CHAPTER 1

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
1

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

2.1 Rice Bran

The source of rice bran is from the rice milling process, which are the conversion
of brown rice to white rice. After paddy is harvested, then the paddy undergo drying process.
After the paddy has been dried, then the hull of the paddy need to be removed by the milling
process. After the hull is being removed by the milling process, the resulted product is called
brown rice. The brown rice has the outer layer that need to be removed. The removal process
is done by the process called abrasive milling. The resulted product is called white rice which
most people commonly know as rice. The rice bran, is the side product which produced from
the separation of the brown layer in the brown rice. (Choo et al, 1999).
According to Hu et al (2009), Rice bran is a by product which is produced from
the outer layer of rice. Rice bran is considered as a good source of nutrients such as protein,
mineral and fatty acids. Rica bran is also rich in dietary fiber content. Although rich in
nutrients, the utilization of rice bran is very minimal. Rice bran is mostly burnt off at the rice
milling facilities and also used as animal feeds.
2.1.1 Qualities of Rice Bran
During the application of rice bran in the food products, rice bran may produce a
mild sweet and bitter taste. Rice bran may produce the bitter taste because rice bran contain
saponin compound which can produce the bitter taste. The sweet taste in rice bran is produces

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.

Rice bran stabilization is essentially needed to inactivate lipase and lipoxygenase

activity, sterilize the bran and reduce color development. Lipoxygenase activity will increases
with the presence of FFA resulting in oxidative rancidity which is responsible for the flavor
and odor rancid of the rice bran. There are many methods of rice bran stabilization. These
methods include dry heating method, wet heating method, and extrusion methods (Shahadi,
2005).
According to Choo et al. (1999) the activity of lipase in the rice bran can be
destroyed by applying short term high temperature treatment to the rice bran, and the thermal
process will produce stabilized rice bran. The application of heat will destruct peroxidases as
well, as stated by Silva (2006). In dry heating methods, the rice bran is dried using hot air and
this drying process will reduce the moisture content of the rice bran to 3 4 %. The rice bran
must be kept in dry condition, moisture proof containers could be used to maintain the
dryness of the rice bran, because rehydration of the rice bran bran will cause it regains its
lipase activity (Shahidi, 2005). Silva (2006) also mentioned several other methods for rice
bran stabilization such as chemical stabilization and stabilization by microwave.
2.1.3 Utilization of Rice Bran
As an agricultural crop by products, rice bran utilization are now widely increase,
research has done to utilize their pharmaceutical or nutraceutical potencial. Rice bran
contains good source of antioxidants including vitamin and oryzanol, high quality oil and
protein, and cholesterol lowering waxes and anti tumor compounds like rice bran saccharide.
In a study on tumor prevention and suppresion of tumor growth in rats, riced bran saccharides
was found to suppress carcinogenesis and to prolong survival rate (Rebecca et al, 2007).
Study also show that when rice bran is added to the prudent diets of moderately
hyperlipidemic individuals, will produces significant reduction in trygliceride levels and
6

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

10

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).

11

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).

12

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

13

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.

14

2.7 Rice Flour

Rice flour is a flour made from rice which has soft taste, colourless,
hypoallergenic properties, low levels of sodium and easy digestible carbohydrate. Because of
this properties, rice flour is the most suitable cereal to make gluten free product. But, when
utilizing rice flour, it cannot be used to produce fermented food products because their
proteins cannot develop viscoelastic network like gluten. According to Hui et al (2006), The
source of rice flour is from rice grain, it could be from long rice grain, medium rice grain,
short rice grain, or waxy rice. The chemical composition of rice flour is affected by different
types of grain, and furthermore it will also affect the starch content. The chemical
composition of rice flour is consist of glucose polymer made of amylose and amylopectin, the
amylose and amylopection has different ratio which depends on the variety of rice. The starch
content in the rice flour is about 80% from carbohydrate content.
2.8 Cassava Flour
Cassava flour is the product prepared from dried cassava chips or paste by a
pounding, grinding or milling process and then followed by sifting to separate the fiber from
the flour. The production of cassava flour is done by milling of the dried raw root, whereas
the starch is obtained by washing and wet milling of the root, followed by multi-stage
purification of the slurry. Cassava flour has been utilized for making gluten free product such
as bread. Flours are fine, powdery materials which is obtained by grinding and by sifting the
starch-containing plant organelles such as grain, seed, root, tuber, fruit and so on. Basically
flours contain almost the same components as the components present in the raw materials,
except the moisture content. Some components that are often found in flours include starch,
non-starch polysaccharide, sugar, protein, lipid, and inorganic materials (Shittu et al, 2009).

