Proceeding NSC 2015 - Unika Soegijapranata

15th National Student Conference on
Food Science and Technology

Integrating Innovative Food Product
Development and Consumer
Preferences through Sensory
Evaluation
ISBN 978-602-6865-06-9
The Committees of 15th National Student Conference

On Food Science and Technology
Penerbit :
Universitas Katolik Soegijapranata
Integrating Innovative Food Product Development and Consumer Preferences through Sensory
Evaluation Department of Food Technology, Soegijapranata Catholic University Semarang Tuesday,
September 1st 2015
15th National Student Conference on

Food Science and Technology
Integrating Innovative Food Product
Development and Consumer
Preferences through Sensory Evaluation
C Universitas Katolik Soegijapranata, 2015
The Committees of 15th National Student Conference

On Food Science and Technology
Editors :
Ivone E. Fernandez
Victoria Kristina Ananingsih
R. Probo Y. Nugrahedi
Ong, Cindy Corazon C
Publisher :
Universitas Katolik Soegijapranata
Jalan Pawiyatan Luhur IV/1, Bendan Duwur, Semarang 50234 - Indonesia
Telepon
: +62-24-8441555(Hunting)
Fax
: +62-24-8445625
Email
: penerbitan@unika.ac.id
Tahun
: 2015
September 1st 2015
TABLE OF CONTENTS
TABLE OF CONTENTS .................................................................................................... i
PREFACE...........................................................................................................................iii
RULES OF CONFERENCE ............................................................................................. iv
LIST OF PRESENTATION .............................................................................................. v
PARALLEL SESSION
Food Product Development ............................................................................................... 2
Infusion Indonesian Traditional Food (Dendeng Balado) Into Puff Pastry Product ............ 3
Guar Gum Effect as Fat Replacer on Sensory Characteristics of Broccoli Brownies .......... 17
Cookies Characteristics by Using of Dehulled and Undehulled Mungbean Flour .............. 26
The Application of Crude Palm Oil as A Replacement for White Margarine in Making
Sourdough Bread ................................................................................................................ 36
The Study of Herb Liquers Production ................................................................................ 47
A Healthy Meatball : Application of Carageenan Toward Albumin Meatball and Arrowroot
Flour Toward Breadfruit Based Meatball ........................................................................... 54
Food Processing and Engineering .................................................................................... 61
The Stability of Betalains In Red Beet (Beta vulgaris L.) As A Natural Food Colorant During
Steaming Of Glutinous Rice Flour Batter.. .......................................................................... 62
Utilization Cocoa Pod Husk Powder for Bread................................................................... 69
Calcium From Shrimp Shell : The Effect of HCl Solubilization Condition .......................... 78
The Growth Model of Bacillus Cereus in Cooked Rice........................................................ 86
Utilization of Kersen (Mutingia Calabura L.) Leaves As Functional Drink.. ...................... 97
Effect of Chromanone Deamine Level on Fresh and Frozen Chicken Broiler Meat Quality109
Food Safety and Quality .................................................................................................. 115
Effect of Rare Sugar D-psicose on Peroxidase Activity Derived from Tomato Leaf ........... 116
Fatty Acid Composition Analysis of Coconut Meat From Different Cultivation Areas by
Using Gas Liquid Chromatography Technique.. ................................................................ 121
15thNational Student Conference
September 1st 2015
Page i
Effect of Chromanone Deamine Dosages on The Quality of Osmomeat Products in Terms of

Physicochemical Characteristic ......................................................................................... 130
The Inhibitory Effect of Bamboo Extracts on Melanogenesis in Melanocytes .................... 136
Food Preservative Tools and Harmful Heavy Metal Reducers on Vegetables and Fruits .. 147
The Effectiveness of Antimicrobial-Biodegradable Packaging to Inhibit Escherichia Coli
Growth.. ............................................................................................................................. 157
The Effect of Chromanone Deamine Level on Water Content, pH and Total Microbes of
Broiler Chicken Meat Pre-Rigormortis and Post Rigormortis.............................................165

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Page ii
PREFACE
The growth of food industries in Indonesia in the last decade can be expected by the
launching of hundreds of new food and beverage products to the market recently. With
regard to these new products, some producers concern on the development of new
innovative products, while others focus on the improvement of their previous products. The
new products have to be accepted by consumers; hence, consumers acceptance and
preference are the answer to the success or failure of a new product. Sensory evaluation
can be integrated in the product development to provide important data on which decision on
a new product should be made and launched.
The 15th National Student Conference (NSC) organized by Faculty of Agricultural

Technology, Soegijapranata Catholic University was conducted on 1st September
2015. This 15th NSC presented a forum for student to improve their knowledge in
food product development and sensory evaluation and how to integrate these
aspects in order to produce an innovative food product that can be accepted by the
consumer.
The organizing committee is grateful to all honorable keynote speakers, oral presenters,
participants and sponsors, for joining this gathering and for their valuable contribution on the
conferences.
Semarang, October 2015
Dr. Victoria Kristina Ananingsih, ST, MSc

Dean
Faculty of Agricultural Technology
Soegijapranata Catholic University

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Page iii
RULES OF CONFERENCE
1.
Plenary Session
a. Every speaker will present his/her paper.

b. All participants are prohibited to use any electronic device that produces loud sound
during speakers presentation.
c. After all presentations, there will be a discussion session
d. The audience should state their personal identity before giving question, suggestions,
etc.
2.
Parallel Session
a. The parallel session will be conducted by the chairman of that session (moderator).
b. Speakers should give a short note with their name, institution, and curriculum vitae
before their presentation.
c. Duration of presentation is maximum 8 minutes and followed by 7 minutes
disccussion.
d. The audience should state their personal identity before giving question, suggestions,
etc.

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Page iv
LIST OF PRESENTATION
FOOD PRODUCT DEVELOPMENT
TITLE/ AUTHOR
Infusion Indonesian Traditional Food (Dendeng Balado) Into Puff Pastry
Product
William Wibowo, A.Nootrude Siriboon, V.Kristina Ananingsih
CODE
FPD-01
Soegijapranata Catholic University, Semarang
Guar Gum Effect as Fat Replacer on Sensory Characteristics of Broccoli

Brownies
Stella Gunawan R, Laksmi Hartayanie, and Probo Y. Nugrahedi
Cookies Characteristics By Using Of Dehulled And Undehulled
Mungbean Flour
Mak Alan Darma Saputra, Sun Shine Moninggianti Siani
Widya Mandala Catholic University,Surabaya
The Application of Crude Palm Oil as A Replacement for White
Margarine in Making Sourdough Bread
Jonathan Alvin, Lindayani, Laksmi Hartayanie
The Study of Herb Liqueurs Production
Hana Melinda, Lindayani, Churdchai Cheowtirakul
Soegijapranata Catholic University &Assumption University
A Healthy Meatball : Application of Carageenan Toward Albumin
Meatball and Arrowroot Flour Toward Breadfruit Based Meatball
Rency Gista Anindya, Vania Eka Cahyani Adidharma, Michael
Ardian Iskandar, Lindayani, Laksmie Hartayanie
FPD-02
FPD-03
FPD-04
FPD-05
FPD-06

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FOOD PROCESSING AND ENGINEERING

TITLE/AUTHOR
The Stability of Betalainsin Red Beet (Beta Vulgaris L.) as A Natural
Food Colorant During Steaming of Glutinous Rice Flour Batter
Metta Meliani, Chaterine Meilani Surono, Nies Mayangsari,
Victoria Kristina Ananingsih, and Alberta Rika Pratiwi
Utilization Cocoa Pod Husk Powder for Bread
Ingrid Tertiana Ivana, Jefry Sugiarto Halim
Widya Mandala Catholic University,Surabaya
Calcium From Shrimp Shell : The Effect of HCl Solubilization
Condition
Angela Irena Wibawa, Saranya Dechapinan, Dr. Nattapol
Tangsuphoom,Dr. Ch.Retnaningsih
Soegijapranata Catholic University & Mahidol University
The Growth Model of Bacillus Cereusin Cooked Rice
Maria Jessica Arta, LaksmieHartayanie, NatpisitChaitchawong and
PatchaneeYasurin
Soegijapranata Catholic University &Assumption University
Utilization of Kersen (MutingiaCalabura L.) Leaves As Functional
Drink
Eugenia PrisciliaKusuma, ShiannePuspitaPutri, Patricia Andika
Universitas Pelita Harapan
Effect of ChromanoneDeamine Level on of Fresh and Frozen Chicken
Broiler Meat Quality
StefanyWidjaya, Sumardi, and Ch. Retnaningsih
CODE
FPE-01
FPE-02
FPE-05
FPE-06
FPE-07
FPE-08

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Page vi
FOOD SAFETY AND QUALITY

TITLE/AUTHOR
Effect of Rare Sugar D-psicose on Peroxidase Activity Derived from
Tomato Leaf
Fina Fitriana Budiman, Rizki Dian Lestariningtyas, Muhammad
Johan Adhibuana, Ahmad Nimatullah Al-Baarri, Anang M Legowo,
Shigeru Hayakawa, Masahiro Ogawa
Diponegoro University & Kagawa University, Japan
Fatty Acid Composition Analysis of Coconut Meat From Different
Cultivation Areas by Using Gas Liquid Chromatography Technique
Ivana Aprilia Pratiwi, Assoc Prof. Dr. Visith Chavasit
Soegijapranata Catholic University & Mahidol University
Effect of Chromanone Deamine Dosages on The Quality of Osmomeat
Products in Terms of Physicochemical Characteristics
Mulyanto Onggo Wibowo, Sumardi, and Alberta Rika Pratiwi
The Inhibitory Effect of Bamboo Extracts on Melanogenesis in

Melanocytes
Fellycia Devi Paramitha, Tsung-Yu Tsai, Laksmi Hartayanie
Soegijapranata Catholic University, & Fu Jen Catholic University
Food Preservative Tools and Harmful Heavy Metal Reducers on
Vegetables and Fruits
Emas Agus Prastyo Wibowo, Heru Setiawan
Universitas Negeri Semarang
The Effectiveness of AntimicrobialBiodegradable Packaging to Inhibit
Escherichia coli Growth
Lisa Widagdo, Shaun Chen, R. Probo Y. Nugrahedi Soegijapranata
Catholic University & Fu Jen Catholic University
The Effect of Chromanone Deamine Level on Water Content, pH and
Total Microbes of Broiler Chicken Meat Pre-Rigormortis and Post
Rigormortis
Cindy Elysia, Sumardi, and Rika Pratiwi
CODE
FSQ-02
FSQ-03
FSQ-04
FSQ-05
FSQ-06
FSQ-07
FSQ-08

September 1st 2015
Page vii
FULL PAPERS

September 1st 2015
Page 1
FOOD PRODUCT DEVELOPMENT

September 1st 2015
Page 2
INFUSION INDONESIAN TRADITIONAL FOOD (DENDENG BALADO)

INTO PUFF PASTRY PRODUCT
William Wibowoa*, A.Nootrude Siriboonb, Dr. V.Kristina Ananingsih, Msca
aTeknologi
Pangan, Fakultas Teknologi Pertanian, Universitas Katholik Soegijapranata

Jl.Pawiyatan Luhur IV/1, Bendhan dhuwur, Semarang, Indonesia
bFood Technology, Faculty of Biotechnology, Assumption University
Hua Mak (ABAC), Bangkok, Thailand
*Email: william.9a@gmail.com
ABSTRACT
The study was aimed to develop the fusion product from Indonesian cuisine into bakery
product. The Indonesian cuisine that was used is dendeng balado,a special dish from west
sumatra.A brainstorming with focus group discussion (FGD)was carried out to find the most
potential and preferable bakery product to be developed in the fusion product using dendeng
balado,and the puff pastry was choosen. A preliminary test was done using Muu sawan (Thai
Fried Sun-dried Sweet Slice Pork) that has similiar characteristics to dendeng as filling in the
puff pastry. The objective was to find whether the filling would become softer after bake and
the fusion puff pastry was feasible. Upon confirming the result from preliminary test, the
Muu sawan was used and mix with balado sauce to make it into dendeng balado. Just About
Right (JAR) test was conducted to see the scores from eight attributes color,crispness,
saltiness, sweetness, sourness, spiciness, the amount and size of filling. The result showed
that crispness, sweetness and sourness required no adjustment but the other attributes need.
The color was adjusted by prolong the baking time or increasing the heat, the saltiness was
varied into 2 levels 2 and 4 grams, the spiciness was increased by 3 levels using 9,13 and
17 gram of chilli and for the amount of fill was measured at 1 teaspoon. A hedonic scale was
caried out to ensure that the fusion product was feasible.
Keywords:Fusion product, product development, puff pastry, dendeng balado.

September 1st 2015
Page 3
Introduction
Bakery is a flour-based processed
productsthat are baked in an oven. Pastry
is one of many bakery products that is
characterized with dry and flaky texture.
Pastry dough is a mixture of flour, fat and
also water that combined together to make
paste that can be made into flexible dough
of different textures by combining
different proportion and mixing methods.
This dough then can be shaped into a
variety of sweet or savory filling. (Baking
Industry Research Trust,2011). The flaky
texture was achieved from the pastry
dough that was layered with solid fat that
melted during baking and resulted in
dough with many layers and fat (Stevens,
1995).
Puff pastry has the characteristics of light,
flaky and tender. Puff pastry is made by
forming the dough from a mixture of flour,
salt, fat and water. The ratio of flour to
water in the puff pastry is 2:1, which will
result in the plastic and elastic dough. Puff
pastry consists of as many as 700 layers of
thin paper-like pastry that are separated by
butter and air. These layers are made by
layered the dough with fat or butter, then
folding and rolling to form hundreds of
layers of fat and dough(Baking Industry
Research Trust,2011). Puff pastry, when
bake, can rise 8 times from its original
height. The water from the dough
vaporizes to steam while baked. This
steam eventually separates each layers and
puff up the pastry (Philips, 2000).
Dendeng is a traditional Indonesian food
made from beef, lamb, chicken and so on
with certain spices. This Indonesian

cuisine has brown color due to the reaction
between meat protein and brown sugar in a
seasoning. Dendeng has low water content
as it is dried. Dendeng can be served as a
condiment
to
add
flavor(IPB,
1982).Dendeng not only can be used as
condiment to add flavor but also it can be
cooked into several traditional dish, for
example dendeng balado.
Dendeng balado is a Indonesian traditional
cuisine from Padang, West Sumatra. It
made from beef that had been cut, dried
and fried before mixed with chillies and
other ingredients.People usually eat
dendengbalado together with rice only and
rarely to be eaten as snack.
With this project to developthe fusion
product from dendengbalado in puff pastry
will create a variety in new puff pastry
product from traditional Indonesian
cuisine.Therefore, the project is aimmed to
generate a new idea of consuming
dendengbalado as a snack by made it into
puff pastry.
1. Materials and Methods
1.1. Materials
The materials used for this project are allpurpose flour (United Flour Mill Co. Ltd),
Muusawan(Thailand sun-dried pork jerky),
sugar (brand: MitrPhol), salt(brand:
PrungThip), pepper, chilly, red onion,
garlic, tomato, and vinegar(5% Distil
Vinegar,
Instant
Food
Product
Organization).
1.2. Equipment and apparatus

September 1st 2015
Page 4
The equipments used for this project are

digital balance, oven, bakery utensil,
mortar, and Cooking utensil.
1.3. Methodology
1.3.1. Determination of bakery for
fusion product
1.3.1.1.Focus Group Discussion
Focus Group Discussion was
conducted to generate new idea
and determine the bakery
product that had the potential to
be developed and also feasible
to
be
fused
with
dendengbalado. The focus
group discussion included12
participants. One moderator
and a secretary helped to record
the ideas.
1.3.2. Preliminary experiment
1.3.2.1.Preparation of puff pastry
dough
The puff pastry dough was
prepared according to the
formula in table 1.
Table 1:Puff pastry dough formula
Ingredients
Amount
Percentage
in grams
500
47.4
All-purpose
flour
75
7.0
Margarine
200
19.0
Puffin
margarine
5
0.5
Salt
250
23.7
Cold water
25 (
2.4
Egg
whole egg)
1,055
100
Total
Source:
FT4114
Bakery
Practical
Handbook, Assumption University
Dough preparation
The all-purpose flour was Stiffen first then
salted butter was rubbedinto the flour
thoroughly.Salt was dissolved in cold
water and addedan egg after that poured
the mixture into the flour slowly and knead
to form smooth dough.The dough then was
put into plastic bag and let it stand for 1015 minutes in a refrigerator.The bench was
dusted with flour and put the dough on
it.The dough was rolled out in rectangle
shape with 1 cm thick.The puffin
margarinewas placed over 2/3of the dough,
leaving 2.5 cm margin along the edge.The
dough then was folded with no margarine
over the margarine layer dough, then, fold
it over the remaining part on the dough.
Again, the dough was put into plastic bag
and let it stand for 10-15 minutes in the
refrigerator.The chilled dough was
rolledouton
the
flour
bench
andfoldedusing 3-fold folding method
(single folding) and repeat the folding two
times. After each folding, the doughwas
rested in the refrigerator for 10-15
minutes. For last folding, the dough was
rolled out and folded by 4-fold folding
method (double folding), then the dough
was put in plastic bag and returned to
refrigerator for further use.
1.3.2.2.Preparation
of
balado puff pastry
dendeng
The filling for puff pastry was

dendengbalado, using Muusawan
mixedwith balado sauce. The formula for
dendengbalado was shown in Table 2.
Table 2:Dendengbaladoformula
Ingredients Amount in
Percentage

September 1st 2015
Page 5
Muusawan
Salt
Pepper
Sugar
Chilly
Red onion
Garlic
Tomato
Vinegar
Lime
Total
grams
300
4
1.6
2.6
9
30
12
100
1
10
470.2
63.8
0.8
0.3
0.6
1.9
6.4
2.6
21.3
0.2
2.1
100
Source:http://www.femina.co.id/kuliner/resep/
hidangan.indonesia/dendeng.balado/004/001/2
34/02
i. Making the balado sauce

Garlic, red onion and chilly were mashed
using mortar then tomato was added into
the mix and mashed again.While heating
the pan,a little bit of oilwas poured in the
wok and then the balado sauce was stired
fry with low fire until nice fragrance
comes out.
ii. Making the dendengbalado
Muusawanwas poured into the sauce and
add salt, sugar, pepper, lime juice and
vinegar and then mixed and stired fry the
Muusawan with the sauce. After that, the
muusawan in balado sauce was cooled
down to room temperature and then put in
a plastic bag and stored in a refrigerator
for further use.
iii. Making the dendengbaladopuff
pastry
The dough was cut into 4 parts,
approximately 260 grams. One part was
rolled to a rectangular sheet with 0.5 cm
thick, then, cut into 7 pieces (40 grams).

The dough was filled with dendengbalado
about 1.5 teaspoon. The dough then was
folded and sealed with eggwash. Puff
pastry was put in lined on a tray with
parchment paper. Egg wash was applied
on the surface of the puff pastry before
bake in the oven at 200oC for 35 45
minutes.
1.3.3. Just About Right Test
The puff pastry with dendengbalado filling
was tested using Just About Right (JAR)
test with 30 untrained panelists who were
Biotechnology students and instructors
Nine attributes pertaining to the
characteristics of dendengbalado puff
pastry were studied.
1.3.4. Preference test
About 30 untrained panelists were used to
score the dendengbalado puff pastry with
9-point hedonic scale of preference test.
Ten attributes of the fusion product were
studied.
1.3.5. Adjusting the filling formula
The result from Just About Right test (1.3)
was used to adjust the formula for the
dendengbalado filling. The same 30
untrained panelists from (1.3) were used to
reduce bias of the score. 9-point hedonic
score preference test was conducted to test
the attributes of dendengbalado puff
pastry.
1.3.6. Sensory Evaluation
1.3.6.1. The Just About Right (JAR)
test was used to determine the
attributes of fusion product that
need to be adjusted.

September 1st 2015
Page 6
1.3.6.2. The preference test using 9point hedonic score was used to
determine the acceptability of
the dendengbalado puff pastry
and also the acceptability of the
adjusted filling formula.
1.3.7. Statistical Evaluation
1.3.7.1. The Randomized Complete
Block Design (RCBD) was
used as the experimental design
where the day of preparation
was a block.
1.3.7.2. SPSS (Statistical Package for
Social
Sciences)
program
version 21 was used to perform
statistical analysis for Analysis
of Variance (ANOVA) at p <
0.05.
2.
Results and Discussions
2.1. Determination of bakery product

used in the fusion product
2.1.1. Focus Group Discussion (FGD)
FGD is a rapid assessment, semistructured method of data gathering in the
which a purposively selected set of of
participants gather to discuss issues and
concerns based on a list of key themes
drawn up by the researcher/facilitator
(Kumar 1987). The number of participant
that recomended for focus group
discussion are six to ten people
(MacIntosh, 1993). For this study, the
focus group discussion was conducted
with12 participants and a moderator before
making the fusion product. The objectives
for the focus group discussion were to:
a. Infuse dendeng(Indonesian
traditional food) into bakery
product
b. Explore and find the type of
bakery product that would
match with dendeng
c. Form new product that
fulfilled
consumers
pallatability
and
acceptability.
In the focus group discussion (FGD),
several questions regarding the topic were
discussed
among
the
participants.
Appendix A shows the questions used in
FGD. The questions were divided into two
categories (a)questions on the bakery
product in general including the
preference, characteristics, and consuming
behavior, (b) questionswith more specific
to the aim of the project including the type
of bakery product that went well with
dendengbalado, the expected attributes for
each type of bakery product, and the
ingredient used for filling.
Figure 1 to Figure 4 summarize the
participants consumption behavior on the
bakery products while Figure 5 shows
their idea of the infusion product with
dendengbalado.
Figure 1 Preference of type bakery product
Figure 1 shows the result in a type of the
bakery product that the participants
preferred to have. Seven bakery products
were drawn. From the figure, cake was the
bakery product that was liked most with
32%, followed by bread with 26%, pizza
16%,
and
croissant
11%
whilepuff,cookies, pie were selected 5%
each.

September 1st 2015
Page 7
Bakery Product
Attributes
Time of consume
soft
8%
6%
smooth
8%
Morning
29%
29%
43%
moist
3%
Evening
Afternoon
6%
17%
17%
crisp
14%
After meal
14%
fluffy
6%
Figure 2 Bakery attributes

Figure 2 shows the attributes of the bakery
product that the participants liked. Nine
attributes
were
drawn
including
appearance,
softness,
smoothness,
sweetness, moistness, creaminess or
buttery, crispiness, aroma and fluffy
texture.Four attributes were texture
characteristics, four for taste and one for
appearance. The attributes of the bakery
product that the participants liked most
were softness (29%), following by
smoothness and crispiness (17%) while the
other attributes got the share below 10%.
Figure 3 Time of consume bakery

product
Figure 3 shows the time in a day that the
participants consumedthe bakery product.
Most of the participants answered in the
morning with 43% percentage. Some
participants answered after meal with 29%
percentage and just few participants
consumed bakery product in the evening
or afternoon with the lowest percentage of
16%.
How often?
8%
8%
Bakery product
preference
5% 5% 11%
16%
26%
5%
32%
Everyday
Twice a day
25%
59%
Twice a week
Once a week
Croissant
Bread
Cake
Cookies

Pizza
September 1st 2015
Page 8
Figure 4 Intensity of consume bakery

product
Figure 4 showed the frequency of
consumption bakery product. With 59%
percentage of the participants consumed
the bakery product which is also true to
most Thai who eat bread in the morning.
From the result, it can be said that bakery
product
is
highly
consumed
everyday.Thus, it seems that bakery
product would be a good choice to develop
as the consumption rate was high.
Type of bakery that

goes well with
dendeng balado
14%
Pizza
Bread/roll
22%
64%
Muffin
Puff-pastry
0%
Figure 5 Type of bakery compliment

with dendengbalado
Figure 5 showed the type of bakery
product that would have the potential to be
fused with Indonesian traditional cuisine
(dendengbalado). From the result, it could
be seen that among the 4 different types of
bakery products, with 64% percentage,
puff pastry was chosen as the most
preferable and most likely to be well
matched for the fusion with Indonesian
traditional food which is dendengbalado.
As the dendengbalado taste spicy and
savory, it seem to go well with puff pastry

that is crispy and salty.
2.2. Preliminary test

2.2.1. Preparation of dough
The puff pastry dough was prepared
following the formula in Table 1. The
pastry margarine added to the dough layer
was distributed evenly, folded and rolled
several times that made the puff many
layers after baking. Therefore the dough
formula was suitable to be used in the
project. The dough was made using ratio
2:1 of flour:water, 500 grams of all
purpose flour in 250 ml of cold water. By
using this ratio, it would make a plasticelastic dough, (Baking Industry Research
Trust,
2011).
According
to
Malgieri,N.(1994), the puff pastry dough
was best used when it is cold. If the dough
became too soft, it would melt the butter
inside the dough and did not rise when
baked.
The folding for puff pastry often used 4-6
single folds but in this project 2 single
folds and 1 double fold was used instead.
The number of fold affects the number of
layer made. According to Yankellow, J.
(2005), the more layers is not better when
folding the dough because the layers of fat
will get thinner and thinner each time the
dough is sheeted. If the fat layer become
so thin, the fat will be absorbed to the
dough and there wont create flaky texture.

September 1st 2015
Page 9
Figure 7 dendengbalado puff pastry

before baking (up) and after baking
(below)
2.2.3. Just About Right test
Figure 6 Pastry dough with 2 single fold

and 1 double fold
2.2.2. Preparation of sample

The dendengbalado sample was made by
baking the sample at 200oC for about 30
45 minutes until the crust became gold
brown color. The heat in the oven was
controled so it wont get too hot. Too high
heat would cause too much fat leaked out
that might cause failure in the product.
(Yankellow, Jeffrey. 2005)
In the preliminary test, there were three

things to do. First, making the dough and
filling, sensory evaluation test, which were
Just About Right test and Preference test,
and adjusting the filling formula based on
Just About Right tests result. For the
formula of the dough used the formula
based on FT4114 Bakery Practical
Handbook, Assumption University, while
the formula of dendengbalado that used as
filling from Table 2. After the preparation
of the pastry dough and dendengbalado,
the the sensory test was conducted with 30
untrained panelists. The result from Just
About Right test was shown in Table 3.
Table 3 Just About Right result in

percentage
Attributes
Color of pastry
Crispness
Saltiness
Sweetness
Sourness
Spiciness
Size of pastry
The amount of
filling
The size of meat
filling
Too
little
0.0%
0.0%
0.0%
0.0%
0.0%
3.6%
0.0%
Somewhat too
little
39.3%
17.2%
3.4%
10.3%
6.9%
28.6%
28.6%
Just
right
60.7%
82.8%
69.0%
89.7%
89.7%
60.7%
64.3%
6.9%
34.5%
51.7%
0.0%
20.7%
58.6%
Nine attributes were asked in the JAR test.

There were five level scales for each
attributes range from too little to too
September 1st 2015
Page 10
much. Based on the JAR test, three

attributes required no adjustment, included
crispness, sweetness, sourness. They
obtained more than 80% just right level.
The color of pastry need to be increased
because there 39.3% of the untrained
panelists gavesomewhat too little. Thus,
to improve the color of the pastry can be
done by increasing the baking time orthe
baking temperature should solve the
color.In addition applying egg wash over
the pastry could help develop more color.
The egg wash is a mixture of egg or part of
the egg that had been thinned with
water,milk or cream to increase the brown
color, to create glossy shine or both.(Fine
Cooking.com).The amount of liquid
added to egg wash to get the best
looking pastry according to Fine
Cooking.com is 1 tablespoon per egg or
tablespoon per yolk or white.
Different makeup of egg wash will give
different brown color and glossy shine.
This project used the type of egg wash
containing whole egg with water in a ratio
1:1. The crust color of the puff pastryis
demonstrated inFigure 6.
Table 4 Customized egg wash and effect

Content of
egg wash
Effect on cooked pastry
Whole egg Nicely browned, slightly

with water
glossy
Whole egg
with milk
Micely browned, more

glossy
Egg white
only
Evenly browned, slightly

less brown than whole egg,
very little shine
Egg yolk
only or egg
yolk with
water
Browned and shiny, but

less than with cream or
milk
Egg yolk
with cream
Very browned and glossy,

but a relatively thick egg
wash that's somewhat
difficult to spread neatly
Egg yolk
with milk
The darkest brown crust

and a touch less shiny than
yolk with cream
Source: Fine Cooking.com
Figure 8. Puff pastry color of crust

From table 3, apart from color of the crust,
first, saltiness got 69% just right level but
27.6% at somewhat too much. The result
indicated that almost one third of the
participants
thought
that
the
dendengbaladopuff pastry sample was too
salty and needed to adjust in the adjusting
step.
Second,
spiciness
of
the
dendengbaladopuff pastry sample was
rated by 60.7% of the untrained panellists
at just right level. 28.6% of the untrained
panellists though that it was somewhat too
little and 7.8% gave too little. Thus, the
amount of salt in the filling needed to be

September 1st 2015
Page 11
adjusted. Next, dealing with the amount

and size of dendengbalado filling in the
puff pastry, only 51.7 and 58.6% of the
panellists rated the sample at the just right
level, respectively. More than 40% of the
panellists thought that the amount of the
filling was too little while the size of
dendengbalado pieces was rated at mix
feeling about 20.7% said that it was
somewhat too small and another 17.2%
rated at somewhat too big and 3.4 gave too
big. Thus, they needed to be adjusted in
the further study.
2.2.4. Preference test
Preference test using 9 point of hedonic
scale was also conducted together with
JAR test. The result was used to support
the JAR test result and to see how well the
score of dendengbalado puff pastry and its
acceptance. The result from the prefence
test was shown below in Table 5.
Table 5. Preference test result for the
preliminary test
Attributes
MeanSD
Appearance
Crispness
Saltiness
Sweetness
Sourness
Spiciness
Size of pastry
Amount offilling
Size of filling
Overallacceptance
7.31.1
7.11.2
7.00.9
7.50.9
7.21.0
7.01.1
7.11.2
6.41.7
6.71.5
7.50.9
The results of preference test for

thedendengbalado puff pastry in the
preliminary test on 10 attributes were
shown in Table 5. From that result, the

dendengbalado puff pastry was accepted
by the untrained panellists with the
average preference scores range from 6.4
to 7.5 out of 9-point scale. The highest
score was found in an overall acceptance
and sweetnessof 7.50.9. The score
indicated the untrained panellists preferred
the dendengbalado puff pastry from
moderately like to very much like score
(Appendix B 3). Most of the attributes
gained the preference scores higher than 7,
except the amount and size of the
dendengbalado in the sample. The result
agreed with JAR test, especially in the
amount and size of the dendengbalado for
filling that need improvement.
2.2.5. Adjusting The filling formula
In the adjustment step, only the attributes
related to the formula were adjusted. As
mentioned earlier, the color of the puff
pastry could be adjusted during baking and
applying of the egg wash. It was not
adjusted. For the amount and size of
filling, only the amount of the filling was
increased from one full teaspoon to one
and a half teaspoon.
Based on JAR test, three attributes,
crispness, sweetness and sourness, were
considered as just right by more than 70%
of the untrained panellists and need no
adjustment in the formula. Two attributes,
saltiness and spiciness which were related
to salt and chilli in the formula were
selected for the adjustment.
First, the amount of chili was increased at
2 levels from 9g or 1.6% in control to 13 g
or 2.6% and 17 g or 3.6% of chilli, as
shown below. The treatments were
formulated as followed:

September 1st 2015
Page 12
Control
: 1.6% chilli
Treatment A
: 2.6% chilli
Treatment B
: 3.6% chilli
Three treatments of samples were prepared
twice and tested by 30 untrained panellists
using 9-point hedonic scale preference
test. The results from the preference test
were shown in Table 6.
Table 6 Average score of 9-point
hedonic scale preference test for
dendengbalado puff pastry
Attribut
es
Appeara
nce
Crispnes
s*
Saltiness
Sweetnes
s
Sourness
Spiciness
Overall
acceptan
ce
Average scores SD of
dendengbalado puff pastry
Control Treatm Treatm
ent A
ent B
7.071. 7.171.6 6.831.7
53
4
0
7.001. 6.601.4 7.001.3
31a
5b
4a
6.631. 6.701.4 6.901.1
33
2
8
7.031. 7.271.0 6.801.4
03
8
9
6.531. 7.001.1 6.901.0
17
4
9
6.601. 7.071.3 7.071.2
30
4
8
7.031. 7.171.1 7.330.9
03
5
2
Note: * There wassignificant difference at

p < 0.05
untrained panellists rated the treatment

with no significant difference even though
the amount ofchilliin the formula was
increased
to
2.6%
and
3.6%.Control,treatment A and treatment B
were rated as 6.601.30,7.071.34,
7.071.28, respectively. As a result
treatment B with 3.6% chilli was selected
for further study.
Second, the amount of salt was adjusted
using Treatment B as the control with
0.8% salt was reduced to 0.4% salt. No
more reduction was carried as the amount
of salt was also considered low in the
filling. The treatments were shown as
below:
Control
: 0.8% salt
Treatment 1 : 0.4% salt
The two samples were prepared and tested
with the same 30 untrained panellists using
the same 9 point hedonic scale preference
test on seven attributes. The results for
preference test after adjusting the saltiness
was shown in table 7.
Table 7 Average score of 9-point

hedonic scale preference test for
dendengbalado puff pastry (saltiness)
Attributes
Appearance
Table 6 showed the average preference

scores of seven attributes of the three
dendengbalado puff pastry samples. With
exception to the crispiness, there were no
significant differences in the preference
scores of the remaining attributes at p
0.05. For the spiciness, it was seen that the
Crispness
Saltiness
Sweetness
Average scores SD of
dendengbalado puff
pastry*
Control
Treatment 1
7.10
7.10 1.27
1.03
6.17
6.47 1.33
1.23
6.87
6.97 1.16
1.31
6.67
6.80 1.49
1.49

September 1st 2015
Page 13
Sourness
Spiciness
Overall
acceptance
6.57
1.38
7.07
1.28
6.90
1.18
Note: * there were

difference at p < 0.05
6.77 1.10
6.83 1.42
7.13 1.01
no
significant
Table 7 showed the average preference

scores of the two dendengbalado puff
pastrysamples. Statistical analysis showed
that there were no significant diference at
p < 0.05 in everyattributes. The saltiness, it
was seen that the untrained panellists rated
the control and the treatment with no
significant difference even though the salt
used was decreased to half of the original
content. It could be that there was salt
from the pastry dough as it was made with
salted butter and 0.5% of salt was added in
the formula.
Based on all of the result, there were no
significant
difference
among
the
treatments given. Therefore, all the
formular could be choosen.From an
economic point of view, the control
formula was the most economic to be
made as the infusion product but it
received
the
lowest
overall
acceptance(7.031.03). The formulation
contained 3.6%chilli might also be used as
the overall acceptance of the sample
received the highest preference score
(7.330.92)but it was also thecostest.
Instead of using the control formula and
treatment B, it might be better to use the
formula treatment A (2.6%chilli) since the
overall
acceptance
was
quite
high(7.171.15), higher than the control
formula. The hedonic scale for each

attributes was quite constant from the
preliminary test to the adjustment tests.
Compared to control and treatment B
formula, the score obtained by using
treatment A, most of the score reached 7
except for saltiness and crispness
attributes. Means that there were less
variance in the scores obtained for each
tested attributes. From economic view, this
formula was less expensive than the
treatment B with the less variance in score
and high overall acceptance score. The salt
used in the formula can use the original
formula that is 0.8% salt or use the
reduced one 0.4% salt. From the result
showed there no significant difference,
therefore the reduced formula (0.4%
salt)might reduce the cost in making the
infusion product.
As shown on the result, the crispness
attribute received was not consistent score
during the testing. The sensory score
tended to decrease and got lower and
lower score in comparison to the score in
the preliminary test. The crispness of the
pastry depends on the layer, therefore
damage on the layer would result in
decreasing crispness attribute. Pressing too
hard the end and edges when rolling would
cause the layers stick together and prevent
them to rise. Cutting the puff pastry dough
would be better to use sharp knife or
pastry cutter and not dragging the knife
when cutting the dough, that also would
make the layer stick and prevent them to
rise. Run down egg wash would make the
edges stick and prevent from rising.
(Pepperidge Farm, 2011). There were also
several factors that cause the puff pastry to
fail(Yankellow, Jeffrey. 2005)

September 1st 2015
Page 14
Too little or too much fat

The dough pressed too hard
Too much fold.
The more folded the dough, the
thinner fat layer would become
that the fat would be absorbed
eventually into the dough,
resulting in bread like interior not
flaky texture which was the
characteristic of puff pastry.
The heat on oven is too high or too
little
Most of the failure in crispness of puff and

pastry depends mainly on the technique in
preparation of the pastry dough and the
pastry units. Thus, care must be taken to
produce uniform pastry dough. As a result
crispness will be not related to the
dendengbalado formulation.
Conclusions
Puff pastry was the most suitable for
fusing with dendengbalado as a
filling.
Crispness of the puff pastry depends
technique to produce the layer of the
dough.
All formula could be used since there
were no significant different in the
preference scores from all treatments
in the study.
Formulation A containing 0.8% salt
and 2.6% chilli might be suitable to
develop as the overall score was high
and more constant for all attributes
and it was also economic to produce.
Dendengbalado puff pastry was well
accepted by the panelists, proven by
the overall acceptance score always
more than 7 out of 9-point scale.
3.
References
Femina.2015.Dendeng Balado.Retrieved
March 8, 2015 from
http://www.femina.co.id/kuliner/re
sep/hidangan.indonesia/dendeng.ba
lado/004/001/234/02.
Hawkins, H. 2004. Puff and Flake: what
can go wrong and how to prevent
it.http://www.nzbakingsociety.co.n
z/journ.html. New Zealand.
Industry and trade interviews; Commerce
Ministry. 2013. Business
Opportunities Studyin Thai Bakery
Sector. Retrieved March 8, 2015.
IPB. 1982. Paket Industri Pangan
untukPedesaan: Dendeng. Bogor.
Kumar, K. (1987). Conducting focus
group interviews in developing
countries. A.I.D. ProgramDesign
and Evaluation Methodology
Report No. 8. Washington, D.C.:
U.S. Agency forInternational
Development.
Malgieri,Nick.1994. Quickest Puff
Pastry.Cooks Illustrated. Retrieved
March 8, 2015 from
http://yvonnehelmer.com/RECIPE
%20MASTER/NEW%20RECIPES
/Puffpastry%20Cooks%20Illustrate
d.pdf
Palsgaard Technical Paper.2011. Puff
pastry margarine- Focusing on
functionality and fat reductions.
Retrieved March 16, 2015 from
www.palsgaard.com.

September 1st 2015
Page 15
Pepperidge Farm. 2011. Puff Pastry Tips.

Retrieved March 12, 2015 from
www.puffpastry.com.
Philips, S. 2000.
http://www.baking911.com. New
York. Retrieved March 14, 2015
Wheat Foods Council.2010. Grains of
truth about PASTRY.Retrieved
March 14, 2015 from
www.wheatfoods.org.
Yankellow, J. 2005. lamination: layers
beyond imagination. San Fransisco
Baking Institue.San fransisco.

September 1st 2015
Page 16
GUAR GUM EFFECT AS FAT REPLACER ON SENSORY

CHARACTERISTICS OF BROCCOLI BROWNIES
Stella Gunawan R.a*, Laksmi Hartayanieb, and Probo Y. Nugrahedib
aStudent
of Food Technology Department, Faculty of Food Agricultural Technology,

Soegijapranata Catholic University, Semarang, Indonesia
bLecturers of Food Technology Department, Faculty of Food Agricultural Technology,
*Email: stella.gun93@gmail.com
ABSTRACT
Brownies is a chocolate pound cake high in fat content that can reach more than 60% of total
batter, but high consumption too much fat and trans fatty acid leads to heart diseases. The
risks can be avoided by reducing the use of margarine in brownies. However, the absence of
margarine in the formula would decrease the brownies quality. Guar gum can be used as fat
replacer to improve the quality of low fat bakery products. The addition of broccoli (Brassica
olaracea L.var italic) in the formula aimed to increase the nutritional and functional properties
of brownies. Broccoli is a perishable food therefore the best way to process broccoli is freezedried and powdered so its easily applied, prolonged the shelf life, and maintained the
nutrient. The purpose of the research was to study the effect of various guar gum
concentrations (0.25%, 0.5%, 0.75%, 1%) on sensory properties of brownies. Broccoli
powder concentration that used to substitute wheat flour was 10%. The product was evaluated
by 30 untrained panelists both rating and ranking in aroma, taste, texture, and overall
attributes. The results showed the most preferred broccoli brownies was control which rating
and ranking tended to reach 4 from scale of 5 in all attributes. On free margarine brownies
formulations showed decrease of rating and ranking except aroma attribute (p>0.05)
compared with control. The increasing guar gum concentration 0.5% and 0.75% had no
significance (p>0.05) in preferences and tended to be accepted (nearly 3 from scale of 5) in
overall attributes.
Keywords: brownies, broccoli, free margarine, guar gum

September 1st 2015
Page 17
INTRODUCTION
Brownies is one of the baked bakery
products, also known as pound chocolate
cake made from wheat flour, sugar, fat,
and chocolate (powder or cooking
chocolate). This cake has high fat content
that can reach more than 60% of total
batter. The main source of fat in brownies
is from shortening usually margarine that
can improve the sensory quality of
product. But, high consumption of too
much fat (>30% from total intake energy)
can cause the narrowing of arteries. Some
of partially hydrogenated margarines can
lead to heart diseases from the presence of
trans fatty acid formation. Recommended
the intake of saturated fatty acid by
American Heart Association (AHA) is less
than 10%. FDA (2004) also recommends
intake level of trans fatty acid is less than
1% from total energy (Sartika, 2008). The
risks from saturated and trans fatty acid
can be avoided by reducing the
consumption of too much fat in food
product and increase the consumption of
fruits and vegetables.
One way to reduce the fat content in
brownies is the use of margarine in
formula should be reduced. But, the
absence of margarine will decrease the
brownies
quality.
The
shortening
(margarine) in bakery products are
important to give tenderness, moisture
mouthfeel, lubricity and entraps air bubble
during mixing (Stauffer, 2005). Therefore,
guar gum can be added as fat replacer in
formula to improve the quality of
brownies. Guar gum is a carbohydrate
based fat replacer (fat mimetic) and one of
the galactomannan hydrocolloid group
from Cyamopsis tetragonolobus seeds.

Guar gum has similar function with fat that
can protect protein from direct interaction
with water, weakens the gluten to soften
the cake (Hazen, 2011).
Guar gum has been applied into breads,
oatcakes, and biscuits to yield baked
products with high soluble fiber content
that has positive effect to decrease the
levels of serum cholesterol and lowdensity lipoproteins (LDLs) (Nussinovitch
& Hiroshima, 2013). Guar gum solution at
low concentration in bakery product can
improve mixing, prolong the shelf life, and
prevent syneresis in frozen dough and pie
filing (Kohajdova, 2009). Guar gum also
has advantages such as economical use
(too much dosage leaves bitter taste), has
lower cost than xanthan gum, and made
from natural plant.
Guar Gum is classified as GRAS
(Generally Recognized as Safe) by FDA.
Maximal permitted concentration of guar
gum in food is 2% by weight found in
vegetable products and in fats and oils.
Guar gum has beneficial effect when its
use is at low concentrations about 0.5%1%. Above this concentration, it will show
negative effects such as higher viscosity
can decreases the protein efficacy and lipid
utilization. Higher viscosity more than 1%
interfere absorption of nutrient and
negative effect in physicochemical of
food, also its not accepted by the
consumer (Mudgil et al., 2011).
Broccoli (Brassica olaracea L.var italic)
which usually consumed as main dishes in
Indonesia, can be applied in bakery
product. Broccoli has higher in vitamin C

September 1st 2015
Page 18
and fiber 89.2 mg and 2.6 mg than other

vegetables (carrot, cabbage, and spinach)
(USDA, 2012). Some compounds in
broccoli have advantages in health such as
glucosinolate, vitamins (E, C, K), minerals
(iron, zinc, selenium), and some
polyphenols (Vasanthi et al., 2009). In this
research, the broccoli was processed by
freeze drying and powdering to produce
dried broccoli powder. Freeze drying is
one of the preservation using vacuum
pressure so the water which already
formed as ice (through previous freezing)
can directly change into water vapor. That
phase change is called as sublimation.
Freeze drying has more advantages than
heat / hot air drying such as no case
hardening, the vapor can diffuse very well
from the still wet part to air / environment
so the dried product has better quality
(Muchtadi & Sugiyono, 2013), maintain
physicochemical and sensory properties,
inhibit the growth of microbes and enzyme
activity or other reactions which affecting
the nutrients of food so the product can
have long shelf life.
The technique of baking brownies in this
study is Au Bain Marie (steam-bake). The
brownies batter in the small container is
put in the bigger baking container which
had been filled by hot water. Au bain
marie can keep the batter temperature
lower than boiling temperature of water
inside big container so this can prevent
overheating. The advantages of this
technique are cake will perfectly baked
until the center of cake and give the softer
texture because of the presence of water
vapor during baking (Foster, 2013). The
texture of brownies from au bain marie is
softer than usual baked brownies.
The objective of the research was to study

the effect of various guar gum
concentrations (0.25%, 0.5%, 0.75%, 1%)
on sensory properties of brownies. The
evaluated attributes in sensory properties
were aroma, taste, texture (hardness), and
overall.
MATERIAL AND METHODS
Broccoli Powder Preparation
Fresh broccoli was bought at traditional
market Pasar Bulu Semarang, grew at
Bandungan. The broccoli was washed
under running water and steam blanched
for 3 minutes in temperature 100C. After
that, broccoli was trimmed at the bottom
part and cut into small pieces. Broccoli
was frozen at
-10C in freezer for one
night then dried in PowerDry LL1500
vacuum freeze drier (temperature
-4
100 C and pressure 410 mbar). The
dried broccoli then was ground into
powder and sieved with mesh size 60.
Broccoli powder was ready to be an
ingredient in brownies formulations. The
powder could be stored in plastic
containing silica gel then was put inside
alumunium foil plastic packaging sealed.
Making of Brownies Procedures
There were 2 steps to make brownies for
first sensory (containing margarine) and
second sensory (no margarine) evaluation.
First two formulations were original
brownies (standard) and brownies with
broccoli powder 10% by weight of wheat
flour and substituted with wheat flour.
This concentration was determined
according to research of Kim & Cho
(2010) about application of commercial
freeze dried broccoli powder in sponge

September 1st 2015
Page 19
cake. Next formulations were 4 brownies

with treatment 100% margarine replaced
with different concentrations of guar gum.
The percentage of guar gum was added by
weight of wheat flour. The ingredients
could be seen in Table 1 and Table 2.
Table 1. Brownies Formulations
speed 1 until homogenous batter obtained.

