Feasibility of Rice Bran Oil PDF

Rice Industrial By-products Management for
Oil Extraction and Value Added Products

By
MIAN KAMRAN SHARIF

B.Sc. (Hons.) Agri. Major Food Technology (UAF)
M.Sc. (Hons.) Food Technology (UAF)
A dissertation submitted in partial fulfillment of requirements for the degree of
DOCTOR OF PHILOSOPHY
IN
FOOD TECHNOLOGY
NATIONAL INSTITUTE OF FOOD SCIENCE AND TECHNOLOGY
UNIVERSITY OF AGRICULTURE
FAISALABAD, PAKISTAN
2009
53
To
The Controller of Examinations,

University of Agriculture,
Faisalabad.
The members of the Supervisory Committee find the thesis submitted by Mr.
Mian Kamran Sharif (Regd. 96-ag-1478) satisfactory and recommend that it be
processed for evaluation by External Examiner(s) for the award of degree.
CHAIRMAN
(Dr. Masood Sadiq Butt)
MEMBER
(Prof. Dr. Faqir Muhammad Anjum)
MEMBER
(Dr. Haq Nawaz)
54
DEDICATED
TO
HAZRAT MUHAMMAD
(Peace Be Upon Him)
&
my parents
who taught me to be responsible and professional in any field
55
ACKNOWLEDGEMENTS
I am extremely thankful to ALMIGHTY ALLAH (The Merciful) who
blessed to complete this piece of research work presented in this study. I present
my humble gratitude from the deep sense of heart to the HOLY PROPHET
MUHAMMAD (Peace Be Upon Him), that without him the life would have
been worthless.
I expand my deepest appreciation to my affectionate supervisor,
Dr. Masood Sadiq Butt, Associate Professor, National Institute of Food Science
and Technology, University of Agriculture, Faisalabad for his great help,
illuminating guidance, and consistent encouragement during planning,
execution, and final presentation of this piece of research work
With a deep emotion of gratitude, I express the sincere thanks to
Prof. Dr. Faqir Muhammad Anjum, Director General, National Institute Food
Science and Technology, University of Agriculture, Faisalabad for his
sympathetic attitude and cooperation in the preparation and finalization of this
manuscript.
I am also grateful to my committee member, Dr. Haq Nawaz, Associate
Professor, Institute of Animal Nutrition and Feed Technology for his
compassionate attitude and kind cooperation provided during my research
project.
I also thank my friends and fellow students, who made my busy and
boring life more interesting. I am also grateful to Mr. Tauseef Sultan, Mr.
Muhammad Nasir, Miss Saima Hafeez Khan, Mr. Akmal Nazir, Mr. Kashif
Khan, Muhammad Issa Khan and Dr. Mumtaz Shaheen who helped me day
and night for final presentation of this dissertation.
Finally I would like to convey my sincere admiration to my father Mian
Muhammad Sharif, mother, brother, Sisters and my wife who were always
very kind to provide moral and financial support during the track of this study.
(Mian Kamran Sharif)

56
TABLE OF CONTENTS
S. No.
Contents
Page #
1.
2.
2.1.
2.1.1.
2.1.2.
2.1.3.
2.2.
2.3.
2.3.1.
2.3.2.
2.3.3
2.3.4.
2.3.5.
2.4.
2.4.1.
2.4.2.
2.4.3.
2.5.
2.5.1.
2.5.2.
3.
3.1.
3.2.
3.2.1.
3.2.2.
3.3.
3.3.1.
3.3.2.
3.3.3.
3.3.4.
3.3.5.
List of Abbreviations
Acknowledgement
List of Tables
List of Figures
List of Appendices
Abstract
INTRODUCTION
REVIEW OF LITERATURE
Rice bran: an overview
Physiology and general characteristics
Anti-nutritional factors
Dietary fiber
Processing of rice bran
Rice bran oil and its components
Current status
General characteristics
Effective components
Utilization
Economic Feasibility
Hypocholesterolemic effects of rice bran and rice bran oil
Rice Bran
Rice Bran Oil
Cholesterol-lowering mechanisms
Supplementation in baked products
Bread
Cookies
MATERIALS AND METHODS
Materials
Rice bran processing
Rice bran stabilization
Denaturation of anti-nutritional factors
Stabilization and anti-nutritional appraisal
Lipase activity
Peroxide value
Thiobarbituric acid no.
Haemagglutinin-lectin activity
Trypsin inhibitor activity
57
i
ii
vii
xi
xii
xiii
1
8
8
8
9
11
12
14
14
15
16
21
22
22
22
25
30
32
33
35
37
37
37
37
38
38
38
38
38
39
39
3.3.6.
2.4.
3.4.1.
3.4.2.
3.5.
3.5.1.
3.5.2.
3.5.3.
3.5.4.
3.5.5.
3.5.6.
3.6.
3.7.
3.7.1.
3.7.2.
3.7.2.1.
3.7.2.2.
3.7.2.3.
3.8.
3.8.1.
3.8.1.1
3.8.1.1.1.
3.8.1.1.2
3.8.1.1.3.
3.8.1.1.4.
3.8.1.1.5.
3.8.2.
3.8.3.
3.8.3.1.
3.8.3.2.
3.8.3.3.
3.8.3.4.
3.8.3.5.
3.8.4.
3.8.5.
3.8.6.
3.8.6.1.
Phytates
Raw Materials Analysis
Proximate analysis
Mineral analysis
Rice bran oil
Extraction
Refining
Yield
Quality of refined rice bran oil samples
Antioxidants potential
39
39
39
40
41
41
41
41
41
42
43
Fatty acid profile

Selection of best treatment
Efficacy studies for safety evaluation
43
43
44
Experimental plan
Analysis of serum profile
Liver function tests
Renal function tests
Lipid profile
Product development
Preparation of rice bran oil cookies
Quality attributes of cookies
Physical analysis
Proximate analysis
Total acidity
Sensory evaluation
Preparation of rice bran supplemented flours
Analysis of rice bran supplemented flours
Proximate analysis
Mineral analysis
Dietary fiber
Dough rheological studies
Preparation of rice bran supplemented cookies
Preparation of rice bran supplemented leavened pan bread
Analysis of rice bran supplemented cookies and bread
Physical analysis
58
45
45
45
45
46
46
46
46
47
47
47
47
47
48
48
48
48
49
49
50
50
51
51
3.8.6.2.
3.8.6.3.
3.8.3.4.
3.9.
4.
4.1
4.1.1.
4.1.2.
4.1.3.
4.1.4.
4.1.5.
4.1.6.
4.2.
4.3.
4.3.1.
4.3.2.
4.3.3.
4.3.4.
4.3.5.
4.3.5.1.
4.3.5.2.
4.3.6.
4.4.
4.4.1.
4.4.1.1.
4.4.1.2.
4.4.1.3.
4.4.1.4.
4.4.2.
4.4.3.
4.4.3.1.
4.4.3.2.
4.4.3.3.
4.4.3.4.
4.4.3.5.
4.4.3.6.
4.5.
4.5.1.
4.5.1.1.
4.5.1.2.
4.5.1.3.
4.5.1.4.
Mineral analysis
Dietary fiber
Sensory evaluation
Statistical Analysis
RESULTS AND DISCUSSIONS
Stabilization and anti-nutrition appraisal
Lipase activity
Peroxide value
Haemagglutinin-lectin activity
Trypsin inhibitor activity
Phytates
Raw materials analysis
Rice bran oil
Refining
Yield
Quality evaluation
Fatty acid profile of RBO
Antioxidants potential
Oryzanol
Tocopherols and tocotrienols
Selection of best sample
Efficacy studies
Physical parameters of rats
Feed intake
Water intake
Gain in body weight
Organ weight
Renal and Kidney functioning tests
Serum biochemical profile
Cholesterol
High density lipoprotein (HDL)
Low density lipoprotein (LDL)
Triglycerides (TG)
Glucose
Serum proteins
Product development
Preparation of rice bran oil cookies
Physical analysis
Proximate analysis
Total acidity
59
51
51
51
52
53
53
54
56
56
57
59
59
60
63
63
63
63
68
71
71
73
74
75
75
75
75
78
79
81
81
83
88
91
91
95
97
99
99
99
102
107
107
4.5.1.5.
4.5.2.
4.5.2.1.
4.5.2.2.
4.5.2.3.
4.5.2.4.
4.5.2.5.
4.5.2.5.1.
4.5.2.5.2.
4.5.3.
4.5.3.1.
4.5.3.2.
4.5.3.3.
4.5.3.4.
4.5.4.
4.5.4.1.
4.5.4.2.
4.5.4.3.
4.5.4.3.1.
4.5.4.3.2.
5.
Sensory evaluation
Preparation of rice bran supplemented flours
Proximate analysis
Mineral analysis
Dietary fiber
Dough rheological studies
Mixographic studies
Farinographic studies
Preparation of rice bran supplemented cookies
Physical analysis
Mineral analysis
Dietary fiber
Sensory evaluation
Preparation of rice bran supplemented leavened pan bread
Mineral analysis
Dietary fiber
Sensory evaluation
External characteristics
Internal characteristics
SUMMARY
RECOMMENDATIONS
LITERATURE CITED
APPENDICES
60
109
115
116
124
127
129
132
132
136
141
142
145
147
147
154
154
157
159
159
165
175
180
181
206
LIST OF TABLES
S. No.
Title
Page #
1.
Utilization of RBO in cookies
46
2.
Rice bran supplemented flours used in study
48
3.
Treatments used for preparation of rice bran supplemented cookies
50
4.
Treatments used for preparation of leavened pan bread
51
5.
Mean squares for FFA, POV and TBA no. of rice bran samples
55
6.
Effect of stabilization on FFA, POV and TBA no. of rice bran

samples
55
7.
Effect of storage on FFA, POV and TBA no. of rice bran samples
55
8.
Anti-nutritional factors in rice bran samples
58
9.
Proximate composition of fullfat rice bran samples and commercial

straight grade flour
62
10.
Mineral analysis of rice bran samples
62
11.
Proximate composition of defatted rice bran samples
62
12.
Yield of rice bran oil from different bran samples
65
13.
Quality characteristics of rice bran oil samples
65
14.
Fatty acid composition of rice bran oil samples
70
15.
Mean squares for antioxidants in rice bran oil samples
72
16.
Mean squares for physical parameters of different groups of rats
76
17.
Mean squares for organs weight of different groups of rats
80
18.
Effect of diets on organs weight of different groups of rats
80
61
19.
Organs weight of different groups of rats during study periods
80
20.
Mean squares for serum kidney and liver function tests
82
21.
Effect of diets on serum kidney and liver function tests in different

groups of rats
82
22.
Serum kidney and liver function tests in different groups of rats

during study periods
82
23.
Mean squares for lipid profile and serum glucose in different

groups of rats
84
24.
Effect of diets on serum lipid profile and glucose (mg/dL) in

different groups of rats
84
25.
Serum lipid profile and glucose (mg/dL) in different groups of rats

during various study periods
84
26.
Mean squares for serum proteins in different groups of rats
98
27.
Effect of diets on serum proteins in different groups of rats
98
28.
Serum proteins in different groups of rats during study periods
98
29.
Mean squares for physical analysis of RBO cookies
100
30.
Physical analysis of RBO cookies
100
31.
Effect of storage on physical analysis of RBO cookies
100
32.
Mean squares for proximate composition of RBO cookies
103
33.
Proximate composition of RBO cookies
105
34.
Effect of storage on proximate composition of RBO cookies
105
35.
Mean squares for total acidity and TBA no. of RBO cookies
108
36.
Total acidity and TBA no. of RBO cookies
108
37.
Effect of storage on total acidity and TBA no. of RBO cookies
108
38.
Mean squares for sensory attributes of RBO cookies
111
39.
Color scores of RBO cookies
112
40.
Flavor scores of RBO cookies
112
41.
Taste scores of RBO cookies
112
42.
Texture scores of RBO cookies
114
43.
Crispness scores of RBO cookies
114
44.
Overall acceptability scores of RBO cookies
114
45.
Mean squares for proximate composition of supplemented flours
117
62
46.
Moisture content of supplemented flours
118
47.
Crude protein of supplemented flours
120
48.
Crude fat of supplemented flours
122
49.
Crude fiber of supplemented flours
123
50.
Ash content of supplemented flours
125
51.
Nitrogen free extract of supplemented flours
126
52.
Mean squares for mineral content of supplemented flours
128
53.
Mineral contents of supplemented flours
128
54.
Mean squares for dietary fiber and TBA no. of supplemented flours
130
55.
Dietary fiber content of supplemented flours
130
56.
TBA no. of supplemented flours
131
57.
Mean squares for mixographic characteristics of supplemented

flours
134
58.
Mixing time of supplemented flours
134
59.
Peak height of supplemented flours
135
60.
Mean squares for farinographic characteristics of supplemented

flours
137
61.
Water absorption of supplemented flours
137
62.
Dough development time of supplemented flours
140
63.
Dough stability of supplemented flours
140
64.
Mean squares for physical parameters of rice bran supplemented

cookies
143
65.
Physical analysis of rice bran supplemented cookies
144
66.
Effect of storage on physical analysis of rice bran supplemented

cookies
144
67.
Mean squares for minerals of rice bran supplemented cookies
146
68.
Mineral contents of rice bran supplemented cookies
146
69.
Mean squares for dietary fiber of rice bran supplemented cookies
148
70.
Dietary fiber content of rice bran supplemented cookies
148
71.
Mean squares for sensory attributed of rice bran supplemented

cookies
149
72.
Color, flavor and taste scores of rice bran supplemented cookies
150
63
73.
Effect of storage on color, flavor and texture of rice bran

supplemented cookies
150
74.
Texture, crispness and overall acceptability scores of rice bran

153
75.
Effect of storage on texture, crispness and overall acceptability of

rice bran supplemented cookies
153
76.
Mean squares for mineral contents of rice bran supplemented

breads
156
77.
Mineral contents of rice bran supplemented breads
156
78.
Mean squares for dietary fiber of rice bran supplemented breads
158
79.
Dietary fiber of rice bran supplemented breads
158
80.
Mean squares for

supplemented breads
81.
Volume and crust color scores of rice bran supplemented breads
162
82.
Effect of storage on volume and crust color of rice bran

supplemented breads
162
83.
Symmetry and evenness of bake scores of rice bran supplemented

breads
164
84.
Effect of storage on symmetry and evenness of bake of rice bran

supplemented breads
164
85.
Character of crust scores of rice bran supplemented breads
166
86.
Effect of storage on character of crust of rice bran supplemented

breads
166
87.
Mean squares for internal characteristics of rice bran supplemented

breads
167
88.
Grains and crumb color scores of rice bran supplemented breads
169
89.
Effect of storage on grains and crumb color scores of rice bran

supplemented breads
169
90.
Aroma and taste scores of rice bran supplemented breads
171
91.
Effect of storage on aroma and taste of rice bran supplemented

breads
171
92.
Texture scores of rice bran supplemented breads
173
93.
Effect of storage on texture of rice bran supplemented breads
173
external
64
characteristics
of
rice
bran
161
LIST OF FIGURES
S. No.
Title
Page #
Feed intake in different groups of rats (per rat/day) during six

weeks
77
Water intake in different groups of rats (per rat/day) during six

weeks
77
Percent decrease in gain in body weight in different groups of

rats (per rat/week) during six weeks
77
Percent decrease of cholesterol in different groups of rats
85
Percent increase of HDL in different groups of rats
89
Percent decrease of LDL in different groups of rats
92
Percent decrease of triglycerides in different groups of rats
94
Percent decrease of glucose in different groups of rats
96
65
LIST OF APPENDICES
S. No.
Title
Page #
Composition of salt mixture
206
II
Composition of vitamin mixture
207
III
Performa for sensory evaluation of cookies
208
IV
Performa for sensory evaluation of leavened pan bread
209
Saturated/unsaturated fatty acids profile of rice bran oil
210
VI
Micro-nutrient profile of rice bran oil
211
66
LIST OF ABBREVIATION
RB
Rice bran
FFRB
Fullfat rice bran
DFRB
Defatted rice bran
Un-RB
Unstabilized rice bran
ES-RB Extrusion stabilized rice bran
PAR-RB
Parboiled rice bran
MW-RB
Microwave stabilized rice bran
RBO
Rice bran oil
PAR-RBO
Parboiled rice bran oil
MW-RBO
Microwave stabilized rice bran oil
ES-RBO
Extrusion stabilized rice bran oil
NS
Normal shortening
CSGF
Commercial straight grade flour
POV
Peroxide value
TBA no.
FFA
Free fatty acids
UC
Unsaponifiable content
Tocols
Tocopherol and tocotrienol
SD-rats
Sprague Dawley rats
NFE
Nitrogen free extract
CVD
Cardiovascular disease
TRF
Tocotrienol rich fraction
HDL
High density lipoprotein cholesterol
LDL
Low density lipoprotein cholesterol
TG
Triglycerides
TC
Total cholesterol
ALP
Alkaline phosphatase
ALT
Alanine amino transferase
AST
Aspartate amino transferase
BRBO
Bioactive components from rice bran oil
SOV
Source of variation
df
Degree of freedom
wk
Weeks
wb
Wet basis
hr
Hour
min
Minutes
rpm
Revolutions per minutes
67
ABSTRACT
Rice bran, one of the main by-products of rice milling industry, has been
recognized as an excellent source of edible oil, protein, dietary fiber and allied
micronutrients. In Pakistan, it is under-utilized and generally used in poultry
feed and fuel purposes. It contains about 15-20% edible oil, which could
efficiently be used for bridging the oil deficiency in the country. Current research
was conducted to utilize indigenous rice bran (RB) for oil extraction as well as
preparation of value-added products. Rice bran samples, stabilized by extrusion
cooking, microwave heating and parboiling; were analyzed for lipase activity
during 60 days storage. On the basis of analysis, microwave (MW) stabilization
was found to be the most effective stabilization technique in controlling lipase
activity. After stabilization, oil was extracted from bran samples and evaluated
for physical & chemical characteristics, fatty acid profile and antioxidant
potential. In current study, microwave stabilized rice bran (MW-RB) was
preferred on the basis of better stability (FFA, POV and TBA no.), color of oil and
high antioxidant potential. MW stabilized fullfat rice bran (FFRB); its defatted
portion (DFRB) and extracted oil (RBO) were used for efficacy studies and
preparation of value added products. The diets prepared from selected
treatments alongwith control were fed to four groups of SD-rats for 45 days and
evaluated for physical and hematological parameters. The rats fed on RBO diet
had the highest feed intake (19.21g/rat/day); water intake (37.81mL/day) and
gain in body weight (7.24g per rat/week). Mean squares for organs weight, renal
and liver functioning tests exhibited non-significant differences with respect to
diets and study periods in different groups of rats. Animals fed on RBO, FFRB
and DFRB resulted significant reduction in serum cholesterol, LDL and
triglycerides. It was concluded that experimental diets imparted no adverse
effects on the animal growth and improved serum profile of SD-rats; showing
suitability of RB and RBO for product development. In the 2nd phase of research,
RBO was supplemented in cookies by replacing normal shortening. It was
concluded that rice bran oil can successfully be used for preparation of cookies
upto 40-60%. Moreover, FFRB and DFRB were mixed separately with commercial
straight grade flour in different proportions and analyzed for chemical
composition and rheological behavior to find out the most appropriate
compositions showing suitability for preparation of cookies and leavened bread.
Later, cookies and leavened breads were prepared from selected FFRB and DFRB
supplemented flours. On the basis of physicochemical and sensory assay, it was
concluded that cookies can be supplemented 10-20% and leavened pan bread
upto 15% with either type of rice bran without affecting nutritional and sensory
quality attributes. From the present investigation, it is concluded that rice bran
has a potential to be used for oil extraction and preparation of value added
products. This will not only be helpful to fulfill the countrys edible oil
requirement but also to cope with the protein deficiency in the communities at
risk through bran supplemented value added products.
68
Chapter-I
INTRODUCTION
Rice (Oryza sativa) is the 2nd leading cereal crop and staple food of half of
the worlds population. It is grown in at least 114 countries with global
production of 645 million tons; share of Asian farmers is about 90% of the total
produce. In Pakistan, rice is the 3rd largest crop after wheat and cotton. During
fiscal year 2007-08, it was cultivated on an area of 2515 thousand hectares with
production of 5563 thousand tons (IRRI, 2008; GOP, 2008).
In the developing countries, budding dilemma of food crisis, arising due
to lower crop yields and escalating population, needs to utilize each pent of
available resources. In order to provide enough food to all people, there is the
holistic approach of using the by-products generated during food processing and
preparations. Rice is being processed in well established industry but the major
apprehension is the utilization of its by-products; rice bran (5-8%) and polishing
(2-3%) that are going as waste. Rice processing or milling produces several
streams of materials including milled rice, bran and husk. In developing
countries, rice bran is considered as a by-product of the milling process and
commonly used in animal feed or discarded as a waste. The potential of
producing rice bran at the global level is 29.3 million tons annually while the
share of Pakistan is worked out to be 0.5 million tons (FAO, 2001; GOP, 2008).
Rice bran, brown outer layer of rice kernel, is mainly comprised of
pericarp, aleuron, subaleuron layer and germ. It contains appreciable quantities
of nutrients like protein, fat and dietary fiber. Furthermore, it contains
substantial amount of minerals like K, Ca, Mg and Fe. Presence of antioxidants
like tocopherols, tocotrienols and - oryzanol also brighten prospects of rice bran
utilization for humans (Gong and Yao, 2001; Moldenhauer et al., 2003).
Although, overall composition and nutritional profile of rice bran holds
significant importance yet presence of anti-nutritional compounds such as
69
phytates, trypsin inhibitors, pepsin inhibitors, hemagglutinins and antithiamine

factors prove to be a major hindrance in its possible food applications. Presence
of some higher quantities of lipases render instability to oil fractions as these
enzymes are released during milling and act upon triglycerides thus increasing
the free fatty acid content (Lima et al., 2002; Ahmed et al., 2007).
The effective utilization of rice bran is possible only by deactivating the
lipase enzyme responsible for the hydrolytic degradation of rice bran
constituents (Martin, 1994). Stabilization is an effective treatment turning rice
milling by-products into valuable dietary constituents. Various stabilization
techniques like heat treatment, low temperature storage, chemical treatment,
controlling storage relative humidity, simultaneous milling & extraction and
microwave heating have evolved to inactivate lipase (Ramezanzadeh et al., 2000;
Lakkakula et al., 2004). These have resulted in emergence of rice bran as an
important by-product of rice milling industry. Heat treatment is effective and
resultant product could be stored at refrigerated temperature upto 16 weeks
without imparting antinutritional effects and allied quality attributes. Microwave
heating is considered as more effective method for the inactivation of lipase,
responsible for rice bran degradation (Ramezanzadeh et al., 1999; Ramezanzadeh
et al., 2000). In rice bran, dipolar water molecules are excited by the
electromagnetic waves and are made to spin. The resultant enhanced kinetic
energy, alongwith friction, produces heat that results in the even distribution of
heat having deleterious effects on lipase activity. Moreover, microwave heat had
little effect on nutritional quality and the functional property of rice bran.
In recent years, rice bran has been recognized as a potential source of
edible oil. It contains 15-20% oil, depending upon degree of milling, variety and
other agro-climatic factors (Marshall and Wadsworth, 1994; Lima et al., 2002).
Globally, during last few decades, efforts were made towards exploiting the nonconventional sources for oil extraction and value addition; special attention has
been paid towards edible oil/food supplements from food processing by70
products. Rice bran oil (RBO) holds unique nutritional profile and is of high
nutraceutical worth. Recently, scientists have also shown tremendous interest in
exploring the cholesterol lowering properties of RBO. It is extensively used in
Japan, Korea, China, Taiwan and Thailand as a Premium Edible Oil (Ghosh,
2007). In Japan and some western countries, it is more popularly known as a
Heart Oil and acquired the status of Health Food (CAC, 2003). Rice bran and
its oil can be utilized for value addition of cereals based food products to attain
multiple benefits.
RBO contains oleic acid (38.4%), linoleic acid (34.4%), and linolenic acid
(2.2%) as unsaturated fatty acids while palmitic (21.5%) and stearic (2.9%) acids
as saturated fatty acids. A potential advantage of RBO over other oils with
similar fatty acid composition is its oxidative stability imparted by high levels of
tocopherols, tocotrienols and -oryzanol and its cholesterol lowering ability (Kim
and Godber, 2001; Wilson et al., 2000). It also contains high amount of
unsaponifiable matter i.e. 4.2% including phytosterols. It improves plasma lipid
and lipoprotein profiles by interrupting absorption of intestinal hydrophobic
compounds. RBO also lowers total serum cholesterol and low density lipoprotein
concentrations without effecting high density lipoproteins (Wilson et al., 2000;
2007; Most et al., 2005). The occurrence of peculiar components such as oryzanol
and tocotrienols in RBO are responsible for its hypocholesterolemic worth
(Vissers et al., 2000; Nagao et al., 2001).
Rice bran oil is rich source of natural vitamin E that is complex of eight
chemically distinct molecules: , , and -tocopherol; , , and -tocotrienol.
Palm oil and RBO represent two major nutritional sources of natural tocotrienol
(Sen et al., 2007). Oryzanol is nutritionally and medicinally important constituent
of rice bran oil that reduces cholesterol oxidation. The percentage of oryzanol in
crude RBO ranged from 1.9 to 2.2%. In Japan it is widely used as natural
antioxidant in foods and cosmetics. It has functions similar to vitamin E in
promoting growth, facilitating capillary growth in the skin and improving blood
71
circulation along with stimulating hormonal secretion (Luh et al., 1991; Xu and
Godber, 1999; Xu et al., 2001).
Although rice bran has been recognized as an excellent source of vitamins
and minerals yet it has been under-utilized in human diet and in Pakistan 90% is
being used primarily in animal feeds (Moldenhauer et al., 2003). Proteins are
more concentrated in the rice bran and are unique in their nutritional value,
which is quite comparable with that of its endosperm protein or protein from
any other cereal or legume. The protein of rice bran is highly digestible and
hypoallergenic food ingredient (Helm and Burks, 1996; Tang et al., 2003).
Supplementation of wheat flour with rice bran or its defatted portion
holds potential to uplift the nutritional profile of cereal based food products with
special reference to protein, lysine and dietary fiber contents. Research
interventions conducted in the past decade highlighted that stabilized full-fat
rice bran up to 20% level and unstabilized full-fat or stabilized defatted rice bran
upto 10% are suitable in various food preparations (Singh et al., 1995). However,
in yeast leavened pan bread formulations, rice bran can be substituted upto 15%
of the wheat flour without affecting loaf volume (Sharp and Kitchens, 1990).
Rice bran is an excellent source of dietary fiber ranging from 20-51%
(Saunders, 1990). Rice bran fiber has laxative effect with increased faecal output
and stool frequencies. Soluble fibers have gained popularity to reduce the
postprandial glycemia in normal and diabetic subjects. It acts like a sponge and
absorbs water in the intestine, mixes the food into gel and there by slows down
the rate of digestion and absorption (Abdul and Yu, 2000). Use of 1g of soluble
fiber can lower total cholesterol by about 0.045mmol/L. Researchers have also
observed 29% less risk of coronary heart disease for each additional intake of 10g
of fiber daily (Rimm et al., 1996; Brown et al., 1999). Possible health claims of
consumption of rice bran and its defatted portion include increased faecal bulk
and reduced blood cholesterol owing to its dietary fiber contents (Abdul and Yu,
2000).
72
The research was carried out using indigenous rice cultivar i.e. Basmati
Super. Although lot of research has been carried out on various aspects of rice
bran abroad but in our study we have focused on the local cultivar to explore its
potential for human as little or no effort has been made earlier in Pakistan on
extraction of oil form rice bran. There are many factors like soil, environment,
cultural and agronomic practices etc. which have pronounced effects on the grain
quality characteristics. Moreover this work will provide information to the
scientific community, farmers and industrialist about the value addition in rice.
In Pakistan, rice is the 3rd largest crop after wheat and cotton and it is one of the
major foreign exchange earning crops mainly in the form of Basmati super (upto
40% export) which is liked throughout the world due to its specific aroma.
Basmati Super is processed in well established modern mills with production of
good quality rice kernel and bran as a by-product. Other fine rice cultivars and
coarse varieties (for local use) are usually processed through conventional
shellers and bran produced is of poor quality due to more contamination of
endosperm and husk in the resultant bran. So bran of Basmati Super was chosen
due to better quality and relatively higher yield of oil.
Pakistan is confronting with the problem of food security especially in
edible
oils.
Some
of
our
conventional
oil
crops
like
cotton
seed,
rapeseed/mustard, sunflower and canola are used for extracting edible oil but
they only account for 29% of domestic requirements (GOP, 2008) and rest is
imported resulting in huge drainage of foreign exchange. During 2007-08,
Pakistan spent US$ 1217 and $ 92.1 millions on the import of palm oil (unsuitable
due to high melting point) and soybean oil from Malaysia and US, respectively.
Rice bran oil (RBO) production is feasible for the region, where bran can be made
available in abundance within stipulated period during rice milling. Rice bran oil
needs no extra land for cultivation. Moreover, its utilization in baked products
will not only explore its functional and nutraceutical role but also contribute
towards value addition in rice sector. Extraction of RBO through solvent and
73
utilization of stabilized rice bran and its defatted portion in cereal based food
products could also play an important role in minimizing current food crisis.
Moreover, RBO as a cooking medium and its meal for supplementation in wheat
flour reckon its prospects in lowering the blood glucose and cholesterol to
improve consumers health.
There are many challenges involved with the utilization of rice bran in
Pakistan. The main challenge is its utilization as feed ingredient due to low price.
Poultry feed mainly comprised of rice bran. This industry procures almost all
barn from rice industry for its utilization throughout the year. As Pakistan is the
6th most populous country of the world and it population is still increasing with
high rate. Now it is very difficult to feed this population. Utilization of rice bran
for human will results in some relief on grain crops because it can be efficiently
supplemented in baked products both in full fat form as well as after oil
extraction. Another challenge was the stability of rice bran during storage.
Although rice bran is considered an excellent source of edible oil but the main
problem is its inherent enzyme system especially lipases which results in
splitting of triglycerides into free fatty acid and make it unfit for human
consumption. Moreover extracted oil will be of poor quality and results in
economic loss during refining process. So in present study, one of the objectives
was to find out the most suitable stabilization technique. For the purpose, rice
bran samples were stabilized through different stabilization techniques like
extrusion, microwave heating and parboiling and stored for two months. On the
basis of lees FFA production, peroxide value and TBA no., microwave technique
was preferred. Microwave heating is considered to be one of the most energyefficient and rapid method for heating foods. In rice bran, dipolar water
molecules are excited by the electromagnetic waves and are made to spin. The
resultant enhanced kinetic energy, alongwith friction, produces heat that results
in the even distribution of heat having deleterious effects on lipase activity.
Moreover, microwave heat had little effect on nutritional quality and the
74
functional property of rice bran. MW heating is now used at household level so it

is feasible in terms of technical and commercial point of view.
The present project was planned to achieve the following objectives:
Utilization of rice industrial by-products for oil extraction and its quality
evaluation
The prospects of blending oil and bran for the preparation of value added
products i.e. cookies and leavened pan bread
Efficacy study; to study the effect of selected compositions on feed, water

intake and body weight of Sprague Dawley Rats
To determine the effects of selected compositions of RBO and

supplemented flours on serum bio-chemical profile with special reference
to lipid profile of Sprague Dawley Rats.
75
Chapter-II
REVIEW OF LITERATURE
Rice bran, a by-product of rice milling industry, is composed of pericarp,
aleurone and subaleurone layers, parts of the germ and small portion of the
starchy endosperm. It is rich in vitamins, minerals, amino acids, dietary fiber,
essential fatty acids and plant sterols like -oryzanol, tocopherol and tocotrienol
having promising health-related benefits. Besides its exceptional nutritional
profile, it is currently used as animal feed and fuel source. The literature
available pertaining to different aspects of the present study has been reviewed
under the following headings:
2.1 Rice bran: an overview
2.2 Processing of rice bran
2.3 Rice bran oil and its components
2.4 Hypocholesterolemic effects of rice bran and its oil
2.5. Supplementation in baked products
2.1. Rice Bran: An Overview

2.1.1. Physiology and general characteristics
Rough rice (paddy) is composed of a white starchy rice kernel tightly
covered by a coating of bran, enclosed in a tough siliceous hull (Lakkakula et al.,
2004). When husk is removed, bran layer comes in direct contact with air,
resulting in the development of off-flavor in brown rice due to its endogenous
lipase. Moreover, the appearance of brown rice is not appealing due to its color
(Saunders, 1990). Hence further processing of rice is required to remove the bran
from brown rice to produce white rice (Hu et al., 1996). It is consumed after
appropriate polishing to give a desired degree of whiteness (Juliano, 1985).
Rice bran constitutes about 10% of the weight of rough rice (Hu et al.,
1996). It is comprised of pericarp, aleurone, sub-aleurone, seed coat, nucellus
along with the germ and a small portion of endosperm (Salunkhe et al., 1992;
76
Hargrove, 1994). The percentage and composition of rice bran vary according to
the rice variety, pretreatment before milling, type of milling system and the
degree of milling (Saunders, 1990). Rice bran is light in color, sweet in taste,
moderately oily and has a slightly toasted nutty flavor (Hu et al., 1996). Texture
varies from a fine, powder-like consistency to a flake, depending on the
stabilization process (Barber and Benedito de Barber, 1980).
Rice bran contains 12-22% oil, 11-17% protein, 6-14% fiber, 10-15%
moisture and 8-17% ash. It is rich in vitamins including vitamin E, thiamin,
niacin and minerals like aluminum, calcium, chlorine, iron, magnesium,
manganese, phosphorus, potassium, sodium and zinc (Sunders, 1990; Hu et al.,
1996; Xu, 1998). It also contains a significant amount of nutraceutical compounds
and approximately 4% unsaponifiables, mainly comprised of naturally occurring
antioxidant such as tocopherols, tocotrienols and oryzanol (Ju and Vali, 2005).
Rice bran proteins are of high nutritional value (Kennedy and Burlingame,
2003) and hypoallergenic (Tsuji et al., 2001). These proteins are rich in essential
amino acids, especially lysine, hence can be used as ingredients in food recipes
(Wang et al., 1999). Stabilized rice bran is also a good source of both soluble and
insoluble dietary fiber ranging from 20-51% (Saunders, 1990), which is almost twice
as much as that of oat bran. Rice bran can be used as a stool bulking agent
(Tomlin and Read, 1988) and for the enrichment of some foods (Burton, 2000).
2.1.2. Anti-nutritional factors
The effective utilization of rice bran is possible only by deactivating the
lipase enzyme, responsible for the hydrolytic degradation of bran constituents
and denaturation of anti-nutritional factors. Successful developments in the use
of various techniques to stabilize rice bran have resulted in the emergence of rice
bran as an important by-product of the rice milling industry. The following antinutritional factors exist in rice bran:
2.1.2.1. Lipases
77
Lipases (E.C. 3.1.1.3) are enzymes that are primarily responsible for the
hydrolysis of triglycerides into glycerol and fatty acids. Rice bran contains
several types of lipases which results in significant increase of the free fatty acids
(FFA) by hydrolyzing the oil. Rapid increase in the free fatty acid occurs within
hours and reaches 7-8% within 24 hours, followed by about 5% increase per day
(Ramezanzadeh et al., 1999; Rukmani, 2002).
Lipase activity is greatly affected by moisture, temperature, pH, time and
water activity (Dunford and King, 2001; Gangodavilage, 2002). The enzyme was
active up to 40C and the activity declined sharply to 65% at 60C and then
gradually decreased (Bhardwaj et al., 2001). In addition to native lipases, the
microbial lipases also deteriorate the nutritional quality of the oil, making it unfit
for human consumption. The hydrolytic rancidity severely affects the nutritive
value and palatability of rice bran (Rajeshwara and Prakash, 1995).
2.1.2.2. Trypsin inhibitors
Trypsin inhibitors are also endogenous enzymes, which can form stable
complex with proteolytic pancreatic enzymes i.e. trypsin and chemotropysin.
Due to complex formation, the activity of these enzymes decreases. Rice bran
contains trypsin inhibitor (Kratzer and Payne, 1977; Deolankar and Singh, 1979).
Approximately 85-95% trypsin inhibitor activity was found in rice embryo. One
mole of rice bran trypsin inhibitor can inhibit two moles of trypsin.
2.1.2.3. Haemagglutinin-lectin
Haemagglutinin-lectins are toxic globulin proteins present in the rice bran
and agglutinate mammalian red blood cells (Ory et al., 1981). Similarly, lectin is a
glycoprotein and is present in germ portion. It comprised of 27% carbohydrate,
predominantly glucose (Takahashi et al., 1973) while another 10% carbohydrate is
mainly in the form of xylose and arabinose (Indravathamma and Seshadri, 1980).
The lectin also contains a large number of glycine and cystine residues (Tsuda,
1979).
2.1.2.4. Phytates
78
Phytates (1,2,3,4,5,6-hexaphosphate of myoinositol) occur in discrete

regions of cereal grains and accounts for 85% of the total phosphorous content of
grains. They reduce the bio-availability and digestibility of nutrients by forming
complexes with minerals, protein, digestive enzymes and amino acids mainly
lysine, methionine, arginine and histidine (Jangbloed et al., 1991; Bird, 1998). It is
a rich source of minerals particularly phosphorous, zinc and ferrous (Farrell,
1994). Phytic acid showed strong chelating properties due to its structure
(Ramzan, 2000). Phytates also affect the solubility, functionality and digestibility
of proteins and carbohydrates.
2.1.3. Dietary fiber
Dietary fiber is the edible parts of plants or analogous carbohydrates
that are resistant to digestion and absorption in the human small intestine
with partial fermentation in the large intestine (CAC, 1998). These are plant
food materials that are not hydrolyzed by enzymes secreted by the human
digestive tract but may be digested by micro flora in the gut. These plant food
materials include non-starch polysaccharides such as celluloses, some hemicelluloses, gums and pectins as well as resistant starches (DeVries, 2001).
The components of dietary fiber include cellulose, hemicellulose, pectins,
hydrocolloids and lignin. These can be classified into two major categories
depending on their solubility in water. In humans, the structural or matrix fibers
(lignins, cellulose, and some hemicelluloses) are insoluble, whereas the natural
gel-forming fibers (pectins, gums, mucilages, and the remainder of the
hemicelluloses) are soluble. Soluble fiber acts like a gel and insoluble fiber adds
bulk or softens stool. Soluble fiber forms a gelatin like substance in the intestine
and increases the water content in stool. It has been shown that soluble fiber
decreases blood cholesterol and sugar after meals in diabetics (Yeager, 1998).
Similarly, insoluble fiber is effective in increasing feeling of fullness, stool size,
bulk and helps to reduce constipation and hemorrhoids. Good sources of soluble
79
fibers include fruits, vegetables, legumes, psyllium seeds and oat bran whereas
whole grains are good sources of insoluble fiber (Matz, 1991).
Fiber supplementation has been used to enhance the fiber content of array
of foods. Traditionally, fiber supplementation has focused on the use of milling
by-products of cereal grains like wheat, corn, sorghum and other grains (McKee
and Latner, 2000). Nowadays, fiber supplementation has focused in cookies,
crackers, snack foods, beverages, spices, imitation cheeses, sauces, frozen foods,
canned meats, meat analogues and many other cereal-based products (Hesser,
1994). The WHO recommendation for total dietary fiber intake is above 25 g/day
(WHO, 2003).
The total dietary fiber content in stabilized rice bran ranges from 25 to 40%
depending on the product (Carroll, 1990). Rice brans fiber comprised of a
relatively low proportion of soluble fiber (713%) and the rest is insoluble fiber
(Anderson et al., 1990). However, rice bran has high percentage of oil (1223%) as
compared to other bran sources, with 4.2% unsaponifiable matter (Sugano and
Tsuji, 1997). Rice bran oil, possibly because of unsaponifiable fraction or its fatty
acid content, lowers cholesterol levels in hamsters, rats, humans and nonhuman
primates (Sharma and Rukmini, 1986; Seetharamaiah and Chandrasekhara, 1989;
Nicolosi et al., 1991; Kahlon et al., 1992; Purushothama et al., 1995).
2.2. Processing of Rice Bran

