are some
issues that limit the application of synthetic colorant such as an adverse effect on the human health. Study showed
that consumer prefers to have naturally derived food colorant in their food because of the trustfulness and safety
perception (Dafallah et al., 2015).
The synthetic colorant can be replaced by using plant natural colorant. In addition, plant natural colorant
usually will have value added properties such as antioxidant effect. This will gives better healty beneficial effect and
restore consumer perception about food colorant. Ultrasound assisted extraction (UAE) is a method widely used in
extracting natural colorant from plant. However no study on the extraction of colorant from Roselle using UAE
reported. The optimum UAE conditions such as percentage of samples, extraction time and pre-incubation are varied
between samples. This observation was in alignment with previously reported study on the sonication of tomato juice.
The study mentioned that sonication process may led to the decreases in Lightness (L*), redness (a*) and yellowish
(b*) values in tomato juice (Adekunte et al., 2010). The color changes occur during UAE also can be explained due to
cavitational effect that governs various physical, chemical and biological reactions (Sala et al., 1995). Thus, the
optimum condition for Roselle should be identified. Besides that, Roselle extract contains anthocyanins. Anthocyanin
is a light and heat sensitive compound. The suitable storage temperature for Roselle red colorant should be
identified. Therefore, the objectives of the study were to optimize UAE conditions for produce high red colorant from
Roselle and to study the stability of red colorant from Roselle at different temperature and packaging materials.

241
Materials and Method
3.1. Raw material. The dried Roselle was bought from Batu Pahat, Johor. The individual flower, without defects and
diseases were selected for further analysis.

Extraction of sample. The samples of Roselle were extracted using ultrasonic water bath (WiseClean, Wisd
Laboratory Instrument).
3.2. Optimization of UAE condition. The quadratic central composite design (CCD) with 3 factors was used to
optimize the UAE condition. The design consisted of 20 experimental points generated by using Minitab 16 software
(Minitab®, United States) with redness and total monomeric anthocyanin as responses.
3.3.The redness was measured by using a Hunter Lab (UltraScan Pro, HunterLab).
3.4. Total monomeric anthocyanin content (TAC) in the samples was measured using pH differential method with
cyanidin-3-O-glucoside as a internal standard to develop standard curve (10, 20, 30 and 40 ppm).
3.5. Stability of the sample. Approximately 5 mL of Roselle extract was transferred into two types of storage materials
(opaque bottle and clear bottle). Synthetic red colorant was used as a control sample. Test and control samples were
kept under room temperature (28°C) and chilled temperature (8°C). Then, the redness was recorded every 4 days
for 20 days.
3.6. All data were expressed as mean±standard deviation. The analyses were done in triplicate. Data were analyzed
using one-way ANOVA using SPSS version 16 (SPSS Inc., Chicago, Illinois, USA) and Minitab 16. .

Results and Discussion

In this study, UAE condition was optimized using response surface methodology (RSM) with three factors namely
percentage of sample, sonication time and pre-incubation time in order to produce a Roselle extract with highest
redness and anthocyanin content. Figure 1 shows the response surface plots for the effect for different ultrasonic
parameter on the redness of Roselle extract. The selected ultrasonic parameters significantly affect the redness of
Roselle and the quadratic model was recommended for optimization. The redness increased with the percentage of
sample. The maximum redness was achieved with 10% sample. Increase sample concentration over 10% decreased
the redness.

Figure 2 shows the effect of ultrasonic parameters on TAC of the Roselle extract. At constant pre-incubation time and
time of sonication, the anthocyanin value in Roselle colorant increased. The increment also was significant. As
illustrated in Figure 2, TAC concentration increased with percentage of sample in combination with pre-incubation
time and sonication time.

Previous studies showed that the degradation of anthocyanin is caused by frequency, temperature and exposure
time of the extract (Vercet et al., 1998). Besides, anthocyanin degradation also because of water sonolysis by
cavitation, inducing the formation of hydroxyl radical which lead to the chemical decomposition (Vercet et al., 1998).
Higher degradation of anthocyanin occurs with increasing of treatment time and temperature due to oxidation,
cleavage of covalent bonds or enhanced reaction of oxidation (Tiwari et al., 2009). Since the current study did not
use higher frequency and higher temperature, the degradation on anthocyanin of Roselle is relatively low.

Optimization of UAE condition to produce red colorant from Roselle was carried out. By using RSM method, the
optimal UAE condition to obtain red colorant from Roselle was as follow: a sample weight (15%), sonication time (10
minutes) and pre-incubation time (20 minutes). At the optimized condition, the redness and TAC of the Roselle was
at 48.97±0.035, and 2.516±0.121 mg/mL, respectively. The experimental value was not significantly different
(P>0.05) from predicted value.

Visually, there was no color changes observed in Roselle extract during twenty days of storage at room and chilled
temperature. However, there was a minimal change on the redness observed by using colorimeter as shown in
Figure 3. The redness values (a*) of Roselle extract decreased from 48.98 to 43.89 at chilled temperature.
Meanwhile at room temperature, the redness reduced from 48.98 at day 0 into 44.05 at day 20.

242
Design-Expert® Software
Design-Expert® Software
Factor Coding: Actual Factor Coding: Actual
redness (a) redness (a)
Design points above predicted value Design points above predicted value
Design points below predicted value Design points below predicted value
61.97 61.97
59.55 62 59.55 62
re d n e s s (a )

re d n e s s (a )
X1 = A: sample weight 61.5 X1 = A: sample weight 61.5
X2 = B: time of sonication X2 = C: pre-incubation
61 61
Actual Factor Actual Factor
C: pre-incubation = 10.5 60.5 B: time of sonication = 20 60.5
60 60
59.5 59.5

30 20

16.2
25

12.4
20 15 15
13 8.6 13
B: time of sonication (min) C: pre-incubation (min)
11 11
15
9 4.8 9
7 7
10 5 1 5
A: sample weight (%) A: sample weight (%)

A B
Figure 5 Response surface plots for the effect of different ultrasonic parameter on redness from Roselle. A: The interaction
between time of sonication (min) and sample weight (%) on the redness. B: The interaction between pre-incubation time (min)
and sample weight (%) on the redness.
Design-Expert® Software
Design-Expert® Software
Factor Coding: Actual
Factor Coding: Actual
anthocyanin (mg/ml)
Design points above predicted value
anthocyanin (mg/ml)
Design points below predicted value Design points above predicted value
2.197 Design points below predicted value
a n th o c y a n in (m g /m l)
a n th o c y a n in (m g /m l)

2.197
0.848 2.4
0.848 2.4
X1 = A: sample weight 2.2 2.2
X2 = B: time of sonication X1 = A: sample weight
2 X2 = C: pre-incubation 2
Actual Factor 1.8
C: pre-incubation = 10.5 Actual Factor 1.8
1.6 B: time of sonication = 20 1.6
1.4 1.4
1.2 1.2
1 1
0.8 0.8

30 20

25 16.2
15
13
20 12.4 15
11
B: time of sonication (min) 13
15 9 8.6
7 C: pre-incubation (min) 11
10 5 A: sample weight (%) 4.8 9
7
1 5
A: sample weight (%)

A B
Figure 2. Response surface plots for the effect of different ultrasonic parameter on anthocyanin content from Roselle. A: The
interaction between time of sonication (min) and sample weight (%) on the anthocyanin content. B: The interaction between pre-
incubation time (min) and sample weight (%) on the anthocyanin content.

Figure 3. Relationship between storage time versus redness of sample in different storage temperature and storage materials for
Roselle and red synthetic colorant. CO = Roselle at chilled temperature (opaque bottle); RCC = Roselle at chilled temperature
(clear bottle); RRTO = Roselle at room temperature (opaque bottle); RRTC = Roselle at room temperature (clear bottle); SCCO =
Synthetic colorant at chilled temperature (opaque bottle); SCCC = Synthetic colorant at chilled temperature (clear bottle);
SCRTO = Synthetic colorant at room temperature (opaque bottle); SCRTC = Synthetic colorant at room temperature (clear
bottle)

243
Interestingly, the Roselle red colorant stored at room temperature spoiled at twelfth day and the similar trend was
observed with commercial colorant. The Roselle extract stored in clear bottle had higher reduction in redness as
compared to the opaque bottle. Besides that, there was no significantly different on type of packaging material at
same temperatures. However, the redness value of Roselle red colorant stored at room temperature was rapidly
decreased as compared with storage at chilled temperature.

The study provides information that the redness value in Roselle is contributed by anthocyanin. Anthocyanin is a light
sensitive compound. Therefore, reduction in redness of the Roselle extract may due to the anthocyanins degradation.
Prevoious study also found that anthocyanins are considered as a good indicator of for redness (Wong et al., 2003).
Another study reported that changes on redness and anthocyanins are also due to the heat and light exposure
(Hewavitharana et al. 2013).

Conclusion
The optimum UAE condition such as sample weight, sonication time and pre-incubation time to produce colorant with
high redness and TAC from Roselle was identified. The stability of red colorant from Roselle was depended on
temperature and storage material. The redness of Roselle red colorant degraded faster at room temperature as
compared with chiller temperature. The Roselle red colorant was stable at chilled temperature as compared with at
room temperature for both types of storage materials. Therefore, it is recommended that Roselle red colorant to be
stored at cold temperature in order to maximize its shelf life.

Acknowledgements
The authors would like to thank UPM for financial support.

Reference
Adekunte, A., Tiwari, B.K., Cullen, P.J., Scannell, A. and O’Donnell, C.P. 2010. Effect of sonication on colour,
ascorbic acid and yeast inactivation in tomato juice. Food Chemistry, 122: 500–507.
Dafallah, A. A., Abdellah, A. M., Abdel-Rahim, E. A., and Ahmed, S. H. 2015. Physiological Effects of Some Artificial
and Natural Food Coloring on Young Male Albino Rats. Journal of Food Technology Research, 2(2): 21–32.
Hewavitharana, A. K., Tan, Z. W. Shimada, R. Shaw, P. N. and Flanagan. B. M. 2013. Between fruit variability of the
bioactive compounds, β-carotene and mangiferin, in mango (Mangifera indica). Nutrition Diet, 70:158–163.
Sala, F.J., Burgos, J., Condon, S., Lopez, P. and Raso, J.1995. Effect of heat and ultrasound on microorganisms and
enzymes. In: New Methods of Food Preservation (edited by G.W. Gould). Pp. 176–204. London: Blackie
Academic and Professional
Tiwari, B.K., O'donnell, C.P. and Cullen, P.J. 2009. Effect of sonication on retention of anthocyanins in blackberry
juice. J. Food Eng, 93:166–171.
Vercet, P., Lopez, A. and Burgos, J. 1998. Free radical production by monothermosonication. Ultrasonics, 36:615–
618.
Wong, P.K., Yusof, S., Ghazali, H.M., and Che, M.Y. 2003. Optimization of hot water extraction of roselle juice using
response surface methodology: a comparative study with other extraction methods. Journal of the Science of
Food and agriculture, 83(12):1273-1278.

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 IFRC 2017: 277-254 Food Processing and Post-Harvest Technology

Rheological Properties, Emulsion and Oxidative Stability of Cocoa Butter Based Salad Dressing During
Storage

1,3*Ishak, I., 1,2Hussain, N. and 2Mohd Hariri, N.A.

1Halal Products Research Institute, Universiti Putra Malaysia, 43400 Selangor, Malaysia.
2Department of Food Science and Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia,
43400 Selangor, Malaysia.
3Innovation Centre for Confectionery Technology (MANIS), Faculty of Science and Technology, Universiti
Kebangsaan Malaysia, 43600 Selangor, Malaysia.

Abstract
This study was carried out to determine the rheological property, emulsion stability, optimal microscope observation
and oxidative stability of the newly formulated cocoa butter salad dressing (CBSD) and its storage study at 18 °C for
28 days. The most stable ratio 20 % CBSD was selected for the storage study due to insignificantly (p>0.05) similar
rheological characteristics with control in terms of flow behavior (0.35±0.00), consistency index (18.78±0.20),
apparent viscosity (3.48±0.02) and emulsion stability (97.9±0.96). The optical observation showed that the droplet
size of 20% CBSD was similar to the control and commercial salad dressing. The iodine and peroxide value also
increased throughout shelf life period comparable to commercial salad dressing. This research may benefit the cocoa
industry to meet the consumer’s demand for values add food products.

Keywords: emulsion, salad dressing, cocoa butter, storage, rheology

*Corresponding author’s email: izzreen99@gmail.com

Introduction
Cocoa butter (CB) usually exists in tons of formulations for chocolate due to unique characteristics and beneficial for
health (Serafini et al., 2003) and in confectionary products blended with hydrogenated soybean oil, palm oil fractions,
palm kernel, and coconut oil (Pe´rez-Martı´nez et al., 2007). However, there is no research done on application of
premium CB as salad dressing (SD). CB has favorable fatty acid distribution; 37.5 % of the fat in CB is unsaturated
fat and about 61.4 % is saturated fat as well as the presence of natural antioxidants, such as tocopherols and
tocotrienols. An added benefit that cocoa butter provides is oxidative stability, which contributes to extended shelf life
for foods (Manach et al., 2004). Also, CB contains melting behavior and texture at different temperatures distinguish
this fat and provide its added value (Maleky and Marangoni, 2008).

The objective of the study were to evaluate the properties and storage study of cocoa butter based salad dressing at
18 °C for 28 days.

Material and Methods

Materials
Soybean oil, egg, Arabic and xanthan gum, sodium benzoate, water, vinegar, sugar, salt and lemon juice were
purchased from local supermarket (Giant, Malaysia). Soybean oil was used as the main ingredient in salad dressing
while cocoa butter was used to value add the SD. Commercial Lady’s Choice salad dressing used in study as
comparison to control and CBSD.

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CBSD Formulations
Different concentration of CB and soybean oil were blended (0%, 10%, 20%, 30%, 40%, and 50%) in the formulation
of CB based salad dressing. Control sample (0 %) is the salad dressing without addition of CB. Amount of salad
dressing used in each of the formulation is 150 g. The best ratio of salad dressing with the best rheological properties
of salad dressing was selected for storage study to determine their oxidative stability.

Methods
Rheological measurements were performed using rheometer (HAAKE RheoStress 6000, USA). This analysis was
done to determine the flow properties based on shear stress to shear rate of all CBSD samples.

Emulsion stability is the method to determine the volume of oil separated from an emulsion during centrifugation. This
method was analyzed by using Thermo Sorvall Legend Micro 17 (USA).

Optical Microscopic Observation was performed using Inverted Microscope Nikon Diaphot, (USA) to develop a clear
picture of the salad dressing microstructure were acquired by a digital camera (Liu et al., 2007).

Storage study to determine oxidative stability based on the method from AOCS Cd 8b-90 (2003) was carried out for
peroxide value (PV) and iodine value (IV) on 20% CBSD compared to salad dressing without cocoa butter and
commercial salad dressing.

All analyses were conducted in triplicate. Data were statistically analysed by one-way analysis of variance procedure,
using SPSS software. Significant differences (p<0.05) between means were determined by Duncan’s multiple-range
test and Tukey’s test.

Results and Discussion

Shear stress to shear rate of the CBSD prepared from different formulations of (0% to 50%) at 20˚C was shown in
Table 1. The apparent viscosity of 0% (3.44±0.03) and 20% (3.48±0.02) CBSD in Herschel-Bulkey equations
showed slightly similar value. The flow behavior index for all samples of CBSD was less than 1. However, the value
of control showed insignificant different with 20% CBSD compared to the other samples. Sample with flow behavior
index of less than 1 indicated pseudoplastic fluid characteristic. The higher flow behavior index of CBSD indicated
more elastic or greater gel-like structure. The most stable ratio of 20% CBSD was selected for the next storage study
due to insignificantly (p>0.05) similar rheological characteristics (0.35±0.00) with control (0.36±0.00) for flow
behaviour, (18.78±0.20) consistency index and apparent viscosity (3.48±0.03).

The oil in-water emulsions of six different samples of CBSD (0 – 50%) were analyzed. In this study, again 20% CBSD
showed insignificant different (p>0.05) characteristic with control during emulsion test (Table 2).

Analysis of optical microscopic observation was carried out to visualize the droplet size distribution of commercial
salad dressing, 0 until 50% of CBSD. The size of droplets varied. As the addition of cocoa butter in salad dressing
increases, droplet size of oils also increases. In 0%, 10% and 20% CBSD, small droplets and close pack structure of
oil were found similar in size as commercial salad dressing (Figures 1a, b, c, d) whereas large droplets were found in
30%, 40% and 50% of CBSD (Figures 1e, f, and g).

During storage at temperature 18°C for 28 days, PV of 20% CBSD increased to 18.63 meqO 2/kg at the end of
storage, insignificantly different with control (14.43) meqO2/kg and commercial (15.96) meqO2/kg (Table 3). Blending
of soybean oil which was high in linoleic acid with cocoa butter shows increased in IV for all samples. The 0% CBSD
has significantly (p<0.05) the highest IV (18.56±0.3) followed by 20% CBSD (18.20±1.1) and commercial salad
dressing (17.30±0.6) as duration of storage extended (Table 4). This result provides a general status of unsaturation
of the oils which caused the increased in IV and PV thus is impossible to point out the position of double bond(s)
which are more vulnerable to be oxidized (Knothe and Dunn, 2003).

246
 Table 1: Shear stress to shear rate for different ratios of cocoa butter in CBSD
Addition of cocoa butter in Power flow behaviour Consistency index, k (Pa Apparent viscosity, ƞ (Pa
salad dressing, (%) index, n (no unit) sn) s)

0 (control) 0.36 ± 0.00abc 19.23 ± 0.19d 3.44 ± 0.03d

10 0.40 ± 0.04a 15.00 ± 0.09e 2.21 ± 0.03e
20 0.35 ± 0.00abc 18.78 ± 0.20d 3.48 ± 0.02d
30 0.37 ± 0.00ab 29.95 ± 0.05 c 4.64 ± 0.00c
40 0.31 ± 0.00c 57.98 ± 0.01 b 7.32 ± 0.00b
50 0.33 ± 0.01bc 90.85 ± 0.30 a 10.32 ± 0.04a
Data were mean ± sd (n=3). Means that do not share similar alphabet within the same column are significantly
different at (p<0.05).
Table 2: Emulsion stability values of the ratio of CBSD
Addition of cocoa butter in salad dressing, % Emulsion stability, (%)
0 96.60 ± 0.51bc
10 96.43 ± 0.57c
20 97.94 ± 0.96bc
30 98.02 ± 0.95ab
40 98.08 ± 0.06ab
50 99.28 ± 0.47a
Data were mean ± sd (n=3). Means that do not share similar alphabet within the same column are significantly
different at (p<0.05).

Table 3: Peroxide Value (PV) of 0 % and 20 % CBSD and commercial one during storage
PV for 28 days of storage (meqO2/kg)
Sample 0 day 7 days 14 days 21 days 28 days
0% 8.87±1.35b 11.66±0.47ab 12.40±0.10a 14.00±1.57a 14.43±1.40a
20 % 11.26 ±0.35 b 11.53±0.55 b 13.70±1.99 b 17.20±0.10 a 18.63±0.11a
Commercial 11.30±0.36 b 11.63±0.85 b 12.46±0.63 b 12.50±1.01 b 15.96±0.46a
Data were mean ± sd (n=3). Means that do not share similar alphabet within the same column are significantly
different at (p<0.05).

Table 4: Iodine Value (IV) of 0 % and 20 % cocoa butter based salad dressing compared with commercial salad
dressing during storage for 28 days
IV for 28 days of storage (meqO2/kg)
Sample 0 day 7 days 14 days 21 days 28 days
0% 11.90±0.72 b 12.86±0.58 b 13.43±1.41 b 18.20±0.34 a 18.56±0.30a
20 % 15.96±0.64 b 16.66±0.20 ab 17.16±0.47 ab 17.57±0.72 ab 18.20±1.15a
Commercial 12.03±0.22b 16.33±0.47a 16.50±0.45a 16.66±0.56a 17.30±0.62a
Data were mean ± sd (n=3). Means that do not share similar alphabet within the same column are significantly
different at (p<0.05).

247
 (a) (b) (c) (d)

(e) (f) (g)

Figure 1: Droplet structure of salad dressing; (a) Commercial salad dressing, (b) 0 % CBSD, (c) 10 % CBSD, (d) 20
% CBSD, (e) 30 % CBSD, (f) 40 % CBSD, (g) 50 % CBSD

Conclusion
The rheological characteristic of 20 % CBSD showed similar properties as control. The emulsion stability of 20 %
CBSD showed that it almost had similar degree of destabilization of oil droplets in the emulsion as control SD.
Moreover, 20 % CBSD could retain its low in oxidative studies until 28 days of storage comparable to commercial
salad dressing. The sensory evaluation is advisable to be conducted to compare the texture and taste by mouthfeel
instead of using only rheometer. It may help to recognize consumer’s preferences on the newly developed salad
dressing.

Acknowledgements
The authors would like to thank Universiti Putra Malaysia for financial support.

References
American Oil Chemists’ Society. (2003). AOCS official method Cd 8b -90 peroxide value acetic acid-isooctane
method. Official methods and recommended practices of the AOCS. Champaign, IL.
Knothe, G. and Dunn R. (2003). Dependence of oil stability index of fatty compounds on their structure and
concentration and presence of metalsJ. Am. Oil Chem. Soc., 80, pp. 1021–1026.
Liu, H., Xu, X., Guo, S., (2007). Rheological, texture and sensory properties of low-fat mayonnaise with different fat
mimetics. LWT – Food Science and Technology 40 (6), 946–954.
Maleky F, Marangoni AG. (2008). Process development for continuous crystallization of fat under laminar shear. J.
Food Eng. 89, 399-407.
Manach C, Scalbert A, Morand C, Remesy C, Jimenez L. (2004). Polyphenols: food sources and bioavailability. Am
J Clin Nutr.;79:727–747.
Pe´rez-Martı´nez, D. , Alvarez-Salas, C. , Charo´-Alonso, M., Dibildox-Alvarado, E. & Toro-Vazquez, J.F. (2007).
The cooling rate effect on the microstructure and rheological properties of blends of cocoa butter with
vegetable oils. Food Research International 40: 47–62.

248
 IFRC 2017: 279-255 Food Processing and Post-Harvest Technology

Effect of Extraction Methods with Different Matrix for Gelatin Recovery and Properties: A Review

Ee, S. C. and *Jamilah Bakar

Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM,
Serdang, Selangor, Malaysia.

Abstract

Gelatin is one of the most versatile and unique hydrocolloid ingredient widely used in food industries, serving multiple
functions in food system such as emulsifier, foaming agent, encapsulating agent, colloid stabilizer and film-forming
ability among others. These advantages led to the continuous exploration of new source of gelatin as well as
optimization of the extracting conditions of gelatin. Major commercial production of gelatin are from the skin and
bones of porcine and bovine (mammalian gelatin) and the rest are from non-mammalian sources such as fish, which
are still far behind the porcine and bovine in terms of production. Poultry derived gelatin is growing in importance as
new potential mammalian gelatin source. The current article entails an overview of recent research in extraction of
gelatin from different matrixs using alkaline, acid, enzymatic aided extraction and these selected methods affect the
functional properties of the extracted gelatin. Various enzymes used to improve the physicochemical properties such
as bloom strength, thermal properties, solubility and pH stability of gelatin of gelatin are also discussed.

Keywords: gelatin, extraction, physicochemical properties

*Corresponding author ‘s email: jamilah@upm.edu.my

Introduction

Gelatin is perhaps one of the most versatile multifunctional ingredient of high interest and value, able to form thermo-
reversible gel and has long been used in the food industry as emulsifier, foaming agent, encapsulating agent, colloid
stabilizer, film-forming ability and so on. Majority of commercial gelatins are manufactured from bovine and porcine
hide and bones. Alternative gelatin from non-mammalian especially fish species had grown in importance but their
production are still very limited. Poultry derived gelatin is commercially viable to be used as new potential mammalian
gelatin source given the projected growth in the industries in years to come.

Apart from the well-known religious practices and safety issues, the increase in demand of the global gelatin market
(Transparency market research, 2014) as well as the rising interest to add value to industrial by-products (the meat
and fish industry), resulted in the need to find alternative sustainable source of gelatin which should be functionally
equal or superior. This scenario has attracted the attention to explore new source of gelatin with innovative and
optimize extracting conditions as well as potential novel applications of gelatin. This review focuses on current
studies of gelatin, the various extraction methods and approaches that have been undertaken to develop gelatin from
different matrix, and evaluation of the physicochemical and functional properties of the resultant extracted gelatins.

Extraction of gelatin

Gelatin is produced by the partial hydrolysis of native collagen. The nature and amount of collagen in animal (skins,
bones and tendons) vary significantly from one tissue to another, the source, age of the animal, and type of collagen,
are all intrinsic factors influencing the properties of the gelatins. Due to this great heterogeneity in collagen raw
material, different extraction parameters in term of chemical reagents, temperature, pH, time, severity of the of
pretreatment and some sequential procedures are specially catered to produce the best gelatin.

249
During gelatin extraction process, acid or alkaline pretreatment is normally used to achieve partial cleavage of some
intra- and intermolecular covalent collagen crosslinks in addition to remove the non-collagenous proteins and other
impurities (Karim and Bhat 2009), resulting in helix-to-coil transition to such an extent that ‘warm water soluble
collagen’, i.e. gelatin, is formed. In converting collagen to gelatin, different types of collagen fragment are created by
acid or alkaline hydrolysis plus the given heat dissolution energy. Breakdown is largely dependent on the severity of
the extraction conditions.

Two types of gelatin are obtainable, Type A gelatin which is acid-extracted and has a broader isoelectric point of pH
7-9, whereas Type B gelatin is produced from alkaline process has an isoelectric point of pH 4.8-5.4 (GMIA, 2013).
Traditionally, hydrogen chloride and lime or sodium hydroxide are used for the manufacturing of Types A and B
methods, respectively. Alkaline treatment is preferred for older animals like bovine hides with more complex
collagens, while for less covalently cross-linked or immature collagens like fish and porcine skin, a short period of
mild acid treatment is all that necessary.

An alternative approach to acid and alkali treatment is the use of proteolytic enzymes, such as trypsin, chymotrypsin,
pepsin, alcalase, properase E, pronase, collagenase, bromelain, papain and crude proteases. Employment of
proteases increase gelatin extractability by cleaving the peptides in the telopeptide region of native collagen, thus
improves yield, shorten processing time and generates less waste compared with the chemical treatments in gelatin
extraction process. However, the used of enzymatic treatment might lower the gel strength of the obtained gelatin
(Ahmad et al. 2017). Numerous studies have investigated enzymatic hydrolysis of collagen or gelatin for the
production of bioactive peptides that play an increasingly important role in various products and applications.

Results & Discussion

Numerous studies have reported on the extraction of gelatin from different matrix using different extraction methods
(Table1). Bloom strength is always one of the important criteria for gelatin selection for applications. It reflects the
gelling quality of gelatin. The bloom value of commercial gelatin range from 50-300 g, with the 200-300 g is
designated as high bloom and most desirable. Higher bloom value contributes to the higher melting and gelling point,
therefore, shorter setting time of the final product. The bloom strength, gelling and melting temperatures of gelatin are
influenced mainly by the amino acid composition (imino acids) and molecular weight distribution (higher α and β
chains) in the gelatin.

Conclusion
Various factors including matrix of raw materials and processing conditions affecting the efficiency of gelatin
extraction and the quality attributes of gelatin are the subjects of this review. With more details studied on gelatin
extraction, it is possible for the gelatin manufacture to customize a gelatin in accordance with the required properties
for specific applications by carry out a controlled partial hydrolysis of the collagen (within a certain scope) in near
future.

Acknowledgements
The authors would like to thank UPM for financial support.

250
Table 1: Extraction procedures, bloom strength, gelling and melting temperatures of gelatin from different matrix
Source Extraction Bloom Gelling Melting Reference
procedures strength (g) temp. (°C) temp. (°C)

Goat skin Alkaline 267 21.2– 25.2 30.7–34.1 (Mad-ali et al. 2016)
Pig skin Acid 133.5-136.1 - - (Sompie et al. 2015)

Poultry parts
Chicken head Acid 200.4-247.9 26.2-28.2 33.7-34.2 (Du et al. 2013)
Turkey head Acid 332.7-368.4 26.2-28.2 33.7-34.2 (Du et al. 2013)
Chicken feet Acid 96.5-294.8 - - (Almeida and Lannes 2013)
Chicken skin Acid 355 24.9 33.6 (Mhd Sarbon et al. 2013)
Chicken deboner residue Acid 520 - - (Rafieian and Keramat 2015)
Duck feet Acid - 26.7 34.4 (Abedinia et al. 2017)
Alkaline - 21.8 33.6 (Abedinia et al. 2017)
Pepsin -- 26.1 36.4 (Abedinia et al. 2017)

Fish
Amur sturgeon (Acipenser schrenckii) skin Acid 105-141 14.2 22 (Nikoo et al. 2014)
King Weakfish (Macrodon ancylodon) Bones Alkaline 200 22 23.5 (Alfaro et al. 2010)
Jellyfish (Rhopilema hispidum) Alkaline - 18.0 22.3 (Cho et al. 2014)
Red tilapia (Oreochromis nilotica) skin Alkaline 384.9 - 27.8 (Jamilah et al. 2011)
Walking catfish (Clarias batrachus) skin Alkaline 147.4 - 25 (Jamilah et al. 2011)
Striped catfish (Pangasius sutchi fowler) skin Alkaline 238.9 - 26.2 (Jamilah et al. 2011)
Sea bream scales Acid 126 - 26 (Akagündüz et al. 2014)
Sea bream bones Acid 87.3 - 22-23 (Akagündüz et al. 2014)
Sea bream bones Alcalase 87.1 22-23 (Akagündüz et al. 2014)
Cuttlefish (Sepia officinalis) skin Acid + 181 - - (Balti et al. 2011)
protease

251
References
Abedinia, A., Ariffin, F., Huda, N., & Mohammadi, A. (2017). International Journal of Biological Macromolecules
Extraction and characterization of gelatin from the feet of Pekin duck (Anas platyrhynchos domestica) as
affected by acid , alkaline , and enzyme pretreatment. International Journal of Biological Macromolecules, 98,
586–594.
Ahmad, T., Ismail, A., Aqlima, S., Khalil, K. A., Kumar, Y., Adeyemi, K. D., & Sazili, A. Q. (2017). Recent advances
on the role of process variables affecting gelatin yield and characteristics with special reference to enzymatic
extraction: A review. Food hydrocolloids, 63, 85–96.
Akagündüz, Y., Mosquera, M., Giménez, B., Alemán, A., Montero, P., & Gómez-Guillén, M. C. (2014). Sea bream
bones and scales as a source of gelatin and ACE inhibitory peptides. LWT - Food Science and Technology,
55(2), 579–585.
Alfaro, A. T., Costa, C. S., Graciano Fonseca, G., & Prentice, C. (2010). Effect of Extraction Parameters on the
Properties of Gelatin from King Weakfish (Macrodon ancylodon) Bones. Food Science and Technology
International, 15(6), 553–562.
Almeida, P. F., & Lannes, S. C. D. S. (2013). Extraction and physicochemical characterization of gelatin from chicken
by-product. Journal of Food Process Engineering, 36(2007), 824–833.
Balti, R., Jridi, M., Sila, A., Souissi, N., Nedjar-Arroume, N., Guillochon, D., & Nasri, M. (2011). Extraction and
functional properties of gelatin from the skin of cuttlefish (Sepia officinalis) using smooth hound crude acid
protease-aided process. Food Hydrocolloids, 25(5), 943–950.
Cho, S., Koo, J. A. J., & Kim, S. (2014). Physicochemical Properties of Gelatin from Jellyfish Rhopilema hispidum.
Fisheries & Aquatic Sciences, 17(3), 299–304.
Du, L., Khiari, Z., Pietrasik, Z., & Betti, M. (2013). Physicochemical and functional properties of gelatins extracted
from turkey and chicken heads. Poultry Science, 92, 2463–2474.
GMIA. (2013). Standard testing methods for edible gelatin. Official Procedure of the Gelatin Manufacturers Institute of
America, Inc. http://www.gelatin-gmia.com /images/ GMIA_Official_Methods_of_Gelatin_Revised_2013.pdf.
Jamilah, B., Tan, K. W., Umi Hartina, M. R., & Azizah, A. (2011). Gelatins from three cultured freshwater fish skins
obtained by liming process. Food Hydrocolloids, 25(5), 1256–1260.
Karim, A. A., & Bhat, R. (2009). Fish gelatin: properties, challenges, and prospects as an alternative to mammalian
gelatins. Food Hydrocolloids, 23(3), 563–576.
Mad-ali, S., Benjakul, S., Prodpran, T., & Sajid, M. (2016). Characteristics and gel properties of gelatin from goat skin
as affected by extraction conditions. Journal of Food Processing and Preservation, 00, 1–10.
Mhd Sarbon, N., Badii, F., & Howell, N. K. (2013). Preparation and characterisation of chicken skin gelatin as an
alternative to mammalian gelatin. Food Hydrocolloids, 30(1), 143–151.
Nikoo, M., Benjakul, S., Bashari, M., Alekhorshied, M., Cissouma, A. I., Yang, N., & Xu, X. (2014). Physicochemical
properties of skin gelatin from farmed Amur sturgeon (Acipenser schrenckii) as influenced by acid
pretreatment. Food Bioscience, 5, 19–26.
Rafieian, F., & Keramat, J. (2015). Physicochemical properties of gelatin extracted from chicken deboner residue.
LWT - Food Science and Technology, 64, 1370–1375.
Sompie, M., Surtijono, S. E., Pontoh, J. H. W., & Lontaan, N. N. (2015). The Effects of Acetic Acid Concentration and
Extraction Temperature on Physical and Chemical Properties of Pigskin Gelatin. Procedia Food Science, 3,
383–388.
Transparency market research. (2014). Collagen Peptide and Gelatin Market (Source: Bovine, Porcine, Chicken,
Marine and Others; Application: Bone and Joint Health, Nutraceuticals, Cosmeceuticals, Food and Beverage,
Pet Food, Photography, Pharmaceuticals and Others) - Global Industry Analysis,. New York.

252
 IFRC 2017: 310-269 Food Processing and Post-Harvest Technology
Comparison of Sensory Quality and Preference Between Fermented Barleys, Glutinous Rice and the
Combination of Barley and Glutinous Rice

Ibrahim, N., Ezani, N. A. B., Ahmad Kamal, N., Mazlan, N., Abu Kassim, N. A., Jipiu, L. B., Abdul Aziz, S. A. and *Mat
Issa, Z.

Faculty of Hotel and Tourism Management, Universiti Teknologi MARA, Kampus Puncak Alam, 42300 Bandar
Puncak Alam, Selangor, Malaysia.

Abstract
Fermented rice or locally known as tapai is one of the traditional fermented foods available in Malaysia. It is
commonly made of either white rice or glutinous rice. The process of fermentation involves the addition of various
types of moulds. Considering such a background, the present study aimed to investigate the sensory quality and the
acceptability of fermented barley as an alternative to fermented glutinous rice. Specifically, barleys, glutinous rice and
a combination of both were fermented and some standard procedures of rice fermentation with the addition of sugar
were followed. Upon three days of fermentation, the fermented grains were then subjected to an assessment of
sensory quality such as texture quality, and moisture content. In addition, a hedonic study was also carried out to
investigate the overall acceptance of the fermented grains. The fermented rice was used as a control throughout this
study. The findings of the study revealed that fermented barleys had a softer texture and higher moisture content in
comparison with the fermented rice and its combinations. Besides, the hedonic study equally indicated that
fermented barley had a good acceptability by the locals. In conclusion, fermented barley has been found to have the
potential to be commercialised due to greater acceptability by the locals along with the health benefits associated
with it.

Keywords: fermentation, barley, glutinous rice, sensory quality, acceptability

*Corresponding author’s email: zurainim@salam.uitm.edu.my

Introduction
Fermentation has widely been defined as the chemical breakdown of a substance by bacteria, yeast, or other
microorganisms. In the course of such a process, it changes the initial characteristics of a food item into a product
that is significantly different and highly acceptable to consumers. In this regard, Holzapfel (2002) described
fermented food items as palatable and wholesome foods, which are prepared from either raw or heated raw
materials. One of the popular traditionally fermented food items in Malaysia is tapai (i.e., the local Malay term
referring to fermented grains). It is usually prepared by means of either glutinous rice (Oryza sativaglutinosa) or
cassava tuber (Manihotutilisima) (Law et al., 2011). Tapai has locally been known as a popular dessert/snack with
both sweet and acidic taste.

In terms of consumption, it can either be consumed as it is or it may also serve as an ingredient for home-cooking
and baking. Depending on individuals’ differences in taste sensitivity, some may prefer having these fermented
grains on its own whereas others have it served with contemporary toppings like ice cream, chocolate and fruit. In the
course of the grains’ fermentation process, a mixed culture of microorganisms is often used as the starter culture. In
this regard, Merican and Yeoh (1989) indicated that Amylomyces rouxii, Saccharomycopsis fibuligera and Hansenula
anomala are required to produce fermented glutinous rice, while A. rouxii and S. fibuligera are necessary for
fermented cassava.

The U. S. Food and Drug Administration (USDA, 2006) has recently issued an endorsement highlighting the benefits
of foods containing barley. In that endorsement, it was evidentially argued that principally due to its soluble fibre
content (β-glucans), which has been proven to keep blood cholesterol at minimal levels. In line with such an
argument, several studies have also reported the benefits of barley in improving the increasing good cholesterol
(Tiwari and Cummins, 2011) and lowering bad cholesterol (Wang et al., 1993; Brennan and Cleary, 2005), which is
also capable of reducing the risk of coronary heart disease in the long run (Satija and Hu, 2012). In addition, it has
253
also been found that barley is capable of reducing the glucose level (i.e., glycemic index), with specific reference to
those who are diagnosed as diabetics (Brennan and Cleary, 2005; Nettleton et al., 2010; Maslowski and Mackay,
2011). Barley also contains major phytochemicals such as phenolics, tocols and folate (Ward et al., 2008). Some
other studies have also confirmed the presence of both lignans and polyphenolic in cereal grains such as barley, oat
and rye. These compounds have also been found to be both bioavailable and capable of reducing the risk of
hormone-dependent cancer (i.e., breast and prostate) and colon cancer (Cotterchio et al., 2006; Qu et al., 2005).
Hence, the present study was carried out to investigate the sensory quality and the acceptability of fermented barley
as an alternative to fermented glutinous rice.

Material and Methods

Preparation of fermented grains
In the present study, both glutinous rice and barley were used as the main ingredients for fermented grains
preparation. Three types of fermented grains consisting of glutinous rice, barley and combination of glutinous rice
(50%) and barley (50%) were prepared. Also, all the three fermented grains were prepared by means of employing
similar procedures.

Prior to the preparation of fermented grains, 250 g of grains were washed (note: for a mixture of grains, equal amount
of each grain was prepared). The washed grains were then soaked overnight and steamed for 30 minutes. Next, the
steamed grains were put at room temperature on a clean, dry stainless steel tray before 0.04% (w/w) of fermentation
starter (i.e., in the form of powder) and 0.4% (w/w) of sugar were sprinkled over the steamed grains and were mixed
well. At the next stage, the grains were then packed in a banana leaf and covered tightly with a clean cloth. At this
stage, the grains were allowed to ferment for 3 days at room temperature. It has to be noted that the control sample
was the fermented glutinous rice.

Determination of water activity

A Awmeter Rotronic HygroPalm AW1 (RotronicInstrument Corp., Basserdorf, Germany) was used to determine the
water activity (Aw) of the fermented grains. The Aw was measured on after the third day of the fermentation process at
room temperature.

Determination of texture
A texture analyser TAXT2 (Stable Micro System, Godalming, UK) with a load cell of 5 kg, cylinder probe (diameter 50
mm) was used on the third day to determine the texture of fermented grains. In this regard, the samples were
compressed at a speed of 10 mm/s and a return strain of 50 mm. In addition, the firmness of the samples was
determined at the peak of the compression force (in g), which was detected in the course of the probe being
proceeded to penetrate into the fermented grains.

Consumer preference
The consumer’s preference towards the fermented grains had to be gauged by means of the hedonic test.

Data analyses
Both the water activity and texture analyses were analysed by means of Excel (Microsoft Inc., version 2010) for
descriptive statistics and one sample t-test. Prior to proceeding with any further analyses, the compression force was
first converted into gram per second (gm/sec). In addition, the Hedonic data were analysed using Excel (Microsoft
Inc., version 2010) for descriptive statistics and a Single Factor ANOVA.

Results and Discussion

A summary of the water activity, the textural analyses, and also the mean preference of consumers towards the
grains after 3 days fermentation is presented in Table 1. It was revealed that all the three types of fermented grains
had a water activity greater than 0.9. Such findings were in line with those of Oduro-Yeboah et al. (2016), in which it
was revealed that fermented grains result in an increase in moisture. The present study also revealed that the
fermented barley had the highest amount of moisture (0.954 ± 0.003). However, one sample t-test on water activity
revealed that there no significant difference was observed between the three types of fermented grains (p > 0.05). In
addition, the textural analysis indicated that the fermented barley has a softer body (26.65 gm/sec) in comparison

254
with the glutinous rice (30.52 gm/sec) and the combinations of barley and glutinous rice (54.26 gm/sec). However,
the one sample t-test revealed that no significant differences were observed between the firmness of the fermented
grains (t = 0.77, n = 3, p = 0.74 > 0.05). It was argued by Dung (2013) that the composition of amylose and
amylopectin and soaking may possibly affect the enzymic hydrolysis of grains. In addition, the findings of the study
carried out by Jomduang and Mohamed (1994) led them to conclude that the moisture content of grains was
positively correlated to amylopectin. Hence, the findings of the present study suggested that the grains used in this
study may have almost similar percentage of amylose and amylopectin.

Table 1: Water activity (Aw), firmness and preference of different types of fermented grains after three days
fermentation process.
Types of tapai Aw Firmness Preference
(gm/sec)
Glutinous rice 0.943 ± 0.003 30.52 6.44 ± 1.11
Barley 0.954 ± 0.003 26.65 5.78 ± 1.45
Glutinous rice + barley 0.937 ± 0.007 54.26 6.46 ± 1.2

The data from the Hedonic test had to be interpreted by means of using a Single Factor ANOVA and the F-test. If the
F > F crit, the null hypothesis would be rejected. Hence, the results of the present study revealed that significant
differences were observed between the three types of tapai (F(2, 147) = 4.717 > 3.058). It was also found that the
fermented barley was preferred less by the consumers than the other two fermented grains due to its non-sticky
texture upon the completion of the fermentation process. This is due to the fact that the soluble fibre of the barley is
not affected by fermentation (Lambo, Oste and Nyman, 2005).

Conclusion
Fermented foods are widely prevalent in the context of Southeast Asia. Such a prevalence is often associated with
processing and preserving the food in order to retain the good flavour, aroma, enhanced nutritive values and
retaining its quality under ambient temperatures. Unfortunately, no extensive study on the fermented barley to the
knowledge of the researchers has been carried out in Malaysia. It is hoped that the present study would help future
researchers to formulate a more full-fledged methodology and to consider the need to look beyond liking and
acceptability of the fermented barley. It is also hoped that the food industry would be able to consider the scientific
and market research evidences highlighted in the present study, in promoting healthy, authentic and traditional
fermented barley to the consumers.

Acknowledgements
A special thanks to Madam Siti Hamidah So’ad and Madam Rusni Saad for technical assistance.

References
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ingredients. Journal of Cereal Science, 42, pp. 1-13.
Cotterchio, M., Boucher, B. A., Manno, M., Gallinger, S., Okey, A. and Harper, P. 2006. Dietary phytoestrogen intake
is associated with reduced colorectal cancer risk. Journal of Nutrition, 136, pp. 3046.
Dung, N.T.P., 2013. Vietnamese rice-based alcoholic beverages. International Food Research Journal, 20(3).
Holzapfel, W. H. 2002. Appropriate starter culture technologies for small-scale fermentation on developing countries.
International Journal of Food Microbiology, 75, pp. 197-212.
Jomduang, S. and Mohamed, S., 1994. Effect of amylose/amylopectin content, milling methods, particle size, sugar,
salt and oil on the puffed product characteristics of a traditional thai rice‐based snack food (Khao Kriap
Waue). Journal of the Science of Food and Agriculture, 65(1), pp.85-93.
Lambo, A.M., Öste, R. and Nyman, M.E.L., 2005. Dietary fibre in fermented oat and barley β-glucan rich
concentrates. Food Chemistry, 89(2), pp.283-293.
Law, S. V., Abu Bakar, F., Mat Hashim, D. and Abdul Hamid, A. 2011. Mini Review: Popular fermented foods and
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Indigenous Fermented Foods, in Steinkraus, K. H., pp. 169-189. Marcel Dekker, Inc., New York.
Nettleton, J. A., McKeown, N. M., Kanoni, S., Lemaitre, R. N., Hivert, M. F., Ngwa, J., van Rooij, F. J., Sonestedt, E.,
Wojczynski, M. K., Ye, Z., et al 2010. Interactions of dietary whole-grain intake with fasting glucose- and
insulin-related genetic loci in individuals of European descent: a meta-analysis of 14 cohort studies. Diabetes
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Oduro-Yeboah, C., Mestres, C., Amoa-Awua, W., Fliedel, G., Durand, N., Matignon, B., Michodjehoun, V.L., Saalia,
F.K., Sakyi-Dawson, E. and Abbey, L., 2016. Steeping time and dough fermentation affect the milling
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grains. Journal of Cereal Science, 69, pp.377-382.
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Tiwari, U. and Cummins, E. 2011. Meta-analysis of the effect of beta-glucan intake on blood cholesterol and glucose
levels. Nutrition, 27, pp. 1008-1016.
Wang, L., Xue Q., Newman, R. K., and Newman, C. W. 1993. Enrichment of tocopherol, tocotrienol, and oil in barley
by milling and pearling. Cereal Chemistry, 70(5), pp. 499-501.
Ward, J. L., Poutanen, K., Gebruers, K., Piironen, V., Lampi, A. M., Nystrom, L., Andersson, A. A., Aman, P., Boros,
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Bioactive compounds of coffee pulp and cocoa pod: Valorisation as food ingredient
*Raseetha, S. and Raudzatul-Adawiyah, M. H.

Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia

The aim of this study were to determine the effect of different extraction methods on the availability of bioactive
compounds i.e. total phenolic content (TPC), chlorogenic acid (CGA) content and antioxidant activity that was
measured by Ferric Reducing Antioxidant Power (FRAP) assay and DPPH radical scavenging activity of coffee pulp
(CFP) and cocoa pod (CCP). Solvent extraction (SE) using ethanol and ultrasound-assisted extraction (UAE) method
was applied, for 2 hour and 30 min at 40°C. The UAE method gives higher TPC in CFP (6.285 mg/g) and CCP
(4.808 mg/g), and higher antioxidant activity measured by FRAP assay for both CFP and CCP with value 3.508 mg/g
sample and 2.454 mg/g respectively. The SE method on the other hand gives higher but no significant different of
CGA content, 27.122 mg/g in CFP and 0.249 mg/g in CCP and higher antioxidant activity measured by DPPH radical
scavenging assay in CFP (73.4275 %) and CCP (87.523 %). UAE method was more effective than SE as it can
recover higher TPC and antioxidant activity (FRAP assay) compared to the SE method for both CFP and CCP
sample.

Keywords: coffee, cocoa, solvent extraction, ultrasound-assisted extraction, antioxidant

*Corresponding author’s email: raseetha@salam.uitm.edu.my

Introduction

Coffee is one the most consumed product in the world. Usually, coffee pulp (CFP) is produced in wet method
processing (Marcel et al, 2011) and thrown as waste material. This coffee waste made up to 45% of the whole coffee
cherry hence might be further treasured especially for the polyphenolic content (Esquivel & Jimenez, 2011). Cocoa
bean (Theobrama cacao) is the source of chocolate and during processing, only cocoa bean are used, leaving the
cocoa pod and husk as a waste product (Karim et al., 2014).

Phenolic content of cocoa beans depends on botanical variety, genetic and agronomical factors such as postharvest
handling, fermentation and drying conditions can also act as an antioxidant (Tomas-Barberan et al., 2007). Cocoa
pod husk measured by FRAP contains 2.00 µM TE/ g sample when extracted by absolute ethanol and 4.69 µM TE/g
sample when extracted using methanol/acetone (Martinez et al., 2012). Chlorogenic acid (CGA) presents within
coffee and contribute to the flavor after roasting. A commercial roasted coffee may contain about 0.5– 6g DW of CGA
depending on the roasting conditions (Ayelign & Sabally, 2012). CGA was claimed to modulate glucose and lipid
metabolism in vivo in both healthy and genetically disordered conditions in rats (Meng et al., 2013). This research
was carried out in an attempt to determine the effect of different extraction methods (solvent and ultrasound-assisted
extraction) on the availability of bioactive compound (phytochemical content, total phenolic content, chlorogenic acid
content and antioxidant properties i.e. FRAP and DPPH) in coffee pulp and cocoa pod.

Materials and methods

Preparation of coffee pulp and cocoa pod

Fresh coffee beans were bought from a grower in Pelabuhan Klang, Selangor and fresh cocoa beans were bought
from Lembaga Koko Malaysia. The coffee pulps (CFP) and cocoa pods (CCP) were freshly separated from the
respective beans. The separated CFP and CCP were rinsed with tap water and dried in cabinet dryer at 60°C for 48
hour until constant weight is obtained. The dried CFP and CCP were ground into powder at 1 mm size using grinding
machine with a sieve. The powders were kept in airtight container prior to analysis.

257
 Extraction using solvent extraction method This page is intentionally left blank

The procedure of solvent extraction (ethanol) was carried out according to Karim et al, (2014).

Extraction using ultrasound-assisted extraction method

The procedure of ultrasound-assisted extraction (UAE) used in this study was developed by Saleh et al, 2016 by
using direct sonication extraction method.

Test for alkaloids, tannins and saponins

Analyses for alkaloid were conducted according to the method of Yadav and Agarwala (2011). The qualitative results
are expressed as positive (+) for the presence and negative (−) for the absence of phytochemicals.

Determination of total phenolic compounds

Total phenolic concentration was measured using the Folin–Ciocalteu method (Singleton & Rossi, 1965).

Chlorogenic acid analysis using High Performance Liquid Chromatography

The procedure following the method of Ayelign and Sabally, (2013). The analysis done by using an external standard
procedure with calibration curve.

Determination of antioxidant activity (FRAP and DPPH)

Ferric Reducing Antioxidant Power (FRAP) assay was conducted according to Benzie and Strain (1996). DPPH
assay was carried out according to Amin et al. (2006).

Statistical analysis

All data were expressed as mean ± standard deviation. All statistical analysis will be conducted by using SPSS 20.
Duncan’s multiple-range test was used to access difference between means.

Result and discussion

Qualitative phytochemical screening

The phytochemical screening of CFP and CCP extract revealed that alkaloid, tannins and saponin were detected in
both CFP and CCP extract from both SE and UAE method. The phytochemical compounds detected in both sample
had correlation with their bioactivity properties and are also known to have medicinal importance. For instance,
alkaloid were derived from medicinal plants show biological activities like anti-inflammatory, antimalarial,
antimicrobial, cytotoxicity and pharmacological effects (Iqbal et al., 2015).

Phenolic compounds and antioxidant properties

The phenolic content observed in UAE method was significantly higher (CFP, 6.285 mg GAE/g) compared to CCP
with 4.808 mg GAE/g. Nevertheless for the SE method, there is no significant difference in both samples.The
measured TPC values were lower that literature values for CFP ranging from 11 – 20 mg GAE/g sample using 70%
methanol/water extract (Ulloa Rojas et al., 2002) and waste coffee spent ground reported by Al-Dhabi et al., (2016)
but higher than CFP extract reported by Duangjai et al., (2016). On the other hand, the measured value for CCP for
both extraction method were higher than reported by Martinez et al., (2012). There were no significant differences for
chlorogenic acid (CGA) content in both samples for both extraction techniques.
258
 Generally, the CFP extract had higher antioxidant activity than the CCP extract based on the FRAP assay. The CFP
sample extracted by UAE exhibited the highest a This page is intentionally left blank

ntioxidant potential among the others, followed by CFP sample extracted by SE method. According to Rice-Evans,
Miller, and Paganga (1997), phenolic compounds have redox properties that allow them to act as reducing agents,
hydrogen donators and singlet oxygen quenchers. Both CCP sample shows higher percentage (%) radical
scavenging compared to CFP. The CCP sample by SE method shows the highest % of radical scavenging, followed
by CCP sample by UAE method which are 87.5235% and 78.2967 % respectively. The higher % or radical
scavenging of CCP compared to CFP might be because of the presence of carboxylic acid, precisely citric acid and
malic acid, which suggested to contribute to high antioxidant value by DPPH in CCP (Karim et al, 2014) besides
phenolic acid and other polyphenols compounds.

Conclusion

This study has shown that the UAE method gives higher value of TPC in CFP (6.285 mg/g sample) and CCP (4.808
mg/g sample), and higher antioxidant activity measured by FRAP assay for both CFP and CCP with value 3.508
mg/g sample and 2.454 mg/g sample respectively. The SE method on the other hand gives higher but no significant
different value of CGA content, 27.122 mg/g sample in CFP and 0.249 mg/g sample in CCP and higher antioxidant
activity measured by DPPH radical scavenging assay in CFP (73.4275 %) and CCP (87.523 %). By comparing the
extraction method with constant time (2 hour and 30 minutes) and temperature (40°C) used, UAE method is more
effective than SE as it can recover higher TPC, antioxidant activity (FRAP assay) and no significant different in CGA
for both sample.

Acknowledgements

The authors would like to thank the Research Acculturation Grant Scheme (RAGS) RAGS/1/2014/STWN03/UITM//2
from the Ministry of Higher Education Malaysia.

References

Amin, I., Norazaidah, Y. and Hainida, K. I. 2006. Antioxidant activity and phenolic content of raw and blanched
Amaranthus species, Journal of Food Chemistry 94: 47–52.
Al-Dhabi, N.A., Ponmurugan, K. and Prakash, M.J. 2016. Development and validation of ultrasound-assisted solid-
liquid extraction of phenolic compounds from waste spent coffee grounds. Journal of Ultrasonics Chemistry
34: 206-213.
Ayelign, A. and Sabally, K., 2013. Determination of chlorogenic acids (CGA) in coffee beans using HPLC. American
Journal of Research Communication 1(2): 78-91
Benzie, I.F. and Strain, J.J. 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”:
The FRAP Assay. Analytical Biochemistry 239: 70-76.
Duangjai, A., Suphrom, N., Wungrath, J., Ontawong, A., Nuengchamnong, N. and Yosboonruang, A. 2016.
Comparison of antioxidant, antimicrobialactivities and chemical profiles of three coffee (Coffea arabica L.) pulp
aqueous extracts. Integrative Medicine Research Journal 324-331.
Esquivel, P. and Jiminez, V. M. 2011. Functional properties of coffee and coffee byproducts. Food Research
International 46(2): 488–495.
Iqbal, E., Salim, K. A. and Lim, L. B. L. 2015. Phytochemical screening, total phenolics and antioxidant activities of
bark and leaf extracts of Goniothalamus velutinus (Airy Shaw) from Brunei Darussalam. Journal of King Saud
University - Science 27(3): 224–232.
Karim, A. A., Azlan, A., Ismail, A., Hashim, P., Salwa, S. and Gani, A. 2014. Phenolic composition , antioxidant , anti-
wrinkles and tyrosinase inhibitory activities of cocoa pod extract. BMC Complementary and Alternative
Medicine 14(1): 1–13.
Marcel, B.K.G., Andre, K.B., Viviane, Z.T. and Seraphin, K.C. 2011. Potential food waste and by-products of coffee in
animal feed. Electronic Journal of Biology 7(4): 74-80.
Martinez, R., Torres, P, Meneses, M. A., Figueroa, J. G., Perez-Alvarez, J. A. and Viuda-Ma

259
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rtos, M. 2012. Chemical, technological and in vitro antioxidant properties of cocoa (Theobroma cacao L.) co-
products. Food Research Journal 49: 39-45.
Meng, S., Cao, J., Feng, Q., Peng, J. and Hu, Y. 2013. Roles of chlorogenic acid on regulating glucose and lipids
metabolism: A review. Journal of Evidence-Based Complementary and Alternative Medicine 2013: 1-11.
Rice-Evans, C. A., Miller, N. T. and Paganga, G. 1997. Antioxidant properties of phenolic compounds. Trends in
Plant Science 4: 304-309.
Saleh, I. A., Vinatoru, M., Mason, T. J., Abdel-Azim, N. S., Aboutabl, E. A. and Hammouda, F. M. 2016. A possible
general mechanism for ultrasound-assisted extraction (UAE) suggested from the results of UAE of
chlorogenic acid from Cynara scolymus L. (artichoke) leaves. Journal of Ultrasonic Sonochemistry 31: 330–
336.
Singleton, V. L. and Rossi, J. A. 1965. Colorimentry of total phenolics with phosphomolybdic-phosphotungstic acid
reagents. American Journal of Enology and Viticulture 16: 144–158.
Tomas-Barberan, F. A., Cienfuegos-Jovellanos, E., Marín, A., Muguerza, B., Gil-Izquierdo, A., Cerdá, B., Zafrilla, P.,
Morillas, J., Mulero, J., Ibarra, A., Pasamar, M. A., Ramón, D. and Espín, J. C. 2007. A new process to
develop a cocoa powder with higher flavonoid monomer content and enhanced bioavailability in healthy
humans. Journal of Agricultural and Food Chemistry 55(10): 3926-3935.
Ulloa Rojas J. B., Verreth J. A. J., van Weerd J. H. and Huisman E. A. 2002. Effect of different chemical treatments
on nutritional and antinutritional properties of coffee pulp. Animal Feed Science Technology 99(1): 195-204.
Yadav, R. N. S. and Agarwala, M. 2011. Phytochemical analysis of some medicinal plants. Journal of Phytology
3(12): 10–14.

260
 IFRC 2017: 018-113 Functional Food

‘Ceri’Terengganu, Lepisanthes fruticosa the rare fruits of Malaysia, with new potential.

*Sukirah Abdul Rahman, Muhammad Anas Othaman, Nur Yuhasliza Abdul Rashid, Nur Diyana Alyas, Hazniza
Adnan, Nor Hazniza Aziz & Musaalbakri Abdul Manan.

Fermentation and Enzyme Technology, Programme, Biotechnology and Nanotechnology Research Centre, MARDI
(Malaysian Agricultural Research and Development Institute), 43400, Serdang, Selangor, Malaysia.

Malaysia possesses a rich diversity of tropical fruits other than the common and commercial fruits and there are
many other fruit species which are grouped under rare fruits. Ceri Terengganu or Lepisanthes fruticosa is one of the
rare fruits in Malaysia. It is not very commonly cultivated thus it can be categorized as a rare fruit tree. The tree is
non-seasonal and produces fruits throughout the year. This tree had been used traditionally for medical purposes by
some communities in Malaysia. MARDI (Malaysian Agricultural Research and Development Institute) had done
ethnobotanical studies on the underutilised fruit to explore the potential of these fruits and fervent to conserve this
tree in our country. In identifying the potential of the fruits, few studies namely antagonistic activities, fermentation
with Saccharomyces cerevisiae and determination of the phenolic compound of unfermented and fermented ceri
Terengganu were done to exhibit the possible potential of ceri terenggganu. The antagonistic activities was tested on
seven bacterial cultures and one yeast namely Escherichia coli, Salmonella typhimurium, Staphylococcus aureus,
Bacillus cereus, Listeria monocytogens, Listeria innocua, Staphylococcus epidermidis and Saccharomyces
cerevisiae. The positive results were exhibited by Staphylococcus epidermidis with inhibition zone at 15mm. The
outcomes of fermentation with Saccharomyces cerevisiae with 10% carbon source showed that the colony forming
unit of the yeast was the highest at day 6, 2.12X107CFU/ml. The total phenolic content of unfermented ‘ceri’
Terengganu extract was detected at 1142.7ppm and increased about 11.93% after being fermented with
Saccharomyces cerevisiae, with the final value of 1297.49ppm.

Keywords: Lepisanthes fruticosa, fermentation, antagonistic activities, phenolic compound

Corresponding author’s email: sukirah@mardi.gov.my; Tel: +603-89536112

Introduction
The allure of the’ceri’ Terengganu tree and fruit tree makes it often used as an ornamental plant for landscaping
purposes. In addition, this unique plant is also often used as street trees and shade trees at public area. The whole
part of ceri Terengganu tree is believed to have efficacy and utility of its own. In traditional medicine, the roots are
said to have high nutrition. A decoction of the roots was drink to reduce itching and lowers high body temperature
due to fever. Terengganu cherry fruit can be eaten fresh and has a sweet flavor when fully ripe. Scientific studies
such as those carried out in the Strategic Resource Research Center, MARDI, showed that ‘ceri’ Terengganu has
high antioxidant activity compared to commercial fruits such as guava, citrus and apples. Phenolic compounds in ceri’
Terengganu also found to be higher compared to the commercial fruits tested. Phenolic antioxidants are natural
compounds in plants key that can kill free radicals in the body which is to be the leading cause of various diseases in
humans. The red color on the skin of a cherry Terengganu also has potential as an antioxidant due to chemical
compound known as anthocyanins. Anthocyanins are naturally red pigment that gives color to the flowers, fruits,
leaves, stems and roots of a plant that can prevent a variety of diseases such as cancer and atherosclerosis. In
addition, studies have shown anthocyanins also works in a variety of biological activity as anti-inflammatory drugs (to
reduce inflammation), anti-microbial (prevents bacterial), anti-diabetic (control of blood sugar levels) and anti-aging
261
(to help maintain healthy skin and the problem of premature aging). The red color also has the potential to be used
as a natural dye that can replace synthetic dyes. This preliminary study was carried out to determine the potential
and benefit of the rare fruit, ‘ceri’Terengganu that can be featured and commercialized as a functional fruit.

Methodology
Collection of selected fruits and preparation of the substrate
Ripe ‘ceri’ Terengganu, Lepisanthes fruticosa fruit have been collected from certain areas that have been targeted in
Malaysia. Fruits that have been received have been washed, chopped and deseeded. After that, the fruit is small
sliced and dried in an oven for 48 hours at a temperature of 55 ° C. After the pulp has dried, it will grind to a powder
form for storage purposes. ‘Ceri ‘Terengganu fruit powder stored in the refrigerator (4°C) until used.Powder of each
part of the plant were tested for antagonistic activity by well diffusion method on Mueller Hinton Agar.

Fermentation Study
Fermentation studies were conducted in the conical flask (500ml) with a total volume of 300ml. The yeast strains
were used in this study, namely Saccharomyces cerevisiae IRNA Nabonne 7013, DSM has been obtained from the
German Collection of Microorganisms and Cell Cultures. Strains of yeast will be cultured in a liquid medium potato
dextrose (PDB) for 24 hours at 30 °C prior to inoculation. For the preparation of the substrate, ‘ceri’ Terengganu
powder was added to distilled water at a concentration of 10% and 10% of sucrose was added. Substrate will be
pasteurized to prevent contamination. The substrate has to be cooled, pasteurized inoculated with 3% yeast strain.
Aerobic fermentation was carried out in a static state for 7 days at 30°C. Sampling will be taken at specific intervals.
Each sampling will be followed by a reading of pH value using a pH meter. Growth in the number of cells was
determined by counting the number of living cells (CFU) using serial dilution technique. Growth plates will be
incubated in an oven at 30°C for 24 hours before counting colonies made. Each sampling will be followed by a
reading of pH value using a pH meter. Growth in the number of cells was determined by counting the number of living
cells (CFU) using serial dilution technique. Growth plates will be incubated in an oven at 30 ° C for 24 hours before
colonies counting were made. pH and total soluble solids of ‘ceri’ Terengganu was measured throughout the
fermentation. Analysis of total phenols used gallic acid as standard and 5 mL of Folin-Cicalteu reagent. The
concentration of total phenolic content was calculated as gallic acid equivalent.

Results

Figure 1 Antagonistic activity of 20% ripe seeded flesh of ‘ceri Terengganu” towards Staphylococcus epidermidis.

262
 Colony Count (log 10 6
5
4 'Ceri Terengganu' seeded
flesh + S.cerevisiae
CFU/mL)
3
'Ceri Terengganu' seeded
2
1 flesh (Control)
0
0 2 4 6 8
Time (Day)
Figure 2

6 5.65 5.6 5.57 5.585

5 5.7
4 3.65 3.39 'Ceri Terengganu'
pH

3 3.33 seeded flesh +

2 S.cerevisiae
1 'Ceri Terengganu'
0 seeded flesh (Control)
0 2 4 6 8
Time (Day)
Figure 3
Total Phenolic Content

1350
1297.49
1300
1250
(ppm)

1200
1142.7
1150
1100
1050
Unfermented 'ceri' Terengganu Fermented 'ceri' Terengganu

Figure 4
Fig: 2 Growth colonies of Saccharomyces cerevisiae in fermented ‘ceri’ Terengganu on Potato Dextrose Broth agar
reported in log10CFU/ml. Fig:3 pH of fermented ‘ceri’ Terengganu. Figure 3: Fig.4: Total phenolic content of
unfermented ‘ceri’ Terengganu and fermented ‘ceri’ Terengganu.

Discussion & Conclusion

It showed that 20% ripe seeded flesh of “ceri “Terengganu showed antagonistic activity towards towards
Saphylococcus epidermidis at 15mm (Fig.1).There is a report that indicated that the root of “ceri “Terengganu has
medicinal properties that was used as poultice to relieve itching and to lower the temperature during fever. The
growth of Saccharomyces cerevisiae in ceri Terengganu was depicted in Figure 2 in log10 CFU/mL. It showed that the
exponential growth started after 0 day and continued until the second day. The growth of the yeast started at
stationary stage after day 2 until day 7 and the highest CFU/ml was at day 6, 2.12X10 7 (Fig. 2). The pH of the
fermented ‘ceri’ Terengganu was 5.7 initially and decrease to 3.3. on day 7 of fermentation (Fig 3) which is regarding
pH requirements for S.cerevisiae fermentations, yeasts thrive in warm and acidic environments with most S.
cerevisiae strains growing well between 20 and 30 ◦C and pH 4.5 and 6.5(Graeme &Graham,2016). In the presence
of sugars, together with other essential nutrients such as amino acids, minerals and vitamins, S. cerevisiae will
conduct fermentative metabolism to ethanol and carbon dioxide (as the primary fermentation metabolites). High
263
water activity is required for it cells which typically possess a minimum aw of around 0.65. Water is absolutely
essential for fermentation, and high sugar-containing media can impose osmotic stress (reduced water availability)
on cells to adversely affect cell physiology. The total phenolic content of ‘ceri’ Terengganu after being fermented by
S.cerevisiae for 7 days compared to the unfermented ‘ceri’Terengganu showed an increment from 1142.7ppm and
increased about11.93% after being fermented with S.cerevisiae, with the final value of 1297.49ppm.The total
phenolic content of fresh ripe ‘ceri’ Terengganu has similar value reported by Mirfat et al.2017 around 1330ppm. It
can be concluded that ‘ceri' Terengganu can be fermented by S.cerevisiae and could be a substrate for the yeast due
to the increasing colony count of the yeast, reduced in pH and increasing in total phenolic compound after the
fermentation process.

Acknowledgement

Author wished to acknowledge MARDI for the RMK 11 Grant to pursue this project and also all the project members
and staffs involved in this research project.

References
Graeme M. W & Graham. G S. 2016. Saccharomyces cerevisiae in the Production ofFermented Beverages, A
Review paper Bv beverages MDPI, Creative Commons Attribution (CC-BY) license
(http://creativecommons.org/licenses/by/4.0/). Abertay University, Dundee, Scotland DD1 1HG, UK Academic
Editor: Edgar Chambers IV
Mirfat A. H. S, Zaulia O, Joanna C.L.Y, Erny Sabrina M. N. & Salma I.2017. Antioxidant Activity and Phytochemical
Content of Fresh Freeze-Dried Lepisanthes fruticosa Fruits at Different Maturity Stages. Journal of Agricultural
Science; Vol. 9, No. 2; 2017.Page 147-153.
Muhammad A. O., Nur Diyana A, Mohd Izwan M. L, Anisah J, Nur Yuhasliza A,R, Hazniza A, Shaiful Adzni S. &
Musaalbakri A.M. 2016. Penghasilan Etanol Dari Fermentasi Yis Di Dalam Medium Berasaskan Ceri
Terengganu. Proceeding Persidangan Kebangsaan Agrobiodiversiti.
Lim T.K. 2013. Edible Medicinal and Non–Medicinal Plants: Volume 6, Fruits, DOI 10.1007/978-94-007-5628-1_8, ©
Springer Science+Business Media Dordrecht.

264
 IFRC 2017: 029-018 Functional Food

Bioactive compounds and nutritional properties of Khao-Mao

1*Singthong, J., 2Oonsivilai, R., 3Oonmetta-aree, J. and 1Onsaard, E.

1Department of Agro-Industry, Faculty of Agriculture, Ubon Ratchathani University, Warinchamrap, Ubon

Ratchathani, 34190 Thailand
2School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Muang,
Nakhon Ratchasima, 30000 Thailand
3Food Science and Technology Program, Faculty of Science and Technology, Nakhon Ratchasima Rajabhat
University, Muang, Nakhon Ratchasima, 30000 Thailand

Khao-Mao is one of popular One Tambon One Product (OTOP) in Ubon Ratchathani, Thailand. The aim of this
research was to investigate bioactive compounds and nutritional properties using 9 varieties of young rice. Four
varieties: Kam, RD6, Jasmine, Dor He were obtained from Phibun Mangsahan (PM) and five varieties: Kam, RD6,
Lee Nok, Dor Hom, E-Tei were obtained from Trakanpuetpon (TP), Ubon Ratchathani, Thailand. Antioxidant activities
and total phenolic content were evaluated using DPPH, ABTS, FRAP assays and the Folin-Ciocalteu method. It was
found that Kam showed the highest total phenolic content. In addition, Kam displayed the highest antioxidant
activities determined by DPPH assay, ABTS assay and FRAP assay. On the other hand, Dor He and E-Tei
presented the highest antioxidant activity measured as %inhibition using hydroxyl (OH) and superoxide ion (O2)
method. Anthocyanins content of Kam variety (pigmented rice) from PM was higher than Kam variety from TP. The
non-pigmented rice varieties provided chlorophyll content between 0.026-0.052 mg/ml. A proximate analysis of
different young flatten rice was also conducted that Dor He was highest content as protein, ash and fiber. Lee Nok
variety provided high value of GABA content (2.84 mg/100g). However, Dor He presented high content of total
gamma-oryzanol (11.35 mg/100g) and derivatives: cycloartenyl ferulate (3.13 mg/100g), 24-methylene-cycloartenyl
ferulate (3.04 mg/100g), campesteryl ferrulate (3.27 mg/100g) and sitosteryl ferulate (1.90 mg/100g). The result of
this study could be used as a basic knowledge in food application, especially in development of food products to
make an economic potential of local community.

Keywords: Khao-Mao, bioactive compounds, nutritional properties

*Corresponding author’s email: jittrawara@gmail.com

Introduction
Rice (Oryza sativa L.) is an important cereal crop consumed both as staple food as well as processed products
(Saikia et al. 2012). Khao-Mao is one of popular One Tambon One Product (OTOP) in Ubon Ratchathani, Thailand.
In Thailand, “Khao” means rice, and “Mao” means immature grains, which are formed between 13th and 19th day after
anthesis (DAA), depending the variety. Khao-Mao made from young flattened glutinous rice. It is roasted sticky rice
with the husks still on and pound it with a pestle in a motar, and it is press flat. The husks can be removed by shaking
and blowing. Khao Mao mixed with sugar, salt and shredded coconut is a traditional rice dessert in Thailand
especially in the Northeastern. Bioactive compounds in foods are important parameter for evaluating its potential
health benefits. The health benefits of rice are attributed to the presence of phenolic compounds that possess
antioxidant, anticarcinogenic, anti-imflammatorry, antiatherosclerosis and hypoglycemic activities. The evaluation of
total antioxidant activity as a way of predicting potential roles of food in preventing degenerative diseases may be an
important factor (Zhou et al. 2014).

The objective of this study was to improve the knowledge about the presence of total phenolics, anthocyanins,
chlorophylls and antioxidant activities in Khao-Mao subjected to different young rice varieties. Moreover, the
proximate composition, GABA and γ-oryzanol were also recorded as a function of young rice varieties.

265
Materials and Methods
Materials
Khao-Mao samples made from the young glutinous rice were purchased from Phibun Mangsahan (PM) and
Trakanpuetpon (TP), Ubon Ratchathani, Thailand. Four varieties: Kam, RD6, Jasmine, Dor He were obtained from
PM and five varieties: Kam, RD6, Lee Nok, Dor Hom, E-Tei were obtained from TP, Ubon Ratchathani, Thailand.
Khao-Mao samples were packed in laminated foil bags, sealed, and kept at 4C until analysis.

Proximate analysis
Moisture, ash, protein, lipid, crude fiber and carbohydrate of Khao-Mao were determined according to the method of
AOAC (2000).

Total phenolic content (TPC) of Khao-Mao was determined using the Folin-Ciocalteu reagent with gallic acid as
standard. (Oonsivilai et al., 2012).

Antioxidant activity was evaluated in accordance with the DPPH method, the FRAP method and the ABTS method
(Oonsivilai et al., 2012). Superoxide radical scavenging activity and Hydroxyl radical scavenging cappacity were
determined according to the method of Singh and Rajini (2004).

Total chlorophyll was carried out using the method of Ramesh and Devasenapathy (2006) with slightly modification.
Total Anthocyanins was carried out using the method of Saikai et al. (2012) with slightly modification.

Gamma Oryzanol and Gamma-aminobutyric acid (GABA)

-Oryzanol content was determined using the method of Chen and Bergman (2005) with slight modification. GABA
content was determined with the method of Varanyanond et al. (2005) with slight modifications.

Results and Discussion

Total phenolic content was determined by used the Folin-Ciocalteu reagent mrethod as shown in Figure 1a. Gallic
acid was used as standard. Antioxidant activity as analyzed using the DPPH method, the FRAP method and the
ABTS method was shown in Figure 1b. Result of the study showed that Kam variety from PM and TP had
significantly higher total phenolic content at 2982.98-3238.23 mg GAE/100 g, compared with other varieties. The
different of total phenolic content may because of the different in varieties of rice and source of production. Khao-Mao
from Kam variety that contain high total phenolic content also show the high antioxidant activity (DPPH+, FRAP+ and
ABTS+). However, in this study, Khao-Mao from Dor He and E-Tei contain high inhibition by hydroxyl (OH) and
superoxide ion (O2-), respectively (Figure 1c). Anthocyanins content of Kam variety (pigmented rice) from PM was
higher than Kam variety from TP (Figure 1d). The non-pigmented rice varieties provided chlorophyll content between
0.026-0.052 mg/ml (Figure 1e).

Proximate analysis for different young rice varieties of Khao-Mao was carried out according to AOAC standard
methods. Table 1 and 2 presents the proximate composition of moisture, ash, protein, lipid, crude fiber and
carbohydrate in 9 young rice varieties of Khao-Mao. In the present study, Dor He was the highest content in protein,
ash and fiber.

Figure 2 presents the content of GABA (Figure 2a) and gamma-oryzanol (Figure 2b) in Khao-Mao from 9 young rice
varieties. Lee Nok variety provided high value of GABA content (2.84 mg/100g). However, Dor He presented high
content of total gamma-oryzanol (11.35 mg/100g) and derivatives: cycloartenyl ferulate (3.13 mg/100g), 24-
mmethylene-cycloartenyl ferulate (3.04 mg/100g), campesteryl ferrulate (3.27 mg/100g) and sitosteryl ferulate (1.90
mg/100g).

266
 a b

c d e
Figure 1 Bioactive compounds of Khao-Mao from young rice varieties.
(a) Total phenolic content, (b) Antioxidant activities,
(c) %inhibition by hydroxyl and superoxide radical scavenging activity,
(d) Total chlorophyll and (e) Total anthocyanins

Table 1 Chemical composition of Khao-Mao from young rice varieties

Chemical PM
composition Kam RD6 Jusmine Dor He
Moisture 21.80±0.12 d 26.79 ±1.06 a 21.38 ±0.09 d 22.95±0.10c
Fat 0.72±0.08 d 0.60 ±0.07 e 1.44 ±0.03 a 1.21±0.05b
Protein 5.30±0.04g 6.89 ±0.16 b 5.44 ±0.06 fg 7.28±0.10a
Ash 1.16±0.02 de 1.09±0.04 e 1.19 ±0.03 cd 1.42±0.03a
Fiber 0.59±0.05 c 0.81±0.03 b 0.70 ±0.06 b 0.95±0.13a
Carbohydrate 71.02±0.17 a 64.63±1.24 f 70.55 ±0.11 ab 67.14±0.03e
Chemical TP
composition Kam RD6 Dor Hom Lee Nok E-Tei
Moisture 21.94±0.09d 22.89±0.14cd 21.66±0.07d 23.83±0.05 b 20.72±0.14 e
Fat 0.95±0.04c 0.90±0.04c 1.24±0.02b 1.24±0.03 b 1.42±0.03 a
Protein 6.39±0.23 c 7.00±0.04 b 5.76±0.09 e 5.59±0.05 ef 6.12±0.03 d
Ash 1.16±0.02 de 1.19±0.09 cd 1.20±0.01 cd 1.26±0.04 bc 1.31±0.08 b
Fiber 0.77±0.06 b 0.33±0.05 d 0.77±0.05 b 0.75±0.08 b 0.58±0.07 c
Carbohydrate 69.56±0.31c 68.03±0.21d 70.14±0.05bc 68.08±0.09 d 70.43±0.15ab
a,b,c,d,e,f,g Values with different letters in the same row are significantly different (p0.05).

Means  SD, each value in the table is the mean of five replications.

267
 (a) (b)

Figure 2 GABA (a) and gamma-oryzanol (b) in Khao-Mao from young rice varieties

Conclusion
The study has shown that Khao-Mao from Kam variety (pigmented rice variety) contained high phenolic content and
antioxidant activity. Khao-Mao from Dor He variety (non-pigmented rice variety) presented high protein, ash and fiber
including gamma-oryzanol. Khao-Mao from Lee Nok variety (non-pigmented rice variety) displayed high GABA
content. The result of this study could be used as a basic knowledge in food application, especially in development of
food products to make an economic potential of local community.

Acknowledgements
The authors would like to thank ARDA and UBU for financial support.

References
AOAC. 2000. Official Methods of Association of Analytical Chemists. Washington, DC: Association of Analytical
Chemists.
Chen, M.H. and Bergman, C.J. 2005. A rapid procedure for analyzing rice bran tocopherol, tocotrienol and -
Oryzanol contents. Journal of Food Composition and Analysis 18: 319-331.
Saikia, S., Dutta, H., Saikia, D. and Mahanta, C.L. 2012. Quality characterization and estimation of phytochemicals
content and antioxidant capacity of aromatic pigmented and non-pigmented rice varieties. Food Research
International 46: 334-340.
Singh, N., and Rajini, P.S. 2004. Free radical scavenging activity of an aqueous extract of potato peel. Food
Chemistry 85: 611–616.
Varanyanond, W., Tungtrakul, P., Surojanametakul, V., Watanasiritham, L. and Luxiang, W. 2005. Effects of water
soaking on -aminobutyric acid (GABA) in germ of different Thai rice varieties. Kasetsart Journal (Natural
Science) 39: 411-415.
Ramesh, T., and Devasenapathy, P. 2006. Physiological response of cowpea in a rainfed alfisol ecosystem to the
impulse of soil moisture conservation pracyives. Gen.Appl.Plant Physiology. 32: 181-190.
Oonsivilai, R., Singthong, J. Oonmetta-aree, J., and Ningsanond, S. 2012. Bioactivity, antioxidant activity, and
cytotoxicity of Yanang, Kreu-Ma Noy, and Rang Chuet extracts. IFT Annual Meeting 2012, Las Vegas, USA,
June 23-29, Poster Presentation.

268
 IFRC 2017: 043-030 Functional Food

Comparative analysis of the nutrient content and antioxidant activity of lagikway (Abelmoschus manihot L),
alugbati (Basella alba L), camote (Ipomoea batatas L), and saluyot (Corchorus capsularis L) leaves

*Algar, A.F.C. and Sediño, D.J.I.

Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines Los
Baños, Laguna, 4031

Several indigenous and endemic plants are given less attention due to limited research studies, thus it is important to
establish their basic nutritional information and functional properties in order to increase its utilization. The
Abelmoschus manihot or lagikway is one of the least well-known and underutilized vegetable in the Philippines thus
its nutritional and antioxidant properties were compared to the commonly consumed leafy vegetables namely the
saluyot, camote tops and alugbati. The proximate composition of lagikway was comparable to the three vegetables
having a relatively high ash content and low fat content. It showed the highest Vitamin A content (365.42±23.42 IU)
but is low in Vitamin C (31.93±2.11 mg/100g). In addition, the iron (45.7mg/100g) and calcium (4449mg/100g)
contents of lagikway were appreciably higher compared with the three vegetables. However, the antioxidant activity
of lagikway (25.15 ± 1.01%) was comparably lower than the other samples but is still significantly present. The
nutritional properties of lagikway as compared with other leafy vegetables showed that it has many benefits and has
the potential to be processed as a high value product.

Keywords: Abelmoschus manihot, indigenous plants, nutritional content, antioxidant activity

*Corresponding author’s email: acalgar@up.edu.ph

Introduction
In the Philippines there are indigenous leafy vegetables which are underutilized due to insufficient information about
its nutritional content and functional properties. However, these indigenous leafy vegetables have the potential to
become an important alternative to the usual agricultural crops being consumed (Linsken and Jackson, 1993).
Lagikway is one of the underutilized indigenous leafy vegetable that should be popularized in the Philippines due to
its nutritional value (Sarian, 2013). This vegetable should be recognized because it has a vital role in alleviating
“hidden hunger” which includes the lack of important minerals and vitamins in the diet. Thus, it is important to
establish its basic nutrient and functional properties. The proximate composition is considered as the most basic and
important information for a food and the antioxidant activity is considered by many consumers as one significant
functional property nowadays.

The objective of this study is to determine the proximate composition and antioxidant activity of Lagikway
(Abelmoschus manihot) and compare it to the commonly utilized indigenous vagetables consumed in the Philippines
which are Alugbati (Basella alba), Saluyot (Corchorus capsularis) and Camote (Ipomoea batatas) leaves.

Materials and Methods

Sample Preparation
Leaves of the samples were collected from the Crop Production and Management Division of the Institute of Crop
Science, College of Agriculture and Food Sciences, University of the Philippines Los Baños, Philippines. The leaves
were washed, dried, and ground.

Proximate analysis was done according to the AOAC Standard Methods; Vitamin A analysis was determined using
spectroscopic method using β-carotene as standard while Vitamin C was determined by titration using dichlorophenol
indophenol; iron and calcium were analyzed using atomic absorption spectroscopy (AAS) at the regional standard
testing laboratory of the Region 4A Department of Science and Technology (DOST) in Laguna, Philippines; and
antioxidant activity was analyzed using DDPH scavenging activity method.
269
All analyses were done in triplicates and the data were reported as mean ± SD and were analyzed using one-way
ANOVA.

Results and Discussion

The proximate composition of the four leafy vegetables is presented in Table 1. It can be observed that lagikway has
the lowest crude fat and highest carbohydrates in the form of NFE, camote leaves has the highest crude fat and
crude protein, saluyot has highest ash content and crude protein, and finally alugbati leaves has the highest crude
fiber but the lowest protein content. Further analysis of its minerals using AAS, as presented in Table 2, shows that
camote leaves have the highest iron content (46.2mg/100g) which is not far from the lagikway leaves (45.7mg/100g)
while the lagikway leaves have the highest calcium content (4449mg/100g). These values are consistent with the
relatively high ash content of lagikway as presented in Table 1. The appreciable amount of minerals in lagikway and
camote indicates that it can be a potential source of mineral requirement in human diet. It can minimize the case of
iron deficiency or calcium inadequacy if consumed efficiently (Asaolu et. al, 2012).

Using acetone-hexane extraction and spectrophotometry at 440nm, Vitamin A content of the four leafy vegetables
were determined and shown in Table 3. Lagikway, having a statistical difference among the samples, obtained the
highest amount of vitamin A (365.42 IU). The obtained value of vitamin A of lagikway is smaller than the reported
vitamin A content in study by Preston (1998), which is 969mg/100g, which may be due to the plant source and
maturity. With the obtained value, consuming 100g of lagikway leaves can provide half of the required daily intake of
vitamin A which was 750mg. On the other hand, Vitamin C was determined by titration using dichlorophenol
indophenol and is also shown in Table 3. Camote tops obtained the highest amount of vitamin C (120.25 mg
ascorbic/100g) and the lowest was the lagikway leaves (31.93mg ascorbic/100g) which is comparable to the results
of a study by Preston (1998) wherein the vitamin C of the lagikway leaves was 39mg/100g. By consuming 100g of
lagikway leaves, it can still provide the recommended daily intake of Vitamin C which is 40mg.

The DPPH assay showed the highest scavenging activity from the camote (76.32%) and saluyot (74.85%) leaves
and lowest in lagikway (25.15%) leaves. According to Vimal and colleagues (2009), the values obtained by camote
and saluyot leaves indicate that is it a good radical scavenger. The high antioxidant activity of camote and saluyot
leaves can be attributed to the presence of high amount of phenolic compounds and flavonoids as well as the
synergistic effects of the different phytochemicals present as reported by Baang and colleagues (2015).

Table 1. Proximate composition of the four indigenous leafy vegetables.

Component, % Lagikway Camote Saluyot Alugbati
(A. manihot) (I. batatas) (C. capsularis) (B. alba)
Moisture 80.75 ± 0.62a 85.53 ± 0.55b 81.85 ± 0.33a 93.49± 0.08c
Ash 2.12 ± 0.07c 1.59 ± 0.04b 2.27 ± 0.04c 0.78 ± 0.19a
Crude fat 0.13 ± 0.12a 0.38 ± 0.12a 0.17 ± 0.12a 0.32 ± 0.13a
Crude fiber 0.56 ± 0.04 a 0.73 ± 0.29 a 0.50 ± 0.07a 1.48 ± 0.04b
Crude protein 4.24 ± 0.11a 5.58 ± 2.93ab 7.92 ± 2.69b 1.94 ± 0.11a
NFE 12.2c 6.19b 7.28b 1.98a

270
 Table 2. Iron and calcium content of the four indigenous leafy vegetables.
Lagikway Camote Saluyot Alugbati
Mineral, mg/100g
(A. manihot) (I. batatas) (C. capsularis) (B. alba)
Iron 45.7 46.2 19.8 14.9
Calcium 4449 558 2951 863

Table 3. Vitamin A and C content of the four indigenous leafy vegetables.

