Rice Bran Utilization Thesis

“PROCESSING OF RICE BRAN AND ITS
UTILIZATION IN FOOD PRODUCTS”
SHWETA BHOSALE
PALB 2220
DEPARTMENT OF FOOD SCIENCE AND NUTRITION

UNIVERSITY OF AGRICULTURAL SCIENCES
BENGALURU-560065
2014
“PROCESSING OF RICE BRAN AND ITS
UTILIZATION IN FOOD PRODUCTS”
SHWETA BHOSALE
PALB 2220
Thesis submitted to the

University of Agricultural Sciences, Bengaluru
in partial fulfillment of the requirements
for the award of the degree of
Master of Science (Agriculture)

in
FOOD SCIENCE AND NUTRITION
BENGALURU JUNE, 2014
Affectionately
Dedicated to
My Beloved Parents,
Brother and my guide
Dr. Vijayalakshmi D.
ACKNOWLEDGEMENT
In every one's life, the day arises when one has to shape the feelings in words.
Sometimes, the words become unable to express the feelings of the mind, because, the
feelings of heart are beyond the reach of the words. When, I come to complete this
manuscript, so many memories have rushed through my mind which is full of gratitude
to those who encouraged and helped me at various stages of this research. It gives me
immerse pleasure to record my feelings at this place.
I wish to express my sincere gratitude and respect to Dr. Vijayalakshmi, D.,

Professor, Department of Food Science and Nutrition, UAS, GKVK, Bengaluru
Chairperson of my advisory committee, for her inspiring, enlightening guidance and
encouragement. I am deeply grateful for her generous help and for having shown keen
interest during the course of my research work without which, I would not reached to
this milestone in my life.
With a sense of pride and dignity, I sincerely thank the members of my advisory
committee Dr. Shivaleela, Professor and Head, AICRP Home Science, UAS, GKVK,
Bengaluru, Dr. Usha Ravindra, Associate Professor, Department of Food Science and
Nutrition, UAS, GKVK Bengaluru, Dr. C. K. Suresh, Professor, Department of Plant
Biotechnology, UAS, GKVK Bengaluru and Mr. R. Chandru, Associate Professor,
Department of Post Harvest Technology, UAS, GKVK Bengaluru, for their
constructive criticism, valuable guidance, providing facilities in carrying out my
research work and timely correction of my thesis manuscript.
A special thanks to Dr. Chowdhary, Gurukumar from NEIST Jorhat Assam. I

would like to thank EU- India NAMASTE Project, UAS Bengaluru (Sponsored by
Department of Bio-Technology, New Delhi.)
I record my sincere thanks to Jyoti Mahesh Sajjan and Ramya for their generous
help, co-operation and inspiration during the course of my research work.
I would like to extend my heartfelt respect and affection to my teachers
Dr. Sunanda Sharan, Dr. Neena Joshi, Dr. Umadevi S. Hiremath, Dr. M.L. Revanna
and Smt. K. V. Jamuna, Department of Food Science and Nutrition, UAS, GKVK,
Bengaluru and non-teaching staff of the Department for their constant support
throughout my course of investigation.I also thank Mr Shivalingaihya, laboratory
assistant and other staff members of Food Science and Nutrition department for their
kind help during my work.
The thesis must surely bear the imprint of the love and affection showered on me
by my family members. I want to extend my appreciation to my parents for their
boundless love, needy inspirations like showers to a drying crop and for their
unshakable confidence in me. I am greatly beholden of vocabulary and owe deep sense
of honor to my beloved parent's shree Piroji Bhosale and Smt. Prema Bhosale for their
love and dedicated efforts in shaping my career since childhood. My deep sense of love
and gratitude also goes to my brother Vinayak Bhosale, sisters Swati, Sakshi and other
family members.
Fruitful results would not have hastened without the moral of my friends,
Priyanka Sajjan, Priyanka, Nayayna, Suchi, Ravi, Nataraj, Akhila H, It is time to
express my gratitude to my beloved childhood friend Shweta. I sincerely thank them for
their timeless support and help. I also would like to thank my seniors Jayalaxmi Baddi,
Shilpa D.H. and Bhaphi for their suggestions and encouragement and my heartfelt
thanks to my junior roommate prasuna and all my juniors. Any omission in this
acknowledgement does not mean indeed.
Bengaluru
June, 2014 (Shweta Bhosale)
"PROCESSING OF RICE BRAN AND ITS UTILIZATION IN FOOD
PRODUCTS"
SHWETA BHOSALE
THESIS ABSTRACT
Rice bran is a by product obtained during rice milling process. Bran is being
wasted during rice milling process, which contains high amount of nutrients and needs to
be exploited. Present study revealed that rice bran was safe from microbes, heavy metals,
pesticide residue and was stabilized by microwave heating. Stabilized rice bran was
inoculated with Lactic Acid Bacillus culture and used as probiotic treated rice bran.
Macronutrient composition of stabilized and probiotic treated rice bran for moisture,
protein, fat, ash and carbohydrate were 4.30 and 5.40, 17.50 and 19.25, 13.10 and 17.20,
4.92 and 4.64, 52.33 and 48.55 g/100g respectively and it contained 7.85 and 4.96g crude
fibre, 21.17 and 13.10g insoluble dietary fibre, 2.17 and 1.80g soluble dietary fibre and
23.34 and 14.90g total dietary fibre. Mineral content of stabilized and probiotic treated
rice bran for calcium, phosphorous, iron and zinc were 52.10 and 49.90, 1185.20 and
1186.50, 28.10 and 30.05, 6.02 and 5.89 mg/100g respectively. Five products were
developed namely, biscuit, bread, muffin, chocolate and chapati. The processed rice bran
was incorporated at 5 to 25 percent level. Sensory scores of the products revealed that
products were well accepted and had higher nutrient content. Storage study revealed that
the microbial load of developed products were within safe limits. Glycemic index of
stabilized and probiotic treated chapati was 68.00 and 64.13. Thus, processed rice bran
was found to be an excellent source of nutrients, hence it can be incorporated in food
products and used as a hypoglycemic functional food.
June, 2014
Department of Food Science and Nutrition (Dr. Vijayalakshmi. D)

University of Agricultural Sciences Major Advisor
Bengaluru
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CONTENTS
CHAPTER TITLE PAGE No.
I INTRODUCTION 1-2
II REVIEW OF LITERATURE 3-13
III MATERIALS AND METHODS 14-24
IV EXPERIMENTAL RESULTS 25-49
V DISCUSSION 50-61
VI SUMMARY AND CONCLUSION 62-64
VII REFERENCES 65-71
ANNEXURES 72-100
LIST OF TABLES
Table Page
Title
No. No.
1 Analysis of rice bran sample for heavy metals and pesticide residue 27
Microbial load of rice bran sample-serial dilution plate count
2 28
technique
3 Percentage of free fatty acid (FFA) in rice bran on storage 28
4 Functional properties of processed rice bran 29
5 Nutrient composition of rice bran per 100g 30
6 Physical properties of biscuit 31
7 Mean sensory scores of biscuit 32
8 Mean sensory scores of biscuit on storage 33
9 Peroxide and free fatty acid (FFA) value of biscuits 34
10 Functional properties of bread 35
11 Mean sensory scores of bread 36
12 Mean sensory scores of bread on storage 37
13 Physical properties of muffin 38
14 Mean sensory scores of muffin 39
15 Mean sensory scores of muffin on storage 40
16 Peroxide and free fatty acid value of muffin 41
17 Mean sensory scores of chocolate 42
18 Mean sensory scores of chocolate on storage 43
19 Mean sensory scores of chapati 44
20 Nutrient composition of the developed products 46
21 Microbial load of food products during shelf life study 48
Mean values for blood glucose level (mg/dl) for standard, control and
22 49
test foods
Mean IAUC for glycemic index (GI) of standard, control and test
23 49
foods
LIST OF FIGURES
Figure Between
Title
No. Pages
1 Research design 15-16
2 Microwave stabilization of rice bran 15-16
3 Probiotic treatment of rice bran 17-18
4 Preparation of biscuit 21-22
5 Preparation of bread 21-22
6 Preparation of muffin 21-22
7 Preparation of chocolate 21-22
8 Preparation of chapati 21-22
9 Percentage of Free Fatty Acid in rice bran on storage 28-29
10 Mean sensory scores of biscuit 32-33
11 Mean sensory scores of bread 36-37
12 Mean sensory scores of muffin 40-41
13 Mean sensory scores of chocolate 42-43
14 Mean sensory scores of chapati 44-45
Mean area under blood glucose curve for standard, control and test
15 49-50
foods (SRB & PRB)
LIST OF PLATES
Plate No. Title Between Pages
(a) Microwave stabilized rice bran

1 15-16
(b) Probiotic treated rice bran
2 Rice bran incorporated biscuit 21-22
3 Rice bran incorporated bread 21-22
4 Rice bran incorporated muffin 21-22
5 Rice bran incorporated chocolate 21-22
6 Rice bran incorporated chapati 21-22
7 Sensory evaluation of products by panel members 23-24
8 Testing of blood glucose in subjects 24-25

LIST OF ANNEXURES
Annexure Page
Title
No. No.
I Estimation of protein 92
II Estimation of fat 94
III Estimation of crude fibre 95
IV Estimation of total dietary fibre 97
V Estimation of total ash 102
VI Estimation of mineral solution 103
VII Estimation of calcium 104
VIII Estimation of phosphorous 105
IX Estimation of iron 106
X Estimation of zinc 108
XI Estimation of antioxidant activity by DPPH method 109
XII Estimation of phytic acid 111
XIII Estimation of trypsin inhibitor 114
XIV Preparation of biscuit 116
XV Preparation of bread 117
XVI Preparation of muffin 118
XVII Preparation of chocolate 119
XVIII Preparation of chapati 120
XIX Score card for the sensory evaluation 121
XX Estimation of total lipids 122
XXI Determination of acid value 123
XXII Determination of peroxide value 124
XXIII Consent form 125
XXIV Calculation of incremental area under the curve 126
XXV Profile of subjects taken for the glycemic index study 127
I. INTRODUCTION
Rice is the staple food of 65 per cent of population in India. The major rice
growing countries are China, India, Indonesia, Bangladesh, Thailand, Burma, Vietnam,
Japan and Philippines. Rice is the largest consumed calorie source among the food grains.
With a per capita availability of 73.8 kg it meets 31 per cent of the total calorie
requirement of the population. India is the second largest producer of rice in the world
next to China. In India paddy occupies the first place both in area and production. Apart
from rice milling, processing of rice bran for oil extraction is also an important agro
processing activity for value addition, income and employment generation (Qureshi et al.,
2000).
Milling of paddy to obtain edible rice grain yields two major by-products of
economic and nutritional importance, namely, paddy husk and rice bran. Paddy husk has
no food value but has several industrial uses. Rice bran is an inexpensive, underutilized
milling by-product of rough rice. Rice bran is the cuticle existing between the rice and the
husk of the paddy and consists of embryo and endosperm of the seeds of Oryza sativa,
family Graminae. Rice bran, on the other hand, can serve as an human food supplement
and as a valuable source of edible oil. Rice bran - both full fat and defatted is a rich
source of nutrients and can serve as a source of nutrient supplement. Both the bran and
oil from rice bran have a range of bioactive phytochemicals with potential for reducing
the risk of chronic degenerative diseases. There is a need to utilize the full potential of the
available rice bran in the country, both as a source of healthy edible oil and as a food
supplement for promoting our population's nutrition and health. The yield of husk bran
and milled rice from 100 kg paddy are 22.8 kg and 73 kg, respectively. The yield of bran
depends upon the degree of milling of the brown rice, it may vary from 5 to 10 per cent.
In India, polishing is restricted to 5 per cent by Government regulation (Narasinga Rao,
1988).
The main drawback of rice bran is a fast oxidation reaction due to the high
content of unsaturated fatty acid in rice bran oil. This is primarily due to the presence of
endogenous enzyme lipase which causes the pro-oxidative mechanisms of oxidation
leading to hydrolytic rancidity on the oil content that hydrolyze the ester bonds of
triacyglycerol, releasing fatty acids and glycerol and forming of hyperoxides (Yin and
Wen, 2011). Within one hour of separating the bran from the grain during milling, the
material turns rancid liberating toxic free fatty acids. These shortcomings have now been
overcome by destroying the lipolytic activity using an advanced stabilizing technology,
the resulting material thus obtained is called "stabilized" rice bran which has a good taste,
readily soluble with a longer shelf life of one year.
Rice bran constitutes 8 per cent of the weight of the whole grain and contains
most of the nutrients (65 per cent), such as vitamins, minerals, oils, trace elements,
antioxidants, phytosterols and phytochemicals. It contains 12-22 per cent oil, 11-17 per
cent protein, 6-14 per cent fibre, 10-15 per cent moisture and 8-17 per cent ash. It is rich
in vitamins including vitamin E, thiamin, niacin and minerals like aluminium, calcium,
Processing of Rice Bran and its Utilization in Food Products 1

chlorine, iron, magnesium, manganese, phosphorus, potassium, sodium and zinc
(Quereshi et al., 2000).
Rice bran is used for the enrichment of foods, due to its high dietary fibre content.
Since the middle of the 1970s, the role of dietary fibre in health and nutrition has
stimulated a wide range of research activities which caught public attention.
Accumulating evidence favours the view that increased intake of dietary fibre can have
beneficial effects against diseases, such as cardiovascular diseases, gastrointestinal
disease, decreasing blood cholesterol, diverticulosis, diabetes and colon cancer. In view
of the therapeutic potential of dietary fibre, more fibre incorporated food products are
being developed. Addition of dietary fibre to a wide range of products will contribute to
the development of value-added foods or functional foods that currently are in high
demand (Hu et al., 2009).
Rice bran also plays an important role in decreasing cholesterol and controlling of
blood glucose level. Stabilized rice bran (SRB), is a powerful source of vitamins,
nutrients, proteins and fibre. The soluble and insoluble fibres are necessary for optimum
digestion, blood sugar regulation, lowering cholesterol and prevention of diabetes and
heart diseases. The stabilized rice bran contains an approximate insoluble versus soluble
fibre ratio of 5 to 1, which exhibits a high digestive tolerance that occurs along the whole
digestive tract with no excessive fermentation in the large intestine. Processed rice bran
contains astounding quality of synbiotics, tocols, oryzanols, polyphenols, sitosterol,
phytosterols and is packed with full of omega-3 and omega-6 fatty acids. Healthy
complex carbohydrates found in processed rice bran have “low glycemic index” which
means they do not cause spikes in blood glucose (Sayre et al., 2007).
Rice bran, a "little known" food is highly nutritious and delivers a powerhouse of
health supporting nutrients which is either thrown away or used for low-level animal
feed. In the view of popularity of rice bran and its therapeutic use, it was proposed to
process the rice bran and to develop food products to enhance the nutrient contents.
Therefore, in the present investigation, an attempt was made to process the rice
bran, development of food products by incorporating rice bran and to test the glycemic
index of the developed product. In view of the above, the present investigation was
undertaken with the following objectives:
1. Processing of rice bran by different methods
2. To analyze the chemical composition of rice bran
3. Development of food products, evaluate the sensory qualities and shelf life
4. Glycemic Index (GI) test of the rice bran food product
2 Shweta Bhosale
II. REVIEW OF LITERATURE
The available literature on stabilization and probiotic treatment of rice bran
reveals that very little research has been done in the area of nutrient composition,
development of products, microbial assay, storage study and glycemic index of rice bran,
hence, in addition to rice bran, literature pertaining to other cereals have been reviewed
and presented under the following headings.
2.1 Processing of rice bran
2.2 Nutrient composition of rice bran
2.3 Development of products and their sensory evaluation
2.4 Shelf life study of the developed products
2.5 Glycemic Index test of rice bran incorporated food product

Tao et al. (1993) reported that that the microwave heating is an effective method
for the inactivation of lipase that is responsible for rice bran degradation and instability.
Rice bran stabilized by microwave heating at 2450 MHz for 3 min, found to be stable up
to four weeks in storage. Free fatty acid content of microwave stabilized increased from
4.0 to 4.9 per cent in long grain rice bran and from 4.6 to 6.25 per cent in medium grain
rice bran, even when stored under unfavorable storage conditions (33º±2ºC, 75±5%
relative humidity). In contrast, untreated bran FFA ranged from 4.0 to 68.3 per cent and
4.6 to 56.8 per cent in medium grain bran respectively.
Qinger et al. (1998) studied the stabilization process of rice bran immediately
after milling by added-moisture heating and dry heating. During experimental storage,
the result showed that the added-moisture heating was very efficient for the lipase
inactivation. The free fatty acid values were all less than 10 percent for the samples
processed at 1100C and 1200C for 3 or 5 mm. after 40-days storage. While the dry-heated
rice bran could only be stored for about 15 days with the free fatty acid value around 15
per cent since the lipase activity was gradually recovered with the absorption of ambient
moisture.
Hong et al. (1998) studied the stabilization process of rice bran immediately after
milling by added-moisture heating and dry heating. During experimental storage the
result showed that added-moisture heating was very efficient for the lipase inactivation.
The free fatty acid values were less than 10 per cent for the samples processed at 1100C
and 1200C for 3 or 5 min after 40 days of storage. While the dry-heated rice bran could
only be stored for about 15 days with the free fatty acid value around 15 per cent, since
the lipase activity was gradually recovered with the absorption of ambient moisture.
Ramezanzadeh et al. (1999) examined the production of free fatty acid (FFA) in
rice bran subjected to microwave heating. Packaging and storage temperature on the
production of free fatty acid (FFA) in rice bran was also examined. Freshly milled raw
rice bran was adjusted to 21 per cent moisture content and heated in a microwave oven at

850W and 2450MHz for 3 mins. Raw and microwave heated rice bran were packed in
zipper-top bags or vacuum sealed bags and stored at 4-5°C or 25°C for 16 weeks. The
FFA level in raw rice bran increased rapidly from an initial value of 2.5-34.4 and 38.8 per
cent during 4 weeks of storage in zipper top and vacuum packs, respectively, when stored
at 25°C. In contrast the FFA level reached 8.9 and 9.3 per cent in zipper top bags and
vacuum packs, when stored at 4-5°C. Results showed that hydrolytic rancidity of rice
bran can be prevented by microwave heating and that the recommended storage condition
for microwave rice bran is 4-5°C in zipper top bags.
Ramezanzadeh et al. (2000) studied the effect of microwave heat, packaging

methods and storage temperatures on proximate and fatty acid compositions of rice bran
during 16 weeks and storage was examined. Freshly milled raw rice bran, adjusted to 21
per cent moisture content and microwave heated for 3 min. Raw and microwave heated
bran were packed in zipper-top bags and vacuum-sealed bags and stored at 4-5°C and
25°C for 16 weeks. The moisture content decreased significantly from an initial 8.4 to 6.4
percent in microwave-heated samples regardless of packaging methods and storage
temperatures. Protein, fat, linoleic and linolenic contents did not change significantly in
all raw and microwave-heated samples during 16 weeks of storage. The microwave-
heated rice bran packed in zipper-top bags can be stored at 4-5°C for up to 16 weeks
without adverse effect on proximate and fatty acid composition quality.
Hettiarachchy (2009) conducted a study on yeast fermentation of rice bran

extracts. Heat-stabilized defatted rice bran (HDRB) is fermented with yeast to afford a
food product having superior prebiotic for probiotic properties. Fermentation of HDRB
with yeast yields a prebiotic composition that can promote the growth or activity of
beneficial intestinal bacteria (probiotic) when consumed by an animal or human.
Inclusion of legumes during fermentation can synergistically enhance the bioactivities of
HDRB. Heat-stabilized defatted rice bran can be a source of growth medium for
yeast/mold/microorganisms.
Rosniyana et al. (2009) evaluated rice bran at 4 per cent and 8 per cent milling
degree was stabilized by either autoclaving or parboiling process. The rice bran was
autoclaved with commercial retort at 120°C for 20 min. For the production of parboiled
rice bran, the harvested paddy was soaked for 2 h, steamed for 20 min then dried and
milled. The free fatty acid levels for both parboiled and autoclaved rice bran were below
the 10 per cent permissible level for 4 months and 6 months respectively for the product
packed in oriented polypropylene/ polypropylene packs, either vacuumed or without, and
stored in ambient temperature room condition. The storage of rice bran by polypropylene
packs, as control packaging material, led to rapid production of free fatty acids. These
findings indicate that rice bran can be stored without risk of deterioration for a substantial
time prior being used for the production of many health-related food products.
Ryan et al. (2011) studied on the development of stabilized rice bran for human
use as a functional food and dietary supplement. A global and targeted metabolomic
investigation of stabilized rice bran fermented with Saccharomyces boulardii was
performed in three rice varieties. Metabolites from S. boulardii fermented rice bran were
4 Shweta Bhosale
detected by gas chromatography-mass spectrometry (GC-MS) and assessed for
bioactivity compared to nonfermented rice bran in normal and malignant lymphocytes.
Global metabolite profiling revealed significant differences in the metabolome that led to
discovery of candidate compounds modulated by S. boulardii fermentation. Fermented
rice bran extracts from three rice varieties reduced growth of human B lymphomas
compared to each variety’s non-fermented control and revealed that fermentation
differentially altered bioactive compounds. These data support that integration of global
and targeted metabolite analysis can be utilized for assessing health properties of rice
bran phytochemicals that are enhanced by yeast fermentation.
Saman et al. (2011) reported that the growth on rice-based media of the probiotic
strain Lactobacillus plantarum NCIMB 8826 isolated from the human gut. Fermentation
broths were obtained from the whole grain brown rice and rice bran of two Thai rice
cultivars, RD6 (glutinous) and RD17 (nonglutinous). The rice used was not germinated
and fermentations were carried out in a single step without growth supplementation. L.
plantarum grew well in all tested broths, and a final biomass value of approx. 10.4 log
CFU/mL was obtained. The results confirm that brown rice and rice bran are suitable
substrates for the culture of the probiotic L. plantarum NCIMB 8826. Rice bran, currently
a by-product of the traditional cereal processing industry, has shown similar
fermentability to brown rice. This indicates that rice bran or rice bran extracts could be
used in new probiotic food developments, while probably still maintaining other
functional properties of the bran.
Abdel-Hady (2013) investigated the effect of some thermal processing

(Microwave, Parboiled, Roasted and Hot air) on stability of rice bran during storage in
polyethylene bags at room temperature for 8 weeks. The results showed that, stabilization
treatment improved oil extraction yield. The moisture content of roasted rice bran lower
than those of stabilized and unstabilized rice bran. In addition, the moisture content of
rice bran samples decreased with increasing storage period. Thermal processing
decreased the free fatty acids of rice bran after 8 weeks compared with unstabilized rice
bran. Thermal processing showed an increase in palmitic and stearic acids, while linoleic
and linolenic acids were decreased. Saturated fatty acids were increased after 8 weeks of
storage period. Parboiled rice bran had comparatively higher levels of protein, fat and ash
contents than unstabilized (Un-RB) and other stabilized rice bran. Thus the microwave
stabilization of rice bran has advantages over the other stabilization method.

Amissah et al. (2003) conducted study on the nutrient composition of 16 bran
samples from new rice varieties. Parameters measured were moisture, fat, crude fibre,
protein, ash and mineral content. Carbohydrate and energy levels were calculated from
the appropriate data obtained. Results showed significant differences in fat, crude fibre
and ash contents of samples. With the exception of GRUG 7, ITA 334 and ITA 402,
significant differences existed in moisture content. Significant variations also existed in
protein content with the exception of ITA 304 and ITA 334. The bran from the different
rice varieties had appreciably high levels of K, P and Ca. Energy levels were within the
300 Kcal/100 g range except that of GRUG 7 and BETA which were lower.