For free margarine brownies formulations,
the making procedure was same but the
salt was beaten together with eggs and
sugar for 3 minutes speed 2. Then guar
gum was added and mixed again for 1
minute speed 1 until blended. Dry
Table 2. Free Margarine Broccoli

Brownies Formulations with
Various Guar Gum
Ingredient
s
Con
trol
63
Treatments
0.25 0.5% 0.75
%
%
63
63
63
Wheat
flour (g)
Broccoli
7
7
powder
(g)
Sugar (g) 105 105
Salt (g)
0,7
Cocoa
14
14
powder
(g)
Eggs (g) 100 100
White
49
margarine
(g)
Guar gum
0,16
(g)
Concentrations
Ingredients
1%
63
105
0,7
14
105
0,7
14
105
0,7
14
100
-
100
-
100
-
0,32
0,47
0,63
First step to make brownies containing

margarine were eggs and sugar were
beaten with mixer for 3 minutes (speed 2)
until light and fluffy, then salted white
margarine was added and mixed again for
1 minute (speed 1) until blended. Dry
ingredients in Table 1 were mixed again at
Formulations
Standard
Broccoli
Powder 10%
70
63
7
Wheat Flour (g)

Broccoli
powder (g)
Sugar (g)
105
105
Cocoa powder
14
14
(g)
Eggs (g)
100
100
White
49
49
margarine (g)
ingredients in Table 2 were added and
mixed at speed 1. The batter was poured to
cake mould which had been greased inside
the mould. Mould containing cake batter
was put inside the oven in top rack. The
bigger container was filled with hot water
and taken inside the oven right under the
top rack. The cake then baked in
temperature 180C set at top heat 35
minutes and bottom-top heat 25 minutes.
Sensory Evaluation of Brownies
(Meilgaard et al., 1999)
The sensory tests were evaluated by 30
untrained panelists from students of Food
Technology Department Soegijapranata
Catholic University Semarang. First
sensory test was to evaluate original
brownies (control) and brownies after
substituted with broccoli powder by rating
hedonic at overall attribute. Panelists rated

September 1st 2015
Page 20
the product using 0 6 scales (0=disliked

a lot, 6=liked a lot).
For the second sensory test, these same
panelists evaluated free margarine
brownies with broccoli and different
concentration of guar gum (0.25%, 0.5%,
0.75%, 1%) together with control
(contained margarine and broccoli) by
rating and ranking hedonic. The attributes
were aroma, taste, texture (hardness), and
overall. The evaluation used 1 5 scales
(1=disliked a lot, 5=liked a lot). Panelists
evaluated each product in individual
booths and illuminated with fluorescent
light. The tests were held at Laboratory of
Sensory
Soegijapranata
Catholic
University, Semarang.
Data Analysis
Sensory data were statistically analyzed
using IBM SPSS statistics version 20.00
program. Data were analyzed by Wilcoxon
non parametric test for first sensory, then
second sensory used Kruskal Wallis to
determine the significance of each
treatment followed by Mann Whitney non
parametric test to determine which
treatment were different at level of
confidence 95%.
RESULTS AND DISCUSSION
The first sensory test was conducted to
know the acceptance of brownies after
substituted with 10% broccoli powder. The
result of overall acceptance at this
brownies compared with original brownies
(control) are shown in Table 3.
Table 3. Sensory Mean Scores of

Brownies
Samples
Overall rating
scores
4.93 0.91a
Original brownies
(Control)
Brownies with 10%
4.27 1.26a
broccoli powder
All values
are mean standard
deviation
Value of each row with different
superscript shows theres significance
difference between both treatments at
confidence level 95% based on
Wilcoxon test
Score :
0 = dislike very much
1 = dislike
2 = dislike slightly
3 = neutral
4 = like slightly
5 = like
6 = like very much
The results showed the overall rating score
of brownies with broccoli powder were
slightly lower than brownies control. Its
also known no significance difference in
rating scores between two brownies at
confidence level 95%. Panelists scored the
preferences in almost similar value, as
shown at score approximately 4 (like
slightly). This could to be expected
because broccoli powder was added at low
concentration (10%) so it didnt affect
overall attributes sensory of brownies.
The second sensory test was conducted to
know which free margarine brownies
replaced with guar gum was preferred by
panelists based rating and ranking scores.
The evaluated attributes were aroma, taste,
texture (hardness), and overall. The results

September 1st 2015
Page 21
both rating and ranking are shown in Table

4, Figure 1 and Table 5, Figure 2.
Table 4.
Rating Hedonic Broccoli
Brownies with Various Guar Gum
Concentrations
Treat
ment
Attribute (Scores)
Taste Textur Overal
e
l
3.931 4.071 3.871
.31a
.17a
.55a
2.931 2.271 2.801
.34bc
.14b
.35b
3.271 3.131 3.50
.20b
.14c
0.90ac
2.731 2.631 3.131
.05bc
.00bc
.20bc
2.471 2.271 2.671
.23c
.17b
.12b
are mean standard
Arom
a
A
3.57
1.33a
B
2.93
1.05a
C
3.00
1.39a
D
2.83
0.99a
E
2.87
1.36a
All values
deviation
difference among the treatments at
confidence level 95% based on Kruskal
Wallis testfollowed by Mann Whitney
test
Score: 1 = dislike very much, 2 =
dislike, 3 = like slightly, 4 = like, 5 =
like very much
Brownie
sE
Brownie
sD
Brownie
sA
6
4
2
0
Brownie
sB
Brownie
sC
Figure 8. Rating Scores at Different

Treatments
A : Substituted with broccoli powder 10%
B : Free margarine broccoli brownies +
guar gum 0.25%
C : Free margarine broccoli brownies +
guar gum 0.5%
D : Free margarine broccoli brownies +
guar gum 0.75%
E : Free margarine broccoli brownies +
guar gum 1%
Table 5. Ranking Hedonic Broccoli

Brownies with Various Guar
Gum Concentrations
Treat
ment
Attribute (Scores)
Taste Textur Overal
e
l
3.901 4.271 3.901
.54a
.34a
.61a
2.901 2.371 2.601
.37bc
.25b
.43bc
3.201 3.331 3.131
.27b
.27c
.11b
2.731 2.731 2.831
.11bc
.11bc
.32bc
2.271 2.301 2.531
.31c
.18b
.22c
are mean standard
Arom
a
A
3.60
1.67a
B
2.97
1.13a
C
2.97
1.38a
D
2.73
1.08a
E
2.67
1.58a
All values
deviation
Aroma
difference among the treatments at
Taste
confidence level 95% based on Kruskal
Texture
Wallis testfollowed by Mann Whitney
Overall
test
Score: 1 = dislike very much, 2 =
dislike, 3 = like slightly, 4 = like, 5 =
like very much

September 1st 2015
Page 22
Browni
es E
Browni
es D
Browni
es A
6
4
2
0
Browni
es B
Browni
es C
Aroma
Taste
Texture
broccoli powder was not detected and

masked by strong chocolate aroma from
brownies. According to Setyaningsih et al
(2010), the aroma is obtained from volatile
compound, slightly soluble in water or
slightly soluble in oil.
Overall
Figure 9. Ranking Scores at Different

Treatments
A : Substituted with broccoli powder 10%
B : Free margarine broccoli brownies +
guar gum 0.25%
C : Free margarine broccoli brownies +
guar gum 0.5%
D : Free margarine broccoli brownies +
guar gum 0.75%
E : Free margarine broccoli brownies +
guar gum 1%
The results can be seen that all brownies
with different concentrations of guar gum
affected rating and ranking scores in all
attributes except aroma. The most
preferred brownies in all attributes was
brownies control, as shown at highest
rating and ranking scores nearly 4 (like).
All free margarine broccoli brownies
showed decrease score compared with
control.
The aroma of control showed higher
scores than all free margarine brownies,
but there were no significance difference
in aroma scores among all 5 treatments at
confidence level 95%, as shown at score
nearly 3 (like slightly). This proved that all
brownies aroma were accepted by
panelists. This could to be expected
because adding guar gum wouldnt affect
the aroma and low concentration of
The most preferred taste of free margarine

broccoli brownies after control was C
(0.5% guar gum). But at 0.25% (B) and
0.75% (D) guar gum treatment, panelists
showed similar preferences with C (0.5%
guar gum), as shown at score nearly 3 (like
slightly). Meanwhile, brownies E (1% guar
gum) had the lowest taste preference, as
shown at rating 2.47 and ranking 2.27.
This would be happened because theres
other attribute like if attributes of texture
was less acceptable, it would affect the
tendency of panelist evaluation on taste
attribute. According to Winarno (2004),
taste of food is combination stimuli of
tasting, odor, and experiences involving
tongue. Changes in texture of product also
change the taste and aroma that arises,
because it can affect the rate of stimulation
on the olfactory receptor cells and saliva
gland.
The next evaluated attribute was texture,
indicating the preference of panelists on
hardness of brownies when chewed. The
results showed the most preferred texture
by panelist was control with nearly rating
and ranking 4 (like). This proved panelists
still prefer brownies containing margarine
(fat). Fat in bakery product can shorten
(tenderizes) the texture of finished product
(Stauffer, 2005). Hence, all without
margarine brownies formulation showed
lower score than control because the
panelist tended to prefer brownies with

September 1st 2015
Page 23
softer texture. The highest rating and

ranking score (3.13 and 3.33) after control
is brownies C (free margarine with 0.5%
guar gum). At brownies D (0.75% guar
gum), the texture score slightly decrease so
it had no significance difference and
tended to reach score 3 (like slightly) so
both brownies could be accepted by
panelists. According to Heflich (1996) in
Koksel (2009), gum can make the crumb
cake elastic and rubbery. Crumb can be
perceived as softer if adding guar gum at
optimum low concentration however it can
be too tough if adding too much gum
concentration. This statement also found in
the result of highest guar gum
concentration in brownies E (1% guar
gum) which less preferable by panelists, as
shown 2.3 in rating and 2.4 in ranking
(dislike). Brownies E also showed no
significance difference with B (0.25% guar
gum) at confidence level 95%. The free
margarines broccoli brownies formulations
which still accepted in texture (softer when
chewed) were 0.5% and 0.75% guar gum
treatments.
At overall attribute showed control was the
most preferred both rating and ranking but
at rating scores, there was no significance
difference with brownies C (0.5% guar
gum). Brownies D (0.75% guar gum) also
showed no difference in overall
preferences with brownies C at confidence
level 95% and still accepted by panelist at
score nearly 3. The lowest rating and
ranking scores was brownies E (1% guar
gum), as shown at 2.6 and 2.5 (nearly
dislike). This score was also similar with
other attribute preferences score which
also had the lowest score in rating and
ranking so this brownies was less
acceptable in overall attribute. Free

margarine broccoli brownies which
replaced with guar gum at 0.5% and 0.75%
were still overall accepted by panelists.
CONCLUSIONS
In free margarine (100% fully replaced)
broccoli brownies formulations, the use of
increasing guar gum as fat replacer at
0.5% and 0.75% were the most preferred
in overall sensory properties by 30
untrained panelists. Broccoli brownies
with 0.5% and 0.75% guar gum showed no
significance difference (p>0.05) that still
accepted because the scores were nearly 3
from scale of 5 in overall attribute.
REFERENCES
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http://www.wisegeek.org/what-is-abain-marie.htm. Accessed on
October 14th 2014.
Hazen, C. 2011. Reduced-Fat Formulating.
Food Product Design 21 (8) 1-4.
www.foodproductdesign.com.
Accessed on October 29th 2014.
Kim, C.H. & K.R. Cho. 2010. Quality
Characteristics of Sponge Cakes
Made with Different Quantities of
Broccoli Powder. Korean Journal of
Food Science and Technology 42 (4)
459-467. Department of Food and
Nutrition,
Hanyang
Womens
College, Seoul 133-793, Korea.
Kohajdov, Zlatica; Jolana Karoviov;
tefan Schmidt. (2009). Significance
of Emulsifiers and Hydrocolloids in
Bakery Industry. Acta Chimica
Slovaca 2 (1) 46 61. Institute of

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Biotechnology and Food Science,

Faculty of Chemical and Food
Technology, Slovak University of
Technology, Radlinskho 9, 812 37
Bratislava, Slovak Republic.
Koksel, H.F. 2009. Effects of Xanthan and
Guar Gums on Quality and Staling
of Gluten Free Cakes Baked in
Microwave-Infrared
Combination
Oven. (Thesis). The Degree of
Master of Science, Food Engineering
Department, Middle East Technical
University. Turkey.
Meilgaard, M., G.V. Civille, T. Carr. 1999.

Sensory Evaluation Techniques 3rd
Ed. 241-247. CRC Press, Boca
Raton, Florida.
Muchtadi, T.R. & Sugiyono. 2013. Prinsip
Proses dan Teknologi Pangan. 180187. Alfabeta, Bandung.
Mudgil,
D.,
S.
Barak,
S.K.
Bhupendar.2011.
Guar
Gum:
Processing, Properties, and Food
Applications-A Review. Jornal of
Food Science and Technology 51 (3)
409-418 DOI 10.1007/s13197-0110522-x. Jambheshwar University of
Science and Technology, Hisar
125001, India.
Nussinovitch, A. & M. Hiroshima. 2013.
Cooking Innovations: Using
Hydrocolloids for Thickening,
Gelling, and Emulsification. 128135. CRC Press, Taylor & Francis
Group, United States.
Sartika, R.A. 2008. Pengaruh Asam

Lemak Jenuh, Tidak Jenuh dan
Asam Lemak Trans terhadap
Kesehatan.
Jurnal
Kesehatan
Masyarakat Nasional 2 (4): 154-160.
Fakultas Kesehatan Masyarakat
Universitas Indonesia, Kampus Baru
UI Depok 16424.
Setyaningsih, D., A. Apriyanto, M.P. Sari.
2010. Analisis Sensori Untuk
Industri Pangan dan Agro.180
halaman. IPB Press, Bogor.
Staufffer, C.E. 2005. Fats and Oils in
Bakery Products. In: Baileys
Industrial Oil and Fat Products 6th
Ed. Edited by Shahidi F. 207-212.
John Wiley & Sons, Inc., Hoboken,
New Jersey.
USDA. 2012. National Nutrient Database
for Standard Reference Release 27:
Broccoli, Cabbage, Carrot and
Spinach.
http://ndb.nal.usda.gov/ndb/nutrients
/. Accessed on June 10th 2015.
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Chemico-Biological
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Winarno, F.G. 2004. Kimia Pangan dan
Gizi. 251 halaman. PT Gramedia
Pustaka Utama, Jakarta.

September 1st 2015
Page 25
COOKIES CHARACTERISTICS BY USING OF DEHULLED AND

UNDEHULLED MUNGBEAN FLOUR
Mak Alan Darma Saputraa*, Sun Shine Moninggianti Siania
aProgram
Studi Teknologi Pangan, Fakultas Teknologi Pertanian, Universitas Katolik Widya

Mandala Surabaya
Jalan Dinoyo 42-44, Surabaya, Indonesia
*Email: alanpooh8@gmail.com
ABSTRACT
Mungbean production is quite high in Indonesia, but the high production of mungbean are
often not balance with the consumption rate of mungbean. Nowadays, cookies product
favored by many people. Seeing the fairly high levels of people who consume cookies in
Indonesia, mungbean is used as substitution of wheat flour in cookies making process. This
substitution is using two kinds of mungbean, dehulled and undehulled. The dehulled process
will decrease the number of fiber that contains in mungbean flour. This study aim to presume
the physical, chemical, and organoleptic characteristics of the cookies substitues by the
mungbean flour. The proportion uses for this study are 85% wheat flour : 15% undehulled
mungbean flour and 85% wheat flour : 15% dehulled mungbean flour. The application of
those two kinds of mungbean flour will affected the cookies characteristics. The application
of those two kind of mungbean flour can minimize gluten content in the dough that can
reduce dough viscoelasticity. Dehulled mungbean cookies has a higher moisture content,
protein content, and calorie than undehulled mungbean cookies, but has a lower fat content,
total ash, fiber, and carbohydrate. Using undehulled mungbean flour will result a cookies
with higher spread factor, darker color, and harder texture than dehulled mungbean cookies.
Keywords:characteristics, cookies, substitution, mungbean flour

September 1st 2015
Page 26
INTRODUCTION
Mungbean (Vigna radinata L.) is one of
legumes which has high protein content,
essential fatty acid, antioksidant, and
minerals. Mungbean production is quite
high in Indonesia, so that it can easily get
and cheap. According to Direktorat
Budidaya Kacang dan Umbi (2013)
research, the production of mungbean
average from year 2003-2011 is 316,76
tons. Indonesia is also one of the biggest
mungbean producer in Asia. High
production rate of mungbean are often not
balance with the consumption rate of
mungbean. Mungbean consumption rate in
the years 2003-2011 is only 278,33 tons
(Direktorat Budidaya Kacang dan Umbi,
2013). Usually, mungbean consume as
sproud, but it can turn into some of food
product such as mungbean porridge , ondeondes filling, and gandas turi or it can be
turn into hunkue flour.
Nowadays, bakery product favored by
many people. For example, cookies.
Consumption rate of cookies is 0,40
million/capital/year (BPS, 2009). Seeing
the fairly high levels of cookies
consumption in Indonesia, mungbean is
used as substitution of wheat flour in
cookies making process, so it can help to
raise
the
number
of
mungbean
consumption rate in Indonesia.
The production of mungbean cookies use
two kinds of mungbean flour which are
dehulled mungbean flour and undehulled
mungbean flour. The making of mungbean
flour from mungbean is aim to extend the
shelf life of mungbean and make the
mixing process easier. Almost all of the
step of flouring process of dehulled and
undehulled mungbean is same such as
washing, oven drying, soaking, boiling,

oven drying, milling, and sieving, but in
dehulled mungbean flours making process
there is a dehulling process after soaking
process.
This literature is aim to know about the
effect of using dehulled and undehulled
mungbean flour as a substitution of wheat
flour towards cookies characteristics. The
proportion of wheat flour:undehulled
mungbean flour which discuss in this
literature is 85:15 and the proportion of
wheat flour:dehulled mungbean flour is
85:15. The application of two kinds of
mungbean flour will give a different effect
to the appearence, texture, chemical
content, and the consumen acceptance of
this mungbean cookies.
MATERIAL AND METHOD
Tools
The tools which use in the making of
mungbean flour are washbowls,
ovendrying, 80 mesh strainer, pot, stoves,
corona manual grinder, blender, and
desiccator.
The tools which use in the making of
cookies are oven, mixer, rolling pin,
refrigerator, and baking trays.
Besides that the tools that use in physical
and chemical analysis are muffle furnance,
porcelain crus, clamp, analitic scale,
soxhlet extraction tools, kjeldahl tools,
titration tools, and ruler
Material
From Blessing (2010), the material that
use in this literature is mungbean (Vigna
radiata L.) which get from the Crop
Science Department of Michael Okpara
University of Agriculture, Umudike,

September 1st 2015
Page 27
Nigeria. All the chemical material which

use in this research get from Joechem
Chemical Store, Nsukka and Hoslah,
Umuahia, Nigeria.
Mungbean seed
Dry cleaning
Oven drying (60oC, 2 hours)
Mungbean Flouring Process

Mungbean flouring process reference
from Blessing (2010). Mungbean in this
processing consist of two batch, the first
batch is for the undehulled mungbean and
the second batch is for the dehulled
mungbean, each of them weighed 800 g
and soaked in aquades (1:4w/v) for 12
hours at room temperature (25oC). In the
dehulled mungbean flouring process there
is a manual dehulling process after the
soaking process for 12 hours with aquades
(1:10w/v). Mungbean is drained and rinse
three times using 600 mL aquades then
boiled with tap water (100oC), 1:10 (w/v)
on a hot plate for 60 minutes. The water
was drained off and the seed is dried in
oven at 65oC and cooled in a desiccator.
The seeds were dry milled, sieved and
packaged. Mungbean flouring process can
be seen in Picture 1.
Soaking (12 hours)
Boiling (60 minutes)
Dehulling
Oven drying
Boiling (60 minutes)
Milling
Oven drying
Sieving
Milling
Undehulled mungbean flour
Sieving
Dehulled mungbean flour
Picture1. Flow Chart of The Mungbean

Flouring Process
Source: Blessing and Gregory (2010)
Cookies Making Process
Cookies making process use reference
from Fine Cooking Cookies (2011). The
formulation can be seen in Table 1.
Table1. Cookies Formula
Ingridients
Composition
Wheat Flour
Salt
Baking powder
Margarine
Sugar
Egg
Vanilla powder
Source: Agen (2011)
405 g
0.10 g
0.05 g
300 g
0.25 g
1 egg
0.3 g
Ingridients weighed use analytical scale.

The sugar and margarine mixed firtsly into
cream. After that add the egg and mix
September 1st 2015
Page 28
again. Add the dry ingridients into the

mixture of cream and eggs, and then mix
again until homogen. Cool the mixture in
refrigerator for 10 minutes (0-4oC). The
cooled dough is cut with cookie cutter and
baked for 9-10 minutes at 150-170oC. The
cookies making process can be seen in
Picture 2.
Sugar, Margarine
Creaming
Eggs
Flour, baking powder,
salt
The crude protein content was determined

using micro kjeldahl method described by
AOAC (1990); Kirk and Sawyer (1991).
The carbohydrate content was determined
using by difference method described by
Onwuka (2005). In this method
carbohydrate content determined by the
other fraction. The energy value was
calculated using Atware factor [(9 x fat) +
(4 x carbohydrate) + (4 x protein)] that
describe by Osborne and Voogt (1978);
Eneche (1991); Chinma and Igyor (2007)
and Nwabueze (2007).
Mixing
RESULT AND DISCUSSION
Mixing
Physical Characteristic Analysis

1. Dough
Cookies dough is a stiff dough that doesnt
need much water inside. The ratio of
water:flour in the cookies making is
approximately 1:6. The most important
component in cookies dough structure is
protein in the wheat flour. That protein are
gliadin and glutenin. Both of them will
create gluten when they meet water.
Gluten matrix looks like three dimensional
nets, strong, and has a viscoelastic
structure.
Refrigerating (10 minutes) at 0-4oC
Cutiing
Baking (150-170oC, 9-10 minutes)
Cookies
Picture 2. Flow Chart of the Cookies

Making
Source: Agen (2011)
Analysis Method
Analysis method use reference from
Blessing (2010). Chemical analysis for
moisture content, ash content, and crude
fat were done in triplicates using the air
oven, dry ashing, and soxhlet extraction
method described by AOAC (1990);
Nielson (2002); Onwuka (2005).
The using of mungbean flour as a

susbtitution of wheat flour predictally will
effect the characteristic of cookies dough.
Dehulled mungbean flour has a soluble
fiber such as pectin and some
hemicelulose fraction. Acoording to
Aykroyd et al. (1982),
pectin and
hemicelulose content in mungbean is
1,5%.
Fiber has the ability to absorb water faster
than protein and starch because fiber has

September 1st 2015
Page 29
more hydrophil group. When soluble fiber

meet water, it will absorb water and
hidrating. Each component of soluble fiber
which hidrated will interact one to another
and create a gel that trapped water inside.
The water that trapped inside the gel will
be hard to release. Fiber in the dough will
absorb the water to create gluten, it makes
gluten hard to create, so that the number of
gluten will decrease.
Undehulled mungbean flour has higher
fiber content than dehulled mungbean
flour. The mungbean hull contain more
unsoluble fiber such as celulose,
hemicelulose, and lignin. According to
Aykroyd et al. (1982), mungbean has 4,6%
celulose content, 2,2% lignin content, and
approximately
7,0%
of
unsoluble
hemicelulose. The higher content of fiber
in the dough the less gluten will created.
The other component in mungbean are
starch and protein. Carbohydrate and
protein component are found more in
cotyledon. The embrio of mungbean also
contain protein. Protein and starch in
mungbean will trapped inside the gluten in
mixing process. Protein from mungbean
cant create gluten because it isnt contain
gliadin and glutenin. That protein in the
mungbean has the ability to absorb water
too. The protein content can decrease the
water that use to create gluten, so that it
can decrease the number of gluten in the
dough. The mungbean starch component
cant create and strengthen the gluten
matrix in the dough.
2. Spread factor cookies
Cookies dough will expand and become
bigger while baking, but the expansion
isnt as big as the expansion of bread.

Fiber component in mungbean will
decrease the number of gluten. Gluten
ability to hold the gas expansion that
happen in baking process will decrease, it
makes cookies harder to expand.
The other component that help to create
the cookies structure is starch. Starch in
cookies dough contain wheat flour starch
and
mungbean starch. The high
temperature during baking will make the
starchs granule expand. Mungbean also
has a protein content. That protein cant
create gluten, but mungbeans protein can
help to create cookies structure. That
protein is in the gluten matrix and
coagulated during the baking time. The
protein that coagulated will help create
cookies structure.
Eventhough it can help to create cookies
structure, starch and protein of mungbean
cant fix or strengthen the gluten matrix in
holding the gas expansion that happen in
the dough or during the baking time. The
addition of mungbean will decrease the
use of wheat flour in the dough.
Mungbeans starch and protein also absorb
water that needed to create gluten. It
decrease the number of gluten and dough
viscoelasticity.
The wheat flour substitution with
dehulluded or undehulled mungbean flour
will effect the physical characteristics of
cookies. Both of mungbean flour will give
the different effect to the diameter,
thickness, and spread factor of the cookies.
The subtitution effect of the cookies
physical characteristic can be seen in
Table 2.

September 1st 2015
Page 30
Table 2. The Effects of Wheat Flour

Substitution with Mungbean Flour to
Cookies Physical Characteristics
Wheat Flour:Mungbean
Flour
Characteristics
100:0
85:15
Diameter (cm)
25.15
25.70
Thickness
5.50
5.75
(mm)
42.99
43.04
Spread Factor
Source: Pasha, et al. (2011)
According to table 2. Is known that the
spread factor of mungbean cookies is
bigger than 100% wheat flour cookies. The
gas expansion in cookies dough will
happen during the baking. The ability of
dough matrix to hold the gas expansion
will effect the spread factor of the final
product. When it baked, it will spread
wider firstly until it reach the maximum
diameter, then it will get thicker. The
setting process in cookies hasnt happen
when it reach the maximum diameter. The
dough will expand thicker, then the protein
will denaturated and the starch will
gelatenize thats called the setting process.
The 100% wheat flour cookies will have
the more elastic and extensible matrix
dough, so that the viscoelasticity will raise.
That thing cause the dough matrix can
hold the gas expansion during baking. That
dough can maintain the shape, so the
expansion of the dough will happen
equivalently. The substitution of wheat
flour and mungbean flour can increase the
number of the component in the dough,
but it will create less gluten. It decrease the
elasticity and extensibilty of the matrix
dough. Dough viscoelasticity will decrease
and make it cant hold the gas expansion
during baking and it break the matrix and

make the gas released. That make the
dough collapse and cookies will be
thinner. The dough ability to maintain it
shape will decrease so it will be wider.
Wheat flour substitution undehulled
mungbean flour predicted will create the
bigger spread factor than the substitution
of dehulled mungbean flour. The higher
fiber content in undheulled mungeban
flour can decrease viscoelasticity. That
will make the ability to maintain the shape
and to hold the gas expansion of the dough
will decrease.
Chemical Analysis
The wheat flour substitution with
mungbean flour can results cookies that
has better nutrition content such as higher
protein content, mineral, and dietary fiber.
Undehulled and dehulled mungbean flour
have a differrent chemical composition.
Chemical composition in dehulled and
undehulled flour can be seen in Table3.
Table 3. Chemical Composition of
Dehulled and Undehulled Mungbean Flour
Undehulle
Dehulled
Compositio
d
Mungbean
n
Mungbean
Flour
Flour
Moisture
10.322.8 10.642.0
Content
7
8
(%)
19.10.30 22.770.0
Crude
4
Protein (%) 1.200.03
Crude Fat
1.800.00 1.150.02
(%)
4.050.05
Ash (%)
0.180.30
Crude Fiber 62.922.7
(%)
4
3.700.00

September 1st 2015
Page 31
Carbohydra
te (%)
Calori
(kkal/g)
341.120.
00
61.602.0
9
347.830.
00
Number above show the averageSD

triplicate
Source: Blessing and Gregory (2010)
The using of those two kinds of mungbean
flour in cookies making process predicted
will results cookies with different chemical
composition. Cookies from substitution of
undehulled mungbean flour predicted will
has higher fat content, minerals, dietary
fiber, and carbohydrate. Dehulling process
will decrease the fiber content in
mungbean flour so the cookies will has
lower fiber content.
Fat content in dehulled mungbean cookies
is lower because dehulling process make
the embrio which has fat content wasted.
Mineral content also become lower
because mungbeans mineral are usually
found in the hull, otherwise in cotiledon is
dominated by protein and starch. The
using of dehulled mungbean flour will also
decrease carbohydrate content in cookies.
Fiber in mungbean hull is also a
carbohydrate component, so dehulling
process will decrease the carbohydrate
content. Dehulling process before milling
usually make some percent cotiledon
wasted and cause the decrease of
carbohydrate content in mungbean flour.
The using of undehulled mungbean flour
predicted will result cookies with lower
moisture content. Water that bound by
unsoluble fiber component is count as
weak bounded water, so there are many

water was evaporate during baking.
Soluble fiber can absorb water stronger
through creating a gel which can trapped
water inside. That gel cause the water in
the dough harder to release even when the
dough is heated, so the moisture content
become higher.
Protein content in dehulled mungbean
cookies predict will be higher because the
protein usually found in cotiledon.
Undehulled mungbean cookies has lower
calori because it has the higher dietary
fiber than the dehulled mungbean cookies.
Fiber cant be digest by the body, so the
consumption cant give more energy to the
body.
Sensory Analysis
The good quality cookies is a crispy
cookies, easy to bite, and has golden
brown appearence. Crispiness in cookies
determined by the size of the small and
uniform pores, also spread equivalently
among the part of cookies. The golden
brown color of cookies is caused by
maillard and caramelized reaction during
baking.
Mungbean cookies predict will has darker
color than 100% wheat flour cookies. The
browning in cookies happen because
theres a reaction between carbonyl group
from the sugar and free amino group from
protein that result melanoidin pigment
which give brown color. The addition of
mungbean flour will increase protein
content in the dough. Mungbean also
contain sugar such as glucose, so when it
baked it will reacted with free amino group
from wheat flours protein.

September 1st 2015
Page 32
Dehulled mungbean cookies predict will

has different color from the undehulled
mungbean cookies. Protein content in
undehulled mungbean flour is lower than
dehulled mungbean flour. It decrease the
number of free amino group in the dough,
so the intensity of brown color will
decrease.
Mungbean cookies predicted will have
lower crispiness than the 100% wheat
flour cookies. Mungbean contain fiber that
can decrease the number of gluten, so it
cant hold the gas expansion during
baking. It cause gas trapping of gluten
cant be optimum, so the pores size
become not uniform and the spread not
equivalent, so it will result cookies with
harder texture and hard to bite.
Undehulled cookies predict harder and less
crisp than dehulled mungbean cookies.
The use of mungbean cookies increase the
number of fiber in the dough, so it will
decrease the quality and quantity of gluten
in the dough and it will cause the
crispiness of cookies becomes lower.
CONCLUSION
The addition of mungbean flour will
decrease the viscoelastisity of cookies
dough. The substitution of undehulled
mungbean flour will result cookies with
higher fat, mineral, fiber, and carbohydrate
content, bigger spread factor, darker color,
and harder texture than dehulled
mungbean cookies. The wheat flour
substitution with dehulled mungbean flour
will result cookies with higher moisture
content, protein, and calori than
undehulled mungbean cookies.
GRATITUDE
Thanks to the Faculty of Agricultural

Technology, Widya Mandala Surabaya
Catholic University and Ms. Anita Maya
Sutedja, S.TP, M.Si as an advisor who
help us during the making of this paper.
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September 1st 2015
Page 35
THE APPLICATION OF CRUDE PALM OIL AS A REPLACEMENT FOR

WHITE MARGARINE IN MAKING SOURDOUGH BREAD
Jonathan Alvin 1) Lindayani2)Laksmi Hartayanie2)
1)
Student of Agricultural Faculty, Department of Food Technology, Soegijapranata Catholic

University, Semarang, Indonesia
2) Lecturer of Agricultural Faculty, Department of Food Technology, Soegijapranata Catholic
University, Semarang, Indonesia
ABSTRACT
In general, Bread has high content of carbohydrate but lack of micronutrient such as vitamin.
Therefore, bread can be used to become healthy food with crude palm oil for replacement of
white margarine as a shortening. One type of bread that can be used i.e. sourdough bread.
Shortening is one of important ingredient to make sourdough bread such as white margarine.
One of alternatives to replaced white margarine is crude palm oil. The objectives of this
research is to know the application of crude palm oil as source vitamin A, vitamin E and
antioxidant for replacement of white margarine in making sourdough bread also, to know the
best formula of sourdough bread with different CPO level that can be accepted by consumer.
The formulation between CPO and white margarine were 0:4(F4), 3:1(F3), 2:2(F2), 1:3 (F1),
and 0:4 (Control). There were sensory analysis, physical analysis, chemical analysis during
storage and total plate count. Physical analysis consists of color measurement, porosity,
volume expansion, pore diameters, textures analysis. Chemical analysis during storage
consists of vitamin E, vitamin A, and antioxidant activity. Four treatments and control were
analyzed by hedonic sensory using 30 untrained panelists with parameters i.e. taste, aroma,
colour, texture, and overall. The three preferred product and control were analyzed for
physical analysis, chemical analysis and total plate count. For physical analysis there were no
significant different for all parameters except color measurement. F4 treatment had the lowest
value of lightness around 63.880.64. For chemical analysis for first day storage, F4
formulation has the highest value of antioxidant activity (32.490.61%), vitamin
E(47.140.74 mg/kg), and vitamin A (31782.72841.80). At the last day of storage, F4 still
had the highest value of antioxidant activity (20.790.65%), vitamin E (32.210.81mg/kg),
and vitamin A (15431.25550.30 IU).In total plate count, 3 formulations and control had no
significant difference during storage.
Keywords : sourdough bread, crude palm oil, vitamin A, vitamin E and Antioxidant

September 1st 2015
Page 36
INTRODUCTION
Today, bread has becomes a trend and
popular food in Indonesia because it is
availability and cheap. Bread has high
content of carbohydrate but lack of
micronutrient such as vitamin. Therefore,
bread can be used to become healthy food
with crude palm oil for replacement of
white margarine as a shortening. Crude
palm oil has high content of vitamin A and
vitamin E so it can overcome nutrition
problem like vitamin deficiency especially
vitamin A. One type of bread that can be
used i.e. sourdough bread. The advantages
of sourdough bread are has long shelf life,
improve aroma, taste and flavor.
to help vitamin A deficiency in Indonesia.

Beside pro-vitamin A content, CPO also
contain high of vitamin E (600-1000 ppm).
Two type of vitamin E on CPO are
tocopherol and tocotrienol.
CPO is widely used as a raw material to
make margarine and cooking oil. CPO
rarely applied for food product because
affected flavor and taste of the food
although can improve the nutrient.
Therefore, research is needed for
application crude palm oil for white
margarine substitution on sourdough
bread.
2. MATERIALS AND METHOD
Shortening is important to form and soften

the texture of the bread. Usually, white
margarine is used for bread making. White
margarine contained 80% of water, 17% of
fat and others compound such as vitamin
and flavor. Fatty acid contents of white
margarine are miristic acid, palmatic acid,
linoleic acid and oleic acid. The
composition of fatty acid on crude palm oil
has similar content with white margarine
so that crude palm oil can replace white
margarine as shortening in bread making.
Crude palm oil (CPO) contains the highest
carotenoid among the other food product.
CPO is extracted from palm fruit which
has a high content of pro-vitamin A,
vitamin E, fiber, saturated fatty acid and
unsaturated fatty acid (Ketaren, 2005).
CPO contains 500700 ppm. -carotene
(500-700 ppm) which is can be a natural
colorant in food application. Based on provitamin A content, CPO can be alternative
2.1. Materials
The materials used in this study were
crude palm oil (CPO) from west borneo,
DPPH solution, FeCl3, 2,2 bipiridin,
acetone, hexane, ethanol, petroleum ether,
toluene, and Potato Dextrose Agar
(PDA),white margarine, baker yeast, bread
improver, wheat flour with high protein
(cakra kembar), water, sugar, salt, full
cream milk powder, standard solution of
beta carotene,
and alpha tocopherol,
Na2SO4 anhydrous.
2.3. Methods
2.3.1. Preliminary Study
2.3.1.1. Making Sourdough Bread
a. Making ofSourdough
All the ingredients were mixed well.
Then, the dough was stored in room
temperature for 12 hours. The
formulation used to make sourdough as

September 1st 2015
Page 37
a material to produce sourdough bread

can be seen at Table 1.
three hours. Last step, It was baked in

2000C for 15-25 minutes.
Table 1. Formulation of ingredient used

for sourdough
2.3.1.2.Substitution of Sourdough Bread

with Crude Palm Oil (CPO)
Sourdough bread was substituted by
margarine with crude palm oil. The
formulation between margarine and CPO
to make the sourdough bread can be seen
at Table 3.
Ingredients
Wheat flour (g)
Sugar (g)
Yeast (g)
Water (ml)
Weight
50
1
2
50
b. Making of Sourdough Bread

The formulation used to make
sourdough bread can be seen at Table 2.
Table 2. Formulation of Sourdough
Bread
Ingredients
Wheat
Flour
(Cakra
Kembar)
Sourdough
Water
Sugar
Salt
Full cream milk powder
White Margarine
Bread improver
Weight
475 g
50 g
225 ml
25 g
7.5 g
15 g
40 g
1.5 g
All the ingredientswerepoured and mixed

with mixer, except the white margarine.
White margarine was added when all
ingredients formed dough. The mixing
process was continued until the dough
became viscous and elastic. The dough
was kneaded to remove the gas and rested
for 30 minutes. The dough was divided
into 50 gram/ each and shaped as ball.
After that, the dough is put in proofer for
Table 3. Formulation of CPO to substitute

white margarine in sourdough bread
Formulation
F Control
F1
F2
F3
F4
White
Margarin (g)
40
30
20
10
-
CPO (g)
10
20
30
40
The method and the other ingredients to

make sourdough bread can be seen in
2.3.1.
2.3.1.3. Sensory Analysis
Sourdough bread with different CPO
content was continued to the sensory
analysis. The sensory analysis was
conducted in Soegijapranata Catholic
University, Semarang. Hedonic ranking
test was used to know the acceptance level
of sourdough bread replaced with crude
palm oil. There were 35 untrained
panelists and 5 attributes for this sensory
test, i.e. colour, aroma, taste, texture and
overall
(Meilgaard,
1999).
As
threeformulation that most preferred by the
consumer would be used for physical,
chemical, and shelf life analysis.
15thNational Student Conference Integrating Innovative Food Product Development and Consumer
Preferences through Sensory EvaluationDepartment of Food Technology, Soegijapranata Catholic
University SemarangTuesday, September 1st 2015
Page 38
Parameter score was 1 (most preferred), 2

(preferred), 3 (rather preferred), 4 (Less
preferred), 5 (Least preferred).
analysis (color measurement, volume

expansion, texture, and porosity), and the
shelf life analysis (Total Plate Count for
Molds)
2.4. Main Study

Based on pre-eliminary study of sensory
analysis found that panelist prefered
control, F2, F3 and F4 then F1. Therefore,
These resultswas used for the chemical
analysisduring storage (vitamin A, vitamin
E and antioxidant analysis), physical
Table 4. Sensory Analysis of Sourdough Bread
Parameter
Texture
Taste
Color
Aroma
Overall
Total
Control
4
3
2
3
3
15
F1
5
5
3
5
4
22
F2
3
1
1
1
1
7
3. RESULT AND DISCUSSION

3.1. Sensory Analysis
Sensory analysis for four samples (F1, F2,
F3 and F4) and control were done. The
result was shown in Table 4.
F3
2
2
4
2
5
15
F4
1
4
5
4
2
16
Key:
Control: sourdough bread 40g margarine

F1
: Sourdough bread with composition 30 g margarine and 10 g CPO
F2
F3
F4
: Sourdough bread with 40 g CPO
Parameter (Taste, Texture, Aroma and Color) :1 Most Preffered, 2 Preferred,3 Rather
Preferred, 4 Less Preferred,5 Least Preferred
Page 39
The level of consumer acceptance is

determinedby sensory method hedonic
ranking test (Meilgaard, 1992).. A food
which has high nutrient content, will not
be consumed by the consumer if the color
cannot be accepted. Based on the sensory
analysis results (Table 6), The most
preferred on color parameter sample was
F2 formula and F4 formula was the least
preferred sample. On the color analysis
result (Table 8) F4 has the most yellowish
color among the others sample. This result
indicated that panelist preferred the color
of sourdough bread in between control and
F2 formula. F4 formula was the least
preferred by the panelist because the color
was too yellow. Aroma and taste (sweet,
salty, sour, bitter, and umami) are an
important parameters for sourdough bread
acceptance because related with the
acceptance of food product (Winarno,
1984).Based on the sensory analysis
results (Table 6), The most preferred on

aroma parameter sample was F2 formula
and F1 formula was the least preferred
sample. Crude palm oil has a unique odor
because it contains fatty acid compounds,
phosphate, carotenoids that give specific
taste and aroma (Muchtadi, 1994). The
acceptance
of
texture
evaluation
isincreasing in line with the the addition of
crude palm oil as a shortening. Shortening
can make soft texture on the bread. In taste
and aroma parameters, there were
decreasing value of panelist evaluation in
line with the addition of crude palm oil.
This result was related with unique odor of
CPO. Based on sensory analysis (Table
6),The most preffered was F2 formula and
the most unpreffered was F1 formula. This
result show that substitution white
margarine with cpo can increasing
consumer acceptance.
3.2 Vitamin A, Vitamin E, and Antioxidant changes during storage

Vitamin A, Vitamin E, and Antioxidant changes of three sourdough bread formulas (F2, F3
and F4) and control during storage can be seen in Table 5.
Table 5. Vitamin A,Vitamin E, and Antioxidant changes During Storage
DayControl
Vitamin A (IU/100g)
0
8641.73546.11a
3
6087.55278.12a
5
3981.68133.61a
Vitamin E(mg/kg)
0
7.220.19a
3
5.110.41a
5
4.960.24a
Antioxidant activity (%)
0
11.990.79a
3
9.690.46a
5
8.160.18a
F2
F3
F4
13077.63565.45b 20362.65663.08c 31782.72841.80d