Lagikway Camote Saluyot Alugbati
Vitamin
(A. manihot) (I. batatas) (C. capsularis) (B. alba)
Vitamin A, IU 365.42 ± 23.42b 60.83 ± 7.49a 68.87 ± 5.08a 98.07 ± 9.27a
Vitamin C, mg/100g 31.93 ± 2.11a 120.25 ± 5.07b 55.04 ± 2.83ab 44.18 ± 0.00 ab

90
80 [VALUE] ± 1.75c [VALUE] ±2.82c
%DPPH SCAVENGING ACTIVITY

70
60
50
40 [VALUE]±1.83b

30 [VALUE]± 1.01a
20
10
0
lagikway camote saluyot alugbati

Figure 1. Percentage DPPH scavenging activity of the four indigenous leafy vegetables.

Conclusion

The four indigenous leafy vegetables contained significant amounts of two important minerals, iron and calcium, as
well as vitamins A and C. It also showed antioxidant activity through the DPPH assay. Lagikway leaves, the most
underutilized among the four, is a very good source of calcium and vitamin A but has a low but still significantly
present antioxidant activity. In general, the results of this study shows that these indigenous leafy vegetables can
provide adequate nutrition and can be further explored for further processing and product development. The basic
nutritional and functional information obtained in this study can raise awareness and increase the propagation,
utilization, and promotion of these indigenous vegetables in the Philippines.

Acknowledgements

The authors would like to thank the Philippine Department of Agriculture – Bureau of Agricultural Research for the
research funding and for the Institute of Crop Science of UPLB as the research collaborator.

271
References

Asaolu, S.S., Adefemi, O.S., Oyakilome, I.G., Ajibulu, K.E., & Asaolu, M.F. 2012. Proximate and mineral
composition of Nigerian leafy vegetables. Journal of Food Research, 1(3), 214.
Baang, R.P., Del Rosario, R.M. and Palmes, N.D. 2015. Phtochemical profiles and antioxidant activity of selected
indigenous vegetables in northern Mindanao, Philippines. International Scholarly and Scientific Research &
Innovation 9(8): 769-774.
Linkens H.F. and Jackson, J.F. eds. Modern Methods of Plant Analysis. Vol.16. Vegetable and Vegetable Products.
p.7.
Preston, S.R. 1998. Aibika/Bele. Abelmoschus manihot (L.) Medik. Promoting the conservation and use of
underutilized and neglected crops.24. Institute of Plant Genetics and Crop Plant Research, Gatersleben
International Plant Genetic Resources Institute, Rome, Italy. pp. 30-42, 37-41.
Sarian, Z.B. 2013. Gikway: An Indigenous Vegetable. Agri Ideas, Agri News, Sarian Farm Press.
http://www.zacsarian.com/gikway-an-indigenous-vegetable/
Vimal, K., Gogoi B.J., Meghvansi, M.K., Lokendra S., Srivastava, R.B. and Deka, D.C. 2009. Determining the
antioxidant activity of certain medicinal plants of Sonitpur (Assam), India Using DPPH assay. Journal of
Phytology, 1(1):49–56.

272
 IFRC 2017: 070-055 Functional Food

Effect of solvents on extraction and bioactive properties of commercial grape cultivars in Taiwan
Sridhar, K. and *Charles, A.L.

Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and
Technology, 1 Shuefu Road, Neipu, Pingtung 91201, Taiwan

Kyoho (Vitis labruscana), a new commercial grape cultivar developed in Taiwan and attractive for its large size
berries, was studied for its potential food functional properties. The effect of the solvents acetone: water (4:1, v/v),
methanol: water (4:1, v/v), methanol: formic acid (9:1, v/v), and water on the extraction of polyphenols and
antioxidant properties were investigated in fresh and freeze-dried Kyoho and Jubilee (an imported commercial)
cultivars, respectively. Methanol: water and water showed higher potential for extracting bioactive compounds from
Kyoho and Jubilee, respectively. Methanol: water solvent extracted the maximum total polyphenols (698.73 and
674.09 µg GAE/mL) and flavonoids (1919.46 and 1898.46 µg QE/mL) in fresh and freeze-dried Kyoho, whereas
water extracts exhibited the highest polyphenols (738.22 and 693.65 µg GAE/mL) and flavonoids (2172.80 and
1628.80 µg QE/mL) in fresh and freeze-dried Jubilee. The methanol: water extracts of Kyoho fresh and freeze-dried
samples showed the highest nitric oxide scavenging activity (67.89 -73.45 %), superoxide scavenging activity (38.12-
43.03 %), DPPH (89.91 - 89.25 %), and total antioxidant inhibition (97.78 – 98.26 %), respectively. Comparatively,
water extracts obtained from Jubilee fresh and freeze-dried samples also exhibited the highest antioxidant properties
except H2O2 scavenging activity of fresh and freeze-dried Kyoho and Jubilee, respectively. Positive correlations
(p<0.05) was determined between total phenolic content and DPPH (r = 0.82, R2 = 0.67) and total antioxidant activity
(r = 0.83, R2 = 0.68) except H2O2 scavenging activity (r = -0.97, R2 = 0.94). The polyphenol and antioxidant activities
of grapes extracts were significantly affected by the type of extracting solvent, and the findings further demonstrated
that consumption of these two cultivars with water and methanol (alcoholic drinks) would provide an alternative to
obtaining antioxidant rich material in the diet.

Keywords: grape cultivars, solvent extraction, total phenolics, antioxidants, radical scavenging activity.
*Corresponding author’s email: alcharles@mail.npust.edu.tw; Tel: +886-8-774-0429

Introduction
Plant based foods have been extensively studied for their antioxidant properties in recent years. It is scientifically
demonstrated that plant based foods have synergistic effects to assist in reducing the risk of various diseases
including cancer and cardiovascular diseases (Pérez-Jiménez et al., 2008). Among phytochemicals, polyphenolic
compounds have gained increased interest due to their antioxidant activity and wide range of potential health
benefits.

The main objectives of this study were (i) to investigate the effect of different solvent systems for the extraction of
polyphenols, (ii) to determine the polyphenols content and in vitro antioxidants properties, and (iii) to examine the
possible correlation between total phenolic and flavonoid content with antioxidant activity of fresh and freeze dried
commercial Kyoho and Jubilee extracts, respectively.

Materials and Methods

Sample preparation

Fresh and freeze-dried (10 g) samples of Kyoho and Jubilee were ground in a kitchen blender (Eupa TSK-9391,
Japan) and added with solvent mixture (Table 1). The homogenized samples were sonicated for 60 min (Delta
Ultrasonic Cleaner, Taiwan). The mixtures were centrifuged (Universal 32 R centrifuge, Germany) at 3000 g for 15
min and the supernatant was evaporated in a rotary evaporator (Eyela, A-1000 S, Japan) at 45 oC under controlled
vacuum. The extracts of different solvents were stored at 4 °C in air tight container for further analysis. All the
extraction processes were carried out in triplicate.
Total phenolic and flavonoid content (Moniruzzaman et al., 2014) in fruit sample extracts were estimated using
colorimetric assays.

273
 Table 1. Composition of solvents used for bioactive compound extraction
Number of extracting solvent mixture Solvent composition
I Acetone: water (4:1, v/v)
II Methanol: water (4:1, v/v)
III Methanol: formic acid (9:1, v/v)
IV Water

Measurements of antioxidant activities

Antioxidant assays such as nitric oxide radical scavenging assay (Boora et al., 2014), superoxide anion radical
scavenging activity (Li, 2012), hydrogen peroxide (H2O2) radical scavenging assay (Sroka and Cisowski, 2003),
DPPH activity (Shimada et al., 1992), and total antioxidant activity (Charles and Huang, 2009) were conducted in
order to determine antioxidant properties of extracts. All data were expressed as mean ± standard deviation and
were done in triplicate independent analyses. Analysis of variance (ANOVA) and Duncan’s multiple range method
were used to compare any significant differences between solvents and samples (p<0.05) using IBM SPSS
Statistics® version 22.0 (Armonk, NY).

Results and Discussion

Total phenolic content (TPC)

For the fresh and freeze-dried samples of Kyoho, solvent II (methanol and water) exhibited the highest total phenolic
content (698.73 ± 0.01 and 674.09 ± 0.01 µg GAE/mL DW, respectively). Solvent IV (water) extracts exhibited the
highest total phenolic content (738.22 ± 0.04 µg GAE/mL) in fresh sample of Jubilee cultivar, whereas for the freeze-
dried sample, solvent I (acetone: water) showed the highest total phenolic content (708.65 ± 0.01 µg GAE/mL).

Total flavonoid content (TFC)

The total flavonoid content from fresh and freeze-dried samples of Kyoho and Jubilee extracted by different solvents
(Table 2) exhibited the same trend as total phenolic content except freeze-dried Jubilee cultivar. Solvent IV extract
from fresh samples of Jubilee cultivar had the highest (2172.80 ± 0.03 µg QE/mL) total flavonoid content, followed by
those from solvent II, solvent III, and solvent I extracts.

Measurements of antioxidant activities

DPPH radical scavenging activity

DPPH activity of 89.91 % for fresh sample and 89.25 % for freeze-dried sample were observed using the solvent II.
Similarly, solvent IV was the most effective DPPH radical scavenging activity in Jubilee fresh (86.95 %) and freeze-
dried (87.76 %) material. The inhibition capacity of ascorbic acid (standard) was recorded as 90 %.
The methanol and water extracts (solvent II) of Kyoho fresh and freeze-dried samples showed the highest nitric oxide
scavenging activity (67.89 – 73.45 %), superoxide scavenging activity (38.12 – 43.03 %), and total antioxidant
inhibition (97.78 – 98.26 %), respectively. Comparatively, water extracts (solvent IV) obtained from Jubilee fresh and
freeze-dried samples also exhibited the highest antioxidant properties except H2O2 scavenging activity of fresh and
freeze-dried Kyoho and Jubilee, respectively.

274
 Table 2. Total phenolic and flavonoid contents of grape cultivars extracted by different solvents1

Total phenolic content (µg GAE/mL) Total flavonoid content (µg QE/mL)
Solvent Fresh Freeze-dried Fresh Freeze-dried
Kyoho Jubilee Kyoho Jubilee Kyoho Jubilee Kyoho Jubilee
I 671.92±0.02b 709.60±0.02c 626.99±0.02b 708.65±0.01b 1860.80±0.02b 458.13±0.01a 1772.80±0.03c 1634.13±0.03d
II 698.73±0.01c 617.93±0.01b 674.09±0.01d 684.60±0.01 1919.46±0.01d 815.46±0.01c 1895.46±0.02d 866.13±0.01c
II 307.06±0.02a 338.22±0.02a 475.90±0.03a 332.42±0.01a 434.13±0.01a 639.46±0.02b 932.80±0.02b 620.80±0.01b
IV 673.36±0.02 b 738.22±0.04 d 632.42±0.01 c 693.65±0.03 b 1916.80±0.01 c 2172.80±0.03 d 239.46±0.01 a 572.80±0.01a
Notes: The values are given as mean ± SD (n=3). I–IV – different solvents used for extraction (see Table 1) and values followed by different superscripts within
1

the same column are significantly different (p<0.05) from each other.

Correlation between total phenolic content and antioxidant activities

For fresh Kyoho, there was a strong positive correlation (p<0.05) was observed between total phenolics and antioxidant activity (DPPH (0.82) and total
antioxidant activity (0.83)). Moreover, total flavonoid content was positively correlated with antioxidant activity in the NO• scavenging activity (0.18), superoxide
(0.61), DPPH (0.80), and lipid peroxidation inhibition (0.80), but no significant difference was observed (Table 3).

Table 3. Coefficients of correlations between total polyphenols and antioxidant activity for tested extracts
Cultivar

Antioxidant activity Kyoho Jubilee

Fresh Freeze-dried Fresh Freeze-dried
TPC TFC TPC TFC TPC TFC TPC TFC
NO• scavenging activity 0.22 0.18 0.24 0.98* 0.76 0.91* 0.88* -0.01
Superoxide scavenging activity 0.64 0.61 0.24 -0.70 -0.32 0.69 0.18 -0.75
H2O2 scavenging activity -0.97* -0.97* -0.81 -0.81 -0.80 -0.56 -0.99* -0.4
DPPH 0.82* 0.80 0.83 0.01 0.62 -0.19 0.94* 0.20
Total antioxidant activity 0.83* 0.80 0.69 0.91* 0.52 0.74 0.62 -0.37
*: Correlation is significant at the 0.05

275
Conclusions

Mixture of methanol and water (solvent II) was the most efficient solvent for extracting polyphenols from Kyoho fresh
and freeze-dried form, while water (solvent IV) was presented the effective solvent in extracting polyphenolic
compounds and antioxidants from the Jubilee fresh and freeze-dried, respectively. The results suggested that the
consumption of Kyoho cultivar with methanol and water, Jubilee cultivar with high amount of water is solubilize the
phenolic compounds. Therefore, consumption of these two cultivars with water and methanol (alcoholic drinks)
would provide an alternative to obtain antioxidant rich material.

Acknowledgements

The authors are grateful to the International Cooperation Development Fund, Taiwan (TaiwanICDF) and National
Pingtung University of Science and Technology, Taiwan for providing fellowship and laboratory facilities.

References

Boora, F., Chirisa, E. and Mukanganyama, S. 2014. Evaluation of nitrite radical scavenging properties of selected
Zimbabwean plant extracts and their phytoconstituents. Journal of Food Processing 2014: 1-7.
Charles, A. L. and Huang, T.C. 2009. Sweet cassava polysaccharide extracts protects against CCl4 liver injury in
Wistar rats. Food Hydrocolloids 23(6): 1494-1500.
Li, X. 2012. Improved pyrogallol autoxidation method: a reliable and cheap superoxide-scavenging assay suitable for
all antioxidants. Journal of Agricultural and Food Chemistry 60(25): 6418-6424.
Moniruzzaman, M. Yung-An, C., Rao, P.V., Hawlader, M.N.I., Azlan, S.A.B.M., Sulaiman, S.A. and Gan, S.H. 2014.
Identification of phenolic acids and flavonoids in monofloral honey from Bangladesh by high performance liquid
chromatography: determination of antioxidant capacity. BioMed Research International 2014: 1-11.
Pérez-Jiménez, J., Arranz, S., Tabernero, M., Díaz- Rubio, M. E., Serrano, J., Goñi, I. and Saura-Calixto, F. 2008.
Updated methodology to determine antioxidant capacity in plant foods, oils, and beverages: extraction,
measurement, and expression of results. Food Research International 41(3): 274-285.
Shimada, K., Fujikawa, K., Yahara, K. and Nakamura, T. 1992. Antioxidative properties of xanthone on the auto
oxidation of soybean in cylcodextrin emulsion. Journal of Agricultural and Food Chemistry 40: 945–948.
Sroka, Z. and Cisowski, W. 2003. Hydrogen peroxide scavenging, antioxidant, and anti-radical activity of some
phenolic acids. Food and Chemical Toxicology 41(6): 753-758.

276
 IFRC 2017: 079-086Functional Food

Quality of dried rice noodles incorporated with differently encapsulated carrot powders

Ismail, H. Karim, R. and *Muhammad, K.

Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan,
Malaysia.

Carrot powders which were differently encapsulated using wall materials such as maltodextrin DE10 (MD), resistant
maltodextrin (RMD), CAPSUL® (CAP) and HI-CAP® 100 (HP) were incorporated into extruded rice noodles. Each
carrot powder at three different levels (5-15 %) was mixed with rice flour and tapioca starch blend (80:20) and the
moisture content of the feed was adjusted to 30 % before extrusion at 80 °C, 100 °C, 100 °C, 100 °C for zones 1, 2,
3 and 4 of the extruder barrel, respectively, with a feed rate of 20 rpm and screw speed of 60 rpm. The noodles were
dried at 30 °C for 16 hours and then evaluated for retention of ß-carotene, colour, cooking quality and textural
properties. The level of carrot powder used and the type of wall material have an effect on the quality of the noodles.
ß-carotene from carrot powders spray dried with HP was retained the highest in the noodles followed by that from
carrot powders encapsulated with MD, CAP and RMD. Rice noodles incorporated with HP and CAP encapsulated
carrot powders were more orange in colour compared to noodles containing MD and RMD encapsulated carrot
powders. Rice noodle containing 15 % RMD encapsulated carrot powder showed the highest rehydration (213.40 %)
and the lowest cooking loss (15.86 %). Gumminess, chewiness and hardness of noodle containing 5 % MD
encapsulated carrot powder were close to that of noodle containing no carrot powder. In conclusion, HP should be
employed as the wall material for encapsulation of carrot juice that is to be incorporated in dried rice noodles.

Keywords: extrusion, retention, β-carotene, noodles, quality

*Corresponding author’s email: kharidah@upm.edu.my

Introduction
Noodles made from cereals and legumes are potential nutrient vehicles since they are well accepted by consumers.
Durum wheat noodle or pasta which has been added with natural pigments is already commercially available and the
natural pigments that have been used are betalains from red beet, carotenoids from carrot, chlorophylls from spinach
and lycopene from tomato. Milled rice contains limited amount of vitamins and minerals due to the removal of bran
during milling, thus adding carrot powder into dried rice noodle could aid in its fortification. Carotenoids, including ß-
carotene play an important role in cancer prevention and provide various other health benefits (Sims et al. 1993).
Carrots are a rich source of ß-carotene and it is reported to be the most stable carotenoids. However, ß-carotene in
carrot powder needs to be encapsulated for better retention during extrusion. Encapsulation envelopes ß–carotene in
a coating material or “wall” to isolate it from the environment (Desobry et al. 1997). This study was therefore aimed at
evaluating the potential of various wall materials namely maltodextrin (MD), resistant maltodextrin (RMD) and OSA
starches CAPSUL® (CAP) and HI-CAP® 100 (HP) on retaining β–carotene from carrot powders which were
incorporated into dried rice noodle produced by extrusion. The effect of level of carrot powders used on the colour
and cooking quality of the dried rice noodles, and textural characteristics of the noodles after cooking would also be
evaluated.
Materials and Methods
Rice flour preparation
Beras Nasional rice was purchased from Padiberas Nasional, Selangor D.E., Malaysia. The rice grains were ground
using a Good and Well grinding machine (Malaysia) and the flour was sieved using a 200 µm mesh size sieve. The
sieved rice flour with particle size ranging from 40 – 200 µm was collected for further processing.

277
Carrot powder preparation
Carrot powder was prepared using the method described by Muhammad et al. (2015). The carrot powders
encapsulated with MD, RMD, CAP and HP were termed as MD carrot powder, RMD carrot powder, CAP carrot
powder and HP carrot powder, respectively.

Extrusion of rice noodles

Rice noodle formulation used was based on Abdul Rahman et al. (1994). Rice flour and tapioca starch were weighed
at 80 g and 20 g, respectively. Each carrot powder was added at 5 %, 10 % and 15 % levels to different batches of
100 g of rice flour and tapioca starch mix. Water was added to the mixture to achieve a final moisture content of 30 %
and the mixture was conditioned at 4 ± 0.5 °C overnight. The conditioned mixture was then sieved using a 500 µm
sieve to avoid clumping. The mixture was subsequently extruded using a Brabender® stand-alone KE19 extruder
(Duisburg, Germany) with a dimension of 1350 mm (width) x 850 mm (height) x 680 mm (diameter) and barrel
diameter of 19 mm, screw length of 25 D, and a die size of 2 mm. Extrusion was conducted at 80 °C, 100 °C, 100
°C, 100 °C for zones 1, 2, 3 and 4 of the extruder barrel, respectively, with a feed rate of 20 rpm and a screw speed
of 60 rpm. The extruded rice noodles were collected and dried at 30 ± 0.5 °C for 16 hours. Dried extruded rice
noodles were packed in aluminium pouches and stored at 21 ± 0.5 °C until further analysis.

Determination of ß-carotene
Extraction of β-carotene from dried rice noodles was conducted according to Desobry et al. (1997).
Spectrophotometric reading was made as described by Georgé et al. (2011).

Cooking quality determination

Cooking loss and rehydration of dried rice noodles were determined in accordance to Bhattacharya et al. (1999).
Texture profile analysis (TPA)
Texture profile analysis of dried rice noodle after cooking was performed based on the method described by
Bhattacharya et al. (1999).

Statistical analysis
All results were statistically analysed using one-way analysis of variance (ANOVA) at a probability level of P<0.05
using SPSS 16 statistical software.

Results and Discussion

ß-carotene in rice noodles added with 5 % HP carrot powder was retained the highest (32.22 %) compared to that in
all the other extruded rice noodles (Figure 1). This was due to the OSA starch present in HP carrot powder which
was able to protect β–carotene during extrusion. OSA starches have been widely utilised in the microencapsulation
of lipophillic bioactive compounds including essential oils and vitamins and have exhibited better retentions
performance than those containing β-cyclodextrin, whey protein as well as maltodextrin (Sweedman et al., 2013; Xie
et al., 2010). Choudhari et al. (2012) stated that microcapsules of lycopene produced by spray drying were relatively
stable to extrusion temperatures up to 187 °C. The retention of β–carotene decreased as the level of carrot powder
incorporated increased. This might be due to the increasing amount of β–carotene exposed to heat during extrusion.
Chromatic results in Table 1 show that noodles incorporated with 5 % RMD carrot powder was the brightest while
noodles with 15% HP carrot powder were the most orange in colour which suggests the highest concentration of ß–
carotene in the dried rice noodles. There was an apparent difference in the chroma values between samples except
for noodles incorporated with 10 % MD and 10 % CAP, while noodles incorporated with 5 % RMD showed the
highest chroma value.

Table 2 shows there were no significant differences in the dried noodle cross sections lengths. Generally, high weight
and volume gains and low cooking loss are the desirable characteristics for high quality Asian noodles (Lee et al.
2002). Rice noodles incorporated with 15% RMD exhibited the highest rehydration (213.4%) and the lowest cooking
loss (15.86%). Increment in the amount of carrot powder added resulted in an increase in the rehydration and

278
decrease in the cooking loss, hardness, gumminess and chewiness of the rice noodles. Noodles incorporated with
5% MD carrot powder showed no significant differences from the control noodles.

40
ß-carotene retention (%)

31.42b 32.22a
28.57c
30 27.00d 26.37e
25.03f 24.53g
23.32h
19.13i 18.90j
20 16.72l
12.82k

10

0
0
Control MD RMD CAP HP
5% 10% 15%

Figure 1: ß–carotene retention in dried rice noodles incorporated with carrot powders prepared using four different
wall materials: maltodextrin (MD), resistant maltodextrin (RMD), Capsul® (CAP) and Hi-CAP® 100 (HP). Values
are means ± standard deviations from triplicate analysis of dried rice noodles produced from two extrusion runs.
Different superscript lettering for different categories indicated significant difference at p<0.05.

Table 1: Chromatic measurements of dried rice noodles incorporated with carrot powders prepared with four different
wall materials
Carrot powder L* H° C*
Control 38.48±0.57d 78.29±0.36i 8.91±0.25a
MD
5% 43.12 ± 0.07i 80.98 ± 0.04k 35.54 ± 0.02g
10% 39.22± 0.19e 76.70 ± 0.09g 33.87 ± 0.14e
15% 40.48 ± 0.20g 74.39 ± 0.04e 36.24 ± 0.17h
RMD
5% 44.66 ± 0.01j 79.17± 0.03j 39.29 ± 0.01l
10% 43.48 ± 0.09i 75.75± 0.05f 38.74 ± 0.07k
15% 36.15 ± 0.37a 72.40± 0.05c 32.08 ± 0.39c
CAP
5% 39.90 ± 0.09f 77.62 ± 0.05h 34.81 ± 0.09f
10% 37.69 ± 0.09c 73.47 ± 0.04d 33.97 ± 0.12e
15% 36.82 ± 0.14b 70.29 ± 0.07b 33.06 ± 0.11d
HP
5% 35.86 ± 0.11a 75.98 ± 0.07f 30.44 ± 0.12b
10% 41.60 ± 0.04h 73.34 ± 0.05d 38.11± 0.04j
15% 40.72 ± 0.08g 69.59 ± 0.03a 37.21 ± 0.08i

Values are means ± standard deviations from triplicate analysis of dried rice noodles produced from two
extrusion runs. Different superscript lettering for different categories indicated significant difference at p<0.05.

279
 Table 2: Cooking quality and textural properties after cooking of dried rice noodles containing carrot powders
encapsulated with MD, RMD, CAP and HP
Sample Cross Rehydration (%) Cooking loss Textural properties after cooking
section (%) Hardness Gumminess (g) Chewiness
length (mm) (g) (g x mm)
Control 2.30±0.06a 172.26±2.42cd 45.57±0.16a 403.69±11.48ab 345.68±10.44a 339.61±6.62a

MD
5% 2.19±0.07a 159.48±0.57f 38.47±0.11b 432.83±18.95a 364.61±11.91a 357.08±10.86a
10% 2.24±0.05a 190.18±1.54b 30.61±0.11e 280.45±10.58e 253.23±10.05cd 227.92±11.70cde
15% 2.20±0.06a 191.02±2.50b 24.65±0.30f 245.18±6.31f 215.12±8.62f 211.35±10.05e
RMD
5% 2.16±0.09a 162.67±3.83ef 37.87±0.11b 372.35±18.71c 382.07±5.60b 301.75±14.16b
10% 2.20±0.08a 169.72±1.98de 18.65±0.33g 289.99±14.89de 268.78±11.55cd 247.53±12.14cd
15% 2.17±0.07a 213.40±3.48a 15.86±0.12h 245.85±12.79f 222.61±0.03ef 224.53±10.49de
CAP
5% 2.22±0.11a 136.70±0.54h 34.08±1.01c 374.95±16.83bc 338.35±16.65b 309.00±15.99b
10% 2.27±0.06a 158.62±1.48f 33.08±1.01cd 312.14±6.91d 250.14±12.04c 250.14±10.99c
15% 2.19±0.05a 161.01±0.64f 31.78±0.48de 291.01±14.81de 248.01±7.68de 221.53±8.40e
HP
5% 2.28±0.09a 142.40±3.53gh 33.68±0.28c 288.40±13.61de 254.32±11.40cd 226.49±6.65cde
10% 2.26±0.05a 144.93±4.90g 31.91±0.09de 220.94±8.35f 200.87±8.05f 179.48±6.74f
15% 2.24±0.07a 179.23±2.58c 18.87±0.78g 185.03±3.36g 171.13±7.39g 153.62±8.41g

Values are means ± standard deviations from triplicate analysis of dried rice noodles produced from two extrusion runs. Different
superscript lettering for different categories indicated significant difference at p<0.05.
Conclusion
The results of this study indicated that the level of carrot powder used and the type of wall material had an effect on
the quality of the rice noodles. The colour of the rice noodles become more orange as the level of the carrot powder
increased. Rice noodles incorporated with more carrot powder showed an increase in rehydration and decrease in
cooking loss, hardness, gumminess and chewiness. The retention of ß–carotene decreased as the level of carrot
powder added increased and was the highest when HP was employed in the encapsulation of the carrot powder.
However, the retention of ß–carotene during storage and cooking of rice noodles fortified with carrot powder is yet to
be evaluated and may show a different trend.
References
Abdul Rahman, H., Zakaria, H., Abdul Rahman, A. O., Wahid, M. A., & Mat Top, O. (1994). Model Perusahaan Makanan Bihun.
Institut Penyelidikan dan Kemajuan Pertanian Malaysia. 3-7
Chi-Ho, L., Cho, J.K., Lee, S.J., & Koh, W. (2002). Enhancing (Beta)-carotene content in Asian noodles by adding pumpkin
powder. Cereal chemistry. 79(4). 593.
Choudhari, S., Bajaj, I., Singhal, R., & Karwe, M. (2012). Microencapsulated lycopene for pre-extrusion coloring of foods. Journal
of Food Process Engineering, 35, 91–103.
Desobry, S. A., Netto, F. M., & Labuza, T. P. (1997). Comparison of Spray‐drying, Drum‐drying and Freeze‐drying for β‐
Carotene Encapsulation and Preservation. Journal of Food Science, 62(6), 1158-1162.
Georgé, S., Tourniaire, F., Gautier, H., Goupy, P., Rock, E., & Caris-Veyrat, C. (2011). Changes in the contents of carotenoids,
phenolic compounds and vitamin C during technical processing and lyophilisation of red and yellow tomatoes. Food
Chemistry, 124(4), 1603-1611.
Muhammad, S. K. S., Amin, H., & Bakar, J. (2015). U.S. Patent No. 9,028,891. Washington, DC: U.S. Patent and Trademark
Office.
Sims. C.A., Balaban. M.O., & Matthews. R.F. 1993. Optimization of carrot juice color and cloud stability. J. Food Sci. 58; 1129-
1191.
Sweedman, M. C., Tizzotti, M. J., Schäfer, C., & Gilbert, R. G. (2013). Structure and physicochemical properties of octenyl
succinic anhydride modified starches: A review. Carbohydrate Polymers, 92(1), 905-920.
Xie, Y. L., Zhou, H. M., Liang, X. H., He, B. S., & Han, X. X. (2010). Study on the morphology, particle size and thermal
properties of vitamin A microencapsulated by starch octenylsucciniate. Agricultural Sciences in China, 9(7), 1058-1064.

280
 IFRC 2017: 079-086 Functional Food

Effect of sago and tapioca starches on the physicochemical properties of expanded rice products coloured
with red beetroot (Beta vulgaris) powder
Abdul Alam, N.A., Karim, R. and *Muhammad, K
Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM, Serdang Selangor Darul Ehsan,
Malaysia
The aim of this study was to determine the effect of two local starches, sago and tapioca starches, at 20 % level of
substitution on the physical properties and betanin content of extruded rice coloured with 7.4 % red beetroot powder.
The moisture of each feed was adjusted to 10 % and it was extruded at 80 ºC, 100 ºC, 120 ºC and 160 ºC for the
first, second, third and fourth zones of the extruder barrel, respectively, with 120 rpm screw speed and 40 rpm feeder
speed. 100 % rice flour coloured with 7.4 % red beetroot powder was also extruded as the control. The results
showed that the expanded rice product with sago or tapioca had better expansion compared to 100 % rice, while the
expansion ratio of extrudates incorporated with tapioca was higher than that with sago. No significant difference was
found in the density of the extrudates. Extrudate substituted with sago had the highest water solubility index but the
lowest water absorption index. The hardness and crispness values for the expanded rice product containing sago
were 16.97 kg and 110.07 kg.sec, respectively, while that containing tapioca were 14.48 kg and 92.77 kg.sec,
respectively. Meanwhile, the hardness and crispness values for 100 % rice extrudate were 18.37 kg and 126.55
kg.sec, respectively. No significant difference was found between the redness, a* values of the extrudates. However,
retention of betanin in the expanded product was the highest when its rice content was substituted with tapioca (36.6
% retention), followed by that substituted with sago (34.1 %) and without substitution (27.8 %). As a conclusion,
addition of tapioca starch or sago starch can help to improve the properties of naturally coloured expanded rice
product.
Keywords: extrusion, rice flour, sago starch, tapioca starch, red beetroot, betalains.
*Corresponding author’s email: kharidah@upm.edu.my
Introduction
Consumer’s awareness on the negative health effects upon consuming synthetic food colourants and the
antioxidative benefits of using natural pigments have increased the market for naturally coloured food products
(Martins et al., 2016). Betacyanins from red beetroot are water soluble pigments and they give off bright red-violet
colour making them good alternatives for similarly coloured synthetic colourants. The European Union and Food and
Drug Administration (FDA) has approved betanin from red beetroot (E162) as a natural food colourant (Martins et al.,
2016). Betanin is one of the betacyanin compounds and together with betaxanthins (yellow pigments), betacyanins
are known as betalains.
Sun and Yoo (2015) found that a blend of tapioca and rice starches was able to improve the physical properties of
rice starch based products. The blending of tapioca starch with other starches has commonly been used in expanded
products as it gives high expansion (Fiorda et al., 2015; Akonor et al., 2016). Other than tapioca starch, sago starch
has also been used in making expanded products by the conventional method (Cheow et al., 2004; Tongdang et al.,
2008). However, very limited studies on the behaviour of sago starch during extrusion cooking have been conducted.
The purpose of this study was therefore, to determine the effect of sago and tapioca starches on the physical
properties of expanded rice products at 20% substitution and to determine the betacyanins (measured as betanin)
and retention in the expanded rice products coloured with 7.4 % red beetroot powder.
Materials and Methods
Sample preparation
Beras Nasional rice was purchased from Padiberas Nasional Berhad, Selangor D.E., and Malaysia. The rice was
ground using Good and Well grinder (Malaysia) and the flour was sieved using a 200 μm mesh size sieve (Test Sieve
Shaker Haver EML Digital Plus, Germany). Sago and tapioca starches were purchased from a local bakery in
Selangor D.E., Malaysia. The red beetroot powder was prepared by spray drying beetroot juice as described by

281
Muhammad et al. (2015). The extrusion feed was prepared by adding 8 g red beetroot powder (7.4 %) to 100 g rice
flour, and rice flour blend with sago or tapioca starch (80:20), each. The feed moisture was adjusted to 10 % and the
feed was pre-conditioned overnight at 4 ºC.
Extrusion process
The extrusion process was conducted using a single screw Brabender extruder KE19 (Duisburg, Germany). A screw
compression ratio of 2:1 and a 4 mm in diameter round die were used during the extrusion process. A feed rate of 40
rpm, a screw speed of 120 rpm and a cutter speed of 115 rpm were set throughout the extrusion cooking. The
temperatures for the first, second, third and fourth extruder barrel were held constant at 80 ºC, 100 ºC, 120 ºC and
160 ºC, respectively. The extrudates were then collected and dried in an oven at 30 ºC, overnight.
Expansion ratio
The expansion ratio of the extrudates was calculated in accordance to Ding et al. (2006).
Density
The density of the extrudates was determined using a solid displacement method following Joardder et al. (2015).
Water absorption index (WAI) and water solubility index (WSI)
WAI and WSI of the extrudates were determined by using AACC method 56 – 20.
Texture analysis
Texture analysis of the extrudates was performed with the TA.XT Plus Texture Analyser (Stable Micro System, UK)
with an Ottawa Cell and the force in compression mode was recorded. Hardness and crispness values of the
extrudates were evaluated.
Colour analysis
The colour of the extrudates was measured using a CR-400 Chroma Meter (Konica Minolta, Inc., Tokyo, Japan). The
hue angle (Hº) and chroma value (C) of the extrudates were calculated as explained by Cai and Corke (1999).
Betacyanin quantification
Betacyanins measured as betanin was quantified using a Perkin Elmer Lamda 35 UV-Vis spectrometer according to
Herbach et al. (2007).
Statistical analysis
The data was analysed for statistical significance by analysis of variance (ANOVA). Pearson correlation was also
performed with SPSS 16.0 (SPSS Inc., Chicago, Ill., U.S.A.).
Results and Discussion
As can be seen in Table 1, the addition of sago and tapioca starches significantly increased the expansion of the
extrudates over the control sample. Even though no significant differences were observed between both expanded
rice with sago starch and with tapioca starch, expanded rice with tapioca starch had a higher expansion ratio
compared to extrudates with sago starch. According to Tongdang et al. (2008), an increase in sago starch in cracker
formulation will increase gel strength, resist bubble growth, and thus reduce the expansion of the crackers. There
was no significant difference in the density of the extrudates. The WAI and WSI of the rice extrudates were negatively
correlated to each other (Table 2). Both expanded rice with 20 % sago and tapioca starches had lower WAI and
higher WSI compared to the control. This might be due to weak bonding force of the sago and tapioca starch
granules causing degradation of amylose and amylopectin through chain splitting that easily occur during the
shearing process.

282
 Expanded rice with 20 % tapioca starch had the lowest hardness and crispness values compared to other
expanded rice products. As shown in Figure 1, the feed for expanded rice with tapioca starch had the lowest
viscosity when gelatinization occurred. The low viscosity of the melt during extrusion process will favour
bubble growth with small and soft extrudates (Ding et al., 2005). According to Paula and Conti-Silva (2014),
the crispness of the extruded snacks analysed using Kramer shear probe was negatively correlated to their
sensory properties. Therefore, expanded rice with low crispness value was more acceptable.

Figure 1: Paste viscosity of feeds

measured using rapid visco analyser.

Although there were no significant differences in the redness of the rice extrudates, the yellowness (b*) and the hue
angle of the extrudates were significantly different from one another. The b* value and Hº of the control sample were
higher than that of the other rice extrudates indicating degradation of betanin (change of colour from red-violet to
yellow) was prominent in 100 % expanded rice. The betanin content and retention were negatively correlated to the
yellowness and hue angle of the extrudates, r = -0.842 and r = -0.846, respectively (Table 3). The obvious
degradation of betanin in 100 % rice extrudates might be due to high mechanical energy that was required to break
down the strong starch-protein bonding of the rice flour which generated more heat during extrusion process (Guy,
2001).

Table 1: The physical properties, colour and betanin content of expanded rice products
Expanded rice with 20% Expanded rice with 20%
Parameters Control
sago starch tapioca starch
Expansion ratio 2.01 ± 0.07 a 2.12 ± 0.08 b 2.15 ± 0.10 b
Density (g/cm3) 0.30 ± 0.12 a 0.24 ± 0.07 a 0.21 ± 0.06 a
WAI (g/g) 5.75 ± 0.12 a 4.46 ± 0.03 b 4.83 ± 0.10 b
WSI (%) 18.36 ± 0.31 a 20.23 ± 0.04 b 18.79 ± 0.16 b
Hardness (kg) 18.37 ± 0.91 a 16.97 ± 0.95 ab 14.84 ± 0.89 b
Crispness (kg.sec) 126.55 ± 3.76 a 110.07 ± 8.64 b 92.77 ± 5.20 c
L* 62.21 ± 0.01 a 60.82 ± 0.38 b 60.79 ± 0.32 b
a* 14.46 ± 0.04 a 14.42 ± 0.07 a 14.49 ± 0.21 a
b* 15.38 ± 0.21 a 14.19 ± 0.10 c 14.60 ± 0.04 b
Hue º (Hº) 46.70 ± 0.13 a 44.56 ± 0.12 c 45.26 ± 0.09 b
Chroma (C) 20.55 ± 0.05 a 20.23 ± 0.11 a 21.13 ± 0.30 b
Betanin content (mg/L) 16.33 ± 0.23 a 18.12 ± 0.01 b 18.16 ± 0.05 c
Betanin retention (%) 27.82 ± 0.41 a 34.14 ± 0.09 b 36.06 ± 0.05 c
The results were calculated as mean values of three replications ± standard deviations. Means that do not share the
same letter are significantly different (p<0.05) as measured by Tukey’s test.

Table 2: Correlation coefficient between physical properties of the expanded rice products
Parameters Expansion ratio Density WAI WSI Hardness Crispness
Expansion ratio 1 -0.217 -0.424 0.054 -0.600 -0.533
Density 1 0.379 -0.238 0.013 0.176
WAI 1 -0.873 0.589 0.580
WSI 1 -0.129 -0.104
Hardness 1 0.942
Crispness 1

283
 Table 3: Correlation coefficient between colour values and betanin retention for expanded rice products
Parameters L a* b* Hue º Chroma Betanin retention
L* 1 -0.248 0.787 0.844 -0.295 -0.847
a* 1 0.207 0.130 0.627 -0.102
b* 1 0.963 0.195 -0.842
Hue º 1 0.148 -0.846
Chroma 1 0.357
Betanin retention 1

Conclusion
As a conclusion, the addition of both sago and tapioca starches during the extrusion process was able to improve the
physical properties of the expanded rice product. However, expanded rice with tapioca starch gave better expanded
product properties and betanin retention compared to expanded rice with sago starch.

References
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284
 IFRC 2017: 092-088 Functional Food

Anti-browning and antioxidant properties of Clinacanthus nutans (Burm. F.) Lindau on “Granny Smith” apple
juice.
Husain N. F., Rahman R.A., *Suleiman N.

1Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor.

The effect of anti-browning in Clinacanthus nutans (Burm. F.) Lindau in “Granny Smith” apple juice at two different
concentration (0.76 % w/w and 1.52 % w/w) has been investigated. The motivation for this work is to aid the
understanding of antioxidant properties in C. nutans, which is important for the anti-browning of apple juice. The
measurements have been performed using two methods; total phenolic compound (TPC) and 2,2-diphenyl-1-
picrylhydrazyl (DPPH) assay which initially extracts the C. nutans using 50% aqueous acetone at 38 ˚C. It
demonstrates these two methods are a suitable and reliable technique to determine the antioxidant activity in both
pure apple juice and apple juice + C. nutans mixtures. The finding shows increases of antioxidant properties with a
high concentration of C. nutans + apple juice mixture, thus, the presence of C. nutans reduces the browning process
of apple juice. This work also provides preliminary indications that the extraction of C. nutans using aqueous acetone
has a higher yield of antioxidant compared to the other solvent (water) extraction based on DPPH result. This could
have implications for understanding the contribution of antioxidant properties contains in C. nutans towards anti-
browning of apple juice and represent important information for the development of healthy drinks.

Keywords: C. nutans, apple juice, antioxidant, TPC, DPPH

*Corresponding author’s email: su_hidayah@upm.edu.my

Introduction
An increasing demand for the consumption of fruit and fruit products is alarming and become a major issue by the
European Union (Picouet et al., 2009). In typical industrial processes for apple juice production, raw apples first need
to cut and diced for further processing. This might result in browning of apple juice if there is no preheating process
has taken place along an apple juice production. Polyphenoloxidase (PPO) is the enzyme that contributes for
browning. It might happen in the presence of oxygen by oxidizing phenols into o-quinone. Browning process can be
reduced using chemicals, pH adjustment, thermal treatment or exclusion of oxygen. In consideration of the consumer
preferences for more “fresh appearing”, healthier, and greener fruit products, and browning enzyme should be
inactivated naturally. Therefore, it is highly necessary to find an alternative way to prevent the browning process. In
preserving conservation of the environment, the research has now shifted towards more sustainable and green
resources to prevent the browning process.
Clinacanthus nutans or known as Belalai Gajah or Sabah Snake Grass has been used in traditional medicine in
Malaysia and Thailand (Roosita et al., 2008; Shim et al., 2013). Traditionally, C. nutans has been consumed as
herbal tea or extracts from fresh leaves since it associated with health benefits. For instance, it can be used for
treating skin rashes and snake bites, lesions caused by herpes simplex virus, diabetic myelitis, fever, and diuretics
(Sakdarat et al., 2006; Tuntiwachwuttikul et al., 2004). It has been reported, the C. nutans can be directly consumed
or mixed with other juices such as apple juice, sugarcane or green tea (Sakdarat et al., 2009). A range of bioactivites
and therapeutically used of C. nutans has stimulated the development of innovative food products, especially in the
fruit juice production. Herein, the focal aim was to investigate the effect of C. nutans on the ‘Granny Smith’ apple
juice browning. In addition, to identify consumer acceptance on ‘Granny Smith’ apple juice with C. nutans based on
the sensory evaluation.

285
Materials and analytical method
Materials
Granny Smith’ apples and Clinacanthus nutans (Burm. F.) were purchased from AEON BIG Putrajaya, Selangor and
Taman Herba YPL, Seremban, Negeri Sembilan, respectively. All chemicals used in this research were of analytical
grade.

Sample pre-treatment and extraction

Fresh C. nutans leaves were sun dried prior to grinding. The extraction was carried out by adding approximately 20 g
C. nutans powder in 200 mL aqueous solution (acetone: water, 50: 50 v/v) for 3 hours in the water bath shaker at a
temperature of ±38 °C. The extract of C. nutans was separated from residues through centrifugation at 1500 rpm.
The supernatant was collected and centrifuged again to remove the leftover residues. The extracts collected were
stored at 4 °C until further used.

Determination of total phenolic compound (TPC)

TPC method will be employed from Singleton and Rossi (1965) and Moein et al. (2007) with a minor modification.
200 µL of C.nutans extracts were mixed with 200 μL of 10 Folin-Ciocalteu reagent prior to vortex for 10 sec. It follows
by the addition of 1 mL of 7.5% sodium carbonate, then continues vortex for another 10 sec. The mixtures were
incubated at room temperature for 2 hours. The absorbance of mixtures was carried out using microplate reader
(Infinite M200, TECAN, Austria) at 765 nm against blank. Deionized water and gallic acid will be used as a blank to
replace the oil and as a standard, respectively. The total phenolic content will be expressed as gallic acid equivalents
per 100 g oil.

Determination of radical scavenging activity using DPPH

Determination of free radical scavenging activity will be performed based on the method described by Moein et al.
(2007) with a minor modification. A C.nutans extracts were diluted in DMSO at different concentration (0.1, 0.05,
0.025, 0.0125, 0.00625 mg/mL). A 1 mL of 0.1mM methanolic DPPH will be added into 2.5 mL of each extracts
dilution. All mixtures were kept for 30 min in a dark room before determine the absorbance reading using microplate
reader at 517 nm. DMSO is used as a blank and gallic acid as a standard.

Color measurement and sensory evaluation

An apple juice with three different concentration of 0.00% (marker), 0.76 % and 1.52 % (w/w) were prepared. The
color measurements of L, a, b and YI-values were measured using HunterLab UltraScan PRO with EasyMatch QC
software in 4 hours time interval at 20±2 °C. The sensor mode was set for RTRAN, sensor mode for observation of
hazy liquid. The degree of browning was expressed by the percent decrease in the L value.
The sensory evaluation with attributes of color, aroma, texture, taste, flavor, and overall acceptability was evaluated
by 30 public judges by 7 points Hedonic scale starting from 1- extramely dislike to 7-extremely like.

Results and discussion

Total phenolic compound (TPC) of C. nutans
A total phenolic compound present in C. nutans extracts can be obtained based on gallic acid standard
calibration curve. The average absorbance of triplicate sample of C. nutans extracts in TPC assay was 0.3963. The
TPC in C. nutans extracts was 0.5014 mg GAE/g dried sample. Lusia Barek et al., (2015) have studied the TPC in C.
nutans leaves with different drying method and soaked in hot boiled water. They found that the TPC in C. nutans
leaves was in the range of 88 – 177 mg TAE/ L. In this study, the finding showed the TPC were in the range of 4.4 –
8.85 mg TAE/g dried sample based on the sample preparation of soaking 2 g sample in 100 mL hot boiled water.
This showed the TPC will increases when the extraction had performed at high temperature. However, further studies
need to be conducted for the effect of extraction temperature on the TPC.

286
Determination of DPPH free radical scavenging activity

Figure 1. The free radical scavenging activity of the aqueous acetone extract of C. nutans and ascorbic acid
examined by DPPH assay. (●: C. nutans; ●: ascorbic acid)
Measuring scavenging of DPPH radical allows the determination of the intrinsic ability of any molecule to donate
hydrogen atom or electron to this reactive species in the homogenous system. As clearly shown in Figure 1, the
percentage differences of scavenging activity between C. nutans and ascorbic acid was only approximately 10 %,
particularly at a concentration of 0.1 mg/ mL. Again, Lusia Barek et al., (2015) have studied the percentage of
scavenging activity for the fermented and unfermented C. nutans soaked in hot boiled water. They found the
percentage of scavenging activity were decreased up to 30 % compared to ascorbic acid. Hence, the finding between
these two studies indicates that the extraction of C. nutans using aqueous acetone will produce a higher yield of
antioxidant compared to the extraction using water.
The effect of C. nutans on the browning process in ‘Granny Smith’ apple juice

Figure 2. Color changes of 0, 0.76, and 1.52 % (w/w) C. nutans in ‘Granny Smith’ apple juice
Two different concentration (0.76% and 1.52%) of C. nutans with one marker (0% C. nutans) were added in ‘Granny
Smith’ apple juice to investigate the effect of C. nutans on the browning process. This observation on the color
changes were performed from 0 min up to 120 min. Figure 2 presents the color changes of ‘Granny Smith’ apple
juice at 0, 45, 60 and 240 min. It clearly shows the browning process had occurred in all samples, particularly for the
marker and 0.76 % sample. It can be noticed the juice still retained its green color with the addition of 1.52 % C.
nutans at minutes 45, but, slightly observable change can be detected at minutes 60 up to 120. This indicates the
presence of C. nutans might slow down the browning process of ‘Granny Smith’ apple juice due to the antioxidant
properties that belong to C. nutans.
Sensory evaluation
Table 1. The average score for sensory evaluation of ‘Granny Smith’ green apple juice with C. nutans leaves
expressed in means ± standard deviation.
Attributes 0 % (marker) 0.76 % 1.52 %
Color 4.83 ± 1.58a 4.33 ± 1.34a 4.20 ± 1.58a
Aroma 4.70 ± 1.15a 4.63 ± 1.25a 4.63 ± 1.25a
Texture 4.00 ± 1.55a 4.67 ± 1.16a 4.50 ± 1.41a
Taste 4.23 ± 1.14a 4.43 ± 1.38a 4.20 ± 1.58a
Flavour 4.27 ± 1.29a 4.27 ± 1.53a 4.30 ± 1.60a
Total acceptance 4.53 ± 1.28a 4.77 ± 1.36a 4.53 ± 1.46a
Different letters indicate statistical differences at α = 0.05.

287
 Table 1 lists the average score for sensory evaluation with the highest mean score for all attributes at a sample
concentration of 0.76 w/w C. nutans. Nevertheless, there are no significant differences between each concentration
of C. nutans. These results might due to the low percentage concentration of C. nutans used in ‘Granny Smith’ green
apple juice.

Conclusion
C. nutans could significantly affect the anti-browning process on ‘Granny Smith’ apple juice. The concentration of C.
nutans and time had shown sensitivity to the browning process of apple juice. Increases of antioxidant properties with
a high concentration of C. nutans slow down the browning process of apple juices as time goes by. In addition, TPC
of C. nutans was 0.5014 mg GAE/g dried sample and it also shows the capability as antioxidant to scavenge DPPH
radical activity as detected through the color changed of DPPH radical. Eventually, the presence of C. nutans in
apple juice were accepted by consumers based on the sensory evaluation even though at different concentrations
level.

Acknowledgement
The authors gratefully acknowledge fundamental research graduate scheme grant from the Ministry of Education
Malaysia.

References
Lusia Barek, M., Hasmadi, M., Zaleha, A.Z. and Mohd Fadzelly, A.B. (2015), “Effect of different drying methods on
phytochemicals and antioxidant properties of unfermented and fermented teas from Sabah snake grass
(Clinacanthus nutans Lind.) leaves”, International Food Research Journal, Vol. 22 No. 2, pp. 661–670.
Picouet, P.A., Landl, A., Abadias, M., Castellari, M. and Viñas, I. (2009), “Minimal processing of a Granny Smith
apple purée by microwave heating”, Innovative Food Science and Emerging Technologies, Vol. 10 No. 4, pp.
545–550.
Roosita, K., Kusharto, C.M., Sekiyama, M., Fachrurozi, Y. and Ohtsuka, R. (2008), “Medicinal plants used by the
villagers of a Sundanese community in West Java, Indonesia”, Journal of Ethnopharmacology, Vol. 115 No. 1,
pp. 72–81.
Sakdarat, S., Shuyprom, A., Na, A.T.D., Waterman;, P.G. and Karagianis;, G. (2006), “Chemical composition
investigation of the Clinacanthus nutans Lindau leaves”, Thai Journal of Phytopharmacy, Vol. 13 No. 2, pp.
13–24.
Sakdarat, S., Shuyprom, A., Pientong, C., Ekalaksananan, T. and Thongchai, S. (2009), “Bioactive constituents from
the leaves of Clinacanthus nutans Lindau”, Bioorganic and Medicinal Chemistry, Elsevier Ltd, Vol. 17 No. 5,
pp. 1857–1860.
Shim, S.Y., Aziana, I. and Khoo, B.Y. (2013), “Perspective and insight on Clinacanthus nutans Lindau in traditional
medicine”, International Journal of Integrative Biology, Vol. 14 No. 1, pp. 7–9.
Tuntiwachwuttikul, P., Pootaeng-On, Y., Phansa, P. and Taylor, W.C. (2004), “Cerebrosides and a
monoacylmonogalactosylglycerol from Clinacanthus nutans.”, Chemical & Pharmaceutical Bulletin, Vol. 52
No. 1, pp. 27–32.

288
 IFRC 2017: 098-066 Functional Food

Prebiotic potential of oligosaccharides derived from Kappaphycus alvarezii using microwave-assisted

hydrolysis

Chan, S.T., *Chye, F.Y. and Siew, C.K.

Faculty of Food Science and Nutrition, Universiti Malaysia Sabah,Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia

The present study aimed to assess the prebiotic potential of Kappaphycus alvarezii var. tambalang, a commonly
cultivated seaweed in Malaysia. The seaweed polysaccharide was undergone microwave – assisted hydrolysis (500
– 800 W) prior to the ethanol precipitation yielding oligosaccharide fraction. The total sugar and reducing sugar
content, degree of polymerization (ADPn) and molecular weight (AMWn) of the oligosaccharides were determined. The
prebiotic effect of the oligosaccharide fractions was evaluated using four selected human origin probiotic strains,
Lactobacillus acidophilus LA-314 (LA314), Bifidobacterium longum (BB46), Lactobacillus plantarum 8014 (LP8014)
and Lactobacillus casei 334 (LC334). Furthermore, the amount of short chain fatty acid (SCFA) and lactic acid
released was assessed by high performance liquid chromatography (HPLC). The highest yield of hydrolyzed
oligosaccharide is obtained from 700 W for 10 minutes (12.48%) with ADPn: 7.48 and AMWn: 1,230.23 Da. Both
microwave hydrolyzed oligosaccharides at 700 W and 800 W for 10 min were able to enhance the growth of all
probiotic strains tested by at least 3 log CFU/g within 48 hours, which are significantly higher than other
oligosaccharide fractions. The prebiotic potential of these seaweed oligosaccharides is comparable to inulin
(commercial prebiotic). The SCFA production, particularly acetate and propionate has significant increased (p<0.05)
in the presence of seaweed oligosaccharide, indicating that they were readily fermented. In conclusion, the
oligosaccharides derived from K. alvarezii could be used as novel prebiotic for the development of functional foods.

Keywords: Tropical seaweed, microwave digestion, oligosaccharide, SCFA, functional foods

Corresponding author’s email: fychye@ums.edu.my

Introduction
Prebiotics are non-digestible components that could selectively stimulate the growth or activity of one or more
microbial species in the gut microbiota that confer(s) health benefits to the host (Rastall and Gibson, 2015). They are
mainly soluble oligosaccharides with 3-10 molecules monosaccharide sugars. Prebiotics are naturally found in fruits,
vegetables, oatmeal and wheat. Currently, identified prebiotics are lactulose, fructo-oligosaccharides (FOS), inulin,
galacto-oligosaccharides (GOS), oligofructose (OF), isomalto-oligosaccharides, fiber gums, lactiol, lactosucrose,
pyrodextrins, soligosaccharides, transgalacto-oligosaccharides and xylo-oligosaccharides (Sekhon & Jairath, 2010).
Recently, the uses of oligosaccharide from terrestrial plants as a source of prebiotics have gained much attention due
to the increasing awareness on health and demand of functional foods. Seaweeds contain significantly higher level of
dietary fibers (Zaporochets et al., 2014; Gupta and Abu-Ghannam, 2011) and rich in prebiotic-like oligosaccharides
that can potentially be incorporated into functional foods. There is emerging evidence to show that some seaweed-
derived compounds have positive effect on gut health as well as a source of prebiotic carbohydrate such as
alginates, fucoidan, laminarin and agar (Ramnani et al., 2012). Thus, the study aimed to evaluate the prebiotic
potential of seaweed-derived oligosaccharides using microwave assisted extraction method.

Materials and Methods

Sample Preparation
Fresh seaweeds were collected from east coast (Semporna) of Sabah, Malaysia. Samples were dried and made into
powder before subjected for further extraction of polysaccharide. Hot water extraction for polysaccharide was carried
out according to the method described by Zha et al. (2012) with slight modification.

289
Microwave treatment
Four different microwave settings (500, 600, 700 and 800 W for 10 minutes) were carried out on the seaweed
polysaccharides obtained and were sequentially precipitated using ethanol (Coelho et al., 2014). Freeze-dried
precipitated hydrolyzed oligosaccharides (HO) obtained were labeled correspond to the microwave power used for
hydrolysis (HO500, HO600, HO700 and HO800).

The total sugar and reducing sugar content was determined spectrophotometrically using phenol-sulphuric acid and
3,5-dinitrodslicyclic acid by the method of Wichienchot et al. (2010).

The degree of polymerization (dpn) was determined by [total carbohydrate (using the phenol sulfuric acid method
64)/the reducing value (DNS methods)] × 1.9. The number average molecular weight (MWn) was determined by (dpn
× 162) + 18 (McIyntyre et al., 2013).

Evaluation of seaweed oligosaccharide as a source of fermentable substrates for probiotic bacteria growth, was
carried out according to the method described by Gullon et al. (2015). Probiotic strains used for prebiotic activity were
Lactobacillus acidophilus LA-314 (LA314), Bifidobacterium longum (BB46), Lactobacillus plantarum 8014 (LP8014)
and Lactobacillus casei 334 (LC334)

Short chain fatty acids were determined using high performance liquid chromatography method (Gullon et al. 2015).

All data were expressed as mean ± standard deviation and analyses were done in triplicate. Data were analyzed
using one-way ANOVA using SPSS version 20 (IBM Corp, Armonk, NY, USA).

Results and Discussion

According to Table 1, it can be inferred that the higher the microwave power used for hydrolysis, the higher the yield
of hydrolyzed oligosaccharides obtained (0.78 - 12.48%). The highest yield was obtained from HO700 and HO800
(12.48%, 11.96%) in which higher than the reported pectic oligosaccharides obtained from enzymatically
depolymerized longan pulp (Thitiratsakul and Anprung, 2014) and Cempedak (Artocarpus integer Merr.) skin (Li et
al., 2016). Thus, it could be cost-effective to use seaweed as potential prebiotic comparable to these fruit by-product.

Both HO700 and HO800 were observed able to proliferate all probiotic strains tested. They were able to enhance the
growth by 3.36 – 3.85 log CFU/g within 48 hours, which is comparable to the commercial prebiotic (Inulin) (Figure 1).
Higher amount of readily available short chain oligosaccharides that could be easily fermented by the probiotics
which might be significant factor justifying the observed results (Ng et al., 2014). According to Rycroft et al., (2011),
production of SCFAs is an ensemble product of the activities of microflora present in the fermentation. Figure 2
shows the highest amount of short chain fatty acids (acetate: 184.58 mM; propionate: 24.84 mM; lactate: 550.58 mM)
obtained after 48 hours fermentation of HO700 with BB46. These results are in agreement with previous study
(Rycroft et al., 2012), SCFAs formed during batch fermentations with bifidobacteria which that is generated via the
bifidus pathway. Hence, both of these hydrolyzed oligosaccharides have the potential to be use as prebiotics.
As anticipated, DP has an impact on effectiveness as a prebiotic which a low degree of polymerization of
oligosaccharides offers better stimulation of probiotic bacteria. According to Table 1, oligosaccharides obtained from
HO700 and HO800 both show ADPn of 7.48 (AMWn: 1,230.23) and 7.24 (AMWn: 1,190.44) respectively. This is
favorable as the production rate of the health beneficial short chain fatty acids (SCFAs) by the bifidobacteria and
lactobacilli in the intestine is rapid for shorter chain oligosaccharides (Slavin, 2013).

290
 Table 1: Extraction yields, total sugar and reducing sugar content of various 80% precipitated hydrolyzed seaweed
samples with different microwave power (W).
Power Yield* (%) Total sugar** Reducing sugar** Degree of Average of molecular
(W) (mg/g) (mg/g) polymerization1 weight (AMWn)2

500 0.78 ± 0.15c 869.91 ± 7.10c 118.53 ± 1.10c 13.94 ± 0.35c 2,277.01 ± 13.38c
600 2.89 ± 0.05b 947.17 ± 34.35b 196.38 ± 3.07b 9.16 ± 0.18b 1,502.54± 28.59b
700 12.84 ± 0.84a 961.70 ± 29.79a 245.43 ± 7.99ab 7.48 ± 0.47a 1,230.23 ± 76.84a
800 11.96 ± 0.39a 965.62 ± 80.58a 252.39 ± 2.15ab 7.24 ± 0.54a 1,190.44 ± 88.28a

Inulin 977.17 ± 51.97a 264.13 ± 6.76a 7.04 ± 0.55a 1,157.88 ± 89.75a

Values are given as mean ± standard deviation from triplicate determinations. Different superscripted letters in the
same column indicate significantly different (p≤0.05).
*Yield determined as percent of dry weight.
**Total sugar and reducing content are expressed as galactose equivalent (mg/g, dry weight basis).
1Degree of polymerization = number average degree of polymerization = (total carbohydrate/the reducing value) x 1.9
2Number average molecular weight = (dp x 162)+18
n

Figure 1: Net growth of LP8014, LC334, LA314 and BB46 in basal media supplemented with various hydrolyzed
oligosaccharides after 48 hours of fermentation. Values are given as mean ± standard deviation from triplicate
determinations. Different letters indicate significant differences.

Lactate

Acetate

Propionate

Figure 2: Chromatogram showing the detected short chain fatty acids in the fermentation medium of Bifidobacterium
longum (BB46) with 1% hydrolyzed oligosaccharides (700 W for 10 min).

291
Conclusion
This work has shown that microwave assisted extraction method was effective in producing oligosaccharides from K.
alvarezii with microwave power at 700 and 800 W for 10 minutes. Therefore, it could be applied as ingredients in the
development of symbiotic products or functional foods. However, selection of operating parameters in optimizing the
microwave-assisted hydrolysis is still going on based on it prebiotic activity.

Acknowledgements
The author would like to thank Ministry of Higher Education for the provision of research grant under ERGS0039-
STWN-1-2013 and MyBrain 15 (MyMaster) scholarship.

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In vitro fermentation and prebiotic potential of novel low molecular weight polysaccharides derived from agar
and alginate seaweeds. Anaerobe 18:1-6.
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and promote intestinal health. Current Opinion in Biotechnology 32: 42-46.
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Thitiratsakul, B. and Anprung, P. 2014. Prebiotic activity score and bioactive compounds in longan (Dimocarpus
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prebiotic properties. Food Chemistry 120(3): 850-857.
Zaporozhets, T.S., Besednova, N.N., Kuznetsova, T.A., Zvyagintseva, T.N., Maarenko, I.D., Kryzhanovsky, S.P., &
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 IFRC 2017: 102-067 Functional Food

Antioxidant and Metabolite Identification of Different Varieties of Dates (Phoenix dactylifera L.)
Hana Kaduma, Azizah Abdul Hamida,*, Faridah Abasab, Abdul Karim Sabo Mohammeda,
Nurul Shazini Ramlia
a Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia
bInstitute of Bioscience, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia.
Date fruits are important for both nutritional and therapeutic properties. They are rich sources of sugars, vitamins,
minerals, fibres and various phytochemicals. The aim of this study is to determine the antioxidant activity and
metabolite profiling of 5 different dates (Phoenix dactylifera L.) varieties including Ajwa, Mariami, Piyarom, Tunisia,
and Anbar. The antioxidant activity of the dates was measured using DPPH, FRAP assay systems. Proton nuclear
magnetic resonance (1H NMR) was used for metabolite profiling. Results of the study revealed that Mariami extract
consisted of the highest total phenolic content (355 mg GAE/g DW), while the most potent DPPH scavenging activity
(IC50 of 16.2 µg/mL) and FRAP (18.38 Mm Fe (II)/g) was demonstrated by Piyarom variety. A discriminatory study on
metabolites responsible for the variation between the samples was successfully performed using 1H-NMR-based
metabolomics. The principal component analysis plots exhibited clear and distinct separations between the dates
studied. The metabolites identified that may attribute to the separation were sucrose, succinic acid, leucine,
glutamine and lysine. The results suggest that all the studied dates serve as a good source of vital nutrients and can
be considered as a premium quality having significantly higher antioxidant activity.
Keywords: Antioxidant activity, Date palm, DPPH, FRAP, Metabolite profiling.
*Corresponding author’s email: azizahah@upm.edu.my
Introduction
In the last few years, the metabolism of oxygen in humans has been investigated by biochemists all over the world. It
is well known that an excess of oxygen leads to the formation of reactive oxygen species, responsible for oxidative
stress in the tissues. Antioxidants have been widely used in different fields of industry and medicine as substances
which interrupt radical-chain oxidation processes, improve general health, help cell rejuvenation, and prevent
(Ghiaba et al., 2013).Moreover, recent Studies have shown that date fruits are an excellent source of phenolic and
therefore possess an extremely high antioxidant capacity. Dates have potent anthocyanins, carotenoids, and
phenolic compounds (protocaechuic, p-hydroxy benzoic, vanillic, syringic, caffeic, coumaric, ferulic, hydroxy benzoic,
mainly cinnamic acids) and flavonoids (flavones, flavonols and flavanones). The Fruit is known as vital to equilibrate
our diet to avert several diseases (Al Harthi et al., 2015).
Metabolomics is a technique being used to discover thousands of non-nutrient constituents which although not
necessary for human body to support life but may affect human health and well-being. It is an approach normally
associated with analytical techniques. Among the analytical platforms being used for metabolomics research, 1H
NMR-based metabolomics is most widely used approach, as it is highly reproducible and non-destructive (Kim et al .,
2010). The Identification of the metabolites is still evolving within the scientific community. Most of the metabolites
reported in metabolomics literature are based on spectral similarity with public/commercial spectral databases
(Goodacre et al., 2007). The present investigation was carried out to evaluate the antioxidant activity of five date
varieties normally consumed in Malaysia .An attempt was also made to identify the main constituents responsible for
the antioxidant activities of date extract 1H NMR.
Materials and Methods
Materials
Five commercially available date fruits, that is, Tunisia imported from Tunis, Ajwa and Anbar imported from Saudi
Arabia, and Piyarom, Mariami imported from Iran, were purchased from a local source in Kuala Lumpur, Malaysia.
The moisture content of cultivars was variable: Tunisia (12.7%), Ajwa (13.4%), Anbar (15.1%), Piyarom (11.8.4%),
and Mariami (14.6%).

293
Sample Preparation
The dates were collected, seeds removed, crushed and cut into small pieces and dried at 40ºC until it reached a
constant weight. They were then blended, for three minutes and extracted with 80% ethanol. The samples were stirred
for 24 h at 20ºC using magnetic stirrer. Extracts were filtered and concentrated using rotary evaporator at 40ºC for (3-
4) hours, and then freeze dried until constant weight (72 hr.) and kept at – 80 °C for further experiments.
Total Phenolic Content.
Determination of TPC was done based on a modified Folin-Ciocalteau colorimetric procedure(Al-Farsi et al.,
2005).Briefly, 0.5 mL of date extract was mixed with Folin-Ciocalteau (0.5 mL) and 7 % sodium carbonate (10 mL) and
permitted to react in the dark for 1 hr. The resulted blue complex was then subjected to absorbance measurement at
725 nm. Gallic acid was used to draw the standard curve with concentration range 0-100 μg /mL and TPC content is
measured.
DPPH Free Radical Scavenging Assay
The antioxidant activity of the date extracts was determined based on their potential to scavenge 2, 2-diphenyl-1-
picrylhydrazyl radicals (DPPH) using a modified procedure Hatano et al. (1988). Briefly, 0.20 mL of different ethanol
date extracts (0.1, 0.2, 0.4, 0.6 ,0.8 and 1 mg/ml) were dissolved in methanol, mixed with 1.80 mL of DPPH reagent (6
× 10-5mol) in a 12 55 well plate and allowed to react at room temperature for 30 min in the dark. The absorbance was
then measured with Elisa plate reader (Biotek, EL 800) at 515 nm. BHA and α-tocopherol were used as positive
controls for synthetic and natural antioxidants, respectively. Percent inhibition is calculated as follows:
% Inhibition = control absorbance − sample absorbance / control absorbance × 1.
The graph of concentration (mg/mL) was a plot against percentage inhibition to determine the concentration of ML
extract required to inhibit 50% DPPH radical scavenging and the results were reported as IC50 (µg/mL).
Ferric Reducing Antioxidant Power (FRAP).
FRAP assay was used for the estimation of ferric reducing activity of the understudy date extract (Benzie & Strain,
1996). Fresh FRAP reagent was prepared by mixing FeCl3, 2, 4, 6-tripyridyl-s-triazine solution and acetate buffer (pH
3.6) in 1:1:10 (v/v/v) proportions. Ten μL methanol sample was taken into a micro-plate consisted of 96 wells. FRAP
reagent (200 μL) was incubated at 37°C for about half an hour and was placed in the same well along with the sample.
The solution is thoroughly mixed, incubated for 30 min at room temperature and absorbance was measured at 593 nm.
A standard curve was prepared using ferrous sulphate solution (FeSO4.7H2O) with concentrations ranging from 0.1 to
1 mM.
1H NMR Measurement and Data Analysis
Date extracts (10 mg) were mixed with 0.375 mL of CH3OH-d4 without any internal standard along with 0.375 mL of
KH2PO4 buffer in D2O (pH 6 adjusted with NaOD) containing 0.1 % TSP. Six full technical replicated were examined
for each sample. The mixture was vortexed for 1 minute and ultra-sonicated for 15 minutes at 30 0C. The solution was
then centrifuged at 13000 rpm for l0 minutes and 600 μL of the supernatant was transferred to NMR tube for 1HNMR
analysis (Mediani et al ., 2012). Spectra were recorded at 26 0C on a Varian Unity INOVA 500 MHz spectrometer
(Varian Inc, CA), with a frequency of 499.887 MHz. For each sample, 64 scans were recorded with an acquisition time
of 193 s, pulse width of 3.75 μs and a relaxation delay of 1.0 s. TMS was used an internal standard and all spectra
were manually phased and bucketed using Chenomyx software, with standard bins of δ 0.05 from the region of δ0.50
to δ10.00. The water region (δ4.70 – 4.96) and residual ethanol region (δ 3.28-3.33) were excluded from the analysis
(5.1, Alberta, Canada). Identification of compounds was done using two-dimensional 1H–1H J-resolved. Principal
component analysis and partial least-squares analysis were performed using the SIMCA-P software (Umetrics,
Sweden).
Statistical analysis
Data was analyzed using MINITAB version 16. Statistical differences between the samples and the controls were
evaluated by one-way analysis of variance (ANOVA) test. Results are expressed as the mean of three determinations
± standard deviation (SD). A difference in the mean values of p < 0.05 was considered to be statistically significant.

294
 Results and Discussion
Total phenolic content
Results showed that TPC of the five dates varied from 34.07to 45.4mg GAE/g DW (Figure 1). The highest TPC was
observed in Mariani whereas the lowest was found in Anbar. Significant differences (𝑝 ≤ 0.05) among the various
date types were noticed except for varieties Tunisia and Anbar. The present results were much higher compared to
that reported by Zineb Ghiaba et al. (2013), indicating the possible effect of location, weather, and agricultural
practices.

Figure 1. TPC of different varieties of date extracts.

Means of different letters are significantly different at p < 0.05; data is presented as mean ± SD of triplicates
Antioxidant Activity of Date Fruit
The results showed that the antioxidant activity of the different date varieties varied to a large extent. DPPH assay
revealed the IC50 of antioxidant values in the range of 5.4 to 20.9 𝜇g/mL (Figure2). Piyarom exhibited the highest
antioxidant activity that is significantly different compared to the other date varieties. Antioxidant activity assayed by
FRAP showed that values varied from 11.93 to 18.38 mM Fe (II)/g DW (Figure3). Similar to DPPH activity, Piyarom
exhibited the highest activity while Tunisia showed the lowest activity.

Figure 2. DPPH scavenging activity of different varieties of Figure 3. FRAP value of different varieties of date
date Extracts Means of different letters are significantly different
Extracts Means of different letters are significantly different At P < 0.05; data are presented as mean ± SD of triplicates.
At P < 0.05; data is presented as mean ± SD Of triplicate
1H NMR Spectra and metabolites identification
In the current study, 1H NMR was used to explore the metabolite profile of different date varieties of ethanol extracts.
1H NMR spectra of the different dates are shown in (Figure 4). The metabolite signals are shown in the entire region:

sugar region (δ 3.0-5.5), and aromatic region (δ 5.5-9.0). The five-different date ethanol extract’s spectra showed clear
variation in the intensity of the signals, particularly those in the aromatic region. It was observed that Tunisia and
Piyarom showed highest intensity spectra in sugar region.

Figure 4. A representative of 1H-NMR spectra of the different varieties of ethanol date extracts.

295
 Multivariate analysis of date extracts.
In the present study, Principal Component Analysis (PCA) score plot (R2 = 0.817 and Q2 =0.808) was performed to
evaluate the variation within the samples (Figure 5). The metabolite profiles of the various ethanol extracts of date
varieties were clearly separated by both principal components, PC1 showed the major sample variation i.e., 81.7 %
and clear separation were observed between Tunisia from other date ethanol extracts. The loading scatters plots
also depicted metabolic variations, attributed to the separation between Tunisia and other for four date ethanol
extracts as shown in (Figure 6). Moreover, the separation related to the six main compounds identified in five date
extract clearly separated the Tunisia from others. Results revealed that sucrose and betaine compounds were found
in the Tunisia, while fructose, ascorbic acid, isoleucine and arginine were found in the other four dates.

Figure 5. The PCA score plot (pc1, Figure 6. The PLS loading column plots of different
pc2) varieties of date fruit
5- Isoleucine 6- Arginine 4- Fructose 3- Ascorbic acid
1-Sucrose 2- Betaine
Conclusion
In the present study, functional properties and metabolites profile of various ethanolic extracts of dates was
evaluated. All the dates showed potent antioxidant activities. The PCA score plots showed clear separation for the
five date ethanol extracts. Sucrose, betaine, ascorbic acid, fructose, isoleucine and arginine were depicted to be the
main constituents of the date extracts that are responsible for the separation.

Reference
Al-Farsi, M., Alasalvar, C., Morris, A., Baron, M., & Shahidi, F. (2005). Compositional and sensory characteristics of
three native fresh and sun-dried date (Phoenix dactylifera L.) varieties grown in Oman. Journal of Agricultural
and Food Chemistry, 53(19), 7586–7591.
Al Harthi, S. S., Mavazhe, A., Al Mahroqi, H., & Khan, S. A. (2015). Quantification of phenolic compounds, evaluation
of physicochemical properties and antioxidant activity of four date (Phoenix dactylifera L.) varieties of Oman.
Journal of Taibah University Medical Sciences, 10(3), 346–352.
Benzie, I. F. F., & Strain, J. J. (1996). The Ferric Reducing Ability of Plasma ( FRAP ) as a Measure of “‘ Antioxidant
Power ’”: The FRAP Assay, 76, 70–76.
Ghiaba, Z., Yousfi, M., Hadjadj, M., Saidi, M., & Dakmouche, M. (2013). Study of antioxidant properties of five
algerian date (phoenix dactylifera l) cultivars by cyclic voltammetric technique. International Journal of
Electrochemical Science, 9(2014), 909–920.
Goodacre, R., Broadhurst, Æ. D., Smilde, Æ. A. K., Kristal, Æ. B. S., Baker, J. D., Beger, Æ. R., … Sjo, M. (2007).
Proposed minimum reporting standards for data analysis in metabolomics, 231–241.
Kim, H. K., Choi, Y. H., & Verpoorte, R. (2010). NMR-based metabolomic analysis of plants.
Mediani, A., Abas, F., & Ping, T. C. (2012). Influence of Growth Stage and Season on the Antioxidant Constituents of
Cosmos caudatus, 344–350. https://doi.org/10.1007/s11130-
Tapas, A. R., A. M. Sakarkar, and R. B. Kakde. 2008. Flavonoids as nutraceuticals: a review. Trop. J. Pharm. Res.
7:1089–1099.
Cho, Hyun-Woo, Seoung Bum Kim, Myong K. Jeong, Youngja Park, Nana Miller, Thomas Ziegler, and Dean Jones.
2008. "Discovery of metabolite features for the modelling and analysis of high-resolution NMR
spectra." International journal of data mining and bioinformatics no. 2 (2): 176-192.

296
 IFRC 2017: 106-069 Functional Food

Effectivity of Ethanol Extract of Purple Sweet Potato Var. Ayamurasaki as Natural Antihypertension in Doca-
Salt Hypertensive Rats

Irma Sarita Rahmawati1, Soetjipto2, Annis Catur Adi3, Aulanni’am4

1Health Science Graduate Program, Faculty of Public Health, Airlangga University, Surabaya 60115, Indonesia
2 Department Biochemistry, Faculty of Medical, Airlangga University,Surabay, 60115, Indonesia
3Department Nutrition, Faculty of Public Health, Airlangga University, Surabaya 60115, Indonesia
4Biochemistry laboratory, Faculty of Life Sciences, Brawijaya University, Malang, 65145, Indonesia.
Abstract

There is an increasing amount of evidence that oxidative stress related to hypertension and damages the function of
aorta and kidney. It is a well-established fact that chlorogenic acid and anthocyanine in purple sweet potato
generates bioactive compound with antihypertensive activities. The present study sought to investigate blood
pressure lowering effect of extract ethanol of purple sweet potato (EP) in Deoxycorticosterone acetate (DOCA)–salt
induced hypertensive rats (Rattus norvegicus). Extraction of purple sweet potato with ethanol 95% used for lowering
blood pressure therapy. The rats were orally administrated with extract ethanol of purple sweet potato (EP) in daily
dose of 100 and 200 mg/kg body weight for 4 weeks. Systole blood pressure (SBP) was examined. Mean of SBP
were lowered in the hypertensive rats following EP therapy from their respective values observed at the time of
administration. This is the first report that demonstrated blood pressure lowering effects of extract ethanol of purple
sweet potato (EP) in DOCA–salt model of hypertension.

Keywords: extract ethanol of purple sweet potato (EP), DOCA–salt, chlorogenic acid, antihypertensive
*Corresponding author’s email: irmas86@gmail.com
Introduction
Hypertension is a major cause of mortality because of its association with cardiovascular disease,cerebrovascular
disease and renal disease. It has a prevalence of 26.4% in the adult population, totaling nearly one billion individuals
and has been estimated to increase up to 29% (1.5 billion) by the year 2025 (Rubattu et al., 2015). The renin-
angiotensin aldosterone system (RAAS) is a hormonal cascade that has funtions in pathogenesis of cardiovascular
diseases. Angiotensin II, a potent vasoconstrictor is the primary active product of the RAAS that play the role in the
development of hypertension (Atlas, 2007). Hypertension also has been associated with stress oxidative which is
resulted from an imbalance between the production of reactive oxygen species (ROS) and the antioxidant defense
system (Vaziri, 2008). In this case, ROS production by NADPH oxidase is increased causes vascular disease and
dysfunction. ROS production in other organs, particularly the kidney likely contribute to blood pressure regulation
(Harrison et al., 2007). DOCA salt induced is an endocrine hypertension model that progress quickly severe
hypertension and oxidative stress (Dornas and Silva, 2011) thus allowing an understanding progression of the
disease and potential use of the extract ethanol of purple sweet potato derived chlorogenic acid as therapeutic agent.

Purple sweet potato is known to have several advantages more than other sweet potatoes. It contain anthocyanine,
dioscorin protein, and chlorogenic acid that functioned with antihypertensive and antioxidative activity. Chlorogenic
acid can inhibit the angiotensin converting enzyme (ACE) that have an important role inconverting angiotensin-I to
angiotensin-II by blocking the active site of the enzyme (Yashimoto et al., 1999). Angiotensin-II is both a potent
vasoconstrictor and stimulator for the synthesis and release of aldosterone which subsequently increases blood
pressure by promoting sodium retention in the distal tubules (Ferrario, 2010). Given the above considerations, the
inhibition of ACE could be useful in the treatment of hypertension (Ramos-Nino and Blumen, 2009). Hence,
antioxidants is considered a useful therapeutic approach in the treatment of high blood presure. To understand dose

297
dependent effects of extract ethanol of purple sweet potato derived chlorogenic acid treatment on systole blood
pressure (SBP), we used DOCA-salt induced hypertension rats (Rattus norvegicus) strains Wistar as animal model.
The current study demonstrated the decrease of SBP after a single oral administration of extract ethanol purple
sweet potato derived to DOCA-salt induced hypertension rats.

Experimental

Chemicals

Purple sweet potatoes var. Ayamurasaki, ethanol 95%, Deoxycorticosterone acetate (DOCA) salt (Sigma, Pcode
1001376001, USA), NaCl, corn oil (Sigma, Pcode 1000925370 C8726-500 ml).

Animal model

All procedures were carried out in accordance with conventional guidelines for experimentation with animals. Twelve-
week old male rats (Rattus norvegicus) strains Wistar were used. The rats were divided into 5 groups, i.e
normotensive-NTN (A), hypertensive-HTN (B), HTN + extract ethanol (EP) ½ standard dose of 100 mg/kg b/w (C),
HTN + extract ethanol (EP) standard dose of 200 mg/kg b/w (D), and were housed in groups of five per cage, as a
number for replication, in a regulated environment with a 12 h light/dark cycle. Hypertensive rats were prepared by
induction of deoxycorticosterone acetate (DOCA), twice a week for 5 weeks (10 injections). DOCA was injected
subcutaneously in the cervical spine with a dose of 20 mg/kg for 5 times, followed by a dose of 10 mg/ kg for 5 times
and were given 1% NaCl in drinking water. DOCA was dissolved in 0.5 ml corn oil (Badyal et al., 2003) (Khorsid et
al., 2012). The treated hypertensive rats received, by oral administration, using a canula, a daily dose extract ethanol
of purple sweet potato (EP) in daily dose of 200 and 100 mg/kg body weight dissolved in reverse osmosis water for 4
consecutive weeks. The control group received a normal diet.

Preparation extract ethanol of purple sweet potatoes (EP)

Purple sweet potatoes sorted and weighted then washed by clean water. After that, purple sweet potato and ethanol
95% with ratio 1:8 (v/v) sliced in small size and blended during 30s. The solution screened and maserated 2x12
hours. After that, the solution screened again by vacuum screening and whatmann paper@40 untill get the filtrate for
evaporating and their residue extracting again untill 4 times. The final filtrate is the result of extraction ethanol of
purple sweet potato (EP).

Measurement of Systolic blood pressure

Systolic blood pressure (SBP) was measured in awake rats using the tail-cuff method with a photoelectric sensor
(blood pressure analyzer, IITC, Model 179, Woodland Hills-USA) as previously described (del Mar Contreras et al.,
2009). SBP was measured weekly before DOCA induction, post induction and also up to 4 weeks post-administration
of extract ethanol of purple sweet potato (EP). The average of at least three readings, taken in a quiescent state.

Statistical Analysis

The results of systole blood pressure (SBP) were expressed as means ± standard deviation (SD). Differences
between trial groups were statistically analysed using analysis of variance (ANOVA), followed by the post hoc Tukey
test for determining significant difference at p < 0.05.

Results and Discussion

Effect of extract ethanol purple sweet potato (EP) on Systolic Blood Pressure (SBP)

Induction of DOCA-salt 2 times weekly for 5 weeks at the dose of 20 mg/kg bb (5x injections) followed by 10 mg/kg
bb (5x injections) produced a gradual elevation of systolic BP (Figure 1).

298
 HTN +EP 100 HTN +EP 200

Figure 1. Bar graph showing systole blood pressure (SBP) in normotensive Wistar rats (NTN), DOCA-salt induced
hypertensive rats (HTN), 100 mg extract ethanol sweet potato (EP) - treated HTN (HTN+EP 100), 200 mg extract
ethanol sweet potato (EP) – treated HTN (HTN+EP 200). Systolic blood pressure was determined by indirect tail-cuff
method. Values not sharing a common superscript indicate statistical significance at p<0.05.

The mean systolic BP of DOCA-salt induced rats (HTN) was significantly higher (201, 25 ± 2, 06 mmHg) in
comparison to normotensive rats (NTN) in basal conditions (108, 00 ± 2, 94). In contrast, the SBP of HTN rats
treated with extract ethanol of purple sweet potato significantly decreased after the administration of extract
ethanolpurple sweet potatoes in a daily dose of 100 mg/kg bw and 200 mg/kgbw. The final SBP at the end of
experiment (week 10) were 144, 00 ± 2, 45 mmHg and 152, 75 ± 2, 36 mmHg, respectively. Treatment of HTN rats
with extract ethanol purple sweet potatoes at the dose of 200 mg/kgbw gave better antihypertensive effect. In this
study, extract ethanol of purple sweet potato var. Ayamurasaki showed blood pressure lowering effect and caused
major changes of the SBP in the treated DOCA-salt hypertensive rats for more than 50 mmHg. In contrast to the
present study, the blood pressure lowering effect of the extract ethanol of purple sweet potato var. Ayamurasaki was
lower (15 mmHg). This effect mainly due to activity of angiotensin converting enzyme inhibitor (ACEI) chlorogenic
acid in extract ethanol of purple sweet potato var. Ayamurasaki. In our study, induction of the DOCA-salt lead to
reabsorption of sodium and water that increased circulating blood volume and resulted in elevated of blood pressure
(Sharma et al., 2010).

Conclusion

Assessment of the antihypertension properties of extract ethanol purple sweet potato derived chlorogenic acid in the
present study showed a significant effect in reducing systole blood pressure in DOCA-salt hypertensive model. These
findings have suggested that extract ethanol purple sweet potato derived chlorogenic acid might be considered as
potential antihypertension that resistant to digestive proteases. However, further investigation is needed to evaluate
bioavailability and efficacy of extract ethanol purple sweet potato derived chlorogenic acid after GI digestion, which
was significant for the preparation of nutraceutical food component.

