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INTRODUCTION
1.1 Background
Agriculture is the biggest business in Indonesia, especially the production of rice
which is also staple food for Indonesian people. The production, processing and distribution
of rice become one of the most important agricultural business in Indonesia which support the
national food security. In 2011 the production of rice in Indonesia reaching the amount of
65.39 millions tons (Badan Pusat Statistik, 2011).
The production of rice bran is proportional with the production of rice, because rice
bran is by product from the milling process of paddy into rice. In the production process of
rice, about 8 12 % of rice bran is produced from the paddy milling process
(Widowati,2001). Indonesia produce a large amount of rice, hence Indonesia also produce a
large amount of rice bran. However, rice bran usually only used as a feed for poultry or cow,
although actually rice bran has a potential value to be processed in food for human and has
many benefits towards human health (Alvita et al,2007).
Rice bran contains very rich in valuable nutrition. Rice bran rich in B complex
vitamins which includes B1,B2,B3,B5,B6 and B12 vitamins. It also rich in vitamin E,
essential fatty acid, dietary fibers and proteins. In the stabilized rice bran about 20 - 27 % of
dietary fibers can be found (Yu et al, 2012). The other benefits from rice bran is that rice bran
is free from gluten, easy to digest and rich in complex carbohydrate. These benefits from rice
bran can be utilized in the food processing. Because of the nutritious value in rice bran,
nowadays rice bran has been applied in the food processing for some food products. Rice
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bran can be used as the substitute of wheat flour in the processing of food product like bread
as studied by Hu et al (2009) or cookies as studied by Fauziyah (2011). The substitution of
rice bran can increase the dietary fiber content in a food product such as frozen pizza whose
the research was conducted by Delahaye (2005). Based on study done by Huang et al (2005),
rice bran can be added to pork meatballs with concentration up to 10% that will produce
meatballs which is still accepted by the panelist.
Coeliac diseases is a life long intolerance to gluten proteins. A decade ago, coeliac
disease has a rate of 1 in 1000 or lower population and only considered as an uncommon
disorder, however nowadays the rates of coeliac disease increase and study shown that it may
affect 1 in 100 population, To avoid the symptomps of coeliac disease, the only effective
method is strict adherence to the diet free of the allergence, which is gluten based product that
are toxic to the small intestine (Korus, 2008).
Therefore in this research, we want to substitute the use of wheat flour in brownies
with several substitutes for wheat flour, such as cassava flour, potato starch, or rice flour, in
order to accomodate a gluten sensitive people. The best formulation will be later enriched
with rice bran flour. Substitution of rice bran flour is expected to increase the amount of
dietary fibers in the brownies.
1.2 Research Problem
Rice bran that contains good source of dietary fiber and othe nutritions components
has very limited usage in Indonesia. People like to produce bread or cookies that made from
wheat flour, although wheat contain gluten that prohibited in the diet of coeliac diseases
patient. On the other hand, Indonesian people lack of dietary fiber in their daily diets. Hence,
replacing the use of wheat flour in brownies making with other sources such as cassava flour,
rice flour and potato starch with rice bran substitution is expected to overcome the problem
above.
1.3 Objectives
1.3.1 General Objectives
The general objectives of this research is to study the utilization of rice bran in the
making of gluten free rice bran brownies.
1.3.2 Specific Objectives
The specific objectives of this research is to :
1. Determine the best wheat flour substitute in the making of gluten free rice bran brownies
based on the consumer acceptance using the sensory evaluation test.
2. Evaluate the effect of rice bran concentration and different baking time on the physical and
chemical characteristics of gluten free rice bran brownies.
3. Observe the consumer preferences by utilizing sensory evaluation method.
4. Determine the dietary fiber and nutritional composition in the best gluten free rice bran
brownies formula.
CHAPTER 2
LITERATURE REVIEW
by the sugar content in the rice bran including glucose, sucrose and fructose. Besides flavor,
color is one of the most important consideration when applying rice bran to the food
products. Rice bran has important functional properties in terms of color which are the
change in color during the processing. The rice bran color can be changed during the
processing because of the heat treatment and increase of the moisture content in the food
product. This will affect the end result of the food product. Rice bran also has the ability to
absorp water and oil, and can be used as emulsifier. Rice bran also has low foaming ability
(Luh, 1980).