The processing of rice bran was carried out to inactivate lipases as well as
other nutritional inhibitors in such a way that their toxicity is ruined without
damaging the protein quality of rice bran. Furthermore, it also destroys the field
fungi, bacteria and insects infestation, so that the bran becomes safe from further
deterioration which alternately enhanced its shelf life.
The greatest restriction to the use of rice bran as a food ingredient is its
instability during storage. Upon milling, the oil is exposed to lipases, causing
rapid breakdown to free fatty acids @ 57% of the weight of oil per day. Hence
due to the naturally occurring enzymatic activity and subsequent hydrolytic
80
rancidity, it is necessary to stabilize the rice bran by suitable techniques for

controlling undesirable reactions. Bran, after proper stabilization, can serve as a
good source of protein, essential unsaturated fatty acids, calories, and nutrients
such as tocopherols and ferulic acid derivatives.
The commonly used stabilization techniques are thermal and chemical
treatments (Randall et al., 1985; Kim et al., 1987). There are different types of heat
stabilization procedures such as retained moisture heating (Lin and Carter, 1973),
added moisture heating (Saunders, 1986), extrusion cooking (Sayre et al., 1982),
microwave heating (Malekian et al., 2000) and Ohmic or electrical heating
(Lakkakula et al., 2004).
Heat stabilization is accomplished commercially by wet or dry heating
methods i.e. hot air, drum drying, dry extrusion and microwave (Prabhakar,
1987; Narisullah and Krishnamurthy, 1989). Although hot air drying is an
effective method of stabilization, the non-uniform heating of material in the tray
driers limits its application. Rice bran was stabilized by fluidized bed drying at
90-130C (Fernando and Hewavitharana, 1993). Although fluidization provides
uniform heating of bran; however, high air velocities are required for the process;
making it uneconomical (Narisullah and Krishnamurthy, 1989). The stabilized
rice bran was obtained by drum drying at 156C (Delahaye et al., 2005).
Parboiling also results in stabilization of rice bran by destroying lipase activity
(Narisullah and Krishnamurthy, 1989). An edible acid (0.1-2.0% acetic acid)
having anti-oxidative properties was added to parboiled rice bran to maintain
the stability of the bran for at least 6 months at ambient conditions (Tao, 2001).
The common drawbacks in heat treatment methods

are: severe processing conditions capable of damaging
valuable components, substantial moisture removal and
inability to achieve irreversible inactivation of enzyme. To
81
cope with these problems, moist heat treatment is

suggested. Extrusion cooking has been found to produce
stable rice bran by holding at 125-130 C for few seconds,
then at 97-99 C for 3 min prior to cooling (Randall et al.,
1985). Heating in the presence of moisture is more effective
for permanently denaturing lipases (Ramezanzadeh et al.,
1999). Long-term storage studies with extrusion cooking
indicate stability against FFA development upto 4 months
(Carroll, 1990; Randall et al., 1985), in contrast to dry heat
methods. Hence steaming is suitable method of bran
pretreatment with respect to decrease in FFA development
and the oil extractability in small-scale (Amarasinghe and
Gangodavilage, 2004). However, less flexibility and higher
initial and operating costs make the process uneconomical.
Furthermore, moist heat results in agglomeration of bran,
resulting in lumpy bran.
To achieve proper stabilization, every discrete bran particle must have
uniform moisture content, depending upon time and temperature. In recent
years, use of microwave energy as an inexpensive source of heat for thermal
processing of foods has offered an alternative energy source for stabilization of
rice bran. It is considered to be one of the most energy-efficient types and a rapid
method for heating food items (Yoshida et al., 2003). Considering other heat
treatments, microwave heating is efficient, economical, shorter in processing
time, minor effect on the nutritional value and has a little or even no effect on the
82
natural color of bran. Microwave heating is an effective method for stabilizing

rice bran with the addition of moisture, which enables heating over 100C to
occur (Malekian et al., 2000). The water molecules in the rice bran are excited to
spin by the electromagnetic waves resulting enhanced kinetic energy along with
the friction. Since water molecules play an important role in this process, the
initial moisture content is a critical factor in the microwave stabilization. Rice
bran was stabilized by heating in a microwave for 4 minutes until the internal
temperature reached 110-115C to denature the enzymes (Zhu, 2000).
2.3. Rice Bran Oil and its Components

2.3.1. Current status
Rice bran oil (RBO) is traditionally consumed in Asian rice producing
countries with growing interest in Western markets (Jariwalla, 2001; Kim and
Godber, 2001; Nasirullah, 2001; Pszczola, 2001). It is in steady demand as
healthy oil in Japan where approximately 80 thousand tons is consumed
annually (Sugano and Tsuji, 1997). Traditionally, rice bran oil has been used for
frying food, due to its oxidative stability and flavor; it is now considered as a
good substitute for vegetable oils (Sayre and Saunders, 1985; Goenka, 1987). It is
widely used in pharmaceutical, food and allied industries due to its unique
properties, high medicinal value & therapeutical applications (Cicero and Gaddi,
2001; Amarasinghe and Gangodavilage, 2004).
RBO is an unconventional vegetable oil believed to be healthy in some
populations (Sugano et al., 1999) due to higher levels of antioxidants (Patel and
Walker, 2004) and phytosterols (Stoggl et al., 2005). It is superior to other
vegetable oils because it contains -3 and -6 fatty acids; particularly due to
oryzanol and higher amounts of unsaponifiables (Krishna et al., 2005). Currently,
efforts are being made to develop RBO with retained non-saponifiable
components, while minimizing levels of problematic free fatty acids (Ginsberg et
al., 1998).
2.3.2. General characteristics
83
Rice bran contains 15-22% oil by weight (Orthoefer, 1996; Patel and
Walker, 2004). Crude rice bran oil contains 90-96% of saponifiable and about 4%
unsaponifiable lipids. The saponifiable lipids include 68-71% triglycerides, 2-3%
diglycerides, 5-6% monoglycerides, 2-3% free fatty acids, 2-3% waxes, 5-7%
glycolipids and 3-4% phospholipids (McCaskill and Zhang, 1999) whereas the
principal component of the unsaponifiable fraction is -oryzanol (Raghuram and
Rukmini, 1995).
Rice bran oil has excellent fatty acid profile. It has oleic acid (38.4 %),
linoleic acid (34.4%) and linolenic acid (2.2%) as unsaturated fatty acids while
palmetic acid (21.5%) and stearic acid (2.9%) as saturated fatty acids (Rukmini
and Raghuram, 1991). The saturated, monounsaturated and polyunsaturated
fatty acids are in the ratio of approximately 1: 2.2: 1.5 (Shin and Chung, 1998;
Krishna, 2002). Three major fatty acids, palmitic, oleic and linoleic make up 90%
of the total fatty acids of the rice bran oil (Amarasinghe and Gangodavilage,
2004).
2.3.3. Effective components
2.3.3.1. Fatty acids
Dietary fat is a crucial factor in the regulation of cholesterol levels and
there is devastating evidence to support the hypocholesterolemic effects of
vegetable oils rich in polyunsaturated fatty acids, mainly linoleic acid (Grundy,
1994). Growing interest in health benefits of polyunsaturated fatty acids has
focused on providing suitable sources of these constituents. Polyunsaturated
fatty acids include linoleic acid (C18:2n6c), -linolenic acid (ALA, C18:3n3), linolenic acid (GLA, C18:3n6), arachidonic acid (AA, C20:4n6), eicosapentaenoic
acid (EPA, C20:5n3) and docosahexaenoic acid (DHA, C22:6n3).
Polyunsaturated fatty acids are required in the body for normal
functioning of nervous, immune & inflammatory, cardiovascular, endocrine,
respiratory and reproductive systems (Certik and Shimizu, 1999). Their presence
on membrane phospholipids can influence cellular activities. Fatty acids also
84
alter membrane fluidity and consequently modulating changes in conformation

or function of receptors, transporters and enzymes (Calder, 2003).
Edible oils rich in polyunsaturated fatty acids have been reported to result
in a decrease in total cholesterol, triglycerides, low density lipoprotein
cholesterol as well as the beneficial high density lipoprotein cholesterol (Schaefer
et al., 1981; Mattson and Grundy, 1985). In rice bran oil, the amount of linoleic
acid is moderate, and proportion of oleic acid is a relatively high. Studies have
indicated that RBO has significant hypocholesterolemic effect in both animals
and humans when compared to other oils, inspite of limited polyunsaturated
fatty acids (Rukmini, 1988; Raghuram et al., 1989). The effect has been attributed
to components like tocotrienols, oryzanol and monounsaturated fatty acid
(Mediterranean diet). The study with rats fed on RBO diet demonstrated a
significant reduction in total serum cholesterol, LDL-cholesterol and an increase
in fecal steroid excretion compared with that of peanut oil diet (Rukmini and
Raghuram, 1991). Different research findings proved that unsaponifiable
fractions in RBO could compensate for its high saturated fats and played a
predominant role in decreasing cholesterol levels (Nicolosi et al., 1991; Wilson et
al., 2000).
2.3.3.2. Unsaponifiable matter
Recently, rice bran oil has received attention because of its unique health
benefits (Nicolosi et al., 1994) attributed by its high level of unsaponifiable matter
(Shin et al., 1997). These are bioactive components with nutraceutical value and
cannot be saponified by caustic treatment (Sugano et al., 1999). The
unsaponifiables are mainly composed of sterols (42-43%), triterpene alcohols (2428%) and less polar components such as squalene or tocotrienols (19%)
depending on the type of rice bran and method used to extract and refine the
lipids (Sugano and Tsuji, 1997; Lloyd et al., 2000; Dunford and King, 2001).
Crude rice bran oil contains an unusually high content of unsaponifiables
(3-5%) several times greater than most commonly used vegetable oils where as
85
refined oil may contain 0.3-0.9%; because most part is removed during refining
(Rong et al., 1997). The content of the unsaponifiable material in refined RBO is
regulated to be 0.5% under the Japan Agricultural Standard; this value is
considerably higher than that of other vegetable oils (Sugano and Tsuji, 1997).
The unsaponifiable fraction in RBO also contains a unique complex of naturally
occurring antioxidant, of which the tocopherols, tocotrienols and oryzanol have
received much attention (Sayre et al., 1988). The amount of -tocopherol is
relatively large (0.1% of the total oil) in rice bran oil compared with other
vegetable oils (Nicolosi et al., 1994).
There are several mechanisms by which unsaponifiables improve serum
bio-chemical profile such as by interrupting the absorption of intestinal
cholesterol rather increasing the excretion of fat and neutral sterols (Kahlon et al.,
1996; Nagao et al., 2001) and increased fecal steroid excretion through
interference with cholesterol absorption (Ikeda et al., 1985; Sharma and
Rukumini, 1986).
2.3.3.3. Antioxidants
Any substance that delays or inhibits the oxidation of substrate, inspite of
low concentrations, is called antioxidant. The physiological role of antioxidants is
to prevent damage to cellular components arising as a result of chemical
reactions involving free radicals (Halliwell and Gutteridge, 1995).
Several important nutraceutical compounds can be extracted from rice
bran which contains high levels of phytochemicals having antioxidant activities
(Chen and Bergman, 2005). These phytochemicals include vitamin E comprised
of four homologs (, , and ) of tocopherol & tocotrienols (Jariwalla, 2001;
Birringer et al., 2002) and the -oryzanol (Akihisa et al., 2000; Jariwalla, 2001).
Vitamin E is considered to be the major chain-breaking antioxidant especially in
biological membranes (Ricciarelli et al., 2001).
Rice bran is a rich natural source of vitamin E and -oryzanol (Shanggong
et al., 2007). It contains over 300 mg/kg vitamin E (Shin et al., 1997). Vitamin E is
86
pale-yellow and viscous oil (Hu et al., 1996). It protects cell membrane by
blocking the oxidation of the unsaturated fatty acids and acting as a scavenger of
free radicals (Komiyama et al., 1992; Nesaretnam et al., 1998). In addition to
health benefits, antioxidants of rice bran and its oil have a potential use as
additives to improve the storage stability and frying quality of foods (Lloyd et al.,
2000; Nanua et al., 2000; Kim and Godber, 2001). RBO is rich in -oryzanol having
antioxidant properties; their structure includes ferulic acid, a strong antioxidant
(Miller and Rice, 1997; Sierra et al., 2005).
2.3.3.3.1. Oryzanol
Oryzanol is a mixture of ferulate (4-hydroxo-3-methoxycinnamic acid)
esters of sterols (campesterol, stigmasterol and -stigmasterol) and triterpene
alcohols (cycloartenol, 24-methylenecycloartanol, cyclobranol); about 9.8gkg-1 is
found in rice bran (Xu and Godber, 2000; Fang et al., 2003; Miller et al., 2003). It
was first isolated from rice bran oil by Kaneko and Tsuchiya in 1954 (Kaneko and
Tsuchiya, 1954) and named because it was first discovered in rice bran oil (Oryza
Sativa L.). The most accessible natural source of -oryzanol is rice (Seitz, 1989). oryzanol is a white or slightly yellowish, tasteless crystalline powder with little
or no odor and has a melting point of 137.5-138.5oC (Xu and Godber, 2000). It is
insoluble in water, slightly soluble in diethyl ether and n-heptane and practically
soluble in chloroform (Bucci et al., 2003). Initially, it was reported as a single
component in rice bran (Kaneko and Tsuchiya, 1954.) but now it is known a
mixture of at least 10 components (Xu and Godbar, 1999; Kim et al., 2001).
The concentration of -oryzanol in rice bran oil ranges from 115 to
780ppm, depending on the degree and method of processing (Rogers et al., 1993).
-oryzanol is 13-20 times (w/w) in rice bran than total tocopherols and
tocotrienols (Bergman and Xu, 2003). It has been observed that about 20% of
unsaponifiable fraction in RBO is oryzanol (Rong et al., 1997). Different extraction
methods can result in different levels of these components because some
87
tocotrienols and tocotrienol-like compounds are bound to cellular components in

the rice bran (Shanggong et al., 2007).
Complete role of -oryzanol as functional ingredient, has not so far
thoroughly been observed whereas health claims like antioxidant activity (Xu
and Godber, 2001), reduction of serum cholesterol (Akihisa et al., 2000; Xu et al.,
2001), reduction of cholesterol absorption (Lloyd et al., 2000), increase of HDL
cholesterol (Cicero and Gaddi, 2001), inhibition on platelet aggregation
(Seetharamaiah et al., 1990), inhibition of tumor promotion (Yasukawa et al.,
1998), and menopausal syndrome treatment (Rogers et al., 1993)
have been
investigated. -oryzanol reduces serum cholesterol in rats and hyperlipidemic

humans (Seetharamaiah and Chandrasekhara, 1989; Yoshino et al., 1989). It has
been proven that -oryzanol has higher antioxidant activity as compared to
tocols; might be due to the similarity between the structure of -oryzanol and
cholesterol (Xu et al., 2001; Godber et al., 1994).
2.3.3.3.2. Tocols (tocopherols and tocotrienols)
Vitamin E consists of tocopherols and tocotrienols collectively known as
tocols. Humans and animals cannot synthesize this vitamin; they primarily
acquire tocols from plants. Tocopherols and tocotrienols differ in number and
positions of methyl groups on the fused chromonol ring, and the absence and
presence of three double bonds in the isoprenoid side chain. The structural
differences of tocotrienols and tocopherols influence their biological activities
(Qureshi et al., 1996).
Tocotrienol has 3 double bonds within the main body of the molecule at
the 3,7 and 11 positions of the hydrocarbon tail. Just like edible oils with high
level of polyunsaturated fatty acids, the presence of these double bonds give
greater fluidity to tocotrienols and make it much easier for the body to
incorporate them into cell membranes (Yap et al., 2001). The major forms of
tocotrienol are -tocotrienol (5,7,8-trimethyltocotrienols), -tocotrienol (7,8-
88
dimethyltocotrienol) and -tocotrienol (8-methyltocotrienol) (Xu and Godber,

1999).
Evans discovered tocopherols in 1922 (Evans and Bishop, 1922). Major
forms of tocopherols in rice bran oil are -tocopherol (5,7,8-trimethyltocol), tocopherol (7,8-dimethyltocol) and -tocopherol (8-methyltocol) (Xu and Godber,
1999). RBO also contains high concentrations of the tocopherols compared with
other oil seeds (Kao and Luh, 1991). Approximately 1.0% (v/v) of the
unsaponifiable fraction of RBO is -tocopherol. HPLC analysis of RBO showed
that 1g of RBO contains 3.02mg of -tocopherol (Qureshi et al., 2000).
Tocotrienols are present in vegetable oils like palm oil and rice bran oil
(Stephens et al., 1996; Qureshi et al., 2000). Barley, oats, palm, and rice brans
contain more than 70% tocotrienols known as tocotrienols/tocotrienol rich
fraction (Raghuram and Rukmini, 1995). Two novel tocotrienols d-P21-T3
(desmethyl tocotrienol) and d-P25-T3 (didesmethyl tocotrienol) have been
identified and isolated from stabilized rice bran (Qureshi and Qureshi, 1993;
Qureshi et al., 2000). RBO is a rich source of tocotrienols ranged from 72-1157ppm
depending upon different bran sources and commercial refining methods.
Approximately 1.7% (v/v) of the unsaponifiable fraction of RBO is tocotrienol
(Deckere and Korver, 1996). HPLC analysis of RBO showed that 1g of RBO
contains 0.5mg of -tocotrienol (Qureshi et al., 2000). It has been observed that
human consumption of 240mg/day of tocotrienols upto two years caused no
adverse effects and they are safe at even much higher levels. The content and
biological activities of tocotrienol are higher than those of tocopherols (Qureshi et
al., 2001).
2.3.3.4. Effect of processing on antioxidants
After stabilization, crude oil is extracted and refined before human
consumption. There was a gradual decrease in sterol content in each step of
refining. It has been reported that 10-70% of the total sterols were lost depending
on processing conditions (Kochhar, 1983). The effect of bleaching, deodorization
89
and hydrogenation on -oryzanol content in refined oil has not yet been cleared;
it is considered that -oryzanol is mainly lost after neutralization. Similarly,
tocotrienols are also lost during each step of refining. In some cases, upto 90%
losses have been reported, which demands advance refining techniques.
2.3.4. Utilization
Rice bran oil with higher thermal and oxidative stability than sunflower
oil can be used for deep fat frying (Krishna et al., 2005). Blending of other oils
with rice bran oil has also been found to improve the stability of the blend during
frying and storage. The high oxidative stability of RBO makes it preferred oil for
frying and baking applications (McCaskill and Zhang, 1999; Semwal and Arya,
2001). The stabilized oil may be useful as spray oil for crackers, nuts, chips and
other snack foods. It extends the shelf-life of snack foods due to high levels of
phytosterols which may impart resistance to thermal oxidation and storage
deterioration (Taylor et al., 1996).
2.3.5. Economic Feasibility
Among the food grains, the production of paddy is the highest next to
wheat. In Asian countries, rice is the principal cereal produced and consumed by
the population. In 2001, the world production of paddy was 597.3 MMTs (FAO,
2001). The worldwide estimated potential of rice bran is 29.87 MMTs. However,
the commercial production of RBO in 2000 was estimated to be about 783
thousand tons, extracted with hexane (Perretti et al., 2003). The total potential of
rice bran oil production in the world worked out to be 4.48 MMTs. The Asian
countries alone contribute about 98.4% (4.41 MMTs) of rice bran oil.
In 2007-08, rice was cultivated on an area of 2515 thousand hectares and
yield was 5563 thousand tons. The estimated production of rice bran oil can be
worked out to be 81577-108760 thousand tons. As for as Pakistan is concerned,
out of the non-conventional oil sources, rice bran oil is the most important in
terms of its potential to augment the availability of oils. Full realization of the
90
potential will help in reducing the gap between demand and availability of
indigenous edible oils in the country to a significant extent (GOP, 2008).
2.4. Hypocholesterolemic Effects of Rice Bran and Rice Bran Oil

2.4.1. Rice Bran
Scientific studies support recommendations to increase dietary fiber as
part of hyperlipidemia treatment. The hypocholesterolemic effects of rice bran
have been demonstrated in experimental animals (Sharma and Rukmini 1987;
Seetharamaiah and Chandrashekhara 1989; Kahlon et al., 1992) and humans
(Hundemer et al., 1991; Sanders and Reddy, 1992; Hakala et al., 2002).
The cholesterol lowering effects of rice bran (fullfat), soybean fiber, oat
and barley bran were compared in mice adding 0.06% cholesterol in their diets.
Both rice bran and soybean fiber diet had significantly lower total blood
cholesterol compared with placebo. Rice bran was found to be the most effective
supplement in reducing liver and plasma total cholesterol compared to the
control diet. Moreover, mice consuming rice bran diet, demonstrated higher
HDL to total cholesterol ratios (Hundemer et al., 1991). In another study, rats fed
on rice and wheat bran showed significant reduction in liver cholesterol and
triglycerides. The rice bran diet also increased LDL receptor activity in the liver
more than the wheat bran, hence, effectively lowering plasma cholesterol levels
(Topping et al., 1990).
The cholesterol lowering effects of fullfat and defatted stabilized rice bran,
parboiled rice bran and rice bran in combination with wheat bran were studied
in hamsters fed on fiber diets with 0.5% added cholesterol. The liver cholesterol
concentrations, in particular, were significantly lower in animals consuming
fullfat stabilized rice bran than all other groups (Kahlon et al., 1990).
Rice bran with extremely low -glucan content is known to be as effective
as high-glucan oat and barley bran in lowering serum cholesterol
(Seetharamaiah and Chandrasekhara, 1989; Kahlon et al., 1990; 1992). The
hypocholesterolemic effects of rice bran may be attributed to the unsaponifiable
91
fraction of rice bran oil, primarily phytosterol, tocols (tocopherols and

tocotrienols), -oryzanol, triterpene alcohol and other minor compounds
(Sharma and Rukmini, 1987; Yoshino et al., 1989; Nicolosi et al., 1991). In a similar
study, benefits of bran addition from rice, oats, corn and wheat in diets fed to
hamsters were evaluated at relatively high cholesterol level (0.3%). Liver
cholesterol concentrations and liver weights were significantly lower for the rice
bran diet than for either the corn or wheat bran diets. Animals fed on rice bran
had significantly lower VLDL levels and the highest HDL to total cholesterol
ratios when compared to all other bran due to greater lipid and sterol excretion
(Kahlon et al., 1998).
Chicks were fed on 60% fullfat rice bran and corn/soy diets with 0.5%
added cholesterol. Significant differences were found in total cholesterol,
triglycerides, high-density and low-density lipoprotein cholesterol. Likewise, in a
second study, chicks were fed on fullfat rice bran, defatted rice bran and
corn/soy diets balanced for 18% protein, 14.47% total dietary fiber and 10.78%
lipid with 0.5% added cholesterol. Total cholesterol and triglycerides were
significantly lower in chicks fed on fullfat rice bran diets. Significant differences
were found in HDL values for all diets with fullfat rice bran exhibiting the
highest (155 mg/dL) and corn/soy exhibiting the lowest mean value (114 mg/dL).
Fullfat rice bran appeared to increase HDL and lower LDL in chicks, but did not
always affect TC (Newman et al., 1992). It has already been concluded that rice
bran might lower cholesterol by increasing short chain fatty acid production in
the cecum by hindering cholesterol absorption due to a change in intestinal fluid
viscosity or by directly inhibiting cholesterol synthesis in the liver (Fukushima et
al., 1999).
Rice bran supplementation has been found effective in lowering total
cholesterol
and
LDL
levels
in
human
subjects
with
moderate
hypercholesterolemia (Hundemer et al., 1991). However, serum cholesterol was

found to be decreased in patients with mild hypercholesterolemia who
92
consumed 300g/d unpolished rice or 100g/d stabilized rice bran (Hakala et al.,
2002). Fullfat rice bran was found to be more effective in lowering cholesterol
than isolated rice bran fractions or their combinations (Kahlon et al., 1992).
Rice bran has been found to be equivalent to oat bran in lowering
cholesterol. Mildly hypercholesterolemic subjects were fed on treatment diets
(100g/day stabilized rice bran or oat bran) with 300mg/day added cholesterol.
Total cholesterol levels were significantly reduced in both bran diets when
compared to the control (Hegsted et al., 1993). Similarly, changes in plasma lipid
levels were studied in men with slightly above the normal cholesterol levels
providing test diets containing 35g/day of wheat bran, 60g/day of rice bran or
95g/day of oat bran. The varying amounts of the different brans provided a
constant amount of total dietary fiber i.e. 11.8g/day. At baseline level, only oat
bran was effective in reducing plasma cholesterol as compared to other
treatments. However, the highest rise in HDL was associated with the rice bran
diet, resulting in an improved HDL to total cholesterol ratio. Moreover, plasma
triglycerides were also lower in case of rice bran compared to wheat bran diet
(Kestin et al., 1990).
2.4.2. Rice Bran Oil
A number of studies in humans and animals have proved that RBO is
effective in lowering plasma cholesterol levels (Rukmini and Raghuram, 1991;
Lichtenstein et al., 1994). In some cases, RBO lowered plasma cholesterol more
effectively than other vegetable oils rich in linoleic acid (Rukmini and Raghuram,
1991); might be due to occurrence of specific components in RBO, -oryzanol and
perhaps tocotrienols (Nicolosi et al., 1994).
Rice bran oil and its components significantly improve the plasma profile
in rats. Rats were fed on diets containing 10% rice bran oil alongwith an equal
amount of groundnut oil as control diet for 8 weeks. Half of the animals in each
group also had 0.1% cholesterol and 0.05% cholic acid added in place of a portion
of starch. Rats fed on 10% RBO showed significantly lower serum cholesterol,
93
LDL-C and VLDL-C plasma levels, both on cholesterol-containing and

cholesterol-free diets. HDL-C was increased, while TG showed significant
decrease (Sharma and Rukmini, 1986). Rice bran oil was found to be superior to
groundnut oil in cholesterol improvement. Significant reduction in serum total
cholesterol and elevated tendency in HDL-cholesterol were found in rats
consuming cholesterol-free diets with 10% rice bran oil as compared to
groundnut oil diets (Seetharamaiah and Chandrasekhara, 1989).
Similarly, in another study, rats were fed experimental diet having 5 and
20% rice bran oil while control group was fed on diets containing same level of
peanut oil (PNO). There was no significant difference with respect to the organ
weights between control and experimental groups. In general, group fed on 20%
oil gained more weight than groups fed on 5% oil. The animals having rice bran
oil in their diet showed comparatively lower levels of cholesterol, triglycerides
and phospholipids. On the other hand, animals receiving 20% rice bran oil in
their diet, showed an increase of 20% in HDL-C, within 18 weeks, when
compared to the animals fed with peanut oil. Moreover, LDL-C and VLDL-C
were also found to be lower in rice bran oil fed groups (Purushothama et al.,
1995).
In animal modeling, rice bran oil was blended with safflower and
sunflower in different ratios and fed to rats fed for a period of 28 days. Rice bran
oil plus safflower oil and sunflower oil in 70:30 ratios showed, significantly,
lower levels of TC, TG and LDL-C and increased HDL-C in animals fed on high
cholesterol diet and cholesterol free diet. Faecal excretion of neutral sterols and
bile acids was increased with the use of rice bran oil blends. The high linoleic
acid content of safflower oil, in combination with the micronutrients of the RBO
unsaponifiable fraction, acts synergistically to lower the serum cholesterol level.
Moreover, high content of tocopherols and tocotrienols in rice bran oil may
improve the oxidative stability of the blends (Sunitha et al., 1997).
94
The cholesterol lowering effects of rice bran oil and safflower oil were
compared, alone and in combinations, both in cholesterol and cholesterol free
diets. Half of the animals were randomly assigned to cholesterol-free diets in
which one of these two oils, alone or in varying combinations, contributed 10%.
The other half were assigned to similar diets with additional 0.5% cholesterol.
Among the animals not fed on dietary cholesterol, total cholesterol levels were
similar between the rice bran oil and safflower oil groups, but HDL levels were
significantly higher in the rice bran oil group, resulting in a higher HDL:total
cholesterol ratio for this test group, although the differences were nonsignificant. Rice bran oil and safflower oil in 7:3 ratios in the diet appeared
optimal with respect to cholesterol levels. The average HDL:total cholesterol
ratio was significantly higher for this group than all other groups (Koba et al.,
2000).
Despite variations in fatty acid profile, rice bran oil has resulted in
reductions in total cholesterol and LDL levels in animals consuming diets
containing rice bran oil. Likewise, LDL levels dropped and HDL levels remained
unchanged or increased in rats fed on diets supplemented with the
unsaponifiable matter from rice bran oil (Sharma and Rukmini, 1987).
Unsaponifiables prepared from rice bran oil were evaluated in
exogenously hypercholesterolemic rats. Animals were maintained for two weeks
on 0.5% cholesterol diet with 10% fat content either rice bran oil, mixture of palm
& safflower oils or palm & safflower oils plus 0.25% of unsaponifiable content
prepared from rice bran oil. Serum and liver total cholesterol concentrations
were significantly lower and HDL levels significantly higher in both groups of
rats consuming the unsaponifiables versus oil without added unsaponifiables.
Higher fecal excretion of cholesterol was noted in the two unsaponifiable groups
as well. It was concluded that the unsaponifiable fraction of rice bran oil acts to
lower cholesterol by interrupting cholesterol absorption in the gut, not by
altering hepatic cholesterol metabolism (Nagao et al., 2001).
95
In a comparative fatty acid study, monkeys were fed on, in random order,
on control diet and three experimental diets having 20% energy content from rice
bran oil, canola oil or corn oil. HDL levels were maintained on the rice bran oil
diet while rest of the diets showed negative effect. The results suggested that
unsaponifiable fraction is critical for oils ability to decrease risk of
cardiovascular disease (Wilson et al., 2000)
Addition of oryzanol to rat diets containing rice bran oil was associated
with lower cholesterol levels compared to rat diets containing rice bran oil alone
(Seetharamaiah
and
Chandrasekhara,
1989).
Oryzanol
administered
to
hyperlipidemic persons showed inverse correlation with that of cholesterol levels

(Yoshino et al., 1989). Oryzanol, extracted from rice bran, was added in
crystalline form to test diets in varying amounts to find out the optimal dosage.
Rats were randomly assigned to diets enriched with 1% cholesterol and 0.15%
bile salts and either devoid of oryzanol or oryzanol enriched @ 0.2, 0.5, 1.0 and
2.0%. A control group was fed on cholesterol free diet. Oryzanol fed animals had
lower cholesterol levels compared to all other groups. It could be helpful to
prevent reduction in HDL level. A significant decrease in plasma triglyceride
levels was seen in the 0.5% oryzanol group. It was concluded that 0.5% oryzanol
was an optimal dosage to lower liver cholesterol, triglycerides, and
phospholipids significantly than rats fed with oryzanol free diet. In a similar
research, 0.5% oryzanol was supplemented with 1% cholesterol in contrast to
10% refined rice bran oil containing traces of oryzanol. The oryzanol
supplemented diet showed lower total cholesterol levels. However, improved
cholesterol status was also observed in rats fed on diet with refined rice bran oil
compared to those maintained on a control diet containing 10% groundnut oil
(Seetharamaiah and Chandrasekhara, 1989).
Intravenous administration of -oryzanol and cycloartenol ferulic acid
ester (10mg/kg) for 6 days significantly inhibited the increases in serum TC and
free cholesterol induced by a high cholesterol diet in male Sprague-Dawley rats.
96
After 12 days, both substances were able to significantly decrease TG as

compared to control animals. It was concluded that the intravenous administered
-oryzanol and cycloartenol ferulic acid ester could accelerate the excretion of
lipids from the blood (Sakamoto et al., 1987).
Besides, rice bran oil and -oryzanol supplementation in diets, bioactive
components from rice bran oil (BRBO), have been shown to play a protective role
against the alteration caused by hypercholesterolemic diet. Male SpragueDawley rats were fed for 4 weeks with normal diet, high-cholesterol diet and
high-cholesterol diet supplemented with the concentrated bioactive components
from rice bran oil (BRBO). The high-cholesterol diet increased serum cholesterol
in rats, compared with those fed on the normal diet. Serum high-density
lipoprotein cholesterol was significantly increased in rats of the BRBO group. In
addition, BRBO recovered the activities of serum aspartate amino transferase
which was elevated in rats by a high cholesterol diet. It was found that BRBO has
significant practical value for protecting against the alterations caused by a
hypercholesterolemic diet, and antioxidative ingredients which suppress lipid
peroxidation (Haa et al., 2005).
The hypolipidemic response of rice bran oil was investigated in nonhuman primates fed on semi-purified diets containing blends of oils including
rice bran oil at 0-35% Kcals as dietary fat. The study demonstrated that the
degree of reduction of serum total cholesterol (TC) and low density lipoprotein
cholesterol (LDL-C) was highly correlated with initial serum cholesterol levels in
monkeys fed on standard diet. Further, rice bran oil supplementation in the diet,
significantly, influenced serum TC and LDL-C, causing upto 40% reduction in
LDL-C without affecting HDL-C levels when rice bran oil was the sole dietary oil
(Nicolosi et al.,1991).
Similar to animal studies, a range of human studies have shown that rice
bran oil (RBO) is an edible oil of preference for improving serum cholesterol
levels and lipoprotein profiles. The first scientific statement about rice bran oils
97
anti-hyperlipidemic property in humans was published in 1970. RBO blended

with corn, safflower and sunflower oil was consumed by healthy young women
for 7 days to evaluate the effect of blending different vegetable oils on serum
cholesterol levels. It was observed that the hypocholesterolemic effect of RBO
was comparable to other vegetable oils such as corn, safflower and sunflower oils
(Suzuki
et
al.,
1970a,b).
Furthermore,
the
blended
oil
still
exerted
hypocholesterolemic effects, even when five eggs were consumed daily for 7
consecutive days. In contrast, there was an increase in HDL-cholesterol after
consumption of the blended oil, and consequently, the atherogenic index was
significantly improved (Tsuji et al., 1989).
The hypocholesterolemic effects of rice bran oil were evaluated in
moderately non-obese hyperlipoproteinemic human subjects fed on rice bran oil
for a longer period. For comparison, the control group continued use of palm or
groundnut oils. The rice bran oil treated patients showed a 16-25% decrease in
plasma total cholesterol and 32-35% in triglycerides after 15-30 days of treatment
as compared to the control group (Raghuram et al., 1989).
The diets of healthy volunteers with normal cholesterol levels were
supplemented with a margarine enriched with rice bran oil sterols to assess the
impact of sterols present in the unsaponifiable fraction of rice bran oil on lipid
profile. The subjects were instructed to continue usual dietary and physical
activities while supplementing their diets with control margarine containing
traces of sterols or one enriched with 2.1g/day of the sterols from rice bran oil for
three weeks each. The enriched margarine, significantly, lowered total and LDL
cholesterol compared to control (Vissers et al., 2000).
Hyperlipidemic subjects were administered -oryzanol (300mg/day) for
three months. A significant decrease in plasma TC and LDL-C was observed in
both hypercholesterolemic and hypertriglyceridemic patients, while a relevant
increase in HDL-C was noted only in the hypercholesterolemic group without
any side effect (Yoshino et al., 1989).
98
It seems that rice bran oil and its components are able to safely improve
the plasma lipid pattern of hypercholesterolemic patients. The available data in
humans suggest that rice bran oil (RBO) is an edible oil of preference for
improving plasma lipid and lipoprotein profiles (Sugano and Tsuji, 1997).
2.4.3. Cholesterol-lowering mechanisms
The mechanism of action of rice bran and its oil on lipid metabolism is not
yet evident. However, the most probable hypothesis of RBO hypolipidemic
action is its specific content of phytosterols, polyphenols (-oryzanol) and tocols
(tocopherols and tocotrienols). The cholesterol-lowering effects of RBO are
possibly attributable to its relatively high unsaponifiables; physiologically
bioactive in controlling cholesterol levels in subjects. These compounds have
been found to work synergistically to exhibit hypocholesterolemic effects.
2.4.3.1. Phytosterols (campesterol, -sitosterol and stigmasterol)
The phytosterols present in crude rice bran oil like campesterol, sitosterol and stigmasterol, have been proven effective in lowering plasma total
and LDL-cholesterol without affecting HDL-cholesterol due to similarities in
structures of plant sterols and cholesterol (Weststrate and Meijer, 1998).
There are several mechanisms through which plant sterols affect
cholesterol concentration in the body like formation of non-absorbable complex
with cholesterol, altering the size and/or stability of the micelles, interferences
with cholesterol esterification in the mucosal cell and interacting with protein
receptors required in cholesterol absorption (Rong et al., 1997). It is generally
assumed that plant sterols inhibit intestinal absorption of dietary and biliary
cholesterol, because of the structural similarities with cholesterol. Some studies
indicated that plant sterols contributed more hypocholesterolemic effects than
unsaponifiables. In addition, some plant sterols may be more active than others
(Wilson et al., 2000). Among the sterols, -sitosterol has been recognized the
99
predominant cholesterol-lowering component (Vissers et al., 2000; Trautwein et

al., 2002).
2.4.3.2. Polyphenols (oryzanol)
There are numerous mechanisms by which oryzanol lowers cholesterol
levels such as: (i) cholesterol-esterase inhibition by cycloartenol or by the
inhibition of the accumulation of cholesterol-esters within macrophages or by the
modulation of cholesterol acid esterase and acyl-CoA-cholesterol-acyltransferase
(Rukmini and Raghuram, 1991); (ii) sterol moiety of -oryzanol is partly split off
from the ferulic acid part in the small intestine by cholesterol esterase (Sugano
and Tsuji, 1997); (iii) effect on biliary secretion resulting in increased faecal
excretion of cholesterol and bile acids (Seetharamaiah et al., 1990); (iv) direct
inhibition of lipid metabolism (Sakamoto et al., 1987); (v) increased fecal
excretion of cholesterol and its metabolites (Wilson et al., 2007) and (vi) oryzanol
exercises its effects on cholesterol metabolism at sites other than the intestine.
2.4.3.3. Tocols (tocopherol and tocotrienol)
In case of tocols, cholesterol lowering mechanisms include: (i) antioxidant
activity that inhibits cholesterol oxidation (Xu et al., 2001); (ii) inhibit HMG-CoAR, a key enzyme in the endogenous synthesis of cholesterol, via increasing the
controlled degradation of reductase protein and decreasing the efficiency of the
translation of HMG-CoA-R messenger RNA (Parker et al., 1993; Khor et al., 1995);
(iii) inhibit the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA)
reductase, the liver enzyme that is critical to the rate at which cholesterol is
synthesized (Khor and Ng, 2000); (iv) inhibit cholesterol synthesis by
suppressing HMG-CoA reductase activity through a posttranscriptional
mechanism in HepG2 cells (Pearce et al., 1992; Parker et al., 1993; Qureshi et al.,
2000) and (v) via decrease in serum total and LDL cholesterol by inhibiting the
hepatic enzymic activity of -hydroxy--methylglutaryl coenzyme A (Qureshi et
al., 2002).
2.5. Supplementation in Baked Products

