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JOURNAL OF FOOD, AGRICULTURE & ENVIRONMENT

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Vol. 12, No. 3&4

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Chu Thanh Chau

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M. Pessarakli (Prof.), (USA)
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Muhammad Zia-Ul-Haq (Dr.), Pakistan
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Konstantinos M. Kasiotis (PhD), Greece
Gunjan Mukherjee (PhD), India
Sunil Pareek (ass. Prof.), India
Yueming Jiang (Prof.), China
Bhaskar C Behera (Dr.), India
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Fnor Casierra-Posada (Dr.), Colombia

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Mirza Barjees Baig (Prof.), Canada
Emad Al-Karablieh (Prof.), Jordan
Tugay Ayasan (Dr.), Turkey
Changhe Lu (Prof.), China
Muhammad Munir (Dr.), UK
P. Duraimurugan (Dr.) India
Shamel M. Alam-Eldein (Dr.),Canada
G. D. Satish Kumar (Dr.), India
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Ketema Tilahun Zeleke (Dr.), Australia


SANJEEV AGARWAL (Prof.), India
Wei Hu (Dr.), Canada
Xiaohua YU (Prof.), Germany
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Hui-Lian Xu (Dr.), Japan
Hongsheng Liu (Ass.Prof.), China
Senthilvel Senapathy (Dr.), India

July-October 2014
Jiban Shrestha (Dr.), Nepal
Adams Sadick, Ghana
Ming Meng (Prof.), China
Mostafa Moradzadeh (PhD.), Iran
Rachna Chandra (Dr.), India
Viktor J. Bruckman (Dr.), Austria
Talal Almeelbi (Dr.), Saudi Arabia,
Lin Du (Ass.Prof.), Denmark
Masayuki Aizawa (Dr.), Japan
Gordana Medunic (Prof.), Croatia

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Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

WFL Publisher

www.world-food.net

Science and Technology


Vol. 12, No. 3&4

CONTENTS

July-October 2014

Chemical composition of pumpkin (Cucurbita maxima D.)


flesh flours used for food

Food & Health


Reduction of foodborne pathogens in parsley by an improved
formulation containing lime and oregano extracts

Jurgita Kulaitien, Elvyra Jarien, Honorata Danilenko, Judita


erniauskien, Agata Wawrzyniak, Jadwiga Hamulka and
Edita Jukneviien

61

Alany Celestino, Brianda Jaime, Ricardo Luvano, Luisa Sols, Santos


Garca and Norma Heredia
6

Training a sensory panel for describing texture in peach and


nectarines

Food security of Northwest China under current water

Loreto Contador, Paulina Shinya, Andrea Bunger, Carmen Senz and


Rodrigo Infante
65

resources and food consumption patterns


Jianping Li, Jing Chen and Zhouping Shangguan

12

Modified atmosphere storage of banana (Musa acuminata)


using diffusion channel and mathematical modelling of steadystate oxygen concentration within the package
Arulselvam Karthiayani, Nachimuthu Varadharaju and
Madasamy Siddharth

19

Anti-inflammatory and anti-cancer effects of -carotene,


extracted from Dunaliella bardawil by milking
24

Effects of some technological parameters on chemical and


sensory qualities and free fatty and amino acids of various
probiotic cultures in Beyaz cheese during ripening process
32

77

The in vitro antibiofilm activity of Rosmarinus officinalis L.


essential oil against multiple antibiotic resistant
Pseudomonas sp. and Staphylococcus sp.

Effect of storage time and temperature on the quality


characteristics of chicken eggs
Yeasmin Akter, Azhar Kasim, Hishamuddin Omar and Awis Qurni
Sazili
87

40

Improving the layout of ventilation ports in packaging for fresh


produce using computational fluid dynamics
Hiroaki Kitazawa and Naoko Hasegawa

Inhibitory effect of gamma radiation in degrading and


preventing fungal toxins

Ozgur Ceylan, Aysel Uur, Nurdan Sara, Filiz Ozcan and Tuba
Baygar
82

The effects of mint (Mentha spicata) essential oil fortified


edible films on the physical, chemical and microbiological
characteristics of lor cheese
Gkhan Kavas and Nazan Kavas

Muhammad T. Sultan, Masood S. Butt, Saeed Akhtar, Atif N. Ahmad,


Mubasher Rauf, Muhammad S. Saddique and Ambreen Naz
71

Amira Hassan Abdullah Al-Abdalall

Abeer M. Badr, Effat F. Shabana, Hoda H. Senousy and


Hend Y. Mohammad

Filiz Yanglar and Salih Ozdemir

Antioxidant and antimicrobial potential of dried cumin


(Cuminum cyminum L.), caraway (Carum carvi L.) and
turmeric powder (Curcuma longa L.)

46

Evaluation of the antioxidant activity of extracts from Psidium


guajava L. and Anacardium occidentale L. leaves obtained by
different extraction methods
Suzara R. C. Sena, Theresa R. F. Dantas and Camila G. Pereira

93

Validation of ELISA-based detection of L. monocytogenes and


E. coli O157:H7 in fresh cut vegetables

Peanut protein isolates improve the nutritional quality of


muffins that can be handy tool to cure protein energy
malnutrition in developing economies
Muhammad Sibt-e-Abbas, Masood Sadiq Butt, Muhammad Tauseef
Sultan, Atif Nisar Ahmad, Muhammad Abrar and Mir Muhammad
Nasir Qayyum
51

Assessment of microbiological quality of ready-to-eat foods in


Istanbul, Turkey
Vecdet z, Sukriye Karadayi, Hseyin akan, Beytullah Karadayi and
Filiz Ekim evik
56

Marina Cavaiuolo, Antonio Ferrante, Spiros Paramithiotis, Agni


Hadjilouka, Periklis Tzamalis and Eleftherios H. Drosinos

98

Composition and content of selected elements of Croatian


blackberry wines
Ivana Alpeza, Tatjana Varga and Veronika Kubanovi

100

Physicochemical properties of honey samples from Ondo state,


Nigeria, and their bioactivity against spoilage and pathogenic
organisms
Funmilola Oluyemi Omoya, Oluwatosin Ademola Ijabadeniyi and
Olayemi Bosede Ogonnoh
104

ii

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Shigatoxin-producing Escherichia coli in raw cow milk from


small farm producers and phylogenetic subtype determination

Genotypic variability for nutrient, antioxidant, yield and yield


contributing traits in vegetable amaranth

Ivo Sirakov, Ralitsa Popova, Hristo Daskalov, Iskra Slavcheva, Eva


Gyurova and Boyko Mitov
108

Umakanta Sarker, Md. Tofazzal Islam, Md. Golam Rabbani and


Shinya Oba
168

Pre-processing glutinous rice effects on textural and


morphological changes

Respiratory activity of rice seeds stored for 10 years at

Viboon Pansa-ead, Kannika Huaisan, Supan Yangyeun and


Songchai Wiriyaumpaiwong

different temperatures
115

Agriculture

Effect of dietary crude palm oil on quality and oxidative stability


of chicken eggs

Assessment of performance ability of Cabernet Sauvignon,


Merlot and Syrah wine cultivars on Southeast region of Turkey
Dilek Deirmenci Karata and Hseyin Karata

122

Dry matter accumulation trend on corn (TWC 647) as affected


by plant density and planting pattern
Ali Reza Saberi and Siti Aishah Hassan

Fabola de Oliveira Krger, Dario Munt de Moraes, Daniel Fernandes


Franco, Caroline Jcome Costa, Chaiane Fernandes Vaz and
Paula Rodrigues Gayer Ribeiro
175

127

Yeasmin Akter, Azhar Kasim, Hishamuddin Omar and Awis Qurni


Sazili
179

Evaluation a polyvalent vaccine against abscess disease of


sheep from pathogenic bacteria isolated from Saudi Arabia
K. B. Alharbi

Performance evaluation of the Telagasari Irrigation Scheme


(TIS) of Karawang Regency, Indonesia

Productivity and gas exchange parameters of selected pasture


grasses under drought stress

Sangam Shrestha, Foyya Yusufu Aquino and Vishnu P. Pandey

Anna Koco and Mariola Staniak

Biodegradation of imidacloprid in an open compost pile

131

Effect of sulphur fertilization on fatty acid composition of faba


bean (Vicia faba L.), white lupin (Lupinus albus L.) and pea
(Pisum sativum L.) grains
Eugenio Cazzato, Vito Laudadio, Edmondo Ceci and
Vincenzo Tufarelli

136

Yield and quality of Cenchrus ciliaris (L.) affected by nitrogen


and phosphorus fertilization
Ihsan Abu-Alrub, Ahmed Aran, Omar Hamad and
Abdelaziz Awaga

139

Meo Maksim, Murrja Arif, Ndregjoni Agim and Cerpja Teuta

143

Improved water use efficiency in rice under limited water


environment through microbial inoculation

Numerical investigation into optimal agricultural water


management for typical soils using HYDRUS-1D model
Po Li, Feiqing Wu and Kefeng Zhang

155

Effects of allelopathic crop water extracts and their


combinations on weeds and yield of rainfed wheat
Shahbaz Hussain, Fayyaz-ul Hassan, Muhammad Rasheed, Safdar Ali
and Mukhtar Ahmed

161

198

Potential methane production from manure of cattle fed diet


supplemented with wet brewery grain
Larissa S. Mallmann, Simone Damasceno, Maximiliane A. Zambom,
Mnica S. S. M. Costa, Douglas G. B. Torres and
Jefferson L. G. Silva
203

Influence of the auxin-like activity of humic acid on bio and


microbiometric parameters of Pisum sativum L. by in vitro
cultures of pea plants
209

Biotransformation of 2-(4-methoxybenzyl)cyclopentanone by
Solanum aviculare and Rheum palmatum plant cells
Petr Soudek, Zdenk Wimmer and Tom Vank

Mohamad Husni Omar, Zulkarami Berahim, Norazrin Ariffin, Mohd


Razi Ismail, Halimi Mohd Saud, Nurul Amalina, S. H. Habib and
H. Kausar
149

187

eljko Herner, Renata Baok and Felicita Briki

Andrzej Gawlik, Danuta Kulpa, Dorota Gobiowska and


Romualda Bejger

Some alternatives of improvement the cow milk production


efficiency in Albania: Cash flow analysis

182

213

Impact of nitrogen fertilisation and irrigation on water utilization


efficiency, N accumulation, growth and yields of Zea mays L.
Waleed Hassan Abou El-Hassan, Emad Maher Hafez, Alaa A. Ahmed
Ghareib, Mohamed Ragab Freeg and Mahmoud Fathy Seleiman 217

Effect of water stress on yield components of sorghum


(Sorghum bicolor)
Cndido Ferreira de Oliveira Neto, Ricardo Shigueru Okumura, Ismael
de Jesus Matos Vigas, Herclito Eugnio Oliveira da Conceio, Lucila
Elizabeth Fragoso Monfort, Raimundo Thiago Lima da Silva, Jackeline
Arajo Mota Siqueira, Luma Castro de Souza, Roberto Cezar Lobo da
Costa and Daiane de Cinque Mariano
223

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

iii

Linseed response to treatment with swine wastewater as


biofertilizer
Jhonatas Antonelli, Cleber Antonio Lindino, Reginaldo Ferreira
Santos, Samuel Nelson Melegari de Souza, Willian Czar Nadaletti,
Paulo Cremonez, Eduardo Rossi and Flvio Gurgacz
229

Thermic sum and crop coefficient of canola (Brassica napus L.)


for the region of Tangar da Serra, Mato Grosso State, Brazil
Kssio De Marco, Rivanildo Dallacort, Adalberto Santi, Ricardo
Shigueru Okumura, Mirian Hiroko Inoue, Joo Danilo Barbieri,
Dejnia Vieira de Araujo, Roberto Antnio Savelli Martinez and Willian
Fenner
232

Soil health sustainability and organic farming: A review


Sudarsan Biswas, Md. Nasim Ali, Rupak Goswami and Somsubhra
Chakraborty
237

Ergonomic assessment of traditional and improved methods of


paddy threshing for drudgery reduction of hill region
Divya Singh and Deepa Vinay

244

Phenology and green leaf yield of coriander at different sowing


dates and harvesting times
Sagarika Guha, Amit Baran Sharangi and Sandip Debnath

251

Applications of xerophytophysiology in plant production:


Potato yield increase induced by drying the cut trace of seed
tuber blocks
Feifei Su, Hui-lian Xu, Fenglan Li, Feifei Qin, Yili Chen, Dianqiu L,
Linshuang Hu, Yong Li, Shaopeng Wang and Ying Shi
255

Hydric and physiological monitoring of soybean in oxisol and


oxisol incorporated with biodegradable waste residue
Alexandre C. Salvestro, Paulo Sergio Loureno de Freitas, Roberto
Rezende, Erci Marcos Del Quiqui, Cludia Regina Dias Arieira, Jos
Carlini Junior, Magnun Rodrigo Silva, Vinicius Hicaro Frederico
Abe and Matheus A. Mendes
265

Sowing dates and plant density of peanut cultivars in different


soil and climatic conditions of Mato Grosso state, Brazil
Joo Danilo Barbieri, Rivanildo Dallacort, Adalberto Santi, Kssio
De Marco, Alcir Jos Modolo, Santino Seabra Jnior, Ronicely
Pereira Rocha and Rafael Cesar Tieppo
269

Effect of composted sewage sludge on durum wheat:


Productivity, phenolic compounds, antioxidant activity, and
technological quality
Antonella Pasqualone, Laura Nunzia Delvecchio, Giovanni Lacolla,
Luciana Piarulli, Rosanna Simeone and Giovanna Cucci
276

Genetic diversity and presence of DREB gene in watermelon


cultivars and wild type of watermelon based on molecular
markers
Abdullah S. Alsohim and Mohamed I. Motawei

281

Impact of PAR interception at different time points on total


dry matter production in rice (Oryza sativa L.) crop
transplanted on different dates
Shrabani Basu, Srijani Maji, Swaraj Kumar Dutta , Sarika Jena,
Rajib Nath and Prodip Kumar Chakraborty
285

Growth and visual symptoms of nutrient deficiencies in young


Mentha piperita plants
Diocla Almeida Seabra Silva, Mrio Lopes da Silva Jnior, Ismael de
Jesus Matos Vigas, Allan Klynger da Silva Lobato, Vnia Silva de
Melo, Snia Maria Arajo Botelho, George Rodrigues da Silva, Joze
Melisa Nunes de Freitas, Cndido Ferreira de Oliveira Neto, Milton
292
Leite Alves da Cunha and Ana Regina da Rocha Araujo

Decrease in photosynthetic pigments promotes negative


consequences on carbon compounds in young Euterpe oleracea
plants submitted to progressive water deficiency
Emilly dos Santos Pereira, Allan Klynger da Silva Lobato, Odyone
Nascimento da Silva, Argemiro Pereira Martins Filho, Carla Leticia
Figueredo de Carvalho Souza, Tiago Rodrigues Ferreira, Gustavo
Antonio Ruffeil Alves, Elaine Maria Silva Guedes, Ismael de Jesus Matos
Vigas, Ricardo Shigueru Okumura, Augusto Jos Silva Pedroso, Roberto
Cezar Lobo da Costa and Benedito Gomes dos Santos Filho
297

Environment
Microbes and dietary values of some major fish sources
in Nigeria
Olajide Adedayo Ajayi, Emmanuel I. Adeyeye and Anthony I.
Okoh
303

Impact of micro credit and training on efficiency of smallscale entrepreneurs: Evidence from National Directorate
of Employment (NDE) loan/training programmes in
Nigeria
Olumide Oyewole Akinrinola, M. M. Fasoranti and Oluyede
Adeleke Aturamu

307

Modulation of micronutrients and antioxidants defenses in


Conocarpus lancifolius under abiotic stress
Amina Redha, Redha Al-Hasan and Mohammad Afzal

312

Heavy metal distribution in Fagonia indica and Cenchrus


ciliaris
native vegetation plant species
Adel M. Ghoneim, Soud M. Al-Zahrani, Salem E. El-Maghraby
and Abdullah S. Al-Farraj
320

Performance of lateral move type sprinkler irrigation


system
Jonathan Dieter, Silvio C. Sampaio, Gisele Vogel , Mrcio A. Vilas
Boas, Elisandro P. Frigo and Alvaro Mari Junior
325

Improving water quality in the Nile Delta irrigation


network by regulating reuse of agricultural drainage
water
Abd Elhamed Khater, Yoshinobu Kitamura, Katsuyuki Shimizu ,
Hiroaki Somura and Waleed H. Abou El Hassan
329

iv

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Antioxidant potential and secondary metabolite content of


grape berries influenced by microclimate

Trophic State Index (TSI) applied in the assessment of anthropic

Mustafa Ozden

Adir Otto Schmidt, Slvio Cesar Sampaio, Ralpho Rinaldo dos Reis, Camila
Jussara Schmidt, Edison Barbosa da Cunha and Lisdefferson Hamann
Andrade
400

338

Tourists perception on local economy of Terengganu state


in Malaysia
Md. Anowar Hossain Bhuiyan, Chamhuri Siwar, Shaharuddin
Mohamad Ismail and Aini Aman
345

Identification of brownfields in China: Concept, procedure


and practice
Hongbin Xie, Mingshui Lin , Changchun Zhou, Yang Zhang and
Liangjin Zhou
349

Evaluation of the microbiological and physicochemical quality


of artesian well water used for irrigation in ArRiyadh
Sulaiman Ali Alharbi, M. E. Zayed, Arunachalam Chinnathambi, Naiyf
S. Alharbi and Milton Wainwright
355

X-ray diffraction (XRD) and x-ray fluorescence (XRF) analysis


of ancient bricks from Sungai Batu Temple (site SB1), Bujang
Valley, Kedah

impacts on the surface water of a watershed

Seasonal elemental variations of Fe, Mn, Cu and Zn and


conservational management of Rastrelliger kanagurta fish from
Karachi fish harbour, Pakistan
Quratulan Ahmed, Farzana Yousuf, Maliha Sarfraz, Nor Kartini Abu
Bakar, Mansour A. Balkhour and Muhammad Aqeel Ashraf
405

The use of urea molasses multinutrient block on pica symptom


of cattle
Haili Li, Keling Wang, Limin Lang, Yindi Xu, Qinxian Zhang, Wenhao
Zhu, Lixian Zhang, Yi You, Feng Xu and Wan Lu
415

Views and attitudes of mulberry cultivators according to the


Regulation (EC) No 1257/99: The case of Evros prefecture
S. Ch. Tsiantikoudis , A. Parissi , Z. Papadopoulou , A-M. Fidani-Mantzoula,
420
G. Kourtelis and Z. Andreopoulou

Zuliskandar Ramli, Nik Hassan Shuhami Nik Abdul Rahman, Abdul


Latif Samian, Muhammad Rizal Razman, Sharifah Zarina Syed Zakaria
and Hossein Sarhaddi Dadian
360

A proposal to standardize herbicide sorption coefficients in


Brazilian tropical soils compared to temperate soils

Estimating residents willing to pay using contingent valuation


for ecological restoration and recreational benefits of AL-Prespa

Kassio Ferreira Mendes, Marcelo Rodrigues Dos Reis, Ana Carolina


Ribeiro Dias, Jos Ari Formiga , Pedro Jacob Christoffoleti and Valdemar
Luiz Tornisielo
424

protected area in Albania


Dorina Grazhdani

365

Jatropha cake as a fertilizer for the growth of Blc. Amy Wakasugi


Yamanashi orchid
Roberto A. Ribeiro, Maria Flvia R. Starling and Luiza A. R.
Rossi-Barbosa
371

In vitro regeneration of Acacia crassicarpa A. Cunn Ex Benth


through organogenesis from juvenile sources
Griffin Akeng, Sures Kumar Muniandi and Nor Aini Ab Shukor

375

Assessing salt-affected degraded soils using remote sensing.


Case study: Al-Qassim region, Saudi Arabia
Abdulla S. Modaihsh, Abdelazeem Sh. Sallam, Adel M. Ghoneim and
Mohamed O. Mahjoub
383

Determination of pond water quality for aquaculture and


ecosystem management
Umme Shahina Khanom, Sabrina Sharmeen, Jannatul Ferdouse, Wahhida
Shumi, Arifin Abdu, Hazandy Abdul Hamid and Md. Aktar Hossain 389

The relocation of undisturbed soil in long-term experiment


impacts organo-mineral complex degree and combined humus of
black soil
Fengqin Chi, Enjun Kuang, Baoku Zhou, Jiiuming Zhang, Qingrui Su
and Shanshan Cai
434

Environmental guidelines of So Francisco Verdadeiro river


according to Brazilian standards
Kayla W. Garmus, Silvio C. Sampaio, Maria Hermnia F. Tavares, Adir
Otto Schmidt and Marcelo Remor
439

Isolation and identification of microorganisms from soil in a


young oil palm plantation
Nur Masirah Mohd Zain, Rosli B. Mohamad, Kamaruzaman Sijam and
Yahya Awang
443

Monitoring larval populations of Aedes aegypti in different


residential districts of Jeddah governorate, Saudi Arabia
Khalid M. Al-Ghamdi , Abbas M. Al-Azab, Hassan M. Khormi, Lalit
Kumar and Jazem A. Mahyoub
448

Impact of pre-sowing treatment on seed germination and seedlings


growth attributes of Calamus longisetus Griff. at nursery and
field conditions
M. Rafiqul Haider, Md. Sah Alam, Md. Aktar Hossain and
Nor Aini Ab. Shukor
395

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

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Science and Technology
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Helsinki, Finland
e-mail: info@world-food.net

Journal of Food, Agriculture & Environment Vol.12 (3&4): 6-11. 2014

www.world-food.net

Reduction of foodborne pathogens in parsley by an improved formulation containing


lime and oregano extracts
Alany Celestino, Brianda Jaime, Ricardo Luvano, Luisa Sols, Santos Garca and Norma Heredia *
Departamento de Microbiologa e Inmunologa, Facultad de Ciencias Biolgicas, Universidad Autnoma de Nuevo Len, Apdo.
Postal 124-F, San Nicols, Nuevo Len, Mxico 66451. *e-mail: norma@microbiosymas.com
Received 18 May 2014, accepted 24 September 2014.

Abstract
Parsley (Petroselinum crispum-Apiaceae) has been reported as a vehicle for Salmonella and E. coli O157:H7; and Shigella sonnei has been responsible
for shigellosis outbreaks. Plant-derived extracts have been proposed as alternatives to reduce food contamination without modifying food properties,
and several extracts have decreased pathogenic bacterial growth in vegetables. The objective of this study was to reduce E. coli O157:H7, Salmonella
and Shigella levels inoculated in parsley, after washing with an improved formulation of edible vegetable extracts. Extracts from five edible plants,
resuspended in ethanol, were tested for antimicrobial activity and the minimum bactericidal concentration (MBC) determined. Extract mixtures were
analysed for synergistic activity. The mixture exhibiting synergism was used to wash parsley samples previously inoculated with 105 bacterial cells/
g. Following 0, 1, 3, 5, and 7 d, viable bacterial counts were determined. Chlorine, Citrol, and ethanol were used as controls. Mexican lime and Mexican
oregano extracts (4.3 - 4.8 and 1.5 - 2.0 mg/ml MBC, respectively) were selected. Synergistic antimicrobial effects were observed under a 1.25:0.19
mg/ml mixture. The mixture exhibited a > 2 log reduction in the bacterial level in parsley on the first day. In this study, we followed a simple, low cost,
and laboursaving extraction system. The antimicrobial efficacy of the improved formulation was clearly demonstrated on parsley. Considering human
health and environmental hazards associated with chlorine use, the Mexican lime:oregano mixture provides a viable alternative to chlorine, and is
equally effective at significantly reducing bacterial pathogens associated with outbreaks stemming from leafy green vegetables.
Key words: Escherichia coli, Salmonella, Shigella, produce contamination, natural antimicrobials, parsley.

Introduction
Foodborne diseases (FD) caused by pathogenic microorganisms
remain a major cause of morbidity and mortality worldwide. Fresh
produce consumption has increased as a result of healthier
lifestyles. However, fresh produce is often consumed minimally
processed or raw, and raw foods are known vehicles for human
disease 1, 2. In 2008, WHO placed leafy greens at its highest priority
for fresh produce safety at a global level due to its potential to
cause widespread FD outbreaks 3. The most notably, Escherichia
coli O157:H7, Salmonella sp. and Shigella sp. have been
associated with FD following contaminated leafy green vegetable
consumption. Parsley (Petroselinum crispum-Apiaceae) has been
reported as a vehicle for Salmonella and E. coli O157:H7 4, 5; and
Shigella sonnei was responsible for eight shigellosis outbreaks
in Canada and the US 6.
A great variety of compounds and intervention measures have
been tested and used to reduce or eliminate foodborne pathogens
from produce. Chlorine is a common disinfectant used to
decontaminate fresh produce 7; however, a chlorine wash cannot
completely remove or inactivate microorganisms, and it can
generate chlorinated organic compounds, which result in safety
concerns for humans 8.
Synthetic chemical compounds have been applied by the food
industry to control microbial contamination, however, the new
trends of consumers include ingesting fewer synthetic food
additives, and consuming more natural or all-natural foods 9, 10.
The antimicrobial activities of many essential oils have been
reported, and studies have demonstrated these compounds exhibit
activity against foodborne pathogens 10. Yet, essential oils can be
6

costly to extract, many are not acceptable for industrial processing


and some alter the organoleptic properties of foods 3, 10. However,
plant-derived extracts have been proposed as alternatives to reduce
food contamination without modifying food properties 11, 12, and
several extracts have decreased pathogenic bacterial growth in
vegetables 3, 11, 13. In the present study, we examined the efficacy
of six edible plant extracts in reducing Salmonella, Shigella, and
E. coli O157:H7 in parsley, developed an improved formulation,
and compared the extract performance to chlorine and a commercial
citrus-base sanitizer.
Materials and Methods
Bacterial cultures: Escherichia coli O157:H7 ATCC 43890 GFP
(modified with green fluorescent protein gen as marker), E. coli
ATCC 43895, E. coli O157:H7 ATCC 43894, Shigella sonnei F2353
(modified with green fluorescent protein gen as marker), S. flexneri
ATCC12022, Salmonella Typhi ATCC19430, and S. Typhimurium
ATCC 14028 (serovars of Salmonella enterica subsp. enterica)
were used in this study. All strains were maintained at -80C in
Brain Heart Infusion (BHI) broth (Difco) containing 20% (v/v)
glycerol. Fresh cultures were prepared by inoculating an aliquot
of stored culture in fresh BHI broth, and subsequently incubated
at 37C for 24 h. An aliquot of this culture was plated onto BHI
agar, and incubated at 37C for 24 h. Colonies were suspended in
saline solution, and adjusted to a 1.5 x 105 CFU/ ml concentration.
Plant extracts: Five edible plant species (Table 1) were purchased
from retail markets in the metropolitan area of Monterrey, Nuevo

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Table 1. Plants analysed, common names and parts used.


Scientific name
Citrus aurantifolia (Christm.) Swingle
Hibiscus sabdariffa L.
Lippia graveolens Kunth
Origanum majorana L.
Tamarindus indica L.

Common name
Mexican lime
Roselle
Oregano
Sweet marjoram
Tamarind

Part used
Peel
Flower
Leaves
Leaves
Peel / Pulp of fruit

Leon, Mexico. Plant material was surface washed with distilled


water and dried for 24 h at 50C. Twenty grams of each sample
were immersed in 100 ml of 96% ethanol, and ground using a mixer
to extract soluble plant material. Extracts were macerated at room
temperature for 48 h 14. Macerated samples were filtered through
Whatman #1 paper, and placed on glass plates to facilitate complete
ethanol evaporation (approximately 48 h at room temperature).
Dried extracts were resuspended in 10-15 ml of 96% ethanol,
centrifuged (Eppendorf Model 5414C) at 14,000 rpm for 15 min,
supernatants removed, and filter-sterilized using nitrocellulose
membranes (Millipore, 0.45-m pore size). Samples were collected
in sterile amber flasks, and maintained at 4C until experimental
procedures were performed (maximum 6 mos) 12. An aliquot of
each extract was used to determine dry weight.
Antimicrobial activity test: Preliminary screening for antimicrobial
activity was conducted using the agar diffusion well test 12. Briefly,
Petri dishes (150 mm) were filled with 25 ml Mueller-Hinton (MH)
agar (Difco), and surface inoculated with aliquots (100 l) of the
bacterial culture (1.5 105 CFU/ ml). Five holes (5 mm in diameter)
were subsequently made on the inoculated agar plate, and each
filled with 100 l of each extract. Ethanol (96%) was used as a
negative control. The plates were incubated at 37C for 48 h.
Inhibitory activity was visualised as the reduction or cessation of
bacterial growth in the area surrounding the holes, and quantified
by measuring the inhibition zone diameter.
Determination of minimum bactericidal concentration (MBC):
A range of extract concentrations (10 to 1 mg/ml) 13, 15 was added
to sterile 96-well polystyrene U-microtiter plates (BD Falcon) filled
with 150 l of 2X MH broth (Difco), then distilled sterile water was
added to reach a final volume of 300 l. Active cultures of each
pathogen (3 l, 1.5 105 CFU/ml) were used to inoculate each
plate, and subsequently incubated at 37C for 24 h. Following
incubation, the content of each well was plated on MH agar and
incubated at 37C for 48 h. The MBC was regarded as the lowest
extract concentration that prevented any visible bacterial colony
growth (total absence of colonies) on the MH agar plate after the
48 h incubation period.
Extract mixture analysis: The efficacy of the extract mixtures
was established by evaluating the natural antimicrobials
individually or in combination with other extracts. The extracts
exhibiting the highest antimicrobial activities were mixed, and the
effects against bacteria were determined by the checkerboard
method following Orhan et al. 16, with minor modifications. Sterile
96-well microtiter plates were filled with 50 l of 2X MH broth,
plus 50 l of an extract mixture (containing lower concentrations
than the MBC of each extract) (Table 2). Plates were inoculated,
incubated, and plated as described above. Mixtures and synergism
effects were evaluated by the fractional inhibitory concentration
index (FIC) 13. The FIC was defined as the sum of the MBC of each

Table 2. Extract combinations at different concentrations


determined by the checkerboard method following Orhan
et al. 16.

Mexican
lime extract
(mg/ml)
lower row

0.05
0
0.05
0.312
0.05
0.625
0.05
1.25
0.05
2.5
0.05
5
0.05
10

Oregano extract
(mg/ml) upper row
0.1
0.19
0.38
0
0
0
0.1
0.19
0.38
0.312 0.312 0.312
0.1
0.19
0.38
0.625 0.625 0.625
0.1
0.19
0.38
1.25
1.25
1.25
0.1
0.19
0.38
2.5
2.5
2.5
0.1
0.19
0.38
5
5
5
0.1
0.19
0.38
10
10
10

0.75
0
0.75
0.312
0.75
0.625
0.75
1.25
0.75
2.5
0.75
5
0.75
10

1.5
0
1.5
0.312
1.5
0.625
1.5
1.25
1.5
2.5
1.5
5
1.5
10

3
0
3
0.312
3
0.625
3
1.25
3
2.5
3
5
3
10

extract when applied in combination, divided by the MBC of each


extract applied individually 16. A synergistic effect was defined as
a FIC value < 0.5, an indifferent effect was defined as a FIC value
between 0.5 and 2, and an antagonistic effect was defined as a FIC
value 2 16.
Determination of leafy green decontamination effectiveness:
The method for assessing decontamination effectiveness
followed Lang and Harris 17, and Orue et al. 13, with minor
modifications.
Inoculum preparation: The assay was performed using E. coli
O157:H7 ATCC 43890, S. sonnei F2353, and S. Typhimurium ATCC
14028. A sterilised loopful of fresh cultures grown on BHI agar
was inoculated into tubes with 10 ml tryptic soy broth (TSB, Difco),
and incubated at 37C for 18 h. Cells were collected by
centrifugation (3000 rpm, 10 min at room temperature), and the
pellet resuspended in 1% peptone water. Similar volumes of each
strain were mixed into a cocktail, and the suspension (1.5 105
CFU/ml) was used as inoculum.
Parsley inoculation: Parsley was purchased from local markets,
and stored at 4C for a maximum of 2 d before inoculation. The
initial microbe content of the purchased samples was determined
by microbiological analyses (total viable counts and E. coli
O157:H7, Shigella sp., and Salmonella sp. presence) according
to the Bacteriological Analytical Manual 18. When these
pathogenic bacteria were detected in the samples, the vegetables
were discarded.
For the assays, 25 g of parsley samples were cleaned with gentle
washing using running tap water followed by gentle rinsing in
sterile distilled water, and dried for 2 h in a class II biosafety
cabinet (Labconco) at room temperature. Twenty-five aliquots of
10 l of the cocktail strains (adjusted al 1.5 x 105 CFU/ml) were
spotted over the previously disinfected parsley surface 17 in a
biosafety cabinet. The inoculated vegetables were subsequently
air-dried for 15 min at room temperature.
Determination of effectiveness of decontamination agents:
Inoculated parsley was submerged in 225 ml of a respective
1.25:0.19 mg/ml (final concentration) lemon peel and oregano for

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

20 min. Chlorine (200 ppm) and a grapefruit-based commercial


disinfectant (Citrol K Ultra, Corpo Citrik, S.A. de C.V. Mxico,
D.F.; [200 ppm dissolved in water]) were used in place of plant
extracts and as positive controls. Non-rinsed and water-rinsed
parsley were also used as controls. Parsley was transferred to
sterile bags and stored at 4C for 7 d.
Parsley samples were removed at 0, 1, 3, 5, and 7 d, and the
pathogenic bacteria presence was determined. A 5 g sample was
placed in 45 ml 0.1% peptone water and homogenised. Following
homogenisation, and decimal dilutions, the samples were streaked
in duplicate on XLD agar (for Salmonella, Difco), TSB
supplemented with 0.05 mM isopropyl--D-thiogalactopyranoside
(IPTG, for E. coli O157:H7 GFP) 19 and MacConkey agar (for S.
sonnei GFP). These plates were incubated at 37C for 24 - 48 h.
The TSB and MacConkey agar plates were examined under
ultraviolet light (356 nm, UV SL-58 Ultra-Violet Products), and
green fluorescent colonies were counted. For Salmonella, pink
colonies with or without black centers on XLD agar were
enumerated.
Statistical analysis: All experiments were performed twice, and at
least three replicates were conducted for all samples. SPSS 17.0
was used to compare and analyse efficacy among treatments (SPSS
Inc. Chicago, Illinois). Non parametrical test and Kruskal-Wallis
test were used. ANOVA and Tukey test (P 0.05) were used to
multiple comparison of means.
Results and Discussion
Plant extracts might serve as important alternatives to develop
antimicrobial formulations to control microorganisms, and as food
preservatives 20. Results of our preliminary antimicrobial activity

testing five extracts against growth of E. coli, Salmonella and


Shigella strains narrowed the panel to four active extracts: Mexican
lime, roselle, Mexican oregano, and thyme, which showed growth
inhibition zones for most bacteria ranging from 1.4 to 2.7 cm in
diameter (Table 3). Mexican lime and oregano activity was
congruent with Orue et al. 13; however, inhibition diameter was
smaller than observed in the present study. The resuspension
solvent (PBS) used in Orue et al. 13 might contribute to these
inconsistencies. Furthermore, differences in antimicrobial effects
of the same vegetable type may be attributed to a variety of factors,
including vegetable quality, growth stage at extraction, and field
growth (ecological) conditions 21, 22.
MBC analyses (Table 4) indicated that the most active ethanolic
extracts were Mexican lime (MBC of 4.3 to 4.8 mg/ml) and oregano
(MBC of 1.5 to 2.0 mg/ml). Orue et al. 13 reported MBCs from 5.3 to
8.1 mg/ml from lime extracts resuspended in PBS against
Salmonella, Shigella, and E. coli strains, and MBC of 5.2 to 6.4
mg/ml from oregano extracts against the same bacteria. MBC values
were lower from the extracts applied in the present study. The
solvents used to resuspend the extract (PBS or ethanol) can
increase the efficacy of plant extracts. For example, ethanol,
methanol, and acetone are superior to water in the extraction of
most plant bioactive compounds 23.
Different mixed concentrations of oregano and lime extracts
were tested based on the checkerboard method (Table 2), and the
respective 1.25:0.19 mg/ml lime:oregano mixture exhibited a FIC
synergistic effect. Subsequently, parsley artificially contaminated
with E. coli O157:H7 was washed with this mixture, Citrol, and
chlorine. At time 0 (immediately after washing), chlorine resulted
in a pathogen reduction of 2.4 log CFU/g, Citrol showed a decrease
of 1.9 log CFU/g, and 1.7 log CFU/g was observed with the

Table 3. Antimicrobial effects of plant extracts against Salmonella, E. coli, and Shigella bacteria determined
by the agar well diffusion method. All extracts were prepared using 96% ethanol.

Scientific name

Citrus aurantifolia
(Christm.) Swingle
Hibiscus
sabdariffa L.
Lippia graveolens
Kunth
Origanum
majorana L.
Tamarindus
indica L.

Fruit/
Vegetable

Part
used

Mexican
lime

Diameter of growth inhibition


(cm standard deviation)
S.
S.
E. coli
Typhi
Typhi
43895
murium
19430
14028

S.
flexneri
12022

S.
sonnei
F2353
GFP

1.90.0

2.21.0

2.40.7

1.60.3

1.70.1

2.00.7

1.60.4

2.40.2

1.50.1

1.70.1

2.70.2

2.10.5

NI

NI

NI

NI

NI

E. coli
43890
(GFP)

E. coli
43894

Peel

2.10.1

1.70.2

2.00.3

1.80.3

Roselle

Flower

1.70.2

1.40.2

1.40.2

Oregano

Leaves

2.60.1

2.50.2

Sweet
marjoram

Leaves

NI

NI

Tamarind

Peel
Pulp of
fruit

NI

NI

NI

NI

NI

NI

NI

1.70.4

1.40.2

1.50.4

1.60.2

1.50.1

2.30.4

2.10.8

* NI indicates no inhibition.

Table 4. Minimum bactericidal concentration (MBC) of the selected extracts against E. coli
O157:H7, Salmonella Typhimurium, and Shigella sonnei.
Plant
(common name)
Roselle
Mexican lime
Oregano
Tamarind (pulp)

E. coli
O157:H7
43890 (GFP)
>10
4.30.3*
2.00.1
>10

E. coli
O157:H7
43894
>10
4.70.3
2.00.1
>10

MBC (mg/ml)
S.
E. coli
Typhimurium
43895
47028
>10
>10
4.80.3
4.50.0
1.50.1
2.00.1
>10
>10

S.
Typhi
19430
>10
4.50.0
2.00.1
>10

S.
flexneri
12022
>10
4.50.0
1.50.1
>10

S. sonnei
F2353
(GFP)
>10
4.30.3
1.50.1
>10

* Standard deviation.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

lime:oregano extract. The control, washing with water alone,


reduced the pathogen by 0.9 log CFU/g (Table 5). S. Typhimurium
was reduced 2.9 log CFU/g with chlorine, 2.7 log CFU/g using
Citrol, 2.4 log CFU/g with lime:oregano extract, and the water
control resulted in a reduction of 0.9 log CFU/g. Results for S.
sonnei showed chlorine decreased the pathogen by 1.9 log CFU/
g, Citrol by 1.8 log CFU/g, lime:oregano extract reduced the
pathogen by 2.2 log CFU/g, and the water control showed a 1.0
log CFU/g decrease. Significant differences (P 0.05) were
observed among all antimicrobial treatments and water controls
(washed and not-washed); however, significant differences were
not detected among antimicrobial treatments. The number of
microorganisms exhibited a decrease over the seven-day test
period, including the not-washed treatments; however, the
reduction in microorganisms was more notable in the chlorine,
Citrol, and lime:oregano extract treatments (Table 5).
The mesophilic microorganism number was reduced immediately
following all treatments, but increased during the study period.
At day five of the study, these species returned to log CFU levels
comparable to the water washed or not-washed samples (Table 6).
Orue et al. 13 reported that no significant difference in mesophilic
microorganism levels were observed at seven days among samples
washed with aqueous extracts of lime or oregano, and the control,
in accordance with our results.
The synergistic effect between both extracts found in this study
(1.25:0.19 mg/ml, lime:oregano mixture), allowed to reduce
significantly the amount of extracts for the washing solution with
similar efficacy. Previous studies used individual extracts dissolved
in PBS at levels > 5.0 mg/ml of each extract and no synergistic
effect was found 13.
In this study, the formulation was tested against
enterohemorrhagic and non-enterohemorrhagic strains of E. coli,
S. sonnei, S. flexneri and two serovars of Salmonella. The results
exhibited the efficacy of these compounds between different
strains/species.
Chlorine (200 ppm) reportedly removes approximately 1.5 to 2
log CFU/g of background or pathogenic microflora on lettuce,
cilantro, and parsley 8, 24. The chlorine efficacy in our study was

Table 6. Mesophilic organism enumeration on parsley following


different washing treatments.
Log CFU/g
Day
0
1
3
5
7

Not washed

H2O

Chlorine

Citrol

4.70.09*
5.70.18
5.70.11
6.10.16
6.10.16

4.20.25
4.50.06
5.10.16
6.00.05
6.10.16

3.20.16
3.90.67
5.20.24
6.00.05
6.40.09

3.80.14
4.40.31
5.40.09
6.20.31
6.00.0

Mexican
Lime-Oregano
mixture
4.20.16
4.40.09
4.70.04
5.00.00
6.20.16

* Standard deviation.

slightly higher (reducing 2.9 log), and interestingly, the


lime:oregano extract mixture was comparable (P < 0.05) to chlorine
in reducing S. Typhimurium, E. coli O157:H7, and S. sonnei.
The antimicrobial activity of different Citrus species has been
documented. Lemon extract showed activity against Bacillus
licheniformis, Saccharomyces cerevisiae, and Pichia
subpelliculosa 10. Citrus sp. rind has antimicrobial activity against
E. coli and S. aureus growth 25. Studies have demonstrated lime
extract activity against several virulent microorganisms 13, 26.The
antimicrobial activity of Citrus sp. extracts has been established,
however, few applications with the exception of commercial oils
have been developed. Valtierra et al. 12 showed lime extracts alone,
or in a mixture with other edible fruits reduced viability of C. jejuni
and C. coli in vitro, and in chicken skin by more than 4 log CFU/
g. Orue et al. 13 also demonstrated that aqueous lime and oregano
extracts reduced Salmonella, Shigella, and E. coli O157:H7
viability by 2 log CFU/g from cilantro, parsley, and spinach.
Several plants that share similar odor and flavor attributes are
known as oregano. Origanum vulgare L. (Lamiaceae) and
Lippia graveolens Kunth (Verbenaceae) (synon. L. berlandieri
Schauer), are the most studied oregano that exhibit antimicrobial
activity. These plants are commonly used spices with medicinal
properties for the treatment of gastrointestinal and respiratory
illnesses 27 . Lippia graveolens exhibits antioxidant and
antimutagenic activities 28, and the essential oil is active against
Gram-positive, Gram-negative bacteria, and phytopathogenic
molds 27, 29, 30. Extracts of L. graveolens contains high amounts of
phenolics compounds, with flavonoids
as the major constituents. Thymol and
Table 5. Enteropathogenic bacteria enumeration in parsley immediately after washing
carvacrol, and the oil precursors, i.e., pwith different agents (Day 0) or washing and storage at 4C and counts at Days
cymene and -terpinene are considered
5
1, 3, 5, and 7. Inoculum 1.5 10 CFU/ml.
the antimicrobial compounds 30, 31. These
Mexican Lime-Oregano
Not
compounds affect the structure and
mixture
Bacteria
Day
H2O
Chlorine
Citrol
washed
functionality of the bacterial membrane,
(1.25:0.19 mg/ml)
provoking changes in intracellular pH, and
Log CFU/g
0
3.90.02* 2.70.06 1.00.0 1.20.14
1.50.02
alterations in membrane potential and ATP
1
3.70.07 2.40.09 1.00.0 1.50.41
1.40.17
synthesis 32, 33.
Salmonella
3
2.40.58 2.20.19 1.00.0
1.00.0
1.10.18
Typhimurium
Formulations containing grape seed
5
1.50.58 1.10.12 1.00.0
1.00.0
1.00.06
and olive extracts have been proposed
7
1.90.03 1.00.06 1.00.0
1.00.0
1.00.0
as alternatives to chlorine-based
0
3.50.07 2.60.01 1.10.16 1.60.22
1.80.09
1
3.50.13 2.50.02 1.00.0 1.50.19
1.60.27
solutions for washing fresh produce 13, 3436
E. coli O157:H7
3
3.30.23 2.50.04 1.00.0
1.00.0
1.00.02
.

Shigella sonnei

5
7
0
1
3
5
7

3.00.12
3.00.02
3.90.03
3.70.19
3.50.15
2.40.76
1.00.0

2.40.06
2.20.12
2.90.0
3.00.19
2.60.11
1.90.14
1.00.0

1.00.0
1.00.0
2.00.10
1.00.0
1.00.0
1.00.0
1.00.0

1.00.0
1.00.0
2.10.22
1.10.16
1.00.0
1.00.0
1.00.0

1.00.0
1.00.0
1.70.09
1.30.4
1.00.0
1.00.0
1.00.0

* Standard deviation.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Conclusions
In this study, we followed a simple, low cost, and laboursaving
extraction system. The antimicrobial efficacy of the improved
formulation was clearly demonstrated on parsley. Considering
human health and environmental hazards associated with chlorine
use, the Mexican lime:oregano mixture provides a viable alternative
to chlorine, and is equally effective at significantly reducing
bacterial pathogens associated with outbreaks stemming from leafy
green vegetables.
Acknowledgements
This research was supported by the Consejo Nacional de Ciencia
y Tecnologa de Mxico (CONACYT) grant #105389.
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of Salmonella, Shigella, and Escherichia coli O157:H7 from leafy


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Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

11

WFL Publisher
Science and Technology
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Helsinki, Finland
e-mail: info@world-food.net

Journal of Food, Agriculture & Environment Vol.12 (3&4): 12-18. 2014

www.world-food.net

Food security of Northwest China under current water resources and food
consumption patterns
Jianping Li 1, Jing Chen 1 and Zhouping Shangguan 2*
1

School of Agriculture, Ningxia University, Yinchuan Ningxia, 750021, P. R. China. 2 State Key Laboratory of Soil Erosion and
Dryland Farming on the Loess Plateau, Institute of Soil and Water Conservation,Chinese Academy of Sciences, Yangling
Shaanxi, 712100, P. R. China. *e-mail: shangguan@ms.iswc.ac.cn

Received 22 June 2014, accepted 24 September 2014.

Abstract
This study investigated the effects of water resources and food consumption patterns on the food security of Northwest China. A regional water
requirement model (RWRM) and a food security model (FSM) were set up to evaluate the water shortage and food security of Northwest China,
respectively. The results showed that the water resource shortage of Northwest China is severe and thus the status of food security is unsafe without
food import; the urban food security of the region is better than the rural food security, and more water and energy are needed for the urban
population; and the water shortage of Northwest China has increased dramatically since 1983 and will continue to increase in the future, having
already reached 170 billion m3 in 2010 and will reach 400 billion m3 in 2050. Finally, some countermeasures that should be taken to safeguard the food
and water securities of Northwest China are as follows: control the population growth according to the local conditions and the population and
structure of the ethnic (minority) peoples; promote calorie-appropriate and energy-efficient diets instead of unhealthy diets; eliminate food wastes;
and develop water-saving agriculture and breed water-saving crop varieties. The models and proposed countermeasures are expected to provide
theoretical foundation and practical guidance for sustainable development and food security of Northwest China.
Key words: Food security, agricultural water resource, food consumption pattern, Northwest China, model.

Introduction
In addition to population growth, industrial development and
uncontrolled economic growth, water shortage is more and more
recognized as a major threat to food security due to its restriction
of agricultural production 1, 2. The changes in consumption
patterns, i.e., the increasing proportion of water-intensive food
(e.g. meat), may become the main cause of water shortage 3.
Currently, approximately one third of the worlds population lives
in countries suffering water shortage, including north China, west
Asia, and Libya and Saudi Arabia, who have used water for
irrigation that greatly exceeds their annual total water resources 4.
Many authors estimate that a large part of the worlds population
- up to two-thirds - will be affected by water shortage over the
next decades 5, 6.
In China, water resources uses can be divided into four forms:
agricultural water (62% of the total amount), industrial water (24%),
domestic water (12%), and eco-environmental water (2%) 7. Nearly
all agricultural water is freshwater 8 and a shortage of freshwater
exists all over the world 9. So water shortage fundamentally results
from insufficient freshwater for food production 3, 10. Generally
speaking, different amounts of water are required to produce
different foods. For example, about 1 - 3 m3 of water is required to
produce 1 kg of cereal, and about 13.5 m3 of water is required to
produce 1 kg of beef in California 11. Consequently, different food
consumption patterns require different amounts of water
resources. With economic development and improved living
standards, the proportion of water-intensive foods has been
growing in food consumption patterns, so that more water
resources are required to meet human food demands. For example,
12

a typical American diet which includes red meat requires twice as


much water as a vegetarian diet to provide the same nutritional
intake 12. Also, climate change can be a significant factor that
affects agriculture and food production, exerting either a positive
or a negative influence on food security. For instance, higher CO2
concentrations can have a positive effect on many crops by
enhancing their biomass accumulation and final yields. However,
extreme weather conditions due to climate change can have
negative effect on food security by blocking food distribution,
and causing food supplies to be unstable and stored foods to
decompose 13.
In arid regions, the difference between water resource supply
and water demand is increasingly becoming acute due to
increasing water requirements and unchanging or decreasing water
supply. China is a drought-prone country suffering severe water
shortage. Although the total water resources amount of China is
2.8 trillion m3, ranking sixth in the world, its per-capita water amount
is only 2300 m3, 1/4th of the average level in the world. However,
China is one of the 13 countries suffering water shortage 14. In
terms of their spatial distribution, the water resources of China
tend to decrease from its northeast coastal area to northwest inland
area. Meanwhile, the agricultural water use efficiency of China is
low and farmland soil salinization and environmental pollution are
severe, particularly in the rural area of China 15. With its population
growth and rapid industrialization, the industrial water and
domestic water needs of China are increasing and their combined
demand restricts the agricultural water use for food production.
In the meantime, the food consumption pattern of China has shifted

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

200'0" N

200'0" N

300'0" N

300'0" N

400'0" N

400'0" N

500'0" N

500'0" N

towards animal product-dominated patterns, particularly meatMaterials and Methods


dominated ones, which mean that the agricultural water needs of Profile of study area: With the longitude ranging within 7341'E China have further increased. The result will be that the water 11115'E and latitude from 3142'N - 4933'N, Northwest China has
supply for food production will be insufficient to meet all demands a total area of 3,045,600 km2, accounting for 30.8% of the total area
which may result in food insecurity. Relevant data collected in the of China. It covers five provinces and autonomous regions from
2010 China Agriculture Yearbook 16 showed that China uses 48% southeast to northwest: Shaanxi, Ningxia Hui Autonomous
of irrigation farmland to produce 75% of grain yield and more than Region, Gansu, Qinhai and Xinjiang Uygur Autonomous Region
90% of cotton and vegetables. Therefore, the agricultural water (Fig. 1). In late 2010, Northwest China had its peak population of
use of China is a key factor that affects its food security.
96.6 million, which accounted for 7.2% of the total population of
Northwest China is the region suffering the most severe water China and of which the minority population accounted for 19%.
shortage, where the water and agricultural land resources are The landscape of Northwest China includes plateaus, basins and
generally spatially unevenly distributed and the ecosystem is mountains. Characterized by low rainfall and high evaporation,
fragile. The data collected from the Main Data Communique of the Northwest China is an arid and semi-arid region. The annual mean
Sixth National Population Census 17 showed that Northwest China precipitation of Northwest China decreases from 400 mm in the
has a population growth rate of more than 10, higher than the east to 200 mm in the middle part to less than 50 mm in the
average national level of 5.7. Also, the food consumption pattern northwest. Northwest China has an annual total water resources
of Northwest China differs greatly from the other regions of China. (surface and aquifer water) amount of 230 billion m3, which
For instance, the animal products consumption of the former is accounts for 9% of that of China. Since a majority of agricultural
higher compared with that in other regions. Northwest China is a irrigation facilities are poorly developed and most of farming
district, where there are many minority ethnic peoples including practice is dry land farming, northwest China depends on
Uygur, Hui, Tibetan, Mongolian and so on. Since these ethnic precipitation for the majority of crop production.
peoples consume more fresh milk and meat-dominated foods, and
only use grains as dietary supplements, Northwest China needs Methods:
more agricultural water than other regions to produce foods in RWRM and its parameters: A RWRM was established, in terms of
order to maintain its food security for its diverse population. water amount necessary to produce a unit of product and per
Meanwhile, with the acceleration of implementing the Great capita food consumption, to calculate the amount of water resource
Western Development Strategy in China where Northwest China that was used in agriculture to produce food, and to calculate the
was zoned as one industrial development region, more and more total amount of water resource requirement using a proportional
freshwater will be used by industry, which will probably rate of agricultural water use. The model is expressed as follows:
n
additionally limit water resources used by agriculture. In a word,
1
people will need more and more water resources to safeguard WRWR = ( M iWi )( Pr + Pu )
i =1
their food security, but there are limited water resources, which
are insufficient to produce food for food security, in Northwest
where WRWR represents the regional water requirement under
China, as well as the limited water resources will
800'0" E
1000'0" E
1200'0" E
1400'0" E
be used by industry and other aspects instead of
agriculture. Therefore, the sharp conflict between
the water resources and food security are
becoming acute, and how to tackle these conflicts
N
is a highly important topic that attracts attention
from all the fields.
Focusing on the water resource and food
consumption patterns of Northwest China, the
objectives of this study were to establish a
regional water requirement model (RWRM) and a
food security model (FSM) to evaluate the status
of its water shortage and food security, respectively;
develop models for predicting its future water and
food security depending on the current status of
water and food security, which were calculated
by RWRM and FSM; and provide sustainable
development-oriented countermeasures to tackle
the conflicts between the water shortage and food
security of the region depending on the models
and analyses. The results will lay a theoretical
Provincial capital
foundation and provide practical guidance for the
Study area
sustainable development and food security of
Provincial boundary
Northwest China.
900'0" E

1000'0" E

1100'0" E

1200'0" E

Figure 1. Location of the study area.


Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

13

the current food consumption pattern, 1/() is ratio of water


consumption for food production to the total water resources,
is the proportion of water supply to total water resource, is the
proportion of agricultural water to total water supply, is
agricultural water use efficiency, M i is annual per-capita
consumption of the i th food (kg), Wi is water amount necessary
for to produce the i th food (m3 kg-1), n is food number, Pr is rural
population, and Pu is urban population. The parameters of Wi are
listed in Table 1 and those of Mi in Table 2.
Wtotal is assumed to represent the total water resources . If WRWR >
Wtotal, then there will be insufficient water resources and insufficient
water supply to produce enough food for consumption under the
current food consumption patterns. Otherwise, food and water
security will be guaranteed. Different regions have different
over time; the average is about 83% from 1980 to 2010. The
average is within 80 - 86%, which comes from the China Statistics
Yearbook 13. The value of is from the published papers 18-20,
ranging from 0.4 to 0.6. Since rural and urban food consumption
patterns differed greatly, the rural and urban populations were
Table 1. Actual water consumptions and energy water
productivities for mostly consumed food items.
Food items
Cereals and roots
Rice
Wheat
Maize
Other
Potatoes and other starchy roots
Sugar and sweetener
Oil corps and vegetable oils
Soybeans and other oil crops
Vegetable oils
Vegetables and fruits
Vegetables
Fruits
Animal products
Beef
Pork
Poultry
Mutton and goat meat
Fish and sea food
Eggs
Milk
Alcoholic

-1

Ei (kcal kg )

1.31
0.98
0.84
1.24
0.23
1.02

3625
2633
2872
2709
699
3481

3.2
5.08

3314
8720

0.19
0.5

188
413

12.56
4.46
2.39
4.5
5
3.55
1
0.18

2021
3500
1708
2005
497
1455
670
490

FSM and its parameters: A FSM was established to convert food


intake into energy intake to estimate food security. It was defined
as:
n

E EI = M i Ei

-1

Wi (m kg )

separately introduced into the RWRM so that the estimation and


evaluation would not be biased.
Food consumption patterns greatly influence water security.
For example, beef production needs 13 times more water than
wheat (Triticum aestivum L.) production to produce same amount
of weight, and 17 times more water than wheat to supply same
amount of energy 21. The study divided food consumption patterns
into two types: rural and urban patterns, because rural people and
urban people have different purchasing powers, dietary habits,
and food consumption habits. Thus, the comparison errors
resulting from ignoring these differences were reduced. Table 2
presents the annual per capita food consumption in the past 30
years (1980 - 2010), which indicates that the per capita annual
consumption of all food items has increased, while the
consumption of cereals has decreased. It also indicates that the
per capita food consumption of the rural population was less than
that of the urban population, again except for cereal consumption.

i =1

where EEI is per capita energy intake per day (kcal), Mi is daily
per-capita consumption of the food (kg), Mi = Mi /365, and Ei is
the energy of the i th food (kcal kg-1), n is foods number (Table 1).
Energy intake, recommended as the main indicator for measuring
food security by FAO 24, consists of four requirements:
i. Basal Metabolic Rate (BMR) for adults: 1300 - 1700 kcal per
person per day.
ii. Allowance for light activities: 1720 - 1960 kcal per person per
day.
iii. Allowance for appropriate activities: 2000 - 2310 kcal per person
per day.
iv. Allowance for labours or activities above the average intensity
or surpassing appropriate activities: 2600 - 2950 kcal.
This study adopted the average-weighted caloric requirement
of 2300 kcal/person/day to measure food security, which was the
calorie number required for appropriate activities. If EEI>2300 kcal,
then the food security was adequate; and if 1700 kcal<EEI< 2300
kcal, the food security was low; and if EEI<BMR, the energy
supply was insufficient and people were in the state of
malnourished and starvation. However, with development of
economic and improvement of the peoples living standard, the

Sources: Actual water consumptions of cereals, soybean, vegetables and fruits from Liu
et al. 21, fish and seafood from Zimmer and Renault 22, other food items from Chapagain
et al. 23.
Note: Wi is water amount necessary for to produce the i th food (m3 kg-1), Ei is the energy
of the i th food (kcal kg-1).

Table 2. Rural and urban food consumption patterns of northwest China over time.
Food items
Cereals and roots
Vegetables
Vegetable oils
Pork
Beef & goat meat
Poultry
Eggs
Fish and sea food
Milk
Fruits
Alcoholic

1980
Urban Rural
130
257
60
40
5
2
15
8
2
0
2
1
4
1
2
0
3
0
18
3
0
2

1985
Urban Rural
135
257
74
47
6
4
17
11
2
2
3
1
7
2
7
1
5
2
25
5
5
4

1990
Urban Rural
131
262
89
68
6
5
18
11
3
2
3
1
7
2
8
2
9
2
34
6
5
6

1995
Urban Rural
97
259
116
85
7
6
17
11
2
3
4
2
10
3
9
3
14
2
40
14
6
7

2000
Urban Rural
82
248
115
86
8
8
17
12
3
3
5
1
11
2
10
0
17
4
46
16
5
2

2005
Urban Rural
76
214
123
95
9
5
23
21
4
5
10
3
8
3
7
1
19
4
52
14
6
9

2010
Urban Rural
70
195
125
99
10
5
28
25
5
5
15
4
8
2
10
1
16
8
51
17
12
10

Source: National Bureau of Statistics (1981-2011)


Notes: China Statistical Yearbooks generally tell consumption data by classifying food items into food groups. One example is the consumption of cereals and roots. So, the study divided the cereal and
roots into there parts: 65% of wheat, 35% of rice, and 10% of roots, according to the food consumption habits of northwest China.

14

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

per capita energy intake should increase as well as the measurement


of food security in underdeveloped countries and developing
countries.
Data sources: The data for this study, including water resources,
population and food consumption patterns, were from China
Statistics Yearbook 16 and China Agriculture Yearbook 25. The
parameters of the RWRM and FSM were extracted out of the
published papers and China Statistics Yearbook 16 and China
Agriculture Yearbook 25. The parameters of the predicted models
(shown in Table 3) come from the regression models, which were
developed by the data (from 1980 to 2010) that were calculated by
RWRM and FSM.

3500
Population (104)

6000
5250

Rural population
Per capita water resource
Urban population

4500
3750

3250
3000
2750

3000

2500

2250
1500

2250
1980

1985

1990

1995
Year

2000

2005

Per capita water resource (m3)

Analysis and Results


Current water resources and population: The annual total water
resources for Northwest China were almost stable around 230
billion m3 in the past 30 years, without much change from year to
year (Fig. 2). Because of the exploding population, the annual per
capita total water resource sharply decreased (Fig. 3) from 3400 m3
at the end of 1980 to 2300 m3 in 2010. In the meantime, the per
capita water supply also decreased dramatically from 1019 m3 in
1980 to only 699 m3 at the end of 2010, which was far below the
water shortage-warning line (1000 m3 per capita).With the increased
water pollution and reduced usable fresh water resource, fresh

2010

Total water requirement and total


water resources in northwest
China (cu.m billion )

Figure 2. Population and per capita water resource reserves


in the different years.
400

water resource for the people was significantly reduced 26.


Figure 3 shows that the urban population has been growing
linearly since 1980. Although it has a large base, the rural
population has increased slowly since 1980. Because the
urbanization and industrialization have caused many rural
residents to migrate to the urban areas, and the natural increase in
the rural population was offset by the amount of rural population
who migrated to urban areas, the rural population has remained at
approximately 53 million in the past 30 years. So, the urban
population growth can be viewed as the population growth of
Northwest China. Meanwhile, Northwest China will be a main
area that has a population growth faster than the other regions of
China, because the population growth rate is 14.05 (average
value in the past 30 years), compared with the national average of
5.7. Therefore, more population, especially the urban population
who consume more energy than rural residents, will threaten water
security and food security.
Status of water security: The regional water requirement is defined
as the amount of water resources required for the present food
consumption patterns and all other water use. The water
requirement of Northwest China has been larger than its supply
since 1982, and the gap between the former and the latter has
increased dramatically year by year (Fig. 2). In 1980, the total
water resources of 240 billion m3 were larger than the water
requirement of 225 billion m3. This indicated that there was
sufficient water for producing food and other uses and water
resources were not a key constraining factor in agriculture and
industry. However, the difference between the water requirement
and the water supply has increased sharply from 5 billion m3 in
1982 to 170 billion m3 in 2010. By 2010, more than 80% of the total
water resource was used for agriculture. Due to water shortage,
the agriculture production will be reduced and food supply will be
insufficient to meet food consumption, and food insecurity will
occur without the importation of food. In the meantime, how to
properly distribute the limited total water resource among
agriculture, industry, life and ecology will be a big problem, and
an irrational distribution will have a negative effect on the regional
economy and society.

Total water requirement


Total water resources

Status of food security: The average per capita energy intakes per
day were obtained by the FSM model. The rural per capita energy
320
intake per day was less than the urban one during the past 30
years (Fig. 4). After 1983, the urban per capita energy intake per
280
day was over 2300 kcal, indicating that the urban food security of
240
Northwest China was safeguarded. The urban per capita energy
intake per day reached a high record of 3100 kcal in 2000 and
1980 1985 1990 1995 2000 2005 2010
generally decreased from 2000 to 2010 but remained above 2700
Year
kcal, surpassing the food security threshold of 2300 kcal. This
Figure 3. Total water resources and total water requirement
decrease resulted from the changes in food consumption patterns,
of northwest China in the different years.
which was the consumption of less cereal and more
meat and vegetables. However, the rural food security
Table 3. Forecast models and water securities in the following 40 years.
was poor. The rural per capita energy intake per day
was 1400 kcal in 1980, which was lower than that
needed for the Basal Metabolic Rate for adults. The
rural population suffered malnutrition and starvation
in 1980s. The per capita energy intake per day has
generally increased from 1400 kcal in 1980 to 2290 kcal
Note: P is population, E is per capita energy intake per day (Kcal), W is amount of total water need for food, e is the base
in 1998 but has remained below the food security
360

of the natural logarithm (approximately 2.7183), x is year, and R2 is determination coefficient.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

15

Per capita energy intake per day


(Kcal/person /day )

3000
2700
2400
2100
1800

Urban area
Rural area
Food security line

1500
1980

1985

1990

1995
Year

2000

2005

2010

Figure 4. Per capita energy intakes per day of northwest


China in the different years.

threshold; the per capita energy intake per day has generally
fluctuated around 2300 kcal after 1998, being 2400 kcal in 2000,
2250 kcal in 2005, and 2330 kcal in 2010. Thus, the food security
for the rural population was nearly average. Therefore, the food
security of Northwest China was safeguarded from 1998 to 2010,
but this sufficient food security may not last long because of the
population growth, water shortage, and living standard
improvement.
Food security and water security in the future: This study
developed the Population Forecast Model (P), the Energy Intake
per Capita per Day Model (E), and the Water Resource Requirement
Forecast model (W) depending on the populations, per capita
energy intakes per day, and total water resources and water
requirement from 1980 to 2010. W Wtotal, the differences between
the water requirements and the existing total water resources, was
used to evaluate the future water situation (Table 3). The forecast
models depended on the economies, population and food patterns
in the past 30 years to forecast the future.
Table 3 shows that the population of northwest China is
projected to grow exponentially from 110.5 million at the end of
2020 to 135.9 million in 2050, a 25.4 million increase, and more than
80% of the population will become urban residents in 2050.
Meanwhile, the per capita energy intake per day and the regional
water requirement will reach 3762 kcal and 625.5 m3 in 2050,
respectively. The water gap (W Wtotal) will increase sharply in the
next 40 years. The total water resources will meet only 50% of the
water requirement of 463.5 billion m3 in 2020 and the gap will reach
400.1 billion m3 in 2050. Consequentially, the population growth,
sharp water gap increase and energy intakes of northwest China
will threaten the food security because of the insufficient water
for producing food in the future.
Discussion
Water shortage and food security have become primary factors
that restrict the national economic progress of China. If water
shortage evolves into a crisis, the effects may be far more severe
than the crisis of oil shortage that we have experienced so far 27.
Water security is the basis for food security, which is the basis for
modern agriculture. Agriculture can save water resources to
safeguard food security by consuming less water to produce more
food 28, 29. At present, water resources for food security of China is
facing challenges resulting from the water shortage, due to
increasing industrial and urban water uses, lack of extensive water
resources management, and water loss and soil erosion.
Agricultural water use is crucial to China, because China has a big
16

population and suffers poverty. Two-thirds of the undernourished


population (not enough food to eat) of the world live in seven
countries (Bangladesh, China, the Democratic Republic of Congo,
Ethiopia, India, Indonesia and Pakistan) and over 40% of the
undernourished population live in China and India 30. Thus, water
for food production cannot be diverted for other uses, especially
in the rural area of Northwest China where much of the
undernourished population lives. The typical water distribution
pattern of China is that it is dry in its northwest and humid in its
southeast, and its water supply differs across regions and time.
Because the water supply of Northwest China represents a much
smaller portion of Chinas total water resource, it faces very serious
problems with agricultural water shortage. Since agriculture is
now the largest branch of water user, it is important to develop
modern water-saving agriculture to safeguard the food security,
water security, and ecology security of China. The measures for
this purpose have been recognized by the scientists and
governments 31, 32. In addition, most reserve cultivated-land
resources of China are located in Northwest; it is difficult to exploit
them because of water shortage. So, the option to safeguard food
security by expanding the amount of cultivated land will be difficult
to achieve.
Insufficient food resulting from water shortage in Northwest
China can be made up through trade 23 or food distribution. Our
study showed that in 2010, the total water requirement of Northwest
China was 390 billion m3, but the total water resources were only
230 billion m3. Also, the agricultural water requirement of 72.5
billion m3 is more than agricultural water supply of 42.5 billion m3,
so that obviously the region does not have enough water to
produce food under the current food consumption patterns.
Nevertheless, both the rural and urban food securities of northwest
China were good in 2010 mainly because reasonable food trade
and grain circulation offset its food shortage. Where trade is
possible at a reasonably low water cost, the crucial food security
issue is whether the monetary and non-monetary resources at the
disposal of the population are sufficient to allow everyone to get
access to adequate food supplies rather than not whether the
foods are sufficient, that is to say, food availability is not a key
factor affecting food security. An important corollary to this is
that regional self-sufficiency is neither necessary nor sufficient
to guarantee food security at the regional level. It is noted that
Hong Kong and Singapore are not self-sufficient (they dont have
agriculture) but their populations are food-secure, meanwhile,
India is self-sufficient but a large part of its population is not
food-secure 13. However, more and more people will suffer from
food insecurity, if there are insufficient food supplies for trading
or circulation. In other words, both food production and trading
are important for safeguarding food security.
The results of this study showed that the water requirement for
food production has almost doubled from 1980 to 2010 (Fig. 3),
largely due to an increase in animal products consumption in the
recent decades. The models indicate that the future total water
requirement for food production will be likely to increase in the
next four decades. Even under the low modernization scenario,
food consumption pattern shifts along with population growth
will probably cause the total water requirement to reach 400 billion
m3 per year in 2050, even taking into consideration relevant
technological advances. This will undoubtedly put enormous
pressure on limited water resources in Northwest China. The

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

RWRM model showed that there were five key factors affecting
the regional water requirement of Northwest China: population,
food consumption pattern, agricultural water, water supply
capacity, and crop water use efficiency. The following
countermeasures in terms of the five factors probably could
guarantee the future water security and food security of Northwest
China.
First, the population growth of Northwest China should be
regulated according to the local conditions. At present, the
population of northwest China is mainly rural with many ethnic
peoples living together, and thus the possibilities of the economic
security, societal security and cultural security should be
considered in a balanced way. Proper population countermeasures
should be taken and timely adjusted according to the population
and structure of the ethnic peoples to ensure common development
and prosperity of all the nationalities concerned. While the
population size will be controlled, the populations quality of life
should be improved.
Second, caloric-appropriate diets and energy-efficient food
consumption patterns instead of unhealthy diets should be
promoted, and food wastes should be prohibited. In general, food
consumption patterns are closely related to increasing affluence.
However, eating habits probably plays a role in affecting the food
preference of Chinas population. For example, the meat
consumption of China now exceeds what is recommended by China
Nutrition Society 33, and Northwest China has higher meat
consumption than other regions. In addition, the current diet shifts
of Northwest China may be detrimental to the populations health,
and could cause higher incidences of diet-related diseases 34.
Raising public consciousness and promoting the diet recommended
by the CNS may help mitigate the future water shortage of China.
Meanwhile, food losses of China resulting from seeding, feeding,
harvesting, food processing, storage, transportation, and cooking
are large, making up 30% of the total food production of the
country. Consequentially, to eliminate all such food losses by
educating the population in healthy diet, food producing and
nutrition are wise measures to save food and water resources.
Third, Northwest China should develop water-saving agriculture
to guarantee the food and water securities. It is necessary for
Northwest China to increase water use efficiency and then water
supply as the total water resources are limited in the arid area. The
key approaches for increasing the water supply of Northwest
China can be summarized as follows: constructing reservoirs and
implementing water diversion projects; reasonably exploiting and
scientifically managing urban groundwater resources under the
precondition of sustainable development economy and society;
and trying to recycle waste water. Water-saving agriculture should
be promoted to safeguard water security. Water-saving agriculture
as an integrated system should include four aspects: rational uses
of agricultural water resources, water-saving irrigation, agronomic
water-saving techniques, and agricultural management 24.
Governmental supports and encouragements will be necessary to
achieve this system.
Fourth, biotechnological as well as traditional breeding methods
should be adopted as the efficient method to develop water-saving
crops for the arid areas of northwest China to feed its growing
population. Water-saving crops developed by modern biotechnology
breeding cannot only improve water use efficiency but also can
increase food production. In other words, more foods will be

produced without additional or with lowered consumption of the


currently existing water resources. Water-saving crop varieties,
such as drought resistant wheat in Australia, drought tolerant
cotton (Gossypium hirsutum L.) in America and drought tolerant
fruit trees in Israeli 35, have good drought tolerance ability, stable
yields and good qualities.
Conclusions
In brief, improving water use efficiency through biological
approaches in combination with other water-saving methods and
projects and establishing sustainable agriculture with limited water
resources have become, and will continue to be, the challenges
for agricultural scientists in the future.
Acknowledgements
This study was funded by the Startup Project of Doctor Scientific
Research of Ningxia University (BQD2012008) and Key Project of
the Knowledge Innovation Program of the Chinese Academy of
Sciences (KZCX2-YW-JC408).
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18

18

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

WFL Publisher
Science and Technology
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e-mail: info@world-food.net

Journal of Food, Agriculture & Environment Vol.12 (3&4): 19-23. 2014

www.world-food.net

Modified atmosphere storage of banana (Musa acuminata) using diffusion channel


and mathematical modelling of steady-state oxygen concentration within the package
Arulselvam Karthiayani 1*, Nachimuthu Varadharaju 2 and Madasamy Siddharth 3
1
Department of Food Engineering, College of Food and Dairy Technology, Chennai-52, India. 2 Department of Food and
Agricultural Process Engineering, College of Agricultural Engineering, Coimbatore-3, India. 3 College of Food and Dairy
Technology, Chennai-52, India. *e-mail: mankarthi@yahoo.com

Received 22 April 2014, accepted 4 September 2014.