2.1.2 Deterioration of Rice Bran Qualities
According to Choo et al (1999), the bran containing lipid and lipase, which make
rice bran is prone to rapid degradation of lipid because of the lipase activity toward the lipid.
The contact between the lipid and lipase in the bran is occur during the milling process of the
brown rice. This lipid degradation will cause the rice bran become unpalatable and so the rice
bran cannot be utilized for human and only utilized for animal feed.
According to the Shahidi (2005), Most lipids in rice bran consists as
lysophospholipids, triacylglycerols and free fatty acids. The nonstarch lipids in the aleurone,
sub aleurone, and germ layers were 86 91 % neutral lipids, 2 5 % glycolipids, and 7 9 %
phospholipids. These percentage amounts of lipids are different and affected by the milling
degrees. The instability of rice bran is affected by the lipase enzymatic activity. When the
kernel of the rice bran is intact, lipase is physically isolated from the lipids. However, when
dehulling process is done, it will disturb the surface structure and the lipase and oil will be
mixed together. As the result, the oil in rice bran will be hydrolyzed by lipase enzyme into
glycerol and free fatty acid.
improvement in the HDL ratio. The study shown that rice bran has some insoluble fiber
including cellulose and hemicellulose which can bind to bile acids (Takakori et al, 2005).
Rice bran also utilzed to produce food which is rich in dietary fiber, because rice bran has
large amount of dietary fiber content (Chotimarkorn and Silalai, 2008). Rice bran has been
processed into several products. There is rice bran beverage which is produced by using rice
bran extract and added strawberry and cocoa flavor (Faccin et al,2009). There is also pizza
which is enriched by using rice bran as studied by Delahaye et al (2005), Bunde et al (2010)
also studied the supplementation of biscuit using rice bran powder. Chortimarkorm et al
(2007) also study the utilization of rice bran powder to prevent the oxidative reaction of fried
dough from riced flour during storage. The other food products that has been utilizing rice
bran are rice bran sponge cake which was studied by Aftasari (2003) and utilization of rice
bran oil in the processing of red bean paste which was studied by Metta (2003). The rice bran
also utilized to produce a low fat frankfurter, which has reduced fat content, reduced
cholesterol and trans fat value (Choi et al, 2010).
2.1.4 Rice Bran Nutritional Value
Rice bran is rich in nutritional value, it contains 12 25 % fat, 10 16% protein,
10 20% starch, 3 8% reducing sugars, 8 11% hemicelluloses, 10 12% celluloses, 6
15% crude fiber and 6.5 10% ash content. Rice bran is abundant in vitamins of the B group
and tocopherols, although it is poor in vitamins A and C (Sharma, 2004).
Rice bran is also source of antioxidant, one of the natural antioxidant found in the
rice bran is gamma oryzanol. Gamma oryzanol is a group of ferulic acid esters of phytosterols
and triterpene alcohols which has been reported to exhibit antioxidant activity and has other
health beneficial properties. Gamma oryzanol exists mainly in bran layers and therefore it is
also found in extracted rice bran oil.Gamma oryzanol has potential in lowering blood
cholesterol (Cicero and Gaddi, 2001).
The component in rice bran which has the highest antioxidant activity is 24methylenecycloartanyl ferulate. It had activities higher than any of the components in vitamin
E. The quantity of oryzanol is up to 10 times higher compared to the quantity of vitamin E in
the rice bran. The oryzanol is the most important bioactive compound of rice bran and has the
ability to reduce cholesterol oxidation. Because of the antioxidant activity of these
components in rice bran, rice bran has potential hypocholesterolemic property (Xu et al,
2001).
Rice bran also contains carbohydrate, mostly in the form of cellulose,
hemicelullose and starch. The endosperm of rice bran is rich in starch. Naturally, the starch is
not present in the outer layer of the bran. But during the milling process, the outer layer
(pericarp) will be released. The endosperm will be broken down during the abrasion process
and causing the starch to be released and developed in the bran. The starch content in the bran
is affected by the degree of milling which determine the amount of breakage (Hargrove,
1994). The chemical composition of rice bran could be seen in Table 2.1.