10464.47618.44b 13755.99103.69c 24185.07409.06d
7810.07454.05b 12564.44552.56c 15431.25550.30d
20.570.54b
12.410.23b
9.590.4b
24.420.68b
12.801.17b
11.130.17b
30.710.35c
26.90.55c
20.10.61c
47.140.74d
42.690.67d
32.210.81d
28.910.54c
17.410.29c
13.110.11c
32.490.61d
26.691.46d
20.790.65d
Page 40
Key :
F2
F3
F4
All values are meanstandard deviation
The value which has different superscript indicated that there is significant difference
with other Formulas in one row (p<0,05) based in one way ANOVA analysis
Table 5. shown that vitamin A content on
the sourdough bread was increasing in line
with the adding proportion of crude palm
oil. The highest beta carotene contained on
F4 as much 31782.72841.8 (IU). and
control as much 8641.737546.11(IU).
Salunkhe et al (1992) reported that, Crude
Palm Oil is one of the carotenoid source.
Beta carotene is antioxidant that has
functionality to reduce free radical, act as
an anticancer agent and stimulates bodys
immune defense mechanism. Dutta et al
(2005) added that carotenoid is an
effective
antioxidant
which
has
functionality to reduce free radical with
give hydrogen bond and as an oxygen
quencher.
Based on vitamin E analysis (Table 5), F4
formula has the highest content of vitamin
E compared the other formulaas much
47.140.74mg/kg. Vitamin E content on
sourdough bread will be higher in line with
crude palm oil addition. This result related
with vitamin E content on crude palm oil
has high contain of vitamin E especially
alpha
tocopherol.Vitamin
E
has
antioxidant activity with give hydrogen
group to lipid radical peroxyl. According
to Salunkhe (1992), The function of
tocopherol is to reduce cellular damage
from free radicals.
According to antioxidant analysis result

(Table 5), F4 formula has the highest
antioxidan activity as much 32.490.61%
and control has the lowest content as
much11.990.79%. High antioxdidant
activity on F4 formula related with beta
carotene and alpa tocopherol as an
antioxidant. Antioxidant can act as a free
radical binder so can protect body from
cancer sickness.
Vitamin A, Vitamin E and Antioxidant
activity was decreasing during five days of
storage in room temperature. Vitamin A
content on F4 formula as much 15431.25
IU and control as much 3981.688 IU at the
day-5. This result related with-carotene
contain during storage. According to
Andarwulan & Koswara (1992), carotene can be oxidiezed to mutachrom
that has no antioxidant activity. On the
day-5 of storage, Vitamin E content on F4
formula as much 32.21 mg/kg and control
as much 4.96 mg/kg. This decreasing is
caused by stability of vitamin E during
storage is easily damaged by oxygen
(oxidation) and light. Antioxidant activity
also was decreased during five day of
storage. On day-5 of storage, antioxidant
activity on F4 formula was as much
20.79% and as much 8.16%. This result is
caused by decreasing of vitamin A and
Vitamin E which has antioxidant activity.
Pokorny et al (2001) reported that,
Page 41
antioxidant activity is affected by fat

composition, antioxidant concentration,
temperature, air pressure, oxygen, and the
other food compound like protein and
water.
Almetsier (2002) reported, recommended
dietary allowances for vitamin A for man
and woman is 7000 IU per day. Based on
average of sourdough consumption (50g
per day), then with consuming F2 formula
of sourdough can fulfill 82% of
recommended dietary allowances of
vitamin A. If consuming F3 formula of
sourdough
can
fulfill
145%
recommended dietary allowances
vitamin A. With consuming F4 formula
sourdough
can
fulfill
227%
recommended dietary allowances
vitamin A
of
of
of
of
of
3.3. Physical Analysis of Sourdough

Bread
Physical analysis results of sourdough
bread with different heat moisture
treatment compared with control are
shown in Tables 6 and 7.
Table 6. Volume Expansion, Total Pores, and Pores Diameter of Three Sourdough
(F2,F3 and F4) Bread Formula and Control
Formula
Volume expansion(%)
Total Pores
Pores diameter
Control
225.9110.82a
1142.13a
0.050.01a
F2
226.5216.10a
1131.56a
0.040.02a
F3
224.7417.53a
1111.43a
0.050.02a
F4
226.1018.51a
1152.32a
0.040.01a
Key :
F2
F3
F4
The value which has different superscript indicated that there is significant
differencewith other Formulas in one row (p<0,05) based in one way ANOVA analysis
Table 7. Color Analysis of Sourdough Bread with Different CPO Content
Formula
Control
F2
F3
F4
L*
75.225 0.89a
68.03 1.32b
66.650.42c
63.880.64d
a*
-1.5970.05
-3.770.33
-3.3850.12
-2.430.64
b*
14.821.33a
33.481,33b
35.9152.12c
39.4730.76d
Key :
F2
F3
F4
Page 42

differencewith other Formulas in one column (p<0,05) based in one way ANOVA
analysis
If L* (Lightness) = 0-100 (dark to light)
If a* is positive, the sample is reddish, but if a* is negative the sample is greenish.
If b* is positive, the sample is yellowish, but if b* is negative the sample is bluish
Table 6 shown that expansion volume
value from control to F4 formula has not
significantly different. This is related with
using of crude palm oil as substitution of
white margarine doesnt affect the volume
expansion of the bread. The function of
crude palm oil was as a softener and
texture formation. Crude palm oil
substitution with white margarine has
function shortening as a softener and help
texture
formation.
Accordingto
Matz(1992),showed the function of crude
palm oil as shortening is to lubricate the
internal structure from dough so the
texture will be softer.
In porosity analysis of sourdough bread
(Table 6), total pores and diameter on each
formuladidnt have significant difference.
This result related with using same type
proportion of wheat flour with high protein
for each formula. According to
Matz(1992), Pores total on sourdough
bread is resulted from CO2 that is trapped
when fermentation process. The uniform
size of pores diameter is caused by gas
retention ability from gluten. (Matz, 1992)
In Color measurement analysis is

determined from L*,a*, and b* (Table 7).
In Lightness (L*) measurement, L* value
is decreased with the increasing of crude
palm oil addition. This decreasing is
caused beta carotene contain on crude
palm oil. According to Salunkheet
al(1992), Crude palm oil has beta carotene
as much 500-1000ppm so it will affect the
lightness value for F2 to F4 formula.The
increasing of b* value is in line with the
adding of crude palm oil. Value of b*
positive will give yellow color. This matter
accordance with Hendry & Houghton
(1996)reported, that showed beta carotene
is one of natural coloring that have
yellowish to reddish as a basic color.
3.4. Hardness Changes During Storage
And
Texture analysis (hardness) results ofthree
sourdough bread formula (F2, F3 and F4)
and controlduring storage can be seen in
Table 8.
Table 8. Texture Analysis During Storage

Day
0
3
Hardness (gf)
Control
F2
a
603.753.15
553.41 3.84b
896.34 4.43a 850.73 3.08b
F3
499.273.58c
792.204.14c
F4
450.213.71d
749.49 2.73d
Page 43
1247.61 3.85a 1180.85 3.13b
1100.17 4.26c 1001.59 3.76d
Key :
F2
F3
F4
Based on thetexture analysis results
(Table10), the lowest hardness value was
contained on F4 formula as much
450.21gf. Hardness value is decreased in
line with the increasing proportion
substitute of crude palm oil. This result
related with function of crude palm oil as a
shortening. Fat content on crude palm oil
is higher than white margarine. According
to Potter & Hotchkiss(1996), The function
of shortening is giving soften effect and
lubricate bread structure.
During storage, hardness value for each
formula of sourdough bread was
increasing. Rosenthal (1999) reported,
texture of the bread will become harder
and elasticity of bread also decreasing

during storage. According to Hoseney
(1994), The increasing of hardness value is
caused
by
starch
retrogradation.
Retrogradation is a recrystallization
process of starch granule which has
through gelatinization process. During
storage, retrogradation process is shown
with water binding lost which is water is
contained on crystal starch will be moved
from gluten to starch.
3.5. Total Plate Count
Total
Plate
Count
results
of
threesourdough bread formula (F2, F3 and
F4) and control substituted are shownin
Table 9.
Table 9. Total Plate Count Analysis

Day
Formula (CFU/ml)
Control
F2
0
0
a
1.880.06
1.820.06a
7.530.22a
7.730.31a
F3
F4
0
0
0
a
3
1.790.05
1.830.07a
5
7.860.35a
7.640.42a
Key :
F2
F3
F4
Page 44
Mold usually grows in bread during

storage which is physically can be seen
from the color of the bread become green
or black. On TPC analysis for five days
storage (Table 11), each formula was
contained mold growth. In the first day of
storage each formula, there is no mold
growth. In the third day to fifth day there
were increasing on mold growth. Usually
mold growth is caused by improper
storage and availability of nutrient need
for molds growth.In low humidity
condition can inhibit mold growth on the
bread, otherwise in high humidity
condition can accelerate mold growth.
Mold can grow on food product at
moisture content around 14 - 15%,
minimum Aw 0.8, and 25-30oC (Frazier &
Westhoff, 1988). In accordance toFardiaz
(1992), the other factor for mold growth
was the availability of oxygen, food
composition, packaging condition and
optimum temperature for mold growth
which is 25-30oC.Sourdough bread can be
keep in storage at room temperature for 6
days. According to SNI 01-2840-1995, the
maximum total plate count that permitted
is 106. On the last day of storage even
though there is mold growth but
sourdough bread is still saves to consume.
4. CONLUSION
Formulation F2 can be accepted by
panelist in overall parameter, also has
the high content of vitamin A as
much13077.63565.45IU, vitamin E
as much20.570.54 mg/kg and
Antioxidant
activity
as
much
24.420.68% during storage.
The application of crude palm oil can

increase vitamin A, vitamin E and
Antioxidant content on sourdough
bread.
Crude palm oil has good potential to
replacewhite margarine in making
sourdough bread.
Vitamin A, vitamin E, antioxidant
activity, and hardness was decreasing
during five days storage at room
temperature. F4 formula has the
highest content of vitamin A
5. REFERENCES
Prasetya, A.P.E.; I.U. Pri.;H. Dwi. (2009).
Pengaruh pemanasan terhadap
kadar vitamin E pada kacang hijau
(Vigna radiata L.) dengan metode
spektrofotometri sinar tampak.
Pharmacy Vol6 : 1-9.
Meilgaard, M., G.V. Civille, and B.T.
Carr. (1999). Sensory Evaluation
Techniques. CRC Press. New
York.
Matz,S.A. (1992). Bakery, Technology
and Engineering, 3rd ed. Van
Nostrand Reindhold. Texas.
Aida, W.M. Wan, W.Y. Kam, A.M.
Sahilah, and M.Y. Maskat. (2011).
Volatile Compounds and Lactic
Acid Bacteria in Spontaneous
Fermented
Sourdough.
Sains
MalaysianaVol.40 (2): 135 138.
Saeed, M., F.M. Anjum, Tahir, Z., Haq N.,
and Sajjad, R. (2009). Isolation and
Page 45
Characterization of Starter Culture

from Spontaneous Fermentation
Sourdough. International Journal
of Agriculture & BiologyVol. 11:
329 332.
Rollan, G., C.L. Gerez, A.M. Dallagnol,
M.I. Torino, and G.Font. (2010).
Update in Bread Fermentation by
Lactic Acid Bacteria. In: MendezVilas A (ed) Current Research,
Technology and Education Topics
in Applied Microbiology and
Biotechnology, 1st edn.Formatex,
Spain
Semic, A., S. Orucevic, I. Bauman, S.
Muminovic, N. Spaho, and B.
Klepo.
(2009).
Effects
of
Increasing Sourness of Bread
Dough on Bread Quality. 5th
International Congress FLOURBREAD 09 & 7th Croatian
Congress of Cereal Technologists :
416 424.
Salunkhe, D.K., J.K. Chavan, R.N.

Adsule,and S.S. Kadom. (1992).World
Oilseeds. Van Nostrand Reinhold. New
York
Potter, N. N. and J. H. Hotchkiss. (1987).
Food Science, 3rd ed. CBS Publishers and
Distributiors. New Delhi
Pokorny, J, N. Yanishlieva, and M.
Gordon. (2001). Antioxidants in Food :
Pratical
Appliactions.
Woodhead
Publishing Limited. Cambridge. Finland
Matz,S.A. (1992). Bakery, Technology
and Engineering, 3rd ed. Van
Nostrand Reindhold. Texas.
Lay, B. W. (1994). Analisis Mikroba di
Laboratorium. PT Raja Grafindo
Persada. Jakarta.
Hendry, G. A. F. and Houghton, J. D.
(1996). Natural Food Colorants.
Blackie Academia and Profesional.
USA.
Standar Nasional Indonesia. (1995). Roti

SNI
01-3840-1995.
Badan
Standarisasi Nasional.
Britton, G., S. Liaaen-Jensen, and H.
Pfander.
(1995).
Carotenoids
Volume 1A: Isolation and Analysis.
Library of Congress. USA.
Dutta, D, U. R. Chaudhuri, and R.
Chakraborty.
(2005).Structure,
Health
Benefits,
Antioxidant
Property and Processing and
Storage of Carotenoids. African
Journal Biotechnology Vol. 4 (13)
: 1510-1520.
Page 46
THE STUDY OF HERB LIQUEURS PRODUCTION

1)
2)
Hana Melinda Lindayani Churdchai Cheowtirakul
3)
1) Students; Food Technology Department; Faculty of Agriculture; Soegijapranata Catholic

University
2) Lecturer; Food Technology Department; Faculty of Agriculture; Soegijapranata Catholic
University
3) Lecturer; Food Technology Department; Faculty of Biotechnology; Assumption University
ABSTRACT
Thai food and beverage is currently attracting considerable interest because their ability in blend of
flavors, aromas and it health benefits. Herb spirit that made from extracts of aromatic and medicinal
herbs has good effects on human especially for human metabolism. Using Tom Yam (one of Thai
cuisine) as a raw material for developing a new product of Herb liqueur would add a great value both
for the Tom Yam and also for Herb liqueur. On the other hand, because of Tom Yam consist of
several kind of Herb ingredient, liqueur will also be made from the ingredient of Tom Yam.
Lemongrass and lemon (Citrus) are some of the basic ingredients for making Tom Yam. From those
three liqueurs (tom yam, lemongrass and lemon liqueur), the preference of some attributes of the
liqueur will be performed. Panelists (n=30) were specified, where they can drink alcohol beverages.
Panelist had to evaluate and express their liking in the questioner to shows their preference of some
sensory attributes (color, turbidity, aroma, sweetness, taste, after taste, and overall) using hedonic
rating method. The data was analyzed with One-Way ANOVAs. The result shows that all of the
liqueurs attribute were significantly different (p<0.05). The highest total preference is in the basic
ingredient of tom yam, lemon. The data shows for tom yam liqueur and lemongrass liqueur panelists
like the color and the turbidity of the liqueur, and for lemon liqueur panelist shows their liking in the
attributes of aroma, sweetness, taste, aftertaste, and overall. From this research can be conclude that
the application of herbs in liqueurs beverage are acceptable. However, panelists did not show their
high preference on the application of tom yam as a Thai cuisine in liqueur.
Key word: tom yam, liqueur, herb, sensory, hedonic rating
Page 47
INTRODUCTION
Herb spirit that made from extracts of

aromatic and medicinal herbs has good
effects on human especially for human
metabolism. Herb liqueurs are a spirits which
have a bitter taste. It made by dilute a basic
spirit containing ethyl alcohol with minimum
15 % volume of alcohol, sugar syrup, fruit
juices and herb extracts. Herb liqueurs are
part of traditional gastronomy in the
southeast Europe (Karabegovia et al.,
2012).
Nowadays, Thai restaurants has enlarging
their area for serving Thai food. Even though
Thai food has just begun to reach the global
market, but it has rapidly gained to be one of
international favorite food. (Sunanta, 2005).
Thai food and beverage is currently attracting
considerable interest because their ability in
blend of flavors, aromas and health benefits.
Thai vegetables especially Thai herbs are
commonly used for flavoring agent or
condiment. Antioxidant properties of Thai
herbs make Thai foods and beverages are not
only rich in plant species which contain a
variety of phytochemicals, but they also
provided nutrient (Tangkanakul et al., 2009).
Using Tom Yam (one of Thai cuisine) as a
raw material for developing a new product of
Herb liqueur would add a great value both for
the Tom Yam and also for Herb liqueur.
Besides, Tom Yam as a Herb material and
liqueur it self have been proven their
medicinal properties and their advantages for
human health. On the other hand, because of
Tom Yam consist of several kind of Herb
ingredient, liqueur will also be made from the
ingredient of Tom Yam.
Lemongrass and lemon (Citrus) are some of
the basic ingredients for making Tom Yam.
The reason of choosing Lemongrass and
Lemon as a raw material for make Herb
liqueur is because of they used to be a blend
component added to increase beverages flavor
(Vanisha & Hema, 2012; Rang el et al.,
2011). Moreover, in this research 95% white
spirit will be used as a basic spirit for making
liqueurs. White spirit containing 95% of
alcohol often used for industrial solvent,

white vinegar, medicines, surgical spirit and
food ingredients. If it will be use as a basic
material as a source of alcohol for beverages,
it has to be diluted with water to decrease the
alcohol contain. The reasons of using white
spirit as a basic spirit are because white spirit
has a plain taste and bland odor so it will not
affect and change the flavor that will come
out from the herb. White spirit is also have no
color (limpid solution), so the resulting color
of the liqueur is pure from the extract of herb
it self (Anonym, 2001).
From those three liqueurs (tom yam,
lemongrass and lemon liqueur), the
preference of some attributes of the liqueur
will be performed to determine the quality of
Herb liqueur that must be produced in the
future based on sensory evaluation. The
attributes of sensory are color, turbidity,
aroma, sweetness, taste, after taste, and
overall. Combination of color, taste and
aroma are used to evaluate the liqueurs
(alcoholic beverages) quality.
When liqueurs is taken into the mouth, odor
will firstly perceived by nose during normal
orthonasal olfaction. Some aroma chemicals
also interact with the trigeminal nerve endings
of the olfactory system to produce a sensory
perception that is a mixture of odor and
trigeminal stimulation. Therefore, flavor is
often a complex perception (Buglass, 2013).
Almost all liqueurs have a bitter taste with a
highly concentrated. Therefore, it has to be
sweetened with several sweetener such as
sugar (Murphy, 2013). Herbs essential oil is
one of the factor that affecting sensory
attribute in food (Chauhan et al., 2012). In
this study, the sweetness attribute will be
determine. The purpose of putting sweetness
as one of sensory attribute is to perceive
whether theres a change perception of
sweetness in the liqueurs that caused by herb
(where each liqueurs is added with the same
ratio of sugar). After that, when it prove
affecting the sweetness attribute, then which
herb is give the high acceptable in it roles on
Page 48
changing sweetness of liqueurs.
liqueur as a functional alcoholic beverage and

to develop herb in the field of food
especially
for
beverage.
The aim of this research are to introduce technology
Furthermore, this research is also
conducted to use Thai cuisine such as tom
yam as a popular cuisine from Thailand
and to use tom yam basic ingredient such
as lemon and lemongrass for making herb
liqueur. After liqueur was made, this
research will determine the preferred
between herb liqueur that made from
various kind of herb (tom yam liqueur)
and herb liqueur that only made from one
herb (lemon liqueur and lemongrass
liqueur), and also to evaluate the consumer
preference between these three herb
liqueur.
MATERIALS AND METHODS
Time and Place of Research: The

research is conducted in the Pilot Plan
Laboratory of Biotechnology Faculty,
Assumption University HuaMak Campus,
Bangkok, Thailand, and take place from
January 16th to March 25th 2015.
Materials: The Materials used for make
herb liqueurs are kaffir lime leafs,
lemongrass, galangal, lemon, white spirit
(95% of alcohol), and drinking water.
Apparatus: The Apparatus for make herb
liqueurs are a large glass jar, analytical
balance,
lemon
squeezer,
vacuum
filtration, refractometer, and alcohol meter
(hydrometer).
Liqueurs Making: Step for making
liqueur shown in diagram as in Figure 1.
a.
Tom Yam Liqueurs
The tom yam liqueur made from the
proportion of 28.1 g (3 pieces) of
lemongrass which has been crushed, 14 g
of kaffir lime leafs, 24.9 g of crushed
galangal and 1 lime that has been
squeezed were added into a large glass jar
that contain 1500 ml of a mix spirit (white
spirit and water). The glass jar sealed with
plastic and rubber. Then, incubated for 2
weeks. After 2 weeks, the solution was
filtered by vacuum filtration to clear the
liqueur up. After filtration, the clear
liqueur was added with 300 ml of sugar
syrup and the liqueur incubated for 3 days.
Then, the alcohol content must be
measured by alcohol meter before the
sensory evaluation.
b.
Lemongrass Liqueurs
Initially, the lemongrass liqueur made by

crushed 56.2 g (6 pieces) of lemongrass
and it added into the 1400 ml of basic
spirit in the jar. The glass jar covered with
plastic and tightened with rubber. Then,
incubated for 2 weeks. After that the
solution was filtered by vaccum filtration
to produce clear liqueur. After filtration,
the clear liqueur was added with 250 ml
Page 49
of sugar syrup. Liqueur incubated for 3

days. Before the sensory evaluation, the
measurement of alcohol content in liqueur
done by alcohol meter.
c.
Lemon Liqueurs
For make a lemon liqueur, 9 pieces of
lemon were squeezed to produce 300 ml
of lemon juice. The lemon juice then
added into 1500 ml of basic spirit in the
glass jar. The glass jar then sealed with
plastic and rubber and incubated for 2
weeks. After 2 weeks, the solution was
filtered by vacuum filtration to clear the
liqueur up. The clear liqueur then added
with 300 ml of sugar syrup. The liqueur
incubated for 3 days Then, the alcohol
content measured by alcohol meter before
the sensory evaluation was conducted.
Sensory Evaluation
Consumer Test: The test were conducted
in Assumption University, Hua Mak
Campus. Panelists (n=30) were specified,
where they can drink alcohol beverages.
The purpose is to obtained the valid data
of liqueurs (alcoholic beverage) sensory
from alcoholic beverages consumer.
Panelist had to evaluate and express their
liking to shows their preference of some
sensory attributes using hedonic rating
method. The samples of liqueur that were
placed in shot glass with a difference 3digit code were observed by the attributes
of color, turbidity, aroma, sweetness, taste,
after taste, and overall. The order of the
samples given was randomized so
consumer would have no idea what is the
first or second sample they evaluated, and
the bias could be minimized.
Statistic Tools and Analysis: The data
from this study were collected and
analyzed using SPSS version 20
(Statistical Package for the Social Science
for Windows, Version 20) to find out the
significance of differences between
samples in herb liqueurs. For Total
preference, the data was analyzed with
One-Way ANOVAs, and if there is a
significantly different, the data will be
analyzed by Duncan Model to assess each

attribute on every sample that significantly
different and find out how much the
difference is of herb liqueurs. Analysis
Performed on each attribute and total
preference that has been counted before.
Alcohol Content: Alcohol content is one

of important characteristic of liqueur
before starting the sensory evaluation. The
alcohol measurement conducted to make
sure the it is not too high to be consume so
it will not be hazard for panelist health.
Karabegovia et al. (2012) reported that
the alcohol content for liqueur is between
15% to 40%. For herb liqueurs that have
been made, the result of alcohol content
measurement shown in Table 1.
Table 1. Alcohol Content in Herb Liqueur
Temperature Scale Alcohol
Sample
(0C)
*
(%)
Tom Yam
26
39
36.6
Liqueur
Lemongrass
30
42
38.0
Liqueur
Lemon
29
38
34.4
Liqueur
* Scale information that read from hydrometer
can be seen on Appendices
Measurement result of alcohol content as

shown on Table 1. According to the
alcohol measurement, tom yam liqueur
contains 36.6% of alcohol, lemongrass
liqueur contains 38% alcohol and lemon
liqueur contains 34.4% alcohol. Reveal
that there is difference alcohol content
between sample bases on herb ingredient.
The highest content of alcohol is in
lemongrass liqueur and the lowest is
lemongrass liqueur.
Sensory Evaluation: The sensory test
was held using measurement of
acceptance
and
measurement
of
preference. The measurement of affective
used prefference hedonic rating test.
Preference test will give data about what
consumer prefer about three products.
Page 50
Panelists (n=30) were specified, where

they can drink alcohol beverages. The
purpose is to obtained the valid data of
liqueurs (alcoholic beverage) sensory
from alcoholic beverages consumer.
Panelist had to evaluate and express their
liking in the questioner to shows their
preference of some sensory attributes
using hedonic rating method (1= dislike
very much, 2= dislike slightly, 3= neither
like nor dislike, 4= like slightly, and 5=
like very much). The result shows that all
of the liqueurs attribute were significantly
different (p<0.05). The result of sensory
evaluation of herb liqueur shown in Table
2.
Table 2. Analysis of Herb Liqueur
on Hedonic Rating (Preference
Test)
Attribute
Color
Turbidity
Aroma
Sweetness
Taste
After Taste
Overall
TomYam
Liqueur
3.5a 1.0
3.4a 1.3
2.9b 0.9
2.7b 0.9
2.8b 1.0
2.9b 1.0
3.1b 1.0
Lemongrass
Liqueur
3,9a 1.1
3.6a 1.2
2.6b 0.9
2.7b 1.0
2.5b 1.0
2.6b 1.1
2.7b 0.9
Lemon
Liqueur
2.8b 1.3
2.5b 1.3
3.7a 1.2
3.6a 1.2
4.2a 0.8
3.9a 1.1
4.1a 0.6
*Same letter means no significant difference

for each sample in every attribute at 95%
confidential level. **The highest the value
means it has a high preference by consumer.
Based on Table 2, shows that both

tom yam and lemongrass liqueur
the result are for every attribute
having significantly different with
lemon liqueur. Tom yam and
lemongrass liqueur have a highest
preference in color and turbidity,
but for another attribute they have
lower preference then lemon
liqueur. Contrarily, for lemon
liqueur has a high preference in
every attribute except color and
turbidity.
Here is the result of herb liqueurs
product that have been bottled.
From this (figure 2), can shown the
comparison between the existing
sensory data.
a.
b.
c.
Figure 1. Product of Herb Liqueur; tom yam

liqueurs (a), lemongrass liqueurs
(b), and lemon liqueurs (c).
(Source: Personal documentation)
From Figure 2 shown that virtually

lemon liqueur is more turbid than tom
yam
and
lemongrass
liqueur.
Therefore, the result shows less
preference in color and turbidity of
lemon liqueur.
For the total preference, all the data was
calculated by Microsoft excel. Each
attribute has percentage to be calculated
by multiplied the data of hedonic scale
from panelist with the percentage of each
attribute. To make 100% of total
preference, it will be consist of 20% of
color, 10% of Turbidity, 10% of aroma,
10% of sweetness, 20% of taste, 10% of
after taste and 20% of overall. Color and
taste have a higher percentage (20%) than
other attribute because color and taste
play an important role to evaluate the
liqueurs (alcoholic beverages) quality
(Buglass, 2013). Overall also has a higher
percentage. It is because overall means
the accumulation of panelist acceptance
in every attribute. For the total preference
(counted by ms. excel) shown in Table 3.
Table 3. Analysis Total Preference of Herb
Liqueurs
Attribute Tom Lemongrass Lemon
Yam
61.8b 59.6b
72.7a
Total
Preference
*Same letter means no significant difference for
each sample in every attribute at 95% confidential
level.
Page 51
**The highest the value means it has a high

preference by consumer.
From Table 3. The result shows that with

the percentage counting on each attribute,
lemon liqueur receive the most
prefer/liking in this study compare with
tom yam liqueur and lemongrass liqueur.
Alcohol Content and Preference
Relation: The result in alcohol content
measurement shows that there is different
percentage of alcohol in every liqueurs.
The difference of alcohol percentage
might be the factor of the panelist
preference. To determine the relation
between alcohol content and the
preference result of the herb liqueurs, the
comparison between alcohol content and
consumer preference shown as follows.
Table 4. Analysis Between Preference
Result and Alcohol Content
Sample
Preference Alcohol
content
(%)
TomYam
61.8
36.6
Liqueur
Lemongrass
59.6
38.0
Liqueur
Lemon
72.7
34.4
Liqueur
As shown in the Table 4, above on this
study, there is inverse relation between
alcohol content and consumer preference.
The result shows that the lower the alcohol
content is the higher preference.
Otherwise, the higher the alcohol content
is the lower of preference. Lemongrass
liqueur has the lowest preference while it
has the highest alcohol content. On the
contrary, lemon liqueur has the highest
preference while it has the lowest alcohol
content.
CONCLUSION
From this research can be conclude that
the application of herbs in liqueurs
beverage are acceptable. However,
panelists did not show their high

preference on the application of tom yam
as a Thai cuisine in liqueur. Instead, the
highest total preference is in the basic
ingredient of tom yam, lemon. Generally
the data shows for tom yam liqueur and
lemongrass liqueur, panelists like the color
and the turbidity of the liqueur. But the
aroma, sweetness, taste, aftertaste, and
overall of both tom yam liqueur and
lemongrass
liqueur
have
a
low
acceptability. The result shows that lemon
liqueur which made only from one herb is
more acceptable than tom yam liqueur
which made from various kind of herb.
SUGGESTION
For the future work, research of other herb
liqueur should be done. Moreover, the
making of lemon liqueur with the color
and turbidity as well as both tom yam
liqueur and lemongrass liqueur can be
done. Also to find out how low of alcohol
percentage of liqueur that acceptable for
panelists.
REFERENCES
Anonym. (2001). The Manufacture Of

Ethanol
From
Whey.
http://nzic.org.nz/ChemProcesses
/dairy/3H.pdf.
Buglass A.
J.(2013).
Instrumental
Assessment of
the
Sensory
Quality of Wine. Korea Advanced
Institute
of
Science
and
Technology, Republic of Korea
and
D.J.
Caven-Quantrill,
Frutarom (UK) Ltd, UK.
Chauhan D. K., Vinita Puranik and G.
K.Rai.(2012).Development
of
Functional
Herb
RTS
Beverage.Open Access Scientific
Reports Vol 1 (12): 1-5.
Karabegovia I. T., Predrag V.
Vukosavljevib, Miroslav M.
Novakovic,
Stanislava
.
Gorjanovid, Ana M. D, and
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Miodrag L. L. (2012). Influence

of The Storage on Bioactive
Compounds
and
Sensory
Attributes of Herb Liqueur.
Digest Journal of Nanomaterials
and Biostructures Vol 7(4): 15871598.
Murphy, J. P. (2013) The Principles and
Practices of Bar and Beverage
Management
The
Drinks
Handbook.Goodfellows
Publishing Ltd, Oxford, England.
Sunanta S. (2005). The Globalization of
Thai Cuisine. University of
British
Columbia,
Toronto.
http://citeseerx.ist.psu.edu/viewd
oc/download?doi=10.1.1.404.193
4&rep=rep1&type=pdf
Tangkanakul,P.,
Auttaviboonkul,
P.,Niyomwit, B., Lowvitoon,
N.,Charoenthamawat,
P.
and Trakoontivakorn,
G.
(2009). Antioxidant Capacity,
Total Phenolic Content and
Nutritional Composition
of
Asian Foods After Thermal
Processing. International
Food
Research Journal
Vol 16: 571-580.
Vanisha S. Nambiar and Hema M.
(2012). Potential Functions of
Lemongrass
(Cymbopogon
citratus) in Health and Disease.
International
Journal
of
Pharmaceutical & Biological
Archives Vol 3(5):1035-1043.
Yusaku,
F. (2008). Tom
Yam
Khung.Department of Food
Science
and
Technology,
Kyushu University.Hakozaki,
Japan
Page 53
A HEALTHY MEATBALL : APPLICATION OF CARAGEENAN TOWARD

ALBUMIN MEATBALL AND ARROWROOT FLOUR TOWARD
BREADFRUIT BASED MEATBALL
Rency Gista Anindya1), Vania Eka Cahyani Adidharma1), Michael Ardian Iskandar1),
Lindayani2), Laksmie Hartayanie2)
1)Student
of Food Technology Department, Faculty of Agricultural Technology,

2)
Lecturer of Food Technology Department, Faculty of Agricultural Technology,
Email : rencygista@gmail.com
ABSTRACT
Meatball is a popular food, used to be served in various food vendors, from restaurant to
street vendors. Commonly, meatball is made from beef, chicken, and fish. Meatball can be
processed into various kind of food or be used as one ingredient in food, hence development
of meatball product without meat is worth evaluated. Potential material to be used as main
ingredient of meatball can divided into two kind, is animal (non-meat) like albumin and plant
like breadfruit. In meatball (non-meat) processing used albumin which is good source of
protein, containing essential amino acids, less expensive and easily obtained. Albumin
contains complete essential amino acid compared to another material such as fish, chicken,
tofu and tempe. Albumin meatball was made with addition of 0,25%, 0,5% and 1%
carrageenan. Carrageenan can bind water and can stabilize the system of an emulsion, so
carrageenan can affect an increase of viscosity, the formation of a gel, a deposition and
stabilization. The evaluation of albumin meatball including hardness testing, protein content,
and water content. In meat-like product processing, used breadfruit that has a meat-like
texture, contains high carbohydrate, low calorie and good dietary fiber. However, breadfruit
processing is poor, indicated by the number of breadfruit abandoned after harvest, so the
utilization of breadfruit as the main ingredient is worth to be evaluated. Another ingredient to
make meatball is tapioca/cassava flour, which can be added by arrowroot powder. Breadfruit
meatball were made with addition of arrowroot powder 10%, 30%, and 50% of total flour
used. Arrowroot powder is a good adhesive and also be known to improve digestion system.
The evaluation of breadfruit based meatball including hardness testing, crude fiber content,
and water content.
Keywords: Albumin, breadfruit, meatball, arrowroot flour, protein, crude fiber
Page 54
INTRODUCTION
Indonesia have a many variance of food
that popular in community, one of the
popular food is meatball. Meatball is a
popular food, used to be served in various
food vendors, from restaurant to street
vendors.Commonly, meatball is made
from beef, chicken, and fish. In the
nutritional value, meatball can meet the
nutritional needs of the community and
highly preferred (Widyaningsih & Murtini,
2007dalam Chernanda, 2008).
Meatball can be processed into various
kind of food or be used as one ingredient
in food, hence development of meatball
product is worth evaluated. One of
potential animal raw material (non-meat)
to be used as main ingredient of meatball
is albumin.Albumin is a good source of
protein, shaped like a gel, containing
essential amino acids, less expensive and
easily obtained. As animal protein,
albumin contains complete essential amino
acid compared to another material such as
fish, chicken, tofu and tempe.Protein
content on albumin are ovalbumin,
conalbumin, ovomucoid, ovoglobulins,
lysozyme,
ovomucin,
ovoinhibitor,
ovoglycoprotein,
flavoprotein,
ovomacroglobulin, ficin inhibitor, and
avidin. The function of ovalbumin as
gellingagent,
ovomucin,
andovoglycoprotein that can affecting
viscosity (Linden & Lorient, 2000).
Substitution the raw material that used in
making meatball will produce meatball
albumin are rich in protein and more
healthy because albumin meatball not
containing fat that can consumed by all of
age groups. This is in accordance with the
community who prefer food with low
cholesterol level (Abubakar et al., 2011).

Nutrient content of chicken egg in 100 g
material can be seen in Table 1.
Table1. Nutrient content of chicken egg
in 100 g material
Composition
Fresh Chicken Eggs

Whole
Yolk
Albumin
egg
12,8
16,3
10,8
11,5
31,9
0,0
Protein (g)
Fat (g)
Carbohydrate
0,7
(g)
Calsium
54,0
(mg)
Phosphorus
180,0
(mg)
Fe (mg)
2,7
Vitamin A
900,0
(SI)
Vitamin
B
0,1
(mg)
Source : Departemen
dalam Sarwono 1996.
0,7
0,8
147,0
6,0
586,0
17,0
7,2
0,2
2000,0 0,0
0,27
0,0
Kesehatan, 1981
The high water content in albumin should

be added food additives such as
carrageenan to form a structure albumin
meatball similar to meatball in general.
Carrageenan serves as a stabilizer,
thickener, gelling agent in food processing
(Winarno, 1996). To know the difference
then need to physical analysis of the
texture and alsochemical analysis of water
content and protein content.
Potential plant as raw material like
breadfruit has meat-like texture, bright
appearance, and good dietary fiber for
digestion system. Breadfruit is potential
raw material because have nutrition,
Page 55
vitamins, and minerals, also less

expensive. Lack of dietary fiber cause
healthy trouble like a cholestrol,
hypertension,
and
cardiovaskular
dissruption. Nutrient content of breadfruit
in 100 g unripe bradfruit, ripe breadfruit,
and breadfruit flour can be seen in Table 2.
Table2. Nutrient content of unripe
bradfruit,
ripe
breadfruit,
and
breadfruit flour in 100 g material
N
o
Composit Unripe
ion
Breadf
ruit
1 Energy
46
(kkal)
2 Water (g) 87,1
3 Protein
2,0
(g)
4 Fat (g)
0,7
5 Carbohy 9,2
drate (g)
6 Fiber (g) 2,2
7 Ash (g)
1,0
Source: Pitojo, 1992
Ripe
Breadf
ruit
108
Breadf
ruit
flour
302,4
69,3
1,3
15
3,6
0,3
28,2
0,8
78,9
0,9
The making process of breadfruit meatball

is using arrowroot flour too. Arrowroot
flour has a higher protein nutritional
content than tapioca flour, and it contents
water soluble fiber as much as 5,03%db
and water non-soluble fibre as much as
8,74%db (Marsono et al, 2005), The
advantages of using arrowroot flour is
having a high fiber content because of this,
arrowroot flour can strengthen digestion
system and it doesnt content purin. Purin
may cause uric acid.Nutrient content of
arrowroot flour in 100 g can be seen in
Table 3.
Table 3. Nutrient content of arrowroot

flour in 100 g
Composition
Energy (kkal)
355
Protein (g)
0,7
Fat (g)
0,2
Carbohydrate (g)
85,2
Calsium (mg)
8
Phospor (mg)
22
Iron (mg)
1,5
Thiamin (mg)
0,09
Water(g)
12
Source : Rukmana, 2000
MATERIAL & METHOD
Albumin meatball
Firstly,chicken eggs washed with water
flowing, then eggs separated into albumin
and yolk. Compasrison albumin with
tapioca flour are 2:1. As much as 1,6 kg
albumin are stirred with 800 g tapioca
flour. Then added with garlic, salt, and
food seasoning (Lindayani, 2014). After
that, the dough weighed then divided into
four parts then added with 0%, 0,25%,
05%, and 1% of carrageenan.
Breadfruit based meatball
Firstly, to make arrowroot flour, arrowroot
peeled and washed with water flow, and
then dipped with natrium metabisulfit
solution 0,3% during one hour. Then
sliced with automatic slicer, arrowroot
slice arranged above tray and put in to
dehumidifier (60-700C) 5-6 hours. Dried
arrowroot crushed with food processor,
and then screened with 100 mesh sieves.
Arrowroot flour used in breadfruit based
meatball production (Widaningrum et al.,
2005).
Breadfruit cleaned and peeled, then
blanched in steam pan and crushed with
Page 56
food processor. Mixed with salt and garlic,

tapioca flour arrowroot flour, and cold
water. Then make a dough and make
round shape like commonly meatball in 10
grams. Meatball boiled (1000C) until
floated (Daniati, 2005).
egg whites causes partial coagulation of

proteins. (Gaman & Sherrington, 1994)
In the Table 2, the moisture content of egg

whites meatball that had been produced
approximately 56-58%. The meatballs
were good that if it does not contain a lot
of moisture content in it. If the moisture
content in the meatball is too high then it
Albumin meatball
Physical and chemical analysis results of
can reduce shelf life product because of
egg whites meatball can be seen in Table
the bacteria and fungi can easily multiply.
4.
Egg whites meatball contains an enough
high protein content because it derived
Table 4. Physical and Chemical Analysis
from the egg white that contains high
Results of Egg Whites Meatball
proteins. The proteins contents of egg
whites
Treatment
Hardness (gf)Water Content (%)
Protein
(%) meatball that had been produced
approximately
0%
529,18
56,36
8,62 8,50%-9,50%. The higher
the concentration
of carrageenan were
0,25%
771,86
57,83
9,08
added, the
0,5%
835,97
56,76
9,29protein content will increase,
because 9,49
carrageenan is a hydrocolloid
1%
887,39
56,74
compounds derived from seaweed
containing approximately 5.4% protein
Based on the research result on table 2, it
(Widyastuti, 2010). Carrageenan having a
is known that the higher the concentration
properties that can bind and trap water in
of carrageenan were added, the texture of
the gel matrix, so the water soluble protein
white eggs meatball that had produced also
is fewer because it binded by carraggeenan
increased. The texture of meatball can be
(Trisnawati & Nisa, 2015). Carrageenan
affected by water holding capacity, the
can interact with charged macromolecules
higher water holding capacity the fewer
such as proteins, which can affect the
water that lost during cooking process so,
increase in viscosity, gel formation,
the springiness of the meatball is more
deposition and stabilization (Winarno,
springy. The formation of the texture of
1996). Carrageenan can enhance the
the meatballs due to the process of the
capability of binding water so it can be
starch gelatinization of the arrowroot flour,
used to improve the texture of the product
coagulation in the egg white protein, and
(Keeton, 2001) and also can enhance
the addition of carrageenan. The gel
proteins holding capacity. (Rust et al.,
shaped egg white can be used as a
1973 in Chernanda, 2008).
thickener and binder for food because of
coagulation in the egg white protein if
Breadfruit based meatball
heated. Protein on the egg white will
Physical and chemical analysis results of
coagulate on the 600C. The presence of
breadfruit meatballs can be seen in Table
mechanical treatment such as stirring the
5.
Page 57
Table 5. Physical and Chemical

Results of Breadfruit Meatballs
Water
Treatmen Hardness
Content
t
(gf)
(%)
1339
0%
54,44
,30
1736
10%
53,84
,53
2377
30%
55,44
,97
2547
50%
56,53
,27
Analysis
Crude
fiber
(%)
12,33
12,90
14,70
15,13
In the breadfruit meatball, the result of the

hardness value is 1339,30-2547,27 gf. The
highest hardness value is taken from
breadfruit meatball with the use of
arrowroot flour 50%. Thehardness value in
the breadfruit meatball is affected by
arrowroot flour. This thing is happened
because of the amylase and amylopectin
content on the arrowroot starch. The
amylase in the arrowroot only 20-25%, so
the texture of the meatball is springy.
While amylopectin in arrowroot assist the
process of the breadfruit meatball dough
bonding(Hakim, 2013). The higher the
concentration of arrowroot flour is added
in making meatballs breadfruit, then the
better gelatinization process that can
produce the springy texture of meatballs.
According
to
Indarmono
(1987)
gelatinization process in the meatball
occur between starch and proteins
gelatinization.
Breadfruit meatballs has a moisture
content of between 53.84% to 56.53%.
The addition of arrowroot flour affects the
moisture content of the breadfruit
meatball. This is due to arrowroot flour
has a good water absorption capability
because it has a very high amylopectin

content. It contents 75-80%. So when
compared with SNI meatballs, meatballs
breadfruit has a moisture content lower
than the meatballs in general. Additionally,
breadfruit meatballs has a rough fiber
which is high at 12.33 to 15.13%. The
highest rough fiber contained in breadfruit
meatball with additional arrowroot flour as
much as 50%. The greater the
concentration of arrowroot flour, then the
higher the rough fiber content. This thing
can be happened because of the arrowroot
flour contains high rough fiber.
Additionally, not only arrowroot flour that
contains rough fiber but also breadfruit. It
contains 6,45% of rough fiber in the
breadfruit. Fiber is needed because it play
a important role in digestion system.
Consumption of fiber 25 g / day is
sufficient to meet the needs of the human
fiber and maintain a healthy body, so that
the meatballs breadfruit rough fiber can be
used as an additional value
CONCLUSION
Albumin meatball with carrageenan
addition and breadfruit based meatball
with arrowroot flour addition can be used
as substitute for meatball because there are
not containing fat but rich of protein
content to albumin meatball and rich of
crude fiber content to breadfruit based
meatball most healthy than commonly
meatball.
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Pengaruh Penambahan Karagenan
Terhadap Sifat Fisik, Kimia, dan
Palatabilitas Nugget Daging Itik
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cucut dengan Bahan Tambahan Jenis
Tepung yang Berbeda.Universitas
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to Food Science,Nutrition and
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Gardjito M, Naruki S, Murdiati A,
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dan Mikrobiologi Edisi Kedua.
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Hakim, U.N, D. Rosyidi, dan A.S.
Widati.2013. Pengaruh Penambahan
Tepung
Garut
(Maranta
arrundinaceae) Terhadap Kualitas
Fisik dan Organoleptik Nugget
Kelinci.Jurnal Ilmu dan Teknologi
Hasil TernakVol.8(2):9-22. Diunduh
18 Maret 2015.
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Pelayuan dan Jenis Daging Karkas
serta Jumlah Es yang Ditambahkan
Ke Dalam Adonan Terhadap Sifat
Psikokimia Bakso Sapi. Institut
Pertanian Bogor, Bogor. [Skripsi]
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Product. Di dalam: A. R. Sham (Ed).
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Lindayani.
2014.
Communication.
Botta
Personal
Linden, G.; D. Lorient. 2000. New