Abstract
Banana (Musa acuminata), variety Poovan (Group AB) fruits of commercial maturity were stored inside the air-tight container under modified
atmosphere conditions using diffusion channels. The gaseous exchange was made through the glass channels called as diffusion channels of different
geometries viz., length 5, 10, 15 and 20 cm and inner diameter 3, 5 and 7 mm. The fruits were packed with rated quantities of moisture and ethylene
absorbents and the containers were stored at three different temperatures (RT-Room temperature, 24C and 14C). Gas samples were drawn from
the container and was analysed for oxygen concentration for every 24 h up to a period of 20, 30 and 40 days for RT, 24C and 14C storage,
respectively. Oxygen concentration was noted for the storage containers with different dimensions of diffusion channel and storage temperature. A
mathematical model was developed using non-linear multiple regression to fit the observed data. The developed model was found to be useful in
determining the length of the diffusion channel for the required steady-state oxygen concentration for banana. The shelf life of banana can be increased
to three to four folds by adopting diffusion channel technique compared to control fruits at all the respective storage temperatures.
Key words: Modified atmosphere storage, diffusion channel, banana, modelling, oxygen.

Introduction
Indias contribution to world production of banana is about
15.02% 9. The Indian banana growers are losing around 20 to 40%
of their production on account of existing handling practices 14.
The post harvest losses in India are much higher due to tropical
climate and also due to lack of technology for handling and
processing 18. Hence an attempt is made to enhance the shelf life
of banana using diffusion channels and to develop a mathematical
model for determining the steady-state oxygen concentration.
The principle behind modified atmosphere packaging is
reduction in respiration rate of fruits by modifying the gas
composition of the storage atmosphere and thus enhancing the
shelf life of the fruits 17. If the availability of oxygen is restricted,
the rate of respiration could be slowed down; thereby quality of
the commodities is preserved with enhancement of shelf life 11.
The appropriate atmosphere is made to evolve within the storage
chamber by the respiration of the produce and maintaining that
atmosphere by selective permeability of the gases through
membrane or by diffusion through channels 12, 19. Diffusion channel
is a hollow tube or channel fitted to an airtight storage chamber in
which produce is stored, and the other end of the tube is exposed
to the ambient air. The diffusion channel storage system is one of
the methods of modified atmosphere packaging in which the
storage chamber is impermeable to gas of concern and the exchange
of gas takes place only through the diffusion channels. The
presence of diffusion channel controls the flow of gases on either
direction (i.e., from package atmosphere to ambient atmosphere
and vice versa), thus maintaining the gas composition.
The gas composition surrounding the fruit material within the
package plays an important role in determining shelf life of fruits

under MA Storage. Low oxygen and elevated carbon dioxide levels


are known to influence flavour by reducing loss of acidity 7, starch
to sugar conversion, sugar inter conversion and biosynthesis of
flavour volatiles. However, levels of oxygen below 2% and carbon
dioxide above 15% have been found to result in irreversible effects
on the fruit quality 4. Elevated carbon dioxide atmosphere inhibited
the activity of ACC synthesis (key regulatory site for ethylene
synthesis) and retarded biosynthesis of carotenoids and
anthocyanins synthesized during fruit ripening 3. This also slowed
down the activity of cell wall degrading enzyme which influenced
softening of fruit tissue 13.
Materials and Methods
Experimental containers for storage study: The containers made
of Poly Ethylene Teraphthalate (PET) with 8.5 litres capacity and
transparent bodies were selected for the study. Three holes were
made in the lid. In the first hole, a silicon septum was fitted using
brass fittings to draw gas samples for gas analysis. The second
hole was used for flushing nitrogen gas into the storage chamber
and the third one was used to fix diffusion channel using rubber
cork. The diffusion channels of rigid type were made up of glass
with the different geometries viz., length 5, 10, 15 and 20 cm and
inner diameter 3, 5 and 7 mm (Table 1), were selected for the study.
The geometry of the diffusion channels was arbitrarily fixed based
on the information available in the literature cited 1, 20. All the
experimental containers were washed well with soap water and
rinsed with chlorinated water (500 ppm) and tested for air tightness
using soap solution to detect any leaks before starting the
experiment. An advantage of this design is the containers provide

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

19

quick and easy opening and closing procedures and good


visibility to assess the product during the storage period. The
schematic view of the experimental container is depicted in Fig. 1.
Table 1. Treatment details for banana and mango.

T1

Channel dimensions
L
d
5
3

T2

10

T3

15

T4

20

T5

T6

10

T7

15

T8

20

T9

T10

10

T11

15

T12

20

Treatment

till the required oxygen concentration of 5% was reached. The


containers were stored in clean and dry place in three different
temperatures viz., room temperature (27 - 33C, 50 - 70% relative
humidity-RH), 24C and 14C. The samples were kept in walk-in
cold room to store at 24C and 14C and RH 90 - 95%. Considering
the recommended cold storage temperature and evaporative cooler
temperature for banana, the storage temperatures of 14C and
24C were chosen, respectively.
Preliminary studies on MA storage of banana: Preliminary
experiments were done to ascertain the position of the diffusion
channel and best fruit to free volume ratio to be kept inside the
container. Three position viz., top (bottom of the channel touching
the top of the lid), middle (middle of the channel touching the top
of the lid) and bottom (top of the channel touching the top of the
lid) and five levels of fruit to free volume ratios viz., 1:2, 1:3, 1:4, 1:5
and 1:6 were investigated. The experimental procedure was
repeated. Visual as well as gas analysis was done at 24 h interval
to check the ripening of fruits and gas composition inside the
package, respectively.

L - length of diffusion channel (cm). d - inner diameter of diffusion channel (mm).

Determination of gas composition within the package and


modelling: Oxygen concentration within the container was found
out using MAP analyser (PBI Dansensor Model, Checkmate).
The needle of MAP analyser was inserted into gas sampling septum
of the storage chamber and the pump button was put on. The
vacuum pump inside the MAP analyser sucks about 5 ml of gas
from the storage container and oxygen concentration was
calculated by the sensor and the percentage was displayed in the
digital screen. The gas concentration was analysed for all the
containers with different treatments as described in Table 1.
Mathematical model for length of diffusion channel: Based on
the review of previous work done, a mathematical model was
developed and represented as follows:

rmsV f
v

yO container
KAc DO2  N 2 DO2  N 2 2
ln(
)
rmsV f
v

yO2 air
KAc DO2  N 2 DO2  N 2

(1)

Equation (1) can be further be simplified as:

Figure 1. Schematic view of diffusion channel storage.

Experimental procedure: Banana (Musa acuminata), variety


Poovan (Group AB) was selected for the study. The fresh banana
bunches with 90% maturity were procured from farm orchard,
Coimbatore, Tamilnadu, India. The bunches were carefully
dehanded and washed in tap water and were dipped in 500 ppm of
benomyl fungicide solution for 2 - 3 min 5 and then shade dried for
about 4 - 5 h to facilitate evaporation of surface moisture. This
could prevent the fruits from fungal infection during storage.
Calcium hydroxide, CO2 and moisture absorbent (@100 g/kg of
fruit) were kept at the bottom of the storage container. A perforated
plastic plate was placed above the absorbent in order to keep the
fruit samples for storage so that there was no direct contact
between the fruits and the absorbent. Known quantity of fruit
was kept over the perforated plastic tray along with the ethylene
absorbent (@20 g/kg of fruit) pouch. The containers were then
closed with the lids and nitrogen gas was flushed into the container
20

DO2  N 2

rm V a  a y
1
ln( s f 2 1 O2 container )
a1
rmsV f a2  a1 yO2 air

where:

a1

a2

(2)

v
DO

N2

1
KAc DO2  N 2

where:
L - Length of the diffusion channel (cm)
DO2 - N2 - Diffusivity of O2 in N2 (cm2 h-1)
Ac - Cross sectional area of the diffusion channel (cm2)
yO2 - Oxygen molar fraction
k - Total mole of gas within the storage container (mole)
Vf - Free volume inside the storage container (cm3)

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

r - Respiration rate of the stored produce (mg O2 kg-1 h-1)


ms - Mass of the stored produce (kg)
v - Velocity gradient of O2 in N2 (cm s-1).
Equation (1) is used to find out the length of the diffusion
channel for given mass of sample and free volume inside the
storage chamber. The model parameters a1 and a2 were obtained
by fitting the experimental data by non-linear regression using
the software package Sigma-plot 8.0.

It is evident from the results that, the steady-state oxygen


concentration was directly proportional to diameter and inversely
to length of the channel. The results obtained are in accordance
with Stewart et al. 16 for the modified atmosphere packaging of
Dwarf Cavendish banana using diffusion channel. From Fig. 3, it
may also be observed that the steady-state oxygen concentration
was higher at lower storage temperature. This may be due to less
respiration of fruits at lower storage temperature.

Results and Discussion


Position of the channel and fruit to free volume ratio: Based on
the preliminary studies, it is found that the top position of the
diffusion channel and fruit to free volume ratio of 1:5 was the most
suitable to maintain the required oxygen concentration of 2 - 5% 5,
6, 10
. Hence, further experiments were conducted on top position
of channel and fruit to free volume ratio 1:5.

Values of constants fitted with the developed mathematical model:


The mathematical model was developed as described. The
parameters viz., a1 and a2 described in the model were determined
using the experimental data by non-linear regression using Sigmaplot 8.0 software and presented in Table 2.

Oxygen concentration in storage container: Oxygen concentration


attained by banana inside the storage chamber was recorded at one
day interval throughout the storage period at different temperatures
and presented in Fig. 2. The initial gas level was kept at 5% oxygen
for all the treatments. In all the cases, the oxygen level increased
initially and stabilised at that level for about three to five days and
the concentration subsequently decreased and attained a steady
state. Also, a sudden decrease in oxygen concentration was seen
during the later period of storage in most of the samples.
From the Fig. 2, it is observed that the attainment of steadystate oxygen concentration took about 5 - 10 days, 6 - 8 days
and 12 - 15 days for the fruit samples stored at RT, 24C and
14C, respectively.

Oxygen concentration (%)

Steady-state oxygen concentration attained by the fruits: The


steady-state oxygen concentration attained by the fruits is
presented in Fig. 3. At ambient storage temperature, the maximum
steady state oxygen concentration of 4.8 and minimum of 2.1%
was noted for T9 and T4, respectively. Also for all the storage
temperatures, the maximum and minimum values of oxygen
concentration were noted for T9 and T4, respectively. Kader 8 and
Kim et al. 2 observed that the optimum modified atmosphere
conditions for most of the tropical fruits were 3 to 8% oxygen
which indicated that all the samples were containing recommended
oxygen concentration.

Ambient
Am

24
24C

14
14C

Table 2. Values of constants a1 and a2 for banana at different


storage temperatures.
Variety
Poovan

d
(mm)
3
5
7

Ambient
a2
a1
0.4927 0.0222
0.3676 0.0165
0.4075 0.0183

Determination of best shelf life of banana: The samples stored


for determining the best shelf life and the data is represented in
Table 3. The oxygen concentration attained by the best treatments
ranged between 2.3 and 5.2% which is in conformation with the
results obtained by Isaak et al. 5, 6 and Kudachickar et al. 10 for the
storage of banana. It is inferred from the above results that the
shelf life of banana can be increased to three to four folds by
adopting diffusion channel technique compared to control fruits
at all the respective storage temperatures.

14C

Table 3. Best treatment for banana at different storage


temperatures.

Sample

Storage
temperature

Banana

Ambient
24qC
14qC

2
1
T1

T2

14qC
a1
a2
0.2208 0.0297
0.1788 0.0217
0.2012 0.0225

The convective flow of oxygen and nitrogen within the chamber


is taken care of the constant a1 which is described as the velocity
of oxygen flow per unit diffusivity coefficient of oxygen in nitrogen
(DO2-N2). The parameter a2 represented the reciprocal of the product
of total mole of gas within the storage container, cross sectional
area of the diffusion channel and the diffusivity coefficient of
oxygen in nitrogen (DO2-N2). Since DO2-N2 is assumed as constant,
the parameter a1 is directly proportional to the velocity of air flow
and a2 is inversely proportional to the cross-sectional area of the
diffusion channel. The length of the diffusion channel may be
determined by substituting the values of a1 and a2 in Equation (2).

24qC
a1
a2
0.1745 0.0128
0.1725 0.0126
0.1724 0.0125

T3

T4

T5

T6
T7
Treatment

T8

T9

T10

T11

T12

Fruits under diffusion


channel storage
Best
Shelf life
treatment
(days)
T 11
15
T4
27
T3
38

Control
(fruits stored in air)
Shelf life
(days)
4
7
11

Figure 3. Effect of different channel diameters, lengths and storage


temperatures on steady state oxygen concentration of banana.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

21

Oxygen concentration (%)

Oxygen concentration (%)

00

88

1010

l = 5cm
cm
I-5

10
10

Storage period (days)

15
15

10
10

1515

l = 10cm
cm
I-10

l = 15cm
cm
I-15

Storage
period (days)
Storage
periods
(days)

55

20
20

l = 20cm
cm
I-20

2020

20
20

d = 7 mm

d = 7 mm

Storage periods (days)

55

d = 5 mm
d = 5 mm

d = 3 mm

d = 3 mm

10
10
1515
55
Storage period (days)
Storage periods (days)

Ambient

00

22

44

66

88

00

0
0

00

1010

00

22

44

66

88

1010

00

22

44

66

88

1010

l I-5
= 5 cm
cm

55

55

15
15

l=
15 cm
I-15
cm

25
25

l =I-20
20 cm
cm

25
25

d = 7 mm

30
30

30
30

30
30

d = 5 mm

d = 7 mm

10
15
20
20
15
10
Storage period (days)
Storage periods (days)

lI-10
= 10cm
cm

25
25

d = 3 mm

d = 3 mm

d = 5 mm

20
20

Storage period (days)

Storage periods (days)

10
10

10
15
20
10
20
15
Storage period (days)
Storage periods (days)

24C

q
8

10 10

0
0

00

0
0

10 10

lI-5
=5
cm
cm

5
5

55

55

10
10

10
10

10
10

14C

15
15

20
20

25
25

20
20

25
25

l I-10
= 10cm
cm

35
35

lI-20
= 20cm
cm

35
35

40
40

40
40

40
40

d = 7 mm

d = 7 mm

30
30

30
30

l I-15
= 15cm
cm

Storage period (days)

Storage periods (days)

15
15

Storage period (days)


Storage
periods (days)

35
35

d = 3 mm

d = 3 mm

d = 5 mm
d = 5 mm

15
20
25
30
20
25
15
30
Storage
(days)
Storageperiod
periods
(days)

g 2: Oxygen concentration attained by Poovan at different channel lengths diameters and temperatures during storage (where d is

00

22

44

66

00

00
00

22

44

66

88

1010

00

22

44

66

88

Oxygen concentration (% )

1010
Oxygen concentration (%)

Figure 2. Oxygen concentration attained by Poovan at different channel lenghts,diameters and temperstures during storage.

Oxygen concentration (%)

O xyg en co n cen tratio n (% )

Oxygen concentration (% )

Oxygen concentration (%)


Oxygen concentration (%)

Oxygen concentration (%)

Oxygen concentration (%)

Oxygen concentration (%)

Oxygen concentration (%)


Oxygen concentration(%)

Oxygen concentration (%)


Oxygen concentration (%)

Oxygen concentration (%)


Oxygen concentration (%)

22

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Conclusions
The smallest channel length of 5 cm attained the steady state
quickly compared to largest channel length of 20 cm at all the
storage temperatures. Also, the variation in attainment of steadystate period was more pronounced for smaller channel diameter of 3
mm compared to 5 and 7 mm. The maximum and minimum values of
steady-state oxygen concentration were noted for T9 and T4,
respectively, for all the storage temperatures which represented
that the steady-state oxygen concentration was directly
proportional to diameter and inversely to length of the channel.
The developed mathematical model will be a useful tool for
determining the length of the diffusion channel for said oxygen
concentration. Also the shelf life of banana can be increased to
three to four folds by adopting diffusion channel technique
compared to control fruits at all the respective storage
temperatures.
Highlights:
The time of attainment of steady-state oxygen concentration
within the package increased as the storage temperature
increased.
The attained of steady-state oxygen concentration was directly
proportional to the diameter and inversely proportional to the
length of the channel.
The developed mathematical model will be a useful tool for
determining the length of the diffusion channel for banana for
the required steady-state concentration.
Acknowledgements
The authors sincerely express their gratitude towards the funding
agency viz., University Grants Commission, New Delhi, India for
carrying out the research work.
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8

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

23

WFL Publisher
Science and Technology
Meri-Rastilantie 3 B, FI-00980
Helsinki, Finland
e-mail: info@world-food.net

Journal of Food, Agriculture & Environment Vol.12 (3&4): 24-31. 2014

www.world-food.net

Anti-inflammatory and anti-cancer effects of -carotene, extracted from


Dunaliella bardawil by milking
Abeer M. Badr 1*, Effat F. Shabana 2, Hoda H. Senousy 2 and Hend Y. Mohammad

Zoology Department, 2 Botany and Microbiology Department, Faculty of Science, Cairo University, Giza 12613, Egypt.
e-mail: abeerbadr@gmail.com, dr-effatshabana@hotmail.com, hodasenousy1@hotmail.com, hendgooonet@hotmail.com

Received 10 July 2014, accepted 22 September 2014.

Abstract
Natural -carotene was continuously extracted from Dunaliella bardawil in a two phase system by milking with the biocompatible solvent dodecane
(20% v/v) at 20,000 lux and 170 rpm for 15 days. The b-carotene yield was 23.30 g/ml at the end of the experiment. High performance liquid
chromatography (HPLC) analysis showed that b-carotene is formed of the two isomers 9-cis and all trans in a ratio of 1.13. We aimed to assess the
protective and therapeutic effects of natural b-carotene at various doses (30, 140 and 350 g/kg) compared with synthetic b-carotene at a fixed dose
(350 g/kg) on lipopolysaccharide (LPS)-induced inflammatory cytokines in CD1 mice, and also to test the cytotoxicity of natural b-carotene on
breast cancer (MCF-7) and hepatoma (HepG2) human cell lines in vitro. In pre- and post-treatment of LPS injected mice, low dose (30 g/kg) of
natural b-carotene treatment caused a significant reduction in the levels of interleukin (IL)-1a, interferon (IFN)-g and tumor necrosis factor (TNF)-a
compared with LPS-treated control group. Administration of natural b-carotene at dose of 140 g/kg exhibited a significant decrease in the levels of
IL-1a and IFN-g as protective and IL-1a as therapeutic. In contrast, high dose (350 g/kg) of natural of b-carotene failed to exert anti-inflammatory
effect either in pre or post-treatment. Synthetic b-carotene pretreatment induced protective inhibition of IL-1a and IFN-g levels while post-treatment
had no influence. IC50 of natural b-carotene was 14.58 and 7.44 g/ml, while IC50 of doxorubicin was 7.51 and 2.67 g/ml for HepG2 and MCF-7 cell
lines, respectively. Hence, natural b-carotene is capable of enhancing anti-inflammatory activity in vivo and is a promising anti-cancer drug.
Key words: Dunaliella bardawil, -carotene, milking, lipopolysaccharides, anti-inflammation, pro-inflammatory cytokines, breast cancer MCF-7 cell
line, HepG2 cell line, anti-cancer, acute inflammation.

Introduction
Drug research in marine environment has shown that algae are an
important source of innovative biochemically active compounds.
Carotenoids biosynthesized by algae have commercial
applications in food science, pharmaceutical industry, cosmetics,
and nutritional health 1. -carotene, as an important carotenoid, is
metabolized in the mammalian cells and acts as a natural source of
vitamin A 2. It is considered as a powerful antioxidant and has
been suggested to inhibit the development of inflammationassociated diseases such as rheumatoid arthritis 3, inflammatory
bowel disease 4, and atherosclerosis 5.
Dunaliella bardawil, a marine unicellular halotolerant green
alga, showed an environmental adaptation through excess
production of -carotene and glycerol to maintain its osmotic
balance 6. Natural -carotene produced by D. bardawil is
composed of equimolar mixture of two stereoisomers, all-trans
and 9-cis -carotene 7. Milking, a kind of in-situ extraction process
in which the starting cell mass is reused for continuous production
to overcome the low productivity of algal cultures in producing
high-value compounds, i.e. the first step in the experiment did not
need to be repeated. Milking has been found successful in carotene production from D. salina 8-10. Production of carotenoids
from D. salina by milking is selective since mainly secondary
carotenoid was extracted when gentle mixing was used 10.
Production of -carotene under stress conditions was higher than
24

under normal conditions 11. A 9-cis stereoisomer in D.bardawil is


still highest among all other natural carotenoid sources 12. A 9-cis-carotene isomer has a valuable metabolic activity as it has a
better antioxidant property, a higher liposolubility than the alltrans isomer, and accumulates more effectively in animal tissue 13.
Early epidemiological evidence has suggested that intake of carotene may introduce protection against certain types of
cancers 14, 15. However, administration of synthetic all trans carotene implied no effects on cancer and cardiovascular
diseases 16 and sometimes affected negatively on smokers with
lung cancer 17 and atherosclerosis 18.
Inflammation is a complex process mediated by action of various
immune cells, such as natural killer cells, neutrophils, and
macrophages. It is characterized with overexpression of
inflammatory mediators (IL-1, IL-6, and TNF-) 19. Lipopolysaccharide (LPS), a bacterial endotoxin, presents in the outer
membrane of Gram-negative bacteria that induces toxic effects 20,
and able to promote inflammatory responses. LPS treatment is a
way to mimic inflammatory state 21.
The present study aimed to assess the efficacy of natural carotene extracted from D. bardawil by milking on beneficial
inhibition of pro-inflammatory cytokines during acute inflammation
of LPS-stimulated mice comparing to synthetic -carotene. We
also aimed at investigating the potential cytotoxicity of natural -

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

carotene on hepatoma (HepG2) and breast cancer (MCF-7) human


cell lines in a dose dependent manner in vitro.
Materials and Methods
Organism and growth conditions: D. bardawil was isolated from
the salt marshes near El-Bardawil Lake, Northern part of SinaiArish Governorate, Egypt. It was grown in batch cultures in MH
nutrient medium 22 with modification of NaCl concentration to 1.5
M (pH 7.5, 251C) and light intensity of 39 Em-2s-1 (2000 lux) by
white fluorescent lamps, the initial algal inoculum was adjusted to
be 5106 cells/ml. Three replicates were used for each treatment.
Extraction ability of different organic solvents to -carotene
and cell membrane integrity of Dunaliella: Some non-polar
organic solvents with log P octane (oct) more than 6 (dodecane, decane,
tetradecane, hexadecane, ethyl oleate) (Sigma-Aldrich) were used
in this experiment to choose the suitable biocompatible one 23.
Each treatment consisted of 80% aqueous phase and 20% organic
phase. D. cultures were incubated at 15,000 lux, 251C and mixing
rate 200 rpm. The extracellular -carotene in the organic phase
was measured after 24 h. Cells were harvested and the chlorophyll
a content, intracellular -carotene and cell count were measured.
The organic solvent phase was protected from light by covering
it with aluminum foil in all solvent experiments. The cell membrane
integrity was expressed as the absorbance of the Evans blue
extraction solution samples at 600 nm 24.
Milking of D. bardawil for continuous production and extraction
of -carotene in two-phase system: After preliminary experiments,
algal culture with initial cell count of 5106 cells/ml was
supplemented with 20% (v/v) dodecane and incubated at
20,000 lux (light intensity) and 170 rpm mixing rate. Every
three days, chlorophyll a content, -carotene (intracellular
& extracellular), and cell count were measured till the end of
the experiment (15 days) 8. Cells were counted by Sedqwick
Rafter cell after the addition of 1-2 drops of Lugols solution.
Chlorophyll a was determined as described previously 25, 26.
-carotene was quantified photometrically using as a molar
extinction coefficient 450 nm 134, 500 L mol-1 cm-1 27.

conditions for 7 days before the experiment. All experimental


procedures in this study were approved (CUFS/S-PHY/11/14) by
the Institutional Animal Care and Use Committee (Faculty of
Science, Cairo University, Egypt).
Induction of inflammation and experimental design: For
induction of inflammation, mice were injected intraperitoneally
(i.p.) with a single acute dose of 2.5 mg/kg body weight of LPS
(Escherichia coli 0.26.B6, Sigma-Aldrich Co, USA) 28, euthanized
after 6 h of LPS injection. The doses of -carotene were chosen
according to Murthy et al. 29. Natural and synthetic -carotenes
were dissolved in olive oil. Mice were divided into 15 groups (n=6
per each) in two sets for assessment of the protective (pretreatment) and therapeutic (post-treatment) effects of -carotene
as shown in Fig. 1. In protective set mice were orally administered
with olive oil once in a day for 15 days as vehicle control (Group
1) followed with LPS injection (Group 2). Mice were orally
administered with synthetic -carotene at only one dose of 350
g/kg (Group 3) and three doses of natural -carotene, 30 g/kg
(Group 4), 140 g/kg (Group 5), and 350 g/kg (Group 6), once in a
day for 15 days. Oral administration of synthetic -carotene (Group
7) and all doses of natural -carotene (Group 8, 9 and 10) once in
a day for 15 days, followed with LPS injection. In therapeutic set
mice were injected with LPS at once and immediately i.p. injected
with a single dose of olive oil as control LPS (Group 11). LPS
injection at once and immediately i.p. injected with a single dose
of synthetic -carotene (350 g/kg, Group 12) and three doses of
natural -carotene: 30 g/kg (Group 13), 140 g/kg (Group 14),

Detection of -carotene by high performance liquid


chromatography (HPLC) analysis: The -carotene extract
from D. bardawil was lyophilized and transferred to hexane
with the addition of water, dried completely under N2, and
re-dissolved in methylene chloride. Then, it was analysed
by HPLC (Agilent 1100, USA, with Thermo Electron
Corporation Hypersil Gold DC8 column, particle size 5 m,
150 mm 4.6 mm and flow rate of 1 ml/min). The HPLC
preparative time for pigment analysis did not exceed 15 min.
The synthetic -carotene (Sigma type I, Sigma-Aldrich,
USA) was used as standard and dissolved prior to
chromatography in methylene chloride 7.
Animals: Male CD1 mice weighing about 20-22 g were
obtained from the breeding animal centre of National
Research Centre (Giza, Egypt). Mice were kept at room
temperature (222C), 555% humidity and a 12 h dark/light
cycle with free access to regular laboratory chow and water
ad libitum. Mice were allowed to acclimate to these

Figure 1. Diagram of experimental design.


LPS injected mice (2.5 mg/kg) euthanized after six hours of injection. (A) Protective set: synthetic
(synth) at single dose (350 g/kg) and natural (nat) -carotene at various doses (30, 140, and 350 g/
kg) were administered for 15 days before LPS injection. (B) Therapeutic set: LPS injected mice were
treated with synthetic -carotene at single dose (350 g/kg) and natural -carotene at various doses
(30, 140, and 350 g/kg).

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

25

and 350 g/kg (Group 15). Mice were euthanized with isoflurane.
The blood was collected by cardiac puncture and centrifuged at
3500 rpm for 10 min. The collected sera were stored at - 20C for
further analysis.
Quantitative determination of cytokines: Levels of cytokines
(IL-1, IFN- and TNF-) were assessed by an indirect enzymelinked immunosorbent assay (ELISA) (eBioscience, GmbH, Vienna,
Austria). ELISA was performed following the instructions of the
ELISA test kits. The levels of cytokines were expressed as pg/ml.
In vitro cytotoxicity assay of -carotene extract: Cytotoxic
activity of natural extract of -carotene was determined by
sulphorhodamine-B assay (SRB) 30. HepG2 of hepatoma cancer
and MCF-7 of breast cancer human cell lines were maintained at
the National Cancer Institute (Cairo, Egypt). -carotene was
prepared in dimethyl sulphoxide (DEMSO) and tested as negative
control. The cells were seeded in 96-well microplates (5104-105
cells/well) for 24 h. The monolayer cells were incubated with serial
dilutions of -carotene (5, 12.5, 25 and 50 g/ml) for 72 h at 37C in
a humidified incubator with 5% CO2. Triplicate wells were prepared
for each concentration. After 72 h, the cells were fixed, washed
and stained with 0.4% SRB stain dissolved in 1% acetic acid.
Excessive dye was washed with 1% acetic acid, the plates were
air-dried, and protein-bound dye was solubilized with 10 mM Tris
base solution. Colour intensity of each well was measured at 564
nm with microplate reader. The anticancer drug doxorubicin was
used as a positive control. IC50 is the concentration necessary to
cause 50% cell inhibition (or 50% cytotoxicity).
Statistical analysis: The present data were analysed by one-way
analysis of variance (ANOVA) using SPSS statistical package
version 20 to test the effect of -carotene on the investigated
parameters. In addition, the comparison between various studied

groups was performed using post ANOVA Duncans test of


homogeneity. The data values were expressed as meanstandard
error of mean (SEM). Regression analysis and correlation
coefficient were used to fit the relationship between various
studied variables.
Results
Extraction ability of different organic solvents to -carotene
and cell membrane integrity of Dunaliella: Table 1 reveals that
decane has the highest extraction ability (extracellular -carotene)
among all solvents used (5.6 g/ml) followed by dodecane (3.3
g/ml) while tetradecane, hexadecane and ethyl oleate have less
and similar ability to extract -carotene from D. bardawil cells.
Measurement of growth parameters showed that most organic
solvents used caused a remarkable decrease in values of growth
parameters after 24 h of extraction at 200 rpm mixing rate when
compared to their initial values. The highest values of chlorophyll
a and cell count were recorded in cells treated with hexadecane
while the least values were recorded in cells treated with decane.
The highest intracellular and total -carotene (intracellular +
extracellular) were observed with dodecane. The cell membrane
integrity measurements (Table 1) showed that optical density (O.
D.) readings increased with increase in log Poct of the solvent
indicating increase in the cell membrane damage. The decane
recorded the least O. D. reading which increased with other
solvents; the highest one was for ethyl oleate. Different organic
solvents have a significant effect on growth, -carotene
production and extraction as well as cell membrane integrity.
Milking of D. bardawil for continuous production and extraction
of -carotene: In milking experiment, chlorophyll a and cell count
decreased drastically by increasing time of continuous extraction
(Table 2). In contrast, both of intracellular and extracellular carotene increased gradually by increasing the time of extraction,

Table 1. Extraction ability of different organic solvents to -carotene; membrane integrity of


Dunaliella bardawil incubated for 24 h at 15,000 lux and 20 % (v/v) solvent at mixing
rate 200 rpm.
Growth
parameters
Solvents
Decane
Dodecane
Tetradecane
Hexadecane
Ethyl oleate

log Poct

Chlorophyll a
(g/ml)

Cell count
(x106 cell /ml)

6.25
6.6
7.6
8.8
8.51

6.050.08a
7.030.01b
8.750.06c
9.310.08e
8.990.07d

2.220.04a
3.890.02b
4.140.03c
5.020.06e
4.900.04d

Intracellular
-carotene
(g/ml)
2.510.20a
5.610.11d
4.080.02b
4.910.05c
4.020.06b

Extracellular
-carotene
(g/ml)
5.600.15d
3.300.05c
1.390.02b
1.030.02a
1.330.04b

Cell membrane
integrity (O.D.)
0.190.01a
0.270.01b
0.280.02b
0.300.01b
0.860.01c

Initials: chlorophyll 9.99 g/ml, cell count 5.31106 cell/ml, -carotene 6.52 g/ml. Means marked with the same superscript letters are not-significant
(P>0.05). Data are average of three replicates; each value represents the mean SEM.

Table 2. Milking of Dunaliella bardawil for continuous production and extraction of carotene using dodecane (20% v/v) at 20,000 lux and 170 rpm.
Growth paramerters
Mixing
rates (rpm)

170

Time
(days)
0
3
6
9
12
15

Chlorophyll a
(g/ml)

Cell count
(x 106 cell /ml)

Intracellular
-carotene
(g/ml)

Extracellular
-carotene
(g/ml )

Total
-carotene
(g/ml)

9.940.03
7.19 0.04
3.580.06
2.010.03
1.910.01
1.280.04

5.840.06
3.110.01
2.340.05
0.790.07
0.620.02
0.190.05

6.400.08
8.820.05
9.610.03
10.350.03
11.290.01
12.340.03

___
6.190.11
8.100.07
9.070.05
9.650.09
10.960.04

6.40
15.01
17.71
19.42
20.94
23.30

Data are average of three replicates; each value represents the mean standard error of mean.

26

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

the highest value of total -carotene was obtained after 15 days


(23.30 g/kg). Since, chlorophyll a and cell count recorded minimum
values after 15 days and so continuous production and extraction
of -carotene in this experiment cannot be completed more than
15 days due to the negative effect on the growth of the cells.
Detection of -carotene by HPLC analysis: HPLC chromatogram
of the standard synthetic -carotene revealed one peak of all trans
-carotene at retention time 3.929 min (Fig. 2A). Chromatogram of
natural -carotene extracted from D. bardawil by dodecane
showed two peaks. These two peaks of stereoisomers 9-cis and
all trans -carotene appeared at retention times 3.480 and 3.882
min, respectively (Fig. 2B). The ratio of 9-cis to all trans -carotene
was 1.13.
mAU

A
2000
1500
1000
500
0
0

mAU
1000

4
6
Retention time (min)

min

800
600
400
200
0
0

min

Retention time (min)

Figure 2. High performance liquid chromatography (HPLC)


chromatograms of (A) synthetic -carotene as standard and (B) -carotene
extracted from D. bardawil. -carotene was detected at 450 nm
absorbance. Rt: retention time.

Protective effect of -carotene: Table 3 shows that either in


presence or absence of LPS, the various concentrations of natural
-carotene (30, 140 and 350 g/kg) have no significant effect on
the levels of IL-1 and TNF-, whereas the level of IFN- was
significantly affected. In absence of LPS, administration of
synthetic (350 g/kg) or natural -carotene at different doses
showed non-significant change on the levels of studied cytokines
compared to vehicle control. Pre-treatment with natural -carotene
in LPS injected groups exhibited a significant reduction in IL-1
and IFN- at both doses of 30 and 140 g/kg. Non-significant
difference was observed in Group 8 compared to LPS control for
all investigated cytokines. Synthetic -carotene administration
for 15 days in the presence of LPS (Group 7) caused a significant
reduction in the levels of IL-1 and IFN- compared to LPS control,

whereas it has no marked effect on the level of TNF-. All


concentrations of natural -carotene showed a positive correlation
with the levels of cytokines (Table 3).
Therapeutic effect of -carotene: One-way ANOVA analysis after
LPS injection showed that various concentrations of natural carotene (30, 140 and 350 g/kg) had no significant effect on the
levels of IL-1, IFN-, and TNF- (Table 4). Administration of low
dose (30 g/kg) of natural -carotene after LPS injection exhibited
a significant reduction in the levels of IL-1, IFN- and TNF-
compared to control LPS. Moreover, the levels of IL-1 revealed a
significant reduction in Group 14 compared to control LPS,
however, the levels of IFN- and TNF- were not affected. Posttreatment with synthetic or natural -carotene at high dose (350
g/kg) failed to induce significant reduction in the levels of IL-1,
IFN- and TNF- compared to control LPS. The levels of IL-1,
IFN- and TNF- were positively correlated with various
concentrations of natural -carotene and their correlation
coefficients were 0.92, 0.70 and 0.94, respectively.
The cytotoxicity of natural -carotene against human cancer
cell lines: Percentages of cell inhibition of HepG2 cell line were
positively correlated with various concentrations of -carotene (r
= +0.90) and doxorubicin (r = +0.76). The value of IC50 was 14.58
g/ml for natural -carotene in comparison with IC50 of doxorubisin
which was 7.51 g/ml (Fig. 3A). The various concentrations of
natural -carotene showed a positive correlation with percentage
of cell inhibition of MCF7 cells (Fig. 3B). The correlation coefficient
of natural -carotene (r = +0.85) was lower than doxorubicin (r =
+0.99). IC50 of natural -carotene mounted 7.44 g/ml while IC50 of
doxorubicin was 2.67 g/ml.
Discussion
A biphasic aqueous/organic two-phase system offers a solution
in which both production and extraction occurs simultaneously.
Two of the most important criteria for solvent selection are high
product recovery capacity and biocompatibility. Many previous
studies mentioned that log Poct is used to predict the activity
retention of biocatalyst in organic media. The retention of the
biocatalyst activity is high in a polar organic solvent having log
Poct > 4 31. It appeared that when log Poct increased, extraction
ability of solvent to -carotene decreased as the solubility of
solvent in water decreased. In the present study, decane has the
highest extraction ability among all solvents used as it recorded
highest extracellular -carotene value, but after more than 24 h
cultures turned white. So, dodecane was chosen as the most
suitable biocompatible solvent for extraction of -carotene from
D. bardawil due to its higher extraction ability (it was the second
after decane) and highest -carotene content (intracellular +
extracellular). Our results were in agreement with Leon et al. 23 as
they found that decane and dodecane were more compatible to D.
salina cells than tetradecane and hexadecane. Most milking
studies have used dodecane as a biocompatible solvent 8, 32. As in
the present study, there is no significant difference between
extraction ability of most of the different biocompatible solvents
used 33. Moreover, -carotene productivity per cell with dodecane
was highest and phase separation was rapid producing -carotene
with high purity.
The cell membrane seems to be the primary target of solvent

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

27

Table 3. The levels of IL-1, IFN- and TNF- (pg/ml) in serum of mice
orally administered an olive oil, synthetic -carotene (synth, 350
g/kg) and various concentrations of natural -carotene (nat, 30,
140, and 350 g/kg) in absence and presence of lipopolysaccharide
(LPS, 2.5 mg/kg).

action. The modification of the cell membrane


characteristics is the more relevant cellular adaptation
mechanism due to stress caused by organic solvent 34.
Solvent toxicity has been associated to the ability of
solvent to penetrate the cell membrane, altering its fluidity
and tampering with normal cellular functions 9. In this
Cytokines levels (pg/ml)
Group
work, decane and dodecane recorded the lowest optical
IL-1
IFN-
TNF-
olive oil
55.089.15a
28.068.28a
80.0022.06a
densities indicating lower cell membrane integrity and so
olive oil+LPS
156.3325.27d
132927.41c
606.16157.23c
lower cell membrane damage.
In absence of LPS
In a two-phase milking experiment, we noticed that the
a
a
a
synth 350
35.104.9
328.22
38.384.54
increase in continuous extraction time caused an increase
nat30
65.055.80a
14.562.57a
55.314.25a
in -carotene content whereas the total amount of nat140
59.513.76a
15.864.24a
86.338.39a
nat350
69.264.36a
56.0912.69a
88.5018.06a
carotene produced (-carotene content of the cells +
ANOVA
F2,15=0.244,P>0.05 F2,15 =9.01, P<0.05 F2,15=2.494, P>0.05
extracted part to the organic phase) is highest at the end
r
+0.80
+0.95
+0.58
of the experiment, which is in agreement with Hejazi et
In presence of LPS
al. 35, whereas higher production of -carotene is caused
synth350+LPS
76.938.53c
898.00144.78b
487.1677.69bc
abc
b
b
nat30+LPS
109.3530.25
615.16196.14
351.6617.41
by the continuous extraction, and the presence of the
nat140+LPS
118.3631.44bc
867.41247.98b
432.16100.93bc
bio-compatible solvent (dodecane) in the culture medium
nat350+LPS
281.809.62d
1297.3323.03c
660.66140.87c
may induce the -carotene production pathway. Mixing

ANOVA
F2,15=14.16, P>0.05 F2,15=6.62, P<0.05 F2,15=2.541, P>0.05
causes some shear stress to the cells and also can increase
r
+0.99
+0.99
+0.96
Data were represented as mean of six mice standard error of mean (SEM). In the columns: Means marked with the
contact of the cells with the organic phase. Our
same superscript letters are similar (insignificant difference, P>0.05).
continuous milking experiment here could not be run for
: the effect of various concentrations of natural -carotene on the levels of cytokines (pg/ml).
P>0.05: insignificant effect of various natural -carotene concentrations on the levels of cytokines (pg/ml).
more than 15 days due to decreased growth levels of
r: correlation coefficient between the concentrations of natural -carotene and the levels of cytokines (pg/ml).
cells (the decreased levels of chlorophyll a and cell count).
It has been found that the mixing of aqueous and organic
Table 4. The levels of IL-1, IFN- and TNF- (pg/ml) in serum of mice
phase may enhance extraction but can also enhance
administered with lipopolysaccharide (LPS, 2.5 mg/kg) followed
damage caused by organic solvent, so a compromise
by olive oil, synthetic -carotene (synth, 350 g/kg) and various
between toxicity and extraction ability has to be taken
concentrations of natural -carotene (nat, 30, 140, and 350 g/
into consideration when choosing the organic solvent 35.
kg) after 6 h of LPS injection.
It was obvious from the present data that extracted Cytokines (pg/ml)
Group
carotene
is composed of two isomers; these are 9-cis IL-1
IFN-
TNF-
carotene and all trans -carotene. The ratio of 9-cis to all
lps+olive oil
127.57.90b
1203.097.01b
959.1615.8c
trans -carotene is 1.13, which is in harmony with
LPS+synth350
120.619.70b
1038.4263.75b
520.0 96.5bc
LPS+nat30
68.49.20a
407.473.57a
321.2 42.8a
Kleinegris et al. 10, who reported that when high light
LPS+nat140
65.023.80a
1129.4193.26b
737.6 95.4bc
intensity was used to stress the cells, the ratio of 9-cis to
LPS+nat350
114.012.50b
1056.4229.11b
966.2 109.9c

ANOVA
F2,12=0.543, P>0.05 F2,12=3.464, P>0.05 F2,12=13.941, P>0.05 all-trans -carotene increased from 0.55 to 1.27. It was
observed that the increase in 9-cis to all-trans -carotene
r
+0.92
+0.70
+0.94
Data were represented as mean of six mice standard error of mean (SEM).
ratio was mainly due to accumulation of carotenoid
In the columns: Means marked with the same superscript letters are similar (insignificant difference, P > 0.05).
containing globules (over 50% 9-cis -carotene) and a
: the effect of various concentrations of natural -carotene on the levels of cytokines (pg/ml).
P > 0.05: insignificant effect of various natural -carotene concentrations on the levels of cytokines (pg/ml).
decrease in thylakoid-bound carotenoids (mainly all-trans
r: correlation coefficient between the concentrations of natural -carotene and the levels of cytokines (pg/ml).
-carotene) 36.

W

y = 5.2237ln(x) + 60.535, r= +0.76

y = 21.134ln(x) - 7.0242, r= +0.90

-carotene
Doxorubicin

12

22

32

42



52

100
90
80
70
60
50
40
30
20
10
0

W

100
90
80
70
60
50
40
30
20
10
0

-carotene

Doxorubucin

y = 32.752ln(x) - 32.246,
r= +0.99

y = 29.433ln(x) - 34.149, r= +0.85

12

22

32

42

52



Figure 3. Dose-response curve of natural -carotene and doxorubicin cytotoxicity against HepG2 and MCF-7 human
cell lines using SRB assay.
Percentages of cells inhibition of (A) HepG2 and (B) MCF-7 cell lines after 72 h of incubation with natural -carotene and doxorubicin at various
concentrations (5, 12.5, 25, 50 g/ml). Each value was a mean of three samples standard error of mean. r: correlation coefficient.

28

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

During inflammation, there is recruitment of various immune


cells to the injury or inflammatory site, the activated cells release
pro-inflammatory cytokines. Hosts defense immune system
functions a pivotal role in controlling the acute inflammatory
response. Overproduction of pro-inflammatory cytokines is
associated with pathogenesis of many diseases 37. Here, treatment
with natural -carotene was evaluated in order to sustain the
preventive efficacy on pro-inflammatory cytokine production in
CD1 mice. Natural -carotene at different doses was investigated
in parallel with synthetic -carotene at only one fixed dose of 350
g/kg.
In the current work, mice treated with either synthetic or natural
-carotene groups at various doses for a period of 15 days showed
no significant effect on the levels of IL-1, IFN- and TNF-
compared to vehicle group. Our findings were in agreement with
Novoselova et al. 28 who reported that antioxidant feeding showed
no considerable effect on cytokine milieu of healthy animals. Upon
induction of inflammation, we observed an inflammatory response
that resulted in enhancement of serum levels of pro-inflammatory
cytokines, whereas the levels of IL-1, IFN- and TNF- were
significantly elevated in LPS control compared to vehicle group.
Within aspects of protective assessment, we implied that serum
IL-1 and IFN- levels were significantly inhibited in natural carotene pretreated inflamed mice compared to LPS control at
doses of 30 and 140 g/kg. This tendency towards normalization
of serum cytokine levels has been previously reported 28. Moreover,
D. bardawil powder rich diet showed reduction in mice liver
inflammation by decreasing the expression of IL-1 18.
TNF- is a pleotropic cytokine implicated in immunomodulation 38. TNF- is a crucial inflammatory cytokine released
by monocytes and macrophages, involved in the pathogenesis of
many inflammatory disorders 39. Pre-treatment of inflamed mice
with natural -carotene caused a marked reduction in TNF- at
low dose of 30 g/kg. It is clear that low dose was accompanied
with beneficial suppression of this key inflammatory mediator,
however, the other doses failed. This result has been demonstrated
by Liu et al.40, whereas low doses of natural -carotene have been
suggested to induce protection on Ferrets lungs exposed to
cigarette smoke. Treatment with D. bardawil alga containing both
all-trans and 9-cis-carotene diet has potential ability to inhibit
atherosclerosis progression, particularly in high-fat diet regime 18.
Additionally, natural -carotene could reduce pro-inflammatory
cytokines in mononuclear cells of Alzheimers disease patients in
vitro 41.
In the present study, we found that high dose of natural carotene pretreatment failed to suppress the release of IL-1, IFN, and TNF-. In addition, levels of IL-1 and TNF- to some
extent showed elevation compared to LPS control. Thereby, high
dose of natural -carotene didnt provide a protective effect and
moreover could provoke inflammatory condition. These results
were confirmed in vitro, whereas the high dose of -carotene
exerted pro-inammatory effects by signicantly increasing the
secretion of TNF- from LPS-stimulated murine macrophage cells 42.
Also, it is suggested as a stimulator of an inflammatory process in
peripheral blood mononuclear cells from healthy donors 43.
In our investigation, when therapeutic efficacy of natural carotene identified in a dose dependent manner, we found that
low dose of natural -carotene post-treatment in inflamed groups,
exerted immunosuppressive effect on IL-1, IFN-, and TNF-

production compared to the control LPS. These findings were


supported by previous study in vivo, which found that elevated
serum levels of TNF- and IL-1 induced by LPS administration
were significantly suppressed by administration of natural carotene 44. Furthermore, dietary administration of rich 9-cis carotene employed inhibition of adipose tissue inflammation in
diabetic mice 45. Recently, the algal carotenoid extract of D. salina
demonstrated a potent suppression of IL-1, IL-6 and TNF-
production in initial inflammatory response of LPS-stimulated
macrophage cells; low doses showed higher effective inhibition
on IL-1 production as compared to all trans--carotene 19. Indeed,
post-treatment with either high dose of natural or synthetic carotene failed to promote significant inhibition of IL-1, INF-
and TNF-. These data explain that with the increase in the
concentration of natural -carotene, its ability to inhibit the release
of pro-inflammatory cytokines decreased.
It has been documented that carotenoids induced antiproliferative activity on cancer cells, introducing alterations in
pathway mechanisms, led to cancer cell death 46. D. bardawil 9cis--carotene is up to ten times more effective at cancer
prevention than all trans -carotene 47. Cytotoxicity of the natural
extract of -carotene was determined in comparison to the
anticancer drug doxorubicin in vitro. Natural -carotene showed
cytotoxicity against HepG2 and MCF-7 cells in a dose dependent
manner. Our results showed that natural -carotene exhibited
cytotoxicity to HepG2 and MCF-7 cell lines, whereas IC50 of carotene was nearly two- and threefolds of IC50 of doxorubicin,
respectively, that was a stronger cytotoxic drug. These findings
were supported by Fujii et al. 48 indicating to the inhibitory effect
of -carotene rich D. bardawil on mammary tumor progression
cell line. IC50 of D. salina algae extracted under stress was lower
than normal condition 11. Moreover, -carotene revealed inhibitory
effect on cell growth 49 inducing apoptosis 50 in MCF7 cells.
Conclusions
Our results suggest that low dose of natural -carotene extracted
from D. bardawil is capable of enhancing beneficial antiinflammatory activity by exerting immuno-suppressive effects on
proinflammatory cytokines in vivo. Hence, natural -carotene may
be a useful therapeutic tool for treatment of various inflammatory
diseases; providing protection against inflammation. Additionally,
this study demonstrates the cytotoxic effect of natural -carotene
on human cancer cell lines in vitro. Further studies will be important
to consider the possible use of -carotene to inhibit cancer cell
survival in vivo.
Acknowledgements
This work was supported by Faculty of Science, Cairo University,
Egypt.
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43

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

31

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Journal of Food, Agriculture & Environment Vol.12 (3&4): 32-39. 2014

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Effects of some technological parameters on chemical and sensory qualities and free
fatty and amino acids of various probiotic cultures in Beyaz cheese during
ripening process
Filiz Yanglar 1* and Salih Ozdemir 2
1

Department of Food, Engineering Faculty, Ardahan University, 75000, Ardahan, Turkey. 2 Department of Agricultural, Food
Engineering, Atatrk University, 25000, Erzurum, Turkey. *e-mail: f_yangilar@hotmail.com, filizyangilar@ardahan.edu.tr

Received 16 May 2014, accepted 2 October 2014.

Abstract
It was aimed in the present study to assess the effects of four probiotic strains on the chemical, physical and organoleptic properties of Beyaz cheese.
For all experimental samples taken into consideration in the study, total chemical parameters were found to increase during ripening (p<0.05) process
in the study. The FFAs concentration in control cheeses was significantly higher than that of made with probiotic dahi cheese samples (p<0.05). In
addition, probiotic B. bifidum BB-12 appeared to increase the production of FFAs (caproic, 1.67; caprilic, 0.16 and capric, 2.57%). Bifidum BB
12+L. acidophilus LA5 increased significantly the rate of nitrogen compounds with low molecular weight and individual free amino acids
(p<0.05). Length of ripening period contributed to a significant increase in the content of free amino acids. For the sensorial characteristics, cheese
sample E was found to the least preferred by the panellists.
Key words: Beyaz cheese, probiotics, free fatty acids, free amino acids.

Introduction
Today consumers are concerned in great majority with not only
food security and its nutritional values, but also its health
benefits 1, 2. Such kind of demands helped shape new concepts in
food industry, e.g. functional food, where probiotic ingredients
take important parts 3. Probiotics are defined in an overview to be
living non-pathogenic microorganisms used in food industry to
be dietary supplements due to their health benefits 4-9, such as
increasing the intake rate of low-fat dairy products and the
reduction of the incidence of cardiovascular diseases (CVD),
preclinical atherosclerosis, and cardiovascular risk factors in
middle-age and older age persons 3, 10-16. Although the number of
probiotic bacteria that provides health benefits has not been
firmly established, levels between 106 and 109 cfu/g have been
suggested 17, 18. Various parameters must be considered when
adding probiotic bacteria to foods: type of culture to use, addition
level required to obtain a physiological effect, survival during
process parameters, stability during storage, and effect on the
sensory properties 8, 19, 20.
Dairy products have been used as carrier foods for probiotic
bacteria, as many of them had already been optimized for survival
of lactic cultures 21, 22 , while new products including milk, yoghurt,
fermented milk, desserts, fruit juice and some cheese types have
also taken their places among probiotic products 23. Cheese owns
a deserved fame to be a good carrier for probiotic bacteria by
allowing them to survive throughout gastrointestinal tract 15, 24-32.
Beyaz cheese (Turkish acronym of white cheese) is a soft or semihard cheese type produced from sheep or cow milk or mixture of
them 26. Cheese may offer several advantages over fermented milk
products such as yoghurt by serving as a delivery system for
32

viable probiotic in gastrointestinal tract; tending to increase fat


content; and offering protection to probiotic bacteria during
storage and passage through the gastrointestinal tract. Cheese
can also exhibit larger buffering capacity than yoghurt 33. Various
chemical and biochemical reactions can be seen during the ripening
process of Beyaz cheese including glycolysis, lipolysis and
especially proteolysis, which is an important process and plays a
direct role in the development of cheese flavour and texture 34.
Being among the most widely used probiotic bacteria, lactobacilli
and bifidobacteria can contain proteolytic and peptidolytic enzyme
types and therefore affect proteolysis 35-40. Addition of lactic acid
bacteria to dairy products was stated to contribute to the
production of free fatty acids (FFAs) causing the lipolysis of milk
fat 41-43.
The present study was conducted to evaluate the implications
of different probiotics [B. bifidum BB-12 (B), B. bifidum BB-12+L.
acidophilus LA-5 (C), B. bifidum (D) and B. longum (E)] in addition
to commercial lactic culture [L. lactis and L. cremoris (A)] in the
chemical and physical composition and sensory performance; and
determine the fatty acid composition and related properties of
Beyaz cheese during storage at 4C.
Materials and Methods
Cultures: Research and Application Farm of Atatrk University
provided cow milk and Lactococcus lactis subsp. lactis and
Lactococcus lactis subsp. cremoris frozen to dry were obtained
from DSM Food Specialties Pty. Ltd. (Moorebank, NSW, Australia)
and used in the preparation of starter culture. Strains of various
probiotic bacteria were used as adjunct cultures, among which B.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

bifidum BB-12 and B. bifidum BB-12+L. acidophilus LA-5 were


obtained from Peyma Hansen (Gayrettepe, Istanbul, Turkey), while
B. bifidum and B. longum were obtained from Christian Hansen
(Christian Hansen, Valinnhos, Brazil). The organisms were
activated using the method by Martensson et al. 44 and
experimental cheese material was produced in the pilot dairy plant
of the Agricultural Faculty of Atatrk University.
Cheese manufacture: In the study, probiotic Beyaz cheese
material manufactured by taking the rules of Demirci and imek 45
into consideration was divided into five experimental groups (one
control and four probiotic containing groups). Cheese
manufacturing process was initiated by taking 500 L raw milk and
adjusting its fat concentration to 3%. Milk sample was pasteurized
at 65C for 30 min, cooled to the incubation temperature of 35C
and divided into five equal parts (batch). A solution of 20 g/100 L
CaCl2 was added to each batch and a control batch was prepared
using 1 mL/100 mL commercial culture mix consisting of L. lactis
and L. cremoris and a four batch mix [B (Bifidobacterium bifidum
BB12), C (B. bifidum BB12+L. acidophilus LA5), D (B.bifidum)
and E (B. longum)] was produced using equal concentrations of
the probiotic and commercial mixes. Experiments on cheese
production were conducted at the beginning with 100 L of raw
milk, which was pasteurized at 65C for 30 min, cooled to 35C,
CaCl2 (20 g/100 L) and a commercial culture mix inoculum (1 mL/
100 mL;1% v/v) of cheese starter (Lactococcus lactis and
Lactococcus cremoris) was added. Probiotic strains were added
into samples B, C, D and E to inoculate at a level up to 107 cfu/mL.
Then 12 mL chymosin (Peyma Hansen, Turkey) dispersed in 100
mL water was added in each cheese vat in a sufficient rate to
coagulate them in 90 min. At the end of curdling process about 1
cm3 small curd blocks were left for storage and compressed, after
which formed cheese material was cut into 8 cm3 cubes salted in
pasteurized brine (12% w/v, NaCl) for 6 h. After brine-salting, cheese
samples in the form of blocks were taken to store at room
temperature for 12 h and then transferred to plastic bags containing
brine water by leaving ripening at 41C. Such manufacturing
process of cheese was triplicated and the samples were left to
ripen for 2 months by analysing at the 2nd, 15th, 30th and 60th days of
ripening.
Chemical and physical analysis: Moisture, fat, dry matter fat,
salt, dry matter salt (%), ash (%), protein (%), water-soluble protein
(WSN; %), ripening degree (%) and titratable acidity (SH) of
probiotic Beyaz cheeses were measured three times according to
Kurt et al. 46. The pH was measured by adding and mixing 20 mL of
distilled water in grated cheese (10 g) using a digital pH meter
(WTW 3401 47).
Nitrogen fractions: Probiotic Beyaz cheese fractions soluble in
12% trichloroacetic acid-soluble nitrogen (TCA-SN) were
determined according to Polychroniadou et al. 48 and the microKjeldahl method by IDF 49. In this process, homogenisation of
grated cheese samples (20 g) was performed by adding and
blending 40 mL of H2O in the samples an Ultraturrax blender (IKA,
Wilmington, NC) for 2 min after which the mixture (homogenate)
was stored at 40C for 1 h and centrifuged at 3000 g for 30 min at
4C. The fatty slick was removed and the supernatant was filtrated
through filter paper (Scleicher & Schuell 589/2). Twenty five mL

extract prepared for WSN was taken in an equal volume of 24%


(w/v) and TCA was added in the mixture to fragment nitrogenous
compounds. The mixture samples were left for incubation for 2 h
at ambient temperature. Precipitates were filtered through white
ribbon filter paper (Schleicher & Schuell, 589/2).
Analysis of free fatty acids (FFAs): Direct transesterification gas
chromatography was used to define FFA profile of milk and dahi
samples as in Akaln et al. 50. One mL of methanol : benzene (3:2
ratio) was added to an aliquot of 100 mL of milk or 100 mg of dahi
samples and freshly prepared 1 mL acetyl chloride : methanol
(5:1000) was also added in the mixture and the tubes were capped
tightly. The mixture tubes were taken in methanolysis process at
100C for 1 h, cooled to room temperature and 1 mL methylated
penta decanoic acid-hexane solution (500 mg dissolved in 1 mL of
hexane) and 1 mL water were added. After that, the tubes were
shaken and stored at 4C until the gas chromatography. GC-Agilent
6890 N (USA) equipment had a glass column of 60 m 0.25 mm ID
packed with 10% DC200 on chromosorb, and a flame ionisation
detector. Nitrogen was the carrier gas with the flow rate of 28 mL
min-1. The injector port temperature was fixed at 100C for 2 min by
reaching gradually up to 250C. Peak rates of each FFA were
identified according to the retention times of the reference
standards (Sigma Chemical Co., St. Louis, MO, USA).
Free amino acid (FAA) analysis: Free amino acids were extracted
from probiotic Beyaz cheese curd slurry samples conveniently to
the method in Standara et al. 51. Cheese slurry (10 g) was added in
90 mL trichloroacetic acid (TCA) by mixing to homogenize the
mixture. Fatty top layer of the mixture was removed and the
remaining sample was stored at 3C to separate cream from the
mixture at more advanced level and then the mixture was centrifuged
at 3C for 10 min at 8000 g to remove fully the cream remnant and
vacuum filtered (No. 1 filter paper, Whatman International Ltd,
Maidstone, UK). The isolated free amino acids were derivatized
according to protocol of EZ: FaastTM kit (Phenomenex, Torrance,
CA, USA) and subjected to gas chromatography (GC-Agilent 6890
N (USA) with a split injection port and flame ionization detector
(FID)). Amino acid samples were injected and separated on a
Zebron ZB-PAAC column (10 m 0.25 mm, Phenomenex) and
helium was carrier gas at 60 kPa and injection port and detector
were set to 250C and 320C, respectively, by increasing oven
temperature 35C in one minute from 110C to 320C and leaving
at 320C for one minute. Amino acid standards included in EZ:
FaastTM kits were used for the identification of amino acids in the
samples.
Sensorial analysis: Experimental samples stored at 41C for 60
days were evaluated by trained and experienced panellists
considering sensorial properties and the principles in Lyne 52.
Each panellist gave scores, ranging from 1 (poor) to 9 (excellent)
to the cheese samples taking five sensory properties into account
including colour, texture, taste and aroma, foreign flavour and
aroma, saltiness and general acceptability. Panellists were
permitted to have water and bread to screen the tastes of each
sample.
Statistical analysis: The randomized complete block design was
adopted for the experiments in the study. Data obtained was

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

33

Results and Discussion


Probiotic Beyaz cheese samples were produced from the skimmed
milk material containing dry matter, fat, protein, ash, % acidity and
pH in the rates of 11.680.45%; 3.210.38%; 3.310.17%;
0.590.20%; 8.880.71SH and 6.400.03, respectively. Mean yields
of cheese samples A, B, C, D and E were 15.94ab, 16.85b, 18.15b,
15.48a and 17.60 b, respectively. Cheese sample D yielded
significantly lower than B, C and E (p<0.05) possibly caused by
the different acidification processes employed in sample D. It was
stated by Kindstedt et al. 54 and Buriti et al. 55 that when the direct
milk acidification process is used in common cheese production,
higher yield rate, higher moisture content, as well as improved
durability can be obtained due to delayed acidification.
Physical and chemical composition: Table 1 gives the changes
observed in the physico-chemical parameters obtained from
probiotic Beyaz cheese samples A, B, C, D and E throughout
during 60-day storage period. Length of the ripening period was
found to have statistically significant effects on the chemical
composition of experimental samples. The highest rate of dry
matter (44.43%) was determined in control sample (Lactococcus
lactis+Lactococcus cremoris) even though the dry matter content
of all cheese samples increased during the ripening period. Such
a situation may be expressed by the positive correlation between
dry matter and salt 56, 57. Differences in the rates of dry matter
content between the samples were found to be statistically
significant (p<0.01) in the present study, while Atasever et al. 58,
Topcu and Saldaml 59 and ksz et al. 60 found mean dry matter
rates of 35.41 to 39.48%, 39.80 to 41.75% and 30 to 61%,
respectively, which are in a similar range with those found in the
present study. The highest fat (22.48%) and dry-matter fat

(52.57%) contents were in B. bifidum BB-12 sample.


In the present study, differences in fat rates between samples
were statistically significant (p<0.01) during ripening period in
which fat contents of all cheese samples increased. A general
decrease in the titratable acidity rates of all probiotic cheese
samples was observed throughout ripening period (Fig. 1). The
acidity rate of B. bifidum BB12+L. acidophilus LA-5 (19.12 SH)
sample was lower than those in others in the present study, which
may be related to the salt contents of C cheese sample. Higher salt
and moisture contents might have reduced the activities of lactic
acid bacteria found in cheese 61, 62. During the ripening period in
the present study, pH rates of the cheese samples showed a slight
increase and the highest increase in pH value was seen in C
probiotic cheese sample (6.44), which is convenient with literature,
e.g. Atasever et al. 58 reporting a mean pH rate ranging from 4.98
to 5.68. The protein content of cheese samples ranged from 13.44
to 16.14% resulting from the hydrolysis of proteins to water-soluble
nitrogenous compounds and to brine 62, 63. Mean protein content
of Beyaz cheese ranged from 12.78 to 17.27% by Hayalolu 64,
being in convenience with the present study.
25
Titratable acidity (SH)

transferred and evaluated in SPSS Statistical Software (version


15.0). Standard deviations of mean chemical and biochemical
values were also calculated and statistically significant differences
in mean values were compared using Duncans multiple range
tests. Each sample was subjected to triplicate analysis 53.

24
23
22
21
20
19
18
2

15
30
Ripening time (day)

60

Figure 1. Changes in titratable acidity (SH) of probiotic Beyaz


cheese samples.

Nitrogen fractions: Table 1 and Fig. 2 summarizes the results of


the assessment of proteolysis in the control and probiotic Beyaz
cheese samples through the determination of water-soluble

Table 1. Some chemical and physical properties of probiotic Beyaz cheese.


Beyaz
cheese
samples
A

Ripening
time
(days)
2
15
30
60
2
15
30
60
2
15
30
60
2
15
30
60
2
15
30
60

Dry
matter (%)

Protein
(%)

Fat (%)

Salt (%)

Ash (%)

Fat in dry
matter (%)

Salt in dry
matter (%)

pH

WSN (%)

40.600.18
41.330.13
42.450.11
44.430.08
40.460.15
41.900.18
42.060.44
43.600.71
37.490.62
38.950.31
40.760.58
41.920.65
39.850.69
41.590.78
42.200.86
43.230.12
39.950.93
39.990.24
41.590.50
42.910.41

13.750.14
14.070.12
14.500.05
15.200.16
13.440.11
13.530.50
13.720.44
15.010.29
14.030.42
14.150.51
15.030.85
15.040.29
16.140.58
15.530.42
15.620.26
15.660.32
15.440.71
15.320.25
15.660.43
15.900.20

19.370.30
20.560.28
21.230.43
22.180.52
20.260.37
21.310.65
22.110.91
22.480.76
18.410.85
19.450.19
20.200.16
20.240.34
18.500.29
20.060.27
19.870.61
20.630.74
18.600.36
18.540.61
19.070.25
19.470.48

4.370.42
5.660.38
6.070.39
6.420.34
4.150.47
4.330.58
5.500.54
6.270.32
4.300.46
4.580.82
5.340.51
5.750.46
4.560.53
5.460.36
6.170.62
6.340.48
5.450.39
5.830.53
6.480.42
7.050.57

4.890.24
6.110.42
6.690.50
6.770.38
4.700.63
4.840.37
5.910.28
6.790.45
4.830.47
5.080.32
5.520.35
6.260.29
5.100.37
6.020.26
6.660.63
6.840.56
5.980.61
6.120.23
6.820.74
7.510.32

47.710.52
49.740.70
49.990.59
49.920.41
50.080.35
50.860.26
52.570.47
51.550.39
49.110.64
49.940.43
49.550.34
48.280.56
46.410.39
48.070.45
47.090.17
47.720.23
46.550.47
46.350.39
45.860.70
45.380.55

10.760.50
13.680.71
14.310.32
14.450.68
10.260.41
10.340.60
13.070.77
14.370.35
11.480.39
11.770.23
13.090.18
13.710.46
11.440.70
13.200.52
16.630.38
14.660.67
13.630.49
14.580.20
15.580.38
16.440.41

5.320.69
5.460.76
5.870.39
6.080.43
5.030.46
5.130.37
5.630.62
5.740.25
6.060.28
6.260.42
6.440.31
6.230.49
5.360.16
5.430.32
5.660.15
5.720.43
5.580.64
5.830.39
5.950.26
6.140.57

1.920.14
2.030.20
2.660.18
3.120.37
2.840.26
2.450.52
2.820.47
2.750.24
2.930.02
2.990.40
2.970.18
3.260.20
2.150.11
2.190.37
2.180.45
2.210.38
2.110.31
2.240.60
2.230.12
2.650.29

Ripening
degree
(%)
13.970.09
14.440.24
18.380.52
20.560.13
21.170.17
18.100.35
20.550.42
18.360.51
20.860.78
21.150.05
19.800.11
21.400.85
13.350.41
14.090.03
13.960.64
14.100.07
13.700.32
14.610.03
14.250.99
16.650.83

** The values presented are the average of three recurrences. A(Control): Only lactic culture. B: B. bifidum BB-12+lactic culture. C: B. bifidum BB-12+L. acidophilus LA-5+lactic culture. D: B. bifidum+lactic culture.
E: B. longum+lactic culture.

34

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Table 2. Free fatty acids (FFAs) composition of probiotic Beyaz


cheeses during storage.

2.5

TCA-SN (%)

Beyaz
Cheese
Samples

1.5
1

A
0.5

B
0

15
30
Ripening time (day)

60

Figure 2. Changes in trichloroacetic acid-soluble nitrogen (TCASN) of probiotic Beyaz cheese samples.

C
D
E

Chemical analysis of all types of cheese samples in the present


study showed that the addition of tested probiotic microorganisms
in Beyaz cheese has no adverse effect on cheese composition
and similar results were reported in previous studies, e.g. Gardiner
et al.29, Ong et al.66 and Kilic et al.26.
Free fatty acid (FFA) composition: Results of free fatty acid
analysis of probiotic Beyaz cheese during ripening period are
given in Table 2. The rate of caproic acid in the present study
ranged from 1.01 to 1.74% (Fig. 3), which is convenient with the
results found in Prandini et al. 67 where this rate was stated to be
1.14% in Alpine cheese samples. Total rate of FFA, capric acid
(1.822.85%), was higher in probiotic dahi in the present study
(Fig. 4). Corbo et al. 19 reported that the rate of capric acid in
Bifidobacterium sp. added Italian cheese was higher than that
found in control samples, which is in agreement with the findings
in the present study. It can be seen in literature that butyric acid is
produced largely through lipolytic activity of LAB 43, 68 and the
butyric acid content of food may contribute to medicinal properties
of the dahi product 43, 69.
Free amino acid (FAA) composition: Proteolysis in ripening
process can contribute to the formation of flavour thanks to
peptides and free amino acids. Presence of free amino acid in

Butiric
(C4)
0.560.63
0.530.32
0.530.56
0.520.35
0.580.50
0.520.42
0.540.28
0.500.45
0.550.29
0.520.64

Free fatty acids (%)


Caproic
Caprilic
(C6 )
(C8)
1.010.70 0.210.16
1.610.52 0.210.21
1.740.34 0.220.10
1.670.43 0.160.16
1.640.56 0.170.39
1.360.17 0.150.25
1.370.25 0.130.13
1.510.41 0.150.34
1.380.53 0.140.18
1.290.59 0.130.31

Capric
(C10)
2.290.32
2.850.24
2.510.32
2.570.22
2.290.30
1.920.24
1.820.43
2.200.29
2.100.50
1.970.21

Caproic acid (%)

** The values presented are the average of three recurrences.

1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8

60
Ripening time (day)

Figure 3. Changes in caproic acid of probiotic Beyaz cheese samples.