Table 2.1 Chemical Composition of Rice Bran
Component
Protein (%)
Fat (%)
Crude Fiber (%)
Carbohydrate (%)
Ash (%)
Calcium (mg/g)
Magnesium (mg/g)
Phosphor (mg/g)
Silica (mg/g)
Zinc (mg/g)
Thiamin (g/g)
Riboflavin (g/g)
Tocopherol (g/g)
Source : Luh et al, 1991
Amount
12 15.6
15 19.7
7 11.4
34.1 52.3
6.6 9.9
0.3 1.2
5.0 13.0
11.0 25.0
5.0 11.0
43.0 258.0
12.0 24.0
1.8 4.0
149 154
The protein in the rice bran is rich in nutrient compared to the milled rice, the
majority of protein in rice bran is lysine. Most protein in rice bran exists in the form of
albumin and globulin with the ratio of albumin-globulin-prolamin-glutelin is 37 : 36 : 5 : 33
(Champagne, 2008). Rice bran is also rich in fatty acid, especially unsaturated fatty acid
which is about 80 %. The palmitic acid, oleic acid and linoleic acid is the main fatty acid
component which contained in the rice bran oil (Gibson, 2009).
Table 2.2 Rice Bran Fatty Acid composition
Type of Fatty Acid
Myristic Acid
Palmitic Acid
Stearic Acid
Oleic Acid
Linoleic Acid
Linolenic Acid
Arachidonic Acid
Behenic Acid
Source : McCaskill and Zhang, 1999
%
0.2
15.0
1.9
42.5
39.1
1.1
0.5
0.2
Rice bran also has an antinutrient compound, which is phytic acid, anti trypsin
and hemaglutinin or lectin (Luh, 1991). These anti nutrient compounds exist in a low amount
in the rice bran and can be inactivated by heat treatment as stated by Hargrove (1994). There
are several enzymes which are contained in rice bran such as amylase, amylase, catalase,
peroxidase, esterase, lipase, glucosidase, glucosidase, maltase, pectinase, phytase,
poliphenoloxidase, and so on. The enzymatic activity in the germs and the outer layer of the
paddy is higher compared to the other part of the paddy. This higher activity of the enzyme in
the outer layer causing rice bran also have high activity of the enzyme (Luh, 1991).
2.2 Dietary Fiber
Dietary ber is a class of compounds which includes a mixture of plant
carbohydrate polymers, both oligosaccharides and polysaccharides, such as cellulose,
hemicelluloses, pectic substances, gums, resistant starch, inulin, it could be associated with
lignin and other non-carbohydrate components such as polyphenols, waxes, saponins, cutin,
phytates, and resistant protein. Resistant starch and resistant protein withstand digestion in
the small intestine. Resistant starch is composed of four groups, which are RS1 as the
physical inaccessible starch, RS2 as the ungelatinised starch granules, RS3 as the retrograded
starch and RS4 as the chemically modied starch as stated by FuentesZaragozaet al (2010).
As stated by Turowski (2007), dietary fiber could be divided into two categories which are
soluble dietary fiber and insoluble dietary fiber. These two categories are distinguished by
their solubility in water.
There are many health benets which associated with an increased intake of
dietary ber. The health benefits including the reduced risk of coronary heart disease,
diabetes, obesity, and some forms of cancer. Some food commodity which are rich in dietary
fiber such as oat bran, barley bran, and psyllium,mostly soluble bre, have earned a healthy
reputation for their ability to lower blood lipid levels. Wheat bran and other more insoluble
bres are typically linked to laxative properties (American Dietetic Association, 2008).
Dietary ber supplementation can result in tness-promoting foods, low in calories,
cholesterol and fat. Food and Nutrition Board, Institute of Medicine (2001) recommend the
average daily requirement of dietary ber is 25 g per day for women younger than 50, 21 g
per day for women older than 50; 38 g per day for men younger than 50, and 30 g per day for
men older than 50. Most nutritionists and diet experts suggest that 2030% of human daily
dietary fiber intake should come from soluble fiber.