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Lubis, Y.M., S. Rohaya, dan H.A.
Dewi.2012. Pembuatan Meuseukat
Menggunakan Tepung Komposit dari
Sukun (Artocarpus communis) dan
Terigu serta Penambahan Nenas
(Ananas
comosus
L.).Jurnal
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IndonesiaVol.4(2):7-14. Diunduh 15
Oktober 2014.
Marsono, Y., P. Wiyono, dan Z. Utama,
2005.Indeks Glikemik Produk Olahan
Garut (Maranta arrundinaceae L) dan
Uji Sifat Fungsionalnya pada Model
Hewan Coba.Laporan RUSNAS
Diversifikasi Pangan Pokok Tahun
2005. Universitas Gadjah Mada.
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Pitojo, S.1992. Budidaya Sukun. Penerbit
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Rukmana, R. 2000. Garut: Budidaya dan
Pasca
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Curring Principle and Modern
Practice.Di dalam: Chernanda, E.
2008. Pengaruh Konsentrasi Natrium
Tripolifosfat Dengan Campuran
Tepung Tapioka dan Tepung Sagu
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Bakso
Sapi.
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Teknologi
Pertanian,
Fakultas
Pertanian, Universitas Sumatera
Utara. [Skripsi].
Trisnawati, M. I.; F. C. Nisa. 2015.
Pengaruh Penambahan Konsentrat
Protein Daun Kelor dan Karagenan
Terhadap Kualitas Mie Kering
Tersubstitusi Mocaf. Jurnal Pangan
dan Agroindustri Vol 3 (1): 237-247.
Widaningrum, S. Widowati, dan S.T.
Soekarto. 2005. Pengayaan Tepung
Kedelai Pada Pembuatan Mie Basah
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yang Di substitusi Tepung Garut.
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Chernanda, E. 2008. Pengaruh
Konsentrasi Natrium Tripolifosfat
Dengan Campuran Tepung Tapioka
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Bakso Sapi. Sumatera Utara:
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Fakultas
Pertanian,
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Sumatera Utara. [Skripsi].
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Kimiawi Karagenan yang Diekstrak
dari Rumput Laut Eucheuma cottonii
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yang Berbeda. Jurnal Agroteksos
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Page 60
FOOD PROCESSING AND ENGINEERING
Page 61
THE STABILITY OF BETALAINS IN RED BEET (Beta vulgaris L.) AS A

NATURAL FOOD COLORANT DURING STEAMING OF GLUTINOUS
RICE FLOUR BATTER
Metta Meliani1), Chaterine Meilani Surono1), Nies Mayangsari1), Victoria Kristina
Ananingsih2), and Alberta Rika Pratiwi2)
1)
Students; Food Technology Department; Faculty of Agricultural, Soegijapranata Catholic

University, Semarang
2) Lecturer; Food Technology Department; Faculty of Agricultural, Soegijapranata Catholic
mettameliani05@gmail.com
ABSTRACT
Red beet (Beta vulgaris L.) powder can be added to food as a natural food colorant that gives
purplish red color. Betalains are pigment contained in red beet that responsible for purplish
red color. It also has high antioxidant activity which gives health benefits. Red beet powder is
added to glutinous rice flour batter for enhancing the color appearance. One kind of glutinous
rice flour heat processing is by steaming, which can affect the stability of betalains pigment
in red beet powder. Change of betalains content contribute to the change of color intensity
and antioxidant activity, so the aim of this research is to know the effect of steaming process
and red beet powder addition on the betalains content, antioxidant activity and color intensity
of glutinous rice flour batter. In this research, the amount of red beet powder added to
glutinous rice flour batter are 0%, 10%, and 20% based on glutinous rice flour weight. The
batter were steamed for 15 minutes. Before and after steaming process, betalains (betacyanins
and betaxanthins) content and antioxidant activity of batter were analyzed. While, the color
intensity of batter was measured at 0, 3, 6, 9, 12, and 15. In this research, the addition of
red beet powder significantly increase betalains content and a* value of sample. Sample with
concentration of 20% red beet powder before steaming had the highest betacyanins and
betaxanthins (33.152.07 mg/L and 16.843.30 mg/L), antioxidant activity (6,240,90%) and
highest a* value which was 22.733.72. Betacyanins and betaxanthins content as well as
antioxidant activity significantly decreased after steaming. The a* value of batter decreased
as long as steaming process.
Keywords:red beet, betalains, color, antioxidant, glutinous rice flour
Page 62
INTRODUCTION
People begins to have concern about the
risk of using synthetic colorants, so the
needs of natural colorants being
increasing. Red beet is one of the natural
colorant that has functional value. The
color comes from betalains pigment that
contribute to give red-purlpe color.
Betalains content in red beet are around
1000mg/100g of total solids or
120mg/100g of wet basis (Nemzer et al.,
2011). Betalains are synthesized into two
structural groups, i.e. the red-violet
betacyanins and the yellow-orange
betaxanthins
(Azeredo,
2009a).
Furthermore
from Agrawal (2013),
betacyanins content in red beet was higher
more than betaxanthin that amount about
0.04-0.21% and betaxanthins amount
about 0.02-0.14% and depends on the type
of it roots.Beside of the colorant properties
of red beet, it has functional value as
antioxidant source that can reduce the risk
of chronic diseases such as cancer and
tumor (Ravichandran et al., 2013).
As natural colorant, red beet can be
applied in the form of powder on many
kinds bakery product for improving the
appearance. Red beet powder has
advantage when compare to liquid form
because their higher stability (Nemzer et
al., 2011). Red beet powder that be
produced by freeze or spray drying have
contains 0,3%-1% of pigment (Azeredo,
2009a). Glutinous rice flour is one of the
basic ingredients for bakery products that
having stickiness when its processing and
also having the white color and turbid
(Codex, 1995). However, during heating
process (steaming process) the glutinous
rice flour had physicochemical changes.
Heating process also can affect the

changes of betalains content that
contribute to the change of color intensity
and antioxidant activity (Ravichandran et
al., 2013). So the aim of this research is to
know the effect of steaming process on the
betalains content, antioxidant activity and
color intensity of glutinous rice flour
batter.
Material
Red beet powder was made from red beet
roots (Beta vulgaris L.) which were
obtained from local farmer in Kopeng,
aquades, ascorbic acid, maltodextrin DE
10. Glutinous rice flour batter was made
from glutinous rice flour (Rose Brand),
water, and red beet powder. DPPH (2,2diphenyl-1-picrylhydrazyl) and methanol
was used for antioxidant activity analysis,
while betalains content analysis used
ethanol 50%.
Methods
Red beet powder preparation
Red beet roots were washed, peeled, and
sliced into small pieces. For extraction of
red beet was done by juicer. Red beet
extract was added aquades (half-part), then
filtered through filter cloths. The pH of
solution was set at 5 by addition of
ascorbic acid (Cai and Corke, 2000).
Maltodextrin DE 10 was used as
microencapsulation agents. 60% of
maltodextrin were added to red beet
extract and homogenized using mixer.
Spray drying process was done using spray
dryer with 1500C inlet and 900C outlet
temperature. Red beet powder which was
obtained was filtered.
Glutinous rice flour batter preparation
Page 63
Sample was prepared by mixing of

glutinous rice flour, water, and red beet
powder. Additional of red beet powder
was variegated to 0%, 10% and 20% of
powder based of flour weight. Material
mixing was done by mixer at first speed
level for 25 seconds. 30 gram of batter
were put on container 9 x 4 cm and
steamed for 15 minutes until gelatinized.
Physicochemical Analysis
Betalain
Content
Analysis(Ravichandran et al., 2013)
Extraction of Betalains
0,1 g of freeze dried sample were
dissolved in 10 ml of 50% ethanol and
agitated for 10 second, then centrifuged at
6000 rpm for 10 minutes. The supernatant
was collected after centrifugation and the
same was repeated for 2 more times to get
better extracted betalains.
Determination of Betalains Compound
with Spectrophotometric Analysis
The betacyanins and betaxanthins content
in extracted betalains were determined
spectrophotometrically at 538 nm and 480
nm by using UV-Vis spectrophotometer.
The betalain content (BC) was calculated
as :
BC = [(ADFMW1000)/(e1)]
The A is absorption, DF the dilution factor
and l the path length (1 cm) of the cuvette.
For betacyanins and betaxanthins, the
molecular weights (MW) and molar
extinction coefficients (e) were applied.
(MW betacyanins= 550 g/mol; e=60,000
L/mol cm in H2O) and (MW betaxanthins=
308 g/mol; e=48,000 L/mol cm in H2O).
Antioxidant Activity Analysis(BrandWilliam et al., 1995)

0.5 g of freeze dried sample was extracted
in 5 ml methanol for 2 hours in dark room.
0.1 ml of extract react with 3.9 ml of
DPPH
solution
(2,2-diphenyl-1-5
picrylhydrazyl) 6 x 10 mol/L (2.9 mg
DPPH in 100 ml methanol) for 30 minutes.
The antioxidant activity analysis used
spectrophotometer at 515 nm. The DPPH
solution with no added extract was
analyzed as At=0.
The calculation of antioxidant activity is
determined in %inhibition:
% inhibition =[1- (
At=30
At=0
)] 100
Color Intensity Analysis(Lebesi &

Constantina, 2009).
Color intensity was measured by
Chromameter. The chromameter was
calibrated before used. The sample was put
in a transparent plastic and shot by
chromameter. The result will be shown as
L* (lightness) for showing brightness, a*
(redness) for showing red or green color,
and b* (yellowness) for showing yellow or
blue color.
Data Analysis
The obtained data from the analysis was
analyzed using Statistical Package for The
Social Science (SPSS) for Windows
version 16.0 with 95% level of confidence.
The analysis type that was used was One
Way Anova with Duncan method.
Betalains content analysis
According to Vargas et al. (2000),
betalains are limited and found in
Page 64
Caryophylalles, for example red beet root.

Betalains are water soluble pigment that
can be analyzed by UV-visible
spectroscopy. Betacyanins absorb the light
at 540 nm wavelength, whereas the
betaxanthins at 480 nm wavelength
(Azeredo, 2009). The spectrophotometry
method were used in this research. Based
on the research, red beet powder addition
contributes
the
betacyanins
and
betaxanthins significantly in sample.
Betacyanins and betaxanthins in sample
increases as higher concentration of red
beet powder added. Sample that added by
20% red beet powder contain the highest
betacyanins (33,152,07 mg/L) and
betaxanthins (16,843,30 mg/L) before
steaming process.It is causedby the
addition of red beet powder, therefore the
betalains content increased.
Betalains content in each sample
significantly decreased after steaming
process of 0%, 10% and 20% red beet
powderconcentration.
According
to
Stinzing & Carle (2004), temperature is
the most affecting factor that decomposed
betalains compared to pH, water activity,
oxygen, light exposure, metal and enzyme
activity. Higher temperature of steaming
would decreases the betalains in sample
(Ravichandran et al., 2013). Reduction of
betalains content in sample after heating
was caused of betanins degradation from
betacyanins group through isomerisation
process and decarboxilation or cleavage
because of heat and indicaxanthin from
betaxanthins group isomerisation (Stinzing
et al., 2007).
Figure 1. Betacyanins changes affected by

the difference addition of red beet powder
and steaming period
Figure 2.
Betacyanins content in
glutinous rice flour batter with addition
of red beet powder variation before and
after steaming
Antioxidant activity
Betalains pigment contained in red beet
root has major contribution on antioxidant
activity of red beet (Nemzer, et al., 2011).
Different addition of red beet powder
affects on antioxidant activity of glutinous
rice flour batter. This research showed that
the addition of red beet result in high
antioxidant activity. In figure 3, there was
increasing on antioxidant activity of
sample as increasing of red beet
concentration on the same heat treatment.
It is caused by the present of hydroxyl
group on betacyanins structure. The
hydroxyl group acts as hydrogen donor of
betanin (Azeredo, 2009a). The addition of
red beet powder in sample result in
Page 65
increasing of betacyanins content,

therefore antioxidant activity was higher.
Figure 3 shows there are decreasing on
antioxidant activity of sample after heat
treatment by steaming. Steaming process
of batter decreases antioxidant activity up
to 43.27%. It is significantly lower than
antioxidant activity of sample before
steaming. Heat is one of factors that affect
stability of betalains. Heating process
causes decreasing of betalains content by
degradation process. Decreasing of
betalains contribute to decreasing of
antioxidant activity (Ravichandran et al.,
2013).
red beet. Betacyanins are group of

betalains that contained in red beet.
Betacyanins content of peeled raw red beet
was 84.26 mg/L (Azeredo, 2009b).The
addition of red beet powder increases a*
value of sample. It is causedby increase
presence of betacyanins. It is proved by
the strong linear correlation of betacyanins
and a* (Table. 1).
Figure4. A* value of glutinous rice flour

batter with different concentration of red
beet powder during steaming process
Table 1. Correlation of a* value and
betacyanins content of glutinous rice
Figure 3. Antioxidant activity of
glutinous rice flour batter with different
concentration of red beet powder before
and after steaming process
Color intensity of glutinous rice flour
batter
Red color of sample was determined by a*
value of chromametry test result. Figure 4
show there were different of a* value
sample with different concentration of red
beet powder. Addition of red beet powder
increases the red color of sample
significantly. The highest concentration of
red beet powder has the highest a* value
was 22.733.72 after 15 minutes of
steaming process. The red color was
affected by the present of betacyanins in
Correlated factors
Correlation
coefficient
a*
value
vs.
Betacyanins
0.871**
content
flour batter
Sign
0.000
**show significance interaction at 0.01

level (99%)
Red color of glutinous rice flour batter
with addition of red beet powder decrease
as long as steaming process (Fig 4). Heat
process such as steaming, effects the
stability of betalains pigment. High
temperature process degraded the betalains
pigment, as well as the red color
(Ravichandran et al., 2013). Betacyanins
Page 66
degraded
by
isomerisation,
decarboxylation, or cleavage mechanism.
Degradation of betalains results in
degradation of red color and appearance of
light brown color as formation of
neobetanin (Azeredo, 2009a). Therefore,
the longest the steaming time, a* color
decrease and form dark color. In figure 5,
there was decrease on lightness sample
during steaming process.
Figure5. L value of glutinous rice flour

batter during steaming process
CONCLUSION
Steaming process of glutinous rice flour
batter with addition of red beet powder
decrease in a* value (red color intensity),
antioxidant activity, and betalains content.
Glutinous rice flour batter with addition of
20% red beet powder had the highest
betalains content, antioxidant activity, and
a* value.
ACKNOWLEDGMENT
The author is very grateful to Research
Team a.n. Dr.V. Kristina Ananingsih
funded by Hibah Bersaing DIKTI No. SK :
052/K6/KL/SP/PENELITIAN/2014.
DAFTAR PUSTAKA
Agrawal, A. (2013). Scope of Betalains As
A Food Colorant. International
journal of advanced scientific and

technical research Vol 3(3).
Azeredo, H.M.C. (2009a). Betalains:
properties, sources, applications, and
stability. A review: International
Journal of Food Science and
Technology 2009, 44, 23652376.
Azeredo, H.M.C.; A.C. Pereira, A.C.R. de
Souza, S.T. Gouveia & K.C.B.
Mendes.(2009b). Study on efficiency
of betacyanin extraction from red
beetroots.International Journal of
Food Science and Technology 2009,
44, 24642469.
Brand-Williams, W., Cuvelier, M. E. And
Berset, C. (1995). Use of a free
radical
method
to
evaluate
antioxidant activity. Food Science
and Technology, 28, 2530.
Cai, Y. Z. & H. Corke. (2000). Production
and Properties of Spray-Dried
Amaranthus Betacyanin Pigments.
Journal of Food Science Vol.65
No.6. USA.
Codex Alimentarius Commission. (1995).
Codex Standard For Rice. CODEX
STAN 198-1995.
Delgado-Vargas, F.; A. R. Jimnez, and O.
Paredes-Lpez. (2000). Natural
Pigments:
Carotenoids,
Anthocyanins, and Betalains
Characteristics,
Biosynthesis,
Processing, and Stability. Critical
Reviews in Food Science and
Nutrition, 40(3):173289.
Lebesi, D.M. and C. Tzia. (2009). Effect
of the Addition of Different Dietary
Fiber and Edible Cereal Bran Source
on the Baking and Sensory
Characteristic of Cupcakes. Journal
Food Bioprocess Technology.
Nemzer, B., Z. Piettrzkowski, A. Sporna,
P. Stalica, W. Thresher, T.
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Michalowski, S. Wybraniec. (2011).

Betalainic and Nutritional Profiles of
Pigment-Enriched Red Bit Root
(Beta Vulgaris L.) Dried Extracts.
Food Chemistry 127 (2011) 4253.
Ravichandran, K., Nay Min Min Thaw
Saw, Adel A.A Mohdaly, Ahmed
M.M.Gabr. Anja Kastell, Heidi
Riedel, Zhenzhen Cai, Dietrich
Knorr, Iryna Smetanska. (2013).
Impact of Processing of Red Bit on
Betalain Content and Antioxidant
Activity.
Food
Research
International 50.
Stintzing, F.C., and R. Carle. (2004).
Functional
properties
of
anthocyanins and betalains in
plants,food, and in human nutrition.
A Review. Trends in Food Science
& Technology 15 (2004) 1938.
Stintzing, F. C., Trichterborn, J., & Carle,
R. (2007). Betalains - emerging
prospects for food scientists. Food
Chemistry.
P.
Thammapat, Meeso
N, and
Siriamornpun S. (2015). Effects of
NaCl and soaking temperature on the
phenolic compounds, -tocopherol,
-oryzanol and fatty acids of
glutinous rice. Food Chem; 175:21824. doi: 10.1016.
Page 68
UTILIZATION COCOA POD HUSK POWDER FOR BREAD

Ingrid Tertiana Ivanaa*, Jefri Sugiarto Halimb
*Program Studi Teknologi Pangan, Fakultas Teknologi Pertanian, Universitas Katolik Widya
Mandala Surabaya
JalanDinoyo No. 42-44, Surabaya, Indonesia
*E-mail: ingridtertiana@yahoo.com
ABSTRACT
Bread is one of the alternative food that can be substituted for rice as a staple food that is practical.
The main ingredient of bread making is wheat flour which has a low fiber content. Indonesia has less
daily fiber intake than it should, so it needs to be improved. One way to increase fiber consumption in
Indonesia is using cocoa pod husk powder. Indonesia is able to produce cocoa with the third level of
productivity in the world and continues to grow each year. The cocoa pod husk waste containing
crude fiber that can help increase the fiber content found in bread. This study studied the effect of
adding cocoa powder to make bread with a proportion of 0%, 5%, 10%, 15%, and 20%. The addition
of fiber in white bread with cocoa pod husk powder can increase the fiber content of bread, but affects
the characteristics of the resulting bread. The parameters examined include volume expansion,
hardness, and color of bread. Allegedly along with the increasing addition of cocoa pod husk powder,
the volume development of bread decreased but the hardness and colour of bread increased
Keywords: bread, cocoa pod husk powder, the characteristicsof bread
Page 69
INTRODUCTION
Food is one of the human needs that must
be fulfill. Along with development of
society, the human activity also increased.
This condition is causing people need food
that is practical, feasting, and able to fulfill
the energy needs. One food that can fulfill
these criteria are bread. Bread is chosen
because it is easy to carry and practical.
Bread is a staple food prepared from a
dough of flour, water, and yeast, usually
by baking. The contents of 100 grams of
bread are 8,0 grams protein, 50,0 grams
carbohydrate, 1,5 grams fat, and 39,0
grams
water
(Gaman
dan
Sherington,1992). Bread usually made
from high protein flour. Wheat flour with
high protein content can absorb large
quantities of water, reach dough
consistency, and have a good elasticity so
the final product has a soft crust, tender
texture, expand the volume and contain
12-13% protein (Astawan, 2004). Bread
has a disadvantages which is low in fiber.
The addition of these fiber can increase
fiber content in bread, also the fiber can be
given from fruits, vegetables, cacao and
some of grains.
One material that can be used as a source
of fiber is cocoa pod husk. Cocoa consist
of three part that is outer layer or husk
(75,6%), placenta (2,59%), and seed
(21,74%). In the past, cocoa pod husk
(CPH) was commonly discarded as a waste
which has large amount if compared with
others parts. According to previous study
from Alemawor (2009), CPH contains
357,4 g/kg (dry basis). Hence, in this
study, cocoa pod husk which is high in
crude fiber was evaluated as an ingredient
(powder) in bread making. The other
reason is cocoa is a plant whose fruit

throughout the year and it is quite
abundant. According to data from the Food
and Agriculture Organization (FAO),
Indonesia produces 574 tons of cocoa in
2010. During the processing of cocoa,
cocoa pod usually not used.In recent time,
cocoa pod husks is used as animal feed and
also as fertilizer. Thus, this agro-industrial
by-product should not become a waste by
expanding its usage in food product. The
cocoa pod husk powder (CPHP) which is
high in fiber content can be supplemented
into high fiber bread thus, create a variety
of high fiber bread.Cocoa pod husk in
powder form chosen because it is easier to
use and has a long self life.These addition
can increase the economic value of cocoa
pod.
The main aim of this study is to determine
the effect of cocoa pod husk powder to the
physical characteristics of the resulting
bread. Addition of cocoa pod husk powder
can provide different effect on the physical
characteristic of the resulting bread.
Equipment
The tools used in the production of cocoa
pod husk powder are stainless steel kitchen
knife, stove, tray, cabinet dryer, warring
blender (warring commercial. Laboratory
blender), sample mill 0,12 mm, and
polyethylene bag.
The tools used in the making of bread are
sieve, mixer, tray, proofer, and oven.
It is also used tools for physical analysis
such as texture analyzer, repeseed,
colorimeter,
electron
microscope,
minitab14 applications, ruler, knife, and
texture Profile Analysis (TPA).
Page 70
Cocoa pod husk
Materials
Materials used by reference to the Amir
(2013). Cocoa Pod Husk (Theobroma
cacao sp) obtained from Kapar, Selangor,
Malaysia. Ingredients to making bread
were obtained from local supplier
(Selangor dan Terengganu).
PREPARATION OF COCOA POD
HUSK POWDER
The cocoa pod husk powder (CPHP) was
prepared by the following steps that is
based on a reference from the Amir
(2013). Firstly, the cocoa pod husk (CPH)
was cleaved by using a stainless steel
kitchen knife. The wet bean (seed)
including the placenta and the outer layer
of the cocoa pod which is known as cocoa
shell was removed from the pod. The CPH
which has white color was cleaned and cut
into smaller pieces. And then, CPH was
cooked for about half an hour in order to
reduce its theobromine content, as well as
practicable in removing the slime layer of
CPH and soften its texture. Next, the CPH
was placed on a tray and dried in a cabinet
dryer at temperature of 60C until the
moisture content was 8-10%.Finally, CPH
was grinded by using warring blender
(Warring
Commercial,
Laboratory
Blender) and continue with milling by
using a sample mill to obtain particle size
of 0.12 mm. Then, CPHP produced was
stored in a polyethylene bag at room
temperature preparation of cocoa pod husk
powder can be seen in Picture 1
Cutting
Separating
Seed, placenta, husk
Cocoa pod husk

cleaned
Cooking30
minutes
Drying ( 60C, MC 8-10%
)
Grinding
(blender)
Milling
(
sample mill)
Cocoa pod husk

powder (0,12mm)
Picture 1.Flow ChartProcessing of Cocoa

Pod Husk Powder
Source : Amir,dkk(2013)
Preparation of Bread
Bread that made is according from Gisslen
(2009),the formulation can be seen
onTabel 1
Ingredients
Grams
Wheat flour
300,00
Instant yeast
3,78
Sugar
10,80
Bread improver
3,00
Non-fat milk solid
15,00
Salt
7,20
Shortening
10,80
Water
192,00
Table 1. The Formulation of Bread
Page 71
Source : Gisslen (2009)

Stages of the process of making bread is
weighing accurately all ingredients
according the formulation. After that, the
flour were mixed together with instant
yeast, and sugar then the process continued
by addition of bread improver, non-fat
solid milk, salt , and shortening. Lastly,
water was added slowly to the mixture.
After kneading, the dough was allowed to
undergone fermentation process for about
30 minutes. Then, the dough was folded to
allow the gas expelled from the dough and
at the same time distribute the yeast for
further growth. Then, the dough was
portioned into the required size. The dough
was rounded to shape it into a smooth
layer. This procedure also may assist in
retaining the gas that was produced by
yeast. The dough was allowed to rest for
about 10-20 minutes before molding and
placed into a baking tin. Then, the dough
was placed into the proofer for the final
proofing stage for about 45 minutes at
37 C. Next, the dough was baked in baking
oven at 200C for 30 minutes. The process
of making bread can be seen on Picture 2
Flour, non-fat solid milk, salt, shortening,

water, sugar
Weighing
Flour, instant yeast,
and sugar
Mixing I
Bread improver, nonfat solid milk, salt,

shortening, andwater
Mixing II
Dough
Fermentation30 minutes
Kneading
Molding
Proofing
(t= 45 minutes,37C)
Baking
(t= 30 minutes),
T=200C
Bread
Picture 2.Flow Chart of Making Bread

Source : Gisslen, 2009
METHODS
Testing methods used reference from
Amir, dkk (2013. Physical analysis tested
on sample of bread is the analysis of
texture, volume and color. The texture
characteristic (hardness) of breads were
analyzed by using TA.XT. Plus Texture
Analyzer (Stable Micro System Ltd., U.K).
The volume of bread was determined
byusing the rapeseed displacement
methods. The profile color of bread crust
and crumb formed were determined by
Page 72
using colorimeter (Minolta Chroma Meter

300, Japan).The microscopic structure of
bread was observed to determine the effect
of incorporation of CPHP towards the pore
size of bread.
1.
Dough
The formation of the bread dough is
depend on the formation of gluten tissue.
The characteristic of glutens matrix can
be vary, it depends on the flour that been
used. Cocoa pod husk powder that use in
bread making can cause decrease number
of glutens matrix that been created and
decrease gluten abilty to hold CO2.
Number of glutens matrix that decrease
can change the characteristic of final
product which is the dough will become
sticky. That kind of characteristic happen
because cocoa pod husk contain fibers.
Fiber has the ability to hold water. This
abilty make the gluten created imperfectly
because there is competition between fiber
and protein to hold water. This
competition cause the gluten that created
has less abilty to hold gas. The final result
of the dough has higher moisture content
compare to the 100% wheat flour product.
2. Volume
Bread volume expansion is very affected
by glutens matrix. Gluten has the abilty to
hold gas expansion from the material
expansion during fermentation. Glutens
matrix with a good quality will also has a
good abilty in holding gas. The addition of
cocoa pod husk cause glutens matrix
imperfectly created because the creating
process of glutens matrix has been
prevented by fiber in cocoa pod husk.
Fiber prevent the creating process of
glutens matrix by absorbing water near

them. The defective glutens matrix that
created cause the glutens matrix has less
abilty to hold gas during baking process,
so many gas released and decrease the
volume of the final product. The volume
expansion of the final product can be seen
in Table 2. According to table 2 we can
see the spesific volume of bread with
substitution of cocoa pod husk with
different concentration.
Table 2. Expansion Bread Volume with
Cocoa Pod Husk Substitution
Volume of Bread
Wheat Flour :
Spesific
Volume
CPHP (%)
Volume
(cm3)
(cm3/g)
100 : 0
1350.00a
2.88a
90 : 5
1300.00a
2.64ab
90 : 10
1225.00a
2.44b
85 : 15
1050.00b
2.09c
80 : 20
990.00b
1.94c
Note: Notation difference in
one column show there are
significant difference with p
5%, CPHP
is
cooca pod husk powder.
Source : Amir, dkk 2013
The more cocoa pod husk powder add it
will decrease the bread volume, because
there is fiber in cocoa husk powder. This
fiber cause gluten formed not optimally
and decrease glutens ability to hold gas.
Bread with 10% cocoa pod husk powder
substitution given result that there is a
significant difference among the 100%
wheat flour bread and cocoa pod husk
powder substitution bread. Picture of fiber
microscopy picture can be seen in picture
3, which show the structure of final bread.
The more cocoa pod husk powder that
Page 73
substitued the more not optimal the gluten

created. That can happen because fiber in
cocoa pod husk powder distract the
glutens creating process, so gluten that
created become not optimal and it cause
airspaces created un-uniformly.
Picture3. Bread Fiber Microscopy

Structure (A: subtitution 0% ,
B:subtitution 5% , C: subtitution
10%,
D: subtitution 15%, E:
subtitution 20% wheat flour with
cocoa pod husk powder).
Source : Amir,et al (2013)
3. Hardness
Good quality bread has soft texture and
elastic. Substitution of wheat flour with
cocoa pod husk powder will affect the
hardness of bread. The higher number of
substitution wheat flour with cocoa pod
husk powder will bring out higher level of
hardness as well. It can occur because of
the fiber content in the cocoa pod husk
powder.Higher the fiber the dough water
content will also raise and cause the
glutens viscoelasticity become worse.
This will make the dough heavier and can
not expand well. Table 3 show the
hardness of bread with cocoa pod husk
powder substitution.
Table 3. Hardness of Bread with Cocoa

Pod Husk Powder
Wheat Flour :
Hardness of Bread
CPHP (%)
(N.sec)
100 : 0
726.60d
90 : 5
845.40cd
90 : 10
945.90c
85 : 15
1174.30b
80 : 20
1407.00a
Note: Notation difference in
one
column
show
significant difference with p
5%
Source : Amir, dkk (2013).
According to table 3 the hardness of the
bread will increase along with the number
of cocoa pod husk powder subtitued. Fiber
in cocoa pod husk powder cause the
increase of the hardness. Fiber has the
ability to hold water tight, so the more
cocoa pod husk powder subtitued the more
water that will tightly hold by fiber and
make the hardness increase. Bread with
10%
substitution
give
significant
difference compared to 100% wheat flour
bread.
4. Color
Color is one of the visual factors that
consumen see to decide the quality of a
food product. Crust and crumb color in
bread is the result of maillard reaction
between sugar and protein. According to
Fennema (1985), maillard reaction is a non
enzymatic browning reaction that caused
by interaction between sugar and amino
acid from protein. Color of bread can be
seen in Table 4.
Page 74
Table 4. Color (Crust and Crumb) of Bread

Color (Crust)
Color (Crumb)
Bread
*
*
*
*
L
a
b
L
a*
b*
A
58,78a 12,23a
32,75a
69,32a
2,09a
18,10a
B
57,58a 10,32ab
26,76a
61,55ab
2,75a
15,69a
C
56,86a 10,19b
24,93a
59,75ab
2,88a
15,41a
D
56,07a
7,21c
23,33a
51,81b
3,48a
14,61a
E
49,60a
7,11c
23,25a
47,53b
3,55a
14,25a
Note: Notation difference in one coloumn show significant difference with p
5%.
Source : Amir, dkk (2013).
According to table 4 we know that the

decreasing of the color is along with the
increasing of cocoa pod husk powder use.
Crust and crumb color turn into golden
brown. This color turn cause by maillard
reaction. More of the cocoa pod husk
powder subtitued more maillard reaction
will happen. Chemical composition of
cocoa pod husk powder can be seen in
Table 5. Acoording to table 5 we know
that the content of reduction sugar and
protein in cocoa pod husk powder is high
enough so the addition od cocoa pod husk
powder will cause maillard reaction
happen more and it will cause the color of
bread become darker.
Table5. Chemical Composition (mg/g) of
Cocoa Pod Husk Powder
Proximate composition
Cocoa Pod
(mg/g)
Husk Powder
Moisture
84.5 3.1
Ash
79.2 3.9
Fat
22.7 2.5
Protein (N x 6,25)
89.1 2.9
NSP
420.7 2.3
Soluble Sugar Total
131.8 1.6
Reduction sugar
84.3 4.1
Soluble fenol
(mg/
68.9 5.6
GAE/g)
Minerals (mg/g)
K
58.2 3.7
P
7.1 0.9
Ca
6.1 0.8
Mg
4.9 0.6
Fe
0.35 0.05
Na
0.16 0.02
Source :Beda, dkk (2013)
CONCLUSION
The addition of cocoa pod husk powder in
bread making will decrease the volume,
increase hardness and increase the
browncolor in bread.
GRATTITUDE
Thank you to the Technology Faculty of
Agriculture, University of Widya Mandala
Surabaya and Ms. Anita Maya Sutedja,
S.TP, M.Si as lecturer who have helped in
the fluency establishment of this paper.
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CALCIUM FROM SHRIMP SHELL : THE EFFECT OF HCl

SOLUBILIZATION CONDITION
Angela Irena Wibawa 1), Saranya Dechapinan 2), Dr. Nattapol Tangsuphoom 3),
Dr. Ch.Retnaningsih 4)
1)
Students; Food Technology Department; Faculty of Agricultural Technology;

Pawiyatan Luhur IV/1 Street, Semarang, Indonesia
2)
Students; Institute of Nutrition; Master of Science Program in Food Science for Nutrition;
Mahidol University
999 Phuttamonthon 4 Road, Salaya, Thailand
3)Supervisor, Institute of Nutrition; Master of Science Program in Food Science for Nutrition;
Mahidol University
999 Phuttamonthon 4 Road, Salaya, Thailand
4)Supervisor, Food Technology Department; Faculty of Agricultural Technology;
Pawiyatan Luhur IV/1 Street, Semarang, Indonesia
angelairena94@gmail.com
ABSTRACT
Calcium is an important macro-mineral to build and maintain bone and teeth. Calcium intake
of people is usually inadequate, especially for women. Therefore, calcium is fortified in food
products to help the consumers meet their requirements. Shrimp shell is the waste from
seafood processing that is an inexpensive sourcefor calcium. Calcium from shrimp shell can
be extracted by solubilizing with acid and the condition, including temperature, and time can
influent the calcium extraction yield. The objective of this study was to determine the effect
of the condition used foracid solubilization on the yield of the calcium extracted from shrimp
shell. The shell of Pacific white shrimp (Litopenaeus vannamei) was deproteinized using a
serine endoprotease prior to calcium extraction using HCl. Extraction was performed at 28oC,
50oC and 70C for 1, 2, and 4 h. The contents of soluble calcium and that remaining in the
shell residue from each condition were analyzed using atomic absorption spectrometry and
were used to determine calcium extraction yield. Extraction at 28oC for 1 h gave the highest
calcium content in the remaining shell, indicating the lowest extraction yield (87,41 %);
while extraction at 70oC for 4 h gave the highest soluble calcium content and thus medium
yield (90,40%). The optimum condition for extraction of calcium from shrimp shell with HCl
was 70oC for 2 h, from which the obtained yield (90,80%) was slightly higher than that of
longer extraction time.
Keywords : shrimp shell, calcium, acid solubilization, extraction yield
Page 78
1. INTRODUCTION
1.1. Background of The Research
Calcium is one of the macro mineral which
is needed for human body for the skeletal
system. Houtkooper (2004) stated that
calcium is needed to build and maintain
teeth and bones, it helps muscles and
nerves to work properly, and also helps to
prevent high blood pressure. Inadequate
consumption of calcium can cause diseases
such as osteoporosis, osteomalacia, and
bone fracture. The calcium needed per day
for every individual is different and it
depends on their ages. Females need more
calcium than males since their mass bone
more decrease rapidly, especially those
who are menopause which reduce the
amount of estrogen hormone. Fikawati
(2005) also reported that female students
need more calcium intake for replenishing
than do male students. Thus the risk of
osteoporosis is also higher in women than
men (Agustin 2009).A study conducted in
Bandung City suggested that calcium
intake of adolescent students does not
meet the requirement yet which is under
75% RDA.
Shell of the crustaceans, which is the
waste from seafood processing can cause
environmental problem because some of
flesh left in the shells is the ideal growth
media for pathogenic bacteria (Sini et al.,
2007). Crustaceans shell consists mainly
of
chitin,
a
polymer
of
Nacetylglucosamine, which naturally exists
in complex structure with minerals,
especially
calcium,
and
protein.
Ravichandran, et al (2009) reported that
shrimp shell contains 32.5% protein,
26.6% minerals which mostly is calcium,
9.8% lipid, and 12.3% moisture. Therefore
the shell waste is usually utilized as the

source for chitin extraction, from which
minerals and protein are removed by using
strong acid and alkali solutions to
solubilize the minerals and protein from
the shell. The obtained protein and calcium
are frequently discarded as by products.
Nowadays, calcium is added into several
products such as fruit juice and dairy
products. It is also fortified in dairysubstitute, cereal based beverages like soy
milk. Therefore, calcium extracted from
shrimp shell can be utilized for food
fortification with very low cost. Different
types of calcium, which have different
solubility and bioavailability, can be
obtained depending on the type of acid
used for extraction. However, extraction
condition factors such as temperature,
time, and pH can influence the yield of the
calcium extracted from shrimp shell.
1.2.Objectives:
The main objectives of this study are
determine the effect of the condition used
foracid solubilization on the yield of the
calcium extracted from shrimp shell.
2. RESEARCH METHODOLOGY
2.1. Materials
Frozen shell of Pacific white shrimp
(Litopenaeus vannamei) was obtained
from a shrimp processing plant of Charoen
Pokphand Food (Public) Co., Ltd. Serine
endoprotease (Alcalase 2.4L) was donated
by Brenntag (Thailand) Co., Ltd. All other
chemicals unless otherwise stated were
obtained from Sigma Aldrich.
2.2.Methods
2.2.1. Deproteinization of Shrimp Shell
Page 79
First of all, shrimp shell was thawed and

then the thawed shell was mixed with deionized water at the weight to volume ratio
of 1:10. After that, the enzyme was added
into them at the concentration of 0.1%
(v/w) of the shell and the mixture was
heated in a temperature-controlled water
bath for 2 hours at pH 9 and 50oC, which
is the optimal condition of the enzyme, to
hydrolyze and remove the protein from the
shell. After that, the mixture was heated at
90oC for 10 minutes in the water bath to
inactivate the enzyme and after that it was
put immediately into the ice bath to cool
down the room temperature (28C). The
deproteinized shell was separated from
protein solution by wire screen and dried
in the hot air oven at 50oC for 6 hours. The
dried deproteinized shrimp shell was
packed and sealed in LDPE bags and kept
in the desiccator for further analysis.
Chemical composition of the deproteinized
shell was analyzed according to AOAC
Official Method.
2.2.2. Calcium Extraction from Shrimp
Shell
Extraction of calcium was performed by
mixing the dried deproteinized shrimp
shell with HCl solution at the weight to
volume ratio of 1:40. The concentration of
acid was calculated to obtain the
stoichiometric equivalence ratio of the
extracted calcium compound. Extractions
were performed at room temperature
(28oC), 50 and 70C for 1, 2, and 4 hours
on a hot plate with magnetic stirrer. After
that, the mixture was cooled immediately
to the room temperature and the remaining
shell was separated from the calcium
solution by wire screen before being kept
frozen for further analyses.
2.2.3. Analysis
2.2.3.1. Ash Content
The ash contents in the shells before and
after calcium extraction were analyzed
according to AOAC Official Method. The
crucible and lid were put in the furnace
prior to heating at 550oC for overnight.
After that the crucible was cooled in the
desiccator for 30 minutes. Then, the
crucible and the lid were weighed. One
gram of the sample was put in the crucible.
After that, the crucible was heated over
flow above Bunsen flame with lid half
covered until the fume was no longer
produced. Then, crucible with lid and
sample was put in the furnace and heated
at 550oC for overnight. During heating, lid
was not covered to prevent loss of fluffy
ash but it was put on after the heating was
complete, which was noticed as the sample
turned to grey. The crucible was then
cooled down in the desiccator. The weight
of crucible with lid and the remaining ash
was recorded and the ash content was
calculated based on the following
equation:
2.2.3.2. Calcium Content

The calcium contents both in the solution
and in the remaining shell from each
extraction condition were analyzed by
using an atomic absorption spectrometry
(AAS) equipped with flame and graphite
furnace. The remaining ash from the shell
was added 5 ml of 4 N nitric acid solution
and diluted with distilled water to obtain
the ash solution of the final volume of 50
ml. For calcium content analysis by AAS,
aliquots (125 l) of the ash solution from
the remaining shell and the calcium
Page 80
solution were added with 2.5 ml of 4 N

nitric acid solution and 5 ml of 5%
lanthanum oxide and diluted with distilled
water to the final volume of 25 ml prior to
AAS analysis.
2.2.3.3. Calcium Extraction Yield
The calcium extraction yield was
calculated from the calcium contents in the
shell before and after extraction based on
the following equation:
2.2.4. Statistical Analysis

All experiment was performed in
triplicates. The results were presented as
means standart deviations. Statistical
software (SPSS 18.0, SPSS, Chicago,
Illinois, U.S.A.) was used to analyze the
data.
3. RESULTS AND DISCUSSION
Hartini et al. (2003) stated that the source
of calcium is widely-available in broccoli,
cabbage, legumes, milk, dairy products
and also fortified products such as soy
milk. Houtkooper (2004) also added that
the calcium needed for individual ages
between 9-18 years old is 1300 miligram
each day. The adults between 19-50 years
old need 1000 miligram calcium per day
and for people 50 years old need 1200
miligram calcium per day. Fikawati (2005)
reported that 76.2% from 1254 students
consume the calcium less than 75% of
RDA and the RDA for Indonesian is 1200
miligram/ day for the teenagers.
Extracted calcium from the shrimp shell
can be utilized for the fortification because
it is cheap. There are several factors that
can influence the percent yield of shrimp

shell such as pH, temperature and time
and this study used that two parameters to
find the suitable condition for the percent
extraction yield and It was obtained from
deproteinization to remove the protein and
get the calcium from the Pacific White
shrimp shell. Then, calcium was extracted
with HCl to get the mixture of calcium
with acid from the shrimp shell. Percon et
al (2003) reported that HCl didnt give the
good effect to the molecular weight in the
demineralization
of
chitin
but
Poeloengasihhernawan (2009) stated it
has high ability to remove ash and protein
than acetic acid. After that, AAS was used
to analyze the extracted calcium.
The result of chemical compostion of the
deproteinized shrimp shell is shown in the
table 1.
Table 1. Chemical Composition of The
Deproteinized Shrimp Shell
Composition
Content (/100 g)*
Wet weight Dry weight
basis
basis
Moisture
9.96 g
90.05 g
Protein (N x 41.79 g
46.41 g
6.25)
Ash
25.48 g
28.30 g
Calcium
8365.805
9290.280
mg
mg
Dietary fiber
30.38 g
33.74 g
*Mean of duplicate analyses of pooled
sample
From the data above, it showed that the
calcium is the highest composition from
the deproteinized shrimp shell based on
the wet weight basis which is 8365.805
mg/100 g and 9290.280 mg/100 g for the
dry weight basis. Ravichandran (2009)
Page 81
mentioned that the calcium content in the

shrimp shell in Indian White Shrimp
(Penaeus indicus) is higher than its flesh
so shrimp shell is the good source of
calcium. It is also supported by Okoye
(2005) that shrimp waste is good source
for calcium but the bioavailability for the
protein is low for the broiler chicken.
Temperature(o
C)
28
50
70
Calcium
(mg/100g)*
1 hour 2
hours
1052.9 1001.2
2
9
89.03
47.89
971.05 941.47
112.71 76.15
954.58 769.50
183.90 51.60
content
4
hours
1010.6
6
188.64
955.44
149.02
803.10
33.00
The result of ash content of the remaining

shell from different extraction conditions
is shown in the table 2.
Table 2. Ash Content of The Remaining
Shell
from
Different
Extraction
Conditions
*Mean standard deviation of triplicate
samples
Table 2 showed that the higher
temperature, the lower ash content and the
longer time, the lower ash content and
extraction at 28oC for 4 hours gave the
highest result. Poeloengasihhernawan
(2009) stated that the ash of the shrimp
shell are higher than those of heads
because of the different chemical
composition of the raw material but
preconditioned shell has lower ash content
than head and after the demineralization

process, shell has higher ash content than
head. Fall (2012) reported that the highest
ash was obtained from 100% shrimp shell
meal for hybrid tilapia and it could
substitute the soybean meal up to 60% for
hybrid tilapias meal. Trung (2012) also
reported that the purified chitosan showed
the higher quality than chitosan especially
lower ash and protein content. Then
shrimp shell powder also has the higher
ash than raw chitin (Khorrami, 2012).
The result of calcium content of the

remaining shell from different extraction
Temperature(oC) Ash
(mg/100g)*
1
2
hour hours
28
3.90
3.81
0.20
0.24
50
3.00
3.12
0.09
0.34
70
3.17
3.14
0.60
0.11
conditions is shown in table 3.
content
4
hours
5.00
0.35
2.94
0.21
2.68
0.47
Table 3. Calcium Content of The

Remaining
Shell
from Different
Extraction Conditions
Page 82
Temperatu Calcium extraction yield

re(oC)
(%)*
1 hour
2 hours 4 hours
28
87.411 87.910 87.922
.06aA
.43bA
.25aA
50
88.391 88.750 88.581
.35aA
.91bA
.78aA
70
88.592 90.800 90.400
.20aA
.62aA
.40aA
samples
From the data above, it showed that the
higher temperature, the lower calcium
content from the remaining shell and the
longer time, the lower calcium content too
but the calcium content increased slightly
at 4 hours for each temperature.
The result of calcium content of the
calcium solution obtained from different
extractions conditions is shown in the table
4.
Table 4. Calcium Content of The
Calcium Solution Obtained from
Different Extraction Conditions
samples
Based on the data above, it showed that the
higher temperature, the higher calcium
content of the calcium solution and the
longer time, the higher calcium content of
the solution too.
The result of calcium extraction yield
obtained from different extraction
conditions is shown in the table 5.
Temperature(oC
)
Calcium
content
(mg/100g)
1 hour 2
4
hours hours
28
214.1 216.1 221.1
2
5
8
3.00
15.60 7.48
50
224.0 227.1 226.8
6

9
7.67
12.61 3.10
70
224.3 227.1 239.3
3
8.62 8
21.00
6.00
Table 5. Calcium Extraction Yield
Obtained from Different Extraction
Conditions
samples
As we can see from above, there was no
difference of temperature in 1 hour and 4
hours in the extraction yield but there was
difference of temperature in 2 hour and
70oC was different than the other
temperatures in the extraction yield. For
the time, we can see that there was no
difference of time in the same temperature
so calcium extraction from the shrimp
shell at 70oC for 2 hours is the optimum
condition to use.
4. CONCLUSION
Temperature and time can affect the

percent extraction yield because the
higher temperature and time, the
higher extraction yield.
The suitable temperature is 70oC
because the highest % extraction yield
was obtained and 2 hours is the
suitable time to obtain the yield.
Page 83
5. REFERENCES
Agustin, R. 2009. The Relationship
between Status Nutrition, Lifestyle, and
Habits of Calcium and Vitamin D with
Genesis Osteoporosis and Osteoponia on
Citizens 45 years in Taman Wisma Asri
North Bekasi. FKMUI.
AOAC. 1990. Official methods of analysis
of the Association of Official Analytical
Chemists. 15th edition. Washington, DC,
Association of Official Analytical
Chemists.
Fall, Jean, Yi-Theng Tseng, Diegane
Ndong, Shin-Shyn Sheen. 2012. The
Effects of Replacement of Soybean Meal
by Shrimp Shell Meal on the Growth of
Hybrid Tilapia (Oreochromis niloticus x
Oreochromis aureus) Reared Under
Brackish Water. International Journal of
Fisheries and Aquaculture Vol.4 (5), pp.
85-91.
Available
from
http://www.academicjournals.org/IJFA.
Fikawati, S., Syafiq, A., Puspasari, P.
2005.The Factors Associated with Calcium
Intake in Young in Bandung. Vol.24 No.1.
Hartini TNS, Winkvist A, Lindholm L,
Stenlund H, Persson V, Nurdiati DS, and
Surjono A & Hakimi M. 2003. Nutrient
intake and iron status of urban poor and
rural poor without access to rice fields are
affected by the emerging economic crisis:
the case of pregnant Indonesian women.
Eur J ClinNutr. 57(5) : 654. Available
from
http://www.ncbi.nlm.nih.gov/pubm
ed/12771966.
Houtkooper, L., Farrell, V. A. Calcium

supplement guidelines. 2004.Arizona
Cooperative Extension.
Khorrami, M, G. D. Najafpour, H.
Younesi,
M.
N.
Hosseinpour.
2012.Production of Chitin and Chitosan
from Shrimp Shell in Batch Culture of
Lactobacillus plantarum. Chem. Biochem.
Eng. Q. 26 (3) 217223.
Okoye, F.C., G.S. Ojewola and K. NjokuOnu. 2005. Evaluation of Shrimp Waste
Meal as A Probable Animal Protein
Source for Broiler Chicken. International
Journal Poult Sci., pp:
458-461.
Poeloengasihhernawan, Crescentiana D.,
Satriyo
K.Wahono
Suharto,
M.
Kismurtono. 2009 Optimization of Chitin
Production from Penaeus monodon Shells
at Ambient Temperature. ISSN 20861931.
Ravichandran, S., Rameshkumar, G.
Prince, A. R. Biochemical composition of
shell and flesh of
the Indian white shrimp (Penaeus
indicus). 2009. American-Eurasian Journal
of Scientific
Research 4 (3), 191-194. Available
from http://idosi.org/aejsr/4(3)09/13.pdf
Sini, T. K., Santhosh, S., Mathew, P. T.
2007. Study on The Production of Chitin
and Chitosan from Shrimp Shell by Using
Bacillus
subtilis
fermentation.
Carbohydrate Research ; 342(16): 2423.
Available
from
http://www.ncbi.nlm.nih.gov/pubmed/177
07781
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Trung, Trang Si, Pham Thi Dan Phuong.