Sudarat et al. (2005) studied the chemical composition of full fat and defatted rice
bran for protein, fat, moisture content, fibre and ash, using AOAC (1995). The results
revealed that full fat rice bran contains 8.5 per cent moisture, 12.6 per cent protein, 21.13
per cent fat, 5.59 per cent crude fibre, 8.97 per cent ash and 43.12 per cent carbohydrate
where as the defatted rice bran contains 12.43 per cent moisture, 13.89 per cent protein,
1.92 per cent fat, 6.03 per cent crude fibre, 10.13 per cent ash and 55.6 per cent
carbohydrate.
Azizah et al. (2007), investigated the four rice-bran milling fractions, after
stabilization by microwave heating, were analyzed for their chemical composition. The
contents of all fractions tested (g/100gm) consisted of 8.5-12.6 moisture, 8.8-15.2
protein, 8.7–18.9 fat, 18.3-30.5 total dietary fibre, 22.2-44.8 total carbohydrates and 4.2-
7.7 ash. This study revealed that the stabilized first fraction of rice milling fractions was
found to contain high levels of energy, fat, ash, fibre, carotenoids and minerals and is also
a very good source of dietary fibre.
Rosniyana et al. (2009) studied on nutritional content and storage stability of

stabilized rice bran - MR 220. The nutritional composition of rice bran was analyzed at 4
per cent and 8 per cent milling degree which was stabilized by autoclaving and parboiling
process. The chemical composition of autoclaved and parboiled bran predominantly had
carbohydrate (25-47 per cent), fat (19.40-30.45 per cent), protein (13.60-15.10 per cent),
crude fibre (7.25-12.68 per cent), dietary fibre (20-23 per cent) and soluble fibre (1.9-2.2
per cent) respectively. Minerals such as calcium (54- 62 per cent), iron (124-30 per cent)
and phosphorus (1710-1830 per cent) respectively. The values of fat, fibre, ash, most
minerals and vitamins in parboiled bran were generally higher than treatment by
autoclave technique.
The antioxidant and antiproliferative activities of methanolic extracts from

Njavara rice bran was studied by Rao et al. (2010). In this study the antioxidant and
antiproliferative potential of rice bran extracted from an important Indian rice variety,
Njavara and compared the same with two commercially available basmati rice varieties.
Rice bran methanolic extract from Njavara showed the highest antioxidant and cell
cytotoxic properties compared to the other three rice varieties. The value for scavenging
DPPH and nitric oxide were in the range of 30.85-87.72 µg/ml and 52.25-107.18 µg/ml
respectively. Thus it is concluded that crude methanolic extract from Njavara rice bran
contains significantly high polyphenolic compounds with superior antioxidant activity as
evidenced by scavenging of free radicals including DPPH. It is conceivable that the
Njavara rice variety could be exploited as one of the potential sources of plant-based
pharmaceutical products.
Abdel-Galeel et al. (2012) investigated the effect of milling degree on nutritive

value of rice bran that stabilized by extrusion process. The hulled rice was milled for
three durations (30, 60 and 90 sec.) to obtain three fractions of rice bran (1st, 2nd and 3rd).
The chemical composition of moisture, protein, lipids, ash and dietary fibre were
evaluated. Results indicated that all fractions of rice bran contain high values of lipids,
ash and dietary fiber. The rice bran is very rich in phosphorus (1635mg) and potassium
6 Shweta Bhosale
(1453mg). In addition, it contains suitable amounts of all essential and nonessential
amino acids. Thus it is concluded that the 1st fraction of rice bran contains higher values
of protein (13.4 per cent), ash (8.1 per cent), phenolic compounds and dietary fiber (29.6
percent) as well as it has more amounts of essential amino acids and all elements in
comparing with those of other rice bran fractions.
Faria et al. (2012) subjected whole rice bran samples to two stabilization methods
to inactivate enzymatic deterioration. Changes in nutritional value in terms of, concerning
chemical composition, minerals and fatty acid content, were evaluated to supplement
existing data and promote the utilization of rice bran in the human diet. The following
homemade heat treatments were applied: roasting on a conventional stove or heating in a
microwave oven. Results showed that different heating methods affected sample
composition, since the levels of some nutrients of treated samples showed significant
changes compared to corresponding raw samples. The rice bran treated on a conventional
stove produced products with lower moisture (5.14±0.10 g/100 g) and nutrients such as
sodium 11.8 per cent, palmitic acid 9.9 per cent and stearic acid 8.1 per cent. The
microwave oven procedure resulted in better nutrient preservation, with slightly higher
moisture content (6.28±0.10 g/100 g), and appears to be a practical and rapid tool for
home heat stabilization of rice bran.
2.3 Development of products and their sensory evaluation

Shaheen et al. (2005) evaluated the suitability of processed and treated rice bran
for the supplementation of bread. The treated and extruded rice bran was supplemented at
5, 10, 15, 20, 25 and 30 per cent in wheat flour for the production of bread. The bread
was analyzed for different physicochemical parameters and subjected to sensory
evaluation. The results showed an increase in crude protein from 11.87 to 12.94 per cent,
crude fat from 3.64 to 8.63 per cent, crude fibre 0.62 to 2.15 per cent and ash 1.52 to 4.18
per cent. The sensory evaluation showed significant differences in the scorers for volume,
colour of crust, symmetry of form and character of crust. The breads supplemented with
15 per cent incorporation increased the sensory scores. It can be concluded from the
results that up to 15 per cent processed rice bran can be successfully incorporated in the
bread to improve the sensory and nutritional attributes.
Quilez et al. (2008) formulated bread, where wheat flour was replaced by 3 per
cent, 6 per cent and 9 per cent of rice bran (RB) previously exposed to two types of heat
treatment: extrusion (EXT) and steam cooking (STC). The aim was to investigate how
RB affected the properties of wheat-flour dough and partially baked wheat bread, as a
final product. The results of the study indicate that neither of the RB treatments had a
negative influence on the rheological characteristics of the dough, but that its
fermentation capacity and gas retention improved, especially in case of EXT-RB, which
was exposed to more intensive heat treatment. In the final bread product, there was also a
positive increase in the specific total volume for the EXT-RB bread, and a decrease for
the STC-RB bread. Nevertheless, sensory analysis showed that the score for the RB-
enriched bread decreased when RB content increased. They concluded that low
concentrations of RB exposed to a more intensive heat stabilization process improves

several rheological properties of the dough and the physical properties of the partially
wheat bread.
Saeed et al. (2009) studied on the impact of adding 2-20 per cent rice bran to
wheat flour on the rheological behaviour of the dough using Farinograph, Consistograph
and Alveograph. The changes in physico-chemical properties of dough were insignificant
even after including 20 per cent of the bran. The wheat flour and rice bran mixtures were
used to prepare cookies and flat bread (chapati) and the sensory evaluation was carried
out for the products. The sensory scores of the product, such as texture and taste,
decreased with increased substitution with the rice bran. It is concluded that cookies and
chapati, up to 10 per cent rice bran replacement were rated acceptable.
Sharif et al. (2009) developed cookies with microwave stabilized defatted rice
bran, it was supplemented in wheat flour at 10, 20, 30, 40 and 50 per cent
supplementation level to prepare fiber and mineral enriched cookies. Cookies were
analyzed for physical analysis, dietary fiber, mineral content (Na, K, Ca and Mg) and
sensory attributes to find out the most suitable compositions for commercialization.
Overall, rice bran supplementation improved dietary fiber content and mineral profile of
the cookies. On the basis of physical analysis and sensory attributes, it was concluded
that defatted rice bran can be substituted up to 10 to 20 per cent in wheat flour to prepare
rice bran supplemented cookies without adversely affecting quality attributes.
Bagheri and Seyedein (2011), conducted study to increase the fibre content of
bread, with 5, 10, 15 and 20 per cent incorporation of rice bran. Baking and rheological
tests on breads showed that an increase in the amount of rice bran leads to a rise in the
extension coefficient as well as water absorption, but it decreases flour resistance to
extension. The sensory evaluation and statistical analysis of the results revealed that the
sample containing 10 per cent rice bran had the best quality and it was significantly
different at 5 per cent level.
Sairam et al. (2011) studied the physico-chemical characteristics, antioxidant

potential of defatted rice bran (Laboratory-LDRB and Commercial-CDRB) and its
utilization in preparation of bread. The effect of incorporation of CDRB at varying levels
(5, 10 and 15 per cent) on the quality characteristics of bread including physical,
rheological and sensory attributes were evaluated and the dietary fibre content and
antioxidant activity were determined. On the basis of physical characteristics, breads with
5 per cent and 10 per cent CDRB were found to be acceptable. The dietary fibre content
and total antioxidant activity of bread increased with increasing levels of CDRB, which
also improved the shelf life.
Younas et al. (2011) formulated cookies from wheat flour with supplementation
of rice bran at 5, 10, 15 and 20 per cent. The rice bran was stabilized with acid and dry
heat treatment before supplementation. Chemical analysis of the cookies revealed that
there was no significant difference in chemical and physical properties of cookies
supplemented with acid stabilized rice bran (ASRB) and heat stabilized rice bran
(HSRB). The moisture, crude protein, fat and mineral contents were significantly
8 Shweta Bhosale
increased with the increment of rice bran. Average width, thickness and spread factor of
cookies also increased with the increase in percentage of rice bran. Sensory evaluation of
cookies showed that scores for colour of cookies decreased significantly with increase in
level of rice bran and sensory scores were significantly higher in the cookies prepared
with HSRB. Hence it is concluded from the results that supplementation of HSRB at 10
percent is more suitable for production of rice bran supplemented cookies.
Premakumari et al. (2012) reported that development of breakfast/ dinner recipes

by substituting cereals with rice bran at different levels and determined the acceptability
of food mixes. Stabilization studies revealed that microwave stabilized parboiled rice
bran had low moisture and low free fatty acid content and hence considered ideal for
product development. Ten standard Indian subcontinent breakfast/dinner recipes namely
chapati, mixed vegetable chapati, wheat dosa, wheat rava idly, adai, rava adai, ragi adai,
rice vermicelli, ragi vermicelli and kolukattai were chosen for incorporating rice bran at
25, 30 and 35 per cent replacing the cereals and pulses in the standard recipe. The
acceptability trials were carried out using 20 semi trained panel members. The results
revealed that, the recipes with 25 per cent incorporation of rice bran had a good
acceptability and on par with standard recipe.
Mishra and Chandra (2012) explored the possibility of fortifying the soya flour
and rice bran to formulate the functional biscuit which have the ability to improve the
quality of food products due to various functional properties. Supplementation of wheat
flour with soya and rice bran was tried at 10, 15, 20 and 25 per cent level each. Prepared
biscuit was subjected to physical, sensory and nutritional analysis to evaluate the
suitability of biscuit for consumption. The width of biscuit decreases from 44 to 36.2 with
increasing in the level of substitution of composite flour of rice bran and soya. Similar
trend was shown by spread ratio. Biscuit thickness increased from 9.2 to 10.6, with
increasing level of substitution. Nine-Point hedonic score system was used for sensory
evaluation of prepared biscuit which is generally decreases with increasing the level of
substitution. Thus, it has been concluded that supplementation of soya flour and rice bran
at 15 per cent level each, would improve the nutritional quality without adversely
affecting the sensory parameters.
Nagi et al. (2012) developed nutritive biscuits by using both full fat and defatted
cereal bran with wheat flour at different levels. Product making, sensory and texture
quality were assessed to find out the most appropriate level of bran incorporation. On the
basis of quality 20 per cent level was selected best. Acceptability of enriched biscuits was
affected with progressive storage, however, the product remained in high acceptability
range up to 3 months. Free fatty acid content of biscuits were within permissible limits
after 3 months of storage except rice bran (full fat) biscuits. Packaging material had
significant impact on biscuit quality. The biscuits were stored safely in both packaging
material i.e. HDPE and laminate. Microbiological study depicted that microbial count
was far below the permissible limits up to 3months of storage of biscuits in HDPE and
laminate at room temperature. Economics of enriched biscuits reveal that wheat bran
enriched biscuits were economically profitable.

Salehi and Bibalan (2012) conducted a study on utilization of indigenous rice bran
(RB) for the preparation of value-added products. The stabilized rice bran is used to
determine the effect of dough rheological and muffin cake sensory properties of one type
of wheat flour (with 75 percent extraction rate). Water absorption, dough development
time (30% rice bran) and valorimeteric value were increased and dough stability and
dough softening were decreased by addition of rice bran in flour than the control. Sensory
evaluation with Friedman test revealed that there is no significant difference between
treatments at 5 per cent level. Cakes containing 20 per cent rice bran flour got the highest
scores for sensory evaluation, it is concluded that the quantity and quality rheological and
sensory properties of muffin cakes was improved with the addition of rice bran flour.
Thus, rice bran could be used for wheat substituting and a good functional ingredient for
value addition of food products. Moreover the present study suggests that T3 (30% RB +
70% wheat flour) can produce superior quality cakes to prove effectiveness of RB as
bakery powder.
Yadav et al. (2012) explored the possibility of utilizing defatted rice bran (DRB)
for making chapati. DRB was ground and blended with wheat flour in the proportion of
0, 5, 10, 15, 20, 25 per cent and blends were evaluated for dough and chapati making
quality. Extensibility of dough and chapati decreased (19.9 ± 0.08 mm to 14.3 ± 0.08 mm,
11.2 ± 0.05 mm to 6.3 ± 0.04 mm, respectively) while peak load to rupture of chapati
increased (3.1 ± 0.04 to 3.6 ± 0.05 N) at 25 per cent replacement of flour with fine DRB.
Water absorption capacity of flour increased from 73 ± 0.4 per cent to 74.6 ± 0.4 per cent
with 25 per cent of large DRB, while the increase was up to 78.4 ± 0.3 and 80.6 ± 0.3 per
cent in case of medium and fine DRB. Overall acceptability score of chapati was also
significantly decreased (8.4 ± 0.2 to 6.8 ± 0.2) with 20 per cent of large DRB, whereas it
was 7.0 ± 0.1 and 7.6 ± 0.1 for medium and fine DRB at the same proportion. The chapati
prepared from the wheat flour containing 20 per cent fine DRB was rated acceptable and
also had significantly higher ash (2.1 ± 0.05) and total dietary fiber (4.3 ± 0.10%) content
than control chapati.
Ameh et al. (2013) studied the effect of rice bran supplementation on physico-
chemical and sensory properties of wheat bread. Blends of wheat flour and rice bran
(95:5, 90:10 and 85:15) were used to bake bread with 100 per cent wheat flour as control.
Thereafter, proximate, vitamin and mineral composition, as well as the physical and
sensory properties of the dough and bread loaves were determined, using standard
methods of analysis. The moisture content, crude protein, crude fat, crude fibre, and ash
of the composite bread loaves increased significantly, while carbohydrate content
decreased with increased level of supplementation. Bread loaf weight increased while
loaf volume and specific loaf volume decreased. There was a significant difference in
physical properties of dough and bread loaves between the composite bread and the
control. Hence, it is concluded that 95:5 blend was better accepted compared to the other
blends and there was a significant improvement in the nutritional composition of the
wheat bread with rice bran supplementation.
Bhaduri (2013) conducted study to produce gluten free healthy cereal based
muffins prepared from two gluten free flours, rice and quinoa flour. Hundred per cent
10 Shweta Bhosale
wheat flour was used as control. Rice flour was replaced by 25, 50, 75 and 100 per cent
quinoa flour to prepare muffin. Physical property measurements including percentage
increase/decrease of crest height, moisture and specific gravity, colour and texture
analyzer was done for the final product. The Sensory attributes, appearance, flavour,
sweetness, texture and general acceptability, were evaluated by using a 9-point hedonic
scale. The study showed that, 100 per cent rice flour and 25 to 75 per cent replacement
with quinoa flour to rice flour formulations for muffin has the better overall consumer
acceptability compared to 100 per cent quinoa flour muffin.
2.4 Shelf life study of the developed products

Delahaye et al. (2004) incorporated high content dietary fiber (26%) stabilized
rice bran flour (SRBF) in the production of pizza dough. The pizza dough was developed
mixing wheat flour with SRBF in a proportion of 5:95 and 10:90; SRF: wheat flour,
water, salt, and yeast. All the pizzas were stored for 60 days at -18°C and their proximal
composition, functional properties, and sensorial characteristics were evaluated at 0, 30
and 60 days. The results showed that the content of dietary fiber increased to 3.8 per cent
and 5.3 per cent as the level of enrichment increased. The farinographic curves of the
pizza dough showed that the development time, water absorption, and stability decreased,
while mixing tolerance index and departure time were not affected by enrichment level.
During storage (60 days), starch content of the three flours were decreased. The sensorial
test results indicate that the pizza dough with an enrichment level of 5 per cent with
SRBF was well accepted by the panel and it was stable up to 60 days at -18°C.
Ajmal et al. (2006) incorporated defatted rice bran (DRB) at different levels with
wheat flour. Five treatments (T0 = control i.e. without DRB; T5 = 5 per cent DRB; T10 =
10 per cent DRB; T15 = 15 per cent DRB; T20 = 20 per cent DRB) were used for bread
preparation. Bread loaves were analyzed for chemical composition and sensory
evaluation at different storage intervals i.e. S0, S24, S48, S72, S96, and S120 hours.
Protein, ash, fiber, and mineral contents of breads were improved and moisture decreased
significantly, whereas fat content showed non-significant effect for increasing levels of
defatted rice bran. Maximum protein, ash, fiber, K, Ca, and Mg contents were found in
T20 while minimum values were observed in T0. Moisture and Na contents were
decreased by the subsequent addition of rice bran. Treatment T5 got maximum scores for
external characteristics (volume, color of crust, symmetry of form, evenness of bake,
character of crust) and internal characteristics (grain, color of crumb, aroma, taste, and
texture) of pan bread. From chemical assay and sensory evaluations, the authors
concluded that the quality bread can be improved by the addition of 5 per cent DRB
having high fiber and mineral content for commercialization.
2.5 Glycemic Index test of rice bran incorporated products

Islampure (2009) conducted a study on nutritional evaluation and glycemic index
of selected varieties of mulberry leaves. Chapati was prepared with mulberry leaf powder
incorporation at 5 per cent level. The glycemic index study was conducted on 10 healthy
volunteers. On the first day of the study white bread was fed to the individuals which
provides 50g of carbohydrate and on the second day of the study mulberry leaf

incorporated chapati has been fed to the individuals which provides 50g of carbohydrate.
The blood glucose level was measured for fasting, 30, 60, 90 and 120 min respectively
and the mean GI was calculated. The results showed that the GI for white bread was 100
and that of mulberry chapati it was found to be 93.66. Thus, it is concluded that mulberry
leaf powder incorporated chapati helps in reducing the blood glucose level.
Teradal (2013) conducted a study on evaluation of grain based wholesome

functional foods for geriatric population. Two composite mix were developed such as
wheat based composite mix and ragi based composite mix. Glycemic index was tested for
these two products. The standard food used was the wheat bread. All these products were
tested for glycemic index in ten healthy volunteers for alternative days. The mean blood
glucose values were measured for every 30 min interval from initial to 2 hours. The
glycemic index for white bread was 100, composite mix-I was 52.95 and composite mix-
II was 50.40 respectively. Hence it is concluded that both the composite mixes helped in
maintaining the blood glucose level in normal and healthy individuals.
Premakumari et al. (2013) developed the recipes incorporated with rice bran and
evaluated their organoleptic properties and estimated their glycemic index. Ten standard
Indian subcontinent recipes namely, chapati, mixed vegetable chapati, wheat dosa, wheat
rava dosa, kozhukattai, ragi vermicelli, rice vermicelli and pulse based preparations
namely adai, rava adai and ragi adai were chosen for incorporating rice bran at three
levels i.e. 25, 30 and 35 per cent replacing the cereals and pulses in the standard recipe.
Ten healthy volunteers were selected for the study of glycemic index of each recipe, 25
per cent was found to be most acceptable than 30 and 35 per cent. Glycemic index of all
the standard and test recipes were compared statistically and the results showed that there
is a significant difference in the test recipes compared to the standard ones. Recipes like
wheat chapati (52.40), mixed vegetable chapati (52.40), wheat dosa (52.81), wheat rava
dosa (46.60), kolukattai (67.68), rice vermicelli (63.70) and ragi vermicelli (59.74) rice
bran incorporated at 25 per cent level showed low and medium glycemic index compared
to the standard recipes.
Srinivasa et al. (2013) conducted study to test the glycemic index of thermally
treated basmati rice variety in healthy volunteers. In this study 70 healthy volunteers were
taken into consideration for arriving at the final GI value. The study procedure was
similar to the recommendations by FAO/WHO. It was observed that reference glucose
curve had the maximum average peak of 166.37 mg/dL while the basmati sample had a
lower peak (136.22 mg/dL). The mean blood glucose incremental area under the curve
for reference food was 5969.64 mins. mg/dL (SEM 95.94) and for rice it was 3267.81
mins. mg/dL (SEM 76.21). The GI of Indian branded basmati rice was found to be < 55
placing it in lower GI category. Thus it is concluded that Indian basmati rice because of
its lower GI can prove to be a healthier rice alternative and it also proved to have
beneficial effect when incorporated into Indian diets to replace high GI rice alternatives.
Singh et al. (2013) conducted a study on utilization of rice bran for development
of chapati and its glycemic response in NIDDM patients. In this study rice bran, a rich
source of carbohydrate and antioxidant was incorporated at 20 per cent level in the wheat
12 Shweta Bhosale
chapati. Its nutritive value, sensory evaluation, glycemic index (GI) and glycemic load
(GL) were calculated. Total 20 subjects, 16 males and 4 females were selected with
average age and BMI of 56.55 and 26 respectively. Overall acceptability scores were in
the acceptable range for controlled chapati (8.99) and rice bran based chapati (8.08). The
GI of rice bran based chapati (68.34) was significantly lower than control chapati (83.92).
It is concluded that the blood glucose concentration was lowest for rice bran chapati.
Wordu and Banigo (2013), tested the glycemic index of different varieties of rice
(Oryza sativa) such as white rice, brown rice and parboiled rice. A group of 22
participants with the mean age, weight, height and body mass index were selected for the
study. The mean fasting blood glucose level of the participants was 84.81 ± 4.37 mg/dl.
The mean blood glucose level at 30 and 60 min after the oral administration of 75 g
glucose were 147.43 ± 11.67 and 125.95 ± 9.30 mg/dl, respectively. The mean glycemic
response of pure glucose at 30 and 60 min were 62.62 ± 11.4 mgdl-1 and 41.14 ± 8.932
mgdl-1 respectively and hence, higher glycemic response for the pure glucose was
obtained at 30 min. To the participants different varieties of cooked rice (white rice,
brown rice and parboiled rice) containing 75 g digestible carbohydrate were
administered, the peak blood glucose response was obtained at 30 min. The mean
glycemic response of white rice, brown rice and parboiled rice were 41.71 ± 6.17, 37.72
± 5.11 and 35.05 ± 3.77 mg/dl, respectively. The glycemic responses after the
consumption of cooked rice sample containing 75 g digestible carbohydrate, showed
significant difference (P > 0.05) between white rice and brown rice, cooked brown rice
and parboiled rice and cooked white rice and parboiled rice. The mean GI values of
cooked white rice, brown rice and parboiled rice were 66.61 ± 9.86, 60.24 ± 8.16 and
55.97 ± 6.01, respectively. Based on these GI values, it can be suggested that among the
three varieties of cooked rice, the parboiled rice is better to reduce the blood glucose
level.