3
2.8
Capric acid (%)

nitrogen (WSN) and trichloroacetic acid-soluble nitrogen (TCASN) over 60-day ripening period at 4C. The ratio of WSN to total
nitrogen (TN) in all samples increased consistently. Cheese
produced from B. bifidum BB-12+L. acidophilus LA-5 (3.26%)
revealed the highest water-soluble protein rate. WSN rates
reported by Ong and Shah 65 in the samples of probiotic Cheddar
cheese are similar to those found in the present study.
The rate of TCA-SN also increased continuously and intensified
more until 60th day (Fig. 2). Ong and Shah 65 found that the levels
of SN-TCA in probiotic Cedar cheeses increased during ripening
period similarly with those in the present study. Reason for such
a situation may be the responsibility of the starter and probiotic
bacteria proteinases for the formation of TCA-SN. Cheese samples
produced from B. longum (2.27%) in general showed the highest
rate of TCA-SN. Such a condition may show that upon the
formation of soluble peptides by rennet and starter culture, the
peptidases and proteinases of probiotic adjuncts hydrolysed these
peptides and released more intermediate- and smaller-size peptides.
This situation was easily seen at the end of 12 weeks when the
primary proteolysis gave products as substrates for the
subsequent proteolysis by the probiotic organisms.

Ripening
time
(days)
2
60
2
60
2
60
2
60
2
60

2.6
2.4
2.2
2
1.8
1.6
1.4

60
Ripening time (day)

Figure 4. Changes in capric acid of probiotic Beyaz cheese samples.

probiotic Beyaz cheese can be attributed to the proteolytic activity


of the bacteria during ripening. Proline was found to increase
until the 30th day and then decreased until the 60th day while glycine,
serine and isoleucine were stable following the 60th day. The
production of free proline was determined to be associated with
flavour Swiss cheese and attributed to the metabolic activity or
the propionibacterium in some studies 70, 71. Such results are
convenient with our study. Free methionine increase in ripening
period may also increase the rate of sulphur volatile flavour
compounds. Free amino acids may exhibit various taste
characteristics depending on their side chains. When the effects
of the presence of probiotic bacteria (Table 3) on free amino acid
rate were taken into consideration, only the effects of lysine and
serine were found to be significant (p<0.05). It was found when
considered 5 sample types treated with 4 different probiotic bacteria
contents (Table 4) that free amino acid rate exhibited a higher
increase in 4 bifidobacteria added cheese curd slurries in 60-day
ripening period compared to B. bifidum BB12+L. acidophilus
LA5, B. bifidum and the B. longum, B. bifidum BB12 and control
sample. In Beyaz cheese curd slurries, proteolytic activity

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

35

Table 3. Effect of ripening time on free amino acid


contents in Beyaz cheese.
Free Amino
Acid a
Glycine
Alanine
Valine
Isoleucine
Methionine
Proline
Phenylalanine
Tyrosine
Tryptophan
Serine
Threonine
Cystine
Lysine
Histidine
Aspartic acid
Glutamic acid
Asparagine
Glutamine

2
216.9a
321.2a
125.6a
78.3a
115.6a
295.7a
139.5a
179.3a
357.6a
1424.2a
175.9
1582.7
479.6a
301.4a
92.5a
318.6 a
392.1a
1729.8a

probiotic cheese. The results of sensory analysis in the present


study showed that there might have been a common effect
between the test culture mix and commercial starter culture and
the combination of test probiotic culture and commercial starter
culture might be suggested to have positive effects on the sensory
characteristics of Beyaz cheese.

Ripening Time (days)


15
30
60
936.1b
1461.2b
1744.0c
2310.5b
3159.4c
3865.9c
b
c
12.260.7
17.100.2
19.213.6d
5114.0b
6635.8b
7064.0b
b
c
5291.7
8276.9
8954.5cd
291.6c
715.3b
827.9d
1578.2b
3891.4c
3759.4b
154.3a
1205.1b
951.6c
914.8b
1890.3c
2162.0c
17.100.2b 17.383.6b 18.940.9b
233.9
321.8
357.6
1374.0
959.2
925.4
5170.3b
7532.9c
5333.7b
1251.7b
2413.6c
3100.9c
1621.2b
2258.0c
2937.4c
b
d
6413.4
9475.3
9978.1b
4720.1b
5465.8b
5873.6d
14.396.1b 20.417.1b 22.645.2b

Conclusions
Objective of this study was to investigate chemical and
organoleptic characteristics of probiotic bacteria added Beyaz
cheese. In B. bifidum BB-12+L. acidophilus LA-5+lactic culture
cheese samples, increase in FFA was found to be higher than that
in probiotics added cheese samples at the end of ripening process.
It was thought by considering the increased content of some free
amino acids in cheese samples that when probiotic bacteria were
added in cheese samples, they might have accelerated the ripening
process. It may be concluded from the results of the study that
Beyaz cheese bears the ability of being one of the most suitable
tools, which can be used to vehicle for delivering the tested strains
of probiotic bacteria via human diet.

a: Values are given in nmoles g-1 cheese slurry and are means of triplicate analyses.

increased with the free amino acid content when bifidobacteria is


presented in the media. Bergamini et al. 2 stated that compared to
control samples larger rate of free amino acid in probiotic bacteria
added semi-hard cheese samples may be caused by higher
proteolytic activity of the samples. These findings are convenient
with the results of the present study.

Acknowledgements
This work was supported financially by the Atatrk University
Research fund (Project No: 2008/64).

Sensory analysis: Table 5 shows the mean scores given to the


sensory parameters for probiotic cheese samples during their
storage period. Panellists gave nine points to all probiotic cheese
groups and the results of sensory analysis indicated that C cheese
batch (B. bifidum BB-12 and L. acidophilus LA-5) received the
highest sensory scores. E samples were given the lowest score
for saltiness parameter (5.72), which might have been due to high
salt concentration of E probiotic cheese. Previous studies (e.g. 26,
33, 72, 73
) reported no negative effects on sensorial properties of
Table 4. Effect of probiotic bacteria treatment on free amino acid contents in
Beyaz cheese samples.
Free Amino
Acid a

Control

Glycine
Alanine
Valine
Isoleucine
Methionine
Proline
Phenylalanine
Tyrosine
Tryptophan
Serine
Threonine
Cystine
Lysine
Histidine
Aspartic acid
Glutamic acid
Asparagine
Glutamine

642.5
2041.0
7149.6
3962.8
4503.4
736.2
1257.0
272.8
959.0
10.143.6a
551.3
2261.9
3125.4a
1252.8
1412.8
4131.5
3511.9
9984.6

Probiotic bacteria treatment


B. bifidum B. bifidum BB12+
B. bifidum
BB12
L. acidophilus LA5
829.1
872.3
1213.5
1627.3
1468.2
2751.0
9359.4
9837.4
10.274.7
4065.7
4261.0
2236.8
4284.1
474.3
5085.4
776.3
752.8
916.3
1860.4
2026.9
2429.5
587.2
607.7
610.2
1492.9
1104.9
1004.7
13.758.2cd
14.861.0bc
13.838.3c
216.9
36.2
272.0
1723.2
1588.7
1492.5
4248.0c
4517.3b
4127.3bc
1142.5
1092.7
1850.4
1698.2
1427.3
1147.2
5254.6
6263.5
5913.1
3227.9
3717.9
4067.2
12.264.7
14.058.0
13.021.9

B. longum
503.9
1167.4
7835.1
3219.0
3606.4
791.2
1245.7
267.4
804.3
7340.6ab
39.4
1522.6
2819.2a
1293.1
1192.4
4629.5
2020.1
8752.0

a: Values are given in nmoles g-1 cheese slurry and are means of triplicate analyses.

36

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Table 5. Results of sensory analysis of probiotic Beyaz cheese samples for 60 days.
Beyaz Cheese
Samples
A

Ripening
time (days)
2
15
30
60
2
15
30
60
2
15
30
60
2
15
30
60
2
15
30
60

Colour

Texture

8.030.28
7.620.45
7.310.37
7.190.63
7.000.74
7.660.32
7.330.21
7.000.61
6.330.47
6.330.35
7.000.29
6.660.32
7.330.19
7.000.31
6.660.03
7.000.15
8.000.56
7.660.63
7.330.37
8.000.26

7.550.21
7.440.14
7.330.08
7.440.32
7.190.18
7.550.32
7.490.63
7.550.07
7.440.49
7.440.24
7.580.12
7.600.57
7.440.84
7.520.29
7.380.33
7.270.69
6.460.28
6.450.12
6.660.55
6.530.19

Taste and
Aroma
7.600.41
7.440.70
7.300.76
7.440.12
7.210.18
7.600.06
7.660.24
7.440.35
7.410.91
7.350.27
7.520.14
7.710.65
7.380.16
7.490.49
7.330.07
6.800.12
6.940.18
6.910.36
6.770.25
6.580.08

Foreign taste
and odour
8.250.80
7.610.43
7.490.62
7.720.54
7.300.10
7.940.13
7.670.05
7.330.11
7.980.62
7.380.56
7.690.32
7.810.45
7.300.33
7.750.68
7.550.16
7.240.64
7.430.44
7.190.30
7.190.55
6.900.39

Saltiness
7.440.98
7.410.63
7.220.76
7.490.15
7.050.80
7.660.63
7.490.43
7.330.49
7.550.24
7.440.77
7.220.90
7.380.21
7.300.52
6.910.43
6.470.28
6.530.78
7.020.27
6.500.19
5.800.63
5.720.19

General
acceptability
7.600.65
7.470.37
7.360.43
7.220.52
7.270.30
7.550.28
7.660.55
7.550.44
7.360.33
7.550.20
7.410.58
7.490.42
7.300.13
7.440.35
7.210.18
7.240.62
6.000.93
6.760.58
6.000.39
6.230.37

** The values presented are the average of three recurrences.

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Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

39

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Journal of Food, Agriculture & Environment Vol.12 (3&4): 40-45. 2014

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The effects of mint (Mentha spicata) essential oil fortified edible films on the physical,
chemical and microbiological characteristics of lor cheese
Gkhan Kavas 1* and Nazan Kavas

Department of Dairy Technology, Faculty of Agriculture, Ege University, 35100 zmir, Turkey.
Dairy Products Programme, Ege Vocational Training School, Ege University, 35100 zmir, Turkey.
*e-mail: gokhan.kavas@ege.edu.tr, nazan.kavas@ege.edu.tr
1

Received 10 July 2014, accepted 16 September 2014.

Abstract
Sorbitol (S) + whey protein isolate (WPI) and 4 different edible films containing mint (Mentha spicata) essential oil were prepared in 1% (v/v) (Me1),
2% (v/v) (Me2), 3% (v/v) (Me3) and 4% (v/v) (Me4) concentrations. These films were used for coating lor cheese (the traditional whey cheese of
Turkey) samples and stored at +4C for 15 days. Microbiological and physico-chemical analyses were carried out on the 1st, 7th, 10th and 15th days
of storage. Coating the lor cheese with edible film had a positive effect on the weight loss. The increase in the concentration of essential oil had a
significant effect on the fat content, titratable acidity, water vapour permeability, weight loss and antimicrobial activity. For the determination of
antimicrobial activity, the cheese samples were artificially contaminated with Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus
aureus, and yeast-mold enumeration was made immediately after the cheese production and accepted as the initial value. E. coli O157:H7 was the
most resistant microorganism to the essential oil, and the most sensitive ones were S. aureus, L. monocytogenes and yeasts and molds, respectively.
The general results showed that addition of mint essential oil to S+WPI based film in 2% (v/v), 3% (v/v) and 4% (v/v) concentrations had a positive
effect on the extension of the shelf life of lor cheese.
Key words: Lor cheese, edible film, essential oil, mint.

Introduction
Whey is revealed during cheese production and its composition
depends on the milk processed to cheese and the cheese
production method, and it is processed to cheese types such as
Mysost, Ricotta, Serac and Requezjao in the world and to lor
cheese in our country. Lor is a traditional cheese type. For the
production of lor cheese, whey is boiled for the precipitation of
serum proteins and separation from whey, kept for 24 hours, and
drained (for 1-4 days) with the help of fine-mesh cloth with weights
placed on. When the desired consistency was obtained, 2-3%
salt was added to the product and offered to consumption as
fresh 1. Despite the elimination of the natural microbial flora with
the heat treatment applied in production, contaminations create
risks after heat treatment and generally the shelf life of the cheese
is limited to 7 days. Microflora of the lor cheese changes even in
storage at low temperatures, microbial load increases in terms of
Enterobacter, yeasts, E. coli and Staphylococcus species. Also,
due to not using lactic acid bacteria in the production, Listeria
monocytogenes, Escherichia coli O157:H7 and Staphylococcus
aureus may grow in the product after contamination 2-4. In this
study, for the extension of the shelf life of lor cheese, which is a
subject that few studies were made on, lor cheese was coated
with Sorbitol (S) + (WPI) based film. WPI films are used in edible
films for their properties such as low oxygen permeability,
gelatinization, thermal stability, foaming and forming polymers
with carbohydrates with covalent binding, as well as their high
nutritional value and functional properties 5, 6. However, due to
their low moisture barrier properties related to their hydrophilic
groups, plasticizers (glycerol, propylene, glycol and sorbitol) are
40

added to WPI based films 7. In many studies, sorbitol is used as a


plasticizer in WPI based films due to its low moisture adsorption
and high-dissolving abilities 8-10. Many studies report that
antioxidant and anti-microbial active compounds may be added
to edible films and coatings. In this regard, many studies show
that essential oils are widely used to take pathogenic bacteria
growth and degradation in foods under control 11-15. In this study,
mint (Mentha spicata) essential oil with antimicrobial (especially
against Staphylococcus aureus, Bacillus cereus, Bacillus subtilis,
Enterococcus faecium, Klebsiella pneumoniae, Escherichia
coli) 16, antifungal (especially against Pseudomonas solanacearum,
Aspergillus niger, Alternaria alternata and Fusarium
chlamydosporum) 17-19, antiviral (against influenza, herpes and
other viruses) 20, 21 properties are used. Menthol found in the
composition of mint essential oil is a potential agent for the
elimination of plasmid resistance of bacteria 22, 23, additionally, it has
an antiviral property 20, and menthone has an antifungal property 24,
25
. Furthermore, eugenol which is found in the oil composition of
some mint species and accepted in GRAS status has medium, and
carvone has high antimicrobial properties against pathogens 26, 27.
Hussain et al. 28 reported that the main components responsible
for the antimicrobial activity of mint essential oil were cis-carveol
and carvone.
In this study, edible films were produced by adding mint essential
oils to S+WPI based film in different concentrations. A portion of
lor cheese was coated with these films in different concentrations,
while a portion is left uncoated (control group). In all cheese
samples, E. coli O157:H7, L. monocytogenes, S. aureus and yeast-

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

mold enumerations were carried out. Lor cheese samples were


stored at +4C and their microbiological and physico-chemical
properties were examined on the 1st, 7th, 10th and 15th days of the
storage.
Materials and Methods
Whey protein isolate, sorbitol and essential oil: For the
preparation of the coating film, WPI was obtained from Bipro,
Davisco Foods International, Le Sueur, MN, USA. Sorbitol, which
was used as plasticizer, was obtained from Merck Company. Oil,
which was obtained from Mentha spicata species by using
Klavenger hydro distillation method contained carvone (40.5%),
1,8-cineole (23.3%), cis-carveol (19.1%), -caryophyllene (3.9%),
-pinene (2.8%), trans-dihydrocarvone (2.6%), carvyl acetate (2.1%),
trans-sabinen hydrate (1%), menthone (0.4%) and mint essential
oil, was obtained from a local business. The active substances of
the essential oils were determined with Shimadzu GC-9A Model,
Thermon 600 T (50 ml, 0.25 mm ID) column, flow ratio of 60:1,
temperature program 70-180C ,10-30 min, split ratio 60:1, injection
temperature 250C, 1 ml injection rate and with the nitrogen as
carrier gas GC-MS (gas chromatography-mass spectrometry).
Lor cheese: In this study, lor cheese produced from whey, which
was left over from a kashar cheese production by a private
company, was used.
Preparation of edible film solution: Edible films were prepared
according to Pintado et al. 29 and Mchugh and Krochta 30, with
some modifications. Accordingly, 5% w/v whey protein isolate
was prepared, and after the addition of 1.5 g/100 ml sorbitol
plasticizer to the homogeneous solution, a homogenization process
was carried out in a homogenizer. The mixture pH was adjusted
to 8 and kept in a water bath at 902C for 30 min in order to
improve the mechanical properties of the film solution. The
solution was cooled to room temperature and sorbitol-amended
PAS protein isolate film (WPI) was obtained. The cooled film was
filtered and divided into five equal parts; mint essential oil was
added to the first four parts in different concentrations (1% (v/v)
(Me1), 2% (v/v) (Me2), 3% (v/v) (Me3) and 4% (v/v) (Me4)), the
fifth part was artificially contaminated but not coated, and it
constituted the control sample (K). Mint essential oils were added
by using Tween 20 15 for the homogeneous distribution of oil in
the solution. Solution was re-homogenized (20,000 rpm/min) 10
and cooled to room temperature. Cheese samples coated with
these films were left to dry at room temperature for 24 hours. For
microbiological examination, 30 ml film solution was moved to
Petri dishes and evaporated at room temperature for 24 hours and
stored at 41C 31.
Preparation and storage of samples: E. coli O157:H7 (ATCC 25922),
L. monocytogenes (ATCC 19118) and S. aureus (ATCC 6538) strains
used for the artificial contamination of kashar cheese samples were
obtained from Hemakim. Yeast-mold enumeration was made
immediately after the cheese production and accepted as the initial
value. Inocula of 106 cfu/g (6 Log cfu/g) were used for the artificial
contamination. Lor cheese samples of 50 g were immersed in
inocula and kept at 4C for 15 min in a sterile cabinet for bacterial
adhesion and absorption of inocula. Artificially contaminated
cheese samples were packaged by immersing in mint essential oil

solutions at different concentrations. Following the immersion


process, the samples were left to dry in a cooled incubator set
to 10C (Nve; ES 500) for 4-5 hours. The prepared samples
were stored at +41C refrigerator conditions for 15 days and E.
coli O157:H7, L. monocytogenes and S. aureus counts were
calculated in terms of Log10 cfu/g on the 1st, 7th, 10th and 15th days
of the storage.
Physico- chemical analysis: The pH values were examined with a
SS-3 Zeromatic pH meter (Beckman Instruments Inc., California,
USA). Titratable acidity (SH) values were analysed according to
AOAC 32. Film thicknesses were measured with a micrometer at
0.001 precision (Digimatic Micrometer/Japan). Water vapour
permeability of films was determined using ASTM E96-80 (1983)
method gravimetrically at 25C.
Microbiological analysis: Enrichments of the bacterial strains
were carried out prior to artificial contamination of the cheese
samples. FDA method for E. coli O157:H7 enumeration 33 and ISO
method 34 for L. monocytogenes enumeration were implemented.
For the enumeration of E. coli O157:H7, Sorbitol MacConkey Agar
(CT-SMAC) containing Cefixime-Tellurite-Supplement (CT) was
used with incubation at 35-37C for 24-48 hours. For the
enumeration of L. monocytogenes, Palcam Listeria Selective Agar
(Base) (Merck 1.11755) was used and inoculated by spreading to
the surface at 37C for 48 hours. For the enumeration S. aureus,
initial enrichment was implemented with Brain Hearth Infusion
Broth (Oxoid CM225) under anaerobic conditions (37C/48 hours).
For the enumeration, 5% egg yolk tellurite emulsion (Oxoid SR 0054)
solution was added to Baird Parker Agar (Oxoid CM 0275
Basingstoke, U.K.) and incubated under aerobic conditions at 3537C for 24-48 hours 35. In yeast and mold enumeration, Yeast-extractglucose chloramphenicol agar (YGC) (Merck 1.16000) was used and
incubated at 25C for 3-5 days.
Statistical analysis: Five different cheese samples were examined
with 3 parallels and 2 repetitions. For this purpose, SPPS version 15
statistical analysis package software was used. Data significance
as a result of analysis of variance (ANOVA) was tested according
to the Duncan multiple comparison test at p < 0.001 level.
Results and Discussion
The pH and titratable acidity (SH) values of cheese samples,
which were coated with mint essential oil fortified S+WPI based
films and K sample, which was left uncoated, are given in Fig. 1,
fat (%) and weight loss values are given in Tables 1 and 2.
The pH values in all samples decreased during storage. The
greatest decrease in pH value was determined in K sample. With
the effect of increase in mint essential oil concentrations, decrease
in pH value was lower in Me3 on the 10th and 15th days and in Me4
on the 7th, 10th and 15th days of storage.
The relationship between film coating and pH values was
significant in terms of both storage and the samples (p<0.01). The
pH results were similar to those of titratable acidity, the highest
increase was in K sample during storage and the increases in Me3
and Me4 samples were close to each other (Fig. 1).
Fat values increased during storage depending on the
concentration of essential oils (Fig. 2A), and the relationship
between the two parameters was significant (p < 0.01). The effect

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

41

Fat (%)
Me1
Me2
Me3
6.562.46 bB 6.592.78c B 6.620.32 dB
6.613.99 bB 6.684.57 cB 6.753.78 dB
6.680.29 bB 6.780.24cB 6.922.54 dB
6.701.00 bB 7.000.08 cB 7.070.02 dB
Weight loss (%)
14.920.23B 13.450.18B 12.050.33B
11.90.32B 10.540.22B 7.680.24B
6.090.41B 5.420.15B 3.450.16B

Day/
sample
K
1
6.52.42 aA
7
6.523.97 aA
10
6.542.70 aA
15
6.553.50 aA
1
7
10
15

15.212.27A
12.453.90A
6.741.72A

Me4
6.6911.8 eB
6.9124.5 eB
7.1116.8 eB
7.2415.7 eB

Fat (%)

Table 1. The average weight loss (%), fat (%) and SH values and
standard deviations in mint essential oil film coated samples
and K sample (p < 0.01), (n = 3).

11.140.29B
7.040.12B
3.250.14B

Me1
Me2
Me3
Me4

Thickness
(mm)
0.179 0.013
0.180 0.010
0.181 0.012
0.182 0.014

Weight loss (%)

Sample

Water vapour permeability


(g mm m-2 h-1 kPa-1)
8.64 A
8.62 B
8.60 C
8.57 D

pH

Me1

Me2
Me2

Me3
Me3

10

Acidity (SH)

K
K

Me1
Me1

Me2
Me2

15

Me3
Me3

7
10
Storage time (days)

Me4
Me4

15

Figure 1. The average pH and SH values in mint essential


oil film coated samples and K sample.

of coating with sorbitol-amended film on fat values was significant


(p < 0.01). The high fat content of samples coated with mint essential
oils in different concentrations are associated with better
dissolution of fat in lipid fraction of the cheese due to its
hydrophobic properties 36 and good barrier properties of whey
based films against fat 37.
Weight losses in film coated cheese samples during storage
42

Me3
Me3

Me4
Me4

7
10
Storage time (days)
Me1
Me1

Me2
Me2

Me3
Me3

15

Me4
Me4

7
10
Storage time (days)

15

Me4

Storage time (days)


159
154
149
144
139
134
129
124
119
114
109

Me2
Me2

Figure 2. The average fat and weight loss values in mint


essential oil fortified film coated samples and K sample.

5.85
5.8
5.75
5.7
5.65
5.6
5.55
5.5
5.45
5.4
5.35
5.3
1

15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0

Me1
Me1

A, B, C, D, E: The differences between the values in the same column are statistically
significant (p < 0.01).

KK

K
K

K
K

a, b, c, d, e: The differences between the values in the same column are statistically significant (p < 0.01).
A, B, C, D: The differences between the values in the same line are statistically significant (p < 0.01).

Table 2. The average film thickness and vapour


permeability values of films containing mint
essential oils in different concentrations
and standard deviations (n = 3).

7.3
7.2
7.1
7
6.9
6.8
6.7
6.6
6.5
6.4
6.3
6.2
6.1

were lower than K sample. The effect of film coating on weight


loss was significant (p < 0.01), and S+WPI based film is a good
water barrier. In all concentrations of essential oil and K sample,
the weight loss was highest on the 7th day of the storage. The
weight losses in K and Me1 were close to each other on the 7th
day (Fig. 2B) and the relationship between increase in the
concentration and the decrease in weight loss was significant
(p < 0.01). In many studies, film coating prevented the passage
of water vapour and thus decreased the economic losses 8, 38, also
using whey based films together with lipids is effective on
preventing the weight losses in products due to their high moisture
permeability 37. The results regarding S+WPI based film in this
study were consistent with those studies.
Film thickness values of mint essential oil fortified films in
different concentrations were 0.179 mm for Me1, 0.180 mm for
Me2, 0.181 mm for Me3 and 0.182 mm for Me4. The differences
between the film thicknesses were not significant (p > 0.001). The
lowest water permeability value was found in Me4, this highest
water permeability value was in Me1, the difference was significant
(p < 0.01). Film thicknesses increased as the essential oil
concentrations increased and vapour permeability and
correspondingly weight losses decreased.
Cheese samples were contaminated with pathogenic microorganisms at 106 (6 Log cfu/g) levels, yeast-mold counts were 6.21
log10 cfu/g. Antimicrobial effects increased related to the increase
in essential oil concentrations, and mint essential oil had
bacteriostatic and bactericidal effects on the chosen
microorganisms. The results obtained in our study showed
similarities with many studies 16-19. The relationship between the
increase in concentration and antimicrobial effect was significant
among the samples in terms of storage (p < 0.01). Moreover,
antimicrobial effect in all concentrations increased with the

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

duration of storage (Table 3).


This result was explained with the slow passage of antimicrobial
substances from film layer to food in edible film systems and the
longer lasting effect of high concentrations of antimicrobial
substance left in film and on food surface against microorganisms 39, 40. Additionally, it was associated with the increase
in hydrophobic properties of essential oil related to the decrease
in pH, thus the increase in antimicrobial activity due to higher
dissolution in lipid phase of cell membrane of food and the
bacteria 36.
Bacteriostatic effects were determined in all concentrations of

essential oils on microorganisms. Bactericidal effect was


determined in Me2 for S. aureus, Me3 for L. monocytogenes and
yeast-mold and Me4 for E. coli O157:H7. The highest
microorganism growth was determined in K sample, while the
bactericidal effect of mint essential oil was effective on all
microorganisms in Me4. Accordingly, the highest increases of the
average storage days in K sample were yeast-mold (7.55 Log10
cfu/g with 1.34 Log10 cfu/g increase), S. aureus (7.65 Log10 cfu/g
with 0.92 Log10 cfu/g increase), E. coli O157:H7 (7.71 Log10 with
0.81 Log10 cfu/g increase) and L. monocytogenes (7.56 Log10 cfu/
g with 0.78 Log10 cfu/g increase), respectively. The highest
antimicrobial activities of the average storage days
Table 3. The average microorganism counts of in mint essential oil fortified
in Me3 sample were S. aureus (0.83 Log10 cfu/g with
film coated samples and K sample (n = 3); (p < 0.01).
5.9 Log10 cfu/g decrease), yeast-mold (1.41 Log10 cfu/
g with 4.8 Log10 cfu/g decrease), L. monocytogenes
Mint essential oil concentration (%)
(2.25 Log10 cfu/g with 4.53 Log10 cfu/g decrease) and
K
Me1
Me2
Me3
Me4
Microorganisms Day
Microorganism counts (Log10 cfu/g)
E. coli O157:H7 (2.89 Log10 cfu/g with 4.01 Log10
1 7.120.11aA 7.090.05aB 6.140.25 aC 3.850.35 aD
cfu/g decrease), respectively. In general, E. coli
7 7.880.23bA 7.631.12bB 5.910.88 bC 3.210.36bD
O157:H7 was the most resistant microorganism to
E. coli O157:H7
cA
cB
cC
cD
7.672.47
5.680.54
2.520.12
10 7.910.09
all concentrations of essential oil, and the most
dA
dB
dC
dD
15 7.930.05
7.711.54
5.280.23
2.010.09
sensitive one was S. aureus. The bacteriostatic effect
1 7.242.36 aA 5.932.87 aB 5.870.99 aC 3.350.01 aD
of essential oil on E. coli O157:H7 was started to
7 7.421.12bA 5.241.47bB 3.920.14 bC
S. aureus
10 7.761.08 cA 5.482.10bB 3.020.03 cC
observe in Me2 and Me3 (in higher concentrations),
15 7.92.05 dA 3.811.15dB
however, no bactericidal effect was observed in these
1 7.090.24 aA 7.010.53 aB 5.860.33 aC 3.800.02 aD
concentrations. Bactericidal effect for E. coli
bA
bB
bC
bD
7 7.030.33
7.050.64
5.470.42
3.040.01
L. monocytogenes
O157:H7 was determined from the 1st day of the
cA
cB
cC
cD
10 7.530.09
7.230.64
4.210.09
2.170.89
dA
dB
dC
storage in Me4 concentration (Fig. 3A).
5.320.10
3.870.08
15 7.710.07
The bacteriostatic effect of essential oil on S.
1 6.881.13 aA 6.740.04 aB 3.541.28 aC 3.120.91 aD
7 7.580.95bA 6.81.49 bB 3.092.87bC 2.520.12bD
aureus
was observed in Me1 from the first days of
Yeast - Mold
10
15

7.760.33 cA 5.210.99 cB 2.851.54 cC


7.870.33dA 5.010.87dB 2.411.54 dC

8.5
8
7.5
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
-0.5

E. colli O157:H7
A

Log10 (cfu/g)

8.5
8
7.5
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
-0.5

7
10
Storage time (days)

15

8
7.5
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
-0.5

Staphylococcus aureus
B

Log10 (cfu/g)

Log10 (cfu/g)

Log10 (cfu/g)

a, b, c, d: The differences between the values in the same column are statistically significant (p < 0.01).
A, B, C, D: The differences between the values in the same line are statistically significant (p < 0.01).

7
10
Storage time (days)

15
K
K

8
7.5
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
-0.5

Me1
Me1

Listeria monocytogenes

10
77
10
Storage time (days)
D

Yeast - Mold

11
Me2
Me2

15
15

10
77
10
Storage time (days)
Me3
Me3

15
15

Me4
Me4

Figure 3. E. coli O157:H7 (A), S. aureus (B), L. monocytogenes (C) and yeast-mold (D) growth in samples coated with
mint essential oil added film and C sample.
Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

43

the storage, related to the increase in concentration, and


bactericidal effect was acquired on the 15th day in Me2 and on
the 7th day in Me3 (Fig. 3B). The bacteriostatic effect of essential
oil on L. monocytogenes was observed on the 10th day in Me1 and
on the 7th day in Me2. The bacteriostatic effect was observed from
the 1st day in Me3 and the bactericidal effect was determined on
the 15th day. The bactericidal effect was observed from the 1st day
of the storage in Me4 (Fig. 3C). All concentrations of essential
oils showed a bacteriostatic effect on yeast-molds. The
bacteriostatic effect was observed on the 7th day in Me1 and on
the 1st day in Me2 and Me3 (in higher concentrations), the
bactericidal effect was determined on the 10th day in Me3 and on
the 1st day in Me4 (Fig. 3D).
The bacteriostatic effect of essential oil on microorganisms was
determined in Me2 and the bactericidal effect was determined in
Me4 from the 1st day of the storage. The results obtained in this
study for E. coli O157:H7 and S. aureus are consistent with those
of many other studies on antimicrobial activity of various essential
oils 6, 15, 41-43.
Conclusions
In our study, fortification of S+WPI with mint essential oil had a
significant effect on extension of the shelf life of lor cheese, and
antimicrobial effect was obtained especially in Me2 and Me3
concentrations. Bactericidal effect was observed on all
microorganisms in Me4 concentration. Additionally, the fat values
increased with the increase in essential oil concentrations and
S+WPI based film proved to be a good water, oxygen and fat
barrier. It was concluded that coating with film decreased weight
losses of lor cheese and created a positive effect in terms of quality.
Acknowledgements
The authors wish to thank O.Turer for expert assistance during
essential oil extraction and Boyacoglu Dairy Factory Co., Turkey,
for assistance during production of lor cheese.
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31

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

45

WFL Publisher
Science and Technology
Meri-Rastilantie 3 B, FI-00980
Helsinki, Finland
e-mail: info@world-food.net

Journal of Food, Agriculture & Environment Vol.12 (3&4): 46-50. 2014

www.world-food.net

Improving the layout of ventilation ports in packaging for fresh produce using
computational fluid dynamics
Hiroaki Kitazawa * and Naoko Hasegawa
Food Engineering Division, National Food Research Institute, NARO, 2-1-12, Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
e-mail: ktz@affrc.go.jp, naohase@affrc.go.jp
Received 2 June 2014, accepted 10 September 2014.

Abstract
Packaging for fresh produce must sometimes include features that ensure uniform and rapid airflow to enable cooling and gas exchange. For this reason,
such packaging usually has ventilation ports. However, attempting to understand cooling and ventilation efficiency by actually manufacturing various
types of packaging in order to develop packaging with maximum ventilation efficiency would require substantial time and effort. Therefore, the size
and layout of the vent ports in the packaging of fresh produce is presumably not determined based on any scientific rationale. Computational fluid
dynamics (CFD) is a method to analyse the behaviour of fluids in space. By setting appropriate parameter values, no great difference was found
between the simulation value and the measured value for temperature change when air passes through the packaging. Consequently, in this study, to
optimise the layout of ventilation ports in packaging made of corrugated fibreboard for fresh produce, we used CFD simulation to propose an
improved vent layout for uniform internal ventilation of one-layer packaging for strawberries. Simulations were conducted in three steps: the first
step involved estimating airflow of packaging with various port layouts; the second step was proposing improved port layouts for the packaging; and
the last step was the estimation of the packagings ventilation ability based on improved port layouts. Our results suggested that, with the improved
port layout resulting from CFD simulation, it is possible to induce large airflow in the lower level of one-layer packaging for strawberries. However,
further study to investigate the shape and number of ventilation ports is needed to achieve uniform ventilation in the upper layer of packaging.
Therefore, in future studies, differences in the number and shape of ports that affect airflow in this space will be considered.
Key words: Computational fluid dynamics (CFD), fresh produce, packaging, ventilation port.

Introduction
Packaging for fruits and vegetables must sometimes include
features that ensure uniform and rapid airflow to enable cooling
and gas exchange 1. For this reason, such packaging usually has
ventilation ports. However, attempting to understand cooling and
ventilation efficiency by actually manufacturing various types of
packaging in order to develop packaging with maximum ventilation
efficiency would require substantial time and effort. Moreover, it
would be difficult to measure the flow of air within each part of a
closed and narrow space. Therefore, the size and layout of the
vent ports in the packaging for fruits or vegetables are presumably
not determined based on any rigorous or tested rationale.
Computational fluid dynamics (CFD) is a method used to analyse
the behaviour of fluids in space. CFD is employed for analysis in
a wide range of fields, including meteorology 2, automotive
engineering 3, and agriculture 4. Therefore, CFD simulation may
be a useful technique for improving ventilation efficiency in fruit
and vegetable packaging. In fact, CFD has been used in several
reports analysing changes in airflow and/or temperature in
packaging containers for certain fruits, such as apples 5, 6 and
strawberries 7-11.
In a previous study 12, the authors used CFD simulation to
study the effect of inlet velocity and port size on airflow in onelayer packaging for strawberries 13; this type of packaging had a
single port in the centre of the boxs long side, and this study
46

revealed factors hindering ventilation efficiency. Furthermore, by


setting appropriate parameter values, no great difference was found
between the simulation value and the measured value for
temperature change when air passed through the packaging 1.
In the present study, we used CFD simulation to obtain port
layouts that enables uniform airflow inside one-layer packaging
for strawberries.
Materials and Methods
Sequence of analysis: The analysis sequence for the present study
is shown in Fig. 1. The scope of this study was to use CFD analysis
to identify issues related to airflow inside packaging with the
normal port layout and, subsequently, to propose an improved
layout. Therefore, evaluation of compression stress and ventilation
efficiency of actual packaging was not performed in this study.
Simulation conditions for CFD: The model for this analysis is a
corrugated fibreboard box for one-layer packaging of strawberries;
the internal dimensions of this box are 210 290 45.25 mm3, as
shown in Fig. 2. In this type of packaging, two trays filled with
strawberries are placed inside the box, and the edges of these
trays touch the boxs inner walls; therefore, the plurality of the
interior space is divided horizontally. A three-dimensional shape
resembling such a space was created using a fluid analysis

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

In this equation, U is the inlet velocity [m s-1] and L is the


representative length [m]. From this equation, if the port diameter
is set to a typical length of 19 mm, Re for the inlet portion in this
analysis will be approximately 260. Consequently, this analysis
assumes a simple laminar flow state, and the numerical
computations were conducted accordingly. Normal calculation
was performed in the simulation analysis, and when the residual
numerical calculations for the inlet velocity became less than 10-3,
the numerical calculation was judged to have converged. A
personal computer (Vostro 260S, Dell Japan CPU: Intel CoreTM
i5-2400 3.10 GHz, RAM: 4.00 GB) was used for the calculation.

Figure 1. Simulation sequence in this study.

Estimation of ventilation ability:


Estimation with various port layouts (Simulation 1): The diameter
of the port was set to 19 mm (which is close to the limiting size),
regardless of whether the port was placed in the upper or lower
layer of the box. There were two ports made on each of the boxs
long sides; a pair of ports was the inlet, and the other was the
outlet. Furthermore, considering the various situations in which
packaging might actually be stored, the boxs centre of gravity
was taken to be the point of symmetry, and ports were then made
at distances of 0, 24, 48, 72, 96, and 120 mm from the centre of the
long side such that the ports were symmetrical with respect to
both the inlet and the outlet sides. Based on these different layouts,
the twenty patterns of port layout that were analysed in this study
are shown in Fig. 3.

Figure 2. Type of packaging for CFD analysis.

preprocessor (GAMBIT 2.4.6, ANSYS, Inc., PA, USA). The number


of meshes in each shape was approximately 130,000. The ratio of
the mesh to the volume of the internal space was equivalent to the
ratio in a previous study 12, and furthermore, it has also been
verified that this ratio matches the actual measurements 1. The
number of ports and their diameter and layout will be described
later. The airflow in the box when air was allowed to flow from one
side was reproduced using CFD analysis software (ANSYS Fluent
14.5, ANSYS Inc., PA, USA). In their CFD analysis of forced
aeration in apple packaging, Zou et al. 5 calculated the inlet velocity
to be 0.9 m s-1. In a previous study 12, the analysed inlet velocity
ranged between 0.3 and 1.5 m s-1. In the present study, to examine
the ventilation capability when the airflow was very small, the
inlet velocity was set to 0.2 m s-1. The physical properties of air
at 15C were used (viscosity = 1.7894 10-5 Pa s and density
= 1.225 kg m-3).
The Reynolds number Re for the flow field in the box is expressed
with the following equation, where Re 2000 is considered to be
turbulent:
Re = U L ( -1) -1

96 48 0 48 96
96 48 0 48 96
120 72 24 24 72 120
120 72 24 24 72 120
Distance from the centre of the
Distance from the centre of the
long side [mm]
long side [mm]
shows inlet ports on the front side.
shows outlet ports on the other side.
shows that the position of inlet ports overlaps with outlet ports.

Figure 3. Basic port layouts for CFD analysis.

This section seeks to determine the port layout in which the


airflow in the box becomes most uniform. First, the effect of
differences in port layout on the average velocity distribution in
lines 1-8 (shown in Fig. 4) was studied. Next, the average and
standard deviation in lines 1-5 in the upper layer and in lines 6-8 in
the lower layer of the box were calculated. Then, the port layout in
which the variation in the average velocity was small and the
average velocity was large in both the upper and the lower layers
was selected. In a previous study 12, researchers concluded that it
was possible to adjust the magnitude of the velocity to a certain
extent by changing the size of the ports. However, it was also

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

47

Estimation of ventilation ability with improved port layouts


(Simulation 3): Minimising port size is essential for maintaining
the strength of the box. In each of the port layouts discussed in
Simulation 1, the air entering the ports is divided in two directions
by the vertical tray placed inside the box. Consequently, to ensure
that the cross-sectional area of the ports located in both the upper
and lower portions were half the size of the port with the 19-mm
diameter, the ports were replaced with smaller ports with a 13.44mm diameter. These ports were then enlarged by factors of 1.05,
1.10, 1.20, 1.30, and 1.40 (which resulted in diameters of 14.10,
14.78, 16.12, 17.46, and 18.80 mm, respectively), and upon placing
the enlarged ports at the positions proposed in Simulation 2, the
airflow in the interior was simulated. The analyses relating to
differences in port diameter were conducted separately for the
lower and upper layers. The analyses of other conditions were
performed according to Simulation 1.

Figure 4. Position for calculating air velocity distribution.

suggested that controlling the airflow distribution by changing


either the size of the ports or the inlet velocity of air through the
ports was very difficult. For this reason, it was decided that, in the
port layout, smaller variation in the average flow velocity would
be prioritised over the maximum velocity, and this reasoning was
followed in subsequent analyses.
Proposing improved port layouts (Simulation 2): From the
results of the analysis in Simulation 1, several port layouts in
which airflow in the upper and lower layers was large and mostly
uniform were selected, and based on these layouts, the airflow in
the interior of the box was simulated. Using the results of
Simulation 1, ports were made in the upper and lower layers.
Therefore, the four ports were made on the inlet and outlet ends
of the long sides of the box. The port diameter and the analysis
method from Simulation 1 were also applied to this simulation.

Results and Discussion


Estimation with various port layouts (Simulation 1): The airflow
distributions in the interior space for several port layouts are
shown in Table 1. Whenever a port was located in the centre of
the long side of the box (Cases 1-5), the variation in the velocity
for both the lower and upper layers was large [hereafter, this
variation is referred to as the standard deviation (SD) value], and
in these port layouts, while some places exhibited strong airflow,
there would presumably be places where only limited airflow
occurred (Fig. 5). Based on Cases 6-10 (in which a port was
located 24 mm from the centre of the long side), despite the trend
toward a greater SD for the average airflow in the upper layer in
Case 8 and in the lower layer in Case 10, the SD in the average
velocity was smaller in both the lower and upper layers in other
cases. In Cases 11-14, in which a port was located 48 mm from the
centre of the long side, both the average velocity and its SD in the
lower layer appeared largely unaffected by the port layout.
However, a trend toward a larger average velocity was observed
in Case 13 in the upper layer, and a trend toward a small SD was
seen in Case 14. In Cases 15-17, in which a port is located 72 mm
from the centre of the long side, despite the large average velocity
in the upper layer, a tendency toward a large SD in velocity was

Table 1. Airflow distribution in packaging with various port layouts.


Analysis
pattern
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Case 7
Case 8
Case 9
Case 10
Case 11
Case 12
Case 13
Case 14
Case 15
Case 16
Case 17
Case 18
Case 19
Case 20

Line 1
0.020
0.015
0.011
0.009
0.006
0.008
0.003
0.004
0.005
0.006
0.008
0.006
0.007
0.007
0.011
0.009
0.010
0.015
0.010
0.039

Upper layer (local velocity [m/s])


Line 2 Line 3 Line 4 Line 5 Average
0.020
0.079
0.012
0.010
0.028
0.034
0.077
0.016
0.004
0.029
0.085
0.079
0.021
0.004
0.040
0.016
0.085
0.010
0.004
0.025
0.012
0.099
0.008
0.003
0.026
0.013
0.013
0.021
0.022
0.015
0.016
0.014
0.031
0.015
0.016
0.018
0.013
0.098
0.010
0.029
0.009
0.021
0.014
0.008
0.011
0.011
0.032
0.011
0.006
0.013
0.032
0.014
0.020
0.020
0.019
0.035
0.015
0.032
0.014
0.020
0.034
0.016
0.109
0.010
0.035
0.014
0.016
0.01
0.007
0.012
0.106
0.011
0.026
0.022
0.035
0.109
0.011
0.034
0.014
0.035
0.110
0.013
0.112
0.010
0.051
0.011
0.008
0.011
0.028
0.015
0.016
0.008
0.015
0.011
0.012
0.010
0.006
0.010
0.039
0.021

Z: Standard deviation of average value.

48

SDZ
0.026
0.026
0.035
0.030
0.037
0.005
0.009
0.035
0.005
0.010
0.008
0.011
0.038
0.004
0.036
0.038
0.049
0.007
0.003
0.015

Lower layer (local velocity [m/s])


Line 6 Line 7 Line 8 Average
SD
0.043
0.108
0.024
0.058
0.036
0.025
0.114
0.022
0.054
0.042
0.024
0.119
0.019
0.054
0.046
0.020
0.116
0.016
0.050
0.046
0.011
0.115
0.011
0.046
0.049
0.033
0.043
0.044
0.040
0.005
0.029
0.051
0.027
0.036
0.011
0.025
0.052
0.026
0.034
0.012
0.022
0.054
0.022
0.032
0.015
0.014
0.069
0.014
0.033
0.026
0.039
0.038
0.046
0.041
0.004
0.034
0.047
0.030
0.037
0.007
0.031
0.049
0.030
0.036
0.009
0.027
0.052
0.028
0.036
0.011
0.041
0.036
0.045
0.041
0.004
0.036
0.04
0.034
0.038
0.005
0.032
0.047
0.033
0.037
0.007
0.042
0.033
0.050
0.042
0.007
0.035
0.040
0.036
0.037
0.002
0.053
0.025
0.052
0.043
0.013

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

0.30
0.27
0.24
0.21
0.18
0.15

(Z = 34.6 mm)

96 48 0 48 96
120 72 24 24 72 120
Distance from the centre of the
long side [mm]

0.12
0.09
0.06

96 48 0 48 96
120 72 24 24 72 120
Distance from the centre of the
long side [mm]

shows inlet ports on the front side.


shows outlet ports on the other side.

0.03

Figure 6. Improved port layouts for CFD analysis.

0.00
Velocity [m/s]

(Z = 10.5 mm)

Figure 5. Simulated airflow in Case 3.


Arrows show the positions of inlet ports.

also observed. Furthermore, a similar trend in both the velocity


and its SD in the lower layer were observed in Cases 11-14. For
Cases 18-19, in which a port was located 96 mm from the centre of
the long side, both the average velocity in the upper layer and the
SD for the average velocity in the lower layer were observed to be
small; this trend was similar to the tendency observed in Cases
11-17. In Case 20, in which all ports were located 120 mm from the
centre of the long side, the observed trend was that the SD for the
average velocity was large in the upper layer.
In summary, these results reveal that, when a port is in a position
24-120 mm from the centre of the long side of the box and is
positioned such that the inlet port and the outlet port do not
overlap, the SD in the velocity was generally reduced (even though
the SD for the average velocity in the upper layer may be assumed
to be greater in some portions of the layout), and the velocity in
the interior space may be considered to approach uniformity.
Based on this fact, ten patterns of port layout (shown in Fig. 6)
were selected to simulate the effect of port layout on the velocity
distribution in the interior space in the next section.
Proposing improved port layouts (Simulation 2): Among Cases
21-30 that were analysed, in Cases 22, 25, 28, and 29, a tendency
toward greater average velocity in the upper layer was observed
(Table 2). However, the SD for the average velocity was also large,
and it was therefore expected that, in areas of large velocity, a bias
may occur in favour of the accompanying port layouts. The next

instances of large average velocity in the upper layer occurred in


Cases 26 and 27. The SD in the upper layer of Case 27 was the
smallest among the cases analysed in this section.
For the lower space, the largest average velocity and the smallest
SD occurred in Cases 27 and 29. Because the SD for the average
velocity in the upper layer was large in Case 29, Case 27 was
deemed to present the best port layout in this section. Therefore,
in the following section, ten patterns with changing port diameters
(shown in Fig. 7) were simulated based on the port layout of Case 27.

96 48 0 48 96
120 72 24 24 72 120
Distance from the centre of the
long side [mm]

96 48 0 48 96
120 72 24 24 72 120
Distance from the centre of the
long side [mm]

shows inlet ports on the front side.


shows outlet ports on the other side.

Figure 7. Various port diameters with improved port layouts


for airflow simulation.

Estimation of ventilation ability with improved port layouts


(Simulation 3): In Cases 31-35, in which the effect of differences
in the lower layers port diameter on average velocity in this layer
was analysed, average velocity increased with an increase in port
diameter. This finding is clear from comparing the 1.4-fold increase
in port diameter with the 1.05-fold increase, which results in a
1.78-fold increase in average velocity, as shown in Fig. 8 and
Table 3.

Table 2. Airflow distribution in packaging with improved port layouts.


Analysis
pattern
Case 21
Case 22
Case 23
Case 24
Case 25
Case 26
Case 27
Case 28
Case 29
Case 30

Line 1
0.003
0.004
0.004
0.005
0.006
0.004
0.006
0.010
0.024
0.026

Upper layer (local velocity [m/s])


Line 2 Line 3 Line 4 Line 5 Average
0.006
0.015
0.008
0.005
0.008
0.019
0.010
0.112
0.007
0.031
0.016
0.010
0.025
0.012
0.013
0.010
0.011
0.012
0.026
0.013
0.030
0.011
0.118
0.007
0.034
0.032
0.011
0.028
0.011
0.017
0.026
0.012
0.017
0.027
0.018
0.030
0.008
0.119
0.006
0.035
0.021
0.009
0.124
0.009
0.037
0.007
0.006
0.008
0.013
0.012

SDZ
0.004
0.041
0.007
0.007
0.043
0.011
0.008
0.043
0.044
0.008

Lower layer (local velocity [m/s])


Line 6 Line 7 Line 8 Average
SD
0.025
0.057
0.02
0.036
0.015
0.028
0.056
0.028
0.037
0.013
0.030
0.054
0.030
0.038
0.011
0.043
0.047
0.033
0.041
0.006
0.032
0.049
0.032
0.038
0.008
0.033
0.047
0.033
0.038
0.006
0.046
0.042
0.037
0.042
0.004
0.036
0.046
0.036
0.039
0.005
0.039
0.039
0.047
0.042
0.004
0.040
0.037
0.047
0.041
0.004

Z: refer to Table 1.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

49

Table 3. Effect of differences in port diameter on the airflow distribution in packaging with improved port layouts.
Port diameter (mm)
Upper
Lower
layer
layer
13.44
14.10
13.44
14.78
13.44
16.12
13.44
17.46
13.44
18.80
14.10
13.44
14.78
13.44
16.12
13.44
17.46
13.44
18.80
13.44

Analysis
pattern
Case 31
Case 32
Case 33
Case 34
Case 35
Case 36
Case 37
Case 38
Case 39
Case 40

Z: refer to Table 1. y: not simulated.

Lower layer

Upper layer (local velocity [m/s])

Lower layer (local velocity [m/s])

Line 1

Line 2

Line 3

Line 4

Line 5

Average

SDZ

Line 6

Line 7

Line 8

Average

SD

0.032
0.035
0.041
0.048
0.053

0.012
0.012
0.012
0.012
0.012

0.019
0.020
0.022
0.025
0.028

0.027
0.027
0.027
0.025
0.024

0.019
0.020
0.022
0.024
0.026

0.009
0.010
0.012
0.013
0.015

0.052
0.057
0.067
0.079
0.091
-

0.048
0.052
0.062
0.073
0.084
-

0.041
0.045
0.054
0.064
0.075
-

0.047
0.051
0.061
0.072
0.083
-

0.005
0.005
0.005
0.006
0.007
-

0.007
0.008
0.009
0.010
0.011

layers) without limiting the strength of packaging, it will be


necessary to conduct strength tests on actual packaging.

0.30
0.27
0.24
0.21
0.18
0.15
0.12
0.09
0.06
0.03
0.00
Velocity [m/s]

Acknowledgements
The simulations using GAMBIT 2.4.6 and ANSYS Fluent 14.5
were performed with the assistance of the Agriculture, Forestry
and Fisheries Research Information Technology Center for
Agriculture, Forestry and Fisheries Research, MAFF, Japan.
References
Kitazawa, H. 2012. Optimisation of packaging for fresh produce by
applying computational fluid dynamics (CFD). Food Packag. 53(5):1923 (in Japanese).
2
Flaherty, J. E., Stock, D. and Lamb, B. 2007. Computational fluid
dynamic simulations of plume dispersion in urban Oklahoma City. J.
Appl. Meteor. Climatol. 46:2110-2126.
3
Fu, L., Ishima, T., Long, W. Q. and Tian, J. P. 2009. Research on the
ignition-chamber GDI engine combustion system. J. Therm. Sci. Tech.
4:53-62.
4
Kacira, K., Short., T. H. and Stowell, R. R. 1998. A CFD evaluation of
naturally ventilated multi-span, sawtooth greenhouses. Trans. ASAE
41:833-836.
5
Zou, Q., Opara, L. U. and McKibbin, R. 2006. A CFD modeling system
for airflow and heat transfer in ventilated packaging for fresh foods: II.
Computational solution, software development, and model testing. J.
Food Eng. 77:1048-1058.
6
Opara, L. and Zou, Q. 2007. Sensitivity analysis of a CFD modeling
system for airflow and heat transfer of fresh food packaging: Inlet air
flow velocity and inside-package configurations. Int. J. Food Eng.
3(5):article No. 16.
7
Ferrua, M. J. and Singh, R. P. 2009a. Modeling the forced-air cooling
process of fresh strawberry packages. Part I: Numerical model. Int. J.
Refrig. 32:335-348.
8
Ferrua, M. J. and Singh, R. P. 2009b. Modeling the forced-air cooling
process of fresh strawberry packages. Part II: Experimental validation
of the flow model. Int. J. Refrig. 32:349-358.
9
Ferrua, M. J. and Singh, R. P. 2009c. Modeling the forced-air cooling
process of fresh strawberry packages. Part III: Experimental validation
of the energy model. Int. J. Refrig. 32:359-368.
10
Ferrua, M. J. and Singh, R. P. 2009d. Design guidelines for the forcedair cooling process of strawberries. Int. J. Refrig. 32:1932-1943.
11
Ferrua, M. J. and Singh, R. P. 2011. Improved airflow method and
packaging system for forced-air cooling of strawberries. Int. J. Refrig.
34:1162-1173.
12
Kitazawa, H., Funaki, T., Nakao, M., Ohshiro, Y., Hiruta, M. and
Ishikawa, Y. 2012. Air flow visualization for fresh produce packaging
by CFD analysis. Food Sci. Tech. Res. 18:525-534.
13
Kitazawa, H., Ishikawa, Y., Lu, F., Hu, Y., Nakamura, N. and Shiina, T.
2010. Alleviation of strawberry bruising due to vibration using 1-layer
packaging with cushioning. J. Packag. Sci. Tech. 19:33-42.
1

Y
X
(Z = 10.5 mm)

Figure 8. Effect of port diameter (d [mm]) on airflow in Cases 31-35.


Arrows show the direction of airflow.

In comparison with Case 1 (analysed in Simulation 1, in which


the average velocity in this space is greatest), when the port
diameter was increased 1.4 times, the average velocity was
predicted to increase 1.43 times. Furthermore, with the increase in
port size, there is, conversely, a tendency for the SD of the average
velocity in this space to decrease. In Cases 36-40, in which the
upper layer was analysed, although the average velocity increased
as the port diameter increased, the maximum increase was only 1.35
times. Therefore, compared with the lower layer, the increase in
average velocity in the upper layer was predicted to be small,
even when the port diameter increased. Furthermore, when
compared with Cases 1-20 in Simulation 1 (Table 1), the average
velocity in most cases was found to be less than the corresponding
values in that space. Furthermore, it was assumed that, in this
space, the SD for the average velocity would increase with
increasing port size. Consequently, in this space, either an increase
or uniformity in average velocity could not be expected to result
from increasing the port size.
Conclusions
According to the aforementioned simulation, it is possible to
create large uniform airflow in the lower layer of produce packaging
by increasing the port diameter in the port layout of Case 27,
which was proposed in Simulation 2. For the upper layer,
increasing the average velocity or eliminating the distribution may
not be possible with this port layout. In future studies, differences
in both the number and the shape of ports that affect the airflow in
this space will be considered. To examine how much the number
and size of ports can be increased (in both the upper and lower
50

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

WFL Publisher
Science and Technology
Meri-Rastilantie 3 B, FI-00980
Helsinki, Finland
e-mail: info@world-food.net

Journal of Food, Agriculture & Environment Vol.12 (3&4): 51-55. 2014

www.world-food.net

Peanut protein isolates improve the nutritional quality of muffins that can be handy
tool to cure protein energy malnutrition in developing economies
Muhammad Sibt-e-Abbas 1*, Masood Sadiq Butt 1, Muhammad Tauseef Sultan 2, Atif Nisar Ahmad 2,
Muhammad Abrar 1 and Mir Muhammad Nasir Qayyum 3
1
2

National Institute of Food Science & Technology, University of Agriculture, Faisalabad 38000, Pakistan.
Bahauddin Zakariya University, Multan 60000, Pakistan. 3 Karakram University, Gilgit 15100, Pakistan.
*e-mail: abbas_fst14@yahoo.com

Received 7 July 2014, accepted 12 September 2014.

Abstract
The developing economies are facing the menace of malnutrition particularly due to inadequate intake of quality proteins. The people using wheat
and rice as staples need to increase the intake of quality proteins, e.g. nuts and animal proteins. In the present research, proteins extracted from
partially defatted peanut flour (DPF) of indigenous varieties, i.e. GOLDEN and BARI 2011, were supplemented with straight grade wheat flour in
various proportions. These flours were subjected to rheological characteristics studies. After rheology muffins were prepared using these flour
blends. Muffins were then tested for physical characteristics including colour, texture and volume. At the end, the sensory evaluation was performed
by trained panellists. Results regarding the rheological properties, i.e. farinograph and mixograph, revealed that peanut protein isolates positively
affected the rheology of dough. Best results for water absorption (%) were shown by T4. Similar results were noted for dough development time (min)
and dough stability time (min). The physical characteristics of muffins indicated an increase in quality and nutritional status by the addition of protein
isolates. T3 showed notable result for colour. Furthermore, sensory evaluation of muffins showed remarkable results on 15% supplementation of
wheat flour with peanut protein isolates. Conclusively, the protein isolates obtained from defatted peanut flour or meal left after oil extraction can be
effectively utilized for the supplementation of bakery products, i.e. muffins. As these bakery products are gaining much popularity in the developing
economies, hence these can play an imperative role to curtail the increasing risks of malnutrition.
Key words: Peanut, protein isolates, composite flour, muffins.

Introduction
Malnutrition is a major nutritional dilemma in the developing
countries. It persists due to insufficient intake of nutrients resulting
in adverse effects on body building and function. Malnourished
people either do not have enough calories in their diet or are
eating a diet that lacks protein, vitamins or trace minerals 1. Protein
malnutrition is one of such example that causes severe effects on
immune functions, growth and development of children, their
learning ability and work efficiency. Approximately 70% of the
worlds malnourished children live in Asia, resulting in the region
having the highest concentration of childhood protein energy
malnutrition 2, 3.
The role of proteins in human nutrition is substantial. According
to Modern Nutrition Recommendations, human beings should
rely mostly on vegetable and legume proteins to meet the protein
requirement in their diet. In addition to their nutritional value,
proteins provide great potential as functional food ingredients
enhancing the useful properties when incorporated into food
commodities. In order to utilize a byproduct as a protein source, it
should contain high protein content and protein value (quality)
based on well-balanced essential amino acids.
Peanuts (Arachis hypogaea) are among the most vital sources
of vegetable oil throughout the world. Peanuts also contain
appreciable quantity of valuable proteins. Peanut protein isolates
generally contain 47-55% high quality protein with high essential

amino acid content, which lends itself being used in many food
applications 4-6. These protein isolates can be utilized as functional
ingredients in various food products to improve the nutritional
and textural properties of the product 7.
The supplementation of various food products with peanut
protein isolates can play a vital role in the reduction of protein
deficiency 8. As the demand of bakery products is increasing at
the rate of 10.07% per annum so these are considered as excellent
vehicle for fortification, value addition and feeding at mass scale.
Among the bakery products, muffins (cupcake) prove to be an
excellent tool for the supplementation of peanut protein isolates
as their consumption in world is more than 46% of all other savoury
foods 9. The present research project explicated the role of peanut
protein isolates as a tool against protein deficiency and potential
source for supplementation of baked products.
Materials and Methods
Procurement of raw materials: Two varieties of peanut (GOLDEN
and BARI 2011) were procured from Barani Agriculture Research
Institute Chakwal. Chemicals and other consumables were
purchased from local market. The protein isolates were prepared
in the post graduate laboratories of National Institute of Food
Science & Technology, University of Agriculture, Faisalabad,
Pakistan.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

51

Preparation of peanut protein isolates supplemented blends:


Peanut protein isolates of the two varieties (GOLDEN and BARI
2011) were supplemented with straight grade flour in various
proportions as given in Table 1.
Table 1. Composition of different composite flours.
Treatments
T0
T1
T2
T3
T4
T5
T6
T7
T8

Straight
grade flour (%)
100
95
90
85
80
95
90
85
80

Peanut protein
isolates (%)
(GOLDEN)
0
5
10
15
20
---------

Peanut protein
isolates (%)
(BARI 2011)
----------5
10
15
20

Sensory evaluation of protein enriched muffins: The muffins


were evaluated for taste, colour, flavour, texture, aroma, and overall
quality on a sensory evaluation Performa. All evaluations were
conducted at room temperature on the same day, in the National
Institute of Food Science and Technology (NIFSAT), University
of Agriculture, Faisalabad according to the procedure described
by Meilgaard et al. 15.
Statistical analysis: The data obtained for each parameter was
subjected to statistical analysis in order to determine the level of
significance as described by Steel et al. 16.
Results and Discussion
The present study was designed to explore the nutritional value
of peanut protein isolates with special reference to supplementation
in bakery products, i.e. muffins.

Rheological characteristics of composite flours: The physical


properties of flour supplemented with different levels of peanut
protein isolates such as water absorption, dough development
time, dough stability time, mixing tolerance index and softening of
dough was studied by Brabender Farinograph (Method No. 54-21)
and mixing time and peak height percentage was determined by
running the flour samples through Mixograph (Method No. 5440A) according to their respective methods as outlined in AACC 10.
Preparation of protein enriched muffins: Muffins were prepared
with supplemented blends and of control treatment as mentioned
in Table 1 by following the method described by Shearer and
Davies 11 with some modifications.
Physical analysis of protein enriched muffins: The colour of
muffins was determined with the help of hand held tristimulus
colormeter II (Mod, Neuhaus Neotec, Germany, Colormeter,
Colortest 11 serial no. 95808) as described by Baixauli et al. 12. The
textural study was conducted by using texture analyser (Model
TA-XT2, Stable Microsystems, Surrey, UK) with a 5 kg load cell
as described by Piga et al. 13. It gives the measurements of the
hardness (firmness) and resistance (fracturability) of the muffins
to bend or snap. Muffin volume was measured after baking by
rapeseed displacement method according to procedure as
described in AACC 10. The muffin was placed in the container
filled with rapeseeds and the volume of rapeseeds displaced by
the muffin was recorded according to Keskin et al. 14.

Rheological analysis: The rheological characteristics of straight


grade wheat flour containing different levels of peanut protein
isolates from two peanut varieties (GOLDEN and BARI 2011) were
studied for the parameters such as water absorption, dough
development time, dough stability time, mixing tolerance index
and softening of dough by using Brabender Farinograph. T1 to T8
treatments were prepared using different concentrations of peanut
protein isolates while T0 was control treatment with no addition of
protein isolates. First four treatments were formulated using protein
isolates from GOLDEN peanut variety (T1 5%, T2 10%, T3 15%,
T4 20%) while the other four treatments were prepared using
protein isolates from BARI 2011 peanut variety (T5 5%, T6 10%,
T7 15%, T8 20%).
The data representing the effects of various levels of peanut
protein isolates on the farinographic characteristics of the dough
are given in Table 2. The data regarding water absorption indicated
that the values are not significantly different from each other. It
was obvious from the results that water absorption was higher in
T8 (66.900.30) followed by T7 (65.400.30) and T4 (65.100.20), while
lowest water absorption was observed in T5 (62.100.30). The
results revealed that the water absorption increased with the
increasing levels of protein isolates. These results are in conformity
with the findings of Azizi et al. 17 and Ravi et al. 18. They observed
that the percent water absorption increases with the addition of
protein isolates.
Dough development time is defined as the time required for the
development of gluten. The present results for this trait indicated
a highly significant difference among the treatments. Mean values
for the dough development time of different treatments (Table 2)

Table 2. Farinographic characteristics of flour blends.


Treatments
T0
T1
T2
T3
T4
T5
T6
T7
T8

Water
absorption (%)
62.760.15a
64.900.20a
63.400.20a
64.100.20a
65.100.20a
62.100.30a
62.300.20a
65.400.30a
66.900.30a

Dough development
time (min)
6.700.10a
5.400.10d
4.700.20e
5.300.20d
5.800.30c
6.500.20ab
5.600.25cd
6.800.30a
6.200.30b

Dough stability
time (min)
11.500.20d
14.500.20a
13.600.20b
12.200.20c
14.200.30a
11.900.20c
13.300.20b
11.300.20d
14.400.20a

Mixing tolerance
index (BU)
30.002.51e
35.002.00d
40.002.08c
50.002.52a
20.002.00f
33.002.00de
40.002.00c
35.002.00d
44.003.00b

Softening
of dough (BU)
47.002.51ef
63.002.52cd
71.002.52b
61.003.05d
48.003.00ef
75.002.00a
50.002.00e
66.003.00c
46.002.00f

* Means sharing the same letter in a column are not significantly different. T0 = 100% straight grade wheat flour (SGF). T1 = 95% SGF and 5% peanut protein
isolate (GOLDEN). T2 = 90% SGF and 10% peanut protein isolate (GOLDEN). T3 = 85% SGF and 15% peanut protein isolate (GOLDEN). T4 = 80% SGF
and 20% peanut protein isolate (GOLDEN). T5 = 95% SGF and 5% peanut protein isolate (BARI 2011). T6 = 90% SGF and 10% peanut protein isolate (BARI
2011). T7 = 85% SGF and 15% peanut protein isolate (BARI 2011). T8 = 80% SGF and 20% peanut protein isolate (BARI 2011).

52

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

indicated that the highest value (6.800.30 min) was found Table 4. Physical characteristics of muffins supplemented with peanut
in T7 followed by T0 (6.700.10) while it was lowest for T2
protein isolates.
(4.700.20 min). These results are in agreement with the
Firmness
Fracturability
Volume
Treatments Colour (CTn)
findings of Azizi and Rao 19, Sim et al. 20. The mean values
(g)
(mm)
(cm3)
T0
159.334.16cd
216.6728.86a
865.2731.24a
86.740.14a
showed that the highest value for dough stability time
T1
162.678.73bcd 116.6714.43de 769.2224.24b 87.610.33b
was observed in T1 (14.500.20 min) and the lowest value
162.674.93bcd 133.3314.43bcd 715.238.07c
84.580.10c
T2
was obtained in T7 (11.300.20 min). The above findings
a
cde
d
181.974.72
116.6714.43
675.3316.94
84.380.15c
T3
are in close agreement with the results of Ravi et al. 18 and
161.674.04bcd
83.33314.43e
589.5610.10e 83.760.10d
T4
Indrani and Rao 21.
159.6719.00d 116.6738.18cde
514.138.15f
83.140.09e
T5
d
bc
g
153.3311.54
141.6738.18
478.1315.13
81.170.13f
T
6
The data for the analysis of variance for mixing tolerance
abc
de
h
174.009.00
91.66728.87
429.418.65
80.140.07g
T7
index indicate a highly significant difference among
176.009.00ab
166.6728.87b
412.3713.36h 79.310.12h
T8
treatments. The mean values for mixing tolerance index * Means sharing the same letter in a column are not significantly different.
show the highest value for T3 (50.002.52 BU) followed
by T8 (44.003.00 BU) and T2 (40.002.00 BU) while it was lowest and fracturability. Hardness (firmness) was calculated in terms of
for T4 (20.002.00 BU). These results are in conformity with the maximum force (g) and fracturability was determined in terms of
values as observed by Ravi et al. 18 and Azizi and Rao 19. The distance (mm). Hardness can be defined as the peak force during
mean values for softening of dough showed a maximum value for the first compression cycle (first bite). It is the force required to
T5 (75.002.00 BU) and a minimum value for T8 (46.002.00 BU). attain a given deformation. The values for the hardness of muffins
The above mentioned results are in conformity with the findings ranged from 412.37 g to 865.27 g. The mean values (Table 4)
indicated maximum hardness value for T0 (865.2731.24 g) and
of Asghar et al. 22.
The results of mixographic studies are shown in Table 3. These minimum value for T8 (412.3713.36 g). The results are in agreement
results showed that maximum mixing time was observed for T3 with the observations of Azizi and Rao 19 and Ashwini et al. 25.
(3.000.20 min) while it was minimum for T5 (1.150.02 min). The Fracturability (also known as brittleness) is the force at first
present results are in harmony with the findings of Indrani and significant break in the curve. It is the force with which the material
Rao 21 and Asghar et al. 22. Mean values for peak height percentage or the product fractures. The mean values for the fracturability of
indicated that ranged between 31% and 50%. The mean values muffins are given in Table 4. It is obvious from the mean values
explicated maximum peak height percentage for T5 (50.003.00%) that maximum fracturability was observed in T1 (87.610.33 mm)
followed by T3 (43.006.55%) and T7 (41.500.20%) while the followed by T0 (86.740.14 mm) and T2 (84.580.10 mm) while
minimum value was observed for T6 (31.500.10%). These results minimum value for fracturability was observed in T8 (79.310.12
mm). These results are similar to the findings of Ashwini et al. 25.
are in concurrence with the findings of Indrani and Rao 21.
Table 3. Mixographic characteristics of flour blends.
Treatments
T0
T1
T2
T3
T4
T5
T6
T7
T8

Mixing time (min)


1.450.02f
2.150.20d
2.300.03c
3.000.20a
1.300.02g
1.150.02h
2.000.02e
2.450.02b
2.000.04e

Peak height (%)


41.500.10b
40.004.00bc
34.500.20cd
43.006.55b
33.002.00d
50.003.00a
31.500.10d
41.500.20b
41.500.10b

* Means sharing the same letter in a column are not significantly different.