2012. Bioactive Compounds from ByProducts of Shrimp Processing Industry in
Vietnam.Journal of Food and Drug
Analysis, Vol. 20, Suppl. 1, Pages 194197.
Available
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t/2014012815143852900.pdf
Page 85
THE GROWTH MODEL OF BACILLUS CEREUS IN COOKED RICE

Maria Jessica Arta1, Laksmie Hartayanie1*, Natpisit Chaitchawong2 and Patchanee
Yasurin2*
1 Department
2Food
of Food Technology, Soegijapranata Catholic University,Semarang, Indonesia
Biotechnology Program, Faculty of Biotechnology, Assumption University,Bangkok, Thailand

Corresponding author : patchaneeysr@au.edu
ABSTRACT
There are number of foodborne outbreaks attributed to Bacillus cereus have been reported
recently and all have been associated with cooked rice. This is experiment was aimed to study
the growth model of B. cereus on three different cooked rice varieties; Jasmine rice, Rice
berry, and Organic Rice berry. Three rice varieties were cooked by using electric rice cooker
in ratio 1:1 (rice:water). The growth of B. cereus on cooked rice was evaluated by using total
plate count method on Mannitol Egg Yolk Polymyxin (MYP) agar every hour for 6 hrs at
room temperature. The 1% of 0.1OD600 B. cereus was inoculated into cooked rice and
incubated at room temperature. The results showed that the specific growth rate of B. cereus
on Jasmine rice; 0.34b was significant lower than on Organic Rice berry; 0.82a hr-1 (P<0.05),
respectively. The log CFU of B. cereus was significant different (P<0.05) since 2nd hour
among two rice varieties. The t-test multiple comparison Duncan has been done by using Rstatistic. However the results helped understanding the growth ofB. cereus on cooked rice
that will be stepping stone for food safety.
Keywords: Bacillus cereus, Jasmine rice, Organic Rice berry
Page 86
Introduction
Foodborne diseases caused by pathogenic
bacteria are still a major threat to public
health. B. cereus is responsible for the
majority of foodborne illness attributed to
Bacillus. During 1993-1997, B. cereus was
linked to 14 outbreaks and caused 691
reported cases of foodborne illness in the
United States (Keith et al., 2004). B.
cereus is a Gram-positive, mobile,
facultative and aerobic spore former. In
fact, 19 of 22 strains of B. cereus is
produce enterotoxin which is known as
diarrheagenic toxin, diarrheal agent, fluid
accumulation factor, vascular permeability
factor,
dermonecrotic
toxin
and
intestinonecrotic
toxin
(Spira
and
Goepfert, 1972). Bacillus cereus is often
present as an intrinsic contaminating
microorganism in Refrigerated Processed
Foods of Extended Durability (REPFED),
pasteurized milk, rice dishes and pastas.
During shelf life it may become a major
part of the microflora. Because of its
resistant spores, significant numbers of B.
cereus have also been found in herbs and
spices, vegetables and dehydrated foods.
The presence of both vegetative cells and
spores in food commodities has been
reported and their role in food safety and
food spoilage elaborated (Priest, 1993).
The main goal of any microbiological
treatmentis to improve food quality and
extend shelf life. The safety and shelf life
of many food products is dictated by the
time and/or temperature required for
pathogenic or spoilage microorganisms to
reach a critical level (OBrien, 1996).
Thus, models are essential tools for
helping assess and manage risk to human
illness from food. Microbial modelingcan
predict
pathogen
behavior
under
conditions
for
which
no
data
exist.Although rice is an extremely
important food in many countries, very
few studies to date have focused on
thecontrol and suppression of B. cereus
contamination in rice (Kim, 2013). Still
fewer
studies
directed
at
ricemicrobiological risk assessment have
focused primarily on prediction of B.
cereus growth, especially that ofspores
during storage.
Because the spores can survive extreme
temperatures, when conditions allow they
willgerminate in contaminated food and
multiply relatively slowly. B. cereus
vegetative cells also can grow andproduce
enterotoxins over a wide range of
temperatures from 25 to 42C. Most
studies investigatingthe growth of B.
cereus on rice have employed spores under
nonstress conditions (Heo, 2009). Rice is a
daily meal for people in the AsiaPacic
region. It is believed to provide more
health benets than other carbohydrate
based foods, since it contains several
nutrients and anti-oxidative compounds.
Thai rice, especially, has gained popularity
worldwide for its nutrients and fragrance.
Rice bran is a by-product from rice
milling. This variety has a crunchy texture
and is used in desserts and salads. The
bran is naturally black (purple) and on
cooking releases a purple color. Riceberry,
a Thai black rice, has been recently
developed with the aim of providing
optimum nutritional benet to general
consumers, as well as supplementation to
anaemic and diabetes mellitus patients,
since it contains high iron and low glucose
(Leardkamolkarn et al., 2010). Jasmine
rice, called Hom Mali or KDML 105
Page 87
(Khao Dawk Mali), originates from the

Isaan region in northeastern Thailand.
Released in 1959. Jasmine rice cropping
increased by 74% from 1990 to 1998,
reaching 28.3% of rice overall acreage in
Thailand(Rahman et al. 2009).Khao Dawk
Mali (KDML) 105 is one of the most
popular rice varieties in Thailand. In
Thailand, Jasmine rice is considered as a
vital crop for domestic consumers and a
primary export commodity for economic
growth
(Sarkarung,
Somrith,
&
Chitrakorn, 2000; Chitrakorn, 2003).
Materials and Methods
Materials
Materials used in this research areOrganic
Rice Berry, Jasmine Rice, bacteria culture
of Bacillus cereus, MYP (Mannitol Egg
Yolk Polymixin), TSB (Tryptic Soy
Broth), and 0,1% Peptone water.
Equipments
Equipments used in this research are,
micropipette, tip, spectrophotometer,
shaker incubator, incubator, rice cooker,
spatula, spoon, bowl, stomacher, laminar
air flow, autoclave, tubes, beaker glass,
loop, alcohol lamp, petri dish, and sterile
cotton bud.
Rice Sample Preparation
Organic Rice Berry and Jasmine Ricewere
obtained from local fresh market in
Bangkok, Thailand. Both of rice were
wash with tap water ratio 1 : 2. Cook
organic rice berry first, because it will take
a longer time than jasmine rice. Three
hundred grams of organic rice berry was
cooked with 1,5 L tap water ratio 1 : 5.
Then, 400 grams of jasmine rice was
cooked with 800 ml tap water ratio 1 : 2. It
would take 30 minutes for jasmine rice and
45 minutes for organic rice berry. After

being cooked, both of rice was put into
sterile bowl and measured 1050 kg each
under aseptic condition. The bowl was
covered with aluminium foil and kept in
room temperature during process.
Inoculum Preparation
Culture
preparation
Biotechnology
Faculty, Assumption Universitys Stock
culture of B. cereus were prepared by
inoculating one loopful of culture into 10
mL fresh Tryptic Soy Broth (TSB) and
shake overnight by shaker incubator.
Shaker incubator used to make aeration
condition as Lawley (2008) said that B.
cereusgrow on a medium containing
oxygen (aerobic) that is also known by the
term aerobic sporeformers although they
can grow under facultative aerobic
condition. Most members of the genus
Bacillus can form endospores formed
intracellularly in response to unfavorable
environmental conditions.
Therefore, members of the genus Bacillus
have a high tolerance to environmental
conditions change. Then 1% v/v of
overnight culture was inoculated into 40
mL of fresh TSB and shake for 100 rpm,
until optical density at 600 nm reach 0.1
(SPECTRONIC,
model
GENESYS
5)which is in their early log phase. After
this, 11 ml of culture was taken by
micropipette (1% v/v of overnight culture
from 1050 kilograms samples) and put on
rice samples in the sterile bowl covered
with aluminium foil, samples were mixed
well while the culture was added.
Bacteria Growth Determination
Rice
Page 88
samples were taken and measured 25

grams each hour, then 225 ml 0,1%
peptone water was poured into plastic bag
sample under aseptic condition. Stomacher
was used to smashed rice and then
dissolved by peptone. The bacteria growth
on the samples were carried out by spread
plate method. In this method, TSB were
used as culture media. Before do spread
plate, serial dilution technique there are 101
, 10-2, and 10-3 has done, where 10-1
dilution is rice sample and peptone water.
100l of culture was swabbed on the MYP
agar by loop. All plates were put on plastic
bag and were incubated at 370C for 24
hours. Number of colony was counted by
colony counter (Stuart Scientific). This
experiment was done from zero hour and
every 1 hour after inoculation up to 6
hours and were repeated 3 times with
duplicate plates.
Statistical analysis
The experiment was done in three
replications
independently.
The
ttestmultiple comparison Duncan has been
done by using R-statistic to study the
growth model of B. cereus in cooked rice.
Results And Discussion
In this research, rice samples were
prepared first. Organic Rice Berry and
Jasmine Ricewere were washed with tap
water ratio 1 : 2. Three hundred grams of
organic rice berry was cooked with 1,5 L
tap water ratio 1 : 5. Then, 400 grams of
jasmine rice was cooked with 800 ml tap
water ratio 1 : 2. It would take 30 minutes
for jasmine rice and 45 minutes for organic
rice berry. After being cooked, both of rice
was put into sterile bowl and measured
1050 kg each under aseptic condition. The
bowl was covered with aluminium foil and

kept in room temperature during
process.Organic rice berry should be
cooked first, because it will take a longer
time than jasmine rice. Rice containing
high amylose produce dry rice product,
otherwise the rice containing low amylose
produce sticky rice and soft. Amylose
content related to the amount of water
absorption and development of the volume
of rice during cooking. The higher the
amylose content, the less sticky rice and
the harder (Juliano, 1982). Furthermore,
proposed by Haryadi (2008), rice
containing higher protein requires more
water and a longer cooking time. This
relates to the structure of the seed, which is
enclosed in a starch granule protein that
blocks the absorption of water by the
protein starch granules, and resulted in
more length of time required for cooking
so that gelatinization can take place
perfectly. In addition, high-protein rice
produce flavorful less tasty.
The Standard Total Plate Count is the most
common method used to quantify bacteria
in foods. The food to be tested is
suspended in liquid and a sample is then
spread over the surface of a solid medium
in a petri dishto perform a standard plate
count. Bacterial cells present will form
colonies that can be counted to determine
the number of cells in the original sample.
When the objective is to estimate the total
number
of
bacteria,
a
complex
mediumcalled Plate Count Agar is
commonly usedsince it will support
growth of many different types of
bacteria.We call the results the number of
Colony Forming Units (CFU), not tot;al
bacteria. This is because no single culture
medium will support all different types of
Page 89
Serial dilution technique used in this study.

The serial dilution technique done by find
the one with 30-300 colonies (Figure
4).After serial dilution technique done,
continued by spread plate method. Spread
plates, also known as lawn plates, the
culture spread evenly over the surface of
the growth medium. The spread plate can
be used for quantitative work (colony
counts) if the inoculum is a measured
volume (Kiiyukia, 2003).In the spread
plate method, 0,1 ml of diluted sample is
pipetted onto the surface of a solidified
agar medium and spread with a
sterilizedglass rod.Sterile spreaders are
used to distribute inoculum over the
surface of agar medium in plates.
Log CFU/ml
B. cereus growth curve in

jasmine rice
6
4
Series2
Linear
(Series2)
0
0
2 3 4
hour (s)
Fig 1. Growth of B. cereus in Jasmine Rice
B. cereus growth curve in organic

rice berry
8
Log CFU/ml
bacteria, we can only count those that do

grow to form a visible colony (Hocking,
2003).
6
Series2
4
2
Linear
(Series2)
0
0 1 2 3 4 5 6
hour (s)
Fig 2. Growth of B. cereus in Organic Rice
Berry
The growth curve of B. cereus in jasmine
rice and organic rice berry can be seen in
Graphic 1. and Graphic 2. Based on
Graphic 2, it appears thatB. cereus in
organic rice berry still through a phase of
adaptation (lag) is at 0 hour, but in graphic
1 different occurs inB. cereus growth in
jasmine rice.In this lag phase, B. cereusare
in a phase of adjustment to the new
environment. On microbial adaptation
phase experiencing a period in which the
cells becomelarger but the numbers remain
the same or very little development
occurred populations despite ongoing cell
metabolism.The next phase is the growth
phase logarithmic (log) achieved by B.
cereus which begins after the 1 hour to
achieve growth in both jasmine rice and
organic rice berry. The maximum on the
5th hours of incubation. This phase is the
final phase lag markedto continue splitting
the microbial cells. During log phase, cells
divide continuously with a constant current
high growth rate numbers and logof the
number of cell groups against time on a
straight line (Shantharam, 1997 in
Nurhajati
et
al.,
2009).Bacillus
cereusstationary phase on jasmine rice has
longer time than organic rice berry
Page 90
whichlook began after the 2 hours up to 5

hours of incubation, whereas in organic
rice berry look began 2 and 3 hours.
Table 3. The Growth of B.cereus(log
CFU/ml) in Jasmine rice and Organic Rice
Berry up to six hour.
Hour
Jasmine Rice
Organic
berry
Rice
3.51 0a
3.55 0.0849a
3.63 0.0306a
3.86 0.0755a
4.95 0.0300a
4.87 0.0208b
5.12 0.1217a
4.97 0.0379b
5.22 0.0265a
5.33 0.0721a
0.0306b
0.0503a
5.33
6.31
6.46 0a
6.46 0.0058a
*Remark : Different superscript within a
row show significant different (P<0.05)
Table 3. showed that the log CFU/ml of
B.cereus in jasmine rice was significantly
higher than in organic rice berry at only
2nd and 3rd hour. On the other hour log
CFU/ml jasmine rice is lower than organic
rice berry. This indicate that growth
ofB.cereus in jasmine rice is lower than
organic rice berry. Content B. Cereus in
rice poisoning causes ranges with an
average of 5 x 107 CFU/g (Supardi and
Sukamto, 1999). Consumption of food
containing more than 106B.cereus / g
(USFDA,2001) has been able to cause
food poisoning, especially in foodwhen the
preparation is left without put in the
refrigerator beforeserved. B.cereus grow
rapidly if the substrates contain

carbohydrates.Meanwhile,
when
the
substrates do not contain carbohydrates,
growth will very slow and can not form
the toxin. B. cereustoxin can reached a
maximumafter 4.5 hours (Supardi and
Sukamto, 1999).
Table 4. Comparison of specific growth
rate of B.cereus in Jasmine rice and
Organic Rice Berry.
Spesific Growth Rate (hr-1)
Repli Replic Replic
catio ation 2 ation 3
n1
Jasmi 0.36
ne
Rice
Organ 0.87
ic
Rice
berry
0.34
0.33
A
ve
ra
ge
0.
34
Stand
ard
Devia
tion
0.015
3
0.95
0.65
0.
82
0.158
1
In table 4, the specific growth rate of

B.cereus in Jasmine rice was lower than in
organic rice berry. This indicate that the
Jasmine rice have inhibitory effect on the
growth ofB.cereus.In organic rice used is
sourced from organic matter and manure
that comes from waste plant and animal
byproducts such as compost or straw or
other plant residue, whereas for the
prevention and eradication of the pest is
used biopesticide active ingredients
derived
from
plant
extracts
(Balasubramanian and Bell, 2003). On the
other hand, non-organic rice in this case
jasmine rice, does not use organic
materials in any eradication of pests, they
use chemical compound on pesticide. That
is why B. cereus growth in jasmine rice
Page 91
take longer time than organic rice berry.

Pesticide in jasmine rice still stricken and
inhibit B. cereus to grow, even though the
rice is cooked. Bacillus cereus is a foodpoisoning bacterium that maycause two
types of gastrointestinal disorders: the
emeticsyndrome, caused by ingestion of a
preformed toxin in thefood, and the
diarrhoeal syndrome, caused by a different
toxinthat can be formed in the food but
also in the small intestine(Granum and
Lund, 1997; Granum, 2001). Due to
itsubiquitous distribution in nature, B.
cereus occurs frequentlyin a wide range of
food raw materials.
Rice-based products andfarinaceous foods
such as pasta and noodles are
frequentlycontaminated and involved in B.
cereus poisoning (Kramer and Gilbert,
1989). Levels of B. cereus greater than 103
cfu/g havebeen found in both cooked and
uncooked rice and in cerealproducts all
over the world.
Natural antimicrobial substances, such as
bacteriocins, are being investigated for
food preservation and to replace chemical
preservatives (Cleveland et al., 2001),
although nisin is currently the only
bacteriocin widely used (Thomas et al.,
2000). Some bacteriocins, such as nisin or
lacticin 3147, prevent spore outgrowth and
enterotoxin production by B. cereus
(Jaquette and Beuchat, 1998).
There are two types of B. cereus food
poisoning. The first type, caused by an
emetic toxin, results in vomiting, while the
second type, caused by enterotoxins, gives
diarrhoea (Kramer, 1989). In a small
number of cases both types of symptoms
are recorded , probably due to production
of both types of toxins. Although the
enterotoxins can be preformed, the number

of B. cereus cells in the food would be at
least two orders of magnitude higher than
that necessary for causing food poisoning,
and such products would no longer be
acceptable to the consumer (Granum,
2001).
The emetic toxin causes emesis (vomiting)
only and its structure has for a long time
been a mystery, as the only detection
system involved living primates(Kramer,
1989). The emetic toxin is resistant to heat,
pH and proteolysis but is not antigenic. It
is not clear if the toxin is a modified gene
product or if it is enzymatically produced
through modification of components in the
growth medium. However, with such a
structure it is most likely that cereulide is
an enzymatically synthesised peptide and
not a genetic product.
Bacillus cereus is ubiquitously present in
soil, vegetation water and dust. It has been
isolated from a large variety of foods,
including vegetables, meat, cereals,
pasteurized fresh milk and powdered milk
and processed foods. Under favourable
conditions, the organism multiplies and
causes gastrointestinal illness. It is
implicated in two different forms of food
poisoning; an emetic illness and a
diarrhoeal illness. The emetic illness is
mediated by a highly stable toxin that
survives high temperature, exposure to
trypsin, pepsin and pH extremes. The
diarrhoeal illness is mediated by a heat and
acid labile enterotoxin. Lecithinase activity
is the key reaction in the differential
identification of B.cereus, the most
commonly encountered and important
species in clinical laboratories, from the
majority of the other Bacillus species
Page 92
(Bergdoll, 1981). If unknown isolate

produceslecithinase, Bacillus cereus can be
presumptively identified by also observing
colonial morphology, hemolytic reactivity
and motility tests.In 1967, Mossel et al
formulated Mannitol-Egg Yolk-Polymyxin
(MYP) Agar, which is recommended by
APHA to isolate and enumerate B.cereus
from foods, whose principles were based
on two factors, there are the lack of
mannitol fermentation by Bacillus cereus
and the presence of a lecithinase in
majority of tested strains.The authors
showed that satisfactory selectivity was
obtained with polymyxin B. When present
in large numbers in certain foodstuffs,
B.cereus
can
produce
metabolites
responsible for the clinical symptoms of
food
poisoning.
This
medium
differentiates B.cereus from other bacteria
based on the basis of lecithinase activity,
mannitol fermentation and resistance to
polymyxin (Rhodehamel, 1995).
MYP Agar contains peptic digest of
animal tissue and meat extract, which
provide nitrogen source. Mannitol
fermentation can be detected by phenol
red, which yields yellow colour to the
mannitol fermenting colonies due to acid
production.
Added egg yolk emulsion helps in
differentiation of lecithinase producing
colonies, which are surrounded by a zone
of white precipitate. Addition of
Polymyxin B Sulphate (FD003)used to
inhibit accompanying microflora when the
tested sample is heavilycontaminatedand
helps to restrict growth of gram-negative
bacteria such as Escherichia coli and
Pseudomonas
aeruginosa.
These
differentiating media allow differentiation
of B.cereus from other Bacillus species by

its inability to ferment mannitol and poor
sporulation. B.cereus dissimilates egg yolk
and gives rise to typical bacilli form
colonies. MYP used as selective media
which are designed to suppress the growth
of unwanted bacteria and encourage the
growth of the desired microorganisms.
Antibiotics, high concentrations of salt, or
high acidity might be used.MYP Agar
could be storage at 6 12Cand have shelf
life 14 weeks.
Conclusion
Spesific growth rate of Bacillus cereus in
jasmine rice is lower than organic rice
berry.Mannitol Egg Yolk Polymixin Agar
is selective media for Bacillus cereus.
Acknowledgements
I would thanks to my Lord Jesus
Christ who is always bless me and
also to my Family which is always
support and pray for me everyday. Big
thanks to Asst.Prof. Dr. Patchanee
Yasurin and Dr. Laksmie Hartayanie
M.P as my advisor who was advising
me for making this paper and Natpisit
Chaitachawong as my partner who
always support and accompanied me
duringresearch and report making from
Assumption University, Thailand. Last
but not least, I thanks to my Dean Mrs.
Dr. Victoria Kristina Ananingsih, Msc.
for picking me to this great opportunity
to participate this internship program.
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Page 96
UTILIZATION OF KERSEN (Mutingia Calabura L.) LEAVES AS

FUNCTIONAL DRINK
Eugenia Priscilia Kusumaa*, Shianne Puspita Putria, Patricia Andikaa
Food Technology Department, Faculty of Science and Technology, Universitas Pelita
Harapan
Jalan MH Thamrin Boulevard Raya 1100, Tangerang, Indonesia
*Email: nia_violin@yahoo.com
ABSTRACT
Kersen (Mutingia calabura L.) leaf is a kind of fruit that has not been widely applied
in food and beverage products. Several studies have shown that kersen leaves actually have
high phenolic content, thus has a great potency to be utilized in functional food products.
This research was aimed to determine the extraction method and number of stages in the
production of kersen tea that yield highest antioxidant activity, total phenolic and total
flavonoid, yet still acceptable. Two different extraction methods and six different number of
extraction stages were used as the treatments. The antioxidant activity, total phenolic and
total flavonoid of the tea resulted were then assessed by using DPPH assay, FolinCiocalteau assay and aluminum chloride method, respectively. Scoring and hedonic test were
performed on each result of extractions. The results showed that there was significant effect
of interaction between the extraction method and the stages (p <0.05) on the antioxidant
activity, total phenolic and total flavonoid of kersen tea. Kersen tea processed using
boiling method and extracted with four stages has the highest antioxidant activity of 195.580
8.540 mg AAE/L, total phenolic content of 608.889 2.143 mg GAE/L and total flavonoid
content of 309.922 5.058 mg QE/L. However, the hedonic test results showed that the
sample had a taste and aftertaste that was less preferred by panelists. The acceptance of
kersen tea was further optimized by the addition of 10% sugar and different concentration
of cinnamon extract. The concentration of 20%, 25% and 30% (v/v) of cinnamon extracts and
10% (w/v) sugar were added into the kersen tea produced by boiling in 4 stages. The addition
of 30% cinnamon extracts has shown to increase the acceptance of panelists to slightly
preferred.
Keywords: antioxidant, Mutingia calabura L. leaves, extraction method, extraction stage, tea.
Page 97
INTRODUCTION
Kersen (Muntingiacalabura L.) is a plant
that could grow up to 5-12 meters and
produces red small fruits with sweet taste.
The leaves of this plant are dark green in
color on the surface part, whilst grey color
on the downside part. It grows and spreads
horizontally with oval shape and simple
lancet that leads to the end. The length of
the leaves ranges from 4 to 14 cm,
meanwhile the width ranges from 1 to 4
cm (Janick and Paul, 2008; Gargiullo,
2008).
Several studies showed that kersen leaves
contain bioactive compounds that could
bring health benefit for human. kersen
leaves have anti-inflammatory, antipyretic,
antiproliferative (Zakaria, et al., 2007;
Zakaria, et al., 2011), antioxidant
(Narayanaswamy and Duraisamy, 2011;
Su, et al., 2003), and anticancer activity
(Kaneda, et al., 1991; Lim, 2012). In
addition, the leaves also have high
contents of phenolics, flavonones, and
flavones (Lim, 2012; Su, et al., 2003).
Previous research conducted by Su, et al.
(2003); Sindhe et al. (2013) found that
methanolic extract of kersen leaves has
high antioxidant activity. However, there
is notmany research conducted to utilize
this leaves into beverage products.This
study was aimed to observe the effect of
extraction methods and stepstowards the
antioxidant activity of the kersen leaves
tea beverage. In this study, the process of
making tea drink was carried out by
extracting kersenleaves using water with
different heating process and different
stage of extraction. The effects of those
treatments were then assessed by
analyzing the antioxidant activity, total

phenolic and flavonoid as well as the
sensory.
RESEARCH METHODOLOGY
Materials and Equipment
The materials used in the making of
Mutingiacalabura L. leaves tea were
kersen leaves (MutingiacalaburaL.) from
Villa Permata, Karawaci, Tangerang,
drinking water and Madu Super
Nusantara honey, cinnamon, and sugar.
Materials used for the analysis were 2,2Diphenyl-1-picrylhydrazyl (DPPH), pro
analysis methanol solution, FolinCiocalteau reagent, Na2CO3 7.5% solution,
aquadest, gallic acid standard solution,
NaNO2 5% solution, AlCl3 2% solution,
NaOH 1 M solution, and quercetin
standard solution.
The equipment used in this research were
oven, digital balance, container, spoon, gas
stove, pan, thermometer, stopwatch, filter,
spatula, stirring bar, funnel, micropipette,
tips, UV/Visible spectrophotometer, and
cuvette.
RESEARCH METHODS
Preliminary Research
Kersen Leaves Drying Methods
The making of kersen leaves tea started
with leaves sortation. This process was
carried out to separate the kersen fruits and
branches from the leaves. Kersen leaves
were subsequently washed 3 to 5 times to
remove the mucilage. The leaves were
then arranged in the tray to be dried at
70C for 2 hours in the oven. The dried
leaves were then ready to be used.
Page 98
Research stage I
Kersen
Leaves
Tea
Production
Procedure
One gram of the dried leaves was weighed
for 60 mL of drinking water. Theleaves
extraction process was carried out by using
boiling and brewing methods. The boiling
method was conducted at the temperature
of 100C and the brewing method was
conducted by using hot water (start at
100C). Both were carried out with the
total time of 540 seconds.The treatments
for this experiment were shown on Table
1. Honey (10% (v/v)) was then added into
the tea. Analysis for kersen leaves tea
consisted of antioxidant activity, total
phenolic and flavonoid content, and
organoleptic (scoring and hedonic test).
Table 1. Thetime and Volume of water
used in the making ofKersen leaves tea by
using boiling or brewing with different
stages
Stages
Time used for each stage (sec)
1 stage
540
2 stages
270
3 stages
180
4 stages
135
5 stages
108
6 stages
90
minutes. Different concentration of

cinnamon extract (20%, 25% and 30%
(v/v)) was then added to the tea. The
additional sugar of 10% (w/v) was then
added to the tea. Hedonic test on kersen
leaves tea was conducted using 70
panelists.
Product Analysis Procedure
Antioxidant Activity (Tariq andReyaz,
2013 with modification)
Antioxidant activity assay was done using
DPPH method. Stock solution of DPPH 1
mM was made by dissolving 10 mg of
DPPH to the methanol solution until the
volume reach 25.4 ml. 10 ml of stock
solution was taken and diluted until 25 ml
to produce 0.4 mM DPPH solution.
Analysis of sample was done by mixing 1
ml 0.4 mM DPPH solution with 0.8 ml
sample. Control solution was made by
mixing 1 ml 0.4 mM DPPH solution with
0.8 ml methanol solution. 1.2 ml of
methanol
solution
used as blank
Water
(ml) for was
each stage
solution for this60analysis. Antioxidant
activity
measured
with
visible
30
spectrophotometer20with 517 nm for the
wavelength. Antioxidant
activity of sample
15
was calculated 12using the following
formula:
10
Inhibition (%) =
Research stage II
In this stage, kersen leaves tea that has
the highest antioxidant activity, total
phenolic and flavonoid content, would be
further developed into a drink by using the
chosen extraction method and stages from
research stage 1. Some amount of sugar
and cinnamon extract was added to
optimize the acceptance of consumers. The
cinnamon extract was made by boiling the
cinnamon in the water with ratio 1:2 for 15
Absorbance control Absorbance sample

Absorbance control
Antioxidant activity of sample was

expressed in mg Ascorbic Acid Equivalent
(AAE) per liter sample. Ascorbic acid
standard curve was made based on the
analysis of some concentration of ascorbic
acid solution (2.5; 5; 10; 20; 25 and 30
ppm). The results then plotted to the
percent inhibition graph as Y axis to the
ascorbic acid concentration as X axis.
Page 99
Based on the graph, line equation

y=3.3121x-1.8782was obtained.
Total Phenolic Content (Anesini, et al.,
2011)
0.3 ml of sample was placed to the test
tube, then 1.5 ml FolinCiocalteu reagent,
that has been diluted 10%, was added to
the sample. After that, 1.2 ml Na2CO3
7,5% solution was added to the test tube,
then stored in the dark room for 60
minutes. The absorbance of solution then
was read using spectrophotometer in 750
nm of wavelength.
Total phenolic content of sample was
expressed in mg gallic acid equivalent per
liter sample. Gallic acid standard curve
was made based on the analysis of some
concentration of gallic acid solution (10,
20, 30, 40, 50, 60, 70, 80, 90 and 100
ppm). The results then plotted to the
absorbance graph as Y axis to the gallic
acid concentration as X axis. Based on the
graph, line equation y = 0.0099x + 0.0417
was obtained.
Total Flavonoid Content (Alimpi, et al.,
2013 with modification)
0.25 ml of sample was diluted using 1.25
ml aquadest in the test tube, then 75 L of
NaNO2 5% was added to the test tube. The
solution then was stored for 6 minutes.
After that, the solution was added with 5
ml of AlCl3 2% and stored for 5 minutes.
0.5 mL of NaOH 1 M and 775 L of
aquadest then added to the solution. The
absorbance of solution then read using
spectrophotometer in 415 nm of
wavelength.
Total flavonoid content of sample
expressed in mg quercetin equivalent (QE)
per 100 gram sample. Quercetin standard

curve was made based on the analysis of
some concentration of gallic acid solution
(50,100, 150, 200, 300 and 350 ppm). The
results then plotted to the absorbance
graph as Y axis to the quercetin
concentration as X axis. Based on the
graph, line equation y = 0.002936x +
0.01257 was obtained.
Organoleptic Analysis
The organoleptic analyses were assessed
by 70 untrained panelists. The parameters
for the analyses were color, aroma, taste
and aftertaste. The scoring test was done
by assigning score of tested parameter
from 1 to 6, where 1 represents extremely
no brown color/ extremely no foreign
aroma/ extremely astringent/ extremely
aftertaste and 6 represents extremely
brown color/ extremely leafy aroma/
extremely not astrigent/ extremely no
aftertaste. The hedonic test was done using
9 points hedonic scale, where 1 represents
extremely dislike the product and 7
represent extremely like the product.
Statistical Analysis.
All measurements were carried out in at
least two replicate experiments and the
results were reported as the means. These
data were analyzed by using one way
ANOVA. Differences between means at
the 5% level were considered as
significant.
RESULTS AND DISCUSSIONS
Effect of Extraction Method and Stage
on Antioxidant Activity, Total Phenolic
and Flavonoid Content
Based the statistical analysis, there
was significant effect of interaction
between the method and stages of
Page 100
(119.345 7.472)e
(77.2273.416)abc
3
4
Extraction Stage
(170.823 2.562)f
(87.7945.978)bcd
(195.580 8.540)h
(97.7579.821)cde
(157.840 5.124)f
(73.60321.349)cde
(153.915 8.967)f
(62.1305.124)a
400
350
300
250
200
150
100
50
0
(108.7781.067)de
(59.715 14.517)a
Antioxidant Activity
(mg AAE/L)
extraction on the antioxidant activity, total phenolic and flavonoid content (p <0.05) of kersen
leaves tea. Results of the antioxidant activity analysis, phenolic content analysis and
flavonoid content can be seen in Figure 1, Figure 2, and Figure 3 respectively
Boiling
Brewing
200
(475.5566.428)f
(250.303 0.714)c
400
(403.83812.142)e
(307.8797.857)d
600
(608.8892.143)h
(316.46518.571)d
800
(418.9909.285)e
(164.949 1.428)b
1000
(405.8592.143)e
(112.9295.000)a
Total Phenolic Content

(mg GAE/L)
1200
(551.8184.285)g
(241.21219.285)c
Note:Different superscript indicates that there is significant difference (p>0.050)

Figure 1 Antioxidant activity kersen leaves tea
Boiling
Brewing
0
3
4
Extraction Stage
200
(193.4376.984)f
(156.1411.927)d
300
(200.2492.649)f
(169.0841.445)e
400
(255.7666.021)i
(144.5612.408)c
500
(224.4310.723)h
(135.8750.241)b
600
(212.1701.686)g
(119.5275.058)a
Total Flavonoid Content

(mg QE/ L)
700
(309.9225.058)j
(172.3190.241)e

Figure 2 Total phenolic content kersen leaves tea
Boiling
Brewing
100
0
3
4
Extraction
Stage

Figure 3 Total flavonoid contentkersen leaves tea
Page 101
The graphs showed that the increase of

antioxidant activity along with the increase
of total phenolic and flavonoid content.
This indicates that the antioxidant activity
was mostly contributed by the flavonoid
(Stankovi, 2011; Alimpi, et al.,
2014).The antioxidant activity, phenolic
and flavonoid content of boiled kersen
"tea" were higher than the brewed kersen
"tea". The kersen leaves in boiled tea
was heated in hot water at a temperature of
100C for a longer time than kersen leaves
in brewed tea. In the brewing process,
the temperature of the water was allowed
to decrease during the extraction process.
According to Sahin (2013), the extraction
process of phenolic and flavonoid content
wasmore efficient at higher temperature of
water. It has also been reported that the
maximum extraction can be achieved at
100C. According to Shi, et al. (2003),
higher temperatures can also cause
softening plant tissues, damaging the
interaction
between
the
phenolic
compound with protein or polysaccharide,
and help hydrolyze phenolic compounds
bonds that enhance the solubility of
phenolic compounds. This process can
increase the rate of diffusion so the
extraction rate is higher (Shonisani, 2010).
Moreover, according to Sathiskumar, et al.
(2008), the higher the temperature, the

speed of movement of molecules are also
higher so that the diffusion of flavonoids
may occur more rapidly from the cell into
the solvent. This causes the boiling process
contains higher phenolic and flavonoid
content thus antioxidant activity was also
higher.
In fact, the antioxidant activity, phenolic
and flavonoid content of kersen "tea"
increased from one stage to the four
stages, then decreased in five and six
stages. The increasing of the antioxidant
activity, phenolic and flavonoid content at
the one stage to four stages of extraction
was related to the characteristic of the
extraction that would be higher when
carried out in multiple stages. According
to Aguilera (2003), the extraction rate is
influenced by the concentration gradient.
Replacement of water resulted in the
concentration gradient between the solvent
and kersen leaves in every stage were
always higher so that the extraction
process was faster. The extraction used 5
and 6 stages resulted in the less water used
for extraction in each stage. The leaves
could not be entirely immersed in less
water, so the extraction process running
would be less effective.
Page 102
Effect of Extraction Method and Stage on Sensory Characteristics
Number of
Method
Extraction
Color
Aroma
Taste
Extraction
Stage
(Stages)
1
(4.71.1)bcd
(4.11.3)ab
(3.01.4)bc
2
(4.81.1)cd
(4.21.4)abc
(2.51.3)b
3
(4.71.2)bcd
(4.21.3)abc
(2.51.3)b
Boiling
4
(4.81.3)cd
(4.71.4)cde
(3.01.5)bc
5
(4.21.2)a
(3.81.3)a
(1.91.0)a
6
(4.21.1)a
(4.01.4)ab
(2.51.2)b
1
(4.91.2)cd
(4.31.5)abcd
(3.21.6)c
2
(4.91.2)cd
(4.01.4)ab
(3.01.4)c
3
(4.81.2)cd
(4.21.4)abc
(3.01.4)c
Brewing
4
(4.61.3)abc
(4.51.4)bcde
(4.31.5)d
5
(4.3 1.3)ab
(4.81.3)de
(4.31.3)d
6
(5.1 1.3)d
(4.91.6)e
(4.41.5)d
Table 3. Scoring result
Table 4. Hedonic result
Number of
Method
Extraction
Color
Aroma
Taste
Extraction
Stage
(Stages)
1
(4.90.9)de
(3.941.4)cde
(2.91.5)de
2
(5.00.8)e
(4.11.2)de
(2.71.6)e
3
(4.80.9)de
(3.761.3)cd
(2.91.6)de
Boiling
4
(3.11.1)b
(2.81.2)a
(4.71.4)b
5
(3.261.1)b
(3.51.3)bc
(5.11.1)b
6
(3.21.0)b
(4.11.7)de
(4.71.4)b
1
(4.41.1)c
(4.11.3)de
(3.31.4)c
2
(4.4+0.9)c
(4.01.3)de
(3.21.4)c
3
(4.60.9)cd
(4.31.2)de
(3.11.5)cd
Brewing
4
(2.71.1)a
(4.31.4)de
(3.91.3)a
5
(3.31.0)b
(3.11.3)ab
(3.81.3)b
6
(2.61.2)a
(3.11.2)a
(3.91.4)a
Aftertase
(2.91.5)bc
(2.61.3)ab
(2.81.5)bc
(2.91.4)bc
(2.21.1)a
(2.21.3)a
(3.01.5)bc
(3.01.4)bc
(3.11.5)bc
(4.41.7)d
(4.71.5)d
(3.21.5)c
After Taste
(2.81.5)a
(2.71.6)a
(2.81.4)a
(4.71.4)bc
(4.81.4)c
(4.51.2)bc
(3.01.4)a
(3.11.5)a
(2.91.5)a
(4.51.3)bc
(2.71.4)a
(4.31.5)b
Page 103
Scoring and hedonic test in term of color,

aroma, taste and aftertaste were conducted
in order to observe effect of extraction
method and stage in term of parameter.
Based on statistical analysis, there was
interaction between extraction method and
stage on color, aroma, taste and aftertaste
(p0.05).
Based on the organoleptic test results,
kersen "tea" obtained by four stages of
boiling method had the highest antioxidant
activity. However, it was less preferred by

consumers. It was mostly because of the
high
astringent
taste
and
aftertaste.Kersenleave
has
epigallocatechingallateor
EGCG
(Surjowardojo, et al., 2014) and tannins
(Zakaria, et al., 2007; Surjowardjojo, et
al., 2014). According to Obst, et al.,
(2013), EGCG can produce long lasting
astringency. In addition, tannin also gives
astringent taste in this tea (Tharayil, et
al., 2011).
Hedonic result for color
Effect of Cinnamon Extract Addition on Panelists Acceptance

7,0
6,0
5,0
4,0
3,0
2,0
1,0
(5.41.1)b
(5.31.1)b
20%
25%
Cinnamon extract concetration
(4.81.4)a
30%
Hedonic result for

aroma

Figure 4. The result of acceptance test for color of Kersen leaves tea
7,0
6,0
5,0
4,0
3,0
2,0
1,0
(4.51.5)a
(4.71.5)a
(4.71.5)a
20%
25%
30%

Figure 5. The result of acceptance test for aroma of Kersen leaves tea
Page 104
Hedonic result for taste
7,0
6,0
5,0
4,0
3,0
2,0
1,0
(5.1 1.2)c
(4.41.4)b
20%
(3.71.4)a
25%
30%
Note:Same superscript alphabets indicate no significant difference (p>0.050)

Figure 6. The result of acceptance test for taste of Kersen leaves tea
Hedonic result for

aftertaste
7,0
6,0
5,0
(5.11.2)c
(4.31.5)b
(3.81.4)a
4,0
3,0
2,0
1,0
20%
25%
30%
Note:Same superscript alphabets indicate no significant difference (p>0.050)

Figure 7 The result of acceptance test for after taste of Kersen leaves tea
Sensory test in term of color, aroma, taste
and aftertaste were conducted in order to
observe effect of cinnamon extract
concentration on Kersenleaves tea.
Based on statistical analysis, there was
significant effect on color, taste and
aftertaste (p0.05) and no significant
effect on aroma.
Based on the results of the hedonic test,
the kersen "tea" with 4 stages of boiling
method modified with 30% of cinnamon
extract and 10% of sugar could increase
the consumers acceptance of kersen "tea"
in three attributes assessed. The addition of
10% table sugar contributed to the increase
of consumers' acceptance in kersen tea,
although the addition of the table sugar
had a similar function to the addition of
honey, i.e. as a sweetener. The results can
be explained by McGee (2004) theory.