100
Nowadays, people are becoming more conscious about their health &
nutrition. Foods that are convenient with good taste, reasonably priced and carry
a favorable nutritional image are in great demand; bakery products especially
cakes and cookies. Wheat flour is the primary raw material which provides a
matrix in which other ingredients are mixed to form dough or batter.
In Pakistan, the predominant use of rice bran is an ingredient in livestock
feed. Considering its importance, value-added processing technologies for rice
bran have been sought by researchers. The products supplemented with rice
bran can play an important role in the existing food crises besides its health
claims.
From a marketing view point, the most available rice bran derived
product is the oil (Orthoefer, 1996). Rice bran oil has an impressive nutritional
quality, which makes it suitable for nutraceutical products. It has industrial
potential particularly in snack food preparation because of great frying stability
and with a good fry life and nutty flavor (Sarkar, 1992). Production of margarine
from rice bran oil has health benefits with reduced saturated fats and trans-fatty
acids.
Rice bran fractions can be used to produce acceptable low fat, high fiber
bakery products (Kennedy et al., 1996). Torticas de Moron, a traditional Cuban
bakery product, was manufactured with 0, 20, 25 or 30% of the usual white flour
replaced by parboiled rice bran. Protein, fat, crude fiber and ash were higher in
the rice Torticas than in the control. It was further observed that 25% replacement
of flour with rice bran resulted in a product with acceptable sensory properties,
chemical composition and shelf life (Zumbado et al., 1997).
Rice bran fiber has been reported to contain high amounts of functional
proteins and fats along with antioxidants, vitamins and trace minerals in
addition to being a concentrated source of fiber. The presence of these nutrients
allows rice bran fiber to be used as both nutritional and functional ingredient.
Chicken coated with stabilized rice bran fiber tend to absorb less fat during
101
frying while the small amount of fat found naturally in rice bran fiber can act as a
carrier for flavors (Hammond, 1994). Later, stabilized rice bran was incorporated
@ 5, 10 and 20% in chutney powder. Addition of the rice bran had more affect on
color and the least on texture with intermediate effects on aroma, taste and
overall acceptability (Prakash and Jyothilakshmi, 1995).
2.5.1. Bread
Bread is considered as one of the prime bakery products. Its large loaf
volume and fine texture require formation of well developed, elastic dough
structure. An essential element in this process is gluten present in the flour
(Eneche, 1999). Rice bran is used by the food industry in the production of baked
goods, snacks, crackers, breads, and cereals (Rukmani, 2002). The functional
properties of fullfat and defatted rice bran were explored by blending rice bran
in wheat flour @ 5, 10 or 15% to prepare leavened pan bread. Addition of any of
the defatted and fullfat rice brans was associated with reduction in loaf volume
and a decrease in overall acceptability of the bread. Breads containing upto 10%
of either type of rice bran was still considered acceptable (Sekhon et al., 1997).
The stabilized rice bran was successfully incorporated upto 20% for the
production of yeast bread. The hygroscopicity of the rice bran improved
moisture retention in the baked products while foaming ability improved air
incorporation and leavening (Carroll, 1990). In similar study, bread volume
decreased with blending of different types of rice bran; however, the decrease
was more pronounced with the defatted bran. Stabilized full fat rice bran up to
20% level and un-stabilized full fat or stabilized defatted rice bran upto 10% was
found suitable in various food products (Singh et al., 1995).
In commercial production of leavened pan bread, wheat flour was
supplemented with rice bran at 15-30%; concluded that rice bran can be
supplemented successfully upto 15% replacement level without affecting loaf
weight, height or volume (Sharp and Kitchen, 1990). Likewise, leavened pan
bread was made by supplementing 10 and 20% processed fullfat and defatted
102
rice bran to study the functional behavior of bread compared with control.
Texture profile analysis showed no significant differences as far as cohesive and
springiness, but bread hardness, gumminess and chewiness increased with
increasing levels of rice bran and were more prominent in bread from defatted
rice bran. Measurements of texture exhibited no detrimental effect of adding
fullfat rice bran upto 10% and slight hardening of loaves with 20% level
compared to control (Lima et al., 2002).
The addition of rice bran to wheat flour increased proteins, lysine and
dietary
fiber
in
bread
and
cookies
proportionately
to
the
level
of
supplementation. In addition to color, flavor, protein extractability, solubility of

bran and other properties like water & fat absorption, emulsifying & foaming
capacity also showed improvement which further enlightens the potential use of
bran in foods (Sharma and Chauhan, 2002). Defatted rice bran increases dough
yield, contributes to an attractive tan crust and crumb, does not disturb
fermentation or mixing tolerance of dough, causes baked products to remain
fresher and more moist (Lynn, 1969). The flour strength and gluten quality
decreased at different levels (5, 10, 15, 20 and 25%) of rice bran. The replacement
of bran with decreasing amount of gluten has inverse correlation with flour
strength. It was concluded that bran supplementation should not exceed to 15%,
in flours containing low gluten content (Chumachenko et al., 1987).
2.5.2. Cookies
Cookie is chemically leavened product, also known as biscuit.
Generally the term biscuit is used in the European countries and cookies in the
USA. Biscuits and biscuit like products have been made and eaten by man for
centuries (Hoseney, 1986). Cookies are ideal for nutrient availability, palatability,
compactness and convenience. They differ from other baked products like bread
and cakes because of having low moisture content, ensure comparatively free
from microbial spoilage and confer a long shelf life of the product (Wade, 1988).
103
Cookies are considered better for supplemented/composite flours due to

their ready-to-eat form, wide consumption, relatively long shelf-life and good
eating quality (Tsen et al., 1973). Cookies with high sensoric attributes have been
produced from blends of millet/pigeon pea flour (Eneche, 1999), raw rice and
wheat (Singh et al., 1989), blackgram and wheat (Singh et al., 1993), chickpea and
wheat (Singh et al., 1991), wheat, fonio and cowpea (McWatters et al., 2003) and
soybean, chickpea or lupine with wheat (Hegazy and Faheid, 1990). Similarly,
cookies with high sensory ratings have been produced from blends of wheat
flour and rice bran.
Nutritional and functional properties of rice bran are well suited for baked
products like cookies, muffins, breads, crackers, pastries and pancakes (Barber et
al., 1981). The fullfat and defatted rice brans were blended in wheat flour @ 5, 10
or 15% to prepare cookies. There was improvement in spread of cookies with the
addition of fullfat rice bran. In contrast, decrease in spread after supplementation
of defatted rice bran. Cookies supplemented with either type of rice bran were
acceptable upto 10% supplementation level (Sekhon et al., 1997). In another
study, cookies were successfully prepared from stabilized rice bran at levels of
20% (Carroll, 1990). In a similar study, stabilized fullfat rice bran upto 20% level
and un-stabilized full fat or stabilized defatted rice bran upto 10% was found
suitable in various food products (Singh et al., 1995). Dry heat and extrusion
stabilized rice bran was supplemented in wheat flour at 5-20% levels for the
preparation of cookies (Sharma and Chauhan, 2002).
104
Chapter-III
MATERIALS AND METHODS

3.1. Materials
Rice industrial by-product i.e. rice bran (Basmati-Super) was obtained
from Reem Rice Mills (Pvt.) Muridke while, parboiled rice bran of the same from
Haji Beer Din Rice Mills (Pvt.) Faisalabad. Commercial straight grade flour
(CSGF) and remaining ingredients for products preparation were purchased
from the local market, Faisalabad. Bran samples were sifted through 20 mesh
sieve before supplementation in CSGF. All reagents (analytical and HPLC) and
standards were from Merck (Merck KGaA, Darmstadt, Germany) and SigmaAldrich (Sigma-Aldrich Tokyo, Japan).
3.2 Rice Bran Processing

3.2.1. Rice bran stabilization
The processing of rice bran was carried out immediately to inactivate

endogenous lipases, responsible for fat deterioration. To achieve this objective,
rice bran was subjected to various stabilization techniques.
Unstabilized rice bran (Un-RB): Rice bran without any stabilization treatment.
Parboiled rice bran (PAR-RB): Rice bran obtained after soaking, steaming and
drying before normal milling.
Extrusion stabilized rice bran (ES-RB): Rice bran was stabilized in extruder at
125-130C for 30 sec, held for 3 min at 97-99C to inactivate lipase and air cooled
to room temperature (Randall et al., 1985).
Microwave stabilized rice bran (MW-RB): A microwave oven with 550 W output
power was used for the stabilization of bran. The moisture content of raw rice bran was
adjusted to 21% before treatment. One hundred gram of sample was packed in a
microwave-safe polyethylene bag and subjected to microwave heating for 3 min

at 120C and then cooled at room temperature (Ramezanzadeh et al., 2000). All the
105
stabilized rice brans along with un-stabilized bran were packed in zipper-top bags and stored at
room temperature (25oC) for 2 months.
3.2.2. Denaturation of anti-nutritional factors
Inspite of excellent nutritional profile, rice bran also contains some antinutrients like trypsin inhibitor and haemaglutinin-lectin, which must be
denatured before its supplementation in food products. Rice bran samples were
uniformly mixed with 20% (w/w) solution of 1% calcium hydroxide to
accomplish the objective.
3.3. Stabilization and Anti-Nutritional Appraisal
Chemical changes in stabilized and un-stabilized rice bran samples were
monitored by analyzing the samples for lipase activity, peroxide value,
thiobarbituric acid no. (0, 30 and 60 days storage interval), haemagglutinin-lectin
activity, trypsin inhibitor and phytates. The brief description of each method is
given below:
3.3.1. Lipase activity
Lipase activity was determined by estimating the amount of free fatty
acids (FFA) in rice bran on monthly basis upto 2 months (AOCS, 1998; AOAC,
2006). Increase in FFA (%) was taken as function of lipase activity in rice bran
during storage.
3.3.2. Peroxide value
Peroxide value of rice bran samples was determined by the method
described in Pearsons Composition and Analysis of Foods (Kirk and Sawyer,
1991) at the above said intervals up to two months.
3.3.3. Thiobarbituric acid no. (TBA no.)
TBA no. of rice bran samples was determined at specified intervals to
evaluate the stability of rice bran (Kirk and Sawyer, 1991). The rice bran (10g)
was taken in distillation flask and heated to obtain 5.0mL distillate in glass
stoppered test tube with 5.0mL TBA reagent (0.2883g/100mL of 90% glacial
acetic acid) and heated in water bath for 35 min with a blank sample. The tubes
were cooled in water for 10 min and absorbance (D) against blank sample was
taken by adjusting spectrophotometer (Cecil CE-7200, UK) on 538nm
wavelength. TBA no. was calculated by using the following expression:
TBA no. (mg malenaldehyde per Kg sample) = 7.8 x D
106
3.3.4. Haemagglutinin-lectin activity

Haemagglutinin activity of raw and processed rice bran samples was
determined by Rabbit Erythrocyte Agglutination Test (Benedito-de and Barber,
1978) at 0, 30 and 60 days storage interval. Lectin activity was measured in
haemagglutinin units (HU) as reported by Tan et al. (1983).
3.3.5. Trypsin inhibitor activity
For the purpose, 1g sample was blended with 15mL of 0.05N HCl in a
Sorvall Omni Mixer (Ivan Sorvall, Inc., Newtown). The extracted slurry was
centrifuged and trichloroacetic acid (TCA) was added to the supernatant and recentrifuged. After neutralization the enzyme inhibitory activities were
determined as described by Decker (1977).
3.3.6. Phytates
Phytic acid content of raw and processed rice bran samples was
determined by following the method of Haug and Lantszch (1983). Sample was
heated with acidic ammonium iron-III sulphate solution of known content. The
decrease in iron content (determined colorimetry with 2,2 bipyridine at 519 nm)
in supernatant was the measure of phytate content.
3.4. Raw Materials Analysis
3.4.1. Proximate analysis
Rice brans (four) and wheat flour samples were taken and analyzed
individually in triplicate on dry weight basis for crude protein, crude fat, crude
fiber, ash, and nitrogen free extract (NFE) according to their respective methods.
The brief description is given below:
3.4.1.1. Crude protein (CP)

The nitrogen content in each sample was determined by using Kjeltech
Apparatus (Technik GmbH D-40599, Behr Labor, Germany) based on Kjeldhals
AACC Method 46-10 (AACC, 2000). The protein percentage was calculated by
107
multiplying nitrogen with respective conversion factors i.e. 5.70 and 6.25 for
wheat flour and rice bran, respectively (Pomeranz and Meloan, 1996).
3.4.1.2. Crude fat (CF)
The crude fat content of each sample was estimated by using Soxtech
System (HT2 1045 Extraction Unit, Hoganas, Sweden) by following the AACC
method 30-10 (AACC, 2000).
3.4.1.3. Crude fiber (CF)
Crude fiber content of each sample was determined by digesting the
sample in 1.25% H2SO4 followed by 1.25% NaOH solution through Labconco
Fibertech (Labconco Corporation, Kansas, USA) as described in AACC Method
32-10 (AACC, 2000).
3.4.1.4. Ash
The ash content in each dry sample was determined by incinerating 3g
sample in a Muffle Furnace (MF-1/02, PCSIR, Pakistan) after charring according
to AACC Method 08-01 (AACC, 2000).
3.4.1.5. Nitrogen free extract (NFE)
The NFE was calculated according to the following expression:
NFE = 100 (% CP+ % CF+ % CF+ % Ash)
Where;
CP = Crude protein
CF = Crude fat
CF = Crude fiber
3.4.2. Mineral analysis
Rice bran samples were analyzed for Na, K, Ca and

Mg after wet digestion through Flame Photometer-410
(Sherwood
Scientific
Ltd.,
Cambridge)
and
Atomic
Absorption Spectrophotometer (Varian AA240, Australia)

by following procedures of AOAC (2006).
3.5. Rice Bran Oil
108
3.5.1. Extraction
Rice bran oil was extracted from all bran samples by slurring with four
volumes of food grade hexane at room temperature for 1 hr. Hexane was
evaporated in a rotary evaporator at 40oC (AOCS, 1998).
3.5.2. Refining
Crude rice bran oil was refined to remove pigments and esters by using
dewaxing, degumming, neutralization and bleaching processes. Dewaxing was
carried out by cooling the oil at 15oC, allowing the waxes to crystallize, settle and
then removed through centrifugation. For degumming, phosphoric acid (0.25%)
was added in dewaxed oil along with water for hydration to ensure the complete
hydroxylation and then centrifuged. Lastly, bleaching of oil samples was carried
out through acid activated bleaching clay (3%).
3.5.3. Yield (%)
The yield of refined oil (%) was calculated by the formula:
Wt. of refined oil (g)
Yield of oil (%) =
x 100
Wt. of sample (g)

3.5.4. Quality of refined rice bran oil samples
The refined oil samples were analyzed for different quality parameters i.e. free
fatty acids (Ca 5a-40), peroxide value (Cd 8-53), acid value (Cd 3d-63), iodine
value (Cd 1d-92), saponification value (Cd 3-25), color (Cc 13j-97), odor, specific
gravity (Cc 10a-25), refractive index (Cc 7-25) and fatty acid profile (IUPAC
Method 2.301+2.302+2.304) by following their respective procedures (AOCS,
1998; AOAC, 2006).
3.5.5. Antioxidants potential

Antioxidant potential of RBO samples with special reference to
tocopherol, tocotrienol and oryzanol was determined by using HPLC. For this
purpose, 0.5g oil was taken in test tube and extraction was carried with 2.0mL of
hexane, vortexing for 15 sec, and centrifuging for 10 min at 4,000 rpm. The
supernatant was transferred into HPLC vials and analyzed by reverse phase (RP)
HPLC. The HPLC system (Perkin Elmer Series 200 HPLC Systems, USA)
109
comprised of analytical pump, vacuum degasser, Series 200 UV/Vis Absorbance

Detector, Scanning Fluorescence Detector, auto sampler, column oven, refractive
index, 600 series link chromatography interface, C18 column (4.6 mm x 25cm
5m particle size) and guard column.
Oryzanol: The sample extract was injected through a guard-column and
separated on a C18 column using the method of Xu and Godber (1999). For the
purpose, 50L of prepared extract was injected using autosampler. A mobile
phase of ethanol: acetonitrile: dichloromethane: acetic acid (50:44:3:3) was used at
a flow rate of 1.4 mL/min and UV/Vis absorbance detector was set at a
wavelength of 330 nm. The total analysis time was approximately 20 min,
oryzanol peaks were appeared around 16-18 min of retention times. Analysis
was performed in triplicate and values were averaged. Pure standard of oryzanol was used for identification and calibration.
Tocopherols and tocotrienols: The sample was injected through a guard-column
and separated on a C18 column using method of Rogers et al. (1993). The
extraction was performed using the initial mobile phase conditions; 45%
acetonitrile, 45% MeOH, 5% IsOH, and 5% of aq. acetic acid (1%), at a flow rate
of 0.8 mL/min, for 6 min. The mobile phase was changed linearly to acetonitrile:
MeOH: IsOH at the ratio of 25:70:5 (v/v/v) over the next 10 min and held there
for 12 min before being returned to the initial conditions. The tocopherols and
tocotrienols were detected by fluorescence at the excitation and emission
wavelengths of 298 and 328nm, respectively.
3.5.6. Fatty acid profile
Fatty acids need to be converted into fatty acid methyl esters (FAME) for
GLC analysis. FAMEs were prepared with borontrifluoride methanol reagent.
Initially, 4.0mg of fatty acids was placed in a Teflon lined screw cap tube and
1.0mL borontrifluoride methanol reagent was added, tube was closed with screw
cap and then heated on boiling water bath for 1 hr. After heating, it was cooled
and mixture was gently transferred to a separating funnel, with small amount of
110
n-hexane. The funnel was shaken gently several times and the upper hexane
layer was removed. Hexane solution was dried over anhydrous sodium
sulphate, filtered and then vacuum distilled to get concentrated methyl ester for
GLC analysis by following IUPAC Method 2.301+2.302+2.304 (IUPAC, 1987).
FAMEs of different RB samples were analyzed on Shimadzu Gas
Chromatograph Model 14A (Shimadzu Co., Japan) fitted with a methyl lignose
rate coated (film thickness = 0.25m), polar capillary column SP-2330
(30mx0.32mm) and a flame ionization detector. Oxygen free nitrogen was used
as a carrier gas at a flow rate of 5mL/min. The temperature programming of the
column oven was set as 180C-2min-4C/min-210C, injector temperature 230C
detector temperature 250C. Identification of fatty acid methyl esters was made
by comparing their relatives retention times with that of known standard
samples using a CSW32 software program and data processor C-R4A
CHROMATOPAC.
3.6. Selection of Best Treatment

Based on quality of extracted oil from various bran samples one best oil
was selected; the respective defatted bran as well as intact full-fat bran of the
same was also used for further efficacy studies and product development.
3.7. Efficacy Studies for Safety Evaluation

Spraque-Dawley rats were fed on diets containing selected samples i.e.
rice bran oil, full-fat and defatted rice bran, along other essential ingredients for
safety evaluation and to explore the health benefits of this valuable source.
3.7.1. Experimental plan
Male Spraque-Dawley (SD) strain rats: One hundred and twenty, male SD rats
were purchased from National Institute of Health, Islamabad. The rats were
initially fed a basal diet for a week to acclimatize. Later, rats were randomly
divided into four groups; thirty rats in each. The diets prepared from selected
rice bran oil, full-fat and defatted rice brans were fed to the rats for a period of 45
days. The air-conditioned incubating room was maintained at 23 2 C, and the
111
relative humidity was controlled at 505%. The rats were nurtured in screenbottomed cages under 12 hr light-dark cycles and had free access to water and
diet during the entire experimental periods. At the initiation of study, some rats
were anaesthetized and sacrificed to measure base line values for respective
parameters. Feed intake and water consumption were measured daily where as
body weight on weekly basis throughout the experimental period. Overnight
fasted rats, from each group (10 rats) were decapitated at 15, 30 and 45 days
under chloroform anesthesia and blood was drawn in lithium heparin tubes
(Becton, Dickinson & Co., US). Organs like liver, heart, lungs, spleen, right and
left kidney were weighed to determine the effect of the individual experimental
diets on these organs.
Experimental diets: The control diet was composed of 65% corn starch, 10% corn
oil, 10% casein, 10% cellulose, 4% salt mixture (Appendix I) and 1% vitamin
mixture (Appendix II). The detail of remaining diets is mentioned below:
Ingredients
Control
(%)
Fullfat RB
Defatted RB
Rice bran oil
Corn Oil
10
Corn Starch
65
Casein
10
Cellulose
10
Salt mixture
4
Vitamins
1
Total
100
Control = Corn oil/ starch
FFRB = Fullfat rice bran (MWSRB)
TDF
= Total dietary fiber
RBO
FFRB
DFRB
50.2
41.75
10
9.65
65
32.92
41.50
10
2.88
2.10
10
4
4
4
1
1
1
100
100
100
RBO = Rice bran oil (MWSRB)
DFRB = Defatted rice bran (MWSRB)
As anticipated from the design of the diets, the protein, fat, total dietary
fiber, salt mixture and vitamins contents of the six diets were identical.
3.7.2. Analysis of serum profile
112
Serum was separated through centrifugal machine (800-Centrifugal

Machine, China) at 4000 rpm for 6 min after allowing the blood samples to stand
for at least 30 min (Uchida et al., 2001). That serum was used for subsequent
parameters using commercial kits through Microlab-300 (Merck KGaA,
Darmstadt, Germany).
3.7.2.1. Liver function tests
Serum was analyzed to estimate activities of liver

functioning enzymes such as ALT (Alanine amino
transferase),
ALP
(Alkaline
Phosphatase)
and
AST
(Aspartate amino transferase) by using their commercial

kits (Tolman and Rej, 1999).
3.7.2.2. Renal function tests
The extracted serum was analyzed for urea and creatinine by using their
commercial kits (Newman and Price, 1999)
3.7.2.3. Lipid profile
Lipid profile was analyzed for Total Cholesterol (TC) by CHODPAP
method (Stockbridge et al., 1989), Triglycerides (TG) by GPO-PAP method
(Annoni et al., 1982), High Density Lipoprotein Cholesterol (HDL-C) by HDL
Cholesterol Precipitant method (Assmann, 1979), low Density Lipoprotein
Cholesterol (LDL-C) as described by McNamara et al., (1990), Total Protein by
Biuret method (Josephson and Gyllensward, 1975), Serum Albumin (A) by
Bromcresol Green method (Webster, 1974), Serum Globulin (G) and A/G ratio
using commercial kits.
3.8. Product Development
3.8.1. Preparation of rice bran oil cookies
Refined rice bran oil form selected bran was further utilized for
preparation of cookies by replacing normal shortening @ 20, 40, 60, 80 and 100%
(Table 1). Cookies were prepared with some modifications in the AACC Method
113
10-50D (AACC, 2000). Vegetable shortening and sugar were mixed in Hobart
Mixer-N50 (Hobart Manufacturing Co., Troy, Ohio, USA) for 20-30 min; water
was added followed by eggs and mixed till homogeneity (approx. 5 min at a
speed setting of 2). Lastly, the commercial straight grade flour (CSGF) and
baking powder were added to form a homogeneous mass. The batter was spread
into sheet; cookies were molded and baked in the baking oven at 1755oC for 2530 min. After baking, the cookies were cooled at room temperature and packed
in polyethylene bags for a period of two months for further analysis.
Table 1. Utilization of RBO in cookies
Treatments
NS (%)
RBO (%)
T0
100
T1
80
20
60
40
T2
T3
40
60
T4
20
80
T5
100
T0 acts as control with 100% normal shortening
3.8.1.1 Quality attributes of cookies

3.8.1.1.1. Physical analysis
Cookies were analyzed for width, thickness and spread factor at 0, 30 and
60 days, according to the methods described in AACC (2000).
Width (W):
Six cookies horizontally (edge to edge) and rotated at 90o

angle for reading.
Thickness (T):
Six cookies were placed one another to compute thickness.
Spread factor (SF): It was calculated according to the following formula:
SF = (W/T) x 10
3.8.1.1.2 Proximate analysis
Cookies were analyzed for moisture, ash, crude protein, crude fat, crude
fiber and nitrogen free extract (NFE) by their respective procedures as mentioned
in previous section at 0, 30 and 60 days interval (AACC, 2000).
3.8.1.1.3. Total acidity
Total acidity was determined at stated intervals by titration against NaOH
(AACC, 2000).
3.8.1.1.4. Thiobarbituric acid no. (TBA no.)
TBA no. of cookies was determined according to the method of Kirk and
Sawyer (1991) at the same intervals upto two months.
3.8.1.1.5. Sensory evaluation
114
The sensory evaluation of cookies for various attributes like color, flavor,
taste, texture and overall acceptability (Appendix III) was carried out at 0, 30 and
60 days by trained taste panel using 9-Point Hedonic Score System (Meilgaard et
al., 2007) with following individual scores: liked extremely-9, liked very much-8,
liked moderately-7, liked slightly-6, neither liked nor disliked-5, disliked slightly4, disliked moderately-3, disliked very much-2 and disliked extremely-1, to find
out the most suitable composition of cookies for commercialization. All
evaluations were conducted at room temperature on the same day in the
National Institute of Food Science and Technology (NIFSAT), University of
Agriculture, Faisalabad. On the day of evaluation, cookies from all compositions
were placed in transparent cups, labeled with random codes. Panelists were
provided with distilled water and unsalted crackers to rinse their mouths
between the samples. The cookies were presented in random order and panelists
were asked to rate their acceptance by giving a score for all the parameters.
3.8.2. Preparation of rice bran supplemented flours

Initially, trials were conducted by blending full-fat and defatted rice bran
with commercial straight grade flour in different proportions for selecting
suitable compositions of supplemented flours (Table 2) for preparation of cookies
and leavened pan bread.
Table 2. Rice bran supplemented flours used in study
Wheat
Stabilized Rice Bran (%)
Treatments
flour
Full fat
Defatted
(%)
T0
100
T1
95
5
T2
90
10
T3
85
15
T4
80
20
T5
75
25
T6
70
30
T7
60
40
T8
50
50
T9
95
5
T10
90
10
T11
85
15
115
T12
T13
T14
T15
T16
80
75
70
60
50
20
25
30
40
50
T0 (100% commercial straight grade flour) acts as control
3.8.3. Analysis of rice bran supplemented flours

3.8.3.1. Proximate analysis
Rice bran supplemented flour samples were analyzed
for moisture, crude protein, crude fat, crude fiber, ash, and
nitrogen free extract (NFE) according to their respective
methods (AACC, 2000).
3.8.3.2. Mineral analysis
Supplemented flours were analyzed for Na, K, Ca and
Mg by following procedures mentioned in previous
section.
3.8.3.3. Dietary fiber
Dietary fiber content of rice bran supplemented flour
samples along with control was determined by using
Megazyme TDF Test Kit (AACC Method 32-05 and AOAC
Method 985.29). Total dietary fiber (TDF) was determined
on duplicate samples of dried and defatted material.
Samples were cooked at 100C with heat stable -amylase
for
30
min
for
gelatinization,
hydrolysis
and
depolymerization of starch; incubated at 60C for same

116
time interval with protease to solubilize and depolymerize

proteins
and
amyloglucosidase
to
hydrolyze
starch
fragments to glucose; treated with four volumes of ethanol

to precipitate soluble fiber and remove depolymerized
protein and glucose from starch. The residue was filtered;
washed with 78% ethanol, 95% ethanol, and acetone; dried;
and weighed. One duplicate was analyzed for protein and
the other incubated at 525C to determine ash. Total
dietary fiber (TDF) content was determined from the
weight of the filtered and dried residue after deducting the
weight of the protein and ash.
3.8.3.4. Thiobarbituric acid no. (TBA no.)
TBA no. of supplemented flours was determined according to the method
of Kirk and Sawyer (1991) at 0, 30 and 60 days storage intervals.
3.8.3.5. Dough rheological studies
Dough rheological properties are important for the preparation of quality
bread due to their significant effect on final loaf volume. The rheological
behavior of rice bran supplemented flours was evaluated by conducting
mixographic and farinographic studies before the preparation of product as
described below:
3.8.3.5.1. Mixographic studies
Rice bran supplemented flour samples used for the preparation of
leavened pan bread (Table 4) were evaluated for Mixographic studies by using
Mixograph equipped with 10g capacity bowl (National Mfg. Co., Lincoln,
Nebraska). All the flour samples were run through Mixograph by adding water
according to the instructions described in AACC Method 54-40 (AACC 2000).
117
The dough development time and peak height were interpreted from each
Mixogram.
3.8.3.5.2. Farinographic studies
The rheological behavior of rice bran supplemented flours was evaluated
by running samples through a Brabender Farinograph (Brabender DUISBURG
380, Germany) equipped with 50g bowl capacity to assess the dough behavior of
each sample (AACC Method 54-21). Farinograms were obtained at 500
Brabender Unit (BU) line with 50g flour under controlled conditions of
temperature (30C). The farinograms were interpreted for the characteristics such
as water absorption, dough development time and dough stability.
3.8.4. Preparation of rice bran supplemented cookies
Cookies were prepared from supplemented flour samples (Table 3) with
some modifications in AACC Method 10-50D (AACC, 2000) and analyzed for
quality attributes.
Table 3. Treatments used for preparation of rice bran supplemented cookies
Wheat flour
Treatments
Full fat
Defatted
(%)
T0
100
T1
90
10
T2
80
20
T3
70
30
T4
60
40
T5
50
50
T6
90
10
T7
80
20
T8
70
30
T9
60
40
T10
50
50
T0 (100% commercial straight grade flour) acts as control for both fullfat and defatted rice bran supplementation
3.8.5. Preparation of rice bran supplemented leavened pan

bread
118
Leavened pan bread was prepared from rice bran

supplemented flours (Table 4) by straight dough method
(AACC, 2000) and analyzed for various quality traits.
Table 4. Treatments used for preparation of leavened pan

bread
Treatments
T0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
Wheat flour
(%)
100
95
90
85
80
75
95
90
85
80
75

Full fat
Defatted
5
10
15
20
25
5
10
15
20
25
T0 (100% commercial straight grade flour) acts as control for both fullfat and defatted rice bran supplementation
3.8.6. Analysis of rice bran supplemented cookies and breads

3.8.6.1. Physical analysis
Rice bran supplemented cookies were analyzed for width, thickness and
spread factor by following the procedures as described in earlier section (AACC,
2000).

Rice bran supplemented cookies and bread were
analyzed for Na, K, Ca and Mg by following procedures
mentioned in previous section.
119

Dietary fiber content of supplemented cookies and bread was determined
by using Megazyme TDF Test Kit (AACC Method 32-05 and AACC Method 3221) as describes in preceding section.
3.8.6.4. Sensory evaluation

Cookies were evaluated for taste, color, flavor, texture and overall
acceptability (Appendix III) according to the procedure described by Meilgaard
et al. (2007). Likewise sensory evaluation of bread samples was carried out for the
external characteristics i.e. volume, color of crust, symmetry of form, evenness of
bake, character of crust and internal characteristics like grain, color of crumb,
aroma, taste and texture (Appendix IV) as described by Matz (1972) to find out
the most suitable compositions of supplemented flours for bread preparation.
3.9. Statistical Analysis

Complete Randomized Design (CRD) was applied and data obtained for
each parameter was subjected to statistical analysis to determine the level of
significance (Analysis of Variance technique) as described by Steel et al. (1997).
Duncans Multiple Range Test (DMR) was used to determine significant
differences. All the statistical analyses were done by using Minitab software
(Minitab V-15).
120
Chapter-IV
RESULTS AND DISCUSSION

The present research project was planned to utilize indigenous rice bran
(RB) for oil extraction and preparation of value-added products. Rice bran
samples were stabilized by extrusion cooking, microwave heating and
parboiling, to inactivate endogenous lipases; making stable against fat
deterioration. Rice bran oil (RBO) was extracted from all bran samples and after
refining, analyzed for various physical and chemical characteristics, fatty acid
profile and antioxidant potential to select one best sample. Later, selected RBO,
its defatted portion as well as intact fullfat rice bran of the same were evaluated
for palatability and safety in animal model. After efficacy studies, the same
treatments were utilized for preparation of value added baked products. RBO
cookies were prepared by replacing normal shortening and analyzed for various
quality attributes. Moreover, fullfat and defatted rice brans were supplemented
in commercial straight grade flour (CSGF) to prepare various blends. Each
combination was analyzed for chemical and rheological characteristics at
prescribed intervals. Finally, rice bran supplemented cookies and leavened pan
bread were prepared to find out the best compositions for commercialization.
The investigated parameters and their respective results are discussed below:
4.1.
Stabilization and Anti-Nutritional Appraisal