III. MATERIALS AND METHODS
The chapter deals with the details of materials used and methods employed on the
research topic entitled “Processing of rice bran and its utilization in food products”. The
present investigation was carried out on processing of rice bran and preparation of food
products from stabilized and probiotic treated rice bran to determine their acceptability by
the consumers. The work was carried out at the Department of Food Science and
Nutrition, UAS, GKVK, Bengaluru during 2012-2014 and has been presented under the
following subheadings.
3.1 Procurement of rice bran
3.2 Analysis of rice bran for heavy metals, pesticide residue and microbial load
3.4 Functional properties of rice bran
3.6 Development of food products by incorporating processed rice bran
3.7 Physical/functional characteristics of developed products
3.8 Sensory evaluation of the developed food products
3.9 Nutrient composition of the developed food products
3.10 Storage and microbial study of the food products
3.11 Glycemic index test of selected food product
3.12 Statistical analysis

3.1 Procurement of rice bran
The rice bran was procured from NEIST (North East Institute of Science and
Technology) Jorhat, Assam, India.
3.2 Analysis of rice bran for heavy metals, pesticide residue and microbial
load
3.2.1 Analysis of rice bran sample for heavy metals and pesticide residue
Analysis for heavy metals and pesticide residue in rice bran was done at NEIST
Laboratory, Jorhat, Assam, India.
3.2.2 Microbial study of rice bran sample-serial dilution plate count technique
(Tate, 1995)
Ten grams of rice bran sample was mixed in 90ml sterile water blank to give 10-1
dilution. Subsequent dilutions up to 10-4 made by transferring serially 1 ml of the dilution
to 9 ml of sterile water blanks. The population of total bacteria, molds and yeast were
estimated by transferring 1 ml of 10-2, 10-3 and 10-4 dilutions respectively to a sterile
petridish and approximately 20 ml of media viz. Nutrient agar, Martins Rose Bengal Agar
and Davis Yeast Extract Agar for bacteria, molds and yeasts respectively. The plates
were rotated twice in clockwise and anticlockwise direction for uniform distribution of
the inoculums. After solidification of the media, plates were kept for incubation in an
inverted position at 30 ± 10C for 2-4 days and emerged colonies were counted.

3.3.1 Microwave stabilization of rice bran
Rice bran sample was sieved to get uniform particle size, 100 grams of rice bran
sample was taken and microwave stabilization was done for 3 minutes (Fig. 2 and plate
1.a), 10 ml of water was added to avoid the char in bran. The stabilized rice bran was
then subjected to determine the percentage of free fatty acid (FFA).
Five grams of stabilized rice bran was taken for which 15 ml of hexane was added
to it and heated up to 600C for 20 min, the mixture was centrifuged and supernatant
collected. This solvent was oven dried to extract the oil. FFA is determined from the oil
extract by AOAC (2000) international method.
3.3.2 Probiotic treatment of rice bran

Sporlac containing 150 million spores of Lactic Acid Bacillus per gram was
purchased from the medical shop and used as starter culture.
100g of stabilized rice bran sample was taken, for which 4g of Lactic Acid
Bacillus culture was added and kept for fermentation (24 hrs at 300C). Fermented rice
bran sample was subjected to dehydration technique viz. freeze drying. Before the sample
is dehydrated it is kept under cold storage for 24 hrs. Then it is subjected to lyophilization
15 Shweta Bhosale
Procurement of the sample from NEIST (North East Institute of Science and Technology)
Microbial and heavy metal analysis Processing of rice bran
Stabilization of rice bran Probiotic treatment of rice bran
Chemical analysis Functional Product Glycemic Index

properties development test of selected
food product
Moisture
Bulk density Biscuit
Protein
Water Bread
Fat
absorption Muffin
Ash
Fat Chocolate
Dietary fibre
absorption Chapati
Calcium
Phosphorous Swelling
Iron power
Zinc Solubility
Antioxidant index Sensory evaluation Shelf life study
activity
 Phytic acid
 Trypsin inhibitor
Sensory evaluation Microbial load
Fig 1: Research design
Rice bran + water
(Moisture % is made up to required conc.)
Microwave heating (2450MHz for 3min)
Oven drying at 1000C for 1 hour
Cooled to room temperature
Stored in refrigerator condition in

air tight zip lock bag
Fig. 2: Microwave stabilization of rice bran

process, where freeze drying was done for 34 hrs at -330C using Lyodel lab model
Lyophilizer. Later stored in the air tight zip bag for further use (Fig. 3 and plate 1.b).
3.4 Functional properties of rice bran

Bulk density (g/ml) (Narayana and Rao, 1982)
Centrifuge tube was weighed and flour samples were filled to 10 ml mark by
constant tapping, until there was no further change in volume. The content was weighed
and from the difference in weight, the bulk density of the sample was calculated and the
results are expressed as g/ml.
Water and oil absorption capacity (Rosario and Flores, 1981)

One gram sample was mixed with 10 ml of either distilled water or in 15 ml of oil
for 30 min. The contents were allowed to stand at 300C in a water bath for 30 min and
then centrifuged at 3000–5000 rpm for 20-30 min. After centrifuging the volume of the
supernatant was recorded and used for determination of water and oil absorption and the
results were expressed as ml/g sample.
Solubility and swelling power (Iyer and Singh,1997)

Two hundred and fifty mg finely ground sample was thoroughly mixed with 15
ml of distilled water and heated to 650C (being the initial temperature at which
gelatinization of starch granules begins). The content was then cooled to room
temperature and centrifuged at 5000 rpm for 10 min. The soluble solid content was
calculated as percentage of sample soluble in water. At the same time this was used to
calculate the swelling power, since the sample has been heated at 650C for 30 min, the
residue was weighed and the increase in weight was calculated as the swelling power of
the sample at that particular temperature.

The rice bran sample was subjected to nutrient analysis, where the rice bran
sample was taken for macro and micro nutrient analysis. Micronutrients such as moisture,
protein, fat, ash, crude fibre and dietary fibre were assessed (AOAC, 1980).
Micronutrients such as Calcium, Phosphorus (AOAC, 1980), Iron, Zinc (Page et al,
1992) were analyzed. Phytic acid (Sadasivam and Manickam, 1992) was analyzed.
3.5.1 Estimation of moisture (AOAC, 1980)

Moisture was determined by taking 10 g of sample in petridish and dried in an
oven at 1050C till the weight of the petridish with its content was constant. Each time
before weighing, the petridish was cooled in desiccators. Moisture content of the sample
was expressed in g/100g of sample.
Initial weight (g) – Final weight (g)
Moisture content (g/100g) = ---------------------------------------------- X 100
Weight of the sample

3.5.2 Estimation of protein (AOAC, 1980)
The protein content of the dried samples was estimated as per cent total nitrogen
by the Micro-kjeldahl procedure. Protein per cent was calculated by multiplying the per
cent nitrogen by the factor 6.25 (Annexure- I).
Titre value X Normality of HCLX14.001X6.25
Per cent protein = -------------------------------------------------------------X 100
Sample weight (g)
3.5.3 Estimation of fat (AOAC, 1980)

Fat was estimated as crude ether extract using moisture free sample. The solvent
was removed by evaporation and the residue of fat was weighed (Annexure- II).
Weight of ether extract
Fat content (g/100g) = ------------------------------------ X 100
Weight of sample taken
3.5.4 Estimation of crude fibre (AOAC, 1980)

Crude fibre of the sample was estimated by using moisture and fat free samples
and expressed as g/100g of the sample (Annexure- III).
[100-(moisture + fat)] x (We-Wa)
Crude fibre (g/100g) = ----------------------------------------------------- X 100
Wt. of sample taken (moisture and fat free)
Where,
We- pre-weighed ashing dish
Wa- weight of the dish after ashing
3.5.5 Estimation of insoluble dietary fibre, soluble dietary fibre and total dietary
fibre (AOAC, 1995)
Dietary fibre and its components including soluble and insoluble were analyzed.
Initially, insoluble and soluble fibre were estimated then total dietary fibre was calculated
by using the formula (Annexure IV).
3.5.5.1 Insoluble dietary fibre

Defatted foods were gelatinized and proteins and starch were removed by
enzymatic digestion. The residue was quantified gravimetrically.
Insoluble dietary fibre = IDF residue – (protein + ash)

Wt. of the IDF residue (g) – {Protein (g) in IDF residue + Ash (g) in IDF residue}
IDF% = ----------------------------------------------------------------------------------------- X 100
Wt of the sample (g)
17 Shweta Bhosale
Stabilized rice bran
Inoculation of Sporolac powder at

1% level
Fermentation for 24 hrs at 300 C
Lyophilization (34 hrs at -330C)
Stored in refrigerator condition in

air tight zip lock bag
Fig. 3: Probiotic treatment of rice bran

3.5.5.2 Soluble dietary fibre
The soluble fibre is estimated in the filtrate obtained after enzymatic digestion of
protein and carbohydrates of defatted food. The soluble fibre is precipitated and
estimated gravimetrically
Soluble dietary fibre = Weight of SDF residue – (protein + ash)

Wt. of the SDF residue (g) – {Protein (g) in SDF residue + Ash (g) in SDF residue}
SDF% = ---------------------------------------------------------------------------------------- X 100
Wt. of the sample (g)
3.5.5.3 Estimation of total dietary fibre

The total dietary fibre is the sum of the insoluble and soluble dietary fibre,
estimated as follows.
Total Dietary Fibre = IDF+SDF values
3.5.6 Estimation of total ash (AOAC, 1980)

The ash content of sample was obtained by dry ashing the samples completely by
heating it over a flame. This was expressed as g/100g of the sample (Annexure V).
Weight of the ash
Ash content (g/100g) = ---------------------------------- X 100
Weight of the sample
3.5.7 Computation of carbohydrate (AOAC, 1980)

Carbohydrate content was calculated by differential method.
Carbohydrate (g/ 100g) = 100 – [Protein (g) + Fat (g) + Fibre (g) + Ash (g) + moiosture
(g)]
3.5.8 Computation of energy (Differential method)

Energy was computed as follows for all the samples.
Energy(Kcal)= [Protein (g) x 4] + [Carbohydrate (g) X 4] + [Fat (g) X 9]
3.5.9 Preparation of mineral solution (AOAC, 1980)

The mineral solution of all samples were prepared by dissolving the ash obtained
after ashing the samples in a muffle furnace in dilute hydrochloric acid (1:1 v/v)
(Annexure VI).
3.5.10 Estimation of calcium (AOAC, 1980)

The calcium content was estimated by precipitating it as calcium oxalate and
titrating the solution of oxalate in dilute acid against standard potassium permanganate.
To an aliquot (25 ml) of the mineral solution was added a few drops of methyl red
indicator and the solution was neutralized with ammonium until the pink colour changed

to yellow. The solution was heated to boiling and 10ml of 6 per cent ammonium oxalate
was added. The mixture was then boiled for a few more minutes and glacial acetic acid
was added until the colour turned distinctly pink. The mixture was kept overnight and
when the precipitate settled down, the supernatant was tested with a drop of ammonium
oxalate solution to ensure the completion of the precipitate. The precipitate was then
filtered through Whatman No.40 filter paper and washed with water until it was free of
oxalate. The precipitate was then transferred along with the filter paper to free of oxalate.
The precipitate was then transferred along with the filter paper to the same beaker and
about 5mL of 2N dilute H2SO4 was then titrated against N/KMNO4 solution (Annexure-
VII).
1ml of N/100 KMNO4= 0.2004 mg of calcium

Titre value × 0.2004 × vol. Of H2SO4
% Calcium (mg) = ------------------------------------------------------------------- X 100
Weight of the sample used for ashing × aliquot taken
3.5.11 Estimation of Phosphorus (AOAC, 1980)

Determination of phosphorus was carried out by measuring colorimetrically the
blue color formed when the ash solution is treated with ammonium molybdate and thus
phosphomolybdate formed is reduced (Annexure- VIII).
To an aliquot of 0.4 ml of micronutrient solution was added 1mL of ammonium

molybdate, 1mL of hydroquinone and 1mL of sodium thiosulphate solutions in this order,
mixing well after each addition. The volume was then made up to 15 ml with water and
the solution mixed thoroughly. After 30 minutes the optical density of this solution is
measured in a Photoelectric calorimetric against a reagent blank prepared in the same
way as the test, except that the test solution is omitted, at 660 nm. The phosphorus
content of sample was obtained from a standard curve prepared with standard phosphate
solution (range 0.01 to 0.1 mg phosphorus).
Graph ppm X volume of digested sample
Phosphorus % = ----------------------------------------------------- X 100 X dilution
106 X weight of sample X aliquot taken
3.5.12 Estimation of Iron (AOAC, 1980)

Iron was determined by colorimetric method. When potassium thiocyanate was
added to the sample it turned red indicating the presence of iron in a sample (Annexure-
IX) to an (1 ml or less) of the micronutrient solution enough water is added (if necessary)
to make up to volume of 6.5 ml followed by 1ml of 30 per cent H2SO4, 1.0 ml of
saturated potassium per sulphate and 1.5 ml 40 per cent KCNS solution. The red color
that develops is measured within 20 min at 540 nm.
3.5.13 Estimation of Zinc (Page et al., 1992)

Zinc content of the groundnut samples were estimated by using atomic absorption
spectrophotometer and the results were expressed as mg per 100 grams of the groundnut
19 Shweta Bhosale
sample (Annexure- X). 100 ppm standard Zn2+ solution was prepared using 1000 ppm
Zn2+ atomic absorption spectrophotometer solution and appropriate dilutions were made
to get standard solution ranging from 0 to 0.6 ppm. These standards were fed to atomic
absorption spectrophotometer as that of sample to get standard curve as a graph was fit.
To this standard curve the sample readings were compared.
3.5.14 Estimation of antioxidant activity by DPPH method (Ranganna, S., 1995)

The antioxidant activity was expressed in terms of ascorbic acid equivalents; so
ascorbic acid is taken as standard. Various concentrations of ascorbic acid were prepared
and added to DPPH solution. The decrease in O.D. is plotted against concentration of
ascorbic acid. The concentration of sample was calculated using the standard curve
(Annexure XI).
0.6
Antioxidant activity by DPPH method
0.5
y = -1.025x + 0.4883
OD at 517nm
0.4 R² = 0.9951
0.3
0.2
0.1
0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
Concentration of Vitamin C in µg
3.5.15 Phytic acid estimation of rice bran (Sadasivam and Manickam, 1992)
The estimation of phytic acid was based on the principle that the phytate is
extracted with trichloroacetic acid and precipitated as ferric salt. The iron content of the
precipitate was determined colorimetrically and the phytate phosphorous content
calculated from this value assuming a constant 4 Fe: 6 molecular ratios in the precipitate.
Phytates were estimated as phytic and phytate phosphorous was obtained (Annexure-
XII).
3.5.16 Trypsin Inhibitors estimation

Trypsin inhibitors activity (TIA) in both stabilized and probiotic treated rice bran
samples were measured by inhibiting the activity of trypsin as per the method followed
by Kakade et al. (1969) as described in detail in (Annexure-XIII).

3.6 Development of food products by incorporating processed rice bran
The products namely biscuit, bread, muffin, chocolate and chapati were
developed by incorporation of stabilized and probiotic treated rice bran at different levels.
Products without the addition of rice bran served as control and subjected to sensory
evaluation (Fig. 4-8, Plate 2-6) (Annexure XIV - XVIII).
3.7 Physical/functional characteristics of developed products

3.7.1 Physical characteristics of biscuit
Physical characteristics like width, thickness and spread factor were determined
according to the AACC (2000) method.
Width (W): Six biscuits were placed horizontally (edge to edge) and rotated at 900 angle
for reading.
Thickness (T): Six biscuits were placed one another to compute thickness.
Spread factor (SF): It was calculated according to the following formula.

SF = (W/T X CF) X 10
Where, CF= Correlation factor (1.0 in this case)
3.7.2 Physical characteristics of bread

Physical characteristics like water absorption, dough development time, loaf
volume and specific volume were determined according to the AACC (2000) method.
Water absorption: Percentage of water required to yield dough consistency of 500 BU

(Brabender Units).
Dough development time (DDT): It is the time to reach maximum consistency in

minutes.
Weight: Determined by electronic weighing balance.
Loaf volume: It was determined using the volume displacement method. Husked rice
was used as medium displacement. The volume of the container used was determined by
filling the container with rice, the bread was then placed inside the container, followed by
the rice. The rice that was not required to fill the container was used to express volume of
the loaf (ml).
Specific volume: It was calculated according to the following formula,

Specific volume = Loaf volume/ loaf weight.
21 Shweta Bhosale
Cream fat and sugar
Add
Rice bran Refined

(SRB/PRB) wheat flour
Knead and make stiff dough
Divide the dough into small portions
Round them and place on baking tray
Baking at 2000C for 15min
Cooling, packing and storing
Fig. 4: Preparation of Biscuit

Control SRB 10% PRB 10%
Plate 2: Rice bran incorporated biscuit

Cream fat and sugar
Add refined wheat flour + rice bran (SRB/PRB)
Addition of yeast
Resting the dough for 1 hour
Baking at 2000C for 20min
Fig. 5: Preparation of bread

Plate 3: Rice bran incorporated bread

Rub sugar powder and fat together
Add beaten egg to the sugar & fat
Addition of refined wheat flour & rice bran
Prepare dough to required consistency
Pour dough into the greased muffin moulds
Baking at 3750F for 20 min
Fig. 6: Preparation of muffin

Plate 4: Rice bran incorporated muffin

Melting of white chocolate +
dark chocolate
Addition of rice bran (SRB / PRB)
Pouring the mixture into

chocolate moulds
Deep freezing for 30min
Packing and storing
Fig. 7: Preparation of chocolate

Plate 5: Rice bran incorporated chocolate

Mix wheat flour with rice bran (SRB/PRB)
Add Salt and Water
Knead to a soft dough
Roll chapati with pin and board
Roast on a hot tawa
Fig. 8: Preparation of Chapati

Plate 6: Rice bran incorporated chapati

3.7.3 Physical characteristics of muffin
Physical characteristics such as water absorption, dough development time and
volume of the muffin were determined according to the AACC (2000) method.
Water absorption: Percentage of water required to yield dough consistency of 500 BU

(Brabender Units).
Dough development time (DDT): It is the time to reach maximum consistency in

minutes.
Volume: It was determined using the 50g bowl capacity container to assess the volume
of the muffin.
3.8 Sensory evaluation of the developed food products

All the products were evaluated by a panel of semi-trained panel (n=21). The
products were evaluated for appearance, texture, taste, colour, aroma and overall
acceptability on nine point hedonic scale (Plate 7). Where 9= Like extremely, 8= Like
very very much, 7= Like moderately, 6= Like slightly, 5= Neither like nor dislike, 4=
Dislike slightly, 3= Dislike moderately, 2= Dislike very much, 1= Dislike extremely
(Annexure XIX). Score sheet used for the evaluation of products is included in Annexure
(Avantina, 2006).
3.9 Nutrient composition of the developed food products

Nutritive value for all the food products were computed per 100g product using
food composition tables (Gopalan et al., 2012).
3.10 Storage and microbial study of the food products

3.10.1 Storage study
Shelf life of all the products were evaluated. Biscuits were stored up to 15 days,
whereas the other products viz. bread and muffin were stored up to 4 days, chocolates
were stored up to 3 months. Each product was stored in high density polythene food
grade pouches of 350 gauze for further analysis.
3.10.1.1 Estimation of total lipids (Bligh and Dyer, 1959)

Total lipids were estimated using the Bligh and Dyer method. The lipids extracted
in a mixture of chloroform and methanol (2:1 v/v) (Annexure XX).
3.10.1.2 Acid Value (Raghuramulu et al., 2003)

Acid value of seed oil was determined according to AOAC Official Method.
Percentage free fatty acids (FFA) were calculated using oleic acid as a factor (Annexure
XXI).

Acid value = Titre value x Normality of KOH x 1000
Weight of the sample (g)
% free fatty = Acid value

1.99
3.10.1.3 Peroxide value (Raghuramulu et al., 2003)

Peroxide value was determined as per the procedure (Bligh and Dyer, 1959) and
expressed as meq/kg of sample.
0.5 to 1 g of clear melted fat was weighed accurately in the boiling flask. To this
30 ml of acetic acid-chloroform mixture was added and the fat was dissolved. 1 ml of
saturated potassium iodide was added. After 5min 100 ml of distilled water was added.
The liberated iodine was titrated against N/1000ml sodium thiosulphate. When the end
point approached 1ml of freshly prepared starch was added and the titration was
completed till the colour disappears. Blank was carried out using all the reagents without
the oil (Annexure-XXII).
Calculation:
Peroxide value of oil (meq/kg of sample) = (Titre – Blank) x N X 1000
Weight of oil (g)
3.10.2 Microbial study of the food products (Tate, 1995)

The microbiological analysis of the developed value added products was carried
out as per the standard method for Coliforms using the Eiosine Methalene Blue Agar
(EMBA), for general bacteria Nutrient Agar was used and for Fungi using Rose Bengal
Agar (RBA). Ten gram of sample was diluted in 90 ml of buffer blanks and subsequent
dilutions were prepared up to 10-6 dilution. Three dilution factors were used for plating of
coliforms, general bacteria and fungi viz., 10-2, 10-3, 10-4, 10-5 and 10-6. the number of
microbial counts was calculated using the following formula.
No. of colonies x dilution factor
No. of Microorganisms (per g/ml) = ----------------------------------------------------
Weight / volume of aliquots taken(g/ml)
3.11 Glycemic index test of selected food product

The glycemic index is defined as the incremental area under the blood glucose
response curve of a 50g carbohydrate portion of test food expressed as a per cent of the
response to the same amount of carbohydrate from a standard food taken by the same
subject (FAO/WHO, 2003).
Subject: The students of UAS Bengaluru aged between 20-25 years were recruited. All
the volunteers were normoglycemic and lead sedentary life style. The purpose of study
was explained to each subject and consent (Annexure XXIII) taken for participation. The
subjects did not take any medication during the experiment. Blood glucose response vary
23 Shweta Bhosale
Plate 7: Sensory evaluation of products by panel members
considerably from day-to-day within subjects. The same subjects were recruited for
assessing the glycemic index of all samples ensuring a wash out period of one day
between the samples.
Standard, control and test food: White bread was used as the standard food. Chapati
which was best accepted by the sensory panel members (20% SRB and PRB) was tested
for its glycemic response on 10 healthy volunteers using whole wheat flour chapati as
control.
Blood glucose response: The peripheral blood glucose was obtained using 28G pricking
lancets and the blood glucose in peripheral blood was estimated using the in vitro
diagnostic kit (XCE 188-1311) of the Abbott Diabetes care Inc. Almeda. CA 94502, USA
(Plate 8). The glycemic index was calculated using the following formula. The protocol is
included in (Annexure XXIV).
IAUC of the test food curve
Glycemic index (GI) =-------------------------------------------------- X 100
IAUC of the standard food curve
Glycemic index of food X Carbohydrate consumed (g)

Glycemic load (GL) =------------------------------------------------------------------------
100
3.12 Statistical analysis (Fisher and Yates, 1963)

One way analysis of variance (F-test) was applied on the sensory mean scores of
21 panel members in order to find the significant difference between the different
characteristics of products, under the study. Complete Randomized Design (CRD)
analysis of variance was applied and the data obtained for each nutrient and functional
property was subjected to statistical analysis to determine the level of significance. Two
way analysis of variance was used for sensory scores of the products. The statistical
analysis was done by using Minitab software (Minitab v1511). Significant difference was
defined as p ≤ 0.05.