Different types of sugar can give a
different impression of sweetness. The
sweet taste of fructose, found in honey,
come up fast and strong, but also
disappears quickly. Sweetness of sucrose,
which is found in table sugar, takes time to
be detected on the tongue and can last
longer.The addition of cinnamon extract
was acted as the natural flavor, meanwhile
the addition of sugar was acted as the
natural sweeteners. The addition of both
flavor and sweetener was proved in
increasing the consumers acceptance
because they could mask the undesirable
taste. These results correspond with
Deepak, et al. (2012) statement that the
addition of the cinnamon extract can mask
the taste and aftertaste with the pleasant
flavor of cinnamon.
Page 105
CONCLUSION
Extraction methods and stages affect
antioxidant activity, total phenolics
andflavonoid content. Kersen tea
processed with four stages of boiling
method had the highest antioxidant
activity, totalphenolics and flavonoid
content, but not preferred by panelists.
Modification of kersen tea with 30%
cinnamon extract and 10% table sugar
could improve panelists acceptance.
ACKNOWLEDGEMENTS
This study was supported by a student
research funding from Food Technology
Department, Faculty of Science and
Technology,
UniversitasPelitaHarapan.
The author would like to thank Ms. Julia
RatnaWijaya, MAppSc and Isabella
Supardi, S.TPfor the guidance, support,
time and advices during the research and
completion of the paper. The author would
also like to thank Nadia Catherine Prima
Onasiefor helping the authorduringthe
completion of the paper.
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Page 108
EFFECT OF CHROMANONE DEAMINE LEVEL ON OF FRESH AND

FROZEN CHICKEN BROILER MEAT QUALITY
Stefany Widjaya1), Sumardi 2) and Ch. Retnaningsih 2)
1)Student
and 2) Lecturer, Department Food Technology, Faculty of Agricultural Technology,

Email : stefanywidjaya@yahoo.co.id
ABSTRACT
A high consumption rate of chicken broiler meat encouraging efforts to improve its quality,
which one is by inducing chromanone deamine. Induction of small amount chromanone
deamine into animal digestion system has been scientifically proven to reduce FCR (Feed
Conversion Ratio), reduce fat content and increase protein content in chicken broiler meat.
Recently most of chicken broiler meat is sold in frozen form. Therefore the aim of this
research is to evaluate the effect of chromanone deamine level on fresh and frozen chicken
broiler meat quality. This research was conducted by inducing day old chick with
chromanone deamine level of 0, 1, 5, 10 and 50 ml for 30 days since chick in period. After 30
days of treatment, the chicken were slaughtered and its quality parameters including pH,
color, texture, water content, protein content and fat content were evaluated. Afterward, the
carcass were frozen at -20oC for 3 weeks and its quality were re-evaluated. The result
indicated that induction of chromanone deamine was effectively reducing pH, improving
texture, increasing protein content, reducing water content and fat content. Overall, the
induction of 10ml chromanone deamine produced the best meat quality, not only the fresh but
also the frozen one.
Keywords: Chicken, Chromanone Deamine, Fresh, Frozen, Quality
Page 109
INTRODUCTION
Chicken broiler meat is one of nutritous
food with good taste, flavor and texture
also relatively cheap (Kasih et al, 2012).
The nutritional value of chicken broiler
meat is shown in following table:
Table 1. Nutritional Value of Chicken
Meat
Parameter
Amount
Water Content
74,70 g
Protein Content
22,25 g
Fat Content
1,60 g
Saturated Fatty Acid
0,52 g
Monosaturated Fatty Acid
0,75 g
Polyunsaturated Fatty Acid 0,32 g
Cholesterol
59,00 mg
Energy
104,65
kcal
(Probst, 2009)
The average number of chicken
consumption in Indonesia is about 8kg per
capita every year. The high consumption
rate of chicken broiler meat encouraging
efforts to improve its quality, which one is
by inducing chromanone deamine.
Induction of small amount chromanone
deamine (0,0025ml per Kg body weight)
into animal digestion system has been
scientifically proven to reduce FCR,
reduce fat content and increase protein
content in chicken broiler meat (Pusparini,
2008).
Recently due to high demand, most
chicken meat is sold in frozen state to
maintain its quality during distribution
(Matulessy et al, 2010). However freezing
process may cause some changes on food
characteristics. Therefore this research is
aimed to evaluate the effect of
chromanone deamine level on fresh and

frozen chicken broiler meat quality.
MATERIAL & METHOD
This research was done in 2 steps of
studies : field study and laboratory study
Field Study
The field study was conducted at SMK
Theresiana Bandungan. 100 day old chick
(DOC) were separated into 5 groups of
treatment. Each group was treated with
different level of chromanone deamine for
30 days. The chromanone deamine level
being treated to the chicken were 0 ml, 1
ml, 5 ml, 10 ml, and 50 ml. After 30 days
of treatment, the chicken were slaughtered
and directly brought to the laboratory for
further analysis.
Laboratory Study
The evaluation of chicken meat quality
was conducted by analyzing its quality
parameters
including pH value, lightness value,
hardness value, water content, protein
content and fat content. After the fresh
chicken meat samples were evaluated, the
rest of chicken meat samples were frozen
at -20oC for 3 weeks. One day before
frozen meat evaluation, samples were
thawed by put it in the refrigerator (4oC).
The thawed meat samples were evaluated
afterward.
RESULT & DISCUSSION
The result shows that induction of
different level of chromanone deamine
significantly
affect
meat
quality
parameters including pH, color, texture,
water content, protein content and ash
content. The data of those parameters are
shown in following tables
Page 110
Table 2. pH Value of Fresh and Frozen

Chicken Broiler Meat
Treatment
0 ml
1 ml
Fresh
Frozen
6,20 0,02 c 6,04 0,04 d
5,91 0,07 a 5,84 0,04 c
6,09 0,12
5 ml
5,75 0,04 b
b
6,08 0,04 5,73 0,04
10 ml
b
ab
6,03 0,04
50 ml
5,70 0,02 a
b
*Figures followed with same letter
indicates not significantly different at 95%
degree of confident
Table 2 shows that induction of
chromanone deamine results in meat with
lower pH value. The reduction of pH value
is due to anti-stress activity of chromanone
deamine (Pusparini, 2008). Pre-slaughter
stress on chicken may increase metabolism
rate, leading to decrease of muscle
glycogen availability. Muscle glycogen
will be converted into lactic acid after
rigor mortis, so the low glycogen
availability results in higher pH value
since the lactic acid formed is also less
(Allen et al, 1998). Therefore induction of
chromanone deamine lead to lower pH
value due to suppression of pre-slaughter
stress.
Furthermore, Table 2 also shows that pH
value of frozen meat is lower than the
fresh one. According to Akhtar et al
(2013), the frozen meat tend to have lower
pH value than the fresh one. This is due to
water loss during freezing and thawing
process that leads to protein denaturation.
Denaturation causes protein to lose its H+
ion, leading to lower pH value.
Table 3. Lightness Value of Fresh and

Frozen Chicken Broiler Meat
Treatment
Fresh
Frozen
51,24 1,76 51,50 2,75
0 ml
a
a
53,36 4,25 54,25 1,81
1 ml
a
a
54,84 1,34 53,85 2,37
5 ml
a
a
56,68 5,14 53,50 1,82
10 ml
a
a
54,41 4,46 54,50 1,12
50 ml
a
a
degree of confident
Table 3 shows that there is no significant
different between control and the treated
ones. However it is shown that induction
of chromanone deamine produce lighter
color on both fresh and frozen meat
compared to the control. According to
Fletcher (1999) the lighter color of meat is
obtained due to lower pH value. The lower
pH value, the higher lightness value will
be.
Table 4. Hardness Value of Fresh and
Treatment Fresh (gf)
4253,7 67,3
0 ml
a
4458,7 70,4
1 ml
a
5121,4
5 ml
310,4 c
4835,5
10 ml
197,9 b
4323,1 46,1
50 ml
a
Frozen (gf)
2140,1
401,8 a
2276,4
251,7 a
2623,9 60,2
b
2307,3
180,6 a
2188,1
136,7 a
Page 111

degree of confident
chromanone deamine leads to harder
texture of meat, both fresh and frozen.
According to Afrianti et al (2013) and
Wattanachant (2008), this result is related
to protein and fat content. Previous
research found out that chicken meat
texture is affected by protein and fat
content. Higher fat content will make the
texture more tender, while higher protein
content will make the texture harder.
Another research prove that induction of
chromanone deamine result in lower fat
content and higher protein content
(Pusparini, 2008). This research also
appear to have the same result as it is
shown in Table 6 and 7. This means that
induction of chromanone deamine lead to
fat content reduction and protein content
increase, so that the meat texture is
become harder.
As it is shown in Table 4, the hardest fresh
and frozen meat is obtained from chicken
with induction of 5ml chromanone
deamine. However there is a significant
different of hardness value between fresh
and frozen meat. The hardness value of
frozen meat is only about a half of the
fresh one. This is because during freezing
process, the formation of crystal ice may
break apart the myofibril protein so its
physical structure is broken and meat
become softer (Akhtar et al, 2013).
77,37 0,58 73,52 0,23

b
c
74,83 2,55 73,15 0,20
1 ml
a
bc
75,40 1,26 72,76 0,44
5 ml
a
b
74,34 0,71 72,18 0,34
10 ml
a
a
74,62 1,26 71,97 0,51
50 ml
a
a
degree of confident
0 ml

chromanone deamine results in lower
water content. This result indicates that
chromanone deamine may cause a diuretic
effect, which mean improving excretion
rate so that the water content in chicken
body is lower (Mubin, 2013). As it is
shown in Table 5, induction of 10ml
chromanone deamine produce the lowest
water content of fresh meat, while the
lowest water content of frozen meat is
obtained from induction of 50ml
chromanone deamine.
Compared to the fresh meat, the frozen
meat tend to have lower water content.
According to Akthar et al (2013) this
result is due to freezing and thawing
process. During freezing, denaturation of
protein is inevitable, resulting in weaken
water holding capacity. As the meat state
remain frozen, the water will remain in the
meat, but when the meat is thawed the
water will exudate, leaving a decrease on
its water content.
Table 5. Water Content of Fresh and

Treatment
Fresh (%)
Frozen (%)
Page 112
Table 6. Protein Content of Fresh and

Treatment
Fresh (%)
Frozen (%)
19,80 0,59 21,52 0,26
0 ml
a
a
20,00 1,28 24,61 0,51
1 ml
a
c
21,28 1,58 25,28 0,64
5 ml
ab
c
23,52 1,05 25,09 0,62
10 ml
c
c
22,52 0,94 22,73 0,31
50 ml
b
b
*Figures followed with
same letter
degree of confident
chromanone
deamine
significantly
increase protein content. The induction of
10 ml produce fresh meat with highest
protein content, while the highest protein
content in frozen meat is found to be in the
one treated with induction of 5 ml. This
result indicates that induction of
chromanone deamine may improve protein
synthesis in living chicken as the same
result appear in Sunaryanto & Sumardis
(2008) research. In addition, the protein
increase may also comes out as the result
of water content and fat reduction.
According to Winarso (2003) water
content, protein content and fat content is
inversely proportional to each other. A
decrease of one component may cause
increase in other ones. Those statement
also explain the increase of protein content
in frozen. As it is shown in Table 6, the
frozen meat has higher protein content
compared to the fresh one. This may also
as the result of water loss during freezing
and thawing process. Even though a
denaturation appear during freezing, it
doesnt significantly affect the protein

nutritional value, therefore the frozen meat
seems to have higher protein content due
to water loss.
Table 7. Fat Content of Fresh and
Treatment
Fresh (%)
Frozen (%)
2,02 0,11
0 ml
3,22 0,12 a
bc
1 ml
2,25 0,42 c 2,18 0,34 b
5 ml
1,8 0,08 ab 1,74 0,20 c
10 ml
1,67 0,12 a 1,39 0,08 d
50 ml
2,14 0,23 c 1,44 0,09 d
degree of confident
chromanone deamine significantly reduce
fat content. The lowest fat content in both
fresh and frozen meat appears to be the
ones treated by induction of 10 ml
chromanone deamine. This result indicates
that chromanone deamine also has a antihyperlipidemic activity, which mean it can
affect the fat and lypoprotein metabolism
(Mubin, 2013).
CONCLUSION
Induction of chromanone deamine is
significantly affect chicken broiler meat
quality. Induction of chromanone deamine
result in meat with lower pH value, lighter
color, harder texture, lower water content,
lower fat content and higher protein
content. Freezing process leads to lower
pH value, lower hardness value, lower
water content and higher protein content.
In general, freezing process is not
significantly
affect
chicken
meat
characteristic except its texture. Overall
Page 113
both fresh and frozen meat which have the

best quality is the ones treated by
induction of 10 ml chromanone deamine.
REFERENCES
Afrianti, M.; B. Dwiloka.; dan B.E.
Setiani.(2013). Perubahan Warna,
Profil
Protein,
Dan
Mutu
Organoleptik Daging Ayam Broiler
Setelah Direndam Dengan Ekstrak
Daun Senduduk. Jurnal Aplikasi
Teknologi Pangan Vol. 2 No. 3: 116120.
Akhtar, S.; M. I.Khan; & F. Faiz. Effect
of Thawing on Frozen Meat Quality:
A Comprehensive Review. PAK. J.
FOOD SCI., 23(4): 198-211.
Allen, C.D.; D.L. Fletcher; J.K. Northcutt
&
S.M.
Russel.(1998).
The
Relationship of Broiler Breast Color
to Meat Quality and Shelf-Life.
Poultry Science 77:361366
Fletcher, D.L. (1999). Broiler Breast Meat
Color Variation, pH, and Texture.
Poultry Science 78:13231327.
Kasih, N.S.; A. Jaelani & N. Firahmi.
(2012). Pengaruh Lama Penyimpanan
Daging
Ayam Segar Dalam
Refrigerator Terhadap pH, Susut
Masak Dan
Organoleptik. Media
Sains, Volume 4 Nomor 2: 154-159.
Matulessy, D.E; E.Suryanto & Rusman.
(2010). Evaluasi Karakteristik Fisik,
Komposisi Kimia Dan Kualitas
Mikrobia Karkas Broiler Beku Yang
Beredar
Di
Pasar
Tradisional
Kabupaten Halmahera Utara, Maluku
Utara. Buletin
34(3):178-185.
Peternakan
Vol.
Mubin, Y.N. (2013). Profil Biokimia

Daging Ayam Broiler Yang Diberi
Pakan Plus Formula Herbal. Skripsi.
Institut Pertanian Bogor.
Probst, Y. (2009). Nutrient Composition of
Chicken Meat. Rural Industries
Research
and
Development
Corporation. Australian Government.
Pusparini, M.R, Sumardi & Laksmie,H.
(2008). Improving Meat Quality of
Milkfish by Induction Chromanone
Deamine.
National
Student
Conference on Food Science &
Technology, Department of Food
Technology, Unika Soegijapranata,
Semarang.
Sunaryanto, L. T. & Sumardi. (2008).
Enhancing Quality of Chicken Broiler
Meat By Inducing Short Chain
Hydrobenzene of Aegle Marmelos,
International Symposium on Food
Technologiests, Faculty of Food
Technology, Unika Soegijapranata,
Semarang.
Wattanachant, S. (2008). Factors Affecting
The Quality Characteristics of Thai
Indgenous Chicken Meat. Suranaree J.
Sci. Technol. 15(4):317-322.
Winarso,
D.
(2003).
Perubahan
Karakteristik Fisik Akibat Perbedaan
Umur, Macam Otot, waktu dan
Temperatur Perebusan pada Daging
Ayam
Kampung.
J.Indon.Trop.Anim_Agric 28(3):119132.
Page 114
FOOD SAFETY AND QUALITY
Page 115
EFFECT OF RARE SUGAR D-PSICOSE ON PEROXIDASE ACTIVITY

DERIVED FROM TOMATO LEAF
FinaFitriana Budiman1, Rizki Dian Lestariningtyas1, Muhammad Johan Adhibuana1,
Ahmad Nimatullah Al-Baarri2, Anang M Legowo2, Shigeru Hayakawa3, Masahiro
Ogawa3
1Food
Technology Department, Faculty of Animal and Agricultural Sciences, Diponegoro

University, Indonesia
2
Food Technology Department, Integrated Laboratory, Diponegoro University, Indonesia
3
Applied Biological Sciences Department, Faculty of Agriculture, Kagawa University, Japan
Correspondent author: albari@undip.ac.id
ABSTRACT
D-psicose has been understood as zero calorie of sugar that are present in a small quantity naturally
and has been received high attention among consumers in making zero calorie of food products.
Along an increase in producing healthy food, the bio-preservation has also high in demand inline with
producing the healthy food. This research has been done to analyze the effect of peroxidase enzyme,
the natural enzyme for making antibacterial agent that may be applied in food, in the presence of Dpsicose. This research has been conducted in Central Laboratory, Diponegoro University, Indonesia
from May to July 2015. D-psicose was provided from Rare Sugar Research Center, Japan and
peroxidase from tomato leaf was purified using SP Sepharose column. Enzymatic reaction of
peroxidase was measured using ABTS as substrate. Data showed that the inhibition of peroxidase
activity was appeared when it was employed in the solution containing 3.0% (w/v) of D-psicose. The
D-psicose of less than 3.0% didnt provide any remarkable inhibition to enzyme activity. This finding
may provide the information for application of peroxidase as bio-preservative in D-psicose containing
food in low concentration.
Keywords: D-psicose, peroxidase, enzyme activity, food
Page 116
INTRODUCTION
Sugar is the largest constituent of a food,
which occur naturally or deliberately
added.
The addition of sugar as a
sweetener is also used to support food
processing such as enhancing browning
reaction and antioxidant properties (Sun et
al., 2006, Sun et al., 2007), increasing the
quality and selling value of foods, and the
most important is sugar as the main energy
source for human body. Some examples of
sugar that can be found on food are
glucose, sucrose, fructose, lactose, and
maltose. Glucose and fructose are found in
fruits and vegetables, sucrose can be found
in sugar cane, lactose can be found in
milk, and maltose can be found in grains.
The excessive consumption of sugar can
cause some health problems such as
diabetes and obesity(Lindquist et al., 2000,
Izumori, 2002, Matsuo et al., 2002).
Diabetes is a condition which blood
contains high level of sugar because the
pancreas cant produce the insulin for
absorbing and aiming sugar into the body
cells(Lindquist et al., 2000). Diabetes may
arise as a result of excessive sugar
consumption. Therefore, the consumption
of low or no calorie sugar such as DPsicose is demanded to hinder the increase
of diabetes patients.
Enzyme
can
be
described
as
macromolecular that catalyze or accelerate
the chemical reaction(Klibanov, 2001,
Shakeel-ur et al., 2002, Breena and
Batista-Viera, 2006, Calkins and Sullivan,
2007). There are several factors that affect
the enzyme activity, including the sugar
(Clausen et al., 2008, Singh et al., 2009a,
Al-Baarri et al., 2011). Enzyme inhibitor is
a molecule that bind itself into active

site(Singh et al., 2008, Singh et al., 2009a,
Singh et al., 2009b). This occurence make
the active site of enzyme blocked that
resulting in decreases its activities(Sheikh
et al., 2009). The previous researcher has
found that D-psicose was one of inhibitor
for bovine lactoperoxidase but no
information was found when peroxidase
from other source was used. This finding
may explain the effect of D-psicose to
peroxidase from tomato leaf. Therefore,
the purpose of this research to find the
effect of D-psicose on enzyme activity of
peroxidase from tomato leaf.
Materials.
Tomato leaves was provided from tomato
garden in Ungaran, Central Java. Dpsicose was provided from Rare Sugar
Research Center Japan,2,2'-azino-bis(3ethylbenzothiazoline-6-sulphonic acid) or
ABTS, H2O2, NaCl 0.5M, Pb-Aquadest
100mM pH 7, Pb-NaCl 0.5 M pH 6.5, PbNaCl 1.0 M pH 6.5, Pb-NaCl 1.5 M pH
6.5, AmmoniumSulphate 0.1 M, and
Aquadest.
Tools.
Spectrophotometer UV-Vis, Shaker, SDS
Column, Electrophoresis, Hand Mixer,
Centrifuge, and Filter syringe.
Extraction of Tomato Leaves.
Four hundred grams of tomato leaves was
extracted
using
300
ml
Ammoniumsulphate 0.1 M in low
temperature. The mixture of tomato leaves
and Ammoniumsulphate was blended
using hand mixer and was filtrated paper
cloth. Filtrate was centrifuged with speed
of 6000 rpm for about 20 minutes and the
Page 117
temperature was increased by 5oC for

every 5 minutes. Supernatant was ready to
use(Rodriguez-Lopez et al., 2000).
Analysis of enzyme activity.

Analysis of enzyme activity was
determined by using spectrophotometer
with wavelength 412 nm. Cuvette was
filled by 300 l of enzyme (has been
diluted 20x by aquadest as solvent), 300 l
of sugar solution (concentration 0%-3%),
1200 l ABTS, and 1200 l H2O2. The
absorbance was measured in 0 and 60
second(Al-Baarri et al., 2011).
Purification.
The resin was washed using 300 ml NaCl
0.5 M, 500 ml aquadest, and tomato leaves
extract. The eluate was collected for 10 ml
on tube (control). A 300 ml Pb-Aquadest
100 mM pH 7 and 500 ml Pb-NaCl 0.5 M
pH 6.5 was added, the eluate was collected
for 10 ml per tube. A 500 ml Pb-NaCl 1.0
M pH 6.5 was added, the eluate was
collected for 10 ml per tube. A 500 ml PbNaCl 1.5 M pH 6.5 was added, the eluate
was collected for 10 ml per tube. The
protein concentration of each fraction was
determined by measuring absorbance at
280 nm. The purified enzyme was stored
below freezing point(Rodriguez-Lopez et
al., 2000).

Table 1.Absorbance at 412 nm of reaction
solution
containing
ABTS,
H2O2,
peroxidase, and 0 -3% D-psicose. D1
means first measurement; D2 means
second measurement.
Concentration (%)
D1
D2
Average SD
0.017
0.015
0.016 0.001
0.017
0.014
0.0155 0.002
0.014
0.012
0.013 0.001
0.009
0.010
0.0095 0.005
Based on Table 1, it can be concluded that

0% addition of D-psicoseshowed the
decrease of enzyme activity which
indicated as 0.0160.001. The addition of
sugar affected to the decrease of
absorbance. The value of absorbance
indicates the enzyme activity. The
reduction of absorbance indicates the
enzyme activity, which can be observed
from the difference of absorbance between
each measurement. The addition of 1%
(w/v) resulted in the decrease of
absorbance
from
0.0160.001
to
0.01550.002. This decrease indicated

3.125% 18.84% reduction. The addition
of 2% (w/v) resulted in the decrease of
absorbance
from
0.0160.001
to
0.0130.001
(18.75%
18.84%
reduction). However 3% (w/v) addition of
D-psicose show the highest reduction of
absorbance valuefrom 0.0160.01 to
0.00950.005
indicating
40.625%18.84%.
The
remarkable
reduction of absorbance when 3% of Dpsicose was employed might be explained
by the attachment of OH and H component
Page 118
in D-psicose into heme of peroxidase.

Peroxidase was also known for its
sensitiveness to other component such as
OSCN, H2O2, BHT, and BHA (Singh et
al., 2008, Singh et al., 2009b). Casein in

milk was also known as inhibitor the
enzyme activity.
Inhition percentage (%)
Figure 1. Percentage of peroxidase activity

inhibition by 1-3% (w/v) D-psicose
70
50
30
10
-10
2
Sugar Concentration (%)
D-psicose was concluded as an inhibitor

which was able to inhibit the activity of
enzyme. Enzyme has an important part
named heme which has helix h2 around it.
Helix h2 is a hydrophilic group which can
attract H+ or OH- ion from sugar (Clausen
et al., 2008, Singh et al., 2009a). Helix h2
has also known as the active side of
enzyme which helps the reaction process
of enzyme. The H or OHbounded to helix
h2 might interfere the enzyme activity.
CONCLUSION
D-psicose at the concentration of 3 %
(w/v) remarkable inhibited peroxidase
activity,
however
at
the
lower
concentration, D-psicose didnt show the
meaningful inhibition of enzyme activity.
REFERENCES
Al-Baarri, A. N., M. Hayashi, M. Ogawa,
and S. Hayakawa. 2011. Effects of
mono- and di-saccharides on the
antimicrobial activity of bovine
lactoperoxidase system. Journal of
Food Protection 74(1):134139.
Breena, B. M. and F. Batista-Viera. 2006.

Immobilization of enzymes: A
literature
survey.
2nd
ed.
Immobilization of Enzyme and Cells.
Humana Press.
Calkins, C. R. and G. Sullivan. 2007.
Adding Enzymes to Improve Beef
Tenderness. National Cattlemen's
Beef Association.
Clausen, M. R., L. H. Skibsted, and J.
Stagsted. 2008. Inhibition of
lactoperoxidase-catalyzed 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic
acid)(ABTS) and tyrosine oxidation
by
tyrosine-containing
random
amino acid copolymers. J. Agric.
Food Chem. 56:86928698.
Izumori, K. 2002. Bioproduction strategy
for
rare
hexose
sugars.
Naturwissenschaften 89:120-124.
Klibanov, A. M. 2001. Improving enzymes
by using them in organic solvents.
Nature 409.
Page 119
Lindquist, C. H., B. A. Gower, and M. I.

Goran. 2000. Role of dietary factors
in ethnic differences in early risk of
cardiovascular disease and type 2
diabetes. American J. Clinical Nut.
71:725-732.
Matsuo, T., H. Suzuki, M. Hashiguchi, and
K. Izumori. 2002. D-psicose is a rare
sugar that provide no energy to
growing rats. Journal of Nutritional
Science and Vitaminology 48:7780.
Rodriguez-Lopez, J. N., J. C. Espin, J.
Tudela, V. Martinez, A. Cerd, and
F.
Garcia-Cnovas.
2000.
Purification
and
Kinetic
Characterization of Peroxidase from
Tomato Cultivated under Different
Salinity Conditions. Journal of Food
Science 65(1):1519.
Shakeel-ur, R., N. Y. Farkye, and R.
Hubert. 2002. Enzymes indigenous
to
milk
lactoperoxidase.
Encyclopedia of Dairy Sciences.
Oxford: Elsevier.:938941.
Sheikh, I. A., A. K. Singh, N. Singh, M.
Sinha, S. B. Singh, and A. Bhushan.
2009. Structural evidence of
substrate specifitiy in mammalian
peroxidases. Journal of Biological
Chemistry 284(22):14849-14856.
Singh, A. K., N. Singh, S. Sharma, K.
Shin, M. Takase, P. Kaur, and A.
Srinivasan. 2009a. Inhibition of
lactoperoxidase by its own catalytic
product: crystal structure of the
hypothiocyanate-inhibited
bovine
lactoperoxidase at 2.3- resolution.

Biophysical Journal 96:646-654.
Singh, A. K., N. Singh, S. Sharma, S. B.
Singh, P. Kaur, A. Bhusan, A.
Srinivasan, and T. P. Singh. 2008.
Crystal Structure of lactoperoxidase
at 2.4 resolution. Journal of
Molecular Biology 376:10601075.
Singh, A. K., N. Singh, M. Sinha, A.
Bhushan, P. Kaur, A. Srinivasan, S.
Sharma, and T. P. Singh. 2009b.
Binding Modes of Aromatic Ligands
to Mammalian Heme Peroxidases
with
Associated
Functional
Implications. Crystal structures of
lactoperoxidase complexes with
acetylsalicylic
acid,
salicylhydroxamic
acid,
and
benzylhydroxamic acid. The Journal
of
Biological
Chemistry
242(30):2031120318.
Sun, Y., S. Hayakawa, H. Jiang, M.
Ogawa, and K. Izumori. 2006.
Rheological characteristics of heatinduced custard pudding gels with
high antioxidative activity. Biosci.
Biotechnol. Biochem. 70(12):28592867.
Sun, Y., S. Hayakawa, M. Ogawa, and K.
Izumori.
2007.
Antioxidant
properties of custard pudding dessert
containing rare hexose, D-psicose.
Food Control 18:220227.
Page 120
FATTY ACID COMPOSITION ANALYSIS OF COCONUT MEAT FROM

DIFFERENT CULTIVATION AREAS BY USING GAS LIQUID
CHROMATOGRAPHY TECHNIQUE
Ivana Aprilia Pratiwia*, Assoc Prof. Dr. Visith Chavasitb
aDepartement
of Food Technology, Faculty of Agricultural Technology, Soegijapranata

Catholic University
Pawiyatan Luhur IV/1 Bendan Duwur, Semarang 50234
bInstitute of Nutrition, Mahidol University (INMU)
999 Phutthamonthon 4 Rd., Salaya, Phutthamonthon, Nakhon Pathom 73170, Thailand
*ivana.aprilia94@gmail.com
ABSTRACT
Coconut (Cocos nucifera L.) is one of the most extensively grown plants in nearly 90 countries of
thetropical region. Among them, Indonesia, Thailand, and Vietnam are the main consumers and
producers of coconut and coconut products, which makes coconut becomes commercial crop that
contributies significantly to their economics. Mature coconut meat can be used as ingredient for
variety of foods and beverages. Coconut oil extracted from copra (dried mature coconut meat) can be
used for making soap, shampoo, cosmetics, cooking oilsand margarine. Fat is the main component in
mature coconut meat, which the majority 92% is saturated fatty acid (mostly in the form of
triglyserides). Up to 70% of saturated fatty acid in the coconut meat is the medium chain (MCFA)
ones (C8-C12). The fatty acid profile of the coconut meat obtained from different growing
environment however can be different, which can consequently affect their impacts on consumers
health. This study aims to compare the fatty acid profiles of the coconut meats obtained from
Thailand, Vietnam and Indonesia by using Gas Chromatography techniques. The analyzing coconut
meat from different cultivar using gas chromatography shows that the major component of fatty acid
are medium chain fatty acid especially C12 (lauric acid). There are some differences in fatty acid
composition, such as C10, C14, C16, and C18:1. Compotition fatty acid of coconut meat would
different because many factor such as varietas, age of coconuts, geographical origin and ecological
conditions.
Keywords : coconut, fatty acid, gas chromathopgraphy
Page 121
INTRODUCTION
Coconut (Cocos nucifera L.) is one of the
most extensively grown plants in nearly 90
countries of thetropical region. Among
them, Indonesia, Thailand, and Vietnam
are the main consumers and producers of
coconut and coconut products, which
makes coconut becomes commercial crop
that contributies significantly to their
economics. Botanically, coconut fruit
consists of 3 layers: exocarp, mesocarp,
and endocarp. The edible part is endocarp,
which is a thin, white, fleshy layer, about
one inch thick at maturity. This layer is
known as coconut meat. Fresh mature
coconut meatconsists an average of 48.0%
moisture, 35.5% fat and 16.5% oil and
moisturefree residue. Coconut meatis the
most valuable part of coconut since it
provides important ingredients of foods
and other applications (Nathanael, 1960;
Solangi and Iqbal, 2011). Mature coconut
meatcan be used as ingredient for variety
of foods and beverages. Coconut oil
extracted from copra (dried mature
coconut meat) can be used for making
soap, shampoo, cosmetics, cooking oilsand
margarine. Coconut and its products have
been the sources of income for almost 10
million families of the tropical area
(Anon., 2004).
Fat is the main component in mature
coconut meat, which the majority 92% is
saturated fatty acid (mostly in the form of
triglyserides). Up to 70% of saturated fatty
acid in the coconut meat is the medium
chain (MCFA) ones (C8-C12) (A.G,
Gopala; et al, 2010). The medium chain
fatty acid is well-known for the health
benefits since it can be directly
metabolized with minimum accumulation
in the body (Mariana, A.m; et al, 2009).
Nevin and Rajamohan (2004) in Mariana,

A.M, et al (2009) said that for commercial
purpose, coconut oil especially virgin
coconut oil is claimed for its ability in
reducing total cholesterol, triglycerides,
phospholipids, low density lipoprotein
(LDL) and very low density lipoprotein
(VLDL) cholesterol as well as increasing
high density lipoprotein (HDL) cholesterol
in serum and tissues. The benefits become
more interesting since nowadays more
people become more aware of the nutrition
and their health. Due to the ASEAN
Economic Community harmonization in
2015, the free flow of coconut among at
least 3 main growers and producers i.e.
Thailand, Vietnam and Indonesia will
certainly increase. The fatty acid profile of
the coconut meat obtained from different
growing environment however can be
different, which can consequently affect
their impacts on consumers health. The
fatty acid profile of the matured coconut
meat obtained from different countries
should therefore be studied.
1.1.1. Objectives:
This study aims to compare the fatty acid
profiles of the coconut meats obtained
from Thailand, Vietnam and Indonesia by
using Gas Chromatography techniques.
Materials
Coconut meat
Dehuskedmature coconuts samples were
obtained different cultivar areas in
Indonesia, Thailand, and Vietnam as 3
batches, 10 fruits per batch. Meat from
each fruit of the same batch was obtained
from the shell and shredded. The meats
from 10 fruits were pooled together. The
analysis was performed for each batch in
Page 122
triplicate.
The
sample
Theppadungporn coconut Co.,LTd.
from
Methods
Fat extraction
The extraction was performed by
following the Folch method. Sample was
mixed with chloroform/methanol (2/1) to a
final volume 20 times the volume of the
tissue sample (1 g in 20 ml of solvent
mixture). The mixture was agitated in a
shaker at room temperature for 30 min,
then filtrated (funnel with a folded filter
paper) or centrifuged to recover the liquid
phase. The removed solvent phase was
added with 4 ml of 0.9% NaCl solution.
After vortexingfor 2-3seconds, the mixture
was centrifuged at 2000 rpm (centrifuge
HERMLE Z400K Denvilie Scientific, Inc.,
Metuchen NJ., USA.) to separate the 2
phases. The upper phase was removed, and
then the lower phase was evaporated under
vacuum in a rotary evaporator.
Saponification
Saponification is the process used for
hydrolyzing fatty acid from glycerol.
Extracted fat is weighed 0.02-0.05 g into
screw-cap test tube. 1 ml of 0.5 M KOH
in Methanol is then added and the tube was
closed and shaken. The tube was placed in
a water bath at 1000C for 5 min. After
boiling, the tube was cooled down in air at
room temperature.
Methylation
After saponification, fatty acid must be
methylated before being analyzed in gas
chromatography. 400 ul of 14% Boron
trifluoride-methanol reagent was added
into the tube, then the tube was placed in
water bath at 1000C, shaken every 5
minfor 15 min, and cooled down at room

temperature. Then, 2 ml of water was
added. Fatty acid was extracted twice with
3 ml PE (petroleum ether). The PE layer
was transferred into the other test tube and
evaporated out to dryness in a hot air oven
at 600C. After dried, the internal standard
C17 (Supleco 37 sigma aldrich, USA) was
added 1 ml into the tube then the tube was
vigorously shaken. 1 ml of methylated
fatty acid was filled intoto the vial for
analyzing on Gas Chromatography (GC).
Gas Chromatography Analysis
A DB-23 capillary GC column (60 m x
0.25 mm I.D., 0.25 m) installed in a
Agilent 9860 gas chromatograph system
equipped with a flame ionisation detector
and a split/splitless injector was used. The
initial temperature was at 500C, hold for 2
min, then increase to 180 0C at a rate of
50C /min, hold for 2 min at 1800C, then
increase at a rate of 80C /min to 2000C and
hold for 5 min at 2000C. Fatty acid methyl
ester (1ul) is injected onto the split
injection port (100:1 split ratio). The flow
rate for the He carrier gas is 50 ml/min.
Supleco 37 (sigma aldrich, USA) is used
as standard samples. Peak identification of
sample is done by comparing relative
retention times and content of fatty acids is
done by comparing with peak area of
internal standard.
Satistical Analysis
Analysis of variance and least significant
difference tests were conducted to identify
differences among means using SPSS
statistical computer package software
version 13. All analyses were conducted in
triplicate.
Page 123
Chromatography techniques. This report

reported the fatty acid compotions in
coconut meat from three samples
(Thailand, Indonesia, and Vietnam). Every
sample from every country has three
samples and every samples are did three
times.

The result shown in this report is part of
the bigger study about fatty acid
composition. The whole studys final aim
is to compare the fatty acid profiles of the
coconutmeats obtained from Thailand,
Vietnam and Indonesia by using Gas
Table 1. Fatty Acid Profiles of Fat in Coconut Meats from 3 Countries

Cultivar area
Percentage of fatty acid
C8
:0
C10:0
C12:0
C14:0
C16:0
C18:0
C18:1
C18:2
Thai
mean SD
(Range)
1.3
31
.29a
5.170.51a
52.272.49a
21.030.42a
9.90.75a
3.51.05a
60.36a
0.70.31a
Indo
mean SD
(Range)
20
.8a
5.90.1ab
54.670.75a
19.30.44a
8.80.2ab
3.770.21a
4.830.12
0.81.4E16a
Viet
mean SD
(Range)
1.4
0.
44a
5.770.04b
53.530.42a
19.430.55b
9.130.32b
3.730.01a
60.05b
1.070.01a
CODEX
Range
4,6
-10
5,0-8,0
45,1-53,2
16,8-21,0
7,5-10,2
2,0-4,0
5,0-10
1.0-2.5
Denote : Thai: Thailand, Indo: Indonesia, Viet: Vietnam. Codex Standard For Named Vegetable Oils Codex
Stan 210-1999. Means within each column with different superscript are significantly different at P<0.05.
The fatty acid composition of all sample is

presented in Table 1. The fatty acid
composition was as expected and
comparable to the fatty composition of
coconut oil according to the Codex STAN
210-1999. From CODEX STAN 210-1999
the most dominant fatty acid is lauric acid
(C12:0) and ranged from 52.27 to 54,67.
The lauric acid content in the coconut meat
from Indonesia was slightly higher than
the ones from Thailand and Vietnam ,

which were 54,67, respectively. There was
no significant difference found in the
content of lauric acid among thailand,
Indonesia, and Vietnam dispite difference
in the cultivar area. Then, lauric acid
values VIET, which was from 53,53 and
lauric acid THAI which was from 52,27%.
The caprylic fatty acids (C8:0) in this
result ranged from 1,33 to 2 %. These
values were much lower than those
Page 124
reported by Codex Stan 210-1999, it

should be between 4,6 and 10,0. Caprylic
fatty acid is shorter chain fatty acid than
the other, thus few caprylic acid might
gone during process such as fat extraction,
saponification and methylation. However
there was no significant difference found
in the content of caprylic acid among
Thailand, Indonesia, and Vietnam dispite
difference in the cultivar area. In this study
capric fatty acid (C10:0) ranged from 5,0
to 8,0 and the result of all samples have
range from 5,17 to 5,9. While sample
INDO showed the highest percentage of
capric acid (C10:0). There was significant
difference found in the content of capric
acid between Thailand and Vietnam
dispite difference in the cultivar area. The
myristic fatty acid (C14:0) ranged 16,821,0 and the result has range from 19,321,03. There was significant difference
found in the content of myristic acid
between Thailand and Vietnam; and
between Indonesia and Vietnam dispite
difference in the cultivar area.
The
palmitic fatty acid (C16:0) ranged 7,5-10,2
and the result has range from 8,8-9,9.
There was significant difference found in
the content of palmitic acid between
Thailand and Vietnam dispite difference in
the cultivar area. The stearic fatty acid

(C18:0) ranged 2-4 and the result has
range from 3,5-3,77. There was no
significant difference found in the content
of stearic acid among thailand, Indonesia,
and Vietnam dispite difference in the
cultivar area. The oleic (C18:1) ranged
5,0-10,0 and the result has range 4,8-6.
There was significant difference found in
the content of oleic acid between Thailand
and Indonesia; and the difference between
Thailand and Vietnam dispite difference in
the cultivar area. The linoleic fatty acid
(18:2) ranged 1-2,5 and the result has
range 0,7-1.07. there was no significant
difference found in the content of linoleic
acid among Thailand, Indonesia, and
Vietnam dispite difference in the cultivar
area.
This result could be due to
geographical origin and ecological
conditions (Mariana, A.M, 2009). Base on
Djatmiko (1983) and Manullang (1983) in
(Puspitasari, 1992), compotition of
coconut meat would different because
many factor, such as varietas and age of
coconuts. Rachel, et al (2009) also
support, they said that cultivars and
maturation would effect fatty acid content.
Percentages of lauric acid increased during
maturation of coconut meat.
Table 2. Percentage of Fatty Acid and Ratio

Cultivar
Percentage of fatty acid
area
P
M
S
Thai
0.73
6.0
93.2
Indo
0.8
4.8
94.4
P
0,07
0.01
0,06
0.05
1
1
Viet
1.1
93.0
0.001
0.07
FAO
6-11%E By different
8-10%E
6-11%E
By different
8-10%E
6.0
Ratio
M
Denote: P= Polyunsaturated Fatty Acid, M= Monounsaturated Fatty Acid S= Saturated Fatty Acid. FAO= Food
and Agriculture Organization of the United Nations, %E = percentage of energy.
Page 125
The recommended range (AMDR) for

PUFA is 611%E. In accepting that the
U-AMDR
(acceptable
macronutrient
distribution range) values of total PUFA
and total n-3 fatty acids are 11%E and
2%E respectively, the resulting AMDR
(acceptable macronutrient distribution
range) for n-6 fatty acids (LA) intake is
2.59%E. In this results PUFA cames
from n-6 fatty acids and have ranges of
percentages 0,73-1,1 it is lower than
monounsaturated fatty acid. Base on
American
Heart
of
Association
Polyunsaturated fats can help reduce bad
cholesterol levels in blood which can
lower risk of heart disease and stroke.
Polyunsaturated fatty acid also provide
nutrients to help develop and maintain
bodys cells.
The determination of intake of MUFA is
unique in that it is calculated by difference,
i.e. MUFA=Total fat intake (%E) SFA
PUFA TFA . Therefore, the resulting
MUFA intake may cover a wide range
depending on the total fat intake and
dietary fatty acid pattern. Replacing
carbohydrates with Mono Unsaturated
Fatty Acic (MUFA) would increases HDL
cholesterol concentrations (FAO, 2009).
There is convincing evidence that
replacing SFA (C12:0C16:0) with MUFA

reduces LDL cholesterol concentration.
There is possible evidence that replacing
carbohydrates with MUFA improves
insulin
sensitivity
(Elmadfa
and
Kornsteiner, 2009). From the results
monounsaturated fatty acid are from 4,8-6,
it was higher than polyunsaturated fatty
acid.
Base on FAO, they said that individual
Saturated Fatty Acids (SFA) have different
effects on the concentration of plasma
lipoprotein cholesterol fractions. For
example, lauric (C12:0), myristic (C14:0)
and palmitic (C16:0) acids increase LDL
cholesterol whereas stearic (C18:0) has no
effect. They recommend for saturated fatty
acids (SFA) 8-10%E, 8%E should be
recommended for 2-18 years old. In an
other researcher believe that any
relationship between SFA consumption
and cancer. Therefore, it is recommended
that SFA should be replaced with PUFA
(n-3 and n-6) in the diet and the total
intake of SFA should not exceed 10%E
(Elmadfa and Kornsteiner, 2009). Hence,
the maximum energy come from Saturated
Fatty Acid should not exceed 200 kkal or
250 kkal (depend on age or sex).
.
Table 3. Percentages of Different Grups of Fatty Acid Potentially Claimed for Their
Health Benefits
Cultivar area
Thai
Indo
Viet
MCFA (C8-C12)
58.772.63
62.570.65
60.71.06
Percentage Of Fatty Acid

Trans-FA n-3
n-6
0
0
0.730.31
0
0
0.81.4E-16
0
0
1.070.06
n-9
60.36
4.830.16
60.53
Denote : MCFA= medium chain fatty acid, Trans-FA= transfatty acid, n-3= omega-3, n-6= omega 6, n-9=
omega 9.
Page 126
From the result show that the major

component of fatty acid are medium chain
fatty acid. One of the major issues
regarding coconut oil consumption is its
effect on the heart and circulatory system.
Because coconut oil (that made from
coconut meat) contains a high amount of
saturated fat, it has been believed to raise
blood cholesterol levels and promote heart
disease. Now days, there are so many
research to discuss it. Base on Babayan,
Vigen, et al. (1982) medium chain fatty
acid, have a many benifit for health.
Because of medium chain fatty acid easier
to absorbed and transported directly to the
liver where they are burned for energy.
Medium chain fatty acid do not circulate in
the bloodstream like other fats did.
Moreover it can use for source of fat in
malabsorption conditions. It is also used to
increase the energy intake in cystic fibrosis
patients.
Base on coconut research center, Mary G.
Enig, Ph.D said the levels of these
antimicrobial fatty acids can be as low as 3
to 4 percent, when nursing mothers eat
coconut products (shredded coconut,
coconut milk, coconut oil, etc.) the levels
of Medium Chain Triglyseride (MCT) in
their milk increase significantly. Consume
40 grams of coconut oil in one meal can
temporarily increase the lauric acid in the
milk of a nursing mother from 3.9% to
9.6% after 14 hours. The content of
caprylic and capric acids are also
increased. This gives an important benefit
because the milk has increased amounts of
the protective antimicrobials lauric acid
and capric acid, which gives even greater
protection to the infant.
In this result show that omega-6 is lower

than omega-9 in the coconut meat. Omega
6 in coconut meats have ranges 0,73331,0667. Otherwise omega-9 in the coconut
meats have ranges 4,844-6. As far as n-6 to
n-3 ratio is concerned, the 2002 Joint
WHO/FAO review had indicated a
balanced intake of n-6 and n-3 PUFAs is
essential for health (WHO, 2003; Reddy
and Katan, 2004). Some oils rich in
polyunsaturated fats also provide essential
fats that body needs unfortunately body
can not produce itself, such as omega-6
and omega-3 fatty acids. Thus must get
essential fats through food. Omega-6 and
omega-3 fatty acids are important for
many functions in the body. In coconut
meat has some omega-6 for constribute
sufficiently.
This table also shown there is not transfatty acid. This result is appropriate with
CODEX STAN 210-1999, they said that in
coconut meat should has not trans-fatty
acid. Trans fatty acid consumption has
unique adverse effects on serum lipids,
including increasing LDL-C, lowering
HDL-C, increasing lipoprotein, increasing
ApoB levels, and decreasing ApoA1 levels
(Katan et al., 1994; Mensink and Katan,
1992; Mozaffarian and Clarke, 2009;
Mozaffarian et al., 2006). The transfatty
Figure 3. Structure of Trans Fatty Acid

acid (TFA) intake from all sources should
be no more than 1%E (Federal Register,
2003).
Page 127
Unfortunately, coconut meat also has

amount of long chain fatty acid (36,2%), it
is turned into triglycerides and then are
taken up by cells and used for energy or
are stored. Thus bile from the gallbladder
is needed to digest long chain fatty acids.
Hence long chain fatty acid can make bad
effect for our health, such us increases
blood pressure, increases LDL, increases
triglycerides, and reduces HDL. American
Heart Association recommends that only
7% of total daily calories come from
saturated fat.
CONCLUSION
The analyzing coconut meat from different
cultivar using gas chromatography shows
that there are some differences in fatty acid
composition, such as C10, C14, C16, and
C18:1. Compotition fatty acid of coconut
meat would different because many factor
such as varietas, age of coconuts,
geographical origin and ecological
conditions. Thus, the major component of
fatty acid are medium chain fatty acid
especially C12 (lauric acid).
REFERENCES
A.G, Gopala; et al. (2010). Coconut Oil:
Chemistry,
Production
and
Its
Applications - A Review. Department
of Lipid Science & Traditional Foods,
Central Food Technological Research
Institute (CSIR), Mysore 570020
American
Heart
Center.
http://www.heart.org/HEARTORG/Ge
ttingHealthy/NutritionCenter/HealthyE
ating/TropicalOils_UCM_306031_Article.jsp.
accessed on 15 March 2015
Andre C Bach and Vigen K Babayan.