15

CHAPTER 3
RESEARCH METHODOLOGY

3.1 Materials and Equipments

3.1.1 Materials
The materials used in the making of rice bran brownies are stabilized rice bran
which are obtained from rice milling unit at Semarang, cassava flour which obtained from PD
Sumberwangi Semarang, rice flour Rose Brand, potato flour which obtained from PD
Sumberwangi Semarang, sugar Gulaku, cocoa powderBensdorp, vanilla powder, egg,
margarine Blueband. The chemical materials used in the proximate analysis are aquadest,
concentrated H2SO4, NaOH Na2SO3, selenium, H2O2, H3BO3, methyl red indicator, HCl,
petroleum benzene, asbestos, K2SO4, ethanol 95 %, sodium phosphate, termamyl, pepsin
enzyme, pancreatin enzyme, dry celite.
3.1.2 Equipments
The main equipments used to produce rice bran brownies in this research are
analytical balance, oven,mixer, wok, frying spatula, dry blender, sifter, aluminum pan, brush,
bowl, spoon, graduated cylinder, and glassware. The equipment used in the analysis are oven,
desicator, texture analyzer, aw meter, furnace, reflux, watch glass, burette, heating bath,
crucible, kjehdahl tube, buchner, fat extractor, stirrer, thermometer, volumetric pippete,
spatula, filter paper, and funnel.

16

3.2 Research Procedure

3.2.1 Preliminary Research
The preliminary research was done to determine the best gluten free flour that
would be used as the wheat flour replacer in the making of rice bran brownies. The rice bran
brownies would be made by using three different types of flour, i.e cassava flour, potato flour
and rice flour. In this preliminary research the addition of 10 % of rice bran concentration
was done as the substitute of each flour used in the formulation. The treatment formulas
could be seen in the Table 3.1. The best gluten free flour that would be used in the making of
Rice bran brownies was determined by sensory evaluation. The sensory evaluation method
used was the hedonic test, the method could be seen in appendix 1. The physical and
chemical parameter of the rice bran brownies was also analysed, the parameter analysed was
texture, in term of hardness, moisture content, and water activity.
Table 3.1 Rice bran brownies formulation
Ingredients
Flour (g)
Rice bran flour (g)
Margarine (g)
Sugar (g)
Eggs
Vanilla Powder (g)
Cocoa Powder (g)
Source : Wulandari (2011), with modification

Amount
90 g flour
10
225
225 (See Table 3.2)
3 (270 g)
1.25
50

3.2.2 Main Research

The procedure of the main research was divided into several steps. The first step
was

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

17

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.

Cocoa powder, margarine,

eggs, sugar and vanilla
powder

Selected flour and

rice bran flour (See
Table 3.2)

Mixing of all of the ingredients using mixer

with medium speed for 5 minutes

Depositing the dough into aluminium pan

and the dough is spread evenly in the pan

The dough is put to the oven in 200o C

temperature and baked according to the
treatment (35, 45 and 55 minutes)

Gluten Free Rice Bran Brownies

Figures 3.1 Flowchart of main research

18

Source : Szafranski et al (2005)

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

19

Hypothesis of the preliminary research :

Ho :
There is no effect of different types of flour towards the sensory acceptance of rice bran
brownies
H1:
There is effect of different types of flour towards the sensory acceptance of rice bran
brownies.
3.3.2 Main Research
The treatment that was done is the main research consists of two treatments which are :
1. Ratio of substituted flour : rice bran flour (R), which consists of five levels :
- 100 % substituted flour : 0 % rice bran flour (R0)
- 90 % substituted flour : 10 % rice bran flour (R1)
- 80 % substituted flour : 20 % rice bran flour (R2)
- 70 % substituted flour : 30 % rice bran flour (R3)
- 60% substituted flour : 40% rice bran flour (R4)
2. Baking time (B), which consists of three levels :
-

35 minutes baking time (B1)

45 minutes baking time (B2)
55 minutes baking time (B3)

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

Rice Bran Concentration

R0
R1
R0B11
R1B11
R0B12
R1B12
R0B13
R1B13
R0B21
R1B21
R0B22
R1B22
R0B23
R1B23
R0B31
R1B31
R0B32
R1B32
R0B33
R1B33

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 randomized factorial design is :