Dietary ber also have effects toward functional properties of foods such as
increase water holding capacity, oil holding capacity, emulsication and/or gel formation.
When dietary ber incorporated into food products (bakery products, dairy, jams, meats,
soups) it can modify the textural properties, avoid synaeresis (the separation of liquid from a
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gel caused by contraction), stabilise high fat food and emulsions, and improve shelf-life
(Elleuch et al, 2011).
2.3 Brownies
Brownies is a type of cookies which is usually has dark brown colour. Brownies
is classified as bar cookies. Bar cookies is the simplest type of cookies to made, the process is
spreading the batter in a pan and bake it. Basically, cookies are made from a batter or dough
that may be similar to some types of cake batter (Suas, 2008).
There are two types of brownies which are steamed brownies and baked
brownies. Similar with cake, brownies has specific structure which are slightly porous and
has soft texture. But different with cake, brownies structure is more compact compared to
cake and does not leaven as in the processing of cake. The main ingredients of brownies is
eggs, fat, sugar, and wheat flour (Sulistyo, 2006). Brownies also can be categorized as fudgy
brownies or cakey brownies, for fudgy brownies, less flour is used during the processing, for
cakey brownies, more flour is used during the processing (Corriher, 2008). Because brownies
is a type of cookies, it can be produced using wheat flour that have relativele weak gluten
strength. In general cookies processing, the gluten development is very low. Minimum gluten
formation is contribute in the crispness and softness of the cookies product, such as brownies
(Hui,2006).
2.3.1 Eggs
Eggs have five major components which is the yolk, albumen, shell membranes,
air cell and shell. Eggs, and especially the egg white are composed of dozens of different
proteins. Each of these proteins has its own characteristics and functions (Brown, 2008).
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In the brownies processing, the function of eggs is as the substitute of water, to form the
brownies structure, contribute to the softness of the brownies structure, aeration and to
distribute the dough. The eggs also contribute to the color, aroma and the flavour of the
brownies.
2.3.2 Wheat Flour
Wheat is the seed of a grass like plant which is cultivated widely in temperate
climates. The grains or seeds consist of about 85% endosperm, 2% embryo or germ and 13%
husk (bran). The seeds are ground to produce a variety of flours where most wheat being used
in this form. The endosperm and hence flour consist mainly of starch and also contains from
7 to 15% protein. The proteins can be divided into four groups, the water soluble albumins
(15%), globulins (7.5%), prolamins which consist of gliadin (32.5%) and glutelins which
consist of glutenin (45%). These last two groups, making up the majority of wheat protein,
interact in the presence of water to form a viscous, colloidal complex, known as gluten. The
elastic, network forming gluten plays a major role in the structure and texture of the food
product (Street, 1991). In the brownies processing the function of the wheat flour is to form
the brownies structure and texture and also to bind the other ingredients evenly (Matz, 1991).
Gluten, or the gluten matrix, is noted for its strong, three dimensional viscoelastic
structure that is created by specific proteins. Specifically, it is the hydrophobic, inslouble
gliadin proteins that contribute sticky, fluid properties to the dough and the insoluble
glutenins that contribute elastic properties to the dough. Not all flours and therefore not all
dough, forms gluten. Nongluten flours contain starch that provides some structure; however,
it is gluten protein that provides the major framework for many batters and dough (Vaclavik,
2007).
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2.3.3 Sugar
Sugar in high concentration can act as a preservative by inhibiting the growth of
microorganisms. The concentration of sugar dehydrates the bacteria or yeast cells to the point
of inactivation or death. The hygroscopic nature of sugars is responsible to their influence on
a foods moistness and texture. The main ability of sugar in the food is act as sweetener
(Brown, 2008). In the brownies making, the function of sugar is to act as sweetener and also
bind the water in brownies.