Plate 8: Testing of blood glucose in subjects
IV. EXPERIMENTAL RESULTS
The present study was undertaken on “Processing of rice bran and its utilization in
food products.” The work was carried out at the Department of Food Science and
Nutrition, UAS, GKVK, Bangalore during 2012-2014. The results obtained in the study
are presented under the following sub headings.
4.3 Functional properties of processed rice bran samples
4.4 Nutrient composition of processed rice bran samples
4.5 Development of products by incorporating stabilized and probiotic treated rice bran
4.5.1 Physical properties, sensory evaluation and shelf life study of biscuit
4.5.2 Functional properties, sensory evaluation and shelf life study of bread
4.5.3 Physical properties, sensory evaluation and shelf life study of muffin
4.5.4 Sensory evaluation and shelf life study of chocolate
4.5.5 Sensory evaluation of chapati
4.6 Nutrient composition of developed products
4.7 Microbial study of the food products
4.8 Glycemic Index (GI) test of selected food product

load
4.1.1 Analysis of rice bran sample for heavy metals
Analysis of rice bran sample for heavy metals was carried out at North East
Institute of Science and Technology, CSIR, Jorhat. The results obtained showed that the
contents of lead, cadmium, arsenic and mercury were less than 0.01 ppm (Table 1).
4.1.2 Analysis of rice bran sample for pesticide residue

Analysis of rice bran sample for pesticide residue was carried out at North East
Institute of Science and Technology, CSIR, Jorhat. The results are presented in Table 1,
which showed that the pesticide residues are not detected in rice bran sample.
4.1.3 Microbial study of rice bran sample–serial dilution plate count technique
Microbial study for total bacterial count, moulds and yeast count was carried out
for rice bran sample and the values are depicted in Table 2. It was observed that there was
no microbial load in all the three group of microorganisms in the sample.
Hence the obtained rice bran sample was safe for further processing, i.e. for
stabilization, probiotic treatment and utilization in food products.

Stabilization of rice bran was carried out by using microwave method. Percentage
of free fatty acid (FFA) of rice bran was calculated, after microwave stabilization at 2450
MHz for 3 minutes. The free fatty acid percentage of microwave stabilized rice bran from
initial, 1st, 2nd, 3rd and 4th week was 4.10, 4.98, 5.20, 6.80 and 7.50 respectively (Table 3,
Fig. 9).
Probiotic treatment of rice bran was carried out by using Lactic Acid Bacillus
(LAB) culture. The free fatty acid percentage of probiotic treated rice bran from initial to
fourth week was 4.35, 5.0, 5.50, 7.05 and 7.95 per cent respectively. The results are
presented in Table 3. There was non significant difference between the control, stabilized
and probiotic treated rice bran during the initial period of storage and significant
difference has been found between the three types of rice bran during the time intervals
i.e. 1, 2, 3 and 4 weeks.
4.3 Functional properties of processed rice bran samples

The functional properties analysed for the rice bran were bulk density, water
absorption, oil absorption, water solubility and swelling power capacity (Table 4).
Bulk density: Statistically significant results (p≤0.05) were noted for the bulk density.
The bulk density of stabilized rice bran was found to be 0.22 g/ml and that of probiotic
treated rice bran was 0.38 g/ml.
26 Shweta Bhosale
Table 1: Analysis of rice bran sample for heavy metals and pesticide residue
Heavy metals
Tests Amounts detected Limit of quantification
Lead (Pb) 0.0054 ppm 0.01 ppm
Cadmium (Cd) 0.0048 ppm 0.01 ppm
Arsenic (As) 0.0058 ppm 0.01 ppm
Mercury (Hg) BDL 0.01 ppm
Pesticide residue
Amounts detected Limit of quantification

Pesticides
(mg/kg) (mg/kg)
Alpha-HCH ND 0.02
Beta-HCH ND 0.02
Gama-HCH (Lindane) ND 0.02
Heptachlor ND 0.02
Aldrin ND 0.02
Heptachlor epoxide ND 0.02
Dieldrin ND 0.02
p,p-DDE ND 0.02
o,p-DDD ND 0.02
p,p-DDD ND 0.02
o,p-DDT ND 0.02
P,p-DDT ND 0.02
ND: Not Detected

BDL: Bellow Detecting Level
Source: Bangalore test house, Rajaji nagar, Bangalore.

Table 2: Microbial load of rice bran sample-serial dilution plate count
technique
Group of microorganisms
Sample Total bacteria Molds Yeast
Population (x 102 CFU / 10g sample)
Rice bran Nil Nil Nil
Table 3: Percentage of free fatty acid (FFA) in rice bran on storage
Storage time (weeks)
Type of rice bran Initial 1 2 3 4
FFA%
Control 4.36 8.48 12.98 16.50 24.70
Microwave stabilized rice bran (SRB) 4.10 4.98 5.20 6.80 7.50
Probiotic rice bran (PRB) 4.35 5.00 5.50 7.05 7.95
F-value NS * * * *
SEm± 0.06 0.58 0.08 0.08 0.08
CD 0.18 1.78 0.25 0.25 0.25
Standard value for free fatty acid: <10%

NS - Non Significant * Significant at 5%
28 Shweta Bhosale
30
Control SRB PRB

25
20
% of FFA
15
10
0
Initial 1st week 2nd week 3rd week 4th week
Storage period
SRB: Stabilized Rice Bran PRB: Probiotic treated Rice Bran
Fig. 9: Percentage of Free Fatty Acid in rice bran on storage

Water and oil absorption capacity: Water absorption capacity ranged from 2-3 ml/g,
where as oil absorption ranged from 1.5-2.5 ml/g. There existed a non significant
difference among the stabilized and probiotic treated rice bran for water and fat
absorption capacity.
Water solubility (%): Highest water solubility per cent was found in stabilized rice bran
7.3, followed by probiotic treated rice bran i.e. 8.0. There was no significant difference
observed between the samples.
Swelling power (%): Swelling power capacity of probiotic treated rice bran was 7.2
followed by stabilized rice bran i.e. 6.7. The difference in swelling power was found to
be non significant.
Table 4: Functional properties of processed rice bran samples
Bulk Water Oil Water Swelling

Type of bran density absorption absorption Solubility power
(g/ml) (ml/g) (ml/g) (%) (%)
Stabilized rice bran

0.22 2.0 1.5 7.3 6.7
(SRB)
Probiotic treated rice

0.38 3.0 2.5 8.0 7.2
bran (PRB)
F-value * NS NS NS NS
SEm± 0.01 1.0 1.0 0.10 0.10
CD 0.03 3.08 3.08 0.31 0.31
NS - Non Significant * Significant at 5%
4.4 Nutrient composition of rice bran samples

Nutrient composition of stabilized and probiotic treated rice bran samples were
shown in Table 5. The macronutrient composition of stabilized and probiotic treated rice
bran were in the order of moisture (4.30 and 5.40 %), protein (17.50 and 19.25 g), fat
(13.10 and 17.20 g ), crude fibre (7.85 and 4.96 g), insoluble dietary fibre (21.17 and
13.10), soluble dietary fibre (2.17 and 1.80), total dietary fibre (23.34 and 14.90),
carbohydrate (52.33 and 48.55 g), energy (398 and 426 Kcal) and ash(4.92 and 4.64)
respectively. The micronutrient composition of stabilized and probiotic treated rice bran
samples were calcium (52.10 and 49.90 mg), phosphorous (1185.2 and 1186.5 mg), iron
(28.10 and 30.05 mg) and zinc (6.02 and 5.89 mg) respectively.
The antioxidant activity for stabilized rice bran was 65 Vit-C Eq. µg/g and
probiotic treated rice bran was 70 Vit-C Eq. µg/g respectively. Antinutritional factors for

stabilized and probiotic treated rice bran were, Phytic acid (23.5 and 22.15mg/g), Trypsin
Inhibitor (10.8 and 10.2mg/g) respectively. Significant difference was observed for all
the nutrients except soluble dietary fibre, phosphorus, iron, phytic acid and trypsin
inhibitor.
Table 5: Nutrient composition of rice bran per 100g
Stabilized Probiotic
Nutrients F-value SEm± CD at 5%
rice bran rice bran
Moisture (%) 4.30 5.40 * 0.07 0.30
Protein (g) 17.50 19.25 * 0.05 0.21
Fat (g) 13.10 17.20 * 0.07 0.30
Crude fibre (g) 7.85 4.96 * 0.00 0.03
Insoluble dietary fibre (g) 21.17 13.10 * 0.05 0.21
Soluble dietary fibre (g) 2.17 1.80 NS 0.70 3.08
Total dietary fibre (g) 23.34 14.90 * 0.05 0.21
Carbohydrate (g) 52.33 48.55 * 0.00 0.03
Energy (KCal) 398 426 * 0.70 3.08
Ash (g) 4.92 4.64 * 0.00 0.03
Calcium (mg) 52.10 49.90 * 0.07 0.30
Phosphorus (mg) 1185.20 1186.50 NS 0.50 2.18
Iron (mg) 28.10 30.05 NS 0.50 2.18
Zinc (mg) 6.02 5.89 * 0.00 0.03
Antioxidant activity (Vit- * 0.70 3.08

65 70
C Eq. µg/g)
Anti-nutrients
Phytic acid (mg/g) 23.50 22.15 NS 0.50 2.18
Trypsin Inhibitor (mg/g) 10.80 10.20 NS 0.70 3.08
NS – Non-Significant * Significant at 5% level
30 Shweta Bhosale
4.5 Development of products by incorporating stabilized and probiotic
treated rice bran
The products were developed by the incorporation of stabilized and probiotic
treated rice bran and subjected to sensory evaluation. The products developed were
biscuit, bread, muffin, chocolate and chapati for which the acceptability and difference in
sensory attributes were tested. The sensory evaluation and acceptability test were carried
out using 9 point hedonic scale by 21 semi trained panel members from the department of
Food Science and Nutrition.
4.5.1 Physical properties, sensory evaluation and shelf life study of biscuits
4.5.1.1 Physical properties of biscuit
Bakery products are becoming increasingly popular in India due to their
convenience, unique taste and easy availability at reasonable cost. Among bakery
products, biscuits/cookies are the most popular and versatile snack foods and widely
consumed to satisfy the occasional 'pangs' of hunger and are an integral part of the
society. As the biscuits are mainly prepared from refined wheat flour the fibre content of
these biscuits are low, hence the use of stabilized and probiotic treated rice bran helps in
improving the fibre content and texture of the biscuit. In the present study biscuits were
prepared in three variations (5, 10 and 15 %). Control samples were prepared without the
addition of rice bran. Effect of stabilized and probiotic treated rice bran sample on the
physical properties of biscuits were studied. Table 6 indicates that there was no
significant difference among weight of biscuits where as significant difference existed in
width, thickness and spread ratio.
Table 6: Physical properties of biscuit

Level of Weight Width Thickness Spread ratio
incorporation (%) (g) (mm) W (mm) T (W/TXCF) X 10
Control 15.00 64.40 24.20 26.61
SRB 5 15.50 62.00 25.15 24.65
PRB 5 15.40 62.15 25.00 24.86
SRB 10 16.10 60.30 26.50 22.86
PRB 10 16.00 60.60 26.35 22.88
SRB 15 17.20 58.00 27.90 20.82
PRB 15 17.00 58.15 27.75 20.90
F-value NS * * *
SEm± 0.41 0.27 0.36 0.14
CD at 5% 1.27 0.83 1.11 0.44
NS- Non Significant * Significant at 5% level CF (Correlation Factor)=1

SRB 15 per cent level of incorporation had highest weight of 17.20g and lowest
was in control biscuit i.e. 15.00g. The control biscuit had highest width 64.40mm and
PRB 15 per cent had lowest width 58.00mm. The width of the biscuits decreased with the
levels of incorporation of rice bran.
SRB 15 per cent biscuit had highest thickness (27.90mm) and control biscuit had
lowest (24.20mm), with increase in rice bran levels there was increase in thickness of
biscuits and the spread ratio was highest for control biscuit (26.61mm) and lowest for
SRB 15 per cent (20.82mm).
4.5.1.2 Mean sensory scores of biscuit

Biscuits were developed by incorporating 5, 10 and 15 per cent level. Sensory
evaluation of the biscuits are presented in the Table 7 and Fig (10). The control biscuits
showed highest scores of 8.9, 9.0, 8.8, 8.7, 8.9 and 9.0 for appearance, texture, colour,
aroma, taste and overall acceptability. The biscuits with SRB 10 per cent (8.7, 8.4, 8.8,
8.5, 8.9 and 8.8) and PRB 10 per cent (8.8, 8.6, 8.7, 8.5, 8.8 and 8.7) were best accepted
for sensory attributes among the stabilized and probiotic treated rice bran variations.
When analyzed statistically, the mean sensory scores were found to be significant at 5 per
cent level.
Table 7: Mean sensory scores of biscuit (N=21)
Sensory attributes
Treatment
Appearance Texture Colour Aroma Taste Overall acceptability
Control 8.9 9.0 8.8 8.7 8.9 9.0
SRB 5% 8.5 8.1 8.6 8.2 8.6 8.5
PRB 5% 8.5 8.2 8.6 8.2 8.4 8.5
SRB 10% 8.7 8.4 8.8 8.5 8.9 8.8
PRB 10% 8.8 8.6 8.7 8.5 8.8 8.7
SRB 15% 7.7 7.6 8.1 7.8 7.6 7.7
PRB 15% 7.9 7.8 8.0 7.8 7.7 7.8
F-value * * * * * *
SEm± 0.11 0.12 0.11 0.12 0.10 0.09
CD at 5% 0.32 0.33 0.32 0.35 0.30 0.27
* Significant at 5% level
32 Shweta Bhosale
10
8
Mean sensory scores
0
Control SRB 5% PRB 5% SRB 10% PRB 10% SRB 15% PRB 15%
Treatments
Fig. 10: Mean sensory scores of biscuit

4.5.1.3 Mean sensory scores of biscuit on storage
Biscuits (Control, SRB 10 and PRB 10 per cent) were kept for storage study. The
samples were observed daily for visual changes and were subjected to sensory evaluation
on 15th day.
The results of the mean sensory evaluation of biscuits from initial day to end of
storage period are presented in the Table 8, which depicts that the control biscuits showed
highest scores of 8.9, 9.0, 8.8, 8.7, 8.9 and 9.0 for all the sensory parameters for the initial
day, however at the end of 15 days the control biscuits had lower scores of 7.8, 7.6, 7.8,
7.5, 7.8 and 7.5 for appearance, texture, colour, aroma, taste and overall acceptability.
The SRB with 10 per cent incorporation showed scores of 8.7, 8.4, 8.8, 8.5, 8.9
and 8.8 for all the sensory parameters for the initial day which are on par with control
values and had shelf life up to 15 days. In PRB with 10 per cent incorporation the sensory
parameters decreased from initial (8.8, 8.6, 8.8, 8.5, 8.9 and 8.8) to 15 days (7.4, 7.5, 7.4,
7.1, 7.0 and 7.2) of storage period.
Statistical analysis revealed a significant difference for all the sensory

characteristics of biscuits between the treatments and also duration of the storage period.
Table 8: Mean sensory scores of biscuit on storage (N=21)
Sensory attributes
Duration
Biscuit Overall
(Days) Appearance Texture Colour Aroma Taste
acceptability
Initial 8.9 9.0 8.8 8.7 8.9 9.0
Control
15 days 7.8 7.6 7.8 7.5 7.8 7.5
SRB Initial 8.7 8.4 8.8 8.5 8.9 8.8
(10%) 15 days 7.2 7.6 7.5 7.2 7.4 7.4
PRB Initial 8.8 8.6 8.8 8.5 8.9 8.8
(10%) 15 days 7.4 7.5 7.4 7.1 7.0 7.2
Treatments
F-value * * * * * *
SEm± 0.080 0.072 0.084 0.091 0.076 0.079
CD at 5% 0.317 0.284 0.332 0.356 0.301 0.310
Duration
F-value * * * * * *
SEm± 0.08 0.07 0.08 0.09 0.07 0.07
CD at 5% 0.31 0.28 0.33 0.35 0.30 0.31

4.5.1.4 Peroxide and free fatty acid value of biscuits
Table 9 represents the peroxide and free fatty acid value of biscuits and are
compared with standard values. Peroxide value of control, SRB 10 per cent and PRB 10
per cent biscuits showed significant difference. Initially control biscuits had 3.2
miliequiv. of peroxide/Kg of sample, SRB 10 per cent had 1.5 miliequiv. of peroxide/Kg
of sample and PRB 10 per cent had 1.3 miliequiv. of peroxide/Kg of sample. The
peroxide content of control biscuit increased to 9.8 miliequiv. of peroxide/Kg of sample
after 15 days, more rapidly than SRB and PRB (4.4 and 4.0 miliequiv. of peroxide/Kg of
sample) after 15 days of storage period.
Free fatty acid value increased from 0.8 to 2.9 per cent in control biscuit, 0.6 to
1.0 in SRB 10 per cent biscuit and 0.5 to 0.9 per cent in PRB 10 per cent biscuit i.e. the
free fatty acid content increased as the storage period increased.
Statistical analysis revealed that the peroxide and free fatty acid value of biscuits
was non significant at initial stage and significant at 5 per cent at the end of storage
period.
Table 9: Peroxide and free fatty acid (FFA) value of biscuits

Sample Initial 15 days
Peroxide value (miliequiv. of peroxide/Kg of sample)
Control 2.6 9.8
SRB 10% 2.0 4.4
PRB 10% 2.1 4.0
F-value NS *
SEm± 0.10 0.10
CD 5% 0.31 0.31
Free fatty acid (FFA) value (% of oleic acid/ 100g of sample)
Control 0.8 2.9
SRB 10% 0.6 1.0
PRB 10% 0.5 0.9
F-value NS *
SEm± 0.10 0.10
CD 5% 0.31 0.31
NS - Non Significant * Significant at 5% level
Standard values: Peroxide value less than 10 milliequiv.of peroxide/kg of sample.
Free Fatty Acids less than 1.0 of oleic acid/100g of sample. (Food Regulation Act)
34 Shweta Bhosale
4.5.2 Functional properties, sensory evaluation and shelf life study of bread
4.5.2.1 Functional properties of bread
Bread is a staple food, it has been popular around the world and is one of
humanities oldest food. To enrich the nutrient content of the bread rice bran is being used
in the present study. Bread was prepared in three different variations (5, 10 and 15 per
cent) and control bread without the addition of rice bran. The effect of rice bran on the
physical properties of bread were analyzed.
Water absorption capacity and weight of the bread were highest in PRB 15 per
cent (76.26% and 245.06g) and lowest in control (62% and 218g) i.e. as the rice bran
incorporation level increases the water absorption capacity and weight of the bread were
increased. The dough development time increased with the addition of rice bran into
bread. Highest loaf volume was found in control (1535cm3) and lowest was found in PRB
15 per cent (1420 cm3). Specific volume of the bread decreased as the addition of rice
bran increased (Table 10). Significant difference was found in all the functional
properties of bread.
Table 10: Functional properties of bread
Level of Water Dough Loaf Specific

Weight
incorporation absorption development Volume volume
(gm)
(%) (%) time (min) (cm3) (cm3/gm)
Control 62.00 10.00 218 1535 7.04
SRB 5 65.80 12.50 228 1505 6.60
PRB 5 66.50 12.30 230.2 1500 6.51
SRB 10 72.56 14.75 235.4 1482 6.29
PRB 10 73.98 14.00 236.3 1478 6.25
SRB 15 75.08 18.60 242.10 1425 5.88
PRB 15 76.26 18.50 245.06 1420 5.79
F-value * * * * *
SEm± 0.74 0.72 0.99 0.93 0.04
CD at 5% 2.27 2.21 3.06 2.85 0.12
Significant at 5% level

4.5.2.2 Mean sensory scores of bread
Bread was prepared by incorporating stabilized and probiotic treated rice bran at
5, 10 and 15 per cent levels. Results of the sensory evaluation of bread are presented in
Table 11 and Fig.11.
The control bread showed highest scores of 8.8, 9.0, 8.8, 8.8, 9.0 and 9.0 for
appearance, texture, colour, aroma, taste and overall acceptability respectively. SRB with
5 per cent showed second highest scores of 8.6, 8.8, 8.8, 8.7, 8.8 and 8.9 respectively for
sensory attributes and the lowest scores of sensory attributes was found in SRB with 15
per cent level (7.9, 7.8, 7.8, 7.6, 7.7 and 7.5). In case of probiotic treatment highest
sensory attributes were found in PRB 10 per cent and the lowest scores were found in
case of PRB with 15 per cent incorporation levels. Statistical analysis revealed a
significant difference for all the sensory characteristics at 5 per cent level.
Table 11: Mean sensory scores of bread (N=21)
Sensory attributes
Treatment
Control 8.8 9.0 8.8 8.8 9.0 9.0
SRB 5% 8.6 8.8 8.8 8.7 8.8 8.9
PRB 5% 8.0 8.2 8.5 8.6 8.5 8.6
SRB 10% 8.1 8.3 8.2 8.0 8.2 8.8
PRB 10% 8.5 8.6 8.5 8.8 8.7 8.9
SRB 15% 7.9 7.8 7.8 7.6 7.7 7.5
PRB 15% 7.7 7.9 8.0 7.8 7.6 7.7
F-value * * * * * *
SEm± 0.12 0.10 0.10 0.11 0.11 0.09
CD at 5% 0.34 0.30 0.29 0.31 0.30 0.26
4.5.2.3 Mean sensory scores of bread on storage

The mean sensory scores showed a decreasing trend during 2nd day of storage as
indicated in Table 12. Control sample showed higher scores compared to the SRB and
PRB at 5 and 10 per cent level. The changes in mean scores from initial to 2nd day of SRB
at 5 per cent for appearance was 8.8 to 7.1, texture 9.0 to 7.0, colour 8.8 to 7.2, aroma 8.8
36 Shweta Bhosale
10
9
Mean Sensory Scores
0
Treatments
Fig. 11: Mean sensory scores of bread

to 7.0, taste 9.0 to 7.4 and overall acceptability 8.9 to 7.8 respectively. The mean sensory
scores of PRB at 10 per cent level on 2nd day was 7.1, 7.0, 7.0, 7.2, 7.2 and 7.0 for
appearance, texture, colour, aroma, taste and overall acceptability respectively.
Statistical analysis revealed a significant difference at 5% level between the

treatments and duration for all the sensory characteristics of control, SRB and PRB bread
from initial to the end of storage period.
Table 12: Mean sensory scores of bread on storage (N=21)
Sensory attributes
Duration
Bread Overall
(days) Appearance Texture Colour Aroma Taste
acceptability
Initial 8.8 9.0 8.8 8.8 9.0 9.0
2nd day 8.1 7.9 8.0 7.9 7.6 7.8
Control
4th day 7.1 7.0 7.2 7.0 7.0 7.4
Initial 8.6 8.8 8.8 8.7 8.8 8.9
SRB
2nd day 7.6 7.5 7.9 7.9 7.7 7.8
(5%)
4th day 7.0 7.2 7.0 7.1 7.2 7.4
Initial 8.5 8.6 8.5 8.8 8.7 8.9
PRB
2nd day 7.5 7.3 7.2 7.5 7.4 7.5
(10%)
4th day 7.1 7.0 7.0 7.2 7.2 7.0
Treatments
F-value * * * * * *
SEm± 0.07 0.06 0.06 0.07 0.07 0.06
CD at 5 % 0.36 0.33 0.33 0.34 0.33 0.33
Duration
F-value * * * * * *
SEm± 0.07 0.06 0.06 0.07 0.07 0.06
CD at 5 % 0.36 0.33 0.33 0.34 0.33 0.33