Rice bran, in spite of being rich in nutrients, has limitations in food
applications and its successful use is only possible after stabilization. Upon
milling, bran is exposed to enzymes from the outer layers of the rice kernel,
resulting in hydrolysis of its oil. This in turn leads to rancidity of the bran,
making it unsuitable for human consumption. In this study, stabilization of rice
bran was carried out to inactivate lipases and other nutritional inhibitors such as
trypsin inhibitor and haemaglutinin-lectin. After processing, there was
significant reduction in these anti-nutritional factors. The chemical changes in
121
stabilized and unstabilized rice bran samples were monitored by analyzing for
lipase activity (in terms of FFA production), peroxide value (PO) and
thiobarbituric acid number (TBA no.) at 0, 30 and 60 days storage.
Mean squares for FFA, POV and TBA no. of various rice bran samples
showed (Table 5) significant variations due to stabilization techniques and
storage intervals whereas interaction was found to be non-significant for these
traits. The mean values (Table 6) explicated that FFA, POV and TBA no. were
significantly affected by the stabilization techniques.
4.1.1. Lipase activity
Rice bran contains lipases, primarily responsible for the hydrolysis of
triglycerides into glycerol and free fatty acids; further oxidized by peroxidases,
provoking brans rancidity. In present study, increase in FFA was used as
criterion of lipase activity. The highest FFA level (Table 6) was observed in
unstabilized rice bran (17.75%) followed by parboiled (6.95%) and extruded bran
(6.12%); however, the minimum value (5.25%) was observed in microwave
stabilized bran samples. After stabilization, there was less formation of FFA in all
stabilized bran samples. Nevertheless, there was a gradual increase in FFA level
in all bran samples during 60 days storage due to residual lipolytic activity that
increased under favorable conditions (Table 7). At the initiation of study, the
mean FFA level was 3.82% which gradually increased to 7.34 and 15.89% after 30
and 60 days storage, respectively. However, the maximum increase was
observed in unstabilized rice bran.
Hydrolysis of triglycerides forms free fatty acids, the principal cause of
deterioration occurring rapidly during the first few days or weeks after milling
(Randall et al., 1985; Ramezanzadeh et al., 1999). After bran separation, the oil is
exposed to lipases, causing its rapid breakdown to free fatty acids (Desikachar,
1974).
122
Table 5. Mean squares for FFA, POV and TBA no. of various rice bran samples
SOV
df
FFA
POV
TBA
no.
Treatments (T)
Storage (S)
SxT
Error
Total
**
3
2
6
24
35
309.197**
462.575**
82.228**
0.110
0.0227**
0.6188**
8.62010-4ns
0.0013
8.99010-4**
1.7510-4**
1.57410-5ns
0.00002
Highly significant at P 0.01; ns Non-Significant
Table 6. Effect of stabilization on FFA, POV and TBA no. of rice bran samples
Bran types
Un-RB
ES-RB
MW-RB
PAR-RB
FFA
POV
TBA no.
17.75a
6.12c
5.25d
6.95b
0.91a
0.81b
0.80b
0.88a
0.083a
0.063c
0.062c
0.074b
Means carrying the same letters in a column are not significantly different
Un-RB
Rice bran (unstabilized)
ES-RB
Extrusion stabilized rice bran
MW-RB
PAR-RB
Parboiled rice bran
Table 7. Effect of storage on FFA, POV and TBA no. of rice bran samples
Storage (days)
0
30
60
FFA
POV
TBA no.
3.82c
7.34b
15.89a
0.63c
0.83b
1.09a
0.0670c
0.0716b
0.0741a
123
Lipase activity results in significant increase in free fatty acid

concentration and reaches 7 to 8% within 24 hours and then subsequently
increases by 4 to 5% per day (Rukmini, 2002); upto 70% FFA has been reported
for a single month storage (Orthoefer, 1996). Rice bran having more than 15% FFA
becomes rancid (Godber et al., 1994). Free fatty acids concentration in rice bran is dependent on
the changes in temperature and moisture content experienced, by the bran during storage
(Fernando and Hewavitharana, 1993). The nutritional quality and palatability of rice
bran deteriorate rapidly as the oil undergoes hydrolytic and oxidative rancidity
(Subrahmanyan, 1977; Tsai, 1982). Hence, stabilizing the bran just after milling can
prevent oil deterioration.
4.1.2. Peroxide value
The means for peroxide value (Table 6) explicated significance of
stabilization techniques. The highest POV (0.91 mEq/Kg) was observed in
unstabilized rice bran followed by parboiled (0.88 mEq/Kg) whereas the
minimum value (0.80 mEq/Kg) was recorded for microwave stabilized rice bran.
Likewise to FFA development, there was comparatively less increase in POV
bran samples after stabilization. Nonetheless, there was a gradual increase in
POV in bran samples during storage (Table 7). At the start, the mean POV was
0.63 which was steadily increased to 0.83 and 1.09 mEq/Kg after 30 and 60 days,
respectively. The maximum increase was noted in unstabilized samples.
4.1.3. Thiobarbituric acid no.
Thiobarbituric acid test is based on the reaction of thiobarbituric acid with
the oxidation products of fats and oils. It is believed that aldehydes are
responsible for the color formation, measured spectrophotometrically. The
development of red color indicates the degree of oxidation in a given sample.
The mean values for TBA no. (Table 6) also highlighted the importance of
stabilization against lipases. The highest TBA value (0.083) was observed in
unstabilized rice bran followed by parboiled (0.074), extrusion stabilized (0.063)
and microwave stabilized bran (0.062mg malenaldehyde per Kg). Extrusion and
microwave stabilized rice bran showed non-significant differences with respect
to TBA no. Similar to FFA and POV, there was less increase in TBA no. of bran
samples after stabilization. However, a gradual increase was observed in all
124
samples during 60 days storage (Table 7). At the start of study, the mean TBA no.
was 0.067 which was gradually increased to 0.071 and 0.074 mg malenaldehyde
per Kg of bran sample, after 30 and 60 days storage, respectively. TBA no. in
unstabilized bran increased progressively during storage and its measurement
was considered as a viable method to analyze oxidative degradation. Refined oil
in good condition has TBA value of 0.02 to 0.08 whereas badly stored oils have
0.1 to 0.2 mg malenaldehyde per Kg (Kirk and Sawyer, 1999).
Several processes have been studied to inactivate lipases (Rajeshwara and
Prakash, 1995). The commonly used stabilization techniques are thermal and
chemical treatments (Randall et al., 1985; Kim et al., 1987). Thermal treatments are
capable of decreasing the oxidative rancidity (Ramezanzadeh et al., 1999). In
present investigation, pretreatment methods showed a significant reduction in
FFA contents as compared to unstabilized bran. Both microwave heating and
extrusion cooking were found equally effective in controlling lipase activity. The
extrusion process can inactivate the lipases by increasing the temperature of the
extruded material (Sanchez et al., 2000). Extrusion cooking showed no significant
increase in free fatty acids during storage. Heating in the presence of moisture is
more effective for denaturing lipases (Ramezanzadeh et al., 1999). Similar to
extrusion, parboiling also effective to reduce the free fatty acids (Godber et al., 1994). The
thermal efficiency of microwave heat stabilization is greater than that of either dry or wet heating
as enhanced kinetic energy of water molecules due to electromagnetic waves,
alongwith friction, produces even distribution of heat having lethal effects on

lipase activity (Tao, 2001).
4.1.4. Haemagglutinin-lectin
Haemagglutinin-lectin is a toxic globulin protein concentrated in rice
bran, when it comes into contact with blood, agglutinate red blood cells.
Table 8. Anti-nutritional factors in rice bran samples
Rice bran types
Haemagglutininlectin activity
/mg
125
Trypsin
inhibitor
activity /mg
Phytates
(%)
Un-RB
ES-RB
MW-RB
PAR-RB
22.850.19
0.960.008
0.810.009
0.100.001
8.010.07
Nil
Nil
Nil
4.130.04
1.300.012
1.250.013
0.950.008
Values are MeanSD for four rice bran samples, analyzed individually in triplicate
Un-RB
ES-RB
MW-RB
PAR-RB
Parboiled rice bran
Means for haemaglutinin-lectin (activity/mg), trypsin inhibitor (activity/mg)

and phytates (%) are presented in Table 8. The haemaglutinin-lectin activity was
maximum (22.85 activity/mg) in unstabilized rice bran, that was significantly
126
decreased after stabilization. Among the stabilized rice bran samples, minimum
activity were observed in parboiled rice bran (0.10) followed by microwave
stabilized (0.81) and extrusion stabilized rice bran (0.96 activity/mg). The results
are in confirmity with the findings of Sayre et al. (1987) and Benedito-de and
Barber (1978). Haemagglutinin activity was reduced by 95% by cooking the moist
rice bran at 1302C. By the addition of acetic acid solution (1%), negligible
activity was observed in extruded rice bran. It can be assumed that these
processes resulted complete denaturation of the toxic globulin proteins. In
another study, haemagglutinin activity was reduced by heating rice bran at 100
C upto six minutes (Rehman and Mahmood, 1996).
4.1.5. Trypsin inhibitor
Trypsin inhibitor is one of the most important anti-nutritional factors
present in rice bran. The maximum activity (8.01 activity/mg) was in
unstabilized rice bran (Table 8). All stabilization techniques resulted in
permanent destruction of trypsin inhibitor from bran samples. The results of the
present investigation are in accordance with earlier findings of Kratzer et al.
(1974) and Kratzer and Payne, (1977). Trypsin inhibitors are readily inactivated
by moist heat treatment (Deolankar and Singh, 1979.)
4.1.6. Phytates
Phosphorus as phytate is relatively unavailable to non-ruminants
resulting in poor absorption of other minerals. The maximum phytate content
(4.13%) was noted in unstabilized rice bran; significantly reduced after
stabilization (Table 8). The minimum phytates were found in parboiled rice bran
(0.95%) followed by microwave stabilized rice bran (1.25%) and extrude bran
(1.30%), respectively. Incubation of rice bran at 55C reduced phytic acid content
upto 80% (Tangendjaja et al., 2006). Heat stabilization of bran significantly reduced the
phytic acid content (Sharma et al., 2004).
The phytate contents of extrusion stabilized rice bran were reduced upto
54.12%. However, the phytates in processed rice bran moist with acetic acid and
127
calcium hydroxide plus extruder cooking, further decreased by 89.88 and 77.65%,
respectively (Shaheen et al., 2005).
4.2. Raw Material Analysis

Rice bran samples were analyzed for moisture, crude protein, crude fat,
crude fiber, ash, NFE and minerals like Na, K, Ca and Mg. After oil extraction,
respective defatted bran samples were subjected to chemical assay. Results
indicated that fullfat rice bran samples contain 14.20 to 16.20% crude protein,
18.22 to 19.30% crude fat, 10.68 to 17.03% crude fiber, 8.30 to 9.03% ash content
and 36.44 to 47.56% nitrogen free extract, on dry weight basis (Table 9). The
parboiled rice bran has comparatively higher levels of protein, fat, fiber and ash
contents. Parboiled rice bran has greater oil content, approximately 20 to 26%
than that of raw rice bran. This might be due to less endosperm contamination,
better extractability of oil by solvents and outward movement of fat from
aleurone and germ cells to the bran layer (Bhattacharya, 1985; Keerthi and
Swarnasiri, 1985; Amarasinghe and Gangodavilage, 2004).
Rice is an excellent source of minerals; mainly concentrated in bran
portion. The results in Table 10 indicated that rice bran samples contain 7.20 to
9.30 mg/100g Na, 1270 to 1820 mg/100g K, 538.57 to 872.50 mg/100g Ca and 935
to 1233 mg/100g Mg, respectively. Commercial straight grade flour (CSGF), used
for cookies and bread preparations, was also analyzed for chemical assay. It was
revealed from Table 9 that CSGF contains 10.23% crude protein, 1.17% crude fat,
0.33% crude fiber, 0.52% ash content and 76.25% nitrogen free extract. The results
regarding chemical composition of wheat flour are in agreement with the earlier
findings of Pasha et al. (2002) and Butt et al. (2006) who reported 9.77-10.29%
crude protein, 1.12-1.21% crude fat, 0.16-0.37% fiber, 0.52-0.60% ash and 76.7076.96% nitrogen free extract in commercial straight grade flour. In Pakistan,
wheat varieties have been found to contain an average moisture 9.69 to 10.35%,
crude protein 9.57 to 14.3%, crude fat 1.47 to 2.93%, ash 1.48 to 2.03% and crude
fiber 0.98 to 1.43% (Khan et al., 1987).
128
Defatted rice bran samples contain 17.41 to 20.16% crude protein, 0.47 to
1.03% crude fat, 13.08 to 21.32% crude fiber, 10.17 to 11.30% ash content and
46.19 to 58.17% nitrogen free extract (Table 11). However, defatted rice bran has
higher levels of protein, fiber and ash content than that of fullfat rice bran
samples.
The results pertaining to proximate composition of processed rice bran
were in conformity with the findings of Farrell (1994) who found 1117% crude
protein, 1118% crude fat, 10% crude fiber and 4565% nitrogen free extract in
rice bran. Similarly, Pomeranz and Oryl (1982) reported 13.217.3% protein, 3.29.5% crude fiber and 9.211.2% ash content in rice bran, on dry weight basis.
Likewise, Warren and Farrell (1990) reported that the crude protein ranged from
13.417.4%, ether extract 20.423.4% and ash 10.5% among several cultivars of
rice bran grown in Australia. The present study is also in concordance with the
findings of Saunders (1990) who reported that stabilized and parboiled rice bran
had 12% moisture, 13% protein, 16% fat, 9% crude fiber and 10% ash. Juliano
(1985) studied proximate composition of rice bran and reported 11.3-14.95%
crude protein and 15.0-19.7% crude fat.
Al-Jasser and Al-Mustafa (1996) conducted proximate analysis of Hassawi rice bran and
reported 12.56% protein, 15.15% fats, 17.74% ash and 45.66% carbohydrates. Similarly, Sharif et
al. (2005) reported that rice bran of Pakistani rice cultivars has 6.68% moisture,
7.89% ash, 15.78% crude protein, 20.55% crude fat, 7.59% crude fiber and 41.51%
nitrogen free extract.
Table 9. Proximate composition (%) of fullfat rice bran samples and

commercial straight grade flour
Composition
Moisture
Protein
Un-RB
ES-RB
MWRB
PARRB
CSGF
7.500.21
14.200.46
8.560.27
14.420.42
7.450.23
14.450.39
8.200.21
16.200.45
11.500.33
10.230.35
129
Fat
Fiber
Ash
NFE
18.220.51
12.20.36
8.300.19
46.082.15
18.670.45
10.680.32
8.670.21
47.562.62
18.850.55
10.750.35
8.700.25
47.253.06
19.300.60
17.030.47
9.030.28
36.442.33
1.170.04
0.330.01
0.520.02
76.254.41
Values are MeanSD for four samples analyzed individually in triplicate

Un-RB
ES-RB
MW-RB
PAR-RB
Parboiled rice bran
CSGF
Commercial straight grade flour
Table 10. Mineral analysis (mg/100g) of rice bran samples

Minerals
Na
K
Ca
Mg
Un-RB
7.500.19
12800.55
543.730.03
9530.43
ES-RB
7.400.23
12830.48
552.450.01
9350.41
MW-RB
7.200.21
12700.52
538.570.03
9690.45
PAR-RB
9.300.25
18200.46
872.500.02
12330.51
Table 11. Proximate composition (%) of defatted rice bran samples

Composition
Moisture
Protein
Fat
Fiber
Ash
NFE
Un-RB
ES-RB
7.450.19
17.410.55
0.890.03
14.950.43
10.170.27
56.583.01
8.400.23
17.660.48
0.470.01
13.080.41
10.620.23
58.172.85
MW-RB
7.390.21
17.690.52
0.550.03
13.150.45
10.650.30
57.963.32
PAR-RB
8.110.25
20.160.46
1.030.02
21.320.51
11.300.29
46.192.56
4.3. Rice Bran Oil

4.3.1 Refining
The degree of oil refining depends upon its intended use. Generally, food
applications require triglyceride portion of the crude oil. Rice bran oil was
130
extracted from bran samples by slurring with food grade hexane and refined to
remove unsaponifiables, pigments and limited quantities of partial esters.
4.3.2 Yield (%)
During refining, there was a substantial decrease in the oil recovery at
each step. The yield of oil after extraction and refining is shown in Table 12.
Before refining, maximum oil content (19.30%) was found in parboiled rice bran
followed by MW-stabilized rice bran (18.85%), extrusion stabilized rice bran
(18.67%) and unstabilized rice bran (18.22%). Parboiled rice bran has
approximately 20-26% greater oil content, than raw rice bran (Amarasinghe and
Gangodavilage, 2004). After refining, maximum recovery was observed in
parboiled rice bran (16.10%) whereas minimum oil content was recorded in
unstabilized rice bran (13.39%); might be due to high initial FFA content.
However, the oil recovery from microwave and extrusion stabilized rice bran
was 15.75 and 15.45%, respectively. No doubt, the yield of parboiled oil was
more but the main hurdle is its dark color that is undesirable.
4.3.3. Quality evaluation
Edible oils were evaluated for physical, chemical, nutritional and
technological properties before their applications in food. The oil samples were
analyzed for different quality parameters i.e. color, flavor & odor, specific
gravity, smoke point, fire point, free fatty acids, peroxide value, acid value,
saponification value, iodine value, unsaponifiable matter (Table 13), fatty acid
profile (Table 14) and antioxidant potential (Table 15) by following their
respective procedures.
The investigated parameters and their respective results are discussed as

below:
4.3.3.1. Color
It is the legitimate factor in determining the worth of edible oils and is
mainly influenced by quality of bran, processing methods, storage conditions
131
and method of extraction. The color of oil extracted from parboiled rice bran was
dark yellow, might be due to processing conditions; whereas oil extracted from
unstabilized, microwave and extruded bran samples was pale yellow.
4.3.3.2. Flavor and odor
The flavor of the oil is an imperative physical property and is influenced
by moisture, temperature, oxidation, light as well as the presence or absence of
antioxidants. Crude oils are normally deodorized to improve their quality. The
oil extracted from rice bran samples possessed agreeable flavor and odor after
refining process.
4.3.3.3. Specific gravity
It is used as a diagnostic criterion in assessing the extent of purity. The
presence of the number of double bonds and increase in chain length of the fatty
acids tend to increase the specific gravity. It is evident from the results (Table 13)
that the specific gravity of rice bran oil samples ranged from 0.910 to 0.935. For
oils, the value of specific gravity is always <1 and normally ranges from 0.850 to
0.950.
4.3.3.4. Smoke and fire point (C)
The smoke and fire points of a fat or oil are measure of its thermal
stability. It is obvious from the results (Table 13) that smoke and fire points of all
bran oil samples were 239 to 251C and 343 to 359C, respectively. Rice bran oil
has comparatively high smoke and fire point which also indicates its stability for
deep-frying purposes.
132
Table 12. Yield of rice bran oil from different bran samples
Yield (%)
Un-RBO
Crude RBO
Refined
RBO
18.220.39
ESRBO
18.670.43
13.390.29
15.450.53
MW-RBO
18.850.61
15.750.69
PARRBO
19.300.63
16.100.
29
Values are MeanSD for four rice bran oil samples, analyzed individually in triplicate
Un-RBO
ES-RBO
MW-RBO
PAR-RBO
Rice bran oil extracted from unstabilized rice bran

Rice bran oil extracted from extrusion stabilized rice bran
Rice bran oil extracted from microwave stabilized rice bran
Rice bran oil extracted from parboiled rice bran
Table 13. Quality characteristics of rice bran oil samples

Parameters
Color
Flavor & Odor

c gravity
Smoke point (C)
Fire point (C)
Free fatty acids (% oleic acid)
Peroxide value (meq/kg)
Acid value (mg/g)
Saponification value (mg/g)
Iodine value (g/100g)
Unsaponifiable matter (%)
UnRBO
pale yellow
agreeable
ESRBO
pale yellow
agreeable
MWRBO
pale yellow
agreeable
PARRBO
dark yellow
agreeable
0.9350.02
2446.17
34310.59
4.0130.12
0.910.05
1.270.05
1915.73
1173.25
2.750.13
0.9150.04
2427.26
35610.68
0.0450.001
0.890.03
1.190.04
1885.44
1093.27
3.100.22
0.9310.02
2396.13
34910.63
0.0470.007
0.860.01
1.210.05
1875.43
1113.27
3.220.12
0.9100.03
2517.53
35910.77
0.0480.003
0.850.02
1.250.06
1895.67
1053.15
3.010.11
133
4.3.3.5. Free fatty acids (%)

The amount of free fatty acids depends on the nature of fat or oil, method
of extraction, storage conditions, humidity, temperature and lipase activity in
rice bran. In present study, oil extracted from unstabilized rice bran possessed
high levels (4.013% as oleic acid) of free fatty acids as compared to oil extracted
from stabilized rice bran samples (Table 13) under identical refining conditions;
might be due to high initial free fatty acids as a result of lipase activity. The free
fatty acid (FFA) content of crude rice bran oil is relatively high, due to lipase
activity in the bran (Nicolosi et al., 1994).
4.3.3.6. Peroxide value (meq/Kg)
Peroxide value is a measure of the oxidative rancidity of oil and expressed
as milliequivalent of peroxide oxygen combined with one kilogram of fat/oil
(meq/Kg). Oils rich in polyunsaturated fatty acids are more susceptible to
oxidation when exposed to light, moisture or heat. Oxygen attaches to the fatty
acid chain to form peroxide, hydroperoxide, aldehydes, ketones and acids
resulting rancidity, discoloration, vitamin destruction thus making edible oils
unacceptable for consumers. The results in Table 13, explored that the peroxide
value of refined rice bran oil samples was 0.85 to 0.91 meq/Kg. Peroxide value is
a measure of the oxidative rancidity of oils. It is one of the most important
chemical characteristic for assessing the degree of deterioration of fats and oils.
Peroxide undergoes cleavage to produce aldehydes, ketones and acids
responsible for off flavor. These products interfere seriously with rancidity,
discoloration, vitamin destruction, nutritional losses and polymerization making
them unacceptable to the consumer. The POV of the rice bran does not remain
low during storage due to presence of enzymes.
4.3.3.7. Acid value (mg/g)
134
The acid value is a measure of the free fatty acid content of oil/fat. It is an
index of the measurement of freshness of oil. Humidity and high temperature
result in an increase of the acid value due to hydrolysis of glycerides into free
fatty acids. Higher values indicate undesirable changes as it not only results in
greater refining losses but also increases susceptibility of oils to rancidity. It is
thus very important in economic of oil refining. The oils intended for dietary
purposes should not contain higher amounts of free fatty acids. The presence of
free fatty acids in oils & fats is not desirable because they impart a sharp and
unpleasant flavor to edible fats & oils. The acid value of refined rice bran oil
samples was 1.19 to 1.27mg/g (Table 13); oil extracted from unstabilized and
parboiled bran samples showed maximum acid value i.e. 1.27 and 1.25mg/g,
respectively; might be due to high moisture, temperature and lipase activity in
bran samples.
4.3.3.8. Saponification value (mg/g)
Saponification value gives the molecular weight of the fatty acids present
in oil. The fat or oil with low molecular weight fatty acids has a higher
saponification value. The results in Table 13 showed that saponification value of
refined rice bran oil samples was 187 to 191mg KOH/g.
4.3.3.9. Iodine value (g/100g)
The iodine value is a measure of the degree of unsaturation of fatty acids
and expressed in terms of the number of grams of iodine absorbed by 100g of fat
or oil. It also indicates the stability of oil towards oxidation. It is observed, higher
the iodine value, greater the degree of unsaturation. It is evident from the results
(Table 13) that the iodine value of refined rice bran oils was 105-117g/100g.
4.3.3.10. Unsaponifiable matter (%)
These are bioactive components with nutraceutical worth mainly
composed of sterols, triterpene alcohols and less polar components such as
squalene or tocotrienols. The results in Table 13 elucidate that unsaponifiable
content of refined rice bran oil samples was 2.75 to 3.22%. Crude rice bran oil
contains 90 to 96% saponifiable and about 4% unsaponifiable lipids (Raghuram
135
and Rukmini, 1995; McCaskill and Zhang, 1999) mainly comprised of naturally
occurring antioxidants such as tocopherols, tocotrienols and oryzanol (Sayre et
al., 1988; Ju and Vali, 2005). Recently, rice bran oil has gained attention because
of its unique health claims (Nicolosi et al., 1994) attributed by its high level of
unsaponifiable matter (Shin et al., 1997).
Crude rice bran oil contains high content of unsaponifiables (3-5%) which
are several times greater than that of commonly used vegetable oils (Rong et al.,
1997). According to CAC specification, the unsaponifiable matter in refined rice
bran oil should not be more than 3.5%; however, Japanese Agriculture Standards
specify it to be less than 5% (CAC, 2003). Currently, efforts are being made to
develop RBO with retained non-saponifiable components, while minimizing
levels of problematic free fatty acids (Ginsberg et al., 1998). There are several
mechanisms by which unsaponifiables improve serum bio-chemical profile such
as by interrupting the absorption of intestinal cholesterol rather increasing the
excretion of fat and neutral sterols (Kahlon et al., 1996; Nagao et al., 2001) and
increased fecal steroid excretion through interference with cholesterol
absorption (Ikeda et al., 1985; Sharma and Rukumini, 1986).
4.3.4. Fatty acid profile of RBO
Rice bran oil samples were analyzed for fatty acid profile through gas
chromatography (GC) and the results are presented in Table 14. The saturated
fatty acids i.e. myristic (C14:0), palmitic (C16:0), stearic (C18:0), and arachidic (C20:0)
acids in oil samples extracted from various rice brans were: unstabilized-RBO:
0.19, 17.70, 0.32, 0.50%; extrusion stabilized-RBO: 0.14, 18.37, 0.19, 0.46%;
microwave stabilized-RBO: 0.18, 16.80, 0.44, 0.50% and parboiled-RBO: 0.15,
19.80, 0.22, 0.47%, respectively. The investigated oils were found to contain a
high level of monounsaturated fatty acids (C18:1) i.e. 42.44 to 44.44%. The contents
of linoleic acid (C18:2) in the bran oils were 34.51 to 36.51% whereas the linolenic
acid (C18:3) contents were found to be 0.87 to 1.10%. The levels of saturated and
136
unsaturated fatty acids in the present analysis were comparable to the values
reported in the literature.
Rice bran oil has an excellent fatty acid profile. It has oleic acid (38.4%),
linoleic acid (34.4%) and linolenic acid (2.2%) as unsaturated fatty acids while
palmetic (21.5%) and stearic acid (2.9%) as saturated fatty acids (Rukmini and
Raghuram, 1991). The rice bran oil contains more than 75% unsaturated fatty
acids of which linoleic acid constitute about 35% (CAC, 2003). The saturated,
monounsaturated and polyunsaturated fatty acids are in the ratio of
approximately 1:2.2:1.5 (Shin and Chung, 1998; Krishna 2002). Three major fatty
acids palmitic, oleic and linoleic make up 90% of the total fatty acids of the bran
oil (Amarasinghe and Gangodavilage, 2004). Rice bran oil with Ideal
SFA/MUFA/PUFA ratio, is closest to WHO recommendation as compared to
other edible oils (Appendix V).
137
Table 14. Fatty acid composition (%) of rice bran oil samples
Fatty acids Un-RBO
C 14:0
C 16:0
C 18:0
C 18:1
C 18:2
C 18:3
C 20:0
0.190.007
17.700.41
0.320.017
43.331.341
36.211.05
1.090.08
0.500.016
S-RBO
0.140.005
18.370.57
0.190.007
44.041.35
35.651.08
1.100.04
0.460.015
MW-RBO
0.180.006
16.800.51
0.440.014
44.441.337
36.511.13
0.870.03
0.500.016
AR-RBO
0.150.003
19.800.41
0.220.011
42.441.312
34.511.04
0.990.01
0.470.011
Saturated fatty acids: C 14:0 (myristic acid); C 16:0 (palmitic acid); C 18:0 (stearic acid); C 20:0 (arachidic)
Mono-unsaturated fatty acid: C 18:1 (Oleic acid)
Poly-unsaturated fatty acids: C 18:2 (linoleic acid); C 18:3 (linolenic acid)
138
4.3.5. Antioxidant potential

A number of bioactive phytosterols have been identified in rice bran,
capable of inhibiting oxidation with additional health benefits (Appendix VI).
These include mainly oryzanol, tocopherol and tocotrienols. Rice bran oil
samples were analyzed for -oryzanol, tocopherols and tocotrienol contents (,
and ). Mean squares (Table 15) explicit significant variation among the
treatments.
4.3.5.1. Oryzanol
Means for oryzanol (Table 15a) showed that maximum level of -oryzanol
was present in oil extracted from microwave rice bran (703.42 g/g), followed by
extruded (690.17g/g) and parboiled rice bran (660.12g/g); whereas minimum
contents were detected in unstabilized rice bran (590.78g/g); might be due to
more refining losses. Among the stabilized rice brans, oil extracted from MWstabilized rice bran showed maximum oryzanol content due to better processing
conditions whereas comparatively lower oil content in extruded rice bran was
might be due to high withholding time during extrusion process (Shin et al.,
1997). Similarly, lower oryzanol content in PAR-RBO was might be due to severe
processing conditions before milling. Overall, oryzanol contents ranged from
590.78 to 703.42g/g in oil samples.
Oryzanol occurs in the unsaponifiable fraction and is named as it was first
isolated from rice bran oil (Oryza Sativa). The -oryzanol is composed of several
kinds of ferulic acids and has an effect similar to that of Vitamin E in promoting
growth, facilitating blood circulation and stimulating hormonal secretions (Rong
et al., 1997). The -oryzanol content in rice bran oil ranged from 1 to 3%
(Seetharamaiah and Prabhakar, 1986; Lloyd et al., 2000), depending on the rice
cultivar and analytical method. The most accessible natural source of -oryzanol
is rice bran. It contains about 9.8gkg-1 oryzanol (Xu and Godber, 2000; Fang et al.,
2003; Miller et al., 2003).
Table 15. Mean squares for antioxidants in rice bran oil samples
SOV
7581.97**
Tocopherol
574.75* 148.75** 425.0**
254.75**
834.062
128.466
20.1803
df
-oryzanol
Treatments
Error
15.036
139
4.488
Tocotrienol
393.0** 254.75**
46.129
5.3081
11
Total
**
Highly significant at P 0.01; * significant at P 0.05
Table 15a. Oryzanol in refined rice bran oil samples (g/g)

Antioxidants
-oryzanol
590.7825.75b
690.1730.08a
703.4230.67a
660.1228.77a
Un-RBO
ES-RBO
MW-RBO
PAR-RBO
Values are MeanSD for four rice bran oils, analyzed individually in triplicate
Un-RBO
Rice bran oil extracted from unstabilized rice bran
ES-RBO
Rice bran oil extracted from extrusion stabilized rice bran
MW-RBO
Rice bran oil extracted from microwave stabilized rice bran
PAR-RBO
Rice bran oil extracted from parboiled rice bran
Table 15b. Tocopherols in refined rice bran oil samples (mg/Kg oil)
Antioxidants
Un-RBO
24310.59b
813.53c
361.57d
ES-RBO
26611.59a
893.88b
431.87c
MW-RBO
27511.99a
984.27a
642.79a
PAR-RBO
25511.11ab
873.79bc
472.01b
Table 15c. Tocotrienol in refined rice bran oil samples (mg/Kg oil)
Antioxidants
Un-RBO
924.01c
1406.10b
411.79d
ES-RBO
1054.58b
1606.76a
502.18c
MW-RBO
1141.57a
1677.28a
632.75a
PAR-RBO
1004.97bc
1556.76a
552.40b
The concentration of -oryzanol in rice bran oil ranges from 115 to

780ppm, depending on the degree and method of processing (Rogers et al., 1993).
The -oryzanol is 13-20 times more than that of total tocopherols and tocotrienols
in rice bran (Bergman and Xu, 2003). It has been observed that about 20% of
unsaponifiable fraction in RBO is oryzanol (Rong et al., 1997); having high
140
melting point (137.5 to 138.5oC) resulting in improved thermal stability (Xu and
Godber, 2000). Initially, it was considered as single component but now proved
to be a mixture of at least 10 components (Xu and Godbar, 1999; Kim et al., 2001).
Complete role of -oryzanol as functional ingredient, has not yet been explored.
However, it reduces serum cholesterol in rats and hyperlipidemia in humans
(Seetharamaiah and Chandrasekhara, 1989; Yoshino et al., 1989).
4.3.5.1. Tocopherols and tocotrienols
Means for , and -tocopherols are presented in Table 15b.
Concentration of tocopherols homologs varied significantly among various rice
bran oil samples. Maximum levels of -tocopherol, -tocopherol and tocopherol were detected in oil extracted from MW-RBO (275, 98, 64 mg/kg),
followed by ES-RBO (266, 89, 43 mg/kg) and PAR-RBO (255, 87, 47 mg/kg),
respectively; whereas minimum level was found in Un-RBO (243, 81, 36 mg/kg).
The content of -, - and -tocopherols ranged from 243 to 275, 81 to 98 and 36 to
64 mg/kg, respectively; exhibited high vitamin E activity in the investigated oil
samples compared with other commonly used vegetable oils (Kao and Luh,
1991). The concentration of tocopherols ranged from 36 to 275 mg/kg, thus,
would be expected to contribute high oxidative stability to the oils during
storage (Rossell, 1991). Approximately 1.0% (v/v) of the unsaponifiable fraction
of RBO is -tocopherol. HPLC analysis of RBO showed that 1g of RBO contains
3.02mg of -tocopherol (Qureshi et al., 2000).
Concentration of tocotrienols differs significantly among various rice bran
oil samples (Table 15c). The highest levels of -tocotrienol, -tocotrienol and tocotrienol were detected in MW-RBO (114, 167, 63mg/kg) followed by ES-RBO
(105, 160, 50mg/kg) and PAR-RBO (100, 155, 55 mg/kg), respectively; whereas
least level was observed in Un-RBO (92, 140, 41mg/kg). The content of -, - and
-tocotrienol ranged from 92 to 114, 140 to 167 and 41 to 63mg/kg, respectively.
The concentration of tocotrienol ranged from 41 to 167mg/kg, in experimental
bran oil samples. The contents of -, - and -tocotrienols have proven their
141
worth against coronary diseases due to their anti-thrombotic properties and have
greater antioxidant potential than the tocopherols (Rossell, 1991; Rukmani, 1995).
RBO is a rich source of tocotrienols ranged from 72 to 1157ppm depending upon
different bran sources and commercial refining methods. Approximately 1.7%
(v/v) of the unsaponifiable fraction of RBO is tocotrienol (Deckere and Korver,
1996). Rice bran is a rich natural source of vitamin E; however, different
extraction methods can affect its recovery (Shanggong et al., 2007).
Rice bran contains over 300 mg/kg vitamin E (Shin et al., 1997). Human
physiological system and animals cannot synthesize this vitamin; they primarily
acquire tocols from plants. The structural differences of tocotrienols and
tocopherols influence their biological activities (Qureshi et al., 1996). Tocols
protects cell membrane by acting as a scavenger of free radicals (Nesaretnam et
al., 1998). In addition to health claims, antioxidants of rice bran oil have a
potential use as additives to improve the storage and frying stability of oils (Kim
and Godber, 2001). It has been observed that consumption of 240mg/day of
tocotrienols upto two years caused no adverse effects and they are safe even at
higher levels (Qureshi et al., 2001).
4.3.6. Selection of best sample

In present study, microwave stabilized rice bran was preferred on the
basis of better stability (FFA, POV and TBA no.), color of oil and high antioxidant
potential (oryzanol, tocopherol, tocotrienol). After selection, microwave
stabilized fullfat rice bran; its defatted portion and extracted oil were used for
efficacy studies and preparation of value added products.
4.4. Efficacy studies

In efficacy studies, selected oil alongwith its defatted portion and fullfat
rice bran samples were tested using experimental rats for safety evaluation and
to prove their health claims. The diets prepared from respective treatments
142
alongwith control were fed to four groups of Sprague Dawley rats (SD-rats) for a
period of 45 days and were evaluated for physical and hematological parameters.
The results are summarized as:
4.4.1. Physical parameters of rats
It is obvious from the mean squares (Table 16) that treatments and study
period showed a significant effect on feed intake, water consumption and gain in
body weight of SD-rats.
4.4.1.1. Feed intake
Means for feed intake in different groups of rats (per rat/day) have been
shown graphically in Fig. 1. It is apparent from the results that rats fed on RBO
diet had the highest feed intake (19.21g/rat/day) followed by rats on control diet
(17.85g/rat/day) and FFRB diet (16.88g/rat/day) while the lowest consumption
was observed in rats fed on diet containing DFRB (14.51 g/rat/day). Study
period (weeks) significantly affected the feed intake; highest consumption was
observed on 6th week (19.84g) while the lowest feed intake was observed in 1st
week (15.42g) followed by 4th week (15.73g) per rat per day. There was an
increasing trend for feed intake during first 3 weeks which slightly diminished
on 4th week and then further increased upto 6th week.
4.4.1.2. Water intake
Means for water intake in different groups of rats (per rat/day) have been
illustrated in Fig. 2. It is apparent from the results that highest water intake
(37.81mL/day) was observed in rats fed on diet containing RBO followed by rats
consuming FFRB (35.98mL/day) and DFRB (35.52mL/day). Lowest water intake
(31.85mL/day) was observed in control group. For water intake almost a parallel
143
Table 16. Mean squares for physical parameters of different groups of rats
SOV
Feed
intake
Water
intake
Gain in body
weight
Treatments
11.7271**
87.9719**
8.40602**
Study period (wk)
23.5655**
37.4444**
3.20624**
Error
15
0.51713
7.7369
0.22927
Total
23
** Highly significant at P 0.01
144
Control
FFRB
DFRB
RBO
Feedintake (g)
25.00
20.00
15.00
10.00
5.00
0.00
1
Weeks
Fig. 1. Feed intake in different groups of rats during 6 weeks (per rat/day)
Control
FFRB
DFRB
RBO
Water intake (mL)
60.00
50.00
40.00
30.00
20.00
10.00
0.00
1
Weeks
Fig. 2. Water intake in different groups of rats during 6 weeks (per rat/day)
Control
FFRB
DFRB
RBO
Weight gain (g)
12.00
10.00
8.00
6.00
4.00
2.00
0.00
1
Weeks
Fig. 3. Gain in body weight in different groups of rats during 6 weeks (per
rat/week)
145
trend was observed in all groups of rats during the entire study period.
However, rats fed on RBO, consumed more water throughout the experiment.
4.4.1.3. Gain in body weight
The means for gain in body weight in different groups of rats (per
rat/week) has been illustrated in Fig 3. It is evident from the results that highest
gain in body weight was observed in rats fed on diets containing RBO (7.24g per
rat/week) followed by control (7.14g per rat/week) and FFRB (6.86g per
rat/week) while the lowest gain was observed in DFRB (5.99g per rat/week)
group. The rats fed on rice bran oil gained higher body weight compared with
other treatments, from the initiation of study, concomitant with its higher feed
intake (Ahmed et al., 2007). The results with respect to study period (week)
showed that highest gain in body weight was observed in 4th week (8.79g)
followed by 5th week (7.61g) per rat per week. Initially, 4.35 to 8.79g (per
rat/week) gain in body weight was observed during 1st four weeks, later,
decreasing from 8.79 to 6.43g (per rat/week) in all groups of rats; however,
decrease was more pronounced in rats fed on defatted rice bran. Rats fed on
DFRB gained less body weight (16.11%) followed by FFRB group (3.21%) than
that of control; might be due to low feed intake from the initiation of the study. It
has already been recognized from various studies that dietary fiber may have
some potential in the management of weight loss. This effect is derived from the
potential influence of fiber on several aspects of food intake and nutrient
availability (Vahouny, 1982). The effects on weight loss are often deduced from
decreased caloric intake, satiety and increased fecal excretion of energy in the
form of fat and nitrogen (Leeds, 1985; Vahouny, 1985; Wisker et al., 1985). Rats
fed on fullfat and defatted rice bran showed less increase in body weight;
defatted rice bran was found to be more effective in weight loss programs.
146
4.4.1.4. Organs weight

The rats were dissected fortnightly and organs like liver, heart, lungs,
spleen, right and left kidney were weighed to determine the effect of the
individual experimental diets. Mean squares for organs weight of different
groups of rats explicit that treatments have non-significant effect on body organs
throughout the experimental period (Table 17). Mean values for liver weight of
different groups of rats (Table 18) explained that liver weight was unaffected by
various treatments. Statistically non-significant variations were observed in
control (4.21g/100g), FFRB (4.13g/100g), DFRB (4.10g/100g), and RBO
(4.07g/100g) group. Mean values for heart weight were found to be 0.38, 0.44,
0.34 and 0.39g/100g in control, FFRB, DFRB and RBO group, respectively. The
non-significant variations in the mean values for lungs weight were established.
Likewise, spleen weight of different groups of rats showed non-significant
differences (Table 18) due to selected diets. The means for spleen weight of rats
were 0.36, 0.34, 0.30 and 0.31g/100g for control, FFRB, DFRB and RBO,
respectively.
The means pertaining to right and left kidney weight showed nonsignificant variations of diets fed to different groups of rats. In case of left kidney,
mean values were 0.48, 0.45, 0.46, 0.49g/100g and for right kidney 0.46, 0.46, 0.46
and 0.49g/100g in rats fed on control, FFRB, DFRB and RBO, respectively.
Similarly, means for effect of study intervals on organ weight of different groups
of rats were non-significant throughout the study (Table 19). In a study, rats were
fed on experimental diet having 5 and 20% rice bran oil alongwith peanut oil as
control, showed non-significant differences with respect to the organ weights
between control and experimental groups (Purushothama et al., 1995).
Hematological clinical chemistry values for male SD-rats of different age groups
for organ weights like lungs, liver heart, spleen, kidney (right) and kidney (left)
were 0.66-1.16, 4.06-4.68, 0.44-0.54, 0.26-0.37, 0.45-0.53 and 0.43-0.55 g/100g,
respectively (TTL, 2008) which are also in harmony with the present findings.
147
Table 17. Mean squares for organs weight of different groups of rats
d
Liver
Hear
t
Lung
s
0.0013ns
0.0060ns
0.0100ns
0.004
0.0224ns
0.0447ns
0.0665ns
0.0499
SOV
Treatments
Study period
TxS
Error
Total
3
3
9
64
79
9.833ns
0.054ns
0.138ns
0.579
Sple
e
n
Kidney
-L
0.007ns
0.002ns
0.004ns
0.003
0.004ns
0.005ns
0.005ns
0.008
Kidney
-R
0.0026ns
0.0033ns
0.0118ns
0.0080
ns = Non-significant; study period (fortnightly)
Table 18. Effect of diets on organs weight of different groups of rats

Treatments
Control
FFRB
DFRB
RBO
Organ weight (g/100g)

Liver
Heart
Lungs
Spleen
Kidney L
Kidney R
4.210.12
4.130.11
4.100.05
4.070.06
0.380.02
0.440.04
0.340.01
0.390.03
1.190.07
1.140.06
1.250.04
1.200.02
0.360.03
0.340.01
0.300.02
0.310.02
0.480.02
0.450.02
0.460.02
0.490.01
0.460.02
0.460.03
0.460.02
0.490.02
Values are meansSEM
Table 19. Organs weight of different groups of rats during study periods
Study
Period
(days)
0
15
30
45
Organ weight (g/100g)

Liver
Heart
Lungs
Spleen
Kidney-L
Kidney-R
4.140.10
4.120.11
4.160.12
4.170.05
0.390.03
0.320.01
0.440.04
0.400.02
1.240.02
1.160.05
1.190.08
1.190.03
0.330.03
0.310.01
0.340.02
0.340.03
0.490.02
0.460.01
0.470.03
0.460.01
0.450.02
0.470.02
0.480.03
0.470.02
148
4.4.2. Renal and Kidney functioning tests