Measurement of physical characteristics of muffins: Colour value


was determined by using colour meter II. It was first calibrated
with the standards having lower and upper limits (51-200),
respectively. The mean values for colour (Table 4) showed that
the highest value was observed in T3 (181.974.72 CTn) and the
lowest in T6 (153.3311.54 CTn). The above mentioned colour
values are in agreement with Azizi and Rao 19 and Abu-Ghoush
et al. 23. Volume of muffins is affected by various factors such as
quality of flour, type of ingredients and processing conditions.
The mean values for the volume of muffins given in Table 4
indicated that the maximum value for volume was observed in T0
(216.6728.86 cm3) followed by T8 (166.6728.87 cm3) and T6
(141.6738.18 cm3) while the minimum value was observed in T4
(83.33314.43 cm3). The current findings are in concord with Azizi
and Rao 19, Kaur et al. 24 and Ashwini et al. 25.
Texture of muffins was measured in terms of hardness (firmness)

Sensory evaluation of muffins: Sensory characteristics are much


significant towards the liking and disliking of product, i.e. muffins.
Muffins with light brown and creamy colour with soft texture are
usually preferred by the consumers. Sensory evaluation of muffins
was carried out by a trained panel for the attributes such as colour,
taste, flavour, tenderness, moistness and overall acceptability.
The results pertaining to all sensory parameters of muffins (Table 5)
are discussed in detail hereunder. The mean values indicated that
the highest score for colour (6.660.51) was assigned to the
treatments T3 and T7 while the lowest score (4.830.75) for this
attribute was given to T4. These findings revealed that the muffins
prepared by using 15% protein isolates from GOLDEN (T3) and
BARI 2011 (T7) showed highest results for the colour score 6.66.
The mean scores for flavour of muffins indicated that the muffins
prepared from T0 and T5 got the highest scores for flavour
(7.000.89) followed by T2 which obtained 6.660.51 score for
flavour. Muffins prepared from T4 were found to be disliked by
the judges regarding their flavour score, i.e. 5.330.51. The current
results are also similar to the findings of McGuire et al. 9 who
declared that addition of protein isolates in any product caused
changes in taste, flavour, texture and overall acceptability scores.
The mean scores assigned by the panellists to the texture of
muffins revealed that the control muffins (T0) got 6.830.75 score
for texture. The muffins prepared from T3 and T7 were considered
more acceptable by the panellists regarding the texture as 6.500.54
and 6.660.51, respectively. Mean scores for tenderness of muffins
indicated that the highest tenderness score (6.660.51) was
assigned to T3 and T7 followed by T1, T2 and T6 (6.330.51). The

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

53

Table 5. Sensory evaluation of muffins.


Treatments
T0
T1
T2
T3
T4
T5
T6
T7
T8

Colour
6.500.54a
6.000.63a
6.500.54a
6.660.51a
4.830.75b
6.160.75a
6.160.40a
6.660.51a
5.000.63b

Flavour
7.000.89a
6.330.81a
6.660.51a
6.500.54a
5.330.51b
7.000.89a
6.330.81a
6.500.54a
5.500.54b

Texture
6.830.75ab
6.500.54ab
6.160.75bc
6.500.54ab
5.500.54cd
7.000.89a
6.500.54ab
6.660.51ab
5.000.89d

Tenderness
6.000.63ab
6.330.51a
6.330.51a
6.660.51a
5.330.51bc
6.160.75a
6.330.51a
6.660.51a
5.000.63c

Moistness
6.500.54a
5.660.51bc
6.160.40ab
6.500.54a
5.660.81bc
6.660.51a
5.500.54c
6.500.54a
5.330.51c

Shape
6.830.98ab
6.330.51ab
6.160.75b
7.160.75a
4.660.81c
6.500.83ab
6.160.75b
7.000.89ab
4.830.75c

Acceptability
5.161.16c
5.500.54bc
6.331.03ab
6.500.54a
5.000.89c
6.160.98ab
5.500.54bc
6.500.54a
5.000.63c

* Means sharing the same letter in a column are not significantly different.

results further indicated that T8 muffins got the lowest tenderness


score 5.000.63. The mean values for this attribute showed that
the highest value (6.660.51) was observed in T5 followed -by T0,
T3 and T7 having moistness score of 6.500.54. The lowest
moistness score (5.330.51) was recorded in T8. Mean values for
shape ranged from 4.830.75 (T8) to 7.160.75 (T3).
Muffins prepared from T4 were assigned the lowest score
4.660.81. Mean values indicated that the overall acceptability
scores of muffins showed a slight variation with the addition of
varying concentrations of peanut protein isolates. The highest
score (6.500.54) was assigned to T3 and T7. The judges slightly
disliked muffins prepared from T4 and T8 and assigned overall
acceptability score of 5.000.89 to both. The current results are in
agreement to the findings of Shearer and Davies 11 and Ramcharitar
et al. 26.
The current results regarding sensory evaluation of muffins
prepared using different concentrations of peanut protein isolates
from two peanut varieties (GOLDEN and BARI 2011) revealed
that the treatments T3 (15% protein isolates from GOLDEN peanut
variety) and T7 (15% protein isolates from BARI 2011 peanut
variety) showed the best results and were assigned maximum
scores by the panellists in terms of likeness.
Conclusions
The overall results that peanut is a vital source of protein and the
protein isolates exhibit remarkable rheological properties when
blended with straight grade wheat flour. In the limelight of these
properties, peanut protein isolates can be utilized for improving
the quality of muffins and value addition. The physical and
sensory attributes of muffins prepared using different levels of
protein isolates revealed that the treatment with 15%
supplementation showed better results. Thus, muffins enriched
with peanut protein isolates can be a handy tool to cope with
protein deficiency among the vulnerable groups.
Acknowledgements
The authors are thankful to National Institute of Food Science
and Technology, University of Agriculture, Faisalabad for
providing instrument facilities to carry out analyses. The authors
are also thankful to Dr. Muhammad Shahid (Assistant Professor)
for their valuable suggestions and inputs regarding the research.
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Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

55

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Journal of Food, Agriculture & Environment Vol.12 (3&4): 56-60. 2014

Assessment of microbiological quality of ready-to-eat foods in Istanbul, Turkey


Vecdet z 1, Sukriye Karadayi 2*, Hseyin akan 1, Beytullah Karadayi 3 and Filiz Ekim evik

Department of Microbiology, Forensic Science Institute, Istanbul University, 34098, Istanbul, Turkey. Department of
Microbiology, Public Health of Agency, 34020, Zeytinburnu, Istanbul, Turkey. 3 Cerrahpasa Faculty of Medicine, Forensic
Medicine Department, Istanbul University, 34098, Istanbul, Turkey. *e-mail: skaradayi2000@yahoo.com
1

Received 4 July 2014, accepted 7 September 2014.

Abstract
The purpose of this study is to assess the microbiological quality of ready-to-eat (RTE) foods produced at catering companies in Istanbul, Turkey.
RTE food was collected from May 2009 to May 2010 in Istanbul and a total of 750 samples including 13 food types were examined for the presence
of coliform bacteria, E. coli, S. aureus, B. cereus, Salmonella spp. and L. monocytogenes. Comparison with the Microbiological Turkish Food Codex
(TFC) shows that 3.6% of 532 meats, 18.3% of 120 salads, and 50% of 12 pastries were at an unacceptable level of microbiological quality. However,
all pasta (32 samples), puddings (41 samples) and patties (14 samples) were at an acceptable level of microbiological quality according to the TFC.
In the samples that were examined coliform bacteria, E. coli, B. cereus and S. aureus, respectively, were isolated to 4.8%, 2.4%, 2.25% and 6.4%.
Salmonella spp. and L. monocytogenes were not detected. RTE food should be served to the consumer in a microbiologically safe form. The results
from this study can be used to assess of microbiological risks in food safety. The present study proposes that to minimize bacterial level in RTE foods
in Turkey regular microbiological quality control programs and good hygiene practices are necessary. The use of microbiologic results of ready-to-eat
foods in the Istanbul will provide an example for similar regions.
Key words: Ready-to-eat foods, foodborne pathogen, microbiological quality, food safety.

Introduction
RTE food production has recently increased due to the fast growth
in population density, which brings rapid, on the go consumption
of food in both in the world and Turkey, especially in Istanbul 1-3.
Catering companies producing RTE food offer cheap, economical
and easily accessed products. Therefore, many private or official
foundations have been using such a service at hospitals, nursing
homes, nursery schools and military posts. The inclination to buy
in services from catering companies brings a new changed lifestyle
and consequently increases the importance of microbiological
analysis in food safety 4. Therefore, assessment of the
microbiological quality of RTE food based on the initial bacteria
level becomes very important 5.
Foodborne diseases due to bacteria occur with the consumption
of food contaminated by pathogen microorganisms 6. At present,
the spectrum of foodborne diseases is increasing and these
diseases create a major health problem 7. Different foodborne
pathogens have been associated with outbreaks of foodborne
diseases 8. Many studies have shown that the most common
bacteria associated with RTE food are Salmonella spp., Listeria
monocytogenes, Campylobacter jejuni, Staphylococcus aureus,
Bacillus cereus and Clostridium perfringens 9, 10. Foodborne
diseases have been studied in many national and international
researches. A study which was done in Taiwan has shown that
the percentage of foodborne diseases, which occurred due to
bacterial pathogens between the years of 1986 and 1995 was 65% 11.
In France in the years of 1999 to 2000, 17,378 people were tested,
and foodborne disease was found in 1,267 people and the deaths
of ten people were determined 12. In Turkey, according to the
56

statistics of the Turkish Ministry of Health, 108,246 people were


hospitalized due to foodborne poisoning in 1993-2005 13.
This study aimed to analyse the bacteriological profile of RTE
food produced by various catering companies, in Istanbul, Turkey.
Furthermore, it was also examined whether catering companies
were compatible according to the Turkish Food Codex 14.
Materials and Methods
Collection of samples: RTE food samples were collected from
May 2009 to May 2010 in Istanbul, Turkey. All food sampled in
the studies was aseptically collected. A total of 750 samples were
transferred from 14 different catering companies to the National
Public Health Association using thermo boxes. Appropriate RTE
food samples were studied at the Istanbul University Forensic
Science Institute and Microbiology Laboratory. The samples
consisted of soup, rice, Turkish kebabs, vegetable meals, food
legumes, meatballs, chicken meals, meat dishes, pastries, puddings,
salads, patties, and pasta. Food samples encountered in the study
contained many various ingredients. For example, 120 soups were
composed of 23 different ingredients (yogurt, rice, spinach, potato,
tomato, and so on) (Table 2).
Sample processing and microbiological analysis: The samples
were examined in terms of the existence of microorganisms such
as coliform bacteria, Escherichia coli, Bacillus cereus,
Staphylococcus aureus, Salmonella spp. and Listeria
monocytogenes. Not all samples were tested for all parameters.
Selection of parameters depended on the type of food and criteria

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

of the TFC. For coliform bacteria, Escherichia coli,


Staphylococcus aureus and Bacillus cereus analysis, 10 g or 10
ml of each of the samples was weighed aseptically into sterile
stomacher bags and homogenized in a stomacher (AES) for about
two min. For Salmonella spp. and Listeria monocytogenes
analysis, 25 g or 25 ml of each of the samples was weighed
aseptically and the process mentioned above was applied. Bacteria
were studied as described in Table 1 15-20. In determining the
microorganisms, biochemical specifications of the bacteria and
identification test kits that were commercially available were used
(Table 1).
Results
In this study, specimens belonging to 14 firms producing RTE in
different regions of Istanbul were used. A total of 750 samples
including 13 food types (soup, rice, Turkish kebabs, vegetable
meals, chicken meals, ready-to-eat salads, and so on) and 218
different kinds of food were examined (Table 2). RTE food whose
sample numbers were highest were 120 soups and salads and 100
vegetable meals (Table 2). Four different micro-organisms were
isolated from the food sampled. These were coliform bacteria, E.
coli, S. aureus and B. cereus (Table 3). Salmonella spp. and L.
monocytogenes were not isolated from any of the samples
analysed.
In the study, coliform contamination was detected in about 4.8%
of samples (Table 3). When 750 RTE foods were researched in 13
food categories, E. coli was determined in 18 of them (2.4% of
samples) (Table 3). The microbiological level changed from 101 to
106 CFU/g (Table 4). In rice samples, the highest E. coli and coliform
bacteria levels were 105-106 CFU/g (Table 4). The pastries were in
the group whose Escherichia coli percentage was highest with
33.33% and that the Escherichia coli percentage of the salads
was 5% (Table 4). E. coli was not isolated in the samples of legumes,
meatballs, meat dishes, pies or pasta (Table 4).
In 718 RTE food samples, 46 (6.4% of samples) contained S.
aureus with levels ranging from 101 to 106 CFU/g (Tables 3 and 4).
The highest values were determined in pastry (33.3% of pastry
samples), chicken meals (6.3% of chicken samples), rice (5.8% of
rice samples) and meatballs (5.8% of meatball samples) (Table 4).
In this study in 13 (2.25% of samples) of the food samples analysed,
B. cereus was detected in 577 samples (Table 3). In the ten
categories studied, B. cereus was detected in all food groups

Table 2. Food types encountered in the study.


Food type
Soup
Rice
Turkish Kebab
Vegetable meals
Food legumes
Meatball
Chicken meals
Meat dishes
Pastry
Pudding
Salad
Patty
Pasta
Total

Number
120
70
23
100
20
52
48
98
12
41
120
14
32
750

Kind
23
8
10
23
4
2
27
32
5
10
34
7
12
218

Table 3. Number and percentage of food samples.


Microorganisms
Coliform bacteria
E. coli
S. aureus
B. cereus

Sample n
709
750
718
577

Positive n
34
18
46
13

Positive %
4.8
2.4
6.4
2.25

except for food legume, patty and pasta.


In this study, in the pastry samples, coliform bacteria ranged
from 102 to 104 CFU/g, E. coli from 101 to 103 CFU/g and S. aureus
from 102 to 105 CFU/g (Table 4). B. cereus was not detected in the
pastry samples. Also, soup and rice samples were the groups
which had the highest levels of B. cereus (104 to 105 CFU/g) (Table
4). Neither indicator nor pathogenic bacteria were detected in the
food legume samples.
In assessing the microbiological results of the RTE foods, TFC
microbiological quality criteria were used (Table 5). When RTE
food was assessed according to the TFC, these results were
obtained: 512 of 532 general meal samples (96.4% of samples), 98
of 120 salads (81.7% of samples), six of 12 pastries (50% of samples),
and all the 32 pasta samples, 41 puddings and 14 cakes were
acceptable in terms of microbiological criteria (Table 6).
Discussion
Recently, the RTE sector which is becoming an important industry
has also brought some problems along with it 1. Every year, the
consumption of RTE has increased and consequently, catering
company sectors have also increased in parallel. Therefore, an
error, which may occur at any step of this sector, can cause

Table 1. Microbiological techniques used for the testing of food samples.


Incubation
Media
Temp (C) Time (h)
Coliform
Violet Red Bile (VRBL) agar (Oxoid CM0968)
Pour plate
37
24
bacteria
purplish-red colonies (ISO 4832, 2006)
Tryptone Bile X-glucuronide (TBX) agar (Oxoid
E. coli
Pour plate
44
18-24
CM0945) blue colonies (ISO 16649-2, 2001)
Baird Parker agar (Oxoid CM0275) black or gray
S. aureus
Spread plate
37
24-48
colonies (ISO 6888-1, 1999)
Mannitol egg Yolk Polymixin (MYP) agar
B. cereus
Spread plate
30
18-24
(Merck 1.05267) pink colonies (ISO 7932, 2004)
Pre-enrichment
37
18
Buffered peptone water (Oxoid CM0509)
Enrichment
41.5
24
Rappaport Vassiliadis (HiMedia 1137)
Salmonella
Xylose Lysine Deoxycholate (XLD) agar (Merck
spp.
Spread plate
37
24
1.05287) black centered colonies
(ISO 6579, 2002)
Pre-enrichment
30
24
Half Fraser Broth
Listeria
Enrichment
37
48
Fraser Broth
monocyt.
Palcam agar (CM0877) Green-gray colonies with
Spread plate
37
24-48
black halo and core (ISO 11290-2, 1998)
Bacteria

Analysis
method

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Confirmation technique
Brilliant green lactose bile broth (BGB) (Oxoid
CM0031)

Coagulation +, catalase +, gram+


Hemolysis+

Biochemical tests: triple sugar iron agar (HiMedia


M021), urea broth (Merck 1.08483) Final test;
VIDAS Identification system (Biomerieux)

API Listeria strip (Biomerieux)

57

Table 4. Microbiological analysis results of coliform, E. coli, S. aureus, B. cereus, Salmonella spp. and Listeria
monocytogenes.
Food type (detailed)
Soup (120)
Rice (70)
Turkish Kebab (23)
Veg. meal(100)
Food legume (20)
Meatball (52)
Chicken meal (48)
Meat dish (98)
Pastry (12)
Pudding (41)
Salad (120)
Patty (14)
Pasta (32)

Coliform
1(%0.8)
4(%5.7)
3(%13)
4(%4.2)
ND
ND
2(%4.2)
ND
4(%33.3)
NT
14(%11.7)
1(%7.1)
1(%3.13)

CFU/g
101-102
105-106
101-102
101-103
103-104
102-104
101-104
102-103
102-103

Salmo. spp.
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND

E. coli
1 (%0.8)
1 (%1.5)
1 (%4.4)
2 ( %2)
ND
ND
2 (%4.2)
ND
4 (%33.3)
1 (%2.4)
6 (%5)
ND
ND

CFU/g
101-102
105-106
101-102
101-103
103-104
101-103
101-103
101-104

L. monoc.
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND

S. aureus
2(%1.7)
4(% 5.8)
1(%4.4)
5(%5)
1(%5)
3(%5.8)
3(%6.3)
4(%4.1)
4(%33.3)
2(% 4.9)
17(%4.2)
ND
NT

CFU/g
102-103
101-106
101-103
102-104
102-103
102-104
101-103
102-103
102-105
102-103
102-105

B. cereus
1(%0.8)
1(%1.4)
3 (%13)
3 ( %3)
ND
1 (%1.9)
2(%4.2)
2 (%2)
NT
NT
NT
ND
ND

CFU/g
104-105
104-105
102-104
102-103
102-103
102-104
101-103

ND: Not detected. NT: Not tested.

Table 5. Microbiological quality of RTE (meal, salad, pasta and pudding) in Turkish Food Codex.
RTE meals (CFU/g)
Microbiological
Criterion
quality
E. coli
<101
S. aureus
103
B. cereus
Salmonella spp.

103
Not detected in
25 g-mL

RTE salads (CFU/g)


Microbiological
Criterion
quality
E. coli
101
S. aureus
103
Not detected in
Salmonella spp.
25 g-mL
Not detected in
Listeria monocyt.
25 g-mL

RTE pasta (CFU/g)


Microbiological
Criterion
quality
E. coli
<101
B. cereus
103
Not detected in
Salmonella spp.
25 g-mL

Table 6. Acceptable/unacceptable RTE foods according to


Turkish Food Codex.
Food type
Meal (n=532)
Salad (n=120)
Pasta (n=32)
Patty (n=14)
Pudding (n=41)
Pastry (n=12)
Total (n=750)

Acceptable
512
98
32
14
41
6
703

%
96.43
81.67
100
100
100
50
93.3

Unacceptable
19
22
0
0
0
6
47

%
3.6
18.33
0
0
0
50
6.7

foodborne disease and great economic loss, even death.


The most common known causes of foodborne diseases are
pathogenic bacteria. In this study, the aim was to analyse the
bacteriological profile of RTE food produced by various catering
companies in Istanbul, Turkey. Therefore, the potential risks, which
RTE products could cause for health care, were studied.
In Korea, coliforms were detected in approximately 50% of the
samples at levels up to 105 CFU/g 21. In our study, coliforms were
detected at levels up 106 CFU/g. The incidence of the fecal indicator
organism E. coli in salad vegetables was found to be 102 CFU/g
(3.7% of samples) in a study prepared in the UK 22. According to
the Public Health Laboratory Service (UK) criteria, the amount of
E. coli in RTE food was defined as <20 CFU/g (satisfactory), 20
<100 CFU/g (acceptable), 100 CFU/g (unsatisfactory) 8. In our
study, E. coli was detected from 10 CFU/g to 106 CFU/g in 18
(2.4%) of the 750 samples tested and was found as 10 CFU/g to
104 CFU/g (5% of samples) in salads. Microbial contamination of
RTE can occur due to the environment, from contact with
contaminated containers, equipments and utensils, hands,
aerosols or pets 23, 24. As a result, the detection of coliform bacteria
and E. coli shows the possibility of fecal contamination. RTE
foods, especially salads have been implicated in foodborne illness
outbreaks because these foods are often prepared by hand 25.
The decontamination activity of the washing system for bacteria
removal is mostly unknown in vegetables 26. We think that
58

RTE pudding (CFU/g)


Microbiological
Criterion
quality
Yeast and mold
103
S. aureus
103
Not detected in
Salmonella spp.
25 g-mL
Not detected in
Listeria monocyt.
25 g-mL

approximately one in five salads is unacceptable for consumption


due to the reasons mentioned above.
The presence of S. aureus in RTE food is an indication of poor
hygiene practices. S. aureus in RTE food is associated with crosscontamination occurring during processing and storage or through
the contamination of raw ingredients 27. In a study in Cyprus in
1991-2000, 1382 RTE samples were analysed and in 2% of them S.
aureus (> 104 CFU/g) was determined 28. In a study in Turkey,
among a total of 512 RTE samples, S. aureus was detected in eight
of 32 Russian salads (25% of samples) and in nine of 75 vegetable
salads (12% of samples) 29. In the present study, S. aureus was
detected in 6.4% of 718 RTE samples and 4.2% of 120 salads.
In a study of sauces in England, B. cereus and/or other
pathogenic Bacillus spp. were detected in 3.8% of 1208 samples
(from 104 CFU/g to 105 CFU/g) 22. In another study in Taiwan,
164 RTE samples were studied and in noodles (a traditional meal),
B. cereus was detected at a high percentage (66.7%) 11. In the
present study, the percentage of B. cereus (from 102 CFU/g to
104 CFU/g) was 13%, in traditional Turkish kebabs. These levels
are significant, potentially hazardous levels. As a result of all our
analysis, the level of B. cereus determined in RTE foods changes
from 101 CFU/g to 105 CFU/g. These results are similar to those
of Meldrum et al. 22 but are much lower in comparison with those
of Fang et al. 11. We consider that these differences possibly
resulted from discrepancies in meal preparation culture in different
geographical regions and hygienic conditions during the
production and storage of the food.
Salmonella spp. and Listeria monocytogenes are the most
important factors in foodborne disease and in our study these
pathogens were not detected in any RTE food. However, in some
examinations, Salmonella spp. wasnt detected in the studies done
on RTE in 2000 and 2004 in the UK 23, 30. The results of our studies
are a positive finding in terms of hygiene conditions.
When 750 RTE samples in total were assessed according to the
TFC, 6.7% of them were not acceptable (Table 6). In the pastry

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

products, the bacterial level was found to be higher when


compared with other RTE food. In this study, 50% of pastries,
18.33% of salads and 3.6% of the meat samples were detected as
unacceptable according to the TFC (Table 6). Despite poor
environmental hygiene, all RTE food samples except for pastry,
salad and meat were detected to be microbiologically safe food
(Table 6). Similar results were reported for soup, rice, general meat
and salad in Ankara 31.
Because the samples were determined to be unacceptable in
half of the pastries analysed, these foods are potentially a big risk
to public health if necessary measures arent taken. Pastries have
the property of increasing bacterial level with their water activity
and specific pH 5. Also, we think that the reason for this finding is
the fact that pastry is exposed to lower heat processes compared
with others as well as the many more steps during the preparation.
S. aureus was isolated from the pudding samples (Table 4).
However, these samples were assessed to be acceptable, because
the borderline limit in S. aureus for pudding samples was
considered to be 103 CFU/g as cited by the TFC (Table 5).
In Turkey, the number of studies about the microbiological level
of RTE foods is very limited. We encourage increasing the number
of studies about this subject and controls according to the TFC in
Turkey. We conclude that companies who provide such a service
should be careful with personnel training and should always be
up to date with all recent developments and innovations in the
field in order to provide the best practice. It is crucial that all
employees including administrators and the people who work in
the kitchen should receive theoretical and practical training.
According to the results of our studies, we think that if RTE food
meets the required criteria, the risk to public health posed by
microorganisms can be reduced.
Conclusions
We reached the conclusion that pastries, salads and chicken meals
served in Istanbul constitute a risk to public health. Crosscontamination and infected food handlers were reported generally
as important factors in the determination of these microorganisms.
RTE food should be served to consumer in a microbiologically
safe manner. The production of safe RTE food is the responsibility
of the producer. At the same time, maintenance of correct
transportation is important for the safety of RTE food as well. The
results from this study can be used to assess microbiological
risks in food safety. The present study proposes that to minimize
bacterial levels in RTE foods in Turkey regular microbiological
quality control programs and good hygiene practices are
necessary. The use of microbiologic results of ready-to-eat foods
in the Istanbul will provide an example for similar regions.
Acknowledgements
The authors wish to thank the Forensic Science Institute
Microbiological Laboratory for technical support and Public Health
Laboratory. This work was supported by the Scientific Research
Projects of the Istanbul University with project number 5601.
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Kotzekidou, P. 2013. Microbiological examination of ready-to-eat foods
and ready-to-bake frozen pastries from university canteens. Food
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5
Bryan, L. F. 1976. Public health aspects of cream-filled pastries. A
review. J. Milk Food Technol. 39:289-296.
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Bhunia, A. K. 2008. Foodborne Microbial Pathogens. Springer Press,
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Mensah, P. D., Yeboah-Manu, K., Owusu, D. and Ablordey, A. 2002.
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Gilbert, R. J., De Louvois, J., Donovan,T., Little, C., Nye, K., Ribeiro,
C. D., Richards, J., Roberts, D. and Bolton, F. J. 2000. Guidelines for
the microbiological quality of some ready-to-eat foods sampled at the
point of sale. Communicable Disease and Public Health 3:163-167.
9
Bean, N. H. and Griffin, P. M. 1990. Foodborne disease outbreaks in the
United States, 1973-1987: pathogens, vehicles and trends. J. Food
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10
Notermans, S. and Borgdorf, M. 1997. A global perspective of foodborne
disease. J. Food Prot. 60:1395-1399.
11
Fang, T. J., Wei, Q. K., Liao, C. W., Hung, M. J. and Wang, T. H. 2003.
Microbiological quality of 18C ready-to-eat food products sold in
Taiwan. Int. J. Food Microbiol. 80:241-250.
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Haeghebaert, S., Le Querrec, F., Gallay, A., Bouvet, P., Gomez, M. and
Vaillant, V. 2002. Les toxi-infections alimentaires collectives en France,
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Turkish Statistical Institute 2006. Available at: http://www.tuik.gov.tr/
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Turkish Food Codex 2010. Official Gazette, Number 27456. Turkish
Food Codex-Mikrobiyolojik Kriterler Teblii (No. 2009/68), pp. 1-9.
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23

60

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

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Journal of Food, Agriculture & Environment Vol.12 (3&4): 61-64. 2014

www.world-food.net

Chemical composition of pumpkin (Cucurbita maxima D.) flesh flours used for food
Jurgita Kulaitien 1, Elvyra Jarien 1, Honorata Danilenko 1, Judita erniauskien 1, Agata Wawrzyniak 2,
Jadwiga Hamulka 2 and Edita Jukneviien 1*
1

Institute of Agriculture and Food Sciences, Agronomy Faculty, Aleksandras Stulginskis University, Studentu str. 11, LT 53361
Akademija, Kaunas distr, Lithuania. 2 Faculty of Human Nutrition and Consumer Sciences, Warsaw University of Life Sciences
(WULS-SGGW), 02-776 Warsaw, 159C Nowoursynowska str., Poland. e-mail: jukneviciene.edita@gmail.com,
judita.cerniauskiene@asu.lt, jurgita.kulaitiene@asu.lt, honorata.danilcenko@asu.lt, elvyra.jariene@asu.lt

Received 28 June 2014, accepted 10 September 2014.

Abstract
Pumpkin flours (Cucurbita maxima D.) are alternative horticultural products and functional properties of food components. The main aim of this
study was to investigate the quality parameters of the pumpkin (Cucurbita maxima D.) fruit flesh flours of different cultivars: Justynka F1,
Karowita and Amazonka. Standard methods were applied to determine dry matter, crude fibre, crude protein, crude fat, crude ash, the neutral
dietary fiber (NDF), modified acid-detergent fibre (MADF), water-soluble carbohydrates (WSC) and carotenoids (-carotene, lutein + zeaxanthin,
lycopene). The pumpkin fruit flours of Justynka F1 accumulated significantly highest content of dry matter, crude ash, crude fiber, water-soluble
carbohydrates (WSC). The highest amount of crude protein, crude fat and lutein + zeaxanthin were in the pumpkin fruit flours of Karowita. The
maximum of neutral detergent fiber (NDF) and modified acid-detergent fibre (MADF) was accumulated in Amazonka pumpkin flour, respectively,
21.37, 20.13% DM, so that the flour of this pumpkin variety is most suitable to enrich food with dietary fiber.
Key words: Pumpkin, flesh, flours, quality, nutritional value.

Introduction
Pumpkins can be processed into flour which has a longer shelflife. This flour can be used for its flavour, sweetness, deep yelloworange color and considerable amount of dietary fiber. It can be
also used to supplement cereal flours in bakery products, soups,
sauces, instant noodles and also as a natural coloring supplement
for food 28, 29.
Currently Lithuanian consumers also buy more vegetables that
are grown in small farms and have exclusive properties (organic
products), vegetables of exceptional quality. In this way changes
are inevitable in the cultivation of raw materials, their processing
and marketing. Lithuanian climate is suitable for growing pumpkins
as well. They grow well in the soil which is sheltered from the
winds, in sandy loam or in clay, warming soil.
Pumpkins produce high yields in comparison with other
vegetables and they are rated for the simple production
technology14. The breeders have already created shrubby type of
pumpkin plants. Cucurbita maxima is cultivated for flesh and
seeds for human nutrition, either for direct consumption or for
preparation of other foods such as syrups, jellies, jams, and purees.
This vegetable can be processed in different ways. It can be baked,
frozen, dried, crystallized, marinated or lyophilized 10. The fruits of
pumpkins have a lot of biologically active compounds : vitamin C,
vitamin E, minerals, pectins and carotenoids. The beneficial
influences of carotenoids on human health have proven by many
researchers. In the human body carotenoids keep same chemical
reactivity as in plants - catching free radicals and active atomic
oxygen 11. Carotenoids also potentially play an important role in
human health by acting as biological antioxidants, protecting cells

and tissues from the damaging effects of free radicals and singlet
oxygen. The protective role of xanthophyll pigments lutein and
zeaxanthin have been recently added to the list of potentially
beneficial nutrients for coronary heart diseases and stroke, cataract
and macular degeneration (AMD) 18. In China, Yugoslavia,
Argentina, India, Mexico, Brazil, and America pumpkins are utilized
in the pharmaceutical industry 30.
Pumpkins are good sources of proteins and fibre. Proteins are
irreplaceable, because other nutrients dont have nitrogen or amino
acids. Many investigations have been reported which concerning
the health benefits or the quantities of the fibre found in fruits and
vegetables 13, 25. Most of the research has concentrated on the
physiological properties of fibres and how they influence the
gastrointestinal tract. The fibre plays an important role in the
prevention and cure of diabetes, obesity, atherosclerosis, heart
diseases and colon cancer 7, 8. The structural polysaccharides are
the major part of plant cell walls. The types of plant material that
are included within the definitions of dietary fiber may be divided
into two forms based on their water solubility: insoluble dietary
fiber, which includes celluloses, some hemicelluloses and lignin
and soluble dietary fiber which includes -glucans, pectins, gums,
mucilages and some hemicelluloses 4.
Minerals play an important role for the human body. They affect
the utilization of dietary vitamins and are integral parts of bones,
teeth, soft tissues, muscles, blood and nerve cells. At least 22
mineral elements are required for the well-being of humans and
these can be supplied by a balanced diet 33.
Pumpkin flour is currently the main processed product of

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

61

pumpkin fruit, because it can be easily stored for long time and
conveniently used in manufacturing formulated foods. Adding
pumpkin flour in the processing of noodles, breads and cakes,
not only enhances the content of various nutrients, but also
improves the flavour of products 6.
The main aim of this study was to investigate the quality
parameters of the pumpkin (Cucurbita maxima D.) fruit flesh flours
of cultivars Justynka F1, Karowita and Amazonka.
Materials and Methods
Three pumpkin (Cucurbita maxima D.) cultivars Justynka F1,
Karowita and Amazonka were grown in the experimental field
of ecological farm (Lithuania, Kaunas distr.). The field was not
fertilized. Pumpkins were sown in the plastic cups in the end of
April, 20122013 (23 seeds were put into one hole of 24 cm
depth) and were considered in the glasshouse. Into the constant
growing place of the field shoots were planted on the middle of
May, 2012-2013. Plants were grown in four replications. Pumpkins
were harvested at the end of September.
Plant material and flour preparation: The pumpkin fruits were
washed, halved and the seeds were removed. The flesh and peel
were sliced, and dried at 60C in the thermostat (Termaks, Norway).
Dried slices of pumpkin were grinded using ultra centrifugal mill
(ZM 200, Retsch, Germany) to produce flours, which were kept
chilled in an air-tight container at 1218C temperature, until the
laboratory analysis. The samples were evaluated in triplicate for
each analysis.

quality and output of the recycled products. Depending on the


type and cultivar, the amount of the above mentioned substances
in pumpkin flesh can fluctuate from 4.15% to 23.1% 16, 27. The fruits
of great pumpkins accumulate higher amounts of dry matter
compared with the amount of the fruits of oil pumpkins. This is
due to the relatively high sugar content in the flesh of Cucurbita
maxima fruits 1, 26. Great pumpkins that are grown in Lithuania can
accumulate 7.4122.20% of dry matter. Plant fertilization with
complex and humus fertilizers increases dry matter content in the
fruits of pumpkins 17.
The amount of dry matter in different great pumpkin fruit
flesh flours ranged from 7.57 to 12.44% (Table 1). The significant
higher quantities of the above mentioned substances have been
found in Justynka F1 12.44%.
Protein is important for tissue repair and cell growth. They
provide the building block for just about every tissue in human
body (i.e. muscle, hair skin, blood, enzymes, etc.). They affect
transport through the cell membranes of various vitamins and
minerals. The content of crude protein in dry matter of tested
pumpkin fruit flesh flours was in the range from 8.35 to 11.33%
(Table 1). Amazonka flesh accumulated the lowest amounts of
crude protein (8.35%). This can be explained by the shortest
vegetation period of this cultivar. In the pumpkin Karowita flesh
flours had twice higher amount of crude protein (Table 1).
Quantities of minerals in pumpkins are influenced by numerous
complex factors including genotype, soil, environmental
conditions and nutrition interactions 32. It is very beneficial to
consume food with sufficient amounts of these substances. The
content of crude ash in the fruit flesh flours of tested pumpkins
was in the range from 6.61 to 8.89% (Table 1). The highest amount
of crude ash was accumulated in the flesh flours of Justynka.
Cucurbits are among the most important plant families supplying
with edible products and useful fibres 2. The fibre is mainly present
as cell-wall polysaccharides, which have cholesterol-lowering
properties. Antioxidative effects of these pumpkin components
have been also reported 24. On the average, most of the crude fibre
in dry matter was accumulated in Justynka F1 flesh flours (6.66%)
(Table 1). The fruit flesh of analysed cvs. accumulated similar
amounts of crude fibre.
Amount of crude fat in pumpkin fruit flesh flours ranged from
2.45 to 3.21%, between cultivars there were insignificant differences
(Table 1). Pumpkin flesh flours have very low amount of crude fat.
Foods produced from plants abounds with natural biologically
active compounds, such as polyphenols, vitamin C or b-carotene,
have antioxidant properties and are the great value to human
health 3. The most notable positive effect of processing on the
overall quality or health capacity of food is the increased
bioavailability of b-carotene resulting in an increased antioxidant
status. According to scientists, Pumpkin varieties with high lutein
content and low carotene content show a bright yellow color in
seeds 19.

Methods of sample preparation and chemical analyses: The


studies were carried out at the laboratories of Lithuanian Research
Centre of Agriculture and Forestry, Agriculture and Food Sciences
Institute of Agronomy Faculty of Aleksandras Stulginskis
University and Faculty of Human Nutrition and Consumer
Sciences, Warsaw University of Life Sciences (WULS-SGGW).
Dry matter (DM) content of the pumpkin flesh flours was
determined by drying samples to the constant weight at 105C 20.
Amounts of crude protein, crude fibre 22, crude ash and crude fat
were also determined 22.
The amount of water-soluble carbohydrate (WSC) was
determined using anthrone method 35. Samples were subjected to
the analyses of fiber components: neutral detergent fiber ((NDF)
cellulose, hemicellulose and lignin) and modified acid-detergent
fibre (MADF) using cell wall detergent fractionation method
according to Faithfull 5 and Van Soest et al. 34.
Carotenoids (-carotene, lutein + zeaxanthin, lycopene) content
were detected according to the methods Konings and Roomans 15
and Helsper et al. 12. Analyses were performed with Shimadzu
HPLC 10A system.
The experimental data was statistically processed by the
analysis of variance (ANOVA), software STATISTICA 7.0
(StatSoft, USA). Arithmetical means and standard
errors of means of the experimental data were Table 1. Dry matter, crude protein, ash, fibre and fat contents (mean s.d.) in the
great pumpkin flesh flours.
calculated. Tukey test (p<0.05) estimated statistical
reliability of mean differences.
Cultivars
Dry matter
Crude protein Crude ash Crude fibre
Crude fat
Results and Discussion
One of the most important chemical content quality
indicator is the amount of dry matter. It ensures the
62

'Justynka F1'
'Karowita'
'Amazonka'

%
12.441.89b*
8.351.33a
7.570.08a

% DM
9.911.1ab
11.331.33b
8.350.16a

% DM
8.890.62b
6.851.61a
6.610.02a

% DM
6.660.82a
6.090.71a
5.610.47a

% DM
2.450.40a
3.211.35a
2.901.19a

*Means in column with different letters are significantly different (p < 0.05) for the different cultivars.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Our results show that great pumpkin flesh flours are rich in
source of carotenoids, especially in lutein and zeaxanthin. Of
course there are other carotenoids that are good precursors of
vitamin A. It was established that the amount of lutein and
zeaxanthin was significantly different in all cvs. pumpkin fruit flours
(Table 2). According to our results significantly highest amount
of lutein and zeaxanthin was in pumpkin flours of Karowita 12.31
mg/100 g.
Murkovic et al. 23 reported that three species of pumpkin (C.
pepo, C. maxima and C. moschata) consisted of beta-carotene
(0.06-7.4 mg/100 g), alpha-carotene (0-7.5 mg/100 g) and lutein (017 mg/100 g).
Lycopene concentration is lower than that of other carotenoids.
The fruit flesh of cv. accumulated lycopene amount varied from
0.72 to 0.81 mg/100 g, and the significant highest was identified in
Justynka F1 flours (Table 2).
Table 2. Carotenoids content (mean s.d.) in great
pumpkin flesh (mg/100 g).
Cultivars
'Justynka F1'
'Karowita'
'Amazonka'

Lutein+zeaxanthin
7.960.07a*
12.310.03c
7.960.02b

Lycopene
0.810.01c
0.790.01b
0.720.02a

- carotene
2.420.02b
1.860.02a
2.440.02b

The content of WSC varied depending on cultivars. Pumpkin


flours displayed high content of total WSC (44.73% DM) in
Justynka F1and lowest content in Karowita (36.40% DM)
(Table 3).
Conclusions
The pumpkin fruit flours of Justynka F1 accumulated significantly
highest content of dry matter, crude ash, crude fiber, water-soluble
carbohydrates (WSC). The highest amount of crude protein,
crude fat and lutein+zeaxanthin was in fruit flours of Karowita.
The maximum neutral detergent fiber (NDF) and modified aciddetergent fibre (MADF) was accumulated in Amazonka pumpkin
flour, respectively, 21.37, 20.13% DM, so the flour of this pumpkin
variety could be most suitable to enrich manufactured food with
dietary fiber.
Acknowledgements
This publicatios is funded by European Social Fund and the
Budget of the Republic of Lithuania (project Eureka E! 6855
ECORAW Higher functionality food products from organic
vegetable raw materials).

*Means in column with different letters are significantly different (p < 0.05) for the different
cultivars.

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Our results show that the highest amount of b-carotene was in


Justynka F1 and Amazonka (accordingly 2.42 and 2.44 mg/100
g) and lowest in Karowita (1.86 mg/100 g) pumpkin flesh flours.
A higher amount of lignin is undesirable in NDF fiber content,
since it reduces the other fiber, hemicellulose and cellulose
degradation 21, 31. According to Nawirska et al. 26, the NDF fiber
content in C. pepo pumpkin flesh ranges 0.23 4.37% fresh matter,
C. maxima pumpkin 1.20 4.37% fresh matter., ADF fiber,
respectively, 0.22 0.47% fresh matter and 0.431.46% fresh matter.
The NDF fiber content is more dependent on the cultivars than
the genotype. The NDF content ranged between 18.80 and 21.37%
DM in the flour of three pumpkin cultivars (Table 3). The NDF
content is high in the pumpkin flour from Amazonka and slightly
lower in Justynka F1 and Karowita.
Table 3. Fractional composition (mean s.d.) of dietary
fiber and water-soluble carbohydrates in
great pumpkin flesh flours% DM.
Cultivars
'Justynka F1'
'Karowita'
'Amazonka'

NDF

MADF
WSC
amount, % DM
18.800.10a* 16.430.06a 44.730.12c
18.870.25a 18.860.28b 36.400.26a
21.370.47b 20.130.15c 39.830.06b

*Means in column with different letters are significantly different (p < 0.05) for the
different cultivars.

The MADF content in the flour of all the pumpkin cultivars was
different. The MADF content varied between 16.43 and 20.13%
DM depending on cultivars (Table 3). The highest MADF content
was in Amazonka flour, the lowest content in Justynka F1.
It was observed that Justynka F1 pumpkin flour contains
maximum amounts of water-insoluble fiber. However, in Amazonka
pumpkins the highest number of NDF and MADF fiber was
indicated.
WSC quickly digested energy-rich compounds are the primary
products of photosynthesis, so their content is highly dependent
on temperature, sunlight and other environmental factors 9.

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14
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20
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(Lithuanian Standard).
21
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24
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activity of water-soluble polysaccharide in pumpkin fruits (Cucurbita
maxima Duchesne). Biosci. Biotechnol. Biochem. 73(6):1416-1418.
25
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26
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27
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p. (in Polish).
28
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33

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

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Journal of Food, Agriculture & Environment Vol.12 (3&4): 65-70. 2014

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Training a sensory panel for describing texture in peach and nectarines


Loreto Contador 1, Paulina Shinya 1, Andrea Bunger 2, Carmen Senz 1 and Rodrigo Infante 1*
1

Facultad de Ciencias Agronmicas, Universidad de Chile, Casilla 1004, Santiago, Chile. 2 Facultad de Ciencias Qumicas y
Farmacuticas, Universidad de Chile, Casilla 223, Santiago, Chile. *e-mail: rinfante@uchile.cl

Received 7 July 2014, accepted 12 September 2014.

Abstract
Analysing the texture of fresh fruit is a fundamental aim in the food industry because it is the main factor that affects consumer acceptance, and it must
therefore be measured objectively. However, as it is a sensory property, human beings should be involved in assessing it. To achieve this goal, panels
of trained judges describe and quantify certain previously defined textural attributes for a particular food. For fresh peaches and nectarines, there are
no established methodologies for training a specific panel to describe texture. Therefore, the aims of this investigation were (1) to select sensory
attributes, develop lexicon and intensity scales for training a sensory panel and (2) to describe peach and nectarine cultivars according to their textural
properties. An appropriate lexicon was generated using five descriptors: hardness, juiciness, melting, crispness and crunchiness. The panel
was able to describe twelve peach cultivars and segregate them according to their textural properties in three groups: melting flesh, non melting flesh,
and an intermediate group formed by MF and NMF genotypes.
Key words: Peach, nectarine, sensory properties, trained panels, texture attributes, lexicon, descriptive analysis, principal components analysis,
hierarchical clustering.

Introduction
The most important attributes that define the quality of a food are
appearance, flavour, aroma, texture and nutritional attributes.
However, from the consumers point of view, texture is the main
quality attribute that influences acceptance or rejection of food 1,
and for most fresh fruit, texture is more important than the aromatic
properties 2. Therefore, when the food satisfies consumers
psychological and physiological expectations, the perception of
texture is placed on a subconscious level, but if there is a defect,
it becomes the main cause for rejection 3.
There are two approaches to study texture of foods: rheological
properties and sensory analysis 1, 4. However, the so-called texture
testing instruments can detect and quantify only certain physical
parameters which must be interpreted in terms of sensory
perception 1. For any kind of food, descriptive profiling is an
essential tool that involves the evaluation of both qualitative and
quantitative sensory characteristics of a product by a panel 5. The
beginning of this process is product familiarization and
development of a lexicon that comprehensively and accurately
describes the product dimension 5. The lexicon of texture can be
so varied that there are studies examining the differences between
languages, such as between English and Finnish 6, English, French,
Japanese and Chinese 7, and a study by Hayakawa et al. 8 that
reports the complexity of Japanese and classifies 445 different
terms to describe components of texture. There are also studies
that go more deeply into the description of the specific language
for texture in red apple 9, tomato 10 and mango 11.
Publishing lexicons is beneficial because it promotes
standardization of sensory vocabulary across multiple panels,
companies and countries. Ideally, a published lexicon has the
complete list of products from which it was developed; all attribute
terms, definitions for every attribute and references for every
attribute 12.

For fresh foods such as fruits and vegetables, textural properties


such as firmness are widely used as indices of readiness to harvest
(maturity) to meet requirements for long-term handling, storage
and assuring acceptability of consumers 13. It should be considered
that fresh fruits are prone to biological heterogeneity, therefore
variations in the data may be due to assessor differences and/or
product heterogeneity 14, 15. For this reason, the analysis of the
fresh fruit texture is more complex than in other processed foods.
Moreover, according to Hampson et al. 16 who studied the apple
genotype differences from a sensory point of view, the
heterogeneity of the fruit may cause difficulty in differentiating
samples. Due to this fact inherent to fresh fruits, it is necessary to
find the most effective methods to homogenize the samples.
The sensory analysis of fresh peaches and nectarines has been
approached using the descriptors appearance, aroma, firmness
sweetness and acid taste 17-20. No researches have delved more
deeply, into the sensory analysis of the texture of these types of
fruits.
The aims of this study were (1) to select sensory attributes,
develop lexicon and intensity scales for training a sensory panel
and (2) to describe peach and nectarine cultivars according to
their textural properties.
Materials and Methods
Training of the panel for descriptive analysis (DA): The training
process was conducted in 16 sessions of approximately 1.5 hours
each. All of the sessions were conducted in a sensory evaluation
laboratory, equipped with individual booths and a conference
room, following the protocols based on ISO 8586-1 (1993) 21.
Recruitment and selection: Twenty-three people were recruited
for the following training process. The participant ages ranged

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

65

between 21 and 35 years. To ensure that all of the participants


were suitable for training, they had to have previously passed a
test of recognition of basic tastes and a test of visual ranking 21.
As the purpose of the training was to quantify textural variables,
it was necessary to determine the selected candidates ability to
correctly define the textural properties of certain foods used in
the reference scales. To focus the scales toward fresh fruits, the
anchors for texture evaluation compiled by Harker et al. 22 were
applied. The assessors had to test pairs of samples and describe
their texture. With this exercise, the skill to discriminate differences
and express textural descriptors with suitable language was
determined. All participants correctly determined the basic
characteristics that differentiated the two samples.
Qualitative stage: Open discussions took place regarding those
descriptors that best define the texture of peaches. Finally, each
participant was asked to write down the parameters that they
considered the most important. The most frequently mentioned
attributes were hardness (13 mentions), juiciness (13 mentions),
smoothness/melting (13 mentions) and crispness/crunchiness
(9 mentions). According to the description of the assessors
smoothness and melting were considered similar, as the sensation
of breakup of the fruit in the mouth without chewing the sample.
Other concepts named less frequently were turgidity,
consistency, fibrousness and compression. During the
discussion turgidity, consistency and compression were
considered less relevant and were attributed to the overall concept
of hardness. The fibrousness of the flesh was defined as an
attribute that is not always present in peach, and therefore, it was
considered a defect that does not define the texture of the flesh.
Quantitative stage: Participants were taught to quantify the 5
textural descriptors defined in the previous stage: hardness,
juiciness, melting, crunchiness and crispness, definitions
and the evaluation protocols are shown in Table 1. Fruit and
vegetable samples were obtained from a supermarket in Santiago,
under normal retail conditions. Sample size for sensory evaluation
was standardised in 2 cm thick slices. Samples were prepared

immediately before tasting.


For each textural descriptor, panellists, according to the ranking
method, scored each product from lowest (-) to highest (+)
intensity on the proper intensity scale 23. This task improves
assessors skill in recognising different intensities and therefore
in developing their ability in building a hierarchy according to a
given characteristic (Table 2).
For hardness, the scale used was proposed by Harker et al. 22.
For crunchiness and crispness, the standard scale for wet
foods created by Chauvin et al. 24 was applied with some
modifications. For the attributes juiciness and melting there is
not reference scales for fresh fruits sensory training, so new scales
are proposed in this work. The scales were constructed in open
discussions 25 and using the products for filling the ends of the
scale suggested by Harker et al. 22.
After finishing each session, an open debate took place where
each panellist could discuss their ranking compared with the panel
average. Scores on the 15-cm-scale were assigned for each product
(Table 1). Sample tasting was repeated as many times as necessary
until reaching consensus. The purpose of this phase was to reduce
the inherent individual differences between assessors 26.
Cultivar selection: Fruits were picked at a pre-climacteric but
physiologically mature stage from an experimental orchard located
near Santiago, Chile (334814.85S; 70406.54W). The cultivars
were the melting flesh (MF) nectarines Artic Snow, Andes Nec 1,
Nectaross, Venus and Mara Dolce; the melting flesh (MF) peach
September Sun; and the non-melting flesh (NMF) peaches Dr.
Davis, Andross, Hesse, Malherbe, Corona and 11A1.
Uniform size fruits were selected when the skin background colour
was green-yellow 17, 27-30.
Immediately after harvest, the fruits were transported to the lab
and kept in a ripening chamber at 20C and 80% RH for 4 days
until the flesh had reached an adequate firmness value for
consumption (approximately 10 20 N). Fruits of the NMF cultivars
were also kept in the same chamber even if a firmness value of 10
20 N was not reached, as NMF genotypes are characterised as
having a slow softening rate 31, 32.

Table 1. Definition, evaluation technique and references for fresh peach and nectarine textural descriptors.
Medium
reference
(medium
intensity = 7.5)

High
reference
(extreme
high= 15)

Ripe banana

Granny Smith
apple

Raw carrot

Ripe banana

Apricot

Raw carrot

Ripe banana

Radish

Green pepper

Ripe banana

Strawberry

Watermelon

Raw carrot

Ripe Nectarine

Canned sliced
mango

Low reference
Descriptor

Definition

Crispness

Unique, strong, clean and acute


sound produced in the first bite of
the food with incisors and open
lips.

Hardness

Force required for compress the


sample between the molars.

Crunchiness

Multiple and full sound perceived


as a series of events, evaluated
with molars and closed lips.

Juiciness

Amount of fluid released during


chewing.

Melting

Ease with which the flesh


disintegrates under a slight
pressure exerted between the
tongue and the palate.

66

Technique
Place the sample between the
incisors (front teeth) and penetrate
it. Evaluate the sound intensity
produced at the first bite.
Place the sample between the
molars and evaluate the force
necessary to compress the food
until the molars are joined.
Place the sample between the
molars and chew it three times, and
evaluate the intensity of the sound
produced.
Place the food between the molars,
chew it 3 times and evaluate the
amount of juice released.
Place the sample on the tongue and
press it against the palate. Evaluate
how the sample flows.

(absent/ extreme
low = 0)

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Table 2. Products used for training each texture descriptor.


Descriptor
Crispness
Hardness
Crunchiness
Juiciness
Melting

ripe banana
ripe banana
ripe banana
ripe banana
raw carrot

red apple var. 'Royal Gala'


mushroom
red apple 'var. Royal Gala'
raw carrot
banana

Descriptor of lower intensity (-) to high (+)


pickle
radish
Peach
apricot
radish
green apple var. 'Granny Smith'
strawberry
grape
ripe nectarine (1-2kg-f)
canned mango

Sensory analysis: The fruit was assessed at consumption ripeness.


Evaluations were performed at normal light and temperature
conditions, in individual booths and following the standard
protocols 26. To reduce the enzymatic reactions caused by cutting
when the samples were prepared, each panellist was instructed to
slice the fruit by cutting with a sharp knife a piece of peach
lengthwise along the seam, extracting a quarter of the fruit. It was
specified that the sample was to be a bite of the equatorial zone of
the quarter. This procedure also homogenised the samples, as the
ripeness of the peaches is not homogenous within the same fruit 15.
Each fruit was provided on a white plate marked with a three-digit
code, with the same code in the score sheet. The attributes
evaluated were hardness, juiciness, melting, crunchiness
and crispness on an unstructured scale from 0 to 15. In all
sessions, assessors had the information of Table 1 available.
Statistical analysis: To analyse the discrimination capacity of
each panellist, the inversion number of the assessor was
calculated using the subtraction (in absolute value) between the
correct rank of the sample evaluated (according to its intensity)
and the rank given by the assessor, and thus, the inversion
number of each assessor was obtained (Table 3).
The inversion number of each assessor must be equal to or
less than n + 1 (n being the number of samples) to be considered
discriminating. This procedure was performed for all attributes.
Likewise, the analytical ability of the sensory panel as a single
entity had to be confirmed. The inversion number of the panel
as a whole was calculated by taking the total sum of the partial
values of each sample tasted by all of the assessors. Then, this
total sum was multiplied by the correct rank value of each sample
within the series. These results were added, and the total inversion
number of the panel was obtained 33. If the alternative hypothesis
is greater than null hypothesis, the panel was considered to
have significantly discriminated the difference in concentration
between the samples evaluated 21.
The panel result consisted on the average of eight trained
panellists whose scores were centred to take into account scale
effects. A principal components analysis (PCA) and hierarchical
clustering using average linkage with Euclidean distance method
were conducted to describe cultivars. The statistical program
InfoStat v. 2011 (InfoStat Group, Crdoba, Argentina) was used 34.

green pepper
red apple var. 'Royal Gala'
raw carrot
watermelon

raw carrot

Results and Discussion


Training the panel for DA: Once the participants had been
recruited and selected, the qualitative stage of this work consisted
of the creation, understanding and unification of the specific
sensory lexicon of texture in peaches. This phase represents an
important challenge in the study of the textural properties of foods
because of the large number of terms associated with the sensory
description of texture, which is also specific to each food and to
each country 35. If creating an adequate texture lexicon for a certain
food is a difficult objective, the task is made even more arduous
when the terms crispness and crunchiness are defined as
descriptors. In this study, both descriptors were associated with
the sound that occurs when biting and chewing a fruit. Although
a diligent definition was not required to create the lexicon, when
unifying the criteria during the discussion sessions, it was
necessary to spend some extra time for understanding and
differentiating both descriptors. The term crispness alone has
more than one meaning and is difficult to define with exactitude 36.
Both crispness and crunchiness are associated with the fracture
properties of the food 37, 38 as they occur in materials that are
essentially non-deformable and that, therefore, are broken with
relative ease 37. Most likely, to determine and quantify crispness,
the initial bite suffices, whereas crunchiness requires a
succession of fracture events that occur during chewing 37, 39. The
correct use of both terms is so important that some studies have
concentrated on analysing the use of these descriptors among
consumers in different Spanish-speaking countries 35, 40. As both
descriptors are relevant in the textural characterisation of fresh
fruit 22, strategies have been developed to correlate the sensory
perception of the sound with the wavelengths produced when
the food is penetrated by a probe 24, 41-45 and also relate to the
analysis of the force-deformation curve to the sensory values
recorded through a sensory panel 46.
One of the most frequently identified descriptors was melting,
which has also been identified as a main attribute in the texture of
mango flesh 25. There is a certain consensus in its definition that
has to do with the way in which the sample disintegrates in the
mouth, often without chewing 22.
The last descriptor defined was juiciness, for which there was
general agreement among the panellists that it is associated with
freshness 22, 47, 48. As a textural parameter, juiciness is related to

Table 3. Inversion number of each panellist and each sample, and L value of the complete panel for the attribute hardness.
ASSESSORS
order

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23
Ripe banana 1* 2 1 1 1 1 1 1 1
1
1
2
2
1
1
1
1
1
1
1
1
3
1
1
X 28
28
Mushroom
3 1 2 2 2 2 2 2 3
2
2
1
1
2
3
3
2
2
2
2
3
2
2
2
X 48
96
Peach
2 3 4 3 3 3 3 3 2
3
3
4
3
4
2
2
3
3
5
3
2
1
3
3
X 67 201
Apricot
4 4 3 4 5 5 5 5 4
5
4
3
4
3
4
5
4
4
3
5
4
5
5
4
X 97 388
Apple
5 5 5 5 4 4 4 4 5
4
5
5
5
5
5
4
5
5
4
4
5
4
4
5
X 105 525
Raw carrot
6 6 6 6 6 6 6 6 6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
X 138 828
Inversion
2 2 2 0 2 2 2 2 2
2
0
4
2
2
2
4
0
0
4
2
2
6
2
L 2066
number
Sample

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

67

PC 2 (17.1%)

5
Juiciness
the free water content 49 or moisture in the sample 1,
and it is often included in the sensory evaluation of
fruits, although the texture might not necessarily be
3
11A1
the main goal of the investigation 50-52.
Nectaross
Corona
Crispness
September Sun
The quantitative stage of the training process
Hardness Hesse
Andross
Andes
Nec
1
0
Crunchiness
began with the descriptor hardness and to obtain
Maria Dolce Malherbe
Melting
Dr Davis
Artic Snow
the association between the definition and the
Venus
physical sensation, the food scale was ordered as
-3
proposed by Szczesniak 3 and adapted to the
availability of local brands. Despite the ease with
-5
which the group correctly ordered the samples during
-5.0
-2.5
0
5.0
2.5
the discussion, this scale was not appropriate for the
PC 1 (76.8%)
aim of this research. This result is extremely important,
Figure 1. PCA biplot for the sensory attributes of texture of 12 varieties of peaches
as for the panels performance to be optimal in terms of
and nectarines.
discriminating ability, consistency, reproducibility and
precision, the products used on the scales during training must be
Table 4. Pearsons correlation coefficients among the sensory
similar to the food being studied 5. Therefore, based on panellist
attributes, based on the results of panel trained to
input and literature on fresh fruit, the scales used for the attributes
evaluate peaches and nectarines.
defined in this investigation were created.
Crispness
Hardness
Crunchiness Juiciness
For hardness, the L value with the L = 2066, number of
Hardness
0.88
products = 6 and judges = 23 was 10 (Table 3). Therefore, HA was
Crunchiness
0.91
0.97
accepted both for 5% and 1%. This means that the panel was
Juiciness
-0.27
-0.4
-0.42
being able to discriminate the intensities of hardness correctly.
Melting
-0.77
-0.91
-0.89
0.27
The scores of the inversion number of each panellist were less
than 7 (n + 1); therefore, all panellists determined the order correctly.
Hierarchical cluster analysis identified 3 groups with similar
This procedure was followed for the rest of the attributes, and for texture sensory characteristics (Fig. 2). The first group is composed
each, it was indicated that each panellist and the panel as a whole of the MF cultivars Artic Snow Nectaross, Andes Nec 1 and
ordered the given samples correctly at random. In total, the training September sun. It was observed that such cultivars are closer to
process was performed in 24 hours, divided into 16 sessions, and the descriptor melting (Fig. 1). A second group was formed by
a panel of trained judges was formed to evaluate texture in fresh the NMF cultivars Dr. Davis and Andross and the MF cultivars
peaches and nectarines. Generally, there is no literature that details Mara Dolce and Venus. Finally, the third group is composed
panel training methodology for DA or quantitative descriptive only by NMF cultivars Corona, 11A1, Hesse and Malherbe
analysis (QDA), which are intimately related 5. There are studies characterised by reaching higher scores for crispness, hardness
that detail training methodologies for mango 25, fibre 53 and bread 54. and crunchiness.
However, knowledge of the textural properties of foods is a goal of
Fruit quality is a complex trait and is not determined by any
primary importance for both the actors of the food industry as single attribute but rather by a combination of sensory
well as researchers in the area 13.
characteristics (e.g., appearance, texture, flavour and aroma),
nutritional value, and chemical, mechanical and functional
Sensory evaluation: PC1 and PC2 explained 93.9% of the total properties 2. This is how the sensory evaluation of fresh peaches
variation in the model with respect to sensory parameters (Fig. 1), and nectarines has become an important goal in research, using
indicating that cloud of data was effectively bi-dimensional.
trained or consumer panels 57. In general, the aim of sensory tests
Crispness, hardness and crunchiness were strongly has been to evaluate the effects of postharvest treatments 30, 58, to
correlated between them and with the PC1 (Table 4), whereas establish differences between states of maturity 20, 59 and to
melting was correlated negatively with PC1 and with hardness determine differences between cultivars 17, 19, 60-64 , with
(r = -0.91), crispness (r = -0.77) and crunchiness (r = -0.89)
Artic Snow
(Table 4).
Nectaross
These results are similar to those obtained by Valente et
Andes Nec 1
25
al. , and therefore, PC1 can be considered to be associated September Sun
with the viscoelastic (elastic and plastic) nature of peach
Dr Davis
flesh, whereas PC2 is associated with (the viscous nature)
Andross
juiciness. High degrees of correlations were also found Maria Dolce
Venus
between the attributes hardness, crispness and
Hesse
55
fracturability in apples and pears , between hardness
Malherbe
and fracturability when evaluating reference scales for
Corona
texture 56 and between firmness, crunchiness and melting
11A1
on mangos 25, which eliminates redundancy in the terms
0
1
2
3
4
used. Furthermore, the definition and evaluation methodology
Dissimilarity
for each of these descriptors are different, so the decision
Figure 2. Hierarchical clustering of peaches and nectarines according to their
was made to keep the three descriptors.
sensory texture characteristics perceived by the panel.

68

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

appearance, aroma, flavour, firmness or hardness,


sweetness, acidity and juiciness being the most frequently
used attributes. There are very few sensory investigations of
texture in peach. It has been approached from the point of view of
the physical behaviour of the fruit, as in the studies of apricots
conducted by Haciseferogullari et al. 65 and Missang et al. 66 or
through the characterisation of the changes in the components of
the cell wall during the softening process 27, 67, 68. Texture has also
been considered a unique attribute 17 , but given its complexity, it
must be disaggregated into its particular components.
Conclusions
An appropriate lexicon was generated for describing the texture
of peaches and nectarines, which considers five descriptors:
hardness, juiciness, melting, crispness and crunchiness.
The scales used for the training process for these attributes allowed
the panel to describe twelve peach and nectarine cultivars and
segregate them according to their textural properties in three
groups: MF and NMF genotypes and an intermediate group formed
with both genotypes.
From the biological standpoint, peach and nectarine flesh are
classified as MF and NMF; however, these categories are
insufficient for describing and analysing the growing supply of
new cultivars released annually to market that exhibit novel flesh
typologies. Due to this dynamism, sensory evaluation is a tool
that allows more efficient classification of the different types of
flesh, which is important, as measuring only the firmness of the
pulp appears to be insufficient.
Acknowledgements
This work was conducted with funding from CONICYT through
the scholarship Doctorado nacional 2010 and FONDECYT
Project 1130198.
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Antioxidant and antimicrobial potential of dried cumin (Cuminum cyminum L.),


caraway (Carum carvi L.) and turmeric powder (Curcuma longa L.)
Muhammad T. Sultan 1*, Masood S. Butt 2, Saeed Akhtar 1, Atif N. Ahmad 3, Mubasher Rauf 3,
Muhammad S. Saddique 1 and Ambreen Naz 2
Department of Food Sciences, Faculty of Agricultural Sciences & Technology, Bahauddin Zakariya University, Multan.
National Institute of Food Science and Technology, University of Agriculture, Faisalabad. 3 Faculty of Veterinary Sciences,
Bahauddin Zakariya University, Multan, Pakistan. *e-mail: tauseefsultan@bzu.edu.pk
1

Received 28 June 2014, accepted 8 September 2014.

Abstract
The process of oxidation is vital for energy metabolism but it is also coupled with the production of oxygen free radicals (OFRs). The excessive
production of OFRs results in oxidative stress and such conditions demand the supplementation of antioxidants. The bioactive components present
in common spices and condiments are of imperative significance as they scavenge OFRs along with acting as antimicrobial agents. The current study
aimed to explore the antimicrobial and antioxidant potential of dried cumin (Cuminum cyminum L.), caraway (Carum carvi L.) and turmeric
(Curcuma longa L.) powders. The results elaborated the importance of aforementioned spices as they all contain significant amounts of cruder
protein, crude fats, fiber and carbohydrates. Cumin seeds contain the highest amounts of fats, while appreciable amounts of carbohydrates were
observed in turmeric (43.87 1.41). Vitamin C was present in turmeric and caraway seeds. The results regarding minerals indicated that the cumin
contains appreciable quantities of calcium, magnesium, sodium and iron. The results regarding antioxidant potential indicated that the maximum total
polyphenol was present in caraway seeds (1016.72 63.68 mg GAE/100 g) that can also be correlated with higher DPPH and -carotene inhibition
activities (57.71 0.77 and 47.65 0.74%, respectively). The caraway seeds were more effective antimicrobial agent as compared to cumin and
turmeric. Overall, the results indicated the potential of dried condiments as natural antioxidants and antimicrobial agent.
Key words: Natural antioxidants, antimicrobial agents, spices, cumin, caraway, turmeric.

Introduction
The process of oxidation is essential for the vitality of humans to
generate sufficient amount of energy from important food
components like carbohydrates, proteins, and lipids. However,
such processes are also associated with the production of free
radicals 1. These highly reactive radicals, especially oxygen free
radicals (OFRs), can disturb the human metabolisms through
destruction of membranous structures. The process of oxidation
can also result in quality deterioration of different food products.
Although, humans inherited immune defense systems but
antioxidants are required to be supplemented through diet to
control the excessive amounts of OFRs. The natural sources of
antioxidants include fruits, vegetables, spices, and herbs of some
medicinal value 2. In the recent past, the old concepts of food
pyramids and dietary guidelines were modified in the light of
modern research that explored the significant association of diet
and health. The dietitians and nutritionists researched the role of
various plants and their metabolites to control the menace of
oxidative stress and allied complications. The trends also
witnessed the increased share of such health foods in global food
chains owing to their higher acceptability by the consumers 3.
Nutraceutical and functional foods are generic terms used to
categorize foods with certain health claims. Most of these foods
possess multiple health benefits, especially against lifestyle related
and degenerative disorders 4. Natural compounds found in fruits,
cereals, vegetables and spices hold antioxidant activity. The
bioactive antioxidative compounds present in them are usually

classified in different categories like carotenoids, tocopherols,


polyphenols, anthocyanins, alkaloids, and aged glycated peptides 5.
Cumin (Cuminum cyminum L.) belongs to the Apiaceae family
and is annual herb that is native to the Mediterranean region. It is
one of the most commonly used condiment to add flavor and
specific aroma to the foods. The cumin seed contains several
bioactive compounds belonging to the categories of polyphenols,
flavonoids, cumin aldehydes, and terpenoids 6. Some of these
bioactive components are antioxidants and might be useful in
health disorders pertaining to the OFRs. Cumin seeds are reported
to be effective against various ailments including diabetes mellitus
and cardiovascular disorders. Caraway (Carum carvi L.) belongs
to the Umbelliferae family and is biennial herb and used as
condiment to add flavor and specific aroma to the foods. The
caraway seeds are also helpful in mitigating the free radical
production and thus controlling some health disorders. Caraway
seeds contain several bioactive components that belong to the
category of terpenoids, limonene, thymol, glycosides, and
flavonoids. The safety and biochemical efficacy of caraway and
its essential oil has been already tested in various research
interventions 7. Turmeric (Curcuma longa L.), belonging to the
family of Zingiberaceae, is perennial herb usually called as golden
spice due to its specific color tone. Although it is native to Indian
continent, various countries are cultivating it for routine use in
cuisines. The turmeric contains more than 500 bioactive
components and most of them hold some medicinal value. Turmeric

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

71

is effective against various ailments including cancer insurgence,


cardiovascular disorders, diabetes mellitus, infectious, and
degenerative disorders 8, 9.
Globally, the aforementioned plants are explored in various
studies and several herbal supplements containing bioactive
components from these plants are available in the global food
chain 10. Although, fewer research studies are undertaken in
Pakistan, yet such sort of studies have certain limitations. The
present study aimed at exploring the antioxidant and antimicrobial
potential of sun-dried cumin, caraway, and turmeric. The results
of this intervention are providing supporting evidences to claim
these spices as health promoting entities. The limitation of the
present research included the utilization of unidentified strains of
microorganisms, thus later studies should be conducted to check
the influence of these nutritionally rich commodities on identified
microbial strains.
Materials and Methods
The present research intervention was completed in the
Department of Food Sciences and Department of Pathobiology,
Bahauddin Zakariya University, Multan. For the purpose, three
different spices (cumin, caraway and turmeric) were collected from
local market of Layyah, Punjab, Pakistan. The cleaning of the
samples was carried out through the operations like sieving and
washing. Later, the samples were ground into a fine powder for
analytical purpose.
Proximate analysis: The cumin, caraway, and turmeric were
examined for their nutritional components that include protein,
fats, ash, fiber, and moisture contents. Nitrogen free extract (NFE)
was calculated using standard equation mentioned in AACC 11
that indicates the presence of carbohydrates. The AACC Method
No. 44 - 01, Method 08 - 01, Method No: 46 - 13, Method 30 - 10,
and Method 32 - 10 were followed for the said purpose 11.
NFE % = 100 (Total ash % + crude protein % + crude fat % + crude fiber %)

The ash collected was further utilized for the analysis of macro
and micro minerals. Sodium, potassium, and calcium were
determined using flame photometry and rests of the minerals were
analyzed using atomic absorption spectrophotometry.
Determination of total sugars and vitamin C: Total sugar is the
sum of reducing and non-reducing sugars and was determined
using the volumetric method (LaneEynon method) mentioned in
AOAC (1990). Vitamin - C content was estimated using 2,6dichlorophenolindophenol dye using the procedures outlined in
the standard method 12.
Determination of antioxidant potential: The antioxidant rich
extracts of selected spices were prepared by slurring the samples
with aqueous ethanol for a period of one hour. The facilities of
National Institute of Food Science and Technology, University of
Agriculture, Faisalabad, were used in this context. Briefly, the
samples were slurred with aqueous ethanol using mechanical
shaker and centrifuged for 15 min at 7000 rpm at 0C. The
supernatant was further filtered with Whatman filter paper No. 1.
The solvent from the supernatant was separated at 50C in a rotary
vacuum evaporator (EYELA, N-N series, Japan). The extracts were
72

further analyzed for their antioxidant potency through different


parameters like total phenolic contents, antioxidant activity and
free radical scavenging activity (DPPH assay).
Determination of total phenolic content: Total phenolic contents
(TPC) were measured according to Mustafa et al. 13. Firstly, 5 ml of
DMSO dissolved 5 mg of dried extracts of spices. Then, 0.5 ml of
the resulting aliquot was added to 1 ml of 50% Folin-Ciocalteau
reagent and incubated for 3 min at room temperature (20 - 25C).
Next, 3 ml of 1% Na2CO3 was added to the mixture, thoroughly
vortex-mixed and incubated for further 30 min. Absorbance of the
mixture was read at 760 nm, using a spectrophotometer (Thermo
Scientific Genesys 20, USA). Results were expressed as mg of
gallic acid equivalents per 100 g of sample (mg GAE/g).