4.5.3 Physical properties, sensory evaluation and shelf life study of muffin
4.5.3.1 Physical properties of muffin
The physical properties of muffins were studied. As the addition of rice bran
increased the dough development time increased significantly and the volume of the
muffins increased gradually (Table 13).
The water absorption capacity is high in PRB 15 per cent (69.30%) and low in the
control muffin (59.2%). Dough development time increased as the level of rice bran
increased from 3.4 to 6.4min. Highest volume of muffin was found in SRB 15 per cent
(110 cm3) and lowest in the control sample (94cm3). Statistical analysis revealed that
there was a significant difference between the treatments.
Table 13: Physical properties of muffin
Level of incorporation Water absorption Dough development Volume

(%) (%) time (min) (cm3)
Control 59.2 3.4 94
SRB 5 63.5 4.0 98
PRB 5 64.0 4.0 97
SRB 10 67.4 5.1 102
PRB 10 68.1 5.2 100
SRB 15 69.0 6.3 110
PRB 15 69.3 6.4 106
F-value * * *
SEm± 1.2 1.0 1.0
CD at 5% 3.12 3.08 3.08
4.5.3.2 Mean sensory scores of muffin

Muffins were prepared by using 5, 10 and 15 per cent of both stabilized and
probiotic treated rice bran. Results of the sensory evaluation of muffin are presented in
the Table 14 and Fig. (12). Control muffin showed higher values for sensory attributes for
appearance, texture, colour, aroma, taste and overall acceptability 9.0, 9.0, 8.7, 8.8, 8.8
and 8.6 respectively.
38 Shweta Bhosale
SRB at 10 per cent levels were best accepted for sensory attributes among
stabilized rice bran variations i.e. 8.6, 8.5, 8.5, 8.8, 8.6 and 8.5 for appearance, texture,
colour, aroma, taste and overall acceptability and the least was found at 15 per cent levels
(7.8, 7.7, 7.5, 7.7, 7.9, 7.7) for the sensory characteristics. Probiotic treated rice bran at 15
per cent attained high sensory attributes for appearance, texture, colour, aroma, taste and
overall acceptability 8.6, 8.5, 8.5, 8.8, 8.6, 8.5 respectively. Statistical analysis revealed a
significant difference for all the sensory characteristics at 5 per cent level.
Table 14: Mean sensory scores of muffin (N=21)
Sensory attributes
Treatment
Control 9.0 9.0 8.7 8.8 8.8 8.6
SRB 5% 8.7 8.6 8.7 8.4 8.5 8.4
PRB 5% 7.8 7.7 7.5 7.7 7.9 7.7
SRB 10% 8.6 8.5 8.5 8.8 8.6 8.5
PRB 10% 8.7 8.6 8.7 8.4 8.5 8.4
SRB 15% 7.8 7.7 7.5 7.7 7.9 7.7
PRB 15% 8.6 8.5 8.5 8.8 8.6 8.5
F-value * * * * * *
SEm± 0.11 0.11 0.12 0.10 0.10 0.12
CD at 5% 0.32 0.31 0.33 0.29 0.29 0.33
4.5.3.3 Mean sensory scores of muffin on storage

The changes in mean sensory scores of muffin decreased from initial to 4th day of
storage period in both stabilized and probiotic treated rice bran muffins. The sensory
parameters in control sample were decreased from initial to 4th day for appearance (9.0 to
7.9), texture (9.0 to 7.8), colour (8.7 to 7.6), aroma (8.8 to 7.5), taste (8.8 to 7.8) and
overall acceptability (8.6 to 7.9) respectively. In the same way the mean sensory scores
decreased significantly from initial to 4th day in stabilized and probiotic treated rice bran
muffin (Table 15).
In stabilized rice bran muffin the sensory scores decreased from 8.6 to 7.9 for
appearance, 8.5 to 7.8 for texture, 8.5 to 7.4 for colour, 8.8 to 7.5 for aroma, 8.6 to 7.3 for
taste and 8.5 to 7.5 for overall acceptability. In probiotic treated rice bran muffin the
sensory scores decreased from 8.6 to 7.9 for appearance, 8.5 to 7.7 for texture, 8.5 to 7.4
for colour, 8.8 to 7.4 for aroma, 8.6 to 7.4 for taste and 8.5 to 7.5 for overall acceptability.

Statistical analysis revealed a significant difference at 5% level between the
treatments and duration for all the sensory characteristics of control, SRB and PRB
muffin from initial to the end of storage period.
Table 15: Mean sensory scores of muffin on storage (N=21)
Sensory attributes
Duration
Muffin Overall
acceptability
Initial 9.0 9.0 8.7 8.8 8.8 8.6
2nd day 8.8 8.7 8.6 8.6 8.7 8.6
Control
4th day 7.9 7.8 7.6 7.5 7.8 7.9
Initial 8.6 8.5 8.5 8.8 8.6 8.5
SRB
2nd day 8.5 8.3 8.4 8.6 8.0 8.1
(10%)
4th day 7.9 7.8 7.4 7.5 7.3 7.5
Initial 8.6 8.5 8.5 8.8 8.6 8.5
PRB
2nd day 8.2 8.1 8.2 8.1 8.2 8.3
(15%)
4th day 7.9 7.7 7.4 7.4 7.4 7.5
Treatments
F-value * * * * * *
SEm± 0.06 0.06 0.07 0.06 0.07 0.06
CD at 5% 0.33 0.29 0.34 0.32 0.34 0.32
Duration
F-value * * * * * *
SEm± 0.06 0.06 0.07 0.06 0.07 0.06
CD at 5% 0.33 0.29 0.34 0.32 0.34 0.32
4.5.3.4 Peroxide and free fatty acid value of muffin

Table 16 represents the peroxide and free fatty acid value of muffin and are
compared with standard values. Peroxide value of control, SRB 10 per cent and PRB 15
per cent muffin showed significant difference. Initially control muffin had 4.2 miliequiv.
of peroxide/Kg of sample, SRB 10 per cent had 2.1 miliequiv. of peroxide/Kg of sample
40 Shweta Bhosale
10
8
Mean sensory scores
0
Treatments
Fig. 12: Mean sensory scores of muffin

and PRB 15 per cent had 2.0 miliequiv. of peroxide/Kg of sample. The peroxide content
of control biscuit increased to 11.25 miliequiv. of peroxide/Kg of sample for 15 days,
more rapidly than SRB and PRB (5.30 and 5.10 miliequiv. of peroxide/Kg of sample)
during 15 days of storage period.
Free fatty acid value increased from 4.2 to 11.25 per cent in control muffin, 2.10
to 5.30 in SRB 10 per cent biscuit and 2.00 to 5.10 per cent in PRB 15 per cent biscuit i.e.
the FFA content increased as the storage period increased.
Statistical analysis revealed that the peroxide and free fatty acid value of muffin
was non significant at initial stage and significant at 5 per cent at the end of storage
period.
Table 16: Peroxide and free fatty acid value of muffin

Sample Initial 6 days
Peroxide value (miliequiv. of peroxide/Kg of sample)
Control 4.20 11.25
SRB 10% 3.10 5.30
PRB 15% 3.00 5.10
F-value NS *
SEm± 0.10 0.10
CD at 5% 0.31 0.31
Free fatty acid value (% of oleic acid/ 100g of sample)
Control 1.0 2.9
SRB 10% 0.7 1.2
PRB 15% 0.6 1.0
F-value NS *
SEm± 0.10 0.10
CD at 5% 0.31 0.31
Standard values: Peroxide value less than 10 milliequiv. of peroxide/kg of sample.
Free Fatty Acids less than 1.0 of oleic acid/100g of sample. (Food Regulation Act)
4.5.4 Sensory evaluation and shelf life study of chocolate

4.5.4.1 Mean sensory scores of chocolate
The effect of incorporation of rice bran on the sensory parameters of chocolate is
presented in Table 17 and Fig. (13). The control chocolate had 8.35, 8.50, 8.65, 8.30, 8.75

and 8.80 scores for appearance, texture, colour, aroma, taste and overall acceptability
respectively, followed by 10 per cent level of incorporation of stabilized and probiotic
treated rice bran which had the scores for appearance (8.30 and 8.30), taste (8.50 and
8.45), colour (8.60 and 8.50), aroma (8.30 and 8.20), taste (8.50 and 8.25) and overall
acceptability (8.60 and 8.20) respectively, 15 per cent of stabilized and probiotic treated
chocolate had low sensory scores for appearance, texture, colour, aroma, taste and overall
acceptability 8.0 and 8.1, 8.2 and 8.2, 8.2 and 8.1, 8.2 and 8.1, 8.3 and 8.1 8.2 and 8.0
The statistical analysis revealed that there is no significant difference between the
treatments for appearance, texture, aroma and taste except for colour and overall
acceptability.
Table 17: Mean sensory scores of chocolate (N=21)
Sensory attributes
Treatment
Control 8.3 8.5 8.6 8.3 8.7 8.8
SRB 5% 8.2 8.3 8.5 8.2 8.4 8.3
PRB 5% 8.2 8.3 8.2 8.1 8.2 8.1
SRB 10% 8.3 8.5 8.6 8.3 8.5 8.6
PRB 10% 8.3 8.4 8.5 8.2 8.2 8.2
SRB 15% 8.0 8.2 8.2 8.2 8.3 8.2
PRB 15% 8.1 8.2 8.1 8.1 8.1 8.0
F-value NS NS * NS NS *
SEm± 0.13 0.12 0.13 0.15 0.14 0.12
CD at 5% 0.36 0.35 0.37 0.41 0.40 0.33
4.5.4.2 Mean sensory scores of chocolate on storage

The rice bran incorporated chocolate at SRB (10%) and PRB (10%) level has
changes in their mean sensory scores from initial to 60th day for all the sensory
parameters. In control chocolate the sensory attributes decreased from initial to 60 days
for appearance (8.35 to 7.10), texture (8.50 to 7.05), colour (8.65to 7.15), aroma (8.30 to
7.0), taste (8.75 to 7.20) and overall acceptability (8.80 to 7.20) respectively.
The mean sensory scores for stabilized rice bran chocolate at 10 per cent level
significantly decreased from initial to 60 days for appearance (8.30 to 7.25), texture (8.50
to 7.20), colour (8.60 to 7.40), aroma (8.30 to 7.30), taste (8.05 to 7.50) and overall
acceptability (8.60 to 7.30) respectively.
42 Shweta Bhosale
10
7
Mean sensory scores
0
Fig. 13: Mean sensory scores of chocolate

The mean sensory scores for the probiotic treated chocolate at 10 per cent level
significantly decreased from initial to 60 days for appearance (8.30 to 7.20, texture (8.45
to 7.70), colour (8.50 to 7.55), aroma (8.20 to 7.10), taste (8.25) to 7.30) and overall
acceptability (8.20 to 7.40) respectively.
Statistical analysis revealed a significant difference for all the sensory

characteristics of chocolate between the treatments and also duration of the storage period
(Table 18).
Table 18: Mean sensory scores of chocolate on storage (N=21)
Sensory attributes
Duration
Chocolate Overall
acceptability
Initial 8.3 8.5 8.6 8.3 8.7 8.8
Control 30 days 7.6 7.6 7.7 7.5 7.8 7.7
60 days 7.1 7.0 7.1 7.0 7.2 7.2
Initial 8.3 8.5 8.6 8.3 8.5 8.6
SRB
30 days 7.6 7.5 7.8 7.6 7.9 7.7
(10%)
60 days 7.2 7.2 7.4 7.3 7.5 7.3
Initial 8.3 8.4 8.5 8.2 8.2 8.2
PRB
30 days 7.6 7.9 7.7 7.4 7.5 7.7
(10%)
60 days 7.2 7.7 7.5 7.1 7.3 7.4
Treatments
F- value * * * * * *
SEm± 0.07 0.08 0.07 0.07 0.08 0.07
CD at 5% 0.36 0.38 0.35 0.36 0.40 0.36
Duration
F- value * * * * * *
SEm± 0.07 0.08 0.07 0.07 0.08 0.07
CD at 5% 0.36 0.38 0.35 0.36 0.40 0.36
4.5.5 Sensory evaluation of developed chapati

4.5.5.1 Mean sensory scores of chapati
Chapati was developed by incorporating rice bran at 15, 20 and 25 per cent level.
The control sample obtained highest scores of 8.8 for overall acceptability as compared to

other variations. The second highest sensory scores were obtained at 20 per cent
incorporation of stabilized and probiotic treated rice bran chapati, the mean sensory
scores of SRB and PRB are 8.5 and 8.7 for appearance, 8.2 and 8.4 for texture, 8.2 and
8.3 for colour, 8.4 and 8.4 for aroma, 8.3 and 8.5 for taste and 8.4 and 8.6 for overall
acceptability and the lowest score were for the 25 per cent rice bran incorporation levels
(SRB and PRB) i.e. 7.5 and 7.6 for appearance, 7.2 and 7.6 for texture, 7.6 and 7.8 for
colour, 7.5 and 7.5 for aroma, 7.2 and 7.8 for taste and 7.4 and 7.5 for overall
acceptability respectively. When analyzed statistically, the mean sensory scores were
found to be significant at 5 per cent level (Table 19 and Fig. 14).
Table 19: Mean sensory scores of chapati (N=21)
Sensory attributes
Treatment
Control 8.8 8.5 8.7 9.0 8.6 8.8
SRB 15% 8.4 8.0 8.1 8.2 8.0 8.0
PRB 15% 8.3 8.1 8.0 8.2 8.2 8.3
SRB 20% 8.5 8.2 8.2 8.4 8.3 8.4
PRB 20% 8.7 8.4 8.3 8.4 8.5 8.6
SRB 25% 7.5 7.2 7.6 7.5 7.2 7.4
PRB 25% 7.6 7.6 7.8 7.5 7.8 7.5
F-value * * * * * *
SEm± 0.12 0.14 0.13 0.12 0.12 0.14
CD at 5% 0.38 0.42 0.41 0.37 0.38 0.42
4.6 Nutrient composition of the developed products

Biscuit
Nutrient composition per 100 g for the control, SRB 10 per cent and PRB 10 per
cent biscuits were computed and are presented in Table 20. The nutrient content of
control biscuit for protein, fat, carbohydrate, energy, crude fibre, calcium, phosphorous,
iron and zinc were 5.26g, 29.04g, 58.85g, 517Kcal, 0.41g, 13.80mg, 57.85mg, 1.32mg
and 0.28mg respectively. The stabilized and probiotic biscuit contained 5.42 and 5.45g of
protein, 29.28 and 30.28g of fat, 58.33 and 58.26g of carbohydrate, 519 and 520 Kcal
energy, 0.70 and 0.50g of crude fibre, 14.50 and 14.45mg of calcium, 83.33 and 83.33mg
of phosphorous, 2.00 and 1.85mg of iron, 0.40 and 0.41mg of zinc respectively.
44 Shweta Bhosale
9
8
Mean sensory scores
7
6
5
4
3
2
1
0
Treatments
Fig. 14: Mean sensory scores of chapati

Bread
Protein, fat, carbohydrate, energy, crude fibre, calcium, phosphorous, iron and
zinc contents were 7.60g, 10.96g, 71.51, 415Kcal, 0.20g, 18.34mg, 83.65mg, 1.89mg and
0.41mg/ 100g respectively in the control bread. Stabilized rice bran incorporated bread
estimated to have 7.62g, 11.03g, 71.44g, 416Kcal, 0.30g, 18.48mg, 87.17mg, 2.00mg and
0.43mg of protein, fat, carbohydrate, energy, crude fibre, calcium, phosphorous, iron and
zinc per 100g of the sample. Probiotic treated bread had shown to have 7.66g, 11.10g,
71.37g, 416KCal, 0.32g, 18.62mg, 91.03mg, 2.04mg and 0.45mg of protein, fat,
carbohydrate, energy, crude fibre, calcium, phosphorous, iron and zinc per 100g of the
sample respectively as shown in Table 20.
Muffin
Muffin (control) had 7.73g, 14.18g, 48.37g, 352Kcal, 0.08g, 30.69mg, 110.80mg,
1.53mg and 0.16mg of protein, fat, carbohydrate, energy, crude fibre, calcium,
phosphorous, iron and zinc per 100g of the sample. Stabilized rice bran muffin estimated
to have 7.76g, 14.23g, 48.27g, 353Kcal, 0.20g, 30.83mg, 115.80mg, 1.64mg and 0.19mg
of protein, fat, carbohydrates, energy, crude fibre, calcium, phosphorous, iron and zinc
per 100g respectively. The probiotic treated muffin had 7.80g of protein, 14.32g of fat,
48.18g of carbohydrate, 353Kcal of energy, 0.18g of crude fibre, 30.88mg of calcium,
118.23mg of phosphorous, 1.68mg of iron, 0.20mg of zinc per 100g of sample
respectively.
Chocolate
Control chocolate had 9.02g of protein, 30.20g of fat, 78.43g of carbohydrate,
622Kcal of energy, 0.86g of crude fibre, 2.83mg of calcium, 110mg of phosphorous,
1.41mg of iron and 0.96mg of zinc per 100g of sample respectively. Whereas stabilized
rice bran chocolate had 9.86g, 28.51g, 75.81g, 600Kcal, 3.10g, 7.75mg, 119mg, 4.07mg
and 0.6mg of protein, fat, carbohydrate, energy, crude fibre, calcium, phosphorous, iron
and zinc per 100g of the sample. Probiotic treated chocolate estimated to have 10.03g of
protein, 28.90g of fat, 75.44g of carbohydrate, 603Kcal of energy, 2.26g of crude fibre,
7.53mg of calcium, 119mg of phosphorous, 3.73mg of iron and 0.6mg of zinc per 100g
of sample respectively.
Chapati
Nutrient composition per 100 g for the control, SRB 20 per cent and PRB 20 per
cent chapati were computed and are presented in Table 20. The nutrient content of
control chapati for protein, fat, carbohydrate, energy, crude fibre, calcium, phosphorous,
iron and zinc were 11.74g, 4.56g, 67.37g, 357Kcal, 1.84g, 46.6mg, 344.70mg, 4.75mg
and 2.31mg respectively. The stabilized and probiotic biscuit contained 11.94 and 12.25g
of protein, 6.15 and 6.95g of fat, 67.56 and 67.00g of carbohydrate, 374 and 380 Kcal
energy, 4.75 and 3.12g of crude fibre, 47.37 and 47.00mg of calcium, 516.50 and
505.82mg of phosphorous, 7.55 and 6.88mg of iron, 2.91 and 2.91mg of zinc respectively
(Table 20).

46
Table 20: Nutrient composition of the developed products

Biscuit Bread Muffin Chocolate Chapati
Nutrients SRB PRB SRB PRB SRB PRB SRB PRB SRB PRB
Control Control Control Control Control
(10%) (10%) (5%) (10%) (10%) (15%) (10%) (10%) (20%) (20%)
Protein (g) 5.26 5.42 5.45 7.60 7.62 7.66 7.73 7.76 7.80 9.02 9.86 10.03 11.74 11.94 12.25
Fat (g) 29.04 29.28 30.28 10.96 11.03 11.10 14.18 14.23 14.32 30.20 28.51 28.9 4.56 6.15 6.95
Carbohydrate
58.85 58.33 58.26 71.51 71.44 71.37 48.37 48.27 48.18 78.43 75.81 75.44 67.37 67.56 67.00
(g)
Energy
517 519 520 415 416 416 352 353 353 622 600 603 357 374 380
(Kcal)
Crude fibre
0.41 1.0 0.50 0.20 0.30 0.32 0.08 0.20 0.18 0.86 3.10 2.26 1.84 4.75 3.12
(g)
Calcium
13.80 14.50 14.45 18.34 18.48 18.62 30.69 30.83 30.88 2.83 7.75 7.53 46.6 47.37 47.00
(mg)
Phosphorus
57.85 83.33 83.33 83.65 87.17 91.03 110.80 115.80 118.23 110 119 119 344.70 516.50 505.82
(mg)
Iron (mg) 1.32 2.00 1.85 1.89 2.00 2.04 1.53 1.64 1.68 1.41 4.07 3.73 4.75 7.55 6.88
Zinc (mg) 0.28 0.40 0.40 0.41 0.43 0.45 0.16 0.19 0.20 0.4 0.6 0.6 2.31 2.91 2.91
Shweta Bhosale
4.7 Microbial study of the food products during shelf life study
The best accepted products from sensory evaluation i.e. biscuit (SRB 10%, PRB
10%), bread (SRB 5%, PRB 10%), muffin (SRB 10%, PRB 15%) and chocolate (SRB
10%, PRB 10%) were stored in polyethylene covers and microbial load was evaluated.
For biscuits the microbial count was observed initially, 15th and 30th day. In bread and
muffin the count was observed for initial, 4th and 6th day. Chocolate microbial load was
evaluated initially, 30th and 60th day (Table 21).
There was no coliform count seen in any of the products. The total bacterial count
of control biscuit was 2 X 103 on 30th day, bread had 4 X 103 during 6th day, muffin 3 X
103 (6th day) where as chocolate had 1 X103, 3 X 103 and 6 X 103 during initial, 30th and
60th day. Total bacterial count of stabilized biscuit was 1 X 103 (30th day), bread 3 X 103
(6th day), muffin 2 X 103 (6th day) and in chocolate it was 14 X 103, 19 X 103, 26 X 103
during initial, 30th and 60th day. The total bacterial count of probiotic treated biscuit was 2
X 103 during 30th day, bread 3 X 103 CFU (6th day), in muffin the TBC counts were 2 X
103 during 6th day and in chocolate it was 11 X 103, 22 X 103, 28 X 103 during initial, 30
and 60th day of storage period. The fungi count of control biscuit was 2 X 102 CFU on
30th day, 2 X 102 CFU on 6th day in control bread, 2 X 102 CFU on 6th day in control
muffin and 2 X 102, 4 X 102 and 7 X 102 CFU during initial, 30th and 60th day in control
chocolate. Fungi count in stabilized biscuit on 30th day was 1 X 102 CFU, in bread it was
1 X 102 CFU, muffin 1 X 102 CFU and in chocolate 1 X 102, 3 X 102 and 5 X 102 during
initial, 30 and 60th day. In probiotic treated biscuit fungi count was 2 X 102 CFU, in bread
it was 1 X 102 CFU and in muffin it was 2 X 102. Chocolate had the fungi count of 1 X
102 CFU, 2 X 102 CFU and 5 X 102 CFU during initial, 30 and 60th day of storage period.
4.8 Glycemic Index (GI) test of best accepted product

Glycemic index: The glycemic index is defined as the incremental area under the blood
glucose response curve of a 50g carbohydrate portion of test food expressed as a per cent
of the response to the same amount of carbohydrate from a standard food taken by the
same subject (FAO/WHO, 2003).
In the present study white bread was used as standard, wheat flour chapati used as
control and rice bran incorporated chapati ( 20%) was used as test food. The rice bran
was incorporated in wheat flour to make chapati. The blood glucose levels and area under
curve for blood glucose of the above three products are given in Table 22 and 23.
The glycemic index and calculated area under curve for blood response after
ingestion of standard and test foods on separate days are given in Fig. (15). Significant
difference were found for the mean blood glucose levels between time intervals for 30
and 60 mins, and there was non significant difference between fasting, 90 and 120 min
for standard, control and test food. The glycemic index of control chapati was 78.50,
stabilized rice bran incorporated chapati was 68.00 and probiotic treated chapati 64.13
compared to standard white bread i.e. 100. The glycemic load for white bread was 50,
control chapati: 39.25, stabilized chapati: 34.00 and for probiotic treated chapati it was
32.06 respectively.