Serum was separated and analyzed for liver and renal functioning tests
using commercial kits. Means squares for serum urea and creatinine (Table 20)
exhibited non-significant differences with respect to diets and study periods.
Means for serum urea and creatinine concentration (Table 21) ranged from 15.40
to 15.55 and 0.61 to 0.64mg/dL, respectively, in rats fed on experimental diets for
a period of 45 days. Means for ALP (Alkaline phosphatase), ALT (Alanine amino
transferase) and AST (Aspartate amino transferase) ranged from 260.57 to 269.60,
107.00 to 108.60 and 83 to 85 U/L, respectively (Table 21). It is apparent from the
results that AST and ALT activities were slightly reduced with fullfat rice bran,
defatted rice bran and rice bran oil as compared to normal basal diet indicate that
these treatments can alleviate the damage induced by serum cholesterol (Ha et
al., 2005). However, an increase in enzymes concentrations was noted during
various study periods, due to progressive aging phenomenon (Table 22).
Petterino and Storino (2006) also observed similar values for these parameters
while conducting pre-clinical toxicity study using SD-rats. All the values were
within safe limits reported for SD-rats.
It was concluded that diets prepared from fullfat & defatted rice bran and
rice bran oil imparted no adverse effects in SD-rats during study, showing their
suitability for product development. Toxicological studies of rice bran oil by the
food safety evaluation protocol of WHO/FDA carried out in rats; indicated the
safety of RBO for human consumption (Rukmini, 1988).
4.4.3. Serum biochemical profile
Mean squares of serum biochemical profile in

relation to cholesterol, LDL, triglycerides and glucose
(Table 23) showed significant variations in rats fed on
various experimental diets except for HDL whereas study
149
intervals were found to be significant only with respect to

cholesterol and LDL.
Table 20. Mean squares for serum kidney and liver function tests
SOV
Treatments
Study period
TxS
Error
Total
ns
df
3
3
9
64
79
Urea
0.34097ns
0.10587ns
1.05822ns
1.79386ns
Creatinine
0.00500ns
0.00735ns
0.00238ns
0.00286
ALP
ALT
AST
15.1496ns
282.212ns
218.359ns
527.276
46.8465ns
10.3185ns
84.1880ns
87.4762
112.083ns
13.7500ns
66.8055ns
53.3752
= Non-significant; Treatment (T); Study period (S)
Table 21. Effect of diets on serum kidney and liver function tests in different
groups of rats
Treatme
nts
Control
FFRB
DFRB
RBO
Urea
(mg/dL)
15.400.09
15.470.23
15.550.24
15.550.24
Creatini
ne
(mg/dL)
0.610.01
0.640.01
0.630.04
0.620.01
ALP
(U/L)
260.573.61
266.512.13
269.603.49
265.073.49
ALT
(U/L)
108.001.97
108.602.12
107.302.12
107.001.32
AST
(U/L)
85.002.65
83.251.55
81.501.94
83.002.38
Values are meansSEM
Table 22. Serum kidney and liver function tests in different groups of rats
during study periods
Study
Period
(days)
0
15
30
45
Urea
(mg/dL)
15.580.26
15.580.13
15.300.11
15.520.26
Creatini
ne
(mg/dL)
0.610.03
0.610.01
0.640.02
0.630.01
150
ALP
(U/L)
266.515.87
265.752.77
264.931.13
264.561.85
ALT
(U/L)
105.800.84
108.802.24
107.201.63
109.102.18
AST
(U/L)
82.502.06
82.502.47
82.501.44
83.252.56
4.4.3.1. Cholesterol
Means for serum cholesterol (Table 24) showed significant variations in
different groups of rats fed on various diets. Maximum serum cholesterol was
found to be 93.77mg/dL in control group followed by 89.10 and 86.72 mg/dL in
rats fed on diets containing defatted rice bran and rice bran oil, respectively.
Lowest serum cholesterol was observed in rats fed on fullfat rice bran (85.84
mg/dL). The results indicated that fullfat rice bran is more effective in
cholesterol lowering than either rice bran oil or defatted rice bran; certainly due
to presence of comparatively high levels of tocopherol, tocotrienol and oryzanol
as well as unsaponifiables which are reduced during refining process of bran oil.
It was further observed that FFRB showed a significant reduction (12.92%) in
serum cholesterol of SD-rats followed by RBO (9.66%) and DFRB (6.44%) (Fig. 4).
From the present research, it was concluded that there was 6.44 to 12.92%
reduction in cholesterol by the addition of these treatments. Normal levels of
serum cholesterol were 100.39 and 102 mg/dL in rats as observed by Khosla et al.
(1995) and Rehman et al. (2001). Hematological clinical chemistry values for male
SD-rats of different age groups for cholesterol, ranged from 75.4 to 134.1mg/dL
(TTL, 2008).
Scientific studies support recommendations to increase dietary fiber as
part of hyperlipidemia treatment. Bran fiber offers a protective effect during
cholesterol metabolism and reduces the circulating cholesterol levels (Gerhardt
and Gallo, 1998). Diets containing 10% total dietary fiber from intact fullfat
stabilized rice bran resulted significant decline in cholesterol compared with
151
cellulosic control diet (Kahlon et al., 1985). Stabilized fullfat and defatted rice
bran supplemented with 0.5% cholesterol, resulted significant reduction in
cholesterol concentrations in animals consuming fullfat stabilized (Kahlon et al.,
1990). In cholesterol-fed hamsters, diet containing 11, 22, 33 and 44% rice bran
resulted in plasma cholesterol reductions of 8, 11, 15 and 21% respectively
(Kahlon et al., 1992).
Table 23. Mean squares for lipid profile and serum glucose in different
groups of rats
SOV
Treatments
Study period
TxS
Error
Total
3
3
9
64
79
Choleste
rol
252.638**
140.601*
47.0561ns
59.0513
HDL
LDL
22.337ns
2.2379ns
1.5422ns
12.664
197.373**
155.895**
33.4068ns
8.34113
Triglycerid
es
791.0339**
48.74435ns
89.26417ns
39.43058
Glucose
318.580*
12.4733ns
130.689ns
88.8308
**Highly significant at P 0.01; * Significant at P 0.05, ns = Non-significant
Table 24. Effect of diets on serum lipid profile and glucose (mg/dL) in different
groups of rats
Treatments
Control
FFRB
DFRB
RBO
Cholesterol
93.771.03a
85.842.61b
89.101.28b
86.722.00b
HDL
39.630.33
40.870.29
41.860.20
41.840.25
LDL
37.850.57a
31.582.49bc
33.421.25b
31.261.03c
Triglycerides
81.462.55a
66.952.15c
71.571.12b
69.891.87bc
Glucose
113.162.61
107.002.36
104.602.05
112.350.83
Table 25. Serum lipid profile and glucose (mg/dL) in different groups of rats during
various study periods
Study
Period
(days)
0
15
30
Cholesterol
92.510.16a
89.101.19b
87.132.48b
HDL
40.640.27
40.990.62
41.130.67
LDL
37.260.31a
33.551.19b
32.120.16b
Triglycerides
Glucose
73.050.78
74.052.42
72.414.98
110.561.08
109.551.13
108.803.25
70.355.46
108.204.52
45
86.693.49b
41.440.47
31.182.76c
152
15 Days
% Decrease in cholesterol
14.00
30 Days
45 Days
12.92
12.00
9.66
9.69
10.00
8.59
8.00
6.00
6.44
4.76
6.43
4.29
4.00
2.15
2.00
0.00
FFRB
DFRB
RBO
Treatments
Fig. 4. Percent decrease of cholesterol in different groups of rats
153
Similarly, total cholesterol was significantly lower in chicks fed on fullfat

rice bran diet (Newman et al., 1992). The removal of fat from rice bran, resulted
reduction in cholesterol-lowering ability of rice bran, suggesting that lipid
fraction is necessary for maximum hypocholesterolemic effects.
There are several cholesterol lowering mechanisms coupled with rice
bran. It has been observed that rice bran lowers the cholesterol by increasing
short chain fatty acid production in the cecum by hindering cholesterol
absorption due to a change in intestinal fluid viscosity or by directly inhibiting
cholesterol
synthesis
in
the
liver
(Fukushima
et
al.,
1999).
The
hypocholesterolemic effects of rice bran may be attributed to the unsaponifiable

fraction of rice bran oil, primarily phytosterol, tocols (tocopherols and
tocotrienols), -oryzanol, triterpene alcohol and other minor compounds
(Sharma and Rukmini, 1987; Yoshino et al., 1989; Nicolosi et al., 1991).
Several studies in laboratory animals and humans, stipulated that
bioactive
components
of
RBO
acted
synergistically
to
induce
hypocholesterolemic effects (Rukmini and Raghuram, 1991; Lichtenstein et al.,

1994; Sugano and Tsuji, 1997; Vissers et al., 2000). Similarly, significant reduction
in serum total cholesterol and elevated tendency in HDL-cholesterol were found
in rats consuming cholesterol-free diets with 10% rice bran oil (Seetharamaiah
and Chandrasekhara, 1989).
A number of studies in humans and animals have proved that RBO is
effective in lowering plasma cholesterol levels. In some cases, RBO lowered
154
plasma cholesterol more effectively than other vegetable oils rich in linoleic acid
(Rukmini and Raghuram, 1991); might be due to occurrence of -oryzanol and
perhaps tocotrienols (Rukmini and Raghuram, 1991; Nicolosi et al., 1994). The oryzanol is a unique natural antioxidant occurring only in rice bran. It lowers
cholesterol by inhibiting activity of cholesterol-esterase; by the modulation of
cholesterol esterase and acyl-CoA-cholesterol-acyltransferase (Rukmini and
Raghuram, 1991); by partial splitting off the sterol moiety of -oryzanol from the
ferulic acid in the small intestine (Sugano and Tsuji, 1997); by increasing faecal
excretion of cholesterol and its metabolites (Seetharamaiah et al., 1990; Wilson et
al., 2007) and in some cases, direct inhibition of lipid metabolism (Sakamoto et al.,
1987).
Tocotrienols are naturally present in rice bran and palm oil and the later
contains a lower level than found in rice bran oil. They inhibit the liver
microsomal enzyme HMGCo-A reductase (Qureshi and Qureshi, 1993), the key
enzyme involved in the endogenous synthesis of cholesterol and helps to lower
circulating cholesterol. Inhibition of enzyme, ACAT (Acyl coenzyme A; Acyl
transferase), by -oryzanol results in lowered LDL-cholesterol synthesis and
enrichment of HDL-cholesterol and increased cholesterol excretion. The enzyme
inhibitions by these unique rice bran phytonutrients, tocotrienols and -oryzanol,
help to reduce the elevated cholesterol and other lipid parameters, thereby
reducing the risk of CVD. In an experimental study, significant reduction in
cholesterol (48-54%) and LDL (39-74%) was observed in a dose dependent
manner (Minhajuddin et al., 2005).
There
are
several
mechanism
of
cholesterol
reduction
through
tocopherols and tocotrienols such as inhibition of cholesterol oxidation (Xu et al.,

2001); inhibition of HMGCo-A-R, a key enzyme in the endogenous synthesis of
cholesterol, via increasing the controlled degradation of reductase protein and
decreasing the efficiency of the translation of HMG-CoA-R messenger RNA
(Parker et al., 1993; Khor et al., 1995); inhibit the activity of 3-hydroxy-3155
methylglutaryl-coenzyme A (HMG-CoA) reductase, the liver enzyme that is

critical to the rate at which cholesterol is synthesized (Khor and Ng, 2000);
inhibition of cholesterol synthesis by suppressing HMG-CoA reductase activity
(Pearce et al., 1992; Parker et al., 1993; Qureshi et al., 2000) and via decrease in
serum total and LDL- cholesterol by inhibiting the hepatic enzymic activity of hydroxy--methylglutaryl coenzyme A (Qureshi et al., 2002).
The phytosterols present in rice bran oil like campesterol, -sitosterol and
stigmasterol, have been proven effective in lowering plasma total and LDLcholesterol without affecting HDL-cholesterol due to similarities in structures of
plant sterols and cholesterol resulting more competition with cholesterol in the
micelles (Weststrate and Meijer, 1998). It is generally assumed that plant sterols
inhibit intestinal absorption of dietary and biliary cholesterol, because of the
structural similarities with cholesterol (Wilson et al., 2000). Unsaponifiables in
rice bran oil act to lower the cholesterol by interrupting cholesterol absorption in
the gut (Nagao et al., 2001).
The present findings and supportive review suggest that fullfat stabilized
rice bran and its oil hold the potential to lower blood cholesterol in
hypercholesterolemic people. There is also a possibility to incorporate defatted
rice bran for the purpose.
4.4.3.2. High density lipoprotein (HDL)
Means for HDL-cholesterol in different groups of rats indicated in Table
24 were 39.63, 40.87, 41.86 and 41.84mg/dL for rats fed on control, FFRB, DFRB
and RBO diets, respectively. There were non-significant variations among the
experimental diets as compared to control. Furthermore, it was observed that
FFRB showed slight improvement (3.11%) in serum HDL of SD-rats followed by
RBO (2.91%) and DFRB (2.15%) (Fig. 5). There were non-significant variations in
different groups of rats with respect to study period (Table 25). Normal levels of
serum HDL were found to be 41.00 and 42.54 mg/dL in rats (Khosla et al., 1995;
Rehman et al., 2001).
156
HDL-cholesterol is considered as a good form of cholesterol (James and

Claude, 1998). It reduces the amount of deposited cholesterol in the endothelium
by retrieving cholesterol from peripheral cells and other lipoproteins to the liver
for excretion in the bile (Ha et al., 2005). High levels of HDL prevent LDL
accumulation in the walls of the arteries.
15 Days
3.50
% Increase in HDL
45 Days
3.11
2.91
2.84
3.00
2.50
2.15
2.08
1.79
2.00
1.50
30 Days
1.50
1.22
1.17
1.00
0.50
0.00
FFRB
DFRB
RBO
Treatments
Fig. 5. Percent increase of HDL in different groups of rats
157
In present research, HDL level was non-significant in rats fed on diets

containing FFRB, DFRB and RBO. In a similar study, Brown et al. (1999)
observed non-significant variations in HDL by feeding 2-10g/day soluble
fiber. In animal model, mice fed on rice bran, soybean fiber, oat and barley bran
with 0.06% added cholesterol demonstrated higher HDL to total cholesterol
ratios in animal consuming rice bran diet (Hundemer et al., 1991). In a similar
study, benefits of bran addition from rice, oats, corn and wheat in diets, fed to
hamsters were evaluated at relatively high cholesterol level (0.3%). Animals fed
on rice bran had significantly lower LDL levels and the highest HDL to total
cholesterol ratios when compared to all other bran diets (Kahlon et al., 1998). In a
study, chicks were fed on fullfat rice bran and defatted rice bran with 0.5% added
cholesterol. Significant differences were observed in HDL values for all diets,
however, fullfat rice bran exhibiting the highest. Fullfat rice bran appeared to
increase HDL and lower LDL in chicks (Newman et al., 1992).
Rice bran oil and its components significantly improve the plasma profile
in rats. Significant reduction in serum total cholesterol and elevated tendency in
HDL-cholesterol were found in rats consuming cholesterol-free diets with 10%
rice bran oil (Seetharamaiah and Chandrasekhara, 1989). Similarly, rats fed on
experimental diet containing rice bran oil, showed 20% increase in HDL within
18 weeks, compared to control (Purushothama et al., 1995). Rice bran oil plus
safflower oil and sunflower oil in 70:30 ratios showed, significantly, increased
HDL in animals fed on cholesterol and cholesterol free diet (Sunitha et al., 1997).
158
Likewise, HDL level was significantly higher in the rice bran oil group, resulting
in a higher HDL to total cholesterol ratio (Koba et al., 2000).
Male SD-rats were fed on normal, high-cholesterol and high-cholesterol
diet supplemented with the concentrated bioactive components from rice bran
oil (BRBO) for 4 weeks. Serum HDL was significantly increased in rats fed on
BRBO group; it recovered the activities of serum aspartate amino transferase
which was elevated in rats by high cholesterol diet (Haa et al., 2005). Despite
some variations in fatty acid profile, rice bran oil resulted increase in HDL in rats
fed on diets supplemented with the unsaponifiable matter from rice bran oil
(Sharma and Rukmini, 1987).
4.4.3.3. Low density lipoprotein (LDL)
Means for LDL in different groups of rats (Table 24) explicated significant
variations among the rat groups fed on selected diets. Maximum LDL was
37.85mg/dL in control group followed by 33.42, 31.58 and 31.26 mg/dL in
groups fed on DFRB, RBO and FFRB, respectively. Percent decline of LDL in
different groups of rats (Fig. 6) showed maximum reduction (29.74%) in rats fed
on FFRB followed by RBO (23.18%) and DFRB (15.95%).
LDL cholesterol is considered as bad form of cholesterol (James and
Claude, 1998). In a study, rats were fed on experimental diet containing rice bran
and peanut oil; LDL was found to be lowered in rice bran oil fed groups
(Purushothama et al., 1995). In a different study, LDL levels dropped in rats fed
on diets supplemented with the unsaponifiable matter of rice bran oil (Sharma
and Rukmini, 1987).
The hypolipidemic response of rice bran oil was investigated in nonhuman primates fed on semi-purified diets containing blends of oils including
rice bran oil. The supplementation of RBO in the diet, significantly, influenced
serum TC and LDL, resulting upto 40% reduction in LDL without affecting HDL
levels (Nicolosi et al., 1991). In a study, hyperlipidemic subjects were
administered with -oryzanol (300mg/day) for three month. A significant
159
decrease in plasma TC and LDL was observed in both hypercholesterolemic and

hypertriglyceridemic patients (Yoshino et al., 1989).
4.4.3.4. Triglycerides (TG)
Means for triglycerides in different groups of rats (Table 24) indicated
significant variations among the rats fed on experimental diets. Maximum
triglyceride concentration was found to be 81.46mg/dL in control group
followed by defatted rice bran (71.57mg/dL) and rice bran oil (69.89mg/dL);
160
15 Days
30 Days
45 Days
35.00
29.74
% Decrease in LDL
30.00
23.99
25.00
21.21
20.00
15.00
15.95
12.21
23.18
17.26
11.54
10.00
6.62
5.00
0.00
FFRB
DFRB
RBO
Treatments
Fig. 6. Percent decrease of LDL in different groups of rats
161
whereas minimum level (66.95mg/dL) was noticed in rats fed on fullfat rice
bran. Percent decrease of triglycerides (Fig. 7) in different groups of rats showed
maximum reduction (13.44%) in rats fed on FFRB followed by RBO (10.63%) and
DFRB (6.93%). Normal levels of serum triglycerides were 78 mg/dL in rats
(Khosla et al., 1995). Rats fed on rice and wheat bran showed significant
reduction in liver cholesterol and triglycerides; rice bran diet increased LDL
receptor activity in the liver more than the wheat bran, hence, effectively
lowering plasma cholesterol levels (Topping et al., 1990).
Similarly, triglycerides were significantly lowered in chicks fed on fullfat
rice bran diet (Newman et al., 1992). In another study, changes in plasma lipid
levels were observed in men with slightly above normal cholesterol levels
providing test diets containing 35g/day of wheat bran, 60g/day of rice bran or
95g/day of oat bran with constant amount of total dietary fiber i.e. 11.8g/day.
The highest decrease in plasma triglycerides were found in rice bran group
compared to wheat bran diet (Kestin et al., 1990).
Rice bran oil and its components significantly improved the plasma profile in
rats. Rats were fed on diets containing 10% rice bran oil showed significant
decrease in TG (Sharma and Rukmini, 1986). Addition of oryzanol to rat diets
containing rice bran oil was associated with lower cholesterol levels compared to
rat diets containing rice bran oil alone (Seetharamaiah and Chandrasekhara,
1989). In animal study, diets were enriched with 1% cholesterol and 0.15% bile
salts and either devoid of oryzanol or oryzanol enriched @ 0.2, 0.5, 1.0 and 2.0%.
A significant decrease in plasma triglyceride levels was seen in the 0.5% oryzanol
group than rats fed on oryzanol free diet (Seetharamaiah and Chandrasekhara,
1989). The hypocholesterolemic effects of rice bran oil were evaluated in
moderately non-obese hyperlipoproteinemic human subjects fed on rice bran oil
for a longer period. For comparison, the control group continued use of palm or
groundnut oils. The rice bran oil treated patients showed 16 to 25% decrease
162
15 Days
% Decrease in TG
16
30 Days
45 Days
13.44
14
10.63
12
10
7.05
6.93
6
4
2
6.53
3.35
1.72
1.29
0.42
0
FFRB
DFRB
RBO
Treatments
Fig. 7. Percent decrease of triglyceride in different groups of rats
163
in plasma total cholesterol and 32 to 35% in triglycerides after 15 to 30 days of

treatment as compared to the control group (Raghuram et al., 1989).
4.4.3.5. Glucose
Means for glucose concentration in different groups of rats have been
presented in Table 24; significant variations were observed among different
groups of rats fed on various diets. Maximum glucose concentration
(113.16mg/dL) was found in control group followed by 112.35 and 107.00mg/dL
in groups fed on rice bran oil and fullfat rice bran, respectively. Lowest glucose
concentration (104.60mg/dL) was observed in rats fed on defatted rice bran.
Percent decrease of glucose concentration in different groups of rats compared to
baseline values (Fig. 8), depicted that fullfat rice bran showed maximum
reduction (9.22%) followed by defatted rice bran and rice bran oil i.e. 8.09% and
1.43%, respectively. From the present investigation, it was concluded that there
was 1.43 to 9.22% decline in glucose by the addition of selected treatments.
Diabetes is a disorder where glucose metabolism in the body is impaired.
Rice bran is rich natural source of B-complex vitamins; niacin and niacinamide
that are important for intracellular energy production and regulate blood sugar
levels in diabetes (Urberg and Zemel, 1987).
Rice bran hemi-cellulose has been shown to reduce the high blood sugar
(Hikino et al., 1988). Tocopherols also improve glucose metabolism as it has been
shown to inhibit glycosylation in diabetic patients (Ceriello et al., 1991). Dietary
rice bran has been proved to improve glycemic responses in rats with
streptozotocin induced diabetes (Lai et al., 2001). Rice bran is rich in minerals like
magnesium, phosphorus and potassium which are required for proper glucose
metabolism. The protein; -6 and -3 fatty acids also contribute to the regulation
of glucose metabolism. Rice bran in the diet improves pancreatic insulin
production (McPeak et al., 2001) and decreases hepatic glucose levels through
increased glucose uptake and increased muscle glycogen synthesis. Increased
insulin receptor activity may also be a reason for glucose modulation in diabetes.
164
15 Days
% Decrease in glucose
45 Days
9.22
10
8.09
7.80
5.88
6
4
30 Days
3.55
1.47
1.43
0
-2
FFRB
DFRB
-4
RBO
-1.43
-2.14
Treatments
Fig. 8. Percent decrease of glucose in different groups of rats
165
Rice bran in the diet appears to improve insulin utilization resulting in

decreased fasting glucose levels. This appears to be the result of phytonutrients,
antioxidants, vitamins and minerals in diabetic subjects. Therefore this natural
product can be used as a diet therapy in diabetic subjects to regulate glucose
metabolism.
4.4.3.6. Serum proteins
Mean squares for serum protein, albumin, globulin and A/G ratio in
different groups of rats (Table 26) showed non-significant variations in rats fed
on experimental diets during different study intervals. Means for serum total
protein concentration in different groups of rats have been shown in Table 27.
The protein content in rats fed on experimental diets ranged from 6.15 to
6.33g/dL. Means for serum protein were 6.15, 6.28, 6.33, and 6.20g/dL for
control, FFRB, DFRB and RBO group, respectively. Serum protein values found
in different groups of rats in the present investigation are in close association
with hematological clinical chemistry values for male SD-rats that ranged from
6.3 to 8.2 g/dL (TTL, 2008). Rehman et al. (2001) also reported 6.28g/dL total
protein in normal rats. There was non-significant increase in protein
concentration with study intervals (Table 28). At initiation of study, total protein
concentration was 6.03g/dL which increased to 6.20g/dL at the end of study.
Maximum serum albumin concentration was found to be 3.09g/dL in rats
fed on defatted rice bran, followed by 3.05g/dL in rats fed on FFRB that differed
non-significantly from each other. Overall, serum albumin concentration in
different groups of rats ranged from 2.93 to 3.09 g/dL. The hematological clinical
chemistry values for male SD-rats ranged from 3.4 to 3.7g/dL, respectively (TTL,
2008). Likewise, globulin (g/dL) and A/G values ranged from 2.88 to 2.97 g/dL
and 1.01 to 1.04, respectively, during a 45 days feeding trial (Table 27). Similar
findings were reported by Petterino and Storino (2006) while feeding SD-rats
with controlled diet for a period of thirteen weeks to establish a clinical
chemistry reference data for researchers and regulatory agencies.
166
Table 26. Mean squares for serum proteins in different groups of rats
SOV
Treatments
Study period
SxT
Error
Total
ns
d
3
3
9
64
79
Prot
ein
0.11975ns
0.09576ns
0.14075ns
0.291
Alb
umin
0.1070ns
0.0686ns
0.0174ns
0.0684
Glo
bulin
0.04581ns
0.09355ns
0.03764ns
0.06325ns
A/G
0.0022ns
7.6667ns
0.00894ns
0.00818
= Non-significant
Table 27. Effect of diets on serum proteins in different groups of rats

Treatme
nts
Control
FFRB
DFRB
RBO
Protein
(g/dL)
6.150.10
6.280.09
6.330.09
6.200.20
Albumin
(g/dL)
2.930.01
3.050.04
3.090.06
2.970.02
Globulin
(g/dL)
2.890.04
2.960.04
2.970.04
2.880.07
A/G
1.010.01
1.030.02
1.030.02
1.040.02
Table 28. Serum proteins in different groups of rats during study periods
Study
Period
(days)
0
15
30
45
Protein
(g/dL)
6.030.05
6.080.13
6.400.07
6.450.05
Albumin
(g/dL)
2.970.02
2.970.03
3.020.03
3.090.07
167
Globulin
(g/dL)
2.860.02
2.900.07
2.930.04
3.020.02
A/G
1.040.01
1.030.02
1.030.02
1.020.23
4.5. Product Development

After efficacy studies, value added baked products were prepared.
Primarily rice bran oil was supplemented in cookies and later fullfat and defatted
rice bran was supplemented in wheat flour to prepare fortified flours; to be used
further for the preparation of supplemented cookies and leavened pan bread.
4.5.1. Preparation of Rice Bran Oil Cookies
Rice bran oil contains high levels of phytosterols, gamma-oryzanol,
tocotrienols as well as tocopherols (Taylor et al. 1996) which impart resistance to
thermal oxidation. The high oxidative stability of RBO makes it preferred oil for
baking and frying applications (McCaskill and Zhang, 1999; Semwal and Arya,
2001). In this study selected rice bran oil (MW-RBO) was evaluated for its baking
performance in various combinations by replacing normal shortening @ 20, 40,
60, 80 and 100% (Table 1) for preparation of cookies. Results regarding physical
& chemical analysis and sensoric attributes are discussed as below:
Physical analysis of cookies is important from both consumers as well as
manufacturers point of view. The spread of the cookies should according to the
specifications set by the manufacturers. Too much elasticity in the gluten and
dough will spring back to give thicker cookies due to smaller diameter. Similarly,
in case of too little elasticity, dough may flow after molding, resulting in thin
cookies with large diameter.
Cookies were analyzed for width, thickness and spread factor at 0, 30 and
60 days storage. Mean squares (Table 29) explicit that treatment has highly
significant effect on physical parameters of cookies due to replacement of
different levels of RBO with normal shortening while storage and interaction had
non-significant effect.
Table 29. Mean squares for physical analysis of RBO cookies

df
SOV
Treatments
(T)
Thickn
Width
ess
45.63432**
2.486833**
168
Spread
factor
148.392**
Storage (S)
SxT
Error
Total
**
2
10
36
53
0.065ns
0.04465ns
6.039179
0.0139352ns
3.0185x10-4ns
0.0398
0.08225ns
0.05056ns
8.599
Table 30. Physical analysis of RBO cookies

Treatments
Width
Thickne
ss
T0
T1
T2
T3
T4
T5
44.080.18a
43.260.01a
40.370.01b
39.360.01b
38.820.01b
39.270.01b
Spread
factor
9.250.02c
9.370.01c
9.360.02c
9.930.01b
9.970.02b
10.620.02a
47.670.13a
46.160.07a
41.470.07b
40.670.04b
39.480.06bc
36.980.08c
Values are meansSEM; Means carrying same letters in a column for each factor do not differ
significantly
T0 = Normal shortening 100%; T1 = RBO 20%; T3 = RBO 40%; T3 = RBO 60%; T4 = RBO 80%;
T5 = RBO 100%.
Table 31. Effect of storage on physical analysis of RBO cookies

Storage
Width
Thickness
factor
(days)
0
30
60
Spread
40.900.94
40.880.94
40.790.88
9.780.22
9.750.21
9.720.21
42.021.69
42.111.67
42.151.63
4.5.1.1.1. Width (W)

The data concerning the means for effect of various treatments on width
of cookies (Table 30) showed decreasing trend with the proportionate increase of
169
RBO throughout the study. The results revealed that at 0 day, T0 (100% NS)
exhibits maximum width 44.08 mm while T5 (100% RBO) showed minimum
width 39.27 mm. The remaining treatments T1 (43.26 mm), T2 (40.37 mm), T3
(39.36 mm) and T4 (38.82 mm) followed the similar decreasing trend. It is quite
clear that enhancement in the level of RBO gradually decreased the width of the
cookies. However, there was non-significant effect of storage on width of cookies
(Table 31). Means for storage showed that width was 40.90 mm at 0 day and
40.79 mm after 60 days. These results are in corroborated with the findings of
Sharif et al. (2005) who reported a decrease in width of cookies as a result of
replacement of normal shortening with RBO. However, increase in width was
noted by the use of different levels of emulsifiers in cookies (Leelavathi and Rao,
1993; Patel and Rao, 1996).
4.5.1.1.2. Thickness (T)
The data regarding the means for effect of various treatments on thickness
of cookies (Table 30) elucidated increasing trend with progressive increase in
RBO. The results showed that at 0 day, T0 (100% NS) exhibited minimum
thickness 9.25 mm while T5 (100% RBO) showed maximum thickness 10.62 mm.
The remaining treatments T1 (9.37 mm), T2 (9.36), T3 (9.93 mm) and T4 (9.97 mm)
followed the same increasing trend. It is quite clear that with increased levels of
RBO, thickness of cookies was improved whereas storage has been found to
exhibit non-significant effect (Table 31). Means for storage indicated that
thickness was 9.78mm at 0 days and 9.72mm after 60 days. These results are
consistent with previous findings of Sharif et al., (2005) who also reported an
increase in thickness by replacing normal shortening with RBO.
4.5.1.1.3. Spread factor

Spread factor is the ratio, depends upon the values of width and
thickness. The data regarding the effect of rice bran oil substitution on spread
factor of cookies is presented in Table 30. There was a decreasing trend in the
spread factor of cookies with the proportionate increase of RBO. The spread
factor of cookies, prepared from different treatments ranged from 36.98 to 47.67.
Means for spread factor demonstrated non-significant differences in T0 (100%
normal shortening) and T1 (20% RBO); T2 (40% RBO), T3 (60% RBO) and T4 (80%
RBO); T4 (80% RBO) and T5 (100% RBO). Maximum value (47.67) for spread ratio
was recorded in cookies prepared from 100% normal shortening whereas
minimum (36.98) in cookies from 100% rice bran oil. However, rice bran oil
replacement upto 60% level was found to be appropriate in cookies. Spread
factor varies from 42.02 to 42.15 at 0 to 60 days storage of cookies (Table 31). The
spread factor is also affected by different recipe ingredients (shortening, sugar,
sodium chloride, sodium bicarbonate and water), however shortening improved
cookies spread factor in the presence of high sugar concentration (Singh et al.,
2002). In another study, Patel and Rao (1996) investigated effects of sugar, fat and
170
emulsifiers on cookies prepared from blends of wheat and blackgram flours and
reported increase in spread factor with increasing addition of sugar and fat.
Similarly, use of emulsifiers results in significant increase in spread ratio of
cookies (Leelavathi and Rao, 1993).
The cookies were analyzed for moisture, crude protein, crude fat, crude
fiber, ash and nitrogen free extract (NFE) to find out the impact of RBO
replacement on composition of cookies during storage. Means squares in Table
32 explicated that RBO substitution had non-significant effect on proximate
composition of cookies; however, during storage, moisture, fat and protein
contents of cookies were significantly affected.
171
The moisture content is of great importance for many scientific and technical
reasons. Means for proximate composition of cookies (Table 33) showed nonsignificant effect of RBO replacement with normal shortening. The moisture
content, crude protein, crude fat, crude fiber, ash and NFE content ranged from
3.77-3.86, 7.49-7.53, 20.72-21.11, 0.25-0.31, 0.51-0.55 and 66.75-67.12%,
respectively. However, during 60 days storage, there was significant increase in
moisture content of cookies (Table 34). In freshly prepared cookies, the average
moisture content was 2.95%, which was gradually increased to 3.64 and 4.87%
after 30 and 60 days of storage, respectively. This phenomenon of moisture
absorption during storage is also supported by Wade (1988), ultimately affecting
sensory attributes of cookies during storage. When cookies are sealed in moisture
proof packaging, the small amount of moisture present in the atmosphere within
the package rapidly come in equilibrium with the product. When cookies are
exposed to outer environment, they quickly absorb moisture. Moreover, wheat
flour is hygroscopic in nature, resulting increase in moisture content of cookies.
Likewise, water is produced as a result of breakdown of chemical constituents of
cookies during storage which also enhanced moisture content of the product. The
results of present study are supported by the previous findings of the various
researchers (Leelavathi and Rao, 1993; Rao et al., 1995; Pasha et al., 2002; Butt et
al., 2004; Sharif et al., 2005).
Means for protein content of different cookies prepared from various levels of
rice bran oil (Table 33) illustrated non-significant differences of RBO substitution in
cookies. The crude protein content ranged from 7.49 to 7.53% in all treatments. Whilst,
during 60 days storage, there was slight decrease in protein content of cookies (Table
34). In freshly prepared cookies, the average protein content was 7.90%, which
reduced to 7.47 and 7.15% after 30 and 60 days storage, respectively. The decrease in
protein during storage was might be due to the increase in moisture content that
accelerates proteolytic activity in cookies. The present investigation is closely in lines
with the findings of (Pasha et al., 2002; Butt et al., 2004; Sharif et al., 2005).
Table 33. Proximate composition of RBO cookies
Treatme
nts
T0
T1
T2
Moist
ur
e
Prote
in
3.800.56
3.810.55
3.850.55
7.520.21
7.500.21
7.510.22
Fat
20.720.64
20.810.60
20.910.57
172
Fiber
Ash
0.290.01
0.290.01
0.270.01
0.540.01
0.530.01
0.550.01
NFE
67.120.30
67.050.26
66.900.23
T3
T4
T5
3.860.58
3.770.57
3.830.57
7.500.21
7.490.22
7.530.23
20.990.56
21.060.52
21.110.49
0.310.01
0.250.01
0.270.01
0.540.02
0.530.01
0.510.01
66.790.21
66.900.16
66.750.14
Table 34. Effect of storage on proximate composition of RBO cookies

Storage
(da
ys)
0
30
60
Moisture
2.950.01c
3.640.02b
4.870.02a
Protein
7.900.01a
7.470.02b
7.150.01c
Fat
Fiber
Ash
NFE
21.760.01c 0.270.01 0.740.01 66.370.03a

21.180.10b 0.280.01 0.740.01 66.690.09ab
20.870.09a 0.280.01 0.730.01 67.100.09b
The fat content of cookies was significantly affected by the storage

interval. In freshly prepared cookies, the average fat contents were 21.76%, which
decreased to 20.87% after 60 days storage (Table 34). It is apparent from the
results that there is significant decrease in fat content of cookies after every 30
days storage interval; might be due to the increase in moisture content of cookies
173
as well as oxidation of fatty acids resulting in free fatty acid formation. It is worth
mentioning that, although, there was gradual decrease in fat content in all the
treatments, yet, decrease was comparatively low in treatments with higher levels
of rice bran oil; might be due to high levels of oryzanol, tocopherols and
tocotrienols in RBO (Patel and Walker, 2004). Blending of other oils with rice
bran oil has also been found to improve the stability of the blend during frying
and storage. The high oxidative stability of RBO makes it preferred oil for frying
and baking applications (McCaskill and Zhang, 1999; Semwal and Arya, 2001).
Rice bran oil with higher thermal and oxidative stability can be used for deep fat
frying (Krishna et al., 2005). It extends the shelf-life of snack foods due to high
levels of phytosterols; may impart resistance to thermal oxidation and storage
deterioration (Taylor et al., 1996). Regarding fiber and ash, non-significant
differences were observed due to various treatments (Table 33) and storage
intervals (Table 34).
Means for NFE showed non-significant differences of treatments while
significant variations due to storage. The NFE contents ranged from 66.75 to
67.12% in all treatments. In freshly prepared cookies, the mean values were
66.37% that increased to 67.10% after 60 days storage (Table 34). The changes in
moisture, fat and protein content of cookies during storage, showed their
cumulative effect with slight increase in nitrogen free extract. The results of
present study are quite comparable to the observations recorded earlier by Sharif
et al. (2005).
4.5.1.3. Total acidity
Means squares for total acidity and TBA no. of RBO cookies have been
presented in Table 36. The total acidity and TBA no. were significantly affected
during storage; various treatments exhibited non-significant differences with
respect to total acidity and significant for TBA no. of cookies. The interaction
between treatments and storage intervals was to be non-significant for acidity
while significant for TBA no. of cookies.
174
The means for acidity of various treatments showed that storage had
significant effect on acidity; ranged from 0.147 to 0.194% from initiation to end of
the study (Table 37). The present findings concur with Rehman and Shah (1999)
who also observed a similar increasing trend in acidity with storage. Anjum et al.
(2003) has reported a significant increase in acidity for flour samples as a
function of 3 months storage. Leelavathi et al. (1984) described decline in acidity
by lowering moisture contents of stored atta. They observed an increase in
acidity value from 0.098 to 0.400% after 5 months storage. In the present
investigation, there was progressive increase in moisture content of cookies with
the passage of time; might be one of the possible reasons for increase in acidity
during 60 days storage. The rise in acidity may also be credited to the
accumulation of linoleic acid during storage that is oxidized later (Kent and
Evers, 1994).
4.5.1.4. Thiobarbituric acid no. (TBA no.)
Thiobarbituric acid number is a commercial test based on the reaction of
2-thiobarbituric acid with the oxidation products of fats and oils to form a red
color. Means for effect of different treatments of rice bran oil (Table 36) showed
that TBA no. was gradually decreased with proportionate increase of RBO in
cookies formulation. Average TBA values were 0.075, 0.063, 0.056, 0.053, 0.046
and 0.033 mg malenaldehyde/Kg for T0, T1, T2, T3, T4 and T5, respectively. It is
obvious from the results that cookies with increased levels of RBO exhibited the
lowest TBA no. compared with control (100% normal shortening); hence
175
Table 35. Mean squares for total acidity and TBA no. of RBO cookies
SOV
df
Treatments (T)
Storage (S)
SxT
Error
Total
**
5
2
10
36
53
Total Acidity
2.4074x10-5ns
0.0106**
2.6296x10-5ns
5.5556x10-5
TBA no.
0.001**
0.009**
9.51810-5**
1.85110-6
Table 36. Total acidity and TBA no. of RBO cookies