299
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Manag. Care. Pharm., 2007, 13(8) (suppl S-b):S9-S20.
Badyal, D.K., H. Lata and A. P. Dadhich. Animal models of hypertension and effect of drugs. Indian J Pharmacol.
2003, 35: 349-362.
del Mar Contreras, M., R. Carro´n, M. J. Montero, M. Ramos and I. Recio. Novel extract ethanolpurple sweet
potatoes-derived peptides with antihypertensive activity. J. Intern. Dairy, 2009, 19: 566–573.
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Harrison, DG. Gongora MC., Guzik TJ., & Widder J., Oxidative stress and hypertension. J. Am.Society of Hyperten.
2007, 1(1): 30–44
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potato (Ipomoae batatas) root. Biosci. Biotech. Biochem. 1999. 63:541-543

300
 IFRC 2017: 110-070 Functional Food

Chemical composition and physicochemical properties of red seaweeds (Kappaphycus Alvarezii, Eucheuma
spinosum and Eucheuma striatum) from Sabah, Malaysia
1*Mohd Subakir, F.N., 1Wan Ishak, W.M.F., 2Mohd Azman, N.A., and 3Ibrahim, A.I.

1Faculty of Industrial Science and Technology, Universiti Malaysia Pahang, 26300 Gambang, Pahang, Malaysia
2Faculty of Chemical and Natural Resources Engineering, Universiti Malaysia Pahang, 26300 Gambang, Pahang,
Malaysia
3Malaysian Agricultural Research and Development Institute (MARDI), 43400 Serdang, Selangor, Malaysia

Three tropical red seaweeds taken from Kunak, Sabah namely Kappaphycus alvarezii (KA), Eucheuma spinosum
(ESp) and Eucheuma striatum (ESt) contains an important source of bioactive compounds for development of
functional foods. The present study was aimed to investigate the nutrient and physicochemical properties of these
red seaweeds. It was found that KA contained the highest level of protein and crude fibre with 3.72% and 12.5%,
DW respectively. Nutrients content showed significant different among the 3 seaweed species, with value of 0.02-
0.05% DW in lipid, ash (14.1-20.2% DW), energy (288-303 kcal) and carbohydrate (70.2-71.8% DW) (p<.05).
Comparing the element contents of these species, KA was rich in K, Mn, Fe and Cu, while ESp was rich in Mg, Ca,
Na and Zn. As for the physicochemical properties of the seaweeds, their swelling capacity (SWC), water retention
capacity (WRC), and oil retention capacity (ORC) ranged from 20.6 to 34.1 ml/g DW, 8.4 to 13.4 g/g DW and 2.3 to
3.0 g/g DW, respectively. SWC and ORC of KA was higher than those of ESp and ESt (p<0.05). This study
suggested that KA showed high nutritional value could be potentially used as ingredients in human food application.

Keywords: red seaweed, proximate composition, physicochemical properties

*Corresponding author’s email: farahnur@mardi.gov.my

Introduction

Seaweed, one of marine macroalgae is widespread around the world either cultivated or existed in wild. They are
categorized according to their colour pigmentation including red (Rhodophyta, brown (Phaeophyta) and green
(Chlorophyta). In Asian countries, seaweeds have become as a source for human food, animal feed, herbal
remedies, fertilizer, fungicides, herbicides and as well as a source of pharmaceutical ingredients (McHugh, 2003).
Many researchers had stated that seaweeds are nutritive food which contain vitamin, protein, mineral, fiber contents,
polyunsaturated fatty acids, essential fatty acids as well as macro and trace elements which are beneficial to human
and animal (Ortiz et al. 2006; Fleurence, 1999). Seaweeds also source of phycocolloids such as agar, alginate, and
carrageenan for various industrial applications (Alberto et.al, 2011; Mabeau et.al, 1993). Since seaweed has
increasingly become an economically important natural resource in Malaysia, seaweed farming has played an
important role in the development of aquaculture sector in Sabah but the information on their mineral and nutritional
composition is still limited and rather fragment. Therefore the present study aimed to determine the proximate
composition, mineral composition and physicochemical properties of red algae Kappaphycus alvarezii, Eucheuma
spinosum and Eucheuma striatum collected from the Kunak, Sabah.

Materials and methods

Sample preparation
The three seaweeds sample (K. alvarezii, E. spinosum and E. striatum) were collected from Kunak, Sabah.The
seaweeds were thoroughly cleaned and dried at 50°C in oven overnight. Then they were ground into fine powder
using a tabletop grinding machine.

301
Proximate composition and mineral elements analysis
The analyses were carried out in triplicate. The proximate compositions (moisture content, crude protein, ash, fat,
crude fibre, carbohydrate and gross energy) of the powder samples were determined according to the standard
method (AOAC, 2000). Major mineral elements (Ca, Mg, Na, K) and trace elements (Fe, Zn, Cu and Mn) were
determined by inductive coupled plasma mass spectroscopy (Agilent 7500a series), in house method based on
AOAC 999.10.

Physicochemical properties analysis

Swelling capacity seaweed samples was measured by the bed volume technique (Kuniak and Marchessault, 1972).
Water rentention and oil retention capacity of seaweed samples were determined following the method conducted by
(Ang, 1991). The results of SWC, WRC, and ORC were expressed as ml/g DW, g/g DW and g/g DW, respectively.

Statistical analysis
All the data collected were subjected to statistical analysis with ANOVA and any difference between the treatments
means was performed by Duncan Multiple Test using SAS 9.1 at P<0.05.

Results and discussion

The proximate composition of moisture content, ash, protein, fat, crude fiber energy and carbohydrate in K. alvarezii,
E. spinosum and E. striatum is shown in Table 1. From results, the crude fiber content from K. alvarezii is higher than
E. spinosum and E.striatum. As for carbohydrate, there was a significant difference among seaweeds samples with
highest value from E. spinosum, followed by K. alvarezii and E. striatum. High content of carbohydrate in red algae
might be due to their phycocolloid content in their cell walls (Kasimala et.al, 2015). The ash content in both K.
alvarezii and E.spinosum species were similar to each other with 14.10 – 14.90%, and there was no significant
difference among them. However, the ash content in E. striatum is higher (20.21% DW) than other two species High
amount of ash in seaweed is associated to its ability to absorb minerals and trace elements from its surrounding and
because of this, some seaweeds species are used as treatment on heavy metal in water sources (Peña-Rodríguez
et.al, 2011). From the analysis, the crude fiber content from K. alvarezii is slightly higher than E. spinosum with
12.50% DW and 12.10 % DW respectively. It is due to their high content of polysaccharides.

The mineral contents of K.alvarezii, E.spinosum and E.striatum are shown in Table 2. Among the macro minerals, K,
Mg and Na were the most abundant in these seaweeds. The results showed that K.alvarezii contained high quantity
of K followed by Mg, Na and Ca, whereas Fe, Mn, Zn and Cu are present in minor quantities. Similar results were
reported on high K content than Na in red seaweed samples (Benjama & Masniyom, 2012).

Table 1. Proximate composition of Kappaphycus alvarezii, Eecheuma spinosum and Eucheuma striatum (% dry
weight of sample)
Seaweeds K.alvarezii E. spinosum E. striatum
Moisture 10.29+0.03a 9.19+0.01b 7.93+0.01c
Protein 3.73+0.01 a 3.20+0.04 b 1.56+0.01c
Ash 14.10+0.07c 14.90±0.00b 20.21+0.01a
Fat 0.05+0.00a 0.06+0.00a 0.02+0.01b
Crude Fibre 12.50+0.07a 8.10+0.07c 12.10+0.14b
Energy (kcal) 303+0.71 a 304 ±0.00a 288+4.24b
Carbohydrate 71.83+0.01b 72.65+0.04a 70.28+0.04c
abc mean value with different superscript within the same column is different (P<.05)

302
 Table 2. Selected mineral content (mg/100 g dry weight of sample) of Kappaphycus alvarezii, Eucheuma spinosum
and Eucheuma striatum collected from east coast of Sabah, Malaysia..
Seaweeds K.alvarezii E. spinosum E. striatum
Elements (mg/100g DW)
Na 109.19±32.31c 311.87±14.28a 266.71±2.63b
K 6993.24±28.03a 4109.22±108.63b 4148.56±22.98b
Mg 231.30±2.31 c 299.46±11.31 a 271.94±0.16b
Ca 186.35±2.35b 202.65±6.44a 195.91±1.07ab
Mn 1.09±0.01a 0.29±0.00c 0.57±0.01b
Fe 50.49±0.49 a 4.73±0.14 c 7.53±0.07b
Cu 0.02±0.00a 0.01±0.00b 0.01±0.00b
Zn 0.68±0.02b 1.40±0.01a 0.63±0.01b
Na/K ratio 0.02 0.08 0.06
abc mean value with different superscript within the same column is different (P<.05)

Table 3. The swelling, water and oil retention capacity of K.alvarezii, E. spinosum and E. striatum

Seaweeds SWC (ml/g DW)* WRC (g/g DW) ORC (g/g DW)
K. alvarezii 34.19 + 0.72a 10.03 + 0.09ab 3.07 + 0.11a
E. spinosum 20.63 + 0.38 c 13.43 + 2.57a 2.34 + 0.22b
E. striatum 22.17 + 0.15 b 8.45 + 0.09b 2.84 + 0.06ab
abc mean value with different superscript within the same column is different (P<.05)

Table 3 showed the physicochemical evaluation on those three red seaweed species. The SWC of the seaweeds
were significantly different with K.alvarezii was the highest (34.19ml/g DW) than E. striatum (22.17ml/g DW) and
followed by E. spinosum (20.63ml/g DW). Water retention capacity (WRC) of E.spinosum was the highest followed by
K. alvarezii and E. striatum with 13.43, 10.03 and 8.45 g/g DW respectively. SWC and WRC properties of seaweeds
are related to their polysaccharide characteristics which indicated that this type of seaweeds may be used as
functional ingredient that will contribute in improving physical properties of food products. In this study, ORC value
was presented high in K. alvarezii followed by E. striatum and E. spinosum. The high ORC reported in this study
suggested that its ability to stabilize food emulsion. Therefore, this seaweeds could be a good alternative as
stabilizers in formulate food products and probably in animal feed pellet production.

Conclusion

From results, it was found that K. alvarezii and E. spinosum contained high levels of carbohydrate and crude fiber
contents. These seaweeds contained high quantity of macro minerals and trace elements which beneficial in food
application. Their nutritional compositions together with their physicochemical properties in terms of swelling
capacity, water and oil retention capacity suggested that K.alvarezii has a potential food to be functional ingredients
in food industry. Further studies concerned fatty acid, amino acid profile, vitamin and toxic elements are required to
deliver more information for safer and more versatile utilization of these red seaweed species.

303
References

Alberto Peña-Rodríguez, Mawhinney, T. P., Ricque-Marie, D., & Cruz-Suárez, L. E. (2011). Chemical composition of
cultivated seaweed Ulva clathrata (Roth) C. Agardh. Food Chemistry, 129(2), 491–498.
Benjama, O., & Masniyom, P. (2012). Biochemical composition and physicochemical properties of two red seaweeds
(Gracilaria fisheri and G. tenuistipitata) from the Pattani Bay in Southern Thailand. Songklanakarin Journal of
Science and Technology, 34(2), 223–230.
FAO. (2014). The state of world fisheries and aquaculture. Food and Agriculture Oraganization of the United Nations
(Vol. 2014).
Fleurence, È., Physico-chimie, Â., & Qualite, L. (1999). Seaweed proteins : biochemical , nutritional aspects and
potential uses. Trends in Food Science & Technology, 10, 26–29.
Kasimala, M. B., Mebrahtu, L., Magoha, P. P., & Asgedom, G. (2015). A Review on Biochemical Composition and
Nutritional Aspects of Seaweeds. Caribbean Journal of Science and Technology, 3, 789–797.
Marsham, S., Scott, G. W., & Tobin, M. L. (2007). Comparison of nutritive chemistry of a range of temperate
seaweeds. Food Chemistry, 100(4), 1331–1336.
Matanjun, P., Mohamed, S., Mustapha, N. M., & Muhammad, K. (2009). Nutrient content of tropical edible seaweeds,
Eucheuma cottonii, Caulerpa lentillifera and Sargassum polycystum. Journal of Applied Phycology, 21(1), 75–
80.
McHugh, D. J. (2003). A Guide to the Seaweed Industry. FAO Fisheries Technical Paper.
Wong, K. H., & Cheung, P. C. K. (2000). Nutritional evaluation of some subtropical red and green seaweeds Part I Ð
proximate composition , amino acid pro ® les and some physico-chemical properties. Food Chemistry, 71,
475–482.

304
 IFRC 2017: 118-077 Functional Food

Quality Attributes of Malaysian Coconut Water (MATAG and MAWA)

Halim, H.H.1, Williams-Dee, E.1, Pak Dek, M.S. 1, Hamid, A.1, Ahmad, N.2 and *Jaafar, A.H.1
1Department of Food Science, Universiti Putra Malaysia, Jalan UPM, 43400 Serdang, Selangor, Malaysia.
2Stesen MARDI Hilir Perak, Peti Surat 25, 36307 Sungai Sumun, Perak Darul Ridzuan
Abstract
Coconut water (CW) is a popular natural hydration beverage since it possess high electrolytes content and adequate
amount of sugar. This study aimed to compare the physicochemical characteristics, electrolytes and sugars in young
and mature CW from two Malaysian hybrid coconut varieties (MATAG and MAWA). As for the results, flesh thickness
of nuts and pH of CW in both varieties increased significantly (p<0.05) while nut weight, total soluble solids (TSS)
decreased significantly (p<0.05) with maturation of the fruits. Majority of electrolytes decreased as maturity increased
in both varieties. Meanwhile for sugar analysis, fructose and glucose decreased significantly (p<0.05) while sucrose
increased significantly (p<0.05) in both varieties as maturation of the coconuts take placed. As conclusion, mature
CW from both varieties possessed higher electrolytes and sucrose as compared to the CW obtained from the young
coconut.

Keywords: coconut water, physicochemical, electrolytes, sugars and ICP-MS

*Corresponding author’s email: a_hanif@upm.edu.my
Introduction

Coconut water (CW) has been consumed widely as a refreshing drink in tropical countries due to its good taste and
hydration potential (high electrolytes) (Medeiros and Paiva, 2013). It has been reported that the electrolytes in CW
resembles intracellular fluid more than extracellular plasma (Prades et al., 2012). In Malaysia, coconut is the fourth most
important crop where MATAG and MAWA are the most preferable hybrid varieties among coconut planters due to its high
yield productions (Said and Abdul Rashid, 2001). There are two maturity stages of coconuts being consumed which are the
young (six to eight months) and mature (11 to 13 months) coconuts. The young nuts were harvested mainly for its water
whereas the mature nuts were utilized to get the copra or flesh (Prades et al., 2012). Nevertheless, both young and mature
coconuts possess a drinkable CW.

Data on the quality attributes of CW especially the mature coconut from Malaysian hybrid coconut varieties were limited.
Therefore, this study aimed to analyze and compare the quality attributes of CW from different Malaysian coconut varieties
and maturities.

Materials and Methods

Sample collection

The coconuts were collected from MARDI Hilir Perak Station. The maturities of coconuts were indicated by bunch number
and were confirmed by checking the flesh thickness.

Quality assessment of coconut fruits and water

The coconuts were analyzed for their weight and flesh thickness. Meanwhile, the parameters measured for CW were
volume, pH and TTS.

Electrolyte contents (K, Na, Mg and Ca) in CW were quantified by using ICP-MS. Meanwhile, sugar components (fructose,
glucose, sucrose) in CW were determined by using normal phase of High Performance Liquid Chromatography (HPLC)
according to the modified method from Lee and Coates (2000).

305
Statistical analysis was analyzed by using Minitab 16 with Tukey Test where p<0.05 is significant.

Results and Discussion

Quality assessment coconut fruits and water

Results showed that weight and volume of CW in all varieties decreased significantly (p<0.05) as maturity increased. All
coconut varieties showed a significant (p<0.05) increase in flesh thickness with maturation of coconuts (Table 1). As maturity
increased, the reduction of weight was due to decrement of water available in the nut which being used to form flesh (Prades
et al., 2012). Flesh thickness was claimed to be an establish characteristic to indicate maturity of coconuts because CW was
utilized to produce flesh within inner section of the fruits as the coconut grows (Terdwongworakul et al., 2009). As maturity
increased, the pH increased significantly (p<0.05) only in MATAG. Based on pH result, all CW were slightly acidic.
Meanwhile, TSS value in CW where seen to decrease significantly (p<0.05) in MAWA with maturation of fruits. The TSS
measurement indicates the sweetness of CW and the value might be attributed by presence of sugars (Prades et al., 2012).

Table 1: Physicochemical properties of coconut fruits and water.

Weight of Flesh thickness

Samples Volume (mL) pH TSS/ Brix (°)
fruits (kg) (mm)

Y-MATAG 2.15±0.09a 7.24±0.44b 435.00±77.91a 5.36±0.09c 5.75±0.14bc

M-MATAG 1.60±0.14b 11.66±0.63a 391.67±52.98ab 6.33±0.05a 5.63±0.15bc

Y-MAWA 2.00±0.10a 5.04±0.36c 249.17±50.93cd 5.32±0.05c 6.32±0.10a

M-MAWA 1.25±0.05c 11.07±1.23a 159.17±28.36d 5.71±0.54bc 5.57±0.14c

The data was expressed as mean ± SD (n=6). Values that do not share the same superscript represent significant
difference (p<0.05). *Young Pandan (Y-PDN), Mature Pandan (M-PDN), Young Gading (Y-MYD), Mature Gading (M-
MYD), Young MATAG (Y-MATAG), Mature MATAG (M-MATAG), Young MAWA (Y-MAWA), Mature MAWA (M-
MAWA)

Functional electrolytes and sugars in coconut water

As maturity increased, potassium, sodium and magnesium increased significantly (p<0.05) in both MATAG and MAWA while
calcium increased significantly (p<0.05) only in MAWA (Table 2). The potassium was the highest electrolytes detected in all
CW followed by sodium, magnesium and calcium. High potassium in CW is good to replenish intracellular fluid since
potassium ion is the main intracellular cation in body hence indicating efficiency of rehydration (Medeiros and Paiva, 2013).
For sugars, fructose decreased significantly (p<0.05) only both MATAG and MAWA whereas glucose decreased significantly
(p<0.05) in MATAG. In contrast, sucrose in CW of MATAG and MAWA elevated significantly (p<0.05) with the maturation of
the fruits (Figure 1). The trend for sugars was in an agreement with previous study conducted by Jackson et al., (2004).

306
 Table 2: Electrolytes in coconut water from different varieties and maturities detected by ICP-MS.
Electrolytes in coconut water (mg/100 mL)
Coconuts
K Na Mg Ca

Y-MATAG 258.84±1.81c 7.31±0.05d 5.01±0.99c 6.24±1.07cd

M-MATAG 361.20±3.63a 23.71±0.14a 15.10±0.53b 15.88±1.05bc

Y-MAWA 216.81±3.37d 12.17±0.86c 0.09±0.67d 4.78±0.56d

M-MAWA 314.11±3.88b 16.32±0.13b 20.39±0.39a 27.77±1.56a

The data was expressed as mean ± SD (n=3). Values that do not share the same superscript represent significant
difference (p<0.05). *Young Pandan (Y-PDN), Mature Pandan (M-PDN), Young Gading (Y-MYD), Mature Gading (M-
MYD), Young MATAG (Y-MATAG), Mature MATAG (M-MATAG), Young MAWA (Y-MAWA), Mature MAWA (M-
MAWA)

2.5 ab b
a b
Amount of sugars in coconut

a
2
water (g/100 mL)

d a
1.5 a
fructose
1
b glucose
bc b
0.5 c sucrose

0
Y-MATAG M-MATAG Y-MAWA M-MAWA
Coconut varieties and maturities

Figure 1: Sugars in coconut water from different coconut varieties and maturities measured by HPLC (n=3).
Values that do not share the same superscript represent significant difference (p<0.05)

Conclusion

Based on this study, flesh thickness of nuts and pH of CW increased while the weight of nuts, TSS and volume of CW
decreased as maturity increased. The mature CW from both MATAG and MAWA has been discovered to have more
electrolytes and sucrose but lesser fructose and glucose as compared to the young CW.

Acknowledgement

This research was financially supported by grant from UPM and MOA.

307
References

Jackson, J. C., Gordon, A., Wizzard, G., Mc Cook, K. and Rolle, R. 2004. Changes in chemical composition of
coconut (Cocos nucifera) water during maturation of the fruit. Journal of the Science of Food and
Agriculture 84(9): 1049-1052.
Lee, H. S. and Coates, G.A. 2000. Quantitative study of free sugars and myo-inositol in citrus juices by HPLC and
a literature compilation. Journal of Liquid Chromatography & Related Technologies 23(14): 2123-2141.
Medeiros, A.C. and de Paiva, V.D.F.L. 2013. Therapeutic use of coconut water. Journal of Surgical and Clinical
Research 3(2): 83-91.
Prades, A., Dornier, M., Diop, N. and Pain, J.P. 2012. Coconut water uses, composition and properties: a
review. Fruits 67(02): 87-107.
Terdwongworakul, A., Chaiyapong, S., Jarimopas, B. and Meeklangsaen, W. 2009. Physical properties of fresh
young Thai coconut for maturity sorting. Biosystems Engineering 103(2): 208-216.

308
 IFRC 2017: 119-235 Functional Food

Proximate composition and vitamins of Mangifera odarata from fruit pulp and peel
*Nur Diyana Alyas, Muhammad Anas Othaman, Hazniza Adnan, Sukirah Abdul Rahman, and Nur Yuhasliza Abd
Rashid and Norhazniza Aziz

Enzyme & Fermentation Technology Programme, Biotechnology & Nanotechnology Research Centre, Malaysian
Agricultural Research & Development Institute (MARDI),
Persiaran MARDI-UPM, 43400 Serdang, Selangor

As part of an effort to explore indigenous fruit in Malaysia, underutilized Mangifera odorata as known as Kuini was
collected and the nutritional compositions were analysed. Kuini pulp and its peel have 0.36% and 2.15% fat, 5.1%
and 5.33% protein, 7.91% and 5.87% moisture, 3.18% and 5.21 ash, 83.45% and 81.43% of carbohydrate and 357
and 366 kcal/100g energy. Furthermore, vitamins availability in Mangifera odorata fruit pulp and peel were evaluated.
The result of this study showed 3171.53 ug/100g of Vitamin A (Beta carotene), but there are no Vitamin B1
(Thiamine) and Vitamin B2 (Riboflavin) and 23.69 mg/100g Vitamin C (Ascorbic acid) in the fruit pulp. Meanwhile, the
fruit peel consisted of 7186.43 ug/100g of Vitamin A (Beta carotene), 3.23 mg/100g Vitamin B1 (Thiamine) and 6.45
mg/100g Vitamin B2 (Riboflavin) and 23.69 mg/100g Vitamin C (Ascorbic acid). The findings suggest this
underutilised fruit may serve as rich source of nutrients and ascorbic acid to significantly impact health of consumers.
M. odorata is clearly hold nutritional and other desirable quality which have yet to be exploited. Thus, it is potentially a
good source of nutrients for human.

Keywords: Mangifera odorata, proximate composition, vitamins.

*Corresponding author’s email: nurdiyana@mardi.gov.my

Introduction
Mangifera odorata is one of the mango species from Anacardiaceae family and natively grown in Philiphines,
Thailand, Vietnam, Indonesia, Malaysia and Singapore. In Malaysia, it is known as kuini by local community,
specifically to West Malaysia. M. odorata is a cross hybrid between the Mangifera indica (Indian mango) and
Mangifera foetida (bachang or macang) proven by studies in molecular systematics (Kiew, 2002). The fruit is an
obliquely ellipsoid-oblong, hardly flattened drupe with green to yellowish-green skin. The flesh is orange-yellow in
colour, firm and fibrous with a sweet turpentine taste and a strong aroma (Brooke and Lau, 2011). The usage of fresh
kuini is limited to due to its fibrous flesh which causes acrid on throat (Wijaya et al., 1995) compared to mango. Little
information is known about the nutritional value of M. odorata. However, there is potential demand for this
underutilized fruit due to its uniqueness. This study aimed to evaluate the proximate composition and vitamins of
underutilised M. odorata.

Methodology

Sample collection
Local M. odorata sample were bought from Pasar Borong Selayang.

Sample preparation
Matured and ripe M. odorata fruits were thoroughly washed in tap water to remove the gummy sap from the peel. The
pulp was then removed from the fruit. The pulp was homogenized and be kept at -80C for further investigation. M.
odorata peel were dried in ventilated oven at 50C before homogenized and kept at room temperature.

309
Determination of proximate composition
The proximate parameters fat, protein, moisture, ash, carbohydrate and energy and vitamins of the M. odorata
sample were determined using the standard analytical method of Association of Official Analytical Chemists, AOAC
(2000).

Results & Disscussion

Fat Protein
0% 5% Moisture
8%
Ash
3%

Carbohydrate
84%

Figure 1. Proximate composition of Mangifera odorata pulp

Fat Protein
2% 5% Moisture
6%
Ash
5%

Carbohydrate
82%

Figure 2. Proximate composition of Mangifera odorata peel

Table 1. Vitamins analysis of Mangifera odorata pulp and peel

Vitamin Mangifera odorata pulp Mangifera odorata peel

Vitamin A
(Beta carotene) 3.1715 mg/100g 7.19 mg/100g
Vitamin B1
(Thiamine) 0.0000 mg/100g 3.23 mg/100g
Vitamin B2
(Riboflavin) 0.0000 mg/100g 6.45 mg/100g
Vitamin C
(Ascorbic acid) 23.69 mg/100g 20.16 mg/100g

310
Result of the proximate composition of M. odorata pulp and peel are presented in figure 1 and 2 the proximate
analysis of M. odorata pulp and peel provided evidences that have a relative amount of nutritive value for human
consumption. Meanwhile, study by Lauricella et al., 2017 found that 0.38% fat of mango flesh and 8.22% fat on
mango peel by Ojokoh (2007). A 0.82% protein of mango flesh (Lauricella et al., 2017) and 6.16% protein on mango
peel by Ojokoh (2007). This result suggested that Mangifera fruits are commonly hold a small amount of protein.
Moisture on mango peel was 6.56% slightly similar to Ojokoh, 2007 findings. However, our results showed a little
amount of ash compared to 6.4-9.81% of ash studied by Mohammed & Yakubu, 2013. Findings of carbohydrate from
mango flesh reported by Wenkam & Miller, 1965 just 15.05-18.92% and mango peel reported by Ojokoh 2007 was
32.6%. The difference in nutritional composition between M. odorata and M. Indica could emerged from
environmental factors, species and cultural practices (Ishu, 2013). Furthermore, vitamins availability in M. odorata
fruit pulp and peel were tabulated in Table 1. Lauricella et al., 2017 has reported that Vitamin A, B1, B2 and C
content of mango pulp were 1082 IU, 28 ug, 38 ug and 36.4 mg/ 100g. The vitamins C in the M. odorata pulp meet
the minimum requirement of 15 mg/100g recommended by EU/WHO for fruit groups. Vitamins C is beneficial for
human health and wellness.

Conclusion

The information obtained from these proximate composition and vitamins analysis would serve as a guide for further
potential utilization of M. odorata.

Acknowledgement

The authors thank the Director General of MARDI, the Director of Biotechnology & Nanotechnology Research Centre
for their support together with Deputy Director of Enzyme & Fermentation Technology for the guidance and useful
advice given. The project was funded under RMK11-407.

References

AOAC, 2000. Association of Official Analytical Chemists. Official methods of analysis, (Vol.II 17th edition) of AOAC
international, Washington DC, USA.
Brooke, P. and Lau. C.Y. 2011. Nutritional value and economic potential of underutilised Mangifera species in Bungai
area, Sarawak, Malaysia. ISHS Acta Horticulture 979: II International Symposium on Underutilized Plant
Species: Crops for the Future-Beyond Food Security.
Ishu, M.O. 2013. Comparative proximate composition of the fruits juice of three varieties of Mangifera indica L.
(Mango). Makurdi, Nigeria: Benue State University, MSc thesis.
Kiew, R., 2002. Singapore Botanic Gardens. Garden Bulletin Singapore. 54:205
Lauricella, M., Emanuele, S., Calvaruso, G., Giuliano, M and Anneo, A. 2017. Multifaceted health benefits of
Mangifera indica L. (Mango): The inestimable value of orchards recently planted in Sicilian rural areas.
Nutrients, 9,525.
Mohammed, S.S.M. and Yakubu, A. 2013. Comparative analysis of nutritional and anti-nutritional contents of some
varieties of mango (Mangifera indica) in Kaduna metropolis-Nigeria. Research Journal of Applied Sciences,
Engineering and Technology 5 (4), 387-391.

311
Ojokoh, A.O. 2007. Effect of fermentation on the chemical composition of mango (Mangifera indica R) peels. African
Journal of Biotechnology 6 (16), 1977-1981.
Wijaya, C.H., Apriyantono, A., May, T., Raharja, H. And Ngakan, T.A. 1996. Flavor of Kweni (Mangifera odorata
Griff), an exotic tropical fruit. In: Flavour chemistry of ethnic foods. Pp119-125.
World Health Organization/Europe. 2000. CINDI dietary guide. WHO Regional Office for Europe, Copenhagen,
Denmark.

312
 IFRC 2017: 121-079 Functional Food

Response Surface Optimization on the Total Phenolic Content and Antioxidant Activities of Sabah Snake
Grass (Clinacanthus nutans) Leaves Peleg Kinetic Modelling Extract
Fazil, F.N.M., Azzimi, N.S.M. and *Zubairi, S.I.
School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan
Malaysia, 43600 UKM Bangi, Selangor, Malaysia
Clinacanthus mutans contain rich in phenolic content and antioxidant. Based on Peleg’s model, the optimum
concentration for orientin (0.720 ± 0.002 mg/g) and vitexin (2.095 ± 0.131 mg/g) were observed at 18 hrs of
extraction (texhaustive). Optimum extraction parameters for recovery of phenolic content and antioxidant activities from
the leaves of Clinachantus nutans were determined using response surface methodology (RSM). The total phenolic
content (TPC) was analyzed using the Folin-Ciocalteu method and antioxidant activities were evaluated through 2,2-
diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS) free radical scavenging activity and the ferric reducing antioxidant power (FRAP) assay. These
optimum conditions yielded TPC of (25.09 mg GAE/g), DPPH (66.85%), and FRAP (9.44 µmol TE/g). The application
of Peleg model was able to determine the extraction exhaustive time at the maximum extract yield.
Keywords: Peleg model optimization Antioxidants, Orientin, Vitexin
*Corresponding author’s email: saiful_cepp@yahoo.co.uk
Introduction
Sabah snake grass (SSG) or scientifically known as Clinacanthus nutans is widely grown in tropical Asia including
Malaysia and Thailand [1]. Leave part of SSG has diverse and potential medicinal uses in traditional herbal medicine
for treating variety of diseases such as diabetes mellitus, fever, skin rashes, snake bites, herpes simplex virus (HSV),
varicella- zoster virus (VSZ) lesion [2] and diarrhea [3]. In Malaysia, traditionally been used to treat diseases and its
potential to be used for cancer prevention and treatment [4]. In rural area they consumed as it is believed to contain
high nutrients and antioxidants. Previously, SSG has been phytochemically and chemically isolated of stigmasterol,
lupeol, b-sitosterol,six known C-glycosyl flavones. They possess important biological activities including antimicrobial
activity, hepatoprotective activity and antioxidant activity [5]. The flavonoid phytochemicals are natural antioxidants
regarded as a powerful antioxidant as a potential protector of various human diseases such as certain cardiovascular
diseases and cancer [6]. However, high extraction production of bioactive compounds from plant is need to be
attained as it can reduce the economic cost. Many parameters have been established to influence the extraction
efficacy, such as extraction methods, particle size, solvent and concentration [7]. Response surface methodology
(RSM) is an effective tool for optimizing complex processes [8] in determination the level of flavonoid phytochemical
content in SSG leave. Further study was aimed at assessing the presence of the flavonoid phytochemical component
(orientin and vitexin) using high performance liquid chromatography (HPLC), determining the optimum yield of
flavonoid and exhaustive time extraction from leaves of SSG using Peleg’s model as well as determining the
optimum extraction parameters (sonication frequency, temperature and ratio solid-liquid) for optimum recovery of
phenolic content and antioxidant activities from the leaves of SSG using RSM.
Material and methods
Plant Material
SSG fresh leaves were collected from Sendayan, Seremban, Malaysia. The samples were dried (40 oC for 48 hrs).
Chemicals and Reagents
Chemical standards vitexin and orientin, methanol, glacial acetic acid, Folin–Ciocalteu phenol reagent, gallic acid,
2,2-diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid , sodium carbonate
2,4,6-tris(2-pyridyl)-s-triazine and ferric chloride.

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Kinetic Model of Solid-Liquid Extraction
The Peleg’s model is mathematical modelling and a useful engineering tool that able to predict the optimize
concentration extract in a short time [9]. The Peleg’s model is shown in Fazil et al. 2016 [10].
Identification of Flavonoids Compounds via HPLC Analysis
Determine flavonoids compounds was performed using a HPLC system (Shimadzu). The chromatographic
separation was performed using a XBridge C18 at 30˚C. The solvent system consisted of 2 solvents: A; 0.1 % acetic
acid and B; MeOH. The flow rate and injection volume were adjusted at 10 µl and 0.8 ml/min, respectively. The
detection was monitored at 280 nm.
Experimental Design
The extraction parameters were optimized using RSM using three-level factorial central composite design (CCRD).
Three selected independent variables were determined, namely frequency sonication (X1: 25 - 40 kHz), temperature
(X2: 40 - 80 ºC) and solid-to-liquid ratio (X3: 10 - 30 ml/g) on TPC (Y1) and antioxidant capacity of DPPH (Y2) and
FRAP (Y3).
Determination of Total Phenolic Content (TPC)
TPCs of the extracts were determined according to the method by Musa et al. (2011) [11]. 0.2 ml extract and gallic
acid was placed in a separate 10 ml vials, followed by the addition of 0.4 ml water and 0.5 ml diluted Folin–Ciocalteu
reagent. The mixtures were swirled and allowed to stand for 5 mins before mixing 1 ml of sodium carbonate (7.5%
w/v). The solutions were allowed to stand for 2 hrs at room temperature and later the absorbance was observed at
765 nm using UV-visible spectrophotometer.
Antioxidant activiy
DPPH free radical scavenging activity was measured by the method of [12]. Ferric reducing antioxidant power
(FRAP) was estimated according to adapted procedure of [11] with minor modifications. FRAP reagent was modified
on HCl preparation (40 mM HCl;20 mM FeCl3 6H2O in the ratio of 10:1:1).
Results and discussion
Flavonoid compound
RP-HPLC was performed for determination of flavonoid compounds in SSG extract which was orientin and vitexin. In
this study, water (a polar solvent) was used as an extraction solvent because it is non-toxic and environmentally-
friendly solvent for natural product extractions [13]. Besides, it was important to determine the optimal conditions for
extracting flavonoids as to mimic the green tea concoction preparation since this plant has been widely used as tea
too for therapeutic application. This study had shown that the concentration of orientin compound in SSG extract was
significant lower (0.720 ± 0.022 mg/g) as compared to vitexin compound which was (2.095 ± 0.131 mg/g).
Yield of Extract and Exhaustive Time of Extraction
The Peleg’s model is mathematical modelling and a useful engineering tool that able to predict the optimize
concentration extract in a short time, reducing energy and the used of solvent. Based on the Table 1 data, the
calculation of Peleg model from sample showed that the orientin compound had lower extraction rate (3.25 ± 0.45
mg/mins) as compared to compound vitexin (3.58 ± 0.35 mg/mins). According to Raymond (2007) [14], the reaction
rate of extraction of a substance will be decreased if the concentration is increasing. However, the maximum
extraction yield for orientin compound (133.97 ± 2.76 mg) was significantly (p<0.05) higher than the vitexin
compound (76.55 ± 1.48 mg). The exhaustive time to produce the highest concentration of orientin compound
extracted from SSG leaves was too long (17.72 ± 0.71 hrs) as compared to vitexin compound (7.56 ± 0.72 hrs)
(p<0.05). However, 18 hrs of extraction has been chosen in optimization study due to the exhaustive concentration of
orientin compound were successfully attained as well as maintaining the exhaustive concentration of vitexin.

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 Table 1: Yield of extract and exhaustive time of Sabah snake grasses leaves extraction containing orientin and
vitexin (72 hrs)
Orientin Vitexin
Extraction rate (mg/mins) 3.25 ± 0.45 3.58 ± 0.35
Maximum yield extraction (mg) 133.97 ± 2.76a 76.55 ± 1.48b
Time exhaustive extraction (hrs) 17.72 ± 0.71a 7.56 ± 0.72b
Mean value a-b. Different alphabet letters in same row indicate significant difference (p<0.05)

Response Surface Optimization of Processing Parameter

To optimize the ultrasound-assisted extraction (UAE) on the TPC and antioxidants content from SSG extracts, a
central composite rotatable design (CCRD) was designed and employed. A fixed extraction time (18 hrs) was chosen
based on the Peleg model calculation. In this study, the lower and upper values for the factors were set at +alpha (+α
= 1.682) and –alpha (-1.682) and thus all the factor levels was chosen within the limits that were desirable and
practical. The experimental values of TPC and total antioxidant activities of SSG extracts under various experimental
conditions were presented in Table 2. The results showed that TPC, DPPH and FRAP of SSG leaves ranged from
(4.74 - 25.10 mg GAE/g), (13.33 - 65.24%) and (0.91-10.44 µmol TE/g), respectively. The response surface
optimization statistical analysis results shows the coefficients are related to coded variables. The parameter for solid-
liquid ratio exhibited the most prominent as a result of the highest F-value from the other parameters. The result
suggested that the solid-liquid ratio had the greatest impact on the extraction of TPC, DPPH and FRAP from SSG
leaves. Analysis of variance (ANOVA) was performed to evaluate the quality of the fitted model. The ANOVA of the
regression model showed that the model was significant (p<0.05) to all the response variables. Model fitting was
decided by the coefficient of determination (R2) and significant of lack-of-fit. The closer the R2 value to unity, the
better the experimental model fits the actual data [15]. The R2 of the models for TPC, DPPH and FRAP were 0.8104,
0.9289 and 0.7908, respectively. The lack of fit-value for the TPC, DPPH and FRAP model were considered
insignificant (p>0.05). The value of lack of fit (p>0.05) indicated the reliability of the model.
Optimum Extraction Processing Parameters
Determination of optimum extraction processing parameters of SSG leaves was based on the highest desirability
value. Table 2 presented the optimum conditions for TPC, DPPH and FRAP, and its predicted and experimental
values. The optimum conditions were found to be: ultrasonic frequency (25 kHz), temperature (80 ºC) and solid-liquid
ratio (30 g/ml). Under these optimal conditions, the model predicted a maximum response of TPC (21.34 mg GAE/g),
DPPH (64.69%) and FRAP (8.64 µmol TE/g) of SSG leaves extract.
Table 2: The predicted result of processing optimum parameter from snake grass leave extracts
Ultrasonic Solid-to-
TPC FRAP (µmol
frequenscy Temp. (oC) liquid ratio DPPH (%)
(mg GAE/g) TE/g) Desirability
(kHz) (g/ml)
25.00 80.00 30 21.34 64.69 8.64 0.870

CONCLUSION
The present study confirmed that orientin and vitexin compounds had been detected in SSG leaves by using water
extraction (time extraction of 72 hrs). The kinetic Peleg’s model showed exhaustive time of extraction (18 hrs) for
SSG extract have been chosen for the response surface optimization to identify the best processing parameters with
respect to maximum yield of extract. The RSM was successfully employed to optimize the extraction and several
experimental parameters have been evaluated. The parameters such as ultrasonic frequency, extraction temperature
and solid-to-liquid ratio have given a good effect on the extraction rate of TPC, DPPH and FRAP. The optimal

315
parameter achieved based on combination of extraction parameters are as follows: X1 = 25 kHz ultrasonic
frequency, X2 = 80 ºC temperature and X3 = 30 g/ml of solid-to-liquid and yielded TPC, DPPH and FRAP of 25.09
mg GAE/g, 66.85% and 9.44 µmol TE/g respectively. The application of both models (RSM and Peleg) have been
effectively be able to provide the optimum extraction parameters with high TPC and antioxidant activity.
Acknowledgments
The authors would like to thank the Ministry of Science, Technology and Innovation (MOSTI) and Ministry of Higher
Education (MOE) Malaysia for providing financial support to this project (06-01-02-SF1271,
FRGS/2/2013/TK04/UKM/03/1 and GGPM-2013-078).
References
Yuann, J.-M. P., J.-S. Wang, H.-L. Jian, C.-C. Lin and Liang J.-Y. 2012. Effects of Clinacanthus nutans. (Burm. f)
Lindau leaf extracts on protection of plasmid DNA from riboflavin photoreaction. MC-Transaction on
Biotechnology. 4(1): 45-58.
Sakdarat, S. S. 2009. Bioactive constituents from the leaves of Clinacanthus nutans. Lindau. Bioorganic and
Medicinal Chemistry. 17(5):1857–1860.
Png, X., Akowuah, G. and Chin, J. 2012. Acute oral toxicity study of Clinacanthus nutans in mice. International
Journal of Pharmaceutical Sciences and Research. 3:4202–4205.
Roosita, K., Kusharto, C. M., Sekiyama, M., Fachrurozi, Y. and Ohtsuka, R. 2008. Medicinal plants used by villagers
of Sudanese community in West Java, Indonesia. Journal of Ethnopharmacology. 115: 72-81
Zhang, Y., J. Jiao, C. Liu, X. Wu and Zhang Y. 2008. Isolation and purification of four flavone C-glycosides from
Antioxidant of Bamboo Leaves by macroporous resin column chromatography and preparative high-
performance liquid chromatography. Food chemistry. 107(3): 1326-1336.
Ren, W., Qian, Z., Wang, H., Zhu, L. and Zhang, L. 2003. Flavonoids: Promising anticancer agents. Medicinal
Research Reviews. 23(4): 519– 534.
Silva, E. R. 2007. Optimisation of extraction of phenolics from Inga Edulis leaves using response surface
methodology. Journal of Separation and Purification Technology. 55: 381-387
Liu, W., Yu, Y., Yang, R., Wan, C., Xu, B., and Cao, S. 2010. Optimization of Total Flavonoid Compound Extraction
from Gynura Medica leaf using response surface methodology and chemical composition analysis.
International Journal Molecule Science. 11:4750–4763.
Turhan, M., S. Sayar and Gunasekaran S. 2002. Application of peleg model to study water absorption in chickpea
during soaking. Journal of Food Engineering. 53(2): 153-159.
Fazil, F.N.M., Azzimi, N. S. M., Yahaya, B. H., Kamalaldin, N. A., and Zubairi, S. I. 2016. Kinetics Extraction
Modelling and Antiproliferative Activity of Clinacanthus nutans Water Extract. The Scientific World Journal
Musa, K. H., Abdullah, A., Jusoh, K. and Subramaniam, V. 2011. Antioxidant Activity of Pink-Flesh Guava (Psidium
guajava L.): Effect of extraction techniques and solvents. Food Analytical Methods. 4(1): 100-107.
Xu, B. J. and Chang, S. K. C. 2007. A comparative study on phenolic profiles and antioxidant activities of legumes as
affected by extraction solvents. Journal of Food Science. 72(2): S159-S166.
Tan, S. P., S. E. Parks, C. E. Stathopoulos and Roach, P. D. 2014. Extraction of flavonoids from bitter melon. Food
and Nutrition Sciences. 5(5): 458.
Raymond, C. 2007. Chemistry. Ed. Ke- 9: New York. McGraw-Hill
Melecchi, M. I. S., V. F. Péres, C. Dariva, C. A. Zini, F. C. Abad, M. M. Martinez and Caramão, E. B. 2006.
Optimization of the sonication extraction method of Hibiscus Tiliaceus L. Flowers. Ultrasonics Sonochemistry.
13(3): 242-2.

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 IFRC 2017: 123-083 Functional Food

Insect Powder: A New Protein Source

Valenzuela, K.M. and *Duque, S.M.
Institute of Food Science and Technology, College of Agriculture and Food Sciences, University of the Philippines
Los Banos, Los Banos, College, 4301 Laguna, Philippines
Abstract
Several studies have pointed out the important role of protein in the human body. Protein is usually consumed
through the following sources: meat, poultry, eggs, and dairy products. Among these, beef is considered as one of
the largest food protein source consumed by human. However, with rapid population increase and indefinite supply of
beef in the future, the need for a new source of protein has emerged. Thus, this study explores the potential of insect
powder as protein source. Insects (Sphenarium purpurascens [grasshopper], Leucopholis irrorata [beetle], and
Acheta domestica [cricket]) were tested for their protein content. The sample with the highest percent protein was
subjected to microbiological test and chemical tests such as glutamic acid, proximate, reducing sugar, and total sugar
content analyses. Protein content of the samples were found to be not significantly different with that of beef. Data
showed that cricket, which was chosen for further tests due to limited sources of beetle and grasshopper, has a
protein content of 46.15%. It is microbiologically safe and has a glutamic acid content of 0.60-0.80 mg/ml. Its
reducing sugar and total sugar content are 2.42 ± 0.14 and 1.58 ± 0.00 mg/ml, respectively. Therefore, it can be
concluded that the cricket powder has the potential to be a new food protein source.
Keywords: Sphenarium purpurascens, Leucopholis irrorata, Achetado mestica, protein, proximate analysis
*Corresponding author’s email: shebadq@gmail.com

Introduction
One of the most needed biomolecules in the body is the protein. Together with carbohydrates and fats, they are the
sources of energy that the body needs, providing 4 kcal/g protein. They function as catalysts by acting as enzymes,
aids in different DNA processes such as such as DNA replication, transcription and repair, and are involved in cell
signalling throughout the body. Common sources of protein include meat, dairy products, eggs, and poultry.
In the food industry, beef and lamb meat serve as the largest source of dietary proteins. When cooked, they provide
16.9-40.6 and 20.91-50.9 grams protein per 100 grams portion of food, respectively, just almost enough to satisfy the
daily protein requirement, 56 grams for men and 46 g for women (Canadian Nutrient File. Health Canada, 2009).
However, the future of the beef market cannot be guaranteed stable due to large efforts and expenses needed just to
raise one cattle. In addition to this problem, continuous population growth also equates to a need for a more efficient
source of protein.
There are emerging studies proposing that insects are good protein source. Different international food organizations
such as the International Network of Food Data Systems (INFOODS) and Food and Agriculture Organization (FAO)
have published in 2012 FAO/INFOODS Food Composition Database for Biodiversity which included the nutrient
composition of insects (Nowak, 2014). This database showed that insects have minerals and trace elements (34.8%),
proximates (24.5%), amino acids (15.3%) and (pro)vitamins (9.1%). The high amount of amino acids caused the
awareness on insects as alternative protein source.

This study mainly aims to determine the potential of insect powder as food protein source.

Materials and Methods

3.1. Sample Preparation

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Insects, Sphenarium purpurascens [grasshopper] and Leucopholis irrorata [beetle] (obtained from Bauan, Batangas,
Philippines) and Acheta domestica [cricket] (collected from Manila, Philippines), were dried (50˚C for 24 hours) using
a cabinet dryer and powdered using a laboratory mortar and pestle. The standard sample, beef, was cut and dried
(60˚C for three days).

3.2. Crude Protein Determination

Crude protein was determined using Kjeldahl method, AOAC (1990) 981.10.

3.3. Microbiological Test

Serial dilutions of sample were prepared. Each dilution was inoculated in Potato Dextrose Agar (PDA), Plate Count
Agar (PCA), and Violet-Red Bile Agar (VRBA) (Appendix B). For PCA and VRBA, pour plating was used while for
PDA, the sample was spread plated. Plates were incubated for 24 hours (VRBA) and 72 hours (PCA and PDA) at 37
°C. Colony forming units (CFU) per gram was calculated.

3.4. Proximate Analysis

AOAC Standard methods were used to determine the moisture, crude fat, crude protein, crude fiber, ash, and
nitrogen-free extract content of the powder.

3.5. Glutamic Acid Content Analysis

Extraction of glutamic acid was done according to Khan et. al. (1955), using paper chromatography..

3.6. Reducing Sugar and Total Sugar Content Analysis

Test for reducing sugar was done according Borel et. al. (1952) using the Dinitrosalicylic (DNS) method while total
sugars determination was done according to Dubois et. al. (1956) using the phenol sulfuric acid method.

3.7. Statistical Analysis

All experiments were carried out with 3 replicates each unless otherwise stated. The samples from each replicate
was tested for normality using Kolmogorov-Smirnov test for normality and normal sets of data were analyzed using
student’s T-test (Appendix H) at 95% level of confidence. Student’s T-test was used to determine if treatments were
significantly different from each other.

Results and Discussion

As seen in Figure 1, among the three samples, beetle has the highest crude protein content (66.59% ± 0.66),
followed by grasshopper (64.18% ± 1.97) and the least was obtained from the cricket (63.04% ± 2.32). When tested
for significance, results showed that there is no significant difference among the average crude protein content of the
samples and beef. Therefore, any of the three insect samples could be subjected to further tests. Cricket was
selected due to lack in sources of grasshopper and beetle.

Results of the microbiological test for cricket show that it had <2500 CFU/g, <1500 CFU/g, and 0 for total bacterial,
mold, and coliform counts, respectively. According to Food and Drug Administration (FDA) circular number 2013-010
or the Microbiological Standard for Processed Food, if spices contained 10000 CFU/g, it can be subjected to rejection
and 1000000 is the count which is maximally allowed, given that the coliform count is negative. Considering the
values from Table 1, the cricket powder can be considered safe.

The proximate content of the cricket powder is shown on Figure 4, with high amounts of crude fat and crude protein.
This high value of fat indicates that the powder can provide a high caloric value and therefore high energy yield for
human. Aside from this, its fat content may contribute to flavor and may affect its acceptability as food in terms of
taste.

318
In addition to these, the reducing sugar and total sugar contents of the sample are 2.42 ± 0.14%and 1.58 ± 0.00%,
respectively. These were tested due to observed powder clumping when subjected to mild heat. Meanwhile glutamic
acid content, an indicator of taste characteristic,lie between 0.60-0.80 mg/ml (Figure 3).

Figure 1. Crude protein content of Beef, Beetle, Cricket and Grasshopper obtained using

Kjeldahl method.

Table 1. Evaluation of the total counts from plate count agar, potato dextrose agar, and violet-red bile agar from 10-7
to 10-2 dilution.
Test for CFU/g Standards (CFU/g) Remarks
Total Bacterial Count <2500 <10000 Passed
Total Mold Count <1500 <10000 Passed
Total Coliform Count 0 0 Passed

Figure 2. Proximate content of cricket powder.

319
 Figure 4. Paper chromatography for glutamic acid content analysis of cricket using 12:3:5 v/v ratio of 1-
butanol:aceticacid:water as solvent.

Conclusion
At the end of the experiment, results showed that the cricket powder has a relatively high crude protein content of
46.15%. The powder is also microbiologically safe when tested for total bacterial, mold, and coliform counts. It also
has considerable amounts of glutamic acid (0.6-0.8mg/ml), reducing sugar (2.42 ± 0.14%) and total sugar (1.58 ±
0.00%). Therefore, the cricket powder can be considered as a new food protein source. Moreover, tests including
toxicity and sensory evaluation are highly recommended to justify further the cricket powder’s capability of
substituting beef. Further tests, such as those done in the experiment and those recommended must also be done to
other insect species.

References
A.O.A.C. 1990. Association of official, chemists, official methods of analysis. 15th Edition, Washington DC, U.S.A.
Bacteriological Analytical Manual. 2016. U.S. Food and Drug Administration.10903 New Hampshire Avenue, Silver
Spring. Retrieved April, 2016 at
http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm2006949.htm
Canadian Nutrient File-Health Canada. 2009. Canada
Dubois M., Gilles K.A., Hamilton J.K., Rebers P.A., Smith F. 1956. Colorimetric method for determination of sugars
and related substances. Anal Chem 28:350-356.
Fao/Infoods Guidelines For Checking Food Composition Data Prior To The Publication Of A User Table/Database –
Version 1.0. 2012a. Rome: FAO. Retrieved from
<http://www.fao.org/fileadmin/templates/food_composition/documents/Guidelines_data_
checking_02.pdf>.
Khan N.A., Baker B.E., Van Horn W.f.. 1955. Journal of Agricultural and Food Chemistry.
Nowak V., et al. 2014. Review of food composition data for edible insects. Food Chemistry.
Retrieved from http://dx.doi.org/10.1016/j.foodchem.2014.10.114

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 IFRC 2017: 124-166 Functional Food

Extracted Water Soluble Polysaccharide from Gum Arabic as Potential Prebiotic

1* Ahallil Hammad., 1Aminah Abdullah., 2Shahrul R. Sarbini and 1Mohamad Yusof Maskat
1School of Chemical Science and Food Technology Faculty of Science and Technology, University
Kebangsaan Malaysia, 43600 Bangi Selangor, Malaysia.
2Department of Crop Science, Faculty of Agricultural and Food Sciences, Universiti Putra Malaysia Bintulu
Campus, Jalan Nyabau, 97008 Bintulu, Sarawak, Malaysia
Abstract:

Gum Arabic (GA) is the dried gummy exudates obtained from stems and branches of trees of selected Acacia
species (Acacia senegal & Acacia seyal). Present study includes extraction of water soluble polysaccharide of
Gum Arabic from two different species. Polysaccharide was obtained by aqueous extraction, this methodology is
simpler to perform than other existing extraction and purification methodologies, and because it avoids the use of
organic solvents (other than ethanol). The characterized of isolated polysaccharide was defined by FTIR
spectrum. The polysaccharide was characterized by the measurement of total sugar and reducing groups using
phenol sulfuric method and DNS assay respectively. The crude polysaccharides were further studied by
measuring protein and fat content. The digestibility of polysaccharides by human gastric juice was also
determined. The vibrational assignment of the bands observed in FTIR spectrum is verified. The broad peak
appeared at 950-1200 range indicate the finger print of polysaccharide. Results revealed the average values of
proteins; 1.8% to 0.78% and fat; 0%. Acacia Senegal and Acacia Seyal showed great resistance towards artificial
human gastric juice. The current study reveals that the extraction using aqueous extraction eliminates the protein
content, thus providing more pure polysaccharide suitable for biological applications. The results also confirm the
suitability of the extracted polysaccharide obtain from (Acacia Senegal & Acacia Seyal) to resist toward acid
digestibility.

Keywords: Gum Arabic, polysaccharide extraction, prebiotic and Acid digestion

*Corresponding author’s email: ahlilhammad@gmail.com

Introduction:

Gum Arabic (GA) or Acacia gum is an edible biopolymer obtained as exudates of mature
trees of Acacia senegal and Acacia seyal which grow principally in the African region of Sahe in Sudan.
Nowadays, there is an increasing trend of consumer awareness towards the demand for functional foods, which
are claimed to enhance the health of the consumer. Apart from other food ingredients, prebiotics are among
those which have attracted much attention recently (Roberfroid. 2000). Therefore, this study was conducted to
reveal medicinal usages of Acacia Senegal& Acacia Seyal as a prebiotic. The objectives of this study are: first to
extract and characterize water soluble polysaccharide from Acacia Senegal & Acacia Seyal; and second to
elucidate the function of extracted water soluble polysaccharide toward acid digestibility.

Material and Methods:

The Gum Arabic samples used in this study were from two different sores (Acacia Senegal & Acacia Seyal) and
they were purchased from a Sudanese market.

2.1- The polysaccharide extraction of Acacia Senegal& Acacia Seyal was performed with ethanol and distilled
water. The extracted samples were dialysis by using (cut off = 10 kDa) (Vidanarachchi et al. 2009).

2.2- Structural Elucidation of Polysaccharides

2.2- Fourier transform infrared (FTIR) spectroscopy was conducted using Perkin-Elmer spotlight 400 imaging
system in the frequency range of 650 to 4000 cm-1(Pavia et al. 2009).

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2.3- Mojonnier method used to measure fat content (Anonymous. 2002). Protein was calculated from nitrogen
(Nx6.25) obtained using the Kjeldahl method. Carbohydrate content: Reducing sugar was determined using
(DNS) assay (Robertson et al., 2001). The total sugar determined using phenol–sulphuric acid assay (Dubois, et
al, 1956).

2.6- Non-digestibility of polysaccharide is defined as the ability to resist towards gastric acidity, hydrolysis by
mammalian enzymes and gastrointestinal absorption (Mandalari et al., 2008).

2.7- Statistical analysis of the data was performed by one-way ANOVA using (SPSS 17 software)

Result and Discussion:

Proteins and Fat present can limit water penetration, thus affect the extraction efficiency (Aida Firdaus et al.
2012). Acacia Senegal and Acacia Seyal samples showed no fat content (0%). Acacia Senegal should slight
higher protein content comparing to Acacia Seyal. The percentage of crude protein showed significant differences
as (p< 0.05) between the pure and extracted sample. As can be seen in Table(1) protein in pure Acacia Senegal
reduce from 2.04% to 1.8% and protein in Acacia Seyal also decline from 1.04% to 0.78%. Total carbohydrate of
Acacia Senegal and Acacia Seyal were as following 88% and 90% respectively, this finding is in agreement with
study done by Goodrum 2000. As can be seen in Table (2), the extraction showed increase in total sugar content
of both gum samples with significant differences (p< 0.05). These results can insure that, the extracted water
soluble polysaccharides are more neutralize.

Table 1. Protein Content of Acacia Senegal and Acacia Seyal, before and after extraction.
Protein Pure Extracted
Acacia Senegal 2.04+0.05a 1.80+0.02b
Acacia Seyal 1.04+0.04a 0.78+0.03b
Values are the mean ± SD (n=3); means that do not share a same letter are significantly different (p<0.05) as
measured by Duncan test.

Table 2. Carbohydrate Content of Acacia Senegal and Acacia Seyal, before and after extraction.
Carbohydrate Pure Extracted
Acacia Senegal 88.07+1.19b 91.09+1.44a
Acacia Seyal 90.33+0.82b 94.55+0.88a
Values are the mean ± SD (n=3); means that do not share a same letter are significantly different (p<0.05) as
measured by Duncan test.

FTIR spectra for Senegal (Sen) and Seyal (Sey) are shown in Figure 1& 2. A characteristic absorption band at
3274.60 and 3296.60 cm-1 for SEN and SEY repressively, demonstrating the presence of hydrogen bonded OH
group was observed. While, the bond absorbed at 2922.08 and 2927.43 cm-1 for SEN and SEY indicate the
presence of sugars, galactose, arabinose, and rhamnose, also the presence of alkane CAH stretch and aldehyde
C-H stretch. The other strong vibrational mode located at 1601.4 and 1415.5 cm-1 in SEN spectrum and bands at
1601.87, 1416.41 in seyal spectrum are mainly assigned to the absorbance of deprotonated carboxylic function of
glucuronic acids. This peak could be contribution with C‚C stretch, amide NH bend, NO2 from both aliphatic and
aromatic galactoproteins. The peaks near 1370 and 1260 cm-1 in SEN spectrum and near 1370.12 and 1260.90
cm-1 in SEY spectrum might be attributed the presence of C-O-C stretching( between two glactose sugars) and
C-H bending vibrations and deformations) amines C-N stretch (glycoproteins), and alkyl due to sugar backbone
showing alkane bend, alcohol stretch. The vibrational modes at 1018.87 and 1016.45 represent C-H alkene of
sugars (polysaccharides) for SEN and SEY, respectively. Furthermore, the small peaks appear at low frequency
with low intensity between 820-700 cm-1 in Fig1 and Fig 2 is belonging to C-C stretching and C-H bonding of
sugars.

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 Figure 1. FTIR spectra for Acacia Senegal (SEN).

Figure2. FTIR spectra for Acacia Seyal (SEY).

In general, the reduction in the intensity of theses peaks (at 1416.41 cm -1) after extracted indicate decrease of
some metals that have been attached to the carboxylic group in glucuronic. The reduction in peaks of 1601.87,
1370.45 and 1260.86 cm−1 is due to elimination of some glycoprotein (amide, NH, NO2). The small reduction of
certain peak reflects chemical environment change of sample after extraction (Pavia et al. 2009).

In this study, Senegal and Seyal found to be resistant towards artificial human gastric juice as compared to Inulin.
Surprisingly, the non-digestibility of Senegal and Seyal were noted to be more than 98%. It showed small
differences in the degree of hydrolysis (Figure 3). From the data, we can see that Seyal was less susceptible
towards gastric juice comparing to Senegal 1.72% and 1.85 respectively, with no significant differences (p< 0.05)
among them. Food is usually retained in the human stomach where gastric juice (pH 2–4) is released within 2 h;
thus, when polysaccharides are consumed, 98% of them are estimated to reach the intestine. The results
gathered gave a good indication that Senegal and Seyal can be regarded as a potential prebiotic, as it meets
prebiotic characteristics, which need it to be non-digestible. This result is comparable to that of
kojiooligosaccharide, which had 100% resistance to artificial gastric acid (Nakada et al. 2003).

323
 2 1.85
1.72 1.64
1.49
1.5

Hydrolysis
1
%
0.5

0
Senegal Seyal Inulin Nigative
Samples
Figure2. Digestibility of Gum Arabic.

Conclusions
the protein content was eliminated using aqueous extraction, thus providing more pure polysaccharide suitable
for biological applications. Acacia Senegal and Acacia Seyal showed the strong suitability to resist toward acid
digestibility. In conclusion, Acacia Senegal and Acacia Seyal can be regarded as a potential prebiotic, as it meets
prebiotic characteristics, which need it to be non-digestible.

References:

Aida, F.N.M.A ., Mustafa, S., Dzulkifly Md. Hashim & Manap, M.Y. 2012. Prebiotic Activity of olysaccharides
Extracted from Gigantochloa Levis (Buluh beting) Shoots. Molecules.17, 1635-1651.
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. 1956. Calorimetric method for determination
of sugars and related substances. Analytical Chemistry, 28, 350–356.
Mandalari, G., Faulks, R.M., Rich, G.T., Lo Turco, V., Picout, D.R., Lo Curto, R.B., Bisignano, G., Dugo, P., Dugo,
G. and Waldron, K.W. 2008. Release of protein, lipid, and vitamin E from almond seeds during digestion.
Journal of Agricultural and Food Chemistry 56: 3409-3416.
Nakada, T., Nishimoto, T., Chaen, H., & Fukuda, S. 2003. Kojioligosaccharides: Application of kojibiose,
phosphorylase on the formation of various kojioligosaccharides. In G. Eggleston & G. L. Cote (Eds.).
Oligosaccharides in food and agriculture. Washington, DC: American Chemical Society. 849: 104–117.
Pavia, D. L., Lampman, G. M., Kriz, G. S.& Vyvyan, J. R. 2009. Introduction to Spectroscopy.USA: Book/cole
Cengage learing.
Roberfroid, M.B. 2000. Chicory fructooligosaccharides and the gastrointestinal tract. Nutrition. 16, 677–679.
Robertson, J. A., Ryden, P., Botham, R. L., Reading, L., Gibson, G. R., & Ring, S. G. 2001. Structural properties
of diet-derived polysaccharides and their influence on butyrate production during fermentation. British Journal
of Nutrition. 81, S219–S223.
Vidanarachchi, J.K., Iji , P.A., Mikkelsen, L.L., Sims, I., Choct, I. 2009. Isolation and characterization of water-
soluble prebiotic compounds from Australian and New Zealand plants. Carbohydrate Polymers. 77: 670–676.

324
 IFRC 2017: 129-212 Functional Food
Effect of Taste Genetic Determinants on Oral Fatty Taste Sensitivity and Perception among Obese and
Non-Obese Subjects
1, 3Ahmad Riduan Bahauddin, 1Roselina Karim, 1Nazamid Shaari and 2Zalilah Mohd Shariff

1 Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43000,
Serdang, Selangor, Malaysia
2 Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia,
43000 Serdang, Selangor, Malaysia
3 Faculty of Food Science and Nutrition, Universiti Malaysia Sabah, Jln. UMS, 88400 Kota Kinabalu, Sabah,
Malaysia

Several studies on oral fat sensitivity and acceptance were associated with variant of CD36 gene, genetic ability
in tasting 6-n-propylthiouracil (PROP) and also obesity risks. However, little is known about those relationship and
function between lean and obese individuals. Therefore, this research is carried out to analyse the relationship
between CD36 gene variants and PROP taster status towards fatty taste sensitivity and preference amongst lean
and obese individuals. A total of 88 obese and 92 lean subjects aged 20-45 were classified into PROP
nontasters, medium tasters, or supertasters by using PROP filter paper screening procedure. Suprathreshold
sensitivity for linoleic acid solutions, intensity and liking rating towards 2 food products (gravy dessert and
pudding) at different fat content was assessed using general/hedonic Labeled Magnitude Scales. All the subjects
were genotyped for CD36 gene variants (SNPs: rs1761667, rs152748 and rs1049673). Overall, obese subjects
had a higher degree of acceptability for higher concentrated fatty foods compared to lean subjects. Oral fatty
sensitivity was correlated to CD36 gene variant (SNPs: rs1761667) and PROP taster status (p<0.05). Subjects
with AA homozygous for rs1761667 and also supertaster had lower acceptability towards high fatty content
product compared to G allele carriers for rs1761667 in both lean and obese subjects (p<0.05). These findings
suggested that PROP taster status and CD36 gene variation could play a significant role in oral fatty sensitivity
and perception in obese and non-obese subjects.

Keywords: PROP taster status, CD36 gene variants, Fatty taste Perception, Obesity
*Corresponding author’s email: ammatduew@gmail.com

1.0 Introduction
Dietary fat is the most energy-dense macronutrient and overconsumption of this nutrient has been attributed to
the rising prevalence of obesity. However, the etiology of those individuals’ characteristic; either who are prone to
like fattier or less fatty food products were remain to be elusive. Taste plays a key role in human daily food
preference and food intake. It was speculated that there was a link between individual’s taste perception, food
intake and also weight gain (Donaldson et al., 2009). It has been investigated the most on the ability to detect 6-
n-propyltiouracil (PROP) chemical which appeared to follows incomplete dominance, and further divided the
taster classification into non taster, medium taster and supertasters . Tasters of 6-n-propylthiouracil (PROP) are
more sensitive to fattiness in foods, and showed lower acceptance of foods that are high in these taste qualities
(Tepper, 2008). Recently, a growing evidence showed that variation of human fatty taste perception caused by
genetic variants in taste receptors genes. The CD36 gene polymorphism in human resulting in its decreased
expression and lowering oral fatty taste sensitivity among obese subjects (Pepino et al., 2012). These novel
findings are changing our view on the understanding of obesity developing mechanisms related to taste.
However, no comparative study on lean and obese subjects is available. It would be interesting to know the role
of CD36 gene and PROP taster status in fatty taste perception in both BMI status. Therefore, this present study
aimed to determine the role of CD36 gene variants and PROP taster status in fatty taste perception amongst lean
and obese adults.

325
Materials and Methods

Subjects
A total of 88 obese (30males; 58females) and 92 lean subjects (24males; 68 females) aged 20-45 years were
included in this study. Subjects must be in good health, no chronic diseases, no food allergies, currently not
taking any medications that interfered with taste or olfactory perception, and not pregnant or lactating. They were
excluded if they have eating restraint (score less than 13 for Restrained Scale) and under depression (score less
than 50 for Zung self-rating Depression Scale).

Anthropometry measurement
BMI was calculated by using the formula; BMI = kg/m2. Subjects who had BMI more than 30 were classified as
obese whereas for those who had in between 18.5 and 25 were categorized as normal weight (WHO, 2014).

Sensory Test

PROP Taster Status determination

The PROP and sodium chloride (NaCl) paper disk were used in this part. Preparation of the paper disks and
subject’s PROP taster status were determined according to Zhao et al. (2003).

Suprathreshold Intensity Rating

Linoleic acid emulsion was used stimuli for suprathreshold intensity rating. All five concentrations of linoleic acid
emulsion were prepared according to Martinez-Ruiz et al. (2014) with slight modification. Subjects were
presented with 5 samples of linoleic emulsions sample in 1 oz. plastic cups. Samples were tasted, spat, and
scored for perceived intensity (oiliness) on a 170 mm general Linear Magnitude Scale (gLMS).

Intensity Rating and Hedonic Test

Two food samples; local creamy porridge (‘Bubur Chacha’) and mango pudding were used in intensity and hedonic
rating. For ‘bubur chacha’, coconut milk was used as fat source while evaporated milk was used in mango pudding.
Preliminary test were used to determine the amount of coconut and evaporated milk to be used in sample preparation.
The final amount of added coconut milk for ‘bubur chacha’ are 32%, 40%, 50%, 65% and 80% (v/v) while 25%,
32%,40%,50% and 65% (v/v) evaporated milk for mango pudding. Subjects tasted the food samples; ‘bubur chacha’
and mango pudding’ and rated creaminess level on 170mm gLMS for intensity rating and their liking towards those
food samples on the hedonic gLMS.

DNA Collection and Genotyping

The disposable cytobrush oral swab (Medical Packaging Corporation, USA) was used to collect the buccal cell
specimens from the subjects. DNA extraction was done by using standard commercialized DNA extraction kit. All
the CD36 variants (rs1761667, rs1527483, rs1049673) were genotyped by using Polymerase Chain Reaction–
Restriction Fragment Length Polymorphism (PCR - RFLP) based on Banerjee et al. (2010) with slight
modification.

Statistical Analysis
Data were analysed using SPSS (version 21.0). Chi-square analysis was used to measure the association between
PROP taster status and CD36 gene variants towards BMI status. Two-way repeated measures Analysis of variance
(ANOVA), controlling for age and sex, was used to test the effect of BMI towards oral fatty suprathreshold rating,
creaminess intensity rating and liking rating. Similarly, 2-way repeated measures ANOVA was used to analyse the
effect of PROP taster status and CD36 gene variants on those sensory measures within obese and lean subjects. A
p-value of 0.05 or lower is reported as significant different.

Results and Discussion

To our best knowledge, this is the first study investigating the relationship between 2 potential tasting markers;
PROP taster status and CD36 gene variants towards fatty taste sensitivity and liking in food products among
obese and non-obese subjects. Most studies published to date focused on total BMI value and also involved
aqueous solutions as taste stimuli. Results from this study revealed that there was significant differences between

326
 obese and non-obese subjects for liking rating for both food samples (p<0.05) but not for fatty (oiliness)
suprathreshold rating and intensity rating (Figure 1a, 1b and 1c). A similar interaction effect pattern; BMI x taste
measures were observed which imply that obesity state does not solely influence individuals fatty taste sensitivity.
Further analysis by repeated measures ANOVA revealed that PROP taster status and rs1761667 of CD36 were
associated with taste measures in this study (p<0.05); controlled for age and sex. Supertaster and AA genotype
of rs1761667 subjects had higher fatty (oiliness) taste sensitivity and rated the food samples creamier compared
to other groups. This findings suggested that both taste genotype and phenotype play significant role in human
fatty taste sensitivity. It also revealed why findings from previous study related to body weight and fatty taste
perception were inconsistent. However, only PROP taster status was associated with fatty taste liking rating
among the subjects. Supertaster dislike high fatty content sample compared to other taster groups (Figure 2a and
2b). Through this results, our data support a direct relationship between fat multimodal oral perception and the
genetic ability to perceive the bitter taste of PROP. Ebba et al. (2012) hypothesized that PROP taster had higher
sensitivity could be attributed by density of fungiform papillae between these two groups which in turn affect the
liking rating. In addition, Melis et al. (2015) speculated that the high expression level of CD36 receptor are more
appeared among those subjects who had low density of taste papillae. This could be the caused we failed to
show that CD36 gene could influence fatty taste liking as our study did not take account this factor.

1 (a)
10
Obese Non-Obese
Oiliness Rating (cm)

0
0.04% 0.50% 1.00% 2.10% 4.30%
Added Linoleic Acid

(b) 1 (c)
12 5
Creaminess Rating (cm)

Obese Non-Obese Obese Non-Obese

10
3
8
Liking Rating (cm)

6
1 * * * *
4
2 -1 32% 40% 50% 64% 80% 25% 32% 40% 50% 65%

0 Local Creamy Porridge Mango Pudding

32% 40% 50% 64% 80% 25% 32% 40% 50% 65% -3
Bubur Chacha Mango Pudding Added Coconut Milk/ Evaporated milk
Added coconut milk/evaporated milk -5
Figure 1a Mean suprathreshold rating (oiliness) for 5 samples with the increasing of added linoleic acid between
obese and lean subjects ; Figure 1b and 1c Mean intensity rating (creaminess) and liking rating for 5 samples
with the increasing of added coconut milk/evaporated milk between obese and lean subjects (*p<0.05). Error bars
are SEM.

327
 2.5
4
2
1.5 3

1 2
Liking (cm)

0.5

Liking (cm)
1
0
32% 40% 50% 64% 80% 0
-0.5 25% 32% 40% 50% 65%
Added coconut Milk -1
-1 OB+ST Added evaporated milk
OB+ST -2 OB+MT
-1.5 OB+MT OB+NT
-2 OB+NT -3 Lean+ST
Lean+ST Lean+MT
-2.5 Lean+MT -4 Lean+NT
Lean+NT

2(a) 2 (b)
Figure 2a and 2b Mean liking rating for 5 ‘Bubur Chacha’ and mango pudding samples with the different fat
stimulus content (coconut milk/evaporated milk) among BMI group with different PROP taster status. (*p<0.05).
Error bars are SEM.
*
Conclusion
*
This work contributed to the understanding of the genetic *
factor underlying fatty taste perception among obese
and non-obese subjects. The differences between obese and non-obese individuals on fatty taste perception *
could attributed by the PROP taster status and CD36 gene variant (rs1761667). In spite, genetic effects were
shown to underlie the variation of individuals taste sensitivity, it was inconsistently appeared in taste liking. Thus,
it showed that fatty taste preference could also be modifiable by other factors such as human physiological
system, psychological system and also sensory cues (e.g. texture, viscosity). Further studies are needed to
elucidate the mechanism of human taste acceptance and food intake (including appetite and satiety regulation)
and their linkage to obesity.

References
M. Banerjee, S. Gautam,M. Saxena, H.K. Bid, C.G. Agrawal. 2010. Association of CD36 gene variants
rs1761667 and rs1527483 with type 2 diabetes in North Indian population, International Journal of
Diabetes Mellitus, 2 : 179–183.
Donaldson, L.F., Bennett, L., Baic, S. and Melichar, J.K. 2009. Taste and weight: Is there a link? The American
Journal of Clinical Nutrition. 90: 800S-803S.
Ebba, S.; Abarintos, R.A.; Kim, D.G.; Tiyouh, M.; Stull, J.C.; Movalia, A.; Smutzer, G. The examination of fatty
acid taste with edible strips. Physiol. Behav. 2012, 106, 579–586
Martínez-Ruiz, N. R., López-Díaz, J. A., Wall-Medrano, A., Jiménez-Castro, J. A., & Angulo, O. 2014. Oral fat
perception is related with body mass index, preference and consumption of high-fat foods. Physiology &
behavior, 129, 36-42.
Melis, M., Sollai, G., Muroni, P., Crnjar, R., & Tomassini Barbarossa, I. 2015. Associations between orosensory
perception of oleic acid, the common single nucleotide polymorphisms (rs1761667 and rs1527483) in
the CD36 gene, and 6-n-propylthiouracil (PROP) tasting. Nutrients, 7(3), 2068-2084.
Pepino MY, Love-Gregory L, Klein S, Abumrad NA. 2012. The fatty acid translocase gene CD36 and lingual
lipase influence oral sensitivity to fat in obese subjects. J. Lipid Res. 53: 561–6.
Zhao, L., Kirkmeyer, S. V.&Tepper, B. J. 2003. A Paper Screening Test to Assess Genetic Taste Sensitivity to 6-
N-Propylthiouracil. Physiology and Behavior. 78(4–5): 625 – 633.

328
 IFRC 2017: 133-091 Functional Food

Antioxidant Activity of Tea Formulation of Leaves of Leucaena Leucocephala (Lam) de Wit, Soursop
(Annona muricata L.) and Bay Leaf (Syzygium polyanthum) with Black Tea

*Rosida, D.F., Putri, C.A., Murtiningsih

Department of Food Technology

University of Pembangunan Nasional "Veteran" Jawa Timur, Surabaya
Jalan Raya Rungkut Madya Gunung Anyar Surabaya 60294 Indonesia
Abstract
Tea drinks contain natural antioxidants, namely flavonoids that can keep the body from the threat of free radical
attack. Flavonoids are antioxidants compound that can help prevent cardiovascular disease. Antioxidants are
beneficial to protect the body from the harmful effects of free radicals. Tea drinks can be produced from plant
leaves that contain many bioactive compounds as phenol and flavonoid. White leadtree or leucaena leaf, soursop
leaf and bay leaf contain many bioactive compounds so widely used for traditional medicine. This study aimed to
determine the ability of antioxidant activity of tea derived from three types of leaves that were formulated with
black tea. This study used a complete randomized design with two factors. The first factor, leaf type: leucaena,
soursop dan bay leaf. The second factor, fermentation time: 1 h; 3 h and 5 h. The best judgment in terms of taste
and aroma was obtained leaf of leucaena with fermentation 1 h was formulated with black tea (70:30) had an
optimal bioactive compound had antioxidan activity 90.83%, total phenol 454.50 ppm , flavonoids 111.83 ppm,
ash 6.19 %, water 8.85%, value L, a, b 39.0; 14.15; 27.45 respectively.

Keywords: functional foods, tea, leucaena, soursop, bay leaf, antioxidant

*Corresponding author’s email: rosy.upnsby@gmail.com; dedin.tp@upnjatim.ac.id

Introduction
Tea is one of the most popular types of functional drinks. Antioxidant ability is useful to protect the body from the
bad effects of free radicals (Cao et al, 1996). Tea can prevent stroke (Weisburger 1996); Lowered the risk of
osteoporosis (Fujita 1994 and Kao et al. 1995); Tea provides protection against liver disease (Imai et al 1995).
Tea drinks can be produced from the leaves of other plants that contain many bioactive components (tannins,
phenols and flavonoids). Leaf leucaena is one of the plants used as a traditional medicine (Radji, 2005).
Leucaena leucophala L. leaf extract had total phenol of 581.64 ppm and antioxidant activity of 76.82% (Rosida et
al 2016), while antioxidant activity of soursop leaf (Annona muricata L.) in tea sample with drying treatment 150
Minute that is equal to 76.06% (Adri and Hersoelistyorini 2013). Ginger tea (Syzygium polyathum) and ginger
filtrate (70:30) with the addition of 10% secang wood filtrate have a total phenol content of 213.13 ppm and
antioxidant activity of 80.63% (Palupi and Widyaningsih 2015). The bay leaf (Syzygium polyanthum) contains
tannins, essential oils (salamol and eugenol), flavonoids, sesquiterpenes, triterpenoids, phenols, strawids, citral,
lactones, saponins and carbohydrates (Fitri, 2007).
Functional beverage can be done by fermentation process or enzymatic oxidation. Enzymatic oxidation is the
oxidation process of polyphenol compounds with the help of polyphenol oxidase enzymes. The substance will
determine the properties of strenght, color, quality, and briskness. The fermentation process results in the
oxidation process in which the catechin changes into simpler compounds are polyphenol flavonoid compounds
(Fulder, 2004). A functional drink of leaves of leucaena, soursop and bay is done using processing black tea by
fermentation orthodox. The purpose of this study was to determine the physicochemical properties and
antioxidant activity of tea formulation (leucaena, soursop and bay leaf formulated with black tea.).

Material and Methods

Sample preparation
fruit peel waste were collected from region of Engineering Faculty, University of
Pembangunan Nasional Veteran Jawa Timur, Indoensia. This research used Completely Randomized Design,
which was arranged with factorial pattern, consist of two factors. The first factor was a leaf type (leaf leucaena,
soursop and bay). The second factor was fermentation time (1 h, 3 h and 5 h). Data obtained from the analysis
results were processed using Analysis of Variance.

329
Production of tea powder from leucaena, soursop and bay leaf
The Leaves (leuceana, soursop, bay) were carried off at room temperature for 8 h. In the leucaena leaf prior to
lavage there was a special treatment with moist heating, ie leucaena leaf inserted into a plastic bag and then
heated in a water bath at 70 ° C for 15 minutes. The reaction was stopped by aerated to reduce mimosin.The
leaves powder were fermented at room temperature with time for (1 h, 3 h and 5 h). The tea powder was dried at
105 ° C for 25 minutes. Dry leaf powder was formulated with black tea with a ratio of 70: 30 (w/w). Tea filtrate
was testing: water and ash content (AOAC), phenol, flavonoids and antioxidant activity.
The antioxidant activity was determined DPPH method Astadi et al. (2009)
Total phenolic content (TPC) was determined spectrophotometrically using Folin–Ciocalteu reagent by the
method of Andarwulan et al. (1999)
Total Flavonoid content was determined spectrophotometrically using katekin as standar (Sahreen et al., 2010)

Results and Discussion

Water content of formulation tea powder from three leaves ranged from 8.45 to 10.52%. The lowest water content
was obtained from leucaena leaf (8.45%). The ash content ranged from 4.06 to 6.98%. The lowest ash content
was obtained from the bay leaf (4.06%), while the highest ash content was obtained from leucaena leaf (6.98%).
The total phenol of soursop leaf was 171,35 ppm (Handayani et al 2016), bay leaves 163,27 μg / ml CGAE
(Palupi et al 2015) and leucaena leaf extract of 581,64 ppm (Rosida et al 2016). Total phenol highest of tea
formulation was obtained from 1 hour fermentation time (438,12 ppm) (Table 1). The longer fermentation
decreased phenol and flavonoids in tea drink. this was caused during the fermentation process occurs enzymatic
oxidation to phenol to change into theaflavin and thearubigin. Kukhtar (2007) stated that fermentation has an
effect on total phenol because the fermentation process resulted in the loss of some components due to the
enzymatic oxidation reaction. Health-beneficial substances change or disappear during the process of producing
black tea (Fulder 2004). The total of flavonoid tea drink ranges from 62.64 to188,78 ppm. Tea drinks of soursop
leaves had a larger flavonoid content than bay and leucaena leaves.

Table 1. Bioactive compound of tea formulation drink from three leaves types
Leaves type fenol (ppm) Flavonoid (ppm) Antioxidant activity (%)
Leucaena Leucocephala 398.41 ± 63.03a 104.00 ± 9.48b 89.82 ± 0.93a
Annona muricata L.(soursop) 431.33 ± 42.95a 188.78 ± 15.29a 90.30 ± 0.88a
Syzygium polyanthum (bay) 308.91 ± 68.27b 62.64 ± 5.34c 89.78 ± 0.73a
Fermentation time (h)

1 438.12 ± 39.27a 128.11 ± 63.31a 90.87 ± 0.31a

3 380.67 ± 75.58b 117.36 ± 56.44ab 89.99 ± 0.34b
5 319.88 ± 66.81c 109.94 ± 53.68b 89.04 ± 0.42c
Values are the mean ± SD (n=3); means that do not share a same letter are significantly different (p<0.05) as
measured by DMRT test.

The antioxidant activity of tea formulation drinks ranged from 89.78 to 90.87%. The highest antioxidant activity
was obtained from soursop leaf treatment (90,30%). High levels of antioxidant activity of tea drinks influenced
from the total of phenol and flavonoids. The greater phenol and flavonoids caused to increased the antioxidant
activity in tea drinks (Table 1). The longer fermentation time decreased antioxidant activity The decreasing of
bioactive components such as tannins in tea during the fermentation process (enzymatic oxidation) caused lower
antioxidant power, because the tannin has oxidized to theaflavin. Furthermore, the antioxidant capacity of food
samples not only depends on the antioxidant level, but also synergy occurring between the antioxidant
compounds and other plant constituents (Zheng and Wang, 2001). The increase in antioxidant capacities
indicated that the formation of new antioxidant compounds after cooking is greater than the degradation of
antioxidant compounds by heat (Hanis Mastura et al 2017).
Formulation of tea drinks with soursop leaf tend a darker color compared to leucaena leaf. The total of
polyphenols that gives the appearance of the color of the steeping tea toward the chocolate to the hammock. The
value a of tea drink ranged from 13.31 to 15.48 (Table 2) showed that the color of tea drink was reddish. The
value b of tea drink ranged from 25.51 to 27.46 (Table 2) which means the yellow color of tea drink. Differences in
the color of the three types of leaves caused by several factors namely age and leaf component content.

330
 Table 2. Color values (L, a and b) on three types leaves of tea drink
Leaves type L a b
Leucaena Leucocephala 37.95 ± 1.16a 13.31 ± 0.96b 26.73 ± 0.85ab
Annona muricata L.(soursop) 35.15 ± 0.86b 15.48 ± 0.64a 27.46 ± 1.01a
Syzygium polyanthum (bay) 35.58 ± 1.08b 14.16 ± 0.66b 25.51 ± 0.92b
Fermentation time (h) L A B
1 37.13 ± 1.59a 15.01 ± 0.95a 27.45 ± 1.10a
3 36.35 ± 1.58ab 14.30 ± 1.06ab 26.58 ± 1.03ab
5 35.20 ± 1.19b 13.65 ± 1.22b 25.68 ± 0.88b
Values are the mean ± SD (n=3); means that do not share a same letter are significantly different (p<0.05) as
measured by DMRT test.

The longer fermentation time, the color of the tea drink tend to be darker, slightly redder and slightly yellowish
than with short fermentation time. The smaller the value of lightness, the brightness will decrease, due to changes
in the color of the tea the more brown. The resulting color due to this enzymatic oxidation process was a brownish
yellow to reddish color. Theaflavin is a compound that contributes to the color of red tea. The reddish and
thearubigin plays a role in determining the steadiness of color steeping brownish red tea a bit dark. Organoleptic
test resulted in the formulation of tea with leucaena leaf most favorable to taste and aroma of tea

Conclusion
The most preferred tea formulation drink was leucaena leaf and black tea (70:30) with 1 h fermentation duration
having antioxidant activity of 90.83%, total phenol 454.50 ppm, flavonoid 111.83 ppm, ash content 6.19%, water
content 8.85%. The value of L, a, b was 39.0; 14.15; 27.45 respectively.

Acknowledgment
The authors would like to express their sincere thanks to the Directorate General of Higher Education, Ministry of
Education and Culture through the research competitive of national strategy (STRANAS) 2015-2017

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Dermatology Case Western Reserve University Cleveland, OH-44106, USA.
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polyantha) with addition of filtrate gingger and secang wood filtrate. Journal of Food and Agro-industry Vol
3 (4): 1458-146.
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Carissa opaca fruits. J. Food Chem 122: 1205- 1211.
Weisburger, J.H. 1996. Tea antioxidants and health. In: Cadenas E, Packer L, eds. Handbook of antioxidants.
New York: Marcel Dekker, pp. 469-486.
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 IFRC 2017: 135-094 Functional Food

Sabah Snake Grass (SSG) Pearls for Food Application

Kong, H. S., Mohd-Kasim, Z. and *Abdullah Sani, N.