1982. Medium-chain triglycerides: an
update. Am. J. clin. Nutr. 36: 950-962.
Anonymous. 2004. Gene flow, a
publication about the earths genetic
resources. Maxtudo, Rome, Italy. 35.
Codex Standard For Named Vegetable
Oils Codex Stan 210-1999
Coconut
Research
Center.
http://www.coconutresearchcenter.org
/savethechildren.htm. accessed on 1
April 2015
Elmadfa, I. & Kornsteiner, M. 2009. Fats
and fatty acid requirements for adults.
Ann. Nutr. Metab., 55: 56-75.
Federal Register. 2003. Part III.
Department of Health and Human
Services.
Food
and
Drug
Administration. 21 CFR Part 101.
Food Labeling; Trans Fatty Acids in
Nutrition
Labeling;
Consumer
Research to Consider Nutrient Content
and Health Claims and Possible
Footnote or Disclosure Statements;
Final Rule and Proposed Rule. Federal
Register, 68: 41434-41506.
Mariana, A. M, et al. (2009). Chemical
Properties of Virgin Coconut Oil. J
Am Oil Chem Soc (2009) 86:301307
Mariana, A. M, et al. (2009). Virgin
coconut oil: emerging functional food
oil.Trends in Food Science &
Technology 20 (2009) 481e487
Mozaffarian, D. & Clarke, R. 2009.
Quantitative effects on cardiovascular
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risk factors and coronary heart disease

risk
of
replacing
partially
hydrogenated vegetable oils with other
fats and oils. EJCN, 63, Suppl. 2:
S22S33.
Rachel, Et Al. 2010.Physicochemical
Characteristics Of Kernel During Fruit
Maturation Of Four Coconut Cultivars
(Cocos Nucifera L.). African Journal
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Papers of the Joint WHO/FAO Expert
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2002) Public Health Nutr. 7: 167186.
Solangi And Iqbal.(2011). Chemical
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Page 129
EFFECT OF CHROMANONE DEAMINE DOSAGES ON THE QUALITY OF

OSMOMEAT PRODUCTS IN TERMS OF PHYSICOCHEMICAL
CHARACTERISTICS
Mulyanto Onggo Wibowo1, Sumardi2, and Alberta Rika Pratiwi2
1
Student; Department of Food Technology; Facullty of Agricultural Technology;

2Lecturers; Department of Food Technology; Facullty of Agricultural Technology;
ABSTRACT
Osmomeat is a processed meat products that use osmotic dehydration process in reducing the water
content of the meat. Processed meat products are often associated with high protein and low fat
content. In order to improve the quality of the product, chromanone deamine applied into chicken
meat is used as main ingredient manufacture osmomeat. Chromanone deamine compound has been
applied in broiler chickens and is proven increasing the protein content, lowering fat content of meat,
and consequently increasing the hardness of the meat. Chromanone deamine compound is able to
improve the physical and chemical quality of osmomeat, but still not known how the effects and the
most appropriate dosage to improve the meat processed product. The purpose of this research was to
determine the effect of osmomeat chromanone dose in osmomeat product in terms of
physicochemical. Methods in this study includes preparation materials, osmotic dehydration process,
and the final drying. Materials research using meat broiler chest with various doses difference
chromanone (without chromanone, 1 ml, 5 ml and 10 ml). The osmotic dehydration process using
60% sugar-salt solution. Drying step is done using a dehumidifier at 60 C for 1 hour. Evaluation of
the osmomeat product include hardness, water content, water activity, protein, and fat content.
Hardness value of the sample by addition of 1 ml dose of chromanone deamine was 3818,186
295.759 gf or showed a decrease compared to the control, then the value of hardness in the sample
with chromanone deamine dose of 5 ml and 10 ml began to increase back. Chromanone deamine
dosage not significantly affect the value of water activity. The water content and fat content of the
samples showed a decrease in line with the increase in chromanone deamine dose given. The protein
content of osmomeat product optimum at 5 ml dose in the amount of 28.387 0.194 %, or increase
4.22 % compared to control.
Keywords : broiler chcken meat, osmomeat, osmotic dehydration, chromanone deamine
Page 130
INTRODUCTION
Meat is a nutritious food that humans
need. This is because high-quality protein
and essential amino acid content of a
complete and balanced. Dagingpun more
digestible protein than vegetable. Chicken
meat is more popular with the public than
other meats and poultry, because it is
easier cooked chicken meat, as well as
nutritional content is high enough. Broiler
chicken is one of the leading commodity
and most in demand by the community
(Brown, 2003 in Mehaffey et al, 2006).
Results of data collection by BPS in 2014
also showed that the Indonesian people
consume more chicken meat compared
with domestic poultry meat, and the level
of consumption of society will continue to
increase each year (Respati et al, 2014).
Osmomeat is processed meat products
involving osmotic dehydration process in
the making. Dehydration is a process of
osmosis water expenses, by immersion /
marinasi groceries in a hypertonic solution
(Misljenovic et al, 2012). Osmotic
dehydration is also a preservation method
without phase change on the product, as
well as to maintain the organoleptic
properties (Barbosa - Canovas and VegaMercado, 1996 in Sunjka and Raghavan,
2004). Osmotic solution that is widely
used
is
salt
or
sucrose
or
kombinasinya.Pada osmotic dehydration
of the fruit usually used sucrose, whereas
the osmotic dehydration of vegetables, fish
and meat are used NaCl (Rahman, 2007).
But the combination of different solutes
can be used to increase the effectiveness of
the osmotic dehydration process (Khan,
2012).
Osmomeat is a new and innovative

products that provide processed meat
products with the texture and quality of
quality. Osmomeat very popular and newly
developed in foreign countries, but so far
only limited to beef and pork. Seeing this,
the processing of chicken meat in
particular types of broiler very potential to
be developed as an innovative food
products, one of which is a product
osmomeat. To improve the quality of the
product
2,6,7-chromanon
deamine
compound applied to the chicken meat is
used as main ingredient manufacture
osmomeat. 2,6,7-chromanon deamine
compounds naturally present in the flesh of
the fruit Maja (Aegle Marmelos L. Corr).
2,6,7-chromanon deamine is cyclobenzene compound, an alkaloid class of
aromatic compounds in the form of two
groups that are bound into one double
bond between two carbon chain
connecting. 2,6,7-chromanon deamine
compound in the flesh of the fruit in the
form 2,6,7-chromanon amine.
Based
on
the
physicochemical
characteristics, 2,6,7-chromanon amine
can be extracted to 2,6,7-chromanon
deamine.
2,6,7-chromanon
deamine.
Extraction is done by drying maja fruit that
is fresh and cooked until the moisture
content reaches 12%. Maja dried fruit are
then crushed and extracted using low
temperature and vacuum conditions
(Indoherb Medical Science, 2008). The
chemical reaction of deamination 2,6,7chromanon deamine maja fruit can be
described as follows:
Page 131
Figure 1. The reaction of 2,6,7chromanone

amine
became
2,6,7chromanone deamine Addition compounds
2,6,7-chromanon deamine aims to increase
the protein content and lower fat content in
the final product. Some studies indicate
that the application 2,6,7-chromanon
deamine can increase as much as 1-3%
protein content and lower fat content as
much as 0.8 to 1.2% in broiler meat
(Sunaryanto and Sumardi, 2008).
Sample Preparation
The study was conducted by using chicken
day old chick (DOC) 999 brands ITB
separated into four treatment groups and
maintained for 30 days. 2,6,7-chromanon
deamine compound applied to the
livestock drinking water with different
doses for each treatment group 0 ml
(control), 1 ml, 5 ml, 10 ml. Applications
2,6,7-chromanon deamine done in the
morning and afternoon. Slaughter carried
out after 30 days of maintenance.
Carcasses taken the chest to be used as a

sample.
Procedure to Make Osmomeat
Chest broiler meat washed and carried
deboning process, then mashed with a
meat blender. Each sample using chicken
meat weighing 100 grams. Then the meat
flattened to a thickness of 2-3 mm.
Furthermore marinasi into the meat in salt
sugar solution at 60% concentrations
respectively for 2 hours as osmotic
dehydration stage. After that osmomeat
dried in a dehumidifier at 60 C for 1
hour. The sample of osmomeat was then
analyzed for Hardness, Water Content,
Water Activity, Protein Content, and Fat
Content (AOAC, 1995)

Table 1. Comparison of Hardness, Water Content, Water Activity, Fat, and Protein of
osmomeat under treatment 2,6,7-Chromanone Deamine and Control
Treatment
1 Treatment 2
Treatment 3
Parameters
Control
(1ml)
(5 ml)
(10 ml)
4870,01
3818,19
4620,55
5240,50
Hardness (gf)
c
a
b
d
233,227
295,759
166,743
349,222
Water Content
27,56 0,874d
26,38 0,772c
22,60 0,617b
21,63 0,503a
(%)
Water Activity
0,67 0,019a
0,68 0,019a
0,68 0,018a
0,67 0,023a
Fat (%)
2,29 0,375b
2,14 0,399ab
2,11 0,377ab
1,97 0,332a
Protein (%)
22,34 0,247a
24,26 0,096b
28,39 0,194c
22,19 0,290a
Note: Figured followed by the same letter, indicates significanlly different at 95% degree of
confidence in column at each parameters
Page 132
Hardness of Osmomeat
The results showed that a dose 2,6,7chromanon deamine influence the change
of the decrease in the level of hardness
compared to the control. 2,6,7-chromanon
deamine sample with a standard dose (1
ml) showed a decrease in the level of
hardness of by 19.39% to 3818.186
295.759, this is caused by 2,6,7chromanon deamine compounds which
cause the texture of chicken meat becomes
more tender. Therefore it will also affect
the texture of the final product. But the test
results showed an increase in dose 2,6,7chromanon deamine texture 5 although
still under the control sample hardness.
Based on the test results showed that the
samples osmomeat with a dose of 10 ml
2,6,7-chromanon deamine have a higher
level of hardness samples compared to
control. This may be caused by the
decrease of water content in the sample
cause the rank of hardness increase.
Increase in violence is due to the increase
in protein osmomeat. The higher the
protein content, the higher the level of
hardness.
Water Activity of Osmomeat
The results showed that 2,6,7-chromanon
deamine given dose did not significantly
affect water activity (Aw) significantly,
whereas the concentration larutanlah
influencing impairment Aw sample. In the
test results of water content is based on the
effect of dose 2,6,7-chromanon deamine
not have a significant influence. It
membuktikkan that 2,6,7-chromanon
deamine given does not affect the water
activity of the product osmomeat. Results
showed that the decrease in water content
osmomeat not followed by a decrease in
the value of Aw. It is said also by Winarno
(2004) which says that each food has a

curve of adsorption and desorption curves
are different. Foodstuffs that have a lower
water content does not necessarily have
the same Aw value low.
Water Content of Osmomeat
Water is an important component in
foodstuffs. All foods contain water in
varying amounts. The water content is
very influential in determining the shelf
life of foods, as these factors will affect the
physical
properties
and
chemical
(Winarno, 2004). Water also reacts with
proteins, polysaccharides, and fat having a
significant effect on the texture. Therefore,
a decrease in water content in food to
extend shelf life and improve the quality of
the food product. The results showed a
significant reduction in the moisture
content of a control sample to a dose 2,6,7chromanon deamine 10 ml. Value
moisture control sample amounted to
27.560 0.874%, and a decline of 1.28%
to 26.383 0.772% in the samples with a
dose 1. Then there is a decrease again on a
sample dose of 5 2,6,7-chromanon
deamine be 22,600 0.617% or 3.94%,
while the water content of the sample
shown by the lowest dose that is equal to
2,6,7-chromanon deamine 10 21.631
0.503.
2,6,7-chromanon
deamine
compounds can trigger the body's
excretion of the chicken so it will affect
the water content in the meat. Chicken
with 2,6,7-chromanon deamine higher
doses would cause the water level is
become lower.
Protein Content of Osmomeat
The results showed a significant increase
in protein content. Value protein content of
a control sample of 22.337 0.247%, and
Page 133
an increase of 1.77% to 24.257 0.096%

in the samples with 1 ml dose 2,6,7chromanon deamine. Then increased again
at doses of 5 ml samples 2,6,7-chromanon
deamine by 4.22% to 28.387 0.194%.
But at 10 ml sample 2,6,7-chromanon
deamine doses actually decreased protein
content of 6.44% compared samples 2,6,7chromanon deamine 5 ml dose. These
results showed the highest protein content
was obtained on samples with 2,6,7chromanon deamine dose of 5 ml, whereas
at a dose of 10 ml begin to decreased
levels of the protein. The results showed
increased significantly 2,6,7-chromanon
deamine also increase levels of a protein to
its optimum limit is 2,6,7-chromanon
deamine 5 ml. Increased protein content,
indicating that the compound 2,6,7chromanon deamine can increase protein
synthesis in the body of the chicken. These
results are also consistent with research
Sunaryanto & Sumardi (2008) which
indicates that the application 2,6,7chromanon deamine able to increase
protein levels.
Fat Content of Osmomeat
The results showed the higher dose given
2,6,7-chromanon deamine the fat content
will decrease. The value of the control
sample fat content of 2.29 0.375, then
continued to decrease with increasing dose
2,6,7-chromanon deamine given. Produced
the lowest fat content in the sample 2,6,7chromanon deamine osmomeat with a dose
of 10 ml which is equal to 1.967 0.332.
This indicates that the reduction in fat may
increase the 2,6,7-chromanon deamine
compounds -oxidation of fatty acids
resulting in lower fat content of broiler
chicken breast meat such as cholesterol
and triglycerides. Fat loss can also be
caused by compounds 2,6,7-chromanon

deamine that increasing the protein content
in chicken meat. Increased protein levels
will lead to decreased levels of fat
(Winarso, 2003).
CONCLUSIONS
1. The application of 2,6,7-Chromanone
Deamine significantly influence on the
texture, moisture content, protein
content, and fat content on osmomeat
product.
2. The higher the dosage of 2,6,7
Chromanone Deamine cause a decrease
in water levels and fat content.
3. The higher the dosage of 2,6,7Chromanone Deamine significantly
increased protein content in osmomeat
until the optimum dosage is 5 ml.
4. 5 ml dose of 2,6,7-chromanon deamine
is the optimum dose in increasing
protein content of osmomeat product.
5. The application of 5 ml dose of 2,6,7chromanon deamine increased protein
content from 22,34%
to 28,39%
(4,22%) and decreased fat content from
2,29% to 2,11% (7,86%).
6. This finding needs further detail
investigation on the processing
technologies suitable to maintains
osmomeat characteristic.
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(2012). Optimization of the
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Dehydration of Foods. In:
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Page 135
THE INHIBITORY EFFECT OF BAMBOO EXTRACTS ON

MELANOGENESIS IN MELANOCYTES
Fellycia Devi Paramithaa, Tsung-Yu Tsaib, Laksmi Hartayaniec
1Students;
Food Tecnology Department; Faculty of Agricultural, Soegijapranata Catholic

2Lecturer of Food Science Department, Fu Jen Catholic University, Taiwan
3Lecturer; Food Tecnology Department; Faculty of Agricultural, Soegijapranata Catholic
Email: fellyciiadevi@yahoo.com
ABSTRACT
Troubled skin cases like acne, pores, sunburn and pigmentation or hyperpigmentation are a skin
trouble that is undesirable for our skin. Hyperpigmentation can make our skin have a black spot and
uneven skin tone that caused by sun damage, inflammation, and also melanogenesis (excess of
melanin pigment). Bamboo have some several bioactive compound like phytosterol (in bamboo
ethanol extract) and phenol (in bamboo ethanol and water extracti) that are known to inhibit
melanogenesis. The aim of this research were to know the potential melanogenesis inhibitor using
bamboo extract (water and ethanol extract).The research methodology were to prepare bamboo
extract, subculture the cell, counting and seeding cells, MTT assay and melanin content test. MTT
assay was used to know the cell viability and melanin content test was used to know the amount of
melanin on the cells. Cells that we used were B16 melanoma cells that was taken on cancer cell on
mouse. The concentration of bamboo extract used in this melanin content test were control, -MSH,
positive control (PC), 20 ppm, 50 ppm and 100 ppm. -MSH or alpha-melanocytes stimulating
hormon was used to stimulate the producing of melanin. Positive control using ascorbic acid was used
as antimelanogenic compound. The results showed that the bamboo ethanol extract were more
significant to prevent melanogenesis than bamboo water extract. The optimum concentration to
reduce melanogenesis of B16 melanoma cell was 100 ppm of bamboo ethanol extract.
Keywords : bamboo extract, cell viability, hyperpigmentation, melanin, melanogenesis
Page 136
1. Background of Research
Hyperpigmentation is the one of
dermatologic cases that can be found in
all skin types. Hyperpigmentation can
make skin have a black spot and also an
uneven skin tone that are caused by sun
damage,
inflammation,
and
melanogenesis
(Lloyd
et
al.,
2011).Bamboo
(Phyllostachys
bambusoides) is a rich natural resources
from China. Bamboo have several
function, especially its leaves can
reduce
the
inflammation,
treat
hypertension,
arteriosclerosis,
and
cardiovascular disease, as antioxidant,
anticancer and antibiotic properties
(Yuan, 1983). The aim of this research
was
to
know
the
potential
melanogenesis inhibitors using bamboo
extracts (water and ethanol extract).
1.1. Bamboo
Bamboo is a woody grass that belong to
angiosperm group (Chapman, 1996) and
the order of monocotyledon (Abd.Latif,
et al., 1990). Grass family Poaceae can
be divided to one small subfamily
(Centothecoideae), and five large
subfamilies (Arundinoideae, Pooideae,
Chloridodeae,
Panicoideae,
and
Bambusoideae)
(Chapman,
1997).
Bamboo can be found in tropical and
subtropical area (Yakubu and Bukoye,
2009).
Bamboo have two main active
compound, there are phytosterols and
phenols. Bamboo that have been
extracted with ethanol have some
bioactive compound such as diterpenes,
phenols,
triterpenes,
saponin,
phytosterols, tannins and flavonoid. In

water extract, bamboo have some
bioactive compound such as saponin,
diterpenes, triterpenes, phenols, tannins
and flavonoids (Jovale, et al., 2014).
Phytosterols are bioactive components
that represent the major part of the
nonsaponifiable fraction of lipids that
have many implication on human
health, such as cholesterol-lowering
effect and anticancer effect in lungs,
stomach, ovaries, and estrogendependent human breast cancer
(Kritchevsky
and
Chen,
2005).
Phytosterols is soluble in ethanol
because of some hydroxyl groups
present in its structure. However,
phytosterols are not soluble in water
because of their large size. Phenols are
more soluble in ethanol than in water.
Different from phytosterols, phenols are
less soluble in water because it cant
break the hydrogen bonds between or
among phenolic compound (Jovale, et
al., 2014).
1.2.B16 cell
The B16 cell is the second most widely
used model for murine melanoma test.
The B16 cell is derived from C57BL/6
mice tumor cell that have been induced
(Fidler and Nicolson, 1976). B16 cell is
produced by mutation of normal
melanocytes.
The
biochemical
metabolism of the cell is also similar
with human melanocytes especially to
its melanogenesis function (Maeda and
Fukuda, 1996). Characteristic of B16
cell is can produce melanin pigment.
Cells may lose ability to produce
melanin in long term culture (Teicher,
2002). The viability of B16 cell can be
15thNational Student Conference Integrating Innovative Food Product Development and

Consumer Preferences through Sensory EvaluationDepartment of Food Technology,
Soegijapranata Catholic University SemarangTuesday, September 1st 2015
Page 137
determined by use of the microculture

tetrazolium technique or MTT assay
(Arung, et al., 2006).
1.3. MTT Assay
MTT assay (3-[4,5-dimethylthiazol-2yl]-2,5 diphenyl tetrazolium bromide) is
based on the convertion of MTT into
formazan crystals by living cell that
determines by mitochondrial activity.
Most cell populations are related to the
number of cell viability. MTT assay
used to measure the in-vitro cytotoxic
effect on cells (Van Meerloo, et al.,
2011). Mosmann (1983) said that cell
viability assay developed for a 96-wells
format that suitable for high throughput
screening (HTS). Marshall, et al.,
(1995) said that viable cells can convert
MTT reagent into purple colored
formazan product that can be detected
by using 570 nm wavelengths. If the
cells die, it will be lose the ability to
convert MTT into formazan. Formazan
can be absorb and precipitated by the
cells surface, so the color of the cells
will be changed into dark purple. Tada,
et al., (1986) said that formazan should
be soluble when recording the
absorbance. There are various liquid to
soluble the formazan such as
dimethylformamide, SDS, acidified
isoporopanol,
DMSO
and
also
combination of detergent and organic
solvent. DMSO or dimethyl sulfoxide is
the one of the most commont solvent
that used to dissolve hydrophobic
substances for in vivo and in vitro
purposes (Bartsch, et al., 1976).
Melanogenesis is a biosynthetic
pathaway to form melanin pigment on
human skin that controlled by
tyrosinase enzyme in melanocytes
(Chang, 2009; Chang, 2002; Hearing
1999). Tyrosinase is an enzyme that
contain copper and can produce oquinones from o-diphenol. O-quiones
are highly reactive compound that can
be polymerize to form melanin and
brown pigments (Seo, et al., 2003).
Tyrosinase activity can be inhibit by
bioactive such as polyphenols in
bamboo. Melanin is a pigment that
responsible for skin and hair color that
have role to predict human skin cancer
risk. Melanin has a role to absorb free
radicals on cytoplasm, and shield the
host from UV light. If the accumulation
of melanin in the skin is in abnormal
quantity, so that can results in
pigmentation patches of skin (Chang,
2009).
-MSH
or
alpha-melanocytestimulating hormone is a hormone that
can stimulate tyrosinase activity in
melanoma cells (Man, et al., 2014).MSH also a peptide derived from
proopiomelanocortin (POMC) that
regulates melanogenesis via a cyclic
adenosine monophosphate (cAMP)dependent pathway (Busca and Ballotti,
2000). Ascorbic acid has known as a
skin whitening agent and anti-browning
agent. Ascorbic acid has an antimelanogenic effect that can inhibit
melanin content (Panich, et al., 2011).
1.4. Melanogenesis

Page 138
2. RESEARCH METHODOLOGY
2.1.Bamboo Sample Extraction
2.1.1. Material
Bamboo powder(bought from Sun-Ling
Bamboo Processing Factory, Taiwan),
water, 95% ethanol, digital scale,
centrifuge tube, centrifuge, speedy
autoclave, shaker, 0.45m membrane
filter, rotary evaporator, and freeze dryer.
2.1.2. Method
2.1.2.1.Bamboo Water Extraction
35 gram of bamboo powder was weight
using digital scale for 3 times. Then, put
the bamboo powder in the centrifuge tube
and added by 250 ml of water and
extracted with hot water at 120oC for 20
minutes using speedy autoclave. Then,
solution was put in the room temperature
to cool down the temperature. Solution
was filtered using 0.45m membrane filter
and freeze dried at -50oC for 48 hours.
2.1.2.2.Bamboo Ethanol Extraction
20 gram of bamboo powder was weight
using digital scale for 6 times. Then, put
the bamboo powder in the centrifuge tube
and added by 150 ml of 95% ethanol and
shake at 200 rpm for 48 hours using
shaker. Ethanol extract were concentrated
using rotary evaporator for 400 rpm until 1
week for all bottle. Recovery of ethanol
extract calculated using formula below.
Recovery =
initial sample weight

final sample weight
100%
2.2. Cell Subculture

2.2.1. Material
75% ethanol, DMEM medium (Dulbeccos
Modified Eagles Medium) with 10%
FBS, B16 cells, PBS, laminar flow,

waterbath, centrifuge tube, centrifuge,
micro pipette, dish plate, suction unit, and
incubator.
2.2.2. Method
Laminar flow was wiped with 75% ethanol
and UV light was opened before 15
minutes to use. DMEM medium was
warmed using waterbath until the
temperature reached 37oC. B16 cells were
taken out from the liquid nitrogen tank and
warmed using waterbath until the
temperature reached 37oC. Cells were
added by 5.5 ml medium and centrifuged
at 1000 rpm for 3 minutes. Dish plate was
prepared and added by 7 ml medium. After
centrifuged, medium was removed using
high pressure and the cells were diluted
using 1 ml medium. The cells were added
to dish plate and incubated at 37oC for 24
hours. The medium was removed after
incubated and washed using 2 ml
phosphate buffer saline (PBS). PBS was
removed and then 7 ml medium were
added to the dish plate. The cells were
incubated at 37oC for 24 hours.
2.3.MTT Survival Cell Test
2.3.1. Material
B16 cells, DMEM medium with 10% FBS,
DMEM medium with free FBS, PBS,
TrypLE, trypan blue stain 0.4%, bamboo
water extract, bamboo ethanol extract,
DMSO, incubator, dish plate, centrifuge,
centrifuge tube, micro tube, micro pipette,
cell counting plate, microscope, vortex,
96-wells, 8 channel pipette, sample cage,
acrodisc syringe filter, suction unit, and
ELISA-reader.
2.3.2. Method
2.3.2.1.Cell Counting and Seeding
Page 139
Cells were taken out from incubator and

removed the medium. Cells were washed
using 2 ml PBS and removed the PBS. 1.5
ml TrypLE were added to the dish and
removed it. The cells were incubated at
37oC for 3 minutes. After 3 minutes, the
dish was knocked and then the cells were
collected using 7 ml medium. The solution
was put into centrifuge tube and then
centrifuged at 1000 rpm for 3 minutes. The
supernatant (medium) was removed and
the cells were collected using 10 ml
medium. 3 micro tubes were prepared and
900l medium were added to second
micro tube. 1 ml of cells were taken and
put into first micro tube. 100l of the first
micro tube were added to the second micro
tube (10 fold dilution).20l of second
micro tube was taken and put into the third
micro tube. 20l of trypan blue stain
(0.4%) were added to third micro tube (2
fold dilution). 20l sample was injected to
cell counting plate using micro pipette.
The cell was counted in 5 squares of two
sides of cell counting plate using
microscope. The number of cells and
preparation proportion was calculated. The
medium and cells were mixed using low
speed vortex.
Preparation
proportion
=1 ml medium with cells x
1 ml medium without cells
Where:
y = Cell number
x = Total medium
a = Two side of counting plate
b = Number of square
c = 10 fold dilution (ml)
d = 2 fold dilution
e = 10 l of sample
f = Coefficient
g = Cell seeding number
The 96 well-plate and 8 channel pipettes
were prepared. The solution of cells was
put into the sample cage. Then, 200 l of
cells solution were added to the 96 wellplate using 8 channel pipettes carefully.
The cells were incubated in 37oC for 24
hours.
2.3.2.2. Sample Preparation
1 gram of each bamboo water extract
powder and bamboo ethanol extract
powder were added by 10 ml water
(100,000 ppm). 10,000 ppm was made by
adding 100l of 100,000 ppm and 900l
serum free DMEM medium, then filtered
using acrodisc syringe filter (blue color
filter for water extract and white color
filter for ethanol extract). 1,000 ppm was
made by adding 400l of 10,000 ppm and
3,600l serum free DMEM medium. 500
ppm was made by adding 1,000l of 1,000
ppm and 1,000l serum free DMEM
medium. 200 ppm was made by adding
400l of 1,000 ppm and 1,600l serum
free DMEM medium. 100 ppm was made
by adding 400l of 1,000 ppm and 3,600l
serum free DMEM medium. 50 ppm was
made by adding 1,000l of 100 ppm and
1,000l serum free DMEM medium. 10
ppm was made by adding 200l of 100
ppm and 1,800l serum free DMEM
medium. 5 ppm was made by adding
100l of 100 ppm and 1,900l serum free
DMEM medium.
2.3.3.3. Sample Adding
After cells were counted, cells were added
to 96-wells using 8 channels pipette. Then,
cells were incubated for 24 hours. Medium
was removed using high pressure suction
Page 140
unit and different concentration of samples

were added to the 96-wells and incubated
for 24 hours.
2.3.3.4. MTT Cell Survival Test
2.5 ml MTT reagent were mixed using
22.5 ml of free serum DMEM medium.
Then, medium in 96-wells was removed
using high pressure suction unit. The
solution was put in the sample cage and
pipetted using 8 channels pipettes to 96wells. 96-wells were incubated in the
incubator for 1 hour. Then, medium was
removed using high pressure suction unit.
200l of DMSO were added to the 96wells to dissolve the purple sediment. 96wells absorbance value was analyzed using
ELISA-reader at 550 nm.
2.4.Melanin Content Test
2.4.1. Material
B16 cells, DMEM medium with free FBS,
PBS, water, ascorbic acid, bamboo water
extract, bamboo ethanol extract, -MSH,
1N NaOH, incubator, dish plate,
centrifuge, centrifuge tube, micro pipette,
micro tube, cell plastic scraper, hot dry
bath, suction unit, and ELISA-reader.
2.4.2. Method
2.4.2.1. Cell Counting and Seeding
The number of the cells needed in melanin
content test was calculated using cell
counting formula. After the determination
is done, then the cells were seeded in 12
dish plates and incubated for 24 hours.
2.4.2.2. Sample Preparation
Medium was removed from the dish and
the cells washed by using PBS twice (first
using 3 ml of PBS and second using 2 ml
of PBS). Then, 5 ml serum free DMEM
medium were added into the dish and
incubated for 3 hours. Then, the 10,000

ppm sample were made by adding 100l
of 100,000 ppm and 900l serum free
DMEM medium, then filtered using
acrodisc syringe filter (blue color filter for
water extract and white color filter for
ethanol extract). 1,000 ppm sample were
made by adding 100l of 10,000 ppm and
900l serum free DMEM medium (3
times). 500 ppm sample were made by
adding 50l of 10,000 ppm and 950l
serum free DMEM medium (2 times). 200
ppm sample were made by adding 200l
of 1,000 ppm and 800l serum free
DMEM medium (2 times). Positive control
was made by adding 5 mg of ascorbic acid
and 500l of H2O. Then, positive control
was filtered using acrodisc syringe filter.
2.4.3.3. Sample Adding
After the sample preparation was done,
medium from dish plate was removed. 7
ml of serum free DMEM medium were
added to control and -MSH dish. Then,
6.3 ml of serum free DMEM medium and
700l of each samples (1000 ppm, 500
ppm and 200 ppm) were added to positive
control, 100 ppm, 50 ppm and 20 ppm dish
(10 fold dilution). Then, dish plate was
incubated for 30 minutes. After incubated,
dish plate (except control) were added 7l
of-MSH and incubated for 72 hours.
2.4.3.4. Melanin Content Test
After incubated for 72 days, medium from
the dish were removed. 2 ml of PBS were
added to the dish plate to wash the cell
from medium and PBS was removed using
high pressure suction unit. Then, 1 ml of
PBS was added to the dish plate and
scrapes the cell using cell plastic scraper.
The cells each dish was collected into the
micro tubes using micro pipette and
Page 141
centrifuged at 12,000 rpm for 15 minutes.

Supernatant was removed using high
pressure suction unit and 500l of 1 N
NaOH were added to the micro tubes.
Micro tubes were put in the 95 oC hot dry
bath for 15 minutes. The cells were put in
the 96-wells 150l for each well and
analyzed using ELISA-reader at 405 nm.
2.5. Statistical Analysis
The experiments were expressed as mean
standard error. Statistical analyses were
performed with SPSS program (SPSS
version 10.0) using one way ANOVA. If
significant by ANOVA, differences in the
means were determined using Duncans
multiple range tests (p > 0.05).
3. RESULTS AND DISCUSSION
bamboo water extract. Whereas, the cell

viability of control group was not
significant different with 1000 ppm, 200
ppm and 10 ppm of bamboo water extract.
The higher bamboo water extract was
added to B16 cells, so the cell viability
will be lower. This means that B16 cells
cannot survive in high concentration of
bamboo water extract. However, all of the
bamboo water extract concentrations had
higher cell viability of B16 cells than
control group. This could be happened
because the number of the cells in each
well was not uniform, so it may influence
the result.
3.1.2. Ethanol Extract
3.1. MTT Cell Survival Analysis

3.1.1. Water Extract
Figure1. Effect of bamboo water extract on

cell viability in melanocytes. Control:
normal cell. 5-1000 ppm: bamboo water
extract at 5-1000 ppm.
Based on Figure 1., cell viability of B16
cells
were
depend
on
different
concentration of bamboo water extract. As
we can see, the cell viability of control
group was significant different with 500
ppm, 100 ppm, 50 ppm and 5 ppm of
Figure 2. Effect of bamboo ethanol extract

on cell viability in melanocytes. Control:
normal cell. 5-1000 ppm: bamboo ethanol
extract at 5-1000 ppm.
Based on Figure 2., cell viability of B16
cells
were
depend
on
different
concentration of bamboo ethanol extract.
As we can see, the cell viability of control
group was not significant different with all
of bamboo ethanol extract concentrations.
This means that B16 cells cannot survive
in high concentration of bamboo ethanol
extract. However, all of the bamboo
Page 142
ethanol extract concentrations had higher

cell viability of B16 cells than control
group. The different concentrations of
bamboo ethanol extract that given in the
cell could influence cell viability in B16
cells.
3.2. Melanin Content Analysis
3.2.1. Water Extract
melanoma cells, so the melanin content

will be increased. Positive control group
has the lowest melanin content than all of
the concentration that given, this could be
happened because positive control that we
used was ascorbic acid. Ascorbic acid is a
skin whitening agent that can prevent
tryosinase activity, so that the melanin
content will be decreased.
3.2.2. Ethanol Extract
Figure 3. Effect of bamboo water extract

on melanin content in melanocytes.
Control: normal cell. -MSH: -MSH at
200M. PC: Positive Control on 100 ppm
of ascorbic acid and H2O. 20-100 ppm:
bamboo water extract at 20-100 ppm.
Based on Figure 3., melanin content of
B16 cells were depend on different
concentration of the treatment that was
gave. As we can see, the melanin content
of control group was not significant
different with 50 ppm and 100 ppm of
bamboo water extract. Whereas, the
melanin content of control group was
significant different with -MSH, positive
control (PC), and 20 ppm. The higher
bamboo water extract was added to B16
cells, so the melanin content will be lower.
-MSH group has the highest melanin
content than all of the concentration that
given, this could be happened because MSH can stimulate tyrosinase activity in
Figure 4. Effect of bamboo ethanol extract

on melanin content in melanocytes.
Control: normal cell. -MSH: -MSH at
200 M. PC: Positive Control on 100 ppm
of ascorbic acid and H2O. 20-100 ppm:
bamboo ethanol extract at 20-100 ppm.
Based on Figure 4., melanin content of
B16 cells were depend on different
concentration of the treatment that was
gave. As we can see, the melanin content
of control group was significant different
with all of the concentrations that given.
The higher bamboo water extract was
added to B16 cells, so the melanin content
will be lower. -MSH group has the
highest melanin content than all of the
concentration that given, this could be
happened because -MSH can stimulate
tyrosinase activity in melanoma cells, so
the melanin content will be increased. The
Page 143
usage of bamboo ethanol extract on B16

cells is more effective than bamboo water
extract on reducing melanin content on
B16 cells.
4. CONCLUSION
Bamboo ethanol extract is more

effective to prevent melanogenesis
than bamboo water extract.
Bamboo that extracted by ethanol has
several active compounds, such as
phenols, diterpenes, phytosterols,
saponins, triterpenes, tannins and
flavonoids that could inhibit or
prevent melanogenesis.
Bamboo that extracted by water has
several active compounds, such as
saponins, diterpenes, triterpenes,
phenols, tannins and flavonoids that
could
inhibit
or
prevent
melanogenesis.
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Fauzidah. (1990). Anatomical features
and mechanical properties of three
Malaysian bamboos. Journal Tropical
Forest Science 2(3): 227-234.
Arung, E.T.; Shimizu, K.; and Kondo, R.
(2006).
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Bartsch, W., Sponer, G., Dietmann, K.,
and Fuchs, G.(1976).Acute toxicity of
various solvents in the mouse and rat.
LD50 of ethanol, diethylacetamide,
dimethylformamide,
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glycerine,
Nmethylpyrrolidone,
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glycol 400, 1,2-propanediol and
Tween 20. Arzneimittelforschung 26:
15813.
Busca, R., and Ballotti, R. (2002). Cyclic
AMP a key messenger in the
regulation of skin pigmentation.
Pigment Cell Res. 13: 6069.
Chang,
T.S.
(2002).
Natural
Melanogenesis Inhibitors Acting
Through the Down-Regulation of
Tyrosinase Activity. pp: 1661-1685.
Chang, T.S. (2009). An updated review on
tyrosinase inhibitors. Int. J. Mol. Sci.
10, 24002475.
Chapman, G.P. (1996). The biology of
grasses. Department of Biochemistry
and Biological Sciences, Wye
College, University of London, U.K.
CAB International. 14-19.
Chapman, G.P. (1997). The bamboos.
Papers presented at an international
symposium organized by the Linnean
Society of London. The Royal Botanic
Gardens Kew and Wye College,
University of London, London.
Linnean Society Symposium Series
No. 19. Academic Press.
Fidler,
I.J.,
and
Nicolson
GL.
(1976).Organ
selectivity
for
implantation survival and growth of
B16 melanoma variant tumor lines. J
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Hearing, V. J. (1999). Biochemical control

of melanogenesis and melanosomal
organization. Journal of Investigative
Dermatology
Symposium
Proceedings, 4, 2428.
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Assays for Cellular Growth and
Survival: Application to Proliferation
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Jovale, V. V. T., Remil, M. A., and

Ramon, A. R. (2014). Proximate
Analysis, Phytochemical Screening,
and Total Phenolic and Flavonoid
Content of Philippine Bamboo
Schizostachyum lumampao. J. Chem.
And Pharm. Res. 6(1): 709-713.
Panich,
U.,
Tangsup-a-nan
V.,
Onkoksoong, T., Kongtaphan K.,
Kasetsinsombat K., Akarasereenont
P., and Wongkajornsilp A. (2011).
Inhibition
of
UVA-mediated
Melanogenesis by Ascorbic Acid
trough Modulation of Antioxidant
Defense and Nitric Oxide System.
Kritchevsky, D., and Chen, S.C. (2005).

Phytosterols Health Benefits and
Potential Concerns A Review. Nutr.
Res. 25:413-28.
Lloyd, H. W., and Jenna, N. K. (2011).
Treatment of Hyperpigmentation.
Elsevier, Inc. Semin. Cutan. Med.
Surg. 30:171-175.
Maeda K, and Fukuda M (1996). Arbutin:
mechanism of its depigmenting action
in human melanocyte culture. J
Pharmacol. Exp. Ther. 276(2):765769.
Man, Kyu Huh., Min, Ho Han., Cheol
Park and Yung, Hyun Choi. (2014).
Inhibitory Effect of Phyllostrachys
bambusoides on Melanin Synthesis
and Tryosinase Activity in Cultured
Human Melanomela Cells.
Marshall, N.J., Goodwin, C.J., and Holt,
S.J. (1995). A Critical Assessment of
the Use of Microculture Tetrazolium
Assays to Measure Cell Growth and
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Seo, S. Y., Sharma, V. K., & Sharma, N.