Treatments
T0
T1
T2
T3
T4
T5
Total Acidity
(%)
0.1720.014
0.1730.013
0.1730.013
0.1740.015
0.1740.015
0.1750.015
TBA
(mg
malenaldehyde/Kg
)
0.0750.018a
0.0630.015b
0.0560.014c
0.0530.015d
0.0460.012e
0.0330.009f
Table 37. Effect of storage on total acidity and TBA no. of RBO cookies
Storage
(days)
0
30
60
Total Acidity
(%)
0.1470.001c
0.1760.005b
0.1940.001a
Values are meansSEM
176
TBA
(malenaldehyde/Kg)
0.0330.004c
0.0510.006b
0.0790.008a
inception of rancidity was delayed. At the start, TBA no. of cookies was 0.033
that gradually increased to 0.079 mg malenaldehyde/Kg during storage (Table
37).
Rice bran oil based products have extended shelf life since it is extremely stable against
the onset of rancidity and oxidative deterioration. During storage, there was increase in TBA
value but the treatment T1 (without RBO) showed maximum increase. Treatments containing
RBO also showed some increase in TBA value but were within limits. Rice bran oil contains
high levels of phytosterols, oryzanol, tocotrienols as well as tocopherols (Taylor

et al. 1996). These naturally occurring components impart resistance to thermal
oxidation and deterioration of the oil. The high oxidative stability of RBO makes
it preferred oil for baking and frying applications (McCaskill and Zhang, 1999;
Semwal and Arya, 2001). It is certainly due to the tocopherols, tocotrienols, and
oryzanols that act as natural antioxidants (Rogers et al., 1993; Lloyd et al., 2000). RBO is
rich in -oryzanol having antioxidant properties; their structure includes ferulic

acid, a strong antioxidant (Miller and Rice-Evans, 1997; Sierra et al., 2005).
Refined oil in good condition has TBA value of 0.02-0.08 mg malenaldehyde/Kg
whereas badly stored oils have 0.1-0.2mg malenaldehyde/Kg (Kirk and Sawyer,
1999).
Sensory evaluation usually performed towards the end of the product
development or formulation cycle and is carried out to assess the reaction of
judges towards the product and they rate their liking on a scale. The cookies
prepared from rice bran oil were subjected to sensory evaluation by taste panel
for various attributes like color, flavor, taste, texture, crispness and overall
acceptability (Appendix III) using 9-Point Hedonic Score System. Mean squares
for sensory evaluation of cookies (Table 38) indicated that color, flavor, taste,
texture, crispness and overall acceptability differed significantly due to
treatments (T) and storage (S), however, all quality attributes showed nonsignificant differences due to the interaction of treatments and storage (T x S).
4.5.1.5.1. Color
177
In baking, color serves as a cue for the doneness of foods and is correlated
with changes in aroma and flavor. The results pertaining to mean score for the
color of the rice bran oil supplemented cookies (Table 39) revealed that T2 (7.61)
was preferred by the judges because it gave excellent color to cookies, followed
by T3 (7.11) as compared to control (6.69). Treatments prepared from 40 to 60%
rice bran oil substitutions were more liked by the judges. Though remaining
treatments got fewer score, yet they were acceptable. There was dullness in color
during storage, resulted lower score in all the treatments. The maximum score
(7.10) was obtained by cookies at the initiation which gradually decreased to 6.61
and 6.00, after 30 and 60 days storage, respectively (Table 39). These results are in
close agreement with the findings of Elahi (1997) who observed a gradual
decrease in color of biscuits made from composite flour of wheat and gram
during 90 days storage. Similar results have been reported by numerous
researchers including Pasha et al. (2002), Butt et al. (2004) and Sharif et al. (2005) in
cookies during storage. The deterioration in color of cookies was might be due to
the absorption of moisture from the atmosphere and oxidation of fats.
4.5.1.5.2. Flavor
Perceptions of flavor are a synthesis of taste and smell impressions, along
with texture and are even influenced by appearance. Means for flavor of cookies
(Table 40) revealed that T2 (40% RBO) and T3 (60% RBO) were much liked by the
judges and got maximum score, 7.31 and 6.87, respectively; whereas T4 (6.27) and
T5 (6.01) got minimum score. As a whole, the maximum score (7.20) was obtained
by fresh cookies (0 day), that was gradually decreased (6.59 and 6.14) after 60
days (Table 40). Bakery products rapidly stale and loose their flavor because
stailing transforms the rich aroma and flavor of the fresh product to a bland or
an off-flavor (Setser, 1996).
178
Table 39. Color scores of RBO cookies

Treatments
T0
T1
T2
T3
T4
T5
Mean
0 Days
30 Days
60 Days
7.45
7.00
8.02
7.48
6.44
6.20
6.56
6.28
7.75
7.10
6.06
5.91
6.05
5.62
7.06
6.76
5.42
5.10
7.100.28a
6.610.29b
Mea
n
6.690.41c
6.300.40d
7.610.29a
7.110.21b
5.970.30de
5.740.33e
6.000.32c
T0 = Normal shortening 100%; T1 = RBO 20%; T3 = RBO 40%; T3 = RBO 60%; T4 = RBO 80%;
T5 = RBO 100%.
Table 40. Flavor scores of RBO cookies

179
Treatments
T0
T1
T2
T3
T4
T5
Mean
0 Days
30 Days
60 Days
Mea
n
7.32
7.34
7.81
7.28
6.84
6.62
7.200.17a
6.73
6.50
7.10
6.93
6.11
6.14
6.590.17b
6.17
6.10
7.02
6.40
5.86
5.27
6.740.33b
6.650.37b
7.310.25a
6.870.26b
6.270.29c
6.010.40c
6.140.24c
Table 41. Taste scores of RBO cookies

Treatments
T0
T1
T2
T3
T4
T5
Mean
0 Days
30 Days
60 Days
7.47
7.13
7.92
7.50
6.64
6.11
6.93
6.68
7.76
7.08
6.20
5.88
6.12
6.08
7.20
7.15
6.02
4.98
7.130.27a
6.760.27b
Mea
6.840.39c
6.630.30c
7.630.22a
7.240.13b
6.290.18d
5.660.34e
6.260.34c
It has been observed that during storage of cookies, moisture absorption

results in deterioration of flavor due to oxidation of fat (Wade, 1988; Sharif et al.,
2005).
4.5.1.5.3. Taste
The taste is a sensation perceived by the tongue and influenced by the
texture, flavor and composition of the foods. The results regarding means for
taste of cookies are given in Table 41. The average score for taste of cookies
ranged from 5.66 to 7.63 among different treatments. Means for taste disclosed
that the judges ranked T2 (7.63) at the first position followed by T3 (7.24) and T0
(6.84), whereas T5 (5.66) was placed at the bottom. Statistically, T0 and T1 showed
non-significant variations with each other while all other treatments exhibited
significant variations. Maximum score (7.13) was obtained by the fresh cookies,
that was gradually decreased (6.76 and 6.26) after 30 and 60 days storage,
respectively. The decrease in cookies score was might be due to the rancidity of
fats during storage. The present results are in accordance with the work of Pasha
et al. (2002), Butt et al. (2004) and Sharif et al. (2005).
180
4.5.1.5.4. Texture
Food texture is extremely important to the consumer. Yet, unlike color
and flavor, texture is used by the consumers not as a sign of food safety but as an
indicator of food quality. Means for texture of cookies (Table 42) reveled that
average score for texture ranged from 6.22 to 6.92 among the treatments. It is
clear from the results that judges placed T2 (6.92) at the top and T5 (6.22) at the
bottom. Highest mean score (7.09) was assigned to fresh cookies (0 day), that was
decreased (6.10) during two months storage. T2 (40% RBO) was preferred by the
judges because it gave the desired texture to cookies, distinguished from others.
The decreasing trend in quality score for texture of cookies was due to
absorption of moisture from the atmosphere that has negative impact on texture
(Pasha et al., 2002; McWatters, 2003; Sharif et al., 2005).
Table 42. Texture scores of RBO cookies
Treatments
T0
T1
T2
T3
T4
T5
Mean
0 Days
30 Days
60 Days
7.10
6.90
7.50
7.50
6.90
6.66
6.12
6.22
6.88
6.34
6.30
6.12
5.96
6.08
6.38
6.22
6.06
5.87
7.090.14a
6.330.12b
Mea
6.390.36bc
6.400.25bc
6.920.32a
6.690.41ab
6.420.25bc
6.220.23c
6.100.07c
Table 43. Crispness scores of RBO cookies

Treatments
T0
T1
T2
T3
T4
T5
Mean
0 Days
30 Days
60 Days
7.06
6.85
7.50
7.50
6.70
6.52
6.56
6.66
7.30
7.23
6.33
6.08
6.02
6.20
6.88
6.74
6.09
5.80
7.020.17a
6.690.20b
Table 44. Overall acceptability scores of RBO cookies
181
6.290.17c
Mea
6.550.30b
6.570.19b
7.230.18a
7.160.22a
6.370.18bc
6.130.21c
Treatments
T0
T1
T2
T3
T4
T5
Mean
0 Days
30 Days
60 Days
Mea
n
7.20
7.30
7.72
7.34
7.06
6.85
7.250.12a
6.76
6.90
7.22
7.10
6.60
6.12
6.780.16b
6.20
6.24
6.90
6.86
6.10
5.82
6.720.29c
6.810.31bc
7.280.24a
7.100.14ab
6.590.28c
6.260.31d
6.350.18c
4.5.1.5.5. Crispness
The quality score in response to crispness of the cookies has been
presented in Table 43. Means for crispness of cookies alluded that T2 got the
maximum score (7.23) followed by T3 (7.16) and T1 (6.57) whereas minimum
score was obtained by T5 (6.13). Cookies prepared from 40 to 60% rice bran oil
substitution got the maximum scores (7.16 & 7.23) while 100% normal shortening
replacement results in significant decrease in crispness. As a whole, the
maximum score (7.02) was obtained by the fresh cookies that decreased
significantly (6.29) after 60 days storage. Cookies lost their crispness during
storage due to moisture absorption (Wade, 1988), might be the reason for this
declining trend.
4.5.1.5.6. Overall acceptability
Overall acceptability was determined on the basis of quality scores
obtained from the evaluation of color, taste, flavor, texture and crispness of the
cookies. The means regarding overall acceptability of cookies are presented in
Table 44. It is evident from the data that the judges placed T2 (7.28) at the top
and T5 (6.26) at the bottom line; where as T3 (7.10) was also favored by the judges.
Collectively, the maximum scores were observed in fresh cookies that gradually
decreased from 7.25 to 6.35 after 60 days storage. The decrease in overall
acceptability was might be due to moisture absorption, increase in peroxide
value and free fatty acid contents in cookies. The present results are in close
agreement with those of Leelavathi and Rao, 1993; Pasha et al., 2002; Butt et al.,
2004 and Sharif et al., 2005. It is concluded from the present study that rice bran
oil can successfully be substituted for preparation of cookies upto 40-60% level,
although higher levels were acceptable with respect to storage stability and
sensoric attributes.
4.5.2. Preparation of Rice Bran Supplemented Flours
182
The main aim of the present investigation was to utilized rice industrial
by-products rice bran for value added products. Initially, oil extracted from
MW-stabilized rice bran was supplemented in cookies by replacing normal
shortening @ 20, 40, 60, 80 and 100%. In the second phase MW-stabilized rice
bran was used in baked products. Fullfat (FFRB) and defatted rice brans (DFRB)
were mixed separately with commercial straight grade flour (CSGF) in different
proportions and were analyzed for chemical composition and rheological
behavior to find out the most appropriate compositions of fortified flour (Table
2) showing suitability for products preparation i.e. cookies and leavened pan
bread. The following parameters of supplemented flours were evaluated before
final product preparation.

Mean squares for moisture, crude protein, crude fat, crude fiber, ash and
NFE in different supplemented flour samples have been presented in Table 45.
All supplemented flours were found to exhibit significant variation with respect
to treatments; however, for storage, the value of moisture, protein and fat
established significant differences while fiber, ash and NFE were found to be
non-significant.
4.5.2.1.1. Moisture
The means for the moisture content of supplemented flours (Table 46)
showed that minimum moisture content (10.64%) was observed at the initiation
of the study while maximum moisture content (11.35%) after 60 days storage.
There was a gradual increase in moisture contents in various supplemented
flours during storage. Increase in the moisture content of various flour samples
was due to the hygroscopic nature of the flour and changes in the relative
humidity during storage. The water content also influences the storage stability
of wheat flour. It should be below 14% to prevent microbial growth and chemical
changes during storage (Pyler, 1971). The absolute moisture content in grain and
flour is less important than relative humidity with which the food is in contact.
Depending on the prevailing relative humidity, the stored samples absorb
moisture from the surrounding. Moisture content of flour samples in the present
183
Table 46. Moisture content of supplemented flours
Treatments
T0
0 Days
(%)
11.50
30 Days
(%)
12.08
60 Days
(%)
12.32
11.970.24a
T1
11.30
11.81
12.03
11.710.22ab
T2
11.10
11.66
11.84
11.530.22bc
T3
T4
T5
T6
10.90
10.70
10.50
10.30
11.40
11.24
11.03
10.79
11.70
11.40
11.18
10.96
11.330.23cd
11.110.21def
10.90f0.21g
10.680.20gh
184
Mean
T7
T8
T9
T10
T11
T12
T13
T14
T15
T16
9.90
9.50
11.33
11.15
10.98
10.80
10.63
10.45
10.10
9.75
10.40
9.98
11.86
11.71
11.40
11.34
11.16
10.97
10.59
10.23
10.56
10.10
12.08
11.89
11.70
11.55
11.31
11.19
10.83
10.39
Mean
10.640.14c
11.150.15b
11.350.15a
10.290.20ij
9.860.18k
11.750.22ab
11.590.22bc
11.360.21cd
11.230.22de
11.030.21ef
10.870.22fg
10.510.21hi
10.120.19j
Values are meansSEM; Means carrying the same letters in a column are not significantly
different
T0 = Commercial straight grade flour
T1 = Fullfat stabilized rice bran 5%
T9 = Defatted stabilized rice bran 5%

T10 = Defatted stabilized rice barn 10%
study ranged from 9.86% to 11.97%, falls within the range as reported by
Whiteley (1970) who observed that moisture content may vary from 11 to 15%
depending upon storage conditions and hygroscopic nature of starch.
4.5.2.1.2. Crude protein
Protein is important in determining the end use quality of flour. Means for
crude protein content of different supplemented flours (Table 47) demonstrated
highest protein content in T16 (13.68%) followed by T15 (12.95%), T14 (12.21%)
whereas T0 showed the lowest value (10.04%). Overall, the crude protein content
in supplemented flour samples ranged from 10.24 to 13.68%. Moreover, the
highest crude protein content was found in treatments containing higher levels
of defatted rice bran due to high initial protein content of defatted rice bran
(17.69%). The present results are in accordance with the previous findings who
reported that protein content in stabilized rice bran ranged from 13.217.3%
(Pomeranz and Oryl, 1982), 11.3-14.95% (Juliano, 1985), 11-17% (Farrell, 1994)
185
and 15.78% (Sharif et al., 2005), depending upon rice bran sources. There was a
significant decrease in protein content during 60 days storage (Table 47). At 0
day, the mean value for protein was 11.57% which decreased to 11.32% and 11.14
% after 30 and 60 days storage, respectively. In the present investigation, the
decrease in protein contents in the commercial flour and remaining samples were
due to the absorption of moisture from the atmosphere that accelerated the
proteolytic activity of the enzymes. The proteases are responsible for the
degradation of protein during storage. This phenomenon is supported by the
findings of Leelavathi et al. (1984) who observed activity of proteases and lipases
in the resultant atta 2 to 2.5 times higher than (Chakki) atta.
4.5.2.1.3. Crude fat
The means for the crude fat content of different flour samples (Table 48)
showed that T8 got the highest value (9.82%) for the crude fat content followed
186
Table 47. Crude protein of supplemented flours

0 Days
30 Days
Treatments
(%)
(%)
60 Days
(%)
Mean
T0
10.23
10.01
9.87
10.040.10n
T1
10.44
10.21
10.05
10.240.11mn
T2
10.65
10.43
10.28
10.450.11lm
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15
T16
10.86
11.07
11.29
11.50
11.92
12.34
10.60
10.98
11.35
11.72
12.10
12.47
13.21
13.96
10.63
10.82
11.02
11.24
11.66
12.07
10.38
10.74
11.10
11.49
11.83
12.18
12.92
13.65
10.48
10.64
10.84
11.06
11.48
11.89
10.24
10.57
10.92
11.33
11.65
11.98
12.71
13.44
10.660.11kl
10.840.12ijk
11.050.13hij
11.260.13gh
11.690.13ef
12.100.13cd
10.410.10lm
10.760.12jk
11.120.12hi
11.510.11fg
11.860.13de
12.210.14c
12.950.14b
13.680.15a
Mean
11.570.26a
11.320.24b
11.140.23c
Means carrying same letters in a column for each factor do not differ significantly
by T7 (8.08%) and T6 (6.34%) whereas the lowest value (0.84%) was found in T16
(defatted rice bran 50%). Fat content in different composite flour samples ranged
from 0.84 to 9.82%. It was observed that as the amount of fullfat rice barn
increased, the crude fat percentage also increased progressively in supplemented
187
flour samples while an inverse correlation exists with the addition of defatted
rice bran. There was significant decrease in fat content of supplemented flours
during storage. At the start, crude fat content was 3.13% that decreased to 3.06%
and 3.01% after 30 and 60 days storage, respectively (Table 48). The results
showed that there was a gradual decrease in fat contents with the passage of
time; may be attributed to the development of rancidity (Leelavathi et al., 1984).
Fat deterioration occurred during storage was probably due to the activation of
lipase enzyme which split off the fat into free fatty acids and glycerol in the
presence of moisture and other proxidants like light and heat. At higher moisture
level, risk of fat oxidation and development of rancidity increases as compared to
flour containing lower levels of moisture (Kent and Evers, 1994). Likewise,
Staudt and Ziegler (1973) reported that fat was gradually split up into glycerol
and fatty acids by lipases and then acids were oxidized by taking up oxygen.
4.5.2.1.4. Crude fiber
The means for the crude fiber content of different supplemented flours
(Table 49) showed that T16 (defatted stabilized rice barn 50%) got the highest
value (6.78%) followed by T8 (5.57%) and T15 (5.49%) whilst T0 (commercial
straight grade flour) exhibited the lowest value (0.33%) for this trait. The results
clearly indicated that wheat flour contained 0.33% crude fiber content. Addition
of fullfat and defatted rice bran upto 50% in commercial wheat flour increased
the fiber level. Storage has non-significant effect on various treatments of
fortified flours. The present study was in line with the findings of Anjum et al.
(2003) who also found non-significant effect of storage in commercial
(unfortified) and fortified flour samples. The findings of Butt et al. (2003) that
188
Table 48. Crude fat of supplemented flours
Treatments
0 Days
(%)
30 Days
(%)
60 Days
(%)
T0
1.17
1.15
1.13
1.170.01i
T1
2.05
2.01
1.98
2.010.02h
T2
2.94
2.88
2.83
2.880.03g
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15
T16
3.82
4.71
5.59
6.47
8.24
10.01
1.14
1.11
1.08
1.05
1.02
0.98
0.92
0.86
3.74
4.60
5.46
6.33
8.06
9.79
1.12
1.08
1.05
1.03
0.99
0.96
0.90
0.84
3.69
4.52
5.37
6.23
7.94
9.64
1.10
1.07
1.04
1.01
0.98
0.95
0.89
0.83
3.750.04f
4.610.06e
5.470.06d
6.340.07c
8.080.09b
9.820.11a
1.120.01ij
1.090.01ijk
1.060.01ijkl
1.030.01jkl
1.000.01klm
0.960.01lm
0.900.01mn
0.840.01n
Mean
3.130.70a
3.060.69b
3.010.68c
189
Mean
Table 49. Crude fiber of supplemented flours
Treatments
0 Days
(%)
30 Days
(%)
60 Days
(%)
T0
T1
T2
0.33
0.33
0.33
0.330.00o
0.85
0.86
0.86
0.860.00n
1.37
1.38
1.38
1.380.00l
T3
1.89
1.91
1.91
1.900.01j
T4
2.41
2.43
2.44
2.430.01h
T5
2.94
2.96
2.96
2.950.01g
T6
3.46
3.48
3.49
3.480.01f
T7
4.50
4.53
4.54
4.520.01d
T8
5.54
5.58
5.59
5.570.02b
T9
0.97
0.98
0.98
0.980.00m
T10
1.61
1.62
1.63
1.620.01k
T11
2.25
2.27
2.27
2.270.01i
T12
2.89
2.91
2.92
2.910.01g
T13
3.54
3.56
3.57
3.560.01f
T14
4.18
4.21
4.22
4.200.01e
T15
5.46
5.50
5.51
5.490.02c
T16
6.74
6.79
6.80
6.780.02a
Mean
3.000.44
3.020.45
3.020.45
190
Mean
fiber has non-significant influence due to storage intervals further confirmed the
present results.
4.5.2.1.5. Ash
Means for ash content of supplemented flour samples (Table 50) revealed
that ash content ranged from 0.52 to 5.77%. The highest value (5.77%) was noted
for T16 (defatted stabilized rice barn 50%) while T0 (commercial straight grade
flour) exhibited the lowest value (0.52%). Higher ash contents were observed in
T16, T8, T15 and T7 i.e. 5.77, 4.79, 4.78 and 3.99%, respectively, are due to presence
of higher fiber in these treatments. The present findings of ash content are in
corroboration with the results stated in the previous study by Farrell (1994) that
fullfat rice bran addition ultimately resulted in higher ash content of the
respective flours. Storage has non-significant effect on ash content of various
treatments of fortified flours (Table 50). Anjum et al. (2003) also observed similar
non-significant effect of storage on ash content of wheat flour samples.
4.5.2.1.6. Nitrogen free extract
191
The means for NFE content of blended flours (Table 51) showed that the
highest NFE content was observed in T0 (75.89%) followed by T9 (74.43%) and T1
(73.96%) while the lowest value (57.87%) was recorded in T8. These results are
supported by the findings of Siddique (1989) who reported 80.43% NFE contents
in whole wheat flour. Means for NFE revealed non-significant differences due to
storage.
These are the inorganic materials present in ash when food or any living
organism is cremated. The mean squares for mineral content of supplemented
flour samples showed that blending of fullfat and defatted rice bran in wheat
flour, significantly improved the mineral content of the fortified flours except for
sodium whereas storage and interaction between treatments and storage showed
non-significant differences (Table 52).
Table 50. Ash content of supplemented flours
Treatments
0 Days
(%)
30 Days
(%)
60 Days
(%)
T0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
0.52
0.52
0.52
0.520.00o
1.21
1.22
1.22
1.220.00n
1.61
1.62
1.62
1.620.00l
2.00
2.02
2.02
2.010.01j
2.40
2.41
2.42
2.410.01h
2.79
2.81
2.82
2.810.01g
3.18
3.21
3.21
3.200.01f
3.97
4.00
4.01
3.990.01c
4.76
4.79
4.80
4.790.01b
1.31
1.32
1.32
1.320.00m
1.80
1.82
1.82
1.810.01k
2.29
2.31
2.32
2.310.01i
192
Mean
T12
T13
T14
T15
T16
Mean
2.79
2.80
2.81
2.800.01g
3.28
3.30
3.31
3.290.01e
3.77
3.80
3.80
3.790.01d
4.75
4.79
4.80
4.780.02b
5.74
5.78
5.79
5.770.02a
2.830.34
2.850.35
2.860.35
Table 51. Nitrogen free extract of supplemented flours
Treatments
0 Days
(%)
30 Days
(%)
60 Days
(%)
T0
T1
T2
T3
T4
76.25
75.61
75.52
75.890.23a
74.14
73.89
73.86
73.960.09b
72.33
72.04
72.04
72.140.10d
70.52
70.30
70.21
70.340.09e
68.71
68.50
68.58
68.600.06fg
193
Mean
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15
T16
Mean
66.90
66.73
66.83
66.820.05h
65.09
64.95
65.05
65.030.04i
61.47
61.35
61.47
61.430.04lk
57.85
57.78
57.98
57.870.06l
74.65
74.34
74.28
74.430.11b
73.35
73.02
73.02
73.130.11d
72.05
71.87
71.75
71.890.09d
70.75
70.43
70.38
70.520.12e
69.45
69.16
69.19
69.270.09f
68.15
67.88
67.86
67.960.09g
65.55
65.30
65.26
65.370.09i
62.96
62.71
62.75
62.810.08j
68.831.22
68.581.20
68.591.18
The means for effect of various supplementation levels on mineral content

in fortified flours have been shown in Table 53. Sodium (Na) is essential in the
194
regulation of water content of the body and in the maintenance of osmotic

pressure of body fluids. Maximum Na content (399.03mg/100g) was observed in
T0 (commercial straight grade flour) whereas least value (203.17mg/100g) was
found in T8 (fuufat stabilized rice barn 50%).
Potassium (K) participates in certain enzyme systems in the body and
controls acid balance alongwith sodium to maintain fluid balance. Maximum K
content (826.66mg/100g) was observed in T16 (defatted stabilized rice barn 50%)
whereas minimum value (90.46mg/100g) was observed in T0 (commercial
straight grade flour).
Calcium (Ca) is essential for bones and teeth formation and also helps in
maintenance of cell membranes, clotting of blood and normal functioning of
nerves, muscles and heart. It participates in the activation of many enzymes.
Maximum Ca content (387.17mg/100g) was observed in T16 while least value
(111.57mg/100g) was found in T0 (commercial straight grade flour).
Magnesium (Mg) is essential for the synthesis of proteins. It is also
involved in maintenance of muscle functions, release of energy from muscle
glycogen
and
conduction
of
nerve
impulse.
Maximum
Mg
content
(607.02mg/100g) was noted in T16 whereas least value (21.61mg/100g) was

found in T0 (commercial straight grade flour). Collectively, there were significant
differences among the supplemented flour samples at different supplementation
levels with respect to different minerals.
The mean squares for dietary fiber content of different flour samples
indicated significant variation due to treatments whereas storage and interaction
between treatments and storage showed non-significant differences (Table 54).
Means for dietary fiber content of various treatments (Table 55) explicated
significant variations Maximum dietary fiber (22.10%) was observed in T16
195
Table 52. Mean squares for mineral content of supplemented flours

SOV
Treatments (T)
Storage (S)
SxT
Error
Total
**
df
Na
Ca
16
32682.817** 54122.193**
2
106.338ns
56.7683ns
32
0.2096ns
0.4316ns
102
62.1698
35.0584
152
Mg
249685.08**
80.0322ns
1.9004ns
60.5029
392739.26**
177.3979ns
3.0221ns
123.871
Table 53. Mineral contents (mg/100g) of supplemented flours
Treatment
Na
Ca
Mg
399.031.04a
90.460.24o
111.570.29o
21.610.06o
379.521.03b
149.790.41n
133.090.36n
69.240.19n
359.840.93c
209.060.54l
154.540.40l
116.840.30l
340.250.88d
268.360.70j
176.03j0.46
164.460.43j
320.840.92e
327.840.94h
197.630.57i
212.190.61h
301.260.88f
387.201.13g
219.140.64h
259.850.76g
281.590.78g
446.431.24f
240.580.67g
307.420.86f
242.350.65h
564.971.52c
283.520.76d
402.621.08d
203.170.55i
683.581.83b
326.500.88c
497.861.34b
379.480.96b
164.040.42m
139.100.35m
80.130.20m
360.010.97c
237.700.64k
166.700.45k
138.690.37k
340.600.93d
311.340.85i
194.270.53i
197.240.54i
320.910.79e
384.750.95g
221.710.55h
255.640.63g
301.550.82f
458.551.24e
249.370.68f
314.310.85f
282.110.80g
532.301.51d
277.000.79e
372.941.06e
243.040.67h
679.481.89b
332.080.92b
489.981.36c
203.990.56i
826.662.26a
387.171.06a
607.021.66a
T0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15
T16
Values are MeanSD for rice bran supplemented flour samples, analyzed individually in triplicate
196
T0 = Commercial straight grade flour
T9 = Defatted stabilized rice bran 5%

(defatted stabilized rice barn 50%) followed by T15 (18.71%) and T8 (18.53%)
whereas lowest dietary fiber content (5.14%) was observed in T0 (commercial
straight grade flour). Means for dietary fiber content in different supplemented
flours showed non-significant effect of storage (Table 55). Maximum increase in
dietary fiber was observed in T16 i.e. 76.74% followed by T15 (72.52%) and T8
(72.26%) as compared to control.
Dietary fiber is the edible parts of plants or analogous carbohydrates
that are resistant to digestion and absorption in the human small intestine
with partial fermentation in the large intestine (CAC, 1998). These plant food
materials include non-starch polysaccharides such as celluloses, some hemicelluloses, gums and pectins as well as resistant starches (DeVries, 2001). The
WHO recommendation for total dietary fiber intake is above 25 g/day (WHO,
2003). Additional daily intake of 10g fiber appeared to lower the risk of coronary
death by 17% (Morris et al., 1977; Khaw and Barrett, 1987). The total dietary fiber
content in stabilized rice bran ranges from 25 to 40% depending on the product
(Carroll, 1990). Rice brans fiber comprised of relatively low proportion of soluble
fiber (713%) and the rest is insoluble fiber (Anderson et al., 1990). Bran
supplementation has been used to enhance the fiber content of array of foods.
Traditionally, fiber supplementation has been focused in the use of milling byproducts of cereal grains like wheat, corn, sorghum etc. and now has been
197
introduced in cookies, crackers, snack foods, functional beverages and many

other cereal-based products (Matz, 1991; Hesser, 1994; McKee and Latner, 2000).
4.5.2.4. Thiobarbituric acid no.
Mean squares for TBA no. (mg malenaldehyde per Kg of sample) of
different flour samples indicated that treatments, storage intervals as well as
interaction between treatments and storage showed significant variations for this
trait (Table 54). The means for TBA no. in different flour samples have been
shown in Table 56. It is observed that with increased levels of fullfat rice
198
Table 54. Mean squares for dietary fiber and TBA no. of supplemented flours
SOV
Treatments (T)
Storage (S)
SxT
Error
Total
**
df
16
2
32
102
152
Dietary fiber
TBA no.
206.231**
0.091ns
8.85010-4 ns
0.105
1.48510-4**
5.30110-4**
1.96510-5*
9.804
Table 55. Dietary fiber content (%) of supplemented flours
Treatments
0 Days
30 Days
60 Days
T0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15
T16
Mean
5.12
5.14
5.15
5.140.01n
6.45
6.48
6.50
6.480.01m
7.79
7.82
7.84
7.820.01k
9.12
9.16
9.18
9.150.02i
10.46
10.50
10.53
10.500.02h
11.79
11.84
11.88
11.840.03g
13.12
13.18
13.22
13.170.03f
15.79
15.85
15.90
15.850.03c
18.46
18.53
18.59
18.530.04b
6.81
6.84
6.85
6.830.01l
8.50
8.53
8.56
8.530.02j
10.19
10.23
10.26
10.230.02h
11.88
11.92
11.96
11.920.02g
13.57
13.63
13.67
13.620.03e
15.26
15.32
15.37
15.320.03d
18.64
18.72
18.77
18.710.04b
22.02
22.11
22.18
22.100.05a
12.061.15
12.111.16
12.141.17
143
Mean
different
Table 56. TBA no. (mg malenaldehyde/ Kg) of supplemented flours
Treatments
T0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15
T16
Mean
0 Days
30 Days
60 Days
Mean
0.082
0.083
0.086
0.0840.001a
0.081
0.082
0.085
0.0830.001a
0.080
0.081
0.084
0.0810.001c
0.079
0.079
0.082
0.0800.001c
0.078
0.078
0.082
0.0790.001c
0.077
0.077
0.081
0.0780.001d
0.075
0.076
0.079
0.0770.001d
0.073
0.074
0.077
0.0750.001d
0.071
0.072
0.075
0.0720.001e
0.082
0.083
0.086
0.0830.001a
0.082
0.083
0.086
0.0840.001a
0.082
0.083
0.086
0.0840.001a
0.082
0.083
0.086
0.0830.001a
0.082
0.083
0.086
0.0830.001a
0.082
0.083
0.086
0.0830.001a
0.082
0.082
0.086
0.0830.001a
0.082
0.082
0.086
0.0830.001a
0.0790.001b
0.0800.001b
0.0830.001a
144
bran supplementation, TBA no. decreased; might be due to high antioxidant

level of fullfat rice bran. Maximum TBA no.
(0.084) was observed in T0
(commercial straight grade flour) followed by T10 (defatted stabilized rice barn
10%) and T11 (defatted stabilized rice barn 15%) whereas the lowest value
(0.072%) was found in T8 (fullfat stabilized rice barn 50%).
Wheat flour supplementation with fullfat and defatted rice bran elicited
significant variations due to initial fat content of the stabilized rice bran. Means
for effect of storage on TBA value of different treatments showed (Table 56), that
minimum value (0.079) was at the initiation of the study that was increased to
0.083 after 60 days storage. TBA test appears to measure deterioration in both
extractable and non-extractable lipids (Kirk and Sawyer, 1999). Thiobarbituric
acid reactive substances (TBARS) values were lower in beef roasts containing (2%) rice bran
oil which improved oxidative stability of food product (Kim et al., 2000; Kim and Godber, 2001;
Kim et al., 2001). In present study, rice barn oil in fullfat rice bran, improved the stability of
supplemented flours. However, all supplemented flours were acceptable even after 60 days
storage as indicated by TBA no. and supported by sensory evaluation.
145
4.5.2.5. Dough rheological studies

Rheological tests are used to predict baking performance and behavior of
the dough before baking. Since dough mixing is the first and most critical stage
of breadmaking, it is important to know optimum water absorption and mixing
tolerance requirements of given flour. The rheological characteristics of wheat
flour form the basis for understanding the dough handling properties and
predict the quality of the bakery products. The rheological behavior of rice bran
supplemented flours (Table 4) was evaluated by conducting mixographic and
farinographic studies, before the preparation of leavened pan bread.
4.5.2.5.1. Mixographic studies
The mixograph measures the physical properties of dough by recording
the resistance of dough to mixing. Mean squares for mixing time and peak height
of supplemented flours (Table 57) revealed variations among the treatments and
storage intervals whereas the interaction between treatments and storage was
found to be non-significant.
4.5.2.5.1.1. Mixing time
The time required for the dough to reach its full development is recorded
as mixing time. It is obvious from the results (Table 58) that mixing time of rice
bran supplemented flours alongwith commercial straight grade flour ranged
from 1.96 to 3.43 min. The highest value (3.43 min) for mixing time was noted in
T6 (defatted stabilized rice barn 5%) followed by T0 (3.19 min) while lowest value
(1.96 min) was recorded for T9 and T10. In case of fullfat rice bran supplemented
flours, the values for mixing time ranged from 2.45 to 2.70 min while defatted
rice bran supplemented flours exhibited 1.96 to 3.43 min. It is obvious from the
results that defatted rice bran supplemented flours exhibit comparatively highest
values certainly due to more protein content in defatted rice bran (17.69%)
compared with that of fullfat rice bran (14.45%). Mixing time of Pakistani wheat
cultivars ranged from 3.5 to 5.0 min (Afzal, 2004).
4.5.2.5.1.2. Peak height
146
Peak height is the highest point on the mixogram showing maximum

dough development. Means for peak height exhibited significant variations
among the treatments (Table 59). The data regarding the mixographic studies
showed that peak height ranged from 58.73 to 68.69%. In present study,
maximum peak height (68.69%) was noted in T3 (fullfat stabilized rice bran 15%)
followed by T2 (fullfat stabilized rice bran 10%) i.e. 68.68%; statistically nonsignificant from each other but differ significantly from rest of the treatments.
The lowest peak height (58.73%) was noted in T5 (fullfat stabilized rice barn 25%).
It is obvious from the results that with increased levels of rice bran
supplementation,
peak
height
was
gradually
increased
upto
15%
supplementation and then decreased at higher supplementation levels, without

discrimination of rice brans, either it is fullfat or defatted.
Table 57. Mean squares for mixographic characteristics of supplemented
flours
SOV
Treatments (T)
Storage (S)
SxT
Error
Total
**
df
10
2
20
66
98
time
Mixing
Peak
height
1.9475174**
0.0843465**
3.198x10-4ns
0.0047657
122.03848**
46.080585**
0.0114737ns
2.625698
Table 58. Mixing time (min) of supplemented flours
Treatments
T0
T1
T2
T3
T4
T5
T6
T7
T8
0 Days
30 Days
60 Days
3.25
2.75
2.50
2.50
2.50
2.50
3.50
3.00
3.00
3.18
2.69
2.45
2.45
2.44
2.44
3.42
2.93
2.93
3.13
2.65
2.41
2.41
2.40
2.40
3.37
2.89
2.89
147
Mean
3.190.03b
2.700.03d
2.450.03e
2.450.03e
2.450.03e
2.450.03e
3.430.04a
2.940.03c
2.940.03c
T9
T10
Mean
2.00
2.00
2.680.14a
1.96
1.96
1.93
1.93
2.620.14b
1.960.02f
1.960.02f
2.580.13c
different
T0
T1
T2
T3
T4
T5
= Commercial straight grade flour