-carotene bleaching assay: The -carotene bleaching assay that


indicates the antioxidant activity based on coupled oxidation of
-carotene and linoleic acid following the procedures was outlined
by Taga et al. 14. For the purpose, 5.0 mg of -carotene was
dissolved in 50 ml of chloroform. Later, linoleic acid (40 mg) and
Tween 20 (400 mg) were added and solvent was removed using
nitrogen gas. The oxidative changes in -carotene emulsion were
measured using spectrophotometer (absorbance at 470 nm) at
specific time intervals. The degradation rate of the extracts was
calculated according to first order kinetics antioxidant activity
(AA) expressed as % inhibition relative to the control.
DPPH free radicals scavenging assay: The DPPH assay was
carried out using the method described by Prabhasankar et al. 15
with slight modification. Briefly, 0.10 mM of DPPH was dissolved
in 100 ml of 99.9% ethanol. Stock solutions of freeze-dried extracts
(0.5 g) were dissolved in 100 ml distilled water on the day before
analysis to allow the extract to be finely dissolved. Serial dilutions
with varying concentrations (50, 100, 500, 1000, 3000 and 5000
ppm) were made from the stock solution of each sample by
adjusting the volume up to 10 ml using volumetric flasks with
distilled water. A 2 ml DPPH solution was mixed with 2 ml of each
dilution in the test tubes and shaken well for at least 15 s. Finally,
2 ml of 99.9% ethanol was mixed with 2 ml of DPPH solution (used
as blank) and test tubes were kept in the dark for 1 hour. The
absorbance of blank, control and all the samples were taken at 517
nm. The scavenging effect (%) was calculated according to the
following equation:
% Inhibition DPPH = (AbsDPPH Abssample) x 100
AbsDPPH

AbsDPPH is the absorbance of the DPPH solution without extracts.


Abssample is the absorbance of sample solution.
Determination of antimicrobial potential of extracts:
Antimicrobial potential of medicinal plant extracts was evaluated
against the bacterial strains, i.e. E. coli, Salmonella,
Campylobacter, Listeria, coliforms, Clostridium and
Staphylococcus, and fungi like Candida. The microorganisms
were isolated and identified by the method described by Koneman
et al. 16. The results are not mentioned in the research project as
this work was carried out in collaboration with Dr. Atif Nisar
Ahmad. Later, the minimum inhibitory concentrations (MIC) of

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

medicinal plants against these bacterial genera were determined


and mentioned as under. Briefly, isolated strains were cultured on
their specific media, i.e. MacConkey agar (E. coli), SalmonellaShigella agar (Salmonella and Shigella spp.), blood agar
(Pseudomonas, Listeria and Streptococcus spp.), mannitol salt
agar (Staphylococcus spp.), modified charcoal cefeperazone
deoxycolate agar (Campylobacter spp.), different reinforced
Clostridium medium (Clostridium spp.), Sabouraud agar (Candida
spp.). The agar disc diffusion method was used to determine the
antimicrobial activity. Sterile discs (6 mm, Hi-media, India) were
loaded with 50 l of (30 mg/ml) cumin, turmeric and caraway powder
extract dissolved in 5% dimethyl sulfoxide (DMSO). Bacterial
suspensions were also diluted to match the 0.5 McFarland
standard scales (approximately 1.5 x 108 CFU/ml). Further, MllerHinton agar (MHA) was poured into Petri dishes to give a solid
plate and inoculated with 100 l of suspension containing 1.5 x 1
08 CFU/ml of bacteria. The plates were incubated at 37C for 24 to
36 h and inhibition zones diameter around each of the discs was
measured and recorded. Minimum inhibition concentrations of
the plant extracts was tested by the checkerboard assay method.
Statistical analysis: The data presented in the paper is the mean
and standard deviation and whole experiment was carried out in
triplicates. The dried spices were further compared through
analysis of variance technique to determine the level of
significance 29. The means were compared through Least
Significance Difference (LSD) test using Statistical Analysis
System (SAS Institute, Cary, NC) version 9.1 and Microsoft Excel
2007.
Results
The mean values regarding moisture contents indicated that
moisture contents varied from 7.91 0.15 to 11.80 0.57% (Table
1). The maximum moisture contents were recorded in turmeric,
while minimum was recorded in cumin seed (7.91 0.15%). The
protein contents in different plants varied significantly and
maximum protein contents of 21.01 1.12% were recorded in
caraway seeds followed by cumin seeds (19.29 1.04%), whilst
minimum protein contents of 7.54 0.05% were observed in
turmeric. Crude fat in cumin seeds (24.90 0.57%) was higher
than in caraway (15.69 0.28%) and turmeric (9.92 0.57%). Cumin
contains the highest amount of ash contents (7.16 0.39%) and
turmeric ranked at the bottom (5.67 0.21%). Caraway and turmeric
contain higher amount of fiber, i.e. 39.83 1.94 and 21.52 0.44%,
respectively. In comparison, nitrogen free extract (NFE) was higher
in turmeric (43.87 1.41%) followed by cumin (31.70 0.21%). The
mean values regarding sugars indicated the total sugars ranged
from 0.62 0.02% to 2.93 0.08% with the maximum in turmeric
and the minimum in caraway 0.62 0.02%. Vitamin C content was
recorded highest in turmeric (24.31 0.45 mg/100 g) followed by
caraway (19.33 0.21 mg/100 g), however, cumin was ranked at
the bottom regarding this trait (Table 1).
The mean values regarding calcium content (Table 2) indicated
that the values for the said trait ranged from 202.45 7.09 to
1004.38 27.58 mg/100 g. The maximum calcium and magnesium
contents were observed in cumin followed by caraway whereas
the minimum was found in turmeric. Overall, phosphorus content
ranged from 270.21 9.12 to 645.56 7.35 mg/100 g. The maximum
phosphorus content was observed in caraway (645.56 7.35 mg/

Table 1. Means for proximate composition of dried cumin,


turmeric and caraway powder.
Selected dried spices
Cumin
Turmeric
Caraway
Moisture (%)
7.91 0.15 11.80 0.57 10.34 0.05
Protein (%)
19.29 1.04 7.54 0.05 21.01 1.12
Fat (%)
24.90 0.57 9.92 0.57 15.69 0.28
Ash (%)
7.16 0.39 5.67 0.21
5.68 0.32
Crude fiber (%)
11.21 0.14 21.52 0.44 39.83 1.94
NFE (%)
31.70 0.21 43.87 1.41 8.07 0.42
Total sugars (%)
2.38 0.07 2.93 0.08
0.62 0.02
Vitamin C (mg/100 g) 8.33 0.04 24.31 0.45 19.33 0.21
Parameter

*NFE = Nitrogen free extract (It represents the carbohydrate fraction.)

100 g) followed by cumin (511.00 11.04 mg/100 g). Mean values


regarding potassium content explicated that it ranged from 1332.57
27.54 to 2574.28 55.10 mg/100 g. The maximum potassium
contents were observed in turmeric, whereas the minimum
potassium contents were recorded in caraway. The sodium
contents varied significantly and the highest sodium contents
were observed in cumin (172.93 4.09 mg/100 g). The turmeric
(34.50 1.51 mg/100 g) and caraway (17.48 0.82 mg/100 g) were
down in the ladder (Table 2).
Table 2. Means for macromineral profile (mg/100 g) of dried
cumin, turmeric and caraway powder.
Selected dried spices
Cumin
Turmeric
Caraway
Calcium (Ca)
1004.38 27.58 202.45 7.09
767.25 3.36
Magnesium (Mg) 406.15 16.45
201.49 6.75
235.16 12.26
Phosphorus (P)
511.00 11.04
270.21 9.12
645.56 7.35
Potassium (K)
1607.71 93.80 2574.28 55.10 1332.57 27.54
Sodium (Na)
172.93 4.09
34.50 1.51
17.48 0.82

Parameter

The results regarding microminerals indicated that spices contain


appreciable amounts but differed significantly from each other
(Table 3). The maximum iron contents were examined in cumin
(60.60 3.71 mg/100 g) and caraway was ranked in the last (15.62
0.50 mg/100 g). Zinc contents were ranged from 4.66 0.22 to
5.29 0.11 mg/100 g in different plants with the maximum in caraway
(5.29 0.11 mg/100 g) and the minimum in turmeric (4.66 0.22 mg/
100 g). Mean values regarding copper depicted that cumin seeds
contain the maximum copper contents (0.92 0.03 mg/100 g), while
the minimum contents were recorded in turmeric (0.66 0.04 mg/
100 g). The mean values regarding manganese varied from 1.30
0.06 to 8.02 0.35 mg/100 g. It is important to write for the readers
that all plants were devoid of fluorides.
Table 3. Means for micromineral profile (mg/100 g) of
dried cumin, turmeric and caraway powder.
Selected dried spices
Cumin
Turmeric
Caraway
Iron (Fe)
60.60 3.71 44.98 2.07 15.62 0.50
Zinc (Zn)
4.98 0.27 4.66 0.22
5.29 0.11
Copper (Cu)
0.92 0.03 0.66 0.04
0.85 0.04
Manganese (Mn) 3.16 0.11 8.02 0.35
1.30 0.06
Parameter

The statistical results regarding TPP indicated that the plants


differed significantly (Table 4). The means indicated that the TPP
contents varied from 329.26 6.41 to 1016.72 63.68 mg GAE/100
g. The maximum amount of TPP was observed in caraway while
least amounts were recorded in cumin. The DPPH inhibition as
perceived through the statistical analysis was significant and all
plants were effective in this respite, however, the percentage varied

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

73

Table 4. Means for antioxidant potential of dried cumin, turmeric


and caraway powder.

free extract (NFE) Moreover, vitamin C contents were recorded


higher in turmeric and caraway seeds. The results regarding
proximate components were quite conclusive that the spices hold
Selected dried spices
Parameter
important nutritional value but their consumption in slight amounts
Cumin
Turmeric
Caraway
TPP (mg GAE/100 g)
329.26 6.41 980.20 47.18 1016.72 63.68 results in their lower worth as energy source. The results regarding
DPPH (% inhibition)
39.01 2.11
47.26 0.32
57.71 0.77
proximate constituents and mineral contents are in agreement with
-carotene (% inhibition) 38.96 0.89
36.17 2.09
47.65 0.74
the scientific literature available. In this regard, Ghosh-Das and
TPP (total polyphenols), GAE: Gallic acid equivalent, DPPH Diphenyl picryl hydrazine
Savage 17 reported that turmeric contains abundance of medicinally
with each plant. The results regarding percent DPPH inhibition, active chemicals. The chemical constituents present in turmeric
the caraway exhibited maximum DPPH inhibition (57.71 0.77%) holds significant antioxidant capabilities. Curcumin as major
followed by turmeric (47.26 0.32%). However, least DPPH ingredient of turmeric reported to be effective antioxidant and
inhibition was recorded in cumin (39.01 2.11%). The percentage helps to fight cancer insurgence and inflammation 8.
Spices are the building blocks of flavor in foods and the presence
inhibition of free radicals through -carotene bleaching method
indicated that antioxidant activity of the plants varied. The means of polyphenols in them is positively correlated with their
regarding the said trait depicted that cumin and turmeric inhibited antioxidant potential that may include DPPH inhibition and lipid peroxidation non-significantly with mean inhibition of 38.96 carotene bleaching assay 18. Various scientists studied the
0.89% and 36.17 2.09%, respectively, whilst the caraway was nutritional profile and antioxidant potential of cumin and overall it
most effective in reducing the extent of lipid peroxidation, i.e. 47.65 can be observed that cumin contains bioactive components like
cymene, cuminlaldehyde, cuminal, -terpinene, -pinene, carveol,
0.74 %.
The results regarding antioxidant potential indicated that and myrtenal as major volatile components. Moreover, cumin
turmeric is most effective against all microorganisms tested in the contains 2.520.11% of essential oil that possesses DPPH
present study (Fig.1). The turmeric powder had MICs of 35.00 inhibition activities of 85.440.50% 2, 6. Hajlaoui et al. 19 also reported
1.77, 21.35 1.21, 41.18 1.01, 37.14 0.96, 32.05 1.18, 38.01 the antioxidant potential in various assay, i.e. -carotene bleaching
1.39 and 42.54 1.56 mm against E. coli, Salmonella, test and DPPH inhibition. Recently, Kedia et al. 2 reported strong
Campylobacter, Listeria, Clostridium, Staphylococcus, and antioxidant potential of cumin essential oil with mean IC50 value of
Candida, respectively. The caraway ranked second but cumin 0.092 l/ml. Lekshmi et al. 20 reported that the antioxidant capacity
seed powder showed minimum MICs for the tested of turmerin (a bioactive compound present in turmeric) is of
significant importance as it inhibits DPPH, superoxide, and ABTS
microorganisms.
radicals with mean IC50 values of 29, 48, and 83 g/ml, respectively.
Caraway seeds essential oil and its bioactive components are
Discussion
The bioactive compounds present in spices make them an ideal effective antioxidants and Kapoor et al. 21 showed their strong
candidate for the extraction of natural antioxidants and antioxidant activity that can be slightly comparable with BHA and
antimicrobial agents and thus can also be effective in lowering BHT. The same authors claimed antioxidant activity through
the risk of various maladies. Amongst different spices, cumin, various assay DPPH assay, -carotene bleaching assay, and total
caraway, and turmeric are important in Asian cuisines and are polyphenols. Samojlik et al. 7 reported that essential oil extracted
globally recognized as condiment and flavor enhancer or taste from caraway seeds inhibits lipid peroxidation and DPPH free
modifier 10. In the present research, sun-dried cumin, caraway, and radical with mean IC50 value of 2.5 l/ml. De Martino et al. 22 reported
turmeric powders were analyzed for their basic proximate and that essential oil of caraway seeds exhibit significant antioxidant
mineral composition, antioxidant potential, and antimicrobial activity owing to the presence of several bioactive compounds
perspectives. The results indicated that the cumin and caraway like anethole, carvacrol, and estragol. Curcumin
contain appreciable amounts of protein and fat. Fiber contents (diferuloylmethane), golden color pigment, is main constitute of
were higher in turmeric and caraway, while turmeric contains turmeric that holds antioxidant and antimicrobial potential 9.
Antimicrobial agents are important to preserve the food products
significant amounts of carbohydrates as indicated from nitrogen
from spoilage. Moreover, some of them are of
Cumin
Caraway
Turmeric
45
significant value for curing infectious disorders.
Kedia et al. 2 reported that cumin essential oil
40
can inhibit aflatoxin production @ 0.6 and 0.5 l/
35
ml, concentrations. Although, cumin seed
30
powder was least effective in inhibiting the growth
25
of microorganisms but several researchers reported
20
its effectiveness against Aspergilus flavus,
Aspergillus niger, Bacillus subtilis, Staphylococcus
15
epidermidis, Saccharomyces cerevisiae and
10
Candida albicans23. Like cumin, turmeric holds
5
several health benefits and its incorporation in
0
different products have been tested, e.g. bread 24
E. coli
Salmonella Campylobacter Listeria
Clostridium Staphylococcus Candida
to enhance the well being of the individuals.
Species
Turmeric and its active ingredients are effective
Figure 1. Minimum inhibitory concentration (mm) of selected spices against various
in inhibiting the growth of Aspergillus niger,
isolated mictobial strains.
Caraway
Caraway

Turmeric
Turmeric

Minimum inhibitory concentration (mm)

Cumin
Cumin

74

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Fusarium moniliformes, S. aureus, Bacillus subtilis and


Fusarium oxysporium 25, 26. Caraway seeds are also effective in
reducing the growth of Staphylococcus aureus, Saccharomyces
cerevisiae and Aspergillus niger in different food products 27.
Previously, De Martino et al. 22 also reported that essential oil of
caraway seed is effective in inhibiting the growth of Bacillus
cereus, Pseudomonas aeruginosa, Escherichia coli and
Penicillium citrinum. Caraway seeds are effective in enhancing
the efficiency of drug to control pathogenic microorganisms 28.
In the nutshell, it can be claimed that spices and their bioactive
components are important in human nutrition as they act as
antioxidants and scavenge free radicals along with acting as
antimicrobial agents to inhibit the growth of pathogenic
microorganisms 4.
Conclusions
The spices evaluated in the present research showed significant
antioxidant activity and all of them could be further explored for
their potential application. Similar to antioxidant potential, the
antimicrobial activities of all spices were of significant importance
and should be tested at industrial level. In the nutshell, the
antioxidant and antimicrobial potential of the selected spices is
of commercial significance, however, safety assessment through
animal modelling is essential for warranting them commercial
antioxidant and antimicrobial agents.
Acknowledgements
Corresponding author is thankful to Directorate of Research,
Bahauddin Zakariya University, Multan, Pakistan, for providing
essential funds for running this project. Moreover, the authors
are thankful to the National Institute of Food Science and
Technology, University of Agriculture, Faisalabad, for providing
instrumental facilities to conduct the experiments regarding
antioxidant potential of selected spices.

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Inhibitory effect of gamma radiation in degrading and preventing fungal toxins


Amira Hassan Abdullah Al-Abdalall
Department of Biology, Faculty of Science, Dammam University El-Dammam, P. O. Box 838, Dammam 31113,
Kingdom of Saudi Arabia. e-mail: dr2000amira@hotmail.com
Received 16 April 2014, accepted 30 September 2014.

Abstract
The aim was to study the effect of ionizing radiation on prevention of fungal growth and degradation of toxins in food materials. Ffungal strains
(Aspergillus niger, A. alliaceus, A . melleus, A. flavus and Fusarium solani) used in this study were isolated from coffee beans. Fungi differed in their
response to different doses of gamma radiation on a decline in the ability to produce biological active materials. Treatment with 3 kGy stopped
production of these materials. F. solani did not lose completely the ability to produce these materials even when using a dose of 10 kGy. Boiling
culture filtrates led to the blocking of active biological materials, or did reduce the inhibitory of bacterial growth. In all the fungi tested in liquid media
and even in the control one, ochratoxin A did not appear, and when exposed to 10 kGy, the fungal cells died and were not able to grow and produce
mycotoxins. On the other hand, the ability to production of aflatoxin by F. solani was increased after treatment with 1 and 3 kGy and decreased
thereafter. Irradiation of culture filtrates with gamma rays may have significantly reduced the inhibitory activity of the growth of bacteria B. subtilis.
The increase of inhibition was affected positively with the dose, and the effect was different with different fungal strains.
Key words: Aspergillus, ochratoxin, aflatoxin, gamma radiation, B. subtilis.

Introduction
Some fungal species belonging to genera Penicillium and
Aspergillus are toxin-producing organisms as food contaminants.
They are leading to health problems for human and animals and
economic loss. Some species of genus Aspergillus are natural
contaminants to medicinal plants and the effect of gamma
radiation on fungal toxin production was studied 1 . Van Dyck et
al. 2 proved that presence of water accelerates destruction of
aflatoxin when gamma radiation is used. Radiation in the presence
of water produces free radicals, which may destroy AFB1 in the
furan ring recycling in products of low biological activities. The
decrease of AFB1 activity in aqueous solution was 34, 44, 47 and
100% after exposure to -radiation doses of 2.5, 5, 10 and 20 kGy,
respectively. Aziz and Abd El-Aal 3 found that -radiation at 10
kGy can stop toxication of coffee seeds and some foodstuffs with
aflatoxin. Gamma-radiation lowered contamination of maize seeds
97.6% at 2 kGy and to 94% at 5 kGy. AFB1 decreased by 68.9 %
using -dose of 2 kGy, 46% at 5 kGy, and at 10 kGy, there was a
complete inhibition of AFB1and AFB2. Aziz et al. 4 studied the
effect of -radiation and maize fats on aflatoxin production by
A.flavus in contaminated maize and found a reversible relationship
between growth of the fungus and amount of -rays. At 3 kGy,
complete inhibition of aflatoxin B1 was recorded, even after storage
of maize until 45 day. Aflatoxin B1 production was activated at 0.1
and 1.5 kGy radiation dosage.
The fungal flora in fruits was sensitive to -radiation, complete
sterilization was obtained at 5 kGy, and there was a reduction of
AFB1 toxins from 380-500 to 20 mg/kg at 20 kGy 5. Complete
degradation of aflatoxin B was found after exposure to 20 kGy radiation 6.

Refai el al. 7 could isolate fungal colonies of about 103-106 CFU/


g in summer from Egyptian pastrami, which is made of meat with
salt and a layer of pepper, garlic, fenugreek and cumin, while in
winter they could isolate 102-105 CFU/g. Then 2.8 - 47 mg/kg of
aflatoxins was recorded from meat, but using 5 kGy radiation led
to complete inhibition of toxin production.
Aziz and Mahrous 8 studied the effect of -rays on aflatoxin
production by A. flavus and the chemical composition of wheat,
common beans and soy beans. They recorded increase in protein
content and decrease in lipids and carbohydrates after infection
by A. flavus. On the other hand, using -radiation at 5 kGy led to
reduction in fungal toxin production.
Kim et al. 9 and Wi et al. 10 proved that exposure to ionic radiation produce free radicals within molecules leading to changes
in shape, anatomy, and biochemical reactions within the plants.
Changes also included cell metabolism as expansion of membranes
and disorders of photosynthesis and accumulation of phenolic
compounds. Aquino et al. 11 mentioned that aflatoxins B1 and B2
decreased to levels that cannot be detected after treatment at 10
kGy.
Seed contamination by spores and fungi from the storage
environment would affect the quality and nutrient contents of
seeds. So, irradiation proved to be effective in crop and seed
production 12.
Irradiation of biological material to keep it from contamination
is important in marketing. Irradiation at 40 kGy kept coffee seeds
sterile from contaminants 13. Also, Chelack et al. 14 and Sajilata
and Singhal 15 recommended irradiation of foodstuffs with the
ionizing radiations.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

77

The aim was to study the effect of ionizing radiation on


prevention of fungal growth and degradation of toxins in food
materials.
Materials and Methods
Test pathogens: The microorganisms (Aspergillus niger, A.
alliaceus, A . melleus, A. flavus and Fusarium solani) used in this
study were isolated from coffee beans. The developing fungal
colonies were identified up to the species level by microscopic
examination according to the keys of several researchers 16-19.
Source of gamma irradiation: The source of irradiation used for
the tested fungal species was Cobalt- 60 gamma Atomic Energy
Research Institute of King Abdulaziz City for Science and
Technology (KACST).
Exposure of the spores of the tested fungal species to gamma
radiation and radiosensitivity test of fungi: Fungi growth from
pure 14 days old culture of each of the tested fungal species were
irradiated by gamma radiation at dose levels of 1, 3, 5, 7 and 10
kGy. Three replicates were used for each dose as well as for the
control (non-irradiated). One cm discs of fungal growth in
irradiated and non-irradiated spore suspension were plated on
Potato Dextrose agar medium in a Petri dish. The plates were
incubated at 27C for 14 days. One-cm discs of fungal species
were used to inoculate yeast extract-glucose medium, incubated
at 25C for 14 day, with 3 replicates, followed by filtration. The
filtrate was incubated in a water bath at 100C for 20 min followed
by centrifugation at 300 rpm for half an hour. The inhibitory effect
of the filtrate on B. subtilis growth was measured. Tubes were
incubated in a water bath at 100C for half an hour to degrade the
active biologically substances. B. subtilis was used for bioassays.
Mycotoxin analysis:
Apparatus: The High Performance Liquid Chromatography (HPLC)
system consisted of Waters Binary pump Model 1525, a Model
Waters 1500 Rheodyne manual injector, a Waters 2475 MultiWavelength Fluorescence Detector, and a data workstation with
software Breeze 2. A phenomenex C18 (250 x 4.6 mm i.d), 5 m from
Waters corporation (USA) for aflatoxins. A HyperClone 5 ODS
column (C18) 120 , DIM: 250 x 4.60 mm (Phenomenex).
Extraction of aflatoxins by VICAM 20 method: Twenty-five g
sample with 5 g sodium chloride was weighed and placed in a
blender jar. Then, 125 ml of methanol: water (70:30) was added,
covered and blended at high speed for 1 min. Cover was removed
from the jar and the extract poured into fluted filter paper. Filtrate
was collected in a clean vessel. Then 15 ml filtered extract was
poured into a clean vessel. Extract was diluted with 30 ml of purified
water and mixed well. Diluted extract was passed through the
glass microfiber filter into a glass syringe barrel using markings
on barrel to measure 4 ml.
Immunoaffinity chromatography: Fifteen ml of filtered diluted
extract (15 ml = 1 g sample equivalent) was passed completely
through AflaTest -P affinity column at a rate of about 1-2 drops/
s until the air comes through the column. Five ml of purified water
was passed through the column at a rate of about 2 drops/s.
Affinity column was eluted by passing 1.0 ml HPLC grade methanol
78

through the column at a rate of 1-2 drops/s and collecting all of


the sample eluate (1 ml) in a glass vial. Eluate was evaporated to
dryness under a stream of nitrogen and sample was determined
by HPLC.
Detection and determination of aflatoxins by HPLC:
Derivatization: The derivatives of samples and standard were
done as follow:100 l of trifluoracetic acid (TFA) was added to
samples and mixed well for 30 s and the mixture stand for 15 min.
900 l of water: acetonitrile (9:1 v/v) was added and mixed well by
vortex for 30 s and the mixture was used for HPLC analysis.
HPLC conditions: The mobile phase consists of acetonitile/water/
methanol (1:6:3). The separation was performed at ambient
temperature at a flow rate of 1.0 ml/min. The injection volume was
20 l for both standard solutions and sample extracts. The
fluorescence detector was operated at an excitation wavelength
of 365 nm and an emission wavelength of 450 nm. AFB 1
concentration in samples was determined from the standard curve,
using peak area for quantification.
Ochratoxin analysis:
HPLC equipment: The HPLC system consisted of Waters Binary
pump Model 1525, a Model Waters 1500 Rheodyne manual injector,
a Watres 2475 Multi-Wavelength Fluorescence Detector, and a
data workstation with software Breeze 2.
Chemicals and reagents: OTA standard, Chartist, microfiber filter
1.5 m, and filter papers were purchased from VICAM, Milford,
MA USA. Acetonitrile, glacial acetic acid HPLC grade were
obtained from BDH, England. Sodium chloride, sodium bicarbonate,
sodium hydrogen phosphate, potassium dihydrogen phosphate
and potassium chloride were purchased from (BDH, Merck
chemicals). Tween -20 was obtained from Sigma (St. Louis, MO,
USA).
HPLC condition: A Symmetry C18 (5 m particle size, 150 mm x 4.6
mm i.d.) from the Waters Corporation (USA) was used along with
a mobile phase of acetonitile/water/ acetic acid (55:43:2). The
separation was performed at ambient temperature at a flow rate of
1.0 ml/min. The injection volume was 50 l for both standard
solutions and sample extracts. The fluorescence detector was
operated at an excitation wavelength of 330 nm and an emission
wavelength of 470 nm. OTA concentrations in coffee extracts were
determined from the standard curve, using peak area for
quantitation.
Statistical analysis: The results obtained in this research were
analysed statistically using the 16th version of SPSS16 program
where transaction averages were compared at the abstract level
(0.05) using the least significant difference test (LSD) designed
by Norusis 21.
Results
Inhibitory effect of -radiations on fungal toxins: Table 1 gives
the effect of -irradiation on fungal toxin production. A. melleus,
A. flavus and A. niger lost ability to produce biologically active
toxins in variable degrees after exposure to 3 kGy, although mycelia
growth was realized. Using 1 kGy reduced toxin production of A.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

melleus by 34.19%, A. flavus by 36.09%, and A.alliaceus by 3.69%.


F. solani did not lose its ability for toxin production even after
exposure to 10 kGy. However, rate of production decreased after
using 1, 3, 5, 7 and 10 kGy, reduction rates were 15.16, 87.31, 96.35
and 75.62%. Boiling of the filtrate after irradiation leads to complete
degradation of fungal toxins at 10 kGy. Aspergillus showed inability
to inhibit bacterial growth after boiling of its filtrate.
Quantitative and qualitative estimates of fungal toxins after
irradiation: Biological effect of fungal filtrate was proved by
B.subtilis growth inhibition. Effects of rays on these substances
are in Table 2. Fungal filtrate of A.niger proved to concentration
8.086 mg/l of G 1 and B1 aflatoxin. After exposure to 1 kGy, G1 toxin
disappeared, while B1 decreased to 1.279 mg/l. Exposure of
A.alliaceus to 1 kGy led to increase in toxin production, from 0.45
mg/l, in control experiment, to 0.616 mg/l. The same finding was
recorded to A. melleus, concentration of all toxins changed after
exposure from 1.255 to 1.275 mg/l. B1 toxin increased with
disappearance of G1.
No fungal filtrates showed ochratoxin production and exposure
to 10 kGy led to death of cells. Table 3 shows that F. solani
production of aflatoxin decreased after exposure to 1 kGy from
1.635 to 0.189 mg/l, but by exposure to 3 kGy toxin production
increased to 9.902 mg/l. Doses at 5, 7 and 10 kGy led to decrease in
toxin production to 0.868, 0.607 and 0.453 mg/l, respectively.
Effect of -rays on the production of biologically active
substances in fungal filtrate: Exposure to -rays affects the active
biological substances in fungal filtrate, which inhibits B.subtilis
growth. The inactivation was directly proportional with dose
increase, it also valued according to type of fungus. The rate of
inactivation reached 35.77% for A. niger, in comparison to control
experiment after exposure to 1 kGy. Inactivation reached 39.41,
45.66, 47.05 and 55.99% after exposure to 3, 5, 7, 10 kGy,
respectively. Exposure of A.alliaceus to 1, 3, 5, 7 and 10 kGy led to
inactivation percentages as 24.995, 34.69, 43.89, 49.75 and 52.70%,
respectively. This fungus was less affected than A.melleus in

Table 3. Effect of gamma rays on the production of aflatoxins


and ochratoxin A by Fusarium solani.
Dose level
(kGy)
Control
1
3
5
7
10
Average
L.S.D

B1
0.972
0.189
3.791
0.734
0.545
0.450
1.114
2.039

Aflatoxin conc. (g/ litre)


B2
G1
G2
0.203 0.460
ND
ND
ND
ND
ND
3.347 1.664
ND
0.084 0.050
ND
0.042 0.020
ND
ND
0.020
0.34 0.665 0.292
1
1.207 1.065

Total
1.635
0.189
8.802
0.868
0.607
0.453
2.092
1.542

Ochratoxin
ND
ND
ND
ND
ND
ND
-

decreasing inhibitory activity of its filtrate until exposure to 10


kGy, while the biological inhibitory substance in A.melleus filtrate
lost its biological inhibitory effect after exposure to 10 kGy followed
by A. alliaceus (1) which showed complete inhibition at 7 kGy,
while A. flavus (2) lost its inhibitory action after exposure to 10
kGy.
Discussion
Results proved the variable decrease in inhibitory action of fungi
after exposure to different doses of -rays. For A.melleus, A.flavus
and A.niger after exposure to 3 kGy stopped the inhibitory activity
and toxin production of these fungi although they can give
mycelia. Exposure to 1 kGy decreased the ability for inhibition of
bacterial growth. Similar finding was recorded by Afifi et al. 22 on
A. flavus, A. fumigatus, A. niger and P. expansum exposed to 0.5,
1, 1.5 and 2 kGy. The dose of 0.5 kGy stimulated toxin production
but 1 and 2 kGy decreased the aflatoxin formation. Although the
doses affected the fungi, no damage was recorded on apple fruits.
The ionizing effect of -rays, leaving free redicals with the
molecules, was explained by Schmidt-Heydt et al. 23 and Boonchoo
et al. 24. These radicals react and do changes in plant morphology,
anatomy and biochemical properties depending on the irradiation
dose. Changes may include in photosynthesis balance,
antioxidant system and accumulation of phenolic compounds.
For F. solani exposure to rays decreased its ability for producing

Table 1. Effect of gamma rays on the production of aflatoxins and ochratoxin A by tested fungi.
Tested
fungi
A. niger
A. alliaceus
A. flavus
A. melleus
F. solani
Average
L.S.D #

Control
41.86

40.37

38.67

33.88

29.61

36.82
16.45

kGy1
33.33
-20.03
41.86
+3.69
25.45
-34.19
21.66
36.09
25.12
-15.16
29.48
8.11

Before boiling
kGy3 kGy5
0
0
-100
-100
0
0
-100
-100
0
0
-100
-100
0
0
-100
-100
20.43 18.99
-31
-35.87
4.09
3.80
1
1

kGy7
0
-100
0
-100
0
-100
0
-100
10.08
-65.96
2.02
1

kGy10
0
-100
0
-100
0
-100
0
-100
7.22
-75.62
1.44
1

Control
29.39
-%
35.78
-%
5.995
-%
3.52
-%
7.92
-%
16.52
2.48

kGy1
22.63
-23
32.5
-9.17
4.33
-27.78
0
-100
7.91
-0.13
13.47
2.21

After boiling
kGy3
kGy5
0
0
-100
-100
0
0
-100
-100
0
0
-100
-100
0
0
-100
-100
5.37
4.52
-32.20 -42.93
1.07
0.90
1
1

kGy7
0
-100
0
-100
0
-100
0
-100
0
-100
-

kGy10
0
-100
0
-100
0
-100
0
-100
0
-100
-

Table 2. Quantitative and qualitative of aflatoxins and ochratoxin A in fungal filtrates before and after irradiation.
Tested
fungi
A. niger
A. alliaceus
A. flavus
A. melleus
Average
L.S.D #

B1
4.739
0.450
0.758
0.806
1.688
1.66

B2
ND
ND
0.094
0.140
0.059
1.67

Control
G1
G2
3.347
ND
ND
ND
0.251 0.126
0.209 0.100
0.952 0.057
1.19
1.71

Total
8.086
0.450
1.229
1.255
2.757
1.54

OTA
ND
ND
ND
ND
-

B1
1.279
0.616
0.569
1.161
0.906
4.95

B2
ND
ND
ND
0.109
0.027
1

1 kGy
G1
G2
ND
ND
ND
ND
ND 0.050
ND 0.005
0.014
1.13

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Total
1.279
0.616
0.619
1.275
0.947
4.98

OTA
ND
ND
ND
ND
-

B1
ND
ND
ND
ND
-

B2
ND
ND
ND
ND
-

10 kGy
G1 G2
ND ND
ND ND
ND ND
ND ND
-

Total
ND
ND
ND
ND
-

OTA
ND
ND
ND
ND
-

79

Table 4. Effect of gamma rays on the active biological substance


in the filtrates of fungi.
Tested fungi
A. niger
A. alliaceus (1)
A. flavus (1)
A. alliaceus (2)
A. flavus (2)
A. melleus (1)
A. melleus (2)
Average
L.S.D #

Control
14.13
-%
49.01
-%
49.01
-%
19.29
-%
29.54
-%
34.90
-%
20.96
-%
34.83
7.51

Inhibition zone (cm2)


kGy1
kGy3
kGy5
26.42
24.92
22.35
-35.77 -39.41 -45.66
36.76
32.01
27.5
-24.995 -34.69 -43.89
27.99
23.04
19.78
-42.89 -52.99 -59.64
10.28
8.38
8.04
-46.71 -56.56 -58.32
11.67
3.98
3.69
-60.49 -86.53 -87.51
0
0
0
-100
-100
-100
12.36
4.65
3.98
-41.03 -77.82 -81.01
17.93
13.85
12.19
3.72
2.93
2.99

kGy7
21.78
-47.05
24.63
-49.75
16.91
-65.50
0
-100
0.695
-97.65
0
-100
3.41
-83.73
9.63
2.31

kGy10
18.10
-55.99
23.18
-52.70
8.81
-82.02
0
-100
0
-100
0
-100
3.35
-84.02
7.63
2.12

biologically active materials, but did not stop it completely even


after exposure to 10 kGy. After exposed the culture filtrate to the
-radiation at 7 and10 kGy. and then boiled it, the mycotoxins
were degradated. The same finding was recorded for filtrate of
Aspergillus.
Control filtrates, without exposure, showed high concentrations
of aflatoxins. These toxins degraded in various degrees after
exposure to 1 kGy. G 1 aflatoxin was highly affected by radiation,
but in case of A.alliaceus, the dosage of 1 kGy stimulated toxin
formation.
In all tested fungi, ochratoxin was not produced even in the
control experiment.
Exposure to 10 kGy led to death of fungal cells. Toxin production
of F.solani increased after exposure to irradiation although this
strain is known not to produce aflatoxin as mentioned by Mokobia
and Anomohanran 25, who could isolate F. kyushuence strain
capable to produce aflatoxin G1 and B1. They used
chromatographic and gene sequence techniques to prove the
sequence of aflatoxin encoded genes which appeared similar to
that of A. flavus. Comparing results of specific microarray covering
oligonucleotide sequencing, they realized similarity in sequence
of toxin nucleotides of A. flavus and F. kynshuense. They also
mentioned that Fusarium fungus had low expression of the toxin
trichothecene in the liquid media. High expression appeared in
solid media. It seems that, the media used is an important factor in
secondary metabolism.
This study proved that pasteurization of coffee seeds using
doses of -rays can sterilize these seeds from any fungal infection.
The recommended doses are 3, 5, 7 and 10 kGy. We could not
isolate any fungal growth even after one month storage period.
Results also showed that at 1 kGy, infection decreased between
Herrary, Habashy, Lokmaty and wild tested types. These findings
are in agreement with Nemtanu et al. 13, who could stop
contamination of green coffee seeds after exposure to 40 kGy
without any damage of seed constituents or its antioxidants. This
technology was applied on malt plant 14, 24, and on rice, food and
drugs 25.
Sterilization using -rays with doses 1-10 kGy is recommended
by FAO, IAEA and WHO 26, 27. Gunckel and Sparrow 28 cleared that
exposure to -rays may affect plant physiology, biochemistry and
morphology. Several reseachers 29-31 mentioned that exposure to
80

low doses of -rays might stimulate growth.


Conclusions
The fungi differed in their response to different doses of gamma
radiation, and a decline in the ability of fungi on the production of
mycotoxins. In all the fungi exposed to a dose of 10 kGy. the
fungal cells died and were not able to grow and produce
mycotoxins, with the exception of Fusarium solani with increased
toxin production. The increase of inhibition is affected positively
with the dose that was used, and in addition it is affected differently
with the different fungal strains used.
Recommendations
This study proved in habitation of fungal toxins after exposure to
-rays. We recommend using this type of irradiation to sterilize
our food, but after more detailed studies about the fate of
degraded.
Acknowledgements
The author deeply thanks for the expert technical assistance of
Atomic Energy Research Institute of King Abdulaziz City for
Science and Technology (KACST).
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The in vitro antibiofilm activity of Rosmarinus officinalis L. essential oil against


multiple antibiotic resistant Pseudomonas sp. and Staphylococcus sp.
Ozgur Ceylan 1*, Aysel Uur 2, Nurdan Sara 3, Filiz Ozcan 3 and Tuba Baygar 3
Apiculture Program, Ula Ali Kocman Vocational School, Mugla Stk Koman University, Ula, Mugla, Turkey.
Department of Basic Sciences, Section of Medical Microbiology, Faculty of Dentistry, Gazi University, Emek, Ankara, Turkey.
3
Department of Biology, Faculty of Arts and Sciences, Mugla Stk Koman University, Kotekli, Mugla, Turkey.
*e-mail: ozgceylan@hotmail.com
1

Received 12 July 2014, accepted 3 September 2014.

Abstract
Rosmarinus officinalis (rosemary) is widely used as a flavouring agent for food and well known medicinally for its chemical composition. The aim of
this study was to investigate the antibiofilm activity of the essential oil from Rosmarinus officinalis against biofilm formation of Staphylococcus
aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Bacillus cereus and Bacillus subtilis. Essential oil was
obtained from the aerial parts of the plant by using a Clevenger apparatus for 4 h. The antibacterial activity of the obtained essential oil from
Rosmarinus officinalis was determined using disc diffusion technique, minimum inhibitory concentrations (MICs) and minimum bacterial concentration
(MBC). Staphylococcus sp. and Pseudomonas sp. were evaluated as the resistant microorganisms in the antibacterial assays. The antibiofilm effect
of MBC, MIC, MIC/2, MIC/4, MIC/8 and MIC/16 concentrations of Rosmarinus officinalis essential oil was assessed by the microplate biofilm
assay. The essential oil exhibited significant antibacterial activity against all tested bacteria. In contrast to the antibacterial activity, MBC and
subinhibitory concentrations of essential oil showed limited antibiofilm activity. MBC concentrations of essential oil attenuated the biofilm
formation at 60.76% and 74.7% for Staphylococcus aureus MU 47 and Pseudomonas aeruginosa MU 187, respectively. Direct observation by
scanning electron microscope (SEM) analysis further revealed an exact reduction for the bacterial biofilm formation in response to the effective
concentration. This study has demonstrated that the Rosmarinus officinalis essential oil may be considered as a potent agent for the prevention of
biofilm-related applications that are increasingly problematic in the food processing environments and medical industries.
Key words: Rosmarinus officinalis, antimicrobial, antibiofilm, Staphylococcus, Pseudomonas.

Introduction
Rosmarinus officinalis L. (rosemary) is a spice and medicinal herb
widely used around the world 1. The fresh and dried leaves are
frequently used in traditional Mediterranean cuisine as an additive.
They have a bitter, astringent taste, which complements a wide
variety of foods. A tisane can also be made from them. They are
extensively used in cooking, and a distinct mustard smell gives
off while they are burned, therefore, they often are used to flavor
foods while barbecuing 2. As medicinal plant rosemary belongs to
the pool of herbs, which probably more than others, lies at the
boundary between myth, superstition and traditional popular
usages, but at the same time, its efficacy is largely acknowledged,
being, in fact, listed in the official pharmacopoeia of several
countries 3. Historically, rosemary has been used as a medicinal
agent to treat renal colic and dysmenorrhea. It has also been used
to relieve symptoms caused by respiratory disorders and to
stimulate the growth of hair. Extracts of rosemary are used in
aromatherapy to treat anxiety-related conditions and to increase
alertness 4.
The latest research related with rosemary essential oil has mainly
focused on its antibacterial 1, 2, 5-18, antioxidant 18-27, antifungal 28, 29,
anticancer 30, 31 and antibiofilm properties 12-14, 32.
82

In many ecosystems, bacteria are growing in surfaces as a layer


that is called biofilm 33. Bacterial biofilms are associated with a
large number of infections. Biofilms are ubiquitous, for example in
dental plaque, endocarditis, lung infections, and infections related
to the use of medical devices, such as catheters and stents. Many
persistent and chronic bacterial infections are now thought to be
linked to biofilm formation, over 60% of all bacterial infections
have been estimated to involve biofilms 34. Also, outside the
medical field, biofilms cause a host of problems such as surface
fouling and blocking of equipment 35. Biofilm-dwelling bacteria
are particularly resistant to antibiotics, making it hard to eradicate
biofilm-associated infections. Earlier investigations on plants and
their active constituents have almost exclusively focused on their
effects on planktonic bacteria with little emphasis on the more
antimicrobial resistant pathogens and difficult to control biofilm
forms 32.
There are a number of studies focusing on the biological
activities of R. officinalis essential oil in recent years, but to our
knowledge, fewer comparative studies on antibiofilm activity
including against human clinical isolates have been reported. In
these studies, there are no study that reveals the antibiofilm activity

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

of test bacteria used in our study. The present study reports the
antimicrobial and antibiofilm activity of R. officinalis essential oil
against multi-antibiotic resistant Staphylococcus spp. and
Pseudomonas spp. that cause clinical problems with high biofilm
formation.
Materials and Methods
Plant material: Leaves and flowers of R. officinalis were collected
from Mugla, Turkey, in May-July 2012 and a voucher specimen
has been deposited in the Herbarium of Mugla Sitki Kocman
Univesity. Samples were air-dried at room temperature for 2-4 days.
Extraction of essential oil: One hundred gram of dried plant was
submitted to hydro-distillation for 4 h using a Clevenger apparatus.
Oil was recovered directly, using a micro-pipette from above the
distillate without adding any solvent, and stored in dark vials at
4C.
Bacterial strains and culture conditions: The antimicrobial
activity of the essential oil was individually tested against a group
of bacteria including Bacillus subtilis ATCC 6633, Bacillus cereus
RSKK 863, Staphylococcus aureus ATCC 25923, Pseudomonas
aeruginosa ATCC 29212 and Pseudomonas aeruginosa ATCC
27853. Five clinically relevant microorganisms (Staphylococcus aureus
MU 38, MU 40, MU 46, MU 47 and Staphylococcus epidermidis
MU 30) and three marine microorganisms (Pseudomonas
aeruginosa MU 187, MU 189 and Pseudomonas fluorescens MU
180) provided from Mugla Sitki Kocman University Culture
Collection (MUKK) were also studied.
The above-mentioned bacteria were cultured in nutrient broth
(NB) (Difco, USA) at 370.1C for 24-48 h except from P. aeruginosa
and P. fluorescens strains which were incubated at 300.1C for
18-24 h. Inocula were prepared by adjusting the turbidity of the
medium to match the 0.5 McFarland Standard Dilutions. The strains
were maintained in their appropriate agar slants at 4C throughout
the study and used as stock cultures.
Disc diffusion assay: The antibacterial activity was based on the
disc diffusion method 36 using a bacterial cell suspension whose
concentration was equilibrated to the 0.5 McFarland standard
dilutions. Of each bacterial suspension 0.1 ml was spread on a
Mueller-Hinton agar plate. Sterile 6 mm paper discs (Schleicher
and Schuell) were impregnated with 10 l of essential oil. The
discs were allowed to dry and were then placed on the inoculated
agar. The plates were incubated at appropriate temperature and
time for the microorganisms, as mentioned above. At the end of
the incubation periods, diameters of no-growth zones around the
disks were measured to the nearest 0.1 mm using vernier calipers.
The experiments were performed in triplicate.
Determination of minimum inhibitory concentration (MIC) and
minimum bactericide concentration (MBC): The inhibitory and
bactericidal activities of the rosemary essential oils were
determined by the tube dilution method 37. The MIC was defined
as the lowest antibiotic concentration that yielded no visible
growth. Mueller-Hinton Broth was used as the test medium and
the density of bacteria was 5105 colony-forming units (CFU)/ml.
Cell suspensions (100 l) were inoculated into the wells of 96-well
microtitre plates (Nunc F96 MKroWell plates; NunclonTM ,

Denmark) in the presence of essential oil with different final


concentrations (0.312-80 l/ml). The essential oil was dissolved in
DMSO (Sigma, USA) and serially diluted 2-fold in MHB to give
final concentrations. Negative controls (bacteria+MHB), positive
controls (bacteria+MHB+essential oil), vehicle controls
(bacteria+MHB+DMSO) and media controls (MHB) were
included. The inoculated microplates were incubated at 37C for
Staphylococcus spp., Bacillus spp. and at 30C for Pseudomonas
spp., for 24 h. All experiments were performed in triplicate.
The MBC was obtained by subculturing 100 l of the culture
from each tube, in which the MIC assay showing no apparent
growth, onto substance-free Mueller-Hinton agar plates. The
plates were incubated at 37C or 30C for 24 h and the MBC was
defined as the least concentration that produced subcultures
growing maximum five colonies on each plate.
Effect of essential oil on bacterial biofilm formation: The effect
of subinhibitory concentration of R. officinalis essential oil on
biofilm-forming ability of bacteria was tested with a microplate
biofilm assay 38. Bacterial strains were inoculated in 2-5 ml of
trypticase soy broth (TSB) and growed up to stationary phase.
Cultures diluted to 1:100 in TSB, and 100 l of each dilution was
pipetted to four wells in a sterile flat bottom micotiterplate. After
incubation at 37C for 48 h, the wells were washed with distilled
water twice to remove the planktonic bacteria. The remaining
bacteria were subsequently stained with 125 l of 0.1% crystal
violet solution (Sigma Chemical Co.) at room temperature. Wells
were washed once again to remove the crystal violet solution that
is not specifically staining the adherent bacteria. The plates were
air-dried and 200 l of 95% ethanol and 33% glacial acetic acid
(Sigma Chemical Co.) were added to each Gram-negative and Grampositive bacteria wells, respectively. Biofilm stains solubilized at
room temperature. After shaking and pipetting of wells, 125 l of
the solution from each well was transferred to a sterile tube and
the volume was made up to 1 ml with distilled water. Finally, optical
density of each well was measured at 550 nm wavelength (Thermo
Scientific Multiskan FC, Vantaa, Finland). Negative controls
(cells+TSB), positive control (cells+TSB+essential oil), vehicle
control (cells+TSB+DMSO), and media controls (TSB) were
included. Positive controls for essential oil of 0.312-80 l/ml were
prepared via serial dilution techniques.
Each strain was tested for biofilm formation in duplicate and the
assay was repeated three times. Replicate absorbance readings
for each concentration were averaged and the average of the media
control was subtracted. This value was divided by the mean
absorbance of the (cell+TSB) and multiplied by 100.

Scanning electron microscopy (SEM): To observe the biofilm


formation, glass coverslips were prepared for SEM observation.
Coverslips at size of 3 mm x 3 mm were placed in Eppendorf tubes
containing 1.5 ml TSB supplemented with 1% glucose and sterilized.
Then 300 l bacterial suspension and 200 l essential oil were
added to reach the final concentration of 0.078-80 l/ml. Eppendorf
tubes were incubated at 37C for Bacillus and Staphylococcus
and at 30C for Pseudomonas, for 48 h. Prior to imaging, the
bacteria were fixed and dehydrated. Briefly, the coverslips were

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

83

gently rinsed twice with 0.01 M PBS and then initially fixed by
2.5% glutaraldehyde at 4C for 2 h. The surfaces were washed
twice with 0.01 M PBS for 1 h. The coverslips were post-fixed with
0.1% osmium tetroxide for 1 h. The bacteria were dehydrated by
replacing the buffer with increasing concentrations of ethanol
(30%, 50%, 70%, 80%, 90%, 95% and 100%) for 10 min for each.
After critical point drying and coating by gold sputter, samples
were examined with a scanning electron microscope (JEOL JSM7600F; JEOL Ltd., Tokyo, Japan).
Statistical analysis: Differences between groups were statistically
analyzed using analysis of variance (ANOVA). All experiments
were performed in triplicate.
Results and Discussion
The antimicrobial activity of R. officinalis essential oil was
evaluated in vitro against 13 microorganisms which are known to
cause human diseases. The measured inhibition zones and MIC/
MBC results of the rosemary essential oil against the test bacteria
are given in Table 1. Based on their MIC and MBC values obtained
from antimicrobial tests and the percentage inhibition of biofilm
formation against the test bacteria are given in Table 2. The
antibiofilm activity of different concentrations of R. officinalis
essential oil for Gram-positive and Gram-negative test bacteria is
shown in Figs. 1 and 2, respectively.

The results indicated that the R. officinalis essential oil showed


anti-bacterial activity mainly against the Gram-positive bacteria
(S. aureus and S. epidermidis), similar to Jiang et al. 2, Jordan et al. 10,
Okoh et al. 11, Zaouali et al. 18. The highest antibiofilm activity for
the Gram-positive test bacteria was determined against S. aureus
MU 47 with 1.25 l/ml essential oil concentration (MBC) with the
inhibition rate by 60.76%. S. aureus MU 47 biofilm formation was
reduced to 48.14% by MIC as 0.625 l/ml. Due to the decrease in
the concentration of essential oil, the biofilm formation of S. aureus
MU 47 was inhibited by 33.45%, 14.11% and 8.07% in MIC/2, MIC/
4 and MIC/8, respectively. The MBC and MIC of S. aureus ATCC
25923 were 1.25 and 0.312 l/ml, respectively. R. officinalis essential
oil in MBC and MIC concentrations reduced the S. aureus ATCC
25923 biofilm formation to 58.02% and 27.6%. For S. aureus MU
38, MBC and MIC were 10 and 5 l/ml and biofilm formation was
reduced to 53.83% and 26.43% in these concentrations. Quave et
al. 12 reported the effect of R. officinalis extract on planktonic
growth, biofilm formation and adherence of methicillin-resistant S.
aureus. In this study, MIC50 value of 512 g/ml and IC50 value of 16
g/ml were reported for R. officinalis extract. Here we provide the
first report, to our knowledge, of the demonstration of the
antibiofilm activity of R. officinalis essential oil against S. aureus
and S. epidermidis.

Microorganism
B.s. ATCC 6633
B.c. RSKK 863
S.a. ATCC 25923
S e. MU 30
S.a. MU 38
S.a. MU 40
S.a. MU 46
S.a. MU 47
P.a. ATCC 27853
P.a. ATCC 29212
P.f. MU 180
P.a. MU 187
P.a. MU 189

Inhibition zone
15
16
14
12
17
13
12
12
11
10

MIC
5
5
0.312
0.625
5
2.5
0.312
0.625
20
1.25
1.25
20
1.25

% Inhibition

Table 1. Determination of MIC, MBC (l/ml) and disc


diffusion (mm) assay of R. officinalis essential oil.
MBC
10
10
1.25
1.25
10
5
0.625
1.25
80
5
2.5
80
2.5

l//ml))
Essential oil concentration (

Figure 1. The percent inhibition of R. officinalis essential oil for biofilm


formation in Gram positive bacteria.

B.s.: B. subtilis; B.c.: B. cereus; S.a.: S. aureus; S.e.: S. epidermidis; P.a.: P.


aeruginosa; P.f.: P. fluorescens; -: No inhibition.

Table 2. The effect of R. officinalis essential oil on tested bacteria


biofilm formation expressed as percentage inhibition.
MBC

B.s. ATCC 6633


B.c. RSKK 863
S.a. ATCC 25923
S.e. MU 30
S.a. MU 38
S.a. MU 40
S.a. MU 46
S.a. MU 47
P.a. ATCC 27853
P.a. ATCC 29212
P.f. MU 180
P.a. MU 187
P.a. MU 189

56
38.77
58.02
35.1
53.88
25.89
32.33
60.76
58.3
22.88
30.69
74.7
46.51

Essential oil concentration


MIC/2 MIC/4 MIC/8
Percentage (%) inhibition
51.3
37.09
19.3
6.1
18.52
27.6
21.29
17.76
16.61
5.2
26.43
3.14
27.22
6.81
48.14 33.45
14.11
8.07
21.83
7.73
6.16
18.55
1.04
39.49 19.91
14.38
5.39
35.57
3.22
MIC

MIC/16
-

B.s.: B. subtilis; B.c.: B. cereus; S.a.: S. aureus; S.e.: S. epidermidis; P.a.: P. aeruginosa; P.f.: P. fluorescens;
-: No inhibition.

84

% Inhibition

Microorganism

Essential oil concentration (


l//ml))

Figure 2. The percent inhibition of R. officinalis essential oil for biofilm


formation in Gram negative bacteria.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Essential oil of R. officinalis showed similar antimicrobial activity


against B. cereus RSKK 863 and B. subtilis ATCC 6633, but the
antibiofilm activity of essential oil was more effective against B.
subtilis ATCC 6633. Our results were similar to those previously
reported by Celiktas et al. 8, Jiang et al. 2, Okoh et al. 11, Zaouali et
al. 18 for the antimicrobial activity of the R. officinalis essential oil
against B. subtilis. A good to moderate antimicrobial activity of R.
officinalis essential oil against B. cereus has been reported by
Genena et al. 1 and Zaouali et al. 18.
The R. officinalis essential oil has also exhibited an
antibacterial effect against the Gram-negative bacteria (P.
aeruginosa and P. fluorescens). However, this effect was less
efficient than that presented against the Gram-positive bacteria,
since a higher MIC value was obtained with the Gram-negative
bacteria. In this study, the highest MBC and MIC were 80 and 20 l/
ml against P. aeruginosa ATCC 27853 and P. aeruginosa MU 187,
respectively. P. aeruginosa MU 187 biofilm formation has been
reduced to 74.71% in MBC and 39.49% in MIC. For the same
bacteria, the biofilm formation reduced by 19.91% in MIC/2, by
14.38% in MIC/4 and by 5.39% in MIC/8. MIC/16 concentrations
of the rosemary essential oil did not reduce the bacterial biofilm
formation of the tested bacteria. Biofilm formation of P. aeruginosa
ATCC 27853 was inhibited by 58.3% in MBC, by 21.83% in MIC
and by 7.73% in MIC/2. The results of the antimicrobial activity
on Gram-negative bacteria are similar to Celiktas et al. 8 and Jiang
et al. 2. In contrast to our results, Zaouali et al. 18 reported that
the essential oil of R. officinalis has no antimicrobial activity
against P. aeruginosa. The antimicrobial activity of R. officinalis
extracts against P. aeruginosa have also been reported by Genena
et al. 1 and Sandasi et al. 14. Sandasi et al.14 showed that R.
officinalis extract inhibited 56% of P. aeruginosa biofilm
formation. Contrary to this, they also reported that R. officinalis
extract was unable to inhibit the growth and development of a
pre-formed biofilm of P. aeruginosa.
To evaluate the relevance of biofilm formation assay, scanning
electron microscopy was employed. Direct observation by
scanning electron microscopy of S. aureus MU 47 showed that,
in the absence of rosemary essential oil (Fig. 3), bacterial cells
formed evident biofilms with matrix material. In the presence of
rosemary essential oil at concentrations of 1.25 l/ml (MBC)
bacterial cells grew as looser colonies, and the amount of biofilm
was reduced to 60.76% (Fig. 4).

Figure 4. Scanning electron micrographs showing reduction in S.aureus


MU 47 biofilm with 1.25 l/ml R.officinalis essential oil.

Conclusions
R. officinalis is a spice and medicinal herb widely used around the
world 1. It is widely found in the lands of Aegean and
Mediterranean regions of Turkey 8. In this study, the antimicrobial
and antibiofilm activities of the R. officinalis essential oil was
evaluated. The results obtained are confirmed by SEM observation.
According to the results of antimicrobial activity, the R. officinalis
essential oil is more active against Gram-positive than Gramnegative bacteria, as evidenced by the lower MIC values for the
former. Data showed that R. officinalis essential oil is not only
able to kill Staphylococcus spp. and Pseudomonas spp. cells
efficiently but also inhibits biofilm formation, but the results also
indicate that good antimicrobial activity against planktonic microorganisms does not imply good antibiofilm activity. However, the
results of this study show that rosemary essential oil is capable of
affecting S. aureus biofilm formation significantly. Here it is
suggested that R. officinalis essential oil may be considered as a
potential substance in the development of novel antimicrobial
and antibiofilm agents that could play a solution oriented role in
the field of food and pharmaceutical industries.
Acknowledgements
The authors would like to thank to Prof. Dr. Nazime Mercan Dogan
and Prof. Dr. Omur Baysal for their constructive comments and
suggestions.
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antibiofilm activity of selected culinary herbs and medicinal plants
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microbial development. Annu. Rev. Microbiol. 54:49-79.
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Lewis, K. 2001. Riddle of biofilm resistance. Antimicrob. Agents
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Coetser, S. E. and Cloete, T. E. 2005. Biofouling and biocorrosion in
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Clinical and Laboratory Standards Institute 2007. Methods for
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analyzing static biofilms. Curr. Protoc. Microbiol., pp. 1-3 (Chapter
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24

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

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Journal of Food, Agriculture & Environment Vol.12 (3&4): 87-92. 2014

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Effect of storage time and temperature on the quality characteristics of chicken eggs
Yeasmin Akter 1, 2*, Azhar Kasim 1*, Hishamuddin Omar 3 and Awis Qurni Sazili

Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
Department of Animal Science and Nutrition, Hajee Mohammad Danesh Science and Technology University, Dinajpur 5200,
Bangladesh. 3 Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
*e-mail: yesakter@yahoo.com, azharkasim@putra.upm.edu.my
1

Received 10 March 2014, accepted 30 August 2014.

Abstract
The experiment was conducted to evaluate the effect of storage time and temperature on external and internal qualities of eggs and lipid peroxidation
in the yolk during refrigeration and room storage. Total of eighty ISA Brown hens were used for this experiment. The diet used for laying hen was
prepared as iso-protein and iso-calorie. Eggs were collected daily and stored in refrigerator (4C) and room temperature (28-31C) for 7, 14, 21 and 28
days. At the designated day, the eggs were processed to evaluate their internal and external qualities. Both refrigeration and room storage increased egg
weight loss, percent yolk weight, yolk pH, albumen pH and lipid peroxidation and reduced Haugh units, percent albumen weight at 28 days of
storage. Yolk colour was unchanged during the whole storage period. However, eggs stored in refrigerator showed better quality up to 28 days and in
room temperature up to 14 days.
Key words: Chicken eggs, storage time, egg quality, lipid oxidation.

Introduction
Eggs are highly versatile and easily available foods for all
categories of people 1. Recently, people are very concerned about
the quality of eggs. So the scientists are giving more emphasis
to maintain the quality of eggs. The quality of eggs would be
deteriorated when the eggs are stored for long time that may be
unsuitable for human consumption. As a result, appropriate
technology for storage of eggs is essential to retain quality. The
main degradation factors for eggs are storage time 2, temperature,
humidity, air movement, and handling 3, 4. Interior quality
deterioration of eggs can be retarded significantly by maintaining
storage temperature, because quality deterioration occurs faster
at high temperatures than at refrigerated temperatures during
storage 5, 6.
The major difference between fresh and stored eggs is in
albumen pH and quality 7. During storage, the pH of the egg
albumen increases, which is related to the deterioration of albumen
quality or Haugh unit 3, 8, 9. The albumen of an AA egg is firm and
thick, the air cell is very small, and the albumen and yolk contain
no blood or meat spots. The increase of albumen pH mainly
depends on the buffering capacity of the albumen, not only this,
it also depends on the temperature, storage duration, gaseous
environment in the storage room and conductance of the
eggshell 7, 10-12. In fresh albumen, the buffering capacity is weakest
in pH 7.0 to 9.0 10. It is impossible to maintain albumen pH within
the range of 8.3 to 8.5 during prolonged storage of eggs without
modification of storage conditions. Another important change
that can be observed during storage is the flattening of the yolk
caused by the weakening of the vitelline membrane 13. After eggs
are laid, water moves from the albumen to the yolk due to
differences in osmotic pressure, and this may change the yolk
index and may cause the weakening of the vitelline membrane.

Fromm 14 reported that yolk with high water content showed high
yolk index, when the albumen pH was maintained at or below pH
8.0. So, the albumen pH is the most important factor that affects
the strength of the vitelline membrane and yolk index. Walsh et al. 7
concluded that one requirement for successful long-term storage
was the prevention of water loss from the egg. During storage,
water from egg is lost through evaporation, and the rate of
evaporation is influenced by the length of storage, temperature,
humidity and the surface and porosity of the shell 15. Lipid oxidation
is also an important deterioration that occurs during storage, and
it may produce toxic compounds. Oxidative products in eggs can
reduce its nutritive value 16. So, it is essential to prevent and
minimize the lipid oxidation in order to maintain egg quality and
fatty acid stability during storage,
Therefore, the present study has been undertaken to determine
the effects of storage time and temperature on some external
and internal characteristics for reducing the deterioration of egg
quality during refrigerated and room storage.
Materials and Methods
Experimental birds and diet: The experiment was conducted
at the poultry unit, Department of Animal Science, University
Putra Malaysia. A total of 80 ISA Brown layers, 44 weeks of age
were used for this study to evaluate egg quality characteristics.
The layer diets were isonitrogenous and isocaloric and formulated
according to NRC 17 recommendations for dietary need of laying
hens for various nutrients. Experimental diets were prepared weekly
to avoid oxidation.
Sampling and storing of eggs: Immediately after collection,
eggs were labeled according to date of production and hen

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

87

Evaluation of egg quality: Characteristics were evaluated in


individual eggs for internal and external quality traits. The external
characteristics of eggs were egg weight (g), egg length and width
(cm), specific gravity, shell weight (g) and shell thickness (mm),
whereas internal quality parameters include albumen weight (g),
albumen height (mm), yolk width (cm), yolk weight (g), and
Haugh unit. Other parameters measured included egg shape index,
albumen pH and yolk pH. Egg weight was measured by weighing
egg individually using sensitive balance. Egg length and width
(cm) and shell thickness were measured by using Digital Vernier
Calipers. Egg shape index was obtained as a ratio of the egg width
to the length. Egg shell thickness was determined by the mean
of three measurements taken from three different sides of the
shell. Specific gravity was determined through saline solutions
method of the same temperature, but with different densities.
The analysed egg is passing, one time each, from a pot to another,
being well drained from the previous solution; the pot in which
the egg rise to the surface shows the specific gravity, by its
solutions; density. Albumen height, yolk colour and Haugh unit
were measured automatically by egg multitester (Orka Food
Technology). The yolk was separated from the albumen and
weighed. Egg yolk width was measured by compass. Egg shells
from individual eggs were cleaned, dried at room temperature and
weight recorded 18. Shell percentage was calculated by dividing
shell weight with egg weight and multiplying by 100 19. Albumen
weight was calculated from the difference between egg weight
and weight of the yolk and shell. The pH of the albumen and yolk
were measured immediately using a pH meter.
Water loss measurements during storage: Eggs were weighed
at each sampling time. The same eggs were weighed over the 4
week study. Egg weight loss was calculated as follows:

thiobarbituric acid, and was further incubated at 100C for 30 min


to develop pink colour. Following incubation, the mixture was
cooled under tap water and centrifuged at 3000 g for 10 min.
Absorbance of supernatant was measured at 532 nm using a
spectrophotometer. The concentration of MDA in analysed
samples (mg/kg yolk) was calculated from a standard curve of 1, 1,
3,3 tetra-ethoxypropane. All measurements were conducted in
triplicate.
Statistical analysis: Analyses were conducted using the SAS
software program 21. Significant differences among the means
of the treatment groups were determined by Duncans multiplerange test. Variability in the data was expressed as standard error
(S.E.) and a probability level of p<0.05 was considered as
statistically significant.
Results and Discussion
Effect of storage time and temperature on external qualities
of chicken eggs: The effect of storage time and temperature on
egg weight loss is shown in Fig. 1. The percent egg weight loss
values showed significant (p<0.05) variations during storage.
Total egg weight loss increased when storage duration increased,
and the highest weight losses were recorded at 28 days of storage
in both temperatures. Weight loss occurs due to loss of solvents
from the egg content through the shell by evaporation. These
results are supported by Altan et al. 22, Fasenko et al. 23, Tilki and
Inal 24, Hassan et al. 25, Reijrink et al. 26, Gonzalez-Redondo 27
and Alsobayel and Albadry 28, who reported that with increase
the length of storage, egg weight losses increase.

Egg weight loss (%)

number and weighed by using sensitive balance. Fresh eggs were


analysed within 2 h of being laid. To study the effect of storage on
egg quality parameters, eggs were stored at 4C and room
temperature (28-31C) for 7, 14, 21 and 28 days. The stored
eggs were identified and analysed at each corresponding storage
time and temperature.

5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0

4C
28-31C

14

21

28

Storage time (days)

Total egg weight loss (g) = Initial weight (g) - final weight (g)

Weight loss percentage was calculated in relation to day 0 egg


weight (g):
Weight loss (%) =

Weight loss (g)


Initial weight
(g)

100

Thiobarbituric acid (TBA) determination: The TBA values were


determined for the malonaldehyde (MDA) formed in fresh eggs
and those that were stored at 4C and room temperature for
different time periods (7, 14, 21 and 28 days of storage). This
MDA value was measured according to the TBA method described
by Botsoglou et al. 20 with some modifications. Yolk samples were
homogenized by Polytron homogenizer in the presence of 8 ml 5%
aqueous trichloroacetic acid (TCA), and 5 ml of 0.8% butyrate
hydroxytoluene in hexane was immediately added and the mixture
centrifuged. The top layer was discarded and a 2.5 ml aliquot from
the bottom layer was mixed with 1.5 ml of 0.8% aqueous 288

Figure 1. Effect of storage time and


temperatures on egg weight loss (%).

Similarly egg weight loss depends on the temperatures. As a


result, eggs stored at 4C showed significantly (p<0.05) lower
weight loss than room temperature. This may be due to the less
loss of solvents (water and other gaseous products) from egg
contents than those in room temperature. These results are in
agreement with Shanawa 29, Samli et al. 4 and Hasan and Okur 30
who noticed a decrease in egg weight within 10 days of storage at
29C.
The effect of storage time and temperature on shape index of
egg is shown in Table 1. Shape index did not affect significantly
(P>0.05) by storage time and temperature. These results are also
supported by Woodard 31, Song et al. 32 and Tilki and Saatci 33,
who observed no effect of storage time and temperature on shape
index of eggs.
Similarly, no significant difference was found in the shell
thickness during storage of eggs at two different temperatures

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Table 1. Effect of storage time and temperature on shape index, shell thickness and shell
weight of chicken eggs.

Shell thickness (mm)


Shell weight (%)

0
76.590.53
76.510.33
0.340.005
0.350.002
11.470.43
11.470.43

Storage time (days)


7
14
21
76.590.53 76.340.34 75.950.76
76.480.31 76.240.34 75.880.95
0.340.004 0.340.004 0.340.003
0.340.005 0.340.003 0.340.003
11.450.35 11.440.27 11.440.23
11.440.35 11.430.35 11.420.35

Specific gravity

(Table 1). These findings on shell thickness are also in agreement


with Dudusola 34 and alayan et al. 35, who did not find any
effect of storage time and temperature on shell thickness in
partridges and Japanese quail eggs.
Shell weight as percentage was not affected significantly
(P>0.05) by storage time and temperature (Table 1). These findings
are in line with Silversides and Scott 36, Tilki and Inal 24, Akyurek
and Okur 37 and alayan et al. 35, who reported no effect of
storage time on egg shell weight. In contrast, Samli et al. 4 noticed
significant (p<0.05) change in shell weight during storage at
different time and temperature.
On the other hand, specific gravity of eggs declined (p<0.05)
gradually with the advancing storage time as a result maximum
(p<0.05) decreases at 28 days of storage in both temperatures, but
the specific gravity of eggs kept at room temperature declined
more rapidly (p<0.05) than that of refrigerated eggs (Fig. 2) . This
might be due to the size of the air cell because with increase of
storage time and temperature the size of the air cell increased.
These results are in agreement with Akyurek and Okur 37, who
observed the size of the air cell exceeded 5 mm in 7 days at all
storage temperatures.
1.09
1.085
1.08
1.075
1.07
1.065
1.06
1.055
1.05
1.045
1.04

4C
28-31C

14

21

28

Storage time (days)

Figure 2. Effect of storage time and temperature


on specific gravity of chicken eggs.