(2003). Mushroom tyrosinase: Recent
prospects. Journal of Agricultural and
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Tada, H., Shiho, O., and Kuroshima, K.
(1986). Re-examination and Further
Development of a Precise and Rapid
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cancer research. In B16 Murine
melanoma. Edited by Alvarez E. New
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Van Meerloo, J., Kaspers. G.J., and Cloos
J. (2011). Cell Sensitivity Assays: The
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Yakubu, M.T., and Bukoye. B.B. (2009).
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aqueous extract of Bambusa vulgaris
leaves in pregnant Dutch Rabbits.
Contraception, 80(3): 308-313.
Page 145
Yuan, J.X. (1983). Research on the

Production and Botanical Origin of
Bamboo Juice in Eastern China.
Zhong Yao Tong Bao. 8:10-12.
Page 146
FOOD PRESERVATIVE TOOLS AND HARMFUL HEAVY METAL

REDUCERS ON VEGETABLES AND FRUITS
EmasAgusPrastyoWibowo*, HeruSetiawan
a
Prodi Kimia, Fakultas Matematika dan Ilmu Pengetahuan Alam, UniversitasNegeri

Semarang
Gedung D6 lantai 2, SemarangIndonesia
b
Prodi Pendidikan Biologi, Fakultas Matematika dan Ilmu Pengetahuan Alam, Universitas
Negeri Semarang
Gedung D6 lantai 1, SemarangIndonesia
*Email: emasagus@ymail.com
ABSTRACT
Vegetables, fruits, and fish are the primary food of the Indonesian people. The food that unclean and
polluted by some substances would be harmful for human health. But the fact showed that marine
organisms such as fish in Indonesia has polluted by harmful heavy metals. When heavy metals such as
Pb, Hg, Cd, Cu and Zn are toxic in the body tissues of marine organisms in high concentrations will be
dangerous. Based on this problems it is needed for innovative research in order decreased levels of
heavy metals in food. The aim of this research is to analyze the decreased levels of heavy metals in the
pickling system based reduction of heavy metals in foods that more energy efficient based on the
photocatalytic reaction by visible light from the sun as a solution to providing safe food. The method
of this research was experimental method: preparation of tools and materials, develop the tools, and
application. The results showed that N-doped TiO2 innovation can reduce levels of heavy metals in
food, that can be used as consideration as a food preservative. The food preservative tool using TiO2
material. TiO2 is a photocatalyst material that has antibacterial activity and can reduce the heavy
metals. With the innovation of the N-doped TiO2photocatalyst reaction can also be initiated with
visible light and photocatalytic reactions so it would be more effectively. The work system of this
tools is Photocatalyst N-doped TiO2 can be generate as the superoxide compounds through fotocatalics
reaction that kill bacteria and reduce dangerous heavy metals.
Keywords: food preservatives tools, N-doped TiO2, Heavy Metal Reducers, Photocatalysis
Page 147
INTRODUCTION
Four of five perfectly healthy motto
proclaimed as an invitation to make a
balance of nutrition in daily consumption,
which consists of staple food, side dishes,
vegetables, fruits and milk. Vegetables are
important food in health. However,
vegetables can cause illness when
contaminated by heavy metals or
microorganisms. In 1994 data showed that
vegetables grown on the roadside contain
contaminants tetra ethyl lead at 28.78 ppm
originating from motor vehicle fumes
(Winarno, 1994). Dissertation research in
1997 against cadmium contamination at
kale, basil, and calsim in the Supreme
Lenteng obtained content of 1.56 ppm;
1.04 ppm; and 1.86 ppm.
Fruits and vegetables are foods that are
very easily available in Indonesia, even
each region has a fruit and vegetable as
typical for the area. Fruits and vegetables
with various types and colors can be
complementary nutritional needs required
by our body. In addition, one of the
foodstuffs that contain lots of fiber found
in fruits and vegetables (Jahari, 2001).
Vegetables and fruits are a source of
vitamins and minerals that the body needs
to regulate processes in the body.
Although the need for a relative small, but
the function of vitamins and minerals can
hardly be replaced so that the requirement
for the consumption of these substances is
essential. If the consumption of vitamins
and
minerals
doesnt
meet
the
requirements, then the body will
experience a deficiency of vitamins and
minerals which may result in reduced
endurance.
According
to
research
conducted, the type of foods that contain
vegetables and fruits in the school cafeteria

not meet enough criteria to be consumed.
Nanotechnology in the world is growing
so rapidly. One example is the
nanomaterial TiO2 (Titanium dioxide) is a
photocatalyst substance economically
valuable (Burgess, 2007). The working
principle photocatalyst TiO2 is a nanosized titanium dioxide when exposed to
UV rays will form a super-oxide
compounds which can oxidize a variety of
organic compounds so as to carbon
dioxide CO2 and water H2 O (Samal et al,
2010). TiO2 that can oxidize organic
compounds such as bacteria (antibacterial)
and economical price makes the nano (1
nm- 100 nm) TiO2 as a suitable material in
the manufacture of preservatives and
reducing harmful metals on vegetables and
fruits.
Based on its utilization as a means of
curing and reducing harmful heavy metals
in fish and shellfish photocatalyst TiO2 has
some weaknesses that can only be initiated
by UV light (Liu et al, 2011), whereas the
UV light can damage the protein content in
fish and shells so necessary modifications
to the material in order to work on a range
of visible light. One solution of this
problem is the coat TiO2 and Nitrogen
(Rani et al, 2010). Coating TiO2 with
nitrogen gas to raise the activity of organic
compounds hinggga pendekomposisian
wavelength of 550 nm (Ashahi et al, 2001)
or can be said to be N-doped TiO2 as a
visible light photocatalyst. With these
modifications, the activity TiO2 as a
photocatalyst as an antibacterial and
reducing heavy metal becomes more
effective.
Page 148
Bacteria and harmful heavy metals are

compounds that can be reduced by ndoped photocatalyst TiO2 . N-doped TiO2
can reduce bacteria up to 98.7% by using
visible light (Wang, 2011). In addition
photocatalyst with N-doped TiO2 can
reduce dangerous heavy metals such as Cr
(VI) to 80% (Slamet, 2003). From these
data it can be concluded when the n-doped
TiO2 superimposed on the glass, the Ndoped TiO2 may provide antibacterial
effect resulting in vegetables and fruits to
avoid the decay process and reduce metal
harmful heavy on vegetables and fruits so
that it would be safe to eat.
Based on the facts above, one of the
innovations that will be the solution to the
problem solving preservation system and
harmful heavy metals in vegetables and
fruits is to create a tool preservatives and
reducing heavy metals be deadly in food
(vegetables, fruits) with N-doped TiO2 .
The tool serves to preserve vegetables and
fruits with their high antibacterial activity
and can reduce dangerous heavy metals in
vegetables and fruits. Tools of N-doped
TiO2 is also energy efficient because of the
preservation system only needs energy
from sunlight usual, another advantage of
this tool also does not spoil the taste,
texture and nutrition because vegetables
and fruits will only be illuminated by
visible light. In addition the tool
preservatives and reducing harmful heavy
metals is easily applied and is not harmful
to the environment because it will only
produce byproduct CO2 and H2 O.
Material
Materials used are TiO2 Degussa P-25,
ethanol, HCl, urea, and distilled, plate

glass, plywood and wooden formwork.
Synthesis method
The initial process of making tools
metals in vegetables and fruits have started
from TiO2 N synthesis process. The
nanomaterial-making process is as follows
(Saragih, 2011):
1. Mixing 5 grams of TiO2 Degussa P25 in 70 mL of absolute ethanol
and proceed with the process of
sonication for 10 minutes
2. Mix 10 mL of absolute ethanol, 10
mL of distilled water and 2 M HCl
to achieve pH 2. Stir the mixture
with magnetuc stirrer for 30
minutes to form sol
3. Stirring the mixture TiO2 Degussa
P-25 and ethanol with a magnetic
stirrer for 30 minutes
4. Incorporating urea into TiO2 sol
mixture and followed by the
stirring with a magnetic stirrer for
60 minutes.
5. Evaporating the sol at a
temperature of 70 under the
hood for 1 hour
6. Heating the sol in the furnace at a
temperature of 500 for 1 hour.
TiO2 - N derived from this method
is the basic material that will be used to
coat a glass plate on the appliance
metals
in
vegetables
and
fruitbuahan.Setelah obtained TiO2 - N is then
performed with a glass plate making
system using the method of spray coating ,
Spray method is intended that the glass is
coated with TiO2 - N equitably. Spray
method can create a thin layer of TiO2 - N
Page 149
on the glass plate. This method can also

save on the use of TiO2 - N so that this
method can be produced TiO2 -coated glass
plate - N with a relatively low cost
(Laksono, 2014) . After obtained TiO2 coated N glass plate is then performed
making tools preservatives and reducing
hazardous wastes on vegetables and fruits

with the outer frame design tool using
metal or plastic polymers. But in order to
minimize the cost of these tools can be
made using waste such as straw and wood
plank formwork.
Fig 1. Preservative Tool Vegetables and Fruits

when the glass plate coated yangg NUse wooden plank formwork on the
doped TiO2 irradiated by sunlight then
device not only reduces the cost of
there will be storage diruang super oxide
production tools but also make the tool
compound. Super oxide compound will
becomes lighter and unique. The design of
oxidize bacteria that are in the storage
the tool preservatives and reducing
room so that the bacteria will die. Nharmful heavy metals can still continue to
doped material TiO2 is catalyst so that the
evolve and is still need for improvements
photocatalytic reaction can continue to
to address weaknesses in these tools.
take place continuously (Laksono, 2014).
Preservation mechanism with this tool is
Page 150
Tools preservatives and reducing harmful

heavy metals in vegetables and fruits is
economically valuable. In addition, this
tool does not produce byproducts that are
harmful to the environment and this tool
requires low energy. So with the
innovation and design of the device is
expected to deal with issues regarding the
preservation system and dangerous
problem of heavy metals in vegetables and
fruits in Indonesia.
Burgess, Kevin David. 2007. Self-Cleaning

Titania-Polyurethane Composites.
CONCLUSION
1. Preparation of preservative tool
framework and reducing harmful
heavy metals in vegetables and
fruits can be done by utilizing the
waste straw and wood plank
formwork so as to reduce the price
on the device manufacturing
process.
2. The solution to the provision of
vegetables and fruits is a functional
utilization of N-doped TiO2
photocatalyst which is a material
that is suitable for use in tool
preservatives and reducing harmful
heavy metals in vegetables and
fruits because of their antibacterial
activity and can reduce dangerous
heavy metals. TiO2 nano materials
can be made using the initial
material TiCl4 with sol gel method.
Method of N-doped TiO2 coating
on glass plates using the method of
spray in order to get a thin layer
and evenly N-doped TiO2 on a
glass plate.
Jahari.2001. Epidemiologi Konsumsi Serat

di Indonesia. PUSLITBANG Gizi
DepKes RI.
REFERENCES
Ashahi, T. Ohwaki, K. Aoki, Y. Taga,
Science 293 (2001) 269271
Liu, Xu; Liu, Zhongqing; Jian Zheng, Xin

Yan, Dandan Li, Si Chen, Wei
Chu, 2011,Characteristics of Ndoped TiO2 nanotube arrays by
N2-plasma
for
visible
lightdrivenphotocatalysis, College
of Chemical Engineering, Sichuan
University: China
Laksono,F.B.
2014.
AlatPengawet
danPereduksi
LogamBerat
Berbahaya pada Ikan danKerang
Hemat Energi Berbasis nano NDoped
Semaran
:
2 .
Universitas Diponegoro
Rani,
S.C. Roy, M. Paulose, O.K.

Varghese, G.K. Mor, S. Kim, S.
Yoriya, T.J.LaTempa,
C.A.
Grimes, Phys. Chem. Chem. Phys.
12 (2010) 2780280
Samal, S.S.; Jeyaraman, P.; Vishwakarma,

V, 2010, Sonochemical Coating of
Ag-TiO2 Nanoparticles on Textile
Fabrics for Stain Repellency and
Self-Cleaning- TheIndian Scenario:
A Review. Sathyabama University:
India
Saragih, Winda J. 2011. Degradasi
Polutan
Udara
Ruanagn
Menggunakan Lampu Hias
dengan
Penutup
Berlapis
Katalis2 Termodifikasi.
Page 155
Skripsi. Depok : Universitas

Indonesia
Wang,
H.; Tang, B.;Li, X.;Ma,Y.,

2011,Antibacterial Properties and
Corrosion ResistanceofNitrogendopedTiO2Coatingson
StainlessSteel.TaiyuanUniversity
of Technology:China
Winarno, F.G, 2004. Kimia Pangan dan

Gizi. Jakarta : Gramedia
Page 156
THE EFFECTIVENESS OF ANTIMICROBIALBIODEGRADABLE

PACKAGING TO INHIBIT ESCHERICHIA COLI GROWTH
Lisa Widagdoa, Shaun Chenb, R. Probo Y. Nugrahedic
1Students;
Food Technology Department; Faculty of Agricultural, Soegijapranata Catholic

2Lecturer of Food Science Department, Fu Jen Catholic University, Taiwan
3
Lecturer; Food Tecnology Department; Faculty of Agricultural, Soegijapranata Catholic
Email: Lisa.widagdo@yahoo.com
ABSTRACT
In this era, the demand for minimally processed, easily prepared, and ready-to-eat food products pose
major challenges in terms of safety and quality. In the ready-to-eat foods that can be an indication of
poor hygiene and sanitation or inadequate heat treatment, Escherchia coli can be grow. Antimicrobial
biodegradable packaging may provide an effective way to control food-borne pathogens and spoilage
microorganisms, thus enhance food safety and decrease product spoilage. Besides, the use of
biodegradable packaging also contibute to reduce the municipal solid waste problem. The research
objective is to know that were thymol and chitosan, as antimicrobial agents, can inhibit E. coli growth
and to find the effective concentration of antimicrobial agent that can inhibit E. coli. There are 3
different antimicrobial agent and 2 different concentrations that were used in project. These are
thymol 3%, thymol 5%, chitosan 3%, chitosan 5%, thymol mixed with chitosan 3% (1,5% thymol and
1,5% chitosan) and thymol mixed with chitosan 5% (2,5% thymol and 2,5% chitosan). The material
was coated onto polylactic acid film and then tested with Escherichia coli. The result shows that
thymol and chitosan at 3% concentration can inhibit E. coli but the value is not significant (P<0,05)
and mix 3% concentration can not show any synergic antimicrobial activities.
Keywords: food, packaging, biodegradable, antimicrobial, chitosan, thymol, Escherichia coli
Page 157
1. BACKGROUND OF RESEARCH
Food packaging can improve the food
quality and safety. In this era, the demand
for minimally processed, easily prepared,
and ready-to-eat food products pose major
challenges in terms of safety and quality.
Therefore, development of antimicrobial
biodegradable films to protect fresh and
processed foods from human pathogens
and extend the shelf life of foods is
becoming the new trend in food safety
research (Du et al., 2011).
Some animal products, such asraw or
undercooked eggs, raw meat, raw poultry,
raw fish are more risky for consumers
because those are most likely to contain
harmful bacteria. Raw products without
cooking or undercooked have an increased
risk
of
contracting
Salmonella,
Campylobacter and Escherichia coli
(Oosterom et al., 1980). In the ready-to-eat
foods that can be an indication of poor
hygiene and sanitation or inadequate heat
treatment, E. coli can be grow. E coli is an
organism that is part of the normal
microflora of the intestinal tract of humans
and warm-blooded animals (NSW Food
Authority, 2009). The risk of the food
contaminated by E. coli can affect human
health, such as enteritis and several
extraintestinal diseases such as urogenital
infections, wound infections, mastitis,
septicaemia and meningitis (Wasteson,
2001).
Antimicrobial biodegradable films may
provide an effective way to control foodborne
pathogens
and
spoilage
microorganisms, thus enhance food safety
and decrease product spoilage (Du et al.,
2011). Besides, the use of biodegradable
packaging also contibute to reduce the
municipal solid waste problem (Song et

al., 2009).The research objective is to
know that were thymol and chitosan, as
antimicrobial
agents,
can
inhibit
Escherichia coli growth and to find the
effective concentration of antimicrobial
agent that can inhibit E. coli.
1.1.Biodegradable Film
Biodegradable film is produced from
biopolymers that can be reused and
recycled (Siracusa et al., 2008 and Ramesh
et al., 2010). Polylactic acid (PLA) is a
synthetic biodegradable polymer that
produced from natural monomer (Richard
and Bhanu, 2002). PLA is a linear
aliphatic polyester that is made from lactic
acid monomers (Lim et al., 2008). PLA
has a significant potential for the
packaging industry because it yields stiff
films of high transparency and can be
processed
using
readily
available
production technologies (Petersen et al.,
2001).
The
characteristics
of
PLA
is
environmentally-friendly,
biocompatibility,
sustainability
and
potentially useful physical and mechanical
properties (Ishida et al., 2006). PLA has a
high strength, good crease-retention,
grease and oil resistance, and excellent
aroma barrier properties. PLA is
decomposed by hydrolysis followed by
biodegradation via bacteria (Nakatsuka,
2011). Currently, PLA is being
commercialised as a food packaging
polymer for short shelf-life products, in
common applications of short shelf-life
products such as containers, drinking cups,
and overwrap and lamination films (Kale
et al., 2006).
Page 158
1.2.Antimicrobial Packaging
Antimicrobial packaging is a packaging
system that can kill pathogenic
microorganisms that contaminate the
foods. Antimicrobial packaging also can
inhibit spoilage caused by mishandling or
faulty packaging. The antimicrobial
function can be achieved by adding
antimicrobial agents into the packaging
system and/or using antimicrobial
polymers that satisfy conventional
packaging requirements (Han, 2000).
Essential oils that containing aldehydes or
phenols show the highest antibacterial
activity, such as cinnamaldehyde, citral,
carvacrol, eugenol or thymol as major
components (Dormans and Deans, 2000).
Thymol natural compound is considered as
a health-promoting ingredient in the food
industry (Shoji and Nakashims, 2004).
Thymol (2-isopropyl-5-methylphenol) can
be found as a component of many essential
oils (Gomez-Carneiro et al., 1998).
Thymol has an antimicrobial effect on
bacteria, yeasts and fungi (Shapiro et al.,
1994 and Manou et al., 1998).
Besides the essential oil, chitosan is also
reported to be an active polymer with
antimicrobial activity. Chitosan poly-b(1/4) N-acetyl-D-glucosamine is a cationic
polysaccharide that obtained from chitin
by deacetylation in the presence of alkali
(Cuero, 1999). The characteristics of
chitosan is decomposable, biocompatible,
exhibit a high sensitivity to mositure, and
low water barrier properties (Coma et al.,
2002). Chitosan has an antimicrobial effect
on yeast, fungi and various bacteria.
Chitosan has been used as an antimicrobial
agent to improve food safety and quality
(Roades & Roller, 2000).
2.
2.1. Making PLA Film

First, 0.33 gram Polylactic acid were put
into an erlenmeyer and mixed with 8 ml
chloroform. Then, the erlenmeyer was
covered with the parafilm and alumunium
foil and shaked until dissolved. After that,
the solution was poured into a petri dish
and dried overnight.
2.2. Making Antimicrobial Agent and
Coating Onto a PLA Film
2.2.1. Thymol
For 3% thymol, 0.0102 gram thymol were
put into a small tube and mixed with 1.02
ml ethanol. For 5% thymol, 0.017 gram
thymol were put into a small tube and
mixed with 1.7 ml ethanol. Then, the
solutions were shaked until dissolved and
coated onto a PLA film. Before the
solution coated onto a PLA film and dreid
overnight, 10 l binder that made from 10
l propylene glycol mixed with 90 l
ethanol were added.
2.2.2. Chitosan
For 3% chitosan, 0.0102 gram chitosan
were put into a small tube and mixed with
1.02 ml acetic acid 1%. For 5% chitosan,
0.017 gram chitosan were put into a small
tube and mixed with 1.7 ml acetic acid
1%. Then, the solutions were shaked until
dissolved and coated onto a PLA film.
Before the solution coated onto a PLA film
and dreid overnight, 10 l binder that
made from 10 l propylene glycol mixed
with 90 l ethanol were added.
2.2.3. Mix 3% Concentration
First, 0.0051 gram chitosan were put into a
small tube and mixed with 0.51 ml acetic
acid 1%. Then, 0.0051 gram thymol were
Page 159

ml ethanol. Both must be shaked until
dissolved. After all solution dissolved,
thymol and chitosan were mixed then 10
l binder that made from 10 l propylene
glycol mixed with 90 l ethanol were
added. After that, the solution was coated
onto a PLA film and dried it 1 day.
2.2.4. Mix 5% Concentration
First, 0.0085 gram chitosan were put into a
small tube and mixed with 0.85 ml acetic
acid 1%. Then, 0.0085 gram thymol were
ml ethanol. Both must be shaked until
dissolved. After all solution dissolved,
thymol and chitosan were mixed then 10
l binder that madefrom 10 l propylene
glycol mixed with 90 l ethanol were
added. After that, the solution was coated
onto a PLA film and dried it 1 day.
2.3. Making Trypticase Soy Agar
(TSA) for Bacteria Growth
First, 40 gram TSA powder and 1 L water
were mixed into a glass beaker and stired
until dissolved. After that, TSA was
sterilized (121oC, 15 min) then poured into
petridishes.
2.4. Film Treatment
First, the non-coated PLA film as a control
and coated PLA film were prepared. Then,
400 l Escherichia coli were coated onto
PLA film (thymol, chitosan and mix) and
non-coated PLA film. The LDPE film was
put onto it and all the sample were
incubated in a incubator (37oC, 1 day) to
optimized the Escherichia coli growth.
2.5. Microbial Analysis
First, PLA film and LDPE film were
separated then 10 ml NaCl 0.85% ware
added and homogenized by hand massage.

1 ml sample was taken and diluted into 9
ml NaCl 0.85% (dilution 10-1). The
dilution were done until 10-8. 10 l each
dilution sample were taken and put onto a
TSA. The dillution sample were taken 3
times repetition. After that, the samples
were incubated in the incubator (37oC, 1
day). After all, the Escherichia coli growth
were counted each dilution.
2.6. Statistical Analysis
The result of Escherichia coli count were
averaged each dilution. After that, the
CFU per milliliter were transformed to
log10 values then analyzed using analysis
of variance with SAS software. Duncans
multiple range test was used to determine
the significant differences between
treatment means. The significance level
was set at = 0,05.
3.
Escherichia coli is one of the bacteria that

easily grow in ready-to-eat product.
Therefore, an antimicrobial agent is used
to inhibit the Escherichia coli growth to
improve food quality and safety. The
antimicrobial agent that used is thymol and
chitosan. During processing of making
antimicrobial agent, chitosan can only be
obtained by solving in acid media and
casting techniques (Vargas et al., 2009,
2011a). Chitosan cannot be processed by
melting (Grande and Carvalho, 2011). The
characteristic of thymol is low solubility in
water (Razzaq and Mohamed, 2014). So,
ethanol was used to dissolve thymol and
acetic acid was used to dissolve chitosan.
After that, antimicrobial agent was coated
onto a polylactic acid film.
Page 160
Escherichia coli is used to compare the

effectiveness of antimicrobial agents and
for Escherichia coli growth, Trypticase
Soy Agar (TSA) was used. TSA is used for
the cultivation of a wide variety of
microorganisms. It can be used for aerobic
and anaerobic microorganisms growth
(Leavitt et al., 1955). Escherichia coli
culture that are incubated at 30-35oC (18
24 hours) under the appropriate
atmosphere will be have good results
(United States Pharmacopeial Convention,
2007; EDQM, 2007 and Japanese
Pharmacopoeia, 2007).
Table 2. Effect of 3% of Antimicrobial
Agents on Escherichia coli
Growth
Escherichia
coli
Treatments
(Log CFU / ml)
Control
7,266 0,30a
Thymol 3%
6,791 0,21a
Chitosan 3%
6,908 0,21a
Mix 3%
7,926 0,18b
The value (mean standard deviation) was
obtained from 2of the closest data of each
measurement. The different superscript
shows the significant value (P<0,05).
The average of log CFU/ml resulted from
thymol 3% and chitosan 3% treatments are
lower than control but not significantly
different (P<0,05). This shows that thymol
3% and chitosan 3% can inhibit
Escherichia coli bacteria but the
concentration of antimicrobial agents are
not enough to kill more E. coli. Thymol is
primarily responsible for antibacterial
activity against E. coli (Gill & Holley,
2006). The result also shows that the mix
3% did not show any synergic
antimicrobial activities. It maybe due to
evaporation of the antimicrobial agents.
During the preparation, thymol and

chitosan that coated onto the PLA film was
left in the incubator overnight. Therefore,
the less concentration of antimicrobial
agents was concluded.
Table 3. Effect of 5% Concentration of
Antimicrobial
Agents
on
Escherichia coli Growth
Escherichia
coli
Treatments
(Log CFU / ml)
Control
Spreader
Thymol 5%
Spreader
Chitosan 5%
Spreader
Mix 5%
Spreader
From table 3,the result of the average of
log CFU/ml are all spreader. These are due
to contamination from external factor,
such as unhygienic equipments or
materials that used. So, we can not
conclude which concentration that is more
effective.
4.
CONCLUSION
Thymol and chitosan at 3%

concentration can inhibit Escherichia
coli but the value is not significant
(P<0,05).
Mix 3% concentration can not show
any synergic antimicrobial activities.
5.
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Page 164
THE EFFECT OF CHROMANONE DEAMINE LEVELS ON WATER

CONTENT, pH AND TOTAL MICROBES OF BROILER CHICKEN MEAT
PRE-RIGORMORTIS AND POST RIGORMORTIS
Cindy Elysia1), Sumardi2) and Rika Pratiwi2)

1)Student
of Department of Food Technology; Faculty of Agricultural Technology;

2)Lecturers; Department of Food Technology, Faculty of Agricultural Technology;
ABSTRACT
Attempts to improve the broiler meat quality could be conducted in several ways such
as by using antibiotics, probiotics and herbs as feed additives in poultry feed. However,
the use of antibiotic had been banned due to the accumulation of antibiotic residues in
animal products and the increasing resistance of pathogens. Herbs contain active
compounds which have pharmacology properties which is potential to replace
antibiotic. Furthermore, the use of herbs as feed additives in animal feed was known to
have antimicrobial actions which expected to improve the hygiene carcass. Deaminated
2,6,7-chromanone extracted from fruit of Baelfruit (Aegle marmelos L. Corr) had been
proved to have antimicrobial actions and was able to improve the protein content and
lower the fat levels in broiler chicken meat. Regarding this, it is necessary to conduct a
research to study the effect of chromanone deamine level towards water content, pH and
total microbes of broiler chicken meat pre-rigormortis and post rigormortis. This
research was conducted using 100 broiler chickens which divided into five groups with
different dose levels of chromanone deamine were 1 ml, 5 ml, 10 ml, 50 ml and control.
The application of chromanone deamine was given in chickens drinking water every
day for 30 days cultivation. The broiler chicken meat was then analyzed the water
content, pH and total microbes on three parts that were breast, thigh and wings.
Keywords : herbs, chromanone, broiler chicken, meat quality

Page 165
INTRODUCTION
Broiler chicken is a food which is often
consumed to meet the needs of animal
protein due to it grows faster with a
shorter life cycle and has a fairly
affordable price compared to other
meat-producing animals such as cow
and goat (Matulessy, 2011). The meat
quality which produced from cattle
cannot be separated from the amount of
nutrients available in feed and feed
quality. The nutritional content of food
comes from animals is related to the
quality and size of the microbial flora in
the digestive tract of cattle. In attempts
to increase the productivity of livestock
products, especially meat, can be
conducted by improving the efficiency
of feed which can also maintain the
health of livestock. Those attempts are
using antibiotics, probiotics and herbs
as feed additives for livestock (Costa et
al., 2013). However, the use of
antibiotics had been banned completely
in the European Union since 2006
because of suspected risks such as
antibiotics residue found on meat due to
the excessive use and the increasing
resistance of pathogens to antibiotics
(Alloui et al., 2014).
One alternative in maintaining the
quality of chicken meat produced can be
conducted by using herbs as a feed
additive for livestock. Herbal plant is
known to contain many active
components
which
have
pharmacological properties and had
long been known to have a role as
antibiotic,
antimicrobial,
antiinflammatory and antioxidant. The use
of herbs incorporated into animal feed
to enhance livestock productivity

through
the
improvement
of
digestibility, nutrient absorption and
elimination of pathogens residents in
the animal gut (Kamel 2001; Balunas
and Kinghorn, 2005; Athanasiadou et
al., 2007).
Javanese and Indian people have long
used the fruit powder of Baelfruit
(Aegle marmelos L. Corr) to reduce
pain and stress. Recent studies reported
that fruit of Baelfruit contains
antioxidant, antibacterial, anti-fungi,
anti-cancer, and anti-depressant agents
which come from 2,6,7-Chromanone
amine which are found mainly in fruit
of Baelfruit (Siddiqui and Farooq,
2001). The chemical structure of 2,6,7Chromanone amine is shown on figure
1.
Figure 1. The chemical structure of

2,6,7-Chromanone amine form Baelfruit
The compound of 2,6,7-Chromanone
amine can be deaminated to produce
2,6,7-Chromanone deamine which aims
to bind the free nitrogen. The
deamination technique can be made by
reacting with some acids including
amino acids (Lim et al., 2005) and by
physical pressure at extreme low
temperature and diurnal vacuum
condition (Patent Indonesia No.
P00200500693). The chemical structure
of 2,6,7-Deaminated chromanone is
shown on figure 2.

Page 166
Figure 2. The chemical structure of

2,6,7-Deaminated chromanone
(ISM, 2008)
The produced deaminated chromanone
induced into animal digestive system
then would bind free ammoniac or other
free nitrogen produced in digestive
system of animal. From previous study,
the
application
of
deaminated
chromanone which mixed into animal
feed had resulted less odour of the feces
(Kusumowardhani & Sumardi, 2006),
increasing protein content of broiler
chicken meat and lower fat content of
broiler chicken meat (Lasmono &
Sumardi, 2006).
The quality of chicken meat is
influenced by several factors, among
others ante mortem, the slaughtering
process and a long treatment for post
mortem. Post mortem phase is the phase
where animals died (Muchtadi &
Sugiyono, 1992). Post mortem phase
consists of three phases namely prerigor mortis, rigor mortis and post rigor
mortis phase (Aberle et al., 2001). The
growth of microorganisms is slow on
the phase of pre-rigor mortis. The
characteristics of meat on the pre-rigor
mortis are still pliable and malleable
that then turned into rigid (Lawrie,
2003) because of actin and myosin form
actomyosin after slaughtering called
rigor mortis phase. Rigor mortis
characterized by glycolysis. Glycolysis
can be increased with increasing
external temperature above ambient

temperature (Lawrie, 2003). Glycolysis
process that occurs during post mortem
is anaerobic glycolysis because after
slaughter will cause the cessation of
blood circulation. The end phase of
rigor mortis characterized by the
relaxation of muscles where the
spoilage process starting to take place.
The pH value also will change during
post mortem. The pH value of post
mortem is determined by the amount of
lactic acid produced from glycogen
during anaerobic glycolysis. The
production of the amount of lactic acid
is limited if glycogen depleted because
of fatigue, hunger or fear in animals
before slaughtering process (Lawrie,
2003). The pH value of post mortem
meat will decline due to the
accumulation of lactic acid in muscle
tissue (Aberle et al., 2001). A decrease
in chicken meat pH value will reach
5,80 5,90 after 2 to 4.5 hours post
mortem (Koswara, 2009). Thus, it is
necessary to study on the effect of
chromanone deamine levels of the
microbiological quality of broiler
chicken meat pre-rigormortis and post
rigormortis by determine the water
content and pH of broiler chicken meat.
Field Study
The animals used for the test were 100
Day Old Chicken (DOC) broilers which
reared for 30 days from 26th February
2015 to 26th March 2015 at SMK
Theresiana, Bandungan, Semarang.
Broiler chickens were divided based on

Page 167
the treatment group. The group was

based on the different treatment level of
chromanone deamine compound as feed
additive (in commercial product named
Vet-i) which added into chickens
drinking water with the levels of 1 ml, 5
ml, 10 ml, 50 ml and 0 ml as control
that given every day in the morning and
afternoon. After 30 days cultivation,
randomly chosen 5 samples were taken
from each treatment and then
slaughtered. The slaughter process was
conducted at the abattoir at 9 oclock in
the morning on 26th March 2015
through the stages of dissolution of
blood vessels, scalding, plucking, and
evisceration to obtain the chicken
carcass. After the chicken carcasses
were obtained then was cut into three
parts were breast, thigh and wings. The
experimented parts then were carried to
the laboratory approximately 20
minutes drives by using ice box which
filled with ice block.
Laboratory Study
The samples of breast, thigh and wings
of broiler chicken meat which obtained
from each treatment then analyzed the
water content (AOAC, 1984) at the
storage time of 0 and 5 hours at room
temperature (Koswara, 2009), pH value
(AOAC, 1984) at the storage time of 0
and 5 hours at room temperature
(Koswara, 2009), and the growth of
microorganisms with total plate count
method (SNI 3932:2008) at the storage
time 0 hour, 2 hours, 4 hours and 5
hours at room temperature (W. H.
Freeman and Company; Judge et al.,
1989; Koswara, 2009). Both of water
content and pH value measurement
were conducted in five times of

repetition, while the measurement of the
growth of microorganisms on chicken
meat was conducted twice of repetition.
RESULTS AND DISCUSSIONS
The Water Content of Broiler
Chicken Meat
It can be seen in Table 1 that the water
content of the chicken meat with
addition of chromanone deamine
treatment in various levels applied in
chickens drinking water resulted in
lower water content compared to
control in any experimented parts at
storage time 0 hours. The addition of
chromanone deamine treatment in
various levels known to affect the water
content of the meat in any experimented
parts although the difference was not
significant, except on the wings with the
level of 50 ml chromanone deamine
compound that resulted in the lowest
water content. The lower water content
in the meat with the addition
chromanone deamine treatment during
cultivation wass affected by the active
compounds of herbal extracts that had a
diuretic effect which could trigger the
excretion from chicken body (Rahardjo,
2010). Furthermore, the lower water
content in the chicken meat with
chromanone deamine treatment also due
to the increasing of protein content in
the meat (Lasmono and Sumardi, 2006).
From Table 1 also can be known if the
water content of wings was significantly
different compared to water content in
breast and thigh at storage time 0 hours
post mortem. The difference of water

Page 168
content in the experimented samples

was due to the difference in the
chemical composition. The lower water
content in wings was due to the higher
fat content. Water content was known to
have a negative relationship with the fat
content (Handria, 2015).
Table 1 also showed a decrease in water
content at storage time 5 hours post
mortem at the room temperature both in
control meat and meat with chromanone
deamine treatment in various levels.
The longer storage time would lower
the water content due to the non
microbial and microbial activity. In
addition, the high temperature of the
storage room temperature also affected
water content in meat because of water
evaporation during storage (Affi and ElNashaby, 2001; El-Shamery, 2007;
Kanatt
et
al.,
2009).

Page 169
Tabel 1. Comparison of water content in chicken meat amongst various chromanone

deamine treatment
Storage time
0 hour
5 hours
Levels
(ml)
0 (control)
1
5
10
50
Breast
76,0580,738c,2
74,0100,670a,1
75,1010,442b,2
74,8660,583b,2
74,8690,585b,2
0 (control)
1
5
10
50
73,5011,221a,12
72,6680,956a,12
73,3400,576a,12
72,3092,069a,1
71,5122,227a,2
Parts of Chicken Meat

Thigh
Wings
76,8320,394c,2
73,7582,212b,1
76,5770,578c,2
73,1690,797b,1
ab,2
75,4330,453
72,4560,954b,1
a,2
74,9271,393
72,8761,173b,1
76,4510,695bc,3 68,4181,613a,1
74,7490,384ab,2
75,2900,803b,2
73,8651,643ab,2
72,8641,334a,1
74,5832,245ab,3
72,1002,009b,1
70,9494,353ab,1
71,2051,509,b,1
71,3542,211b,1
67,4751,438a,1
Notes:
abc at different superscripts in column indicate significantly different at 95% degree of
confidence in each storage time
123 at different superscripts indicate significantly different at 95% degree of confidence in
row
pH Value of Broiler Chicken Meat

Table 2 showed the pH value of the
experimented parts and the change of
the pH value during storage time. The
average pH value at storage time 0
hours post mortem on the chicken
breast meat ranged from 6,03 6,20; on
the thigh part ranged from 6,33 6,52;
and on the wings ranged from 5,98
6,28. This result was similar with the
previous study by Schilling et al.
(2010), the average pH value of chicken
breast ranged from 5,80 6,20. From
these results, it can be seen if the
highest average pH value of chicken
meat was on thigh part. The differences
in the pH value on the meat due to the
differences in motor activity during ante
mortem. The higher the motor activity
during ante mortem, the energy needs
will be higher so that increased the
oxidative respiration rate which resulted
in low energy reserves of glycogen in
the muscles. Low glycogen in the

muscles will result in the low of lactic
acid formation so that results in higher
pH value (Souza et al., 2010;
Kokoszynski et al., 2013). Thus, the pH
value of thigh and wings part was
higher than the pH value of breast part.
Due to these result, it can be concluded
that the chromanone deamine treatment
in various levels was no significantly
affecting on the pH of chicken meat.
During 5 hours post mortem storage at
room temperature known that pH value
of chicken meat decreased in any
experimented parts of chicken body
which is shown in Table 2. The pH
value of the meat after slaughtering will
decline due to the cessation of blood
circulation and respiration that caused
anaerobic glycolysis process began to
take place. The process of anaerobic
glycolysis produced lactic acid in the

Page 170
muscles which declined the pH value.

The longer the storage time, the pH
value will decrease until the ultimate
pH between 5,40 5,80 reached

(Rahardjo & Santosa, 2005).
Tabel 2. Comparison of pH value in chicken meat amongst various chromanone

deamine treatment
Storage time
0 hour
5 hours
Levels
(ml)
0 (control)
1
5
10
50
Breast
6,200,02c,1
6,020,05a,1
6,110,10b,1
6,050,05ab,1
6,030,05ab,1
0 (control)
1
5
10
50
6,110,04c,1
5,920,05a,1
6,020,07b,1
5,960,07ab,1
5,910,02a,1
Parts of Chicken Meat

Thigh
Wings
a,2
6,370,08
6,280,04c,2
a,2
6,330,07
5,980,04a,1
6,520,06b,3
6,280,07c,2
ab,3
6,440,15
6,260,05c,2
6,360,04a,3
6,190,02b,2
6,210,02a,2
6,190,07a,2
6,350,02c,3
6,300,11bc,3
6,230,05ab,3
6,170,03c,2
5,880,10a,1
6,140,03ab,2
6,160,03c,2
6,080,02b,2
Notes:
abc at different superscripts in column indicate significantly different at 95% degree of
confidence in each storage time
123 at different superscripts indicate significantly different at 95% degree of confidence in
row

Page 171
The Total Number of Microbes on

Broiler Chicken Meat
Based on Table 3, it can be known that
the total microbes on the chicken meat
with chromanone deamine treatment in
various levels obtained the total number
of microbes which was less than the
control in any experimented parts
(breast, thigh and wings) at storage time
0 hours. The addition of chromanone
deamine applied in chickens drinking
water probably maintained the stability
of the microflora in the gastrointestinal
tract of chicken (Windisch et al., 2008;
Li et al., 2012). The use of chromanone
deamine as feed additives might
improve the carcass hygiene (Windisch
et al., 2008). In addition, from Table 3
can also be seen if the highest total
number of microbes found on wings at
storage time 0 hours. In accordance
with Astorga-Alvarez et al. (2012) also
found that the highest number of
microbes on the wings. Sampers et al.
(2008), chicken wings belonging to the
group that have higher probability of
allowing
the
microorganism
contamination occurred because they
contain almost exclusively skin.
Contamination might originate from
follicles opened during the scalding
process (Cason, 2004). Chantarapanont
et al. (2003) found that the presence of
contamination at the bottom of feather
follicle.
increased
the
number
of
microorganisms which are shown in
Table 3. The chicken meat will soon
enter the rigor mortis phase after
slaughter which generally lasts for 2 to
4.5 hours at room temperature. Rigor
mortis phase will end characterized by
muscle relaxation which is the
beginning of the spoilage process. At
the end of rigor mortis phase, the
spoilage process will start to increase as
a result of the decomposition of the
more
macromolecular
compounds
(Moueljanto, 1992). Increasing the
number of microbes on meat during
storage starts experiencing normal
growth of tha lag phase to the
logarithmic phase. Fardiaz (1992), the
growth of microorganisms is affected
by intrinsic factors (nutrition meat,
water content, pH, oxidation-reduction
potential, whether there is any
inhibiting substances) and extrinsic
factors (temperature, relative humidity,
oxygen availability, and the shape or
condition of the meat). Referred to the
microbiological quality standard of
chicken meat of Indonesian National
Standardization (2009), the number of
microorganisms during storage 5 hours
in chicken meat was still below
standard set 1x106 CFU/g.
Meat has the high water content about

68 75%, rich in substances that
contain nitrogen, pH ranges of 5,30
6,50 which is also beneficial for the
growth of microorganisms (Soeparno,
1994). Along with the storage time
Page 172
Tabel 3. The effect of chromanone deamine treatment amongst various

total number of microorganisms (CFU/ml) on chicken meat
Storage Time (Hours)
Parts of
Levels (ml)
Chicken Meat
0
2
4
2
3
Breast
0 (control)
6,25x10
5,05x10
4,60x104
2
3
1
5,70x10
4,50x10
4,00x104
5
5,00x102
4,10x103
3,95x104
2
3
10
5,30x10
4,15x10
3,40x104
2
3
50
6,10x10
5,35x10
5,50x104
levels on the
5
4,05x105
3,65x105
3,80x105
3,30x105
6,20x105
Thigh
0 (control)
1
5
10
50
7,95x102
7,75x102
6,90x102
7,05x102
7,40x102
6,50x103
6,05x103
5,20x103
5,45x103
5,85x103
5,30x104
5,00x104
4,00x104
4,40x104
5,10x104
4,65x105
4,20x105
3,90x105
4,00x105
4,70x105
Wings
0 (control)
1
5
10
50
8,40x102
8,15x102
7,70x102
7,90x102
7,75x102
6,45x103
5,80x103
6,30x103
5,90x103
6,20x103
5,40x104
4,30x104
5,25x104
4,75x104
5,15x104
4,80x105
3,75x105
4,35x105
4,55x105
5,10x105
CONCLUSIONS
The application of chromanone
deamine compound in various levels
into chickens drinking water lower
the water content of chicken meat.
The treatment of chromanone
applied, was no significantly
affecting the pH value of chicken
meat.
The longer storage time, the lower
water content and pH value, but
increase the total number of
microorganisms on chicken meat.
The application of chromanone
did not affect the growth of
microorganisms in chicken meat
during storage.
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Page 175
THE COMMITTEE OF 15th NATIONAL STUDENT

CONFERENCE
Steering Committee
1. Dr. V. Kristina Ananingsih, MSc.
2. Dr. Probo Y. Nugrahedi, S.TP, MSc.
3. Ivone Fernandez, SSi, MSc.
Organizing Commitee
Chairperson
Secretary
Treasurer
Event
Paper
Sponsorship
Donation
Documentation and Publication
Decoration
Equipment
Consumption
Person in Charge
1. Lukas Terry Boedianto
2. Sherly Putri
3. Rr.Panulu PM
:Yohanes Kristo S.U.

:Caecilia Eka Putri
Elizabeth Gracia E.
:Petra Adventia P.J.
:Cecilia Noviani
Ivo Sidauruk
Maria Margareta Suprajogi
Keshia Devina Wijaya
Ananta Levina Savitri Tarigan
:Cindy Corazon
Riana Natalia S
Monica Vresti C
Ruth Jeane
Beatrix Riski Restiani
:Herliansa Chrisnasari Puspita
Ade Putra Haryono
Jessica Astelia
Monica Ratna Suminar
Yanuar Adi Wijaya
Elia Novita
:Angela Lauvina
Stella Auberta N.
Ivana Suprayogi
:Denny Saputra
Monica Andreina K.
Christina Hanny S.
Carolina Santoso
Verlencia Anggrian K.
:Onny Shelvia
Jessica Novia S.
Bernadeta Pingkan Larasati
Yandika Suharto
:Sandra Meliana
Hendra
Rainer Ravian Z.
Hans Christian P.S.
Kukuh Ody Ariyo B.
:Liem Pamela Lukito
Raissa
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Proceeding NSC 2015 - Unika Soegijapranata

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15th National Student Conference on

Food Science and Technology

The Committees of 15th National Student Conference

15th National Student Conference on

The Committees of 15th National Student Conference

Effect of Chromanone Deamine Dosages on The Quality of Osmomeat Products in Terms of

15thNational Student Conference

The 15th National Student Conference (NSC) organized by Faculty of Agricultural

Semarang, October 2015

Dr. Victoria Kristina Ananingsih, ST, MSc

15thNational Student Conference

a. Every speaker will present his/her paper.

15thNational Student Conference

Soegijapranata Catholic University, Semarang

Guar Gum Effect as Fat Replacer on Sensory Characteristics of Broccoli

15thNational Student Conference

FOOD PROCESSING AND ENGINEERING

15thNational Student Conference

FOOD SAFETY AND QUALITY

The Inhibitory Effect of Bamboo Extracts on Melanogenesis in

15thNational Student Conference

15thNational Student Conference

FOOD PRODUCT DEVELOPMENT

15thNational Student Conference

INFUSION INDONESIAN TRADITIONAL FOOD (DENDENG BALADO)

Pangan, Fakultas Teknologi Pertanian, Universitas Katholik Soegijapranata

15thNational Student Conference

with certain spices. This Indonesian

15thNational Student Conference

The equipments used for this project are

The filling for puff pastry was

15thNational Student Conference

i. Making the balado sauce

thick, then, cut into 7 pieces (40 grams).

15thNational Student Conference

Results and Discussions

2.1. Determination of bakery product

15thNational Student Conference

Figure 2 Bakery attributes

Figure 3 Time of consume bakery

15thNational Student Conference

Figure 4 Intensity of consume bakery

Type of bakery that

Figure 5 Type of bakery compliment

savory, it seem to go well with puff pastry

2.2. Preliminary test

15thNational Student Conference

Figure 7 dendengbalado puff pastry

Figure 6 Pastry dough with 2 single fold

2.2.2. Preparation of sample

In the preliminary test, there were three

Table 3 Just About Right result in

Nine attributes were asked in the JAR test.

much. Based on the JAR test, three

Table 4 Customized egg wash and effect

Effect on cooked pastry

Whole egg Nicely browned, slightly

Micely browned, more

Evenly browned, slightly

Browned and shiny, but

Very browned and glossy,

The darkest brown crust

Figure 8. Puff pastry color of crust