Food Science Programme, School of Chemical Sciences and Food Technology,

Faculty of Science and Technology, Universiti Kebangsaan Malaysia,
43600 UKM, Bangi, Selangor, Malaysia.

Abstract
This study focused on market survey of eating behaviour, product development and sensory evaluation of a
product based on Clinacanthus nutans (Sabah Snake Grass).The market survey was conducted in Universiti
Kebangsaan Malaysia (UKM) amongst 286 respondents. Three types of SSG pearls were produced and sensory
evaluation took place at the Food Pilot Plant, Faculty of Science and Technology, UKM, which is certified with
Good Manufacturing Practice (GMP). Acceptance test (line scale) was carried out. Sixty untrained panellists were
selected to evaluate the SSG pearls according to a hedonic scale ranging from 1 (dislike very much) to7 (like very
much) for colour, aroma, taste, elasticity, juiciness, foreign flavour and overall acceptance. SSG pearls were
labelled with control, A and B according to their formulations. Based on the survey results, C.nutans were not
well-known among the respondents. However, respondents were willing to try on new product because it has
benefits toward health. For acceptance tests, there was higher score in colour, aroma, elasticity, foreign flavour
and overall acceptance for sample B. However, sample A scored higher in taste and juiciness. The SSG pearls
have great potential to be commercialized as a functional food productand applied in healthy drinks.

Keywords: Clinacanthus nutans, survey, acceptance test, sensory evaluation, Sabah Snake Grass
*Corresponding author’s email: norrasani@ukm.edu.my, zalifah.kasim@ukm.edu.my

Introduction
Clinacanthus nutans (Sabah Snake Grass; SSG) is a traditional medicinal herb, which is found in Malaysia. C.
nutans has been listed as research target in NKEA Malaysian Herbal Monograph 2012 and EPP#1 Research
Grant Scheme. C. nutans (Burm.f.) Lindau is used for home decoration, tea and bath (Siew et al., 2014). The
washed leaves of C. nutans (Burm.f.) Lindau can be freshly eaten or blended with apple and drink as fruit juice.
C. nutans is also used to treat skin infections, insect and snakebites and swellings due to fall or boils
(Chiwapreecha et al., 2014). Some of the local companies have developed commercial products such as drinks
(tea bag, mix herbs, fruit mixed) or concentrated extract. A new approach on applying SSG extracts in food is
being considered. Tapioca pearl with SSG extract was proposed. The raw material for producing tapioca pearl is
tapioca starch, which is commonly used in food industry for thickener due to its high viscosity, clear appearance
and low production cost (Pongsawatmanit et al., 2007). Tapioca pearl is known as boba, or bubble (in bubble
tea). It is a common ingredient which is found in desserts and drinks such as sago soup, Taro coconut, tapioca
desserts, milk tea with tapioca pearls or tapioca pudding. Several flavours for pearl products that can be found in
current market are red bean and yam. There is a great window of opportunity on producing pearl with herbal
extract because of its uniqueness. This study focused on market survey of eating behaviour, product
development and sensory evaluation of the new product based from SSG.

Material and Methods

Survey questionnaire
The survey was conducted in UKM. There were eleven questions regarding to the eating behaviour of tapioca
pearl such as frequency of consumption, aspects of pearl, interest towards new product and reason of
purchasing. The analysis was based upon 286 responses received from members of UKM with the age range of
16 and 43 years old. Among 286 valid responses, there were 73.4% of the responses from female.

New product development

The SSG extract was received from You Dun Chao Healthcare Products Sdn. Bhd., Malaysia. The fresh leaves
and stems of SSG were dried up using drum-dried method and extracted with water in the ratio of 50:50. Then,

333
the water extract of SSG was mixed with maltoglucidex in the ratio of 3:7 and stored in powder form. Three
treatments were applied in SSG pearl making: without extract and two different concentrations of SSG extract.
Tapioca starch (Brand: CAP KAPAL ABC), water and rock sugar (Brand: Giant) were prepared in the ratio of
11:8:1 and mixed well. The dough was rolled into spherical beads and placed in the pot with flour for avoiding
them from sticking to each other. Then, they were placed into boiling water. The SSG pearl was cooked until soft
and ready to eat. They were stored in cooled sugar syrup to prevent the pearls from adhering to one another.

Sensory evaluation
Sensory evaluation took place at the Food Pilot Plant, Faculty Science and Technology, UKM, which are certified
with Good Manufacturing Practice (GMP). Acceptance test (line scale) was carried out. A sixty untrained panelists
were selected to evaluate the SSG pearls according to a hedonic scale ranging from 1 (dislike very much) and 7
(like very much) for colour, aroma, taste, elasticity, juicy, foreign flavour and overall acceptance. The SSG pearls
were labelled with control, A and B.

Statistical analysis
The data obtained were statistically analysed using SPSS Version 22 (Chicago, Inc.).

Results and Discussion

About 97.2% of the UKM members heard about tapioca pearl before (94.7% male and 98.1% female). A 68.9% of
them like to consume pearl, which can be found in drink (72.4% male and 67.6% female). Respondents were
focusing more on taste (81.8% in total), followed by price, healthiness, ingredient, appearance and aroma with
39.2, 35.0, 32.5, 23.8 and 17.5% in total respectively (Figure 1). Fruits were preferred by 86.4% of respondents,
followed by herbal-based (25.2% in total) and vegetables (7.7% in total) to be added into pearl (Figure 2). About
52.8% of the respondents would like to try on pearl with herbs (55.3% male, 51.9% female). If there is pearl with
herbs, respondents were concerned more on taste (85.0% in total), followed by aroma, colour and appearance
with 50.0, 18.2 and 15.0% in total respectively (Figure 3). About 29.0% of respondents did not know about C.
nutans herb. However, 38.5% of the respondents were willing to try and purchase pearl with C.nutans extract
because of wanting to try new things, to taste new flavour and health issue. Control sample scored higher in
colour, taste, elasticity, juiciness and overall acceptance than sample A and B. Sample B scored higher in aroma
and foreign flavour than others. In comparison between sample A and B, there were higher scores in colour,
aroma, elasticity, foreign flavour and overall acceptance for sample B. While, sample A scored higher in taste and
juiciness (Figure 4).

Figure 1. Important aspects of pearl on consumers’ preferences (%)

334
 Figure 2. Preferred ingredients to be mixed with pearl (%)

Figure 3. Concerns of consumers on pearl with herbs (%)

Figure 4. Result of acceptance test of the SSG pearl

Conclusion
Based on the results, C. nutans is not well-known by society, which can be considered as under-cultivated on its
usage. However, respondents were willing to try on new product because of its benefits towards health. For
acceptance tests, there were higher score in colour, aroma, elasticity, foreign flavour and overall acceptance for
sample B. While, sample A scored higher in taste and juiciness. Further analysis should be carried on the
identification of phytochemical compounds which contributed on the antioxidant activity of SSG pearl.

335
Acknowledgements
The authors would like to thank the Ministry of Education and Universiti Kebangsaan Malaysia for the financial
support towards this research via FRGS/1/2014/STWN03/UKM/02/1 and DIP-2014-007 respectively.

References
Chiwapreecha, B., Janprasert, K. and Kongpakdee, C. 2014. Comparative Anatomy of Three Medicinal Plants in
Acanthaceae. Acta Horticulturae1023: 229-232.
Pongsawatmanit,, R., Temsiripong, T. andSuwonsichon, T. 2007. Thermal and Rheological Properties of Tapioca
Starch and Xyloglucan Mixtures in the Presence of Sucrose. Food Research International40( 2): 239–248.
Siew, Y.Y., Zareisedehizadeh, S., Seetoh, W.G., Neo, S.Y., Tan, C.H. and Koh, H.L. 2014. Ethnobotanical
Survey of Usage of Fresh Medicinal Plants in Singapore. Journal of Ethnopharmacology155(3): 1450-
1466.

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 IFRC 2017: 137-122 Functional Food
Development of Convenient Fruit Bars as Sources of Dietary Fiber and
Potassium from Thai Fruits

Racha Saiprasongsin, *Visith Chavasit and Aurawan Kettawan

Institute of Nutrition, Mahidol University, Salaya, Nakhon Pathom 73170,Thailand

Abtract

Healthy diet should include at least 400 grams of fruits and vegetables per day, which are the main sources of
dietary fiber, potassium and antioxidants. To fulfill such requirement, it may not be convenient for most people of
these modern competitive societies. The objective of this study was to develop fruit bars using dried Thai fruits
available in the market as sources of dietary filber, potassium and antioxidants with no sugar added. Nam-wa
banana (Musa sapientum Linn.), Leb-mu-nang banana (Musa sapientum Linn. (AA group)), longan (Dimocarpus
longan), lychee (Litchi chinensis Sonn.), tamarind (Tamarindus indica L.), desiccated defatted coconut meat
(Cocos nucifera L.), tomato (Psidium guajava Linn.) and mulberry (Morus nigra L.) were used in the formulation.
Fruit bars of 6 formulas were produced after the dried fruits had been disintegrated, shaped and dried in a hot air-
oven at 70oC for 3 h. The developed fruit bars contained 7.71-8.24% moisture with 0.49-0.51 water activity and
618-679 g hardness. Per 50 g serving, they contained 5.40-5.62 g total dietary fiber, 25.23-27.38 g sugar, 361-
488 mg potassium and antioxidant activities as ORAC and FRAP at 1946-4506 and 671-1671 µmolTE/100g DW,
respectively. Sensory overall acceptability scores on 9-point hedonic scale (n=50) were 7 (like moderately) and
not significantly different (p < 0.05). Per one serving (50 g), a fruit bar contained solid content and antioxidant
activity equilibrium to approximately 200 g and 50 g of fresh fruits, respectively. The products were high in fiber
and good source of potassium.

Keyword: fruit bar, Thai fruit, dietary fiber, potassium, antioxidant activity
*Corresponding author’s email: vchavasit@gmail.com

Introduction

Food and nutrition are the key strategy for reducing the risk of NCDs. For healthy life, WHO (2013) recommended
to take at least 400 gram of fruits and vegetables per day since they are the main sources of dietary fiber,
antioxidants, vitamins and minerals especially potassium. Unfortunately, most people in this world still cannot
follow this recommendation including Thailand, a fertile land of fruits and vegetables. The 4th National Health
Survey of Thai people in 2008-9 also indicated that up to 76.2% of the Thais aged > 15 years old could not
consume adequate fruits and vegetables (Aekplakorn et al., 2011).
Fruit bar can be a convenient source of fruits and vegetables for the expanding urbanized society since it is
ready-to-eat and shelf stable at room temperature. Fruit bar can be produced from substandard fruits and
vegetables as well as their by-products, thus it is also quite economical. The feasibility in developing fruit bars by
using available dried Thai fruits in the market as convenient sources of dietary fiber, potassium and antioxidants
was therefore studied.

Material and Methods

Ingredients
Commercial dried Thai fruits with no sugar-added i.e. Namwa banana, Leb Mu Nang banana, tomato, mulberry,
longan, lychee, tamarind were obtained from stores in Bangkok and suburban areas. Desiccated defatted
coconut meat was by-product from coconut milk production (provided by Theppadungporn coconut Co., Ltd.,
Nakhon Pathom, Thailand). The fruits were analysed for dietary fiber, potassium, sugar, moisture content,
antioxidant activity as well as defined for their unique sensory characteristics (no data reported).

337
Formulation and production
The formulation aimed for no sugar-added fruit bar of weight 50 g that could be good sources of dietary fiber and
potassium as well as having antioxidant activity. Totally 6 formulas were developed by using Microsoft ExcelTM
program. The selected and weighed dried fruits were mixed, disintegrated in an electrical blender, spread onto a
tray and cut into rectangular shape of 11.0 cm long, 3.0 cm wide and 1.0 cm thick. The fruit bars were then dried
in a hot air oven at 70 °C for 3 h, cooled down to room temperature, packed in heat-sealed aluminum laminated
plastic bag, and kept at 4°C for further analysis. The products were analysed for chemical properties (as shown in
Table 1) as well as water activity, color, texture and sensory acceptability.

Analysis
Moisture, total dietary fiber and potassium were analyzed by using the methods of AOAC, 2005. Sugar content
was determined as total reducing sugar (AOAC, 2005). Antioxidant activities were determined as Oxygen radical
absorbance capacity (ORAC) and Ferric reducing antioxidant power (FRAP) in the methanol/water extract of the
sample (Ou et al., 2002). Water activity was determined on a water activity meter (Novasina MS1 AW, Novasina
AG, 8853, 118 Lachen, Switzerland) at 25 ± 1°C. Color was measured using the Hunter Lab system with a
colorimeter. Texture was measured as hardness using a three-point bend rig (HDP/3PB) probe on the Texture
Analyzer model TA.TX Plus (Stable Micro Systems, Ltd., UK). Sensory acceptability was conducted with 50
untrained panelists on 9-point hedonic scales (9, like extremely; 5, neither like nor dislike; 1, dislike extremely)
for overall acceptability, general appearance, color, taste, odor and texture. Statistical analyses were performed
by using SPSS software version 19. Significance difference (p = 0.05) was determined by using One way
analysis of variance (ANOVA) and Duncan’s multiple range for all tests except for sensory analysis that was
analyzed by Kruskal-Wallis test.

Results and Discussion

Thai dried fruits available in the market that were added with sugar in candied form were not selected for this
study. The selected dried fruits as shown in the materials and method can be dominated in different beneficial
characteristics. During the dehydration process, moisture contents of the fresh fruits were reduced from
approximately 90% down to approximately 10-15%, which concentrated all nutrients. However, plant’s secondary
metabolites could be lost due to oxidation (Kliebenstein, 2004). Most of the selected dried fruits had brown color
of different tones due to the browning reaction. However, certain fruits provided unique colors such as red from
dried tomato, deep purple from mulberry. Each fruit had its unique aroma and taste, which after mixing together
was expected to provide unique palatability to the product. Difference from others, desiccated defatted coconut
meat had bright white color, bland flavor and provided dried irritating mouthfeel from the high dietary fiber content.
Six formulas of fruit bars were developed as shown in Table 1. Three main ingredients were included in all
formulas i.e. (i) banana of either kind as source of potassium, (ii) dried coconut meat as a source of dietary fiber
and (iii) tomato (containing lycopene) or Mulberry (containing anthocyanin) as a source of antioxidant. As
considering the solid contents,

338
 Table 1 Physical properties, certain nutrients, antioxidant activities and sensory acceptability of the developed fruit bars 1,2,3

Physical and chemical properties Sensory acceptability5

Formulas6 Hard1,2 Color2 TDF1,4 K1,4 TS1,4 ORAC1,4 FRAP1,4 OA1 GA1 Color Taste Odor Texture
AW
(N) L* a* b* (g) (mg) (g) (µmolTE) (µmolTE)
(F1) Nam Wa banana
30%,Longan 23.3%, 6.18 27.2 4.98 7.94 6.94 6.68 7.00 7.06 7.10 6.84
0.49a 5.60 360.71b 27.38 3251.56b 1123.06c
Lychee 30%,Tamarind (0.03)a (0.11)a (0.02)b (0.09)a (1.11)a (1.12)a (1.03)a (1.20)a (1.02)a (1.36)a
10%,Coconut meat 6.7%
(F2) Leb Mu Nang banana
30%,Longan 23.3%, Lychee 6.06 28.19 4.56 8.28 7.12 6.6 6.84 7.06 7.04 7.14
30%,Tamarind 10%,Coconut 0.51a 5.40 423.00a 26.92 1946.35c 767.26d
(0.04)a (0.02)a (0.01)b (0.10)a (1.22)a (0.99)a (1.24)a (1.15)a (1.23)a (1.23)a
meat 6.7%
(F3) Nam Wa banana
23.3%,Longan 23.3% Lychee
6.15 27.05 5.56 7.51 7.12 6.74 6.96 7.10 7.04 7.12
23.3%,Tamarind 0.51a 5.62 366.00b 26.00 3028.04b 948.03c
(0.02)a (0.14)a (0.13)a (0.15)a (0.96)a (1.06)a (1.01)a (0.90a (1.18)a (1.04)a
10%,Coconut meat
6.7%,Tomato 13.4%
(F4) Nam Wa banana
23.3%,Longan 23.3% Lychee
6.40 22.11 1.73 0.98 6.78 5.92 5.66 6.98 6.42 6.86
23.3%,Tamarind 0.50a 5.58 364.50b 25.59 4506.41a 1671.05a
(0.06)a (0.08)b (0.05)c (0.04)b (1.31)a (1.55)b (1.69)b (1.15)a (1.49)a (1.40)a
10%,Coconut meat
6.7%,Mulberry 13.4%
(F5) Leb Mu Nang
banana 23.3%,Longan
23.3%,Lychee 6.66 27.66 5.70 7.89 7.02 6.94 7.08 6.66 7.02 7.06
0.50a 5.58 396.00b 25.59 1946.01c 671.24d
23.3%,Tamarind (0.04)a (0.14)a (0.04)a (0.03)a (1.19)a (1.04)a (0.97)a (1.24)a (1.04)a (1.15)a
10%,Coconut meat
6.7%,Tomato 13.4%
(F6) Leb Mu Nang banana
23.3%,Longan 23.3%,Lychee
6.37 21.33 1.21 0.48 6.92 6.08 6.00 6.68 6.66 6.96
23.3%, Tamarind 0.49a 5.53 488.44a 25.23 3424.39b 1394.27b
(0.03)a (0.01)b (0.05)c (0.03)b (1.68)a (1.47)b (1.85)b (1.72)a (1.72)a (1.72)a
10%,Coconut meat
6.7%,Mulberry 13.4%
1Hard = Hardness, TDF = Total Dietary fiber, K = Potassium, TS = Total Sugar, ORAC = Oxygen Radical Absorbance Capacity, FRAP = Ferric reducing antioxidant power,
OA = Overall acceptability, GA = General appearance
2Mean (SD) (n=5); 3Means within the same column of different superscripts are significantly different (p<0.05).

4Number in column refers to per serving of 50 g.

5Nine-points hedonic scale (9,like extremely; 5, neither like nor dislike; 1, dislike extremely).

6Weight (g) of fresh fruits per serving (50 g) : F1 = 171.71, F2 = 172.72, F3 = 251.45, F4 = 199.86, F5 = 252.46 and F6 = 200.87

339
one serving (50 g) of the developed fruit bars was equivalent to approximately 200 g of fresh fruits, which was half of
what recommended for daily intake by FAO/WHO (FAO, 2009; WHO, 2013). Pectin in bananas also functioned as
natural binder to the fruit bar. Dried longan lychee and tamarind were mainly used for flavoring; however they
stillcontributed reasonable amounts of potassium and antioxidant activity. Water activities of the developed products
were < 0.6, which did not allow growth of any living microorganism (Lenovich,1992).
The hardness values of all fruit bars were comparable to cereal/nut bar with dried fruits, which were 6-7 N (Ryland
et al., 2010). Colors of most products were light brown to brown except for the products added with mulberry that had
dark purple color. Even no added, sugar in one serving contributed up to 50% of limit recommended for daily
consumption of FAO/WHO (50 g per day). Per serving, the fruit bars provided about 5.5 g of dietary fiber and 400
mg of potassium, which could also be claimed as “High Fiber” and “Source of Potassium” (FAO, 2009). The highest
antioxidant activities were found in the products added with Mulberry, which even dehydrated were only comparable
to the levels found in the fresh fruits of the same weight (Ou et al., 2002). Overall, the developed fruit bars were
moderately acceptable with the score of 7 and not significantly different among the products (p<0.05). The dark color
of mulberry lowered scores for general appearance and color of their products. Taste, odor and texture were also
preferred at the moderate level and not significantly different among the products (p<0.05).

Conclusion

Dried Thai fruits could be used for producing fruit bars as convenient sources of dietary fiber, potassium and
antioxidant with no sugar added. The products were moderately acceptable by consumers. By using substandard
fruits and by-products, costs of the developed fruit bars should be affordable for most people.

References

Aekplakorn V., Satheannoppakao W., Kasemsap R., Pakcharoen H., 2011. National health examination survey IV
2008-2009: Food consumption of Thai population. Health System Research Institute and Bureau of Policy and
Strategy. Ministry of Public Health. Bangkok, Thailand.
AOAC. 2005. Official Methods of Analysis. 18th ed. Washington, D.C. USA.
Food and Agriculture Organization (FAO) of the United Nations, 2009. Retrieved on January 20, 2016. Guideline for
use of Nutrition and Health claims from Website: www.fao.org/input/download/standards/351/CXG_023e.pdf
Kliebenstein, D. J. 2004. Secondary metabolites and plant/environment interactions: a view through Arabidopsis
thaliana tinged glasses. Plant, Cell & Environ. 27, 675–684
Ou B., Huang D., Hampsch-Woodill M., Flanagan AJ. and Deemer KE. 2002. Analysis of Antioxidant Activities of
Common Vegetables Employing Oxygen Radical Absorbance Capacity (ORAC) and Ferric Reducing
Antioxidant Power (FRAP) Assays:  A Comparative Study. J. Agric. Food Chem. 50(11): 3122–3128.
Lenovich L. M. 1992. Water activity: microbiology. In: Hui YH (ed) Encyclopedia of Food Science and Technology.
John Wiley & Sons, New York.
Ryland D., Vaisey-Genser M., Arntfield DS., Malcolmson JL. 2010. Development of a nutritious acceptable snack bar
using micronized flaked lentils. Food Research International. 43: 642-649.
World Health Organization, 2013. Global action plan for the prevention and control of noncommunicable diseases
2013-2020. Geneva, Switzerland.

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 IFRC 2017: 160-117 Functional Food
Profile of Antioxidant in Dark Chocolate Product that Enriched with Herbs

Suprayatmi,M1*., Hutami, R1., Tiastadia, I.P2., Purnamasari, D1

1 Department of Food Technology, Faculty of Halal Food Science, Djuanda University, Jl. Tol Ciawi no. 1
Bogor, Indonesia
2Saraswanti Indo Gentech (SIG) Laboratory, Jl. Rasamala 20, Taman Yasmin, Bogor
.

Abstract
This study was conducted to determine the profile of antioxidant from three processed products of Indonesian dark
chocolate compounds which were powdered with herbs. The herbs were mint (Mentha cordifolia), green tea
(Camellia sinensis), and lime (Citrus aurantifolia) with 4 percent concentration respectively. The capacity of
antioxidant was analyzed using DPPH analysis. Mint chocolate indicated the highest activity of antioxidant with an
IC50 value of 68.63 ppm. The profile of antioxidant was analyzed using Liquid Chromatography - Mass Spectrometry
with the Mass Analyzer Manifold Time of Flight (LC-MS ToF) methods. At this stage a total of 27 antioxidant
compounds have been identified in dark chocolate control and 73 antioxidant compounds in chocolate powdered with
herbs. The determination levels of catechins and epicatechins compounds was done through quantitative analysis
using LC-MS ToF instruments. The highest content of catechins as well as epicatechins was found in dark chocolate
powdered with green tea in amount of 17.70 mg/kg of catechins and 37.08 mg/kg of epicatechins respectively.

Keywords: antioxidants, herbs, LC-MS ToF, catechin, epicatechin

*Corresponding author’s email : mira.suprayatmi@unida.ac.id, mirasuprayatmi@yahoo.com

Introduction

Cacao (Theobroma cacao is known to contain antioxidant compounds including high flavan-3-ols (flavanol) either
as monomers of epicatechin and catechin as well as flavanols polymerization, or procyanidins (Payne, 2010).
However, the antioxidant content may be reducing during processing. To maintain the content of antioxidant, the
chocolate products may be enriched with other natural sources of antioxidants, namely herb. S ome type of herb
contain high antioxidant compounds and enrich flavor of chocolate products (Carlsen, et al., 2010). To address
the needs of the identification and quantitative analysis of specific antioxidants in food samples, test method with
high ability to identify and quantify such as Liquid Chromatography Mass Spectrometry a Time of Flight (LC-M
ToF) was to be applied (Calderon et al, 2009; Tao et al., 2015). The research aims to know the capacity of
antioxidants, antioxidant levels and antioxidant profile relatively refined products from chocolate with the addition
of several types of natural antioxidants in the form of powdered mint leaves (Mentha cordifolia), powdered green
tea leaves (Camellia sinensis) and lime (Citrus aurantifolia).
Methodology
A. Materials and Equipments
Materials used in this research were the dark chocolate of local products, powdered mint leaves ( Mentha
cordifolia), powdered lime leaves (Citrus aurantifolia) and powdered green tea leaves (Camellia sinensis). The
chemicals used is methanol, n-hexane, filter paper, cotton, DPPH (2.2-diphenyl-1-picrylhydrazyl), the standard
solution is a mixture of compounds catechins and their derivatives, acetonitrile, aquabidest, membrane filters of
0.22 µm GHP, formic acid, methanol, isopropanol, heksan, and leucine enkephaline 400 µ g/mL. The main tool
used in the study is a UV spectrophotometer, Liquid Chromatography Mass Spectrometry with mass analyzer at
a Time of Flight (LC-MS ToF) Xevo G2 QTof-MS S from waters corporate equipped with UNIFI Scientific
Information, ACQUITY UPLC T3 HSS column 2.1 x 100 mm 1.8 μm, turbovap.

341
B. Method
Melted dark chocolate was added with powdered mint leaves, powdered green tea leaves, and powdered lime
leaves, each at 4% (w/w). The antioxidants capacity (radical scavenging assay) of the samples was analysed
using DPPH (1, 1-Diphenyl-2-Picrylhidrazyl) with a Spectrophotometer (Patel et al., 2010).
Determination of Antioxidant Profile: qualitative analysis is done by the identification of the molecular mass or
often referred to as the screening analysis using the results of the sample readings on the instrument LC -MS
ToF. Antioxidants are expressed on a sample of unidentified or positive if the results of the reading of the
identification of eligible analytes or criteria: Mass error reading of analytes ≤ 5 ppm error; Isotope match MZ
RMS% ≤ 6%; The intensity of the analytes ≥ 300; There is a fractional value of the brake system on 4 elusidasi <
Fragment match.
Calculation of the weighting of the relative response factor: the relative weights of the response is the ratio
of the standard and sample area multiply by the ratio of standard and sample weighing.
Calculation of catechins compound levels or epicatechin: Ratio of the sample and standard area multiplied
by the standard concentration (ug/kg) multiplied by the ratio of final volume (ml) and the weight of the sample(g)
multiplied by factor dilution.
The process of data reading of the chromatogram results was carried out using the software of UNIFI Scienti fic
Information. It inculded peak integration at chromatogram and the analytes identification comprising of
antioxidants library on LC-MS ToF instruments and also based on the literature of some dominant antioxidants.

Results and discussions

Identification of Antioxidant Compounds
Based on testing using LC-MS ToF instruments, some antioxidant compounds in chocolate were successfully
detected in accordance with the terms or the criteria of identification. Common compounds were detected on all
sample testing, while the typical compounds were detected in chocolate without the addition of herb (chocolate
control). 27 compounds were indentified on the chocolate control, among others were compounds of
procyanidin A2, procyanidin B1, procyanidin B2, B4, procyanidin Procyanidin C-1, catechin, epicatechin, and
quercetin. There were 13 typical compounds identified in chocolate added with powdered mint leaves, such as
hesperetin, hesperidin, luteolin, and flavonoid compound such as clerodendrin, kaepferol, and yuank anin. The
chromatogram of the spectrum is shown in Figure 1.

control mint

Figure 1. The Chromatogram of Chocolate (Control) and Mint Chocolate

On chocolate added with powdered lime leaves were 20 specific compounds identified. Some compounds such
as complanatuside, isovitexin-3-O-glucopyranoside, flavone, 5,7-dihydroxy-4’-O-α-D-glucoside, and kaemferol
were a flavonoid compounds as well. Approximately 67 compounds were identified on chocolate products with

342
the addition of powdered green tea leaves, 40 of which were typical compounds, among others were 1-galloyl-
glucose; 3,8-di-C-glucosylapigenin; kaempferol-3,7-di-O-β-D-glucoside; 7-O-β-D-(6”'-acetyl) glucopyranoside;
patuletin 7-O-[6”-(2-methylbutyryl)]-glucoside; and undulatoside A. The typical chromatogram spectrum is shown
in Figure 2. Through the identification of typical compounds showed that the addition of herb like mint leaves,
lime leaves, and the green tea leaves, may increase its diversity of antioxidant compounds in processed
chocolate products added with herb. This is important, given that the benefits of antioxidant compounds are very
numerous and diverse.

lime green tea

Figure 2. The Chromatogram of Lime Chocolate and Green tea Chocolate

The Relative Weights Of The Response Of Antioxidant Compounds

Based on the calculation of the relative total weight response, the relative weighting response of the three
chocolate products with the addition of herb was increased. The relative weights of the response can directly
indicate the magnitude of the factor of the increase or decrease of compounds that are generally identified in
chocolate plus herb. Total relative weighting of responses was 133.71 in green tea leaves; 31.27 in lime leaves
and 28.32 in mint leaves. The increase in the relative weight of the compounds was occured because the added
material contained compounds such as catechins in chocolate on the addition of green tea leaves (Victoria et al.,
2013). The decrease in the relative weights can be caused by several things such as the presence of i nteraction
on matrix in chocolate against matrix in a spice or herb and the heat influence during chocolate processing
(Brewer, 2011).

The levels of the catechins and epicatechin compounds

Catechins is one of the antioxidants components in chocolate. Testing result of catechins and epicatechin
compounds was presented in Table 1.

Table 1. The content of Catecin and Epicatecin as well as the values of IC 50 Chocolate

Chocolate Chocolate mint Chocolate green Chocolate lime

No Charateristic
control leaves tea leaves leaves

1 Catechin (mg/kg) 8.94cd 8.75 bc 17.70e 8.39a

2 Epicatechin (mg/kg) 13.42bc 13.23b 37.08d 12.75a

3 IC50 (ppm) 399.15bc 68.63a 675.3d 432.59bc

note: different letters in the same row indicate significant differences (p<0.05)
The catechins and epicatechin compounds on chocolate green tea showed a significant difference against
chocolate control. Increased levels of catechin and epicatechin occurred on the addition of powdered green tea
leaves. While the decline in levels of catechin and epicatechin occurs on the addition of powdered lime leaves.

343
This evidenced showed that there is a decrease in the relative weights of chocolate plus lime leaves i.e. 0.02 for
catechin and epicatechin to 0.1.
The IC50 of antioxidant capacity in chocolate plus herb showed different from catechin or epicatin content (Table
1). Chocolate added with powdered mint leaves has shown the lowest IC 50, namely 68.63 ppm. The IC 50 value of
50-100 ppm categorized as a powerful antioxidant whereas from 200-1000 ppm considered as less active
ingredients but still potentially as antioxidants (Molyneux, 2004). Carlsen et al (2010) concluded that mint leaves
contain antioxidants of 116.4 mmol/100 g of material, while green tea leaves 1.5 mmol / 100 g of material.
Special compounds of mint leaves might have strong antioxidant capacity.

Conclusion

The results of the identification using LC-MS ToF showed that chocolate products with the addition of powdered
mint leaves, powdered green tea leaves and the powdered lime leaf were experiencing an increase in the
number of types of antioxidant. Catechins and epicatechin content of the chocolate products with the addition of
powdered leaves of green tea gives the highest when compared to other products i.e. 17.70 mg/Kg cat echins
and 37.08 mg/kg epicatecin. However, chocolate added with 4 percent of powdered mint leaves classified as a
powerful antioxidant with IC 50 values between 50-100 ppm, namely 68.63 ppm that analyzed with the DPPH
method. Further research related to the powerful antioxidant of various herbs is needed to be done.

References
Brewer, MS. 2011. Natural Antioxidants: Sources, Compounds, Mechanisms of Action, and Potential Applications.
Journal Comprehensive Reviews in Food Science and Food Safety Volume 10 : 221–247.
Calderon, Angela I, Brian J Wright, W Jeffrey Hurst dan Richard B. Van Breemen. 2009. Screening Antioxidants
Using LC-MS: Case Study with Cocoa. J. Agric. Food Chem. 57: 5693–5699. Chicago : Department of
Medicinal Chemistry and Pharmacognosy.
Carlsen, M.H., Blomhoff, R. and Andersen, L.F. 2010. Intakes of culinary herbs and spices from a food frequency
questionnaire evaluated against 28-days estimated records. Nutrition Journal 2011, 10:50. Open Access
Journal
Molyneux, P.
 2004. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant
activity. 
Songklanakarin J. Sci. Technol., 2004, 26(2) : 211-219
Patel et al, 2010. Determination of polyphenols and free radical scavenging activity of Tephrosia purpurea linn leaves
(Leguminosae)
. Parmacognosy Reseach Volume : 2, Issue : 3 : 152-158.
Tao Wu, Haiyang Lv, Fengzhong Wang dan Yi Wang. 2015. Characterization of Polyphenols from Lycium
ruthenicum Fruit by UPLC-Q-TOF/MSE and Their Antioxidant Activity in Caco-2 Cells. Journal of Agricultural
and Food Chemistry. China: Key Laboratory of Food Bio-technology
Victoria K. Ananingsih, Amber Sharma, dan Weibiao Zhou. 2013. Green tea catechins during food processing and
storage: A review on stability and detection. Singapore : Food Science and Technology Programme,
Department of Chemistry, National University of Singapore

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 IFRC 2017: 162-119 Functional Food

Effect of Thermal Treatment on Total Phenolic Content and Antioxidant Activity of Garcinia atroviridis and
Fenugreek Seed

*Ummi Kalthum Ibrahim, Umirah Rashidah Dalip, Suzihaque, M.U.H., Syafiza Abd Hashib and Siti Fatma Abd Karim

Faculty of Chemical Engineering, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia

Abstract

Garcinia atroviridis and Fenugreek seed are well known as spices condiment in South East Asia especially in
Malaysia, Thailand and Indonesia. Garcinia atroviridis (GA) is known as ‘asam gelugor’ or ‘asam keping’ while
Fenugreek seed (FS) is traditionally known as ‘halba’. This study aims to identify the thermal treatment effect on total
phenolic content and antioxidant activity in Garcinia atroviridis and Fenugreek seed. Garcinia atroviridis and
Fenugreek seed aqueous extract were subjected to extraction temperature ranged from 54°C to 96°C at various
concentration of methanolic solvent in percentage of 20% to 80% v/v. The total antioxidant activity and total phenolic
content in both samples were measured using 2,2-diphenyl-1-picylhydrazyl (DPPH) radical scavenging assay and
Folin–Ciocalteu reagent, respectively. The highest antioxidant activity (97.48%) was found in Garcinia atroviridis at
extraction temperature of 75ºC and 80% v/v methanolic concentration. While for Fenugreek seed, highest antioxidant
activity (94.58%) was observed at extraction temperature of 75ºC and 50% v/v methanolic concentration. The total
phenolic content values were found highest at extraction temperature of 75ºC and 80% v/v methanolic concentration.
The values were reported as 455.53 mg GAE/g and 17.553 mg GAE/g for Fenugreek seed and Garcinia atroviridis,
respectively. Results from the experiment were optimized using Response Surface Methodology (RSM). The yield of
antioxidant activity and total phenolic content of both samples were found closed to the predicted values. Thermal
treatment significantly affects the values of antioxidant activity for both Garcinia atroviridis and Fenugreek seed
aqueous extracts. Nevertheless, total phenolic content was found unaffected by thermal treatment.

Keywords: Thermal treatment, Antioxidant activity, Total Phenolic Content, Garcinia atroviridis, Fenugreek seed.
*Corresponding author’s email: ummi985@salam.uitm.edu.my

Introduction
Thermal treatment has been one of the significant method employed in food processing technology either to sterilize
or to inhibit the growth of enzyme hence lengthen the shelf lives of the food. However, the nutritional integrity of the
food such as many consumers, nutritionist and scientists are questioning its antioxidant activity and phenolic content.
Some studies illustrated that the degree of thermal processing affects content of phenolic compounds and antioxidant
activity in certain type of vegetables and fruits (Tiwari and Cummins 2013). Balanced diet and consuming supplement
containing antioxidants may contribute in delaying the oxidation of the reactive free radicals. Garcinia atroviridis
familiarly known as Asam Gelugor have crude extract that contained significant antifungal activity against
Cladosporium hebrum while other parts indicated higher antioxidant activity (Mohamed Mackeen, Ali et al. 2000).
While Trigonella foenum-graecum L is a scientific name of Fenugreek seed commonly known as methi (in Hindi) or
‘halba’ has been used in food as spice condiment, flavouring agent and as a medicinal plant from primitive time.

Hence, the purpose of this study is to investigate the thermal treatment effect on the total phenolic content and
antioxidant activity of natural antioxidant sources that are Garcinia atroviridis and Fenugreek seed as the potential
substitute for synthetic antioxidants in food industry. This study will benefit the food industry in discovering
alternatives to synthetic antioxidant that is safer to health.

Material and Methods

3.1. Garcinia atroviridis and Fenugreek seed is obtained from nearest market in Shah Alam. Both items will first be
grinded to fine powder prior to solvent extraction process. After grinding process the samples is dissolved in varies

345
solvent concentration and are exposed to thermal treatment (extraction) of temperature ranges from 60ºC - 90ºC for
30 minutes.

3.2. Antioxidant activity (AA) was determined spectrophotometrically using DPPH assays procedure described by
Akter, Ahmed et al. (2010) but was adjusted to fit the requirement of this experiment.

3.3 Total phenolic content (TPC) was determined spectrophotometrically using Folin–Ciocalteu reagent by the
method as described in Belguith-Hadriche, Bouaziz et al. (2013) with minor modification.

3.4. Statistical analysis of all data was carried out using one-way analysis of variance (ANOVA) by Design Expert
version 6.0.8 where the significant difference was considered at p < 0.01 to be statistically significant.

Results and Discussion

The antioxidant activity of GA and FS were determined by using DPPH radical scavenging activities. DPPH assay is
a simple and favourable method in screening the antioxidant activity of samples extract because it can be analysed
through UV-Vis spectrophotometer. The total phenolic content was determined in mg GAE/ g dried samples by using
Follin-ciocalteu reagent assays. The highest antioxidant activity that is measured as percentage of inhibition and total
phenolic content in mg GAE/ g dried sample for both samples was recorded as 97.48% and 17.553 mg GAE/g,
respectively for Garcinia atroviridis sample extract and 94.575% and 455.53 mg GAE/g, respectively for Fenugreek
seed sample extract.
In this study, the dried fruit of GA were exposed to thermal treatment of temperatures ranging from 60ºC to
90ºC showcase results that proved the thermal treatment affect the antioxidant activity of the Garcinia atroviridis
extracts. The results also portrayed increases concentration of methanolic solvent causes increment in AA. As for
TPC of both GA and FS extracts no distinct trend can be concluded as the data recorded are randomized. No
significant trend in TPC content of both samples may be associated to the instability of the phenolic compounds itself
as stated in Abdullah, Bakhari et al. (2013). From analysis of variance (ANOVA), the Model F-value of 10.33 and
11.91 for antioxidant activity and total phenolic content of both samples implies that the model is significant with a low
p value ‘p < 0.001’ suggesting only a 0.01% chance that a "Model F-Value" this large could occur due to noise hence
the model is suited well with the experimental data. "Lack of Fit F-value" of 3.54 (AA) and 107.41 (TPC) infers the
significance for both responses. The best model shall have an insignificant lack of fit however the R-squared values
should also be diagnosed in determining the goodness of a model. The model generated that is most fitted for AA
response is 2FI (two-factor interaction) whereas for TPC response is quadratic model. The model is suggested as it
has the highest order polynomial which focus in maximizing the R-Squared values. The 2FI model recorded the
values of “Prediction R-Squared" of 0.4299 is in practical agreement with the "Adjusted R-Squared" of 0.5232 whilst
the quadratic models displayed “Prediction R-Squared" values of 0.4826 and "Adjusted R-Squared" of 0.6312. This
represents the coefficient determination of AA and the TPC that illustrated how good a model is from the optimized
responses, resulting in final equation for antioxidant activity and total phenolic content of GA and FS is given as
follows;

Garcinia atroviridis
Antioxidant activity = 8.507 + 0.366X1 + 0.67X2 + 8.518E-4 X3
Total phenolic content = 82.312 + 3.421X1 – 9.624X2 - 0.014X12 + 0.117X2 -0.0254X1X2
Fenugreek seed
Antioxidant activity = 78.368 – 0.449X1 + 0.574X2 + 8.518E-4 X3
Total phenolic content = -72.292 + 2.985X1 – 4.713X2 – 0.014X12 + 0.117X2 -0.0254X1X2

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 DESIGN-EXPERT Plot DESIGN-EXPERT Plot

Antioxidant Activity Antioxidant Activity

X = A: Ext Temperature X = A: Ext Temperature
Y = B: Methanol Conc Y = B: Methanol Conc

Actual Factor 95.4053 Actual Factor 93.7115

C: Type of Herbs = Fenugreek Seed C: Type of Herbs = Garcinia Atroviridis
86.013 83.3085

76.6207 72.9054
Antioxidant Activity

Antioxidant Activity
67.2283 62.5023

57.836 52.0993

70.00 70.00
90.00 90.00
60.00 60.00
82.50 82.50
50.00 50.00
75.00 75.00
B: Methanol Conc
40.00 67.50 B: Methanol Conc
40.00 67.50
A : Ext Temperature A : Ext Temperature
30.00 60.00 30.00 60.00
DESIGN-EXPERT Plot DESIGN-EXPERT Plot

Total Phenolic Content Total Phenolic Content

X = A: Ext Temperature X = A: Ext Temperature
Y = B: Methanol Conc Y = B: Methanol Conc

Actual Factor 191.815 Actual Factor 28.7932

C: Type of Herbs = Fenugreek Seed C: Type of Herbs = Garcinia Atroviridis
137.439 14.3371

83.0638 -0.119055
Total Phenolic Content

Total Phenolic Content

28.6883 -14.5752

-25.6871 -29.0314

70.00 70.00
90.00 90.00
60.00 60.00
82.50 82.50
50.00 50.00
75.00 75.00
B: Methanol Conc
40.00 67.50 B: Methanol Conc
40.00 67.50
A : Ext Temperature A : Ext Temperature
30.00 60.00 30.00 60.00

Figure 1: 3D Response Surface model graph of antioxidant activity and total phenolic plots of the effects of two
variables that is extraction temperatures (60ºC-90ºC) and methanolic solvent concentration (30%-70%)

The graphs of both samples were depicted in 3D surface and contour as in Figure 1 were analyzed. It is
observed in the 3D contour plot the interaction between extraction temperature (X1), solvent concentration (X2) and
type of herbs (X3) on the yield of antioxidant activity and total phenolic content. Fig. 1 shows increasing in methanolic
concentration from 30% to 70% improve the AA yield and is supported by the fact that increasing water in organic
solvent cause decrease in antioxidant activity of the extracts (Do, Angkawijaya et al. 2014). Compared to FS extract
that experienced inverse linear correlation of AA yield and the extraction temperature, GA extract proved that higher
extraction temperature shows positive effect on extraction of antioxidant compound which is aligned with the study by
Zeković, Cvetanović et al. (2014). It exemplifies a similar trend of increment in TPC yield as the methanolic
concentration increases from 30% to 70%. Nevertheless, a constant unchanged trend was found in the correlation of
extraction temperature to TPC yield hence, it concludes TPC in Fenugreek seed is not affected by the changes in
extraction temperature.
From the modelling, the AA yield targeted is 67.10% and TPC yield 227.1 mg GAE/g while the optimization
responses were conducted to determine the optimum condition and yield of both responses. Therefore, the optimum
condition and optimum yield were recorded at 90ºC, 70% methanolic concentration with AA yield of 83.85% (FS) and
63.85% (GA) and TPC of 24.45 mg GAE/ g GA and 164.94 mg GAE/ g FS. A verification experiment has been done
to verify the optimum yield at optimum condition for both samples. It is found that the percentage error of suggested
condition and the verified data is below 10 percent which illustrates the reliability of the model designed.

Conclusion
This study highlighted that Fenugreek seed have higher total phenolic content while Garcinia atroviridis display
higher antioxidant activity. The TPC were reported as 455.53 mg GAE/g and 17.55 mg GAE/g for Fenugreek seed
and Garcinia atroviridis, respectively. The antioxidant activity of 97.48% was found in Garcinia atroviridis while for

347
Fenugreek seed, antioxidant activity was found 94.58%. The optimum condition and yield was found at 90ºC, 70%
methanolic concentration for Fenugreek seed with AA yield of 83.85% and TPC yield of 164.94 mg GAE/ g. While for
Garcinia atroviridis is at 90ºC, 30% methanolic concentration with AA yield of 63.84% and yield of TPC 24.45 mg
GAE/g. The optimum yield has been verified to be having less than 10% error.

Acknowledgements
Thanks to Institute of Research Management & Innovation (IRMI), UiTM for providing internal grant [File No: 600-
IRMI/MYRA 5/3/LESTARI (0145/2016)], Ministry of Education (MOE) Malaysia for research funding and all the
contributions in completing the research study.

References
Abdullah, A. R., N. A. Bakhari and H. Osman (2013). "Study on the relationship of the phenolic, flavonoid and tannin
content to the antioxidant activity of Garcinia atroviridis." Universal Journal of Applied Science 1(3): 95-100.
Akter, M. S., M. Ahmed and J.-B. Eun (2010). "Solvent effects on antioxidant properties of persimmon (Diospyros
kaki L. cv. Daebong) seeds." International Journal of Food Science & Technology 45(11): 2258-2264.
Belguith-Hadriche, O., M. Bouaziz, K. Jamoussi, M. S. J. Simmonds, A. El Feki and F. Makni-Ayedi (2013).
"Comparative study on hypocholesterolemic and antioxidant activities of various extracts of Fenugreek
seeds." Food Chemistry 138(2-3): 1448-1453.
Do, Q. D., A. E. Angkawijaya, P. L. Tran-Nguyen, L. H. Huynh, F. E. Soetaredjo, S. Ismadji and Y.-H. Ju (2014).
"Effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of
Limnophila aromatica." Journal of food and drug analysis 22(3): 296-302.
Mohamed Mackeen, M. M., A. M. Ali, N. Lajis, K. Kawazu, Z. Hassan, M. Amran, H. Mohamad, L. Y. Mooi and S. M.
Mohamed (2000). Antimicrobial, antioxidant, antitumour-promoting and cytotoxic activities of different plant
part extracts of Garcinia atroviridis Griff. ex T. Anders, Elsevier.
Tiwari, U. and E. Cummins (2013). "Factors influencing levels of phytochemicals in selected fruit and vegetables
during pre-and post-harvest food processing operations." Food Research International 50(2): 497-506.
Zeković, Z., A. Cvetanović, B. Pavlić, J. Švarc-Gajić and M. Radojković (2014). "Optimization of the polyphenolics
extraction from chamomile ligulate flowers using response surface methodology." International Journal of
Plant Research 4(2): 43-50.

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 IFRC 2017: 176-140 Functional Food

Inhibitory effects of mungbean soup on the enzymes and regulator related to

type 2 diabetes

Saeting, O. and *Sae-tan, S.

Department of Food Science and Technology, Faculty of Agro-Industry, Kasetsart University, 50 Ngamwongwan
Road, Ladyao, Chatuchak, Bangkok 10900 Thailand

Abstract

Diabetes is a disease associated with abnormal carbohydrate metabolism resulting in hyperglycemia. Many synthetic
drugs are currently used to treat diabetes but patients experience several side effects from them. Therefore, plants
and herbs became alternative approaches for diabetes prevention and treatment. Mungbean is one of the potential
legumes that have been studied its biological activities due to the high amount of phytochemical compounds known
for beneficial effects to human health and also have the preventive effects against several diseases. Vitexin and
isovitexin are flavones found with high amounts in mungbean. Both are known to have many biological activities
including antioxidant, anti-proliferation, anti-inflammation and anti-diabetes. Mungbean soup (MBS) is one of the
popular healthy drinks in Asia. Here, the effects of MBS on diabetes prevention were currently studied. The results
showed that MBS rich with vitexin and isovitexin (9.12 mg/g, 17.93 mg/g MBS powder, respectively). MBS exhibited
α-amylase inhibitory activity with IC50 of 37.86 mg/mL, α-glucosidase inhibitory activity with IC50 of 3.34 mg/mL and
protein tyrosine phosphatase 1B (PTP-1B) inhibitory activity with IC50 of 0.01 mg/mL. In addition, MBS could inhibit
the formation of advanced glycation endproducts (AGEs) with IC50 of 2.26 mg/mL. This study shows that MBS rich
with vitexin and isovitexin has the potential anti-diabetic activities by inhibiting carbohydrate- metabolizing enzymes,
the negative regulator of insulin signalling pathway and the formation of AGEs.

Keywords: mungbean, diabetes, vitexin, isovitexin

*Corresponding author’s email: fagists@ku.ac.th

Introduction
Type 2 diabetes mellitus, is a condition of hyperglycemia resulting from defects in insulin signalling and impaired beta
cell function. Chronic high blood sugar levels of diabetes causes many long-term damages, including abnormalities
and functional failure of several organs. The control of diabetes normally involves exercise, diet, and chemotherapy
(American Diabetes, 2010, Manukumar et al., 2017).

Many medications have been used to treat diabetes. However these drugs were reported about adverse side effects
(Farsi et al., 2011). Therefore herbs and natural products are getting more attention for type 2 diabetes treatment.
Grains and cereals, the main source of energy in Asia, have been recommended for diabetic patients to control blood
sugar (Yao et al., 2008). Mungbean is one of the recommended food for diabetics for long time. In particular, vitexin
and isovitexin in mungbean have been mentioned as the active compounds in mungbean that could help diabetic
patients by controlling blood sugar level and reducing the formation of advanced glycation endproducts (AGEs),
which are the factors that cause diabetic complications (Peng et al., 2008, Yao et al., 2011).

Now there is no clear study on activities of anti-diabetic mechanisms of mungbean and no study about anti-diabetic
activities of mungbean soup extracted with water, a popular healthy drink in Asian. Therefore, the purpose of this
study is to investigate the anti-diabetic effects and possible mechanisms in diabetes prevention of water-extracted
mungbean soup.

349
Materials and Methods
1. Extraction, identification and standardization
Mungbean seeds were cleaned and rinsed with tap water. Mungbean flavonoid was extracted with some
modifications from the method described by Li et al. (2012). After that the filtrate was evaporated some water out with
rotary evaporator, then centrifuged at 3500 rpm for 30 min, and freeze-dried. The freeze-dried sample was kept at -
80C for further analysis.

Vitexin and isovitexin in mungbean soup was detected by using HPLC equipped with diode array detector (DAD)
according to Li et al. (2012). Standards of vitexin and isovitexin were used to calculate the concentration of vitexin
and isovitexin in mungbean extract. Mungbean extract in each replication was standardized the concentration of
vitexin and isovitexin before testing anti-diabetic activities.

2. Inhibitory effects of vitexin and isovitexin from mungbean soup against α-amylase, α-glucosidase,
protein tyrosine phosphatase 1B (PTP-1B) were determined according to modified methods of Fei et al. (2014),
Lekshmi et al. (2012) and Choi et al. (2014), respectively. The inhibition of advanced glycation endproducts formation
was evaluated by bovine serum albumin (BSA)-glucose assay according to modified methods of Peng et al. (2008).

3. In statistical analysis, all data were expressed as mean ± standard deviation and were done in triplicate
independent analyses. Data was analysed using one-way ANOVA using IBM SPSS Statistics version 23 (IBM Corp.,
Armonk, N.Y., USA).

Results and Discussion

Mungbean is a legume rich in phytochemicals, including flavonoids. According to previous report, the flavonoids
mostly found in mungbean are vitexin and isovitexin (Yao et al., 2011, Li et al., 2012). Here we identified and
determined the amount of vitexin and isovitexin with two major peaks in chromatogram of water-extracted mungbean
soup (MBS), which agreed with previous study (Li et al., 2012). MBS contained vitexin 9.12 mg/g and isovitexin 17.93
mg/g of MBS.

Inhibition of carbohydrate-metabolizing enzymes helps to reduce postprandial blood sugar, which benefits diabetic
patients. MBS inhibited α-amylase and α-glucosidase in a dose-dependent manner with IC50 of 37.86 and 3.34
mg/mL, respectively (Figure 1A, 1B). These activities corresponded with the study of Farsi et al. (2011) showed the
inhibitory activities against α-amylase and α-glucosidase of Ficus deltoidea leaves, rich with vitexin and isovitexin.

PTP-1B is an enzyme that negatively regulates insulin signalling. The inhibition of PTP-1B increases phosphorylation
of insulin receptor and insulin receptor substrate and results in the activation of insulin signalling (Tang et al., 2017).
Here we found the inhibitory activity of MBS against PTP-1B in dose-dependent manner with IC50 of 0.01 mg/mL
(Figure 1C). This suggests that MBS could increase the activation of insulin signalling.

During long-term hyperglycemia, glucose forms adduct with plasma protein through a non-enzymatic reaction called
glycation. The formation of advanced glycation endproducts (AGEs) from this reaction plays an important role in
pathogenesis of diabetic complications (Ahmed, 2005). Therefore the inhibition of AGEs is one of the approaches in
prevention of diabetic complications. This study found that MBS inhibited AGEs formation in a dose-dependent
manner with IC50 of 2.26 mg/mL (Figure 1D). The inhibitory effects of MBS against PTP-1B and AGEs formation in
this study agreed with the anti-diabetic activities of C-glycosides including vitexin ang isovitexin reported by Choi et
al. (2014) and Peng et al. (2008).

350
 A B

C D

Figure1. Inhibitory effects of mungbean soup rich with vitexin and isovitexin against α-amylase (A), α-glucosidase
(B), PTP-1B (C) and formation of advanced glycation endproducts (D)

Conclusion
This study shows that MBS rich with vitexin and isovitexin is an alternative natural approach that has the potential to
prevent type 2 diabetes and its complications. The possible anti-diabetic mechanisms of MBS are the inhibition of
carbohydrate-metabolizing enzymes including α-amylase and α-glucosidase, the inhibition of PTP-1B,
negative regulator of insulin, which may help to control blood sugar and the inhibition of AGEs formation, which may
prevent diabetic complications.

Acknowledgements
We would like to thank Thailand Research Fund (MRG5980111) for financial support and Department of Food
Science and Technology, Faculty of Agro-Industry, Kasetsart University.

References
Ahmed, N. 2005. Advanced glycation endproducts—role in pathology of diabetic complications. Diabetes Research
and Clinical Practice 67(1):3-21.
American Diabetes, A. 2010. Diagnosis and Classification of Diabetes Mellitus. Diabetes Care 33(Suppl 1):S62-S69.
Choi, J. S., M. Nurul Islam, M. Yousof Ali, E. J. Kim, Y. M. Kim, and H. A. Jung. 2014. Effects of C-glycosylation on
anti-diabetic, anti-Alzheimer's disease and anti-inflammatory potential of apigenin. Food and Chemical
Toxicology 64:27-33.
Farsi, E., A. Shafaei, S. Y. Hor, M. B. Khadeer Ahamed, M. F. Yam, I. H. Attitalla, M. Z. Asmawi, and Z. Ismail. 2011.
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351
 methanolic extract obtained from Ficus deltoidea leaves. African Journal of Biotechnology 10(67):15184-
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Fei, Q., Y. Gao, X. Zhang, Y. Sun, B. Hu, L. Zhou, S. Jabbar, and X. Zeng. 2014. Effects of Oolong Tea Polyphenols,
EGCG, and EGCG3″Me on Pancreatic α-Amylase Activity in Vitro. J Agr Food Chem 62(39):9507-9514.
Lekshmi, P. C., R. Arimboor, P. S. Indulekha, and A. Nirmala Menon. 2012. Turmeric (Curcuma longa L.) volatile oil
inhibits key enzymes linked to type 2 diabetes. International Journal of Food Sciences and Nutrition
63(7):832-834.
Li, H., D. Cao, J. Yi, J. Cao, and W. Jiang. 2012. Identification of the flavonoids in mungbean (Phaseolus radiatus L.)
soup and their antioxidant activities. Food Chemistry 135(4):2942-2946.
Manukumar, H. M., J. Shiva Kumar, B. Chandrasekhar, S. Raghava, and S. Umesha. 2017. Evidences for diabetes
and insulin mimetic activity of medicinal plants: Present status and future prospects. Critical Reviews in Food
Science and Nutrition 57(12):2712-2729.
Peng, X., Z. Zheng, K. W. Cheng, F. Shan, G. X. Ren, F. Chen, and M. Wang. 2008. Inhibitory effect of mung bean
extract and its constituents vitexin and isovitexin on the formation of advanced glycation endproducts. Food
Chemistry 106(2):475-481.
Tang, D., Q. B. Chen, X. L. Xin, and H. A. Aisa. 2017. Anti-diabetic effect of three new norditerpenoid alkaloids in
vitro and potential mechanism via PI3K/Akt signaling pathway. Biomedicine and Pharmacotherapy 87:145-
152.
Yao, Y., F. Chen, M. Wang, J. Wang, and G. Ren. 2008. Antidiabetic activity of Mung bean extracts in diabetic KK-Ay
mice. J Agr Food Chem 56(19):8869-8873.
Yao, Y., X. Z. Cheng, and G. X. Ren. 2011. Contents of D-chiro-Inositol, Vitexin, and Isovitexin in Various Varieties of
Mung Bean and Its Products. Agricultural Sciences in China 10(11):1710-1715.

352
 IFRC 2017: 188-157 Functional Food

Ovarian Histomorphological Changes in Rats Supplemented with Edible Bird’s Nest

1,3Albishtue, A. A., 1*Yimer, N., 2Zakaria, M.A.,1Haron ,A.W.,1Rosnina, Y
1Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400Serdang,
Selangor, Malaysia.
2Department of Veterinary Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia,
43400Serdang, Selangor, Malaysia.
3Department of Anatomy and Histology, Faculty of Veterinary Medicine, University of Kufa, Najaf, Iraq.

Abstract

Edible Bird’s Nest (EBN) is an animal product from the saliva secretion of male swiftlet birds (Aerodramus fuciphagus
and Aerodramus maximus). It is traditionally consumed among Asians for its nutritional and medicinal values. The
objective of this study was to evaluate the effect of EBN supplement on the ovarian activities of female rats through
histomorphometric analysis. A total of 24 Sprague dawley rats, divided into 4 equal groups (G): G1 as untreated
control group, G2, G3 and G4 as treated groups with EBN at graded concentrations of 30, 60 and 120 mg/kg of body
weight per day for 8 weeks, respectively were used. During the treatment period, body weight of each rats were
recorded weekly. After sacrificing the rats under general anaesthesia at proestrus, the ovaries excised and weighed
and measured in length. The samples at the level of the ovary of similar site fixed in 10% formalin for 24 hrs,
sectioned and stained using Haematoxylin and Eosin (H&E) stain and observed under a microscope for histological
changes. Results showed increased length and weight of ovary in G4 (P<.05) compared to other groups. A parallel
increase in the number of all types of surviving follicles across treatment groups with increase in the dose of EBN
supplemented were recorded. Similarly, thickness of the ovarian surface epithelium as well as the number of
interstitial cells in G4, were higher (P< 0.05) than the other groups. In conclusion the study revealed that EBN
supplement enhances proliferation of ovarian follicular structures and ovarian-body weight ratio in rats.

Keywords: Edible Bird’s Nest, follicular development, interstitial cells, rat.

*Corresponding author’s email : nurhusien@upm.edu.my

Introduction
Beginning with Tang dynasty (618-907 AD), the edible bird’s nest has become an important item in Chinese cuisine
and pharmacy, consumed as a delicious tonic food known as “bird's nest soup”, which is regarded as a luxury food
item (Hobbs, 2004; Marcone, 2005) .It’s one of the most expensive animal products of saliva secretion produce from
male swiftlet’s two sublingual salivary glands (Saengkrajang, 2013; Ma & Liu, 2012). It is consumed for a number of
reasons and its aphrodisiac property (reflection of reproductive role) is one of the traditionally believed benefits
unexplored scientifically.
Material and methods
A total of 24 Sprague dawley rats, divided into 4 equal groups (G): G1 as untreated control group, G2, G3 and G4 as
treated groups with EBN at graded concentrations of 30, 60 and 120 mg/kg of body weight per day for 8 weeks,
respectively. During the treatment period, body weight of each rats were recorded weekly. After sacrificing the rats
under general anaesthesia at proestrus, the ovaries excised and weighed and measured in length. The samples at
the level of the ovary of similar site fixed in 10% formalin for 24 hrs, sectioned and stained using Haematoxylin and
Eosin (H&E) stain and observed under a microscope for histological changes.

353
Result and discussion
During the experimental period, there were alterations in body weight in treated groups with higher (P<0.05)
change resulted from G4. Moreover, ovarian body weight ratio had significant increase in G4 (P<0.05) followed by
G3, while G1and G2 were comparable.This study was conducted to describe ovarian morphology to elucidate the
mechanism of EBN action as a tonic food. the results demonstrated that EBN administration in adult (12 weeks of
age) rats is effective in promoting development, maturity of ovarian follicles and increase in number of surviving
follicles as well as number of interstitial cells. Considering the composition of EBN, the observed result might be
attributed to one of its major component, sialic acid, which is known to participate in an ovarian development
(Thavamanithevi et al., 2014). Sialic acid is considered as the most important sources of nutrients for the
development of ovarian follicles (Utarabhand et al., 2017).
The present findings confirmed that EBN supplement increases thickness of ovarian epithelium and this
might be be attributed to activation of gonadotrophic cells of pituitary glands with a subsequent increase in LH from
the pituitary. Likewise, in insulin- like growth factor 1 action may also contribute to increased thickness of the
epithelium (Ostrowska et al; 2001) that will be be verified with further experimental work. Notably, this growth factor
has been related to the production of sex steroids (Tamura et al., 2009).
In conclusion, the present study confirms that EBN supplement has impact on reproduction through increased
proliferation of ovarian structures and ovarian-body weight ratio of rats.

References
Hobbs, J. J. (2004). Problems in the harvest of edible birds' nests in Sarawak and Sabah, Malaysian Borneo.
Biodiversity & Conservation 13, 2209-2226.
Ma F., & Liu, D. (2012b). Sketch of the edible bird's nest and its important bioactivities. Review. Food Research
International 48 (2012) 559–567.
Marcone, M. F. (2005). Characterization of the edible bird’s nest the “Caviar of the East”. Food research international
38, 1125-1134.
Ostrowska Z, Kos-Kudla B, Swietochowska E, Marek B, Kajdaniuk D, Ciesielska-Kopacz N(2001). Influence of
pinealectomy and long-term melato- nin administration on GH-IGF-I axis function in male rats. Neuro
Endocrinol Let 22: 255–62.
Saengkrajang, Warasri; Narumol Matan, Nirundorn Matan, (2013). Nutritional composition of the farmed edible bird’s
nest (Collocalia fuciphaga) in Thailand. Journal of Food Composition and Analysis 31 (2013) 41–45.
Tamura H, Nakamura Y, Korkmaz A, Manchester LC, Tan DX, Sugino N, et al(2009). Melatonin and the ovary:
physiological and pathophysiological implications. Fertil Steri 92:328–43.
Thavamanithevi, S, R. Sarifah, C.G. Lim, M. Theanmalar, M.S. Aidawati, M. Durgah Devi and A.A. Saleha.(2014).
Characterization And Standardization Of Edible Birds Nest (EBN)-Determination of Sialic Acid. Proceedings of
the Edible Bird Nest Industry Conference, 25-26 November 2014, Putrajaya.
Utarabhand, P., Rittidach, W., Rattanaporn, O., Runsaeng, P., & Hedrick, J. L. (2017). Sialic acid-specific lectin
participates in an immune response and ovarian development of the banana shrimp Fenneropenaeus
merguiensis. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 203,
132-140.

354
 IFRC 2017: 189-158 Functional Food

Effects of pH and Storage Temperature on the Stability of Encapsulated Anthocyanins from Red Dragon Fruit
(Hylocereus polyrhizus (Weber) Britton & Rose)

*Zaidel, D.N.A., Makhtar, N.A., Mohammad, N.A., Mohd Jusoh, Y.M. and Muhamad, I.I.

Department of Bioprocess & Polymer Engineering, Faculty of Chemical & Energy Engineering, Universiti Teknologi
Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia.

Abstract

The study was conducted to determine the effects of pH and storage temperature on the stability of encapsulated
anthocyanins from red dragon fruit (Hylocereus polyrhizus). Anthocyanins were prepared from concentrated extract
of the flesh solution of red dragon with water. The encapsulated anthocyanins were produced in the form of powder
using maltodextrin as the wall material. The encapsulation process was varied at 1 min, 1.5 min, 2 min, 2.5 min and 3
min minutes of drying time and fixed at 330 W using microwave assisted technique. The encapsulated anthocyanins
were then analysed in effect of pH with various pH value (pH 1-8) and storage temperature (4°C, 28°C and 37°C).
pH stability of 2 min capsule showed encapsulated red dragon fruit anthocyanins stable at most of the pH from pH1-8
excluding pH4. Storage stability at 3 different temperatures (4°C, 28°C and 37°C) for 4 weeks exhibited that
encapsulated anthocyanins degrade faster as the temperature increased. Thus, the stability of anthocyanins
compound could be highly achieved at short time of microwave heating, at low pH during processing and at lower
storage temperature. Microwave assisted technique is a promising encapsulation approach in extending the shelf life
of product by protecting the core material, anthocyanins.

Keywords: Hylocereus polyrhizus, pH, storage temperature, encapsulated anthocyanins.

*Corresponding author’s email: dayang@cheme.utm.my

Introduction
Anthocyanins are bioactive flavonoid constituents that is used as natural colorants of red, blue and purple in food
products. The exploration of anthocyanins from various fruits and vegetables has continuously emerged due to their
safety, functionality and pharmacological effects, antioxidant in particular. During preparation, processing, and
storage of foods, some factors such as pH, light, oxygen, metal ions, enzymes, sugars, thermal treatment, and
storage temperature may accelerate anthocyanins degradation (Patras et al., 2010). Encapsulation is an excellent
technology in entrapping bioactive food ingredients in a polymeric matrix to improve their stability and prevent them
from physical and chemical deterioration (Quirós-Sauceda et al., 2014).

The objective of this study is to investigate the effects of pH and storage temperature of encapsulated anthocyanins
from red dragon fruit (Hylocereus polyrhizus).

Material and Methods

3.1. Encapsulation of anthocyanins
The encapsulation process used maltodextrin as wall material. Maltodextrin was mixed with concentrated red dragon
fruit anthocyanins at ratio 1:10 (v/v) and stirred until homogenized using homogenizer for 20 min. The mixture was
put in a petri dish for drying process in a domestic microwave oven. For drying process, a variable parameter was
used that is 1 min, 1.5 min, 2 min, 2.5 min and 3 min minutes of drying time and 30% from 1100W of constant
heating power was used (Zaidel et al., 2015). Then, stability analyses were carried out in duplicate.

3.2. pH Stability Analysis of Encapsulated Powder

The pH stability of encapsulated powder was studied using method described by Fan et al. (2008). Capsule of 2 min
drying time was used (Zaidel et al., 2015). For the preparation, 0.5g of encapsulated powder was dissolved in 9mL of

355
different buffers (pH 1.0 to pH 8.0) and homogenised using vortex. Wavelength of maximum absorbance spectra and
absorbance value was recorded for each pH value using UV/VIS Lambda 25 Spectrophotometer at 200-700
wavelengths with water as blank. Then, the solutions were kept at 4°C and stored for 7 days. Absorbance reading
was recorded at Day 1 and Day 7.

3.2. Storage Stability Analysis of Encapsulated Powder

The storage stability of encapsulated powders was also studied using method described by (Kirca et al., 2006). The
stability was determined at temperatures 4°C, 28°C and 37°C on capsules from different drying time. Sample was
stored in refrigerated incubator for temperatures at 4°C and at 28°C and 37°C. The total anthocyanin content was
analysed weekly for 4 weeks. For total anthocyanins content determination, samples were mixed with 1mL distilled
water to release the anthocyanins from the microcapsules and 9mL 95% ethanol was added to destroy the
microcapsule membrane. Then sample was filtered using filter paper. The anthocyanins content was calculated in
mg/L.

Results and Discussion

pH stability was investigated for 2 min drying time of powder because of its high efficiency compared to other powder
(Zaidel et al., 2015). The stability was determined by the changes shown in the absorption spectrum and also colour
of the powder. The powder and also extract were mixed in 8 different buffers and stored for 7 days at 4°C. The
readings were taken at Day 1 and Day 7. The colour of buffer solution at pH 1-8 containing red dragon fruit
anthocyanins extract did not varied much from each other. The maximum absorbance is in the range of 490-550nm
for all pH at Day 1 where the maximum absorbance is detected at range of 500-560 for all pH at Day 7. Figure 1
shows absorption spectrum of 2 min capsule at buffer pH 1-8. From the graph, the maximum absorbance readings
are in the range of 500-550nm for all pH at both Day 1 and Day 7. From the above figure, the encapsulated
anthocyanins are not stable at pH 4 where the value decreased significantly compared to other pH.

Patras et al. (2010) had summarized that the thermal burden during processing of anthocyanins food may further
degrade them during storage. There are many factors that can affect the storage stability of anthocyanins such as
oxygen, ascorbic acid and enzymes. According to Buckow et al. (2010) existing fruit enzymes such as polyphenol
oxidase (PPO), peroxidase (POD) and β-glucosidases caused oxidation of anthocyanins rapidly. In this study, the
degradation rate of the encapsulated anthocyanins was examined at 3 different temperatures (4°C, 28°C and 37°C)
for 4 weeks of storage (Figure 2). From the observations, anthocyanins content decrease for all capsule types at all
storage temperature. However, storage temperature at 4°C, degradation rate of anthocyanins is the lowest where the
degradation rate at 37°C is the highest compared to others. Degradation of anthocyanins for every capsule type
could not be sorted but 2 min capsule shows the highest anthocyanins content for all the end of storage temperature.
This is supported by a study where anthocyanins of blackberry in juice and concentrate degraded more quickly with
increasing temperature during heating and storage Wang and Xu (2007). Thus, higher stability of the blackberry juice
and concentrate could be achieved at lower temperature and short time heating during processing and storage.

356
 pH 1 pH 2 pH 3 pH 1 pH 2 pH 3 pH 4
(a) (b)
pH 4 pH 5 pH 6
pH 7 pH 8 pH 5 pH 6 pH 7 pH 8
0.25
0.25

0.2 0.2

Absorbance
Absorbance

0.15 0.15

0.1 0.1

0.05 0.05

0 0
400 500 600 700 400 500 600 700
Wavelenght, nm Wavelenght, nm
Figure 1. Absorption spectrum of capsule at buffer pH1-8 (a) Day 1 & (b) Day 7.

(a) 1 min 1.5 min 2 min 2.5 min 3 min

(b) 1 min 1.5 min 2 min 2.5 min 3 min
0.6 0.6
Total anthocyanin content (TAC), mg/L
Total anthocyanin content (TAC), mg/L

0.5 0.5

0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

0 0
1 2 3 4 1 2 3 4
Week of storage Week of storage

(c) 1 min 1.5 min 2 min 2.5 min 3 min

0.6
Total anthocyanin content (TAC), mg/L

0.5

0.4

0.3

0.2

0.1

0
1 2 3 4
Week of storage

Figure 2. Degradation of anthocyanins content at storage temperature of (a) T=4°C, (b) T=28°C &
(c) T=37°C.

357
Conclusion
The findings of the present study emphasize the promising feasibility of encapsulated anthocyanins by using
microwave drying technique. This study showed the relationship between drying time of the encapsulation with the
effectiveness of study and also stability analysis. The process for accuracy of yield can also be done in several levels
of power intensity to convince which level obtained the optimum drying rate. It is recommended if there are variety
uses of wall material like gum Arabic and etc. different wall material has different dielectric constant which will affect
the coating process of anthocyanins.

Acknowledgements
The authors would like to acknowledge Universiti Teknologi Malaysia and Ministry of Higher Education Malaysia for
the financial support under Research University Grant Scheme (Q.J130000.2544.06H39) and Fundamental
Research Grant Scheme (R.J130000.7844.4F447).

References
Buckow, R., Kastell, A., Terefe, N. S., and Versteeg, C. 2010. Pressure and Temperature Effects on Degradation
Kinetics and Storage Stability of Total Anthocyanins in Blueberry Juice. Journal of Agricultural and Food
Chemistry 58(18): 10076–10084.
Fan, G., Han, Y., Gu, Z., and Gu, F. 2008. Composition and Colour Stability of Anthocyanins Extracted from
Fermented Purple Sweet Potato Culture. LWT - Food Science and Technology 41(8): 1412–1416.
Kirca, A., Özkan, M., and Cemeroǧlu, B. 2006. Stability of Black Carrot Anthocyanins in Various Fruit Juices and
Nectars. Food Chemistry 97(4): 598–605.
Patras, A., Brunton, N. P., O’Donnell, C., and Tiwari, B. K. 2010. Effect of Thermal Processing on Anthocyanin
Stability in Foods; Mechanisms and Kinetics of Degradation. Trends in Food Science & Technology 21(1): 3–
11.
Quirós-Sauceda, A. E., Ayala-Zavala, J. F., Olivas, G. I., and González-Aguilar, G. A. 2014. Edible Coatings as
Encapsulating Matrices for Bioactive Compounds: A Review. Journal of Food Science and Technology 51(9):
1674–1685.
Wang, W. D., and Xu, S. Y. 2007. Degradation Kinetics of Anthocyanins in Blackberry Juice and Concentrate.
Journal of Food Engineering 82(3): 271–275.
Zaidel, D. N. A., Makhtar, N., Mohd Jusoh, Y. M., and Muhamad, I. I. 2015. Efficiency and Thermal Stability of
Encapsulated Anthocyanins from Red Dragon Fruit ( Hylocereus polyrhizus ( Weber ) Britton & Rose ) using
Microwave-assisted Technique. Chemical Engineering Transactions, 43(2005): 127–132.

358
 IFRC 2017: 191-162 Functional Food

Total Phenolics, Flavonoids and Antioxidant Activity of Sudanese Baobab (Adansonia digitata) Fruit Pulp

*Idris, Y.M. A., Ibraheem, S. A., Mustafa, S. E.and Kabeir, B. M.

Department of Food Science and Technology, College of Agricultural Studies, Sudan University of Science and
Technology, P.O. Box 71, Shambat, Khartoum North, Sudan.

ABSTRACT‫‏‬

This study aimed to determine phenolics, flavonoids content and antioxidant activity of Sudanese baobab fruit pulp.
Baobab fruit (Adansonia digitata) samples were collected from four locations in Sudan (El Obeid, Um Ruwaba, Niyala
and Damazin). Aqueous and methanolic extracts of fruit pulp were prepared and analyzed for total phenolics and
flavonoids contents and their antioxidant activities were measured with DPPH and FRAP. Total phenolics results
showed significant difference (p≤ 0.05) between extracts. The methanolic extracts values were 99.66, 99.43, 84.49
and 41.37 mg of gallic acid equivalent(GAE) /g for Damazin, Um Ruwaba, El Obeid, and Niyala sample,
respectively, while aqueous extracts values were 22.62, 21.38, 17.16 and 15.50 mg GAE/g for Damazin, El Obeid,
Um Ruwaba and Niyala sample, respectively. Results for flavonoids content showed significant difference (p≤ 0.05)
between samples. Flavonoids content of methanolic extracts were 21.53, 11.66, 4.62 and 4.07 mg of Catechin (CA)
/g for Damazin, El Obeid, Niyala and Um Ruwaba respectively, and for aqueous extracts were 2.00, 1.62, 1.46 and
1.03 mg of CA/g for Niyala, Um Ruwaba, Damazin and El Obeid, respectively. Results for antioxidant capacity
evaluated by DPPH for methanolic extracts were 232.70, 232.70, 229.22 and 223.20 mg ascorbic acid equivalent
antioxidant capacity (AEAC)/ g in Damazin, Um Ruwaba, Niyala and El Obeid sample, respectively, that for aqueous
extracts were 221.30, 200.17, 193.67 and 184.70 mg AEAC /g for Um Ruwaba, Damazin, El Obeid and Niyala
samples, respectively. Results of FRAP for methanolic extracts were 222.28, 217.24, 215.10 and 203.90 mmol /g of
Fe2+ for Damazin, Niyala, El Obeid and Um Ruwaba sample, respectively, while for the aqueous extracts were
209.50, 206.83, 205.80 and 191.64 mmol /g of Fe2+ for El Obeid, Niyala, Um Ruwaba and Damazin sample,
respectively. The methanolic extracts values were significantly higher (p ≤ 0.05) than aqueous extracts. These
findings suggest that Sudanese baobab fruit pulp is rich in phenolics, flavonoids, has high antioxidant activity and
could be a potential functional foods ingredient.

Keywords: Baobab fruit, Phenolics, Flavonoids, Antioxidant activity, DPPH, FRAP.

*Corresponding author’s email: yousifidris@yahoo.com, yousifidris@sustech.edu

INTRODUCTION

Baobab (Adansoia digitata ) is a large deciduous tree originally found in Africa ( Sidibe et al.,1998).In Sudan it is
found in belts in Kordofan, Blue Nile, and Darfur (Elamin, 1990). Its fruit pulp has high contents of pectin, low protein,
low fat, very little iron and is a relatively poor source of manganese, but contains exceptionally high calcium content
(Osman, 2004) and high amounts of vitamin C, a powerful antioxidant (Sidibe and Williams, 2002). Antioxidants are
closely related with the prevention of degenerative illness such as cardiovascular, neurological diseases, and cancer
(Diplock, 1995). Several studies were carried out with the objective of evaluation of antioxidant potential of many
botanicals and herbs, potentially useful as nutriceutical ingredients (Farrukh and Mukhtar, 2002).
The aim of this study is to determine the antioxidant activity, total phenolic and flavonoid contents of Sudanese
Baobab (Adansonia digitata) fruit pulp.

359
Materials and methods

Materials

Baobab fruits were obtained from El Obeid, Um Ruwaba, Niyala and Damazin, areas in Sudan.

Preparation of baobab fruit pulp

Fruit pulp was obtained by breaking the capsules manually, seeds were removed and pulp powder was sieved using
appropriate mesh. The resulting fruit pulp was stored in a dark polyethylene bag at -18ºC until used.

Preparation of fruit pulp extracts

Baobab fruit pulp aqueous and methanolic extracts were prepared by the method of Kim and Lee (2002) with some
modification. The resulting extracts were freeze-dried and kept in dark glass bottles at -18°C until used for analysis.
Methods

Total phenolics content (TPC) was determined spectrophotometrically using Folin–Ciocalteu reagent according to
the method of Tuberoso et al. (2010) with some modifications.

Total flavonoids content was determined spectrophotometrically using aluminum chloride by the method of Kim
et al. (2003).

Antioxidant activity assays of the extracts was evaluated using DPPH (1, 1-dipheny1-2-picrylhydrazyl radical)
and FRAP (ferric reducing antioxidant power) according to the method described by Tuberoso et al. (2010) with
some modifications.

Statistical analysis

Data were mean ± standard deviation of four measurements. Data were analyzed using one-way ANOVA using
MINITAB16 Statistical Software for Windows (State College, PA. USA).

Results and Discussion

Total Phenolics and Flavonoids Contents

The total phenolics and flavonoids contents of Baobab fruit pulp are shown in Table1 and expressed as mg of gallic
acid (GA)/g) and catechin equivalent (CA) mg /g of extracts, respectively. There are significant differences (p≤ 0.05)
in total phenolic contents; which ranged from 15.50 to 99.66 mg GA/g. These values were higher than that reported
by Lamien–Meda et al. (2008).The concentration of phenols extracted in methanol was higher than the aqueous
extracts in for all samples. That may be due to effect of methanol polarity. High solubility of phenols in polar solvents
provides high concentration of these compounds in the extracts obtained using polar solvents for the extraction
(Mohsen and Ammar, 2008). No significant difference between Um Ruwaba and Damazin samples in phenolic
contents of the methanolic extracts.

The concentration of flavonoids in extracts ranged from 1.03 to 21.53 mg of CA/g showing significant difference (p≤
0.05).These values were higher compared to those reported by Lamien–Meda et al. (2008).The highest
concentration of flavonoids was in samples from Damazin. Variations in concentration of flavonoids in fruit pulp
extracts could be due differences in polarity of solvents used in of these compounds. The variations in phenolic, and
Flavonoids content could probably be due to genetic variation, and soil chemical composition.

360
 Table (1): Phenol and Flavonoi contents expressed as mg/g equivalents of Gallic acid

Phenolics content Flavonoids content

Extracts Aqueous Methanolic Aqueous Methanolic

Samples

El Obeid 21.38 ± 0.20b 84.49 ± 0.34 b 1.03 ± 0.08c 11.66 ± 0.10b

Niyala 15.50 ±0.30d 41.37 ± 0.13c 2.00 ± 0.10a 4.62 ± 0.07c

Um Ruwaba 17.16 ± 0.05c 99.43 ± 0.80a 1.62 ± 0.02b 4.07 ± 0.11d

Damazin 22.62 ± 0.07a 99.66 ± 0.18a 1.46 ± 0.02b 21.53 ± 0.12a

Values are means±SD(n=4);means with different superscripts in the same column are significantly different (p≤
0.05).

Antioxidant activity assays

The DPPH and FRAP antioxidant capacity results are shown in Table2. DPPH assay results of the antioxidant
capacities showed significant difference (p≤ 0.05) between the four samples.

The FRAP assay values obtained revealed significant difference (p≤ 0.05) between the four samples of baobab fruit
pulp. Moreover FRAP antioxidant activity of the aqueous extracts revealed that of Damazin sample was significantly
different (p≤ 0.05) from that of the other three locations, these differences could be due to the fact that Damazin
area in which Baobab trees grow is mountainous with heavy rain fall resulting in different characteristics of baobab
fruits compared to that of other locations. The variation of antioxidant activity obtained from baobab fruit pulp from
different locations in Sudan and different solvent systems might be affected by types and structures of phenolic
compound.

Table (2): Antioxidant activity expressed as mmol /g equivalents of Ascorbic acid and Feso4 .7H2 O.

Methanolic Extract Aqueous Extract

Samples DPPH FR AP DPPH FR AP

mmol/g mmol/g mmol/g mmol/g
223.20±0.99a 215.10±0.00c 193.67±0.04c 209.50±0.00a
El Obeid
229.22±0.01a 217.24±0.01b 184.70±0.00d 206.83±0.01a
Niyala
211.22±0.03b 203.90±0.00d 221.30±0.00a 205.80±0.00a
Um Ruwaba
232.70±0.00b 222.28±0.04a 200.17±0.03b 191.64±0.70b
Damazin
Values are means±SD(n=4);means with different superscripts in the same column are significantly different (p≤
0.05).

361
Conclusion
Based on the finding of this study, Sudanese baobab fruit pulp is rich in phenolics, flavonoids, a good source of
natural antioxidant and could be a potential functional foods ingredient.

Acknowledgements
We are grateful for Deanship of Scientific Research, Sudan University of Science and Technology for the financial
support of this research project.

References
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Elamin, H. M. 1990.Trees and Shrubs of the Sudan. Ithaca Press, UK.
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15:297-306.
Kim, D. O. and Lee, C.Y. 2002. Extraction and isolation of polyphenolics. In R. E. Wrolstad (Ed.) Current protocols in
food analytical chemistry. New York: John Wiley and Sons, pp: I1.2.1–I1.2.12.
Kim, D.O., Jeong, S.W.and Lee, C.Y. 2003. Antioxidant capacity of phenolic phytochemicals from various
cultivars of plums. Food Chemistry, 81(3): 321-326.
Lamien-Meda, A., Lamien, C. E., Compaoré, M. M. Y., Meda, R.T.N., Kiendrebeogo, M., Zeba, B., Millogo, J.F and
Nacoulma, O.G. 2008. Polyphenol content and antioxidant activity of fourteen wild edible fruits from Burkina
Faso. Molecules 13: 581–594.
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Food Chemistry, 112(3): 595-598.
Osman, M.A. 2004. Chemical and Nutrient Analysis of Baobab (Adansonia digitata) Fruit and seed Protein Solubility.
Plant Foods for Human Nutrition, 59(1): 29-33.
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1251.

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 IFRC 2017: 223-210 Functional Food

Comparison of Sensory Quality and Preference between Fermented Barleys, Glutinous Rice and the
Combination of Barley and Glutinous Rice

Ibrahim, N., Ezani, N. A. B., Ahmad Kamal, N., Mazlan, N., Abu Kassim, N. A., Jipiu, L. B., Abdul Aziz, S. A. and *Mat
Issa, Z.

Faculty of Hotel and Tourism Management, Universiti Teknologi MARA, Kampus Puncak Alam, 42300 Bandar
Puncak Alam, Selangor, Malaysia.
Abstract
Fermented rice or tapai is one of the traditional fermented food in Malaysia. It is commonly made of white rice or
glutinous rice. The fermentation is performed by the addition of various types of molds. The aim of this study was,
therefore, to investigate the sensory quality and the acceptability of fermented barley as an alternative to fermented
glutinous rice. In this study, barleys, glutinous rice and combination of the two grains were fermented by following the
standard procedures of rice fermentation with ad addition of sugar. After three days of fermentation, the fermented
grains were then subjected to sensory quality such as texture quality, and moisture content. A hedonic study was
also conducted to investigate overall acceptance of the fermented grains. Fermented rice had been used as a control
throughout this study. The study revealed that fermented barleys had a softer texture and higher moisture content
than the fermented rice and its combinations. The hedonic study showed that fermented barley had a good
acceptability by the locals. In conclusion, fermented barley has a potential to be commercialised due to greater
acceptability by the locals and also due to health benefits provided by barley.