48
Table 21: Microbial load of food products during shelf life study
Control Stabilization process Probiotic treatment

Storage
Total Total Total
Products period
bacterial E.Coli Fungi bacterial E.Coli Fungi bacterial E.Coli Fungi
(Days)
count (X102 CFU) (X102 CFU) count (X102 CFU) (X102 CFU) count (X102 CFU) (X102 CFU)
(X103 CFU) (X103 CFU) (X103 CFU)
Initial-15 Nil Nil Nil Nil Nil Nil Nil Nil Nil
Biscuit
30 2.00 Nil 2.00 1.00 Nil 1.00 2.00 Nil 2.00
Bread
6 4.00 Nil 2.00 3.00 Nil 1.00 3.00 Nil 1.00
Muffin
6 3.00 Nil 2.00 2.00 Nil 1.00 2.00 Nil 2.00
Initial 1.00 Nil 2.00 14.00 Nil 1.00 11.00 Nil 1.00
Chocolate 30 3.00 Nil 4.00 19.00 Nil 3.00 22.00 Nil 2.00
60 6.00 Nil 7.00 26.00 Nil 5.00 28.00 Nil 5.00

Shweta Bhosale
Table 22: Mean values for blood glucose level (mg/dl) for standard, control
and test foods
Food products Fasting 30 min 60 min 90 min 120 min
White bread 83.1 105.4 116.7 106.3 100.3
Control chapati 86.3 123.6 100.2 99.6 95.0
SRB (20%) chapati 81.5 105.9 101.9 94.0 86.0
PRB (20%) chapati 78.6 107.2 95.3 88.2 81.3
F- value NS * * NS NS
SEm± 2.93 3.81 4.32 4.62 3.98
CD 9.04 11.73 13.32 14.25 12.25
Table 23: Mean IAUC for glycemic index (GI) of standard, control and test
foods
Incremental area under the curve (IAUC)

Food products GI GL
AUC-1 AUC-2 AUC-3 AUC-4 IAUC
White bread 334.5 838.5 852.0 606.0 2631.0 100.00 50.00
Control chapati 559.5 768.0 408.0 330.0 2065.5 78.50 39.25
SRB (20%) chapati 366.0 672.0 493.5 255.0 1786.5 68.00 34.00
PRB (20%) chapati 429.0 679.5 394.5 184.5 1687.5 64.13 32.06
0-55 : Low glycemic index, 56-70 : Medium glycemic index, >70 : high glycemic index
(FAO/WHO, 2003).

140
Standard (white bread) Control (Wheat flour chapati)
SRB (20%) PRB (20%)
120
Blood glucose level (mg/dl)
100
80
60
40
20
0
0 30 60 90 120
Time (mins)
Fig. 15: Mean area under blood glucose curve for standard, control and test
foods (SRB & PRB)
V. DISCUSSION
Rice bran is the cuticle existing between the rice and the husk of the paddy and
consists of embryo and endosperm of the seeds of Oryza sativa, family Graminae. It
constitutes 8 per cent of the weight of the whole grain and contains most of the nutrients
(65 per cent). During milling process rice bran containing nutrients is completely
removed. Rice bran, a "little known" food is highly nutritious and delivers a powerhouse
of health supporting nutrients which is either thrown away or used for low-level animal
feed (Qureshi et al., 2000). The nutritional composition of rice bran has led the discovery
of the varied health benefits. Also rice bran is used for the enrichment of some foods, due
to its high dietary fibre content.
The results are presented under the following subheadings:

5.2 Processing of rice bran by different methods
5.3 Functional properties of different rice bran samples
5.5 Product development, sensory evaluation and shelf life study of products
5.8 Glycemic index test of product developed from rice bran
load
It was observed that the heavy metals were below detection limit and pesticides
were not detected in the rice bran sample and also found that there was no microbial load
in the sample (Table 1 and 2).
5.2 Processing of rice bran by different methods

Rice bran is a component of raw rice that is obtained when it is removed from the
starchy endosperm in the rice milling process. It has been used as a feedstock and has the
potential to be used as a food ingredient, since it has good amounts of nutrients.
However, bran must be stabilized immediately upon production due to the presence of
lipase, an enzyme that rapidly hydrolyzes oil to free fatty acid (FFA) and glycerol, which
results in a drastic quality reduction of the rice bran. The primary means for rice bran
stabilization includes deactivating the enzyme through heat treatment such as microwave
heating.

Rice bran is a very rich source of nutrients containing protein, fat, carbohydrates,
dietary fibre, vitamins, minerals, oils, trace elements, antioxidants, phytosterols and
phytochemicals. The abundant dietary fibre of rice bran is now being explored as an
important ingredient of health foods. The main drawback of rice bran is a fast oxidation
reaction due to the high content of unsaturated fatty acid in its oil content. This is
primarily due to the presence of endogenous enzyme lipase which caused the pro-
oxidative mechanisms of oxidation leading to hydrolytic rancidity on the oil content that
hydrolyze the ester bonds of triacyglycerol, releasing fatty acids and glycerol and
forming of hyperoxides (Yin and Wen, 2011). Within one hour of separating the bran
from the grain during milling the material turns rancid liberating toxic free fatty acids.
These shortcomings have now been overcome by destroying the lipolytic activity using
an advanced stabilizing technology, the resulting material thus obtained is called
"stabilized" rice bran which has a good taste, readily soluble with a longer shelf life of
one year.
Tao et al., (1993) reported that microwave heating is an effective method for the
inactivation of lipase that is responsible for rice bran degradation and instability. Rice
bran is stabilized by microwave heating at 2450MHz for 3 minutes was found to be stable
for up to four weeks in storage. The results showed that free fatty acid content of
microwave stabilized bran increased from 4.0 to 4.9 per cent in long grain rice bran and
4.6 to 6.25 per cent in medium grain rice bran from initial to 4 weeks of storage period,
which were stored in polyethylene zipper top bags at 40C. In contrast, there was increase
in the untreated bran, the free fatty acid ranged from 4.0 to 68.3 per cent and 4.6 to 56.8
per cent in long and medium grain rice bran.
In the present study the free fatty acid content for microwave stabilized rice bran
was found to be 4.10, 4.98, 5.20, 6.80 and 7.50 per cent during initial, 1st, 2nd, 3rd and 4th
week of storage period. In probiotic treated rice bran free fatty acid value ranged from
4.35 to 7.95 per cent from initial to fourth week of storage period. The values were found
within standards i.e. less than 10 per cent, as reported by Tao et al. (1993). Whereas in
case of control or untreated rice bran sample the percentage of free fatty acid increased to
3 folds in the second week i.e. from 4.36 to 12.98 per cent which is more than the
standards.
Raw and microwave heated rice bran were packed in food grade polythene bags,
stored at 4-50C for 16 weeks. Free fatty acid content of rice bran was measured at 4
weeks interval. The total free fatty acid increased rapidly over the 16 week period from
the initial value of 2.5 to 25.4 per cent in raw rice bran. Total free fatty acid of
microwave heated rice bran increased from 2.8 to 6.9 per cent respectively
(Ramezanzadeh et al., 1999).
Increased levels of free fatty acid percentage i.e. greater than 10 per cent in the
rice bran sample was unfit for human consumption. Enochain et al. (1981) reported that
the FFA percentage below 10 per cent is acceptable for human consumption.
51 Shweta Bhosale
Hence from the Table 3, it is evident that the microwave stabilization for 3
minutes is best suitable method for rice bran. The heat used in microwave has
significantly inactivated the enzymatic activity in rice bran by denaturation of lipase
enzyme, and further slow down the oxidation process and lowers the free fatty acid value
and hydroperoxide produced. Microwave heating did not adversely affect other
components like proximate composition or colour.

Probiotics are one of the fastest growing sectors within functional foods. Probiotic
foods are fermented products containing sufficient number of a certain live
microorganisms that favourably modifies the intestinal microbiota of the host. Stabilized
rice bran is a unique whole food that naturally contains protein, vitamins, minerals,
complex carbohydrates, phyto-nutrients, phospholipids, essential fatty acids and more
than 120 antioxidants. Rice bran contains the main nutrients for microbial growth.
Therefore, it may serve well as a substrate for microbial fermentation to add value to rice
bran. Rice bran has a relatively high amount of α-linolenic acid and linoleic acid which
could be advantageous to the microbial production. Probiotic food products are regarded
as an important group of 'functional foods'.
Mortazavian et al. (2011) revealed that the minimum viable probiotic cells per
gram or milliliter of probiotic product generally, 106 and 107 to 108 cfu mL-1 (cfu g-1)
respectively, have been accepted as the minimum and satisfactory levels. The optimum
temperature for growth of most probiotics is between 370C and 430C. Temperature is also
a critical factor influencing probiotic survival during storage period. Probiotic food
products usually, should be stored at a refrigerated temperature, preferably 4-50C. The
storage temperature of probiotic food products affect the viability of the probiotics via
effect of temperature on the cells survival.
In the present study microwave stabilized rice bran was inoculated with Lactic
Acid Bacillus culture and kept for fermentation at 300C for about 24 hours. The
fermented sample was lyophilized (freeze drying) at -33.80C for 34 hours and was packed
in air tight zip-bag then stored in the refrigerator at 40C. The viability count of the
probiotic treated rice bran sample was found to be 7.26X107. Freeze drying is the best
process for maintaining the viability of the bacterial cells used for preparing starter
culture cells. Probiotic treatment is carried out for the rice bran to enhance properties
such as taste, aroma, shelf-life, texture and nutritional value.
5.3 Functional properties of different rice bran samples

Functional properties for both stabilized and probiotic treated rice bran were
determined, which includes bulk density, water absorption, fat absorption, water
solubility and swelling power. The functional properties are broadly defined as those
properties other than nutritional attributes that affects its utilization. The overall
functional properties of a food system are a result of composite properties of individual
protein components as they interact with one another and with non-protein component
also. The study on functional property is important as it is necessary to know the different

functions and behaviour of the bran while formulating different products. In the present
study stabilized and probiotic treated rice bran were used for functional property analysis
and the results are expressed in Table 4.
Bulk density:
Each type or variety of grain has a characteristic bulk density. This is defined as
the weight per standard volume, measured in a standard manner. In the present study the
bulk density of stabilized rice bran was found to be 0.22g/ml and for probiotic treated rice
bran it was high when compared to stabilized rice bran i.e. 0.38g/ml. The results are on
par with the reported values of Chandi and Sogi (2006). The differences between
stabilized and probiotic treated rice bran were found significant. The two rice bran
samples were found to be suitable for product development.
Water and oil absorption capacity:

Water absorption capacity is kinetics of water movement under controlled
condition. The amount of water retained by the solids was measured. The oil absorption
capacity was measured by fat retained by the solids. Dietary fibre present in the bran is
known to bind water. Water absorption capacity of stabilized and probiotic treated rice
bran were 2 and 3 ml/g. The hydrophyllic nature of crude fibre might have contributed to
the increased water absorption in the probiotic treated rice bran samples. The variation in
water absorption may be attributed to the source of bran and their processing conditions.
Probiotic treated rice bran have higher water absorption capacity than stabilized rice bran.
Oil absorption is the ability of flours to retain oil and it is important in food applications
because if oil absorption capacity of bran is high it makes the flours suitable in
facilitating enhancement in flavour and mouth feel when used in food preparations. Oil
absorption capacity was found high in probiotic treated rice bran i.e. 2.5 ml/g when
compared to the stabilized rice bran i.e. 1.5 ml/g respectively. Stabilized rice bran
absorbed less oil as compared to probiotic treated bran. It must be noted that there were
no significant differences in water and oil absorption among stabilized and probiotic
treated rice bran samples. The results are in harmony with the results obtained by Sairam
et al. (2011) the reported values found between 2.0 to 2.60 ml/g for water absorption and
2.20 to 2.70 ml/ g for oil absorption capacity.
Water solubility:
Solubility per cent measures the amount of product sediment after the application
of low centrifugal process under specified condition (Iyer and Singh, 1997). This quality
has implications in product development to improve the quality traits. In the present study
solubility index was found to be 7.3 and 8.0 per cent for stabilized and probiotic treated
rice bran samples. No significant difference was found between these two rice bran
samples. The results were within range as reported by Sharma et al. (2004) i.e. 9.34 per
cent.
53 Shweta Bhosale
Swelling Power:
Swelling power is the volume occupied by a known weight of the sample under
controlled conditions. The sample is hydrated with water for a particular time with no
external stress except gravity (Ocheme and Chinma, 2008). Softness of cooked product is
an important variable. Retention of water in the swollen starch granules plays an
important role. Swelling power of stabilized rice was 6.7 per cent and that of probiotic
treated rice bran was found to be 7.2 per cent respectively. Abdul-Hamid (2000) reported
that swelling power of dietary fibre from rice bran ranged between 3.46 to 14.43 per cent
respectively.

The nutrient composition of the stabilized and probiotic treated rice bran were
analyzed and the results are presented in Table 5.
The macronutrients content in the stabilized rice bran such as moisture, protein,
fat, crude fibre, insoluble dietary fibre, soluble dietary fibre, total dietary fibre,
carbohydrate, energy and ash were 4.30 per cent, 17.50g, 13.10g, 7.85g, 21.17g, 2.17g,
23.34g, 52.33g, 398Kcal and 4.92g respectively. Micronutrient content in the stabilized
rice bran were: calcium- 52.10mg, phosphorous- 1185.2mg, iron- 28.10mg and zinc-
6.02mg. Rao (1998) reported similar results i.e. the macronutrient composition of 100g of
stabilized rice bran for protein, fat, crude fibre, soluble dietary fibre, total dietary fibre,
carbohydrate, energy and ash were 16.5g, 21.30g, 11.4g, 2.1g, 25.3g, 49.4g, 359Kcal and
8.3g respectively. The micronutrient composition of the stabilized rice bran were similar
to the results reported by Rabbani and Ali (2009) which were 40mg, 1591mg, 25mg and
5.50mg/100g for calcium, phosphorous, iron and zinc respectively.
The macro and micronutrient contents in the probiotic treated rice bran were
moisture-5.40 per cent, protein-19.25g, fat-17.20, crude fibre-4.96, insoluble dietary
fibre-13.10, soluble dietary fibre-1.80g, total dietary fibre-14.90g, carbohydrate-48.55g,
energy-426Kcal and ash-4.64g, calcium- 49.90mg, phosphorous-1186.5mg, iron-
30.05mg and zinc-5.89.mg respectively. The content of carbohydrate, fibre, ash and
calcium were decreased in the probiotic treated rice bran compared to the stabilized rice
bran because these compounds are the principal energy source of fermenting
microorganisms, so that the level of these compounds decreased during the microbial
fermentation. Certain amino acids may be synthesized during the fermentation process.
The protein, fat, phosphorous and iron contents were increased in the probiotic treated
rice bran as the availability of these nutrients will increase by the probiotic treatment
process (Robert Nout, 2010).
The antioxidant activity of stabilized rice bran was 65 Vit-C Eq. µg/g and for
probiotic treated rice bran it was 70 Vit-C Eq. µg/g, the results are similar to the study
conducted by Rao et al., 2010. The antinutritional factors such as phytic acid (23.50 and
22.15 mg/g) and trypsin inhibitor (10.8 and 10.2mg/g) respectively, were found in
stabilized and probiotic treated rice bran. The results found were within the safe limit and
are in correlation with the study conducted by Kaur et al. (2011).

5.5 Product development, sensory evaluation and shelf life study of products
Stone and Sidel (1993) defined sensory evaluation as "A scientific discipline used
to evoke, measure, analyze and interpret those responses to products that are perceived by
the senses of sight, smell, touch, taste and hearing". Sensory analysis aims to determine
the probable product acceptance by consumers in the early development stages. The
sensory characteristics of biscuit, bread, muffin, chocolate and chapati were evaluated for
acceptance test with 9- point hedonic scale.
5.5.1 Biscuit
Biscuits are most widely consumed bakery product in many parts of the world.
Healthy baking is an vogue these days. Biscuits are widely consumed, relatively long
shelf life, ready-to-eat form and have excellent eating quality (Tsen et al., 1973).
Therefore, biscuit is selected for the present study for incorporation of rice bran.
5.5.1.1 Physical properties of biscuit

The effect of replacing 5, 10 and 15 per cent refined wheat flour with stabilized
and probiotic treated rice bran on physical properties of biscuit were studied and the data
presented in Table 6. The results explicated that control biscuits exhibited maximum
width (64.40mm), followed by PRB 10 per cent (62.15mm) and 10 per cent (60.60mm)
while minimum width was measured in SRB 15 per cent biscuit (58.00mm). The results
elucidated that SRB 15 per cent biscuit exhibited maximum thickness 27.90mm, followed
by 10 per cent (26.50mm), 5 per cent (25.15mm) and control (24.20mm) level of
incorporation of rice bran. The spread ratio of biscuits, prepared from different treatments
ranged from 20.82 to 26.61. The maximum value (26.61) for spread ratio was observed in
control biscuit where as minimum (20.82) in biscuits prepared from SRB 15 per cent rice
bran. Sharma and Chauhan (2002) also reported that physical properties of biscuits like
width, thickness and spread ratio were affected significantly with the increase in the level
of bran and also by method of stabilization.
5.5.1.2 Sensory evaluation of biscuit

Mean sensory scores of biscuits are presented in Table 7 reveals that biscuits were
acceptable till 15 per cent incorporation of stabilized and priobiotic treated rice bran but
the best accepted was 10 per cent level SRB(8.8) PRB (8.7) which is next to the control
biscuits. As the rice bran incorporation level increased from 5 to 15 per cent the sensory
scores decreased due to hard texture and dark in colour. These values are similar to the
values reported by Sharif et al. (2009) and Younas et al. (2011).
5.5.1.3 Mean sensory scores of biscuit on storage

Shelf life is a major consideration in developing, producing and marketing of food
product, it refers to the time during which a product remains 'acceptable' to a consumer in
terms of sensory characters. Many factors influence the shelf life of a product like,
moisture loss, spoilage due to microorganisms, enzymatic changes and oxidation
(Adegoke et al. 1998).
55 Shweta Bhosale
Mean sensory scores of shelf life study of SRB and PRB depicted in Table 8.
Maximum scores (9.0) were assigned to fresh biscuits, which were gradually decreased
(7.5) after 15 days storage. The SRB scores decreased from 8.8 to 7.4 and PRB scores
decreased from 8.8 to 7.2. The decrease in biscuit scores was due to the rancidity of fats
during storage. Similar results were found by Sharif et al. (2009). The declining trend in
quality scores for texture was due to absorption of moisture from the atmosphere that has
inverse correlation with texture (Sharif et al., 2005).
5.5.1.4 Peroxide and free fatty acid value of biscuit

Peroxide values measures the content of hydro peroxides and are often used as an
indicator of primary products of lipid oxidation. Changes occurring in the peroxide and
free fatty acid values of biscuits during storage are presented in Table 9. The increase in
peroxide value was observed in all the three biscuit samples, however control biscuit had
higher value of 2.6 to 9.8 milli equiv. of peroxide per kg of sample, after 15 days and
peroxide value of SRB 10 per cent level biscuit ranged from 2.0 to 4.4 milli eqvi. and
also the PRB 10 per cent which ranged from 2.1 to 4.0 milli eqvi. of peroxide per kg of
sample from initial to 15 days of storage the values obtained are within the acceptable
range and significantly lower in SRB and PRB compared to the control biscuit. The
values are in comparison with the study conducted by Reddy et al. (2005).
The free fatty acid content was maximum in case of control biscuit (0.8 2.9 per
cent of oleic acid/ 100g of sample) from initial to 15 days of storage period. Free fatty
acid content in SRB and PRB were significantly lower compared to control biscuit, which
ranged from 0.6 to 1.0 per cent oleic acid /100g in SRB and 0.5 to 0.9 per cent oleic acid
/100g in PRB samples. These values are comparable to study conducted by Nagi et al.
(2012). The increase in free fatty acid content was due to greater increase in their
moisture content which promoted fat hydrolysis during storage.
5.5.2 Bread
Bread is a staple food, it has been popular around the world and is one of
humanities oldest food. To enrich the nutrient content of the bread rice bran is being used
here. In this study bread was prepared in three different variations (5, 10 and 15 per cent)
of stabilized and probiotic treated rice bran. A traditional recipe without rice bran served
as control.
5.5.2.1 Functional properties of bread

The effect of replacing 5, 10 and 15 per cent refined wheat flour with stabilized
and probiotic treated rice bran on physical properties of bread were studied and the data
presented in Table 10. The results revealed that water absorption capacity of bread
increased as the rice bran incorporation level increased i.e. it was maximum in the PRB
15 per cent bread (76.26 %) and minimum was found in control bread (62 %). The dough
development time and weight of bread also increased as the level of incorporation
increased in both stabilized and probiotic treated bread. Bread dough volume and specific
weight of the bread in response to fermentation and proofing decreased significantly with
increasing proportion of rice bran. The loaf volume decreased from 1535 (cm3) to 1420

(cm3) and specific weight decreased from 7.04 to 5.79 (cm3/gm) respectively. The results
were on par with the results reviewed by Bagheri and Seyedein (2011).
5.5.2.2 Sensory evaluation of bread

Ameh et al. (2013) studied the acceptability of stabilized rice bran in bread
preparation and found to be highly acceptable. In the present study control bread showed
highest scores for all sensory characters. The results are depicted in Table 11. In the
stabilized rice bran incorporated bread at 5 per cent level (8.9) attained high scores
compared to other treatments. The probiotic treated bread at 10 per cent level (8.9) was
best accepted because of the probiotic treatment the texture of the product improved and
intern it improved the quality of the bread. It can be concluded from the results that up to
10 per cent processed rice bran can be successfully incorporated in the bread to improve
the sensory and nutritional attributes.
5.5.2.3 Mean sensory scores of bread on storage

The increasing level of bran particles in formulations had significant effect on
both internal as well as external attributes of bread consequently lowering its overall
acceptability. The mean sensory scores of bread on storage are presented in Table 12. The
sensory scores decreased significantly from initial to 4th day of storage period (9.0 to 7.4).
SRB bread decreased from initial (8.9) to 4th day (7.4) of storage period. PRB bread
decreased from 8.9 to 7.0 from initial to fourth day of storage period respectively. Similar
results were found by Ajmal et al. (2006) who reported that evaporation and gas loss
from bread surface has inverse correlation with symmetry with bread showing declining
trend towards acceptability.
5.5.3 Muffin
The term muffin typically refers to an individual sized quick bread product which
can be sweet or savoury.
5.5.3.1 Physical properties of muffin

The physical properties of muffin are presented in Table 13. The water absorption
capacity, dough development time and volume were increased as the rice bran level
increased from 5 to 15 per cent. The results are in comparison with the study conducted
by Salehi and Bibalan (2012). This increased parameters are due to the high fibre content
of rice bran. Fibre is characterized by its high water holding capacity as reported by
Holloway and Grieg (1984).
5.5.3.2 Sensory evaluation of muffin