2.3.4 Fat
All baked products contain lipids. Fat has versatile function in baked products,
the major function of fat are affecting the richness and tenderness in bakery product,
improving the flavor and eating characteristics, enhancing aeration for leavening and volume,
promoting desirable grain and texture qualities, providing flakiness in pastry product, provide
lubrication for wheat gluten, affecting the moisture retention of the bakery product and also
providing structure for cakes. Product like cake is highly dependent on fat to gain proper
aeration that will affect the quality of the final product. Fat will contribute to the texture,
mouthfeel and lubricity of the cake. In cookies making, fat acts as lubricant, it keeps the
dough from sticking to the feeding and forming equipment. It also facilitates mixing by
lubricating with other ingredients (Hui et al, 2008).
2.4 Brownies Processing
The making process of brownies is almost like the making process of cake. There
are several steps in the making of brownies which are mixing, depositing, baking, cooling
and packaging. There are several methods of mixing, such as sugar batter method, flour batter
method and single stage mixing method. In the flour batter method, the mixing process is
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done by mixing the flour and shortening together, but the egg and sugar is mixed together
with medium speed mixer in a separated container. Then after all of the ingredients have been
mixed together, all of the ingredients is mixed together. In the sugar batter method, the
shortening, sugar, and the dry ingredients is mixed in low speed until the ingredients are
mixed properly, after that the addition of eggs, milk and flour is done. In the single stage
mixing, all of the ingredients is mixed together in a container, and mixed together until the
mixture is properly homogenized (Suas, 2008). After the mixing process is done, the next
step in the making of brownies is to pour the mixed dough into the baking pan. After that the
pan is put into the oven. The baking is the main factor that determine the quality of the cake.
The improper baking time can result in the lower quality of the end product. The improper
temperature during baking can affect the color, the texture, and the volume of the brownies
product.
2.6 Potato Flour
Potato Flour is the oldest commercial potato product and it can be used in several
processed food products, such as bakery product. Potato flour has long been used in baking,
and it could be used to impart the potato flavor and also improve retention of freshness in
bread. Potato has the ability to increase the growth of yeast cells and also increase the activity
of sugar fermentation. Potato flour also has a distinctive flavor while incorporated in bakery
product, and also could reduce product firming and staling and also helps in the leavening of
the product (Preedy et al, 2011). Misra et al (2003) stated that potato is not an rich source of
protein, but contain good quality protein, dietary fiber, several minerals and trace elements. It
also contains essential vitamins and little or no fat.
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15
CHAPTER 3
RESEARCH METHODOLOGY
16
Amount
90 g flour
10
225
225 (See Table 3.2)
3 (270 g)
1.25
50
making the gluten free rice bran brownies utilizing the best wheat flour replacer
obtained from the preliminary research. The next step was evaluating the brownies produced
by observed the physical and chemical parameter, including texture, moisture content and
water activity. The next step was sensory evaluation test of gluten free rice bran brownies
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using the hedonic test. After sensory qualities of the brownies were evaluated, then the best
gluten free rice bran brownies formulation could be determined. Proximate analysis was
done for the best gluten free rice bran brownies formulation including moisture content,
protein content, ash content, fat content, and dietary fiber content. Proximate analysis for the
rice bran flour was also conducted.
The formula which was used in the making of the gluten free rice bran brownies
was modified from formula stated by Wulandari (2011). The modification done was the
substitution of wheat flour with potato starch, rice flour or cassava flour and the use of palm
sugar. The formula of brownies could be seen in Table 3.1, while the modification according
to the treatment could be seen in Table 3.2.
Table 3.2 Formula modification
Flour and rice bran flour ratio
100 % selected
90 % selected flour + 10 % rice bran flour
80 % selected flour + 20 % rice bran flour
70 % selected flour + 30 % rice bran flour
60% selected flour + 40% rice bran flour
The flowchart of the main research activities can be seen in Figures 3.1.
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The selected flour and rice bran flour (see Table 3.2), sugar, cocoa powder,
margarine, eggs, and vanilla powder were mixed using mixer with medium speed for 5
minutes. After that the dough was deposited in the aluminium pan and spread evenly. The
dough was put to the oven in 200o C temperature and baked according to treatment (35, 45
and 55 minutes). After the baking process was done, the gluten free rice bran brownies was
produced and ready to be further analysed.