Keywords: fermentation, barley, glutinous rice, sensory quality, acceptability

*Corresponding author’s email: zurainim@salam.uitm.edu.my

Introduction
Fermentation can be defined as the chemical breakdown of a substance by bacteria, yeast, or other microorganisms.
Throughout this process, it changes the initial characteristics of a food into a product that is significantly different and
highly acceptable to consumers. Holzapfel (2002) described fermented foods as palatable and wholesome foods
prepared from raw or heated raw materials. One of the popular traditionally fermented food in Malaysia is tapai (a
Malay term for fermented grains). It can be prepared using either glutinous rice (Oryza sativa glutinosa) or cassava
tuber (Manihot utilisima) (Law et al., 2011). Tapai is a popular dessert/snack with a sweet and acidic taste. In
Peninsular Malaysia, tapai from cassava or rice is very popular and sometimes black glutinous rice is used (Merican
and Yeoh, 1989). They can be consumed as such or used as an ingredient in home-cooking and baking. These
fermented grains can be eaten on its own or served with contemporary toppings like ice cream, chocolate and fruit.
During the grains fermentation process, a mixed culture of microorganisms is used as the starter culture. Merican
and Yeoh (1989) indicated that Amylomyces rouxii, Saccharomycopsis fibuligera and Hansenula anomala were
necessary to produce fermented glutinous rice), while A. rouxii and S. fibuligera were required for fermented
cassava.

The U. S. Food and Drug Administration (USDA, 2006) issued an endorsement of benefits of foods containing barley
principally due to its soluble fibre content (β-glucans) which has been shown to lower blood cholesterol levels.
Several studies have also reported the benefits of barley in improving the increasing good cholesterol (Tiwari and
Cummins, 2011) and lowering bad cholesterol (Wang et al., 1993; Brennan and Cleary, 2005), which later can
reduce the risk of coronary heart disease (Satija and Hu, 2012). Barley also seems to lower glucose level (glycemic
index) with implication for those suffering from diabetes (Grando and Macpherson, 2005; Brennan and Cleary, 2005;
Newman and Newman, 2008; Nettleton et al., 2010; Maslowski and Mackay, 2011). In addition, barley also contained
major phytochemicals such as phenolics, tocols and folate (Ward et al., 2008). Lignans are polyphenolic substances
are also found in cereal grains such as barley, oat and rye. These compounds are bioavailable and have
demonstrated to reduce the risk of hormone-dependent cancer (i.e., breast and prostate) and colon cancer

363
(Cotterchio et al., 2006; Qu et al., 2005). Hence, this study was conducted to investigate the sensory quality and the
acceptability of fermented barley as an alternative to fermented glutinous rice.

Material and Methods

Preparation of fermented grains
In this study, glutinous rice and barley were used as the main ingredients for fermented grains preparation. Three
types of fermented grains were prepared consisting glutinous rice, barley and combination of glutinous rice (50%)
and barley (50%). All the three fermented grains were prepared using similar procedures.

Prior to preparation of fermented grains, 250 g of grains were washed (note: for a mixture of grains, equal amount of
each grain was prepared), then soaked overnight and steamed for 30 minutes. Then, the steamed grains were
cooled to room temperature on a clean, dry stainless steel tray before 0.04% (w/w) of fermentation starter (in a
powder form) and 0.4% (w/w) of sugar were sprinkled over the steamed grains and mixed well. The grains were then
packed in a banana leaf and covered tightly with a clean cloth. The grains were allowed to ferment for 3 days at room
temperature. The control sample was fermented glutinous rice.

Determination of water activity

The water activity (Aw) of the fermented grains was determined using a Aw meter Rotronic HygroPalm AW1 (Rotronic
Instrument Corp., Basserdorf, Germany). The Aw was measured on day 3 after the fermentation process at room
temperature.

Determination of texture
The texture of fermented grains on day 3 was determined using a texture analyser TAXT2 (Stable Micro System,
Godalming, UK) with a load cell of 5 kg, cylinder probe (diameter 50 mm). The samples were compressed at a speed
of 10 mm/s and return strain of 50 mm. The firmness of the samples was determined as the peak of compression
force (in g), which was detected when the probe proceeded to penetrate into the fermented grains.

Consumer preference
The hedonic test was used to determine the consumer’s preference towards the fermented grains.

Data analyses
The water activity and texture analyses were analysed using Excel (Microsoft Inc., version 2010) for descriptive
statistics and one sample t-test. Prior to further analyses, the compression force was converted to gram per second
(gm/sec). The Hedonic data were analysed using Excel (Microsoft Inc., version 2010) for descriptive statistics and
a Single Factor ANOVA.

Results and Discussion

Table 1 summarises the water activity, the textural analyses, and also the mean preference of consumers towards
the grains after 3 days fermentation. The results showed that all the three types of fermented grains have a water
activity greater than 0.9. Similar findings were recorded by Oduro-Yeboah et al. (2016) where fermented grains would
result in an increase in moisture. Fermented barley had the highest amount of moisture (0.954 ± 0.003). However,
one sample t-test on water activity showed that there was no significant different between the three types of
fermented grains (p > 0.05). The textural analysis showed that the fermented barley has a softer body (26.65 gm/sec)
compared to glutinous rice (30.52 gm/sec) and the combinations of barley and glutinous rice (54.26 gm/sec).
However, one sample t-test showed that there were no significant different between the firmness of the fermented
grains (t = 0.77, n = 3, p = 0.74 > 0.05). According to Dung (2013), the composition of amylose and amylopectin and
soaking can affect the enzymic hydrolysis of grains. Jomduang and Mohamed (1994) concluded that the moisture
content of grains was positively correlated to amylopectin. Hence, the findings suggested that the grains used in this
study may have almost similar percentage of amylose and amylopectin.

364
 Table 1: Water activity (Aw), firmness and preference of different types of fermented grains after three days
fermentation process.
Types of tapai Aw Firmness Preference
(gm/sec)
Glutinous rice 0.943 ± 0.003 30.52 6.44 ± 1.11
Barley 0.954 ± 0.003 26.65 5.78 ± 1.45
Glutinous rice + barley 0.937 ± 0.007 54.26 6.46 ± 1.2

Data from the Hedonic test was interpreted using a Single Factor ANOVA and the F-test. If the F > F crit, the null
hypothesis would be rejected. Hence, the results of this study showed that there were significantly different between
the three types of tapai (F(2, 147) = 4.717 > 3.058). Fermented barley was preferred less by the consumer than the
other two fermented grains due to its non-stick texture after the fermentation process. This is due to the fact that the
soluble fibre of the barley is not affected by fermentation (Lambo, Oste and Nyman, 2005).

Conclusion
Fermented foods are prevalent in Southeast Asia especially in processing and preserving the food in keeping the
good flavour, aroma, enhanced nutritive values and retaining keeping quality under ambient temperatures.
Unfortunately, there has not been any extensive study on the fermented barley in Malaysia. It is hoped that
throughout this study, researchers are able to formulate a more full-fledged methodology and the need to look
beyond liking and acceptability of the fermented barley. It is also hoped that the food industry can consider the
scientific and market research evidence presented from this study, and also in promoting healthy, authentic and
traditional fermented barley to the consumers.

Acknowledgements
The authors would like to thank Universiti Teknologi MARA for the support and encouragement throughout this study.

References
Brennan, C. S. and Cleary, L. J. 2005. The potential use of cereal (1→3, 1→4)-β-D-glucans as functional food
ingredients. Journal of Cereal Science, no. 42, pp. 1-13.
Cotterchio, M., Boucher, B. A., Manno, M., Gallinger, S., Okey, A. and Harper, P. 2006. Dietary phytoestrogen intake
is associated with reduced colorectal cancer risk. Journal of Nutrition, no. 136, pp. 3046.
Grando, S. and Macpherson, H. G. 2005. Food barley: importance, uses and local knowledge. Proc. International
Workshop of Food Barley Improvement, Hammamet, Tunisia, January 14-17, 2002, ICARDA, Aleppo, Syria.
Holzapfel, W. H. 2002. Appropriate starter culture technologies for small-scale fermentation on developing countries.
International Journal of Food Microbiology, no. 75, pp. 197-212.
Law, S. V., Abu Bakar, F., Mat Hashim, D. and Abdul Hamid, A. (2011). Mini Review: Popular fermented foods and
beverages in Southeast Asia, International Food Research Journal, no. 18, pp. 475-484.
Maslowski, K. M. and Mackay, C. R. 2011. Diet, gut microbiota and immune responses. Nat Immunol, no. 11, pp. 5-9.
Merican, Z. and Yeoh, Q. L. 1989. Tapai proceeding in Malaysia: A technology in transition. In Industrialization of
Indigenous Fermented Foods, in Steinkraus, K. H., pp. 169-189. Marcel Dekker, Inc., New York.
Nettleton, J. A., McKeown, N. M., Kanoni, S., Lemaitre, R. N., Hivert, M. F., Ngwa, J., van Rooij, F. J., Sonestedt, E.,
Wojczynski, M. K., Ye, Z., et al 2010. Interactions of dietary whole-grain intake with fasting glucose- and
insulin-related genetic loci in individuals of European descent: a meta-analysis of 14 cohort studies. Diabetes
Care, no. 33, pp. 2684-2691.
Newman, R. K. and Newman, C. W. 2008. Barley for food and health science, technology, and products. John Wiley
and Sons, Hoboken, NJ.
Qu, H., Madl, R. L., Takemoto, D. J., Baybutt, R. C. and Wang, W. 2005. Lignans are involved in the antitumor
activity of wheat bran in colon cancer SW480 cells. Journal of Nutrition, no. 135, pp.598.
Satija, A. and Hu, F. B. 2012. Cardiovascular benefits of dietary fiber. Curr. Atheroscler. Rep., no. 14, pp. 505-514.

365
Tiwari, U. and Cummins, E. 2011. Meta-analysis of the effect of beta-glucan intake on blood cholesterol and glucose
levels. Nutrition, no. 27, pp. 1008-1016.
Wang, L., Xue Q., Newman, R. K., and Newman, C. W. 1993. Enrichment of tocopherol, tocotrienol, and oil in barley
by milling and pearling. Cereal Chemistry, vol. 70, no. 5, pp. 499-501.
Ward, J. L., Poutanen, K., Gebruers, K., Piironen, V., Lampi, A. M., Nystrom, L., Andersson, A. A., Aman, P., Boros,
D., Rakszegi, M., et al., 2008. The HEALTHGRAIN Cereal Diversity Screen: concept, results, and prospects, J
Agric Food Chem, no. 56, pp. 9699-9709.
Lambo, A.M., Öste, R. and Nyman, M.E.L., 2005. Dietary fibre in fermented oat and barley β-glucan rich
concentrates. Food Chemistry, 89(2), pp.283-293.
Oduro-Yeboah, C., Mestres, C., Amoa-Awua, W., Fliedel, G., Durand, N., Matignon, B., Michodjehoun, V.L., Saalia,
F.K., Sakyi-Dawson, E. and Abbey, L., 2016. Steeping time and dough fermentation affect the milling behaviour
and quality of white kenkey (nsiho), a sour stiff dumpling prepared from dehulled maize grains. Journal of Cereal
Science, 69, pp.377-382.

366
 IFRC 2017: 231-216 Functional Food

Glycemic Index of Chocolate Fortified with Pumpkin (Cucurbita moshata) and Taro (Colocasia esculenta)
Powder and its Effect on Mood and Cognitive Functions of Female students

*Shahidan, N., Salleh, N.Z., Rois Anwar, N.Z., Zakaria, Z.

Faculty of Bioresources and Food Industry, University Sultan Zainal Abidin, Besut Campus, 22200, Besut,
Terengganu, Malaysia

Abstract
Pumpkin and taro are underutilised local crops which have benefits in vitamin and mineral supplement that can be
incorporated into food product. At present moment, there is limited number of fortified product with local crops in
confectionary market. Thus, this study aimed to determine the Glycemic Index of chocolate fortified with pumpkin and
taro powder and observe its effect on mood and cognitive functions of UniSZA female students. Two groups of
female students (n=30) were asked to consume control chocolate (Group A) and fortified chocolate (Group B) for four
weeks. Their mood and cognitive functions were observed through the four weeks and the results were represented
in mean of three replications. Their 2-hours postprandial blood sugars also were assessed by finger-prick blood in
order to determine their Glycemic index value. The results showed an increased in mean value of 5-points mood
scale in both groups from not at all happy (1.81) to moderately happy (3.38) after 15 minutes of its consumption.
Meanwhile, respondents in group B also able to score the highest mean value (95%) in memorizing 10 listed items as
compared with respondents in group A (90%). Besides, respondents in group B also gave a steady rise in their blood
sugar and insulin levels (5.14±0.47, 5.42±0.43, 5.63±0.39, 6±0.38, 5.86±0.53, 5.58±0.73, 5.3±0.58, 5.07±0.45) as
compared with group A who gave a result in fluctuation of blood glucose levels. Thus, it was suggested that inclusion
of this food in diets of human can enhance the positive mood, increase the cognitive development and prevent
diabetes mellitus.

Keywords: Fortified chocolate, pumpkin, taro, glycemic index, cognitive function, mood
*Corresponding author’s email: norshazila@unisza.edu.my

Introduction

Chocolate confectionaries form a great part of the sweet’s market and produce diverse products. However, certain
food products especially confectionaries have negative reputation due to the high content of calories what is the main
reason for obesity and diverse health issues. Concerning this fact, market should provide more products with
additional nutritional value which is good taste and also ensure good functional properties. In this study, fortification
of pumpkin and taro powder into chocolate is an effective way to overcome deficiency beside give a lot of beneficial
health to human due to it higher mineral and vitamin content.Studies that support a link between chocolate and good
health are very popular with readers. However, the reality is that the most chocolate studies are observationally in
nature and therefore limited in what they can tell us about its supposed benefits. At present moment, there are limited
number of fortified products with underutilized crops such as pumpkins and taro tubers. Hence, fortification of the
chocolate with these crops is an effective strategy to prevent or overcome deficiency. Apart of it, it is an excellent
approach to diversify functional foods using local crops. In this study, glycemic Index of chocolate fortified with
pumpkin and taro powder was determined and the effect of fortified chocolate consumption on mood and cognitive
function among UniSZA female students were observed.

367
Materials and Methods

2.1 Materials

Two different chocolate flavours, white chocolate and dark chocolate brand Beryl’s were purchased at supermarket in
Terengganu. The Glycemic Index (GI) of the students was measured using a glucometer that was assessed by
finger-prick blood samples. The portion of chocolate was 35 grams equivalent to one small bar for both the control
and enriches chocolate as the test foods. Others materials used in this study including pumpkin and taro powder that
was obtained from the various laboratories in the Faculty of Bioresources and Food Industry, University Sultan Zainal
Abidin.

2.2 Methods

2.2.1 Preparation of control and fortified chocolate

The chocolates were chopped into small pieces and 200 grams of each chocolate were weight. Then, white and dark
chocolates were melted in the tempered machine separately at temperature 35°C. After melted, 1 gram of pumpkin
powder was added into white chocolate compound and another 1 gram of taro powder was added into dark chocolate
compound. The chocolates were stirred continuously at constant temperature until mixed. For the fortified chocolate
sample, 1.2 ml of white chocolate mixed with pumpkin powder was filled into the mould bar and kept in chiller for 10
minutes. White chocolate will acts as coating. After 10 minutes, another 2.5 ml of extra dark chocolate mixed with
taro powder was filled into the same mould bar and allowed the chocolate to cool in chiller for another 10 minutes.
After cool, filled another same 1.2 ml of white chocolate into the same mould bar and kept it chill for 10 minutes. The
same procedures were conducted for the control sample by excluding pumpkin and taro powder.

2.2.2 Selection of subjects

In this study, all the subjects were divided into two groups. Group A (n=30) will consume 35 grams of control
chocolate. While, another Group B (n=30) will consume 35 grams of chocolate fortified with pumpkin and taro powder
for 4 weeks in order to determine their Glycemic value, mood and cognitive function. Subjects were selected based
on Nutrition and Health Questionnaire.

2.3 Design and procedure

2.3.1 Glycemic Index test

This study was conducted as described by Marangoni and Poli (2007) with slightly modifications.

2.3.2 Consumption of chocolate and its effect on mood

This study was conducted as described by Macht and Dettmer (2006) with slightly modifications.

2.3.3 Consumption of chocolate and its effect on cognitive function

This study was conducted as described by Lemos et al., (2015) with slightly modifications.

Results and Discussions

3.1 Effect of Chocolate Consumption on Mood

368
Mood of the respondents were examined throughout four weeks. The data was presented in total mean score value
of three replications. Mean score values were calculated and represented scale from 1 (not at all) to 5 (extremely
much). Table 1 showed the respondents mood effect after consumption of control and fortified chocolate. From the
results, it showed a change in respondent’s mood after 15 minutes consuming control chocolate by an increase the
mean score of happy mood from 1.86 (not at all) to 2.85 (slightly). Besides, after 15 minutes consumption of control
chocolate, it showed again an increase in happy mood of respondents from 2.82 (slightly) to 3.23 (moderately).
Meanwhile, for the fortified chocolate, the results clearly showed that after 5 minutes of it consumption, mood of the
respondents increase from 1.81 (not at all) to 2.51 (slightly) happy. Then after 15 minutes of the consumption it again
rise the mean score of their happy mode from slightly to moderately happy. For both chocolate samples, during 15
minutes to 90 minutes of its consumption, most of the respondent mood remain moderately happy.

Table 1: Mean score of mood at difference time points after consumption of test foods
Minutes
Samples Mood
0 5 15 30 60 90
Happy 1.4 1.86 2.82 3.23 3.18 3
Control Sad 3.27 2.23 1.34 1.31 1.21 1.1
Neutral 3.03 2.4 1.51 1.13 1.38 1.15
Happy 1.81 2.5 3.38 3.38 3.47 3.3
Fortified Sad 3.29 2.51 1.57 1.59 1.21 1.1
Neutral 3.06 2.38 1.27 1.43 1.12 1.48
*values in the column showed total mean score of 5-point mood scales, *1-1.9=not at all, *2-2.9=slightly,*3-
3.9=moderately,*4-4.9=very much,*5-5.9=extremely much

3.2 Effect of Chocolate Consumption on Cognitive Function

Cognitive data of the respondent was determined before and after 30 minutes of chocolate consumption. The data
was expressed in percentage mean value of the score for three replications. In control group, the result showed that
respondents able to memorize 52.5% of the score list items at trial 5. It shows an increase in the total score as
compared at trial 1, they only able to memorize 22.5% out of 100%. However, in the fortified group, the result
showed a bit increse in the respondents memory test. At trial 5 they able to memorize 55% as compared at trial 1
they only can memorize 27.5%.Aafter 30 minutes of the chocolate consumption, it also clearly showed an increase
in the total percentage mean score of list item the respondents able to memorize in each trials. For control group, the
respondents able to memorize up to 90% score of items at trial 5 as compared during trial 1, they only can memorize
only 45% score of items. However, respondents who consume fortified chocolate showed a higher score, which is
95% at trial 5. From the overall result, it showed that an increase in the percentage mean score of item in each trial
for both groups. This was happened because regular intake of cocoa flavanols may have a beneficial effect on
cognitive function and possibly protect against normal age related to cognitive decline (Crichton et al., 2016).

3.3 Effect of Chocolate Consumption Glycemic Index data

GI was measured the carbohydrate in a food to raise blood sugar which is glucose levels after being eaten. In
particular, GI value of the control and fortified chocolate group was significantly (p<0.05) different with glucose group.
Glucose is a simple sugar that is easily broken down by the body and transport into cell for the energy. Consumption
of glucose solution give a fluctuation in respondents blood sugar levels because it is easily absorb into bloodstream
and cause a dramatically rises in their blood sugar level after 15 min consumption. According to Jenkins et al.,
(2002), blood sugar levels can start increasing after 15 minutes after meals and food with high GI will rise highly due
to the type of carbohydrates found in food.

369
 Table 2: Blood glucose level (mmol/L; mean ± standard deviation) at different time points after consumption of the
test foods
Minutes
Sample 0 15 30 45 60 75 90 120
5.10a,B ± 8.92f,B ± 8.16e,f,B ± 7.15e,B ± 6.37d,B ± 5.7c,B ± 5.14b,B ± 4.86a,B ±
Glucose 0.43 0.64 0.67 0.5 0.45 0.41 0.31 0.21

5.14a,A ± 5.36f,A ± 5.69e,f,A ± 5.99e,A ± 6.00d,A ± 5.78c,A ± 5.36b,A ± 5.12a,A ±

Control chocolate 0.46 0.34 0.41 0.47 0.38 0.31 0.36 0.29
5.14a,A ± 5.42f,A ± 5.63e,f,A 6.00e,A ± 5.86d,A ± 5.58c,A ± 5.3b,A ± 5.07a,A ±
Fortified chocolate 0.47 0.43 ±0.39 0.38 0.53 0.73 0.58 0.45
Note: values in the same column with difference alphabet are statistically significant from each other (p<0.05),
*alphabets A,B,C represented samples, *alphabets a,b,c,d,e,f represented minutes. Presented data was mean value
± standard deviation

Conclusion
Product made in this study with addition of pumpkin and taro powder had increase positive mood of the respondents
after the consumption. Besides, the result also showed that cognitive performances of the respondents who consume
fortified chocolate increase in term of their memory test. Hence, fortification of chocolate with these two local crops
can be a good food source for students who suffer from stress that can lead to poor cognitive function. Consumption
of this product showed a steady rise in blood sugar and insulin levels.

Acknowledgement
We would like to thank Food Analysis Lab, Universiti Sultan Zainal Abidin for the facilities provided and R0001-R291
Precommercialization grant for the financial support

References

Crichton, G., E., Elias, M., F. & Alkerwi, A. (2016). Chocolate intake is associated with better cognitive function: the
maine-syracuse longitudinal study. Appetite, 10: 1010-1016.
Jenkins, D., J., A., Kendall, C., W., Augustin, L., S., Franceschi, S., Hamidi, M., Marchie, A., Jenkins, A., L. &
Axelsen, M. (2002). Glycemic index: overview of implications in health and disease. Journal of clinical
nutrition, 76(1): 2665-2735.
Lemos, R., Simoes, M., R, Santiago, B. & Santana, I. (2015). The free and cued selective reminding test: validation
for mild cognitive impairment and Alzheimer;s disease. Journal of neuropsychocology, 9(2): 242-257.
Macht, M. & Dettmer, D. (2006). Everyday mood and emotions after eating a chocolate bar or an apple. Appetite, 46:
332-336.
Marangoni, M. & Poli, A. (2007). The glycemic index of bread and biscuits is markedly reduced by the addition of a
proprietary fiber mixture to the ingredients. Nutrition, Metabolism & Cardiovascular Disease, 18: 602-605.

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 IFRC 2017: 240-220 Functional Food

Chemical Composition of Mesocarp and Exocarp from Borassus flabellifer

abRodiah M. H., a*Jamilah B., Russly A. R., and aSharifah Kharidah S. M.

aUniversiti Putra Malaysia, Faculty of Food Science and Technology, 43400 Serdang, Selangor, Malaysia
bUniversiti Selangor, Faculty of Engineering and Life Sciences, Department of Science and Biotechnology, Bestari
Jaya Campus, Jalan Timur Tambahan, 45600 Bestari Jaya, Selangor, Malaysia.

Abstract
Palmyra palm (Borassus flabellifer L.) belonging to the family Palmae has been reported to be the source of fiber, me
dicinal, food and beverage. The immature, soft, juicy seed nuts are popularly prepared as natural drink, but
approximately 40-55% of the skins (mesocarp and exocarp) are discarded. Negligible literature is available on the
composition of these parts of the fruit. Hence, the chemical composition of the mesocarp and exocarp from Borassus
flabellifer were determined with the objective of evaluating the potential of utilizing these two fruit components into a
value-added product. Proximate, total phenolic content and antioxidant activity analysis were carried out for both
mesocarp and exocarp. Both mesocarp and exocarp had high crude fiber (23.92% and 28.20%, respectively) but low
in ash, protein and fat contents. The carbohydrate content of all samples exhibited moderate amount of carbohydrate
ranging from ~53 to 56%. Phenol and tannins were present in both mesocarp (5.58 mg GAE/g and 3.23 mg GAE/g,
respectively) and exocarp (0.007 mg TAE/g and 0.003 mg TAE/g, respectively). Nevertheless, saponin was only dete
cted in mesocarp at 16.75%. Alkaloids, cardiac glycosides, flavonoids, steroids, terpenoids and anthraquinone were n
ot detected. Radical scavenging activity (157.05 mM TE/g) and reducing power (213.05 mM Fe2+) of exocarp were
significantly higher (p< 0.05) than mesocarp. On the overall, this study indicates the present of significant amount of
fiber and antioxidative properties in the mesocarp and exocarp of Borassus flabellifer which could be a potential
source as a functional ingredient.

Keywords: Borassus flabellifer, exocarp,fiber and mesocarp.

*Corresponding author’s email: jamilah@upm.edu.my

Introduction
Plants are supplied with different phytochemical particles, for example, vitamins, terpenoids, phenolic acids, lignins,
stilbenes, tannins, flavonoids, quinones, coumarins, alkaloids, amines, betalains, and different metabolites, which are
rich in antioxidant activity (Shahid and Mohammad, 2013; Samanta and Konar, 2011). Phytochemicals are natural
bioactive compounds which are available in plants. These natural compounds work with nutrients and dietary fibres
to protect animals and man against diseases. Since time immemorial, which are gotten from plant parts such as stem
bark, leaves, fruits and seeds have been part of phyto-medicine, in this manner demonstrating that any part of a plant
may contain essential active compounds (Soladoye and Chukwuma, 2012).

Borassus flabellifer Linn of the Arecaceae family is well known as Palmyra palm. Palmyrah palm has great economic
potential due to its multipurpose uses. In Malaysia, the immature soft juicy seed nuts are very popular to be eaten
raw or prepared as natural drink. However, only the seed nut of the fruit is used as food and beverages and the skins
of the fruit are thrown away. The discarded portion from these palm fruits have not received much attention to be
used or recycled. In fact, there are no conclusive report on the chemical composition, nutritional value, and antioxida
nt properties of fruit discards (mesocarp and exocarp). Keeping this in view and due to awareness of eco-friendly
environment, the chemical composition (e.i; proximate, phenolic content, antioxidant activities etc) of mesocarp and
exocarp residues were explored.

371
Materials & Methods
2.1 Sample preparation
The young fruit of Borassus flabellifer were collected from Cameron Highland, Pahang, Malaysia The exocarp and
mesocarp were cut into smaller pieces, dried (50˚C for 24 hours) and ground. The samples were sieved to pass
through a 0.5 mm sieve and kept at 30˚C in an air tight plastic container in a dessicator prior to conducting the
experiments.

2.2. Proximate analysis

Moisture, protein, lipid, crude fiber and ash contents of mesocarp and exocarp were determined according to official
methods (AOAC, 2000).

2.3 Phytochemical Analysis

Active component of mesocarp and exocarp was extracted using microwave assisted in an experimental microwave
oven (Samsung, Korea) according to method Asma et al. (2016). Phytochemical analysis of mesocarps and exocarps
was conducted according to standard of procedures with minor modification (Obidoa et al., 2010; Aiyegroro and Oko
h, 2010; Obadoni and Ochuko (2001).

2.4 Antioxidant Activity

The ability of mesocarp and exocarp extract to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was measured
by the reported method Chu et al. (2000) with slight modification. The results were expressed as mol of Trolox equiva
lent DPPH radical scavenging activity per g of sample. The antioxidant capacity was also determined using a modific
ation of the FRAP assay described by Langley-Evans (2000) with a minor modifications and the results were express
ed as milimol ferrous per g of sample.

2.5 Statistical Analysis

All experiments were carried out in triplicates, and the results were expressed as means ± SD (standard deviation).
One way ANOVA was used to analyze the experimental data and Turkey test was applied to determine the significant
difference (p<0.05) of the samples.

Results and Discussion

Proximate Composition of Mesocarp and Exocarp of Borassus flabellifer

The results of chemical composition in mesocarp and exocarp of Borassus flabellifer is summarized in Table 1.
Proximate analysis showed the moisture and ash content of mesocarp and exocarp was between 9.17-10.3% and
2.76%-3.66%, respectively. In addition, protein in exocarp showed significantly higher (5.27%) than mesocarp (4.20%
). The crude lipid content obtained for both samples were very low and ranged from 1.52 till 1.86. As reported by Ven
gaiah et al. (2015), the percentage of ash, protein and lipid content of palmyrah fresh pulp from matured fruit were 1.
2%, 1.23% and 0.8%, respectively. In comparison to this study, the young mesocarp and exocarp have slightly higher
ash, protein and lipid content than palmyrah matured pulp.

The crude fibre obtained for exocarp was 28.20% (DM) which was significantly higher than mesocarp (23.92%).
Although crude fibre enhances digestibility, its presence in high level can cause intestinal irritation, lower digestibility
and decreased nutrient usage (Oladiji et al., 2005). However, this low level of crude fibre is considered appropriate,
because it aids absorption of glucose and fat. In addition, the carbohydrate content of all samples exhibited fairly
higher amount of carbohydrate ranged from 52.68%–56.35%. On the contrary, the carbohydrate content of palmyrah
fresh pulp from matured fruit was only 22.5% (Vengaiah et al., 2015).

372
 Table 1: Proximate Composition of Palmyrah palm (Borassus flabellifer)
Borassus flabellifer
Composition (%)
Mesocarp Exocarp
Moisture 10.349a±0.215 9.197b±0.514
Ash 3.664a ±0.122 2.794b ±0.208
Protein 4.201b ±0.469 5.273a ±0.375
Fat 1.522a ±0.507 1.861a ±0.620
Crude fibre 23.917b ±1.703 28.196a ±1.545
Carbohydrate (by difference) (%) 56.347a 52.678 b
Results are the means from triplicates. a–b Different superscript letters between mesocarp and exocarp denote signific
ant differences (Tukey's test, p < 0.05).

Phytochemical Analysis
Phenols and tannins were present in all the extracts but only mesocarp was contained with saponin. Mesocarp
extracts are slightly lower in total phenolic content (5.58 mg GAE/g) but rich in tannin (3.23 mg GAE/g) and saponin
(16.75%).) as compared to exocarp extracts (data not shown). In contrary, alcohol extract of root from B. flabellifer
showed higher phenolic content of 707.08 mg GAE/g and petroleum ether extract showed minimum total phenol
content of 28.75 mg GAE/g (Lina et al., 2013).

Antioxidant Activities
Figure 1 displays the radical scavenging activity of the mesocarp extract (157.05 mM TE/g) was significantly higher
than exocarp (29.90 mM TE/g). The result showed that mesocarp extract had a better scavenging activity or antioxid
ant potential as compared to exocarp. Mean while, mesocarp extract had significantly higher reducing power (213.05
mM Fe2+ /g) as compared to exocarp extract. As points out by Jun et al. (2016), the reducing power of a compound
may serve as a significant indicator of its potential antioxidant activity.

Figure 1. Antioxidant activity of mesocarp and exocarp of Borassus flabellifer.

Note. There are significant differences among values marked with the different superscript letters between mesocarp
and exocarp (Tukey's test, p < 0.05).

Conclusion
This study revealed that the mesocarp and exocarp analysed in this study has considerable amount of antioxidant act
ivity associated with fiber. Mesocarp and exocarp have constituents of phenols and tannins except saponin (only mes
ocarp has positive result). It could be seen that mesocarp contained higher radical scavenging activity and reducing
power ability than exocarp. Overall, all these results indicate that the mesocarp and exocarp of Borassus flabellifer
contains nutrients and bioactive compounds that makes it a potential source for the production of functional food
products/food supplements and various nutraceuticals.

373
Acknowledgment
The authors would like to express their sincere thanks to Ministry of Higher Education, Malaysia for the post graduate
fellowship (MyBrain15-MyPhD).

References
Aiyegroro, O.A., and Okoh, A.I. 2010. Preliminary phytochemical screening and in vitro antioxidant activities of
aqueous extract of Helichrysum longifolium DC. BMC compementaryl. and Alternative. Medicine., 10:21
AOAC International .1993. Methods of Analysis for Nutrition Labelling. Chapter 33. Sugar (Mono and Di), Glucose,
Fructose, Sucrose and Maltose in Presweetened Cereals Liquid Chromatographic Method (982.14), Sugar
(Mono and Di), Separation of sugar in Honey Liquid Chromatographic Method (977.20)
AOAC . 2005. In W. Horwitz, & G.W. Latimer (Eds.), Official methods of analysis of AOAC
International (pp. 2200)., 18th ed. Gaithersburg: AOAC.
Asma, F.Z., Rodiah, M.H., & Aziah, M.Y. 2016. Microwave-assisted extraction of natural colorant extracted from
mesocarp and exocarp of Cocos nucifera (coconut palm). European Journal of Biotechnology and Bioscience,
4(4),1-5.
Association of Official Analytical Chemists Official methods of analysis (17th ed.). 2000. AOAC International,
Gaithersburg
Chu, Y. H., Chang, C. L., & Hsu, H. F. 2000. Flavonoid content of several vegetables and their antioxidant activity. Jo
urnal of the Science of Food and Agriculture. 80:561–566.
Jun, J. S., Jian, G. Z., Yu, H. S., Jie, Q., Ling, L., Chandan, P., & Zhao,J. W. 2016. Physicochemical properties and
antioxidant activities ofpolysaccharides sequentially extracted from peony seed dreg. International Journal of
Biological Macromolecules, 91:23–30.
Langley-Evans, S. C. 2000. Antioxidant potential of green and black tea determined using the ferric reducing power
(FRAP) assay. International Journal of Food Sciences and Nutrition, 51(3), 181–188.
Lina, S. M. M., Mahbub, K. M. M., Ashab, I., Al-Faruk, M., Atanu, S. H., Alam, M. J., and Sahriar, M. 2013. Antioxidant
and Cytotoxicity potential of Alcohol and Petroleum ether extract of Borassus flabellifer Linn. International
Journal Pharmaceutical Science Resource, 4(5); 1852-1857.
Mongeau, R. 2003. Dietary fibre. In R. Macrae, R. K. Robinson, & M. J. Sadler (Eds.), Encyclopaedia of food
science and nutrition (pp. 1362−1387).NewYork:Academic Press.
Obadoni, B. O., Ochuko, P. O. 2001: Phytochemical studies and comparative efficacy of the crude extracts of some
homeostatic plants in Edo and Delta States of Nigeria. Glob.J. Pure Appl. Sci., 86: 2003-2008 2.5 Antioxidant
Analysis
Obidoa O., Joshua P. E., Eze N. J. Phytochemical analysis of Cocos nucifera L. Journal of Pharmaceutical Research.
2010;3(2):280–286.
Oladiji, A. T. & Mih, F. O. 2005. Proximate composition, mineral and phytochemical constituents of Eleusine coracana
(finger millet). African Journal Biotechnology., 4 (12): 1440- 1441.
Samanta A. K., & Konar, A. 2011. Dyeing of Textiles with Natural Dyes, Natural Dyes, Dr. Emriye Akcakoca Kumbasar
(Ed.), InTech, Available from:
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Shahid, M., & Mohammad, F. 2013. Recent advancements in natural dye applications: a review. Journal of Cleaner
Production, 53, 310–331.
Soladoye, M., O. & Chukwuma, E. C. 2012. Quantitative phytochemical profile of the leaves of Cissus populnea Guill.
& Perr. (Vitaceae) – an important medicinal plant in central Nigeria. Archives of Applied Science Research. 4
(1):200-206.
Vengaiah, P. C, Vijaya Kumara, B, Murthy, G. N., and Prasad, K. R. (2015) Physico-Chemical Properties of
Palmyrah fruit Pulp (Borassus flabellifer L). Journal Nutrition Food Science, 5: 391.

374
 IFRC 2017: 244-224 Functional Food
Physico-chemical Properties of Spray Dried Powders from Two Varieties of Amaranth (Amaranthus viridis)

Siti Faridah, M.A., Tan, L.Y. and * Muhammad, K.

Faculty of Food Science and Technology, Universiti Putra, Malaysia, 43400 UPM, Serdang, Selangor Darul Ehsan,
Malaysia

Abstract
The increasing demand for natural colorant had led to the search for new sources of various colour pigments.
Amaranthus viridis which is locally known as ‘bayam hijau’ is found in abundant in the Malaysian market and hence
this green leafy plant has the potential to become a new source of colorant due to its significant content of
chlorophylls. A study was, therefore, carried out to determine the chlorophyll content of 5 local green leafy vegetables
including two species of green amaranth (round and oval leaves). The effect of water extraction with and without prior
blanching on the yield of amaranth powder, chlorophyll content, antioxidant activity and powder properties of the
spray-dried amaranth of both varieties were evaluated. The extracts were microencapsulated using 20% maltodextrin
of DE 10 at an inlet temperature of 150 ± 50C and a feed rate of 300 mL/h. The highest yield (69.8%) of spray-dried
powder was obtained from the extract of unblanched amaranth round leaves. In addition, this powder possessed the
highest 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity and showed the highest chlorophyll
retention which was 1536.8 µM Trolox Equivalent (TE)/g and 95.6%, respectively. It was also found that this powder
had the highest bulk density and solubility compared to the powder from unblanched amaranth oval leaves which
were 0.51 g/mL and 77.9%, respectively. However, the spray-dried powder from unblanched amaranth oval leaves
appeared to be more green than the powder from unblanched round leaves with L* value of 76.7, a* value of -3.8 and
b* value of 14.0. The chlorophyll content of the former powder was higher than that of the latter powder which was
8.87 mg/100 g. The findings of this study indicated that blanching did not improve the antioxidant activity and colour
of spray-dried amaranth powder. Meanwhile, spray-dried powder from unblanched amaranth round leaves had better
physico-chemical properties compared to that from unblanched oval leaves except for its chlorophyll content and
green colour.

Keywords: Amaranthus viridis, chlorophylls, colorant, microencapsulation, maltodextrin, antioxidants.

*Corresponding author’s email: kharidah@upm.edu.my

Introduction
There is an increasing awareness of diets rich in vegetables to ensure an adequate intake of most vitamins and other
micronutrients, dietary fibers, and phytochemicals (Joao Silva Dias, 2012). Epidemiological studies have shown that
a high intake of vegetables can lower the occurrence of stroke, cardiovascular diseases, and certain cancer (Steffen
et al., 2003). There is also demand for the green colour from chlorophylls by the food industry (Sengklang and
Anprung, 2010). As a local and cheap vegetable in Malaysia, amaranth has been reported to be significantly higher in
minerals (Ebert et al., 2011) than most vegetables. Amaranthus spp. contains 3 times more vitamin C, calcium, iron
and niacin when compared to spinach, and 18 times more vitamin A and 7 times more iron when compared to
lettuce, cabbage and Chinese cabbage (Mnkeni et al., 2007). In Malaysia, amaranth vegetables are blanched in
water before being consumed. However, very limited information is available on the antioxidant activity and total
chlorophyll content of this vegetable after blanching. Furthermore, thermal processing and chlorophyll extraction
would cause brownish or discoloration of vegetable tissues (Gunawan and Barringer, 2000) as chlorophylls are easily
degraded by light, heat, pH, oxygen and certain chemicals (Koca et al., 2007). Thus, spray drying is used to convert
vegetables into dried powders for use as functional ingredients in food systems. Moreover, encapsulation with
maltodextrins entraps the sensitive compounds in a coating material which functions to create a physical barrier from
the environment (Ozkan and Bilek, 2014). The aim of this study was therefore, to evaluate the effects of blanching
and spray drying on the physico-chemical properties of amaranth vegetable powders.

375
Materials and Methods
Sample collection, extraction and blanching
Amaranthus viridis with oval and round leaves were purchased in the early morning from a local market in Kuala
Lumpur and placed in a covered container to avoid degradation of pigments by light. After washing, amaranths were
separated into two parts weighing 300 g each. One part was subjected to blanching prior to the extraction process
while the other part remained unblanched. The blanching process was carried out at 800C for 5 minutes with 4 parts
of water to the weight of amaranth. Amaranths were then chopped and the roots were discarded before being
subjected to homogenization using a laboratory Waring blender together with 4 parts of water. The homogenization
process was carried out at a speed of 5 for 5 minutes. Each homogenized amaranth was then filtered using a Muslin
cloth and the filtrate was then collected and stored at -200C in an aluminium pouch which was sealed tightly until
further analysis.

Microencapsulation of amaranth extracts

The frozen amaranth extract was thawed in the refrigerator at 4 ± 20C overnight and then homogenised with 20%
maltodextrin DE 10 (San Soon Seng Food Industries, Selangor, Malaysia) in a laboratory Waring blender for 10
seconds at maximum speed. The homogenized sample was then spray dried using a pilot scale spray drier (Niro A/S,
GEA, Germany) at an inlet temperature of 150 ± 50C and outlet temperature of 90 ± 50C. The feed rate was set at
300 mL/h. The powder was collected and stored in an aluminium pouch, which was refrigerated at 4 ± 2 0C until
further analysis. The yield of spray-dried powder was calculated according to Bustos-Garza et al. (2013).

Analysis of physico-chemical properties

Hunter Lab Ultra-Scan Colorimeter (Sphere Spectrocolorimeter, Hunter Association Inc., USA) was used to
determine L*, a*, b* (CIELAB system) values. Antioxidant activity was measured using the free radical, 2, 2- diphenyl-
1-picrylhydrazyl (DPPH) according to Brand-Williams et al. (1995). Measurement of chlorophyll content was done in
accordance to Vernon (1960). The chlorophyll content of the supernatant was calculated using absorbance values at
663 nm and 645 nm with 70% acetone as the blank using Perkin Elmer UV/Vis Spectrophotometer (Shelton, USA).
Chlorophyll retention (%) was calculated by the formula: (chlorophyll content of spray-dried amaranth) x 100 /
(chlorophyll content of amaranth extract). Results were expressed as means ± standard deviations of triplicate
determinations and then analyzed by MINITAB 17 (Minitab Inc., Sydney). One-way analysis of variance (ANOVA)
with Tukey’s test was carried out to determine the significant differences of means at the 5% level.

Results and Discussion

Chlorophylls a, b and total chlorophylls in local green leafy vegetables
Chlorophyll a, chlorophyll b and total chlorophyll content of fresh leaves of selected vegetables were estimated and
tabulated in Table 1. The results show that amaranth with oval leaves (Amaranthus viridis) had the highest content of
chlorophyll a (33.54 mg/100g), chlorophyll b (34.37 mg/100g) and total chlorophyll (67.91 mg/100g) among the five
green vegetables followed by amaranth with round leaves and brassica (Brassica alboglabra). Therefore, the two
varieties of amaranth were further converted into vegetable powders as sources of micronutrients and
phytochemicals especially chlorophylls.

Table 1: Estimation of chlorophyll a, chlorophyll b and total chlorophyll in acetone extract of local green leafy
vegetables (mg/100g fresh weight)
Vegetables Chlorophyll a Chlorophyll b Total chlorophyll
Amaranth oval leaves (Amaranthus viridis) 33.54a ± 1.77 34.37a ± 1.93 67.91a ± 3.66
Amaranth round leaves (Amaranthus viridis) 28.90b ± 1.47 29.04b ± 1.52 57.94b ± 2.99
Brassica (Brassica alboglabra) 17.22c ± 0.20 32.44a ± 0.35 49.66c ± 0.55
Chinese kale (Brassica oleracea) 15.72c ± 0.92 29.40b ± 1.75 45.11d ± 2.67
Water spinach (Ipomoea aquatica) 13.45d ± 0.17 26.19c ± 0.27 39.64e ± 0.09
Mean ± standard deviation values in the same column followed by the same superscripts are not significantly
different (p>0.05). All values are averages of three determinations.

376
Chlorophyll content, chlorophyll retention, antioxidant activity of spray-dried powders of amaranth
The yield of powder from spray drying of the two varieties of amaranth is shown in Table 2 with the highest yield
obtained from unblanched amaranth with round leaves (69.82%). The chlorophyll contents of extracts and powders
from blanched and unblanched amaranth of both varieties were significantly difference (p<0.05). The results
indicated that not only spray-dried powder from unblanched amaranth with oval leaves had the highest content of
chlorophylls (8.87 ± 0.02 mg/100g) but also its extract (9.53 ± 0.02 mg/100g). However, the chlorophyll content of
both varieties of amaranth decreased after spray drying. It was due to the degradation of chlorophylls during spray
drying to form derivatives (Ryan-Stoneham and Tong, 2000). A study by Weemaes et al. (1999) indicated that
chlorophylls started to degrade at a temperature range of 80ºC to 120ºC to form chlorophyll derivatives. They found
that chlorophyll a was more heat sensitive than chlorophyll b. Nevertheless, blanched and unblanched amaranth
powders of both varieties showed a significantly high percentage of retention of chlorophylls which were above 80%.

Table 2: Yield, chlorophyll content and chlorophyll retention of spray-dried powders of amaranth
Chlorophyll Powder
content
Samples Chlorophyll content Chlorophyll
(mg/100g) of Yield (%)
(mg/100g) retention (%)
extract
Unblanched round leaves 4.11 ± 0.01d 69.82 ± 0.36a 3.92 ± 0.01d 95.57 ± 0.25a
Blanched round leaves 4.76 ± 0.02c 64.82 ± 0.16b 4.12 ± 0.02c 86.46 ± 0.82c
Unblanched oval leaves 9.53 ± 0.02a 63.01 ± 0.19c 8.87 ± 0.02a 93.01 ± 0.24b
Blanched oval leaves 7.28 ± 0.03b 62.51 ± 0.19c 6.91 ± 0.05b 94.91 ± 0.99a
Mean ± standard deviation values in the same column followed by the same superscripts are not significantly
different (p>0.05). All values are averages of three determinations.

Physico-chemical properties of spray-dried powders of amaranth

As shown in Table 3, blanching had a significant effect (p<0.05) on the colour of amaranth powders from both
varieties. In terms of lightness (L*), spray-dried powders from blanched amaranth with oval and round leaves were
lighter (higher L value) than powders from unblanched oval and round leaves although the results were only
significantly (p<0.05) different for the oval leaves. However, spray-dried powder from unblanched with oval leaves
possessed the lowest a* value (-3.79 ± 0.11), indicating a more greenish colour. The powder from unblanched
amaranth with round leaves showed higher DPPH radical scavenging activity (1536.75 ± 2.70 µM TE/g) as
compared to unblanched with oval leaves (1424.75 ± 6.55 µM TE/g) as shown in Table 3. It was also observed that
spray-dried powders of blanched amaranth from both varieties had lower DPPH radical scavenging activity as
compared to the unblanched amaranth. According to Chu et al. (2000), blanching can lead to the reduction of
antioxidant activity due to degradation and loss of antioxidative components into water during the blanching process.

Table 3: Physico-chemical properties of two varieties of amaranth powders

Colour DPPH radical
scavenging activity
Spray-dried powder
L* a* b* (µM Trolox Equivalent,
TE/g)
Unblanched round leaves 78.92 ± 0.04b -0.85 ± 0.06b 10.67 ± 0.32c 1536.75 ± 2.70a
Blanched round leaves 79.47 ± 0.38ab -0.43 ± 0.03a 11.11 ± 0.02b 1254.50 ± 4.58c
Unblanched oval leaves 76.66 ± 0.48c -3.79 ± 0.11c 14.02 ± 0.03a 1424.75 ± 6.55b
Blanched oval leaves 80.36 ± 0.51a -1.03 ± 0.07b 10.68 ± 0.07bc 1256.25 ± 4.56c
Mean ± standard deviation values in the same column followed by the same superscripts are not significantly
different (p>0.05). All values are averages of three determinations.

Conclusion
Amaranth with oval leaves is recommended for the production of spray-dried amaranth powder due to the higher
chlorophyll content and greener colour of its powder than powder from amaranth with round leaves. Blanching of the

377
amaranth prioor to its water extraction would negatively affect the antioxidant activity and colour of amaranth powder
from both varieties of amaranth.

References
Brand-Williams, W., Cuvelier, M. and Berset, C. 1995. Use of a free radical method to evaluate antioxidant activity,
LWT-Food Science and Technology 28(1): 25-30.
Bustos-Garza, C., Yanez-Fernandez, J. and Barragan-Huerta, B.E. 2013. Thermal and pH stability of spray-dried
encapsulated astaxanthin oleoresin from Haematococcus pluvialis using several encapsulation wall materials.
Food Research International 54: 641-649.
Chu,Y.H., Chang, C.L. and Hsu, H.F. 2000. Flavonoid content of several vegetables and their antioxidant activity.
Journal of the Science of Food and Agriculture 80: 561-566.
Ebert, A. W., Wu, T. H., and Wang, S. T. 2011. Vegetable amaranth (Amaranthus L.). AVRDC Publication. 11-754.
Gunawan, M. I. and Barringer, S. A. 2000. Green color degradation of blanched broccoli (Brassica oleracea) due to
acid and microbial growth. Journal of Food Processing and Preservation 24(3): 253-263.
Joao Silva Dias. 2012. Nutritional Quality and Health Benefits of Vegetables: A Review. Food and Nutrition Sciences
3: 1354-1374.
Koca, N., Karadeniz, F. and Burdurlu, H. S. 2007. Effect of pH on chlorophyll degradation and colour loss in blanched
green peas. Food Chemistry 100(2): 609-615.
Mnkeni, A., Masika, P., and Maphaha, M. 2007. Nutritional quality of vegetable and seed from different accessions of
Amaranthus in South Africa. Water SA 33(3): 377-380.
Ozkan, G. and Bilek, S.E. 2014. Microencapsulation of natural food colourants. Internationak Journal of Nutrition and
Food Sciences 3 (3): 145-156.
Ryan-Stoneham, T. and Tong, C. 2000. Degradation kinetics of chlorophyll in peas as a function of pH', Journal of
Food Science 65(8): 1296-1302.
Senklang, P. and Anprung, P. (2010). Optimizing Enzymatic Extraction Of Zn–Chlorophyll Derivatives From Pandan
Leaf Using Response Surface Methodology, Journal of Food Processing and Preservation, 34(5), pp. 759-
776.
Steffen, L. M., Jacobs, J. D. R., Stevens, J., Shahar, E., Carithers, T. and Folsom, A. R. 2003. Associations of whole-
grain, refined-grain, and fruit and vegetable consumption with risks of all-cause mortality and incident
coronary artery disease and ischemic stroke: The Atherosclerosis Risk in Communities (ARIC) study.
American Journal of Clinical Nutrition 78: 383−390.
Vernon, L.P. 1960. Spectrophotometric determination of chlorophylls and pheophytins in plant extracts. Analytical
Chemistry 32: 1144-1150.
Weemaes, C. A., Ooms, V., Van Loey, A. M., and Hendrickx, M. E. 1999. Kinetics of chlorophyll degradation and
color loss in heated broccoli juice. Journal of Agricultural and Food Chemistry 47(6): 2404-2409.

378
 IFRC 2017: 246-228 Functional Food

Investigation of Nutritional and Bio-active Properties of Selected Sri Lankan Marine Macro-algae

1*Warnasooriya, S.G.V.B., 1Jayawardana, B.C., 2Liyanage, N.L.B.R. and 3Nirooparaj, B.

1Department of Animal Science, Faculty of Agriculture, University of Peradeniya, SriLanka.

2National Fundamental of Fundamental Studies, Hantana road, Kandy, Sri Lanka.
3National Aquaculture Development Authority of Sri Lanka, Battaramulla.

Abstract

The present study aimed at elucidating the proximate composition and antioxidant properties of eleven selected Sri
Lankan seaweed species collected from four coastal sites (Erukkalampiddy, Madiha, South Bar and Wellamadama).
The Ulva rigida (green algae) showed the highest crude protein content (23.67+0.73% DM). The Cladaphora
herpestica (green algae) and Turbinaria ornate (brown algae) were significantly (p<0.05) high in crude fat content.
Crude fiber level was highest in Sargassum cinereum and Gracilaria corticata. Among crude ethanol extracts of
samples, two species of brown algae showed significantly (p<0.05) high total phenolic content (51.32+0.61 and
49.92+2.68 mg/GAE g). The DPPH radical scavenging activity was high in Sargassum crassifolium (brown algae).
Caulerpa racemose (green algae) showed the highest inhibitory activity on Fe3+ (420.19+6.78 mM Fe2+/ g of crude
ethanol extract). Present results suggest that the selected Sri Lankan seaweeds contain high nutritional and
antioxidant properties.

Keywords: seaweeds, proximate composition, antioxidant activity, crude ethanol extract

*Corresponding author’s email: viraji_bandara8091@yahoo.com

Introduction
Marine macro-algae are primitive type of plants, flourish wherever rocky, coral or suitable substrata are available for
their attachment. Seaweeds are valuable sources of dietary protein, lipid, fiber, vitamin and some essential minerals
(Mabeau and Fleurence, 1993; Darcy-Vrillon, 1993; Fleurence, 1999; Novaczek, 2001; Ortiz et al., 2006). Sri Lanka
has a coastline of approximately 1700 km along which many varieties of seaweeds are found. However, bioactivities
of the Sri Lankan seaweeds have not been studied extensively and only a few reports have been found in the
literature. Among them, Premekumara et al. (1995) have reported the post-coital contraceptive mechanism of the
crude extract of Sri Lankan marine red algae, Gelidiella acerosa.

The overall objective of this study was to determine the nutritional and functional properties of some selected macro-
algal species found in different locations of Sri Lanka and to ensure their safety in utilization.

Materials and Methods

3.1 Sample preparation
Eleven seaweed species were collected from 4 different areas of Sri Lanka. Samples were then brought to the
laboratory and washed 3 to 4 times with fresh water to remove the debris, epiphytes and adhered sand/dirt particles.
All these samples were dried at room temperature for 5-7 days followed by 60 oC in the hot air oven for 2 days. The
samples were ground to particle size less than 2 mm and these powdered samples were used to estimate the
proximate composition and crude extract of the samples were used for the antioxidant activity of the species.

3.2 Proximate analysis was done in triplicate for moisture, ash, crude protein, crude fat and crude fiber according to
AOAC Standard (2005).

379
3.3 Ferric reducing antioxidant power (FRAP) assay was determined by the method of Al-Farsi et al., (2005).

3.4 DPPH radical scavenging assay was done according to Brand-Wiiliams et al., (1995).
3.5 Total phenolic content was done according to Folin-Ciocalteu method by Singleton, V.L. and Rossi, J.A. (1965).
3.6 All data were expressed as mean + standard deviation and were done in triplicate independent analysis. Data
were analyzed using SAS version 9.1 statistical software.
Results and Discussion
Table 1 shows the percentages of moisture, ash, crude protein, crude fat and crude fiber of the 11 species of
seaweeds. The nutritional and antioxidant properties of the seaweeds vary significantly among the species (p<0.05).
The Ulva rigida (green algae) showed the highest crude protein content (23.67+0.73% DM). The Cladaphora
herpestica (green algae) and Turbinaria ornate (brown algae) were significantly (p<0.05) high in crude fat content.
Crude fiber level was highest in Sargassum cinereum and Gracilaria corticata.
Phenolic compounds are regarded for their important dietary roles as antioxidants and chemo preventive agents
(Bravo et al., 1998). As shown in figure 1 significantly high (p<0.05) total phenolic content was recoded in Sargasuum
cinereum (55.38 ± 0.61 mg/ GAE g) and the lowest value was in Codium tomentosum (2.26 ± 0.28 mg/ GAE g).
As shown in figure 2 Caulerpa racemose showed significantly high (p<0.05) ferric reducing antioxidant power (420.19
± 6.78 mM Fe 2+/g of crude ethanol extract of sample) followed by Sargassum cinereum (212.52 ± 6.54 mM Fe 2+/g
of crude ethanol extract of sample) and Turbinaria ornate (200.34 ± 8.39 mM Fe 2+/g of crude ethanol extract of
sample) (p<0.05).
DPPH is a free-radical compound, which is widely used to determine the free radical scavenging ability of samples
(Qi et al., 2005; Wang et al., 2009). In this study significantly high (p<0.05) DPPH radical scavenging activity was
showed by Sargassum crassifolium (figure 3). The concentration of Sargassum crassifolium, which needs to
scavenge 50% DPPH radical was 437.16 ppm (p<0.05). The lowest DPPH radical scavenging activity was in
Gelidiopsis variabilis and IC50 value was 4421.67± 94.80 ppm (figure 3).
Table 1: Proximate composition of selected 11 seaweed species
Types of seaweed MC% Ash% (DW) Crude Crude fat% Crude fiber%
(FW) protein% (DW) (DW)
(DW)
Ulva rigida 80.84 20.26±0.11cf 23.67±0.73a 0.09±0.02c 11.64±0.31cd
Codium tomentosum 91.80 21.43±0.73c 13.85±0.48d 1.31±0.23cd 10.66±0.39d
Caulerpa racemose 92.89 26.78±0.04dc 18.61±0.82bc 0.54±0.66c 12.33±0.08c
Cladophora herpestica 93.76 28.18±0.61d 17.53±1.90bc 3.89±0.40a 15.42±0.26b
Sargassum crassifolium 89.02 31.18±0.05bc 10.44±0.51c 2.65±0.01b 15.78±0.11b
Padina antillarum 87.97 33.39±0.11bc 18.44±0.51bc 2.33±0.00b 16.32±0.04b
Sargassum cinereum 88.02 12.96±0.37h 8.30±0.24f 2.47±0.22b 17.46±0.38a
Turbinaria ornate 87.02 21.11±0.12c 4.87±0.85g 4.13±0.12a 15.33±0.56b
Gracilaria corticata 85.68 35.20±0.17a 19.43±0.27b 2.68±0.09b 18.73±0.39a
Gelidiopsis variabilis 91.31 16.69±0.00g 13.66±0.04d 1.46±0.09cd 9.94±0.13c
Pterocladiella caerulescens 87.36 22.58±0.19fc 22.58±0.39ab 2.09±0.14bc 10.49±0.04d
Values are expressed as means ± S.D (Standard deviation); means that do not share same latter are significantly
different (p<0.05)

380
 60 a b
50
TPC (mg/ GAE g)
40 c
30
20 d d d d de
10 ef f
g
0

Types of seaweeds
Figure 1.Total phenolic content of collected 11 species of seaweed.Mean values with different superscript letters
were significantly different (p<0.05).

500 a
400
FRAP (mM Fe2+/g of

300 b b
200 c c
d
extract)

100 e
f f
0

Types of seaweeds
Figure 2. Ferric reducing antioxidant power of selected seaweeds. Mean values with different superscript letters were
significantly different (p<0.05).
5000
a
Concentration (ppm)

4000
b b
3000
bc
2000 cde cde
1000 de de e de
f
0

Type of seaweeds
Figure 3. Concentrations of the samples which scavenge the DPPH radicals. Mean values with different superscript
letters were significantly different (p<0.05).

381
Conclusion
In conclusion, the study revealed that selected 11 species of Sri Lankan marine macro algae species could be
employed as a potential human food source and may be used in food industry as a source of ingredients with high
nutritional value. Among green algae species three species were significantly higher (p<0.05) in crude protein, crude
fat and Fe3+reducing antioxidant power than other eight seaweed species. Two brown algae species were
significantly higher in crude fibre content and DPPH radical scavenging activity. Ash was significantly high (p<0.05) in
two red algae species than other 9 species of seaweeds.

Acknowledgment
The authors would like to thank all staff members of the Department of Animal Science, Faculty of Agriculture,
University of Peradeniya for their enormous support and encouragement given throughout research project.

References
Al-Farsi, M., Alasalvar, C., Morris, A., Baron, M. and Shahidi, F., 2005. Comparison of antioxidant activity,
anthocyanins, carotenoids, and phenolics of three native fresh and sun-dried date (Phoenix dactylifera L.)
varieties grown in Oman. Journal of agricultural and food chemistry, 53(19), pp.7592-7599.
Brand-Williams, W., Cuvelier, M.E. and Berset, C.L.W.T., 1995. Use of a free radical method to evaluate antioxidant
activity. LWT-Food science and Technology, 28(1), pp.25-30.
Bravo, L., 1998. Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutrition
reviews, 56(11), pp.317-333.
Darcy-Vrillon, B.1993. Nutritional aspects of the developing use of marine macroalgae for the human food
industry. International Journal of Food Sciences and Nutrition (United Kingdom).
Fleurence, J. 1999. Seaweed proteins: biochemical, nutritional aspects and potential uses. Trends in Food Science &
Technology, 10(1), pp.25-28.
Mabeau, S. and Fleurence, J. 1993. Seaweed in food products: biochemical and nutritional aspects. Trends in Food
Science & Technology, 4(4), pp.103-107.
Novaczek,I. 2001. A guide to the Common and Edible and Medicinal Sea Plants of the Pacific Island, University of
the South Pasific, 40pp.
Ortiz, J., Romero, N., Robert, P., Araya, J., Lopez-Hernández, J., Bozzo, C., Navarrete, E., Osorio, A. and Rios, A.
2006. Dietary fiber, amino acid, fatty acid and tocopherol contents of the edible seaweeds Ulva lactuca and
Durvillaea antarctica. Food chemistry, 99(1), pp.98-104.
Premakumara, G.A.S., Ratnasooriya, W.D. and Tillekeratne, L.M.V.1995. Studies on the post-coital contraceptive
mechanisms of crude extract of Sri Lankan marine red algae, Gelidiella acerosa. Contraception, 52(3),
pp.203-207.
Qi, H., Zhao, T., Zhang, Q., Li, Z., Zhao, Z., & Xing, R. 2005. Antioxidant activity of different molecular weight sulfated
polysaccharides from Ulva pertusa Kjellm (Chlorophyta). Journal of Applied Phycology, 17(6), 527–534.
Singleton, V.L. and Rossi, J.A. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid
reagents. American journal of Enology and Viticulture, 16(3), pp.144-158.

382
 IFRC 2017: 253-240 Functional Food

Comparative Evaluation of Total Phenolics, Total Flavonoids and Antioxidant Capacity of Dried Shrimp and
Fermented Shrimp Products

1Kaida, S.T., 2Rahmat, A. and 3*Ramli, N.S.

1Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400
UPM Serdang, Selangor, Malaysia
2Faculty of Science, Technology and Human DevelopmentUniversiti Tun Hussein Onn, Malaysia (UTHM), 86400
Parit Raja, Batu Pahat, Johor, Malaysia
3Department of Food Science, Faculty of Food Science and Technology, Universiti Putra, Malaysia, 43400 UPM,
Serdang, Selangor, Malaysia

Abstract

The planktonic shrimp (Acetes indicus) is known as ‘geragau’ by local people in Malaysia. The fermented shrimp
products, ‘belacan’ (shrimp paste) and ‘cincalok’ (fermented shrimp sauce) have attracted considerable attention due
to their delicacy. The present work aimed to compare total phenolic content, total flavonoid content and antioxidant
capacity of dried shrimp and its fermented products, ‘belacan’ and ‘cincalok’. Total phenolic (TPC) and flavanoid
content (TFC) were determined using Folin-ciocalteu and aluminium trichloride colorimetric method respectively.
Antioxidant capacity were determined using beta-carotene bleaching method and 2,2-diphenyl-1-picylhydrazyl
(DPPH) radical scavenging assay. Results showed that fermented shrimp products (‘belacan’ and ‘cincalok’) had
higher TPC content (p<0.05) compared to dried shrimp. However, ‘cincalok’ had the highest TFC and antioxidant
capacity followed by ‘belacan’ and dried shrimp (p<0.05). The present study suggested that fermented shrimp
products exhibited excellent antioxidant capacity compared to dried shrimp which might be due the preservation of
the bioactive compounds and/or production of new antioxidant compounds during the fermentation process of
‘belacan’ and ‘cincalok’.

Keywords: Shrimp paste, ‘belacan’, fermented shrimp sauce, ‘cincalok’, antioxidant, phenolic.
*Corresponding author’s email: shazini@upm.edu.my

Introduction
Belacan or fermented shrimp paste is made of small shrimps through salt-controlled anaerobic fermentation
(Steinkraus, 1996). It is one of the main condiments in Malaysian cuisine and is normally added as a flavoring
ingredient in most spicy foods, especially seafood (Sobhi et al., 2013). Cincalok is another traditional Southeast
Asian food made by fermenting tiny shrimps in salt and cooked rice. These fermented shrimp products impart
delicacy and have high nutritional value. Several studies have proven that food products from marine sources
displayed significant antioxidant activities especially from the astaxanthin pigment in the shrimp (Pongsekul et al.,
2014). This study is carried out to determine the total phenolic content, total flavanoid content and antioxidant activity
of dried shrimp (Acetes indicus), belacan (shrimp paste) and cincalok (fermented shrimp sauce).

Materials and Methods

Sample preparation and extraction

All the samples were sliced into small pieces and homogenized before being subjected to freeze drying for three
days. The samples were then ground into a fine powder using laboratory grinder. One gram of each sample was
mixed with 25 mL of 80% (v/v) methanol. The mixture was shaken using shaking incubator at 200 rpm for 120
minutes at 50°C and centrifuged at 3000 rpm for 15 minutes. The supernatant was saved for further analysis.

383
Determination of Total Phenolic Content (TPC)
Total phenolic content was determined by using Folin-Ciocalteu’s reagent based on method proposed by Singleton
and Rossi (1999).

Determination of Total Flavonoid Content (TPC)

The aluminium trichlroride (AlCl3) test was conducted to evaluate the TFC of samples by previously described
method (Quettier-Deleu et al., 2000).

Determination of β-carotene bleaching method

The β-Carotene-Linoleate bleaching method was done according to Velioglu et al. (1998).
Determination DPPH radical scavenging method

Determination of 2,2-diphenyl-1-picryhydrazyl (DPPH) radical Scavenging Method

The DPPH assay was done according to the method proposed by Brand–Williams et al. (1995).

Statistical Analysis
All data were analyzed using one-way ANOVA using SPSS version 21(SPSS Inc., Chicago, Illinois, USA).

Results and Discussion

In the present study, quantification of TPC of the samples was based on the linear calibration curve of gallic acid with
r2 value of 0.9544 (not shown). Figure 1 shows mean TPC of the samples measured using the gallic acid equivalent
(GAE) equation of y = 4.6551x + 0.1384, whereby y = absorbance at 750nm and x = concentration of total phenolic
compounds in mg per 100 g extract. Among the sample, cincalok and belacan had higher TPC compared to the dried
shrimp (p< 0.05). Flavonoids are the most common and widely distributed plant phenolic compounds. TFC was
determined based on the development of colored flavonoid-aluminium complex formation using aluminium trichloride
in alkaline condition which was measured at 430nm using spectrophotometer. The TPC was expressed as rutin
equivalent (RE) in mg per 100g of dried extract. Result in Figure 2 showed that cincalok had the highest TPC
followed by belacan and dried shrimp.

Figure 3 shows mean antioxidant activity of the samples. Cincalok had the highest antioxidant activity which was
69.30 ± 1.97%. The mean antioxidant activities of dried shrimp and belacan were 14.14 ± 1.35% and 26.49 ± 1.59%
respectively. Analysis of variance revealed a significant difference (p<0.05) in mean of antioxidant activity of all the
samples. The mean antioxidant activity of samples according of samples according to descending
order was cincalok, belacan and dried shrimp.

Figure 4 shows the mean scavenging activity of dried shrimp, belacan and cincalok assayed by DPPH radical
scavenging method. The ascorbic acid was used as the standard. Results showed that the scavenging activity was
increased when the concentration of the sample extracts increased. At low concentration (0.32 mg/ml), the
scavenging activity of dried shrimp, belacan and cincalok were 4.12%, 8.62& and 6.05% respectively. When the
concentration of sample extracts increased to 10.24 mg/ml, the scavenging activity of dried shrimp, belacan and
cincalok were 34.11%, 49.63% and 53.17% respectively. Therefore, the scavenging activity of the samples extracts
according to descending order was cincalok > belacan > dried shrimp. The mean IC50 values of cincalok and
standard ascorbic acid were 5.08±0.06 and 2.50±0.02 mg/ml.

384
 a a

Dried shrimp Belacan Cincalok

Figure 1. Mean Total Phenolic Content of samples assayed by Folin-Ciocalteu Method. The values were expressed as
mean ± standard deviation (n=3). Mean with different letters were significantly different at the level of p<0.05

a
Total Flavonoid Content (mg
rutin/100g)

Dried shrimp Belacan Cincalok

Figure 2. Mean Total Flavonoid Content of samples assayed by Aluminium trichloride Method. The values were expressed as
mean ± standard deviation (n=3). Mean with different letters were significantly different at the level of p<0.05.
Antioxidant activity
(%)

Gallic acid Cincalok Belacan Dried shrimp

Figure 3. Mean Antioxidant Activity (%) of samples assayed by β-carotene Bleaching Method. The values
were expressed as mean ± standard deviation (n=3). Mean with different letters were significantly different
at the level of p<0.05.

385
 80
70

Scavenging Activity (%)

60
50
Ascorbic acid
40 Shrimp
30 Belacan
20 Cencalok
10
0
0 2 4 6 8 10 12
Concentration (mg/ml)

Figure 4. Scavenging activities (%) of samples assayed by DPPH radical Scavenging Activity.The values were
expressed as mean ± standard deviation (n=3). Mean with different letters were significantly different at the
level of p<0.05.

Conclusion
Fermented shrimp products exhibit excellent antioxidant activity compared to dried shrimp. Different fermented
shrimp products had varying antioxidant capacity depending on the phenolics and carotenoid composition, and the
nutritional state of the raw material during the fermentation process. Further study on the identification of antioxidant
compounds should be carried out.

Acknowledgement
The authors wish to thank Universiti Putra Malaysia for financial support.

References
Brand-Williams, W., Cuvelier, M.E. and Berset, C. 1995. Use of a free radical method to evaluate antioxidant activity.
Lebensmittel-Wissenschaft und-Technologie/Food Science and Technology 28: 25-30.
Pongsetkul, J., Benjakul, S., Sampavapol, P., Osako, K. and Faithong, N. (2014). Chemical composition and physical
properties of salted shrimp paste (Kapi) produced in Thailand. International Aquatic Research 6(3):155-166.
Quettier-Deleu, C., Gressier, B., Vasseur, J., Dine, T., Brunet, C., Luyckx, M., ... and Trotin, F. 2000. Phenolic
compounds and antioxidant activities of buckwheat (Fagopyrum esculentum Moench) hulls and flour. Journal
of Ethnopharmacology 72(1):35-42.
Singleton, V. L. and Rossi, J. A. 1965. Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid
reagents. American Journal of Enology and Viticulture 16(1): 144-158
Steinkraus, K.H. 1996. Handbook of Indigenous Fermented Foods. Marcel Dekker Inc. New York, NY, USA. 671 pp.
Velioglu, Y. S., Mazza, G., Gao, L. and Oomah, B. D. 1998. Antioxidant Activity and Total Phenolics in Selected
Fruits, Vegetables, and Grain Products. Journal of Agriculture and Food Chemistry 46 (10):pp 4113–4117.
Sobhi, B., Adzahan, N. M., Shamsudin, R., AbKarim, S., Rahman, R. A., Bakar, J., & Ghazali, Z. 2013. Development
of instrumental methods for textural evaluation of chili paste. Kasetsart Journal-Natural Science 47:122-131.

386
 IFRC 2017: 281-257 Functional Food
Effect of Germination Treatment in Amino Acids and Proteins Content of Jackfruit Seeds

I.Zuwariah, H. Hadijah, I.Aida Hamimi and R. Rodhiah

Food Science and Technology Research Center, Malaysian Agricultural Research and Development Institute,
MARDI. G.P.O. Box 12301, 50774 Kuala Lumpur.

Abstract

Jackfruit seeds were germinated for 0, 6, 12, 18, 24, 48, 72, and 96 hours of germinating period then quantitative
changes in amino acids and proteins were investigated. The amino acid profile of germinating seeds were better than
ungerminated seeds and the improvement would provide the specialty benefits for plant based protein source as an
functional ingredient in food applications. Results showed that, amino acid profile was significantly increased
(p>0.05) in the germinating jackfruit seed for 24 hours of germination time. However, after 96 hour of germination
process, a marked decreased of amino acid content were found but protein content remained constant. The
germination process did not affected the amount of crude protein (9%) after 96hr of germination treatment. During 24
hours of germination, the essential amino acid histidine, threonine, valine, lysine, isoleucine and leucine were
increased more than 50% while phenylalanine increased to 49% compared to ungerminated jackfruit seed.

Keywords: Amino acids, germination, jackfruit seeds, protein

*Corresponding author’s email: fawwazody@gmail.com

Introduction
Sprouting or germinating is the process whereby seeds are germinated to manipulate the nutritional composition of
plant seeds. The technology of sprouting has been employed over many decades in beer making industry in order to
modify nutritional and functional composition of cereal grains. According to Srilakshmi (2008), seed sprouting of
legumes improves the protein and other nutritional contents and also reduces the anti-nutritional composition. In
Malaysia, the raw jackfruit seeds have been consumed in various form but seed sprouting were not realized to have
active enzymes that increase bio-chemical and nutrients of seeds. Many researchers, Kayahara et al. (2001),
Bau et al.(1997), Dhaliwal and Aggarwal (1999) have been reported that soaking allowed water uptake into seeds
and promotes germination, then there are certain changes in seeds not only existing nutrients are increased but new
components are also released from the inner change due to germination.
This present study was undertaken to provide scientific data on the effect of germination treatment in amino acids
and proteins content of jackfruit seeds

Material and Methods

Jackfruit seeds were obtained from minimally processed jackfruit industries and some fruits were purchased from
Lanchang Pahang. The jackfruit seeds were further treatment while separation of seed from the pulp continued
manually.
The seeds were washed with water several times then samples were soaked in clean water for 24 hours. Next, the
water was drained off and was then spread on towel inside the trays to germinate for 0, 6, 12, 18, 24, 48, 72, and 96
hours.During 96hr seeds were sampled each time, then the seeds were sliced into thin layers, dried and used for the
analysis. The chemicals used for the studies were of analytical grade.

Amino acids analysis

Amino acids was extracted according to the modified method of Abe et al. (2000). The identification and
quantification of amino acids were determined using the AccQtag Method (Waters, U.S.A), scanning fluorescence
detector. The mobile phase consists of eluent A and eluent B. Eluent A was borate buffer while the Eluent B was 60
% acetonitrile (v/v). All separation was carried out on a 4 µm AccQ. Tag C18 column (150 x 3.9 mm) at 36ºC.
Analysis of Amino acids was done in duplicate.

387
Protein analysis
The Kjeldahl method was used for the analysis of total nitrogen content, which then was utilized to calculate the
protein content (N×6.25) (Association of Official Analytical Chemists, 1990b).

Results and Discussion

The amino acid variation of germinated jackfruit seeds are presented in Graph 1 and 2.
During the first twelve hours of germination, the amino acids content was reduced. However, in the eighteen hours,
the amino acid content was started increased until reached the optimum germination period at 24 hours for jackfruit
seeds. The amino acids graph also showed that the last two period (72hr and 96hr) the amino acids profile were
decreased. During 24 hours of germination, the essential amino acid histidine, threonine, valine, lysine, isoleucine
and leucine were increased more than 50% while phenylalanine increased to 49% compared to ungerminated
jackfruit seed. From the graph bar (Graph 1 and 2), 24hour of seed germination showed, in descending order, that
aspartic acid, glutamic acid, serine, glycine and lysine were the major amino acids. Generally, the graph trend
showing the decreasing amount of amino acids profile in the first three period germination time, then the amino acids
content reached the optimum value at 24hr of germination and lastly the amount of amino acids reduced gradually.
This is due to hydrolysis of protein at certain stages of seed germination and the amino acids produced by hydrolysis
of the protein reserves are not used solely in synthesizing new components, but may also be used as an energy
sources (Chen et al. 1975).

2
1.8
1.6
1.4 Asp
1.2
g/100g

Ser
1
Glu
0.8
0.6 Gly

0.4 His
0.2 Arg
0
Fresh 0 6 12 18 24 48 72 96
Germination time (hr)

Graph 1: Amino acids variations during germination of jackfruit seeds

388
 1.2

1
Thr

0.8 Ala
Pro
g/100g

0.6 Tyr
Val
0.4 Lys
Ile
0.2
Leu
Phe
0
Fresh 0 6 12 18 24 48 72 96
Germination time (hr)

Graph 2: Amino acids variations during germination of jackfruit seeds -continued

The protein content of jackfruit seeds during germination was showed in Graph 3. Although the amino acid profile
showed significant variation, the protein content showed little significant changed (Graph 3). The protein content did
not significantly increased from fresh seed until 18 hours of germination time. It was observed that the protein graph
bar showed decreasing amount of protein after 18 hour until 96hour of germination period. However the variation in
the protein content was little affected by germination process, the protein content was ranged between 9.3 to 10.6
g/100g. These results are in agreement with other works where only a small increase in crude protein content during
48 hours of soy bean germination (Donangelo et al., 1995) while crude protein remained almost unchanged
throughout the germination period in sesame (Hahma et al., 2009).

11.00
ab a
10.50 ab bc cd
10.00 d
e
g/100g

ef
9.50 f
9.00
8.50
8.00
Fresh 0h 6h 12h 18h 24h 48h 72h 96h
Protein 10.40 10.47 10.24 10.16 10.62 9.66 9.95 9.50 9.29

Graph 3: Protein content variations during germination of jackfruit seeds

Values are the mean ± SD (n=3); means that do not share a same letter are significantly different (p<0.05) as
measured by Duncan test.

389
Conclusion
In conclusion, during germination process, the amino acids profile increased after 24 hours of germination time. The
formation of the seedling during germination process lead to increasing amount of amino acid where aspartic acid,
glutamic acid, serine, glycine and lysine were the major amino acids. In general, crude protein was slightly affected
during germination process resulted , 9.3 to 10.6 g/100g protein content. This finding was conclude that germinated
jackfruit seeds could be potential nutritional sources of amino acids and possibility to be nutritious functional
ingredients.

Acknowledgements
The authors would like to thank Projek Pembangunan MARDI for financial support.

References
Abe, T., Matsuo, T., Sasahara,T. (2000). Varietal difference of free amino acids and GABA contents in rice
endosperm. Breeding Sci. 2: 158
Association of Official Analytical Chemists – AOAC. (1990b). Official Methods of Analysis of AOAC International
(15th ed., Method 979.09). Arlington:AOAC.
Bau, H. M., Villanme, C., Nicolos, J. P. and Mejean, L.( 1997). Effect of germination on
chemical composition, biochemical constituents and antinutritional factors of soybean (Glycine max) seeds. J. Sci.
Food Agric. 73(1): 1-9.
Chen L. H., Wells., C.E. and J.R. Fordham (1975). Germinated seeds for human consumption. J.Food Sci.40:1290-
1294
Dhaliwal, Y. and R. Aggarwal. (1999). Composition of fat in soybeans as affected by duration of germination and
drying temperature. J. Food Sci. Technol. 36(3): 266-267.
Donangelo, C. M., Trugo, O. L. C., Trugo, N. M. F., & Eggumb, B. (1995). Effect of germination of legume seeds on
chemical composition and on protein and energy utilization in rats. Food Chemistry, 53(1), 23-27.
http://dx.doi.org/10.1016/0308-8146(95)95781-Z.
Hahma, T. S., Park, S. J., & Martin, L. Y. (2009). Effects of germination on chemical composition and functional
properties of sesame (Sesamun indicum L,) seeds. Bioresource Technology, 100, 1642-1647. http://
dx.doi.org/10.1016/j.biortech.2008.09.034.
Kayahara H, Tsukahara K, Tatai T (2001) In: Spanier AH (ed) Flavor, health and nutritional quality of pre-germinated
brown rice. Royal Society of Chemistry, Cambridge
Srilakshmi (2008). Nutrition Science, New Age International (P) Limited Publishers, New Delhi (2008), pp. 108–125

390
 IFRC 2017: 284-261 Functional Food
Hypocholesterolemic Effect Of Dietary Fibre Powder From Pink Guava By-Product

1*Ibrahim A. H,. 2Hassan H., 3Ismail A., 4Samad A.N., 1Nordin N.

1 Food Processing and Packaging Program, Food Science and Technology Research Centre, MARDI, 43400
Serdang, Selangor
2 Food Security and Forensics Program, Food Science and Technology Research Centre, MARDI, 43400 Serdang,
Selangor
3 Department of Nutrition and Health Science, Faculty Medicine and Health Science, Universiti Putra Malaysia,
43400 UPM Serdang, Selangor
4 Laboratory Management Program, Technical Service and Technology Commercialization Centre, MARDI, 43400
Serdang, Selangor

Abstract
This study analysed the hypocholesterolemic effect of dietary fibre powder (DFP) from pink guava by-product in Male
Sparague-Dawley rats. In studying the hypocholesterolemic effect of DFP, 3 groups of 8 rats each was used. The
three groups in this study were normal rats (basal diet), control rats (basal diet + 1% cholesterol and 0.2% cholic
acid), and treated rats (10% DFP + 1% cholesterol and 0.2% cholic acid). The experimental results showed that
there was no significant difference (p>0.05) in the mean body weight gained and food efficiency between the rat’s
feds with 10% DFP and those rats in control and cholesterol diet. As expected, 10% DFP had remarkable fecal
bulking effect with fresh weights of feces were significantly (p<0.05) higher than in control and cholesterol group.
Supplementation of 10% DFP had reduced total serum cholesterol (43%) and low-density lipoprotein levels (51%),
however the high density and triglycerides levels were not affected within 30 days of experiments. From the lipid
profiles analysis, it shown that DFP from pink guava by-product has potential to reduce total cholesterol, LDL-
cholesterol and decrease the risk of cardiovascular diseases by decreasing the atherogenic index in rats. A potential
cholesterol-lowering DFP could offer an opportunity to explore new product formulation utilizing pink guava
processing by-product.

Keywords: pink guava, dietary fibre, hypocholesterolemic effect.

*Corresponding author’s email: aidahamimi@gmail.com

Introduction
The designed of food products that confer a health benefit is relatively new trend, and recognized the growing
acceptance of the role of diet in disease prevention, treatment and well-being. This change in attitude or product
design and development has forced industry involved in formulating food for health benefit (Sangeetha et al., 2005).
There is an interest in the food industry in developing functional foods to modulate blood lipids such as cholesterol
and triglycerides. It is widely believed that elevated cholesterol levels in the blood represent a risk factor for coronary
heart disease, with low-density lipoproteins (LDL) being of most concern (Delzene and Kok, 1999). The long-term
cholesterol lowering effects of several sources of dietary fibres have been documented (Lairo, 2001). The objectives
of these studies were to determine the hypocholesterolemic effect of the dietary fibre powder (DFP) prepared from
pink guava by-products.

391
Materials and Method

3.1 Sample Preparation

Samples used in this study were refiner waste from pink guava by-products which collected from Sime Darby
Beverages Sdn. Bhd. Manjung, Perak, Malaysia. The sample was treated with hot water, dry, dry milled and pack in
high density polyethylene bag and kept at room temperature for further analysis.

3.2. Subject and location of the study

Male Sparague-Dawley rats, age approximately 10 weeks with average initial body weight in the range of 150 ± 20 g
were used in this study. The rats were grouped by stratified allocation, based on body weight, and placed into 3
groups (control, cholesterol, and 10% DFP) of 8 rats each.

3.3. Preparation of diet

Three different diets were prepared in this study, normal diet (control group), cholesterol diet (hypercholesterol group)
and 10% DFP diet (10% DFP group) (Anderson et al., 1994; Chau et.al., 2004). All diets were introduced in the form
of powder.

3.4. Animal study

The animal study was carried out according to method developed by Anderson et al. (1994) and Chau et al. (2004).

3.5. Statistical analysis

The results were expressed as mean of values ± standard deviation of three separate determinations. Comparison of
means was performed by one-way ANOVA followed by LSD and T-test. Statistical analyses were run using computer
SAS V. 9.1 software (SAS, USA).

Results and Discussion

After 30 days of feeding, the body weights of the rats increased from 19 – 19.1 g (initial weight) to 32.6 – 35 g (final
weight) among the three diets groups (Table 1). At the initial experiment, there was no significant difference in feces-
fresh weight among the three diets group. As expected, cholesterol-10% DFP group had remarkable fecal bulking
effect, with the fresh weights of feces significantly (p<0.05) higher than in the control and cholesterol groups
respectively at 30 days experimental.

The present study showed that total dietary fibre and its soluble and insoluble fractions of DFP resulted in an
increase in fecal weight. From the preliminary study, 48.7% of IDF and 7.9% of SDF present in dietary fibre powder
from pink guava processing waste. Due to that, dietary fibre powder had an ability to retain water in large intestine
which contributes to the increase of weight stool.

The group fed with the cholesterol-rich diets had altered serum lipid concentrations, causing a marked hyperlipidemia
(Table 2). Total serum cholesterol concentration averaged 2.15 mmol/L when cholesterol and choline acid were
added to the basal diet (Table 2). Supplementation of 10% DFP had reduced total serum cholesterol and low-density
lipoprotein levels (Table 2) within 30 days of experiment.

The supplementation of 10% DFP was significantly (p < 0.05) lowered the serum total cholesterol by 43% which
normalized to the level of control group. This showed that the inclusion of DFP in diets could effectively decrease the
serum total cholesterol concentration, with the hypocholesterolemic effect.

There was a significant increase (p<0.05) of the LDL concentration within 30 days experiment for all the rats.
However, the supplementation of 10% DFP diet could decrease in LDL concentration (51%) compared to
hypercholesterol diet, while there was no significant difference

392
in the LDL concentration between the control and 10% DFP groups (0.68 mmol/L versus 0.84 mmol/L respectively)
after 30 days experimental (Table 2). The effect of DFP in lowering serum LDL was in agreement with other
researches (Lecumberri et al. 2007; Martinez-Flores et al., 2004).

The present study showed that supplementation with 10% DFP had significantly (p<0.05) lowered antherogenic index
(Figure 1). It has been shown that there was a positive correlation between antherogenic index and risk of coronary
heart disease, the lower antherogenic index obtained with consumption of DFP will beneficial for heart patients.

Table 1: Effects of diets on body weight gain, food intake (g), fecal weight (g)
Groups
Control Hypercholesterol 10% DFP
Initial Final Initial Final Initial Final

Body weight gain (g/30d) NA 13.1a NA 14.3 a NA 16.1 a

Food efficiency NA 0.86 a NA 0.58 a NA 0.73 a
Feces- fresh weight 4.4 7.7c* 6.0 10.3 b* 7.2 12.6 a*

Values are presented as mean ± SD (n = 8). Values with same with superscripts ( a,b,c) are not statistically different at
level p < 0.05 according to LSD test. Asterisk (*) indicates there were significant difference at level p<0.05.