= Fullfat stabilized rice bran 5%

Table 59. Peak height (%) of supplemented flours
Treatments
T0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
Mean
0 Days
64.00
64.00
70.00
70.00
60.00
60.00
65.00
65.00
65.00
60.00
60.00
63.911.12a
30 Days
60 Days
62.65
62.59
68.53
68.53
58.60
58.58
63.53
63.58
63.59
58.76
58.69
61.73
61.63
67.52
67.53
57.65
57.62
62.53
62.62
62.62
57.92
57.79
62.511.11b
61.561.10c
148
Me
an
62.800.66b
62.740.69b
68.680.72a
68.690.72a
58.750.68c
58.730.69c
63.690.72b
63.730.69b
63.740.69b
58.900.60c
58.830.64c
It was observed that there was prompt increase in peak height of fullfat
supplemented rice bran samples, might be due to inherent oil content, resulting
in better emulsion formation. The peak height percentage in Pakistani wheat
cultivar, Auqab-2000, was 53 to 56% (Afzal, 2004). It was concluded that rice bran
supplementation in wheat flour is suitable upto 15% for the preparation of
leavened pan bread, beyond this level; there was deformation in peaks of
mixograms.
4.5.2.5.2. Farinographic studies
The rheological behavior of rice bran supplemented flours was evaluated
by conducting farinographic studies, before the preparation of rice bran
supplemented leavened pan bread. Mean squares for water absorption, dough
development time and dough stability of different flour samples have been
presented in Table 60. The results indicated significant variations among
different supplemented flour samples due to storage and treatments for different
farinographic characteristics.
4.5.2.5.2.1. Water absorption
149
The amount of water added to achieve a required consistency is known as

water absorption. It refers to the ability of flour to hold maximum amount of
water without additional mixing for development of dough. It is the amount of
water required to center the farinograph curve on a 500-Brabender Unit (BU)
line. Means for water absorption of different flour samples are shown in Table
61. The results indicated significant variations among the treatments due to
different level of supplementation; ranged from 60.20% to 72.30%. Highest value
for water absorption (72.30%) was noted in T5 (fullfat stabilized rice bran 25%)
followed by T4 (72.00%) and T3 (70.90%) while lowest water absorption (60.20%)
was recorded for T10 (defatted stabilized rice bran 25%). The water absorption in
fullfat rice bran supplemented flours ranged from 69.42 to 72.3% whereas
defatted rice bran supplemented flours exhibited 60.20 to 69.28% which is
comparatively lower than fullfat bran supplementation.
Table 60. Mean squares for farinographic characteristics of supplemented
flours
SOV
Treatments
(T)
Storage (S)
SxT
Error
Total
df
DT
10
133.632**
22.468**
109.993**
2
20
66
98
26.092**
0.088ns
8.816
21.233**
0.983**
0.069
43.039**
0.630**
0.170

WA = Water absorption (%); DDT = Dough development time (min); DS = Dough stability (min)
**
Table 61. Water absorption (%) of supplemented flours
Treatments
T0
0 Days
67.90
30 Days
60 Days
68.15
68.99
ean
68.350.3
3bc
T1
68.40
69.50
69.420.5
70.35
6abc
T2
69.20
70.45
71.30
70.320.6
1abc
150
T3
69.90
71.00
71.80
70.900.5
5ab
T4
71.00
72.05
72.95
72.000.5
6a
T5
71.40
72.40
73.10
72.300.4
9a
T6
68.20
69.40
70.23
69.280.5
9abc
T7
66.60
67.45
68.54
67.530.5
6cd
T8
64.50
65.20
66.00
65.230.4
3de
T9
61.80
62.80
63.60
62.730.5
2ef
T10
59.40
60.20
61.00
60.200.4
6f
Mean
68.901.72a
67.121
.14b
68.051.
72ab
T0
T1
T2
T3
T4
T5
= Commercial straight grade flour


Similar findings have been recorded by Nurul Islam and Johnsen (1987)
that water absorption in commercial wheat flour of Indo-Pak wheat varieties
ranged from 60-76%. Flour from strong wheat varieties had the ability to absorb
and retain larger quantities of water (Pyler, 1988). The rheological behavior of
blended flours is greatly influenced by the level of rice bran in wheat flour (Singh
et al., 1995). The results alluded that there was an increase in water absorption of
151
fullfat rice bran supplemented flour samples with subsequent increase in the
amount of rice bran (Barber et al., 1981; Sudha et al., 2007). The increase in water
absorption was might be due to greater number of hydroxyl groups which exist
in the fibre structure and allow more water interaction through hydrogen
bonding (Rosell et al., 2001).
Similarly, enhanced protein contents result in an increase in pentosans,
especially ribose and deoxyribose which have higher water holding capability
(Matz, 1972). Similar findings have been reported by Shahzadi et al. (2005) that
water absorption increases with increased levels of protein in flours. In contrary
to previously reported findings, decrease in water absorption (69.28% to 60.2%)
was observed in defatted rice bran supplemented flours with proportionate
increase in defatted rice bran supplementation, suggesting role of fat besides
protein, for better water absorption and might be due to the reason, better bread
can be prepared from fullfat rice bran supplemented flour even with higher
levels compared with defatted supplemented flours. Storage has significant effect
on water absorption of different fortified flours. At the initiation of study (0 day),
67.12% water absorption was recorded which was increased to 68.05 and 68.90%,
after 30 and 60 days storage, respectively. In previous studies, it has been
reported that water absorption varies (54-65%) based upon the weight of flour
and the way flour is to be used. If too much water is added, dough with low
consistency will be obtained. It is difficult to handle such dough due to sticky
nature and if too little water is added, the dough will be stiff and difficult to
process (Cauvain, 2003).
4.5.2.5.2.2. Dough development time
The time interval in minutes from addition of

water to the point of maximum consistency, before
start of weakening process, is recorded as dough
152
development time (min). Means for dough

development
time
(min)
supplemented
flour
samples
of
rice
bran
showed
highly
significant variations among the treatments. The

results
in
Table
62
indicate
that
dough
development time ranged from 3.49 to 8.01 min

among different treatments. The highest dough
development time (8.01 min) was observed in T10
(defatted stabilized rice bran 25%) followed by T9
(7.90) and T8 (7.79) min while the lowest dough
development time (3.49 min) was noted in T1. The
dough development time in fullfat rice bran
supplemented flours ranged from 3.49 to 5.80 min
whereas defatted rice bran supplemented flours
showed 4.79 to 8.01 min among different
treatments. Higher value for this parameter was
observed in defatted rice bran fortified flours.
There was increase in dough development time,
with increased levels of stabilized fullfat and
153
defatted rice bran supplementation (Sudha et al.,

2007).
Present results are consistent with previous
findings of Kailasapathy and MacNeil (1985) who
observed an increase in peak time with an
increase in winged bean flour in wheat flour. In
another study, increase in dough development
time was observed in composite flours prepared
from different levels of chickpea (Shahzadi et al.,
2005). The results of present investigation are
comparable with the findings of Singh et al. (1995)
who reported improved dough strength as a result
of blending of stabilized fullfat and defatted bran
in wheat flour. However, high fibre doughs have
limited tolerance to over-mixing, and care is
necessary to develop a thin, elastic gluten sheet
and to avoid over-mixing (Stauffer, 1993). Dough
development time increased during 60 days
storage from 4.96 to 6.55 min (Table 62). The
154
results are supported by the previous findings of

Leelavathi et al. (1984) who reported 6 to 10
min
155
Table 62.
Dough development time (min) of
supplemented flours
Treatments
T0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
Mean
0 Days
30 Days
60 Days
4.00
3.00
3.85
3.80
3.50
5.00
4.00
6.00
7.00
7.12
7.25
4.98
3.97
4.65
4.70
4.40
5.97
4.97
6.95
7.92
8.02
8.12
5.50
3.50
5.05
5.18
7.95
6.42
5.40
7.45
8.45
8.55
8.65
5.880.48b
Me
an
4.830.44e
3.490.28h
4.520.35g
4.560.40fg
5.281.36d
5.800.42c
4.790.41ef
6.800.43b
7.790.42a
7.900.42a
8.010.41a
6.550.53a
4.960
.48c
Table 63. Dough stability (min) of supplemented flours
Treatments
T0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
Mean
0 Days
30 Days
60 Days
7.90
6.40
4.70
4.40
3.90
2.50
8.30
10.00
10.00
11.00
14.15
9.15
7.55
8.85
5.60
5.00
3.70
9.45
11.24
11.20
12.30
15.35
10.00
8.35
8.70
6.50
5.85
4.45
10.35
12.05
12.10
13.10
16.55
7.571.07c
9.041.04b
9.821.07a
156
Me
an
9.020.61d
7.430.57e
7.421.36e
5.500.61f
4.920.56g
3.550.57h
9.370.59d
11.100.60c
11.100.61c
12.130.61b
15.350.69a
increase in dough development time during four

months storage of atta. The increase in dough
development time may be due to the presence of
higher moisture content of the bran particles in
the atta which interfere the quicker development
of gluten (Rao et al., 1983).
4.5.2.5.2.3. Dough stability
Dough stability, which indicates the dough strength, is the difference
between arrival and departure time. It is obvious from the results (Table 63) that
dough stability time ranged from 3.55 to 15.35 min among different treatments.
The highest value (15.35 min) was observed in T10 followed by T9 (12.13 min) and
T8 (11.10 min) that were statistically non-significant from each other while lowest
dough stability (3.55 min) was noted in T5. The dough stability in fullfat rice bran
supplemented flours ranged from 3.55 to 7.43 min., whereas defatted rice bran
supplemented flours showed 9.37 to 15.35 min at different levels of
supplementation. High dough stability time was observed in defatted rice bran
supplemented flours whereas significant decrease was observed in fortified
flours with increased levels of stabilized fullfat rice bran supplementation (Sudha
et al., 2007). At the beginning, dough stability was 7.57 min which subsequently,
increased to 9.04 and 9.82 min at 30 and 60 days storage, respectively (Table 63).
Dough stability increased with storage of the flour samples might be attributed
157
to the difference in the protein content and quality (Holas and Tipples, 1978;
Finney, 1984; Hoseney, 1986; Abdel-Kader, 2000; Shahzadi et al., 2005). The
rheological behavior of blended flours is greatly influenced by the level of rice
bran in commercial wheat flour (Singh et al., 1995).
4.5.3. Preparation of Rice Bran Supplemented Cookies
In present research, cookies were prepared from fullfat and defatted rice
bran supplemented flours (Table 3) and evaluated for physical, chemical and
sensoric attributes to find out the effect of rice bran supplementation.

Mean squares (Table 64) exhibited that supplementation of various levels
of fullfat and defatted rice bran has significant effect on width, thickness and
spread factor of cookies whereas storage and interaction was found to be nonsignificant. The data regarding means for effect of various treatments on width of
cookies (Table 65) showed an increasing trend with the proportionate increase of
either type of bran supplementation. The results elucidated that T0 (cookies with
0% RB) exhibits maximum width 44.15 mm, followed by T1 (43.09 mm) and T2
(40.23 mm) while minimum width (38.67 mm) was observed in T5 (cookies with
50% FFRB). However, there were non-significant differences with respect to
storage (Table 66).
The results pertaining to the means for thickness of cookies (Table 65)
revealed increasing trend with proportionate increase of fullfat and defatted rice
bran in commercial straight grade flour. The results explicated that T5 (cookies
with 50% FFRB) exhibited maximum thickness (10.60 mm) followed by T4 (9.95
mm) and T3 (9.90 mm) while minimum thickness (9.23 mm) was measured in T0
(cookies with 0% RB). The similar increasing trend of thickness was observed in
cookies prepared from defatted rice bran supplemented flours. The thickness of
cookies remained unaffected during 60 days storage (Table 66).
The statistics regarding the effect of rice bran oil substitution on spread
factor of cookies is presented in Table 65. There was a decreasing trend in the
spread ratio of cookies with the proportionate increase of supplementation. The
spread factor of cookies, prepared from different treatments ranged from 36.83 to
47.80. The maximum value (47.80) for spread ratio was observed in T0 (cookies
with 0% RB) whereas minimum (36.83) in cookies prepared from 50% rice bran
supplementation. Rice bran replacement upto 20-30% was found to be
appropriate in cookies. On spread ratio, there were non-significant variations
during two months storage (Table 66).
158
159
160
The results of present study are quite close to the observations reported
earlier by numerous researchers. Wheat flour was supplemented with 5-25%
defatted soy flour samples. Increasing levels of defatted soy flour reduced
diameter and increased thickness of cookies resulting in a significantly reduced
spread ratio (Grover and Singh, 1994). The fullfat and defatted rice brans were
blended in wheat flour @ 5, 10 or 15% to prepare cookies. Spread of cookies
decreased with the addition of rice bran (Sekhon et al., 1997). Cookie spread
decreased with the addition of the various rice brans (Sudha et al., 2007) however, the
decrease was more pronounced in flours containing defatted bran. Stabilized full fat rice bran up
to a level of 20% and stabilized defatted rice bran upto a 10% level was considered suitable for
use in various bakery products (Singh et al., 1995).

The mean squares for mineral content (Table 67) depicted that blending of
fullfat and defatted rice bran, significantly improved the mineral content of the
cookies. The means for mineral content in supplemented cookies (Table 68)
represented that maximum Na content (196.51 mg/100g) was observed in T0
(cookies with 0% FFRB) followed by T1 (177.22) and T2 (157.92) whilst the lowest
value (100.04mg/100g) was observed in T5 (cookies with 0% FFRB). Likewise,
maximum K content (336.60mg/100g) was recorded for T5 (cookies with 50%
FFRB) followed by T4 (278.19) and T3 (219.78), while the lowest value (44.55
mg/100g) was found in T0 (cookies with 0% FFRB).
The highest value for Ca content (160.77 mg/100g) was recorded for T5
(cookies with 50% FFRB) followed by T4 (139.60) and T3 (118.44); however, the
minimum value for Ca (54.95 mg/100g) was in T0. The magnesium content of
fullfat rice bran supplemented cookies ranged from 57.54 to 245.15mg/100g.
Similarly, in defatted rice bran supplemented cookies, the mineral contents were
improved except for sodium, with proportionate increase of supplementation.
Table 67. Mean squares for minerals of rice bran supplemented cookies
SOV
df
Treatments
Na
FFRB- Cookies
DFRB-Cookies
FFRB- Cookies
DFRB-Cookies
3909.1593**
3876.877**
35823.145**
55178.08**
161
Error
Total
12 43.836839
17
SOV
df
43.93233
125.9768
Ca
FFRB- Cookies
Treatments 5 4703.4242**
Error
12 24.585227
Total
17
**
87.912438
Mg
DFRB-Cookies
FFRB- Cookies
DFRB-Cookies
7732.022**
32.72592
23097.136**
43.269008
34891.56
63.91906
Table 68. Mineral contents (mg/100g) of rice bran supplemented cookies

Na
Treatments
FFRBCookies
T0
T1
T2
T3
DFRBCookies
196.518.57a
177.227.22b
157.926.68c
138.636.04d
196.518.57a
177.307.73b
158.086.89c
138.876.05d
FFRB- Cookies
DFRBCookies
44.551.94f
102.964.49e
161.377.03d
219.789.58c
44.551.94f
117.045.10e
189.538.26c
262.0311.4
2c
T4
119.335.20e
119.655.22e
278.1912.13b
334.5214.5
8b
T5
100.044.36f
100.444.38f
336.6014.67a
407.0117.7
4a
Ca
Treatments
T0
T1
T2
T3
T4
T5
Mg
FFRBCookies
DFRBCookies
FFRB- Cookies
DFRBCookies
54.952.39f
76.113.32e
97.274.24d
118.445.16c
139.606.09b
160.777.01a
54.952.39f
82.083.58e
109.224.76d
136.355.94c
163.497.13b
190.68.31a
10.640.46f
57.542.51e
104.454.55d
151.356.60c
198.258.64b
245.1510.69a
10.640.46f
68.292.98e
125.935.49d
183.588.00c
241.2210.51b
298.8713.03a
Values are MeanSD of RB supplemented cookies, analyzed individually in triplicate
162
Mean squares for dietary fiber content of different treatments of cookies

revealed significant variation due to supplementation of fullfat and defatted rice
bran (Table 69). In defatted rice bran supplemented cookies, maximum dietary
fiber (11.01%) was observed in T5 (cookies with 50% FFRB) followed by T4
(cookies with 40% DFRB) and T3 (cookies with 30% DFRB) i.e. 9.32 and 7.63%,
respectively; whereas lowest dietary fiber content (2.56%) was found in cookies
prepared from straight grade wheat flour (Table 70). Similar increasing trend for
dietary fiber was observed in fullfat rice bran supplemented cookies, with
proportionate increase of rice bran supplementation.
Mean squares for sensory evaluation of cookies (Table 71) indicated that
color, flavor, taste, texture, crispness and overall acceptability differed
significantly due to treatments and storage, nevertheless, non-significant
differences were established due to their interaction.
The results pertaining to mean score for the color of fullfat and defatted
rice bran supplemented flour cookies (Table 72) revealed that in case of fullfat
rice bran supplemented cookies, T3 (7.55) ranked at the top due to excellent
appearance, followed by T2 (7.48) as compared to control (6.65). Treatments
prepared from 20 to 30% fullfat rice bran and upto 40% defatted rice bran
substitution were acceptable by the judges for this trait. By the progressive
increase in supplementation, color of cookies turned darker, leading to lower
acceptance (Sudha et al., 2007). There was a declining trend in color score during
storage. The maximum score (7.06) was assigned to fresh cookies that decreased
to 6.67 and 6.31, after 30 and 60 days, respectively (Table 73). The deterioration in
color of cookies was possibly due to the absorption of moisture from the
atmosphere and oxidation of fats. Color changes are might be due to
caramelization, dextrinisation of starch or Maillard reaction involving the
interaction of reducing sugars with proteins.
163
Table 69. Mean squares for dietary fiber of rice bran supplemented cookies
SOV
df
Treatments
Error
Total
5
12
17
**
Dietary fiber
FFRB-Cookies
DFRB-Cookies
18.682**
0.0756
29.989**
0.1037
T0
T1
T2
T3
T4
T5
= Cookies with 100% wheat flour act as control for both fullfat and defatted supplementation
= Cookies with 10% FFRB
T1 = Cookies with 10% DFRB
Table 70. Dietary fiber content of rice bran supplemented cookies
Treatments
T0
T1
T2
T3
T4
T5
FFRB-Cookies
2.560.14f
3.900.18e
5.230.21d
6.560.32c
7.900.40b
9.230.37a
164
Dietary fiber (%)

DFRB-Cookies
2.560.14f
4.250.21e
5.940.27d
7.630.38c
9.320.49b
11.010.52a
165
166
There is natural trend of color fading with progressive storage which

affects the appearance (Manley, 2002). These results are in close agreement with
the findings of Elahi (1997) who observed a gradual decrease in color of biscuits
made from composite flour during storage.
Means for flavor of cookies (Table 72) revealed that T2 (cookies with 20%
FFRB) and T3 (cookies with 30% FFRB) were liked by the judges and obtained
maximum score i.e. 7.82 and 7.78, respectively; whereas T4 (6.13) and T5 (5.51) got
minimum score. However, in case of defatted rice bran supplemented cookies,
comparatively higher supplementation levels were acceptable. As a whole, the
maximum score (7.13) was obtained by fresh cookies (0 day), that was gradually
decreased (6.83 and 6.51) after 60 days storage (Table 73). It has been observed
that during storage of cookies, moisture absorption results in deterioration of
flavor due to oxidation of fat (Wade, 1988; Sharif et al., 2005) and stailing (Setser,
1996). The development of off-flavor as a result of oxidation of fats is particularly
related to the presence of moisture, further accelerated by metal ions and light
(Manley, 2002).
Means for taste (Table 72) unveiled that the judges ranked T2 (7.85) at the
top followed by T3 (7.82) and T1 (6.93), whereas T5 (5.48) was placed at the
bottom. Statistically, T2 and T3; T0 and T1; showed non-significant variations with
each other. Similar, declining trend for taste score was noted in cookies prepared
from defatted rice bran supplementation. Maximum scores (7.18) were assigned
to fresh cookies, that were gradually decreased (6.84 and 6.53) after 30 and 60
days storage (Table 73). The decrease in cookies score was might be due to the
rancidity of fats during storage.
The mean values for texture of cookies (Table 74) explicated that judges
placed T0 (7.58) at top, followed by T1 (7.15) and T2 (6.80). Highest mean score
(6.97) was obtained by fresh cookies (0 day), that was decreased (6.33) during 60
days storage (Table 75). The declining trend in quality score for texture might be
due to absorption of moisture from the atmosphere that has an inverse
correlation with texture (Sharif et al., 2005).
The quality score in response to crispness of the cookies has been
presented in Table 74. Means for crispness of cookies alluded that T0 got the
167
maximum score (7.67) followed by T1 (7.52) whereas minimum score was

obtained by T5 (5.40). Collectively, the maximum score (7.22) was obtained by the
fresh cookies that decreased significantly (6.38) after 60 days storage (Table 75).
Deterioration during storage can be manifested itself by changes in physical and
chemical characteristics, referred as spoilage mechanism. In case of physical
changes, loss of crispiness occurs due to moisture uptake, as biscuits are
hygroscopic in nature (Wade, 1988; Manley, 2002).
The means regarding overall acceptability of cookies are presented in
Table 74. It is evident from the results that T2 (cookies with 20% RB) was more
appealing (7.30) for judges followed by T1 (cookies with 10% RB) and T0 (cookies
with 0% RB) i.e. 7.08 and 6.78, respectively. Similar trend of acceptance was
observed in case of cookies prepared from defatted rice bran supplemented
flours. As a whole, the maximum scores were obtained by fresh cookies, which
gradually decreased from 6.95 to 6.14 during 60 days storage (Table 75). The
decrease in overall acceptability was due to decrease in color, flavor, taste,
texture and crispness scores. These results were in close agreement with those of
observed in earlier studies (Pasha et al., 2002; Butt et al., 2004; Sharif et al., 2005).
From the present research work, it was concluded that replacement of wheat
flour with fullfat and defatted rice bran upto 10 to 20% is possible without any
adverse change in the sensory characteristics of cookies.
Cookies are ideal for nutrient availability, palatability, compactness and
convenience. They differ from other baked products like bread and cakes
because of their low moisture content, comparatively free from microbial
spoilage and confer a long shelf life (Wade, 1988). Cookies are considered better
for supplemented/composite flours due to their ready-to-eat form, wide
168
169
consumption, relatively long shelf-life and good eating quality (Tsen et al., 1973).
Cookies with high sensoric attributes are manufactured from blends of
millet/pigeon pea flour (Eneche, 1999), raw rice and wheat (Singh et al., 1989),
blackgram and wheat (Singh et al., 1993), chickpea and wheat (Singh et al., 1991),
wheat, fonio and cowpea (McWatters et al., 2003) and soybean, chickpea or
lupine with wheat (Hegazy and Faheid, 1990). Similarly, cookies with high
sensory ratings have been prepared from blends of wheat flour and rice bran.
Nutritional and functional properties of rice bran are

well suited for baked products like cookies, muffins,
breads, crackers, pastries and pancakes (Barber et al., 1981).
Cookies supplemented with fullfat rice bran were
acceptable upto 10% supplementation level (Sekhon et al.,
1997). In another study, cookies were successfully prepared
from stabilized rice bran at levels of 20% (Carroll, 1990). In
a similar study, stabilized fullfat rice bran upto 20% and
stabilized defatted rice bran upto 10% was found suitable
in various food products (Singh et al., 1995). In similar
study, stabilized rice bran was supplemented in wheat
flour at 5-20% levels for the preparation of cookies
(Sharma and Chauhan, 2002).
4.5.4. Preparation of Rice Bran Supplemented Leavened Pan Bread
The increasing awareness of the potential benefits of

high fibre diets has promoted interest for the preparation
of its allied value added products like bran breads. The
206
simplest way for achieving nutritional enrichment of

cereal based products like bread is through the addition of
different ingredients in the flour at the beginning of bread
making process. Numerous ingredients have been added
to wheat breads with the main aim of increasing their
protein content or to augment the dietary fibre supply. In
present study, leavened bran bread was prepared from
fullfat and defatted rice bran supplemented flours (Table
4) and analyzed for various quality traits. The detail of
each is mentioned below:
The mean squares for mineral content of leavened pan bread showed that
blending of fullfat and defatted rice bran in wheat flour, significantly effect the
mineral content (Table 76). The means for effect of treatments on mineral content
in bread have been shown in Table 77. It is obvious from the results that
significant variations exist within various supplemented breads for Na, K, Ca
and Mg concentrations (mg/100g).
Maximum Na content (358.55mg/100g) was observed in T0 (Bread with
100% commercial straight grade flour) followed by T1 (bread with 5% stabilized
fullfat rice bran) and T2 (bread with 10% stabilized fullfat rice bran) i.e. 340.17
and 320.88mg/100g, respectively; whereas least value (269.42mg/100g) was
found in T5 (bread with 25% stabilized fullfat rice bran). It is obvious from the
results that Na content was significantly decreased, with proportionate increase
of rice bran supplementation. Likewise, decreasing trend of Na content was
observed in pan bread prepared from supplemented flour samples. The decrease
in Na was due to its low initial levels in rice bran compared to straight grade
207
flour. Maximum K content (349.16mg/100g) was observed in T5 (followed by T4

and T3 i.e. 293.87 and 236.93mg/100g, respectively; whereas minimum value
(80.93mg/100g) was observed for T0. It is clear from the results that K content
was significantly increased, with gradual increase of rice bran supplementation.
The increase in K content of bread was mainly due to high levels of K in rice bran
samples i.e. 1270mg/100g compared to commercial straight grade flour
(90.46mg/100g).
Maximum Ca content (195.40mg/100g) was observed in T5 followed by T4
and T3 i.e. 176.70 and 157.03mg/100g, respectively; whereas minimum value
(100.57mg/100g) was noted in T0. It is clear from the results that Ca content was
significantly improved with rice bran supplementation. Similar increasing trend
of Ca content was observed in pan bread prepared from defatted rice bran
supplemented flour samples. The increase in Ca content of bread was mainly due
to its high level in rice bran samples i.e. 538.57mg/100g compared to commercial
straight grade flour (111.57mg/100g). In case of fullfat rice bran supplemented
bread, maximum value for Mg content (233.98mg/100g) was observed in T5
followed by T4 and T3 i.e. 191.17 and 146.79mg/100g, respectively; whereas
208
Table 76. Mean squares for mineral contents of rice bran supplemented breads
SOV
df
Treatments 5
Error
12
Total
17
SOV
df
Na
FFRB-Bread
DFRB-Bread
FFRB-Bread
DFRB-Bread
3292.4867**
241.0441
3193.9309**
227.2736
46237.665**
112.8474
2.8054**
0.0808
Ca
FFRB-Bread
Treatments 5 3777.8979**
Error
12 56.06101
Total
17
**
Mg
DFRB-Bread
FFRB-Bread
DFRB-Bread
6396.011**
56.9393
19372.254**
34.15073
29159.351**
69.7736
Table 77. Mineral contents (mg/100g) of rice bran supplemented breads

Na
Treatments
T0
T1
FFRB-Bread
DFRB-Bread
FFRB-Bread
DFRB-Bread
358.5518.53a
340.1714.70ab
358.5518.53a
341.7318.60a
80.933.21f
134.214.94e
80.933.21f
146.687.65e
320.3914.02b
186.977.24d
212.669.64d
304.3011.41c
236.938.62c
276.7713.68c
289.2013.66d
293.8714.43b
347.8913.97b
271.3413.67e
349.1613.97a
411.8612.07a
T2
320.8816.58bc
c
T3
303.6315.83cd
d
T4
286.7614.21de
e
T5
269.4212.62e
Ca
Treatments
T0
T1
T2
T3
T4
T5
Mg
FFRB-Bread
DFRB-Bread
FFRB-Bread
DFRB-Bread
100.574.37f
119.424.92e
139.176.96d
157.036.12c
176.709.34b
195.4010.94a
100.574.37f
123.836.11e
148.297.53d
173.727.86c
197.838.58b
223.339.55a
19.450.89f
62.032.94e
103.794.23d
146.796.69c
191.176.35b
233.989.61a
19.450.89f
71.742.50e
124.916.30d
176.628.60c
230.539.10b
282.7214.65a
Values are MeanSD for rice bran supplemented breads, analyzed individually in triplicate
209
minimum value (19.45mg/100g) was found in T0. Rice bran supplementation

significantly improved the mineral profile of wheat flour and ultimately in the
final product.

Recently, the importance of dietary fibre has increased owing to its
relation with the reduction of blood cholesterol and colon cancer. There have
been different approaches to escalate the dietary fiber content of the wheat bread;
presently concerned with rice bran. Mean squares for dietary fiber content of
fullfat and defatted rice bran supplemented flour bread indicated that various
supplementation levels significantly affect dietary fiber of breads (Table 78). The
results in Table 79 depicted that in case of fullfat rice bran supplemented bread,
maximum dietary fiber (10.63%) was observed in T5 followed by T4 (9.42%) and
T3 (8.16%) whereas lowest dietary fiber content (4.62%) was found in T0 (straight
grade flour bread). Maximum increase in dietary fiber content (62.28%) was
observed in T5 followed by 56.98% in T4 and 49.51% in T3 as compared to bread
prepared from 100% commercial straight grade flour. The similar increasing
trend in dietary fiber content was observed in breads prepared from different
levels of fullfat rice bran.
In the present study, commercial straight grade flour was used showing
dietary fiber content 5.14% whereas dietary fiber contents of fullfat rice barn and
defatted rice bran were 31.80 and 38.92%, respectively. The supplementation of
fullfat and defatted rice bran significantly improved the dietary fiber contents of
bread ranging from 24.50 to 62.26%.
Fiber supplementation has been used to enhance the fiber content of array
of foods. The importance of dietary fiber has been demonstrated in many studies
(Newman et al., 1989; Anderson and Siegel, 1990). The effects of fibre may be
mechanical or physiological. It may reduce the incidence of gall bladder disease
210
by reducing bile saturation; help to control diabetes by rendering glycaemic

dietary compounds unabsorbable and reduce symptoms of diverticular disease
(Katina,
211
Table 78. Mean squares for dietary fiber of rice bran supplemented breads
SOV
df
Treatments
Error
Total
5
12
17
Dietary fiber (%)

FFRB-Bread
DFRB-Bread
15.1556**
0.12473
24.5812**
0.1537
** Highly significant at P 0.01

FFRB = Fullfat rice bran supplemented bread; DFRB = Defatted rice bran supplemented bread
Table 79. Dietary fiber of rice bran supplemented breads

Dietary fiber (%)
Treatments
T0
T1
T2
T3
T4
T5
T0
T1
T2
T3
T4
T5
FFRB-Bread
DFRB-Bread
4.620.21f
5.810.24e
6.990.32d
8.160.35c
9.420.43b
10.630.49a
4.620.21f
6.120.29e
7.630.29d
9.150.41c
10.740.45b
12.250.53a
= Bread with 100% wheat flour acts as control for fullfat and defatted rice bran supplementation
= Bread with 5% stabilized fullfat rice bran T1 = Bread with 5% stabilized defatted rice bran
212
2003). A typical Western diet contains less than 20g/day whereas the
recommended daily intake is 25-30g (Drehner, 1987; WHO, 2003). Abdul-Hamid
and Siew Luan (2000) reported that use of defatted rice bran as a source of
dietary fibre in bread, significantly reduced loaf volume and increased crumb
firmness.
Sensory evaluation of bread loaves was carried out for external
characteristics i.e. volume, crust color, symmetry, evenness of bake, character of
crust; and internal characteristics like grain, crumb color, aroma, taste and
texture (Appendix IV) to find out the impact of rice bran supplementation on
these characteristics.
4.5.4.3.1. External characteristics
Mean squares for external characteristics of rice bran supplemented
breads explicated significant differences in volume, crust color, symmetry,
evenness of bake and character of crust due to treatment and storage whereas
interaction was found to be non-significant (Table 80).
4.5.4.3.1.1. Volume
Volume is an important consideration in consumer acceptance and
provides valuable information about product quality. Means for loaf volume in
leavened pan bread (Table 81) showed significant variations within treatments
due to supplementation of fullfat and defatted rice bran in commercial straight
grade flour, used for the bread preparation. In case of fullfat rice bran
supplemented bread, maximum score for volume (7.82) was observed in T0
followed by T1 (7.71) and T2 (6.64) whereas the lowest score (4.67) was found in
T5 with rough grain and honey comb texture. It is obvious from the results that
there was significant decrease in loaf volume with proportionate increase of
supplementation level. The similar decreasing trend in loaf volume was also
observed in bread prepared from different levels of defatted rice bran
supplementation. However, addition of fullfat rice bran produced bread with
improved volume than that of defatted rice bran supplemented flours. Breads
prepared with 100% wheat flour, were comparatively soft when pressed. There
213
was concomitant decrease in gluten protein with an increase in rice bran level
during fermentation. Moreover, by increasing bran portion, there was a decrease
in gas retention resulting lowered bread volume as obvious from the present
results. Means for effect of storage on volume of bread (Table 82) showed
significant decrease in score for volume from 6.68 (0 hour) to 5.74 (120 hours).
The decrease in volume might be due to loss of moisture resulting deleterious
effects on crumb grain and ultimately on volume. The addition of cereal bran,
especially in such amounts that health benefits can be expected, cause problems
in bread quality resulting in flavor changes depending on type of fiber (Seibel,
1983). In previous studies, the deleterious effects of fibre supplementation on
dough structure have been attributed to dilution of gluten network, which in
turn impairs gas retention rather than gas production (Pomeranz et al., 1987).
Bran supplementation usually weakens the structure thus decrease bread
volume and the elasticity of the crumb. Wheat bran upto 20% substitution has
been reported to decrease volume by 19% (Salmenkallio et al., 2001). In another
study, there was 20% decrease in volume with 15% addition of bran (Krishnan et
al., 1987).
4.5.4.3.1.2. Crust color
Bread crust is a dry thin layer that encloses a soft, sponge like cellular
structure. Crust color normally ranges from a deep golden brown of the top to
the light golden brown of the side and bottom. However, the desired crust color
is one with an appetizing golden brown shade. Means for crust color showed
significant variations among the treatments (Table 81). In case of fullfat rice bran
supplemented bread, maximum score for crust color (6.81) was observed in T1
followed by T0 (6.03) and T2 (5.88); whereas the lowest score (3.96) was found in
T5, designated as dark. Means for the effect of storage on bread crust color
showed significant decrease in score (Table 82) from 5.95 (0 hours) to 5.15 (120
hours). Bread prepared from defatted bran also followed similar trend due to
214
treatment and storage. The color of the crust is greatly affected by temperature at
which loaf is baked and the level of residual sugars present in the dough.
Table 80. Mean squares for external characteristics of rice bran supplemented
breads
SOV
df
Treatments 5
Storage
5
TxS
25
Error
144
Total
179
SOV
df
Treatment 5
Storage
5
TxS
25
Error
144
Total
179
SOV
df
Treatment 5
Storage
5
TxS
25
Error
144
Total
179
**
Volume
Color of crust
FFRB-Bread
FFRB-Bread
DFRB-Bread
DFRB-Bread
57.6282**
3.57841**
0.03718ns
0.07087
31.38776**
2.943544**
0.60965ns
0.071460
29.1606**
1.2347**
0.00681ns
0.05404
38.15416**
1.36105**
0.00872ns
0.05987
Symmetry
Evenness of bake
FFRB-Bread
DFRB-Bread
FFRB-Bread
DFRB-Bread
23.3483**
0.97569**
0.02630ns
0.02207
18.16432**
0.817032**
0.016856ns
0.021995
6.15959**
0.09521**
0.02238 ns
0.01068
3.954940**
0.27395**
0.01293 ns
0.00669
Character of crust
FFRB-Bread
DFRB-Bread
8.18317**
0.64756**
0.14452ns
0.0785
8.56903**
0.20910**
0.002203ns
0.009718

Treatments (T); Storage (S)
215
Table 81. Volume and crust color scores of rice bran supplemented breads
Volume
Treatments
T0
T1
T2
T3
T4
T5
FFRB-Bread
Crust color
DFRBBread
7.820.16a
7.710.18a
6.640.17b
5.680.12c
4.830.11d
4.670.11e
7.820.16a
7.670.13b
6.620.13b
6.410.33c
5.750.10d
4.660.10e
FFRB-Bread
6.030.09b
6.810.10a
5.880.09c
5.160.08d
5.020.08e
3.960.06f
DFRBBread
6.030.09b
6.850.10a
6.810.10a
5.880.09c
5.020.08d
3.910.06e
significantly
T0 = Bread with 100% wheat flour acts as control for fullfat and defatted rice bran supplementation
T1 = Bread with 5% stabilized fullfat rice bran T1 = Bread with 5% stabilized defatted rice bran
Table 82. Effect of storage on volume and crust color scores of rice bran
supplemented breads
Storage
(Hours)
0
24
48
72
96
120
Volume
Crust color
FFRB-Bread
DFRB-Bread
FFRB-Bread
DFRB-Bread
6.680.59a
6.540. 59a
6.440. 59a
6.130.56b
5.930.54c
5.740.52d
6.680.45a
6.660.45a
6.300.41b
6.260.52bc
6.090.39c
5.900.39d
5.950.48a
5.660.42a
5.570.41ab
5.460.40bc
5.350.39c
5.150.38b
5.950.48a
5.940.48a
5.850.47ab
5.730.46bc
5.610.45c
5.400.43d
216
These sugars result in caramelization and Maillard reaction due to

interaction of proteins and reducing sugars. Too much bran tends to cause the
final bread to be darker; at low levels as in case of 5-10% rice bran, it improves
the pale color without requiring additional time or temperature. The results are
in line with the previous findings of Lynn (1969) that defatted rice bran can
contribute towards attractive crust and crumb formation. In some bread recipes,
the color may be darker, when wholemeal, brown or non-wheat flours are used
during production (Khetarpaul et al., 2005). There are certain limits for enriching
baked products with brans due to their detrimental affects on sensory attributes
like appearance, mouthfeel, flavor and chewing characteristics especially at
higher concentrations (Salmenkallio et al., 2001).
4.5.4.3.1.3. Symmetry
Means for symmetry showed significant variations among the treatments
as a result of fullfat and defatted rice bran supplementation (Table 83). In case of
fullfat rice bran supplemented bread, maximum score for symmetry (4.29) was
observed in T0 followed by T1 (bread with 5% stabilized fullfat rice bran) and T2
(bread with 10% stabilized fullfat rice bran) i.e. 4.22 and 4.13, respectively;
whereas the lowest score (2.18) was found in T5 (bread with 25% stabilized fullfat
rice bran). Slightly low levels of rice bran may strengthen dough to get a more
symmetrical form and withstand oven spring and avoid collapse. However, at
higher level, there was a decrease in bread volume, deforming the shape of the
bread. Similar decreasing trend in symmetry was observed with defatted rice
bran. Means for effect of storage on symmetry showed significant decrease in
score (Table 84) from 3.64 (0 hours) to 3.18 (120 hours). There was non-significant
effect of storage upto 48 hours in both types of supplemented flours. The rest of
changes might be due to evaporation and gas loss from bread surface that has
inverse correlation with symmetry of bread.
217
4.5.4.3.1.4. Evenness of bake