Effect of storage time and temperature on internal qualities of


chicken eggs: The results of Haugh unit (HU) of eggs are
presented in Fig. 3. HU of fresh eggs declined significantly (P<0.05)
with the increasing storage time. Haugh unit reduction was
happened due to the decrease in thick albumen height, because
during storage, the ovomucin-lysozyme complex breaks down,
which helps to increase the pH of eggs. The results of this study
were supported by Morais et al. 38 who observed the reduction in
the Haugh unit of eggs at 21 days of storage. Similarly storage
temperatures can affect the HU of eggs. High storage
temperatures promote the breakdown of ovomucin-lysozyme
complex. As a result, HU of eggs stored at room temperature was
reduced significantly (P<0.05) compared to refrigeration. The results
are in agreement with Campos and Baio 39, Sauveur 40 and Samli

28
75.930.43
75.710.45
0.340.003
0.340.002
11.430.27
11.410.27

90

4C

80
Haugh unit (HU)

Shape index (%)

Temperature
(C)
4
28-31
4
28-31
4
28-31

28-31C

70
60
50
40
30
20
10
0

7
14
21
Storage time (days)

28

Figure 3. Effect of storage time and temperature


on Haugh unit (HU) of chicken eggs.

et al. 4, that storage time and temperature adversely affected HU


of eggs. Consequently, other researchers like Tona et al. 41 and
Akyurek and Okur 37 reported that the rate of water loss from egg
is influenced by the rate of evaporation from egg content.
Figure 4 also indicates that storage of eggs at different time
intervals showed a significantly (P<0.05) higher pH value than
fresh eggs, which may occur due to evaporation and exchange of
carbon dioxide from eggs; the equilibrium of the carbonatebicarbonate buffer system is thought to be shifted towards
production of CO2. Maximum increase occurred during the first 7
days of storage followed by progressively slower rate of increase
throughout the rest of the storage period. These findings are
supported by Scott and Silversides 18, Samli et al. 4 and Akyurel
and Okur 37. Along with storage time, the pH of egg albumen can
also be influenced by temperature. As a result the albumen pH
was significantly (P<0.05) higher at room temperature than in
refrigeration during the whole storage period, which may be due
to higher amount of evaporation from eggs. Increase in albumen
pH with storage was also reported by Moula et al. 42 and Silversides
and Budgell 43. In contrast, Walsh et al. 7 revealed that neither
temperature nor storage time influenced the pH of egg albumen.

Albumen pH

Parameter

9.4
9.2
9
8.8
8.6
8.4
8.2
8
7.8
7.6
7.4

4C
28-31C

14

21

28

Storage time (days)

Figure 4. Effect of storage time and


temperature on albumen pH of chicken eggs.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

89

Albumen weight (%)

64
4C

62

28-31C

60
58
56
54
52

14

21

28

Storage time (days)

Figure 5. Effect of storage time and temperature


on albumen weight (%) of chicken eggs.

The effect of storage time and temperature on the pH of egg


yolk is shown in Fig. 6. The results revealed that the pH of egg
yolk increased significantly (p<0.05) at 28 days of storage, the pH
of the yolk was significantly higher (p<0.05) in room temperatures
than in refrigeration. This may occur due to the loss of carbon
dioxide from egg by diffusion, which helps to increase the yolk
pH. The present results are in agreement with Samli et al. 4 and
Akyurel and Okur 37, that increases in yolk pH were significantly
affected by storage time. This increase in pH value was lower
than the value of Samli et al. 4 who found that yolk pH differed
from 5.75 to 6.08 during 10 d of storage at 29C. This may be due
to lower diffusion rate of carbon dioxide from the egg.
6.5

Yolk pH

Yolk weight (%)

28
27
26
4C
28-31C

25
24
23

14

21

28

Storage time (days)

Figure 7. Effect of storage time and temperature


on yolk weight (%) of chicken eggs.

and Barbosa et al. 47. On the other hand, when the storage
temperature was higher, the rate of increase in yolk weight was
significantly (p<0.05) higher than in refrigeration (4C). These
results are supported by Davis and Stephenson 48, Morais et al.38
and Leandro et al. 49, who reported that the most important factors
that affect egg quality during storage are temperature and relative
humidity.
From Fig. 8, it is noticed that the duration and temperatures of
storage significantly (P<0.05) increased the value of yolk width.
The increase in yolk width observed in this study could be due to
decrease of the strength of vitelline membrane. When eggs are
stored under room temperature for long periods, the strength of
vitelline membrane breaks and makes the yolk to spread into the
albumin. These results are in agreement with Kirunda and McKee 50,
who reported that vitelline membrane strength (VMS) decreases
during storage and makes the yolk more susceptible to breaking,
as a result, water slowly enters into the yolk from the albumen, so
this creates a mottled appearance in yolk, and the yolk becomes
flattened. The yolk width values were higher in room temperature,
because at a very high temperature, the amount of water migration
from the albumen to the yolk is high, which helped to increase
yolk width 51.
40.5
40
39.5
39
38.5
38

4C

37.5
37

28-31C

14

21

28

Storage time (days)

6.3

Figure 8. Effect of storage time and


temperature on yolk width of chicken eggs.

6.2
4C

6.1

28-31C

6
0

14

21

28

Storage time (days)

Figure 6. Effect of storage time and


temperature on yolk pH of chicken eggs.

Yolk weight and yolk weight as % of egg weight (Fig. 7) showed


significant (p<0.05) changes during storage, and increased linearly
with storage time, which might be due to the diffusion of water
from the albumen to the yolk. These results concur with Haugh 46
90

30
29

36.5
36

6.4

5.9

31

Yolk width (mm)

Effect of storage time and temperature on albumen weight (%)


of eggs is presented in Fig. 5. The albumin weight was significantly
(p<0.05) affected by storage time. As a result highest albumen
weight losses were recorded for 28 days of storage in both storage
temperatures. This loss of albumen weight happened due to loss
of solvents from albumen, which may decrease the weight of the
albumen in egg by increasing the weight of yolk. These results
are inconsistent with Siyar et al. 44 and Tabidi 45, who reported that
the loss in albumen weight is attributed to loss of humidity from
inside the egg due to evaporation. Consequently, albumen weight
loss was significantly (p<0.05) higher at room temperature than in
refrigeration at 28 days of storage. This may be due to the higher
amount of water loss from the albumen to the yolk. Similar results
were demonstrated by Tona et al. 41 and Akyurek and Okur 37,
that water loss from eggs may be influenced by storage time,
temperature, relative humidity and porosity of the shell.

In this study, the pigmentation (Fig. 9) was observed for both


temperatures at different storage periods, but no significant
(P>0.05) difference was for any period and temperature studied;
this proves that the temperature did not influence the yolk
pigmentation. This might be due to the antioxidant content of
layer ration, because layer diet was formulated by using crude
palm oil (CPO), which was rich in carotenoids and vitamin E, which
delayed or prevented the oxidation of carotenoid pigments
contained in feed and yolk 52; while in another study, it was
demonstrated that the addition of vitamin E (20 and 40 mg/kg) to
hen diets reduced yolk colour intensity, in comparison with the

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Yolk colour score

5.4

4C
28-31C

5.35
5.3
5.25
5.2
5.15

7
21
14
Storage time (days)

28

Figure 9. Effect of storage time and temperature on


yolk colour score of chicken eggs.

control group 53. Spada et al. 54 found increased pigmentation of


the red in yolks after 28 days of storage at room temperature
(25C) and stabilizing on the 36th day. No recent data was found
regarding the relationship between yolk colour and storage
temperature or time.
Yolk lipid oxidation increased significantly (p<0.05) with
storage time and temperature (Fig. 10), because storage of egg
for longer periods at different temperatures may reduce the
antioxidant activity. This result agrees with Franchini et al. 55 and
Lakins et al. 56, that the TBARS values increased when the eggs
were stored for 30-90 days at 4C. On the contrary, the storage at
room temperature increased TBARS due to reduction in the
amount of vitamin E in stored eggs 55.
1.4
TBARS (mg/kg)

1.2
1
0.8
0.6

4C

0.4

28-31C

0.2
0

14

21

28

Storage time (days)

Figure 10. Effect of storage time and


temperature on TBARS value (mg/kg of
yolk) of chicken eggs.

Conclusions
This study has confirmed that the quality characteristics of eggs
were not adversely affected when eggs were stored in refrigerators,
but room temperatures negatively affect some egg quality
characteristics by increasing weight loss, yolk weight, yolk pH,
albumen pH, yolk lipid oxidation and by reducing HU and albumen
weight during storage for different time intervals. It has been
concluded that egg should be kept in refrigerators up to 28 days
and at room temperature up to 14 days.
Acknowledgements
The authors would like to thank Universiti Putra Malaysia for
providing financial support for the publication of this manuscript.
The authors are also grateful to Organization for Women in
Science for the Developing World (OWSD) for providing the
scholarship for PhD student.

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Journal of Food, Agriculture & Environment Vol.12 (3&4): 93-97. 2014

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Evaluation of the antioxidant activity of extracts from Psidium guajava L. and


Anacardium occidentale L. leaves obtained by different extraction methods
Suzara R. C. Sena, Theresa R. F. Dantas and Camila G. Pereira *
Laboratory of Separation Process in Foods, Department of Chemical Engineering (Federal University of Rio Grande do NorteUFRN), CEP 59072-970 Natal, Brazil. *e-mail: camila@eq.ufrn.br
Received 20 February 2014, accepted 28 September 2014.

Abstract
The presence of bioactive compounds in foods presents the possibility to improve public health through the diet. However, the obtaining of active
compounds from food for posterior application depends on the processing and fractionating methodologies used. Several functional properties have
been attributed to compounds presented in different parts of the plant, including the leaves and fruit. Parallel, the discovery of new applications of
by-products improves regional economy and makes value on the regional products. The aim of this work was to evaluate the antioxidant activity of
extracts from guava (Psidium guajava L.) and cashew (Anacardium occidentale L.) leaves and quantify the total phenolic compound concentrations
of different extracts. Extracts were obtained by employing three different techniques: low-pressure solvent, Soxhlet, and ultrasound extraction
methods. The effect of extract concentration on the percentage of inhibition (antioxidant activity) was analysed. Higher total phenolic compound
concentrations were obtained through Soxhlet extraction for guava leaf extracts (44.02 mg GAE/g extract) and low pressure solvent for cashew leaf
extracts (29.21 mg GAE/g extract). The results indicated that all extracts had good antioxidant activity due to the presence of phenolic compounds and
other compounds present in the extracts, with greater than 70% inhibition for all guava leaf extracts at concentrations greater than 5 mg/ml. The
percentage inhibition was also dependent on extract concentration, which can be attributed to differing concentrations of phenolic compounds and the
possible presence of other compounds that enhance or decrease the antioxidant activity of the extracts.
Key words: Guava leaves, cashew leaves, phenolic compounds, antioxidant activity.

Introduction
Antioxidants are now being included in functional diets due to
their potential beneficial characteristics for human physiology.
The ingestion of food products high in antioxidants is important
not only to combat malnutrition, but also to protect against
degenerative diseases caused by free radical-mediated oxidative
reactions. It may be possible to improve the nutritional value of
traditional food products and protect against damage caused by
free radicals by manufacturing new food products with
supplemental antioxidant compounds.
Northeast Brazil is a region rich in natural products. Some natural
products are locally consumed. However, the use of them in large
scale, commercial applications, such as food, cosmetic or medicinal
industries, is uncommon. These products could be used to
formulate new commercial products. In fact, the use of food as a
source of bioactive compounds has increased in recent years.
The bioactivity of the foods arises due to several classes of
compounds, including essential oils, flavonoids, carotenoids and
alkaloids. These compounds could be used industrially in different
formulations, thereby creating a demand for regional natural
products. In addition, novel compounds may be discovered,
potentially resulting in new products. Hence, tropical food species
are important in Northeast Brazil not only for the high nutritional
value as a food source, but as a potential contributor to the local
economy.
Special attention has been paid to natural products with
antioxidant activity as antioxidants play a role in the maintenance
of healthy organisms. Studies have revealed that antioxidant

activity is mainly due to the presence of phenolic compounds 1-4.


Among regional species of great economic importance in
Northeast Brazil is Psidium guajava L. (Myrtaceae), commonly
known as guava. Guava trees are native to Mesoamerica and are
found in Southeast and Northeast Brazil. The fruit of guava
trees is tasty and highly nutritious, and has important activities
such as anti-inflammatory, hydrophilic and lipophilic antioxidant
activities 5, 6. In addition, the leaves of this species exhibit
intestinal anti-spasmodic, hypoglycaemic, hypotensive,
antimicrobial, anti-nociceptive, anti-diabetic and hepato-protective
effects 7-12.
Another economically important species in Northeast Brazil is
Anacardium occidentale L. (Anacardiaceae), commonly known
as cashew. This tree is originally from tropical America, and is
native to Northern and Northeast Brazil 13. This species produces
fruits (nuts) and pseudo-fruits that are widely consumed by local
populations. Although the principal use of this species is as a
food source, A. occidentale can also be used for other purposes.
Previous studies have found that the branches of A. occidentale
have antimicrobial activity against different microorganisms 14-16.
The cashew nut has demonstrated pharmacological effects, such
as antibacterial activity, mutagenicity, antioxidant potential, and
anti-mutagenic activity against hydrogen peroxide activities 17, 18.
In addition, extracts from the leaves of A. occidentale are also
effective for the treatment of leishmanial ulcers due to the presence
of flavonoids and tannins 19 and antibacterial action against some
selected microorganisms 20.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

93

Although several investigations had been done with these


species in last decades, recent studies concerning on their
pharmacological effects and applicability studies continue being
made 21-27, indicating that several information still needs to be
revealed about these species.
In order to use these species in the industry, it is necessary to
evaluate what kind of process is more appropriate to obtain the
desired product. The recovery of active compounds in plant
extracts depends on the type of extraction and fractionation
methodologies used in the extraction process. Optimisation of
the extraction of specific compounds is essential for the
development of commercial uses for these species. In this scenario,
the objective of this study was to evaluate the concentrations of
phenolic compounds present in extracts from cashew (Anacardium
occidentale L.) and guava (Psidium guajava) leaves obtained
using different extraction techniques, and to determine the
antioxidant activity of these extracts.
Materials and Methods
Preparation and characterisation of the raw plant materials:
Cashew and guava leaves were collected in the Lagoa Nova region
in Natal, Brazil. The material was dried at ambient conditions (1720C) in the shade with air circulation for 25 days and subsequently
packed in plastic bags before storage in a refrigerator (Cnsul,
Modelo VU28A0, Santa Catarina, Brazil). The dried leaves were
ground using a knife mill (Tecnal, Model TE-631/2, Piracicaba,
Brazil). The processed material was classified according to size
using an agitator (Bertel, model NOVO 110/220, So Paulo, Brazil)
with standard Tyler series sieves.
Methodology of the analysis: Extracts obtained in previous studies
in the Laboratory of Separation Processes in Foods (LAPSEA/DEQ/
UFRN) were analysed to determine the concentration of phenolic
compounds and antioxidant activity 28, 29.
The extracts were obtained using the following methods: low
pressure solvent extraction (LPSE) at 60C, Soxhlet extraction at
78C, and ultrasound extraction at 40C. The temperatures were
chosen based on optimised conditions reported in previous
studies 28, 29. In all extraction processes, ethanol was used as the
solvent using a solid: solvent ratio of 1:10. Table 1 shows the
global yield of the extracts used in this study.
Table 1. Global yield of extracts from guava leaves
(P. guajava) and cashew leaves (A.
occidentale) obtained by different
extraction methods.
Extraction method
LPSE
Soxhlet
Ultrasound

Global yield (%)


Guava leaves 28 Cashew leaves 29
12
10.3
21
14.3
5.9
18.5

Quantification of total phenolic compounds: The total


concentration of phenolic compounds was determined using
the methodology described by Singleton and Rossi 30 modified
as described below. Approximately 1 ml of the extract and 1
ml of Folin-Ciocalteau reagent were mixed. After 5 min, 1 ml
of a saturated solution of Na2CO3 (approximately 35% in
methanol; PA ACS, Lot 0904809, Vetec) was added and the
94

total sample volume was made up to 25 ml with methanol. The


mixture was protected from light for 90 min before reading the
absorbance at 725 nm using a spectrophotometer (Varian 50, UVvis). A standard curve was generated using gallic acid standard
(Lot 0806387, Vetec) diluted in methanol at 20, 40, 60, 80, 100 and
120 mg/l with 1 ml of Folin-Ciocalteau reagent 31.
Measurement of antioxidant activity: Antioxidant activities of
the extracts were determined using the methodology of BrandWilliams et al. 32 as modified by Herrero et al. 33. In this method,
free radicals of DPPH (2,2-difenyl-1-picryl-hydrazil, Lot 07717TH,
Sigma-Aldrich) are neutralised with antioxidants in the extracts
resulting in a decrease in absorbance at 516 nm. To carry out the
reaction, a solution of 0.0214 mg/ml DPPH in methanol (Lot
0904809, Vetec) was prepared. Of the DPPH solution 3.9 ml was
mixed with 0.1 ml of the extract solutions at concentrations from
10 to 0.5 mg/ml. The total reaction volume was 4 ml. The reaction
was left to proceed for 4 h at ambient temperature (21C). The
absorbance was then determined at 516 nm using a
spectrophotometer. The antioxidant activities (AAs) were
calculated in terms of percentage of inhibition using Eq. 1:
AA (%) = 100 {[(Abssample Abscontrol) x 100] / AbsDPPH}

(1)

where Abssample is the absorbance of the sample, Abscontrol is the


absorbance of the control solution and AbsDPPH is the absorbance
of the DPPH.
Results and Discussion
Characterisation of the raw plant materials: The results of
characterisations of the raw plant materials are shown in Table 2.
After the initial preparation stage, the raw plant materials had
humidity, particle diameters (Dst), real and apparent densities,
granulometries and porosities suitable for use with extraction
processes to obtain bioactive compounds from natural products34.
The choice of the best technique should not only be based on the
global yield. An analysis of the chemical profile is also essential.
Total phenolic compounds: Quantification of the total phenolic
compounds was performed for each extract using a standard curve
from measurements of gallic acid standards (20-120 g/ml), and
expressed as mg of GAE (gallic acid equivalent) per g of extract.
The total phenolic compounds found in the guava and cashew
leaf extracts are shown in Table 3. Guava leaf extracts obtained by
Soxhlet extraction yielded higher amounts of total phenolic
compounds (44.02 mg GAE/g extract) than the other extraction
methods. For cashew leaves, the extracts obtained by LPSE yielded
higher concentrations of total phenolic compounds (29.21 mg
GAE/g extract).

Table 2. Characterisation of guava (P. guajava) and cashew (A.


occidentale) leaves.
Properties
Granulometry-in mesh (%)
Dst (mm)
Humidity (%)
Real density (g/cm)
Apparent density (g/cm)
Porosity (H)

Guava leaves
24 (52.0%), 28 (16.9%)
32 (4.1%), 48 (27.0%)
0.657
10.4 r 0.4
0.439 r 0.004
0.424 r 0.005
0.035

Cashew leaves
24 (70.4%), 28 (12.1%)
32 (2.8%), 48 (14.7%)
0.682
14 r 3
0.295 r 0.001
0.218 r 0.004
0.263

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Extraction method
LPSE
Soxhlet
Ultrasound

Total phenolic compounds


(mg GAE/g extract)
Guava leaves Cashew leaves
12.72
29.21
44.02
25.36
11.74
23.87

100
90
80
Inhibition (%)

Table 3. Total phenolic compounds present in


extracts from guava (P. guajava)
and cashew (A. occidentale)
leaves obtained by different extraction
methods.

70
60
50
40
30
20
10
0

Antioxidant activity of the extracts: The antioxidant activity of


each guava and cashew leaf extract was determined using DPPH
radicals and is expressed in terms of inhibition percentage, which
indicates the antioxidant activity of the extract. Figs. 1 and 2 show
the percentage of inhibition as a function of the concentration of
the diluted extracts.
As shown in Fig. 1, extracts from guava leaves obtained by
Soxhlet extraction had more than 70% of inhibition at low extract
concentrations. The inhibitory effect increased slightly with
increasing concentration (73.6-79.0%). These results indicate that,
for these extracts, the AA is independent of the concentration of
bioactive compounds. In other words, the AA of the extract is
associated with the presence of an unidentified antioxidant
compound, in a manner independent of the concentration of the
antioxidant present in the extract. This is consistent with results
from a previous study 35. In contrast, extracts obtained by
ultrasound extraction had a low inhibition (31.5%) at a low extract
concentration but increased to 72.1% inhibition as the extract
concentration was increased from 0.5 to 5 mg/ml. In this case, the
inhibitory effect is dependent on the concentration of bioactive
compounds. These results are consistent with the total phenolic
compound contents shown in Table 3, as the extracts with higher

4
6
8
Concentration (mg/ml)

10

12

Figure 1. Inhibition (%) by guava leaves extracts obtained


by LPSE (z), Soxhlet (*) and ultrasound (S) extraction
methods.
100
90
80
Inhibition (%)

Previous studies have presented only qualitative data for


extraction methods studied here 28, 29. In these studies, TLC analysis
showed that the amount of flavonoids obtained by Soxhlet
extraction and LPSE were similar to that for other techniques
studied. Flavonoids are a sub-group of phenolic compounds.
Based on these results, it could be inferred that the amount of
phenolic compounds in the Soxhlet and LPSE extracts may also
be similar. However, the results of this study show that this is not
the case. In our study, the total phenolic content was determined
quantitatively so it was possible to determine that there are
significant differences (p<0.05) between extraction methods,
especially for the guava leaf extracts. This indicates that nonflavonoid phenolic compounds are also present in the extracts.
It is also important to notice that the global yield (Table 1) is
useful only as a first analysis to guide the choice of extraction
methods for further study. Although the global yield and amount
of total phenolic compounds were highest for the Soxhlet extracts
of guava leaves, the opposite was true for the cashew leaf extracts
where the highest total phenolic compound content was obtained
for the LPSE extract, which had the worst global yield. These
results reinforce the importance of evaluating more than the global
yield of an extraction method in order to avoid selecting an
extraction method that does not give a high yield of the desired
compounds.

70
60
50
40
30
20
10
0

4
6
8
Concentration (mg/ml)

10

12

Figure 2. Inhibition (%) by cashew leaves extracts obtained


by LPSE (z), Soxhlet (*) and ultrasound (S) extraction
methods.

total phenolic compound concentrations, such as Soxhlet extracts,


showed higher AA activity. In general, AA was observed for all
guava extracts, with observed inhibitions higher than 70% for all
extracts at concentrations greater than 5 mg/ml. It is important
also to note that LPSE extracts showed high AA (60.0-72.5%)
even though the LPSE extracts had low concentrations of total
phenolic compounds (12.72 mg GAE/g extract). Based on these
results, there must be non-phenolic compounds in the LPSE extract
with AA.
Figure 2 shows that the cashew leaf ultrasound extract had an
AA effect different from that observed for guava leaf ultrasound
extract. The percentage of inhibition increased (21.73 to 74.96%)
as the extract concentration increased from 0.5 to 5 mg/ml, reaching
a plateau above 5 mg/ml. The effect of concentration on the AA
was not evident for the Soxhlet extracts of cashew leaves. However,
the inhibition percentage was high (74.6%) for the lowest extract
concentration (0.5 mg/ml). Interestingly, for cashew leaf LPSE
extracts, high concentrations of the extract yielded lower inhibition
percentages than low concentrations. This may occur if other
compounds present in the extracts counteract the AA of the
bioactive compounds. For instance, the results showed that for
guava extracts obtained by Soxhlet extraction, the extract
concentration had little effect on AA, indicating the presence of a
bioactive compound for the AA independent of concentration.
The AA of the bioactive compound may increase with increasing
extract concentration, but other compounds may also be present

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

95

that react with the antioxidant compound, thereby decreasing the


observed AA. Another possibility is the presence of compounds
in the extracts that promote oxidation, thereby opposing the AA
of the extract.
Figure 2 also shows that all extracts exhibited AA over the range
of extract concentrations studied here. LPSE and Soxhlet extracts
had high AA at low concentrations (0.5-1.0 mg/ml), while
ultrasound extracts had high AA for concentrations higher than 5
mg/ml.
Conclusions
In our study, it was observed that when developing methods for
the extraction of specific compounds from natural products, the
global yield should only be used as an initial guide as high global
yields do not necessarily guarantee efficient extraction of specific
target compounds. Higher total phenolic compound concentrations
were observed in extracts obtained by Soxhlet extraction and LPSE
for guava and cashew leaves, respectively. Antioxidant activity
was observed for all extracts, with greater than 70% inhibition for
all guava leaf extracts at concentrations greater than 5 mg/ml. The
percentage inhibition was also dependent on extract concentration
for cashew leaf extracts. Variability in AA with extract concentration
can be attributed to differing concentrations of phenolic
compounds and the possible presence of other compounds that
enhance or decrease the AA of the extracts.
Acknowledgements
The authors are grateful to FAPERN (PPP003/2007) for financial
support.
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Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

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Validation of ELISA-based detection of L. monocytogenes and E. coli O157:H7 in


fresh cut vegetables
Marina Cavaiuolo 1, Antonio Ferrante 1*, Spiros Paramithiotis 2, Agni Hadjilouka 2, Periklis Tzamalis
and Eleftherios H. Drosinos 2

Dept. Agricultural and Environmental Sciences, Universit degli Studi di Milano, via Celoria 2, 20133 Milano, Italy.
Laboratory of Food Quality Control and Hygiene, Department of Food Science and Human Nutrition, Agricultural University
of Athens, Iera Odos 75, GR-118 55 Athens, Greece.
e-mails: marina.cavaiuolo@unimi.it, antonio.ferrante@unimi.it, sdp@aua.gr, agni_xatz@aua.gr, pertzam@otenet.gr,ehd@aua.gr
1

Received 12 June 2014, accepted 20 September 2014.

Abstract
Innovative diagnostic methods were developed for the detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in
minimally processed fresh cut fruits and vegetables. The aim of the present study was to validate the technical efficiency of these methods and
evaluate their efficacy and viability for routine analysis. To this purpose, ready-to-eat fresh fruits and vegetables were collected throughout the
production chain. A multidisciplinary approach, including a newly developed ELISA method compared to ISO procedures, was applied to detect the
pathogenic bacteria after harvesting, processing and shelf-life. Results obtained exhibited the technical efficiency of the developed methods showing
similar sensitivity, specificity, negative predictive values and negative likelihood ratios.
Key words: Leafy vegetables, melon, vegetables, ELISA, ISO.

Introduction
Ready to eat fresh-cut vegetables (RTEs) are convenient foods
that have increased the volume and value of commercialisation
among different European countries. Nevertheless, the economic
crisis in the recent years has slightly affected the fresh-cut fruits
and vegetables market. This trend can be explained considering
the higher quality of the products, which must be convenient and
safe 1. Most of the leafy vegetables used for the fresh-cut
preparation are grown in the soil and bacteria contamination can
easily occur. Moreover, their short growing cycles require an
higher supply of organic matter in order to keep the soil fertility.
The organic matter supply is usually performed by manure and as
such represents a possible source of contamination of human
pathogenic bacteria like Escherichia coli O157 L. and Listeria
monocytogenes L. 2.
Washing treatments are able to reduce the bacteria load and
allow to preserve the RTE products for long time 3. The official
standard procedures for the detection of bacterial pathogens
require 4-5 days. Unfortunately, the shelf life of leafy vegetable
products is limited to 5-7 days that include two days of processing
and five days of shelf life. Therefore, faster and reliable methods
are required to detect the presence of pathogens without losing
the period of commercialisation. In the framework of the EU project
QUAFETY - Comprehensive Approach to Enhance Quality and
Safety of Ready to Eat Fresh Products different detection methods
based on ELISA approach was developed and compared with the
standard official ISO procedures.
Materials and Methods
Samples: Rocket and mixed salads were provided by Agronomia
s.r.l., (San Paolo dArgon, BG, Italy) and EuroCatering S.A. (Greece),
98

while Piel De Sapo melons were provided by NoviFruits (Portugal).


Samples were collected from raw materials, processed materials
(immediately after packaging) and three days shelf-life materials.
Thirty-nine samples were analysed corresponding to 2 different
batches of melons (Portugal), 7 different batches of rocket (Italy
and Greece) and 4 different batches of mixed salad (Italy and
Greece).
ELISA-based detection of L. monocytogenes and E. coli
O157:H7: Five g of material was homogenised in 1PBS (1 mM
KH2PO4, 154 mM NaCl, 3 mM Na2HPO4, pH 7.2). Isolation of
bacteria, ELISA detection and data analysis were performed
according to the protocol described in Cavaiuolo et al. 4. For the
detection anti-E. coli O157 (ab20976), anti-L. monocytogenes (LZA2)
(ab11439) and Polyclonal Goat anti-Mouse IgG+IgM H&L (HRP)
antibody (ab47827) were purchased from Abcam (Cambridge, UK).
ISO procedures for L. monocytogenes and E. coli O157:H7
detection: Twenty-five g of each sample was used to detect the
presence of L. monocytogenes and E. coli O157:H7 according to
ISO 11290-1:1996/Amd 1:2004 and ISO 16654:2001 5, 6, respectively.
A sample was characterised as positive for the presence of L.
monocytogenes when the identity of typical colonies isolated from
ALOA agar was verified by series of biochemical tests (Gram stain,
catalase test, oxidase reaction, hemolysis, fermentation of
rhamnose, xylose, mannitol and methyl -D-mannopyranoside,
and a motility test). In any other case (i.e. identity not verified,
absence of typical colonies), the sample was characterised as
negative for the presence of L. monocytogenes. A sample was
characterised as positive for the presence of E. coli O157:H7 when

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

the identity of typical colonies was verified by successive


subculturing in BH agar and submittion to E. coli O157:H7 Latex
Test (Remel, Lenexa, USA). In any other case (i.e. identity not
verified, absence of typical colonies) the sample was characterised
as negative for the presence of E. coli O157:H7.
Results and Discussion
While all melon samples were negative for both bacteria (average
OD450nm 0.09, negative control (NC) OD450nm 0.06), in fresh cut
salads the ELISA method identified few suspected samples. Based
on the NC absorbance values (average OD450nm 0.101), on the
coefficient of variation (CV) among samples and assays and on
the OD450nm values of different concentrations of E. coli O157:H7
and L. monocytogenes reported in the work of Cavaiuolo et al. 4,
the thresholds were set for classifying the samples as sure
negative, suspect and sure positive. Samples with and OD450nm0.3
were considered sure negative and samples with OD450nm0.5 were
considered sure positive for E. coli O157:H7. Only three samples
were considered as suspect: Rocket raw (OD450nm 0.36), mix raw
(OD450nm 0.321) and mix three days-shelf (OD450nm 0.322). Samples
with and OD450nm0.5 were considered sure negative and samples
with OD450nm0.6 were considered sure positive for L. monocytogenes
(Table 1). Based on these thresholds, two suspected samples were
isolated: Mix salad raw (OD450nm 0.52) and mix three days shelf
(OD450nm of 0.566).
Table 1. Detection of E. coli O157:H7 and L. monocytogenes in
rocket and mix salad samples using the ELISA method.
E.coli 0157:H7
L. monocytogenes
OD450nm
OD450nm
Stdev
Stdev
Sample
mean
mean
Rocket raw
0.360 0.204 Suspect
0.449 0.181 Negative
Rocket processed 0.152
0.08 Negative 0.203 0.138 Negative
Rocket shelf-life
0.202 0.105 Negative 0.285 0.171 Negative
Mix raw
0.321
0.08 Suspect
0.519 0.148 Suspect
Mix processed
0.242
0.05 Negative 0.419 0.202 Negative
Mix shelf-life
0.322
0.06 Suspect
0.566 0.228 Suspect
Negative
0.101 0.017
0.095 0.030
Melon raw
0.091 0.002 Negative 0.076 0.025 Negative
Melon processed
0.087 0.001 Negative 0.088 0.002 Negative
Melon shelf-life
0.094 0.001 Negative 0.092 0.001 Negative
Negative
0.061 0.003
0.063 0.001

The use of classical microbiological approaches verified the


presence of E. coli O157:H7 in mixed salad raw and rocket three
days shelf-life and absence in the rest of the samples. Absence of
L. monocytogenes was verified in all samples.
If the samples classified as suspects are considered positives,
then a 22 contingency table summarising the results obtained
by the ELISA can be formed and the samples can be differentiated
into true positives, true negatives, false positives and false
negatives (Table 2). Furthermore, the performance indices of the
newly developed methods used to detect L. monocytogenes and
E. coli O157:H7 can be calculated (Table 3).

Table 3. Performance indices of the newly ELISA method


used to detect L. monocytogenes and E. coli
O157:H7.
Sensitivity
Specificity
Positive predictive value
Negative predictive value
Positive likelihood ratio
Negative likelihood ratio

L. monocytogenes
1
0.94
0.5
1
19.5
0

E. coli O157:H7
0.75
0.94
0.06
0.97
13.87
0.26

Conclusions
Results obtained exhibited the technical efficiency of the
developed methods. More accurately, all methods compared had
similar sensitivity, specificity, negative predictive values and
negative likelihood ratios. False positive results obtained by the
ELISA method resulted in the reduction of positive predictive
values. Regarding their efficacy and viability for routine analysis
it is mostly dependent upon available equipment and technical
expertise.
Acknowledgements
The research leading to these results has received funding from
the European Union Seventh Framework Programme (FP7/20072013) under grant agreement no. 289719 (Project QUAFETY). All
authors contributed equally to this publication.
References
Rico, D., Martn-Diana, A. B., Barat, J. M. and Barry-Ryan, C. 2007.
Extending and measuring the quality of fresh-cut fruit and vegetables:
A review. Trends Food Sci. Tech. 18(7):373-386.
2
Oliveira, M., Usall, J., Vinas, I., Anguera, M., Gatius, F. and Abadias,
M. 2010. Microbiological quality of fresh lettuce from organic and
conventional production. Food Microbiol. 27(5):679-684.
3
Goodburn, C. and Wallace, C. A. 2013. The microbiological efficacy of
decontamination methodologies for fresh produce. Food Control
32(2):418-427.
4
Cavaiuolo, M., Paramithiotis, S., Drosinos, E. H. and Ferrante, A. 2013.
Development and optimization of an ELISA based method to detect
Listeria monocytogenes and Escherichia coli O157 in fresh vegetables.
Anal. Methods 5(18):4622-4627.
5
International Organization for Standardization (ISO) 1996. Microbiology
of Food and Animal Feeding Stuff - Horizontal Method for the
Detection and Enumeration of Listeria monocytogenes. Part 1:
Detection Method. Geneva, Switzerland.
6
International Organization for Standardization (ISO) 2001. Microbiology
of Food and Animal Feeding Stuffs - Horizontal Method for the
Detection of Escherichia coli O157. Geneva, Switzerland.
1

Table 2. The 2 2 contingency table obtained from the


application of the newly ELISA method used to
detect L. monocytogenes and E. coli O157:H7.
L. monocytogenes
E. coli O157:H7

TP
2
3

TN
37
35

FP
2
2

FN
0
1

TP: True positive. TN: True negative. FP: False positive. FN: False negative.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

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Journal of Food, Agriculture & Environment Vol.12 (3&4): 100-103. 2014

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Composition and content of selected elements of Croatian blackberry wines


Ivana Alpeza *, Tatjana Varga and Veronika Kubanovi
Croatian Centre for Agriculture, Food and Rural Affairs, Institute of Viticulture and Enology, Jandrieva 42, 10000 Zagreb,
Croatia. *e-mail: ivana.alpeza@hcphs.hr
Received 6 February 2014, accepted 20 September 2014.

Abstract
The blackberry wine is recognised as a natural source of many bioactive molecules and essential elements that play an important role in health
promotion and disease prevention. The wine is traditionally popular medicine for anemia and iron deficiency. The aim of this work was to evaluate
quality physical and chemical characteristics and concentration of macro elements Ca, K, Mg, Na, essential elements Cu, Fe, Mn, Zn, soil associated
elements Li, Rb, Sr and toxic elements Al, Co, Pb of selected 22 blackberry wines from different regions of Croatia. Basic quality characteristics of
all fruit wines were determined as follows: alcoholic strength (% vol), total sugars (g L-1), total extract (g L-1), ash (g L-1), pH, total acidity (g L-1, malic
acid) and volatile acidity (g L-1, acetic acid). The results indicate that blackberry wines represent high quality beverage. For determination of elements,
fast and precise method of inductively coupled plasma optical emission spectrometry (ICP-OES) was used. Following elements were detected in
investigated blackberry wines in different amounts: macro elements Ca, K, Mg, Na (mg L-1), essential elements Cu, Fe, Mn, Zn (mg L-1), soilassociated elements Li, Rb, Sr (g L-1) and toxic elements Al, Co and Pb (g L-1). It can be concluded that moderate consumption of blackberry wines
may contribute to daily dietary intake of essential elements and wines can be considered as health safe, because potentially toxic elements are kept
under allowable limits.
Key words: Blackberry, fruit wine, elements, ICP-OES.

Introduction
Blackberry (Rubus fruticosus L.) does not get enough credit when
it comes to its health benefits. Ancient civilisations prised
blackberries as a food and traditionally used them for medicinal
and health purposes as well. The ancient Greeks used blackberries
and their juice to treat gout and the Chinese used blackberries to
treat kidney and urinary problems 5. Blackberries were used in
Europe during the16th century as a medicinal plant to treat
infections of mouth and eyes 1. Blackberries are notable for their
health benefits based on high nutritional contents of dietary fiber,
vitamins, folic acid, the essential mineral manganese and other
bioactive compounds 2. Blackberry fruit contains high level of
anthocyanins and other phenolic compounds, mainly flavonols
and ellagitannins, which contribute to its high antioxidant potential
and other biological activities 3, 4. In sense of nutritional value,
daily consumption of blackberry wine in recommended quantities
(about 250 ml) can be a significant dietary source of essential
minerals and could improve health 5, 6. Influence of fermentation
process on aroma composition 7, phenolic, antioxidant capacity
and volatile compounds in blackberry wine 8 were studied. Not
only blackberries, but also their fruit wines rank highly for
antioxidant strength 9, particularly due to their high contents of
phenolic compounds, such as quercetin, gallic acid and
anthocyanins 10. Potent in vitro antioxidant and vasodilatory
effects of Croatian commercial blackberry wines that are roughly
comparable to those of red wines were confirmed 11. On the other
hand, several metals in wine and fruit wine, such as cadmium, lead
and arsenic, are toxic and harmful 26. There is not much available
data on mineral and heavy metal contents in blackberry wines.
Therefore, metal content in wine is regulated according to the
100

national legislation and the legislation of the European Union. As


the production of blackberry wine has been increasing in Croatia
for the last few years, the aim of this study was to determine the
content of macro elements Ca, K, Mg, Na, essential elements Cu,
Fe, Mn, Zn, soil associated elements Li, Rb, Sr and toxic elements
Al, Co, Pb in 22 blackberry fruit wines from different Continental
and Coastal sub-regions of the Republic of Croatia by ICP-OES
method.
Materials and Methods
Samples: In this study, 22 market ready blackberry wines from
Continental and Coastal region of Croatia were analysed. Ten
wines were in the category of fruit wine and twelve in the category
of dessert fruit wine 12. The vintage comprised the years 2011 and
2012.
Physical and chemical analysis: Alcoholic strength (% vol), total
sugars (g L-1), total extract (g L-1), ash (g L-1), pH, total acidity (g L-1
malic acid) and volatile acidity (g L-1 acetic acid) were determined
by methods described by International Organization of Vine and
Wine OIV 13.
Sample preparation for ICP-OES determination: Four mL HNO3
60 % (Ultra-pure, 101518, Merck, Darmstadt, Germany) was added
to 50 mL of fruit wine sample. This solution was evaporated in a
water bath at 90-95C to reduce the volume to approximately 30 mL,
in order to remove ethanol and therefore, minimise matrix
interferences during analysis and diminish plasma instability,
caused by introduction of organics into the plasma. The sample

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

residue was quantitatively transferred to volumetric flask and


volume was set to 50 mL with 2% nitric acid 14.
Blanks and standards: HNO3 ( 2%) solution was prepared from
60% HNO3 and ultra-pure water 18 M/cm (obtained from Easy
pure RF, Barnstead, Dubuque, IA, USA), which was used as blank.
Standard solutions containing 2% HNO3 were prepared with
appropriate dilutions to cover the concentration range of each
element in the wines. Standard solutions of Al (13-119), Co (1410), Cu (13-145), Fe (14-17), Mn (13-146), Mg (13-119), Pb (14-18),
Zn (14-47), Li (15-31), Sr (14-90), Rb (14-134), K (14-102), Ca (14-37)
and Na (14-44) were PerkinElmer (Shelton, USA).
ICP-OES method: The validation parameters of method used for
determination of 14 elements is fully described 15 and samples
were analysed using a direct calibration curve.
Instrumentation: Multi-element determinations were carried out
with Perkin-Elmer Optima 2000 DV instrument equipped with a
Meinhard spray chamber, nebulizer and peristaltic sample delivery
system. The instrument was controlled by the ICP WinLab 1.35
Perkin Elmer software. The operating conditions and the
wavelength used for the analysis of each element are given in
Table 1. The flow conditions for plasma gas, auxiliary gas and
nebulizer gas were 15.0, 0.2 and 0.8 L/min, respectively. Power was
set at 1300 W.
Table 1. Operating conditions and wavelengths used
for quantification of each element by ICP
OES method.
Element
Al
Ca
Co
Cu
Fe
K
Li
Mg
Mn
Na
Pb
Zn
Rb
Sr

Wave length (nm)


396.153
317.933
228.616
327.393
238.204
766.490
670.784
285.213
257.610
589.592
220.353
206.200
780.023
407.771

Plasma view
Axial
Radial
Axial
Axial
Axial
Radial
Axial
Radial
Axial
Radial
Axial
Axial
Axial
Axial

Results and Discussion


The quality physico-chemical characteristics of investigated
blackberry wines are presented in Table 2. The alcoholic strength
for all wines varied between 10.5 and 15.6% vol, which corresponds
to the range published for other Croatian blackberry wines 6, 11.

Values of alcoholic strength were in accordance with the Croatian


Regulation on fruit wine 12, where the fruit wine is defined as a
food product produced by fermentation of fruit juice or must with
a minimum of 1.2% by volume alcoholic strength and dessert fruit
wine is allowed to have addition of sugar or alcohol of fruit origin,
to extent the actual alcoholic strength from 13% vol to 22.0% by
volume for total alcoholic strength.
The content of total sugars in all wines varied between 19.1 and
146.2 g L-1 that was within range published for other Croatian
blackberry wines 16. The content of total extract for all wines ranged
from 44.3 to 174.9 g L-1 while the content of ash ranged from 1.6 to
4.7 g L-1. The pH values for all wines varied between 3.11 and 3.99.
The range of pH values published for the blackberry wines
produced in Croatia is 3.11-3.56 with an average of 3.33 5. The pH
values of Croatian blackberry wines obtained by fermentation
using commercial yeasts (Fermol Rouge and Fermol Mediterranee)
were 3.34 and 3.33 17, respectively, which are the same as published
values 16. The total acidity expressed as g L-1 of malic acid for all
wines varied between 8.0 and 16. Published data of Croatian
blackberry wines total acids 6 ranged from 6.0 to 16.2 g L-1 and
were within the range obtained in this study. The volatile acidity
expressed as g L-1 acetic acid for all wines varied between 0.3 and
1.5 g L-1. Croatian Regulation on fruit wines 12 has a requirement
that fruit wines can contain maximum of 1.5 g L-1 volatile acidity,
meaning that wines submitted to this study had about two times
lower volatile acidity.
Metals are unique nutrients because of their important role in
metabolism. They are essential part of many important enzymes
and they also play roles as catalysts and antioxidants. The majority
of literature data refers to K, Na, Ca and Mg as major elements of
wine and fruit wine. As expected, in this study K was the most
predominant element present in blackberry wines at an average of
1250 mg L-1 (Table 3).
Potassium is known to play an important role in the regulation
of osmotic pressure and neuromuscular, enzymatic, hormonal and
other metabolic activity 18. The concentration of K varied over the
range from 615 to 1760 mg L-1, which is rather wider than range 924
to 1507 mg L-1 published in the earlier study 6. All considered
samples contain a high amount of Ca and Mg, but little quantities
of Na. The concentration of Ca varied from 115 to 555 mg L-1 and
was higher than range from 86.4 to 457.1 mg L-1 obtained in the
earlier study 6. Magnesium is an important element, because it is
essential for many biosynthetic processes, as well as nucleic acid
and protein metabolism. Together with Ca, Mg is known to be
associated with the regulation of heart muscles 19. The
concentration of Mg in blackberry wines ranged from 77 to 238
mg L-1 that was much narrower than published 6 range from 183.2
to 381.2 mg L-1. Furthermore, detected Na concentrations varied
from 3.0 to 13.5 mg L-1. Those concentrations were much lower

Table 2. Quality physico-chemical characteristics of fruit wines (n=10), dessert fruit wines (n =12) and all wines (n=22).
Physico-chemical parameter
Alcoholic strength (% vol)
Total sugars (g L-1)
Total extract (g L-1)
Ash (g L-1)
pH value
Total acidity (g L-1 malic acid)
Volatile acidity (g L-1 acetic acid)

Fruit wine
MeanSD
Min
12.60.9
10.5
97.834.9
44.3
134.944.2
75.8
3.40.6
2.5
3.370.22
3.18
11.71.8
10.0
0.80.3
0.5

Max
13.9
146.2
174.9
4.7
3.62
13.6
1.4

Dessert fruit wine


MeanSD
Min
Max
14.10.9
13.0
15.6
78.941.8
19.1 140.3
112.437.2
44.3 175.0
3.00.6
1.6
4.6
3.420.11
3.11
3.99
11.31.1
8.0
16.0
0.80.2
0.3
1.5

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

All wines
MeanSD
Min
13.41.2
10.5
87.139.3
19.1
122.241.2
44.3
3.20.6
1.6
3.400.18
3.11
11.41.51
8.0
0.80.3
0.3

Max
15.6
146.2
174.9
4.7
3.99
16.0
1.5

101

Table 3. Concentrations of macro elements Ca, K, Mg and Na in blackberry fruit wines (n=10),
dessert fruit wines (n=12) and all wines (n=22).
Element
(mg L-1)
Ca
K
Mg
Na

Fruit wine
MeanSD
Min
13419
115
1333238
955
16424
122
9.82.3
6.1

Dessert fruit wine


MeanSD
Min
155129
51
1181269
615
14841
77
8.87.0
3.0

Max
171
1760
207
13.3

Max
555
1585
238
13.5

than range from 11.81 to 120.10 mg L-1 obtained for blackberry


wines originated from three different Croatian regions Slavonija,
Prigorje-Bilogora and Zagorje-Medimurje 6. Sodium is required in
human body for the regulation of osmotic pressure and the acidbase balance.
Moreover, as it can be seen in Table 4, detected concentrations
of essential elements Cu, Fe, Mn and Zn in blackberry wines were
0.051-3.83 mg L-1, 0.093-5.49 mg L-1, 0.472-11.3 mg L-1 and 0.3041.96 mg L-1, respectively.
Copper and iron are essential in blood formation and copper is
involved in normal carbohydrate and lipid metabolism. Copper
acts as a cofactor of enzymes involved in various metabolic
processes within living cells 20. Reported range 6 of copper 0.0580.767 mg L-1 was much narrower than results of this study, while
concentrations of iron ranged from 0.082 to 6.273 mg L-1 and Mn
from 1.47 to 11.53 mg L-1 and they were very close to the values
obtained in this study. Zinc is an important microelement, because
it acts as a cofactor of enzymes involved in metabolic reactions.
The published data 21 of Zn concentration that ranged from 0.557
to 3.569 mg L-1 were much higher in comparison to our results.
The concentration of three elements that are soil-associated
elements, Li, Rb and Sr in investigated blackberry wines were 140, 90-1470 and 165-1445 g L-1, respectively (Table 5).
Red wines from the Province of Chieti and the Province of
Teramo 22 had Rb concentrations of 1815.36 and 1599.7 g L-1 ,
respectively, that are much higher than those obtained for
blackberry wines. At the same time, concentration of Sr in red

All wines
MeanSD
Min
14695
115
1250261
615
15635
77
9.235.3
3.0

Max
555
1760
238
13.5

wines varied from 395.58 in the Province of Chieti to 458.36 g L-1 in


the Province of Teramo 22 and were close to the concentration
obtained for investigated blackberry wines (439 g L-1). The
concentration of Sr for red wines from two wine producing Spanish
regions (Ribera del Guardiana and Mntrida) 23 were 379 and
981 g L-1. The concentration of Sr of blackberry wines was closer
to that for red wines from region Ribera del Guardiana. The
concentrations of Li in red wines from two regions 23 were 48 and
69 g L-1, which is much higher than concentration of 21 g L-1 in
blackberry wines.
In general, the toxic effects of aluminium result from its
competition with other metal ions in enzymes and proteins. As the
aluminium ion substitutes the metal at its binding site, the function
of the protein is changed and the metabolism of the cell is altered,
consequently affecting the organism gravely 24. The concentration
of toxic elements Al, Co and Pb in the investigated blackberry
wines were 37-1110, 2-40 g L-1 (six samples had concentration
below the detection limit of the method) and 17-154 g L-1,
respectively (Table 6). The concentration of Al found in the
previous study 25 ranged from 6.01 to15.73 mg L-1 , much higher
than concentration obtained in this study. Concentration of Co
varied from 1.29-4.43 g L-1, which is far below concentration found
in this study and concentration of Pb varied from 13.56 to 52.81 g
L -1 , where maximum value was three times higher than
concentration obtained in this study. It should be noted that
concentration of Pb was below the internationally established
maximum allowed values.

Table 4. Concentrations of essential elements Cu, Fe, Mn and Zn in blackberry fruit wines (n=10), dessert
fruit wines (n=12) and all wines (n=22).
Element
(mg L-1)
Cu
Fe
Mn
Zn

Fruit wine
MeanSD
Min
0.6551.127
0.076
1.8391.412
0.731
5.7483.069
1.12
0.9440.425
0.304

Max
3.83
5.49
10.56
1.96

Dessert fruit wine


MeanSD
Min
0.2540.155
0.051
1.0580.628
0.093
4.3623.483
0.472
0.8310.375
0.360

Max
0.53
2.22
11.3
1.74

All wines
MeanSD
Min
0.4360.774
0.051
1.4131.104
0.093
4.9923.299
0.472
0.8820.393
0.304

Max
3.83
5.49
11.3
1.96

Table 5. Concentrations of Li, Rb and Sr in blackberry fruit wines (n=10), dessert fruit wines (n=12) and all
wines (n=22).
Element
(g L-1)
Li
Rb
Sr

Fruit wine
MeanSD
Min
2618
2
414173
90
385114
240

Max
40
716
576

Dessert fruit wine


MeanSD
Min
Max
1815
1
40
631415
160
1470
483345
165
1445

MeanSD
2116
532340
439266

All wines
Min
1
90
165

Max
40
1470
1445

Table 6. Concentrations of toxic elements Al, Co and Pb in blackberry fruit wines (n=10), dessert fruit wines
(n=12) and all wines (n=22).
Element
(g L-1)
Al
Co
Pb

102

MeanSD
290138
139
5020

Fruit wine
Min
136
4
30

Max
490
30
100

Dessert fruit wine


MeanSD
Min
297290
37
1411
2
6143
17

Max
1110
40
154

MeanSD
294229
1411
5634

All wines
Min
37
2
17

Max
1110
40
54

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Conclusions
The selected quality physical and chemical characteristics indicate
that blackberry wines were in accordance with the Croatian fruit
wine legislation. Therefore, they represent high quality beverage.
The concentration of most of the investigated elements is different
among blackberry wines. Results of Cu, Fe, Mn and Zn, the most
important essential elements, lead to the conclusion that blackberry
wines can be considered as good source of these elements. The
concentrations of toxic elements, such as Co and Pb, did not
exceed the limits given by the Croatian regulations, which leads
to the conclusion that organic and mineral fertilizers, inorganic
pesticides and other means of blackberry growing and winemaking
practice are safe. All these parameters suggest that Croatian
blackberry wine is safer than the wines mentioned above. One
explanation might be the absence of environment pollutants, such
as heavy industry and automobiles exhaust gases around orchard
where blackberry was grown. It can be concluded that moderate
consumption of blackberry wines may contribute to daily dietary
intake of essential elements and blackberry wines can be
considered as health safe, because potentially toxic elements are
under allowable limits.
Acknowledgements
The authors would like to thank to the producers for the support
of this study.
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11

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

103

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e-mail: info@world-food.net

Journal of Food, Agriculture & Environment Vol.12 (3&4): 104-107. 2014

www.world-food.net

Physicochemical properties of honey samples from Ondo state, Nigeria, and their
bioactivity against spoilage and pathogenic organisms
Funmilola Oluyemi Omoya 1, Oluwatosin Ademola Ijabadeniyi
1

2, 3

* and Olayemi Bosede Ogonnoh

Department of Microbiology, Federal University of Technology, Akure, Nigeria. Department of Biotechnology and Food
Technology, Durban University of Technology, South Africa. *e-mail: tosynolu@yahoo.com
2, 3

Received 22 January 2014, accepted 6 September 2014.

Abstract
Honey can be defined as the natural sweet substance produced by honeybees (Apis mellifera) from the nectar of blossoms or from the secretion of
living parts of plant or plant sucking insects living on parts of plants. The medicinal property of honey has been an area of interest to researchers in
recent times. This study focused on assessing the physicochemical components of honey samples and their bioactivity on some food spoilage
organisms. One hundred samples of honey were collected from different locations in Ondo state, Nigeria. Their physicochemical components which
include conductivity, ash content, moisture content, pH, mineral contents and colour were determined. There was variation in the physicochemical
components of some of the honey samples with reference to international standards. The assessment of the honey samples as an antibacterial agent
revealed it inhibitory potency on both bacteria and fungi isolated from food sample. The inhibitory effect was compared with that of standard
antibiotic. The honey samples were seen to display a higher inhibitory effect on the tested organisms than the employed antibiotic.
Key words: Bioactivity, honey, physicochemical, food spoilage organisms, antibiotics.

Introduction
Honey is a sweet food made by bees using nectar from flowers 18.
The variety produced by honey bees (the genus Apis) is the most
commonly referred to and is the type of honey collected by
beekeepers and consumed by humans 12. Honey produced by
other bees and insects has distinctly different properties. Honey
bees form nectar into honey by a process of regurgitation, and
store it as a primary food source in wax honeycombs inside the
beehive. Beekeeping practices encourage overproduction of
honey so the excess can be taken from the colony. Honey gets its
sweetness from the monosaccharides fructose and glucose, and
has approximately the same relative sweetness as that of
granulated sugar 18. It has attractive chemical properties for baking,
and a distinctive flavor that makes some people to prefer it over
sugar and other sweeteners. Most microorganisms do not grow
in honey because of its low water activity of 0.6 13. However,
honey sometimes contains dormant endospores of the bacterium
Clostridium botulinum, which can be dangerous to infants, as
the endospores can transform into toxin-producing bacteria in
the infants immature intestinal tract, leading to illness and even
death 22.
Honey has a long history of human consumption, and is used
in various foods and beverages as a sweetener and flavoring 12. It
also has a role in religion and symbolism. Flavors of honey vary
based on the nectar source, and various types and grades of
honey are available. It is also used in various medicinal traditions
to treat ailments. The study of pollen and spores in raw honey can
help to determine floral sources of honey. Furthermore, because
bees carry an electrostatic charge, and can attract other particles,
the same technique of its pollen and spores study can be used in
area environmental studies of radioactive particles, dust or
particulate pollution 16. The term traditional medicine (indigenous
104

medicine or folk medicine) essentially represents a natural form of


health care which has been used through generations 1.
Traditional medicine practices existed in Africa and other cultures
for centuries since man came into being but until recently, has
been neglected or even outlawed in some cases due to undue
pressure from practitioners of modern medicinal practice and
unscientific background of its method of operation 8. The 21st
century is witnessing serious scientific effort to discover major
active ingredients in medicinal plants through research and
development. According to Osermene et al. 20, this could help
orthodox medicine to comprehensively address most disease
conditions plaguing mankind or it may be a response to the clarion
call by the World Health Organization 17 that developing countries
should endeavour to develop and utilize local medications that
are most appropriate to their local circumstances especially for
Primary Health Care (PHC) in order to reduce the cost associated
with incessant drug importation 17. Traditional medicine is the
sum of all knowledge and practical application, whether explicable
or not used in diagnosis, prevention and elimination of physical,
mental or social imbalances and relying exclusively on practice
and experience and the sociological environment 5, 23. Most of
these medicines produced traditionally entail herbs from different
plants, honey, Aloe vera, and from bitter tasted plants 15. This
present study was aimed at investigating the physico- chemical
properties of honey samples in Ondo state, Nigeria and their
inhibitory potency on spoilage bacteria isolated from food samples.
Materials and Methods
Study area: The Ondo state of Nigeria is one of the states in
south west Nigeria (7100" N and 5 50"E). It covers 15,500 km2,
with an estimated population in the 2006 census of 3,440,000

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

inhabitants. The climate condition of this state is classified as


tropical, with a mean temperature ranging from 25C to 29C year
round. It has a minimum and maximum temperature between 21.6
and 33.8C respectively; and a mean relative humidity of 85%.
Sample collection: The honey samples were purchased from
different honey sellers from various locations in the Ondo state of
Nigeria namely; Akure, Owo, Akungba, Ile-oluji, Ondo town, IkareAkoko, Ilara, Igbara Oke, Itaogbolu and Iju. They were taken to
the laboratory and screened for their microbiological and
physicochemical properties. The local food samples namely rice,
beans moin-moin, pounded yam and amala were collected into
sterile Petri dishes from selected cafeteria in Akure and were taken
to the laboratory for microbiological examination.

Antibacterial activity of honey samples and antibiotic sensitivity


tests: An agar diffusion method was used to access the
antibacterial activity of the selected honeys against the 5 isolated
bacterial strains. A cell load of 107-108 cfu/ml in their log phase of
isolated bacteria were inoculated in Mueller- Hinton broth and
incubated for 10 h. Petri dishes containing 1ml of each bacterial
isolate in the broth and Mueller-Hinton agar were prepared. Wells
were bored with a sterile cork borer on the seeded agar plates. The
honey, water and antibiotics were aseptically pipetted into the
bored well and the plates were incubated uninverted at 37C for 24
h. The water and antibiotics served as negative and positive
controls, respectively. The sensitivity of the tested organisms
was indicated by zones around the wells and diameter was taken
as an index of degree of sensitivity.

Microbiological examination of honey and food samples: The


fresh honey samples were conveyed to microbiology research
laboratory of Federal University of Technology, Akure. The honey
samples were filtered separately with sterile Seitz filter and the
obtained filtrates were aseptically streaked on nutrient agar and
potato dextrose agar plates and incubated at 37C for 24 h and
272C for 72 h, respectively, to observe for bacterial and fungal
growth. After incubation, the honey plates where no growth were
observed were dispensed into sterile Pyrex sample bottles and
kept at room temperature prior to further analyses. One g of each
of the food samples was weighed and with the use of sterile spatula
homogenized inside 9 ml of sterile distilled water. This was serially
diluted and 0.1 ml was pour plated on nutrient agar and potato
dextrose agar plates and incubated at 37C for 24 h and 272C for
72 h, respectively, to observe for bacterial and fungal growth.

Results and Discussion


Bacteria and fungi isolated from the tested food samples included
Bacillus cereus, Bacillus subtilis, Escherichia coli, Aspergillus
fumigates, Salmonella typhi and Varicosporium elodea (Table 1),
most of which are pathogenic microorganisms. However, Bacillus
subtilis is also a spoilage bacterium. Varicosporium elodea is
spoilage fungus while Aspergillus fumigates, a fungus, has a
potential to produce mycotoxins.
Most foods contain viable bacteria and fungi, which could be
result of improper handling, exposure to dust, air, flies or result of
the food being under heated. These food spoilage organisms can
also be pathogenic to consumers. Endospores of Bacillus species
are more resistant to heat which could be the reason why this
species occurred most in the food samples. Physiologically, these
organisms could produce chemical changes in foods, such as
breaking down of proteins to polypeptides, amino acids, fats to
Physico-chemical analyses of honey samples: The pH of the glycerol and fatty acids, and hydrolysis of complex carbohydrates
samples was measured using a pH meter according to the method to simple ones. Unpleasant odours resulting from gases formation
described by Gonnet 9. The moisture content was obtained by may occur. Spoilt rice is by nature slimy and rotten. This nature
drying the granulated pulp in a hot air oven under vacuum at readily provides a veritable and suitable environment for the
105C until a constant weight was attained and the ash content growth of fungal isolate like Aspergillus fumigates. This was in
by incinerating dried samples in a muffle furnace at 550C for 6 h. agreement with the work of Leveen et al. 14. Vit et al.26 reported
They were then cooled in a desiccator and weighed immediately 4. that the pH and temperature of food substrate readily support the
The mineral contents were analyzed using the atomic absorption growth of mesophilic food spoilage organisms such as fungal isolates.
and spectrophometer method as described by Paulwels et al. 21.
The physicochemical properties of the different samples of
The minerals analyzed included calcium, potassium, sodium and honey obtained from different locations in Ondo, Nigeria, are
phosphorus. Vitamin C content was also analyzed. Electrical reported in Table 2. The color ranged from light amber to completely
conductivity was determined by measuring 20 g dry matter of dark. The pH values were in the range of 2.90 to 4.40. Although
honey in 100 ml of ultra pure water. This was mixed thoroughly to there was significant variation between honey obtained from
form a solution. The electrical conductivity cell was immersed at Itaogbolu and the remaining locations. The values were within
20C while reading was expressed in mhos. The colour of the the reference value and in agreement with White 11 who reported
honey samples was determined by using the P- fund scale (mm). that honey was characteristically quite acidic. The pH of honey is
Two ml of the honey samples was poured in a beaker, the instrument low enough to inhibit the growth of many species of bacteria but
was calibrated, dipped into the sample and the reading was taken. this acidity is neutralized in the body by buffering fluids. The
moisture content investigated varied from 13.28-17.98%. The
lowest was recorded in Igbara Oke sample while the highest was
from Ikare Akoko samples. This could
Table 1. Frequency of microorganisms isolated from selected food samples.
be as a result of the composition and
floral origin of the honey samples. The
Food samples
Bacillus
Bacillus Escherichia Aspergillus Salmonella
Varicosporium
low moisture content property of
cereus
subtilis
Coli
fumigatus
typhi
elodea
honey serves as a protection from
Beans
+
+
+
+
Yam
+
+
+
+
attack by microorganisms 25. When the
Moin- moin
+
+
+
+
+
moisture content is high, it is an
Amala
+
+
+
indication of adulteration. According
Rice
+
+
Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

105

106

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

15.21.03

0.55.01

0.1801

0.28.02

0.11.02

0.13.02

0.05.01

0.60.0.2

0.10.0.1

0.70.0.1

0.30.0.1

Ash
(%)

38.50.02

3.60.0.1

21.00.0.2

4.00.0.1

17.60.0.1

19.20.0.1

45.00.0.2

12.50.0.1

51.50.0.2

23.00.0.2

Electrical
conductivity
(mhos)

1.50.01

2.00.01

1.60.02

2.90.02

2.60.01

2.10.01

1.00.03

1.40.02

1.80.01

1.90.03

Ca2+

0.68.01

0.19.01

1.32.02

0.70.02

1.60.01

0.20.01

1.36.02

0.65.01

1.11.03

0.50.01

K+

22.451

21.241

20.012

22.981

22.012

32.422

50.431

18.611

44.901

50.002

Mineral content(g/g)

20.28

9.68

20.30

20.28

17.69

40.22

42.515

75.26

30.91

30.31

Na+

NR

102
mm

NR

102
mm

NR

NR

NR

C
112
mm
122
mm
152
mm

B
132
mm
102
mm
162
mm

A
142
mm
132
mm
182
mm
21
mm
162
mm
NR

NR

NR

NR

Selected honey samples


D
E
F
G
122
132
122
112
mm
mm
mm
mm
132
122
142
102
mm
mm
mm
mm
152
202
182
152
mm
mm
mm
mm
21
NR
NR
NR
mm
102
152
102
102
mm
mm
mm
mm
NR

102
mm

NR

H
132
mm
122
mm
152
mm

NR

I
152
mm
162
mm
212
mm
102
mm
182
mm
NR

102
mm

NR

J
92
mm
112
mm
182
mm

NR

102
mm

NR

AM
102
mm
102
mm
112
mm

NR

182
mm

NR

CPX
132
mm
102
mm
172
mm

NR

112
mm

NR

51
mm

NR

202
mm
NR

71
mm

NR

NR

NR

GRI

NR

PEF
122
mm
122
mm
202
mm

35.121

32.341

30.323

31.331

32.422

32.422

33.453

30.234

35.542

34.231

32.422

Glucose

Key: NR= No zone of inhibition A=Akure, B= Owo, C= Akungba, D= Ile-oluji, E= Ondo town, F=Ikare- Akoko, G=Ilara, H=Igbara Oke, I=Itaogbolu, J=Iju, AM=Ampiclox, CPX=Ciprofloxacin, S=Streptomycin,
PEF=Pefloxacin, GRI= Griseofulvin.

Tested
organisms
Bacillus
Cereus
Bacillus
subtilis
Escherichia
Coli
Aspergillus
fumigatus
Salmonella
typhi
Varicosporium
elodea

Light
Amber
Light
Amber

Amber

Amber

Amber

Amber

Amber

Dark
Amber
Dark
Amber
Light
Amber

Color

Antibiotics
S
102
mm
122
mm
132
mm

1.40.02

4.30.01

3.40.01

1.80.02

2.35.03

1.90.02

3.40.01

1.25.01

3.75.01

2.00.02

Vitamin C
(mg/10ml)

Table 3. Antibacterial activity of honey samples and antibiotic sensitivity test on food spoilage organisms.

Reference values from Tchoumboue et al. 24, Vinda-Martos et al .2,Anon., 3; Crane 6,7.

3.12.20

Iju

13.28.03

3.89.11

14.14.02

13.61.01

4.00.21

2.90.07

17.98.01

3.21.12

Itaogbolu

16.00.02

16.75.01

16.21.01

3.27.05

4.40.01

Ile-oluji

Ondo
town
IkareAkoko
Ilara
Igbara
Oke

4.40.02

Akungba

17.00.01

13.40.02

4.35.15

3.28.02

Moisture
(%)

pH

Owo

Akure

Honey
samples

Table 2. Mean valuesSE of physicochemical properties of selected honey samples.

32.422

33.661

30.121

28.422

27.524

29.123

31.003

31.225

30.112

29.421

33.122

Fructose

Color=light to
amber

Vitamin C = ___

Na+ = 0.6- 40

P=2.0-60.0

K =1.0-47.5

Ca2+ =4.0-30.0

Electrical
conductivity= __

Ash=0.020-1.030

Moisture(%)=13.426.6

pH=3.4-6.1

Glucose=
22.0-40.7
Fructose= __

Reference values

to the international regulatory standard for honey 10, honey with


high water content aids fermentation or deterioration. Values for
the ash content ranged from 0.10% to 0.70%. This variability in
ash content could be explained by the floral source of the honey 26.
Obtained values are within the reference value. The mineral content
from Akungba Akoko and Ondo town turned out to be in
decreasing order of Na, P, Ca and K while in samples from the
remaining locations, the phosphorus content is highest followed
by sodium and the least mineral content is potassium. The presence
of these minerals makes it nutritionally suitable for both children
and elderly people. Variation in mineral content was recorded from
the reference value in some honey samples for instance the sodium
ion content samples from Akungba Akoko and Ile Oluji did not
agree with reference value. The electrical conductivity values
varied from 3.60 to 51.50 mhos. According to International Standard
of honey (2002), the electrical conductivity of honey is near zero
hence higher conductivity indicates adulteration of the honey sample.
The bioactivity of honey samples compared favourably with
the tested conventional antibiotics. The honey sample showed
more inhibitory potency against the tested bacteria than the fungal
isolates (Table 3). Generally honey sample from Itaogbolu
displayed high inhibitory potency against all the tested organisms
with the highest zone of inhibition recorded against Escherichia
coli (212 mm), this in no doubt could be because of the
physicochemical quality this sample possessed, although its
bioactivity against fungal isolates was low compared to the
conventional antibiotics. In all the honey samples tested none
was able to inhibit Varicosporium elodea. The antibiotic
griseofulvin, was able to inhibit Varicosporium elodea with 52
mm zone of inhibition while (71mm) was recorded for Aspergillus
fumigatus, however, honey samples from Itaogbolu showed
greater zone of inhibition than this antibiotic (10 2 mm). This
study revealed that the honey samples possess different
antimicrobial activities, which agreed with the study conducted
by Omoya and Akharaiyi 19 that honey in its saturated solution of
sugar will cause osmotic effect on bacteria.
Conclusions
The results confirm that the physicochemical parameters, such as
moisture content, pH level and sugar content of different honey
samples, influence their bioactivity against microorganisms. The
honey samples were able to establish variable inhibitory zones in
vitro. Favourably comparison was established between the honey
samples and the conventional antibiotics employed on the tested
spoilage organisms. Honey may therefore be a potential eco-friendly
intervention to control food spoilage and pathogenic microorganisms.
Acknowledgements
Acknowledgements to Omoya and Associates for funding of this
study. Acknowledgements also to Fred Akharaiyi of Microbiology
Department FUTA and Department of Biochemistry FUTA for their
technical assistance.
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3

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

107

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e-mail: info@world-food.net

Journal of Food, Agriculture & Environment Vol.12 (3&4): 108-114. 2014

www.world-food.net

Shigatoxin-producing Escherichia coli in raw cow milk from small farm producers
and phylogenetic subtype determination
Ivo Sirakov, Ralitsa Popova, Hristo Daskalov *, Iskra Slavcheva, Eva Gyurova and Boyko Mitov
National Diagnostic and Research Veterinary Institute, Blvd. Pencho Slaveykov 15, 1606 Sofia, Bulgaria.
*e-mail: hdaskal@hotmail.com
Received 30 April 2014, accepted 10 September 2014.