Table 2: Effect of diets on total cholesterol, high density lipoprotein, low density lipoprotein, triglycerides.
Groups

Control Hypercholesterol 10% DFP

mmol/L Initial Final Initial Final Initial Final

TC 0.63 1.12 b* 1.06 2.15 a* 0.88 1.28 b*

HDL 0.17 * 0.28 0.31 0.30 0.27 0.24
LDL 0.39 b* 0.68 0.60 1.7 a* 0.49 0.84 b*
TG 0.15 0.35* 0.30 0.32 0.27 0.42*

Values are presented as mean ± SD (n = 8). Values with same with superscripts (a,b,c) are not statistically different at
level p < 0.05 according to LSD test. Asterisk (*) indicates there were significant difference at level p<0.05. *Data are
presented as mean ± (n = 8). Data in a row with different superscript letters are satistically different (p ≤ 0.05). TC=
total cholesterol, HDL= high density lipoprotein, LDL = low density lipoprotein, TG = triglicerides.
0.7
a
0.6

0.5
Antherogenic index

b 0.4
b
0.3

0.2

0.1

0
Control Hypocholesterol 10% RW

Figure 1: Effect of Diets on Antherogenix Index of Rats within 30 days

393
Values are presented as mean ± SD (n = 8). Values with same superscripts (a,b,c) are not statistically different
between groups at level p < 0.05 according to LSD test.

Conclusions
Dietary fibre powder from the pink guava by-products had very pronounced hypocholesterolemic effects as it could
significantly (p<0.05) decrease the levels of serum total cholesterol and LDL levels in rats. Thus, dietary fibre from
pink guava by-products could be a potential cholesterol-lowering ingredient in human diets, and offer industry an
opportunity to develop new formulation of fibre-rich functional foods from pink guava processing waste.

Acknowledgements
The authors would like to thank MARDI for financial support.

References

Anderson, J.W., Jones, and Riddel-Mason, S.1994. Ten Different Fibers Have Significantly Different Effects On
Serum And Liver Lipids Of Cholesterol-Fed Rats. Journal of Nutrition, 124:78 – 83.
Chau C.F., Huang, Y.L., Lin, C.Y. 2004. Investigation Of The Cholesterol-Lowering Action Of Insoluble Fibre Derived
From The Peel Of Citrus Sinensis L. Cv. Liucheng. Food Chemistry,87(3), pp. 361-366.
Delzenne, N.M., and Kok, N.N. 1999. Biochemical Basis Of Oligofructose Induced By Hypolipidaemeia In Animal
Models. Journal of Nutrition, 129: 1467S-1470S.
Lairo , D. 2001. Dietary fibre and dietary lipids. In B.V. McCleary and L. Prosky (Eds.), Advanced Dietary Fibre
Technology (pp. 177 - 185). Oxford: Blackwell Science
Lecumberri E, Mateos R, Izquierdo-Pulido M, Ruperez P, Goya L, Bravo L. 2007. Dietary Fibre Composition And
Antioxidant And Physico-Chemical Properties Of A Fibre-Rich Product From Cocoa (Theobroma cacao). Food
Chemistry (in press)
Martinez-Flores, H.E., Chang, Y.K., Martinez-Bustos, F., Sagabieri, V. 2004. Effects of tuber products on blood lipids
and lipoproteins in harmster. Nutrition Research, 24:85-93.

394
 IFRC 2017: 140-097 Halal Food

Perception of Food Sellers towards Halal Labelled Fish Ball in Kelantan

Zul Ariff Abdul Latiff, Mohamad Izwani Halim & Mohamad Amizi Ayob

Faculty of Agro Based Industry, Universiti Malaysia Kelantan, Jeli Campus, 17600 Jeli, Kelantan, Malaysia.

Abstract
Fish ball are one of the popular meat products in Malaysia. In recent years, many manufacturers have invested in
modern machinery to escalate the production of fish balls. Food manufacturers are required to get the halal
certification from Department of Islamic Development Malaysia (JAKIM) for them to be able to use the official halal
logo on their product. This includes fish ball production; manufacturers need to obtain official halal logo by JAKIM on
the fish ball packaging. Food sellers commonly use fish ball in their cooking dish and even sell it directly as snacks.
Thus, a survey was conducted in Kelantan area to determine 58 food seller perception towards halal labelled fish
ball. The study showed that food sellers’ perception influences their intention. Data were used to analysed by using
descriptive analysis of mean and percentage to see whether knowledge, attitude and perception has impact toward
halal labelled fish ball or not. The result indicates that knowledge, attitude and practice of food sellers has impact
toward halal labelled fish ball. This showed that food sellers have the knowledge about the ingredient used in the
halal labelled fish ball. Factor analysis was used to determine among the factor that influences the decision of food
sellers in purchasing halal labelled fish ball. Result indicate that knowledge is the major factors that influence the
decision of food sellers in purchasing halal labelled fish ball products.

Keyword: Fish ball, halal, knowledge , attitude and practice

*Corresponding author’s email: zulariff@umk.edu.my

Introduction
Fish balls are the popular value added products in Malaysia. Fish ball production is on second place after fish cracker
production for processed fish-based production in Malaysia. Department of Fisheries shows that fish ball production
contributes roughly 15-20% for total fish-based processed food products in Malaysia. Malaysia is moving near to
becoming the core of “Halal” food industry and a substantial portion of study on halal food consumption. Muslim show
a positive perception towards the halal logo and the ingredients on the products label (Talib et al., 2010). In Islam
“Halal” commonly refers to all such manners and acts that are in unity with the sayings of Allah and the last prophet.
Mohamed, Rezai, Shamsudin, & Chiew (2008) stated that certifying the halalness of food products is the factor that
influence the confidence of respondents on official halal logo by JAKIM and the important part of halal logo. Fish ball
production in Malaysia is commonly started by minor family-based enterprises. In recent years, many manufacturers
have devoted their capital in modern machinery to escalate the production of fish balls (Huda et al., 2010). In South
Korea, buyers were very concern about preservative, colorants, and artificial sweeteners in foods and most of them
voiced that evidence on food additives was unsatisfactory (Shim et al., 2011). The food label degree appears
adequate to present the ongoing standards of halal logo, nutritive value and ingredient use by Malaysian to go
through food items before buying (Zulariff et al., 2016). In recent years, fish ball producers substitute the use of fish
meat with surimi (Huda et al., 2010). According to Huda et al. 2010, surimi is a steadied myofibrillar proteins gained
from mechanically deboned fish meat that cleaned with water and mixed with cryoprotectants. Few surimi in the
market can be non-halal due to the process of using non-halal plasma transgutaminase to increase the gelling
characteristics of surimi (Alina et al., 2012). High level of knowledge and halal awareness indicated by Muslim
respondents shows that the respondents have knowledge related to halal concept (Elias, Othman, & Saifudin,
2016).Packaging act as the protective layer to protect the foods and carry the food labels and other important
information about the foods. The amount of useful information given on food labels and packaging is a role of the
level of caution, which is frequently higher for people who show more apprehension about the food they consume
(Coulson, 2000). The Asian Food Information Centre (AFIC) has explored buyers’ reactions to nutrition evidence on
food packaging as well as relative factors that influenced consumer feedback in China and Malaysia. Based on the

395
study, most people fixed that the nutrition evidence on packaging should be included for all manufactured foods.
However, buyers acknowledge their least knowledge relating to food product information. Buyers are more concern
of the price tag and expiration date on the packaging more than about the details of the ingredient used. Kordnaeij,
Askaripoor, & Bakhshizadeh, (2013) stated that factor such as advertising, halal product quality, religious,
consumption difficulties, subjective norm and attitude toward halal product are the factor that determine attitude
toward products carrying halal brand.

Material and methods

Conceptual Framework
The KAP conceptual framework (knowledge, attitude and perception) is used in this study to construct the
questionnaire. The data has been collected from the population to determine the perception of food sellers towards
halal labelled fish ball in Kelantan.. The Figure 1 below shows the conceptual framework used in conducting this
study.

Independent Variable Dependent Variable

Knowledge Perception of Food Sellers

towards Halal labelled fish ball
Attitude in Kelantan
Perception

Figure 1: Study of Perception of Food Sellers towards Halal labelled fish ball in Kelantan
(Adopted and modified from KAP Model ).

Data Collection and Sampling Frame

A convenience sample of 58 food seller in Kelantan area was obtained using face-to-face interviews. The primary
data was collected randomly using a structured questionnaire based on several sections related to objective of this
study and was said to be reliable due to the entire variable obtained was more than 0.6, as stated by Coakes and
Ong (2011) (Table 1).

Table 1: Reliability Statistic

No Variables Reliability Statistic Number of Items
(Cronbach’s Alpha)
1 Knowledge 0.697 7
2 Attitude 0.657 9
3 Perception 0.662 6

The first section of the questionnaire was information on demographic characteristics of respondents. The second
section of the questionnaire covered on impact of food sellers’ knowledge toward halal labelled fish ball. The next
section was covered about impact of food sellers’ attitude toward halal labelled fish ball. The last section of the
questionnaire focuses on impact of food sellers’ perceptions toward halal labelled fish ball. Type of question in the
questionnaire used was 1-5 Likert scale, dichotomous questions and ordinal questions.

Descriptive analysis was used to describe the demographic information of the food seller and to investigate the
perception of food sellers towards halal labelled fish ball in Kelantan. For demographic factor relationship with
consumer preference based on knowledge, attitude and perception, chi-square test was used to analyze the data.
Then factor analysis was run to determine the most inclination factor on knowledge, attitude and perception.

396
Results and discussion
Based on the study, 22 statements relating to the food sellers’ perception towards halal labelled fish ball were
analysed using the Varimax rotation with the factor loading of 0.6. Factor analysis was used to analyze the inclination
factor on perception, attitude and knowledge of food sellers towards Halal labelled fish ball in Kelantan. The Kaiser-
Meyer-Olkin (KMO) sampling adequacy test and Barlett’s test of Sphericity were firstly performed to confirm whether
the factor analysis can be carried out as proper analysis or not. For this study, the Kaiser-Meyer-Olkin (KMO) got
values of 0.802 which was more than 0.6, thus the factor analysis can be run as a proper analysis (Tabachnick,
2007). As shown in Table 2, the most influencing factor between knowledge, attitude and practice toward halal
labelled fish ball was recognised. Three factors which have 74.624% of total variance explained are summarised as
follow.

First factor is food sellers’ knowledge about halal labelled fish ball. This factor consisted of six sub-variables and has
a total variance explained of 33.290%. From the result, it indicates that food sellers have knowledge about halal
labelled fish ball and ingredients used in the fish ball. Thus, knowledge influences food sellers’ decision in purchasing
halal labelled fish ball.Next factor as second factor is attitude toward halal labelled fish ball. This factor comprises of
eight sub-variables and has total variance explained of 26.524%. The results of this factor showed that the perception
of food sellers towards halal labelled fish ball can be influenced by attitude.
Last factor is perceptions toward halal labelled fish ball. This factor consists of six sub-variables which have total
variance explained of 14.810%. Base on the results indicated that food sellers got several practices toward halal
labelled fish ball when they made a choice.

Table 2: Factor Analysis

No Variables Factor Variance (%) Number of Items
1 Knowledge about Halal Labelled Fish Ball 33.29 6
2 Attitude toward Halal Labelled Fish Ball 26.52 8
3 Perception toward Halal Labelled Fish 14.81 6
Ball
Total Variance (% of explained) 74.624 20
(Source: Survey, 2016)

Conclusions
The food sellers’ knowledge on halal labelled fish ball should be promoted more through television and social media
as this lead their attitude when purchasing halal labelled fish ball. Food sellers also should be expose more about the
official halal logo by JAKIM and other country halal logo that are certified by Malaysia government as this can
increase their confident level toward halal labelled fish ball. This study also showed that food sellers have more
confident to purchase fish ball that have halal label on the packaging. Thus, manufacturers should include halal logo
on their fish ball packaging to influence food sellers in purchasing the fish ball.

References

Alina, A. R., Nur Illiyin, M. A., Juriani, J., Salmah, Y., Siti Mashitoh, A., & Imtinam, A. K. (2012). Detection of Non-
Halal Plasma Transglutaminase in Selected Surimi-Based Products by using Sandwandwich ELISA Method.
World Applied Sciences Journal, 17, 39–44.
Coakes, J.C., & Ong, C. (2011). SPSS Version 18.0 for Windows Analysis Without Anguish. 1st Edition. Dougall
Street, Milton: John Wiley & Sons Australia, Ltd
Coulson, N. S. (2000). An application of the stages of change model to consumer use of food labels. Bristish Food
Journal, 102(9), 661–668.
Huda, N., Shen, Y. H., Huey, Y. L., & Dewi, R. S. (2010). Ingredients , Proximate Composition , Colour and Textural
Properties of Commercial Malaysian Fish Balls. Pakistan Journal of Nutrition, 9(12), 1183–1186.

Elias, E. M., Othman, S. N., & Saifudin, A. M. (2016). Halal awareness and knowledge among Muslim’s student

397
 entrepreneurship program : A ... In 2nd International Conference on Business Global & Social
Entrepreneurship (ICoGBSE2016), 1–9.
Kordnaeij, A., Askaripoor, H., & Bakhshizadeh, A. (2013). Studying Affecting Factors on Customers’ Attitude toward
Products with Halal Brand ( Case study : Kuala lumpur , Malaysia ). International Research Journal of Applied
and Basic Sciences, 4(10), 3138–3145.
Mohamed, Z., Rezai, G., Shamsudin, M. N., & Chiew, E. (2008). Halal logo and consumers’ confidence : What are
the important factors ? Economic and Technology Management Review, 3, 37–45.
Shim, S., Hee, S., Lee, Y., Moon, G., Kim, M., & Park, J. (2011). Consumers’ knowledge and safety perceptions of
food additives : Evaluation on the effectiveness of transmitting information on preservatives. Food Control,
22(7), 1054–1060.
Tabachnick, B.G. & Fidell, L.S. (2007). Using multivariate statistics (5th Edition). Pearson Publisher. Boston.
Talib, Z., Zailani, S., & Zainuddin, Y. (2010). Conceptualization on the Dimension for Halal Orientation for Food
Manufacturers: A Study in the Context of Malaysia. Pakistan Journal of Social Sciences, 56–61.
Zulariff, A.L, Mohamed, Z.A., Rezai, G. & Mohamadamizi, A. (2015).“Food Labels Impact Assessment on Consumer
Purchasing Behavior in Malaysia”, Journal of Food Products Marketing, Volume 21-4. Page 137-146

398
 IFRC 2017: 153-110 Halal Food

Halal Malaysia Brand Equity Mishap: False Recognition of Brand Mere Recognition using Implicit
Association Test.

Wan Rusni Wan Ismail 1*, Mohhidin Othman 2, Russly Abdul Rahman 3, Nitty Hirawaty Kamarulzaman 4 and
Suhaimi Ab. Rahman 5

Halal Products Research Institute, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia
1 Faculty of Hotel and Tourism, Universiti Teknologi MARA, Malaysia
2 Department of Food Service and Management, Universiti Putra Malaysia
3 Department of Food Technology, Universiti Putra Malaysia
4 Department of Agribusiness and Bioresource Economics Universiti Putra Malaysia
5 Department of Management and Marketing, Universiti Putra Malaysia

Abstract
Brand mere recognition is the fundamental step in the brand awareness and the first hurdle that any brand need to
achieved before brand equity can come into the picture. Thus, consumers’ ability to recognize brand through its
symbol or logo is very crucial and despite of its significant it is considered to be the lowest order in brand equity
assessment but it does reveal the initial clue on how well the brand is performing in the market. Consumers ability to
recognized the right Halal logo is also important to protect themselves against false logo that still plaguing Malaysian
market. Therefore, this study will test on how well consumers able to identify the right Halal Malaysia brand using
Halal semiotic cues. This study is an exploratory in nature using true experimental design methodology and implicit
association test (IAT) as the measurement tool. A total of 33 Malay Muslim respondents participated in this
experiment where respondents are required to response to the stimulus that consist of various Halal logo and
response time are recorded and analyse according to the IAT procedure. Findings from this study clearly showed that
there is a hesitation in response when facing the current Halal Malaysia logo which provide a clear indication of lack
of mere recognition for this brand. In reality mere recognition is very important because it help consumers to choose
the right product with the right Halal endorsement. Unfortunately the proliferation of other Halal logos that flooded the
market consists of both recognized and unrecognized logo by JAKIM often adopted similar logo design strategy only
add to the confusion. Clearly, the findings on Halal Malaysia logo mere recognition also revealed that there are some
problems during the transition of old JAKIM Halal logo and current logo where the information related to new logo
failed to reached targeted audiences.

Keywords: Halal Malaysia, Brand Equity, Implicit Association Test, Brand Recognition, Attentional Saturation
*Corresponding author’s email: suhaimiabrahman@upm.edu.my

Introduction
Although consumers ability to recognize brand symbols does not sound significant to an establised brand such as
Halal Malaysia, however it is still worth considering due to the neverending problems related to the Halal
endorsement (“PPIM Gesa Pihak Berkuasa”, 2017). For instance, proliferation of Halal logo from non-certified Halal
agencies is only adding salt to the injury especially when Halal Malaysia Brand is the one that have to pay heftily
resulting from the controversies related to Halal endorsement, which is definately not from JAKIM. Thus it is crucial
for the consumers to be able to distinguished the right logo in order to protect them from the false one that might not
be able to delivere the Halal assurance promise. Apart from that, Halal Malaysia logo has undergone one major
change over the year, however the impact of the changes on consumers awareness has never been investigated
(Abdul Khalek & Mohd Mokhtar, 2017). Thus, the objective of this paper is to identify consumers ability to recognize

399
the right Halal Malaysia brand using Implicit Association Test (IAT) that require respondents to respond to the
stimulus given in a quick manner that is very much resemble consumers actual behavior when dealing with low
involvement products.

Literature Review
Renowned Brand name with Failed Symbol Recollection
Logo play a significant role in marketing, in fact business organization often made a significant investment just to
ensure that their logo stand out from their competitors. Unfortunately having renowned and recognizable brand name
does not guaranteed that consumers able to memorize and recalled the logo whenever they need it. Such setback is
not only experienced by regular brand, in fact renowned brand such as Apple also experience similar problem as
90% of the respondents found to be facing difficulties in recalling the Apple logo design despite of regularly using
Apple products (Blake, Nazarian & Castel, 2015). Mental shortcut or availability heuristic is a common human traits
that allow us to recall the important information based on our own assumption and valence (Tversky & Kahneman,
1973; Park & Lessing, 1981). Such trait allow us to make quick decision particurlarly when facing with low
involvement purchase, thus it is not impossible when certain features in the logo for instance is not accurately
memorize by consumers, a conditioned known as “attentional saturation” (Blake, Nazarian & Castel, 2015, pp12). In
the case of Halal logo the problem of confusion and attentional saturation is clearly shown through the consumers
inability to recognize the right Halal Malaysia logo (Shafiq, Haque & Omar, 2015).

Failed Recognition
Brand awareness reflects the importance of brand in the consumers mind, however the levels vary as different brand
rank differently in consumers mind (Aaker, 1996). Given to the sensitivity of Halal issues among Muslim consumers,
Halal brand recognition will definitely place at the highest ranking order as most Muslim consumers agreed that Halal
logo does help to improve their purchase confident (Rezai, Mohamed & Shamsudin, 2012). The indication that a
brand have the power to influence consumers purchase is a good indication that the brand have strong equity and
posessed commercial value (Aaker, 1996). On the other hand, mere recognition is in fact the lowest form of brand
awareness way before equity can come into the picture that reflect on consumers ability to recognize the brand
symbol immediately once they sees them. Thus, consumers ability to recall and identify brand symbol is considered
to be the highest form of awareness when taking into account problem related to attentional saturation and availabity
heuristic (Aaker, 1996; Tversky & Kahneman, 1973).

Methodology
While most of the previous studies on Halal Malaysia logo perception carried out by Rezai et al (2012) and Shafiq et
al (2015) rely on self-reported questionaire and interview, this study on the other hand will take a different approach
in measuring consumers attitude by using Implicit Association Test or IAT. The strength of IAT lies on its ability to
detect respondents valence toward the attitude object through latency response and this quick response set below
3000 millisecond is the most importance features of IAT that set this test apart from other instruments. Apart from
that, Maison, Greenwalds and Bruin (2001) also suggest that this test also has striking similarity to consumers
automatic decision and low involvement purchase behavior as these decisions are often made in a quick manner
thus respondents ability to choose the right Halal Malaysia logo will reflect on brand mere recognition. This research
is based on true experimental design approach and respondents are subjected to a control and another two stages of
experiments. There was no intervention given at the first and second stages of the experiments instead its only
served as a baseline and counterbalancing purpose while the final stages involvement with intervention that consists
of previous informations on Halal Malaysia. A total of 33 respondents participated in this study and it was carried out
in the laboratory setting to overcome with confounding variables problem that might interfere with the findings of this
study as interference might influence respondents ability to response accurately (Greenwald & Farnham, 2000).

400
Results and Discussion
Data from IAT was analyzed using D algorithm and according to the guidelines proposed by Greenwald, Nosek &
Banaji (2003). The final D algorithm data is subjected to exploratory Data Analysis (EDA) using frequency distribution
and was carried out to identify the number of respondents that are able to response correctly in all three experiments.

Findings showed the percentage of the total correct response on three different Halal logo. For Halal logo 1 only
42.2% of total respondents able to recognize this as the current Halal Malaysia during the control and treatment 1
stage which is also equivalent to been recognized by only 14 out of 33 respondents. However, the percentage of
respondents able to pair the logo with the stimulus “JAKIM” are drastically reduced into 21.1% after the intervention
which indicate that some respondents are no longer convinced that logo 1 is the correct logo. This occur right after
respondents are exposed to the various Halal logo image that commonly found on the food products, in fact similar
confusion was also documented in previous research by Shafiq et al (2015). Reduction in correct response time are
also occurred for logo 2 and 3, even though the changes for logo 2 is not that signifantly differ in all stages however it
still show that there are almost half of the respondents still repeatedly pair the word “JAKIM” with this logo. Similar
with logo 2, logo 3 is also no longer valid however there are still large number of respondents out there that still
convinced these are valid Halal logos (Ismail et al, 2016; “Logo Halal yang tak diiktiraf, “ 2014).

Conclusion
Problem with failed mere recognition is a serious issue especially when involving the validity of Halal endorsement
logo such as Halal Malaysia. In this case, both consumers and Halal endorsement agency will suffer the negative
consequences as consumers are exposed to the risk of using falsely Halal endorsed products. Meanwhile, the name
“JAKIM” are quite predominant in the Halal endorsement arena thus any negative publicity is not beneficial towards
Halal Malaysia brand equity. Furthermore, one also should not ignore the practicality issues that originally causing
this problem in the first place. Ineffective promotion during the introduction stage of the logo and the transition of old
JAKIM halal logo with current Halal Malaysia obviously does not goes smoothly as planned as many still convinced
that old logo is still valid. Which showed that there is an urgent need to promote public awareness on the current
Halal Malaysia logo which allow consumers to recognize this logo even at their first glance. This is crucial because
there are still a lot of Halal logo in the market that are not being fully endorsed by JAKIM that carry identical design
that can easily be mistaken by consumers. As the attentional saturations taking place in consumers mind they might
think that the products they purchase as being endorsed by Halal Malaysia but in fact, it is a totally different Halal
logo.

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102-120.
Abdul Khalek, A. & Mohd Mokhtar, R., A. (2017). With or without Halal logo? The descriptive analysis of the
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concept. Journal of personality and social psychology, 79(6), 1022.

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 IFRC 2017: 210-195 Halal Food

Do SMEs Halal Food Products Measure Up to Customer Expectation? : An Empirical Investigation

1*Abdul Salam, S. S., 1,2Othman, M., 2Ungku Zainal Abidin, U. F., and 1,3Kamarulzaman, N. A.

1Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
2Department of Food Service and Management, Faculty of Food Science and Technology, Universiti Putra Malaysia,
43400 UPM Serdang, Selangor, Malaysia
3Department of Agribusiness and Bioresource Economics, Faculty of Agriculture, Universiti Putra Malaysia, 43400
UPM Serdang Selangor, Malaysia

Abstract
In order to increase and maintain their competitiveness and profitability, manufacturers of Halal food products from
small and medium-sized enterprises (SMEs) need to continuously improve their products. Thus, in order to identify
the aspects of the products that need improvement and to meet the changing expectation of Muslim consumer,
manufacturers should constantly monitor their consumer satisfaction. This study reports an application of the
expectancy-disconfirmation theory (EDT) in examining the gap (disconfirmation) between customer expectation and
perceived performance on SMEs halal food products. Halal confectionery products produced by SMEs were chosen
as the focus of the study. A total of 403 usable questionnaires consisted of 45 determinants measuring expectation
and perceived performance were collected from Muslim consumers within the Klang Valley area. The results of the
analysis indicate that all attributes experienced negative disconfirmation which points toward the conclusion that in
overall SMEs Halal food products unable to measure up to customer expectation. Specially, in term of features
related to safety, labelling and marketing of the products. Findings of this study reaffirm the manufacturers need to
continuously examine their business strategies in tandem with the increasing demands of halal food products and
also to accommodate the shift in the preferences and standard in halal food selection among Muslim consumer.

Keywords: Halal food, consumer expectation, consumer satisfaction, SME, Expectancy-Disconfirmation Theory
(EDT).
*Corresponding author’s email: solehasalam@gmail.com

Introduction
Over the years, driven by the growing Muslim population, economic growth and increasing awareness in halal
product consumption, the halal market has experienced considerable growth. In tandem with the growth of the sector
and changes in consumer lifestyles, attitude, taste and sociocultural variables, come together the changes in
consumer preferences and standard in halal food selection (Mohayidin and Kamarulzaman, 2014; Abdul Latiff et al.,
2015). Even though religion still plays the most important part in making food decision, there has been an increasing
demand in higher quality, healthy, safe, natural, convenience and even environmental friendly product and halal at
the same time. (Abdul Latiff et al., 2015; Baharuddin et al., 2015). However, recent research conducted on halal food
product especially on SMEs reported that the products are still lacking in various aspects including product
attractiveness, labeling, packaging and popularity (Machfud et al., 2011; Mohd Daud et al., 2011; and Bohari et al.,
2013). There are also several issue reported on halal logo authenticity, halal food adulteration (Fadzlillah et al., 2011)
and hygiene violation (Ghazali, 2015). Therefore, by using Expectancy Disconfirmation Theory (EDT) as a foundation
of the research, the objective of this study is to examine the extent to which the performance of SMEs Halal
confectionery products has met Muslim consumer expectation.

403
Methodology
3.1 Sampling
A self-administered questionnaire was distributed in Klang Valley area by using convenience sampling method
among 430 respondents. Before being selected, the respondents have to meet several criteria, (1) Muslim, (2) aged
13 years old and above, and (3) had consumed halal confectionery product of SMEs in Malaysia at least once.
Overall, 403 completed questionnaire were returned and analysed.
3.2 Research Instrument
A questionnaire was developed based on an extensive literature review and previous instruments that was modified
to fit the context and objectives of this study. To measure customer expectation, 45 satisfaction drivers based on the
aspects of ‘Food Quality’, ‘Food Safety and Hygiene’, ‘Labeling’, ‘Packaging’, ‘Halalness’ and ‘Marketing’ of halal food
products were listed and respondents were asked to indicate their anticipation towards this attributes in 5-points
Likert scale. Next, the performances of SMEs Halal confectionery products were assessed by the respondents
against these same 45 attributes in order to yield the discrepancy between customer expectation and perceived
performance.
3.3 Data Analysis
To measure the disconfirmation or discrepancy (gap) between customer expectation and perceived performance,
inferred approach (subtractive approach) was used, where the mean of expectation is subtracted from the mean of
perceived performance to form disconfirmation value (Yuksel and Yuksel, 2008; Lee et al., 2008).The following
formula depicts the calculation used in this study:
Disconfirmation = Mean of Perceived Performance – Mean of Expectation

Results and Discussion

The respondents were 63.3% female and 36.7% male. Majority of the respondents were below 31 years old.
Substantial portion of the respondents were highly educated: 44.2% percent had a bachelor’s degree and 23.6%
were diploma holders. As for monthly income, 48.1% of the respondents had no income since most of the
respondents were students, followed by those with RM1501-3000 income at 17.1% and below RM1500 at 14.6%.
About two-thirds of the respondents were from urban area and one-third from rural areas.

Figure 1 presents the overall mean of customer expectation and perceived performance of SMEs Halal confectionery
products based on attributes category. In all attributes category, expectation yielded higher mean scores compared
to performance, signalling that all attributes category experienced negative disconfirmation. Negative disconfirmation
occur when a product had lower performance than expected (Churchill Jr. and Suprenant, 1986). This result
suggested that, SMEs Halal confectionery products failed to meet customers’ expectation in all parameter tested. As
a whole, ‘Safety & Hygiene’ attributes category shows the highest gap, suggesting that SMEs Halal confectionery
product unable to meet consumer expectation specifically in term of ‘Safety & Hygiene’. Aside from meeting religious
requirements, Muslim consumer also demand their food to be safe, free from harmful ingredients and clean, and
Muslim consumers believe that Halal certified products represent safety and hygienic production (Badruldin et al.,
2012)

Table 1 shows the attributes of SMEs halal confectionery with the biggest disconfirmation value. The attributes with
the biggest disconfirmation was ‘Healthy’ at -0.59. This indicates that customers anticipated healthier halal
confectionery products than what was being produced by the SMEs. Lately, there is an increasing awareness of the
importance of healthy lifestyle and eating that heavily changed consumer preference in food consumption
(Euromonitor, 2013).

The smallest disconfirmation was on product ‘Islamic Feature’ at -0.04, which is almost close to positive
disconfirmation and this indicate that manufacturer had already fulfilled customer expectation in term of this feature.
This results also suggest that compared to other attributes, ‘Islamic feature’ was the least anticipated attributes to be
possessed by SMEs halal confectionery products. This might be due to Muslim consumer increased confidence and
trust in halal logo (Saabar and Ibrahim, 2014) that eliminate the need for features such as Islamic/ Malay brand
name, image or Arabic words.

404
 Mean

Expectation Performance
Figure 1. SMEs Halal confectionery products expectation and perceived performance based on attributes category.

Table 1: Attributes of SMEs Halal confectionery with the biggest gaps between expectation and perceived
performance

ATRRIBUTES EXPECTATIONS PERFORMANCE GAP

Mean SD Mean SD
1 Safety and Hygiene (Healthy) 4.26 .821 3.67 .947 -0.59
2 Labelling (Clear Logo) 4.59 .661 4.00 .933 -0.58
3 Marketing (Availability) 4.38 .731 3.80 .956 -0.58
4 Safety and Hygiene (Quality Assurance’s 4.46 .688 3.90 .882
-0.56
Certificate)
5 Safety and Hygiene (Fresh) 4.35 .782 3.79 .906 -0.56

Table 2: SMEs Halal confectionery attributes with the smallest gaps between expectation and perceived performance

ATRRIBUTES EXPECTATIONS PERFORMANCE GAP

Mean SD Mean SD
1 Packaging (Technology) 3.95 .770 3.56 .913 -0.39
2 Food Quality (Aroma) 4.22 .751 3.87 .772 -0.35
3 Food Quality (Fit for Consumption) 4.50 .632 4.15 .718 -0.35
4 Packaging (Appropriate Image) 4.30 .806 4.06 .805 -0.24
5 Packaging (Islamic Feature) 3.85 .984 3.81 .915 -0.04

Conclusion
As a conclusion, in overall SMEs halal food products didn’t measure up to customer expectation as proven by the
overall negative disconfirmation experienced by the attributes. Customer deemed that the products failed to measure
up to their expectation specifically in term of the ‘Healthiness’, while in contrast the products’ ‘Islamic feature’ is
almost close to meeting their expectation. Meeting customer expectation is crucial since this may trigger positive
post-purchase behavioural intention such as repeat purchase and positive word-of-mouth. Hence, producer should
continuously examine their customer satisfaction and try to adapt their products to customer needs. Future research
on other halal food product category such as bakery, frozen food, poultry or halal food service such as casual dining
or fast food can be carried out. Mixed method approach can also be used to extract richer and more subjective
information from the respondents regarding the subjects.

405
References
Abdul Latiff, Z. A., Rezai, G., Mohamed, Z., and Ayob, M. A. 2015. Food Labels’ Impact Assessment on Consumer
Purchasing Behavior in Malaysia. Journal of Food Products Marketing 1–12.
Badruldin, B., Mohamed, Z., Sharifuddin, J., Rezai, G., Abdullah, A. M., Abd Latif, I., and Mohayidin, M. G. 2012.
Clients’ perception towards JAKIM service quality in Halal certification. Journal of Islamic Marketing 3(1): 59–
71.
Baharuddin, K., Ahmad Kassim, N., Nordin, S. K., and Buyong, S. Z. 2015. Understanding the Halal Concept and the
Importance of Information on Halal Food Business Needed by Potential Malaysian Entrepreneurs.
International Journal of Academic Research in Business and Social Sciences 5(2): 170–180.
Bohari, A. M., Cheng, W. H., and Fuad, N. 2013. An analysis on the competitiveness of halal food industry in
Malaysia : an approach of SWOT and ICT strategy. Malaysian Journal of Society and Space 9(1): 1–11.
Churchill Jr., G. A., and Suprenant, C. 1986. An investigation into the determinants of customer satisfaction. Journal
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Fadzlillah, N., Che Man, Y., Jamaludin, M. A., Ab. Rahman, S., and Al-Kahtani, H. 2011. Halal Food Issues from
Islamic and Modern Science Perspectives. In 2nd International Conference on Humanities, Historical and
Social Sciences, p. 159–163.
Ghazali, R. May 2015. Restaurant chain’s halal certificate revoked over sanitary issues. Retrieved on August 20,
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chain-halal-cert/
Lee, S. E., Johnson, K. K. P., and Gahring, S. A. 2008. Small-town consumers’ disconfirmation of expectations and
satisfaction with local independent retailers. International Journal of Retail & Distribution Management 36(2):
143–157.
Machfud, A. K., Khatib, J., Haji-Ahmed, A. A., and Ahmad Dahlan, A. R. 2011. Halal Product Price Indicator Portal :
Promoting Malaysia’s Local Halal SMEs. In ICSECS 2011, p. 254–262.
Mohayidin, M. G., and Kamarulzaman, N. H. 2014. Consumers’ Preferences toward Attributes of Manufactured Halal
Food Products. Journal of International Food & Agribusiness Marketing 26(2): 125–139.
Mohd Daud, N., Ramli, L., Jemahadi, N., and Razalli, R. H. 2011. Examining Critical Success Factors of Consumers’
Attitude towards Nutritional Labelling of SMEs Products in Malaysia. Australian Journal of Basic and Applied
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Saabar, S. S., and Ibrahim, S. 2014. The Knowledge of Halal and Advertising Influence on Young Muslims
Awareness. In International Proceedings of Economics Development and Research, p. 36–39.
Yuksel, A., and Yuksel, F. 2008. Consumer Satisfaction Theories: A Critical Review. Tourist Satisfaction and
Complaining Behavior. Measurement and Management Issues in the Tourism and Hospitality Industry 1984:
65–88.

406
 IFRC 2017: 224-217 Halal Food

Revisiting the Theory of Planned Behaviour (TPB) In Halal Food Purchasing: After the Case of Cadbury

*Mohd Helmi Ali, Azman Ismail, Syed Shah Alam, Zafir Mohd Makhbul

School of Management, Faculty of Economics and Management, Universiti Kebangsaan Malaysia, 43600 UKM
Bangi, Selangor, Malaysia.

Abstract
The aim of this paper is to revisit the theory of planned behaviour (TPB) in halal food purchasing especially after the
case of Cadbury Chocolate scandal. Using survey responses from 132 respondents, we compare the TPB generated
with our sample to the original study of TPB in halal food, rationalize the differences, and assess the impact of halal
scandal on the consumer purchasing behaviour. In doing so, we validate the impact of the food scandal on the
purchasing intention of the halal customer with more recent and under a new uncertainty. The results provide in-
depth insights into the halal purchasing behaviour and intended to be used: (a) to assists an understanding of the
food scandal impact towards purchasing behaviour, (b) to clarify whether food scandal has a real effect onto the
customer, and (c) to compare prior and post-scandal effect of a food scandal. Additionally, the investigation provides
evidence that food scandals have a specific effect on the product before the general conclusion can be drawn.

Keywords: halal, TPB, food scandal

*Corresponding author’s email: mohdhelmiali@ukm.edu.my

Introduction
The recent case of Cadbury controversy scandal has made a significant impact towards the halal food consumption
(Ali et al., 2017). The origin of the controversy is from a lab test by an independent body, i.e. Ministry of Health
claiming a few of Cadbury products are not fulfilling the necessary halal condition in carrying the halal logo. The claim
of the Ministry of Health was then rebutted by the Malaysia halal certification body known as JAKIM. The
contradicting information between two reliable government’s sources has perplexed the consumers and eventually
their purchasing behaviour. Likewise, the case has created a chaotic situation among Muslims globally.

The common practice in the halal industry is that, halal logo is a necessary condition for the firm to display on the
packaging. The importance of the halal logo is imminent in the halal industry which has been the main source of
reference to consumers, especially Muslims. From the view of the Muslim consumers, a product with a halal logo is
safer to be consumed physically and spiritually. However, the Cadbury case has shaken Muslim consumers’ belief
towards the reliability on the halal certification. Earlier, the study on the halal purchasing intention was fuelled with
confidence on the halal certification. Nevertheless, little research has been done on the halal purchasing intention
impact in different settings, i.e., shaken confidence after food scandals. In particular of this research, an investigation
is carried out to answer whether the determinant of purchasing intention is similar between pre and post food
scandal?

The objective of the research is to revisit and extend the important findings of the theory of planned behaviour (TPB)
in the halal food that was discussed by Alam and Sayuti, (2011). Since the investigation on the TPB in halal settings
were carried out in time without food scandals, the cross-validation, empirical corroboration and extension are crucial
for its generalizability (Schoenherr and Swink, 2012). A validated theory’s predictive validity and generalizability is an
important aspect in determining the quality of the research and for further theory development (Douglas and Craig,
2006). Alam and Sayuti, (2011) themselves noted that their findings could be of potential value for future researches
in different settings so as to increase it generalizability. The research is a pure application of (Alam and Sayuti, 2011)
work, it is to answer whether there are differences in purchasing intention of halal food purchasing prior and post
halal scandal. The paper contribution is to provide empirical evidence of pre and post effect towards customer
purchasing behaviour in halal food using the TPB.

407
Methodology
Adapted from Alam and Sayuti (2011) conceptual framework as depicted in Figure 1. The attitude, subjective norm
and perceived behaviour control are hypothesized to have a significant and positive relationship with halal food
purchase intention (H1, H2, and H3 respectively). In addition, in answering the research question whether the
purchasing behaviour of halal food is similar to between pre and post scandal, it is expected the analogous value to
be obtained in the hypothesis testing.

Attitude
H1

Halal food purchasing

Subjective Norm
intention
H2

Perceived Behaviour H3
Control

Figure 1. Schematic diagram of the conceptual framework

The similar questionnaire instrument of Alam and Sayuti (2011) were personally administered to the respondents.
The instrument used a six-point Likert ranging from the scale that 1 indicated “strongly disagree” to 6 indicated
“strongly agree”. In ensuring the comparability with Alam & Sayuti 2011 findings, the targeted respondent’s
characteristic is controlled as follows: a) adult consumers who are older than 21 years old, b) halal food consumer;
and c) have experienced consuming Cadbury chocolate. The non-probability convenience sampling is used in this
study. As shown in Table 1, the highest contributors were female (74 percent). Age group of respondents is
dominated by the adult between the ages of 21-40 years old. In total, there are 153 questionnaires returned,
however, only 132 is found fit for further analysis.

Table 1. Demographic characteristic (n=132)

Frequency %
Gender
Male 34 26
Female 98 74
Age group
21-30 years old 42 32
31-40 years old 51 39
41-50 years old 39 29

Data reliability evidenced by the Cronbach’s alpha value (Nunnally, 1978). In specific, the Cronbach’s alpha value for
behavioural control, attitude, and perceived behaviour control were 0.841, 0.677, and 0.751 respectively. In addition,
the data is free from the multicollinearity issue. As shown in Table 2, the value of variance inflation factor (VIF) that is
less than 10 and the value of the tolerance level of more than 0.01 (Kleinbaum et al., 2013). ). Moreover, the value of
Durbin-Watson is 1.618 that falls in the acceptable range. Similarly, it shows that there are no auto- correlation
problems in the data.

408
Results and Discussion
Multiple regression analysis was used to test the model and the hypothesis following the guidelines established by
Hair et al. (2014). Halal food purchasing intention was used as the dependent variable in the multiple regression
analysis. As shown in Table 2, only relationships between subjective norm (β=0.259, p<0.01) and perceived
behaviour control (β=0.708, p<0.001) were found significant with the dependent variable. Thus supporting H2 and
H3. However, there is no significant relationship was found between attitude and the dependent variable that lead to
the rejection of H1.

Table 2. Regression result

Variable β T Tolerance VIF
Attitude 0.060ns 1.481 .813 1.230
Subjective Norm 0.259* 2.902 .564 1.772
Perceived Behaviour Control 0.708** 11.792 .564 1.773
Notes: Significance at: *p <0.01 and **p<0.001; ns: not significant; R2 0.737; adjusted R2 0.731; Dependent variable:
halal food purchasing intention

Alam & Sayuti 2011 argued that his work on TPB model facilitates prediction of intention to purchase halal food
products in Malaysia. However, the result of the replication of the similar research in this paper suggesting otherwise.
In their work, all relationships in the TPB models were found positive with high significant value. Contradicting with
their result, the relationships testing suggesting that not all of the relationship is significant. The apparent result in this
research, the relationship between attitude and halal food purchasing intention is not significant. In addition, the
relationship of the subjective norm and perceived behaviour control to the halal food purchasing intention is
strengthened. In short, the generalizability of the application of TPB in halal food purchasing is also depending on the
time and external pressure. For example, the research has shown that food scandal that impacted the industry
played a major role in determining the purchasing intention of halal food. Similarly, acknowledging the contribution of
the TPB in assisting understanding halal food purchasing intention, this research suggesting that the TPB discussion
should be extended by including additional constructs in measuring different uncertainties.

In a closer context, this research (β=0.708, p<0.001) that investigate post-scandal purchasing intention has shown
the stronger relationship of perceived behaviour control compared to Alam & Sayuti (β=0.205, p<0.001). By definition,
perceived behavioural control is the extent to which a person feels able to engage in the behaviour (Ajzen, 1991;
Alam and Sayuti, 2011). The high relationship in this study can be related to the situation where the consumer belief
on the product is dented by the food scandal. Consumers now put higher effort to control their behaviour such as
impulsive buying. Consumers now demanded more information prior purchasing after learning that despite halal logo
is presented on the product label, there are still possibilities of the logo being misused (Tan et al., 2017). In another
word, the information conveyed through halal logo is not sufficient in solely determining consumers’ purchasing
intention.

The result of the relationship between social behaviour and halal purchasing intention can be regarded similar to the
findings of Alam & Sayuti 2011. This suggesting that the perceived social pressure is similar in the Malaysian culture
that is collectivism in nature. However, the main finding in this research is the attitude of halal food consumer in
purchasing food that has involved in the scandal, which in this case a Cadbury chocolate. The non-significant
relationship shows that how a food scandal can impact the consumers trust in the halal status of the particular
product (Ali et al., 2016). It is now empirically evidenced that the attitude has a strong effect in influencing the
consumer to finally commit to purchasing.

Theoretically, it is clearly shown that generalization of TPB in halal food context should be carefully done. Since halal
food consumption is highly related towards spiritual and religious element, the purchasing behaviour is more prone to
the uncertainties. The interpretation of the results of TPB model in halal research also should also take into account
of the marketplace situation. In practice, a lesson can be learned that getting involved in any food scandal should be

409
avoided. Regardless the source of the scandal, let it be on the product or the management issue can lead to a
negative impact on the business. It does not need a concrete evidence of involvement but a speculation is just
enough to do the damage.

Conclusion
Because the generalizing TPB in halal purchasing intention after food scandal is perplexing, this research deemed it
crucial to revisit Alam & Sayuti (2011) suggestion and findings. Therefore, this paper offers another perspective of
interpretation of TPB in different uncertainty. The paper argued of the generalizability of key variables in the TPB
model. Our findings, in general, corroborates with the TPB model, however, it is found that the not all key variables in
the TPB model provide similar effect to the purchasing intention especially in halal food. Moreover, it is empirically
evidenced by the effect of the food scandal into the attitude of the halal consumers.

References

Ajzen, I. (1991), “The theory of planned behavior”, Organizational Behavior and Human Decision Processes,
Elsevier, Vol. 50 No. 2, pp. 179–211.
Alam, S.S. and Sayuti, N.M. (2011), “Applying the Theory of Planned Behavior (TPB) in halal food purchasing”,
International Journal of Commerce and Management, Vol. 21 No. 1, pp. 8–20.
Ali, M.H., Makhbul, Z.M., Tan, K.H. and Ngah, A.H. (2016), “Augmenting Halal Food Integrity through Supply Chain
Integration”, Jurnal Pengurusan, Vol. 48 No. 2016, pp. 21–31.
Ali, M.H., Tan, K.H. and Ismail, M.D. (2017), “A supply chain integrity framework for halal food”, British Food Journal,
Vol. 119 No. 1, pp. 20–38.
Douglas, S.P. and Craig, C.S. (2006), “On improving the conceptual foundations of international marketing research”,
Journal of International Marketing, Vol. 14 No. 1, pp. 1–22.
Hair, J.F., Black, W.C., Babin, B.J. and Anderson, R.E. (2014), Multivariate Data Analysis, Pearson Education
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Multivariable Methods, Nelson Education.
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of Operations Management, Vol. 30, pp. 99–115.
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at:http://doi.org/http://dx.doi.org/10.1108/SCM-05-2016-0171.

410
 IFRC 2017: 229-232 Halal Food

Muslim Consumers’ Awareness and Perception of Halal Food Fraud

2Ruslan, A.A.A, 1,2,3,4*Kamarulzaman, N.H, and 5,6Sanny, M.

1Department of Agribusiness and Bioresource Economics, Faculty of Agriculture, Universiti Putra Malaysia, 43400
UPM Serdang, Selangor, Malaysia.
2Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
3Institute Tropical Forestry and Forest Products, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
4Institute of Plantation Studies, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
5Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor, Malaysia.
6Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor,
Malaysia.

Abstract
Halal food represents up to 20% of the entire global halal food industry is one of the most profitable and influential
market areas with the increasing of world Muslim population. Halal is an important aspect in the selection of food as it
is one’s part to obey with religious obligations and commandments. Since halal food industry covers ‘farm to table’
operations, the issue of food fraud along the food supply chain become a major concern. Food fraud, in general, is
defined as a substitute, adding, misrepresenting of food label or a food ingredient or misleading statement about the
products intentionally for economic gain. Thus, this study was carried out with the objectives of this study are 1) to
determine Muslim consumers’ awareness and perception of Halal food fraud, 2) to identify the association between
socio-demographic profiles and consumers’ awareness of Halal food fraud products and 3) to investigate factors that
influence consumers’ perception of Halal food fraud. The conceptual framework used for this study was adapted from
Ambali and Bakar (2014). A total of 352 respondents participated in this study was selected using a simple random
sampling in Klang Valley. Data were analysed using descriptive analysis, Chi-square analysis, and factor analysis.
The results indicated that majority of the respondents in Klang Valley were aware of Halal food fraud. However, there
were some respondents who have previous experience on buying Halal food fraud products. Gender, age, education
level and occupation were important factors that have significant relationships with Halal food fraud issues. Factor
analysis identified three factors that influence the consumers’ perception towards Halal food fraud which included
consumer attitude, authorities exposure and control and Halal logo, labelling and packaging. This study also found
that Halal food fraud was sold in the Malaysia market but Muslim consumers were found to have difficulty to still
differentiate between original products and fraud products.

Keywords: Food fraud, Halal, awareness, perception

*Corresponding author’s email: nitty@upm.edu.my

Introduction
Halal food industry covers activities such as processing, preparing, preservative, distributing and serving food and
beverages according to Shariah Law. It is also a complex, global collective of diverse business that supplies most of
the Halal food products to consumers. Halal food processing combines all Halal raw food materials or ingredients to
produce food products that can easily cooked and served by the consumers. With the Muslim world showing rapid
population growth, from 1.8 billion in 2012 to 2.2 billion expected in 2030, the economic development, as well as
increased disposable income, are also increased. The global Halal market now accounts for 16% of the world food
industry and predicted to rise to 20% in the future. According to a recent report by World Halal Forum global trade in

411
halal food and beverages is estimated worth about USD 1.4 Trillion annually (2014). The Halal marketplace has
emerged as one of the most profitable and influential market areas in the world business today as the increasing of
world Muslim population.

Halal food industry involves from ‘farm to table’ operations, the issue of integrity along food chain become a major
concern. Halal food fraud is the fast gaining public interest. However, its rises Muslim consumers concerns in
Malaysia as most of the halal food products nowadays being produced by non-Muslim countries rather that produced
by Muslim countries. As an example, most of the halal meat exporter in the world come from non-Muslim countries
such as Argentina, Australia, Brazil, Canada, France and New Zealand (Ambali and Bakar, 2009). A number of
reported cases of fraudulent halal certification and physical contamination of halal food products is increasing year by
year. Food fraud in general term defined as as cautious and intentional substitution, additional tampering or
misrepresentation of food, food ingredient or even food packaging, and misleading statements in labelling a product
for reason of economic gain (Spink and Moyer, 2011). The main objectives of the study are 1) to determine Muslim
consumers’ awareness and perception of Halal food fraud, 2) to identify the association between socio-demographic
profiles and consumers’ aware Halal food fraud products and 3) to investigate factors that influence consumers’
perception of Halal food fraud.

Methodology
a. Sample and Questionnaire
A total of 352 respondents in Klang Valley area were interviewed via simple random sampling to test their awareness
and perception on Halal food fraud. A survey was conducted through a face-to-face interview which all the questions
were easily understood by the respondents. The questionnaire was divided into four sections to include relevant
statements and questions. The questions were measured using a five-point Likert scale. The first section of the
questionnaire covered questions on respondents’ socio-demographic profiles such as gender, marital status, age,
education level, household size, and income. Then followed with consumers’ knowledge of Halal food fraud. Next
section was establised which includes consumers’ awareness of Halal food fraud. Finally, the last section asked for
the consumers’ perception of Halal food fraud.

b. Method of analysis
The data collected were analysed by using descriptive analysis, Chi-square analysis, and factor analysis were used
to analyse the data.

Results and Discussion

a. Socio-demographic profiles

As shown in Table 1, most of the respondents were female with 66.2% and age between 21-30 years old with 59.7%.
The majority of the respondents marital status were single with 51.7%, followed by married with 46.3%. In terms of
household size, 53.7% of the respondents’ household size were between 4-6 persons. For education level, 81.8%
respondents had tertiary education, followed by 12.8% had secondary school education level. Furthermore, 36.1% of
the respondent's occupation were from the private sector. Lastly, most of the monthly income were between RM
1001- RM 3000 and only 1.4% respondents had monthly income above RM 9001.

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 Table 1: Demographic profile of respondents
Characteristics Number Percentage (%)
Gender
Male 119 33.8
Female 233 66.2
Age
≤ 20 years 3 0.9
21-30 years 210 59.7
31-40 years 86 24.4
41-50 years 38 10.8
51-60 years 12 3.4
≥ 61 years 3 0.9
Marital Status
Single 182 51.7
Married 163 46.3
Divorce/Widow 7 2.0
Household Size
≤ 3 persons 103 29.3
4-6 persons 189 53.7
7-9 persons 53 15.1
≥ 10 persons 7 2.0
Education Level
Never been to school 0 0
Primary school 4 1.1
Secondary school 45 12.8
Pre-University 14 4.0
Tertiary Education 288 81.8
Other 1 0.3
Occupation
Government sector 104 29.5
Private sector 127 36.1
Self-employed 31 8.8
Housewife 12 3.4
Student 72 20.5
Other 6 1.7
Monthly Income
≤ RM1000 48 13.6
RM1001-RM3000 202 57.4
RM3001-RM5000 63 17.9
RM5001-RM7000 27 7.7
RM7001-RM9000 7 2.0
≥ RM9001 5 1.4
Note: n = 352

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 b. Chi-square analysis
Table 2: Association between respondent Socio-demographic profile and aware about Halal food fraud issues
Variable Chi-Square df Significant Result
Gender 24.254 1 0.000 *** Reject Ho
Age 20.138 4 0.000 *** Reject Ho
Marital Status 2.786 2 0.248 Fail to reject Ho
Household Size 0.728 3 0.867 Fail to reject Ho
Education Level 6.933 3 0.074 * Reject Ho
Occupation 9.547 5 0.089 * Reject Ho
Monthly Income 4.539 5 0.475 Fail to reject Ho
*Significant at 10% level of significance
***Significant at 1% level of significance
Note: n = 352

Table 2 shows that gender, age, education level and occupation have significantly related to the awareness of Halal
food fraud. Gender and age were significant at 10% level of significance. While education level and occupation were
significant at 1% level of significance.

c. Factor analysis
Table 3: Factors that influence Muslim consumers’ perception on Halal food fraud
Factor 1 Factor 2 Factor 3
Factor Halal Logo, Labelling Authorities Exposure Consumer Attitude
and Packaging and Control
Cronbach’s Alpha 0.861 0.894 0.761
Number of items 6 4 5
Eigenvalue 6.378 2.038 1.279
Percentage of Variance 37.518 11.990 7.526
Cumulative Percentage 37.518 49.508 57.034
Note: n = 352

The Cronbach’s Alpha value for this analysis was 0.842. The Kaiser-Mayer-Olkin (KMO) must be greater than 0.500
for the satisfactory factor analysis to proceed. As shown in Table 3 below, the KMO test is 0.893 was adequate for
this study because it falls in an acceptable range greater than 0.500. The factors influence perception on Halal Food
Fraud were Halal Logo, Labelling and Packaging, Authorities Exposure and Control, and Consumer Attitude.

Conclusions
This study concluded that consumers were difficult to differentiate between the original Halal logo that certified by
JAKIM with the fake Halal logo. Other than that, Muslim consumers were aware of Halal food fraud issues and they
have a negative perception of Halal food fraud products. Thus, improvement in knowledge among Muslim consumers
about Halal food fraud products is deemed important to reduce the possibility of consuming the fraud products.

Acknowledgements
The authors would like to thank UPM for financial support.

414
References

Ambali, A. R., & Bakar, A. N. (2014). People’s Awareness on Halal Foods and Products: Potential Issues for Policy-
Makers. Procedia - Social and Behavioral Sciences, 121, 3–25. https://doi.org/10.1016/j.sbspro.2014.01.1104
Ardyanti, N., Ahmad, B., Bin, T. N., Abaidah, T., Bin, M. H., & Yahya, A. (n.d.). A STUDY ON HALAL FOOD
AWARENESS AMONG MUSLIM CUSTOMERS IN KLANG VALLEY.
Badrie, N., Gobin, A., Dookeran, S. and Duncan, R. (2006). Consumer awareness and perception of food safety
hazards in Trinidad, West Indies’, Food Control 17 (2006) 370-377.
Fadzlillah, N. A., Man, Y. B. C., Jamaludin, M. A., Rahman, S. A., & Al-Kahtani, H. A. (n.d.). Halal Food Issues from
Islamic and Modern Science Perspectives.
Spink, J., & Moyer, D. C. (2011). Defining the Public Health Threat of Food Fraud. Journal of Food Science.
https://doi.org/10.1111/j.1750-3841.2011.02417.x
Tähkäpää, S., Maijala, R., Korkeala, H., & Nevas, M. (2015). Patterns of food frauds and adulterations reported in the
EU rapid alarm system for food and feed and in Finland. Food Control.
https://doi.org/10.1016/j.foodcont.2014.07.007
Zhang, W., & Xue, J. (2016). Economically motivated food fraud and adulteration in China: An analysis based on
1553 media reports. Food Control. https://doi.org/10.1016/j.foodcont.2016.03.004

415
 IFRC 2017: 230-227 Halal Food

Muslim Consumer’s Awareness and Acceptance on Halal Genetically Modified Food Labelling

2Md Rapi, N.R., 1,2,3,4*Kamarulzaman, N.H. and 5, 6 Ismail, N.W.

1 Department of Agribusiness and Bioresource Economics, Faculty of Agriculture, Universiti Putra Malaysia, 43400
UPM Serdang, Selangor, Malaysia
2 Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
3Institute Tropical Forestry and Forest Products, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
4Institute of Plantation Studies Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
5Department of Economics, Faculty of Economics and Management, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, Malaysia.
6Institute of Agricultural and Food Policy Studies, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor,
Malaysia.

Abstract
Genetically modified (GM) foods have been produced from the development of modern biotechnology and gene
combination of same or different species. GM foods have risen many concerns arisen due to religious, ethical, moral,
health, environment effect, virus, and toxin. This has become an important issue among Muslim consumers in
consuming halalan toyyiban food produced from GM foods. Therefore, having such labelling is an important element
to provide a clear declaration or information on the ingredients of the food products to consumers particularly
Muslims to make right purchasing choices and decisions. In the light of the above discussion, the objectives of the
study are 1) to determine Muslim consumers’ awareness and acceptance on halal GM foods, and 2) to investigate
GM foods labelling that influence Muslim consumers’ awareness and acceptance of halal GM foods. The conceptual
framework of the study was based on Attitude Model of GM Food. A survey was carried out with 320 respondents
using a simple random sampling technique across Klang Valley and was measured using structured questionnaires
with a 5-point Likert-scale related to awareness and acceptance towards halal GM foods labelling. The data was
analysed using descriptive analysis, chi-square analysis and factor analysis. Descriptive analysis showed that
majority of the respondents were not aware of the existence of halal GM foods labelling. Chi-square results revealed
that that there are association between age, education level and household income towards Muslim consumers’
awareness on halal GM foods labelling. Factor analysis identified health concern, religious belief and labelling have
influenced Muslim consumers’ acceptance on halal GM foods. This study emphasized that labelling could help
Muslim consumers to improve their awareness and acceptance on halal GM foods.

Keywords: Genetically Modified (GM) foods, labelling, halal food, awareness, acceptance, Muslim consumers
*Correspondence author’s email: nitty@upm.edu.my

Introduction
Modern biotechnology help cut and splice DNA chain and insert it into the DNA chain of other species. The
technology possibly changes the nature of the food at the most fundamental level is genetic modification, whereby
the actual physical blueprint of the food is changed from its original state. Genetic modification aggressively disrupts
the natural development of the genes, thereby preventing the modified plant and animal from functioning as a
wholesome form of life (Khattak et al., 2011).

Food not only serves to protect the health and survival, but the food was also influenced in shaping the personality
and character of a person Based on the Food Act 1983 and Food Regulations 1985, the food and drinks that are
obtained through modern biotechnology genetic resources need to be stated on the packaging labelling. Apart from

416
that, a statement such as "(name of ingredient) genetically modified" should be clearly stated on the label to provide
information to consumers. It is important for Muslim consumers to make food choices that are only allowed in Islam.
Muslim consumers are very particular regarding halal and haram food status. Consumption of GM foods by Muslim
consumers debates on morality, risks and benefits and implication on health (Hassan et al., 2016).
The objectives of the study is to determine Muslim consumer’s awareness and acceptance on halal GM foods
labelling.

Methodology
The data was collected among 320 Muslim consumers in Klang Valley area. Klang Valley was chosen as the
targeted population because it is the centre of economic and social development of Malaysia such as research
centres, universities and industry related to biotechnology (Amin et al., 2011). The sample was selected using a
simple random sampling and face-to-face interview. The questionnaire was developed to measure Muslim
consumers’ awareness and acceptance on halal GM foods labelling using structured questionnaires with 6 sections.
The questions included were about socio-demographic profiles, GM foods labelling, awareness on GM foods and
acceptance on GM foods. The analysis technique used are descriptive analysis, chi-square analysis and factor
analysis.

Results and Discussion

1. Descriptive Analysis
Table 1 shows the result of the respondents’ awareness of existence of Halal GM foods labelling. About 74.1% of the
respondents do not aware on existence of GM foods labelling while 25.9% of the respondents are aware on the GM
food labelling. The majority of respondents did not aware that the Malaysia’s Ministry of Health has issued guidelines
labelling of halal GM foods on 8 July 2014.

Table 1: Respondent’s Awareness of the Existence of Halal GM Foods Labelling

Frequency Percentage (%)

Yes 83 25.9
No 237 74.1
Total 320 100

2. Chi-Square Analysis
The cross-tabulation survey showed that within 320 Muslim respondents, there were three variable that have
association with Muslim consumers’ awareness on halal GM foods labelling. The variables were age, education level,
estimated household monthly income with significant values 0.076, 0.022 and 0.069 respectively related to
awareness on GM foods labelling. On the other hand, gender, marital status, household number, occupation and
origin were not statistically significant with awareness on GM foods labelling. The decision was summarized in Table
2.
Table 2: Chi-Square Analysis between Demographic Profile and Muslim Consumers’ Awareness on Halal GM Foods Labelling
Variable P-value df Significant Decision
Gender 1.592 2 0.451 Fail to reject Ho
Age 8.480 4 0.076* Reject Ho
Marital Status 4.740 6 0.578 Fail to reject Ho
Household Number 6.797 8 0.559 Fail to reject Ho
Education Level 23.719 12 0.022** Reject Ho
Occupation 12.239 12 0.427 Fail to reject Ho
Estimated Household 22.501 14 0.069* Reject Ho
Monthly Income
Origin 28.413 26 0.338 Fail to reject Ho
**Significant at 5% level of significance
* Significant at 10% level of significance

417
3. Factor Analysis
Factor analysis identified health concern, religious belief and labeling have influenced Muslim consumers’
acceptance on halal GM foods. The KMO measure was 0.782, showed that the sample was suitable for measure
sampling adequacy.

Table 3: Factors that influenced Muslim consumers’ acceptance on halal GM foods

Items Factor Loading
F1 F2 F3
Labelling
I will buy halal GM foods if the label is easily understood in terms of the 0.788
font type and size
I will buy halal GM foods if the label is easily seen and read. 0.824
Label serves as an identification card for halal GM foods. 0.807
Label 'Genetically Modified' important for me to distinguish between foods 0.675
based on biotechnology and natural food.
Eigenvalue 2.877
Percentage of variance 26.155
Cronbach’s Alpha 0.787
Religious Beliefs
I am sure that the GM foods that has JAKIM halal logo was halal to eat 0.781
even if it has the label 'Genetically Modified'.
I believe food products that have the label 'Genetically Modified' is 0.859
allowed in Islam.
As Muslim consumers, I believe that GM foods are halal to eat. 0.751
The selection of food products which are lawful and good is important for 0.734
Muslim consumers as outlined by Islam.
Eigenvalue 2.362
Percentage of variance 21.477
Cronbach’s Alpha 0.752

Health Concern
I would buy halal GM foods if it is good for the health of my family. 0.750
I need a label 'Genetically Modified' to prevent allergies (allergic to nuts, 0.723
seafood, milk, eggs).
Label 'Genetically Modified' important for me to ensure it is from a source 0.704
that is clean, healthy and holy.
Eigenvalue 1.421
Percentage of variance 12.921
Cronbach’s Alpha 0.716

Conclusion
In conclusion, this research shows that Muslim consumers are not aware of the existence of labelling requirement for
GM foods. Labelling, religious belief and health concern are the factor influenced Muslim consumer acceptance on
halal GM food. Great effort should be made by the relevant authorities to improve the awareness and acceptance of
Muslim consumers before purchasing halal GM foods that are available in the market.

418
References

Amin, L., Ahmad, J., Md. Jahi, J., Md. Nor, A. R., Osman, M., & Mahadi, N. M. (2011). Factors influencing Malaysian
public attitudes to agro-biotechnology. Public Understanding of Science, 20(5), 674–689.
http://doi.org/10.1177/0963662510369622
Hassan, S. H., John Kua, S. B., & Harun, H. (2016). Muslim consumers’ perception and purchase intention toward
GM food. International Food Research Journal, 23(2), 806–815.
Khattak, J. Z. K., Mir, A., Anwar, Z., Wahedi, H. M., Abbas, G., Khattak, H. Z. K., & Ismatullah, H. (2011). Concept of
Halal food and biotechnology. Advance Journal of Food Science and Technology, 3(5), 385–389.
http://doi.org/10.1017/CBO9781107415324.004

419
 IFRC 2017: 258-244 Halal Food

Halal Assurance System (HAS) Cost Analysis Using Descriptive Quantitative Methods and Prevention,
Appraisal, Failure (PAF) (Case Study at the Chicken Slaughterhouse Mitra Karya Unggas Batu East Java
Indonesia)
Sucipto Sucipto1,2*, Riska A. Novita1, Danang T. Setiyawan 1,2),
Mas’ud Effendi1, Retno Astuti1
1Department of Agroindustrial Technology, Faculty of Agricultural Technology, University of Brawijaya, Veteran Street
No.1, Malang, 65145, Indonesia
2Halal and Qualified Industry Development (HAL-Q ID), University of Brawijaya,Veteran Street No.1, Malang, 65145,
Indonesia.
Abstract
The charges against halal certified of chicken products has not been proportional to the amount of chicken
slaughterhouse halal certified. One of them is due to the unclear of the cost components to control halal assurance
system (HAS). This study purpose to determine the estimated cost of HAS in Mitra Karya Unggas chicken
slaughterhouse consisting of halal prerequisite costs, halal certification costs and HAS implementation costs. The
study was conducted by observation, field studies, interviews, and documentation. The calculation of halal
prerequisite cost and halal certification cost using quantitative descriptive method. HAS implementation costs are
calculated using Prevention Appraisal Failure (PAF) method. Calculation of HAS implementation costs consist of
prevention costs, appraisal costs and internal failure costs in April to June 2016. The results of this study indicated
that the poultry slaughterhouse of Mitra Karya Unggas needs the halal prerequisite cost of IDR 45.919 million,
covering facility prerequisite cost of IDR 33.838 million and management prerequisite cost of IDR 12.081 million.
Halal certification cost is IDR 15.14 million. HAS implementation costs of chicken carcasses IDR 980,477 per month.
Costs of prevention, appraisal and internal failure having an average of 69.08%, 23.77% and 7.13% with an average
allocation of total quality cost of 0.24% from total sales.
Keywords: Poultry Slaughterhouse, Halal Assurance System Cost, PAF
*Corresponding author’s email: ciptotip@ub.ac.id

Introduction
Needs chicken meat halal certified in Indonesia increased considerably. On the other hand, poultry slaughterhouses
assume halal certification is expensive and complicated procedure. Poultry slaughterhouse is a complex of buildings
with specific design and requirements are used to cut the chicken for public consumption (Dirkesmavet, 2010).
Indonesia regulation of halal assurance system (HAS) poultry refered to HAS 23103 (LPPOM MUI, 2012). One
small-scale of chicken producers that poultry slaughterhouse of Mitra Karya Unggas in Batu city, East Java,
Indonesia, which produces about 600 chicken carcass manually per day. This chicken slaughterhouse is not yet halal
certified. Implementation HAS is mandatory before every company propose halal certification (LPPOM MUI, 2008).
Halal certification costs approximated using the Cost of Quality (CoQ). CoQ is used to find a level of quality that
minimizes the total cost of quality (Schiffauerova and Thomson, 2006). Setiyawan, Sucipto and Khairunnisa (2016)
propose halal and safety assurance cost in noodle processing. This study is to obtain components and estimated
costs of preparation, certification, and implementation of HAS in small-scale of poultry slaughterhouse.
Methodology
The research conducted at poultry slaughterhouse of Mitra Karya Unggas, Batu East Java Indonesia. The study used
descriptive quantitative analytic for examining the feasibility of facilities and halal management systems. A
quantitative approach through empirical study to collect, analyse, and display data in numeric form (Given, 2008).
The following is calculation formula of halal prerequisite cost:
∑ HPC = HPFC + HPMC … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … . … … … . . (1)
Where:

420
 ∑ HPC = Sum of halal prerequisite cost
∑ HPFC = Sum of halal prerequisite facilities cost (the cost of procurement of wire netting, the separation of clean
and dirty areas, septic tanks, water content test, hand washing, appliances)
∑ HPMC= Sum of halal prerequisite management cost ( the cost of procurement personnel attributes, Standard
sanitation operational procedure (SSOP) and HAS training, Standard operational procedure (SOP)
documentation).

The following calculation formula of halal certification cost:

∑ HCC = HC1 + HC2 + HC3 + HC4 + HC5............................................................................................................. (2)
Where:
∑ HCC = Sum of halal certification cost
∑ HC1= Sum of preparation cost of internal audit
∑ HC2 = Sum of external audits cost
∑ HC3 = Sum of auditor fee
∑ HC4 = Sum of auditors accommodation (cost of travel, hotel and consumption)
∑ HC5 = Sum of halal certification payment
Halal Assurance System Implementation Cost (HASIC) the following calculation:
∑ HASIC = HASIC1 + HASIC2 + HASIC3................................................................................................................ (3)
Where:
∑ HASIC = Sum of HAS implementation cost ∑ HASIC2 = Sum of appraisal cost
∑ HASIC1 = Sum of prevention cost ∑ HASIC3 = Sum of internal failure cost

Results and Discussion

1. Halal Prerequisite Cost
Halal prerequisite cost is the cost to improve facilities and management system. Program prerequisites for
slaughterhouse should to control of hygiene and minimize contamination of carcasses (Howlett, Declan and Ciaran,
2005). Halal prerequisite costs are in Table 1.

Table 1. Calculation of halal prerequisite cost

Halal Prerequisite Cost Variable Component Cost (IDR
Halal Prerequisite 1.Location - -
Facilities Cost 2.Building and layout - Ventilators 7.500.000
- Separate of dirty area and 1.800.000
3. Infrastructure and clean area
sanitation - Sewer Waste
1.900.000
- The water content test
- Hand washing facilities 300.000
4.Equipment
- Chicken Hanger 638.000
- Container / drainer blood 1.500.000
- Tables clean up 9.500.000
- Desk packaging 7.500.000
3.200.000
Sub Total 33.838.000
Halal Prerequisite 1. Personal 881.000
Management Cost 2. Prerequisite program 10.700.000
3. Registration of Veterinary Health Number 0
4. SOP (Standard Operating Procedures)
500.000
Sub Total 12.081.000
Total of Halal Prerequisite Cost 45.919.000

421
The high preparation cost because the facility of this poultry slaughterhouse is not yet standardized. Therefore, it
need investments in equipment, facilities and sanitation.

2. Halal Certification Cost

Halal certification cost is the cost of external audit to obtain halal certificates. Audit is to access conformity of
production system with the halal requirements. General audits are conducted in the food industry (Dillon, 2001).
Table 2 shows the halal prerequisite cost.

Table 2. Calculation of halal certification cost

Variable Cost (IDR)
- Establishment of halal management team and internal auditor 8.300.000
- The cost of external audit (the halal control point, complexity of processes) 500.000
- Honorarium of auditor 700.000
- Auditor needs (cost of travel, hotel and consumption) 4.140.000
- The cost of halal certification (cost of internal court, commissions fatwa, LPPOM MUI) 1.500.000
Total of halal certification cost 15.140.000

The high cost of formation of the management team needs to be established halal as halal management structure
responsible for the halal production process.
3. Halal Assurance System Implementation Cost (HASIC)
HASIC is the cost for implementing HAS at poultry slaughterhouse. The allocation of the costs of implementing HAS
and the percentage in Table 3.
Table 3 Allocation of HASIC in 2016
Halal Cost April (IDR) (%) May (IDR) (%) June (IDR) (%) Average (IDR) (%)
Prevention cost 677.335 69.36) 677.335 (69.36) 677.335 (68.52) 677.335 (69.08)
Appraisal cost 233.142 (23.87) 233.142 (23.87) 233.142 (23.58) 233.142 (23.77)
Internal failure cost 66.000 (6.75) 66.000 (6.75) 78.000 (7.89) 70.000 (7.13)
Total HASIC 976.477 976.477 988.477 980.477

Based on Table 3 obtained appraisal cost in April-June amounted to 23.87%, 23.87%, 23.58%, and an average of
23.77%. Appraisal costs decreased in June. Internal failure costs increased in June due to inadequate number of the
product is defective, and make a significant increase in the cost of prevention. Total sales of chicken carcasses in
April to June and the percentage halal cost on the value of product sales in Table 4 and Figure 1.
Table 4 Total of product sales
Month Total (head) Price (IDR) Total Sales (IDR)
April 18,000 22,000 396,000,000
May 18,000 22,000 396,000,000
June 18,000 26,000 468,000,000

Figure 1 Halal cost on the value of product sales

422
Based on Table 4, chicken prices in April and May amounted to IDR 22,000, while in June IDR 26,000 per head. It
affects the sales turnover, in April and May respectively IDR 396 million and IDR 468 million in June. Figure 1 shows
prevention cost, appraisal cost and internal failure costs in April and May are compared in June is decrease due to
the increase in total sales. The increase was due to the chicken price in June increased to IDR 26,000 per head.
HAS total cost allocation in Table 5.
Table 5. Halal assurance system (HAS) cost allocation
Cost Component Estimation (IDR)
1.Halal Prerequisite Cost:
a. Halal Prerequisite Facilities Cost 33.838.000
b. Halal Prerequisite Management Cost 12.081.000
Sub Total 45.919.000
2.Halal Certification Cost 15.140.000
3.HASIC 980.477

HAS cost based on Table 5 in poultry slaughterhouse consists of a halal prerequisite cost (cost of facilities and
management system) is IDR 45.919 million, halal certification cost is IDR 15.14 million for one-time certification is
valid for 2 years, and HASIC is IDR 980,477 per month. Estimated of halal prerequisite cost and halal certification
cost performed one cycle of halal assurance, while being HASIC once a month.
Conclusion
This study propose halal assurance system (HAS) cost consists of the preparation cost, halal certification costs, and
HAS implementation cost. Poultry slaughterhouse of Mitra Karya Unggas Batu has halal prerequisite of facilities cost
of IDR 33.838 million and halal prerequisite management cost of IDR 12.081 million. The prerequisite costs are
relatively height because this slaughterhouse is below standard and it needs investment and management systems.
Halal certification costs to establish halal management team and audit process. It need cost of IDR 15.14 million.
HAS Implementation Cost (HASIC) of chicken carcasses using PAF is IDR 980 477 per month. The average of
prevention cost of IDR 677 335 (69.08%), appraisal cost of IDR 233 142 (23.77%), internal failure cost of IDR 70,000
(7.13%) per month. Total of halal costs of 0.24% is below the standard of quality cost of 2.5%, because the
slaughterhouse is not yet implement halal certification.
Acknowledgements
The authors would like to thank University of Brawijaya for financial support.
References
Dillon, M. 2001. Food Standards and Auditing, in Auditing in the Food Industry from Safety and Quality to
Environmental and Other Audits, ed. Mike Dillon and Chris Griffith. Cambridge England: Wood head
Publishing Limited.
Direktorat Kesehatan Masyarakat Veteriner dan Pascapanen. 2010. Pedoman Produksi Dan Penanganan Daging
Ayam yang Higienis. Direktorat Jenderal Peternakan dan Kesehatan Hewan. Kementrian Pertanian Republik
Indonesia.
Given, L.M. 2008. The Sage Encyclopedia of Qualitative Research Methods. Thousand Oaks: Sage.
Howlett, B., Declan, J.B., Ciaran, O.S. 2005. Development of Pre-Requisite Programmes and HAACCP Principles for
Irish Beef Slaughterhouse. Food Safety Authority of Ireland. The National Food Centre.
LPPOM MUI. 2008. General Guidelines of Halal Assurance System (online). Available:
http://www.halalcertifiering.se/newwebsiteimages/ebookhashaki.pdf (Accessed 20 June 2017)
LPPOM MUI. 2012. Pedoman Pemenuhan Kriteria Sistem Jaminan Halal di Rumah Potong Hewan (HAS 23103).
Schiffauerova, A. and Thomson, V. 2006. A Review of Research on Cost of Quality Models and Best Practices.
International Journal of Quality & Reliability Management 23(6): 647-669.
Setiyawan, D.T., Sucipto, Khairunnisa, S. 2016. Analysis of Halal and Safety Assurance Cost in Mie Jogja Pak
Karso Restaurant. Jurnal Teknologi Pertanian Universitas Brawijaya 17(2):105-118.

423
 IFRC 2017: 273-252 Halal Food

Comparative Study of Acid and Alkaline Pre-Treatment Process Prior To Gelatin Extraction from Rohu
(Labeo rohita) Scales

1Khairulnizam, A.B., 1*Jamilah, B., 1Nur Hanani, Z.A., 1Russly, A.R. and 2Kharidah, M.

1Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra, Malaysia, 43400
Serdang, Selangor, Malaysia.
2Department of Food Science, Faculty of Food Science and Technology, Universiti Putra, Malaysia, 43400 Serdang,
Selangor, Malaysia

Abstract
Gelatin was extracted from rohu (Labeo rohita) scales using direct hot water extraction after different chemical pre-
treatment using acid and alkaline. The aim of the study was to compare the extraction yield and physicochemical
characteristics of gelatin obtained. The yield of gelatin extracted from acid pre-treated scales was 10.0 % (w/w) with
gel strength 207.8 g, and viscosity 15.5 mPa·s, respectively whereas the yield of gelatin obtained from alkaline pre-
treated scales was 7.1 % (w/w) with gel strength 180 g, and viscosity 7.7 mPa·s, respectively. The Fourier transform
infrared (FTIR) spectra of both extracted gelatin showed similar absorption band characteristics.

Keywords: Fish scales, gelatin, rohu, halal, extraction

*Corresponding author’s email: jamilah@upm.edu.my

Introduction
The exploitation of agriculture waste in order to produce new added-value products has been developed within the
optimization of by-products utilization and improvement of waste management. For example, by-products from fish
and surimi processing are produced in huge amounts annually and this wastes are often use for animal feed or
discarded which causing environmental issues and income lossess for their valuable components. Fish processing
by-products consist of scales, skins and bones are collagen-rich materials that are abandoned and abundantly
available. They may play as attractive and alternative source of protein such as gelatin which is a multi-functional
ingredient use in numerous areas including food, pharmaceutical and other industrial field as agent for gelling,
stabilizing, emulsifying, film forming and viscosity enhancing additives (Schrieber and Gareis, 2007). Most of the
world’s gelain supply is commercially derived from skins and bones of mammalian such as pig and cattle which are
considered as non-halal or doubtful. Thus, production of gelatin from alternative source has received much attention
due to its high demand for halal industry especially. The use of gelatin have become a controversial issue for Jew
and Muslim consumers because consumption of products derived from pig is strictly prohibited for both Jew and
Muslim regarding to kosher and halal principal, respectively.

In recent decades, various industrial by-products have been used for producing gelatin such as from duck feet
(Abedinia et al., 2017), fish skin (Kittiphattanabawon et al., 2016), and catfish bone (Sanaei et al., 2013). Previous
researches has also reported the gelatin extraction from fish scales of big head carp (Hypophtalmichthys nobilis) (Tu
et al., 2015), sea bream (Akagündüz et al., 2014), bighead carp (Aristichthys nobilis) (Tong and Ying, 2013; Sha et
al., 2013), grass carp (Ctenopharyngodon idella) (Zhang et al., 2011), lizardfish (Saurida spp.) (Wangtueai and
Noomhorm, 2009) and silver carp (Hypophthalmichthys molitrix) (Wang and Regenstein, 2009). In the best of our
knowledge, there is less study on gelatin extraction from scales of rohu (Labeo rohita). Rohu is a freshwater fish
species belongs to the carp family which contribute significantly to the aquaculture production in Malaysia. Therefore,
this study was conducted to investigate the effect of different pre-treatments on extraction yield and physicochemical
properties of rohu scales gelatin.

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Materials and methods
3.1 Raw materials and chemicals
Rohu (Labeo rohita) scales were obtained from wet market in Serdang, Selangor. The scales were washed with tap
water to remove impurities and dirt, dried at 60 °C for 8 hr in air force oven and then kept in air tight container until
further use. All the chemicals used were of analytical grade.

3.2 Extraction of gelatin

Gelatin extraction procedures were carried out with acid and alkaline pre-treatment according to the method of Wang
and Regenstein (2009) and Wangtueai and Noomhorm (2009), respectively, with slight modification. The fish scales
were treated with 0.5 M hydrochloric acid and 0.5 M NaOH in ratio of scales/solution (1:5, w/v) at room temperature
for 3 hrs. After pre-treatment, the scales were neutralized through washing with tap water. The pre-treated fish scales
were rinsed and drained before subjected to extraction process in distilled water (1:2, w/v) for 5 hrs at 80 °C. The
control samples (fish scales without pre-treatment) were also extracted in distilled water (1:2, w/v) at 70 °C, 80 °C,
90 °C and 100 °C for 5 hr, respectively. After extraction process, the mixture was filtered through cheese cloth
followed by Whatman no. 4 filter paper and the filtrate collected was oven dried overnight at 60 °C. The dried gelatin
sample was then ground and kept in an air tight bottle for further analysis. The yield of gelatin was calculated as
follows:
Gelatin yield, % (w/w) = (Dry weight of gelatin)/(Wet weight of scales) x 100.

3.3 Physicochemical properties characterization

The gel strength of gelatin was determined according to Gelatin Manufacturers Institute of America standard test
method (GMIA, 2013).The viscosity of gelatin solution (6.67 %, w/v) was measured using viscometer (Model LVDV-
II, Brookfield, Stoughton, USA). Fourier transform infrared (FT-IR) spectroscopy analysis of gelatin sample was
conducted using a FT-IR spectrophotometer (NicoletTM 6700, Thermo Scientific). All data were expressed as mean ±
standard deviation and were done in triplicate independent analyses. Data were analyzed using one-way ANOVA
using SPSS version 16 (SPSS Inc., Chicago, Illinois, USA).

Results and discussion

The yield of gelatin from acid pre-treatment, Ac-RSG (10 %, w/w) was higher than the yield of gelatin from alkaline
pre-treatment, Al-RSG (7.1 %, w/w) and other samples (Table 1). The low gelatin yield might be caused by
insufficient denaturation of soluble collagen during pre-treatment and extraction process. Akagündüz et al. (2014)
reported the yield of gelatin from sea bream scales was 9.55 % (w/w) whereas Wangtueai and Noomhorm (2009)
reported the yield of gelatin from lizardfish scales was 10.6 % (w/w).

The gel strength of gelatin from acid pre-treatment, Ac-RSG (207.2 g) was higher than the gel strength of gelatin from
alkaline pre-treatment, Al-RSG (179 g) and other samples (Table 1). Gel strength is one of the most important
measurement used to assess the grade and quality of gelatin. The gel strength of commercial gelatin has been
reported ranged from 50 to 300 g (Schrieber and Gareis, 2007). The viscosity of Ac-RSG and Al-RSG was 15.5
mPa·s and 7.7 mPa·s, respectively were higher than the viscosity of control samples. Viscosity is the second most
important physical property of gelatin and the viscosity of commercial gelatin has been reported ranged from 2 to 13
mPa·s (Schrieber and Gareis, 2007).

Table 1. Extraction yields and physicochemical properties of acidic (Ac-RSG), alkaline (Al-RSG) and control gelatin
samples (C1, C2, C3, and C4).
Gelatin samples Ac-RSG Al-RSG C1 C2 C3 C4
Yield (%) 10.6 ± 0.7b 7.1 ± 0.3a 5.4 ± 0.7c 5.7 ± 0.4c 4.6 ± 0.8d 3.1 ± 0.6e
Gel strength (g) 207.2 ± 1.2e 179 ± 1.0d 6.3 ± 0.4a 56.6 ± 0.5c 15.9 ± 0.5b 15.2 ± 0.3b
e d a c b b
Viscosity (mPa·s) 15.5 ± 0.2 7.7 ± 0.1 3.5 ± 0.4 6.4 ± 0.4 5.5 ± 0.4 5.2 ± 0.2
Values are means ± standard deviation (n=3). Means within the same row with different letters are significantly
different at p<0.05 as measured by Duncan test.

425
 Figure 1. Fourier transform infra-red (FT-IR) spectra of acidic (Ac-RSG), alkaline (Al-RSG) and control gelatin
samples (C1, C2, C3, and C4).

In general, Ac-RSG and Al-RSG showed similar FTIR profile as control samples (Figure 1). As a protein, gelatin
structure is build up of carbon, hydrogen, hydroxyl group (OH), carbonyl group (C=O) and amine group (NH). In all
spectra, the broad absorption band found in the range of 3500-3400 cm-1 and 2900-3000 cm-1 represent amide A and
amide B band, respectively (Wang et al., 2008). All gelatin spectra also demonstrated the characteristic pattern
reflecting the amide I band (in region of 1700-1600 cm-1), amide II band (in region of 1550-1500 cm-1) and amide III
band (in region of 1500-1200 cm-1).

Conclusion
Gelatin was successfully extracted from rohu (Labeo rohita) scales and according to results, acid pre-treatment was
identified and selected as more appropriate method based on highest yield and gel strength of the gelatin obtained.
In addition, the results shows a potential prospect of utilizing the fish scales from rohu as alternative source of
gelatin.

Acknowledgement
The authors thanks UPM for the support. One of the author, Khairulnizam, A.B. ackowledges the scholarship
received from MyBrain 15 from MOSTI, Malaysia.

426
References

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Pekin duck (Anas platyrhynchos domestica) as affected by acid, alkaline, and enzyme pre-treatment.
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Akagündüz, Y., Mosquera, M., Giménez, B., Alemán, A., Montero, P., and Gómez-Guillén, M. C. 2014. Sea Bream
Bones and Scales As a Source of Gelatin and Ace Inhibitory Peptides. LWT - Food Science and Technology
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physico-chemical properties of catfish (Clarias gariepinus) bone gelatin, International Food Research Journal
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properties on gelatin from fish scale. Advanced Materials Research, 647: 352–356.
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carp (Aristichthys nobilis). Journal of Food, Agriculture and Environment, 11 (1): 146–150.
Tu, Z. C., Tao, H., Wang, H., Sha, X. M., Shi, Y., Huang, X. Q., Man, Z. Z. and Li, D. J. 2015. Physico-chemical
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427
 IFRC 2017: 205-192 Halal Food

Halal practices integrity and halal supply chain trust in Malaysian halal food supply chain

*Kamisah, S., Mokhtar, A., and Hafsah, A.

Faculty of Business and Accountancy, Universiti Selangor, Jalan Zirkon A 7/A, Seksyen 7, 40000 Shah Alam,
Selangor, Malaysia.