Evenness of bake means that loaf has been made with a uniformity of bake
on all side. In case of fullfat rice bran supplemented bread (Table 83), maximum
score
218
Table 83. Symmetry and evenness of bake scores of rice bran supplemented
breads
Symmetry
Treatments
T0
T1
T2
T3
T4
T5
FFRB-Bread
DFRBBread
4.290.10a
4.220.09a
4.130.10b
2.940.04c
2.940.06c
2.180.05d
4.290.10a
3.930.06b
3.880.07b
3.720.06c
3.070.06d
2.060.06e
Evenness of bake
FFRB-Bread
2.270.04a
2.200.06b
2.170.03b
1.990.19c
1.650.03d
1.500.02e
DFRBBread
2.270.04a
2.190.04b
2.160.03b
1.960.03c
1.500.07d
1.450.02e
Table 84. Effect of storage on symmetry and evenness of bake of rice bran
supplemented breads
Storage
(Hours)
0
24
48
72
96
120
Symmetry
Evenness of bake
FFRB-Bread
DFRB-Bread
FFRB-Bread
DFRB-Bread
3.640.38a
3.610.38a
3.550.38a
3.450.37b
3.300.33c
3.180.32d
3.640.38a
3.630.32a
3.530.34b
3.450.33c
3.340.31d
3.220.30e
2.520.19a
2.460.10b
2.410.21bc
2.400.21bcd
2.400.21cd
2.350.20d
2.520.19a
2.000.15a
1.980.15a
1.930.14b
1.860.14c
1.760.17d
219
for evenness of bake (2.27) was observed in T0 followed by T1 (2.20) and T2 (2.17)
whereas the lowest score (1.50) was found in T5. The loaves were still acceptable
upto 10-15% supplementation levels because crust was still attractive, with
uniform thickness and acceptable color. A similar decreasing trend was observed
with defatted rice bran. However, T1 (bread with 5% stabilized defatted rice
bran) and T2 (bread with 10% stabilized defatted rice bran) were statistically nonsignificant. Means for effect of storage on symmetry showed significant decrease
in score (Table 84) from 2.52 (0 hours) to 2.35 (120 hours) in fullfat rice bran while
2.52 to 1.76 in defatted rice bran supplemented bread.
4.5.4.3.1.5. Character of crust
Generally consumers show a decided preference for a thin, tender crust in
the conventional white pan loaf. The results in Table 85 explored that in case of
fullfat rice bran supplemented bread, maximum scores (2.96) were observed in T0
followed by T1 (2.72) and T2 (2.55); whereas the lowest scores (1.39) were found
in T5. At higher bran levels, crust tends to be too hard, hence unacceptable for the
consumers. It is apparent that there was significant decrease in crust character
with increased supplementation levels and same decreasing trend was also
observed with defatted rice bran supplementation. Means for effect of storage on
character of crust showed significant decrease in score (Table 86) from 2.39 (0
hours) to 1.83 (120 hours) in fullfat rice bran breads while 2.39 to 2.18 in defatted
rice bran supplemented bread.
4.5.4.3.2. Internal characteristics
220
Mean squares for internal characteristics of rice bran supplemented bread

explicated significant differences in grain, crumb color, aroma, taste and texture
due to treatment and storage whereas interaction was found to be non-significant
(Table 87).
4.5.4.3.2.1. Grain
Grain of the crumb has been defined as the cell structure as it is exposed
when the loaf of bread is sliced. A grain is designated as open if it consists of
221
Table 85. Character of crust scores of rice bran supplemented breads

Character of crust
Treatments
T0
T1
T2
T3
T4
T5
FFRB-Bread
DFRB-Bread
2.960.41a
2.720.31b
2.550.20bc
2.300.04c
1.480.03d
1.390.03de
2.960.03a
2.570.22ab
2.360.03b
2.120.03bc
2.020.03c
1.520.03d
Table 86. Effect of storage on character of crust of rice bran supplemented

breads
Storage
(Hours)
0
24
48
72
96
120
Character of crust
FFRB-Bread
DFRB-Bread
2.390.22a
2.180.25a
2.150.25a
2.010.19b
1.950.19c
1.830.18d
2.390.22a
2.380.22a
2.340.22ab
2.290.22b
2.220.21c
2.180.22c
222
Table 87. Mean squares for internal characteristics of rice bran supplemented
breads
SOV
df
Treatment 5
Storage
5
TxS
25
Error
144
Total
179
SOV
df
Treatment 5
Storage
5
TxS
25
Error
144
Total
179
SOV
df
Treatment 5
Storage
5
TxS
25
Error
144
Total
179
**
Grains
Crumb color
FFRB-Bread
DFRB-Bread
FFRB-Bread
DFRB-Bread
213.5894**
2.16205**
0.26190ns
0.16594
118.3269**
1.047453**
0.00266ns
0.19020
55.4996**
1.74840**
0.01291ns
0.07582
33.54116**
1.89932**
0.007803ns
0.081061
Aroma
Taste
FFRB-Bread
DFRB-Bread
FFRB-Bread
DFRB-Bread
33.27018**
2.22143**
0.00940ns
0.08103
39.47838**
1.93551**
0.00936ns
0.082247
90.0967**
4.3833**
0.02067ns
0.19574
44.75885**
4.504027**
0.008439ns
0.234556
Texture
FFRB-Bread
DFRB-Bread
61.02292**
1.52251**
0.00492ns
0.18667
84.17245**
1.66116**
0.00677ns
0.204542

Treatments (T); Storage (S)
223
large individual cells, and as close if it consists of minute cells. In case of fullfat
rice bran supplemented bread (Table 88), maximum score (11.94) was observed
in T0 followed by T1 (11.50) and T2 (11.02); whereas the lowest score (4.91) was
found in T5. Rice bran at higher levels adversely affects grains of crumb resulting
in thick walled coarse grained crumb structure due to poor gluten development
during mixing. However, at lower supplementation levels, better mixing
stability, resulted in strengthening of dough for better crumb or grain structure.
Similar decreasing trend in quality score was also noted in defatted rice bran
supplementation. In case of defatted rice bran supplementation, T0 and T1; T2 and
T3 were statistically non-significant. Means for effect of storage on crumb grain
showed significant decrease in score (Table 89) both in fullfat and defatted rice
bran supplemented breads. During storage, the phenomenon of starch
retrogradation pick up the pace with the passage of time, leading to recrystallization of starch particles that collapsed the crumb grain, hence decreased
score for this characteristic.
4.5.4.3.2.2. Crumb color
The results in Table 88 elucidated that in fullfat rice bran supplemented
bread, maximum score (7.90) was observed in T0 followed by T1 (7.85) and T2
(7.08) whereas the lowest score (4.86) was recorded for T5 (bread with 25%
stabilized fullfat rice bran), because breads with more bran content are coarse
grained and tend to be darker than those without bran or with low levels of rice
bran (Sharma et al., 2004). The similar decreasing trend in quality score was also
noted in defatted rice bran supplemented breads. However, T0 (bread with 100%
commercial straight grade flour) and T1 (bread with 5% stabilized defatted rice
bran); likewise, T2 and T3 were statistically non-significant. Means for effect of
storage on crumb grain showed momentous decrease in score (Table 89) from
6.98 (0 hour) to 6.07 (120 hours) for fullfat and 6.98 to 6.32 in defatted rice bran
supplemented bread.
224
4.5.4.3.2.3. Aroma
The aroma is the quality perceived by the sense of smell. It plays a
primary role in coining consumer appeal. The means for aroma (Table 90)
showed significant
Table 88. Grains and crumb color scores of rice bran supplemented breads
Grains
Treatments
T0
T1
T2
T3
T4
T5
FFRB-Bread
11.940.09a
11.500.30b
11.020.08c
9.080.07d
8.060.06e
4.910.06f
Crumb color
DFRBBread
11.94a
11.92a
10.98b
10.93b
8.90c
6.90d
FFRB-Bread
7.900.12a
7.850.12a
7.080.11b
6.020.09c
5.010.08d
4.860.07e
DFRBBread
7.900.12a
7.800.12a
6.790.10b
6.740.10b
5.970.09c
5.150.08b
Table 89.
Storage
(Hours)
0
24
48
72
96
120
Effect of storage on grains and crumb color of rice bran

supplemented breads
Grains
Crumb color
FFRB-Bread
DFRB-Bread
FFRB-Bread
DFRB-Bread
10.430.82a
9.631.12a
9.581.12a
9.491.12a
9.151.04b
9.021.03b
10.430.82a
10.420.81a
10.370.81a
10.280.81ab
10.110.81bc
9.970.80c
6.980.45a
6.880.39a
6.550.56ab
6.440.55bc
6.300.54c
6.070.52b
6.980.45a
6.960.45ab
6.820.44bc
6.710.43cd
6.570.42d
6.320.41e
225
variations in fullfat rice bran supplemented bread, maximum score (7.97) was
observed in T1 (bread with 5% stabilized fullfat rice bran) followed by T2 (7.78)
and T0 (7.07) whereas the lowest score (5.38) was found in T5 (bread with 25%
stabilized fullfat rice bran); might be due to the presence of too much bran in
bread which alter its aroma towards branny than wheaty. However, bran at low
levels resulted improve aroma, as liked by the judges, especially in rice
consuming regions. In bread, 15% defatted rice bran supplementation was more
appealing as compared to fullfat supplemented breads at same level. Means for
effect of storage on aroma (Table 91) showed momentous decrease in score from
7.02 (0 hour) to 6.29 (120 hours) and 7.07 to 6.36 for fullfat and defatted rice bran
supplemented breads, respectively. Fresh bread loaves had highest aroma due to
presence of more aromatic compounds which were lost with progressive storage.
4.5.4.3.2.4. Taste
Taste is the major component of flavor detected by the taste buds of the tongue
and mouth membranes. Means for taste showed significant variations in fullfat rice
bran supplemented bread (Table 90), maximum score (16.67) was observed in T2
followed by T1 (bread with 5% stabilized fullfat rice bran) and T0 (bread with 100%
straight grade flour) i.e. 15.66 and 15.16, respectively; whereas the lowest score (11.83)
was assigned to T5. Breads supplemented with defatted rice bran at 10 and 15%
supplementation level were statistically non-significant but significantly different
from rest of the treatments; might be due to the presence of higher levels of bran that
alter the taste.
The results of present study are in confirmity with the observations recorded
by previous researchers. There are limits to enrich the baked products with cereal
226
bran, as it has detrimental affects on sensory attributes (Salmenkallio et al., 2001).

Means for effect of storage on taste of bread (Table 91) showed significant decrease in
score from 13.86 (0 hours) to 12.80 (120 hours) for fullfat and 14.86 to 13.86 in defatted
rice bran supplemented breads.
Table 90. Aroma and taste scores of rice bran supplemented breads
Aroma
Treatments
T0
T1
T2
T3
T4
T5
Taste
FFRB-Bread
DFRB-Bread
FFRB-Bread
DFRB-Bread
7.070.11c
7.970.13a
7.780.13b
6.300.10d
5.860.10e
5.380.09f
7.070.11c
6.790.10d
7.800.12b
8.000.12a
5.970.99e
4.960.08f
15.160.15c
15.660.18b
16.670.19a
13.880.15d
13.470.15e
11.830.12f
15.160.15c
15.740.16b
16.810.18a
16.660.17a
15.250.15c
13.460.13b
Table 91.
Storage
(Hours)
0
24
48
72
96
120
Effect of storage on aroma and taste of rice bran supplemented

breads
Aroma
Taste
FFRB-Bread
DFRB-Bread
FFRB-Bread
DFRB-Bread
7.020.49a
6.980.45ab
6.840.44bc
6.710.43c
6.550.42b
6.290.40e
7.070.49a
7.010.48ab
6.860.47bc
6.750.47cd
6.600.46d
6.360.44e
13.860.73a
13.770.73a
13.660.72ab
13.450.71b
13.210.69c
12.800.67d
14.860.51a
14.840.51a
14.720.51ab
14.520.50bc
14.280.49c
13.860.47d
227
228
Fresh loaves obtained the highest score due to presence of flavoring

compounds formed during fermentation of dough. When bread is stored for longer
periods, these volatile compounds are gradually decreased due to chemical and
biochemical changes taking place in bread and thus ultimately affect the taste.
4.5.4.3.2.5. Texture
It is considered as a major quality factor associated with product
freshness. In fullfat rice bran supplemented bread (Table 92), maximum score for
texture (12.02) was observed in T0 (bread with 100% commercial straight grade
flour), followed by T1 (10.85) and T2 (10.80) whereas the lowest score (8.01) was
found in T5. Similar decreasing trend in texture score was observed in defatted
bran supplemented bread at various supplementation levels. The texture of
bread significantly decreased from 10.94 (0 hour) to 9.91 (hours) in fullfat rice
bran supplemented bread while 10.99 to 10.35 in defatted rice bran
supplemented bread (Table 93). This was probably due to retrogradation of
starch leading to crumb firmness. At 0 day, retrogradation of amylose causes the
crumb structure to be established. After that, amylopectin retrogradation is the
main reason for bread firming and badly affects its texture. Bread heating may
cause the bread to retain its freshness as the energy applied may serve to break
the bonds between starch polymers formed during retrogradation (Pyler, 1988).
The present results are supported by the previous findings that increasing
level of bran particles in formulations had significant effect on both internal as
well external attributes of bread, consequently lowering its overall acceptability
(Gujral et al., 2003). The firmness of fresh fiber-enriched bread has been reported
to be 41% higher as compared to bread without bran (Laurikainen et al., 1998).
The results of present work are within the range recorded earlier by numerous
researchers. Rice bran can be successfully supplemented upto 10 to 15%
replacement without affecting sensoric attributes (Chumachenko et al., 1987;
Sharp and Kitchen, 1990; Sekhon et al., 1997).
229
Table 92. Texture scores of rice bran supplemented breads

Texture
Treatments
T0
T1
T2
T3
T4
T5
FFRB-Bread
DFRB-Bread
12.020.11a
10.850.10b
10.800.10bc
10.610.10c
9.140.08d
8.010.07e
12.020.11a
11.830.11ab
11.780.11b
11.390.10c
8.990.08d
8.160.07e
Table 93. Effect of storage on texture of rice bran supplemented breads

Texture
Storage
(Hours)
FFRB-Bread
DFRB-Bread
0
24
48
72
96
120
10.940.70a
10.750.59a
10.340.59ab
10.210.58bc
10.060.57cd
9.910.56b
10.990.70a
10.910.70a
10.810.69ab
10.670.68bc
10.510.67cd
10.350.66d
230
However, in similar studies, stabilized rice bran was successfully incorporated

upto 20% for the production of bread (Carroll, 1990; Singh et al., 1995; Lima et al.,
2002).
It is an established fact that high-fibre diets, especially those containing
cereal fibers, have definite health benefits in reducing the risk of chronic diseases
such as diabetes, cancer and coronary heart disease (Katina, 2003). The
supplementation
of
rice
bran
increased
the
dietary
fiber
in
breads
proportionately to the level of supplementation. It is concluded that leavened

pan bread can be prepared from stabilized fullfat and defatted rice bran
supplemented flours upto 15% level without deleterious effects on its quality.
231
Chapter-V
SUMMARY
Rice bran has been recognized as an excellent source
of
edible
oil,
protein,
dietary
fiber
and
allied
micronutrients like minerals, vitamins and antioxidants.

Inspite of excellent nutritional profile, the main hindrance
in exploring its full potential is inherent enzyme system
especially lipases causing rancidity; hence successful use is
only possible after stabilization. In present study, rice bran
samples
stabilized
by
extrusion
cooking
(ES-RB),
microwave heating (MW-RB) and parboiling (PAR-RB);

were analyzed for lipase activity (in terms of FFA
production), peroxide value (POV) and thiobarbituric acid
no. (TBA no.) during two months storage. The maximum
FFA level was observed in unstabilized rice bran (17.75%)
followed by PAR-RB (6.95%) and ES-RB (6.12%); however,
the minimum value (5.25%) was found in MW-RB. The
highest POV (0.91 mEq/Kg) was in unstabilized rice bran
followed by PAR-RB (0.88 mEq/Kg) whereas the minimum
value (0.80 mEq/Kg) was recorded for MW-RB. Similarly,
the highest TBA value (0.083) was observed in unstabilized
rice bran followed by PAR-RB (0.074), ES-RB (0.063) and
232
MW-RB
(0.062
mg
malenaldehyde/Kg).
Stabilization
techniques resulted significant reduction in phytates,

haemagglutinin and trypsin inhibitors. It was observed
that MW stabilization is effective in controlling enzyme
activity without affecting the nutritional quality of rice
bran.
After stabilization and antinutritional appraisal, oil was extracted from bran
samples. The maximum oil was recovered form PAR-RB (16.10%) after refining;
followed by MW-RB (15.75%) and ES-RB (15.45%). Extracted oils were evaluated
for physical & chemical characteristics, fatty acid profile and antioxidant
potential. Overall, the concentration of oryzanol, tocopherols and tocotrienols in
experimental bran oil samples ranged 590.78-703.42g/g, 36-275 mg/kg and 41167mg/kg, respectively. The chemical analysis reveled that fullfat rice bran
(FFRB) samples contain 14.20-16.20% crude protein, 18.22-19.30% crude fat,
10.68-17.03% crude fiber, 8.30-9.03% ash content, 36.44-47.56% NFE, 7.209.30mg/100g Na, 1270-1820mg/100g K, 538.57-872.50mg/100g Ca and 9351233mg/100g Mg; whereas comparatively higher values were noted in defatted
rice bran (DFRB) except for crude fat.
In current study, microwave stabilized rice bran (MWRB) was selected for
oil extraction and product development owing to better stability, color of oil and
high antioxidant potential. Microwave stabilized fullfat rice bran; its defatted
portion and extracted oil were used for efficacy studies and preparation of value
added products. Sprague Dawley rats (SD-rats) were fed on diets comprised of
FFRB, DFRB and RBO alongwith control for 45 days and evaluated for physical
and hematological parameters. The rats fed on RBO diet showed highest feed
intake (19.21g/rat/day); water intake (37.81mL/day) and gain in body weight
(7.24g per rat/week). No significant effect of treatments was observed on organs
233
weight, renal and liver in experimental animals. Mean squares of serum

biochemical profile in relation to cholesterol, LDL, triglycerides and glucose
showed significant variations whereas HDL, serum protein, albumin, globulin
and A/G ratios exhibited non-significant differences in rats fed on various
experimental diets. Animals fed on FFRB, DFRB and RBO resulted significant
reduction in serum cholesterol, LDL, triglycerides and glucose with nonsignificant improvement in HDL. Thus, present results suggested that feeding of
experimental diets has no adverse effects on the growth and blood profile of SDrats, showing their suitability for product development.
After efficacy and safety evaluation, RBO was supplemented in cookies
while fullfat & defatted rice brans were blended in wheat flour to prepare
fortified flours. Cookies were prepared by replacing normal shortening @ 20, 40,
60, 80 and 100%, with RBO. There was gradual reduction in spread of cookies
with the proportionate increase of RBO. During 60 days storage; moisture
content was increased from 3.64 to 4.87% whereas protein and fat content
decreased from 7.90 to 7.15 and 21.76 to 20.87%, respectively. There was an increase
in total acidity and TBA no. from 0.147 to 0.194% and 0.033 to 0.079 mg
malenaldehyde/Kg, respectively; cookies containing RBO showed better shelf might be
due to natural antioxidants present in RBO. Sensory evaluation of cookies revealed
significant variations due to treatments and storage. The judges preferred T2
(RBO 40%) due to better color, flavor, texture and crispness. Collectively, the
maximum scores for overall acceptability were assigned to fresh cookies that
gradually decreased during storage. It was concluded that rice bran oil can
successfully be substituted with normal shortening for preparation of cookies
upto 40-60%, although higher levels were still acceptable.
Fullfat (FFRB) and defatted rice brans (DFRB) were mixed separately with
CSGF in different proportions and were analyzed for chemical composition and
rheological characteristics to find out the most appropriate compositions
showing suitability for preparation of cookies and leavened bread. Rice bran
supplementation significantly improved crude protein (10.24-13.68%), crude fat
(0.84-9.82%), crude fiber (0.86-6.78%) and ash content (1.22-5.77%). The
supplementation of either type of bran significantly improved the mineral
content except sodium. There was significant increase in K (149.79-826.66), Ca
(133.09-387.17) and Mg (69.24-607.02) mg/100g, respectively. Maximum dietary
fiber (22.10%) was observed in T16 (defatted stabilized rice barn 50%) whereas the
lowest (5.14%) was found in T0 (CSGF). Maximum increase in dietary fiber was
observed in T16 (76.74%) followed by T15 (72.52%) and T8 (72.26%) as compared to
control. The rheological behavior of the supplemented flours demonstrates
234
highly significant effect of treatments on the mixographic and farinographic

characteristics. The mixing time and peak height of supplemented flours ranged
1.96-3.43 minutes and 58.73-68.69%, respectively. Defatted rice bran
supplemented flours exhibited comparatively high values (2.45-2.70 vs. 1.96-3.43
min) for mixing time, probably due to more protein in defatted rice bran. The
peak height was gradually increased upto 15% supplementation and then
decreased at higher levels with either type of bran. Farinographic parameters like
water absorption, dough development time and stability of supplemented flours
ranged 60.20-72.30%, 3.49-8.01min and 3.55-15.35min, respectively. The
maximum water absorption was observed in FFRB supplemented flours (69.4272.3 vs. 60.20-69.28%); whereas the highest dough development (4.79-8.01 vs.
3.49-5.80min) and stability (9.37-15.35 vs. 3.55-7.43min) were recorded for DFRB
supplemented flours.
Cookies were prepared form supplemented flours and

analyzed for physicochemical characteristics. There was a
gradual decrease in the spread of cookies with the
proportionate increase of supplementation. The maximum
value (47.80) was observed in T0 (cookies without RB)
whereas minimum (36.83) in cookies prepared from 50%
rice
bran
supplementation.
There
was
significant
improvement in dietary fiber (3.90-11.01%), K (102.96407.01 mg/100g), Ca (76.11 to 190.6 mg/100g) and Mg (57.54298.87mg/100g)
content
of
cookies
with
bran
supplementation. Sensory evaluation explicated more

likeliness of judges for cookies supplemented with 20%
FFRB (T2) followed by T1 (cookies with 10% FFRB) and T0
(cookies without RB). Similar trend was observed in case
of cookies prepared from defatted bran supplemented
flours. It was concluded that cookies can be supplemented
235
with either type of rice bran upto 10-20% without affecting

sensoric attributes.
Leavened pan bran bread was also prepared from
fullfat and defatted rice bran supplemented flours and
analyzed for various quality traits. There was momentous
increase in mineral content with supplementation; K, Ca
and Mg contents were improved from 134.21 to 411.86,
119.42 to 223.33 and 62.03 to 282.72mg/100g, respectively.
Likewise, dietary fiber content of supplemented breads
increased from 5.81 to 12.25%; maximum increase (62.28%)
was observed in T5 (25% DFRB) followed by T4 (20%
DFRB) and T3 (15% DFRB) i.e. 56.98 and 49.51% as
compared
to bread prepared
from
CSGF.
Sensory
evaluation of rice bran supplemented breads was carried

out for external and internal characteristics. Maximum
score for volume (7.82) was observed in T0 (100% CSGF)
followed by T1 (5% FFRB) and T2 (10% FFRB) i.e. 7.71 and
6.64, respectively. There was gradual decrease in bread
quality with increased levels of supplementation resulting
in reduced volume, darker crust, shape deformation and
rough grains. It is suggested that leavened pan bread can
236
be prepared form bran supplemented flours upto 15%

level.
It is concluded from the present investigation, that rice bran has a great
potential to be used for oil extraction as well as preparation of value added
products. This will not only be helpful to minimized import of edible oil in
Pakistan but also ensure food security by introducing bran supplemented
products. Moreover, the outcomes derived from present investigation would be
supportive for the scientists, researchers and stakeholders dealing with food for
better understanding of the compositional, nutritional and nutraceutical role of
rice bran.
237
RECOMMENDATIONS
Stabilized rice bran should be declared as food grade

ingredient in Pakistan; the government needs to devise ways to
properly utilize the rice milling by-products for value addition in WTO
scenario.
Rice bran should be explored as source of edible oil; if

fully utilized, can become one of the major
indigenous non-conventional edible oil in the
country.
Rice bran oil should be advised as cooking medium to
get additional hypocholesterolemic benefits.
Defatted rice bran should be included in daily diet to
improve protein, dietary fiber and minerals for their
allied health claims.
Phytochemicals and unsaponifiable fraction from rice
bran should be extracted and used for the preparation
of nutraceutical/functional foods.
238
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264
Appendix I
Composition of salt mixture
Calcium citrate
308.2
Ca (H2PO4)2 H2O
112.8
H2HPO4
218.7
HCl
124.7
NaCl
77.0
CaCO3
68.5
3MgCO3. Mg (OH) 2. 3H2O
35.1
MgSO4 anhydrous
38.3
Ferric ammonium citrate
91.41
CuSO4. 5H2O
5.98
NaF
0.76
MnSO4. 2H2O
1.07
KAl (SO4)2. 12H2O
0.54
KI
0.24
100.00
265
16.7
1000.00
Appendix II
Composition of vitamin mixture
Thiamin hydrochloride
0.060
Riboflavin
0.200
Pyridoxin hydrochloride
0.040
Calcium pentothenate
1.200
Nicotinic acid
4.000
Inositol
4.000
p-aminobenzoic acid
12.000
Biotin
0.040
Folic acid
0.040
Cyanocobalamin
0.001
Choline chloride
12.000
Maize starch
966.419
1000.00
266
Appendix III
Performa for sensory evaluation of cookies
Name of the judge
Character
Color
Flavor
Taste
Texture
Crispness
Overall acceptability
T0
Date..
T1
T2
T3
T4
T5
Signature..
INSTRUCTIONS
Chew a sample of cookies and score for color, flavor, taste, texture,
crispness and overall acceptability using the following 9-point Hedonic Scale:
Extremely poor
Very poor
Poor
Below fair above poor
Fair
Below good above fair
Good
Very good
Excellent
1
2
3
4
5
6
7
8
9
Note:
1.
2.
3.
4.
Chew a sample of cookies and score for color, flavor etc.

Before proceeding to the next sample, rinse mouth with water.
Make inter comparison of the sample and record the score.
Don't disturb the order of samples.
267
209
Appendix V
Saturated / unsaturated fatty acids profile of rice bran oil
Oil
Mustard / Rapeseed
Cottonseed
Sunflower
Safflower
Soybean
Palm
Olive
Canola
Corn
SFA
6
28
12
10
16
51
14
6
13
Fatty Acid % weight

MUFA
67
22
21
15
24
39
77
58
20
PUFA
27
50
67
75
60
10
9
36
62
210
Coconut
Palm Kernel
Groundnut
Rice Bran
WHO Recommended
92
86
20
18
28.6
(Below 33%)
6
12
50
45
42.8
(Above 33%)
2
2
30
37
28.6
(Below 33%)
Rice Bran Oil with Ideal SFA/MUFA/PUFA ratio, which is the closest to WHO recommendation as compared to other edible oils (CAC, 2003).
Appendix VI
Micro-nutrient profile of rice bran oil
211
Micro-nutrient
Amount %
Advantages
1.2 1.7
Increases high density lipoproteins &

decreases low density lipoproteins,
antidandruff and anti-itching agent,
retards aging
Tocopherol
0.02 0.08
Antioxidant, antitumor, decreased

risk of coronary heart diseases and
arthritis
Tocotrienol
0.025 0.170
Hypocholesterolemic,
anticancer,
antioxidant,
protection
from
atherosclerosis
Oryzanol
Squalene
0.3 0.4
Skin toning
(CAC, 2003)
212
Appendix IV
Performa for sensory evaluation of leavened pan bread
Name of the judge..
A
External Characteristics
Max.
Score
Volume
10
Color of crust
Symmetry
Evenness of bake
Character of crust
Sub total
30
Internal Characteristics
Max.
Score
T0
T0
T1
T1
T2
T2
T3
T3
T4
T4
Date..
Treatments
T5
T6
T7
Treatments
T5
T6
T7
T8
T8
T9
T9
T10
T11
T10
T11
213
Grain
15
Color of crumb
10
Aroma
10
Taste
20
Texture
15
Sub total
70
Grand total
100
Signature....
214
Table 32. Mean squares for proximate composition of RBO cookies

SOV
df
Moisture
Protein
Fat
Fiber
Ash
Treatments (T)
Storage (S)
SxT
Error
Total
5
2
10
36
53
0.0117ns
17.024**
0.0034ns
0.0220
0.001ns
2.528**
0.004ns
0.039
0.195ns
16.97**
0.054ns
0.090
0.003ns
3.62910-4ns
4.96310-4ns
0.002
0.001ns
0.001ns
7.34010-4ns
0.002
**
104
NFE
0.189ns
2.419**
0.048ns
0.372
Table 38. Mean squares for sensory attributes of RBO cookies

SOV
df
Color
Flavor
Taste
Texture
Crispness
Treatments (T)
Storage (S)
SxT
Error
Total
5
2
10
36
53
7.539**
9.045**
0.132ns
0.207
3.143**
8.580**
0.128ns
0.195
7.313**
5.719**
0.249ns
0.199
0.957**
8.173**
0.104ns
0.194
2.844**
4.048**
0.047ns
0.206
**
50
1.981**
5.965**
0.084ns
0.130
Table 45. Mean squares for proximate composition of rice bran supplemented
flours
SOV
df
Moisture
Protein
Fat
Fiber
Ash
NFE
Treatments (T)
Storage (S)
SxT
Error
Total
16
2
32
102
152
3.294**
6.894**
0.004ns
0.0742
8.776**
2.349**
0.001ns
0.086
72.636**
0.170**
0.009ns
0.011
30.282**
0.012ns
2.82810-5 ns
0.008
17.642**
0.009ns
1.57210-4 ns
0.006
219.80
0.909n
0.019n
0.635
**
Table 64. Mean squares for physical parameters of rice bran supplemented
cookies
SOV
df
eatments
orage
xS
ror
tal
5
2
10
72
89
Width
Thickness
FFRB-Cookies
DFRB-Cookies
FFRB-Cookies
DFRB-Cookies
48.5929**
0.36501ns
0.00961ns
5.89832
67.5369**
0.3243ns
0.0087ns
0.1138
2.4765**
0.0298ns
4.9778x10-4ns
0.0319
2.0897**
0.0359ns
4.9963x10-4ns
0.0214
**
Spread ratio
FFRB-Cookies
155.2586**
0.016679ns
0.01125ns
7.732122
Highly significant at P 0.01; ns Non-Significant; Treatments (T); Storage (S).
DFRB
158.43*
0.0616n
0.0158n
0.6908
Table 65. Physical analysis of rice bran supplemented cookies

Treatments
T0
T1
T2
T3
T4
T5
Width
Thickness
Spread ratio
FFRB-Cookies
DFRB-Cookies
FFRB-Cookies
DFRB-Cookies
FFRB-Cookies
DFRB-Co
44.150.06a
43.090.09a
40.230.09b
39.170.05b
39.050.12b
38.670.08b
44.150.06a
41.250.08b
39.580.10c
39.110.09d
37.490.09e
9.230.02c
9.340.02c
9.350.02c
9.900.02b
9.950.02b
9.230.02c
9.390.03c
9.400.02c
9.430.03c
9.950.03b
47.800.08a
46.100.01a
41.390.01b
40.620.01b
39.410.04bc
47.800.08
45.550.06
41.470.07
38.860.01
38.300.02
36.530.06f
10.600.03a
10.340.03a
36.830.01c
37.670.01
significantly
T0 = Cookies with 100% wheat flour, act as control for both fullfat and defatted rice bran
supplementation
T1 = Cookies with 10% FFRB
Table 66. Effect of storage on physical analysis of rice bran supplemented

cookies
Storage
(days)
0
Width
Thickness
Spread ratio
FFRB-Cookies
DFRB-Cookies
FFRB-Cookies
DFRB-Cookies
FFRB-Cookies
DFRB-C
40.880.86
39.831.02
9.770.20
9.620.18
42.001.54
41.541.5
30
60
40.680.86
40.610.88
39.651.02
39.571.02
9.720.20
9.690.19
9.560.18
9.530.18
42.021.54
42.061.56
41.621.5
41.661.5
Table 71. Mean squares for sensory attributes of rice bran supplemented cookies
OV
df
eatments
orage
xS
ror
tal
OV
5
2
10
72
89
df
eatments
orage
xS
ror
tal
5
2
10
72
89
Color
Flavor
FFRB-Cookies
DFRB-Cookies
FFRB-Cookies
DFRB-Cookies
9.8286**
4.2255**
0.0058ns
0.0791
4.3269**
5.1145**
0.0156ns
0.0828
12.024**
2.9188**
0.0080ns
0.0823
3.6601**
3.1716**
0.0032ns
0.0829
Texture
Crispness
Taste
FFRB-Cookies
DFRB
12.8208**
3.1682**
0.0152ns
0.0835
3.2393*
2.9323*
0.0055n
0.0824
FFRB-Cookies
DFRB-Cookies
FFRB-Cookies
DFRB-Cookies
FFRB-Cookies
DFRB-
8.4316**
3.0798**
0.0063ns
0.0781
4.6557**
4.0340**
0.0095ns
0.0867
13.2185**
5.2091**
0.0115ns
0.0822
5.2263**
4.9946**
0.0078ns
0.0828
7.6539**
4.8925**
0.0096ns
0.0758
2.8054*
4.2157*
0.0117n
0.0808
**
Highly significant at P 0.01; ns Non-Significant; Treatments (T); Storage (s).
Table 72. Color, flavor and taste scores of rice bran supplemented cookies
Color
Treatments
T0
T1
T2
Flavor
Taste
FFRB-Cookies DFRBCookies
FFRB-Cookies
DFRB-Cookies
FFRB-Cookies
DFRBCookies
6.650.20b
6.800.23b
7.480.23a
6.780.19b
6.880.16b
7.820.17a
6.780.19b
6.850.19c
7.100.20b
6.870.16b
6.930.16b
7.850.17a
6.870.16
6.830.19
6.900.19
6.650.20bc
6.570.25c
6.810.25b
T3
T4
7.550.23a
6.170.19c
7.480.25a
7.470.26a
7.780.22a
6.130.17c
7.060.19b
7.450.17a
7.820.22a
6.170.19c
T5
5.410.22d
6.120.23d
5.510.17d
5.980.19d
5.480.23d
7.100.17
7.420.20
6.020.17
significantly
T0 = Cookies with 100% wheat flour, act as control for both fullfat and defatted rice bran
supplementation
Table 73. Effect of storage on color, flavor and texture of rice bran
Storage
(days)
0
30
60
Color
FFRB-Cookies
7.060.34a
6.670.33b
6.310.32c
Flavor
DFRB-Cookies
7.260.23a
6.860.21b
6.430.22c
FFRB-Cookies
7.130.37a
6.830.37b
6.510.36c
DFRB-Cookies
Taste
FFRB-Cookies
7.190.20a
6.840.20b
6.540.21c
7.180.37a
6.840.38b
6.530.38c
DFRB-Co
7.160.20
6.870.19
6.530.19
Table 74. Texture, crispness and overall acceptability scores of rice bran
Texture
Treatments
T0
T1
T2
T3
T4
T5
Crispness
Overall acceptabilit
FFRB-Cookies DFRBCookies
FFRB-Cookies
DFRB-Cookies
FFRB-Cookies
DFRB-Co
7.580.22a
7.150.17b
6.800.17c
6.800.20c
6.050.17d
5.520.17e
7.670.25a
7.520.26a
7.170.27b
7.180.23b
5.850.23c
5.400.20d
7.670.25a
7.020.23b
7.100.20b
6.720.25c
6.800.23c
5.870.26d
6.780.25c
7.080.20b
7.300.25a
6.750.25c
5.920.32d
5.450.23e
6.780.25
7.050.20b
7.280.22
6.770.23
6.780.23
6.000.17
7.580.22a
7.330.22b
7.320.25b
7.050.25c
6.820.22d
6.010.17e
Table 75. Effect of storage on texture, crispness and overall acceptability of rice
bran supplemented cookies
Storage
(days)
0
30
60
Texture
Crispness
FFRB-Cookies
DFRB-Cookies
FFRB-Cookies
DFRB-Cookies
6.970.32a
6.660.30b
6.330.30c
7.380.24a
7.030.22b
6.650.22c
7.220.40a
6.790.38b
6.380.37c
7.270.24a
6.870.24b
6.450.25c
FFRB-Cookies
6.950.30a
6.550.28b
6.140.30c
DFRB-Coo
7.150.19
6.780.18
6.400.17

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Rice Industrial By-products Management for

Oil Extraction and Value Added Products

MIAN KAMRAN SHARIF

A dissertation submitted in partial fulfillment of requirements for the degree of

The Controller of Examinations,

(Mian Kamran Sharif)

Fatty acid profile

Utilization of RBO in cookies

Rice bran supplemented flours used in study

Treatments used for preparation of rice bran supplemented cookies

Treatments used for preparation of leavened pan bread

Effect of stabilization on FFA, POV and TBA no. of rice bran

Anti-nutritional factors in rice bran samples

Proximate composition of fullfat rice bran samples and commercial

Mineral analysis of rice bran samples

Proximate composition of defatted rice bran samples

Yield of rice bran oil from different bran samples

Quality characteristics of rice bran oil samples

Fatty acid composition of rice bran oil samples

Mean squares for antioxidants in rice bran oil samples

Mean squares for physical parameters of different groups of rats

Mean squares for organs weight of different groups of rats

Effect of diets on organs weight of different groups of rats

Organs weight of different groups of rats during study periods

Mean squares for serum kidney and liver function tests

Effect of diets on serum kidney and liver function tests in different

Serum kidney and liver function tests in different groups of rats

Mean squares for lipid profile and serum glucose in different

Effect of diets on serum lipid profile and glucose (mg/dL) in

Serum lipid profile and glucose (mg/dL) in different groups of rats

Mean squares for serum proteins in different groups of rats

Effect of diets on serum proteins in different groups of rats

Serum proteins in different groups of rats during study periods

Mean squares for physical analysis of RBO cookies

Physical analysis of RBO cookies

Effect of storage on physical analysis of RBO cookies

Mean squares for proximate composition of RBO cookies

Proximate composition of RBO cookies

Effect of storage on proximate composition of RBO cookies

Total acidity and TBA no. of RBO cookies

Effect of storage on total acidity and TBA no. of RBO cookies

Mean squares for sensory attributes of RBO cookies

Color scores of RBO cookies

Flavor scores of RBO cookies

Taste scores of RBO cookies

Texture scores of RBO cookies

Crispness scores of RBO cookies

Overall acceptability scores of RBO cookies

Mean squares for proximate composition of supplemented flours

Moisture content of supplemented flours

Crude protein of supplemented flours

Crude fat of supplemented flours

Crude fiber of supplemented flours

Ash content of supplemented flours

Nitrogen free extract of supplemented flours

Mean squares for mineral content of supplemented flours

Mineral contents of supplemented flours

Dietary fiber content of supplemented flours

TBA no. of supplemented flours

Mean squares for mixographic characteristics of supplemented

Mixing time of supplemented flours

Peak height of supplemented flours