Abstract
Cases of unauthorised direct sale of raw cow milk from small farms do exist in Bulgaria. In 2012, we tested 80 samples of raw cows milk from small
farms in south-western Bulgaria for the presence of shigatoxin-producing Escherichia coli strains. Twenty-three of these samples were taken from
cows with subclinical mastitis. The tests included isolation on selective solid medium, biochemical detection and conventional PCR for detection of
the shigatoxin genes (stx1, stx2), the intimin gene (eae) and the enterohemolysin gene (hlyA). The stx1 gene was detected in three Escherichia coli
isolates from three cows milk samples from animals with subclinical mastitis. An Escherichia coli strain with the intimin eae gene was isolated from
a sample of normal cows milk from a small farm. Two of the isolates with the stx1 gene were also found to harbour the enterohemolysin hlyA gene.
The presence of virulence factors in the four Escherichia coli isolates was additionally validated using Real-time PCR. The amplified virulence genes
were sequenced and a phylogenetic analysis based on amino acid sequences was performed. The cytotoxic effects of the E. coli isolates were studied
on Vero cells. The potential role of shigatoxin-producing E.coli strains for provoking food-borne illness is discussed.
Key words: Escherichia coli, raw cow milk, mastitis milk, PCR, virulence factors.

Introduction
Raw milk produced in very small farms which do not meet the EU
quality and safety requirements could be a risk factor for consumer
health. Raw cows milk from such farms is on the market in violation
of the regulations for the sale of raw milk. There is also a hazardous
practice of mixing milk from healthy animals and subclinical mastitis
milk. Escherichia coli is a microbial agent which is often found in
raw milk, especially in cases of poor hygiene during milking and
primary processing. E. coli causes mastitis or concomitant
infections in lactating cows. According to Schoonderwoer 1, E.
coli is naturally found in the gastrointestinal tract in animals, but
some strains, such as shiga-toxin-producing E. coli, are associated
with diarrhea in humans and animals. Other authors 2, 3 emphasise
that verotoxin-producing E. coli (VTEC) can cause serious illness
in humans, beginning with mild diarrhea and leading to hemorrhagic
colitis and hemolytic uremic syndrome. Mainil 4 point out that a
key factor for E.coli infections in humans are ruminants excreting
E.coli in their feces. VTEC may be transmitted through
consumption of undercooked meat, unpasteurised milk products,
vegetables and water contaminated with the feces of carrier animals.
Cases of person-to-person transmission are also known 3, 5, 6. Animals
intended for food are considered to be the main source of VTEC/
EPEC (enteropathogenic E.coli) strains. In humans, life-threatening
infections associated with consumption of milk and milk products
contaminated with VTEC/EPEC have been reported 7. The main
etiological agent in infections with enterohemorrhagic E.coli is
generally thought to be Escherichia coli O157:H7. It is still unclear,
which other factors in addition to verotoxin production are
involved in the transformation of shigatoxin-producing-E.coli
(VTEC) into enterohemorrhagic E. coli (EHEC). Each VTEC isolate
should be considered potential EHEC and the detection methods
108

should be targeted at detection of verotoxin rather than of the


serotype 8. Another key factor in the virulence of VTEC isolates is
enterohemolysin (Ehly), which is coded by the enterohemolysin
ehxA gene, also known as EHEC-HlyA. The relation of EHEC
hemolysin and verotoxin production in pathogenic E.coli
describes the possible role of EHEC hemolysin in bacterial
virulence 9. Subclinical mastitis caused by E. coli brings about
substantial economic losses in dairy cattle herds because of
reduced amount and quality of the produced milk, incurred
veterinary treatment costs and in some cases death of the diseased
animal 10. Milk can be contaminated with pathogenic microorganisms
through fecal contamination as well as from colonized teats or
from an infected udder in the case of clinical or subclinical mastitis
during the milking process from the environment or from
contaminated water 11. Polymerase chain reaction (PCR) is an
effective method for detection of extremely low quantities of
nucleic acids in various types of samples. That is why PCR is a
common technique used for detection of various food-borne
pathogens.
The aim of the present study was to analyse samples of raw
cows milk, including milk taken from cows with subclinical mastitis,
for the presence of E. coli isolates with specific virulence markers
(stx1, stx2 and eae) by means of PCR and to evaluate their possible
role in provoking food-borne illness in consumers.
Materials and Methods
A total of 80 raw cow milk samples were analysed. The milk was
collected from January to December 2012 from unauthorised
sources (small farms or individual sellers).

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Mastitis assay: All milk samples were analysed according to an


official reference method, the microscopic method for counting
somatic cells described in the International Standard ISO 133661|IDF 148-1 12.
Isolation of Escherichia coli: To obtain Escherichia coli
isolates, 10 g of each sample were weighed and added to 90 ml
of buffered peptone water and the resulting suspensions were
incubated at 37C for 24 h. Then, the broth was used for plating
on selective nutrient media: Sorbitol McConkey Agar (SMAC,
Merck, Darmstadt, Germany), Tryptone Bile X-Glucuronide Agar
(TBX, Merck, Darmstadt, Germany) and McConkey Agar
(HiMedia, India). The plates were incubated for 18 h to 24 h at 37C.
Following cultivation, the selective media were observed for
typical growth. Escherichia coli isolates were simultaneously
analysed for the presence of the stx1, stx2 and eae genes.
Biochemical identification: For biochemical identification typical
colonies of Escherichia coli from the selective media were plated
on nutrient agar and further identified with Kovacs reagent
(Merck, Darmstadt, Germany) for the presence of indole and with
the oxidase test (Oxidase test, HiMedia, India). The isolates which
were positive for stx1 and eae genes by conventional and RT PCR
were also characterised based on 21 specific biochemical
reactions 11 MICRONAUT-E plates (Merlin, Germany).
Identification of serotype O: Whether the isolates identified as
E. coli belong to serotype O was determined with E. coli OK
Pool 1 (O26, O103, O111, O145 and O157) sera obtained from the
Staten Serum Institute (Copenhagen, Denmark) based on
agglutination reaction.
DNA extraction: A minimum of 10 colonies with typical E.coli
morphology were selected from the selective agar plates and were
re-plated on nutrient agar (HiMedia, India) for 18 h to 24 h at 37C.
For conventional PCR amplification of the stx1, stx2 and eae genes,
DNA extracted from the isolated colonies, was resuspended in
100 l of sterile distilled water and heated to 100C for 5 min. DNA
was extracted again from the three stx1 positive colonies and the
eae positive colony with InstaGene DNA Purifica Matrix (BioRad,
Hercules, CA, USA) to validate the positive amplification results
by means of real-time PCR.
Conventional PCR: DNA extracted form E. coli isolates was
amplified by conventional multiplex PCR with primers specific for
the stx1, stx2 and eae gene described in previous research 13.
The PCR reaction was performed with the HotStart-ITFideliTaq
USB Mastermix (Affymetrix, Santa Clara, CA, USA), according to
the manufacturers instructions. The final reaction mixture volume
was 25 l. The primers were added at a final concentration of 10
pmol/l each. All samples were compared with a positive control
strain C210-03 provided by WHO in Copenhagen, which harbours
the three genes. The samples were proved to be contamination
free with a negative control containing distilled water instead of
DNA.
The amplification of stx1, stx2 and eae genes was performed as
described by Paton and Paton 13 with some modifications for this
study. Amplification of the stx1, stx2 and eae genes was performed
in a Techne thermocycler as follows: 95C/5 min, 40 cycles of

95C/1 min, 62C/1.30 min, 72C/1.30 min and 72C/7 min. The
amplification for HlyA was according to Schmidt et al. 9. The
electrophoresis of the amplified products was run in 2% Agarose
(Genshun Biotech, Guangzhou, China), 1Tris-EDTA buffer,
pH 8.1, 120 V, 40 mA for 30 min. A 100 bp molecular weight
marker (Genshun Biotech, Guangzhou, China) was used. Ethidium
bromide 1 g/ml (Sigma-Aldrich, EU) was used for staining.
Real time PCR: The sequences of the primers and probes used in
the real-time PCR assay are according to Perelle et al. 14 and Nielsen
and Andersen 15.
A 5 nuclease PCR assay was used with probes labeled with
FAM as a reporter dye at the 5-end and TAMRA as a quencher
dye at the 3-end. The Maxima Probe/RoxqPCR Master Mix (2)
(Fermentas, Lithuania) was used, following the manufacturers
instructions. The reaction mixture was with a final volume of 20 l.
For amplification of the stx1 and the stx2 gene the following
reagents were used: 4 mM MgCl2, 1 M of each primer, 200 nM
probe and 2 l of template DNA, according to Perelle et al. 14. For
amplification of the eae gene, the final concentration of the probe
was 200 nM and that of each primer was 600 nM, according to
Nielsen and Andersen 15. The pUC19 plasmid (New England
BioLabs, MA, USA) was employed as an internal amplification
control with the primers and the probe described by Fricker et al. 16
and IAC concentration of about 100 copies per PCR reaction.
Amplification was performed in a Step One Plus apparatus
(Applied Biosystems, Foster City, CA, USA), according to Perelle
et al. 14 for the stx genes and Nielsen and Andersen 15 for the eae
gene.
Cell culture assay: Stocks of Vero cell line purchased from Robert
Koch Institute were used after trypsinisation and growth as a
monolayer at 37C in rotation. The procedure by Konowalchuk et
al. 17 was used with some modifications. MEM-Eagle in Earles
BSS (GIBCO) was used as a medium for Vero cells with addition of
0.2 M L-glutamine, 0.075% NaHCO3 for pH 7.4 and fetal calf serum,
10% for growth medium and 2% for supporting medium (GIBCO)
and Gentamycine 50 mg/mL. A quantity of 0.05 mL of bacterial
filtrate from a 24 h bacterial culture was added to the cells for toxin
activity assay. Cultures were incubated at 37C. Morphological
effects of the cultures were screened on 18, 24, 48, 72 and 96 h.
16S rRNA gene amplification, sequencing and data processing:
As additional identification we used 16S rRNA gene sequencing
as described before 18. Nucleotide sequence alignment was
performed with the Muscle algorithm 19. The sequencing data of
the 16S rRNA gene were analysed for close homology using the
Basic Local Alignment Search Tool (BLAST) available at the
National Center for Biotechnology Information (NCBI, Bethesda,
MD) (http // :www .nbi .nlm. nih. Gov /BLAST) 20, 21.
Only nonsynonymous substitutions could lead to changes in
the amino-acid structure of proteins. Because of that, for the
purpose of amino-acid analysis of the PCR products, we used the
nucleotide sequences with a length of 180 bp (between 746-925
bp determined by reference sequence AF461169.1) and 357 bp
(between 40-396 396 bp determined by reference sequence
AJ877229.1), respectively, as previously described 22, corresponding
to the stx1 and eae genes, the number for the eae gene nucleotide
sequence in GenBank being KC196849. The predicted amino-acid

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

109

sequences were obtained using the MEGA 5 software 23.


The protein sequencing data was compared with sequences
deposited in NCBI GenBank (Table 1). These sequences were
used to build amino acid phylogenetic trees.
To find the best model for the construction of phylogenetic
trees based on amino acid sequences, ProtTest 2 24 was used.
PhyML 3.0 software 25 by Phylemon 2 26 and 1000 bootstrap
replications were used in building the phylogenetic trees. The
graphical view of the phylogenetic trees was generated with
FigTree1.4.0 software (http://tree.bio.ed.ac.uk/software/figtree/).
The amino acid sequence of the eae gene region of isolate
number 7 has an ID: AGC29881.1 for access in NCBI. The amino
acid sequence of the stx1 gene was not sent to NCBI due to its
short length.
Results
Escherichia coli was isolated from 56 of the 80 analysed raw
cows milk samples, whereas typical growth was not observed
in 24 samples. An increased somatic cell count (500,000 SC/
cm3) was scored in 23 cows milk samples. These 23 samples were
also shown to contain E. coli. The appearance of the milk and the
results from the microscopic reference method confirmed that the
animals were suffering from subclinical inflammation of the
mammary gland.
The 56 isolates showed the indole-positive and oxidasenegative biochemical profile typical for Escherichia coli. Next,
the 56 Escherichia coli isolates were assayed by conventional
PCR for amplification of the stx1, the stx2 and the eae gene. None
of these three genes was detected in 52 of the isolates. A specific
band for the stx1 gene, however, was obtained for three Escherichia
coli isolates originating from three different cows milk samples
(lab numbers 6, 298-3 and 298-4). The eae gene was detected in an
Escherichia coli strain isolated from a sample of normal cows
milk (lab number 7) (Fig. 1). In two of the E. coli strains positive
for stx1, presence of the gene for enterohemolysin hlyA (lab
numbers 6, 298-3) was found. The positive results in the
conventional PCR assay were validated by real-time PCR. The
isolates of Escherichia coli that were positive for stx1 and eae
genes were further validated with the MICRONAUT-E plates
(Merlin).
In addition, the four Escherichia coli strains that were found to
be positive for virulence factor genes (stx1 or eae) were tested for
the presence of serogroups O111, O145, O103, O157 and O26 most
frequently related to human disease. The isolates were not found
to belong to any of these serotypes.
Some differences were observed in the MICRONAUT-E
biochemical profiles of the four Escherichia coli strains positive
for virulence factors (stx or eae). The eae-positive isolate was
esculin-positive and ornithin decarboxylase-negative. The three
stx1-positive strains (6, 298-3, 298-4) were esculin-negative and

Figure 1. Amplification of vtx1 (180 bp) and eae (384 bp) by


conventional PCR.
A: Lane 1: 100 bp DNA molecular weight marker. Lane 2: Negative control. Lane 3:
Sample 6. Lane 4: Sample 2398-3. Lane 5: Sample 298-4. Lane 6: Sample 7. Lane 7:
Control strain harbouring the stx1, the stx2 and the eae gene.

ornithin-decarboxylase-positive. The stx1-positive strain 298-4


was arginine-dehydrogenase-positive, whereas the other three
isolates (6. 298-3 and 7) were arginine-dehydrogenasenegative.
The first destructive effects of the bacterial filtrates tested on
Vero cells were obvious at different sampling times for the different
isolates. The first evident effect on the monolayer was for the
reference strain that was also included on 24 h. The effect of our
isolates on the monolayer was visible at 48 h after inoculation:
two of the strains positive for the stx 1 gene (6, 298-3) showed the
strongest effect and the third strain positive for stx1 (298-4) had a
slightly weaker effect on the Vero cells. The monolayer inoculated
with the strain positive for the eae gene (lab number 7) appeared
normal after 48 h and showed effect on the cells at 72 h (Fig. 2).
After sequencing of the PCR fragments for the 16S rRNA gene,
sequencing products with a length of 600 to 632 bp were obtained.
After alignment of the sequences, a product with a length of 590
bp was used for analysis. BLAST analysis gave an identity value
of 99% with E. coli. The sequences of the 16S rRNA gene were
deposited in GenBank with the following accession numbers:
KF429755, KF 429756, KF 429757 and KF 429758 for isolates 7, 6,
298-3 and 298-4, respectively. Our results from sequencing 16S
rRNA as well as the biochemical characterisation of the strains
confirmed that the isolates were E. coli.
The part of the eae gene which was examined covers codons
14-132. In comparison with the reference strain 210-03 for the
eae gene our isolate with number 7 showed amino acid differences
(in the part examined by us) in codons 20, 24, 25, 26, 36, 48, 56, 57,
58, 59, 61, 75, 76, 81, 83, 86, 105, 118, 119 and 126. The amino acid

Table 1. NCBI reference numbers for amino-acid sequences of E. coli stx1 and eae proteins included in this study.
NCBI protein ID
stx1 protein source
subtype
NCBI protein ID
stx1 protein source
subtype
NCBI protein ID
eae protein source

CAC85628.1
E.coli ovine
stx1 c
AFM85307.1
E.coli feces
stx1 a
CAI46996.1
human

CAC85553.1
E.coli human
stx1 c
AAO19475.1
E.coli bovine
stx1 d
ABF69117.1
no data

CAA85370.1
E.coli Australia
stx1 c
BAD08527.1
E.coli bovine
stx1 d
AAC32028.2
rabbit

ABE02587.1
E.coli Hungary
stx1 c
AAY43857.1
E.coli shellfish
stx1 d
AAB97764.1
bovine

AAM70037.1
E.coli human
stx1 a
AAY43852.1
E.coli shellfish
stx1 d
BAL47133.1
Hirundo rustica

AAA26538.1
S.dysenteriae
stx1 a
BAD08529.1
E.coli bovine
stx1 d
AAK48432.1
goat kids

AAA98347.1
S.dysenteriae
stx1 a
C210-03
BAL47187.1
human

Positive control strain C210-03 kindly provided by WHO in Copenhagen.

110

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Figure 2. Destructive effect of strains 298-3, 298-4, 7 on the


monolayer at 24 h and monolayer control of Vero cells.

sequence in these codons for the reference strain was


LRCRQGHDSYNDLDNTAPFL and for our isolate was
FSAGNSQNAADNIGSVGSVV.
The part of the stx1 gene which was examined, covers codons
152-211 of SubunitA. Our data from a previous study showed that
in our isolates only the point mutation at position 791 is
nonsynonymous, where C is replaced with T. In this region, the
protein analysis revealed that our isolates differ from the reference
strain C210-03 at codon 167, where there was Y (tyrosine) in
contrast to H (histidine) in the reference strain.
The ProtTest 2.4 software, considering the length of the
analysed fragments, their number and existing amino-acids showed
that the most appropriate models for constructing amino acid
phylogenetic trees for the eae and stx1 genes were MtArt+F and
JTT, respectively.
The amino-acid based phylogenetic analysis differentiated three
groups based on the sequences predicted from the eae gene (Fig. 3)
and two groups based on the sequences predicted from the stx1
gene (Fig. 4).
Discussion
Verotoxigenic Escherichia coli can cause serious illness in
humans, such as hemorrhagic uremic syndrome and hemorrhagic
colitis. However, it is still not clear whether all VTEC variants are
equally pathogenic to humans, having in mind that the presence
of stx1 alone may not be sufficient for VTEC to cause illness in
humans. It has been considered that eae-negative VTEC are not a
serious threat to human health, unlike classical EHEC 27. The results
from our study showed four isolates with virulence factors: Three
stx1-positive strains and an eae-positive one. Both these genes
together, which would pose a higher potential risk to consumer
health, were not found in any of the isolates studied by us. Some
of the infectious potential of E.coli is due to its ability to survive
for a few hours in low acidic conditions (pH<3.0) and thus to
survive in the stomach and further pass in the intestine 28-30. It has
been determined that the high acid resistance of E.coli depends
on genes for decarboxylation of arginin AdiA 31. These data may
suggest that our strain 298-4 (arginine-dehydrogenasepositive)
could in certain conditions show higher pathogenic potential in
humans. During cheese ripening, the pH decreases and pathogens

Figure 3. Phylogenetic tree of the eae gene sequence from the milk
isolate (Protein ID in NCBI AGC29881.1) in comparison with Escherichia
coli reference strains.
The tree was calculated from 119 amino acid aligned positions in the final data set, using the
MUSCULE algorithm. One thousand bootstrap replications were used to build a
phylogenetic tree.

Figure 4. Phylogenetic tree of the vtx1 gene sequences from the milk
isolates in comparison with Escherichia coli reference strains and
Shigella dysenteriae.
The tree was calculated from 60 amino acid aligned positions in the final data set, using the
MUSCULE algorithm. One thousand bootstrap replications were used to build a
phylogenetic tree.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

111

which can survive in low acid environment may become a real


threat when consumed by humans. For example, Muehlherr et
al.32 reported that 16% of the raw goats milk in Switzerland is stxpositive. The results of Orden et al. 33 show that stx1 is the
predominant stx genotype in healthy goats. Interestingly, in our
experiments with raw cows milk, three stx1-positive isolates were
obtained from milk with subclinical mastitis, whereas the eaepositive strain was isolated from a sample of raw cows milk that
met the somatic cell count standards. Since the stx1-positive
isolates were obtained from milk samples with subclinical mastitis,
it could be suggested that bacteria from the teats probably
contaminate the milk following colonisation of the milk duct or
from the infected mammary gland in cases of clinical or subclinical
mastitis. Another possible scenario is for contamination to happen
at some stage of the milking and milk processing technology 11, 34.
There is evidence that mastitis in cows can be caused by E.coli 35,
but there are few data about mastitis caused by E. coli O157 or
VTEC, although they could also contaminate the milk from the
udder of cows with mastitis 36. In dairy herds, fecal contamination
of the udder poses a risk for pathogen contamination of milk. That
is why, it is important to prevent fecal contamination of the udder
in order to reduce VTEC and other pathogens in raw milk. There
are data that other dairy domestic animals apart from cows, e.g.
goats and ewes, also excrete these pathogens with their feces,
which could also lead to milk contamination 37.
The Escherichia coli isolates obtained from raw cows milk in
our experiments are in agreement with the results of Hassan and
Elmalt 38, who isolated Escherichia coli from 38 out of 50 samples.
The authors carried out a PCR assay and identified stx2 in three of
the isolates, stx1 in one of the isolates and the intimin-coding eae
gene in none. Unlike their results, our experiments did not reveal
any isolates harbouring the stx2 gene, but showed one with the
eae gene. Pradel et al. 7 also observed a prevalence of stx1-positive
isolates in dairy products. Since most STEC can be found in the
intestines of ruminants, it is possible for fecal contamination to
happen during the milking process or as a result of crosscontamination from other animals in the farm, which could become
a means for VTEC transmission 39.
Some reports 7 indicate that shiga-toxin production may be
associated with production of hemolysin as a virulence factor,
which is confirmed by our results.
The eae gene, which codes for the outer membrane protein
intimin, was amplified in only one of our raw cows milk samples,
whereas the stx1 gene was identified in three of the mastitis milk
samples. The eae gene was detected in 14 Escherichia coli isolates
from raw yaks milk in India 40. Six of these 14 eae-positive isolates
were found to harbour only the eae virulence factor gene without
stx genes, identifying the strains as enteropathogenic Escherichia
coli (EPEC). In an earlier study, the same authors determined a
considerably lower EPEC counts in rectal swab samples collected
from animals 41.
The phylogenetic groups determined on the basis of aminoacid sequences predicted from the nucleotide sequences of the
stx1 and the eae gene differ from those obtained in our previous
study, where nucleotide phylogenetic analysis was carried out 22.
The results of the amino acid phylogenetic analysis differed from
those of the nucleotide phylogenetic analysis for the stx1, where
two main groups were formed as well as 2 and 3 subgroups. One
of the branches included a group of isolates with stx1a and one
112

group with stx1c. The other branch included three groups with
isolates possessing stx1d (our isolates were placed in a separate
group). This is due to the fact that 8 mutations were determined
(compared to the reference strain). The difference in the topology
of our isolate and the reference strain C210-03 regarding the eae
gene on the basis of the amino acid sequences was determined by
20 nonsynonymous mutations out of 47 mutations in the
nucleotide chain of the examined part. Moreover, a change in the
primary amino acid sequence is a prerequisite for a change in the
secondary and tertiary structure of the protein molecule (the toxin).
This, in turn, incurs a phenotypic change, which in our particular
case affects probably the toxicity of the isolates. Our suggestion
could be supported by the phylogenetic analysis of Scheutz et al. 42,
who demonstrated how specific sequences of stx2 variants could
affect the biological activity of the toxin. The authors determine
Shiga toxin 1 and their nomenclature as stx1a, stx1c and stx1d.
They establish higher percentage similarities between stx1a and
stx1c in contrast with stx1d. This could be confirmed with the
phylogenetic analysis which proved stx1d to be the most distant
and different outbranch of the tree, compared to other two variants.
The nonsynonymous substitutions are important for the structure
and function of the amino-acid chain, as they change the primary
structure of proteins. That is why we performed amino acid
phylogenetic analysis. The result corresponds to Scheutz et al. 42
data; however, there were some differences as compared with the
nucleotide analysis: particularly, two branches were formed with
no additional groups. Stx1a and stx1c were part of one of the
branches and stx1d formed a separate branch. These data show
that the nucleotides in the region of the stx gene could be
appropriate for subtyping of isolates after sequencing and
phylogenetic analysis, while amino acid analysis could clearly
differentiate only stx1d. According to these data, we can conclude
that our isolates belong to stx1d.
Conclusions
Our study showed occurrence of the stx1 gene in Escherichia
coli isolates in milk from cows with subclinical mastitis (3 out of
20 samples). The obtained results indicate that some Escherichia
coli may harbour the intimin-coding eae gene. The existing
unauthorised sales of raw cows milk (this is how the samples
analysed by us were collected) raise the question as to the safety
of the consumed milk and dairy products. Escherichia coli is a
well-known causative agent of mastitis in ruminants. Our study
showed that stx1-positive E. coli strains are also isolated and
could apparently be involved in mastitis development. The results
obtained by us also throw more light on the genetic diversity of
the four isolates and on their phenotypic changes, especially those
associated with the toxicity of the isolates.
The amino-acid phylogenetic analysis separated the studied
strains into two branches on the basis of nonsynonymous
substitution in codon 169 of stx1. This clearly shows that this
mutation is a key factor that determines the stx1d subtype and
differentiates this subtype from stx a and stx1c, but does not
differentiate the latter two subtypes from each other.
Acknowledgements
The authors thank EU RL VTEC, Rome, Italy and WHO in
Copenhagen for providing reference strain and competent support.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

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Pre-processing glutinous rice effects on textural and morphological changes


Viboon Pansa-ead 1, Kannika Huaisan 2, Supan Yangyeun 1 and Songchai Wiriyaumpaiwong 1*
Faculty of Engineering, Mahasarakham University, Khamrieng, Kantarawichai, Mahasarkham, 44150, Thailand.
Faculty of Agro Industrial Technology, Rajamangala University of Technology Isan, Kalasin Campus, Kalasin 46000, Thailand.
*e-mail: songchai.w@msu.ac.th
1

Received 12 May 2014, accepted 30 September 2014.

Abstract
Glutinous rice is frequently enjoyed at snack and meal time in Southeast Asia. Khaowong Kalasin is a cultivar of glutinous rice in Thailand, and its
name is a geographical indicator of Kalasin province in Northeast Thailand. Its quality can be described as outstandingly soft and sticky. This study
addresses the soaking and cooking methods that can be used for Khaowong Kalasin glutinous milled rice to achieve a uniform texture. Textural and
sensory evaluations were compared between Khaowong Kalasin and RD6 cultivars in a container stored overnight. More specifically, the effects of
drying and temperature conditions on rehydration, texture, and external visualization for instant Khaowong Kalasin rice were investigated. Soaking
times were tested from 30 to 90 min. Three cooking methods were used: firewood with bamboo basket and earthenware steamer, LPG with bamboo
basket, and aluminium pot steamer and a modern programmable rice cooker. Temperatures from 40 to 60C with hot air drying and freeze drying were
evaluated. To reduce variations of hardness and stickiness, glutinous milled rice was soaked for 60 min and cooked in a programmable rice cooker
instead of using two traditional methods: firewood with bamboo basket and earthenware and LPG with bamboo basket and aluminium pot.
Khaowong Kalasin cooked rice could be stored in a container for a longer period of time than the RD6 cultivar. A 9-point hedonic test showed
differences between both varieties in softness and colour, but adhesiveness, odour and overall acceptance were very similar. An evaluation of the
effects of drying and temperature conditions on the rehydration and texture of Khaowong Kalasin showed hardness of the rehydrated rice after hot
air drying at 40C was closer, and the stickiness higher, than the control (fresh cooked rice). Rehydrated rice after hot air drying between 50 and 60C
and freeze drying (-80C) was too soft. The instant rice after drying at 40C needed the longest rehydration time but had no significant change in
rehydration maximum weight gain. On external microscope visualization, testers specifically preferred hot air drying below 60C because the higher
temperature led to more fractures inside the kernel and they were hidden by the opaque surface after freeze drying.
Key words: Glutinous rice, soaking, drying, instant glutinous rice, rehydration, sensory evaluations.

Introduction
Glutinous rice (Oryza sativa var. glutinosa) is a cultivar widely
found in Southeast Asia. The Khaowong District of Kalasin
Province in Northeast Thailand is home to one of the most popular
glutinous rice cultivars called Khaowong Kalasin. It originally
derived from RD6, which is cultivated in many parts of the upper
and middle provinces of North-eastern Thailand 1. It has very low
amylose content resulting in a sticky and dense quality when
cooked 2. Glutinous rice after husking is generally opaque and
white. In traditional glutinous rice cooking, the rice is soaked for
approximately 1 hour in water and then steamed using a straining
cloth, bamboo basket and earthenware steamer. During steaming,
the opaque and white kernels gradually become transparent and
clear. In conventional steaming by firewood, the rice becomes
soft and fragrant but needs more time to cook, and it is more
difficult to control the cooking temperature. Thai people still use
traditional steaming techniques rather than modern techniques 3.
Recently, farmers, growing RD6 in Khaowong, has found that
after cooking their rice was softer and more fragrant than the
original cultivar. Even stored in a woven bamboo container for
several hours until it cooled down, its texture remained stable. To
date, no scientific studies have confirmed the better properties of
the Khaowong Kalasin cultivar.

In todays modern world, competitive social pressures cause


people in large cities to turn to meals that save time and can be
eaten quickly. Many of the most favourite meal types are the
ready to eat and instant meals, for instance, rice ready meals,
instant rice and instant hot beverages. These commercial ready to
eat meals require an efficient method of cooling quickly. Vacuum
cooling, air blast cooling and cold room cooling are examples of
quick cooling processes that cool immediately to reduce the weight
loss of cooked rice 4. Similarly, ready to eat glutinous rice loses
weight in the quick cooling processing and often it becomes harder
and less fragrant. However, the instant process probably improves
its texture.
Sung et al. 5 found that storing glutinous rice (often referred to
as waxy rice) over six months was more effective toward increasing
hardness than using gamma radiation. Cooked rice stored at 37C
was harder and less adhesive than cooked rice stored at 4C 6.
Recent research with quick cooking rice 7, cooked rice 8, 9 and
sticky rice 10 has involved soaking and processing using high
hydrostatic pressure (or HHP soaking). HHP soaking leads to
lower hardness and higher cohesiveness for all kinds of rice. The
texture after cooking Jasmine rice, at various pressures and
temperatures 11, revealed that temperature significantly affected

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

115

pore size and texture, whereas pressure had little or no effect. For
Thai glutinous rice starch, pressure and temperature on the
gelatinization rate followed the Arrhenius and Eyring equations 12.
The effects of spray drying 13 and microwave heating 14 on
properties of glutinous rice starch have been determined. Spray
dried glutinous rice starch was almost amorphous and formed a
hydrophilic matrix barrier while microwave heating had a high
effect on starch granules re-aggregation. In addition, changes of
head rice yield and textural and digestive properties of glutinous
rice during hot air fluidized-bed drying have been investigated 15,
16
. Higher temperature drying led to higher head rice yield, and
cooked glutinous rice had reduced hardness and higher stickiness
and more easily digested starch.
Cooking, drying and pre-treatment methods play important roles
in achieving the desired quality for preparing instant rice. It was
found that instant rice, after rehydration in a rice cooker, was
harder and less sticky than freshly cooked rice while rehydrated
rice that had passed a freezing pre-treatment was insignificantly
different 17. Several models were developed to predict drying and
rehydration of instant rice kinetics using combined microwave
and hot air drying 18. It was recommended that a combination of
300 W microwave power and 80C hot air temperature was optimal
in terms of drying time, rehydration time and colour. Moreover,
the V-type pattern amylase-lipid complexes were developed by
instant jasmine rice processing 19.
In this study, we focused on cooking methods and instant
processing of glutinous rice cv. Khaowong Kalasin to determine
optimum conditions for cooking, storage and drying preparation.
The cooked rice, i.e., moisture, texture (hardness and stickiness),
and sensory qualities were evaluated. In addition, texture and
visualization of rehydrated rice was assessed.
Materials and Methods
Premium grade glutinous milled rice cv. Khaowong Kalasin was
collected from Khaowong rice mill at Kalasin Province,
Northeastern Thailand. This rice variety has a special characteristic
and is registered as a Geographical Indication (GI) by the Thai
Department of Intellectual Property (DIP). Supreme RD6 glutinous
milled rice was purchased from a common market. Both varieties
of rice were to be stored for approximately six months.
Experiments:
Rice soaking and cooking: Firstly, the effects of soaking time
and cooking method on the textural properties of cooked glutinous
milled rice (Khaowong Kalasin) were studied. Milled rice (500 g)
was soaked in water at Thai ambient conditions (30C and 50-60%
RH) with soaking times of 30, 60 and 90 min. The three cooking
methods used were firewood earthenware steamer, LPG aluminium
pot steamer and programmable cooker. In the firewood
earthenware steamer and the LPG aluminium pot steamer, rice was
soaked in a straining cloth and then steamed above boiling water
in a bamboo basket until kernels appeared clear. In the
programmable cooker, rice was soaked and cooked with 1:1 rice
and water ratio. Cooked rice samples from all cooking methods
were mixed well and stored in woven bamboo containers until
they cooled down before texture measurement.
Storage tests in a woven bamboo container: Samples of 500 g
glutinous rice cultivars, Khaowong Kalasin and RD6, were soaked
116

in pure water at ambient temperature for about 60 min until


absorbed a constant amount of water. Then, they were cooked in
a programmable cooker (SHARP model KS-ZT18) with rice and
water 1:1 by volume. Khaowong Kalasin glutinous rice acquires
its reputation from storage in a bamboo container. Therefore, the
two varieties were compared after storage in woven bamboo
containers for times from 0 to 24 hours. After the grain cooled
down to room temperature, it was hypothesized that the Khaowong
Kalasin rice texture remained softer. The textural and sensory
properties of cooked glutinous rice were evaluated.
Drying processing: After cooking, the rice became agglomerated
by gelatinization. The rice was spread on a wire mesh tray in a
single layer to facilitate washing in water at room temperature for
about 30 s. After that, it was taken out and left until almost dry
prior to drying in a hot air oven (Memmert ULE500, Germany) at
40, 50 and 60C temperatures compared to -80C freeze drying
(HetoPowerDry PL6000, UK). The rice from the drying process
was instant rice with a final moisture content of 10-12% dry basis
for hot air drying and 2-3% dry basis after freeze drying. Instant
rice samples were kept in desiccators pending rehydration.
Rehydration: Instant glutinous rice was rehydrated in a
transparent glass beaker with rice: water 1:2 by weight in an 850
W microwave oven (SANYO model EM-S 2088W). Samples were
weighed every minute until the weight reached a maximum. Weight
gain on rehydration (WGR) was calculated using equation 1 as
modified allowing for use of a microwave oven. Textural properties
of the rehydrated rice were measured and also visually examined
by microscopy.
WGR = (Wt Wi) / Wi

(1)

where Wt and Wi are weight at any time (t) compared to initial


state (i).
Moisture content determination: Moisture content of cooked,
instant and rehydrated glutinous rice was measured. A 50 g sample
was placed on a disposable aluminium foil pan and was then placed
in a digital moisture analyser (A&D company, Limited, MX-50,
Japan). Triplicates were analysed.
Texture measurement: Hardness and stickiness of cooked and
rehydrated glutinous rice were tested using a texture analyser
(Stable Micro System, TA AT Plus, and UK). Ten rice kernels were
randomized and put on a base plate in 2 rows and pressed by a 30
mm diameter cylindrical probe. The compressive test deformed
rice to 90% at 0.5 mm/s and a post-test speed of 1 mm/s. Ten replicates
were measured for each sample. Hardness of glutinous rice was
recorded from the maximum compressive force of the first peak
and stickiness for the negative force when the probe pulled up.
Sensory evaluations: Five attributes assessed taste (softness),
smell (aroma), touch (adhesiveness) and sight (colour) and overall
acceptance. Softness, aroma, adhesiveness, colour and overall
acceptance were evaluated by a 9-point hedonic scale using 30
trainees as explained in Table 1. Each trainer tested both cultivars.
Fifteen grams warm rice samples stored in woven bamboo
containers were served to trainees who scored on each attribute.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Table 1. Terminology, definitions, and how to evaluate sensory level.


Attributes
Softness

Definition
Force required to press
the sample with molars

Adhesiveness

Smell of cooked rice


seeds
Stickiness

Colour
Overall acceptance

Clarity
-

Aroma

Assessment
Place the samples between
the molars and evaluated
force to bite
Sniffing samples
Moulding samples by hand
Visualizing inside a cup of
sample
-

External visualization: The sample was prepared by the paraffin


section method before external visualization. The external surfaces
and cross sectional area appearances of dried and rehydrated rice
samples, prepared by hot air or freeze drying, were examined
using stereomicroscopy (Meiji Techno EMT-1-P, USA) at 200X
magnification.

1
2
3
4
5
6
7
8
9

=
=
=
=
=
=
=
=
=

Scale
Dislike extremely
Dislike very much
Dislike moderately
Dislike slightly
Neither like nor dislike
Like slightly
Like moderately
Like very much
Like extremely

and stickiness. In order to cook uniformly, the programmable


cooker was used in the following sections. In addition, a 60 min
soaking time was used because it was close to the equilibrium
moisture (Fig. 1).
100

Statistical analysis: Variance analysis (ANOVA) data processed


the means and deviations. Duncans multiple range tests were
applied to determine significant differences between each
treatment 20.
Results and Discussion
Effects of soaking time and cooking method on textural
properties: Even though the tested rice was stale, cooked
Khaowong Kalasin rice had better softness than the other
varieties. Soaking and cooking were important factors which
affected the textural properties. To ensure cooking uniformity, the
variations of soaking time and cooking method were investigated
prior to drying and rehydration. Table 2 shows the hardness and
stickiness of cooked Khaowong Kalasin rice at various soaking
times and cooking methods. The results in all cooking methods
showed that the hardness and stickiness were reduced when the
soaking time increased. The longer soaking time seems to lead to
higher water uptake into the rice which, in turn, leads to the cooked
rice being softer and less sticky. However, replacement of steam
by water inside the kernel, by different steaming rates and cooking
times, may be the cause of a reduction of hardness and stickiness.
Visual observation, both of firewood and LPG combined with a
traditional cooking steamer, showed that these cooking methods
lead to non-uniform cooked rice which, in turn, leads to a wide
range of values of hardness and stickiness. In contrast, the rice
cooked by the programmable cooker was quite uniform in both
visual observation and had a narrow range of values of hardness
Table 2. Hardness and stickiness of steamed glutinous rice at
various soaking times and cooking methods.
Cooking methods

Charcoal earthenware

LPG earthenware

Programmable rice cooker

Soaking
times
(min)
30
60
90
30
60
90
30
60
90

Hardness
(kg force)

Stickiness
(kg force)

2.9 + 0.1a
2.4 + 0.1b
1.3 + 0.0c
5.4 + 0.7b
1.7 + 0.5c
6.7 + 0.6 a
3.7 + 0.1a
3.5 + 0.0b
3.4 + 0.0c

-0.20 + 0.20b
-0.40 + 0.04c
-0.02 + 0.01a
-0.30 + 0.2a
-0.40 + 0.4b
-0.50+ 0.3c
-0.04 + 0.02b
-0.40 + 0.01c
-0.03 + 0.01a

Means with the different letters in the same column are significantly difference (p0.05) by Duncans
multiple range tests.

Moisture content (%)

90
80
70
60
50
40
30
20
10
0
0

30

60

90
120
150
Soaking time (min)

180

210

240

Figure 1. Relationship between moisture content and soaking time.

Textural and sensory evaluations of two cultivars during storage


in a container: Storage was an important motive for achieving a
textural change of the cooked rice 6, 22. Cooked rice texture also
directly affected consumer sensory evaluation. In this section,
RD6 and Khaowong Kalasin glutinous rice effect textural and
sensory evaluations during storage in a woven bamboo container
as listed in Tables 3 and 4. Also shown in Table 3, the hardness of
cooked rice during storage at 0 to 24 h significantly changed at 6
h in RD6 and 12 h in Khaowong Kalasin cultivars. For 0-12 h
storage, the cooked rice was clear and warm, but the cooked rice
kernel left 24 h overnight was more opaque and cool. However,
the hardness of the Khaowong Kalasin cooked rice at 12 and 24 h
storage was significantly lower than the hardness of RD6. The
stickiness of cooked rice changed significantly at 6 h storage for
both cultivars. However, when comparing stickiness after 24 h
storage, the RD6 value was higher than that of Khaowong Kalasin.
These results indicate that the cooked glutinous rice of Khaowong
Kalasin had constant hardness until 12 h in a container - far longer
Table 3. Results of the period keeping in bamboo container shift
towards grain hardness and stickiness in both cultivars.
Storage
duration (h)
0
1
6
12
24

Hardness (kg force)


Khaowong
RD6
Kalasin
b
3.5+0.3
2.6+0.8 c
3.7+0.8 b
3.6 +0.7 c
3.5+0.3 b
3.5+0.4 c
3.7+0.2b
5.6+0.2 b
5.2 +0.3a
14.1 +0.5 a

Stickiness (kg force)


Khaowong
RD6
Kalasin
b
0.03+0.00
0.03+ 0.00 c
0.04+0.01 b 0.04 +0.01 c
0.03+0.00 b 0.04+0.01 c
0.04+0.00 b 0.06+0.00 b
0.20+ 0.01a 0.40+ 0.02 a

Means with the different letter in the same column are significantly difference (p0.05) by Duncans
multiple range tests.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

117

Table 4. Sensory evaluation at different storage durations in a container of both varieties.


Time
(h)
0
1
6
12
24

Khaowong Kalasin

RD6

Colour

Softness

Aroma

Adhesiveness

7.81.3g
7.51.3fg
6.71.2ef
6.11.4cde
5.41.4bc

7.81.0e
7.11.0de
5.71.4c
4.51.6b
3.21.8a

7.01.5f
6.91.2f
6.31.4def
5.61.3ed
4.71.5ab

6.91.8cd
7.31.3cd
6.41.8bc
5.61.6b
4.31.8a

Overall
acceptance
7.61.4d
7.41.1d
6.31.5c
5.71.3bc
4.31.9a

Colour

Softness

Aroma

Adhesiveness

6.41.5de
6.31.5de
5.81.5bcd
5.11.5ab
4.41.6a

7.01.5de
6.41.9cd
5.51.7c
4.62.0b
3.21.8a

6.81.3ef
6.61.4ef
6.01.6cde
5.51.3bc
4.61.8a

7.41.6d
7.11.5cd
6.61.5bcd
5.71.8b
4.31.9a

Overall
acceptance
7.61.0d
7.21.0d
6.41.4c
5.41.5b
3.91.9a

Means with the different letter in the same column are significantly difference (p0.05) by Duncans multiple range tests.

KW = -0.62x + 8.56
R2 = 0.985

10

4
2

6
Storage duration (h)

12

KW = -1.18x + 9.2
R2 = 0.990

Softness

6
Storage duration (h)

12

24

Aroma

Figure 4. Aroma of cooked RD6 and Khaowong Kalasin rice during


storage in bamboo rice containers.
KW = -0.69x + 8.17
R2 = 0.841

10

RD6 = -0.76x + 8.5


R2 = 0.921

KW
RD6
KW
RD6

8
6
4
2
0

6
Storage duration (h)

12

24

Figure 5. Adhesiveness of cooked RD6 and Khaowong Kalasin rice


during storage in bamboo rice containers.
KW = -0.83x + 8.75
R2 = 0.949

KW
RD6
KW
RD6

RD6 = -0.92x + 8.86


R2 = 0.953

8
6
4
2
0

6
Storage duration (h)

12

24

Figure 6. Overall acceptance for cooked RD6 and Khaowong Kalasin


rice during storage in bamboo rice containers.

KW
RD6
KW
RD6

RD6 = -0.66x + 7.6


R2 = 0.945

8
6

testing increased. It is likely that manually sensed adhesiveness


was based on bulk cooked rice which had a little deformation. On
the contrary, stickiness from the texture analyser was from the
compression test which was set at the 90% deformation of 10
kernels.

4
2
0
0

6
Storage duration (h)

12

24

Figure 3. Softness of cooked RD6 and Khaowong Kalasin rice during


storage in bamboo rice containers.
118

24

Figure 2. Colour evaluation of cooked RD6 and Khaowong Kalasin


rice during storage in bamboo rice containers.
10

4
2

Overall acceptance

Colour

RD6

KW
RD6
KW
RD6

10

RD6 = -0.55x + 7.55


R2 = 0.957

KW = -0.59x + 7.87
R2 = 0.940

KW
RD6
KW

RD6 = -0.52x + 7.16


R2 = 0.945

10

Adhesiveness

than RD6. In other words, Khaowong Kalasin cooked rice lost


water at a slower rate than RD6. Hardness and stickiness, in later
periods of storage, increased because water was expelled from
retrograded amylose and amylopectin molecules, which acquired
a stronger structure due to retro gradation. The amylose structure
rearranged and formed hydrogen bonds during retro gradation
leading to the hardness and stickiness.
Table 4 shows the sensory evaluations for different storage
durations in containers of both varieties. To assess textural
properties, sensory evaluation was based on a 9-points hedonic
scale for five attributes - colour, odour, adhesiveness, softness
and overall acceptance. Figs. 2-6 show the scores for each
attribute for both varieties at various elapsed storage times in a
woven bamboo container.
All attributes of sensory evaluations in Figs. 2-6 declined with
storage duration for both varieties. Comparing both varieties, we
found that both colour and softness were different throughout
storage (Figs. 2 and 3) while the other attributes of aroma,
adhesiveness and overall acceptance differed insignificantly (Figs.
4-6). This indicated that human senses can differentiate the
softness and colour, but the overall acceptance for both varieties
appeared similar. The measured softness during storage correlated
with the hardness from the texture analyser. However, adhesiveness
decreased during storage whereas stickiness from compressed

Effects of drying temperature and method on instant rice


rehydration and textural properties: Instant glutinous rice was
made by using hot air drying at different temperatures and freeze
drying processes compared to cooked glutinous rice. Both drying
temperature and drying methods probably play an important role
on the final instant glutinous rice properties. Effects of drying

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

temperature are shown in Table 5. The moisture content of cooked


rice decreased from 121 down to 10.1-11.8% d.b. by hot air drying
and 2.7% d.b. by freeze drying. The drying time using hot air
decreased when the temperature increased. It was particularly
noticed that hot air drying at 50C can reduce drying time by half
compared to 40C. The higher temperature resulted in a higher
heat transfer rate into the rice kernel as well as a higher water
removal rate from the kernel.
All drying temperature and methods insignificantly affected
the maximum weight gain on rehydration (WGR) but had some
effect on the rehydration time. Rehydrated glutinous rice had a
maximum weight gain at 7 min for 40 and 50C hot air drying and
the maximum weight gain at 6 min for 60C hot air drying and -80C
freeze drying process. However, the final moisture content after
rehydration significantly increased with the drying temperature
and method. Drying at higher temperature made more rice kernels
crack leading to increased moisture content after rehydration.
Therefore, the cracking results were confirmed by external
visualization (Fig. 7 (a-c). Freeze-dried rice was opaque and had
loose structure (Fig. 7d) whereas the dried rice was clear with
dense structure and cracking (Fig. 7b). Therefore, the rehydrated
freeze-dried rice had a moisture content and WGR close to that by
60C hot air drying.
From the point of view of textural properties, the rehydrated
rice was considerably harder in hot air drying at 40C for 14 h.
Little cracking appeared due to the length of time for drying. Higher
cracking densities and lower hardness occurred with higher drying
temperature and shorter drying time. In addition, the hardness of
rehydrated rice from the freeze drying process was lower than
cooked rice but insignificantly different from hot air drying at 50
and 60C. For the freeze drying process, the loose structure was
caused by rapid water sublimation inside the rice kernel leading to
a lower hardness value. The stickiness after rehydration lay in the
range of 34 to 370 g force for all drying temperatures and methods.
Only 50C hot air-dried rice was similar in stickiness to freshly
cooked glutinous rice. The remarkable stickiness (370 g force)
was obtained from treatment of drying at 40C. This may be a
consequence of heat treatment over a long period of time, which
is an important key to attaining the remarkable hardness and
stickiness due to retrograde action.
External visualization: The external surface and cross sectional
area of dried and rehydrated Khaowong Kalasin rice in all treatments
were examined (Figs. 7 (a-d) and 8 (a-d). The hot air-dried rice was
glassy and clear whereas freeze-dried rice seemed white and
opaque. However, almost all external surfaces and cross sectional

200 m

200 m

(a) Cooked glutinous rice dried by hot air at 40C

200 m

200 m

(b) Cooked glutinous rice dried by hot air at 50C

200 m

200 m

(c) Cooked glutinous rice dried by hot air at 60C

200 m

200 m

(d) Cooked glutinous rice dried by freeze drying at -80C

Figure 7. Overall and cross-sectional views of dried kernels at


200X magnification by stereomicroscope.

Table 5. Weight gain after rehydration and textural properties of instant glutinous rice in
different drying temperatures and methods.
Glutinous rice
Khaowong Kalasin
Dried by HA at 40oC (14 h)
Rehydrated (7 min)
Dried by HA at 50oC (7 h)
Rehydrated (7 min)
Dried by HA at 60oC (6 h)
Rehydrated (6 min)
Dried by FD at -80oC
Rehydrated (6 min)
Cooked (control)

Moisture content
(% d.b.)
11.8 0.9e
134.4 1.2c
10.7 0.3e
141.7 0.8b
10.1 0.8e
149.3 1.4a
2.7 1.1f
150.8 0.8a
121.0 3.4d

Maximum WGR
(decimal)
NA
1.1 0.1ns
NA
1.2 0.2 ns
NA
1.2 0.4 ns
NA
1.1 0.3 ns
NA

Textural properties (kg force)


Hardness
Stickiness
NA
NA
2.2+0.7b
0.4+0.1a
NA
NA
0.4+0.2c
0.06+0.03bc
NA
NA
0.5+0.2c
0.08+0.02b
NA
NA
0.4+0.2c
0.05+0.01b
3.4+0.3a
0.03+0.00c

Note: HA denotes hot air drying and FD freeze drying process. Values are mean and standard deviation. Different superscripts (a, b, c, d, e and
f) in the same column imply that the values are significant different (p0.05) by Duncans multiple range tests.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

119

200 m

Conclusions
Traditional glutinous rice (cv. Khaowong Kalasin) cooking
techniques resulted in a large variation of hardness and stickiness.
The variation of these textural properties could be reduced by
using a programmable rice cooker and soaking for at least 60 min.
If it is necessary to leave the cooked rice overnight in a woven
bamboo container, the textural properties of Khaowong Kalasin
and RD6 cultivars significantly change at 12 and 6 h, respectively.
A paired test between both cultivars based on a 9-point hedonic
test showed that softness and colour had distinct differences
whereas adhesiveness, odour and overall acceptance were not
significantly different. The cooked rice was processed by drying
to produce instant glutinous rice. The best conditions for
rehydration, using the microwave at the highest power (850 W)
and water and rice ratio of 2:1, was hot air drying at 40C. The
rehydrated rice had a low fracture density and hardness similar to
the control - fresh cooked rice. The rehydrated rice at other
conditions was too soft and less sticky. However, this best
condition had a longer rehydration time than the fastest conditions
of approximately 1 min.

200 m

(a) Rehydrated glutinous rice after dried at 40C

200 m

200 m

(b) Rehydrated glutinous rice after dried at 50C

Acknowledgements
We would like to thank the Thai Research Fund and Higher
Education Commission for financial support.
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Meullenet, J.-F., Marks, B. P., Hankins, J. A., and Daniels. M. J. 2000.
Sensory quality of cooked long-grain rice as affected by rough rice
moisture content, storage temperature, and storage duration. Cereal
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14

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

121

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Journal of Food, Agriculture & Environment Vol.12 (3&4): 122-126. 2014

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Assessment of performance ability of Cabernet Sauvignon, Merlot and Syrah wine


cultivars on Southeast region of Turkey
Dilek Deirmenci Karata * and Hseyin Karata
Department of Horticulture, Faculty of Agriculture, Dicle University, 21210 Diyarbakr, Turkey.
*e-mail: degirmencidilek@yahoo.com
Received 18 April 2014, accepted 26 September 2014.

Abstract
In this study we evaluated the viticultural performance of Cabernet Sauvignon, Merlot and Syrah wine cultivars on dry-hot climate conditions in the
Southeast Region of Turkey. Vine yield components (bud survival (%), cluster number/vine (n), cluster number/shoot ratio (n), cluster weight (g),
yield/vine (kg) and berry weight (g), fruit composition as Brix (soluble solids %), and pH and titratable acidity (TA g/l) were measured during two
growing seasons between the years 2009 and 2010. The maximum mean bud survival rate (0.96%) was measured in the Cabernet Sauvignon grape
cultivar in 2009, and the minimum rate (0.77%) was measured in the Syrah grape cultivar in 2010. The highest yield in 2009 was found in the Cabernet
Sauvignon grape cultivars trained with Guyot system, with 7.63 kg. Mean Brix ranged between 24-27%, and mean pH between 3.64 and 3.86,
whereas TA was between 12.9 and 14.5 g/l. Based on the results, it can be concluded that Cabernet Sauvignon, Syrah, and Merlot are appropriate for
the Southeastern Anatolian conditions in Turkey.
Key words: Vitis vinifera, grapevine, yield components, fruit composition, training system.

Introduction
Grapevine (Vitis vinifera L.) is one of the oldest and most important
perennial crops in the world. Turkey is an important centre of
origin of the cultivated grapevine. The cultivation of grapes in
Anatolia began approximately 7000 - 8000 years ago 1, 16. Anatolia
has long been linked with the origin of viticulture and wine making,
especially in its eastern and southeastern regions, which are
commonly referred to as the epicenter of grapes 2. Canopy
management is widely accepted as an important tool for highquality wine production, however, little information is available
on its effectiveness under warm, dry climatic conditions 12. Jackson
and Lombard 10 argue that each cultivar and training system in a
region should be investigated to obtain the optimum yield that
will produce quality wine. Temperature is one of the primary
microclimatic factors in the driving rates of growth 3, 14, 17. Light
and temperature are the most important climatic factors for
inflorescence induction and differentiation. Environmental
regulations of fruitfulness are the most important climatic factors
for inflorescence induction and differentiation. High temperatures
have been found to promote fruitfulness in developing grapevine
buds 16. It is known that the terroir (the combination of soil, sub
soil, and climatic conditions) has a direct effect on the quantity
and quality of the grapes and on the wines produced 6, 9, 15.
In this study, we aimed to evaluate the viticultural performance
of Cabernet Sauvignon, Merlot and Syrah wine cultivars under
dry-hot climate conditions in the Southeastern Region of Turkey.
Vine yield components (bud survival, cluster number/vine, cluster
number/shoot ratio, cluster weight, yield/vine and berry weight),
and fruit composition (Brix, pH, titratable acidity) were measured
during two growing seasons between 2009 and 2010.
122

Materials and Methods


Experimental site: The experiment was conducted during 2009
and 2010 in Turkey (elevation 677 m, 3755.2' N- 40 13.8E). The
trial site was located within a commercial vineyard in the Province
of Diyarbakr. The study was carried out on wine grape cultivars
of Syrah, Merlot and Cabernet Sauvignon that were grafted on
110R rootstock. Bilateral cordon and Guyot canopy training
systems were characterized. The vines were spaced at intervals of
1.5 m, in north-south oriented rows with 2.0 m spacing.
The experiments were carried out on 25 vines for each grape
variety and studied in four replications. Trial data were taken from
the experimental area in 2009 and 2010. The soil in this region
shows clayey texture. In terms of soil properties, the soil consists
of 22% silt, 56.6% clay, 21.4% sand, 14% lime, and 1.8% organic
material. The vines were trained in two trunks and in bilateral
cordons at a height of 80 cm and winter-pruned to two-bud spurs,
leaving ~20 buds (excluding basal buds) per vine. Two trunks
were Guyot trained at a height of 80 cm and winter-pruned to 7-8
buds, leaving ~25 buds (excluding basal buds) per vine. The shoots
were loosely positioned between two pairs of foliage wires placed
at 40 cm and 50 cm in both systems.
Shoot thinning, hedging, and other canopy management
practices were applied. The vineyard was drip irrigated using
pressure-compensated emitters with a flow rate of 3 L/h, spaced
90 cm apart. Irrigation was applied, as needed, between the bud
break and bloom to avoid plant water stress. The water was
withheld through mid-July to achieve the control of shoot growth.
Irrigation was applied approximately once a week throughout
summer and less frequently during the cooler ripening period.
Nitrogen fertilizer (NH2NO3 x (NH2)2 CO) was applied annually at a

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

rate of ~15 kg N/ha by fertigation. All fertilizer applications and


pest and disease management practices were commercially applied
across the vineyard as uniformly as possible.
The growing season in the Province of Diyarbakr, Turkey, is
characterized by an absence of rainfall and dry-warm days. During
the 2009 and 2010 growing seasons, the phenological stages (bud
break, bloom, fruit set, veraison, and harvest) were recorded
(Table 1). Fig. 1 presents the long-term mean values of mean
temperature (C), mean temperature max. (C), and mean
temperature min. (C) at Diyarbakr conditions. Accordingly, longterm mean values of mean temperature max. (C) from the fruit set
period until the harvest period in June, July, August, and September
were 33.6 C, 37.2 C, 37.6 C, and 31.2 C, respectively.
Table 1. Phenological stages of 2009 and 2010 growing seasons
of the trial site.
Cultivar/Season
Cabernet Sauvignon
2009
2010
Syrah
2009
2010
Merlot
2009
2010

Budbreak

Bloom

Veraison

Harvest

10 April
08 April

26 May
29 May

01 July
03 July

08 August
07 August

30 March
03 April

20 May
22 May

28 June
05 June

01 August
06 August

02 April
05 April

20 May
23 May

02 July
04 July

05 August
07 August

50
40

Mean temperature

30
20

Mean temperature max.

10
0
-10

10

11

12

Mean temperature min.

-20
-30

Figure 1. Long-term average values of mean temperature (C), mean


temperature max. (C), mean temperature min (C) at Diyarbakr conditions.

Statistical analysis: Data were analyzed using IBM SPSS 20.0.


The analysis of variance was performed separately for each
variable, and the significance of each factor was evaluated. A
three-way (year x training system x cultivar) analysis of variance
(ANOVA - GLM Univariate procedure) was used to analyze the
effects of year, training system, and cultivar. When significant
effects (p<0.05) were detected, Duncans multiple comparison tests
were used to separate the means.
Determination of yield components: Yield components (bud
survival (%), cluster number/vine (n), cluster number/vine, cluster
number/shoot ratio (n), cluster weight (g), yield/vine (kg) and
berry weight (g) were measured for each year.
Bud survival (%): Data on bud survival were collected based on
per vine bud break each year, and the percentage of bud survival
was calculated by dividing the number of live nodes by the total
number of nodes and multiplied by 100.
Cluster number/shoot ratio (n): The cluster number/shoot ratio
was studied calculating the clusters on each shoot of each vine
on the experiment field.
Cluster weight (g): Cluster weight (g) was determined for each

vine, randomly selected and weighed by a balance.


Yield/vine (kg): The yield was found by measuring the product
of each vine on the experiment field.
Berry weight (g): A subsample of 100 berries was randomly
removed and weighed by a balance.
Determination of fruit composition: Samples were collected from
each grape cultivar for analyzing fruit compositions (Brix (%), pH,
and titratable acidity (TA) (g/l). Soluble solids (Brix%) were
measured using a refractometer. The pH was determined with a
temperature-compensated pH meter. TA was determined using 0.1
N NaOH. Ten ml of juice was mixed with 20 ml of distilled water,
and titrated to a pH 8.2. With this titration, the TA (g/l) was
calculated and reported as the amount of tartaric acid (g)/L juice.
Results
Yield components:
Bud survival (%):Survival of buds was high both in the training
systems and in all cultivars (Table 2). Bud survival decreased in
the Syrah cultivar in 2010. This result may be due to the fact that
the Syrah cultivar is susceptible to winter bud necrosis 5. The
maximum bud survival rate (0.98%) was measured in the Cabernet
Sauvignon grape cultivar on Cordon training system in 2009, and
the minimum rate (0.70%) was measured in the Syrah grape cultivar
on Guyot training type in 2010. Statistical difference was observed
among cultivars (Fcultivar 2.218, df 1, p>0.114). However, differences
were not significant among the training systems (Ftraining 7.523, df
1, p<0.007) and years (Fyear 12.876, df 1, p<0.000) (Table 2). The
statistical data revealed that the survival rate differed depending
on year and training system. The effects of other factors and their
interactions are not important with an exception of interaction
between training systems and years (Fyear x training 0.931, df 1, p>0.337)
(Table 2).
Cluster number/vine (n): While Merlot grape cultivar on Cordon
training system was found to show the highest production with
42.90 clusters per vine in 2009, the Cabernet Sauvignon grape
cultivar produced the highest number of clusters per vine in 2009,
with 51.60 on Guyot trellis type. From this comparison, it could be
said that the Cabernet Sauvignon grape cultivars gave the highest
values for both years. All of the factors cultivar (Fcultivar 34.792, df
1, p<0.000), training system (Ftraining4.928, df 1, p<0.029) and year
(Fyear121.595, df 1, p<0.000) and interactions were not statistically
significant (Table 2).
Cluster number/shoot ratio (n): In the cluster/shoot ratio, the
highest rate was obtained in the Syrah grape cultivar on Cordon
training system in 2010. The minimum value of cluster number/
shoot (1.94) was observed in Merlot grape cultivars on Cordon
trellis type in 2009. In Syrah grape cultivars, the cluster number/
shoot ratio was higher when it is on the Cordon training system.
No statistical significant differences were established in training
system (Ftraining 2.826, df 1, p<0.096) and year (Fyear48.356, df 1,
p<0.000); however, cultivar (Fcultivar 0.954, df 1, p>0.388) was found
effective on the number of clusters (Table 2). The interaction
between year and training system (Fyear x training0.357, df 1, p>0.551)
was significant (Table 2).

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

123

3.53 2.263
3.61 2.666
2.54 1.377
2.35 0.703
4.87 2.228

Means within columns followed by different letters differ significantly at p < 0.05 by Duncans new multible range test.

92.22 22.117

3.87 2.134
3.21 2.358
5.47 2.575 b
1.75 0.827 b

131.66 9.047
98.62 11.217
86.72 7.166

2.48 0.842 ab
2.22 0.544 ab

63.05 3.797

6.33 2.095 ab
3.42 1.177 ab

2.44 0.959 b
2.65 1.750 b

96.74 23.445
87.70 19.885
138.02 8.137 b
125.19 4.065 b
105.23 12.645 b
92.01 2.921 b
90.45 3.305 ab
82.99 8.162 ab
63.24 3.842 ab
62.86 3.948 ab

180.90 18.908 c
190.99 3.672 c
185.94 14.230
161.60 27.152 b
179.31 22.820 b
170.45 26.046
149.50 10.179 b
129.81 22.842 b
139.65 19.957

253.23 59.429 c 166.97 52.156


177.99 52.325 c 161.09 36.138
215.61 66.780
157.58 33.790

2.57 0.719
2.40 0.686
2.49 0.705
2.39 0.604 a
1.58 0.743 a
1.99 0.779
2.75 0.391 a
2.88 0.672 a
2.82 0.539
1.94 0.359 a
1.97 0.206 a
1.95 0.286

3.19 1.118 a
2.93 0.448 a
3.06 0.840

24.12 13.265
21.03 16.187
22.58 14.817
13.50 4.882 c
13.80 9.090 c
13.65 7.103..
15.20 4.517 b
12.40 2.459 b
13.80 3.820..
42.90 14.813 b
26.40 8.822 b
34.65 14.576 ..

0.94 0.110 a
0.85 0.232 a
0.90 0.183
0.86 0.111 a
0.92 0.161 a
0.89 0.137..
0.91 0.058 a
0.91 0.108 a
0.91 0.084 .

21.50 9.132 c
10.40 4.648 c
15.95 9.064..

0.91 0.116
0.84 0.188
0.87 0.160
0.85 0.114 a
0.70 0.204 a
0.77 0.178

Mean
2010
Syrah
Merlot

2009
2010
2009

Cultivars
Cabernet Sauvignon
Years
2009
2010
Bud survival (%)
Cordon
0.98 0.032 a
0.90 0.132 a
Guyot
0.93 0.078 a
0.70 0.270 a
Mean
0.96 0.064 ..
0.80 0.196..
Cluster numer/vine
Cordon
33.20 5.391 a
18.40 6.022 a
Guyot
51.60 8.996 a
11.60 3.627 a
Mean
42.40 11.883
15.00 5.965..
Cluster number/shoot ratio
Cordon
2.39 0.282 a
2.76 0.625 a
Guyot
2.46 0.208 a
2.57 0.497 a
Mean
2.42 0.244
2.67 0.559
Cluster weight (g)
Cordon
117.25 14.672 a 139.37 21.008 a
Guyot
148.15 16.903 a 140.33 32.410 a
Mean
132.70 22.101
139.85 26.587
Berry weight (100) (g)
91.20 4.365 a
92.29 3.421 a
Cordon
77.60 7.018 a
85.44 3.880 a
Guyot
Mean
84.41 9.000
88.87 5.003
Yield / vine (kg)
3.88 0.767 a
2.60 0 .921 a
Cordon
7.63 1.542 a
1.60 0.509 a
Guyot
Mean
5.75 2.256
2.10 0.888

Table 2. Main effect of training system on yield components of Cabernet Sauvignon, Merlot and Syrah grapevines in Diyarbakr, Turkey, 2009 to 2010.
124

Cluster weight (n): The highest mean cluster weight (253.23


g) was observed in 2010 in the Syrah grape cultivar by
Cordon training system. This is a highly optimal result for
the Syrah grape genotype in the climatic conditions in the
Southern Turkey. The lowest mean cluster weight (139.37 g)
was observed in the Cabernet Sauvignon grape cultivar in
2009. No statistical difference was established among the
varieties (Fcultivar 25.577, df 1, p<0.000) and the training
systems (Ftraining 6.615, df 1, p<0.012) effective on the cluster
weight; however, year was (Fyear 0.274, df 1, p>0.602)
effective on the cluster weight (Table 2).
Yield (kg/vine): The highest mean yield (5.75 kg) was
observed in the Cabernet Sauvignon grape cultivar in 2009.
However, the same cultivar produced mean yield of 2.10 kg
in 2010. The highest mean yield (kg /vine) in 2009 was found
in the Cabernet Sauvignon grape cultivar trained with Guyot
system, with 7.63 kg. In the same year, Cabernet Sauvignon
with Cordon training system produced only 3.88 kg. In 2009,
the highest yield (5.75 kg/vine) was revealed in the Cabernet
Sauvignon grape cultivar. However, according to 2010 data,
yield per grape vine obtained by the Cordon training
system was 2.60 kg and 1.60 kg by the Guyot training system.
The highest yield in the Merlot grape cultivars (6.33 kg)
was obtained in 2009 when it was trained with the Cordon
system. While mean yield was 4.87 kg in 2009, it was 2.35 kg
in 2010. The Cordon training system was applied in the
Syrah grape cultivars in 2010 and significant results were
obtained. In 2010, the Cordon training system produced
5.47 kg, but the Guyot training system yielded 3.42 kg of
production. This result can be explained by the fact that
the lowest buds failed to shoot when leaving many buds
with Guyot training system. Statistical differences were not
significant among cultivars (Fcultivar 4.001, df 1, p<0.021),
training systems (Ftraining 6.990, df 1, p<0.009) and years
(Fyea46.925, df 1, p<0.000). Also, the interactions among
varieties (p<0.000), training systems (p<0.000) and years
(p<0.000) were not statistically significant (p<0.000) (Table
2).
Berry weight (g): Berry weight was based on the weight of
100 berries. The Syrah cultivars produced the highest value
with 138.02 g in 2010 with the Cordon training system. The
weight of 100 berries in the Syrah cultivar was higher in
both years compared to other cultivars. The Syrah grape
variety has a genetic tendency to produce bigger berries
and bigger clusters compared to other cultivars. No statistical
difference was found among cultivars (Fcultivar 452.881, df 1,
p<0.000), training systems (Ftraining 64.776, df 1, p<0.000) and
years (Fyea 329.577, df 1, p<0.000). Only the year x training
system interaction (Table 3) (Fyearxtraining 0.001, df 1, p>0.980)
was found significant (p<0.000) (Table 2).
Fruit composition: Mean Brix (%), pH, and TA values for
grape cultivars in both years are presented in Table 3. Mean
Brix (%) ranged beetween 24.13% and 27.01%. No statistical
difference was established among the cultivars (Fcultivar
16.578, df 1, p<0.000) and years (Fyear 4.097, df 1, p<0.045).
However, the statistical difference between the training

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Table 3. Main effect of training system on fruit composition of Cabernet Sauvignon, Merlot and Syrah grapevines in
Diyarbakr, Turkey, 2009 to 2010.
Years
Brix
Cordon
Guyot
Mean
pH
Cordon
Guyot
Mean
TA (g/l)
Cordon
Guyot
Mean

Cabernet Sauvignon
2009
2010

Merlot

Syrah

2009

2010

2009

2010

Mean

25.19 0.997 b
26.51 1.218 b
25.85 1.278

23.95 1.703 b
24.31 1.552 b
24.13 1.596

26.37 1.247 a
25.08 1.379 a
25.72 1.441

26.80 1.250 a
27.21 1.020 a
27.01 1.131

25.75 0.916 b
25.07 1.057 b
25.41 1.025

24.08 0.978 b
24.81 1.533 b
24.45 1.307

25.36 1.585
25.50 1.620
25.43 1.597

3.53 0.156 c
3.77 0.111 c
3.65 0.182

3.56 0.161 c
3.59 0.325 c
3.58 0. 250

3.97 0.169 a
3.73 0.087 a
3.85 0.182

3.85 0.306 a
3.86 0.137 a
3.85 0.232

3.71 0.451 b
3.57 0.131 b
3.64 0.331

3.82 0.096 b
3.90 0.115 b
3.86 0.112

3.74 0.290
3.74 0.206
3.74 0.250

13.57 0.501 a
13.02 0.622 a
13.29 0.618

14.59 0.956 a
14.47 1.191 a
14.53 1.0538

13.00 0.895 c
13.58 0.426 c
13.29 0.744

12.79 0.501 c
12.98 0.721 c
12.88 0.613

13.44 1.103 b
13.81 0.720 b
13.62 0.926

14.03 0.663 b
13.53 1.160 b
13.78 0.955

13.57 0.983
13.56 0.961
13.57 0.968

Means within columns followed by different letters differ significantly at p < 0.05 by Duncans new multible range test.

systems (Ftraining 0.383, df 1, p>0.537) was significant (Table 3). The


survival rates differed depending on the training systems. Year
and training interactions (Fyear x training 2.455, df 1, p>0.120) and
training and cultivar interactions (Ftraining x cultivar 2.628, df 1, p>0.077)
were both statistically significant (Table 3).
Mean pH ranged between 3.64 and 3.86. No statistical difference
was found among the cultivars (Fcultivar 12.075, df 1, p<0.000).
However, significant differences were observed between the
training systems (Ftraining 0.002, df: 1, p>0.963) and among the years
(Fyear 1.548, df 1, p>0.216). Year and training interactions (p>0.266)
were also significant, but the other interactions were not.
TA (titratable acidity) (g/l) values ranged between 12.88 and
14.53 g/l among cultivars. No statistical difference was found
among the cultivars (Fcultivar 10.681, df 1, p<0.000) and the years
(Fyear 4.694, df 1, p<0.032). However, statistical differences between
years and the training system interactions (Fyear x training 0.838, df 1,
p> 0.362) were significant (Table 3).
Discussion
In this study, we evaluated the viticultural performance of Cabernet
Sauvignon, Merlot and Syrah wine cultivars under dry-hot climate
conditions in the Southeastern Region of Turkey. Mean clusters
per vine ranged between 11.60 and 33.20 for Cabernet Sauvignon,
12.40 to 42.90 for Merlot and 10.40 to 21.50 in Syrah. From these
results, it is wise to claim that better yielding results were obtained
in the Syrah grape cultivars because they were trained by the
Cordon system. Mean yields (kg/vine) in all three cultivars were
examined. The yields of Cabernet Sauvignon (5.75 kg) and Merlot
(4.87 kg) were highest in 2009, whereas similar yields were obtained
in the Syrah cultivars in 2009 (2.54 kg) and 2010 (3.61 kg). The
training methods produced different yield capacities in the Syrah
grape cultivars in 2010. Bilateral Cordon training gave best yield
(5.47 kg) per vine in 2010. We found that the Cabernet Sauvignon
produced 5.75 kg/vine in 2009 and 2.10 kg/vine in 2010. Similar to
our study, Keller et al. 12 found that the crop level (kg/vine) changes
by years (3.54-9.81 kg) in the Cabernet Sauvignon grape cultivar
in Yakima Valley, Washington. Analyses showed that the lowest
cluster weight was observed in 2009 and 2010 as 132.70 g, 139.85
g in Cabernet Sauvignon grape cultivar and 139.65 g at Merlot
grape cultivar in 2009. The differences in cluster weights were
mainly due to the differences in berry weights.
Snhez and Dokoozlian 16 reported that the buds of Cabernet
Sauvignon increases with high-temperature climates. Similarly, we

also found higher values of cluster number/shoot ratio for Cabernet


Sauvignon. The vines had high numbers of clusters per shoot.
Climate was shown to exert an important influence over both the
timing of initiation and the extent of IP (inflorescence primordia)
differentiation, with IP development being far more advanced in a
hotter climate, compared with a cooler climate 7, 13, 21. In the same
way, in our study, the mean cluster number per vine was high
(42.40) in Cabernet Sauvignon and Merlot (34.65) in 2009. This
data is critical for improving the understanding of the
environmental effects on the formation of yield potential in
grapevines and how these may be modified by the temperature
increases related to climate change 21. Additionally, statistical
analyses showed that the cultivars were not effective on the pH
and TA (g/l) levels but the training systems were found effective
on the pH and TA (g/l) levels (Table 3). Brix (%), pH, and TA (g/l)
may show different values depending on environmental factors.
The warmth of the environment over the period of fruit set
could be related to the seasonal differences in the relative
proportions of seeded and seedless berries and live green ovaries
within the bunches in the Merlot grapes at New Zealand conditions
8
. In our study, we also observed low rates of shoot berry formation
in the Merlot grape variety, and we considered this as an effect of
high temperatures (38C) which caused live green ovary during
fruit set in June, 2009. Similar to our study, the differences in
vegetative growth, yield formation and fruit composition within
the cultivars were mostly due to the season (including weather
and soil moisture) rather than to yield 11-13, 19.
Conclusions
High temperatures have been found to promote fruitfulness in
developing grapevine buds. Cabernet Sauvignon grape cultivar
produced highest number of clusters for per vine on Guyot trellis
type with 51.60 in 2009 year. Our results for the Cabernet
Sauvignon, Syrah, and Merlot support the studies conducted in
some other countries where grape farming is rather common. It
can be concluded that Cabernet Sauvignon, Syrah, and Merlot
are appropriate for the Southeastern Anatolian conditions in
Turkey.
Acknowledgements
The authors would like to thank DUBAP (Dicle University Scientific
Research Project Coordination) for financial support of this work.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

125

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in New Zealand can alter yield components of Merlot grapevines.
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Galet, P., 1993. Prcis de viticulture. Dehan, Montpellier, France.
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Jackson, D. I. and Lombard, P. B. 1993. Environmental and management
practices affecting grape composition and wine quality: A review.
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deficit-irrigated winegrapes in a dry climate: Vigor, yield formation, and
fruit ripening. American Journal of Enology and Viticulture 63:29-39.
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Keller, M., Mills, L. J., Wample, R. L. and Spayd, S. E. 2005. Cluster
thinning effects on three deficit-irrigated Vitis vinifera cultivars.
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alter reproductive development in grapevines. Australian Journal of
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development controls leaf area dynamics in grapevine (Vitis vinifera)
growing in drying soil. Annals of Botany 98:175-185.
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new vineyards and fruit orchards in Bulgaria. University Press, UASG,
Sofia.
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fruitfulness in Vitis vinifera L. American Journal of Enology and
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Vanden Heuvel, J. E., Proctor, J. T. A., Sullivan, J. A. and Fisher, K. H.
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1

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Journal of Food, Agriculture & Environment Vol.12 (3&4): 127-130. 2014

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Dry matter accumulation trend on corn (TWC 647) as affected by plant density and
planting pattern
Ali Reza Saberi 1* and Siti Aishah Hassan
1

Agricultural & Natural Resources Research Center of Golestan, Gorgan, 4915677555, Iran. 2 Department of Crop Science,
University Putra Malaysia, 43400 Serdang, Selangor, Malaysia. *e-mail: alireza_sa70@yahoo.com

Received 16 June 2014, accepted 28 September 2014.