Halal food industry has grown substantively in the last few years globally and become crucial to the Muslim
consumers due to its safety, hygiene and quality assurance of what is consumed and used. Muslim consumers
demand for the products that have gone through the process of halal certification. In Islam, Muslim consumers follow
the Islamic dietary law and the food they consume should be halalan-tyoyyiban, i.e. lawful or permissible,
wholesome, authentic and safety. Proper halal food description is accepted by the Muslim consumers as well as
many non-Muslim consumers in the world. Any improper description and inappropriate preparation of halal food
provide a huge impact to the demand for halal food as well as to retain the buyers’ trust in consuming the halal food.
Trust is an essential element in the food production and required along the halal food supply chain by all parties
involved. Given the significant role of trust in the halal production over the Muslim consumers’ life, this study aims to
examine the influence of halal practices integrity on halal supply chain trust and supply chain performance. The
questionnaires distributed to the halal food manufacturers during the Malaysian halal food events and festivals.
Structural Equation Modeling with Partial Least Square was used to analyze the collected data. The findings show
that halal practices integrity has a significant relationship with halal supply chain trust, and supply chain performance.
Halal supply chain trust mediates the relationship between halal practices integrity and supply chain performance.
The results also provide a better understanding of factors influencing the halal supply chain trust in the halal food
industry.

Keywords: Halal practices integrity, halal supply chain trust, halal food, supply chain, Malaysia
*Corresponding author’s email: kamisah@unisel.edu.my

Introduction

Nowadays, Muslim consumers are demanding for halal certified products. Halal is an Arabic or Quranic word that is
associated with permissible or lawful. The concept of halal is not merely about the sources of food and beverages,
the use of alcohol, but it is about processes and standards that related to cleanliness, safety, and quality assurance
(Yusaini et al., 2016). Halal certification is a process of certifying that the products produced have met the Islamic
dietary guidelines (Riaz and Chaudry, 2004). In Malaysia, Department of Islamic Development Malaysia (JAKIM) has
been entrusted to govern any issues related to halal practices and production of halal products by manufacturers. To
be in line with the implementation of global quality standard such as ISO, JAKIM has embarked on the development
of halal assurance system known as MS1500:2009 General Guidelines on the Production, Preparation, Handling and
Storage of Halal Food. The development of the MS1500:2009 standard was based on ISO methodologies and in
compliance with other quality standards such as MS1514:2009 and MS1480:2007. This is to ensure that the
MS1500:2009 halal standard also addresses issues related to cleanliness, hygiene and food safety aspects of the
processing and preparation of halal food.
However, JAKIM has found that claims made by manufacturers that their products are halal are just not good
enough. Many businesses have been found to use confusing statement and halal logo regarding their products and
services. Besides that, there are several issues such as detection of pig-DNA, and non-compliance to halal
requirement have shaken the confidence of the Muslim consumers (Kamisah, 2016). These issues show that having
halal standards regulation (i.e. MS1500:2009) does still not guarantee that the product is halal at the point of
consumption. Effective from 1st January 2013, JAKIM has enforced that legal actions will be taken on any businesses
that involve in misusing halal logo or non-compliance with halal certification including imported products.

428
 In Malaysia, sustaining the integrity of halal products have become a priority for the government and the
consumers (Sarah et al., 2011; Tieman, 2013). However, there are various obstacles, misconceptions, and fallacies
in the supply chain with regards to halal practices integrity (HPI). Some manufacturers and suppliers did not realize
the particularity in handling the halal food process. The production of halal food needs a very meticulous
understanding throughout the supply chain in sustaining the integrity of halal food. Furthermore, it is an obligation for
Muslim consumers to have trust or confidence that the goods they consume are halal, hygienic, and safe that comply
with the Islamic principles (Manzouri et al., 2013). For the Muslim consumers, trust in halal food relates to the
certainty about the process attributes of food processing, handling, and the safety regarding food wholesomeness.
HPI refers to the means of ensuring that the people, process, and resources that deliver integrity of halal
products are Shariah-compliant along the supply chain. HPI requires trust from both buyer-supplier sides and the
halal supply chain is based on trust (Tieman, 2011). Thus, the elements of HPI can be successfully established
through the halal supply chain trust (HSCT) process, which was defined by Morgan and Hunt (1994) as the
confidence in an exchange partner’s reliability and integrity. Trust plays a significant role in inter-firm relationships
which is the desire on the part of each party to a business relationship to maintain and strengthen those relationship
(Morgan and Hunt, 1994). Furthermore, trust facilitates a firm’s ability to respond to customers and global market
conditions (Petrick and Quinn, 2001). However, HSCT has not been tested in the environment of halal integrity.
Specifically, the purpose of this study is to examine the influence of HPI on HSCT and supply chain performance
(SCP) in Malaysian halal food industry.

Materials and Methods

This study adopted cross-sectional research design, quantitative research approach and purposive sampling
technique was performed to select a group of target respondents. The respondents were approached during the
Malaysian halal food events in January 2016 to April 2016 by distributing questionnaires. The sampling unit in this
study was the personnel at managerial level and expected to provide a valid and accurate view of their halal business
as halal is a critical matter in the halal manufacturing.
A seven-point likert scale was used to measure HPI, HSCT and SCP. Many previous studies have used the
seven-point scales to measure integrity, trust and SCP which considered as a valid and appropriate measurement
(Chen and Paulraj, 2004; Green et al. (2008). The questionnaire items were adapted from Chen and Paulraj (2004),
Wilson and Nielson (2000), Chen (2013), Mayer et al. (1995), Svensson (2001) and Panayides and Lun (2009).

Results and Discussion

Based on the survey conducted, out of 350 questionnaires distributed only 212 responses were collected. Structural
Equation Modeling (SEM) with Partial least squares (PLS) being adopted as the method of estimation of the model.
This PLS-SEM approach gained widespread interest as a method of analysis due to less dependent on normality
assumption of the data and for small sample problems (Hair et al., 2017).
Reliability and validity are used in assessing reflective outer models by using PLS-SEM. Composite reliability
(CR) is used to evaluate the construct measures’ internal consistency reliability, whereas construct’s convergent
validity and discriminant validity are used in assessing construct validity. Convergent validity is the extent to which a
measure correlates positively with the alternative measures of the same construct. Hair et al. (2014) suggested that
each item has outer loadings above 0.70, and average variance extracted (AVE) is 0.50 or higher to support
convergent validity of reflective outer models. The results have exceeded the recommended values. Fornell and
Larcker (1981) criterion was employed to assess discriminant validity after confirming the convergent validity by
comparing the square-roots of the AVE values with the correlations. The discriminant validity measures the extent to
which items can be differentiated among constructs. Hence, the results confirmed that the measures used in this
study are distinct and provides adequate discriminant validity.
The results of the structural model analysis were found that HPI (β = 0.708, t = 15.283, p < 0.01) was positively
related to HSCT. HSCT (β = 0.460, t = 4.889, p < 0.01) was positively related to SCP. HPI (β = 0.492, t = 7.807, p <
0.01) was positively related to SCP. Also, the mediating effect of HSCT in the HPI-to-SCP relationship was tested. As
suggested in the literature, the bootstrapping procedure was used to test the indirect effect. The results show that

429
indirect effect (β = 0.495, р < 0.01) was significant and there was a mediating effect in this analysis. The variance
accounted for (VAF) was calculated as suggested by Hair et al. (2013, 2017) which determine the size of the indirect
effect in relation to the total effect. The VAF was 0.50 and classified as partial mediation.
The study aims to examine the influence of HPI on HSCT and SCP. The result shows that HPI has a significant
relationship with HSCT. This finding is consistent with the previous studies by Colquitt et al. (2007) and Albrecht
(2002) that integrity have unique relationships with trust which they are controlling for one another. The relationship
between HSCT and SCP was significant and support the work done by Yu and Choi (2014) that trust had a
significant effect on performance. Moreover, trust is considered as a delicate but vital source of a sustainable
competitive advantage in a successful business-to-business and business-to-consumer relationships (Panayides and
Lun, 2009; Morgan and Hunt, 1994). The findings also indicated that the relationship between HPI and SCP was
significant which are consistent with the previous studies done by Shi and Yu (2013) and Sundram et al. (2011).
Hence, the elements of HSCT and ability to understand halal practices in a competitive of the halal market by the
halal food manufacturers is essential to enhance their SCP.

Conclusion

Although the results of this study contribute to the further understanding of existing literature, the findings cannot be
generalized due to the small sample size and only confined to the food industry. The proposed research model can
motivate further research in the various type of industry to generalize the results. Also, there is a possibility about the
significant contribution of moderating factors such as the managerial level and size of the companies which can act
as a control variable to develop the findings more comprehensively in the future research.

Acknowledgement

The authors are thankful to Universiti Selangor for providing financial support for this research.

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 IFRC 2017: 034-031 Food Bioprocessing
Survival, physicochemical properties and digestive stability of microencapsulated Lactobacillus spp.
strains 21C2-10 in probiotic ice cream.
1,2 1,2*Oonsivilai,
Sengsaengthong, S., R.
1
School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon
Ratchasima, Thailand
2Postharvest Technology Innovation Center, Commission on Higher Education, Bangkok 10400, Thailand
Abstract
The objectives of this study were to compare the survival and physicochemical properties of probiotic ice cream
containing free and encapsulated Lactobacillus spp. strains 21C2-10 during storage for 120 days at -20 °C. In
addition, the investigation of bacterial survival after exposure to simulated gastro-intestinal juices for 5 hours
(simulated gastric, 1 h and intestinal juices, 4 h) carried out. The results showed that % after storage for 120 days at
-20 °C, the probiotic ice cream containing encapsulated cell showed significantly (P<0.05) higher survival
percentage (96.76%) than probiotic ice cream containing free cell (85.13%). Also, the pH value and acidity of
probiotic ice cream containing encapsulated cell were significantly (P<0.05) lower than probiotic ice cream containing
free cell after storage for 120 days at -20 °C. Moreover, after exposure to simulated gastro-intestinal juices for 5
hours, the probiotic ice cream containing encapsulated cell increased the survival of Lactobacillus sp. 21C2-10 when
compared to those containing free cell. Finally, the scanning electron microscopy images evidenced cells with
morphological differences was carried out and the results showed that probiotic ice cream containing cell could
prevent physiological change under GI stress more than probiotic ice cream containing free cell.In conclusion, the
survival and physical properties including the digestive stability of probiotic ice cream containing encapsulated cell
were higher than probiotic ice cream containing free cell during days at -20 °C and the passage through the
simulated gastro-intestinal conditions.
Keywords: encapsulation, probiotic, survival, ice cream, digestion
*Corresponding author’s email: roonsivi@sut.ac.th
Introduction
Probiotics are live microorganisms which confer a health benefit on the host (FAO/WHO, 2002). Probiotics have
been shown to be effective in the stimulation of the immune system, anti-mutagenic, anti-carcinogenic (Zhu et al.,
2011). However, probiotic products must have a microbial count higher than 6 log CFU per mL or per g until the shelf
life has expired to claim benefits (Roy, 2005). Encapsulation in gelatin-maltrodextrin (G-MD) microspheres cross-
linked with TGase increased the survival of Lactacillus spp. strain 21C2-10 during exposure to simulate during GI
tract condition, indicating the protective effect by encapsulating (Nawong et al., 2016).
The objectives of this study were to compare the survival and physicochemical properties of probiotic ice cream
containing free and encapsulated Lactobacillus spp. strains 21C2-10.
Materials and Methods
3.1 Lactobacillus spp. strain 21C2–10 was isolated from cassava pulp and identified using the API 50 CHL test kits
(Nawong, 2013). Preparation of probiotic cells was inoculated in MRS-cys broth and was incubated at 37 °C for 24 h
until cell densities of approximately 10 log CFU/ml and washed twice in peptone saline.

3.2. Encapsulation of probiotic cells was encapsulated by an emulsion technique using a modified encapsulation
method reported by Nawong et al. (2016) which TGase concentration of 10 U/g with gelatin A 300 bloom.
Encapsulated probiotic was released by collagenase using a modified method reported by Nawong et al. (2016). The
measure the diameter of 200 capsule was applied using Laser particle size.

432
3.3. The ice cream product was prepared by using a modified method reported by Corvitto (2011). Probiotic
inoculation with free cell and encapsulated cell were about 10 log CFU/g. The acidity was determined according to
AOAC, 2000. Proximate analysis of ice creams were according to AOAC, 2000.
3.4. The probiotic viability in the products during storage carried out for 10 g of each ice cream. The survival of
probiotic in ice cream were enumerated on MRS-cys agar. Also, the survival of free and encapsulated ice cream in
simulated gastro-intestinal juice using a modified encapsulation method reported by Nawong et al. (2016). Cell
viability of free and encapsulated cell after exposure with gastro-intestinal juice for 5 hr.
3.5. The morphological of probiotic after simulated gastric-intestinal juice using scanning electron microscopy (SEM).
3.6 Statistical analysis for all data carried out by one-way analysis of variance (ANOVA) using SPSS 16.0 software
package for Windows with the Tukey post hoc test at the 5% significance levels.
Results and Discussion
The size reported diameters of 39.234-101.460 µm. SEM images show that spherical shape. Hansen et al. (2002)
suggested that microcapsules with a size below 100 µm could avoid impact on sensory in food products. The
chemical composition of encapsulated cell were shown in Table 1. The encapsulated probiotic was significantly
(P<0.05) higher survival than free cells after exposure with gastro-intestinal juice for 5 hr. Nawong et al. (2016)
suggested that TGase produced iso-peptide that protected from pepsin degradation. Its cause increase cell viability
of encapsulated cells from the gastrointestinal condition.
The chemical composition of probiotic ice creams were shown in Table 1. At 120 day of storage time, the ice cream
containing encapsulated cells showed lower acidity and higher pH, survival significantly (P<0.05) than free cell. The
wall material could protect the cell from Crystallized water (Martin et al., 2013). After 5 hours exposure to simulated
gastro-intestinal juice, the population of probiotic in encapsulated probiotic ice cream higher survival significantly
(P<0.05) than probiotic ice cream. SEM image of probiotic showed morphological changing in the cells surface. Bile
salt has damaging effects on bacterial cell membrane lipids, therefore affecting the cell wall integrity (leverrier et al.,
2003).The reduction of probiotic after exposure possibly due to the breakdown of cell wall.
5
4
loss (Log
Viability

3
CFU/g)

2 Encapsulated cell (21C2-10)

1 Free cell (21C2-10)
0
0 2 4 6
during exposure (hrs)
Figure 1. Microstructure as observed by SEM (A) and Survival of Lactobacillus 21C2-10 after exposure to SGJ (1 h)
and SIJ (4 h) at 37 ºC (B). Average log CFU/g reduction (n=4) are show with error bars.

Table1. Chemical composition of encapsulated probiotic and probiotic ice cream on day 1.
composition Encapsulated probiotics Free cell ice cream Encapsulated cell ice cream
Ash 0.92±0.03a 0.89±0.02b 0.97±0.02c
Protein 43.10±2.06a 10.84±0.50b 10.51±0.36c
Fat 20.27±0.20a 23.05±0.52b 25.23±0.15c
Crude fiber 12.18±0.28a 0.06±0.01b 0.16±0.22c
Values are the mean ± SD (n=4); means that do not share a letter are significantly different (P<0.05) as measured
by Turkey test.

433
 100.0 7.00
Free cells (21C2-10)
Survival rate (%) 95.0 6.90 Encapsulated cells (21C2-10)
6.80

pH
90.0
6.70
85.0 Free cells (21C2-10)
6.60
Encapsulated cells (21C2-10)
80.0 6.50
0 20 40 60 80 100 120 0 20 40 60 80 100 120
Storage time (Days) Storage time (Days)
0.40 2.50

Viability loss (Log

0.30 2.00
Titrable acidity

1.50
0.20

CFU/g)
(%)

1.00
0.10 Free cells (21C2-10)
0.50
Encapsulated cells (21C2-10)
0.00 0.00
0 20 40 60 80 100 120 0 2 4
Storage time (Days) During exposure (hrs)
Figure 2. Survival of Lactobacillus 21C2-10 (A). pH (B) and Acidity (C) during storage time. Survival of Lactobacillus
21C2-10 during exposure to SGJ (1 hr) and SIJ (4 hr) at 37 ºC (B). Average log CFU/g reduction, pH, Acidity (n=4)
are show with error bars.

A B C

D E F

Figure 3. Microstructure of free cell (A-C) and encapsulated cell (D-F) after exposure 0 (A,D) 1 (B,E) and 4 (C,F)
hours.
Conclusion
Microencapsulation in G-MD microsphere was effective maintaining the stability of encapsulated probiotic ice cream
after exposure to GI tract condition and during storag time. In the futher recommended analysis in the part of
sensory analysis of encapsulated probiotic ice cream.

Acknowledgements
This study was supported by Suranaree University of Technology, Thailand.

434
Reference
A.O.A.C. 2000. Official Method of Analysis. (15th ed). The Association of Official Chemists, Arlington, Verginia.
Corvitto, A. 2011. The secrets of ice cream = los secretos del helado ice cream without secrets = El helado sin
secretos. (2nd ed). Sant Cugat del Valles, Vilbo. Spain.
FAO/WHO. 2002. The evaluation of probiotics in food. Food and Agricultural Organization of the United Nations.
Rome: FAO.
Hansen, L. T., Allan-Wojtas, P. M., Jin, Y. L., and Paulson, A. T. 2002. Survival of Ca-alginate microencapsulated
Bifidobacterium spp. in milk and simulated gastrointestinal conditions. Food Microbiology 19(1): 35-45.
Leverrier, P., Dimova, D., Pichereau, V., Auffray, Y., Boyaval, P., and Jan, G. 2003. Susceptibility and adaptive
response to bile salts in Propionibacterium freudenreichii: physiological and proteomic analysis. Appl Environ
Microbiol 69(7): 3809-3818.
Martin, M. J., Lara-Villoslada, F., Ruiz, M. A., and Morales, M. E. 2013. Effect of unmodified starch on viability of
alginate-encapsulated Lactobacillus fermentum CECT5716. LWT - Food Science and Technology 53(2):
480-486.
Nawong, S., Oonsivilai, R., and Boonkerd, N. 2013. Isolation and selection of probiotic lactic acid bacteria from
cassava pulp for cholesterol lowering property. Proceeding of the 13th ASEAN Food Conference, Singapore.
Nawong, S., Oonsivilai, R., Boonkerd, N., and Truelstrup Hansen, L. 2016. Entrapment in food-grade
transglutaminase cross-linked gelatin–maltodextrin microspheres protects Lactobacillus spp. during exposure
to simulated gastro-intestinal juices. Food Research International 85: 191-199.
Roy, D. 2005. Technological aspects related to the use of bifidobacteria in dairy products. Lait 85(1-2): 39-56.
Zhu, Y., Michelle Luo, T., Jobin, C., and Young, H. A. 2011. Gut microbiota and probiotics in colon tumorigenesis.
Cancer Letters 309(2): 119-127.

435
 IFRC 2017: 045-039 Food Bioprocessing

Effects of different processing methods on hydroxycitric acid content of “batuan” [Garcinia binucao
(Blanco) Choisy] fruits
1*Bainto, L.C., 1Dizon, E.I., 1Israel, K.A.C. and 2Laurena, A.C.
1Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines
Los Baños, College, Laguna, Philippines 4031
2Institute of Plant Breeding, College of Agriculture and Food Science,University of the Philippines Los Baños,College,
Laguna, Philippines 4031

Abstract

Hydroxycitric acid, a compound with anti-obesity property, is commonly extracted from Garcinia cambogia. Recent
studies show that the same compound can be found in batuan (Garcinia binucao), a close relative of Garcinia
cambogia that is indigenous in the Philippines. This study was conducted to determine the effects of different
processing methods on hydroxycitric acid content of batuan. The fruits were steam blanched at different time interval
including 10, 15 and 20 mins. The effect of drying was determined by subjecting the samples at different drying
temperatures such as 50°C, 60°C and 70°C. Whole fruits were submerged in 5% brine solution for almost 3 weeks
to allow fermentation then samples were collected every 4 days. To determine the effect of freezing, whole fruit
samples were stored in freezer at -18°C. Sampling was done every week for 2 months. Hydroxycitric acid was
isolated from fresh and processed fruit rinds using water extraction method. Spectrophotometric analysis was
employed to quantify the isolated acid from the samples. Results revealed that fresh batuan fruits contain 4.81 + 0.12
g hydroxycitric acid/100 g sample. It was also found out that steam blanching can reduce the hydroxycitric acid
content of batuan fruit rinds by 14.55% up to 21.41%. On the other hand, drying can decrease the hydroxycitric acid
content of samples by 7.28% up to 16.22%. Also, brine fermentation for 3 weeks significantly reduced hydroxycitric
content of batuan fruit by 39.08%. Freezing for 2 months decreased the hydroxycitric acid content of samples by
13.30% maximum. Based on the results of the study, it was concluded that freezing and drying at 60°C are the most
effective methods in preserving hydroxycitric acid of batuan.

Keywords: hydroxycitric acid, steam blanching, drying, brine fermentation, freezing

*Corresponding author’s email: lcbainto@up.edu.ph

Introduction

Due to alarming increase in cases of obesity around the world, many studies focused on finding ways on how to
prevent it. One breakthrough is the discovery of hydroxycitric acid. It inhibits citrate cleavage enzyme, a key factor in
fatty acid synthesis, thus, preventing fat accumulation (Watson and Lowenstein, 1970). Batuan (Garcinia binucao), is
a Philippine indigenous, under-utilized crop that is well-known in the Visayas region as a souring agent (dela Cruz,
2012). In Palawan, the Bureau of Agricultural Research, Department of Agriculture (DA-BAR) is studying the export
potential of batuan to give the indigenous groups in the province an alternative source of living. Recent findings
confirm the presence of hydroxycitric acid in batuan. With this findings, the economic significance of this fruit
increases. However, batuan is a seasonal fruit. It is most abundant during summer, from March to June. To make the
fruit available all year round, there is the need for processing batuan into stable products. Changes during
processing though may affect the availability of hydroxycitric acid.

436
Hence, this study was conducted to determine the stability of hydroxycitric acid once the fruit is subjected to different
processing conditions.

Methodology

3.1. Sample collection. Fresh and unripe batuan fruits were obtained from a farm located at Brgy. La Granja, La
Carlota City, Negros Occidental. The fruits used in the study have a characteristic green color, hard covering and
about 3.7-5.5 cm in diameter.

3.2. Processing of batuan fruits. The effect of heating time on hydroxycitric acid was determined. Fresh batuan fruits
were heated using steam at 100°C for 5, 10 and 15 minutes without any pre-treatments. The effect of drying on the
hydroxycitric acid content of batuan was also determined. Fresh samples were dried until the moisture content is
reduced to 10% using different drying temperatures such as 50°C, 60°C and 70°C. To determine the effect of
freezing, fresh batuan were frozen at -18°C and then samples were collected every week for 2 months. Fermentation
in 5% brine solution is another processing method that was studied. Samples were obtained during the fermentation
period every 4 days for 3 weeks for analysis.

3.3. Isolation and quantification of hydroxycitric acid. Hydroxycitric acid was isolated from all samples using water
extraction method based on published protocol by Krishnamoorthy et al. (1982). Fresh, untreated batuan fruits were
used as control. Isolated extracts were then quantified using spectrophotometry with 0.5% sodium metavanadate
based on modified protocol by Antony et al. (1999).

3.4. Statistical analysis. All analyses were done in triplicates. The gathered data were analyzed statistically using
Analysis of Variance (ANOVA) to determine if samples significantly differed from one another, followed by Tukey’s
Honest Significant Difference (HSD) Test to know which among the samples are significantly different. All data were
analyzed using STAR software.

Results and Discussion

Quantification using spectrophotometric analysis revealed that the control has 4.81 + 0.12 g hydroxycitric acid/100 g
batuan pulp. As shown in Figure 1, the effect of different time intervals of steam blanching showed that hydroxycitric
acid content of batuan decreases as heating time increases up to 15 minutes. Negligible change in acid content was
observed when heating continues from 15 minutes up to 20 minutes. The observed decrease was due to incurred
cytoplasmic damage of cells during heating (Lin and Brewer, 2005). As a result of this phenomenon, nutrients
including hydroxycitric acid leached out from the tissues of batuan fruits. Statistical analysis revealed that heated
samples did not differ significantly from the control.

On the effect of different drying temperatures, the pulp from fresh batuan samples were dried up to 10% moisture
content using 50°C, 60°C and 70°C. The result of the experiment showed that drying reduced the amount of
hydroxycitric acid from the batuan fruits because of degradation, volatilization and oxidation (Brenan, 2006). Among
the three drying temperatures, samples dried at 60 °C yielded the highest concentration of hydroxycitric acid followed
by 70 °C and the least was 50 °C as shown in Figure 2. Statistical analysis revealed that there is no significant
difference among the treatments and control.

437
The effect of brine fermentation period was also determined. The result of the experiment showed that there was a
continuous decrease in hydroxycitric acid content of batuan samples until day 20 (Figure 3). Osmotic exchange
between the samples and brine solution is the explanation for this observation (Piga and Aggabio, 2003).
Hydroxycitric acid migrates from the fruit tissues to the brine solution where it is submerged. Oxidation of the acid is
also one possible factor. As such, the obtained sets of data showed that the samples collected at days 12, 16 and 20
were significantly different from the control.

Results of the analyses showed that frozen samples tend to have lower hydroxycitric acid content compared to
control (Figure 4). When water in the cells freezes, an expansion occurs and ice crystals cause the cell walls to
rupture (Desrosier and Desrosier, 1977). Due to cell wall damage consequence of the freezing process, this water
does not return to the cells causing drip loss during thawing. Water that translocate from the cell contain nutrients
and other compounds including hydroxycitric acid. Despite the observed decrease in hydroxycitric acid content, no
significant differences among control and treatment groups were noted.

6 6
Concentration of hydroxycitric acid

Concentration of hydroxycitric acid

5 5

4 4
(g/100 g pulp)

3 (g/100g pulp) 3

2 2

1 1

0 0
0 5 10 15 20 25 Control 50 60 70
Time of heating (min) Drying temperature (°C)

Figure 1. Effect of steam blanching on hydroxycitric acid Figure 2. Effect of different drying temperatures on
content of batuan fruit pulp hydroxycitric acid content of batuan fruit pulp.

6
Concentration of hydroxycitric

Concentration of hydroxycitric acid

5
acid (g/100g pulp)

4.9
4
4.8
(g/100g pulp)

3 4.7
4.6
2 4.5
4.4
1
4.3
0 4.2
0 5 10 15 20 25 4.1
Fermentation period (Day) 4 Freezing time (Week)
0 5 10
Figure 3. Hydroxycitric acid content of batuan fruit pulp as
Figure 4. Hydroxycitric acid content in batuan fruit pulp
brine fermentation progresses.
as freezing progresses

438
Conclusion

Based on the results of the study, several conclusions were made. Steam blanching at 100 °C for 10 minutes, drying
at 60 °C and brine fermentation for 8 days are the processing conditions that must be applied to maintain the
hydroxycitric acid content of batuan fruits. Freezing up to 2 months can be considered as acceptable in terms of
hydroxycitric acid content. Of all the processing methods used in this study, freezing can be considered as the best
method in preserving hydroxycitric acid.

Acknowledgement

The authors would like to thank Department of Agriculture – Bureau of Agricultural Research (DA-BAR) and
Department of Science and Technology – Accelerated Science and Technology Human Resource Development
Program (DOST-ASTHRDP) for financial assistance.

References

Antony, B., Elias, M. and Varghese, W. 1999.Spectrophotometric determination of hydroxycitric acid. Indian Journal
of Pharmaceutical Sciences 5, 316.
Brennan, J. 2006. Food Processing Handbook. Germany: WILEY-VCH Verlag GmbH & Co.
Dela Cruz, R.T. 2012. BAR Chronicle. “Binucao: The underutilized souring agent”. September 2102 Issue. Vol. 13.
No.9. Retrieved on July 10, 2015 from http://www.bar.gov.ph/chronicle-home/archives-list/42-september-
2012-issue/1354-binucao-the-underutilized-souring-agent.
Desrosier, N.W. and Desrosier, J.V. 1977. Principles of Food Freezing. The Technology of Food Freezing, 4th ed.,
AVI Publishing Co. Inc. Wesposrt CT, USA.
Krishnamoorthy, N., Lewis, Y.S and Ravindranath. 1982. Chemical constituents of kokkum fruit rind. Journal of Food
Science and Technology 19: 97-100.
Lin, S. and Brewer MS. 2005. Effects of blanching method on the quality characteristics of frozen peas. Journal of
Food Quality.
Piga, A. and Aggabio, M. 2003. Quality improvement of naturally green table olives by controlling some processing
parameters. Italian Journal of Food Science 2:259-262
Watson, J.A and Lowenstein, J.M.1970. Citrate and the conversion of carbohydrate into fat. Journal of Biological
Chemistry 245: 5993-6002.
 IFRC 2017: 125-085 Food Bioprocessing

Physical properties of heat treated purple potato (Solanum tuberosum cv. Shadow-Queen) flour

1*Santiago, D.M.O., 2Yamauchi H., 2Koaze H.

1Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines
Los Baños, College, Laguna, Philippines 4031
2 Department of Food Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro,
Hokkaido 080-8555, Japan

Abstract

In Japan, Hokkaido accounts for 80% of potato production, of which 77% are used for making starch, chips and other
products, whereas 16% go to fresh markets. The Shadow-queen purple potato, a newly developed variety, is
produced as potential natural source of food and cosmetic colorant, and antioxidants. But, because of color
instability, specific utilizations in food industry are limited and not yet fully explored. To make it suitable for
processing, the anthocyanin pigment which is prone to enzymatic oxidation has to be preserved. Thus, processing
conditions that produce a stable color in flour are important for bread and noodle production. This research compared
the physicochemical properties of purple potato flours to determine the most effective treatment for color
preservation. Shadow-queen purple potato tubers were either boiled or steamed, refrigerated, and analyzed for
starch damage, color, rheological and thermal properties. Results showed that boiling (B) and steaming (S) caused a
higher peak viscosity and breakdown in flours, but with lower pasting temperature and enthalpy (ΔH) of gelatinization
compared with no-heating (NT). This indicates that the heat-treated or pre-gelatinized potato flour required less heat
energy for re-gelatinization and had more viscous paste. In contrast, refrigeration of boiled or steamed tubers
resulted in flour with higher ΔH of gelatinization compared with those heated without refrigeration. This can be
attributed to the retrogradation or re-crystallization of the starch component in flour which may be advantageous for
bread and noodle processing. Moreover, boiling brought a higher setback compared with the steamed and control
indicating good gelling property in flour upon cooling. In conclusion, flours which were boiled and refrigerated (BR5),
and steamed and refrigerated (SR5) may be suitable for bread and noodle processing, thus, adding value to the
colored potato.

Keywords: Shadow-queen purple potato, pre-gelatinization, anthocyanin, enthalpy of gelatinization, viscosity profile
*Corresponding author’s email: dosantiago1@up.edu.ph

Introduction
In Japan, most potatoes are not colored and 80% are produced in Hokkaido, of which 77% is used for processing
starch, chips and other potato products, whereas 16% goes to fresh markets. The newly developed Shadow-queen
potato variety is now widely produced and was found to have antioxidant properties (Noda et al. 2011; Jayawardana
et al. 2012). However, possible uses of Shadow queen potato in the food industry have not yet been fully explored
because of color instability. Preservation of anthocyanin pigment which is prone to enzymatic oxidation resulting in
browning would make it suitable for processing. In this regard, development of appropriate processing conditions that
will make flour with stable color that is suitable for bread and noodle production is necessary.

This research aimed to compare the physicochemical properties of purple potato flours to determine the most
effective treatment that may preserve color and will make them suitable for processing. Results of this research will
provide information on the potential of purple potatoes as functional ingredient in existing food products and in
developing new products as well. This will lead to utilization and value addition of colored potato, and increased
availability in the market.

440
Materials and Method
3.1. Sample Preparation
Shadow queen purple potato tubers acquired from Memuro market in Hokkaido were washed, peeled, diced and then
either boiled (B) or steamed (S) for 10 min and refrigerated for 5 days (BR5 and SR5). All heat treated tubers
including the non-heat treated tuber (NT) which serve as the control were dried at 55oC for 24 hours, ground and
analyzed for their starch damage, amylose and total starch composition, and color, rheological and thermal
properties.

3.2. Total starch, amylose, and damaged starch content were determined using the Megazyme assay kits based on
the methods of McCleary et al. (1997), Yun and Matheson (1990), and Gibson et al. (1991), respectively.

3.3. Color properties specifically the L, a* and b* values of the flours were determined using a colorimeter (CR-400;
Konica Minolta Sensing, Inc., Tokyo, Japan).

3.3. Viscosity profile of flours were determined by heating a 10% dry weight basis (dwb) slurry from 50 to 95℃, held
at 95℃ for 2.5 min, cooled to 50℃, and then kept at 50℃ for 2 min using a Rapid Visco Analyzer (RVA) (RVA-4,
Newport Scientific, Inc.). The peak viscosity, breakdown, setback and pasting temperature were determined
(Limpisut and Jindal, 2002; Yadav et al., 2006).

3.4. Thermal properties were determined using a differential scanning calorimeter (DSC) (Micro DSC II, Setaram,
Inc.) according to the method by Murayama et al., 2014.

3.5 All data were expressed as mean ± standard deviation and were done in triplicate. ANOVA and Tukey’s multiple
range test were performed to compare means at a 5% confidence level using SPSS ver. 17.0 (SPSS Inc., Chicago,
Illinois, USA).

Results and Discussion

As shown in Table 1, boiling and steaming of purple potato tuber resulted in higher damaged starch and amylose
content which can be attributed to the effect of heat treatments. This observation agrees with the report of Yadav et
al. (2006) and Murayama et al. (2014) on the increase in amylose content of potato and cassava flours, respectively,
due to degradation of the crystalline region of starch granules subjected to hydrothermal treatments. Moreover, it
may have resulted in hydrolysis of branched amylopectin into straight chain of glucose and mechanical damage to
the heat-treated starch. On the other hand, total starch of the heat treated purple potato flours were not significantly
different with the control and found within the range of total starch of different potato varieties reported by Bhat
(2015).

Table 1. Total starch, amylose and damaged starch content 1 of purple potato flours
Treatments % Damaged Starch % Amylose % Total starch
NT 3.36±0.14 a 17.29±0.28 a 64.78±1.34 a
B 29.47±0.54 c 21.20±0.15 c 67.05±1.90 a
BR5 29.46±1.25 c 19.96±0.46 b 65.98±1.41 a
S 26.55±1.19 b 22.14±0.32 c 64.82±1.33 a
SR5 25.84±0.53 b 22.77±0.22 c 66.79±1.78 a
Abbreviations: NT, no heat treatment; B, boiled; BR5, boiled and refrigerated for 5 days; S, steamed; SR5, steamed and refigerated for 5 days
1)Each value is the mean ± SD. The values followed by different letters within column are significantly different

Table 2 presents the color properties of purple potato flours. Results showed that the L* and b* values of heat treated
flours were significantly lower than the control NT indicating darker color and higher degree of blueness, respectively
(Hunterlab, 2008). Moreover, the a* value of all heated flours were significantly higher than the control specifying
higher degree of redness. Ultimately, the higher degree of blueness and redness, and darker color of heat treated
flours show that the inherent dark purple color of cv. Shadow queen potato was preserved which can be due to the
denaturation of enzymes that degrades the anthocyanin pigment (Mori et al. 2010).

441
Table 2. Color properties1 of purple potato flours
Color Properties
Treatments
L* a* b*
NT 60.45±0.78 bc 9.75±0.32 a -4.71±0.20 d
B 62.00±0.93 c 10.88±.19 b -12.00±0.19 bc
BR5 59.70±0.28 b 11.51±0.22 c -13.015±0.22 a
S 57.97±0.55 a 11.27±0.18 bc -11.505±0.19 c
SR5 56.61±0.62 a 10.96±0.28 b -12.08±0.28 b
Abbreviations: NT, no heat treatment; B, boiled; BR5, boiled and refrigerated for 5 days; S, steamed; SR5, steamed and refigerated for 5 days
1)Each value is the mean ± SD. The values followed by different letters within column are significantly different

Table 3 shows the rheological and thermal properties of purple potato flours. Results showed that boiling (B) and
steaming (S) of tubers resulted in flours with higher peak viscosity and breakdown but with lower pasting temperature
and enthalpy (ΔH) of gelatinization compared with the non-heat treated flour (NT). This indicates that the heat treated
or pre-gelatinized potato flour requires less heat energy for re-gelatinization and has more viscous paste. On the
other hand, refrigeration of boiled or steamed tuber resulted in flour with higher ΔH of gelatinization compared with
those heated without refrigeration. This result can be attributed to the retrogradation or re-crystallization of the starch
component of the flour which may be advantageous for bread and noodle processing (Rodríguez-Sandoval et al.
2008). Boiling, on the other hand, resulted in higher setback compared with the steamed and the control which
indicates that this flour has good gelling property upon cooling and higher potential for retrogradation (Newport
Scientific, 1998). Based from these results, those flours which were boiled and refrigerated (BR5), and steamed and
refrigerated (SR5) may be suitable for bread and noodle processing.

Table 3. Rheological and Thermal properties 1 of purple potato flours

Peak Viscosity Breakdown Setback Pasting Enthalphy
Treatments
(cP) (cP) (cP) Temperature (oC) (Jg-1)
NT 3522.00±8.54 a 389.67±15.50 a 1720.67±28.01 e 70.18±0.08 c 13.42±0.32 c
B 4853.00±34.70 d 2332.67±15.37 d 944.00±21.70 c 62.20±0.95 a 6.39±0.06 a
BR5 6322.67±28.75 e 3415.33±14.29 e 1107.33±7.02 d 63.58±0.10 a 7.39±0.21 b
S 3820.67±43.88 b 1609.66 b 708.33±16.26 a 64.80±0.44 b 6.62±0.04 a
SR5 4027.00±34.87 c 1884.67± c 803.33±10.41 b 64.50±0.05 b 7.75±0.09 b
Abbreviations: NT, no heat treatment; B, boiled; BR5, boiled and refrigerated for 5 days; S, steamed; SR5, steamed and refigerated for 5 days
1)Each value is the mean ± SD. The values followed by different letters within column are significantly different

Conclusion
This study showed that the boiling and steaming resulted in significant change in rheological and thermal properties
compared with the non-heat treated control. These significant changes can be attributed to the degradation of the
crystalline region of starch granules, and significant increase in damaged starch and apparent amylose content.
Moreover, heat treatments preserved the natural purple color of cv. Shadow queen potato by denaturizing the
enzymes that degrade its inherent anthocyanin pigment. On the other hand, refrigeration of heat-treated tubers
resulted in significant improvement in rheological and thermal properties of purple potato flours compared with the
unrefrigerated heat-treated flours which indicate its suitability in processing of food products like bread and noodles.

References
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Sweden: Swedish University of Agricultural Sciences, Master Thesis.
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damage in wheat flour. Journal of Cereal Science 15, 15-27.
HunterLab. 2008. Hunter L, a, b, color scale. Applications note, 8, 1-4.
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442
 anthocyanin-rich colored potato flakes on lipid oxidation, instrumental color evaluation and sensory
characteristics in cooked pork sausages. Food Science Technology Research 18 (3): 455-460.
Limpisut, P. and Jindal, V. K. 2002. Comparison of rice flour pasting properties using Brabender Viscoamylograph
and Rapid Visco Analyser for evaluating cooked rice texture. Starch-Stärke 54, 350-357.
McCleary, B. V., Gibson, T. S., and Mugford, D. C. 1997. Measurement of total starch in cereal products by
amyloglucosidase-α-amylase method: Collaborative study. Journal of Association of Official Analytical
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Starch properties of various colored potato cultivars frown for 8 consecutive years. Journal of Food,
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 IFRC 2017: 104-093 Food Bioprocessing

Comparative study on the phytochemical and antioxidant properties of fermented jackfruit leaves
(Artocarpus heterophyllus L.) leaves using single and mixed starter cultures

Norhazniza, A.,1 Koh, S.P.,1*, Rosmawati A.,1Nur Syazwani, A.H.,1 and Razali, M.2
1Biotechnology and Nanotechnology Research Center, MARDI, P.O. Box 12301, 50772 Kuala Lumpur
2Horticulture Research Center, MARDI, P.O. Box 12301, 50772 Kuala Lumpur
Abstract
The objective of the present study was to evaluate the phytochemical and antioxidant properties of fermented
jackfruit leaves, using yeast and acetic acid bacteria, individually or in combination during 6 days of fermentation.
Changes in pH, total reducing sugar, ethanol, acetic acid, total phenolics and flavanoids content were examined. A
number of antioxidant activities such as ferric reducing antioxidant power (FRAP) and DPPH free radical scavenging
activities were also analyzed to determine the differences in the respective properties during the fermentation.
Results obtained demonstrated that there were differences in the phytochemical and antioxidant properties of
fermented jackfruit leaves, depending on the fermentation starters. Fermented leaves using acetic acid bacteria
alone, exhibited higher total reducing sugar (6.89 mg glucose/ml) compared to others. The ferric-reducing activities of
fermented jackfruit leaves showed an increasing trend in the yeast fermentation process (5.96-10.12 mg ascorbic
acid equivalent/mL). Generally, all extracts of fermented jackfruit leaves exhibited stronger DPPH radical scavenging
activity except in the total phenolics and flavonoids content, whereby it showed a decrease trend throughout the
fermentation process.

Keywords: jackfruit leaves, acetic acid bacteria, yeast, fermentation, antioxidant

*Corresponding author’s email: karenkoh@mardi.gov.my

Introduction
Jackfruit (Artocarpus heterophyllus L.) tree is widely grown in India, Bangladesh and several parts in Southeast Asia,
mainly in Thailand, Vietnam and Malaysia. Jackfruit leaves are abundant and commonly used by farmers as an
animal feed. In some native area in India, the leaves were used as traditional medicine to cure fever, healing wounds
and ulcers (Gupta and Tandon, 2004). In general, the antioxidant activity in plants is due to the presence of phenolic
and flavonoid compounds, which offer various health benefits for human being. Fermentation has been an effective
and valuable method to preserve food and beverages for longer shelf life, improved flavour and retaining the
nutritional properties. Moreover, fermentation also can enhance the antioxidant properties due to the hydrolysis of
plants cell wall by microorganisms, which lead to the secretion of assorted antioxidant compounds (Hur, 2014).This
current work was conducted to explore the potential of jackfruit leaves as a fermentation substrate. In this
fermentation process, the jackfruit leaves were subjected to pure yeast, acetic acid bacteria and in combination of
these two cultures for 6 days and the phytochemical and antioxidant properties of the fermented leaves were
determined.

Materials and methods

3.1 Jackfruit leaves preparation and fermentation process
Leaves of Artocarpus heterophyllus L. were collected from a local jackfruit plantation in Lanchang, Pahang. The
leaves were thoroughly washed before air dried (45oC) and ground to produce powder. The dried powder were
packed in the sealed container and stored in a chiller (5oC) until further use.
Two types of microorganisms selected from the Collection of Functional Food Cultures (CFFC), MARDI were yeast
(Dekkera bruxellensis) and acetic acid bacteria (Gluronacetobacter sp.). The jackfruit leaf powder (10g) was
inoculated individually with yeast and acetic acid bacteria at a concentration of 5% (weight of powder/volume of
starter culture) with the colony count of 1 x 108 cfu/mL for a duration of 6 days at 30°C. The mixed cultures
fermentation was conducted by mixing the acetic acid bacteria and yeast mother stock with colony count 1 x 108
cfu/mL at the ratio 1:4 for the same period of fermentation time. Non fermented jackfruit leaves powder suspension

444
was prepared as a control. During fermentation, sampling was performed at day 1,2,3,4 and 6. The supernatant was
collected after centrifuged the jackfruit leaves suspension at 10,000rpm for 10 min and subjected to pH, Brix, total
reducing sugar content and antioxidant properties analysis.
3.2 pH , total soluble solids (Brix) and total reducing sugar
The pH measurement were made using a pH meter (Mettler Toledo, Model: Seven Easy GMBH 8603, Switzerland)
and the total soluble solids of all samples were measured using a refractometer (Atago, PAL-3, Japan) whereby the
results were expressed in Brix value. Total reducing sugars were estimated using the dinitrosalicylic acid (DNS)
method developed by Miller (1959). Pure glucose was used as sugar standard.
3.3 Total phenolics content (TPC) and total flavonoids content (TFC)
Total phenolics content (TPC) were determined using the Folin-Ciocalteu method according to Singleton and Rossi
(1965). Gallic acid was used as standard and results were expressed in mg gallic acid equivalent (mg GAE/mL).
Total flavonoids content (TFC) was determined using aluminium chloride method adopted from Jia et al. (1999). The
quercetin was used as a standard.
3.4 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method and ferric reducing antioxidant
power (FRAP) assay
DPPH radical scavenging activity was carried out based on the modified method proposed by Thaipong et al. (2006).
FRAP assay was performed according to Benzie and Strain (1996). For both assay, ascorbic acid was used as the
standard and results were expressed as mg ascorbic acid equivalents per mL of sample (mg AAE/ mL).
Results and discussion
Different starter culture inoculated in the jackfruit leaves powder exhibited different pH behaviour changes during
fermentation. Generally, the mixed cultures fermentation showed the drastic reduction of pH value when compared to
pure yeast and acetic acid fermentation after 6 days fermentation as summarized in Table 1. The lowest pH value
(2.95) was recorded in the mixed cultures fermented jackfruit leaves whereas the pH value for yeast and acetic acid
bacteria fermented samples were 5.38 and 3.33, respectively. The declination of pH value in all fermentation
processes could be due to the rapid growth of yeast and acetic acid bacteria that utilized sugar as carbon source and
convert it into other metabolites that contributing to the acidity of fermented samples. The combination of these two
cultures fermentation may provide the synergic effect, whereby the yeast will produce the ethanol for enhancing
acetic acid production by acetic acid bacteria. Total soluble solid was measured using refractometer to estimate the
content of sugar, soluble organic acids, soluble phenolic acid and other soluble solid compound. Generally, the total
soluble solid content in both single culture fermentation processes were decreased as the fermentation time
increased.At the end of the fermentation process, the total soluble solid value was found to be lowest (9.28 oBrix) in
the yeast fermentation process compared to others.

Table 1 Changes of pH, total soluble solids and total reducing sugar in fermented jackfruit leaves during 6 days
fermentation.
Yeast fermentation Acetic acid bacteria fermentation Mixed culture fermentation
Total Total Total
Fermentation Total reducing Total reducing Total reducing
time soluble sugar soluble sugar soluble sugar
(day) pH pH pH
solid (mg solid (mg solid (mg
(oBrix) glucose/m (oBrix) glucose/m (oBrix) glucose/
L) L) mL)
5.70 ± 10.47 ± 10.47 ±
Control 10.5 ± 0.10 4.98 ± 0.02 5.76 ± 0.02 4.98 ± 0.02 5.76 ± 0.02 4.82 ± 0.01
0.00 0.06 0.06
4.35 ± 10.47 ± 19.71 ± 13.40 ±
1 9.77 ± 0.05 2.92 ± 0.01 3.76 ± 0.01 4.02 ± 0.00 1.59 ± 0.01
0.39 0.06 0.66 0.06
4.11 ± 10.30 ± 11.70 ±
2 9.65 ± 0.05 2.19 ± 0.06 3.37 ± 0.01 8.50 ± 0.91 3.33 ± 0.00 1.73 ± 0.07
0.00 0.00 0.06
4.36 ± 10.40 ± 11.20 ±
3 9.53 ± 0.12 1.50 ± 0.01 3.35 ± 0.00 8.03 ± 0.18 3.10 ± 0.00 1.75 ± 0.09
0.07 0.00 0.00
5.33 ± 10.17 ± 11.30 ±
4 9.53 ± 0.05 1.57 ± 0.01 3.33 ± 0.00 7.43 ± 0.61 2.95 ± 0.01 1.81 ± 0.08
0.15 0.06 0.00
5.38 ± 10.17 ± 10.90
6 9.28 ± 0.04 1.57 ± 0.05 3.33 ± 0.00 6.88 ± 0.07 2.95 ± 0.01 1.77 ± 0.04
0.04 0.06 ±0.00

445
Total reducing sugar in yeast and mixed cultures fermentation also showed a decreased trend at the end of the
fermentation. However, acetic acid bacteria fermentation exhibited an inverted trend, whereby it produced higher
amount of reducing sugar after 6 days of fermentation. A drastic reduction of reducing sugar (from 4.98 to 1.57 mg
glucose/ mL,) was shown in the yeast fermentation process, most probably due the sugar uptake by yeast for
production of other metabolites and active compounds. Galafassi et al. (2011) has reported that the yeast Dekkera
sp. can utilize various sugar such as glucose, fructose and arabinose during fermentation. In contrast, the increment
of total reducing sugar in acetic acid fermentation sample may due to the breakdown of complex sugar present in the
jackfruit leaves into simple sugars like glucose and fructose via bacterial hydrolysis action. For further confirmation, it
is important to conduct the sugar composition analysis for both fermented and non fermented jackfruit leaves product
in the next study.

A Y A MC B Y A MC
5.0
2.0

Total flavonoid content

Total phenolics content

4.0

(mg quercetin/mL)
1.5
(mg GAE/mL)

3.0
1.0
2.0
0.5 1.0
0.0 0.0
Control D1 D2 D3 D4 D6 Control D1 D2 D3 D4 D6
Fermentation day Fermentation day

Figure 1 Total phenolics content, TPC (A) and total flavonoids content, TFC (B) of jackfruit leaves during 6 days of
fermentation.Values are the mean of triplicates ± SD. Y- yeast, Dekkera Bruxellenxis , A- acetic acid bacteria,
Gluronacetobacter and MC- mixed culture
Total phenolics and flavonoids in all fermented samples throughout 6 days of fermentation were summarized in
Figure 1. As illustrated in Figure 1A and 1B, all fermented jackfruit leaves exhibited a decrease trend of TPC and
TFC. Particularly, the mixed cultures fermented jackfruit leaves showed a drastic reduction of total phenolics content
(from 1.70 to 0.78 mg GAE/mL) as compared to other samples. Based on the findings, we hypothesized that the
presence of microorganisms (yeast and acetic acid bacteria) utilized polyphenol compounds in jackfruit leaves for
their growth to produce other metabolites that contributing to the improvement of taste, aroma and other functional
bioactive metabolites during fermentation.
DPPH method is based on free radical scavenging activity of specific compound or extracts, indicates the ability of
antioxidant to suppress lipid oxidation (Jayanta, 2016). As shown in Figure 2A, the scavenging effect for all
fermented leaves samples were increased compared to non fermented (control) sample. Mixed culture fermented
leaves gave the highest DPPH free radical scavenging activity (1.16 mg AAE/mL) after 2 days of fermentation and
then remained the same antioxidant activity even subjected to 6 days of fermentation periods. In contrast with DPPH,
the FRAP assay (Figure 2B) exhibited a different trend between all the samples. The FRAP value was found to be
the lowest for mixed cultures samples, whereby it showed a drastic drop after 6 days of fermentation (5.96 mg
AAE/mL) when compared to control sample (9.05 mg AAE/mL). However, the FRAP value for yeast fermented
leaves showed an increasing trend during the fermentation period and exhibited approximately 10.12mg AAE/mL
sample after 6 days of fermentation.

446
 Y A MC Y A MC
A B
DPPH (mg AAE/mL)

1.60 12.0

FRAP (mg AAE/mL)

1.20 9.0
0.80 6.0
0.40 3.0
0.00 0.0
Control D1 D2 D3 D4 D6 Control D1 D2 D3 D4 D6
Fermentation day Fermentation day

Figure 2 Radical scavenging activity of fermented jackfruit leaves by (A) DPPH, (radical scavenging activity, %RSA)
and (B) Ferric-reducing antioxidant power (FRAP) during 6 days of fermentation. Values are the mean of triplicates
± SD. Y- yeast, Dekkera Bruxellenxis and A- acetic acid bacteria, Gluronacetobacter and MC- mixed culture

Conclusion
In general, single and mixed cultures fermentation on jackfruit leaves have exhibited different behaviour changes in
terms of pH value, total soluble solids, total reducing sugar and antioxidant properties. The differences in antioxidant
activities were highly related to the specific metabolites produced by different microorganisms which contribute to
different antioxidant mechanism as observed in this study. Further investigation will be conducted to identify the types
of metabolites produced in single and mixed cultures fermentation of jackfruit leaves.
References
Benzie, I.F.F. and Strain, J.J. 1996. The ferric reducing ability of plasma (FRAP) as a measure of
“antioxidant power”: the FRAP assay. Analytical Biochem. 239:70 -76
Galafassi, S., Merico, A. and Pizza, F. 2011. Dekkera/Brettanomyces yeast for ethanol production from
renewable sources under oxygen-limited and low pH conditions. J Ind Microbiol Biotechnol.
38(8):1079-1088.
Gupta, A.K. and Tandon,N. 2004. Review on Indian Medicinal Plants. New Delhi, India : Indian Council of
Medical Research.
Hur, S.J., Lee, S.Y., Kim, Y-C., Choi, I and Kim, G-B. 2014. Effect of fermentation on the antioxidant activity in plant-
based foods. Food Chemistry, 160: 246-356.
Jayanta, K.P., Sameer, K.S., and Manas, R.S. 2016. Biochemical composition and antioxidant potential of fermented
tropical fruit juices. Agro food Ind Hi-tech. 27(4): 29-33
Jia, Z.S., Tang, M.S. and Wu, J.M. 1999. The determination of flavanoid contents in mulberry and their
scavenging effects on superoxide radicals. Food Chemistry. 64: 555 -559.
Miller, G.A.I.L. 1959. Use of dinitrosalicylic acid for detection of reducing sugars. Analytical Chemistry.
31:3.
Singleton, V.L. and Rossi, J.A. 1965. Colorimetry of total phenolics with phosphomolybdic -phosphotungstic
acid reagents . Am J Enol Vitic. 16:144-158
Thaipong,K., Boonprakob, U., Crosby,K., Cisneros-Zevallos, L. and Byrne,D.H. 2006. Comparison of
ABTS, DPPH, FRAP and ORAC assays for estimating antioxidant activity from guava fruit extracts.
J Food Comp Anal. 19: 669-675

447
 IFRC 2017:107-167 Food Bioprocessing

Changes in Phenolic Content and Antioxidant Activity of Rice Bran by Aspergillus oryzae as Influenced by
Different Initial Moisture Content

*Jamaluddin, A., Abd. Rashid, N., Abd. Razak, D.L., Abd. Ghani, A., Mansor, A., Abdul Manan, M., Md. Saad, A.Z.,
Sani, N. and Jonit, M.J.
Enzyme and Fermentation Technology Programme, Biotechnology and Nanotechnology Research Centre, MARDI
Headquarters, Persiaran MARDI-UPM, 43400 Serdang Selangor, Malaysia
Abstract

The aim of the present work was to evaluate the effect of initial moisture content (IMC) on the growth, phenolic
content and antioxidant activity in rice bran (RB) by Aspergillus oryzae. In order to study the IMC influence, RB
containing 27%,47%, 52% and 54% of IMC were tested in the experiment. The results showed that the fungal
growth as indicated by glucosamine content and total phenolic content (TPC) were increased with the increase of
IMC. The antioxidant potential evaluated via FRAP also exhibited a parallel trend with values ranging from 57.48 to
89.75 mM Fe(II)/g dry mass. Different trend was observed in DPPH analysis as the activity decrease with the
increase of IMC. Changes in phenolic acid content was also observed in this study.

Keywords: Solid-state fermentation, rice bran, moisture content, Aspergillus oryzae, phenolic
*Corresponding author’s email: anisahzubir@yahoo.com

Introduction

Rice bran (RB) is one of main the main by-products of rice processing industry. Traditionally, most RB production
was used in production of animal feed, fertilizers and the cosmetic industry. Several studies have been carried out to
assess its potential for human consumption.

Fermentation process such as solid-state fermentation (SSF) has been used for many decades in production of
various fermented products and it is a simple technique for the production of bioactive compounds. A number of
studies have been done in using the SSF to increase the synthesis of phenolic compounds in agro-industrial residues
(Martins et. al. 2011). In term of type of microbial used in the SSF, among the major microorganisms known for their
ability to produce enzymes that degrade the cell wall of plants, fungi comprise the most interesting group. Therefore,
Aspergillus oryzae has been the candidate to be used in this study.

Among important aspects to enhance the efficiency of SSF are the selection and optimization of process
variables including initial moisture content (IMC). Moisture content has a significant role in SSF as bacteria and
fungi have different moisture content requirements. Fungi need lower moisture content around 40%–60%
(Singhanis et al. 2009) whereas bacteria can require high moisture content (60% –85%) (Martins et al. 2011). The
optimum moisture content in a solid substrate is closely related to the correct nutrient and oxygen/carbon di oxide
diffusion during fermentation (Orzua et al. 2009).

Therefore, the aim in this study was to assess the effect of different IMC used in the RB on the growth of A.
oryzae as well as on total phenolic content and antioxidant activity in RB.

Materials and Methods

Microorganisms and fermentation procedure

The culture of Aspergillus oryzae was maintained on potato dextrose agar (PDA). For the SSF, 30g of rice bran (RB)
was weighed in 100ml Erlenmeyer flasks. The substrates were then autoclaved at 121oC for 15 min. For the

448
inoculation procedure, 1% of fungal spores were used for the fermentation and appropriate amount of sterile distilled
water was added into substrate to gain IMC of 27%, 47%, 52% and 54%. The samples were mixed well and
incubated at 32oC. Samples were incubated for 12 days. Samples were then dried at 50oC for 24 hours, ground and
kept at -20oC for further analysis.

Preparation of water extracts

One gram of unfermented and fermented RB was mixed with 5ml of distilled water and shake for 2 hours. After
centrifuge at 10 000 rpm for 15 min, each supernatant was filtered through Whatman No. 1 filter paper. The filtrates
were then kept at -20oC for further analysis.

Total phenolic content

The Folin-Ciocalteu methodology was according to the method of Thaipong et al. (2006). Briefly, 1ml aliquot of the
samples was allowed to react with 5ml of diluted Folin-Ciocalteu reagent and 4ml of the 7.5% sodium carbonate
solution for 2 hours at room temperature. Absorbance was measured at 765nm using spectrophotometer and the
results were expressed as ug gallic acid equivalent (GAE)/ gram sample.

Radical scavenging activity (DPPH)

The determination of antioxidant activity through DPPH scavenging system was carried out according to the method
of Thaipong et al. (2006) with slight modification. The extract (150ul) was allowed to react with 2850ul of the DPPH
solution for 30 minutes in the dark. Percentage of DPPH scavenging activity was determined as follow:DPPH
scavenging activity (%) = [(Ablank-Asample)/Ablank] x 100% whereby A represents the absorbance.

Ferric reducing antioxidant power (FRAP)

The FRAP assay was done according to Benzie and Strain (1996) with some modifications. The fresh working
solution was prepared by mixing 25mL of 300mM acetate buffer (pH3.6), 2.5mL of 10mM TPTZ solution, and 2.5mL
of 20mM FeCl3.6H2O solution. The sample extract (150ul) was allowed to react with 2850 ul of the FRAP solution for
30 min in the dark condition. Readings of the coloured product (ferrous tripyridyltriazine complex) were then taken at
593 nm. Results were expressed in mg ascorbic acid equivalent (AAE)/g sample.

Phenolic acids content analysis

Free phenolic acids in samples were determined using HPLC Alliance Separation Module (Waters 2695), equipped
with a diode array detector (Waters, 2996) according to Robbins and Bean (2004) with some modifications. Peak
identification was made by comparing retention times and UV spectra at 280nm and 325nm with authentic
compounds. Quantification was made using calibration curves obtained by injecting known amounts of pure
compounds as external standards.

Statistical analysis
All data were expressed as mean ± standard deviation. Analysis of variance was performed by ANOVA procedures
and P≤0.05 was considered to be statistically significant.

Results and Discussion

Biomass is known as a crucial parameter in the characterisation of microbial growth and estimation of fungal biomass
in RB during 12 day-incubation with A. oryzae indicated by glucosamine content is depicted in Figure 1. The level of
glucosamine was found higher in substrate with higher IMC. Glucosamine is an essential and stable component in
chitin of mycelial cell wall (Schmidt and Furlong, 2012) and this result indicates A. oryzae favours a higher IMC to
support its growth in RB in this studied moisture content. The SSF with A. oryzae successfully enhanced the phenolic
content in RB in comparison with its unfermented counterpart (Figure 2). The same trend was observed in TPC
analysis where the TPC of A. oryzae-fermented RB improved as the value of IMC increased which might be
attributed to higher biomass observed in the higher IMC (Figure 2).

449
 7
14
6

TPC (mg GAE/g sample)

12
(mg glucosamine/g sample)
Glucosamine content

10 5
8 4
6 3
4 2
2
1
0
Control 27% 47% 52% 54% 0
UF 27% 47% 52% 54%
Initial moisture content (%) Initial moisture content (%)

Figure 1. Glucosamine content of A. oryzae-fermented Figure 2. Total phenolic content of A. oryzae-fermented

RB as influenced by different initial moisture RB as influenced by different initial moisture
content. content.

Fungi are known to have two types of extracellular enzymatic systems comprise of (i) hydrolytic system which
produces hydrolases that responsible for polysaccharide degradation and (ii) oxidative and extracelluar ligninolytic
system that degrades lignin and opens phenyl rings, increasing the free phenolics content (Martins et al., 2011;
Sánchez, 2009). Thus, the augmentation of phenolic content in the A. oryzae-fermented RB could be attributed to
these extracellular enzymatic systems.

It has been suggested that the phenolic content of plant materials is correlated with their antioxidant activities
(Velioglu et al. 1998). Due to the complex nature of phenolic phytochemicals, it is beneficial to evaluate the
antioxidant potential of the studied extracts using two different methods. Hence, in this present study, antioxidant
activities of the studied extracts were measured by DPPH radical scavenging activity and ferric reducing antioxidant
power (FRAP) as illustrated in Table 1. FRAP analysis shows the ability of A. oryzae in enhancing antioxidant
potential in RB as the activity was enhanced in comparison with the unfermented RB. The result exhibited that the
antioxidant potential was in order of 52%>54%>47%>27%. The activity was in line with the TPC as the difference
between 54% and 52% was not statistically significant (P≤0.05). A contradictory result was observed in DPPH
analysis where the percentage was decrease as the IMC increased. The discrepancy in the antioxidant activities
indicates that both DPPH and FRAP assay determine different aspects of antioxidant capacity. The difference might
be attributed to different mechanism involved in the radical- antioxidant reactions compared to the FRAP assay
mechanism (Lizcano et al. 2010).

Table 1. Antioxidant potential and radical scavenging activity of A. oryzae-fermented RB as influenced by different
initial moisture content.
Initial moisture
FRAP (mM Fe(II)/g sample DPPH (%)
content (%)
UF 49.27 ± 2.12 93.62 ± 0.01
27% 57.48 ± 6.86 91.76 ± 0.29
47% 65.74 ± 7.28 90.38 ± 0.41
52% 89.75 ± 7.49 89.05 ± 0.45
54% 85.00 ± 5.94 88.31 ± 1.50

Chromatography analysis on free phenolic acids content in fermented RB was also carried out using HPLC analysis.
Results in Table 2 indicate that four types of phenolic acids were found in the fermented RB namely caffeic, p-
coumaric, ferulic and sinapic acid. Overall, it shows the phenolic acids content in each sample were enhanced by the

450
fungal fermentation. Nevertheless, only one type of phenolic acid was found in the IMC of 47% and 52%. The
enhanced TPC observed earlier might also be contributed by other groups of phenolic compounds. Other than that,
the non-phenolic compounds also might contribute the antioxidant activity detected in these studied samples.

Table 2. Phenolic acids content in A. oryzae-fermented RB as influenced by different initial moisture content.
Initial moisture Ferulic acid Sinapic acid
Caffeic acid p-coumaric acid
content (%)
UF nd 3.99 ± 0.18 0.62 ± 0.03 nd
27% 0.880 ± 0.53 3.14 ± 0.04 6.12 ± 0.097 3.82 ± 0.183
47% nd nd 4.14 ± 0.185 nd
52% nd nd 2.14 ± 0.419 nd
54% 1.35 ± 0.80 4.41 ± 1.00 1.78 ± 2.30 2.36 ± 0.90

Conclusion
Based on the results of biomass, TPC and antioxidant analyses, the IMC of 54% is the most appropriate for the SSF
of RB with A. oryzae.

Acknowledgements

This study was supported by a Developmental Fund research grant (No. P2100 300125 0001) from the Malaysian
Agricultural Research & Development Institute (MARDI).

References

Benzie, I. F. and Strain, J. J. 1996. The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power":
the FRAP assay. Analytical biochemistry 239(1): 70-76.
Lizcano, L.J., Bakkali, F., Ruiz-Larrea, M.B. and Ruiz-Sanz, J.I. 2010. Antioxidant Activity and Polyphenol Content of
Aqueous Extracts from Colombian Amazonian Plants with Medicinal Use. Food Chemistry. 119: 1566–1570.
Martins, S.; Mussatto, S.I.; Martínez-Avila, G.; Montañez-Saenz, J.; Aguilar, C.N.; Teixeira, J.A. 2011. Bioactive
phenolic compounds: Production and extraction by solid-state fermentation. A review. Biotechnol.
Adv. 29: 365–373.
Orzua, M.C.; Mussatto, S.I.; Contreras-Esquivel, J.C.; Rodriguez, R.; de la Garza, H.; Teixeira, J.A.; Aguilar,
C.N. 2009. Exploitation of agro industrial wastes as immobilization carrier for solid-state fermentation. Ind.
Crops Prod. 30: 24–27.
Singhania, R.R.; Patel, A.K.; Soccol, C.R.; Pandey, A.2009. Recent advances in solid -state
fermentation. Biochem. Eng. J., 44: 13–18.
Thaipong, K., Boonprakob, U., Crosby, K., Cisneros-Zevallos, L., Byrne, D.H., 2006. Comparison of ABTS, DPPH,
FRAP and ORAC assaya for estimating antioxidant activity from guava fruit extracts. J. Food. Comp. Anal.
19:669-675.

451
 IFRC 2017: 221-206 Food Bioprocessing

Selection of Acetobacter species isolated from fermented cocoa beans in Dong Nai Province for their
potential use as starter cultures

*Vu T.L.A., Nguyen M.H., Phan T.H.

Faculty of Food Science and Technology, Nong Lam University HCMC, Linh Trung, Thu Duc District, Ho Chi Minh
City,Vietnam
Abstract

The main objective of the recent study was to select Acetobacter strains capable of affecting the cocoa fermentation
in Dong Nai Province, Vietnam based on their tolerance to alcohol, acid, and temperature and their oxidation capacity
as well. Acetobacter pasteurianus (A. pasteurianus) NH6 was selected with its high oxidation capacity, tolerance to
alcohol at 10% and strong acid production at 35 oC. Cocoa bean fermentation with and without the inoculation of the
isolated and selected A. pasteurianus NH6 were compared based on the cocoa pulp pH and fermentation index
changes in the fermentation process. The fermentation with the A. pasteurianus NH6 addition was seen to dominate
the spontaneous fermentation. The isolated and selected A. pasteurianus NH6 is capable of being applied as starter
cultures in cocoa bean fermentation.

Key words: Acetobacter spp., Acetobacter pasteurianus, starter culture, cocoa bean fermentation
*Corresponding author’s email: laman.vu@gmail.com

Introduction
Acetic acid production produced by acetic bacteria is essential in the cocoa bean fermentation because the acid
diffuses into the nibs, resulting in the nib death, decrease in pH, and the endogenous biochemical reactions that give
chocolate flavor precursors (Thompson et al., 2013).
The recent study aimed to select Acetobacter strains capable of affecting the cocoa fermentation in Dong Nai
Province, one of the large cocoa growing and fermenting areas in southern Vietnam, based on their tolerance to
alcohol and temperature and their acidification and oxidation capacity as well. The selected strains will be applied in
cocoa bean fermentation as starter cultures in large scale cocoa bean production.

Materials and Methods

Materials
A. pasteurianus strains, which were previously isolated from spontaneously fermented cocoa beans collected during
fermentation in Nguyen Loc Company, Dong Nai Province and identified, were used. Nine A. pasteurianus strains
were inoculated in acetic acid medium broth (1% D-glucose, 0.5% ethanol, 0.3% acetic acid, 1.5% peptone, 0.8%
yeast extract) and incubated at 30 oC for 20-24 h. Cocoa pods were supplied by Nguyen Loc Company.

Selection of the A. pasteurianus strains

The selection of the A. pasteurianus strains was based on the analysis of acidification capacity, the tolerance to
temperature (30, 35, 40, and 45 oC) and alcohol (ethanol of 5, 10, 15, and 20% (v/v)), carbon metabolism
(catabolizing glucose, fructose, sucrose, and citrate) and the oxidation capacity as described by Aydin et al. (2009);
Yao et al. (2014). Pure culture of each strain was inoculated on an agar dish and all the assays were carried out in
triplicate. Negative controls were carried out in the same conditions, but with no bacteria inoculated. The tested
strains were selected to be starter culture if they could produce acid at 35-40 oC, grow in medium with up to 15%
alcohol, catabolize carbohydrates and oxidize acetic acid Yao et al. (2014).

452
Comparison of the cocoa bean fermentation with and without the inoculation of A. pasteurianus strains
Cocoa pulps were removed from the pods under sterile condition and put in sterile Erlenmeyer flasks, 100 g/flask.
The fermentation was carried out both with and without inoculation (control) of acetic bacteria and in triplicate. The
bacteria of the selected A. pasteurianus strain(s) suspended in NaCl 0.9% were added to the flasks containing cocoa
pulps to reach the inoculation level of approximately 105 CFUs/g cocoa. The fermentation temperature was controlled
by putting the flasks in an incubator (Memmert, Germany) during the 5-day fermentation as described by Stoll (2010).
After the fermentation, cocoa beans were sun dried to reach the moisture content of 6-8%. Pulp pH and fermentation
index (FI) were determined every 24 h during the fermentation time based on the descriptions reported by Meyer et
al. (1989) and by Gourie and Tserrevitinov (1979), respectively. The cocoa fermentation is considered to be complete
when the FI is more than 1 (Gourie and Tserrevitinov, 1979).

Statistical analyses
Analyses of the variance were performed with Statgraphic Centurion XV 15.1.02. Values were considered
significantly different when the p value was less than 0.05 (p<0.05).

Results and discussion

Selection of the A. pasteurianus strains

The all nine strains tested showed the ability to grow and produce acid at temperatures of 30 and 35 oC with different
levels (Table 1). The strongest acidification was observed in the strain ABB2. The acidification capacity of the tested
strains was reduced when the temperature was increased. In cocoa bean fermentation the acidification influences the
fermented bean quality and consequently the chocolate quality (Schwan and Wheals, 2004).

Table 1: Growth and acidification capacity of the tested strains under different temperatures
Trains Temperature (oC)
30 35 40 45
AAB01 1/2 Petri surface* 1/4 Petri surface -** -
AAB02 1/2 Petri surface 1/4 Petri surface - -
AAB04 1/2 Petri surface 1/4 Petri surface - -
AAB05 Whole Petri surface 1/4 Petri surface - -
AAB06 Whole Petri surface 1/4 Petri surface - -
ABB1 1/2 Petri surface 1/4 Petri surface - -
ABB2 Whole Petri surface Whole Petri surface - -
ABB3 1/2 Petri surface 1/4 Petri surface - -
ABB4 1/2 Petri surface 1/4 Petri surface - -
*, Bacterial growth and yellow zone on Petri dish; ** absence of growth and yellow zone

453
The all tested strains displayed the alcohol tolerance at the ethanol concentration of 5% with different levels (Table
2). Higher ethanol concentration reduced the growth of the tested strains. Only strain AAB2 showed to be tolerate to
ethanol concentration of 15%.
Table 2: Ethanol resistance of the tested strains
Strains Ethanol concentration (%)
5 10 15 20
AAB01 1/2 Petri surface* -** - -
AAB02 1/2 Petri surface - - -
AAB04 1/2 Petri surface - - -
AAB05 1/2 Petri surface - - -
AAB06 1/3 Petri surface Some yellow pots - -
ABB1 1/2 Petri surface - - -
ABB2 >1/2 Petri surface 1/3 Petri surface Some yellow areas -
ABB3 1/2 Petri surface - - -
ABB4 1/2 Petri surface - - -
*, Bacterial growth and yellow zone on Petri dish; ** absence of growth and yellow zone
The all nine strains also possessed the ability of oxidation and utilization of glucose, fructose, sucrose, and citrate.
The strain ABB2 was chosen to be a potential starter culture because of its strong resistance to temperature and
alcohol stresses, and acidification capacity.

Comparison of the cocoa bean fermentation with and without the inoculation of A. pasteurianus strains
The pulp pH values of cocoa pulps in the fermentation with and without inoculation of the A. pasteurianus NH6 (strain
ABB2) varied during the fermentation (Figure 1). Differences in pH values of the pulp with the bacterial inoculation
and those of the pulp fermented spontaneously were observed after 36 h. The microbial activity and metabolites
produced during the cocoa fermentation lead to the increase of temperature and pH value (Moreira et al., 2017).

Figure 1. Changes of pH value of cocoa pulp during fermentation

After 36 h significant differences in FI of cocoa pulps were observed between the fermentation with and without
inoculation of the strain ABB2 (Figure 2). The addition of the strain ABB2 shortened the fermentation time, indicating
that the selected strain could be used as a starter culture in cocoa fermentation. The fermentation process with the
shortened fermentation time is considered a significant improvement.

Figure 2. Changes of fermentation index of cocoa pulp during fermentation

454
Conclusion

The A. pasteurianus strains isolated from fermented cocoa beans indicated the acidification capacity and the
tolerance to temperature and ethanol at different levels. The inoculation of the selected A. pasteurianus NH6
accelerated the fermentation process. The strain could be used as a starter culture for further studies on
improvement of the local cocoa production.

Acknowledgments
This research was financed by Dong Nai Department of Science and Technology.

References
Aydin, Y.A. and Aksoy, N.D. 2009. Isolation of cellulose producing bacteria from wastes of vinegar fermentation.
Proceedings of the World Congress on Engineering and Computer Science 2009 Vol I, WCECS 2009,
October 20-22, 2009, San Francisco, USA.
Gourieva, K.B. and Tserrevitinov, O.B. 1979. Method of evaluating the degree of fermentation of cocoa beans. USSR
Patent no. 646254.
Meyer, B., Biehl, B., Mamot, S., and Rodney, J.S. 1989. Post-harvest pod storage: A method for pulp preconditioning
to impair strong nib acidification during cocoa fermentation in Malaysia. Journal of the Science of Food and
Agriculture, 48(3), 285-304.
Moreira, M.V.I., Vilela, F.L., Miguel, C.P.M.G., Santos, C., Lima, N., and Schwan, R.F. 2017. Impact of a microbial
cocktail used as a starter culture on cocoa fermentation and chocolate flavor. Molecules 22(5), E766.
Schwan, R.F and Wheals, A.E. 2004. The microbiology of cocoa fermentation and its role in chocolate quality. Critical
Review in Food Sciences Nutrition, 44(4), 205-221.
Stoll, L. 2010. Biochemische Indikatoren für Keimung und Fermentation in Samen von Kakao (Theobroma cacao L.)
PhD thesis, University of Hamburg, Hamburg, Germany, 42-44
Thompson, S.S., Miller, K.B., Lopez, A., and Camu, N. 2013. Cocoa and coffee. In: Doyle, M.P., Beuchat, L.R., and
Montville, T.J. (Eds.), Food Microbiology: Fundamentals and Frontiers, 4th ed. ASM Press, Washington DC,
USA, pp. 881-899.
Yao, W., Ouattara H., Goualie B., Soumahoro S., and Niamke S. 2014. Analysis of some functional properties of
acetic acid bacteria involved in Ivorian cocoa fermentation. Journal of Applied Biosciences, 75, 6282- 6290.

455
 IFRC 2017: 114-215 Food Bioprocessing

Fermentation characteristic of kuini (mangifera odorata) and its potential as substrate to acetic acid bacteria

*Adnan, H., Othaman, M.A. and Alyas, N.D.

Biotechnology and Nanotechnology Research Centre, MARDI Headquarters,

Persiaran MARDI-UPM, 43400 Serdang, Selangor.
Abstract
Kuini (Mangifera odorata) is known by its strong smell and attractive orange-yellow colour of flesh. The fruit taste
sweet and slightly sour. The flesh is juicy and less fibrous. Kuini however is lack of market demand, therefore it is
important to identify other nutritional property of kuini to increase its market value. Since ages, kuini has been used in
folk medicine and research has proven that kuini contains prophylactic measures on radical scavenging activity. In
this study, kuini flesh and peel are fermented using acetic acid bacteria and the fermentation characteristics are then
observed. Thus the objective of this study is to identify the optimum conditions for kuini fermentation using
Gluconacetobacter sp. Results indicated that both kuini flesh and peel are able to produce suitable environment for
the growth of Gluconacetobacter sp. The fermentation had changed the biochemical property of fermented kuini as
such pH, acidity and soluble content. In addition, more intense yellow colour was obtained from fermented kuini flesh
whereas more pleasant odour was produced from fermented kuini peel. Fermentation had affected the nutritional
property of the both fermented kuini products. At a similar condition, kuini flesh able to enhance the growth of
Gluconacetobacter sp. up to 4 folds while kuini peel with only 2 folds. In conclusion, kuini flesh and peel are potential
substrates for acetic acid fermentation. Both substrates are able to enhance the growth of Gluconacetobacter sp. and
enhanced the nutritional property of their fermented products. The information gained from this study could provide
an important data for the study of kuini bioactive property in near future. In next study, the fermentation process
would be optimised and the health-related compounds of interest will be identified.

Keywords: kuini, peel, flesh, Gluconacetobacter sp. and acetic acid fermentation
*Corresponding author’s email: hazniza201@yahoo.co.uk

Introduction
Kuini is a seasonal fruit as the plant only produces fruits twice a year during a dry season. There is a market demand
on kuini fruits however lack interests of kuini planters exhibit less kuini production in market. Thus this study intends
to explore the beneficial property of kuini using the bioprocessing approach as such fermentation. Kuini was used as
substrate and underwent batch fermentation using Gluconacetobacter sp., an acetic acid bacterium. The chemical
and microbiological changes during fermentation process were observed. Kuini flesh and peel could be a potential
substrate to acetic acid fermentation and would produce beneficial fermented products. This research would be able
to increase demands for kuini consumption and offer new niche market for fruit planter and agriculture base industry.
Therefore, the objective of this study is to identify the optimum fermentation condition using Gluconacetobacter sp. in
different kuini substrates.

Materials and Methods

3.1 Preparation of samples
Kuini fruit at maturity stage was washed, air dried and peeled off. The flesh was sliced and blend as puree. Both flesh
puree and peel were dried separately in a dryer at 40ºC for 2 days with approximately 10 % moisture content. Dried
samples of kuini flesh and peel were blend as fine powder and separately packed in a plastic bag, sealed and
labelled.

3.2 Preparation of inoculum

The Gluconacetobacter sp. (GAB) inoculum was obtained from Collection of Functional Food Culture (CFFC)
MARDI. The culture was revived on a media alcohol (MA) agar and incubated for four days at 30°C and then
subculture in a MA broth to form an inoculum.

456
3.3 Fermentation of kuini
Briefly, samples of kuini flesh and peel were poured into a conical flask and dissolved in distilled water, separately.
The mixture was pasteurised and let it cool prior for inoculation. Samples were inoculated with GAB at required
concentrations and shake slowly. The flask was covered with sterilised cotton wool, tight using rubber bands and
then incubated at 30°C.

3.4 Analyses
Sample was analysed at interval period for 14 days. The analyses includes cell viability, growth profile, acetic acid
content (%, v/v), pH, total soluble solid (°Brix) and total growth weigh (g). The cell viability was measured using
viable cells count and calculated as colony forming units (log 10 CFU/mL). Growth profile was observed in term of
optical density (OD) at 600nm absorbance using cell density meter (WPA Biowave, UK). The acetic acid content was
estimated using titration method against 0.1N of NaOH and pH was determined using a calibrated pH meter (Mettler
Toledo, Switzerland). The total soluble solid was measured using refractometer (0-32°Brix, Atago ATC-1, Japan),
while total growth (%, w/w) of bacterial cellulose (BS) was weighed at the last day of fermentation period.

Results and Discussion

Figure 1(a) showed growth curve of Gluconacetobacter spp. (GAB) in fermentation of 3 and 5 % (w/v) for both kuini
flesh (KF) and peel (KP). The growth profiles of GAB in kuini samples can be explained by the growth phases which
include the lag, log, stationary and death phases (Maier, 2008). Each phase exhibited a distinct period of growth
represent the physiological changes of GAB in samples (KF and KP). The lag phase initially was at day 0-3 with the
growth rate essentially zero, and no noticeable lag phase was observed on the graph. The GAB cells are in
physiological adapts to the culture conditions at this phase. The lag phase is a transition to the log phase after the
initial population has double (Yates and Smotzer, 2007). The second is log phase and characterised by the most
rapid growth. Log phase is also known as exponential phase due to its exponential growth period. At this phase the
growth rate of GAB cells increased in the culture proportional to the number of cells present at any particular time
which is probably between days 3-6. The viability of GAB in both 5KF and 5KP increased rapidly from 0-4 CFU/mL,
unfortunately no noticeable log phase was observed for 3KF and 3KP at this period.

The third is stationary phase which is probably between days 6-10. The stationary phase is a period of no net growth
at which cells are still grow and divide but the growth of cells is balanced by an equal number of cell dying. At this
phase, the viability profile of GAB in all samples is constant, only that viability profile in both 5KF and 5KP are higher
than in 3KF and 3KP. The final is death phase at which cells of GAB tends to decline and characterised by a net loss
of culturable cells. The death phase is probably between days 10-14. Results indicated that only small number of
cells decrease which showing that the rate of GAB cell death is slower. The viability profile of GAB in all samples at
this phase is constant. A distinct viability of GAB was clearly shown in 5KF and 5KP, but less noticeable in 3KF and
3KP.

Figure 1(b) showed the growth of GAB in different kuini samples. Rapid, automated method such as turbidimetry
using optical density (OD) has been used widely for growth parameters, however does not directly provide the
microbial growth curve (Mytilinaios et al., 2012). OD is very useful to obtain precise growth kinetic parameters (Pla et
al., 2015) and such study is to compare the growth different of the same culture but in different conditions. Results
indicated that both 3KF and 3KP able to highly enhance the growth of GAB. Sample 3KF able to promote the growth
of GAB gradually until day 14, unfortunately the growth was slowly declined in 3KP from day 6-14. Results indicated
that 3% kuini concentration was able to increase the growth of GAB greater than 5% kuini. There is no distinct
difference of growth was observed for 5KF and 5KP, thus 5% concentration of 5KF and 5KP does not affect the
growth of GAB.

Figure 1(c-d) showed the behaviour of GAB in fermentation of kuini to pH and acidity. The changes in pH indicated
that initial pH in both 5KP and 3KP were slightly higher than in both 5KF and 3KF (Figure 1c). Results revealed that
initial pH for kuini peel are less acidic than pH in kuini flesh. Upon fermentation, pH was gradually reduced until the
end of process, probably due to incorporation of undissociated acid components in kuini. GAB was able to reduce pH
in kuini samples to approximately at pH 3.6 at which the optimum pH for GAB growth is 2.5 – 6.0 (George, 2005).

457
Acidity in kuini fermentation was also monitored by the production of acetic acid (Figure 1d). The acetic acid content
increased dramatically from 4–9.3% at day 0 to 61–82 % at day 14. Results indicated that all kuini samples were able
to produce a significant amount of acetic acid during the fermentation period. A dramatic increment of acetic acid was
probably due to the conversion of sugar to acetic acid as sugar content in kuini act as carbon source to the growth of
GAB. Figure 1e exhibited the profile of total soluble solid (TSS) content in kuini fermentation. All samples showed
nearly a similar TSS profile along the fermentation. The TSS content in all kuini samples was gradually decreased
from day 0-14. Results indicated that GAB probably consumed sugar in TSS as its carbon source then converted the
sugar to acetic acids (Yassine et al., 2016).

Figure 1f showed the total growth of GAB cellulose measured at the end of fermentation period. Results indicated
that the most favourable environment for the growth of GAB cellulose was found in KF3, followed by KF5, KP5 and
the least KP3. Flesh of kuini could provide an optimum environment to the growth of GAB cellulose at concentration
of 3 % (w/v). Results also revealed that acidity do effect the growth of GAB in kuini fermentation. The increased on
acetic acid content and decreased in pH had created an acidic environment that favour the growth of GAB in the
fermentation of kuini.

6 7

Growth (OD600nm)
6
Viability(log10

5
4 5
cfu/ml)

4
3 3
2 2
1 1
0 0
Day 0 Day3 Day 6 Day 10 Day 14 Day 0 Day3 Day 6 Day 10 Day 14
5KP 5KF 3KP 3KF 5KP 5KF 3KP 3KF
(a) (b)

7.0 100
Acetic acid (%)

6.0 75
5.0
pH

50
4.0
3.0 25
2.0 0
Day 0 Day3 Day 6 Day 10 Day 14 Day 0 Day3 Day 6 Day 10 Day 14
5KP 5KF 3KP 3KF 5KP 5KF 3KP 3KF
(c) (d)

10.0
KP5
Total Soluble
Solids (°Brix)

8.0
6.0 KF5
4.0 KP3
2.0
KF3
0.0
Day 0 Day3 Day 6 Day 10 Day 14 0 50 100 150
5KP 5KF 3KP 3KF final (g) initial (g)
(e) (f)

Figure 1: Characteristic and behaviour of Gluconacetobacter spp. (GAB) in kuini fermentation which include (a) cell
viability; (b) growth profile; (c) pH; (d) acetic acid content; (e) total soluble solid and (f) total weigh.

458
Conclusion
Kuini is a potential fermentation substrate to Gluconacetobacter sp. (GAB) which provides beneficial property to its
end-products. Characteristic and behaviour of GAB in fermentation of kuini flesh and peel could trigger the optimal
parameters for kuini fermentation and concerning cellulose yield by simple technique for laboratory and industrial
scale.

Acknowledgements
The author would like to thank MARDI for the financial support in conducting this research.