3.3 Experimental Design
3.3.1 Preliminary Research
The treatment that was done in the preliminary research is the replacement of
wheat flour (A1) with cassava flour (A2), rice flour (A3), and potato flour (A4). Based on the
treatment, the experimental design in the preliminary research was complete random design
with one factorial. Factors observed were the different types of flour used for making the rice
bran brownies, which consists of A1, A2, A3 and A4. The preliminary research was done in
six replications. The randomized factorial design is :
Yij = + Ri + 1 (ij)
Where :
Yij = Random variable denoting the (ij)th variable
= real mean value
Ri = effect of different types of flour on level i
1 (ijk) = Galat factor
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Based on the treatment, the experimental design used in this research is complete random
design with two factorials, R x B (5x3). The main research was conducted in three
replications. Factor which observed were :
20
1. The concentration of rice bran which was added to the formulation, which consist of
R0, R1, R2, R3 and R4.
2. The different baking time, which were B1, B2 and B3.
The combination of the factors can be seen in Table 3.3.
Table 3.3 Combination of Factors
Baking Time
B1
B2
B3
R2
R2B11
R2B12
R2B13
R3B21
R3B22
R3B23
R2B31
R2B32
R2B33
R3
R3B11
R3B12
R3B13
R3B21
R3B22
R3B23
R3B31
R3B32
R3B33
R4
R4B11
R4B12
R4B13
R4B21
R4B22
R4B23
R4B31
R4B32
R4B33
The sensory qualities of the gluten free rice bran brownies was determined by using sensory
evaluation test which was the hedonic test (Meilgaard, 2007). The hedonic test was conducted
to determine the gluten free rice bran brownies formula which was most preferred by the
panelist. The panelist in this sensory evaluation procedure was untrained panelist. The
method for the hedonic test could be seen in Appendix 1.
3.4.2 Physical Characteristic
The physical characteristic of the gluten free rice bran brownies was determined by using
texture analyzer towards the hardness parameter. The method of determining the texture of
the gluten free high rice bran brownies could be seen in Appendix 2.
3.4.3 Chemical Characteristic
The chemical characteristic of the gluten free rice bran brownies which was evaluated
consists of moisture content and water activity. The water activity was measured using Aw
meter while the method of determining moisture content could be seen in appendix 3.
3.4.4 Proximate Analyses for the best gluten free - high dietary fiber brownies
formulation
The proximate analyses of the gluten free rice bran brownies included the oven method to
determine the moisture content of the rice bran (AOAC, 2005), ash content using the dry
ashing method (AOAC, 2005), protein content using the micro Kjehdahl method (AOAC,
2005),fat content using the soxhlet extraction (AOAC, 2005) and carbohydrate content using
by difference method. The proximate analyses methods could be seen in Appendix 3. The
dietary fiber content of the gluten free - high dietary fiber brownies was also analyzed by
using enzyme analysis (AOAC,2005). The method for dietary fiber analysis could be seen in
the Appendix 4.
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APPENDICES
29
panelists are required to taste and evaluate the sample. The panelists are informed not to
compare the sample with the other samples.
be put in the texture analyzer table, and then the proper probe should be used based on the
texture characteristic of the food product. The measurement result in terms of number and
graph are shown by CNS Farnell software.
x y
x
100%
Where:
x = initial weight of the sample before drying in gram
y = final weight of the sample after drying in gram
b. Ash content determination, using Dry ashing method (AOAC, 2005).
1. Ash content determination is done by weighing five grams of sample using
analytical balance, the sample is put into crucible. The crucible should have constant
weight before used for weighing.
2. After the sample is put in the crucible, the sample is burnt on the burner until the
white smoke disappears.
3. The next step is ashing the sample in the furnace at 450C for 1 hour and then after
1 hour the temperature should be raised up to 550C. The ashing process is finished
when the color changed from grey to white.
4. The resulted ash is then weighed using analytical balance and the ash content is
calculated using the formula below.
Ash content (%) =
x y
z
100%
Where:
x = weight of evaporating dish and the sample after ashing in gram
y = weight of evaporating dish in gram
z = weight of the sample in gram
c. Protein content determination using Micro Kjeldahl method (AOAC, 2005).