Abstract
To examine the effect of plant density and sowing pattern on some characteristics of corn (hybrid T. W. C. 647), a field experiment was conducted
at Agricultural Research Station of Iranshahr. This experiment was laid out in a randomized complete block design arranged in a factorial with four
replications. This experiment had four levels of plant densities (D1 = 70,000, D2 = 80,000, D3 = 90,000 and D4 = 100,000) with three levels of
planting arrangements (p1 = single row, p2 = double row 15 cm space apart and p2 = double row 20 cm space apart). The results showed increasing
accumulation trend of leaf dry matter on different levels of planting arrangement till receiving 1560 degree day and fixation and after that decreasing.
With increasing density and using double row planting arrangement till before grain milking stage, the most leaf dry weight produced and after that
decreased. With increasing plant density amount of leaf dry matter will increase, as high plant density (100,000 plant ha-1) has the highest leaf dry
matter. The results showed till douching stage (1658 degree day) logarithmical increasing of assimilate accumulation reach to its highest amount and
after a time fixes and later that because of the leaves become old and transferring assimilates from source to sink, amount of leaf dry matter decrease.
Stem dry weight changes started from 607 growth degree day and continued till milking-doughty stage (1357 - 1560 GDD), and decreased later
because of assimilate repeated transferring during seed becoming old. Trend of changes in stem dry weight such as leaf is sigmoid. It means with
passing time stem dry weight have increased and decreased safter reaching to maximum weight. With plant entering to reproductive stage and after
getting 1123 GDD emergence of flowering started which ended to ear production. Study of ear dry matter accumulation trend at different levels of
planting arrangement showed there was significant difference between double row planting pattern and single row, but there is no significant difference
on two levels of planting arrangement. It means at minimum and medium plant density, especially on one double-row pattern, the plants can grow
better and produce a good ear. The changes on husk dry weight from 1123 GDD started and after getting 1357 GDD reached to its maximum very fast
and later there was gradually reduction at the growth season because of grain formation and filling. With increasing plant density amount of husk in
the unit area increased. Husk has chlorophyll and due to closing to grain plays effective role to filling, at single row planting pattern it is able to transfer
its assimilates to ear store sinks more than in other planting patterns. During growth season after pollination and grain formation grain dry weight
increased. Its trends at initial stage after getting 1123 - 1357 GDD is very fast and at the end because of losing assimilate and filling sinks and loading
becomes slow. The highest grain yield (1400 g m-2) was got from 90,000 plants h-1 density and double row with 15 cm space apart treatment at 1797
GDD.
Key words: Sowing density, planting arrangement, hybrid T. W. C. 647, grain yield.

Introduction
With the increase in world population, demand for food
consequently will grow. It is expected that human population will
increase to over 8 billion by the year 2020 and this will worsen the
current scenario of food security. Improved crop productivity over
the past 50 years has resulted in increasing world food supplies
up to 20% per person and reducing proportion of food-insecure
people living in developing countries from 57% to 27% of total
population 5. It is predicted that at least 10 million people will be
hungry and malnourished in the world by the end of this century 5.
Thus, to reduce the food insecurity, crop production will have to
be doubled, and produced in more environmentally sustainable
ways 2. This can be achieved by expanding the area of crop
production, increasing per hectare yield and improving crop
quality. Furthermore, during the second half of the past century,
rise in per hectare crop productivity was due to improved or high
yield potential 1.
The relationship between growth of corn under different

planting pattern and plant density is not well understood. Many


changes take place in plants to enable them to compete and
maintain photosynthetic activity. A consideration of the
adaptation mechanisms by which density affects photosynthesis
would aid the improvement of growth conditions and crop yield
and would provide useful tools for future genetic engineering.
Research in the late 1980s demonstrated that yields can be raised
two to three-fold by using available improved varieties and
appropriate agronomic techniques. However, these findings need
to be refined, improved and tested for local climatic, soil and crop
conditions 8.
These include in the aspects of to what extent of planting pattern
and plant density affect the yield and morpho-pysiological
parameters of corn. In addition, no comprehensive database is
available on corn under combination of pattern and density at
north of Iran. Thus, studies are still needed to improve
understanding of the effects of pattern and density for corn.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

127

Hence, the present study was designed with the following


objectives:
1. To determine dry matter accumulation rate of corn at different
levels of plant density.
2. To study the effect of planting arrangement on dry matter
accumulation trend of corn.
3. To identify how interaction of planting pattern and plant
density affect dry matter accumulation trend of corn.

Results and Discussion


The results of comparing morphological parameters of corn at
four plant densities showed, that most of the corn characters
including leaf dry weight, stem dry weight, ear dry weight, husk
dry weight and grain dry weight were significantly different (P < 0.05)
between plant densities. Generally corn forage parameters increased
with increasing plant density but increasing of ear and grain is
limited and depended to planting arrangement could be increased.
The highest leaf dry weight, stem dry weight, ear dry weight and
grain dry weight were got from double row planting pattern (Fig. 1),
while the greatest husk dry weight was related to single row
planting pattern.
Growth indices and accumulation rate of dry matter were
analyzed based on GDD.
Accumulation rate of leaf dry matter: Fig. 2 shows increasing
accumulation trend of leaf dry matter on different levels of planting
arrangement till receiving 1560 degree day and fixation and after
that decreasing. With increasing density and using double row
planting arrangement till before grain milking stage, the most leaf
dry weight produced and after that decreased. At the end season
there was no significant difference between treatments.
Figure 3 shows with increasing plant density amount of leaf dry
matter increased, as high plant density (100,000 plant ha-1) has the
highest leaf dry matter. Till douching stage (1658 degree day)
logarithmical increasing of assimilate accumulation reached to its
128

Figure 1. General pictures from experiments, showing canopy


of double row planting pattern.
300

250

L - DM (g/m2)

Materials and Methods


A field experiment was conducted at Agricultural Research Station
of Iranshahr, Southern Iran. The experiment was laid out in a
randomized complete block design arranged in a factorial and
replicated four times. The experiment consisted of 12 treatments
outlined as follows: four levels of plant densities (D1 = 70,000, D2
= 80,000, D3 = 90,000 and D4 = 100,000) with three levels of planting
arrangements (p1 = single row, p2 = double row 15 cm space apart
and p2 = double row 20 cm space apart). The inter row spacing
was fixed at 75 cm while within row spacing was adjusted
according to plant densities and planting arrangement. Each
treatment combination was replicated in four blocks using a
randomized complete block design. Each plot comprised of six
raised beds of 6 m length and plants were harvested at the
physiological reach stage.
Sufficient number of plants was sown for each treatment to
facilitate destructive sampling for determining relative growth rates
at the various growth stages. The field was previously under
wheat which was harvested on 15 June. The land was plowed to a
depth of 20 - 25 cm followed by harrowing before planting. Data
were analyzed using the analysis of variance (ANOVA)
procedure 10 and means between the treatments were compared
using Duncan Multiple Range Test at P < 0.05.

200
A1
150

A2

100

A3

50

607

880 1123 1357 1560 1657 1797 1908


GDD

Figure 2. Effect of planting arrangement on leaf dry matter


accumulation trend of corn at different growth stages (average
of 2 years results).

highest amount and after a time fixes and later that because of the
leaves become old and transferring assimilates from source to
sink, amount of leaf dry matter decreased (Fig. 3).
Accumulation rate of stem dry matter: Stem dry weight changes
started from 607 growth degree day, continued till milking-doughty
stage (1357 - 1560 GDD), and later decreased because of assimilate
repeated transferring during seed becoming old (Fig. 4). Trend of
changes in stem dry weight such as leaf is sigmoid. It means with
passing time stem dry weight have increased and after reaching
to maximum weight, decreases.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

350

L - DM (g/m2)

300
250

D1

200

D2

150

D3

100

D4

50
0
607

880 1123 1357 1560 1657 1797 1908

plant density amount of husk in the unit area increased. Husk has
chlorophyll and due to closing to grain plays effective role to
filling, at single row planting pattern is able to transfer its
assimilates to ear store sinks more than in other planting patterns
(Figs. 6 and 7).
200

H - DM (g/m2)

400

150

Figure 3. Effect of plant density on leaf dry matter accumulation


trend of corn at different growth stages (average of 2 years results).
800

A3

607

880 1123 1357 1560 1657 1797 1908


GDD

Figure 6. Effect of planting pattern on ear husk dry matter


accumulation trend of corn at different growth stages (average
of 2 years results).

700
600
500

D1

400

D2

160

300

D3

140

200

D4

120

100
0
607 880 1123 1357 1560 1657 1797 1908
GDD

H - DM (g/m2)

ST - DM (g/m2)

A2

50

GDD

100

Figure 4. Effect of plant density on stem dry matter accumulation


trend of corn at different growth stages (average of 2 years results).

Accumulation rate of ear dry matter: With entering plant to


reproductive stage and after getting 1123 GDD emergence of
flowering started which ended to ear production. Fig. 5 shows
bigger angle that means high speed of ear dry matter increasing,
but gradually with close to end season because of limited
assimilate and fill out sinks lead to decrease angle of curve. Study
of ear dry matter accumulation trend at different levels of planting
arrangement (Fig. 5) shows there is significant difference between
double row planting pattern and single row, but there is no
significant difference on two levels of planting arrangement. It
means at minimum and medium plant density especially on one
double - row pattern, the plants can grow better and produce a
good ear 7, 9, 11.
2000
1500
A1

1000
500

D2

60

D3

40

D4

0
607 880 1123 1357 1560 1657 1797 1908
GDD

Figure 7. Effect of plant density on ear husk dry matter


accumulation trend of corn at different growth stages (average of
2 years results).

Accumulation rate of grain dry matter: During growth season


after pollination and grain formation grain dry weight increased.
Its trend is very fast at initial stage after getting 1123 - 1357 GDD
and at the end because of losing assimilates and filling sinks and
loading become slow. The highest grain yield (1400 g m-2) got
from 90,000 plant h-1 density and double row with 15 cm space
apart treatment at 1797 GDD. Increasing the yield at high plant
density due to double row pattern is because of closing to square
planting arrangement. The yield at low plant density due to lacking
number of plants per surface and at high plant density because of
competition for absorption growth elements and interference of
male and female flowers become limited 3, 4, 6 (Fig. 8).

A2

1600

A3

1400

0
607

D1

80

20

880 1123 1357 1560 1657 1797 1908


GDD

Figure 5. Effect of planting pattern on ear dry matter


accumulation trend of corn at different growth stages (average
of 2 years results).

Accumulation rate of husk dry matter: The changes on husk dry


weight from 1123 GDD started and after getting 1357 GDD reached
to its maximum very fast and later gradually reducted at the growth
season because of grain formation and filling. With increasing

GR - DM (g/m2)

E - DM (g/m2)

A1

100

1200
1000

A1

800

A2

600

A3

400
200
0
607 880 1123 1357 1560 1657 1797 1908
GDD

Figure 8. Effect of planting pattern on grain dry matter


accumulation trend of corn at different growth stages (average
of 2 years results).

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

129

Conclusions
It might be concluded that by using double row planting pattern
the interplant competition could be decreased and higher yield
might be produced.
Acknowledgements
The authors would like to thank the Agricultural & Natural
Resources Research Center of Blochestan and the Seed & Plant
Improvement Institute of Iran for the financial support of this
research.
References
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Brown, R. H., Beaty, E. R., Ethedge, W. J. and Hages, D. D. 1970.
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Duncan, W. G. 1984. A theory to explain the relationship between corn
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FAO 2007. Fertilizer use by crop in Uzbekistan. FAO, Rome. http://
www.fao.org/DOCREP/006/Y4711E/Y4711E00.HTM. Last accessed
12.02.2007.
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Lutz, J. A., Comper, H. M. and Jones, C. D. 1971. Row spacing and
plant population effects on corn yield. Agron. J. 63:12-14.
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Proter, P. M. and Hicks, D. K. 1997. Corn response to row width and
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Qureshi, A. S., Qadir, M., Heydari, N., Turral, H. and Javadi, A. 2007.
A review of management strategies for salt-prone land and water
resources in Iran. International Water Management Institute, Colombo,
Sri Lanka, IWMI Working Paper 125, 30 p.
9
Saberi, A. R., Mazaheri, D. and Heidari Sharif Abad, H. 2006. Effect of
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characteristics of maize (hybrid T. W. 647). Agricultural and Natural
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10
SAS Institute 2004. SAS/STAT Users Guide. Release 9.0. 4th edn.
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1

130

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Journal of Food, Agriculture & Environment Vol.12 (3&4): 131-135. 2014

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Productivity and gas exchange parameters of selected pasture grasses under


drought stress
Anna Koco and Mariola Staniak *
Department of Forage Crop Production, Institute of Soil Science and Plant Cultivation State Research Institute, Czartoryskich
Str. 8, 24-100 Puawy, Poland. e-mail: Mariola.Staniak@iung.pulawy.pl, Anna.Kocon@iung.pulawy.pl
Received 16 May 2014, accepted 8 September 2014.

Abstract
The objective of the study was to determine the effects of water stress on productivity, leaf greenness index and gas exchange parameters of selected
pasture grasses. A pot experiment was performed in a greenhouse of the Institute of Soil Science and Plant Cultivation, State Research Institute in
Puawy, at two soil moisture levels: 70% (optimum moisture content) and 40% (water stress) of field water capacity to study the responses of four
pasture grasses: Lolium multiflorum (Lam.), Festuca pratensis (L.), Festulolium braunii ((K. Richt.) A. Camus) and Dactylis glomerata (L.) to water
stress. The study showed that water stress caused a significant reduction of yield of all tested grasses. The greatest decrease in the yield of dry matter
was observed in L. multiflorum, while the lowest one was found in D. glomerata. Among the studied species of grasses, a higher resistance to stress
was recorded for the D. glomerata and F. braunii (drought susceptibility index (DSI) < 1.0). The water content in the soil modified the relative level
of chlorophyll in the leaves of tested grasses. Under the conditions of soil drought, greenness index in all species of grasses was higher on average by
19% compared to control objects. The lowest value of greenness index was found in the F. pratensis. Under water stress conditions, all studied species
were characterized by lower net photosynthesis (PN) and transpiration (E) rate as compared to control. The mean values of water-use efficiency
(WUE) were statistically higher under stress conditions in D. glomerata and F. pratensis than in L. multiflorum and F. braunii.
Key words: Grasses, drought stress, dry matter yield, net photosynthesis, transpiration rate, drought susceptibility index, leaf greenness index, wateruse efficiency.

Introduction
Crop performance and yield are the results of genotypic expression
as modulated by continuous interactions with the environment.
Water is one of most widely limiting environmental factors for
crop production on a global basis. Drought is strongly affecting
the growth processes and productivity of plants 1-4. The reaction
of forage grasses to drought is first of all strong inhibition of
growth and developmental perturbations, which leads to reduction
of yield. Grass species differ in tolerance for moisture conditions
of habitat and in adaptability to changeable conditions 5.
Chlorophyll is the most common photosynthetic pigment. It
can be also considered an indicator of the annual yield of grasses
and grass regrowth, since there exists a positive correlation
between its content in the leaf blades and dry matter yield 6-8.
Chlorophyll concentration is a reliable indicator of plant vigor
and resistance to environmental stressors. The chlorophyll
contents of leaves depend on species, variety, climatic and soil
conditions, nutrient availability and development stage of plant 9.
Plant growth and yield are the outcome of numerous processes,
photosynthesis being the key one. A rapid decrease in water supply
makes plants to close their stomata. This enables rapid reduction
of water losses during transpiration, but this process is
accompanied by increased diffusion resistance for CO2, leading
to photosynthesis inhibition. This allows the plant to survive
drought, but leads to yield decrease 4, 10, 11.
Droughts in Poland are hardly predictable. It is difficult to
forecast the term of their occurrence, duration, territorial range
and intensity 12. Therefore, searches for greater utilization of forage

plant tolerance to drought are desirable. In this study, an attempt


was made to verify the hypotheses: productivity and physiological
processes of grasses depend on soil moisture conditions. The
objective of the study was to determine the effects of water stress
on productivity, leaf greenness (SPAD) and gas exchange
parameters of selected pasture grasses.
Materials and Methods
A pot experiment was performed in a greenhouse of the Institute
of Soil Science and Plant Cultivation, State Research Institute in
Puawy (5125'N, 2158'E), at two soil moisture levels: 70%
(optimum moisture content) and 40% (water stress) of field water
capacity to study the responses of four pasture grasses: Lolium
multiflorum (Lam.) cultivar Gisel, Festuca pratensis (L.) cv.
Fantazja, Festulolium braunii (K. Richt.) A. Camus cv. Sulino
tetraploid hybrid between Lolium multiflorum and Festuca
pratensis and Dactylis glomerata (L.) cv. Amera to water stress.
Soil moisture content was differentiated 6 weeks after sowing. In
order to maintain the appropriate soil moisture, water losses were
made up on a daily basis, to achieve a specified weight of the pot
with soil. Mitscherlich pots were filled with 7 kg of mineral soil.
The available nutrient contents (mg/100 g) of the soil were P 32.0,
K 12.3 and Mg 5.1. Soil pHKCl was 6.2. The experiment was
performed in four replications. Two to three seeds were sown at
30 points of each pot. After outcrop poorly developed seedlings
were removed, leaving fifteen plants per pot. Nitrogen fertilization,
in the amount of 3.6 g per pot, was applied at three rates, before

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

131

Table 1. Dry matter yield of grasses species [g pot-1].


Species
D. glomerata
F. pratensis
F. braunii
L. multiflorum
D. glomerata
F. pratensis
F. braunii
L. multiflorum
70%
40%

III
26.3 a
11.9 d
32.2 b
20.7 e
33.0 b
22.6 e
41.8 c
25.2 e

Total
yield
111.8 a
72.0 c
121.7 a
74.8 c
121.6 a
77.3 c
152.0 b
89.5 d

19.1 a
26.4 b
27.8 b
33.5 c

91.9 a
98.2 ab
99.5 b
120.7 c

33.4 a
20.1 b

126.8 a
78.4 b

0.422
0.41
0.404
0.399
0.388
0.377
0.366
0.355
0.344
0.33
0.322

DSI = [1 (Dn Kn-1)] [1 (Dx Kx-1)]-1

D. glomerata

YR = 1 (Yry Ypy-1)

where Yry is the reduced yield due to water deficiency, while Ypy is
the potential yield under optimal soil moisture.
The results presented in the paper are means of particular cuts.
They were analysed statically using STATISTICA 6.0 software.
The significance of differences was verified by the Tukey test at
a significance level p < 0.05
Results
Soil moisture is an important factor shaping the size of crop yield.
Under optimal moisture conditions, the highest yield was obtained
from L. multiflorum, while the lowest one from D. glomerata. F.
pratensis and F. braunii yielded at a similar level (Table 1). All the
compared species responded to lower soil moisture with a
significant yield decrease. The smallest decrease in the total yield
(35%) was observed at D. glomerata, while it was greatest at L.
multiflorum (41%). Hybrid F. braunii reacted to drought with a
lower decrease in the total yield than both parent species, L.
multiflorum and F. pratensis (Fig. 1). Considering individual cuts,
D. glomerata and L. multiflorum yielded best in the first cut,

F. braunii

F. pratensis

L. multiflorum

Grasses species

Figure 1. Yield Reduction (YR) of grasses species in condition of water


deficit in the soil.

while F. pratensis and F. braunii in the second. The yield of the


third cut was smallest in all studied species of grasses. The
calculation of drought susceptibility index (DSI) allowed for
ranking the tested grasses according to their susceptibility to
drought. The lowest sensitivity to water shortages in the soil was
recorded for D. glomerata, while L. multiflorum was least resistant
to stress conditions. F. braunii hybrid proved to be more drought
tolerant than the two parent species, which is proved by a less
than one value of the DSI index (Fig. 2).
The water content in soil was a factor modifying the chlorophyll
index in the leaves in all tested species of grasses (Table 2). Under
the conditions of soil drought, leaf greenness index (SPAD)
1.101
1.088
1.066
1.044
DSI

where Dn and Kn are the dry matter of the stressed and non-stressed
object, while Dx and Kx are the mean dry matter averaged over all
objects under stress and non-stress conditions, respectively. Thus,
a low DSI-value (< 1.0) is indicative for a relative tolerance, while
a high DSI-value (> 1.0) for its stress sensitivity.
Yield Reduction (YR) was calculated by abdzkis method 12
using the following equation:

132

Regrowth
Soil
moisture
I
II
70%
49.8 a* 35.6 a
40%
33.2 df 28.9 d
70%
40.1 b 48.0 b
40%
24.5 e 30.4 d
70%
42.5 b 46.1 b
40%
28.3 ef 27.3 d
70%
56.9 c
53.2 c
40%
34.9 f
30.9 d
Mean for species
32.3 a
41.5 a
32.3 b
39.2 b
35.4 b
36.7 b
45.9 c
42.1 c
Mean for soil moisture
47.35 a 45.8 a
30.22 b 29.4 b

* Values marked with the same letter did not differ statistically.

YR

sowing, after the first and second cuttings. Nitrogen was applied
in the form of NH4NO3 solution. Phosphorus, potassium and
magnesium were applied pre-sowing, in the forms of solutions:
KH2PO4, K2SO4 and MgSO4, respectively, at the following rates
[g pot-1]: P 1.0, K 1.5 and Mg 0.5.
Over the vegetation season, the leaf greenness index was
measured with an optical chlorophyll meter SPAD-502Plus (Konica
Minolta, Japan). This device measures the difference between
light absorption by leaf at wavelengths of 650 and 940 nm, and the
quotient of these values represents indexed leaf greenness, i.e.
indexed relative chlorophyll content. The obtained results are
shown in SPAD units (Soil and Plant Analysis Development) in
the range from 0 to 800. The measurements were taken for the first
time 8 weeks after sowing (2 weeks after water stress). Four
measurements were performed for each regrowth and readings
were taken at one-week intervals. The rate of photosynthesis and
transpiration was measured two times over the growing season
(first and third regrowth) with a LI6400XT portable gas analyser
(LI-COR Environmental, USA). The measurements were performed
at a constant concentration of CO2 390 ppm, photosyntetic active
radiation (PAR) of 1200 mol m-2 s-1 and temperature between 23
and 27C. The chlorophyll concentration and the rate of
photosynthesis and transpiration were measured on the youngest,
fully developed leaves selected randomly of each pot. The plants
were defoliated three times over the growing season.
Drought Susceptibility Index (DSI) was computed by Fisher
and Maurer 13 method using the following equation:

1.022
1.001
0.988
0.966
0.944
0.922
D. glomerata

F. braunii

F. pratensis

L. multiflorum

Grasses species

Figure 2. Drought susceptibility index (DSI) of grasses species.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Table 2. Leaf greenness index (SPAD) of grasses species.


Species
D. glomerata
F. pratensis
F. braunii
L. multiflorum
D. glomerata
F. pratensis
F. braunii
L. multiflorum
70%
40%

Regrowth
Soil
moisture
I
II
III
70%
460 a* 574 ad 596 ac
40%
556 b 591 abd 598 ac
526 a
529 ab
70%
469 a
40%
622 b
638 bc
632 c
70%
452 a
503 a
515 b
40%
548 b
652 c
613 c
70%
415 a
515 a
508 b
40%
546 b
604 cd
626 c
Mean for species
583 a
597 a
508 a
545 ab
582 a
581 a
500 a
578 a
564 a
481 b
560 a
567 a
Mean for soil moisture
449 a
530 a
537 a
568 b
621 b
617 b

Table 3. Intensity of net photosynthesis (PN) of


grasses species [mol (CO2) m-2 s-1].

Mean

Species

543 a
582 ce
508 a
631 d
490 b
605 de
479 b
592 e

D. glomerata
F. pratensis
F. braunii
L. multiflorum

563 a
569 a
547 ab
536 b

D. glomerata
F. pratensis
F. braunii
L. multiflorum

505 a
602 b

70%
40%

* Values marked with the same letter did not differ statistically.

Regrowth
Soil
moisture
I
III
70%
11.20 c* 7.40 e
40%
7.03 a
4.17 c
70%
13.80 e 10.27 g
40%
7.87 b
2.53 a
8.87 f
70%
12.53 d
40%
7.03 a
3.37 b
70%
14.08 f
9.37 f
40%
7.93 b
6.80 d
Mean for species
5.78 a
9.12 a
10.83 c
6.40 b
9.78 b
6.12 ab
11.00 d
8.08 c
Mean for soil moisture
12.90 b
8.46 b
7.47 a
4.89 a

Mean
9.33 c
5.57 a
12.03 e
5.23 a
10.70 d
5.17 a
11.73 e
7.40 b
7.45 a
8.63 c
7.93 b
9.57 d
10.95 b
5.84 a

* Values marked with the same letter did not differ statistically.

increased on average by 19% compared to plants grown under


optimal moisture conditions. The smallest increase was recorded
for D. glomerata - an average of 7%, with significant differences
occurring only in the first cut. In other species, leaf greenness
index increased on average by 24%, and the individual cuts ranged
from 17 to 32%. Under conditions of optimum soil moisture, lower
chlorophyll index was observed in L. multiflorum and F. braunii,
and significantly higher in D. glomerata and F. pratensis. In water
deficiency conditions, F. pratensis showed the highest values of
leaf greenness factor. In this study, the lowest chlorophyll index
were recorded in the first regrowth. The calculated values of the
correlation coefficient and regression equations showed a
significant negative correlation between dry matter yield and leaf
greenness index (Fig. 3).
Measurements of photosynthetic activity revealed significant
differences between species of grasses. The mean PN was highest
in L. multiflorum and lowest in D. glomerata. This process was
more intensive in the leaves of all grasses in the first regrowth
than in the third one (Table 3). Water stress considerably reduced
F. braunii

40
30
20

DM yield (g pot-1)

DM yield (g pot-1)

y = 84.526 - 0.0936 x
R 2 = 0.59

50

y = 85.474 - 0.0914 x
R 2 = 0.52

50
40
30
20
10

10
400

450

500

550 600
SPAD

650

400

700

500

550

600

650

700

F. braunii

40
30
20

DM yield (g pot-1)

60

y = 105.464 - 0.1213 x
2
R = 0.66

50

450

SPAD

L. multiflorum

60
DM yield (g pot-1)

F.p pratensis

60

60

photosynthetic activity (on average by 46%). The strongest


response was recorded in F. pratensis, where PN decreased by
56%, as compared with the control treatments (70% FWC). The
decrease in photosynthetic rate, caused by water deficit in the
soil, was accompanied by reduced transpiration in the experimental
species, since both processes are interrelated, as confirmed by
high correlation coefficients. On average, drought stress reduced
E by 54%. Among the tested species, D. glomerata showed the
lowest E under moisture optimal and deficiency conditions (Table
4). The heaviest water losses were recorded in the first regrowth.
In all tested species, the values of water use efficiency were
significantly higher under stress conditions in the first regrowth
(ranged from 5.45 to 7.98) but in the third regrowth it was generally
lower in such conditions (Table 5). The mean values of WUE were
significantly higher under stress conditions in D. glomerata and
F. braunii.

y = 84.526 - 0.0936 x
R 2 = 0.59

50
40
30
20
10

10
400

450

500

550 600
SPAD

650

700

400

450

500

550 600
SPAD

650

700

Figure 3. Relationship between leaf greenness index (SPAD) and dry matter yield of grasses
species.
Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Discussion
The results showed that the grass grown
under water deficit conditions yielded on
average by about 38% lower compared to
those grown under optimal moisture
conditions. According to Olszewska 14, the dry
matter yield of L. perenne and D. glomerata
was on average by 35% lower compared to
control treatments. A significant decrease in
yield, caused by water deficit in forage
grasses was also reported 15, 16. A typical
response of plants to water stress is to reduce
the yield, because there are limitations to the
intensity of photosynthesis and plant growth
processes 10, 11. According to Starck 17 and
Rawson et al. 18, crop yield is also affected
by other factors, such as the efficiency of
transport and distribution of assimilates in
plants. Ardiani et al. 19 reported, that changes
in soil moisture during the season have a
greater impact on the productivity of forage
grasses than the photosynthetic efficiency
of individual species.
133

Table 4. Intensity of transpiration (E) of grasses


species [mmol (H2O) m-2 s-1].
Species
D. glomerata
F. pratensis
F. braunii
L. multiflorum
D. glomerata
F. pratensis
F. braunii
L. multiflorum
70%
40%

Regrowth
Soil
moisture
I
III
70%
2.49 c* 1.77 bc
40%
0.88 a
1.12 a
2.36 d
70%
4.26 f
40%
1.23 b 1.57 abc
4.02 e
70%
3.15 d
40%
1.28 b
1.32 a
70%
3.63 e
2.03 cd
40%
1.41 b
1.85 bc
Mean for species
1.44 a
1.68 a
2.43 c
1.96 b
2.22 b
2.67 c
2.52 c
1.94 b
Mean for soil moisture
3.38 b
2.84 b
1.34 a
1.19 a

chlorophyll content in leaves of grasses in each regrowth. The


lowest leaf greenness values were recorded in the first regrowth,
confirming studies by Gbork 6 and Olszewska 14 of higher relative
chlorophyll concentrations in the second and third regrowth
compared to the first one. The level of chlorophyll in leaves is a
reliable indicator of plant life and their response to changing
habitat conditions 7. It can be assumed that larger leaf greenness
index in the leaves of crops grown in stressful conditions is the
result of defence against stress. Under the conditions of water
deficit, there is a decrease in the density of cells and tissues of the
leaf. Therefore, there is an increase in their concentrations of microand macromolecules, including chlorophyll 5.
Decrease in the photosynthesis and transpiration rate in grasses
and cereals resulting from water stress were also reported by other
authors 19, 24, 25. Olszewska 14 showed that a decrease in soil moisture
from 80% to 40% of FWC in L. perenne and D. glomerata reduced
the rate of photosynthesis by on average 42% and the rate of
transpiration by on average 66%. According to Jones et al. 10,
reduced transpiration rate of grasses exposed to moisture stress
is related to changes in leaf structure. Under water deficit
conditions leaves are shorter, their surface is smaller, tissue is
more compact, and stomata are shorter, but densely arranged.
Such leaf morphology limits transpiration, which makes plants
more resistant to drought.

Mean
2.13 d
1.00 a
3.00 e
1.40 bc
3.59 f
1.30 b
2.83 e
1.63 c
1.56 a
2.20 b
2.44 c
2.23 b
2.88 b
1.33 a

* Values marked with the same letter did not differ statistically.

Table 5. Water use efficiency (WUE) of grasses


species [mol (CO2) mmol (H2O) m-2 s-1].
Regrowth
Soil
moisture
I
III
D. glomerata
70%
4.51 b* 4.21 d
40%
7.98 e 3.72 cd
4.36 d
F. pratensis
70%
3.80 a
40%
6.38 d
1.66 a
F. braunii
70%
3.99 a 2.20 ab
40%
5.45 c 2.76 bc
L. multiflorum
70%
3.87 a
4.63 d
40%
5.64 c 3.67 cd
Mean for species
3.96 b
D. glomerata
6.24 c
F. pratensis
5.09 b
3.01 a
4.72 a
2.48 a
F. braunii
L. multiflorum
4.76 a
4.15 b
Mean for soil moisture
70%
4.04 a
3.85 b
40%
6.36 b
2.95 a
Species

Mean
4.36 bc
5.85 d
4.08 b
4.02 b
3.10 a
4.11 b
4.25 bc
4.65 c
5.10 d
4.05 b
3.60 a
4.45 c
3.95 a
4.66 b

* Values marked with the same letter did not differ statistically.

A quick and easy method to determine the content of the green


pigment in leaves is to use a chlorophyllometer. A close correlation
between the readings of a chlorophyllometer and relative
chlorophyll content calculated by the traditional laboratory method
was found. This is shown by Jordi et al. 20 (r = 0.974), Gregorczyk
and Raczyska 21 (r = from 0.947 to 0.973) and Kozowski et al. 7 (r
= 0.970). Soil water deficit was a factor which significantly affected
the level of chlorophyll in the leaves of forage grasses. All tested
species showed a higher chlorophyll index in conditions of water
deficit in the soil. Also, Olszewska 14 showed that under water
stress conditions, relative chlorophyll content in leaves of L.
perenne and D. glomerata was significantly higher compared to
control. At the same time L. perenne varieties were characterized
by higher leaf greenness index than varieties of D. glomerata.
The author demonstrated similar correlations in the case of F.
pratensis and P. pratense 22. A significant influence of weather
conditions on the increase of chlorophyll content in leaves was
also reported by Michaek and Sawicka 9, who showed that, in the
conditions of lower amount of rainfall and higher air temperatures
in July and August, the plants accumulate more of this pigment in
leaves. Goliski and Xi 23 showed significant differences of relative
134

Conclusions
Water stress caused a significant reduction of yield of D.
glomerata, F. pratensis, L. multiflorum and F. braunii. The
greatest decrease in the yield of dry matter was observed in
L. multiflorum, while the lowest one was found in D. glomerata.
Among the studied grass species, a higher tolerance to stress
was recorded for D. glomerata and F. braunii (DSI index < 1.0). F.
pratensis and L. multiflorum were more sensitive to drought index
(DSI) > 1.0.
The water content in the soil modified the relative level of
chlorophyll in the leaves of tested grasses. Under the conditions
of soil drought, leaf greenness index in all species of grasses was
higher compared to control object. The lowest values of
chlorophyll index were found in F. pratensis.
A decrease in moisture from 70% to 40% of field water capacity
reduced the activity of photosynthesis, on average 46% and the
rate of transpiration 54%. The mean values of water-use efficiency
(WUE) were statistically higher under stress conditions in D.
glomerata and F. braunii.
Acknowledgements
The studies have been supported by Ministry of Agriculture and
Rural Development of Poland within the multi-annual program of
Institute of Soil Science and Plant Cultivation, State Research
Institute, task 3.4. Analysis and evaluation possibilities of
shaping the quality of plant materials taking into account the
different directions of use and regional conditions and task 3.1
The support activities in the field of fertilizer in Poland.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

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Olszewska, M. 2006. Effect of water stress on physiological processes,
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Szoszkiewicz, J., Madziar, Z. and Zbierska, J. 1991. Effect of soil
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22

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

135

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Journal of Food, Agriculture & Environment Vol.12 (3&4): 136-138. 2014

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Effect of sulphur fertilization on fatty acid composition of faba bean (Vicia faba L.),
white lupin (Lupinus albus L.) and pea (Pisum sativum L.) grains
Eugenio Cazzato 1*, Vito Laudadio 2, Edmondo Ceci 3 and Vincenzo Tufarelli
1

Department of Agro-Environmental and Territorial Sciences, University of Bari Aldo Moro, 70125 Bari, Italy. 2 Department of
DETO, University of Bari Aldo Moro, 70010 Valenzano, Italy. 3 Department of Veterinary Medicine, University of Bari Aldo
Moro, 70010 Valenzano, Italy. *e-mail: eugenio.cazzato@uniba.it

Received 8 April 2014, accepted 12 September 2014.

Abstract
Winter legume grains are suited as a significant source of vegetable protein for both human and livestock species, due to high protein content and the
high level of unsaturated fatty acids. This research reported the effect of S fertilization on the quality of three different pulses (faba bean, lupin and
pea) in terms of lipid content and fatty acid profile. For each species, randomized complete block design with three replicates was used, and three S
doses (0, 30 and 60 kg ha-1, respectively) were applied. The S fertilization was split in two times: 50% before sowing and 50% in the early of March
as K2SO4. Our findings indicated that the S fertilization in faba bean, lupin and pea grains led to a significant improvement of the fatty acid profile.
Furthermore, the S fertilization enhanced the legume grains oil composition through the increase of unsaturated fatty acids, and in particular the
remarkable decrease of the erucic acid in lupin grains.
Key words: Faba bean, lupin, pea, sulphur fertilization, fatty acids.

Introduction
Grain legumes are important crops in Mediterranean area and in
other countries of the world. Legume seeds exhibit high levels of
protein, essential amino acids, and important dietary minerals.
They are used in a popular breakfast food, and also used as a
vegetable green, fresh or canned. Also, they are important crops
for soil improvement and used as break crop in cereal rotation to
keep the soil fertile and productive 1, 2. Sulphur (S) is one of the
elements known to be essential for the legume-rhizobium system
with specific physiological and biochemical roles 3. The S demand
of legume crops is higher than that of cereal crops. Studies on
different legumes have shown that the concentration of the Scontaining amino acids was markedly declined with decreasing S
supply 4. Sulphur fertilization was also found to increase N
accumulation and yield of legumes on S-deficient soils. Among
the winter legume grains, the seed of lupin contains 9 - 14% oil,
whose composition includes in particular oleic acid, linoleic acid
and -linolenic acid 5. The increasing selection of varieties has
recently widened the possibility of increase the use of the legume
grains in human or livestock nutrition as a replacement for either
animal/vegetable proteins 6-8. However, to date studies on the
effects of S fertilization on grain legumes fatty acid contents are
limited. Therefore, the present field trial was conducted in Southern
Italy under Mediterranean conditions to evaluate the effect of S
fertilization on the fatty acid composition of faba bean, lupin and
pea grains.
Materials and Methods
A field trial was carried out in Southern Italy at Gaudiano di Lavello
Potenza (4106' N; 1551' E; 145 m above sea level) on a sandyclay soil, characterised as sub-alkaline, low in total N (0.77
136

Kjeldahl method) and high in available P (83 mg kg-1; Olsen


method), exchangeable K (482 mg kg-1; BaCl2; TEA method) and
low soluble-S as SO4-S (4.4 mg kg-1; KH2PO4 as extractant). The
experimental site was characterised by a summer-dry climate with
a total annual rainfall of 560 mm distributed from autumn to spring
and a mean temperature of 16C. During the experimental period
(November - June), the total rainfall was 402 mm and temperatures
did not show any significant variation from the average. Faba
bean (Vicia faba L. cv. Prothabat 69), low alkaloids variety of
lupin (Lupinus albus L. cv. Multitalia) and pea (Pisum sativum L.
cv. Spirale) were sown on November maintaining a row distance
of 20 cm for each species at seed rate of 300 kg ha-1 (faba bean and
lupin) and 270 kg ha-1 (pea), respectively. For each legume species,
a randomized complete block design with four replicates and plot
area of 15 m2 were utilized. Three S fertilization levels (0, 30 and 60
kg/ha) were applied. The S fertilization was split in two times: 50%
before sowing and 50% in the early of March as K2SO4. The K
contained in K2SO4 was compensated with KCl for the plots not
treated with S. Crops were grown under rainfed conditions and no
irrigation was used. At physiological maturity, plants were
harvested. These plant parts were oven-dried at about 70C for 48
h. The plant material was ground to pass through a 0.2 mm sieve.
Total lipids were extracted 9. In preparation for the analysis of FA
composition, samples of the different legume species (1 g each)
were freeze-dried and ground. Methyl heptadecanoate (No. 51633,
Fluka, USA) was dissolved into n-hexane (1 mg/ml) as an internal
standard. Methyl esters of the FA were prepared; samples (300
mg each) and 5 ml internal standard were incubated with methanolic
acetyl chloride in a total volume of 9 ml. After cooling to room
temperature, 7 ml of 7% (w/v) K2CO3 was added with mixing, and

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

the organic phase was collected after centrifuging at 1500 g for 2


min at 4C. Fatty acid methyl esters were fractionated over a CPSIL883 column (100 m 0.25 mm i.d., film thickness 0.20 m fused
silica; Varian, Palo Alto, CA, USA) in a Shimadzu (Model 2GC17A)
gas chromatograph with a Hewlett-Packard HP 6890 gas system
and using flame ionization detection. Helium was used as carrier
gas at a constant flow rate of 1.7 ml/min. The oven temperature
was programmed as follows: 175C, held for 4 min; 175 - 250C at
3C/min; and then maintained for 20 min. The injector port and
detector temperature were 250C. Samples (1 l) were injected
with an auto-sampler. Output signals were identified and quantified
from the retention times and peak areas of known calibration
standards. Composition was expressed as percentages of the total
FA.
Data was analysed by analysis of variance using (ANOVA)
CoStat version 1.03 Software (CoHort Software Inc., Monterey,
CA, USA). The statistical analysis was applied to data following
the one-way ANOVA design. Unless otherwise stated, significance
was declared at P < 0.05.
Results
The results of total lipids and FA analysis (% on total FA) of lupin,
faba bean and pea grains obtained under different S fertilization
are reported in Table 1. Our results indicated that the different S
fertilization rates did not influence the total lipid content of faba
bean (1.83%) and pea grains (1.09%); whereas, the different S
fertilization improved significantly the total lipid content in lupin
grain (from 8.13 to 8.75%; for S0 to S60, respectively). The results
of FA analysis (% on total FA) of lupin seeds obtained under
different S fertilization are presented in Table 1. Overall, total
monounsaturated FA (MUFA) decreased significantly from 67.3
to 65.5% (S0 and S30, respectively). In particular, among the
individual MUFA, a significant decrease was observed for erucic
acid, whereas palmitoleic, oleic and eicosenoic acids resulted
higher when S fertilization increased up to S30. Particularly, in our
cultivar of lupin the main MUFA was the cis9 n-9 oleic acid
representing about the 86% of total MUFA. Total polyunsaturated
FA (PUFA) content increased significantly from 21.9 to 23.1% (S0
and S30, respectively); the higher S application rate did not result
in further increase of PUFA level compared to the intermediate S
dose. The main PUFA was the linoleic acid that represented 93%
of total PUFA. In faba bean grains, the overall total saturated FA
(SFA) decreased significantly from 24.41 to 18.86% (S0 and S30,
respectively), whereas S60 application did not lead further variation
(18.47%) compared with S30 rate. In particular, among the
individual SFA, significant decrease was observed for myristic,
heptadecanoic, stearic and tetracosanoic acids when S
fertilization increased up to S30. No effects of S fertilization were
reported for total monounsaturated FA in faba bean grains that
in overall represented about 25% of total FA. Conversely, total
polyunsaturated FA (PUFA) content increased significantly from
51.35 to 56.12% (S0 and S30, respectively); the higher S application
rate did not result in a further increase of PUFA level compared
with the intermediate S dose. Particularly, in our cultivar of faba
bean seeds, the main PUFA was the n-6 linoleic acid representing
about the 90% of total PUFA. Data on FA content of pea grains
grown under different S fertilization levels are reported in Table 1. In
overall, the total saturated FA (SFA) resulted 16.33% and no
significant differences were reported among S fertilization levels

Table 1. Effect of sulphur fertilization on fatty acid (FA) in lupin,


faba bean and pea grains.
Fatty acids (% on total FA)
14:0 Myristic
16:0 Palmitic
16:1 n-7 Palmitoleic
18:0 Stearic
18:1 cis9 n-9 Oleic
18:2 n-6 Linoleic
18:3 n-3 -Linolenic
18:3 n-6 -Linolenic
20:1 n-11 Eicosenoic
22:1 n-9 Erucic
SFAa
MUFAa
PUFAa
Total lipids
12:0 Lauric
14:0 Myristic
15:0 Pentadecanoic
16:0 Palmitic
16:1 n-7 Palmitoleic
17:0 Heptadecanoic
18:0 Stearic
18:1 cis9 n-9 Oleic
18:2 n-6 Linoleic
18:3 n-3 -Linolenic
18:3 n-6 -Linolenic
20:1 n-11 Eicosenoic
20:4 n-6 Arachidonic
22:0 Docosanoic
24:0 Tetracosanoic
SFAa
MUFAa
PUFAa
Total lipids
16:0 Palmitic
18:0 Stearic
18:1 trans9 n-9 Elaidic
18:1 cis9 n-9 Oleic
18:3 n-3 -Linolenic
18:3 n-6 -Linolenic
SFAa
MUFAa
PUFAa
Total lipids

Treatment
S30
Lupin
0.19a 0.13b
6.59c 7.57a
0.33
0.37
3.98a 3.77ab
55.01b 58.04a
19.96b 21.89a
0.23a 0.14b
1.73
1.42
4.34b 4.85a
7.64a 2.22c
10.76b 11.47a
67.32a 65.48c
21.92c 23.05b
8.13b 8.72a
Faba bean
0.02
0.02
6.61a 0.50b
0.10
0.27
11.93b 13.26a
0.01
0.04
0.52a 0.19b
3.01
2.83
22.31c 23.24b
44.85c 49.82b
0.46a 0.11b
4.94
5.03
1.94
1.75
1.10
1.16
1.01a 0.80b
1.21
0.99
24.41a 18.86b
24.26 25.03
51.35b 56.12a
1.83
1.82
Pea
11.50a 11.38a
5.27
5.16
0.10b 0.26b
15.76b 17.20b
53.16a 51.90b
14.21a 14.10a
16.77 16.54
15.86c 17.46b
67.37a 66.00b
1.11
1.07
S0

S60

Mean SEM P-value

0.11c
7.30b
0.36
3.06b
58.46a
21.90a
0.12b
1.30
4.93a
2.46b
10.47c
66.21b
23.32a
8.75a

0.14
7.15
0.35
3.47
57.17
21.25
0.16
1.48
4.71
4.11
10.90
66.34
22.76
8.63

0.03
0.32
0.05
0.17
0.95
0.41
0.05
0.10
0.15
0.18
0.22
1.10
0.43
0.05

0.045
0.035
0.076
0.037
0.027
0.019
0.034
0.055
0.022
0.005
0.018
0.025
0.009
0.047

0.01
0.49b
0.26
13.30a
0.09
0.17b
2.80
23.87a
50.07a
0.01c
4.96
1.65
0.87
0.84b
0.60
18.47b
25.61
55.91a
1.84

0.02
2.53
0.21
12.83
0.05
0.29
2.88
23.14
48.25
0.19
4.98
1.78
1.04
0.88
0.93
20.58
24.97
54.46
1.83

0.01
0.11
0.05
0.21
0.02
0.04
0.09
0.38
0.45
0.04
0.12
0.08
0.05
0.13
0.15
0.28
0.37
0.49
0.09

0.255
0.014
0.101
0.042
0.095
0.052
0.069
0.045
0.014
0.011
0.052
0.069
0.051
0.039
0.058
0.021
0.117
0.012
0.115

10.25b
5.42
4.37a
22.76a
44.77b
12.43b
15.67
27.13a
57.20c
1.11

11.04
5.28
1.58
18.57
49.94
13.58
16.33
20.15
63.52
1.09

0.15
0.12
0.09
0.29
0.41
0.22
0.15
0.16
0.50
0.04

0.047
0.185
0.009
0.011
0.007
0.013
0.053
0.005
0.006
0.196

applied for both total and individual SFA. Significant effects of S


fertilization were reported for total MUFA in pea grains that in
overall represented about 20% of total FA, and in particular the
MUFA detected (elaidic and oleic acids) increased with the increase
of the S fertilization level. The total polyunsaturated FA (PUFA)
content decreased significantly from 67.37 to 57.20% (S0 and S60,
respectively). Particularly, in our cultivar of pea, the main PUFA
was the -linolenic acid representing about 50% of total PUFA.
Discussion
We could not locate any literature concerning the effects of S
fertilization on FA composition of faba bean, lupin and pea grains;
thus, this subject should be considered in new investigations. In
the present study, the experimental treatments affected FA profile
of the legume species investigated. Our findings are confirmed by
Abramovic and Abram 10 that the differences in the composition
of the FA in seed oil can be caused not only by the different
cultivars, but also by environmental conditions. Similarly, Zubr 11

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

137

concluded that the variations are due to the combined effects of


climatic and soil conditions on the crop, and the same study
revealed an effect of S fertilizer application on FA composition.
The S fertilization caused the highest contents of both palmitic
and oleic acids. The concentration of linolenic acid, which is the
important FA with respect to nutrition in humans and animals,
remained unaffected by the fertilizer treatments applied. This
indicates that oil of our faba bean variety had a good level of
unsaturation and a high linoleic content 12. In the present study,
the experimental treatments affected FA profile of lupin. We could
not locate any literature concerning the direct effect of S fertilization
on fatty acid composition of white lupin seeds. Our findings are
confirmed by Boschin et al. 5 who reported differences in the
composition of the FA in lupin oil content. In our study S
fertilization caused the highest contents of palmitic, oleic and
linoleic acids. Moreover, a significant reduction of erucic acid
content in lupin oil was found in fertilized crops. This work indicates
that a drawback of lupin is the presence of significant levels of
erucic acid 1, 2, 5. Using the same definition already applied to
rapeseed oil, only lupin oil from fertilized crops could be classified
as low-erucic acid oil (erucic acid 2%). However, this negative
feature appears to be marginal since lupin oil is not a commercial
product; on the other hand, the selection of erucic-free or lowerucic acid genotypes would be certainly desirable 5. The lipid
content of peas is low and it was in overall 1.1%; however, the low
crude oil content in pea may be of importance in the flavour of
peas 13. This general composition in pea coincides with the results
obtained by Coxon and Wright 14. Our findings in terms of fatty
acid composition of pea grains are in agreement with those reported
by Murcia and Rincn 15.

albus L. cv. Multitalia) as the main protein source for broilers: Influence
on growth performance, carcass traits and meat fatty acid composition.
J. Sci. Food Agric. 91:2081-2087.
7
Laudadio, V. and Tufarelli, V. 2012. Effect of treated field pea (Pisum
sativum L. cv Spirale) as substitute for soybean extracted meal in a
wheat middlings-based diet on egg production and quality of early
laying brown hens. Arch. Geflgelkd. 76:1-5.
8
Laudadio, V. and Tufarelli, V. 2010. Treated faba bean (Vicia faba var.
Minor) as substitute for soybean meal in diet of early phase laying
hens: Egg-laying performance and egg quality. Poult. Sci. 89:22992303.
9
Folch, J., Lees, M. and Sloane-Stanley, G. H. A. 1957. A simple methods
for the isolation and purification of total lipids from animal tissues. J.
Biol. Chem. 226:497-507.
10
Abramovic, H. and Abram, V. 2005. Physico-chemical properties,
composition and oxidative stability of Camelina sativa oil. Food
Technol. Biotech. 43:63-70.
11
Zubr, J. 2003. Dietary fatty acids and amino acids of Camelina sativa
seed. J. Food Quality 26:451-462.
12
Hossain, M. S. and Mortuza, M. G. 2006. Chemical composition of
Kalimatar, a locally grown strain of faba bean (Vicia faba L.). Pak.
J.Biol. Sci. 9:1817-1822.
13
Mccurdy, S. M., Drake, S. R., Swanson. B. G., Leung, H. K. and
Powers, J. R. 1983. Influence of cultivars, soak solution, blanch method
and brine composition on canned dry pea quality. J. Food Sci. 48:394399.
14
Coxon, D. T. and Wright, D. J. 1985. Analysis of pea lipid content by
gas chromatographic and microgravimetric methods. Genotype
variation in lipid content and fatty acid composition. J. Sci. Food
Agric. 36:847-856.
15
Murcia, M. A. and Rincn, F. 1991. Fatty acid composition of pea
(Pisum sativum L., var. Citrina) during seed growth. Grasas Aceites
42:444-449.

Conclusions
Under Mediterranean environment and sub-alkaline soil, the S
fertilization in winter legume grains led to an improvement of the
fatty acid profile. Furthermore, the S fertilization enhanced the
legume grains oil composition through the increase of unsaturated
fatty acids, and in particular the remarkable decrease of the erucic
acid in lupin grains.
Acknowledgements
The author would like to thank the laboratory technicians involved
in the chemical analysis.
References
Cazzato, E., Tufarelli, V., Ceci, E., Stellacci, A. M. and Laudadio, V.
2012. Quality, yield and nitrogen fixation of faba bean seeds as affected
by sulphur fertilization. Acta Agric. Scand. Sect. B-Soil Plant Sci.
62:732-738.
2
Cazzato, E., Laudadio, V., Stellacci, A. M., Ceci, E. and Tufarelli, V.
2012. Influence of sulphur application on protein quality, fatty acid
composition and nitrogen fixation of white lupin (Lupinus albus L.).
Eur. Food Res. Technol. 235:963-969.
3
Scherer, H. W. 2009. Sulfur in soils. J. Plant Nutr. Soil Sci. 172:326-335.
4
Gaylor, G. C. and Sykes, G. E. 1985. Effect of nutritional stress on the
storage proteins of soybeans. Plant Physiol. 78:582-585.
5
Boschin, G., DAgostina , A., Annicchiarico, P. and Arnoldi, A. 2007.
The fatty acid composition of the oil from Lupinus albus cv. Luxe as
affected by environmental and agricultural factors. Eur. Food Res.
Technol. 225:769-776.
6
Laudadio, V. and Tufarelli, V. 2011. Dehulledmicronised lupin (Lupinus
1

138

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

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Journal of Food, Agriculture & Environment Vol.12 (3&4): 139-142. 2014

www.world-food.net

Yield and quality of Cenchrus ciliaris (L.) affected by nitrogen and phosphorus
fertilization
Ihsan Abu-Alrub *, Ahmed Aran, Omar Hamad and Abdelaziz Awaga
Research and Development, Abu Dhabi Food Control Authority, P. O. Box 52150, Abu Dhabi, United Arab Emirates.
*e-mail: ihsan.joma@adfca.ae
Received 20 February 2014, accepted 20 April 2014.

Abstract
Forage and nitrogen content responses of common buffelgrass (Cenchrus ciliaris L.) to nitrogen (N) and phosphorus (P) fertilization were evaluated
for two successive years in the UAE. Treatments consisted of five rates of N (0, 250, 500, 750 and 1000 kg ha-1 yr-1) and four rates of P (0, 200, 400
and 600 kg ha-1 yr-1), applied via weekly fertigation. All N fertilizer treatments resulted in significant increase in forage dry yield, relative to control.
However, no yield increase was observed from N treatments greater than 250 kg N ha-1. There were neither significant yield responses to P
fertilization treatments over the two years of the study, nor significant N-P treatment interactions for yield. Forage N concentration progressively
increased with increasing levels of N fertilizer, reaching maximum levels under the 1000 kg ha-1 N treatment. A seasonal pattern of forage N
concentration occurred, being higher in winter and lower in summer.
Key words: Cenchrus, nitrogen, phosphorus, fertilizer rate, UAE, forage yield, N concentration.

Introduction
Cenchrus ciliaris (buffelgrass) is a perennial grass native to dry
areas of the African continent, West Asia, and India, and has
been widely introduced to arid and semi-arid regions worldwide 1.
Indigenous to the UAE, it occurs in areas having average annual
rainfall as low as 100 mm 2. However, natural species distribution
is in decline, largely due to selective overgrazing.
Currently, there is huge demand for forage production in the
UAE to support 3.7 million head of livestock, with inadequate
available forage supplies to maintain the livestock population 3. It
has been reported that Cenchrus ciliaris is a valued forage grass
for dry areas, due to its high biomass production and tolerance to
low moisture conditions 4, 5. Therefore, Cenchrus ciliaris is among
species having the greatest potential for forage production in
the UAE.
Soil of the UAE is predominately sandy textured, with very low
organic matter content, resulting in deficiencies in essential plant
nutrients. A number of studies have found forage yields to be
increased by fertilization on sandy lands 6. Nitrogen (N) fertilization
typically increases grass dry matter 7, 8, forage nitrogen
concentration 9, and seed yield 10. Generally, phosphorus (P)
fertilization alone does not sustainable increase forage yields 9, 11,
but combined application of P with N often does 12.
Nutritional quality of unfertilized buffelgrass in rangeland varies
seasonally 13. Improvements in forage quality with fertilization
have been shown in many studies 14-17. Significant increase in
forage N content, digestibility, and mean daily live mass gain per
sheep was found as nitrogen fertilization rates increased 18.
Fertilization has also been shown to increase soil water extraction
by forages, and improve water use efficiency 19, 20.
Limited data are available concerning production of irrigated
buffelgrass forage as affected by the fertilizer applications under
UAE conditions. The objective of this research was to evaluate

the influence of a range of N and P fertilization applications on


forage yield and quality.
Materials and Methods
Field plots were established 23 January 2011 in Al Ain, UAE (24
00'N, 5400' E). Climate is hot and arid, with daily high temperatures
ranging from 24 to 45C, and average annual rainfall 100 - 150
mm. Soil of the study area was classified as sandy. Nitrate,
available P, and extractable K were 164.2, 21.6 and 128 ppm,
respectively. Soil pH was 7.8, electrical conductivity of the
saturation extract (ECe) 10.2 dSm-1, organic matter 1.0%, and CEC
7.1 meq/100 g.
The experiment was laid out in a split plot arrangement within a
randomised complete block design with four replications. The
main plot was assigned to N treatments, with P treatments as
subplots. Individual plot size was 3 2.5 m, consisting of 4
rows, with distance between rows 75 cm. Seeding rate was 40 kg
ha-1 of Cenchrus ciliaris cv. Laredo.
N treatments were 0, 250, 500, 750 and 1000 kg N ha-1 yr-1 and P
treatments 0, 200, 400 and 600 kg P ha-1 yr-1. K fertilization was
constant for all plots at rate of 250 kg ha-1 yr-1. N was applied as
urea (N 46%), P as triple super phosphate (P2O5 46%), and K as
potassium sulphate (K2O 50 %). N and K fertilizers were applied
weekly via fertigation, and P was top dressed once before planting.
All plots were harvested 5 times during 2011, and 7 times during
2012, when the grass reached maturity. After cutting and weighing
the whole plot, 500 g green samples of grass from each plot were
dried at 85C for 24 h for dry matter determination. Prior to cutting,
the lengths of 10 randomly selected plants were measured from
the ground to the tip of the shoot. Forage samples were analysed
for N and P. Plant N concentration was determined by Kjeldahl
digestion and P concentration determined colorimetrically in a

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

139

Results
Forage yield: The effect of fertilizer treatments on forage yield is
presented in Table 1. For both years, all N treatments significantly
increased yields, relative to control. Maximum yields were achieved
with the 250 kg N ha-1 yr-1 treatment, with no further increase in
yield resulting from higher N rates. Yield of treatments N 250 kg
ha-1 yr-1 were 55 and 57% higher than control for the first and
second years, respectively. During the first year, N treatment of
1000 kg ha-1 yr-1 resulted in yield decrease, relative to 500 and 250
kg ha-1 yr-1; however, in second year there was no significant
difference among N application rates. Neither phosphorus
treatments, nor N P treatment interactions were significant in
either year for forage yield or plant height. Average forage yield
per harvest for 2011 was higher than for 2012 (data not shown), as
seasonal harvest progressed average yield per harvest declined.
The reduction in yield per harvest in 2012 for N treatment 0, 250,
500, 750 and 1000 kg ha-1 yr-1 was 22.4, 19.1, 15.4, 4.5 and 1.5,
respectively.
Table 1. Total dry matter yield and average
plant height as affected by N and P
treatment level.
Treatments
-1

Plant height
at harvest (cm)
2011
2012

Dry matter
yield (t ha-1)
2011 2012

64.2
92.4
92.5
96.2
88.7

56.9
87.1
89.7
89.9
88.6

16.3
36.5
34.7
32.3
30.3

17.7
41.3
41.1
43.2
41.8

84.4
87.4
86.9
88.5

80.614
83.747
81.905
83.675

28.5
30.1
31.1
30.6

35.6
38.9
37.8
36.0

3.5
ns
ns

3.3
ns
ns

2.4
ns
ns

3.4
ns
ns

N concentration (%)

0 N (kg/ha)

500 N (kg/ha)

750 N (kg/ha)

1000 N (kg/ha)

2
1.5
1
0.5
0

May

Jul. Aug. Oct. Dec. Feb. Apr. Jun.


Harvest date
2011

Jul. Aug. Oct. Nov.


2012

Error bars represent standard errors of the mean.

harvest dates. N concentration averaged 1.1, 1.5 and 1.6% at 0, 250


and 500 kg N ha-1 yr-1, respectively. In general, 1000 kg ha-1 yr-1 of N
produced significantly higher forage N concentration, relative to
other N treatments.
A seasonal pattern of forage N level occurred, with higher N
concentrations during the winter season. Summer depression in
N concentration was observed from May till August. With
increasing P treatment rates resulting in significantly increased
forage N contents. Application of 600 kg ha-1 yr-1 P producing the
highest forage N content in most harvests (Fig. 2).
1.54

a
b

1.52

1.5
1.48
1.46

1.44
1.42
1.4
0

-1

200
400
P fertilizer level (kg ha-1 yr-1)

600

Figure 2. Forage average N concentrations of buffelgrass at different P


application rates.
Bars with the different letters are significantly different based on LSD test (P < 0.05).

Forage P concentration: The largest significant differences in


forage P content were generally observed between control and
200 kg ha-1 yr-1 P application rate (Fig. 3). Application of 600 kg
ha-1 yr-1 P, relative to lower rates, resulted in significantly higher
forage P content on 5 harvest dates. N application rate had no
significant effect on forage P content. N-P treatment interaction
for forage P concentration occurred only at the June, July and
October harvest dates.
0 P (kg/ha)

200 P (kg/ha)

400 P (kg/ha)

600 P (kg/ha)

Plant height trends followed yield, with all N treatments


significantly increasing height, relative to control. Maximum height
was generally achieved at 250 kg N ha-1 yr-1, with little effect of
higher N rates.
During both years there were a highly significant linear and
quadratic relationship between forage yield and the amount of N
applied (2011, DM Yield = 17.31 + 0.61 (N) 0.0051 (N2), R2 = 0.68,
2012, DM Yield =18.94 + 0.75 (N) 0.0054 (N2), R2 = 0.76).

P concentration (%)

N (kg ha yr )
0
250
500
750
1000
P (kg ha -1 yr-1)
0
200
400
600
LSD (P<0.05)
Nitrogen
Phosphorus
Interaction

250 N (kg/ha)

2.5

Figure 1. Forage N concentration of buffelgrass at different N application


rates averaged across P application rates for the various harvest dates
(averaged across P application rates).

N concentration (%)

nitric acid system 21. Weeds were manually controlled.


An analysis of variance for balanced data with GLM model
(SAS, 1994) was used to determine differences between treatments.
Interaction that did not make a significant contribution to the
variance was omitted in subsequent analyses. Comparison among
treatment differences was determined by Least Significant
Difference (LSD) at P < 0.05. The regression procedure was used
to determine the linear and/or quadratic effects of N fertilizer.

Forage N concentration: Fig. 1 illustrates the influence of five


levels of N fertilizer on forage N concentration. Total N
concentration increased with increasing levels of N fertilizer at all

Figure 3. Forage P concentration of buffelgrass at different P application


rates averaged across N application rates for the various harvest
dates(averaged across N application rates).

140

0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

May

Jul. Aug. Oct. Dec. Feb. Apr. Jun.


Harvest date
2011

Jul. Aug. Oct. Nov.


2012

Error bars represent standard errors of the mean.

Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

Discussion
The response pattern of forage yield to N and P fertilization was
similar in both growing seasons. Though N fertilization increased
yield in both seasons, maximum yield occurred at 250 kg ha-1 yr-1,
with no further yield increase with higher N application rates,
suggesting that forage production had peaked. Quadratic forage
yield response to increasing N fertilization rate has been reported
in several studies 8, 22.
Nitrogen fertilization consistently had a marked influence on
the N concentration of forage in both years. Increased N
concentration in forage associated with increasing N fertilization
has been previously reported 8, 17. However, Rai 9 reported a 17.5%
increase in N concentration of buffelgrass with a single application
of 60 kg ha-1 year-1.
A summer depression in N concentration occurred is consistent
with findings of Johnson et al. 16, who reported a 15% depression
in forage N concentration in July, the hottest month of the
climatological area of the study. Though forage yields peaked in
summer in this study, forage quality declined. This trend may be
related to higher environmental temperature, which encourages
lignifications, rapid physiological development and metabolic
activity, resulting in decline in forage quality 23, 24.
The lack of response of yield to P fertilization was likely due to
the moderate level of P pre-existing in the soil, as indicated by soil
analysis. That N, and not P, was limiting to forage yield is consistent
with several previous studies 8, 25. Ocumpaugh et al. 22 reported
insignificant N-P interactions for forage yield over three years.
However, other research has shown that combined application of
N and P may significantly enhance forage yield 12. Though P
fertilization did not affect yield in this study, it did improve forage
quality by increasing N content. Thus, to maintain maximum yield
responses to N, as well as improved forage quality, P application
may be necessary in the long term under our conditions.
Our results suggest no effect of N application on forage P
content of buffelgrass. Conversely, Wiedenfeld et al. 8 reported
that P content of buffelgrass decreased significantly with
increasing N fertilization, but not observed for Pretoria 90. In
general, plant responsiveness to nutrients is related to genetic
potential for growth 26.
Conclusions
Nitrogen fertilisation had significant effects on the yield and
quality of buffelgrass grown under irrigated condition. The study
shows that buffelgrass can perform well at annual applications of
250 kg ha-1 N. Though P fertilization had no effect on yield, it did
improve forage quality by increasing forage N concentration.
Further studies, on the role of P as related to plant growth
specifically in the area where soil P levels are low, are needed
before concrete fertilizer recommendations can be made.
Acknowledgements
The authors would like to thank Abu Dhabi Food Control Authority
for funding and supporting this research. The authors are grateful
to Dr. Kenneth B. Marcum, United Arab Emirates University, for
reviewing and valuable suggestions.

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Seed and spike traits from remnant populations of Cenchrus ciliaris
L. in South Tunisia: High distinctiveness, no ecotypes. Journal of Arid
Environments 50:309-324.
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Karim, F. M. and Dakheel, A. J. 2006. Salt tolerant plants of the United
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