References
George, M.G. 2005. The proteobacteria. In Don, J.M., Noel, R.K. and James, T.S. (Eds). Bergey’s Manual of
Systematic Bacteriology, 2nd edition, p.72-74. USA: Springer
Maier, R.M. 2008. Bacterial growth. In Environmental Microbiology, p. 37-43. Academic Press Inc.
Mytilinaios, I., Salih, M., Schofield, H.K. and Lambert, R.J.W. 2012. Growth curve prediction from optical density data.
International Journal of Food Microbiology. 154:169-176
Pla, M.L., Oltra, S., Esteban, M.D., Andreu, S. and Palop, A. 2015. Comparison on primary models to predict
microbial growth by the plate count and absorbance methods. BioMed Research International. Vol. 2015,
Article ID365025. Hindrawi Publishing Corporation.
Yassine, F., Bassil, N., Flouty, R., Chokr, A., El Samrani, A., Boiteux, G. and El Tahchi, M. 2016. Culture medium pH
influence on Gluconacetobacter physiology: Cellulose production rate and yield enhancement in presence of
multiple carbon sources. Carbohydrate Polymers. 146:282-291
Yates, G.T. and Smotzer, T. 2007. On the lag phase and initial decline of microbial growth curves. Journal of
Theoretical Biology. 244:511-517

459
 IFRC 2017: 271-250 Food Bioprocessing

Development of GABA Malted Milk Drink from Germinated Brown Rice

*I.Zuwariah, I.Aida Hamimi, R. Rodhiah, and H. Hassan

Food Processing and Packaging, Malaysian Agricultural Research and Development Institute, Food Science &
Technology Research Center, MARDI. G.P.O. Box 12301, 50774 Kuala Lumpur.

Abstract

GABA Malted milk drink has been developed from MR 219 GABA ingredient and germinated brown rice. The
processing technology of GABA functional ingredient involved soaking, germination process and fermentation of
brown rice, germinated soy and tomato under anaerobic condition to optimize the GABA production. Seven
formulation has been developed consisted of three formulation of control malted milk drink and incorporation of
various range (27.5 - 44%) of MR 219 GABA ingredient. The optimal formulation of GABA malted milk drink
consisted of 7.5% of germinated brown rice and 44% of MR 219 GABA. The physico-chemical result showed that,
44% incorporation of MR 219 GABA ingredient in malted milk drink formulation gave GABA content 19 mg/100g,
protein 15.4 g/100g, dietary fiber 2.1 g/100g, calcium 88.9 and vitamin A 392.1 ug/100g. Result also showed that
incorporation of GABA functional ingredient in various formulation, significantly increased the amino acid content
such as lysine, aspartic, serine, glutamic, glycine, threonine, arginine, alanine, proline, isoleucine, leucine and
phenylalanine.

Keywords: GABA, Malted Milk Drink, Germinated brown rice and Amino Acid
*Corresponding author’s email: zuwariah@gmail.com

Introduction
Generally,malted milk drink is defined as drink made from hot or cold milk flavored with a powder containing malted
barley, wheat flour, and evaporated whole milk. The most common malt is barley but any of a variety of cereal grains,
including rice, wheat, oats and rye can be used to make malt. Thus, germinated brown rice could be used as an
alternative to barley malt, in addition it has increased synergetic effect in terms of functional ingredient and nutritional
value. In recent years, the interest on consumption of brown rice is increasing due to the increased awareness about
its health benefits mainly due to the increased levels of GABA (gamma amino butyric acid). Germinated brown rice
utilizes the physiologically active
substances that are present in bran, and the soaking process improves the texture of brown rice, whereas the
nutrients in the seed become easier to digest and absorb during germination (Tian, Nakamura, & Kayahara, 2004).
These processes lead to increasing amounts of simple sugars, peptides and amino acids in germinated seeds. In
addition, many enzymes are activated, which may produce minor nutrients such as GABA and vitamins.
Incorporation of germinated brown rice in malted milk drink will value added the beverages with GABA functional
ingredient. GABA is a four carbon amino acid and provides many beneficial effects for human health such as
decreasing blood pressure, controlling stress (Chung et al., 2009), diuretic effect, tranquilizer effect, while germinated
brown rice with enhanced levels of GABA can inhibit cancer cell proliferation (Oh & Oh, 2004).

The objective of the study is to develop malted milk drink with enhanced nutrients and GABA from germinated brown
rice.

Material and Methods

MR 219 was obtained from Padi beras Nasional Berhad (BERNAS) at Jalan Langgar Kedah. The paddy rice was
dehusked using a Satake rice machine (Satake Engineering Co.LTD, Tokyo, Japan) followed by paddy separation by
paddy separator where the unhulled paddy rice is separated from the brown rice. The soy bean sample and tomato
was bought from TS Trading Sdn. Bhd. in Seri Kembangan Selangor.

460
GBR functional ingredient preparation
In the preparing germinated formulation, brown rice / soy bean seeds samples were soaked in clean water for 8 to 24
hours. Next, the water was drained off and was then spread on towel inside the trays to germinate for 3 days. Then
the germinated brown rice and germinated soy bean were combined with tomato (1:1:1 ratio) and then dried.

Malted milk drink from germinated brown rice

Raw materials which were used for development of malted milk drink were germinated brown rice, germinated soy
bean, wheat flour, malted barley, sweet whey, sugar, non dairy creamer, skimmied milk and salt. In order to develop
malted milk drink, GABA ingredient was mixed with the GBR. Various types of combinations were made and the best
suitable combinations were made and showed in tables 1.

Table 1: Combination of malted milk drink

Malted Milk drink GABA ingredient Germinated brown rice Malted Barley (%)
(%) (%)
Drink C1 - - 15
Drink C2 - 15 -
Drink C3 - 7.5 7.5
Drink A 27.5 7.5 7.5
Drink B 37 7.5 7.5
Drink D 41 7.5 7.5
Drink E 44 7.5 7.5

Amino acids and gamma amino butyric acid (GABA) analysis

Amino acids and GABA was extracted according to the modified method of Abe et al. (2000). The
identification and quantification of amino acid and GABA were determined using the AccQtag Method (Waters,
U.S.A), scanning fluorescence detector. The mobile phase consists of eluent A and eluent B. Eluent A was borate
buffer while the Eluent B was 60 % acetonitrile (v/v). All separation was carried out on a 4 µm AccQ. Tag C18
column (150 x 3.9 mm) at 36ºC. Analysis of amino acids and GABA was done in duplicate.

Results and Discussion

Malted milk drink were developed with various percentage (Table 1) of GABA ingredient, germinated brown rice and
malted barley. Due to this combination, the GABA content had been increased by several folds. The combination of
GABA ingredient with several grain to obtain a synergetic effect in terms of nutritional and amino acid content.
According to Table 2, the highest GABA content (19mg/100g) in malted milk drink was drink E (44% GABA
ingredient, 7.5% germinated brown rice and 7.5% malted barley) compared to another drink formulation.
The result showed that the amino acid profile of drink C1, C2 and C3 was very low if compared with drink E. This
may be because, drink C1 consist of 15% malted barley and without combination of GABA ingredient and germinated
brown rice. Germination process involved in GABA ingredient and germinated brown rice resulted an increasing of
nutrients and also released of new component in the rice. Kayahara et al.(2001) showed that, the nutrients which
have increased significantly include GABA, lysine, vitamin E, dietary fiber, niacin, magnesium, vitamin B1, and
vitamin B6. In particular, the amount of GABA in drink E was notice to be fourteen times more as compared to drink
C1, eight and twelve times more than that of C2 and C3.

461
 Table 1: Amino acid and GABA content GABA Malted Milk Drink functional ingredient

Amino Drink A Drink B Drink C1 Drink C2 Drink C3 Drink D Drink E

acid
Asp 89.81 87.37 4.27 17.19 6.56 61.70 73.00
Ser 24.14 40.15 9.77 10.10 8.91 47.02 60.29
Glu 22.77 29.78 9.94 14.47 10.42 25.87 32.23
Gly 12.88 13.56 2.64 1.65 2.36 11.96 16.14
His 69.76 90.49 48.85 43.10 48.52 71.54 113.88
Arg 19.68 24.55 7.83 9.77 7.79 23.83 31.34
Thr 22.48 20.76 16.68 14.95 15.46 16.69 18.40
Ala 24.86 35.63 6.02 6.13 4.56 30.35 42.50
Pro 19.81 30.97 8.81 9.11 8.73 19.41 30.06
GABA 13.99 14.88 1.34 2.30 1.52 13.82 19.07
Ty r 15.83 23.50 3.28 6.42 3.86 23.88 28.39
Val 15.17 20.98 4.11 5.67 4.26 18.52 25.59
Met 7.28 9.74 1.54 2.55 1.29 9.13 11.87
Ly s 14.62 19.75 1.39 1.43 1.81 14.88 25.04
Ile 11.24 15.16 3.09 4.30 3.16 13.10 17.61
Leu 33.63 54.47 7.64 12.12 7.39 45.34 66.37
Phe 20.12 24.73 3.53 5.23 3.26 25.90 31.08
The addition of 44% GABA ingredient into drink formulation was increased the amino acid content ; aspartic, serine,
glutamic glycine, histidine, arginine, threonine, alanine, proline, GABA, tyrosine, valine, methionine, lysine,
isoleusine, leusine and phenylalanine. However, when addition of 27.5%, 37% and 41% of GABA ingredient into
drink formulations, the GABA content were keep constant around 14 mg/100g. The combination of 44% GABA
ingredient and 7.5 germinated brown rice caused synergetic action in terms of GABA and amino acids content if
compared to drink C3, which is only combination of germinated brown rice and malted barley.

Table 3: The nutritional analysis of drink C3 and drink E

Nutritional Analysis Drink C3 Drink E
moisture,g/100g 5.1 5.9
ash, g/100g 8.3 0.9
crude protein,g/100g 15.4 19.8
crude fat, g/100g 9.3 2.2
Total sugar, g/100g 6.2 0
Starch, g/100g 45.9 75
TDF, g/100g 2.1 12.3
Carbohydrate,g/100g 61.9 71.2
Energy, kcal/100g 397 408
Vitamin A, ug/100g 392.1 334
Vitamin B1, mg/100g 0.2 0.1
Vitamin B2, mg/100g 0.2 0.1
Calcium, mg/100g 88.9 94.3
Pottasium, mg/100g 473.1 503.4
Magnesium, mg/100g 8.9 9.1
Sodium, mg/100g 234.3 231.9
Drink C3: 0% GABA ingredient, 7.5 % germinated brown rice, 7.5% malted barley
Drink E: 44% GABA ingredient, 7.5 % germinated brown rice, 7.5% malted barley

462
The nutritional analysis for drink E and GABA functional ingredient was carried out. From Table 3, it was clearly
observed that GABA functional ingredient were rich source of protein, total dietary fiber, calcium and potassium while
their fat content is quite low. However, the present of 44% GABA ingredient which is combination of germinated
brown rice, germinated soy bean and tomato in drink E resulted lower in protein, total dietary fiber and starch. By
contrast, Mohan et al.(2010) found that malting caused decrease in protein and starch contents but increased in
dietary fiber contents of rice.

Conclusion
The GABA malted milk drink (Drink E) contained high level (19mg/100g) of GABA, increased the amino acid content,
rich source of protein, total dietary fiber and lower in fat content. The processing technology in developing GABA
ingredient and germinated brown rice were increased synergetic effect in terms of nutritional value, amino acid and
functional ingredient GABA. Further studies should be carried out to investigate the effect of germinated brown rice
and GABA ingredient on physiological functions and metabolic disorders such as anti-hypertensive, anti-depressant
effect and suppression of postprandial blood glucose in order to implement scientific based evidence studies.

Acknowledgements
The authors would like to thank Projek Pembangunan MARDI for financial support.

References
Chung, H. J., Jang, S. H., Cho, H. Y., & Lim, S. T. (2009). Effects of steeping and anaerobic treatment on GABA (γ-
aminobutyric acid) content in germinated waxy hull-less barley. LWT-Food Science and Technology, 42(10),
1712-1716. http://dx.doi.org/10.1016/j. lwt.2009.04.007.
Kayahara H, Tsukahara K, Tatai T (2001) In: Spanier AH (ed) Flavor, health and nutritional quality of pre-germinated
brown rice. Royal Society of Chemistry, Cambridge
Mohan, B.H., Malleshi, N.G., Koseki, T.(2010). Physico-chemical characteristics and non-starch polysaccharide
contents of Indica and Japonica brown rice and their malts. LWT - Food Science and Technology 43:784–791
Oh, C. H., & Oh, S. H. (2004). Effect of germinated brown rice extracts with enhanced levels of GABA on cancer cell
proliferation and apoptosis. Journal of Medicinal Food, 7(1), 19-23.
http://dx.doi.org/10.1089/109662004322984653. PMid:15117548.
Oh, S. H., & Choi, W. G. (2001). Changes in the levels of γ-aminobutyric acid and glutamate decarboxylase in
developing soybean seedlings. Journal of Plant Research, 114(3), 309-313.
http://dx.doi.org/10.1007/PL00013992.
Tian, S., Nakamura, K., & Kayahara, H. (2004). Analysis of phenolic compounds in white rice, brown rice and
germinated brown rice. Journal of Agricultural Food Chemistry, 52, 4808–4813.

463
 IFRC 2017: 157-115 Food Service and management

Pilot interviews of job satisfaction with offshore catering employees

1*Majid, M. A. A., 1Othman, M., 1Mohamad, S. F., and 2Lim, S. A. H.

1Department of Food Service and Management, Faculty of Food Science and Technology, Universiti Putra Malaysia,
43400 UPM Serdang, Malaysia

2Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM
Serdang, Malaysia

Abstract
Previous research indicates that employee’s job satisfaction plays a significant role in organizational performance. In
this paper, we draw upon qualitative inquiry alongside offshore catering employees; the overall goal was to explore
their satisfaction with the job. Although there have been a significant number of studies, the field continues to
experience diversity of understandings and ambiguity in this discourse. Shifting our focus from hypothetical job
context where job satisfaction is commonly studied, we draw on the findings to gain insights into these differing
understandings of offshore catering employees as they engaged in isolated workplace. The pilot interview was
conducted as inducement for a dissertation in developing a job satisfaction instrument in a particular context. In this
small-scale pilot study, we explore the job satisfaction experiences of Mark and Karl, two offshore catering
employees. Data in the form of in-depth, semi-structured interviews identified 44 codes, indicating 10 dimensions of
job satisfaction derived from the emerging themes. Evidently, the qualitative findings present important attributes of
job satisfaction among offshore catering employees. Results of this study shed light on the need for future
investigation to extend the scope of the study, bringing different cultures in order to enhance the findings on job
satisfaction across a broader spectrum.

Keywords: Qualitative, pilot study, interview, job satisfaction, offshore catering

*Corresponding author’s email : mh.aliff@gmail.com

Introduction
The paper explores how offshore catering employees viewed job satisfaction. Notwithstanding the occurrence of
previous studies on job satisfaction in various disciplines, little is known about employees' satisfaction with their job
working in offshore catering sector (Majid, Othman, Mohamad & Lim, 2016). Moreover, as part of unusual and
challenging work environment, there is a relatively limited amount of research on the behavior of people working in
these environments (Zimmer, Cabral, Borges, Coco & Hameister, 2013; Suedfeld, 1998). Hence, this study seeks to
address the lack of research in this area. This study was carried out as inducement for a dissertation in developing a
job satisfaction instrument in the particular context (Majid, Othman, Mohamad, Lim & Yusof, 2017).

Design and Method

This study was primarily based on a qualitative inquiry using in-depth, semi-structured interviews as primary data
collection was employed. The informants were identified through word of mouth and referrals from one offshore
catering company located in Shah Alam, Malaysia. An effort was made to interview the two employees, Mark and
Karl, whom work as Western cook and Asian cook, respectively. Both informants had experiences of working in the

464
sector for more than ten projects. The recruitment of the informants was based on purposive sampling technique and
willingness to participate. Both informants were willingly to participate in the study and were contacted to confirm the
date, time and place of interview.

First author conducted the interviews in March 2017. Prior to the interview, research purpose was explained to each
informant and they were provided with the letter of informed consent. Permission was obtained from both informants
to audiotape the interview using IC recorder and both interviews lasted approximately 32 and 37 minutes,
respectively. The interviews were guided by seven open-ended questions and were based on the previous studies on
job satisfaction. Before piloting the interview, the supervisory committee members reviewed the questions in terms of
its language, wording and relevancy. The main question was: “How do you generally feel about your current job?”
The interviews were carried out in Malay language with occasional use of English, as it was perceived as more
casual for both interviewer and interviewees to have comfortable discussions. Interviewer asked the same set of
questions to both informants, however interviewer did not ask the questions in orderly form but rather follow the flow
of the conversation. The interviewees were given a flexibility to freely discuss around each question. That is to say, it
was almost impossible to determine precisely how the interviewees were going to answer the questions. However,
interviewer asked probing questions in order to obtain further clarification on the issue discussed.

Data Analysis
The interviews were transcribed verbatim and analyzed using six-phase of thematic analysis proposed by Braun and
Clarke (2006). The process of coding was carried out with two doctoral course mates in order to ensure transparency
and reliability. The printed transcripts were handed to each course mate. The process was completed in several
iterations. First, the transcriptions were read several times to obtain a general sense of the whole material. Second,
the important phrases were independently marked next to each line or paragraph to reflect our initial coding. The
coders identified codes of all perspectives that the informants described or referred in the interviews that pertained to
their job satisfaction. The codes were then carefully checked back against the transcripts to ensure they demonstrate
the concept. Then, the coders mutually discussed their independent coding until a consensus was reached. Third, a
number of codes that were created then were collated together to search and address the similar themes, thereafter
referred to as dimensions. The first author conducted the process of collating the codes. Fourth, the dimensions were
reviewed and were later presented and discussed with all co-authors to discuss the relevancy of the codes to the
particular dimensions. Re-coding of the data was made and dimensions were revised to ensure the collated codes
form a coherent pattern. Fifth, the identified dimensions were further refined and authors jointly named and finalized
them into 10 dimensions of job satisfaction. Lastly, the evidence from the data analysis is presented in relation to the
research purpose. 10 dimensions emerged based on 44 codes namely remuneration, working condition, freedom,
fringe benefit, flexibility, social support, promotional opportunity, reward, competence and involvement. Each distinct
dimension linked back to the research aim and therefore, is discussed.

Discussions
Remuneration included a-month salary for two-week job, adequate salary, bring home large sum of money and good
salary relative to off day provided. Karl, an Asian cook in the department, asserted that even he was paid daily, he
was able to get a higher salary than onshore’s. Mark, a Western chef, further elaborated on this issue. "Because one
thing, we are working for two weeks and off for two weeks. But, we are able to obtain salary like a-month salary."
Noting that, remuneration was critical to satisfaction with their job.

465
Working condition was the second dimension of job satisfaction. Both informants described that no cost was spent
when going to work because they live at their workplace. Moreover, accommodation, food and uniform are provided.
Hence, no expenses were made while they are working offshore. Apart from that, Mark mentioned that he enjoyed
the challenge of working offshore. He said that even working for 12 hours a day, he enjoyed it. He also identified a
defining moment when he feels happy with the job. "First time I celebrate [Raya festive] there, it was not something to
be sad of but happy. Because of the variety of food, and our working hour was shortened."

Freedom was labeled as the third dimension. According to the informants, being able to feel and live independently
was an important part of feeling satisfied as an offshore catering employee. Mark said that from his experiences,
temporarily separation from family and going to work offshore to calm him from onshore environment made him
happy with the job.

The fourth dimension was fringe benefit that included provision of accommodation, meal, uniform, transportation and
entertainment. According to Mark, "...company which provides the work to the employees takes care of the benefits
that should have. In terms of travelling fare, hotel, accommodation, travelling and so on, I think they have no
problem."

Mark suggested that he was happy with flexibility of the job where he was able to earn more if he stayed back after
he completed his 2-week duty. He mentioned, "If I stay back, I will earn more." Apart from that, he said he also did
part time job onshore after he signed off from offshore duty and earned extra income from that.

The authors labeled the sixth dimension as social support. According to Mark, being able to mix around as very
important, whether with employees or superiors. The feeling that the others interact and doing social activities with
him was a significant part of feeling satisfaction with the job. He proudly remembered, "...I was able to mix around
with them, eat at the same table with them. That makes me feels happy, even their level are different." Both
informants had been making friends from other departments and deriving social support from the catering and other
departments.

Promotional opportunity, or getting promoted, was the seventh dimension of job satisfaction reported in this study.
Karl said he was promoted from steward to Asian cook whom his Camp boss (termed as catering supervisor) gave
the promotional opportunity. He said that he took that opportunity to learn the job tasks and he was happy with the
promotion.

According to Karl, there were rewards provided by the company, for both individual and the team. He mentioned that
the rewards were in the forms of cash or gift. He was rewarded for the best comments and suggestions for safety
improvisation. He also able to defined a moment where he was rewarded. He said, "There was a drilling team came
and they drilled the well. When they were able to get the first oil, they gave reward to all employees at the rig." The
result found not mentioned by Mark, but Karl stated it was one of the factors that made him happy.

Competence was apparently another important dimension of satisfaction with the job. Mark suggested that when
employees are satisfied with the job, they seek ways to perform extra work and help others. Mark mentioned, "To
me, there was time I used to do preparation for next day's product." He explained that the extra work he performed
was not payable, but for the sake to reduce the work tasks. Karl concluded by mentioning that he knows what to do
with the job and his competency precedes when no complains were made during his duty.

466
The tenth dimension emerging from the interviews was involvement. Satisfied employees apparently known to be
involved with their job including the community and the system. Karl described the situation that influenced his
involvement. "There is a program, which they called as unsafe condition. So, anyone could contribute their views,
advices." The best views selected during the program might have been an evidence of successful involvement.

Conclusion
The research was conducted using basic qualitative technique to elicit information that involves the use of in-depth,
semi-structured interview. This paper is concerned with an investigation of what are the attributes of job satisfaction
among offshore catering employees in Malaysia. The results can be regarded as having varying degrees of job
satisfaction attributes and this study might provide a first step towards understanding these employees' satisfaction
with their job. However, due to the small number of informants involved in the study, it is important to note that the
saturation of data was not reached. Thus, there is need for further investigation to extend the scope of the study,
bringing different cultures in order to enhance the findings on job satisfaction across a broader spectrum.

Acknowledgement
The authors would like to thank the respective organization for facilitating this research by enabling access to the
organization and the offshore catering employees for their willingness to participate. The authors also would like to
thank Aziz and Mohd Nazri whom had been contributing efforts and insights during the process of data coding.

References
Braun, V., & Clarke, V. (2006). Using thematic analysis in psychology. Qualitative Research in Psychology, 3(2), 77-
101.
Majid, M. A. A., Othman, M., Mohamad S. F., Lim, S. A. H., & Yusof, A. (2017). Piloting for interviews in qualitative
research: Operationalization and lessons learnt. International Journal of Academic Research in Business and
Social Sciences, 7(4), 1073-1080.
Majid, M. A. A., Othman, M., Mohamad S. F., & Lim, S. A. H. (2016, November). Development and validation of job
satisfaction instrument for offshore catering employees in Malaysia. Paper presented at International
Foodservice Graduate Research Colloquium and Workshop 2016, UPM Serdang, Malaysia.
Suedfeld, P. (1998). What can abnormal environments tell us about normal people? Polar stations as natural
psychology laboratories. Journal of Environmental Psychology, 18, 95-102.
Zimmer, M., Cabral, J. C. C. R., Borges, F. C., Côco, K. G., & Hameister, B. D. R. (2013). Psychological changes
arising from an Antarctic stay: Systematic overview. Estudos de Psicologia (Campinas), 30(3), 415-423.

467
 IFRC 2017: 222-207 Food Service and management

Identifying Possible Factors of Job Stress and Employees Intention to Leave a Job: A Case of Casual Typed
Restaurant Employees in Johor Bahru.

Majid A., N., *Ghazali, H. and Farahwahida, A.

Department of Food Service and Management, Faculty of Food Science and Technology, UPM Serdang, 43400
Serdang, Selangor, Malaysia.

Abstract

Stress relating to the workplace has been special focus of role stress research for many industries including in
restaurant industry. In restaurant industry work stress related identified as noisy environment, strenuous job,
excessive working hours, etc. This study aims to identify the foremost factors that influences employee’s intention to
leave a job in the restaurant industry. It also aims to investigate the relationship between job stresses towards
employee’s intention leave a job in the restaurant industry. Besides that, it is also done to indicate the relationship
between employee’s demographic factors and intention to leave a job in the restaurant industry. The factors used in
this study were working condition, work overload, work ambiguity towards employee’s intention to leave a job in the
restaurant industry. This study used quantitative approach through a self-administered survey questionnaire. A total
of 160 questionnaires were distributed and only 150 useable responses were returned for an 80 percent response
rate. The statistical analyses that used in this study were Descriptive analysis, Multiple Regression analysis, Pearson
Correlation analysis, Independence T-test analysis and One-way ANOVA. Based on the results, it showed that work
overload and working condition was the dominant factor in predicting employee’s intention to leave a job. In terms of
demographic characteristics such as gender, education level, and ethnic the result of the analysis indicated that there
were no significant relationships of those groups with the employee’s intention to leave a job but age group were
significant. The findings of this study hoped to provide useful information about the employee’s intention to leave a
job in the restaurant industry and it is also hoped to help the restaurant manager or supervisor to monitor their
employees before they leave the job.

Keywords: working condition, work overload, work ambiguity, casual dining restaurant, Johor Bahru
*Corresponding author’s email: hazrina@upm.edu.my

Introduction
Employees working in foodservice industries faced particularly stressful work situations. For examples they are
challenged with multiple unexpected situations that arise on the job. They are also expected to meet monthly and
yearly sales goals, maintain a positive image for the restaurant and meet multiple government regulations. These
stressful situations can wear on individuals over time, causing them to become exhausted and frustrated. Thus, it is
important to both individuals and organizations that employees find ways of approaching and managing the stressful
situations they encounter. The most critical intangible costs are the loss of employee morale for those employees
who choose to remain in the organization. Thus, an understanding of employees and the possible causes why they
leave is important to better prevent labor turnover from happening (Ghazali, 2010).

Although Malaysian food service industry continues to expand both in size and diversity (Ingram and Jones, 1998)
and experiences continuous growth throughout the year here in Malaysia (Kueh and Boo, 2007). Unfortunately,
general public view this industry poorly because working in food service industry is always associated with low pay,
long working hours, menial work, lack of benefits, hard work, physically exhausting and underappreciated (Ingram
and Jones, 1998; Wildes, 2005). These phenomenon and perception eventually lead to negative view of food service
industry and as a consequence, restaurants face difficulties in attracting new employees. Malaysian Employers
Federation (MEF) stated that hotel, restaurant industry is placed third as the industry with the highest annual average
turnover rate at 32.4 percent only behind IT/Communication (75.72) and Associations/Societies (33). Previous study

468
by Aon Hewitt, a human capital consulting and outsourcing firm, placed Malaysia as the sixth in Asia-Pacific for staff
turnover 2011 at the percentage of 15.9 (Goh, 2012). Another example for Malaysian fast food industry, it is reported
that turnover rate for managers was 100 percent (Ryan et al., 2011). Therefore, lowering turnover of hourly
employees has been considered one of the major challenges that food service managers have faced over recent
years and this challenge is likely to continue as sales continue to grow (National Restaurant Association, 2002).

This study aims to identify the foremost factors that influences employee’s intention to leave a job in the restaurant
industry. It also aims to investigate the relationship between job stresses towards employee’s intention leave a job in
the restaurant industry.

Methodology
The participants of this research are the employees of casual dining restaurants in Johor Bahru. The locations of the
outlets covered the urban to suburban area around Johor Bahru, Malaysia. A self-administered questionnaire was
designed for present study. The questionnaire design was based on previous study. This study used convenience
sampling method, which is a kind of non- probability nonrandom sampling in which members of the target population,
as Dornyei (2007) mentions, are selected for the purpose of the study if they meet certain practical criteria, for
example geographical proximity, availability at a certain time, easy accessibility, or the willingness to volunteer.
Hence, the questionnaire is adapted, and modified in design for the casual dining restaurant industry in Malaysia.
The questionnaires are available in both Malay and English languages. Prior to conducting actual data collection,
pilot test was done in order to test the reliability of the constructs in the questionnaire. 40 sets of complete
questionnaires were collected from employees of selected casual dining restaurants. For the actual data collection, a
total of 160 questionnaires were distributed and only 150 useable responses were returned for an 80 percent
response rate.

Results and Discussion

Prior to conducting actual data collection, pilot test was done in order to test reliability of the constructs in the
questionnaire. 40 sets of complete questionnaires were collected from employee’s three different outlets of casual
dining restaurants. All of the responses were used reliability analysis measured by Cronbach’s alpha using SPSS 23.
From the result, all the constructs reached Cronbach’s alpha 0.6 and above which is the acceptable level for
reliability (Hair et al., 2006). Hence, all of the construct were included in the questionnaires for the actual data
collection.

Table 2 showed that the Pearson correlation coefficient was used to analyze the relationship between the factors of
job in the selected casual restaurants. For working condition variables, r = - 0.324, N= 150, p = 0.000 which mean
there is a negative moderate relationship between working condition and intention to leave a job and it is significant
since the p value was less than 0.01. Working condition has negative relationship with intention to leave a job
because there is negative (-) sign in front of r values which mean the increase of intention to leave a job correlate
with decrease level of working condition. For work overload variables, r = 0.287, N = 150, p = 0.000 which mean
there is positive weak relationship between work overload and intention to leave a job and it is significant since the p
value was less than 0.01. Work overload has positive relationship with intention to leave a job meaning that the
increases of intention to leave a job correlate with increase work overload. For work ambiguity variables, r = - 0.141,
N = 150, p = 0.085 which mean their negative weak relationship between work ambiguity and intention to leave a job
and it is not significant since the p value was more than 0.01. Work ambiguity has negative relationship with intention
to leave a job meaning that the increases of intention to leave a job correlate with decrease level of work ambiguity.

Based on the result in Table 3, it could be justified that the work overload and working condition variable was the
main factor in affecting the employee’s intention to leave job in selected casual restaurants in Johor Bharu.
Therefore, it could be concluded that when the work overload increase and working condition decrease, the
employees intention to leave job will increase. This is because the effect of working condition and work overload was

469
significant because of the p value was 0.005 and 0.002 which was less than significant level, 0.05. It indicated that
working condition and work overload was significant in predicting the employee’s intention to leave a job. However,
other variable which is work ambiguity were not significant and unrelated to the dependent variable (intention to leave
a job). This was because the p value was 0.845 respectively. This would seem to indicate that the percentage of
work ambiguity were not essential factors that influence employees to leave a job.

Table 1: Reliability result (Pilot Study)

Variables Reliability (Cronbach’s alpha, α)
Working condition 0.832
Work overload 0.801
Work ambiguity 0.756
Intention to leave a job 0.714

Table 2: Pearson Correlations Analysis between Job Stress and Intention to Leave
Variables Intention to leave a job
r Sig(2-tailed) N
Working Condition -0.324** 0.000 150
Work Overload
Work Ambiguity 0.287** 0.000 150

-0.141 0.085 150

**. Correlation is significant at the 0.01 level (2-tailed)

Table 3: Multiple Linear Regression Analysis on Job Stress and Intention to Leave a Job
Model Unstandardized Coefficients Standardized t Sig.
Coefficients
B Std. Error β
1 (Constant) 3.753 0.393 9.543 0.000
Working -0.261 0. 091 -0.282 -2.854 0.005
Condition
Work Overload 0.259 0.080 0.258 3.214 0.002
Work -0.091 0.095 -0.020 -0.196 0.845
Ambiguity

Conclusion

In conclusion, this study is able to contribute a better understanding of possible causes of employee’s turnover in
Malaysia’s restaurant industry. This is because most of the previous studied focused on employee turnover for
overall hospitality and tourism industry, but limited research was specifically focused on restaurant industry only.
Moreover, those researchers were done in western context and not applicable in Malaysia due to the culture of
working environment and behaviours of employees are different in foreign country. This study also is able to provide
valuable information in terms of managerial perspective. It is significantly contribute to restaurant management on
understanding the factor influencing employees to leave. The result of this study hopefully could help managerial
level to retain employees by better monitoring over the factors leading to turnover intention and take appropriate
actions to control the employee turnover intention in restaurant.

Acknowledgements
The authors would like to thank UPM for financial support.

470
References
Dornyei, Z, (2007), Research methods in applied linguistics. New York: Oxford University Press. 366 pages.
Ghazali, H. (2010), Employee intention to leave a job: A case of Malaysian fast food industry (Doctoral dissertation,
University of Wakaito).
Goh, L. 2012, February 19. Why Job-hoppers Hop, The Sunday Star, p. 24. Retrieved April 1, 2012 from Malaysian
Employers Federation (MEF) News database.
Hair, J.F., Black, W.C., Babin, B.J., Anderson, R.E., and Tatham, R.L. (2006). Multivariate Data Analysis 6th Edition.
New Jersey: Pearson Prentice Hall.
Ingram, H. and Jones, S. (1998), Teamwork and the management of food service operations. Team Performance
Management 4 (2): 67–73.
Kueh, K. and Boo, H. V. (2007), Culture and service quality expectations - Evidence from Generation Y consumers in
Malaysia. Managing Service Quality 17 (6): 656–680.
Wildes, V. J. (2005), Stigma in food service work: How it affects restaurant servers’ intention to stay in the business
or recommend a job to another [Abstract]. Tourism and Hospitality Research 5 (3): 213–233.

471
 IFRC 2017: 222-242 Food Service and management

Initial Findings of Possible Factors Contribute to Job Stress among Casual Dining Restaurant Employees in
Klang Valley, Malaysia.

Farahwahida, A. and *Ghazali, H.

Department of Food Service and Management, Faculty of Food Science and Technology, UPM Serdang, 43400
Serdang, Selangor, Malaysia.

Abstract

Despite research in job stress has been actively done in many industries, factors contributed to job stress were still
investigated. Job stress in restaurant industry was happened due to several factors such as excessive working hours,
strenuous job, shift hours and unattractive benefits. The purpose of this study is to investigate factors that affect job
stress among employees in casual dining restaurants. This study focused only on three variables that most used in
the literature identified as working environment, workload and job autonomy. Preliminary study was done and a total
of 30 respondents participated in this study. Questionnaires were distributed to employees in selected casual dining
restaurants at The Mines shopping complex, Seri Kembangan. Reliability results showed that all the variables were
reliable and can be proceed to actual data collection. The reliability results were α = 0.781 (working environment), α
= 0.674 (workload) and α = 0.861 (job autonomy). Additionally, based on descriptive results, it showed that workload
was ranked the first placed as factor contributes to job stress with 15 respondents agreed. Then followed by job
autonomy factor (9 respondents) and lastly working environment with 6 respondents. In term of frequency of feeling
stress at workplace, 19 respondents agreed that they felt 1-2 times in a week followed by 7 respondents’ stated felt
stress at least 3-4 times. Initial findings of this study showed some indication on possible antecedents that affect job
stress among employees working in restaurant industry.

Keywords: job stress; working environment, workload, job autonomy, casual dining, restaurants
*Corresponding author’s email: hazrina@upm.edu.my

Introduction
Job stress is snowballing globally, be it in all countries, organizations, professions, and among employees,
employers, families and society in general. Job stress happened due to several reasons such as work dissatisfaction
and hectic working environment. It is agreed that when employee felt stress at workplace it will affect their job
performance and productivity. In the worst case, employee left the organization and turnover occurred. Lu and
Gursoy (2013), explained that there is positive relationship between employee job stress and employee turnover and
this was supported in previous literature. To support this logic, it is possible that job stress applied in food industry
may give positive results and employees are vulnerable to high job stress level. This is because, in most restaurants
frontline employees have frequent interaction with customers compared to other industries, and thus it makes them
more susceptible to emotional burnout (Karatepe, 2015). Equally, employee at back of the house may also feel stress
when faced job related problems. Despite numerous studies on job stress among restaurant industry workers, few
have focused on the casual dining restaurant employees and particularly the predictors of the job stress which are
working condition and workload.

The objective of the study is to investigate antecedents that affect job stress among employees in casual dining
restaurant.

Methodology
The participants of this research are the employees of casual dining restaurants in Klang Valley area. The locations
of the outlets covered the urban to suburban area around Klang Valley, Malaysia. These outlets located in various
locations including shopping malls, housing areas, and shop lots around business area. A self-administered
questionnaire was designed for this study. The questionnaire design was based on previous studies. Hence, the

472
questionnaire is adapted, and modified in design for the casual dining restaurant industry in Malaysia. The
questionnaires are available in both Malay and English languages. The questionnaire is composed of 3 part; Section
A (employees’ job stress), Section B (influence of working environment, workload and job autonomy towards job
stress) and Section C (demographic profile of employees). Prior to conducting actual data collection, pilot test was
done in order to test the reliability of the constructs in the questionnaire. 30 sets of complete questionnaires were
collected from employees of selected casual dining restaurants. All of the responses were used for reliability analysis
measured by Cronbach’s alpha using SPSS 22. From the result, all of the constructs reached Cronbach’s alpha 0.6
and above which is the acceptable level for reliability (Hair et al., 2006).

Results and Discussion

In order to obtain preliminary result, the Cronbach’s alpha (α) was done. Basically, the Cronbach’s alpha (α) is a
measure of internal consistency, that is, how closely related a set of items are as a group. It is considered to be a
measure of scale reliability. The internal reliability of the items was verified by computing the Cronbach’s alpha (α)
(Nunnally, 1978). Nunnally (1978) suggested that a minimum alpha of 0.6 sufficed for early stage of research. Result
for the pilot study is presented in Table 1. For employees’ job stress the Cronbach’s alpha (α = 0.805), working
condition (α = 0.781), workload (α = 0.674), job autonomy (α = 0.861) and perceived stress scale (α = 0.885). As the
Cronbach’s alpha in this study were all much higher than 0.6, the constructs were therefore deemed to have
adequate reliability.

On top of that, the descriptive results showed that workload was ranked the first placed as factor contribute to job
stress with 15 respondents. Then followed by job autonomy factor (9 respondents) and lastly working environment
with 6 respondents. Excessive workload is one of the sources of stress at work (Faulkner and Patiar, 1997; Lo and
Lamm, 2005) and of emotional exhaustion (Karatepe, 2013). Overload may become rebound, which will resulting in
negative outcomes (Barnabas et al., 2013).

In term of frequency of feeling stress at workplace, 19 respondents agreed that they felt stress 1 – 2 times in a week
followed by 7 respondents stated felt stress at least 3 – 4 times a week. Initial findings of this study showed some
indication on possible factors that affect job stress among employees. Stress can gives adverse effects to an
individual’s life be it in productivity, creativity, economic as well as the healthcare. This was supported by (Tehrani et
al., 2013) stated that higher level of job stress can lead to lower mental and (Mohamadi, Nourollahi, & Latifi, 2013)
added that is also could lead to physical health, thus, it will make the workers abandons their own healthcare.

For this current study, a pilot study was conducted among casual dining restaurant employees in casual dining
outlets in Klang Valley. A self-administered questionnaire was distributed to selected casual dining restaurant
employees in The Mines shopping complex in Seri Kembangan, Selangor. A total of questionnaires distributed were
thirty. The questionnaires were collected on the same day. During the distribution, the employees of each outlet were
given an hour for them to complete the questionnaires. The questionnaires were distributed during non-peak hours
which are before the lunch hours between 10.00 am to 11.30 am. Subsequently, some changes were made in order
to make sure the questionnaires would be clear to the employees.

Even though, the questionnaire seemed long but it was not too-time consuming. Finally, the questionnaire was
reviewed several times by this current study’s supervisory committee before being distributed to employees in the
casual dining restaurant industry.
Table 2: Reliability result
Variables Reliability (Cronbach’s alpha, α)
Employee’s job stress 0.781
Working environment 0.861
Workload 0.674
Job autonomy 0.861
Perceived stress 0.885

473
Conclusion
In conclusion, initial findings of this study give some views on possible factors that affect job stress. This study will
provides linkages between job stress and its antecedents. The role of the antecedents also will be exhibited. On the
other note, as employees work closely with their immediate supervisors, it will create connections and understanding
between the two sides; managerial and non-managerial. Eventually, it will help in reducing the turnover rate that has
been dashingly increase as well as it is time consuming and costly. Through the results of the relationship between
variables, this study also will contribute to the development of a model to suit the social exchange between the
employers and employees of casual dining restaurant in Klang Valley. This study also is hoped to fill the gap in the
body of knowledge of casual dining restaurant scenario.

Acknowledgements
The authors would like to thank UPM for financial support.

References
Barnabas, N. E., Kanu, G. C., Obi, T. C., Aboh, J. U., & Agu, S. A. (2013). Influence of Job Autonomy on ethical
behaviour of nurses in south eastern Nigeria. Journal of Organisation and Human Behaviour, 2(3), 32.
Faulkner, B. and Patiar, A. (1997), Workplace induced stress among operational staff in the hotel industry,
International Journal Hospitality Management, Vol. 16 No. 1, pp. 99-117.
Hair, J.F., Black, W.C., Babin, B.J., Anderson, R.E., and Tatham, R.L. (2006). Multivariate Data Analysis 6th Edition.
New Jersey: Pearson Prentice Hall.
Karatepe, Osman M., (2015). Do personal resources mediate the effect of perceived organizational support on
emotional exhaustion and job outcomes? International Journal Contemporary Hospitality Management. 27 (1),
4–26.
Karatepe, Osman. M. (2013). The effects of work overload and work-family conflict on job embeddedness and job
performance: the mediation of emotional exhaustion. International Journal of Contemporary Hospitality
Management, 25(4), 614-634.
Lo, K. and Lamm, F. (2005), Occupational stress in the hospitality industry – an employment relations perspective,
New Zealand Journal of Employment Relations, Vol. 30 No. 1, p. 23.
Singleton, V. L. and Rossi, J. A. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid
reagents. American Journal of Enology and Viticulture 16(1): 144-158.
Lu, A., and D. Gursoy. (2013). Impact of Job Burnout on Satisfaction and Turnover Intention: Do Generational
Differences Matter?.Journal of Hospitality & Tourism Research -: 1-26.
Mohamadi, A., Nourollahi, M., & Latifi, S. (2013). The effect of risk factors of occupational stress on general health of
the fire fighters of Ahvaz city. Jundishapur Health Science, Vol. 5(No. 3), 167-33.
Nunnally, J.C., (1978). Psychometric Theory. McGraw-Hill, New York, NY.
Tehrani, H. Rakhshani , T., Shojaee, D. Z., Hosseini, S., & Bagheriyan , S. (2013). Aanalyzing the relationship
between job stress to mental health, personality types and stressful life events of the nurses occupied in
Tehran 115 emergency. Iran Red Crescent Med Journal, Vol. 15(No. 3), 272-3.

474
 IFRC 2017: 256-264 Food Service and Management

Internship satisfaction factors and instruments: A review and research directions for the undergraduate
hospitality programs
Ruslan, S., *Mohamad, S.F., and Othman, M.
Department of Food Service and Management, Faculty of Food Science and Technology, Universiti Putra Malaysia,
43400 UPM, Serdang, Selangor, Malaysia

Abstract

An internship is professional learning experiences that integrates knowledge and theory learned in classrooms with
practical application. Most of the local universities embrace internship in their curriculum design as it has been
viewed as the platform to enhance students’ skills. However, little study has been done on the internship satisfaction
whereby this area should be the focus in assessing an internship. There is a need to study on internship satisfaction
in order to maximize the outcomes of the experiential learning that provide benefits to students, universities and
organizations. This paper reviewed different factors that contribute to the internship satisfaction, where the factors
derived from different contexts of studies. The common factors of the internship satisfaction despite of different
contexts are individual factors, university support, job characteristics, organizational environment and also contextual
factors. There are several unique factors that are rarely noticed: academic program; program handling; intern’s
quality; intern’s expectations on: future employability, career enhancement and the importance of the internship
program for the completion of studies; intern’s perceptions- on career related experience, relevant to the field of
study, relevant to interests, flexible schedule, communication skills and peer relationship. On the other hand, this
paper also reviewed key components from different internship satisfaction instruments, which were conducted in
various industries. The common components probed in the instruments are factors influencing internship satisfaction,
demographic information and overall satisfaction towards internship experience. While, the distinctive components
incorporate general internship satisfaction, assessment of criteria: career skills and career focus, students’
employability options and comments from students on their internship experience. A direction of research for the
internship satisfaction instrument comprising its factors, specifically within the context of the undergraduate
hospitality program for local universities, is presented in this paper.
Keywords: Internship, satisfaction, hospitality, university, student, instrument.
*Corresponding author’s email: s_fatimah@upm.edu.my

Introduction
Hospitality education plays an important role of supporting hospitality development and ensuring the continuous
supply of quality workers to meet the industry requirements. In regards to the issues, institutions that provide
hospitality courses make a great effort to develop internship programs. Consequently, students increasingly demand
well-organized internship programs in order to acquire professional skills, work experience and gain a greater
understanding of the industries’ requirements. According to Chinomona and Surujlal (2012), one of the internship
goals is to provide and develop necessary skills to students to be effective in the work environment. Furthermore,
given the fact that internship is the first working experience for many college students, they tend to improve career
decision making and perceptions of self-efficacy obtained from beneficial learning experience (Brooks et aI., 1995).
Undoubtedly, the benefits and positive outcomes of internships are aplenty with many previous academic
researchers had discussed on it. However, the essence of successful internship experiences lies in the satisfaction
of students with their internships. Internship satisfaction is one area of research that has been largely neglected and
little research has been done on it (Klee, 2011). Stansbie (2011) agreed that, although a number of studies had
emphasized on the topic of internships, only few studies have seen to examine internship design as a satisfaction
factor. Also, Silva (2013) asserted that, despite most university programs currently offer a variety of internship
experiences, little work has been done in the measurement of the internship quality. Therefore, this study aims to

475
provide relevant literatures that have been reviewed in the relation to the various factors contributing to internship
satisfaction. Besides, this paper attempts to revise a number of internship instruments that were used by previous
researchers to measure the satisfaction of internship experiences in their studies.

Literature Review

Satisfaction with an Internship Experience

Nelson (1994) conducted a study to investigate the factors related to students’ satisfaction with their hospitality
internship experiences. The literature revealed that students were most satisfied with internships that provide
relevant work, some autonomy, and timely feedback. Faruk and Yirik (2014) assessed the internship satisfaction
among the hospitality students by looking at the five dimensions in which these factors might contribute to internship
satisfaction: better salaries for interns, paying for overtimes, not having excessive working hours, fair management
behavior with respect to interns and training interns throughout the internship period. On the other hand, Wen (2010)
focused on identifying and evaluating the determinants of internship effectiveness to study the degree to which
characteristics of student interns and organization practices contribute to internship success which in turn leads to
internship satisfaction. Three individual factors were identified as important aspects of an effective internship
program: academic preparedness, positive attitude and self-initiative.

Simmons (2006) utilized the factor analysis and identified five factors relevant to students’ satisfaction with internship
experiences. These five factors are length of internship, pay, industry supervision, weekly work hours and internship
preparedness. On the other hand, Stansbie (2011) asserted that, there are nine factors contribute to internship
satisfaction which are skill variety, task identity, task significance, task autonomy, feedback from job, feedback from
agents, dealing with others, pay and benefits obtained from internship experience. Meanwhile, Malekpour (2014) has
highlighted that intern’s quality, designed academic program, hotel program handling and feedback were the most
crucial in evaluating internship satisfaction.

Furthermore, a study explored by Sassnett and Ross (2016), found that internship performance, skills, preceptor
responsibilities, faculty coordination and quality of internship plays a crucial, important and significant role to
satisfaction. Kipreos and Dimitropoulos (2016) analyzed, students ranked nature of the placement, cooperation with
the host organization, future employability, career enhancement, obtaining career related experiences and the
importance of the internship program for the completion of their studies on top when evaluating their internship
satisfaction. In addition, Marinakou and Giousmpasoglou (2013) identified that professional environment, learned a
lot, interesting work, good supervision, made valuable contacts, felt like part of a team, made good money, flexible
schedule, new skills, self-knowledge and meaningful task do influence interns’ satisfaction with the internship
experience.

According to Gupta and Burns (2010) proposed that, positive internship experience, positive work environment,
improved job prospects, new skills, comforts with work environment and communication skills contibute to students’
satisfaction towards their internship experience. However, from the findings of their study, there were no significant
relationship observed among compensation, duration of internship experience, hours worked per week as an intern
and Grade Point Average (GPA) with internship satisfaction. Moreover, Bao and Fang (2008) found that, the job
itself, supervision, training & development, pay and peer relationship have been an invaluable aspect to interns.
Additionally, based on a study conducted by Jean et al., (2012) the findings revealed that individual factors, university
support, job characteristics, organizational environment are imperative factors contributing towards internship
satisfaction. However, contextual factors are the only variable that does not have an impact towards the variation of
internship satisfaction.

476
Review of Internship Satisfaction Instruments

Based on a study conducted by Jean et al., (2012), the survey instrument was adopted from several researchers :
D’Abate et al., 2009; Hackman & Oldham,1980; Gupta et al., 2010; Klee, 2011 and Wen, 2010. The instrument
consists of three sections that are Section A, Section B and Section C. Section A is related to the factors influencing
internship satisfaction among business undergraduates. There are five main factors which include individual factors,
university support, job characteristics, organizational environment and contextual factors. Section B seeks to
understand the general internship satisfaction among business undergraduates and Section C requested
demographic information from the students. Here, their gender, year of study, type of degree program majors they
were studying,current Cumulative Grade Point Average (CGPA), duration of their internship, the nature of business of
their internship companies, whether their internship job is related to their major of study and their range range of
salaries received during internship were requested.

Meanwhile, a scientific reseach done by Bao and Fang (2008) investigated students’ satisfaction level toward
internship experience in the hospitality and tourism industry. The survey instrument adopted a questionnaire based
on an extensive review of literature. The questionnaire consisted of three sections. The first section evaluated
respondents’ satisfaction towards their internship experience: the job itself, superior, training and development, pay
and welfare and peer relationship. The second section measured respondents’ overall satisfaction towards
internship. The third section collected socio- demographic data respondents such as gender, grade and the name of
school.

Through a study investigated by Wen (2010), the author identified and evaluated the determinants of internship
effectiveness in the nature of business prospects. The survey instrument is consisted of three main parts. Section A
contained an assessment of predictors which are academic preparedness, positive attitude, self-initiative, challenge
job, effectiveness of supervision, task clarity and compensation. While, section B measured career skills and career
focus. Section C collected personal information. For instance, respondents’ major, gender, current year of study,
cumulative GPA, length of internship, business nature of internship organization, whether the job nature related to
their majors and whether they received academic credits were included.

Marinakou and Giousmpasoglou (2013) study identified factors that contribute to student satisfaction from this
working and learning experience. A questionnaire was designed based on extensive review of the literature on
student satisfaction and expectations from internships in the hospitality and tourism industry. The questionnaire
included five sections. Section one included questions on demographic information include gender, institution, hotel
location and year of study. In section two, students were asked to evaluate work experience during their internships.
There are meaningful tasks, relevancy to students’ studies, relevancy to students’ interest, supervision, availability of
staff, new knowledge, new skills and self-knowledge. Section three examined the student’s employability options and
section four measured the overall internship experience, for example the students’ likes, dislikes and expectations
from their internships. The students’ satisfaction factors or students’ likes comprise of professional environment,
learned a lot, interesting work, good supervisor, made valuable contacts, felt like part of the team, made good money,
liked co-workers and flexible schedule. Meanwhile, the students’ dissatisfaction factors or students’ dislikes consists
of not paid well, too much work, disorganized work environment, not enough to do, work was boring, not enough
supervision, did not learn anything, work was not well defined and disconnected from co-workers. Additionally, the
students’ expectations from their internships included learn a lot, professional environment, feel like part of the team,
make valuable contacts, interesting work, good supervisor, like co-workers, receive a job offer, receve school credit
and make good money. Finally, section five asked the students to comment on their internship experience in order to
identify any other factors that were not included in the previous sections.

Research Directions

Overall, most of the common factors contribute to internship satisfaction indicate individual factors, university support,
job characteristics, organizational environment and contextual factors. However, the unique factors that create
internship satisfaction of interns are found to be: designed academic program; hotel program handling; expectation

477
and perception of intern’s quality; intern’s expectation on: future employability, career enhancement and the
importance of the internship program for the completion of studies; intern’s perception on career related experience,
relevant to field of study, relevant to interests, flexible schedule, communication skills and peer relationship.

On the other hand, the common components addressed in the internship satisfaction instrument are: factors
influencing internship satisfaction, demographic information and overall satisfaction towards internship experience.
Whereas, the distinctive components incorporate general internship satisfaction, assessment of criterions including:
career skills and career focus, students’ employability options and comments from students on their internship
experience.

Looking at the factors that affects the internship satisfaction, this study suggests that individual factors, job
characteristics, organizational environment, university support and contextual factors plays a dominating roles in the
internship effectiveness. Also, these components are suggested to be added in the instrument according to the
nature of the hospitality program.

References

Bao, Y. and Fang, G. 2008. A study on hospitality students’ satisfaction towards their internship: A case from Hang
Zhou, China. School of Tourism and Health Zhejiang Forestry University
D’Abate, C. P., Youndt, M. A., & Wenzel, K.E. 2009. Making the most of an internship: An empirical study of
internship satisfaction. Academy of management Learning & Educational, 8(4), 527-539
Gupta, P. B. and Burns, D. J. 2010. An exploration of student satisfaction with internship experiences in marketing.
Business Education & Administration, 2(1), 27-37
Hackman, J. R., and Oldham, G. R. 1980. Work Redesign, Addison- Wesley, Massachusetts
Klee, C. R. 2011. Recent school psychology graduates: A preliminary survey of their internship experience,
satisfaction and support. University at Albany, State University of New Work.
Marinakou, E. and Giousmpasoglou, C. 2013. An investigation of student satisfaction from hospitality internship
programs in Greece. Journal of Tourism and Hospitality Management, 1(3), 103-112.
Nelson, A. A. 1994. Hospitality internships: The effects of job dimensions and supportive relationships on student
satisfaction. Wayne State University
Wen, K. P. 2010. Determinants of internship effectiveness for university students in Hong Kong. Hong Kong Baptist
University

478
 IFRC 2017: 276-251 Food Service and management

A qualitative study on factors influencing older consumer dining out behaviour

Ganesan, L., Abu Bakar, A.Z., and Othman, M.

Department of Food Service and Management, Faculty of Food Science and Technology, Universiti Putra Malaysia,
43400 UPM, Serdang Selangor, Malaysia.

ABSTRACT
Aging or growing old is an inevitable, rather than viewing this as another life stage, this transition period are not easily
welcomed by many. The main reason is because with age we are getting more susceptible to disease and disability.
This mind-set is changing slowly thanks to the progress in healthcare and government policy that not only care for
their health but also helps the older people to lead a better life in their golden age. It is important to acknowledge this
age group not just because they are living longer; it is also because they are getting bigger in size and economic
power. The purpose of this study is to find out what would be the factor for older adults in Malaysia to dine out. Semi-
structured in-depth interviews were done with Malaysian older people ages 55 and above (focusing mainly on those
with education up to secondary school). 10 informers were interviewed for this study. The result of this study were
analysed using the Atlas.ti software. Result of this study suggests that Malaysian older adults choose to dine out
based on the cleanliness followed by food taste. This study will be beneficial in providing more insight to this
untapped market segment.

Keywords: older consumer, dining factor, dining out

*Corresponding author’s email: ainulz@upm.edu.my

Introduction
It is expected that Malaysia will reach the status of ‘aged nation’ by the year 2020 (Hamid, 2015). The term ‘aged
nation’ will be achieved when the total older adults (by definition those who are 65 and above) population size
reached 7% of the total population (Hamid, 2015). Even though it is said that older populations in developing
countries like Malaysia are getting older before getting rich, the growth of this population size should not go
unnoticed. This people are also an active consumer group (Gunter, 1998). Some researchers had suggested that
older adults are people who are 55 years and older (Moschis, Curasi, & Bellenger, 2003; Parks & Fu, 2016; Wildes,
Demicco, & Seo, 2001; Yamanaka & Almanza, 2003). Some suggest that mature adults should be defined according
to the American Association of Retired Person (AARP) which classify those who are 50 years or over as seniors
(Knutson, Elsworth, & Beck, 2006). In this paper, we consider mature adults as those 55 years and older. The aim of
this paper is to investigate the factors influencing the older adults (those 55 years and above) in Malaysia to dine out
(specifically those with maximum education up to secondary school) and the importance of this age segment to the
restaurant industry.

Methodology
A qualitative methodology was employed for this study with in-depth interview used as the method of data collection.
A semi-structured interview questions were used to allow flexibility to how and when the researcher can ask
questions and how the interviewee can answer it (Edwards & Holland, 2013). Informants for the research were
chosen based on purposive sampling. 10 informants above the age of 55 years old were chosen from around Klang

479
Valley. The interviews were conducted in Tamil, Malay and English and recorded using a digital voice recorder. The
obtained data were first transcribed verbatim and translated to English manually. The final data is then content
analysed using Atlas.ti software. The analysis involved both inductive coding and deductive coding.

Results and discussions

Five factors influencing older adults or older consumer in Malaysia to dine out has been identified in this study. Figure
1 shows the visual representation of the factors.

Figure 1: Atlas.ti Visual representation of factors influencing older consumer dining out behaviour

Most of the respondents interviewed have mentioned that the number one factor that influence them to decide to visit
a restaurant are the cleanliness of the restaurant. According to Melorose, Perroy, & Careas (2015), Yamanaka &
Almanza (2003), and Harris & West (1995), cleanliness plays a big role as the criteria in choosing a restaurant.
Below are few of the excerpts from the interview talking about cleanliness:

“I only like if it is clean. It is okay if the food doesn’t taste good but it must be clean. The items used must be
clean as well. Only then I will go. If it is very dirty, I will say that I won’t come there next time. If the place that
we can view itself is dirty, imagine what can happen at the places that are hidden from our view. The service
area itself is not clean and who knows how it is in the back (kitchen). As for that reason I will reject the shop.
“-(Informer 3, 54)

“We might have seen something at certain places. I have seen a bottle of soy sauce that is full of maggot
once in a restaurant. I have not visited the restaurant since. My wife would never go there again. “–
(Informer 1, 63)

Some even do not go to a restaurant when someone who had experienced some incidence there. Word of mouth
could influence an individual’s decision to dine (Ahmad, 2014).

“Yes, if other has complains about the shop, for instance they saw a fly in the food; I will not go to the
restaurant.”- (Informer 4, 67)

The appearance of service staff plays a role in determining the visiting intention of older adults.
“I will see the staff, if their attire is not very (pleasing)… the appetite will go away.” – (Informer 8, 56)

480
Food quality is an important determinant in selecting a restaurant (Yamanaka & Almanza, 2003). These are some of
the interview transcription that supports this statement:

“Haa, clean first then service. Then will order food. Will see how it tastes. If we could accept the taste, we
can visit it again next time. Sometimes the taste…about it being expensive I don’t complain. The taste is
important.”- (Informer 8, 56)

“It is because the food does not taste nice. Certain shop is not clean. If you see, the cutleries are not kept
clean. The food does not taste nice. There was an Indian shop that we went and none of the food tasted
nice. At that time I thought of not coming to this shop ever again.” – (Informer 3, 54)

Service quality is another factor that plays quite an important role in the visiting intention of older consumers (Sun &
Morrison, 2007). There are few sub-themes under service quality as shown in Figure 1; one of it is language barrier.

“I have said that it is better for us to visit a different restaurant rather than trying to explain what we want to a
person that does not understand what we want. That person will not understand what we are saying and we
would not understand what that person is saying. Next time we do not need to go there again. If we order
something, the person will bring something else.” - (Informer 3, 54)

A service staff with good communication skills and good command of language can encourage older consumer into
trying new foods.

“If we go to a new restaurant, we do not know how to order. We do feel like eating few things but we do not
know how to order it. Because of this we do not go to a new restaurant.” - (Informer 1, 63)

Another prominent factor in influencing older consumer in their dining out decision is their social factor. Social factor
is an important determinant in food intake among elderly (Popper & Kroll, 2003). Most of the respondent had
mentioned that they go out frequently with their family member to eat and some prefer going out with friends.

“When we go to a restaurant, we can sit as a family and eat.”- (Informer 1, 63)

“I prefer going with family rather than with friends”- (Informer 7, 56)

“If my son and daughter are with me, we will all go out and eat together.”– (Informer 4, 67)

“Other reasons would be friends. Sometimes friends will invite, say there is a new place. They will say
“come let’s have a drink at this new place”.” – (Informer 5, 65)

As people age, location becomes more important as they “lose some agility, dexterity, eyesight, and speed” in doing
things (Gray & Lane, 2002). Most of the informers prefer eating nearby their residential area. For example:

“There is a shop in front of our house. Since it is nearby our house, we would go there at least once. We
would go there since it is nearby. ”- (Informer 2, 59)

Conclusion
The factors identified in the study provide information to the food operators about the older consumer in Malaysia.
Both local and international food establishment can benefit from this research result to form their marketing plan to
target this untapped segment.

481
Acknowledgement
This research work is financially supported by Universiti Putra Malaysia.

References

Ahmad, N. (2014). Impact of Word of Mouth on Consumer Buying Decision. European Journal of Business and
Management, 6(31), 394–404. http://doi.org/10.5901/mjss.2011.v2n3p497
Edwards, R., & Holland, J. (2013). What is Qualitative Interviewing? “What is?” Research Methods Series (Vol. 7).
http://doi.org/10.5040/9781472545244
Gray, L. P., & Lane, C. W. (2002). Profiling the Silent Generation. Journal of Hospitality & Leisure Marketing, 9(1–2),
73–95. http://doi.org/10.1300/J150v09n01
Hamid, T. A. T. A. (2015). Population ageing in malaysia. Universiti Putra Malaysia Press. Serdang.
Harris, K. J., & West, J. J. (1995). Senior Savvy: Mature Diner’s Restaurant Service Expectations. Hospitality Review,
13(2).
Knutson, B., Elsworth, J., & Beck, J. (2006). Restaurant Discounts for Seniors: Perceptions of the Mature Market.
Cornell Hotel and Restaurant Administration Quarterly, 47(1), 61–74.
http://doi.org/10.1177/0010880405277904
Melorose, J., Perroy, R., & Careas, S. (2015). The Kano’s Method: Evaluation Of Restaurant Service Attributes And
Their Contribution To The Mature Customers’ Satisfaction. Statewide Agricultural Land Use Baseline 2015, 1.
http://doi.org/10.1017/CBO9781107415324.004
Moschis, G., Curasi, C. F., & Bellenger, D. (2003). Restaurant-selection preferences of mature consumers. Cornell
Hotel and Restaurant Administration Quarterly, 44(4), 51–60. http://doi.org/10.1016/S0010-8804(03)90258-8
Parks, S. C., & Fu, Y.-Y. (2016). the Relationship Between Restaurant Service Quality and Consumer Loyalty Among
the Elderly, 25(3), 320–336.
Popper, R., & Kroll, B. J. (2003). Food preference and consumption among the elderly. Food Technology, 57(7), 32–
40.
Sun, Y. H. C., & Morrison, A. M. (2007). Senior citizens and their dining-out traits: Implications for restaurants.
International Journal of Hospitality Management, 26(2), 376–394. http://doi.org/10.1016/j.ijhm.2006.03.003
Wildes, V. J., Demicco, F. J., & Seo, W. (2001). Understanding Mature Customers in the Restaurant Business :
Inferences from a Nationwide Survey. Journal of Restaurant & Foodservice Marketing, 4(3), 81–98.
http://doi.org/10.1300/J061v04n03
Yamanaka, K., & Almanza, B. (2003). Older Americans’ dining out preferences. Journal of Foodservice Business
Research, 6(1), 37–41. http://doi.org/10.1300/J369v06n01

482
 IFRC 2017: 275-253 Others

Hemicellulose Extraction and Characterization of Oil Palm Empty Fruit Bunches

1Nor Nadiha, M.Z., 2Russly, A. B.2*Jamilah, B.

1Halal Products Research Institute, Putra Infoport, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
2Faculty of Food Science and Technology, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia.

Abstract
In Malaysia, local production of oil palm produces huge amount of empty fruit bunches during oil processing.
However, it is not fully utilized and cause a serious environmental issue because of the burning of this by-product.
Thus, the study on utilization of this lignocellulosic biomass is important to improve their added value and
subsequently decrease the agriculture waste. In general, oil palm empty fruit bunches consist of cellulose (30-40%),
hemicellulose (20-30%) and lignin (10-20%). In this study, empty fruit bunches (EFB) have been treated with alkaline
using 4% and 13% of sodium hydroxide in different ratio (1:10 to 1:50). The highest yield of hemicellulose (6.35%)
was obtained from the treatment using 13% sodium hydroxide with 1:10 ratio. Proximate and chemical composition of
oil palm empty fruit bunches also has been determined.

Keywords: Empty fruit bunches, hemicellulose, sodium hydroxide, alkali

*Corresponding author’s email: jamilah@upm.edu.my

Introduction
In recent years, scientist has highlighted agro-industrial waste as important renewable sources to be explore due to
their beneficial and promising potential. Many researchers show interest in agricultural biomass study because its
practical applications in various agro-industrial process, for example, the conversion of hemicellulosic biomass into
fuel or chemicals (Rabetafika, et al., 2014), renewable fuel (Said, et al., 2013), bioethanol (Moradi, et la., 2013; Park
et al., 2010) and biobutanol (Ranjan, et al., 2013). Malaysia produce substantial amount of agricultural biomass
because this country located in the region where biomass productivity is high. The major agricultural crops grown in
Malaysia are rubber (39.67%), oil palm (34.56%), rice (12.68%), cocoa (6.75%) and coconut (6.34%). From this
figure, most of the residues has been disposed by burning and only 27% of agriculture residue are used as fuel for
the kiln drying of timber, manufacture of bricks, curing of tobacco leaves, drying of rubber-sheets and manufacture of
products such as particleboard and fibreboard (Zafar, 2015).

Generally, about 20% of the empty fruit bunches processed for oil extraction is discarded (Astimar et al., 2002). As
one of the world largest oil palm producers, an average of 83.8 million tonnes of empty fruit bunches, EFB was
produced in Malaysia yearly from 2007 to 2011 (MPOB, 2012). Cellulose, hemicellulose and lignin are the main
component of EFB where the estimated compositions are of 39.4% of cellulose, 24.1% of hemicellulose and 13.2%
of lignin (Feria, et al., 2011; Hassan, et al., 2013; Karimi, et al., 2006). Hemicellulose is the second most abundant
biopolymer available in the world and xylan is the main component in hemicellulose (Ebringerová and Thomas,
2000). Hemicellulose are attractive biopolymer because of their structural diversity which can be utilized in their
native and modified forms in various areas including food and non-food applications such as film, adhesives, gelling,
stabilizing and viscosity enhancing additives in food, pharmaceutical and other industrial field (Rabetafika, et al.,
2014). Since there are many alternative methods of isolating hemicellulose from plant cell walls of lignocellulosic
materials such as organic solvent extraction (Sun, et al., 2011), hydrothermal treatment (Sun, et al., 2013) and ionic
liquid extraction (Froschauer et al., 2013), unfortunately, the yield of the extracted hemicellulose are limited. Hence,
this study attempts to investigate the best parameters that could be used for obtaining hemicellulose from EFB.

483
Materials and Methods
3.1 Sample Preparation
Oil palm empty fruit bunch (EFB) was donated by Laboratory of Biopolymer and Derivatives, Institute of Tropical
Forestry and Forest Products, University Putra Malaysia (INTROP, UPM). All samples were dried and chopped into
smaller size before ground and milled using Wiley Mill machine. The samples were sieved through 100-mash size
sieve and kept in airtight container until further analysis.

3.2 Chemical composition of samples

Chemical composition of samples was determined according to TAPPI T264 cm-97 method (TAPPI, 1997).
Proximate analysis was adopted from AOAC method (AOAC, 2005).

3.3 Extraction Process

Prior to extraction process, the samples were going through defatting process to remove the fat in the samples. EFB
powder was treated with 80% ethanol (1:10 w/w) for 18 hrs. The sample was then wash with tap water and dried in
the oven at 45°C overnight. The isolation of hemicellulose was conducted according to the method of (Chimphango,
et al., 2012) with slight modification. De-fatted EFB sample was soaked in 4% and 13% sodium hydroxide in ratio of
1:10, 1:20, 1:30, 1:40 and 1:50 and agitated in water bath at 65°C for 1 hr. The sample was then wash with warm
water (2L) and the supernatant was collected. The solution was then centrifuged at 6000g for 5 min to remove the
large particle in the solution before filtered using disc filter funnel and the solution was adjusted to pH 5 using acetic
acid. Rotary evaporator was used to concentrate the solution before 500 ml of 95% ethanol was added to the solution
to precipitate the hemicellulose. The sample was centrifuged at 6000 g for 5 min and the precipitate was collected
and oven dried overnight at 55°C before ground and kept for further analyses.

Results and discussion

Table 1 shows the proximate composition of EFB samples before pre-treatment. The moisture and fat content of EFB
was 11% and 12.5%, respectively. Higher content of fat and extractive in samples could interfere the extraction
process. Thus, the fat content in samples need to be remove from the sample prior to extraction. After defatting
process, fat content of each sample decreased to 3-4%. Other interference in extraction of hemicellulose is also
could come from extractive free content. However, it is assumed that the extractive free from subjected samples were
leached out during defatting process. Extractive free is a small component in wood, which consists of lipid, phenolic
compound, alkanes, proteins, monosaccharides and derivatives. Empty fruit bunches (EFB) represent approximately
52.6% of cellulose, 23.6% of hemicellulose, 19.1% of lignin and 1.60% of extractive free (Table 1). The result
obtained is in agreement with other researcher (Sreekala, et al., 1997).

Table 1. Lignocellulosic composition and proximate value of empty fruit bunches.

Sample Empty Fruit Bunches

Moisture (%) 11.7 ± 0.5
Ash (%) 4.4 ± 0.7
Fat (%) 12.5 ± 0.1
Cellulose (%) 52.6 ± 2.1
Hemicellulose (%) 23.6 ± 6.0
Lignin (%) 19.1 ± 1.8
Extractive free (%) 1.6 ± 0.9

484
 7 13%
6 4%
5
Yield (%) 4
3
2
1
0
1:10 1:20 1:30 1:40 1:50
Sample:NaOH ratio

Figure 1. Yield of EFB hemicellulose extracted with 13% and 4% sodium hydroxide at different ratio.

Higher concentration of sodium hydroxide will affect the dissolution of hemicellulose and lignin (Figure 1). The
highest yield of hemicellulose is obtained from treatment with 13% sodium hydroxide at 1:10 ratio. The yield of
hemicellulose from 13% NaOH pre-treatment is higher than 4% NaOH pre-treatment. Cabrera et al., (2014) also
found the increment of hemicellulose yield when higher concentration of NaOH has been subjected to rice straw.
Increasing the ratio of NaOH to the sample also decrease the yield of hemicellulose. EFB are from softwood family
and has lower lignin and loose structure hence the extractability of their hemicellulose is higher compared to
hardwood structure (Ebringerova and Heinze, 2000).

Conclusion
Different yield of hemicellulose was obtained from EFB when subjected to pre-treatment with different concentration
of sodium hydroxide at different ratio. From the results, hemicellulose obtained from EFB treated with 13% of NaOH
concentration and ratio, 1:10 sample to NaOH could be the potential sample for further study because of the higher
yield obtained when compared to others.

Reference

AOAC. 2005. Official methods of analysis of AOAC International 17th ed. AOAC International, Arlington, VA, USA.

Cabrera, E., Muñoz, M. J., Martín, R., Caro, I., Curbelo, C., and Díaz, A. B. 2014. Alkaline and alkaline peroxide
pretreatments at mild temperature to enhance enzymatic hydrolysis of rice hulls and straw. Bioresource
Technology, 167: 1–7.
Chimphango, A. F. A., Van Zyl, W. H., and Görgens, J. F. 2012. Isolation, characterization and enzymatic
modification of water soluble xylans from Eucalyptus grandis wood and sugarcane bagasse. Journal of
Chemical Technology and Biotechnology, 87(10): 1419–1429.
Ebringerova, A., and Heinze, T. 2000. Xylan and xylan derivatives – biopolymers with valuable properties , 1
Naturally occurring xylans structures , isolation procedures and properties, 556(9): 542–556.
Feria, M. J., López, F., García, J. C., Pérez, A., Zamudio, M. A. M., and Alfaro, A. 2011. Valorization of Leucaena
leucocephala for energy and chemicals from autohydrolysis. Biomass and Bioenergy, 35(5): 2224–2233.
Froschauer, C., Hummel, M., Iakovlev, M., Roselli, A., Schottenberger, H., and Sixta, H. 2013. Separation of
hemicellulose and cellulose from wood pulp by means of ionic liquid/cosolvent systems. Biomacromolecules,
14(6): 1741–50.
Hassan, O., Ling, T. P., Maskat, M. Y., Illias, R. M., Badri, K., Jahim, J., and Mahadi, N. M. 2013. Optimization of

485
 pretreatments for the hydrolysis of oil palm empty fruit bunch fiber (EFBF) using enzyme mixtures. Biomass
and Bioenergy, 56(0): 137–146.
Karimi, K., Kheradmandinia, S., and Taherzadeh, M. J. 2006. Conversion of rice straw to sugars by dilute-acid
hydrolysis. Biomass and Bioenergy, 30(3): 247–253.
MPOB. 2012. Sectoral status: Fresh fruit bunch (FFB) processed by mill. Retrieved on 3 Jun 2015 from website
http://bepi.mpob.gov.index.php/statistics/sectoral-status.html
Moradi, F., Amiri, H., Soleimanian-Zad, S., Ehsani, M. R., and Karimi, K. 2013. Improvement of acetone, butanol and
ethanol production from rice straw by acid and alkaline pretreatments. Fuel, 112: 8–13.
Park, J. Y., Shiroma, R., Al-Haq, M. I., Zhang, Y., Ike, M., Arai-Sanoh, Y., Ida, A., Kondo, M. and Tokuyasu, K.
(2010). A novel lime pretreatment for subsequent bioethanol production from rice straw - Calcium capturing by
carbonation (CaCCO) process. Bioresource Technology, 101(17): 6805–6811.
Rabetafika, H. N., Bchir, B., Blecker, C., Paquot, M., and Wathelet, B. 2014. Comparative study of alkaline extraction
process of hemicelluloses from pear pomace. Biomass and Bioenergy, 61(0): 254–264.
Ranjan, A., Khanna, S., and Moholkar, V. S. 2013. Feasibility of rice straw as alternate substrate for biobutanol
production. Applied Energy, 103: 32–38.
Said, N., Bishara, T., García-Maraver, A., and Zamorano, M. 2013. Effect of water washing on the thermal behavior
of rice straw. Waste Management, 33(11): 2250–2256.
Sreekala, M. S., Kumaran, M. G., and Thomas, S. 1997. Oil palm fibers: Morphology, chemical composition, surface
modification, and mechanical properties. Journal of Applied Polymer Science, 66(5): 821–835.
Sun, S. L., Wen, J. L., Ma, M. G., and Sun, R. C. 2013. Successive alkali extraction and structural characterization of
hemicelluloses from sweet sorghum stem. Carbohydrate Polymers, 92(2): 2224–2231.
Sun, Y. C., Wen, J. L., Xu, F., and Sun, R. C. 2011. Organosolv- and alkali-soluble hemicelluloses degraded from
Tamarix austromongolica: Characterization of physicochemical, structural features and thermal stability.
Polymer Degradation and Stability, 96(8): 1478–1488.
Tappi. (1997). Preparation of wood for chemical analysis. Standard by Technical Association of the Pulp and Paper
Industry, 1–3.
Zafar, S. (2015) Agriculture Biomass in Malaysia. September 2015. Retrieved on 10 Jun 2017 from website
www.bioenergyconsult.com/agricultural-biomass-in-malaysia.

486
 Halal Food

Effect of Heating time and temperature to the dielectric properties of pork and chicken in the range of 0.5 –
50 GHz
Zurina Zainal Abidin*, Fatin Nordalila Omar, Dayang Radiah Awang Biak
Department of Chemical and Environmental Engineering, Faculty of Engineering,
Universiti Putra Malaysia, 43400 Serdang, Selangor.
Tel: 603-8946 4371, Fax: 603-8656 7120

Abstract
The interaction of materials with electromagnetic energy in microwave range provides dielectric constant (ε′) and loss
(ε″) values that can be used to characterize food quantitatively. It offers a rapid, easy to use and low cost detection
compared to current lab-based method. In this work, the effect of changing the heating time and temperature of the
pork and chicken samples towards the dielectrics properties were investigated. All meat samples were mixed in a
water and samples were heated (stewing) at different time and different temperature. After subjected to the heating
process, the dielectric measurements for all samples were taken using VNA machine. ε′ values of chicken samples
decreased with increasing frequency and increased with heating time. The higher ε′ values of 120 min chicken
samples means longer time of heating gives higher potential for electrical energy storage in the flesh. The spectra of
ε′ and ε′’ are dependent on frequency. On the other hand, different results were seen for pork samples. The
significant peak was obtained at 31.19 GHz (ε′) and 32.18 GHz (ε″). Values of ε′ and ε″ of pork are highest at 120 min
and lowest at 30 min. The dependency with frequency are similar to chicken samples. At different temperature, the
dielectric constant of chicken show increment with the rising in temperature and decrease smoothly (with no
significant peak) with the increasing of frequency. Pork exhibits similar trend to chicken whereby ε′ values decrease
with increasing of frequency. Similarly a distinct peak like before was observed at 31.19 GHz (ε′) and 32.18 GHz (ε″).
The above results indicated that at different temperature and different heating time, dielectric properties changed.
Furthermore, only for pork sample, a distinct peak can be seen clearly and this help to further differentiate the
samples from one another. These results show promising development in using dielectric properties as potential
detection methods.

Keywords: Dielectric Properties, Halal, Microwave and Heating

*Corresponding author’s email: zurina@upm.edu.my

Introduction
Determination of halal consumer products has been a global issue for a long time and remains largely unsolved.
Traditionally, the verification of halal status of products was made through field monitoring processes that can be
costly, time-consuming and cumbersome. Researchers have striven to develop techniques in detecting and
identifying species origin in products since last few decades due to the economic, health and religious issues.
Murugaiah et al. (2009) reported that some of the currently existing methods such as PCR based detection (Che Man
et al, 2007), Fourier Transform Infrared (FTIR) spectroscopy (Syahariza et al., 2005) and High-Performance liquid
chromatographic (HPLC) analysis (Marikkar et al., 2005) are expensive, not suitable for routine analysis and
complex. The efforts in finding alternative new and improvised methods had been done continuously. Microwave
dielectric is very attractive and powerful method for characterizing food and some of its components in a non-invasive
way. Here, the use of dielectric technique within electromagnetic radiation in MW range on food was studied. It is
closely related to the storage and dissipation of electric and magnetic energy in materials at radio-frequency (RF) and
microwave (MW) heating (Venkatesh and Raghavan, 2004) which give data on dielectric constant and loss. This
technique offers a rapid, easy to use and low cost detection. Although dielectric is not a new technology, however,
early works mostly focus on the characterization of a material properties such as aqueous ionic solutions, water
solvent, pharmaceutical powders and meat (Lyng et al., 2005). Most of the previous works related to food were also
performed in low frequency range (< 25 GHz). Abidin et al 2016 has investigated the use of dielectric properties for
the detection of pork, chicken and beef samples at high frequency range. In this work, the effect of temperature and

487
heating time to the dielectric properties of meat samples will be investigated.

Materials and Methods

Meat Sample Preparation

Two types of fresh raw meat (pork loin and chicken breast) were obtained from local supplier in Seri Kembangan,
Selangor. Chicken represent halal sample (undergone Islamic ritual) slaughtering process and pork represents the
haram sample. Each different meat type (pork, and chicken) was ground separately with a household blender
attachment for 1 min.

Variation of Heating Time to the Sample

First part of experiments studied on the effect of heating time to the dielectric properties of samples. The grinded
meat was filled in a 500 mL beaker containing water with a ratio of 1:3. The top of the beaker was covered with
aluminium foil that had small holes. The beaker was then placed on the hotplate and started the heating in moderate
mode. A small amount of meat was taken out at 30, 60, 90 and 120 min. After treatment, samples were equilibrated
in room temperature about 1 h to 2 h before subjected to dielectric measurement.

Variation of Heating temperature of Sample

The second parameter involves heating within 1 h using hotplate at controlled temperature. The hotplate was set at
temperature of 60 °C, 70 °C, 80 °C and 90 °C.. When the samples reached the temperature setting value, the value
was maintained at ± 2 °C of accuracy. A temperature probe (attached to hotplate) was immersed into the samples in
order to monitor the set temperature.

Dielectric Properties Measurement

The dielectric response was measured using Agilent N5245A PNA-X which consists of a signal source, a receiver,
open-ended probe, a screen and software to analyze data. After auto calibration, the prepared sample was placed in
100 mL beaker and the probe was attached slowly to the sample to measure the complex permittivity in 0.5–50 GHz
range. The values of dielectric constant (ε′) and dielectric loss factor (ε″) were calculated by the system and
displayed.

Results and Discussion

Variation of Heating Time

ε′ 30 min Figure 1 shows ε′ values of chicken samples
ε′ 60 min systematically decreased with increasing frequency.
ε′ 90 min An exception was shown in the beginning of
ε′ 120 min frequency which there is small increment values from
50 ε″ 30 min 0.5 GHz to 4.46 GHz. ε′ values increase with
increasing time of heating. The higher ε′ values of
ε

120 min chicken samples explained the longer time

of heating gives higher potential for electrical energy
storage in the flesh. The meat undergone more
transformations and permanent changes in its
0 structure as longer heating time was imposed. The
0 10 20 f, 30 40 50 longer heating time may give more time for the water
Figure 1: The measured spectra G of dielectric constant and to seep into the meat.
loss factor for chicken samplesHz
in different time of heating.

Thus, the water content as well as water binding capacity in meat influences the increment values of ε′. The spectra
of ε′ indicate the frequency dependence of ε′ was apparent at any of the heating time as well as in loss factor spectra.

488
 Values of ε″ decreased shortly in the beginning of frequency, then started to increase until frequency around 30 GHz
and slowly decreased until final measured frequency. The dependence on heating time shows loss factor decreases
with increasing time of heating. These results are similar to the description made by Kent et al. (2001). Dielectric loss
factor, that depended on polar and ionic conduction was influenced by these factors and caused a fall in loss factor
values.

Figures 2a and 2b show the spectra of heated pork. A different trend for both dielectric constant and loss factor can
be seen whereby significant Peak B obtained at 31.19 GHz (ε′) and 32.18 GHz (ε″) is similar to Abidin et al., 2016
was observed as in sterilized pork. The appearance of the peaks shows possibility of some substances in pork that
cannot be eliminated even heated in a long time. Values of ε′ and ε″ of pork are highest at 120 min and lowest at 30
min. Laycock et al. (2003) suggested the products that cooked in water bath had tendency to lose more fat than
water content. Heat melts the fat in meat, causes the meat fiber shrinks, and hardens with intense heat. The water
level in meat increases through absorption and proteins undergo denaturation process. Denatured protein gives less
solubility effect and increases the viscosity of the food. The ability of meat to bind its own water and added water
increases during heating but after a while it becomes constant or slowly decreases due to protein denaturation
(Davidek et al., 1990).

Variation of Heating Temperature

In this part, the sample was heated to a particular temperature and maintained throughout the process for 1 h of each
meat samples before it stabilized and subjected to dielectric measurement. Dielectric constant of chicken show
increment with the rising in temperature and decrease smoothly (with no significant peak) with the increasing of
frequency. Meanwhile, ε″ values indicate the increment until one point called relaxation frequency (20.3 GHz) and
start to decrease until 50 GHz. Figures 3a and 3b present the dielectric spectra of pork samples. Both ε′ and ε″ at
90°C sample has the highest values among other samples. Nevertheless, it is inconsistent at the beginning of
frequency. As expected, Peak B emerges at similar points at frequency of 31.19 GHz (ε′) and 32.18 GHz (ε″). The
dielectric constant of pork ε′ values decrease with increasing of frequency, similar to chicken sample. Dielectric loss
factor meanwhile, shows unstable waves upon the occurrence of fluctuations at certain frequency. Other than effect
of water content, the rising in temperatures also is closely related to protein denaturation occurring in sarcoplasmic
proteins (Davidek et al., 1990). Coagulation of meat protein starts at ~35°C and reaches a maximum between 60°C
– 65°C

80 30 min 30
60 min
60 90 min
Peak B
Peak B 20
120 min
″

raw pork
ε

40
ε′

30 min
10
20 60 min
90 min
0 120 min
0
0 10 20 30 40 50 0 10 20 30 40 50
f, GHz f, GHz
Figure 2. The measured spectra of dielectric constant (a) and dielectric loss (b) for pork samples in different time of
heating.

489
80 .60 oC 30
.70 oC
.80 oC 25 Peak B
60
. o

″
ε
90 C
raw pork 20
Peak B
ε
′

40 60 oC
15
70 oC
20 10 80 oC
90 oC
5 raw pork
0
f, GHz30 0 10 20 30 40 50
0 10 20 40 50 f, GHz
Figure 3. The measured spectra of dielectric constant (a) and dielectric loss (b) for pork samples in different
temperature of heating.

Conclusion
Heating time and heating temperature affect the dielectric properties of samples. However, pork sample showed
significant distinct peak that is not available in other chicken samples that could serve as potential biomarker.
This dielectric property promises a feasible detection and discrimination technique between halal and non-
halal products.

Acknowledgements
Universiti Putra Malaysia (UPM) for grant (05-05-10-1079RU), HRPI, UPM and Agilent.

References
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pork, chicken and beef using dielectric properties in the frequency of 0.5 Ghz-50Ghz, International Journal of
Food Properties, 19(5),
Davidek, J., Velisek, J. and Pokorny, J. (1990). Chemical Changes During Food Processing. Volume 21. New York:
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meat product manufacture. Meat Science 69: 589-602.
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microwave dielectric spectra. Food Control 12: 467-482.
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and Halal authentication analysis using mitochondrial DNA. Meat Science 83: 57-61.
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palm olein using an electronic nose. Food Chemistry 90: 829-835.
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agri-food materials. Biosystems Engineering 88(1): 1-18.
Syahariza, Z. A., Che Man, Y. B., Selamat, J. and Bakar, J. (2005). Detection of lard adulteration in cake formulation
by Fourier transform infrared (FTIR) spectroscopy. Food Chemistry 92: 365-371.
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animal fats in admixtures of some vegetable oils using liquid chromatographic data coupled with multivariate
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