Salehi and Bibalan (2012) developed muffin cake by incorporating rice bran and
products were found to be acceptable. In the present study, the mean sensory scores for
all characteristics and at all the levels studied did not differ from control (Table 14). The
control (8.6) muffin was best accepted and followed by SRB 10 per cent (8.5) and PRB
15 per cent (8.5) respectively. Hence rice bran can be incorporated to improve nutritional
and sensory characteristics of the muffins.
57 Shweta Bhosale
5.5.3.3 Mean sensory scores of muffin on storage
The mean sensory scores of muffin are depicted in Table 15. The sensory scores
of all the samples decreased as the storage period increased. In control muffin the sensory
scores decreased from initial (8.6) to 4th day of storage (7.9), SRB 10 per cent
incorporated muffin decreased significantly from 8.5 to 7.5 from initial to 4th day and in
the same way PRB 15 per cent incorporated muffin also decreased from initial to the end
of storage period.
5.5.3.4 Peroxide and free fatty acid value of muffin

The peroxide value of muffin are depicted in Table 16. Increase in peroxide value
and free fatty acid value observed in all the muffin samples from initial to 6 days of
storage period. In the control muffin the peroxide values increased greatly from initial
(4.20) to 6th day (11.25). In SRB muffin it increased from initial 3.10 to end period i.e.
5.30. The peroxide value increased from 3.00 to 5.10 in the PRB muffin.
The free fatty acid content increased significantly from initial to 6th day of storage
period. In the control sample it ranged between 1.0 to 2.9, SRB- 0.7 to 1.2 and in PRB
from 0.6 to 1.0 respectively. The peroxide and free fatty acid value increased gradually
because of the presence of high fat content in the product (Abdel-Razik et al., 2012).
5.5.4 Chocolate
Chocolate, a complex emulsion, is a luxury food that during consumption evokes
a range of stimuli that activate pleasure centres of the human brain. Chocolate acts as a
commercial and novel product which helps in improving the health benefits. Use of rice
bran increases the fibre content in chocolate. Stabilized and probiotic treated rice bran
were used in 5, 10 and 15 per cent incorporation level. Chocolate is selected for the
product development because it can be stored up to longer period compared to other
products.
5.5.4.1 Sensory evaluation of developed chocolate

The results of mean sensory scores of chocolate are presented in Table 17. In the
present study the control (without incorporation of rice bran) had showed highest sensory
scores for overall acceptability. However, it must be noted that chocolate is consumed for
its unique texture and its good taste. It is noticed that sensory scores for texture and taste
of chocolate with rice bran incorporation remain on par with the control. Both the
stabilized and probiotic treated rice bran were best accepted at 10 per cent incorporation
level. Thus, stabilized and probiotic treated rice bran can be easily incorporated into this
product (Chetana et al., 2013).
5.5.4.2 Mean sensory scores of chocolate on storage

In chocolate between treatment and storage period for all sensory attributes there
was a significant difference (Table 18). The sensory characters of all the products
decreased as the storage period increased from initial to 60 days of storage period. In
control chocolate the sensory scores decreased from 8.8 (initial) to 7.2 (60th day), SRB

10 per cent the scores decreased from 8.6 to 7.3 from initial to 60 days and in PRB 10 per
cent the scores decreased significantly from 8.2 (initial) to 7.4 (60 days) respectively. The
results are on par with the study revealed by Huchchannanavar (2013). Hence, we can
say that the rice bran incorporated chocolate can be stored up to 60 days in the
refrigerated condition.
5.5.5 Chapati
Chapati is one of the most common forms in which wheat is used, is a staple food
of South Asia. Stabilized rice bran had low moisture and low free fatty acid content and
hence considered ideal for product development. In the present study wheat flour is
substituted with stabilized and probiotic treated rice bran at 15, 20 and 25 per cent level.
5.5.5.1 Sensory evaluation of developed chapati

In the present study sensory evaluation of developed chapati is reported in Table
19. The viability count for probiotic treated chapati was 16X107 which helped in
improving the acceptability of chapati. Control chapati scored highest acceptability i.e.
8.8, followed by SRB (8.4) and PRB at 20 per cent (8.6) rice bran respectively. Among
the three incorporation levels (15, 20 and 25 per cent) the 20 per cent incorporated
stabilized and probiotic treated rice bran chapati were best accepted. Hence, the resulted
values were on par with the Yadav et al. (2012) who developed chapati by incorporating
stabilized rice bran and the products were found to be acceptable up to 20 per cent.

The products like biscuit, bread, muffin, chocolate and chapati were developed for
which the best accepted level of incorporation values for nutrients were calculated and
presented in Table 20. It is important to know the nutrient composition of the products for
the conformity of their richness in terms of nutrition and to incorporate them in our daily
diet and value addition in particular product preparation.
The highest protein, fat, energy, phosphorus and zinc were in 10 per cent
probiotic treated biscuit (5.45g, 30.28g, 520 Kcal, 83.33mg and 0.40mg) respectively and
the highest crude fibre, calcium and iron were in 10 per cent stabilized biscuit (1.0g,
14.50mg and 2.0mg) respectively. The nutritive value of stabilized and probiotic treated
biscuit were higher compared to the control biscuit. Thus the incorporation of the rice
bran resulted in a substantial improvement in the nutritive value of biscuit.
All the nutrient components were found highest in probiotic treated bread (7.66g
protein, 11.10g fat, energy 416Kcal, crude fibre 0.32g, calcium 18.62, phosphorus
91.03mg) because the acceptance level was 10 per cent in this and that of stabilized bread
it was 5 per cent level. The nutritive value of stabilized and probiotic treated biscuit were
higher compared to the control bread.
The nutrient composition of muffin were highest in probiotic treated followed by

stabilized and control. Nutrient composition in PRB 15 per cent were protein (7.80g), fat
(14.32g), energy (353Kcal), calcium (30.88mg), phosphorus (118.23mg), iron (1.68mg)
59 Shweta Bhosale
and zinc (0.20mg) respectively. Thus rice bran can be used as value added ingredient in
the preparation of muffin.
Probiotic chocolate had highest nutrient composition i.e. protein, fat, energy,
phosphorus and zinc (10.03g, 28.90g, 603KCal, 119mg and 0.6mg). Whereas, crude
fibre, calcium and iron contents (3.10g, 7.75mg and 4.07mg) were highest in SRB
chocolate. Hence the incorporation of processed rice bran resulted in a substantial
improvement in the nutritive value of chocolate.
Rice bran in the chapati was best accepted up to 20 per cent incorporation level.
The nutrient contents such as protein (12.25g), fat (6.95g), energy (380KCal) and zinc
(2.91mg) were found to be high in PRB chapati and the other nutrients such as crude
fibre (4.75g), phosphorus (516.50mg) and iron (7.55mg) respectively. Both SRB and
PRB chapati attained high nutritive values compared to control chapati.

In the present study the colony forming units (CFU) in the developed products i.e.
biscuit, bread, muffin and chocolate were stored in 350 gauze polythene pouch at ambient
temperature were assessed. The total bacterial count, coliform and fungi were analyzed
and are presented in Table 21. There was no coliform observed in any of the products
throughout the study period, the reason may be due to the high temperature applied to the
products through microwave processing. The total bacterial and fungi count were
observed in the products at the end of storage period i.e. in biscuit at 30th day, bread and
muffin on 6th day. In case of chocolate there was presence of total bacteria and fungi from
initial to 60th day of storage period this may be due to the storage condition and the heat
applied while preparation of chocolate was less compared to the other food products. The
total bacterial and fungi count was more in control sample than that of the stabilized and
probiotic treated samples. The increase in microbial load as the storage period lengthened
might have been due to a corresponding increase in moisture content during storage
period but the microbial load for total bacterial count and fungi were within the
permissible limits (Nagi et al., 2012).
5.8 Glycemic index of product developed from rice bran

Chapati was the best accepted product (20 per cent SRB and PRB) by the sensory
panelists, hence this was selected to test Glycemic Index on ten healthy volunteers.
Glycemic index (GI) can be used in conjunction with information about food
composition to guide food choices. The glycemic response for different foods are
markedly different in diabetic and normal subjects (Anon., 1998; Itagi et al., 2012).
Several factors influence glycemic responses. There are choices of standard food,
methodology of testing, subject characteristics, amount of carbohydrate, nature of the
monosaccharide components, nature of starch, cooking or food processing and other food
components like fat and protein, dietary fibre, anti nutrients and organic acids. Meals
containing low GI foods reduce both postprandial blood glucose and insulin responses.
Clinical trials in normal, diabetic and hyperlipidemic subjects show that low GI diet

reduces mean blood glucose concentrations, reduce insulin secretion and reduce serum
triglycerides in individuals with hyperglyceridemia (Cherbut et al., 1995, Cummings et
al., 1993 and Salvador et al., 1992). In addition the digestibility of the carbohydrate in
low GI foods is generally less than that of high GI foods. Thus, low GI foods increase the
amount of carbohydrate entering the colon and increase colonic fermentation and short
chain fatty acid production. In the present study the GI was measured in capillary whole
blood. The differences between foods are larger and easier to detect statistically using
capillary blood glucose (Gibson et al, 1995). Therefore in the present study WHO
recommended protocol was used to get standard results.
In the present study GI response to the SRB chapati and PRB chapati were
compared with the standard test food i.e. white bread and with control chapati. The
results found that GI for white bread was 100, control chapati: 78.50, stabilized rice bran
chapati: 68 and for probiotic treated chapati it was 64 (Table 22 and 23 and Fig. 15). The
glycemic load of wheat bread found to be 50, control chapati as 39.25, stabilized chapati
34 and for probioticated chapati it was 32.06 respectively. Therefore, it is concluded that
stabilized and probiotic treated rice bran chapati acts as a medium gycemic index food,
which helps in regulating cholesterol and blood glucose level. Rice bran being higher in
fibre could be a contributing factor in lowering the GI. These results are similar to the
study conducted by Premakumari et al. (2013). Hence, rice bran plays an important role
in decreasing cholesterol and controlling of blood glucose (Singh et al., 2013).
Stabilized rice bran is a powerful source of vitamins, nutrients, proteins and fibre.
The soluble and insoluble fibres are necessary for optimum digestion, blood sugar
regulation, lowering cholesterol and prevention of diabetes and heart diseases. The
stabilized rice bran contains an approximate insoluble versus soluble fibre ratio of 5 to 1.
Rice bran exhibits a high digestive tolerance that occurs along the whole digestive tract
with no excessive fermentation in the large intestine. Healthy complex carbohydrates
found in processed rice bran have "low glycemic index" which means they do not cause
spikes in blood glucose (Sayre et al., 2007).
61 Shweta Bhosale
VI. SUMMARY AND CONCLUSION
Rice is a staple food for more than half (65%) of population in India. Rice bran is
obtained as a by-product during the rice milling process and the outer layer is removed at
the time of polishing of husked rice. Rice bran have a range of bioactive phyto-chemicals
with potential for reducing the risk of chronic degenerative diseases. Rice bran is a good
source of protein, mineral, fatty acids, and dietary fibre content. Also rice bran is used for
the enrichment of some foods, due to its high dietary fibre content. Addition of dietary
fibre to a wide range of products will contribute to the development of value-added foods
or functional foods that currently are in high demand. Processing of rice bran has
improved the nutritional, functional and keeping quality of rice bran. Healthy complex
carbohydrates found in stabilized rice bran have "hypoglycemic effect".
The rice bran was procured from NEIST (North East Institute of Science and
Technology) Jorhat, Assam, India. The abundant dietary fibre of rice bran is now being
explored as an important ingredient of health foods. The main aim of present study was
processing and nutritional evaluation of rice bran, development and sensory evaluation of
value added products and assessment of glycemic index of a selected product. The
summary of the study entitled "Processing of rice bran and its utilization in food
products" is presented here.
The scientific finding of the study are summarised as follows:

The procured rice bran was subjected to microbial study, heavy metal and pesticide
residue analysis. The results showed that there was no microbial load and the contents
of heavy metals were less than 0.01ppm and the pesticide residues were less than 0.02
mg/kg hence the rice bran obtained was safe for further processing i.e. for
stabilization and probiotic treatment.
The free fatty acid (FFA) percentage of microwave stabilized and probiotic treated
rice bran was less than 10 per cent during 4 weeks of storage study, and statistical
analysis revealed a significant difference between control and processed rice bran.
Among the functional properties analyzed bulk density for SRB and PRB fell in the
range of 0.22-0.38 g/ml, water absorption capacity was in the range of 2.0-3.0 ml/g,
oil absorption capacity ranged from 1.5-2.5 ml/g. Water solubility and swelling power
capacity were in the range of 7.3-8.0 per cent and 6.7 and 7.2 per cent.
The macro nutrient composition of SRB and PRB indicates that the moisture, protein,
fat, crude fibre, total dietary fibre, carbohydrate, energy and ash were 4.30 and 5.40
per cent, 17.50 and 19.25g, 13.10 and 17.20g, 7.85 and 4.96g, 23.34 and 14.90g,
52.33 and 48.55g, 398 and 426 Kcal, 4.92 and 4.64g respectively.
The micro nutrient composition of stabilized rice bran (SRB) and probiotic treated
rice bran (PRB) indicates that the calcium, phosphorus, iron and zinc were 52.10 and
49.90mg, 1185.2 and 1186.5mg, 28.10 and 30.05mg, 6.02 and 5.89mg respectively.
The antioxidant activity of stabilized rice bran was found to be 65 and for probiotic
treated rice bran it was 70 Vit-C Eq.µg/g.

Phytic acid content in the SRB and PRB was 175 and 172mg/100g. Trypsin inhibitor
was 10.8mg (SRB) and 10.2mg/100g (PRB) respectively.
Five products were developed by incorporation of 5-15 per cent of SRB and PRB
(biscuit, bread, muffin and chocolate), whereas chapati was prepared with the range
of 15-25 per cent. All the products were found to be acceptable by the panel members
throughout the shelf life study.
Mean sensory scores revealed that biscuit and chocolate were best accepted at 10 per
cent level of SRB and PRB incorporation. Bread was best accepted at 5 per cent in
SRB and 10 per cent in PRB respectively. Muffin was accepted at 10 per cent in SRB
and 15 per cent in PRB, chapati was best accepted at 20 per cent in both SRB and
PRB. Statistically the mean sensory scores were found be significant at 5% level.
Nutrient composition of product was computed in the developed products. The
protein, dietary fibre, calcium, phosphrous, iron and zinc contents were enhanced
compared to control products.
There was a decreasing trend of sensory scores for appearance, texture, colour,
aroma, taste and overall acceptability for control, stabilized and probiotic treated rice
bran, during shelf life study, and significant difference was found at 5% level
between the treatments and duration for all the sensory characteristics.
There was no coliforms, total bacteria and fungi found in the biscuit, bread and
muffin but chocolate had total bacteria and fungi count with permissible limit and
there was no coliforms, thus the products were microbiologically safe throughout the
storage period.
Glycemic index of stabilized and probiotic treated chapati in comparison with white
bread showed significant differences. The glycemic index of stabilized chapati was
68.00 and probiotic treated chapati was 64.13 compared to standard white bread (100)
and control chapati (78.50).
Hypoglycemic effect was observed in the normal subjects when fed with stabilized
and probiotic treated rice bran chapati.
According to WHO standards, the stabilized and probiotic treated rice bran had
medium GI (Glycemic Inex).
CONCLUSION
From the research work carried out for the processing of rice bran and its
utilization in food products, the following conclusion can be made. Use of dietary fibre
from rice bran need more exploitation as it is a by-product from food processing industry,
discarded as waste or used as animal feed. The results on processing of rice bran with
stabilization and probiotic treatment are suitable for the development of products since it
was acceptable organoleptically and also rich in macro and micro nutrients, having good
functional properties. The dietary fibre has been claimed as a functional ingredient which
is useful in nutraceutical formulations in the management of life style disorders. The
findings of the present study strongly support that the processed rice bran can be
63 Shweta Bhosale
successfully incorporated in products as it enhances the nutritional quality, functional
properties and shelf life. Therefore, the product developed from stabilized and probiotic
treated rice bran can be recommended as a region specific wholesome hypoglycemic
functional food.
FUTURE LINE OF WORK

Rice bran could be incorporated into other Indian food preparations to replace high
GI foods.
Long term effect of feeding processed rice bran can be undertaken on diabetic
patients.

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ANNEXURE – I
Estimation of protein
Principle
Organic nitrogen digested with sulphuric acid in the presence of catalyst is
converted to ammonium sulphate. Ammonium liberated by making the solution alkaline
is distilled into a known volume of standard acid, which is then back titrated. Protein per
cent was calculated by multiplying the nitrogen present by the factor 6.25.
Reagents
1. 2 per cent boric acid solution: 20g of boric acid was dissolved in some distilled water.
The solution was then transferred to a 1000ml volumetric flask and made up to the
mark.
2. 40% NaOH(w/v)
3. 0.1N HCl: 8.33ml of fuming HCl was dissolved in 1000ml of distilled water.
4. Mixed indicator: Was made by mixing methyl red (0.2%) and Bromocresol green (0-
2%) in a 1:2 ratio (v/v) respectively.
5. Digestion mixture: Anhydrous sodium sulphate and copper sulphate.
6. Concentrated sulphuric acid (H2SO4)
Procedure
Digestion: 0.5g of the samples was weighed into the digestion tubes of the Gerhard
digester in duplicate and two heaped spatulas each of sodium and copper sulphate were
added to each tube. 10ml of concentrated sulphuric acid was also added and samples
digested until the contents of the tubes were sea green in colour. Each of the digested
materials was dissolved in distilled water and transferred into a 100ml volumetric flask
and then brought to the mark.
Distillation: 10ml of each sample was transferred into the distillation tube of the
automatic Gerhard unit and 20ml of 2 per cent boric acid to which was added 3-4 drops
of the mixed indicator was placed in the collecting conical flask to trap the liberated
ammonia. The unit was furnished with 40 per cent NaOH and distilled water to facilitate
operation. Distillation was done for 5 minutes and the ammonia collected and trapped by
the boric acid in between of samples, the unit was rinsed with distilled water for two and
half minutes. The boric acid turned from reddish pink to green as it collected the
ammonia.
Titration: The green coloured boric acid was titrated against the 0.1N HCL until its
colour turned to pink. A blank was run simultaneously. The titre values obtained were
incorporated in the equation below to obtain the per cent nitrogen present in the sample
which, in turn, was multiplied by the factor 6.25 to obtain the per cent protein.

V1 100
Per cent nitrogen (% N) = (Va- Vb) × 0.0014 ×----- x -------
V2 W
Where:
VA=Titre value of sample
Vb= Titre value of blank
V1=Volume to which digested sample was made up (100ml)
V2=Volume of aliquot used in distillation (10 ml)
W= Weight of sample taken for digestion (0.5g)
% Protein = % N X 6.25
73 Shweta Bhosale
ANNEXURE – II
Estimation of fat
Principle
The extraction of fat from substances is often tedious and requires thorough
contact and heating with the solvent. This is done in the soxhlet apparatus in which fresh
solvent continuously comes into contact with the material to be extracted over a relatively
long period of time.
Procedure
Five gram of sample was weighed into a thimble and plugged with fat free cotton
wool. The thimble was placed in the soxhlet apparatus attached to a pre-weighed flask
and extracted for about 14-26 hours. Thereafter, the flask was retrieved from the
apparatus with as little solvent in it as was possible. It was then transferred into an oven
to evaporate the remaining solvent, leaving behind only the residue or extract. The flask
was then cooled in desiccators after which it was weighed to estimate the fat.
Weight of the ether extra (g)

Percent Fat content (g/100g) = --------------------------------------- x 100
Weight of the sample taken (g)

ANNEXURE – III
Estimation of crude fiber

Principle
During the acid and subsequent alkali treatment, oxidative hydrolytic degradation
of native cellulose and considerable degradation of lignin occurs. The residues obtained
after final filtration is weighed, incinerated, cooled and weighed again. The loss in weight
is the crude fiber content.
Reagents
0.255± 0.005 N Standards H2SO4
0.313± 0.005 N Standard NaOH
Method
1. Two g of dry fat- free sample previously extracted with petroleum ether was boiled
with 200ml of H2SO4 for 30 minutes with the help of bumping chips.
2. Thereafter, the mixture was filtered through a muslin cloth and then washed with
boiling water until the residue was free of acids.
3. The residues were then boiled with 200ml NaOH solution for 20minutes.
4. Again, the mixture was filtered through a muslin cloth but this time, was washed
25mL of alcohol.
5. The residue was then transferred to a pre- weighed ashing dish (W1g)
6. Therefore, it was dried for 2 hours at 130±2ºC, cooled in a desiccator and then
weighed (W2g).
7. The dry dishes containing the sample were then ignited for 30 minutes at 600±15ºC.
8. Finally, the sample was cooled in desiccator and then weighed again (W2g)
Calculation
Loss in weight on ignition
Per cent of crude fiber (g/100g) = -------------------------------------
Weight of the sample used (g)
(W2-W1) - (W3-W1)
= ------------------------------------- x 100
Weight of the sample used (g)
75 Shweta Bhosale
ANNEXURE – IV
Estimation of total insoluble dietary fibre (AOAC, 1995)

Principle:
Defatted foods are gelatinized and proteins and starch are removed by enzymatic
digestion. The residue was quantified gravimetrically.
Reagents:
• Ethanol (95%)
• Ethanol (78%)
• Phosphate Buffer(0.08M), pH 6.0
• NaOH (0.275 N)
• HCl (0.325 N)
• α- Amylase heat stable solution
• Protease solution: suspended 50 g protease in 1 ml phosphate Buffer pH 6.0
• Amyloglucosidase solution
Sample Preparation:
Homogenisation of sample
Dry overnight in hot air oven at 105ºC. Cool in desiccator.
Dry mill portion of sample to 0.3 to 0.5mm mesh.
Note: If sample cannot be heated, freeze-dry before milling. If high fat content (75per
cent) prevents proper milling defatted with petroleum ether before milling.
Determination:
Run the blank through entire procedure along with samples to ensure any
contribution from reagents residue.
Weigh 1g of sample in duplicate, accurately to 0.1 mg, in to 500ml beakers. Sample

weight should not differ by ±20 mg.
Add 50 ml of phosphate buffer and adjust the pH to 6.0, if necessary.
Add 0.1 ml heat stable α–amylase solution.
Cover the beakers with aluminium foil and place in boiling water bath.
Ensure that the contents of the beaker reach 100ºC.