32
1. The protein analysis is conducted by weighing two grams of sample and then the
sample is put into Kjeldahl tube and the addition of 7 grams of K2SO4, 5 mg of
selenium, 15 ml of 96% H2SO4, and 10 ml of 35% H2O2 are done.
2. And then, the next step is the destruction of the sample at 420C, the sample is
destructed until the solution became clear and then sample is cooled down.
3. The next step is placing the tube the Kjeldahl distillation equipment. After that, the
addition of 25 ml of 4% saturated boric acid and 3 drops of mixed indicator are done.
The mixed indicator is mixture of methyl red and methylene blue and should be put
into erlenmeyer flask. The flask is placed below the condenser and the tip of
condenser pipeline should be soaked in boric acid solution.
4. The next step is the distillation process. The distillation process is done by the
addition of NaOH 35% for 5 minutes.
5. 0.2 N HCl is used to titrate the result of distillation until slightly pink color
appeared. The protein content is calculated as percent of nitrogen as showed by the
equation below.
%N=
100%
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flask is put under Soxhlet equipment. The extraction process of fat from sample is
required for about 6 hours.
3. The extraction result is heated in oven at 105C. Then the sample should be cooled
down in desiccators and weighed until reaching the constant weight. The fat content
can be calculated using the equation below.
Fat content =
100%
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2. One gram of dry fat-free samples and 25 ml of 0.1 M buffer sodium phosphate is added to
the erlenmeyer.
3. Termamyl enzyme with amount of 0.1 ml is added to the mixture and then covered with
aluminium foil. The mixture is then incubated in the water bath at temperature 100 oC for 15
minutes.
4. The mixture in the erlenmeyer is cooled down, after cooling, 20 ml aquadest and HCl 1 M
is added to the mixture. The pH of the mixture should reach 1.5
5. After the pH of the mixture reach 1.5, 100 mg of pepsin enzyme added the the erlenmeyer
covered again with alumunium foil and then incubated inside the water bath at temperature
40 oC and agitated for 60 minutes. After that, the addition of 20 ml of aquadest and NaOH
was done, to adjust the pH of the mixture to reach 6.8
6. Then, 100 mg of pancreatin is added to the mixture. The erlenmeyer is closed again, and
incubated in the waterbath at temperature 40 oC and agitated for 60 minutes. Addition of HCl
is done to adjust the pH to reach 4.5.
7. It was filtered with dry crucible (pores 2) which has been weighed, and contains 0.5 gram
dry celite.
8. The mixture is then washed with 10 ml aquadest twice.
9. The filtrate and the precipitate are used to differentiate insoluble fiber and soluble fiber.
The filtrate is used to determine the soluble fiber, while the precipitate is used to determine
the insoluble fiber.
35
10. The precipitate need to be washed with 10 ml ethanol 95 % and 10 ml acetone twice in
order to get the insoluble fiber. After that, the precipitate is dried in 105 oC until it reaches
constant weight (12 hours). (A)
11. Then, the precipitate ia ashed in furnace 550 oC for 5 hours. It was cooled in dessicators
and weighed until the weight is constant. (B)
% Insoluble fiber = (A B C )/W x 100%
A = weight after being dried (gram)
B = weight after being ashed (gram)
C = weight of fat free blank (gram)
W = sample weight
12. The soluble fiber can be determined by adding water to the filtrate until reaching volume
100 ml. The 400 ml of 95 % ethanol is added and the mixtures is cooled down for 1 hour.
13. It then filtered with dry crucible (pores 2) which has been weighed and contains 0.5 gram
dry celite.
14. The filtrate then washed with 10 ml ethanol 78 %, 10 ml ethanol 95 % and 10 ml acetone,
each was done twice. Then it was dried in temperature 105 oC until the weight is constant (12
hours ) (A)
15. The filtrate is then ashed in furnace at 550 oC for 5 hours, and cooled down in desicator to
be weighed after reaching constant weight. (B)
% Soluble fiber = (A B C )/W x 100%
A = weight after being dried (gram)
36
37