Yijk = + Ri + Sj + Rsij + 1 (ijk)
Where :
Yijk = value of observation at level one, with factor of concentration of rice bran flour on level
i and different type of sugar on level j
= real mean value
Ri = effect of concentration of rice bran flour on level i
Sj = effect of different type of sugar used on level j
Rsij = effect of interaction between factor of concentration of rice bran flour on level i and
factor of different type of sugar used on level j
1 (ijk) = Galat factor
21

Hipotesis of this research :

Ho :
1. There is no effect on concentration of rice bran flour towards the quality and sensory
acceptance of gluten free high dietary fiber brownies
2. There is no effect on concentration of different types of sugar used towards the quality
and sensory acceptance of gluten free high dietary fiber brownies
3. There is no interaction on concentration of rice bran flour and different types of sugar
used towards the quality and sensory acceptance of gluten free high dietary fiber
brownies
H1 :
1. There is effect on concentration of rice bran flour towards the quality and sensory
acceptance of gluten free high dietary fiber brownies
2. There is effect on concentration of different types of sugar used towards the quality
and sensory acceptance of gluten free high dietary fiber brownies
3. There is interaction on concentration of rice bran flour and different types of sugar
used towards the quality and sensory acceptance of gluten free high dietary fiber
brownies

3.4 Analysis Procedure

The parameters which were observed in this research were the sensory qualities, physical,
and chemical characteristic of the gluten free rice bran brownies. The proximate analysis and
the dietary fiber analysis was done for the best gluten free rice bran brownies formulation.
3.4.1 Sensory Qualities
22

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.
23

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27

28

APPENDICES

APPENDIX 1 Method of Sensory Evaluation

Hedonic Test (Meilgaard et al, 2007)
The hedonic test is a sensory evaluation method to determine the consumer acceptance of the
product based on the overall sensory qualities of the food product. The hedonic test is done
using scale from 1 to 7 (1 = extremely dislike, 2 = dislike, 3 = slightly dislike, 4 = neutral, 5 =
slightly like, 6 = like, 7 = extremely like). This test is performed by 70 untrained panelist. The

29

panelists are required to taste and evaluate the sample. The panelists are informed not to
compare the sample with the other samples.

APPENDIX 2 Method of Determining Physical Characteristic

Analysis of Physical Characteristic of Gluten Free High - Dietary Fiber Brownies
The physical characteristic of the rice bran brownies is done by measuring the hardness,
chewiness, cohesiveness and adhesiveness of the brownies. The equipment which is used to
measure the texture of the brownies is LFRA texture analyzer. The texture is measued by
using cylindrical probe. The measure the texture of the brownies, the brownies sample should
30

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.

APPENDIX 3 Method of Proximate Analyses

A. Moisture content analysis using Oven method (AOAC, 2005).
1. Moisture content analysis is done by weighing five grams of sample and put the
sample into the evaporating dish which has constant weight.
2. The sample is then dried in the oven for 6 hours at 105C. Before weighed in
analytical balance, the sample should be cooled down first in the desicators, and
then the sample should be dried again until the constant weight is obtained. The
moisture content calculated is wet basis moisture content.
31

Moisture content (%) =

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=

( ml HClml blank ) N HCl 14.007

weight of sample(mg)

100%

The percentage of nitrogen is multiplied by specific conversion factor based on type

of sample used to obtain the percentage of protein content.
d. Fat content, using Soxhlet method (AOAC, 2005).
1. In the fat content analysis using soxhlet method, the sample used is five grams of
water-free sample, the sample is wrapped using filter paper (thimble) and then it is put
into Soxhlet equipment.
2. After that, the condenser should be put above Soxhlet equipment. The round bottom
flask is filled with petroleum benzene (solvent) and boiling chips. The round bottom

33

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 =

weight of fat extracted( g)

weight of sample (g)

100%

e. Carbohydrate content, using By difference method

Carbohydrate content (%) = 100% - (% moisture + % ash + % protein + % fat)

APPENDIX 4 Method of Determining Total Dietary Fiber

Total Dietary Fiber Analyses (AOAC, 2005)
1. The fat contained in the samples need to be extracted with petroleum eter for 15 minutes.

34

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

B = weight after being ashed (gram)

C = weight of fat free blank (gram)
W = sample weight
Total Dietary Fiber (%) = Insoluble fiber (%) + Soluble Fiber (%)

37