Incubate for 15 min at this temperature (Total of 30 min in water bath should be
sufficient).
Cool the solution to room temperature and adjust pH to 7.5 with NaOH solution.
Add 0.1 ml of protease solution to each beaker.
Cover beaker with aluminium foil and incubate for 30 min at 60º C with continuous
agitation.
Cool and adjust pH to 4.0-4.6 with HCl.
Add 0.3 ml amyloglucosidase, and incubate for 30ºC at 60º C with continuous agitation.
Weigh crucible with a fritted disc containing 1 g celite to constant weight. (The celite in
the crucible is made into bed by using a stream of 78per cent ethanol and applying
suction)
Maintain suction and quantitatively transfer precipitate from enzyme digest to crucible,
using filtration module.
Wash residue successively with 3 times 20 ml portions of 78per cent ethanol, two 10 ml
portions of 95per cent ethanol and two 10 ml portions of acetone.
Dry crucible containing residue overnight at 60ºC in hot air oven.
Cool in desiccator and weigh to nearest 0.1mg.
Subtract crucible and celite weight from the above to obtain the insoluble dietary fibre
residue (IDF residue).
Analyse residue from one sample of set of duplicates for protein by Kjeldahl method
using N ×6.25 as conversion factor and subtract from the IDF residue value.
Incinerate second residue sample of duplicate for 5 h at 525º C.
Cool in desiccator and weigh to nearest 0.1 mg and subtract from the IDF residue value.
Insoluble dietary fibre= IDF residue – (protein + ash)
Wt. of the IDF residue(g) – {Protein(g) in IDF residue + Ash(g) in IDF residue}
IDF% = ------------------------------------------------------------------------------------------ Х100
Wt. of the sample (g)
77 Shweta Bhosale
Estimation of total soluble dietary fibre (AOAC, 1995)
Principle:
The soluble fibre is estimated in the filtrate obtained after enzymatic digestion of
protein and carbohydrates of defatted food. The soluble fibre is precipitated and
estimated gravimetrically.
Reagents:
• Ethanol 95 per cent
• Ethanol 78 per cent
• Phosphate Buffer (0.08M ), pH 6.0
• NaOH (0.275N)
• HCl (0.325 N)
• α- Amylase heat stable solution
• Protease solution: suspended 50 g protease in 1 ml phosphate Buffer pH 6.0
• Amyloglucosidase solution
Sample preparation: Homogenise sample and dry overnight in hot air oven at 105ºC,
cool in desiccator, and dry mill portion of sample to 0.3 to 0.5mm mesh. If sample cannot
be heated, freeze-dry before milling. If high fat content (75%) prevents proper milling
defat with petroleum ether before milling.
Determination: Follow the steps of digestion with α-amylase, protease and

amyloglucosidase in IDF and quantitative transfer the digest and collect the filtrate. Add
4 volumes of pre-heated (60ºC) 95% ethanol. Allow the precipitation to complete for 60
min. filter through an accurately weighed crucible with celite. Follow the procedure given
under insoluble fibre to obtain soluble dietary fibre (SDF) residue. Duplicate samples run
similarly are analysed for protein and ash.
Soluble dietary fibre= Weight of SDF residue-(protein + ash)
Wt of the SDF residue (g) – {Protein (g) in SDF residue +Ash (g) in SDF residue}
SDF % = --------------------------------------------------------------------------------------- Х 100
Wt of the sample (g)
Estimation of total dietary fibre (AOAC, 1995)

The total dietary fibre is the sum of the insoluble and soluble dietary fibre,
estimated as follows,
Blank % = Wt of the blank residue (g) - { Protein (g) in the blank residue
+ Ash (g) in blank residue}
Total Dietary fibre = IDF + SDF values

ANNEXURE - V
Estimation of total ash

Total ash was estimated in the sample by weighing about 5 g of dried samples
into a crucible. The crucible was placed on a wire gauge and heated over a low flame till
the material was completely carried and then the crucible was heated in the muffle
furnace for about 4 hours at 6000 C. It was then cooled in a desiccator and weighed. To
ensure the completion of ashing, the crucible was again heated in the furnace. For one
hour cooled and weighed. This was repeatedly done till two consecutive weights were the
same and the ash was almost white or greyish colour. The estimation was done in
duplicate.
Calculation
Weight of the crucible – W g
Weight of the crucible + sample – W1 g
Weight of the crucible + ash – W2 g
∴ Weight of sample (W1 – W) g
Weight of the ash (W2 – W)
(W2 – W) g
∴ % Total ash= ----------------------- x 100
(W1 - W) g
Weight of the ash

Per cent of Total ash = ------------------------------------------ x 100
Weight of the sample taken
79 Shweta Bhosale
ANNEXURE – VI
Preparation of mineral solution

To the ash that was obtained was added 5 ml of 1:1 solution of distilled water and
fuming HCl. This mixture was then heated over a water bath to dryness before another 5
ml of the solution was added. It was heated further over the water bath until it started
fuming and at this point, the crucible was retrieved and its contents filtered in to a 100 ml
volumetric flask using Whatman No. 44 filter paper. After thorough rinsing of the
crucible and the filter paper, the volume was made up to the mark with distilled water.
Aliquots of this mineral solution were taken for the estimation of all the minerals in this
study.

ANNEXURE – VII
Estimation of calcium
Procedure
To an aliquot (25 ml) of the mineral solution was added a few drops of methyl red
indicator and the solution was neutralized with ammonium until the pink colour changed
to yellow. The solution was heated to boiling and 10ml of 6 per cent ammonium oxalate
was added. The mixture allowed was then to boil for a few more minutes and glacial
acetic acid was added until the colour turned distinctly pink. The mixture was kept
overnight and when the precipitate settled down, the supernatant was tested with a drop
of ammonium oxalate solution to ensure the completion of the precipitate. The precipitate
was then transferred along with the filter paper to free of oxalate. The precipitate was
then transferred along with the filter paper to the same beaker and about 5mL of 2N
dilute H2SO4 was then titrated against 0.01N/KMNO4 solution.
1ml of N/100 KMNO4= 0.2004 mg of calcium

Titre value × 0.2004 ×vol. of H2SO4
% calcium (mg) = ----------------------------------------------------------------------- x100
Weight of the sample used for ashing × aliquot taken
81 Shweta Bhosale
ANNEXURE – VIII
Estimation of phosphorous
Determination of phosphorous was carried out by measuring calorimetrically the
blue colour formed when the ash solution is treated with ammonium molybdate and thus
phosphomolybdate formed is reduced.
To an aliquot, 0.4 ml of mineral solution was added with 1mL of ammonium

molybdate, 1mL of hydroquinone and 1mL of sodium thiosulphate solutions in this order,
mixing well after each addition. The volume was then made up to 15 ml with water and
the solution mixed thoroughly. After 30 minutes the optical density of this solution is
measured in a Photoelectric calorimetric against a reagent blank prepared in the same
way as the test, except that the test solution is omitted, at 660nm. The phosphorous
content of sample was obtained from a standard curve prepared with standard phosphate.

ANNEXURE – IX
Estimation of iron
Iron is determined calorimetrically making use of the fact that form iron gives a
blood red color with potassium thiocynnate.
Reagents
1. 30 % sulphuric acid: 30 ml concentrated H2SO4 diluted t 100ml.
2. Saturated potassium per sulphate solution: 7 gm in potassium per sulphate is
dissolved in glass distilled water and the solution made upto 100 ml.
3. Potassium thiocyanate 40 % solution : 4 g KCNS dissolved in 90 ml glass distilled
water, 4 ml acetone added and the volume made upto 100ml.
4. Standard iron solution : 0.702 g ferrous ammonium sulphate is dissolved in 100 ml
glass distilled water and after addition of 5 ml o f1.1 HCL, the solution is made
upton1 liter and mixed thoroughly (1 ml 0.1 mg FC) the standard solution is prepared
once in six months. Working standard solution (0.01 mg Fe/ml) is prepared by
diluting the above solution ten fold.
Procedure
To an aliquot (1 ml or less) of the mineral solution enough water is added (if
necessary) to make up to volume of 6.5 ml followed by 1ml of 30 per cent H2SO4, 1.0
ml of saturated potassium per sulphate and 1.5 ml 40 per cent KCNS solution. The red
color that develops is measured within 20 min at 540 nm.
Note
For iron estimation all the reagents used should be free from iron. Use of glass
distilled water preferred. Use of reagents containing races of iron cannot be avoided it
should be seen that the final solution of standard and test contain identical quantities of
those containing iron as impurity.
Calculation – eg.
Standard OD = 0.322 contains 0.03 mg
Sample OD = 0.209 contains 0.02 mg
0.209X0.03 = 0.019472
0.322
5 ml 0.019472
100 ml = 0.389
Sample wt. / 4.322 5 g 0.389
83 Shweta Bhosale
ANNEXURE – X
Estimation of zinc
Zinc was estimated in samples using chemito atomic absorption
spectrophotometer 201.
Standard
100 ppm standard Zn2+ solution was prepared using 1000 ppm Zn2+ atomic
absorption spectrophotometer solution and appropriate dilution were made to get
standards solution ranging from 0 to 0.6 ppm. These standards were fed to atomic
absorption spectrophotometer as that of sample to get standard curve and a graph was fit.
Against this standard curve the sample readings were compared and corresponding
readings were recorded
Protocol
Suitable dilutions were made from the extract with deionized triple distilled water
so as to fit their absorbance with the range of standard curve.

ANNEXURE – XI
Estimation of Antioxidant Activity by DPPH method

Principle:
The 2, 2- diphenyl (dpph) radical was the oxidizing radical to be reduced by the
antioxidant (AH) present in the given sample. The whole reaction was indicated as,
DPPH + AH DPPH + A
The disappearance of the DPPH radical absorption at 517nm by the action

antioxidants is measured spectrophotometrically in a methanolic solution until the
absorbance remains constant.
Reagents required :
1. 80% methanol
2. DPPH (Diphenyl picrylhdrazyl) in methanol: 10 mg of 1, 1 - Diphenyl - 2- picryl
Hdrazyl was added with 250 ml of methanol (0.1mM).
3. Standard: Different concentrations of Ascorbic acid was taken as standard.
Procedure:
1. 0.1 ml of the freshly prepared sample was taken in test tube.
2. 6 ml of DPPH solution was added.
3. The test tube kept in BOD for one hour at 350C.
4. The O.D. of the solution was read spectrophotometrically at 517nm.
5. The O. D. of DPPH solution without sample addition was also read.
6. The difference in O. D. of DPPH solution and DPPH solution + sample was
calculated.
7. The decrease in O. D. with sample addition is used for calculation of the
antioxidant activity.
Standard curve:
The antioxidant activity was expressed in terms of ascorbic acid equivalents; so
ascorbic acid is taken as standard. Various concentrations of ascorbic acid were prepared
and added to DPPH solution. The decrease in O. D. is plotted against concentration of
ascorbic acid. The concentration of sample was calculated using the above standard
curve.
85 Shweta Bhosale
ANNEXURE – XII
Estimation of phytic acid
The estimation of phytic acid was based on the principle that the phytate is
extracted with trichloroacetic acid and precipitated as ferric salt. The iron content of the
precipitate was determined colorimetrically and the phytate phosphorous content
calculated from this value assuming a constant 4 Fe: 6 molecular ratios in the precipitate.
Phytates were estimated as phytic and phytate phosphorous was obtained.
Estimation of phytic acid

Reagents
1. 3 per cent trichloroacetic acid (TCA)
2. 3 per cent sodium sulphate in 3 per cent (TCA)
3. 1.5 N NaOH
4. 3.2 N HNO3
5. FeCl3 solution: Dissolve 583mg FeCl3 in 10ml at 3 per cent TCA
6. 1.5M potassium thiocynate (KCSN) : Dissolve 29.15g in 200ml water
7. Standard Fe(NO3)3 solution
Methods
1. Weigh a finely ground (40 mesh) sample estimated to contain 5 to 30mg phytate
P into a 125ml Erlenmeyer flask.
2. Extract in 50ml 3 per cent TCA for 30 min with mechanical shaking or with
occasional swirling by hand for 45 mins.
3. Centrifuge the suspension and transfer a 10ml aliquot of the supernatant to a
40ml conocal centrifuge tube.
4. Add 4ml of FeCl3 solution to the aliquot by blowing rapidly from the pipette.
5. Heat the contents in a boiling water bath for 45 mins. If the supernatant is not
clear after 30 mins. Add one or two drops of 3 per cent sodium sulphate in 3 per
cent TCA and continue heating.
6. Centrifuge (10-15min) and carefully decant the clear supernatant.
7. Wash the precipitate twice by dispersing well in 2 to 25 mol 3 per cent TCA,
heat in boiling water for 5-10 mins and centrifuge.
8. Repeat washing with water.
9. Disperse the precipitate in few ml of water and add 3ml of 1.5N NaOH with
mixings.
10. Bring volume of approximately 30ml with water and heat in boiling water for
30mins.
11. Filter hot (quantitatively) through a moderately retentive paper Whatman No. 2
filter paper.
12. Wash the precipitate with 60-70ml hot water and discard the filter.

13. Dissolve the precipitate from the paper with 40ml hot 3.2 N HNO3 into a 100ml
volumetric flask.
14. Wash the paper with several portions of water, collecting the washing in the
same flask.
15. Cool flask and contents to room temperature and dilute to volume with water.
16. Transfer a 5 ml aliquot to another 100ml volumetric flask and dilute to
approximately 70ml.
17. Add 20ml of 1.5M KSCN dilute to volume and read colour immediately (with
1ml) at 480nm.
18. Run a reagent blank with each set at samples.
Standard:
Weigh accurately 433mg Fe (NO3)3 and dissolve in 100ml distilled water in a
volumetric flask. Dilute 2.5ml at this stock standard and make up to in a volumetric flask.
Pipette out 2.5, 5, 10, 15, 20 ml of this working standard into a series at 100ml volumetric
flasks and proceeded from step 16.
Calculation
Find out of the µg iron present in the test from the standard curve and calculate
phytate P as per the equation.
µg Fe X 15 X 1 1
Phytate P (mg/100g)=--------------------------- X -----------
Wt. of sample (g) 100
87 Shweta Bhosale
ANNEXURE-XIII
Estimation of trypsin inhibitor

Principle
The activity of enzyme trypsin assayed using casein as substrate. Inhibition of this
activity is measured in the extracts. (Kakade et al., 1965)
Reagents
1. a) Trypsin standard stock solution: 20 mg of trypsin was dissolved in 100 ml of
0.01 M and stored at 4°C.
b) Trypsin working standard solution: One (ml) of the stock containing 200µg trypsin
/ml was diluted to 2 ml with 0.1 M sodium phosphate buffer, pH 7.6.
2. One % (w/v) Casein solution: One (g) of casein was dissolved in 100 ml of 0.01 M
sodium phosphate buffer, pH 7.6. The suspension was incubated in boiling water bath
for 15 to 20min, cooled and filtered before use.
3. Five % (w/v) Trichloroacetic acid (TCA)
Preparation of enzyme inhibitor extract

100 mg of acetone powder was defatted with 20 ml of water saturated n- butanol
for 2-3 h at 4° C. The extract was centrifuged at 10,000rpm for 15min and residue was
air-dried at room temperature. The defatted flour was stirred with 10 ml of ice-cold 0.1 M
sodium phosphate buffer, pH 7.6 containing 0.15 M NaCl and 0.1 M per cent PVP for 1-
2hr on a magnetic stirrer at 4°C . The extract was used for protein estimation (Lowery et
al., 1951) and the trypsin inhibitor assay.
Assay of trypsin activity

The trypsin working standard solution (containing 10µg protein) was taken in a
mixture containing 0.1 M phosphate buffer, pH 7.6 in a final volume of 2 ml. The
reaction was initiated by the addition of 1 ml of casein at 37° C. After 30 min, the
reaction was terminated by the addition of 3 ml of 5 per cent TCA, incubated for one
hour at room temperature and filtered through Whatman No.1 filter paper. The
absorbance of the filtrate was recorded at 280 nm in spectrophotometer. The blank was
prepared similarly excepting extract.
One unit of trypsin is defined as the amount of enzyme that increases the
absorbance by 0.001 under the defined assay conditions and the activity is expressed as
units/g acetone powder.

ANNEXURE-XIV
Preparation of biscuit (per 100 g)
Ingredients Quantity (g)
Refined wheat flour 50
Sugar powder 25
Fat 30
Baking powder Pinch
Procedure:
Cream fat and sugar till light and pluffy.
Sieve refined wheat flour.
Add sieved flour to the cream and make stiff dough.
Divide the dough into small equal portions.
Round them and place on baking trays.
Bake them at 2000C till golden brown and crisp.
89 Shweta Bhosale
ANNEXURE-XV
Preparation of bread (per 100 g)
Sugar powder 20
Fat 5
Yeast 0.5
Oil 5
Salt Pinch
Procedure:
Sieve refined wheat flour.
Add yeast to the refined wheat flour
Knead in fat and sugar
Rest the dough under thick cloth for an hour or till it becomes double in size.
Divide the dough into pieces of 400g each.
Mould into bread tin as desired.
Bake at 2000C for 20 mins.

ANNEXURE-XVI
Preparation of muffin (per 100 g)
Sugar powder 8
Egg Medium size egg (30g)
Oil 2
Vanilla essence 1 tea spoon
Procedure:
Rub the sugar powder and fat together.
Take a separate vessel and beat egg adding vanilla.
Sieve the refined wheat flour.
Add the mixture and rub together.
Prepare dough to required consistency.
Pour the dough into the greased muffin moulds and bake them at 3750F for about
20min.
91 Shweta Bhosale
ANNEXURE-XVII
Preparation of chocolate (per 100 g)
White chocolate 75
Dark chocolate 25
Procedure:
Mix white chocolate with dark chocolate.
Melt the chocolate.
Pour the mixture into chocolate moulds.
Deep freezing for 15min.

ANNEXURE-XVIII
Preparation of chapati (per 100 g)
Wheat flour 100
Oil 5
Salt Pinch
Procedure:
Sieve wheat flour.
Add required quantity of water and salt.
Knead the above into soft dough.
Prepare chapati with rolling pin and board.
Roast chapati on hot tawa
93 Shweta Bhosale
ANNEXURE-XIX
SCORE CARD FOR THE SENSORY EVALUATION
Name of the judge: Date:

Name of the product:
Instructions:
• Please evaluate each of the following samples using scoring system given below.
• Write the preferred number score in the column as per evaluation.
• Rinse your mouth in between evaluating each sample.
Aroma/ Overall
Sample Appearance Texture Colour Taste
odour acceptability
Scoring system:-
9-like extremely: 6-like slightly: 3-dislike moderately:
8-like very much: 5-neither like nor dislike: 2-dislike very much:
7-like moderately: 4-dislike slightly: 1-dislike extremely:
Remarks:
Signature

ANNEXURE - XX
Estimation of total lipids (Bligh and Dyer method)

In this method, a mixture of chloroform and methanol (2:1 V/V) was used. The
tissue (about 1 g wet weight) was first ground in a pestle and mortar with about 10 ml of
distilled water. The pulp was transferred to a conical flask (250 ml capacity) and 30 ml of
chloroform – methanol mixture was added and mixed well.
For complete extraction, it was kept overnight at room temperature, and in dark.
At the end of the period, 20 ml chloroform and 20 ml of water was added. The
resulting solution was subjected to centrifugation, and three 3 layers were seen. A clear
lower layer of chloroform containing all the lipids, a coloured aqueous layer of methanol
with water soluble material and a hick pasty interface was seen.
The methanol layer was discarded and the lower layer was carefully collected by
filtering through glass wool. The organic layer was taken in a pre-weighed beaker or vial
and carefully evaporated. The sample was kept in warm water (around 50⁰C).
When the solution was free of organic solvents, the weight was taken again. The
difference in weight gives the weight of the lipids. The results were expressed in terms of
milligrams of total lipids per gram of the sample.
95 Shweta Bhosale
ANNEXURE-XXI
Determination of acid value

Principle: The acid value is the number of milligram of KOH required to neutralise the
free acid in 1 g of the substance.
Reagents:
1. A mixture of equal volume of alcohol (95%) and ether
2. 1% phenolphthalein in alcohol
3. 0.1 N KOH
Procedure:
About 10gm of the oil was weighed accurately into a 250 ml conical flask t which
was added 50 ml of a mixture of equal volume of alcohol and ether previously neutralised
after the addition of 1 ml of phenolphthalein solution. The contents were warmed in a
water bath until the substance is completely dissolved. The solution was titrated with 0.1
N KOH with constant shaking until a pink colour persists for 15 sec. The titre value in ml
(a) was noted.
a x 0.00561 x 1000
Acid value = --------------------------------
Weight in g of substance

ANNEXURE-XXII
Determination of peroxide value

Principle: In the oxidative rancidity, oxidation of fat due to the combination of oxygen
with unsaturated fatty acid takes place and results in the formation of compounds with a
peroxide structure. These are detected by the liberation of iodine from an acid solution of
potassium iodide. There is another type of rancidity is called hydrolytic rancidity, which
is caused by the formation of low molecular weight fatty acids like butyric acid, caproic
acid and caprylic acids. This can be estimated by alkali titration method mentioned under
acid value of ghee and is expressed in terms of butyric acid.
Reagents:
1. Acetic-acid chloroform mixture (Composed of glacial acetic acid and chloroform
in the ratio 2:1)
2. Saturated potassium iodide solution
3. N/1000 sodium sulphate
4. Starch indicator
Procedure:
0.5 to 1 g of clear melted fat was weighed accurately in the boiling flask. To this
30 ml of acetic acid- chloroform mixture was added and fat was dissolved. 1 ml of
saturated potassium iodide was added. After 5min 100ml of distilled water was added.
The liberated iodine was titrated against N/1000ml sodium thiosulphate. When the end
point is approached 1ml of freshly prepared starch was added and titration was completed
till the blue colour disappears. Blank was carried out using all the reagents without the
oil.
Calculation:
Peroxide value of oil (meq/kg of sample) = (Titre – Blank) x N x 1000

Wt of oil (g)
97 Shweta Bhosale
ANNEXURE - XXIII
CONSENT FORM
DEPARTMENT OF FOOD SCIENCE AND NUTRITION
GKVK, UAS, BENGALURU - 65
Name of the student : Shweta Bhosale ID. No : PALB2220
Research Topic : " Processing of rice bran and its utilization in food products"
Name of the product: Chapati (SRB and PRB)
CONSENT FORM
I am voluntarily accepting the dietetic Low glycemic chapati for the study period.
Date: Name:
Sign:
Ph. No :

ANNEXURE - XXIV
Calculation of incremental area under the curve (IAUC)

The IAUC equals the sum of the triangles and trapezoids:
For example,
The area of triangle A=2.0x15/2=15.0
The area of trapezoid B=(2.0+3.6)x15/2=42.0
The area of trapezoid C=(3.6+1.0)x15/2=34.5
The area of triangle D=1.6xt’/2
Since: t’/15=1.0/(1.0+0.2)
Therefore t’=15x1.0/1.2=12.5
Therefore area of the triangle D=1.0x12.5/2=6.25
The area of triangle E=0.3xt,/2
Since: t”/30=0.3/(0.3+0.2)
Therefore: t”=30x0.3/0.5=18
Therefore the area of the triangle D=0.3x18/2=2.7
The area of trapezoid F=(0.3+0.6)x30/2=13.5
Therefore, IAUC=15.0+42.0+34.5+6.25+2.7+13.5=114mmol.min/L
IAUC of the test food
Glycemic index of the test food= ---------------------------------------x 100
IAUC of the standard food
Whereas IAUC: Incremental area under the curve
99 Shweta Bhosale
ANNEXURE - XXV
Profile of subjects taken for the glycemic index study
Height Weight
Sl. No. BMI Age (yr) Gender Occupation
(cm) (kg)
1 159 45 18.02 23 F Student
2 155 50 21.08 23 F Student
3 159 52 20.82 23 F Student
4 165 68 25.28 23 F Student
5 151 62 27.55 23 F Student
6 153 45 19.47 23 F Student
7 161 52 20.31 23 F Student
8 175 61 20.14 23 M Student
9 167 56 20.32 23 M Student
10 168 48 17.21 23 M Student
F: Female
M: Male
BMI: Body Mass Index

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“PROCESSING OF RICE BRAN AND ITS

UTILIZATION IN FOOD PRODUCTS”

DEPARTMENT OF FOOD SCIENCE AND NUTRITION

Thesis submitted to the

Master of Science (Agriculture)

I wish to express my sincere gratitude and respect to Dr. Vijayalakshmi, D.,

A special thanks to Dr. Chowdhary, Gurukumar from NEIST Jorhat Assam. I

Department of Food Science and Nutrition (Dr. Vijayalakshmi. D)

DºÁgÀ «eÁÕ£À ªÀÄvÀÄÛ ¥ÉÆÃµÀuÉ qÁ. «dAiÀÄ®Qëöä r.

CHAPTER TITLE PAGE No.

II REVIEW OF LITERATURE 3-13

III MATERIALS AND METHODS 14-24

IV EXPERIMENTAL RESULTS 25-49

VI SUMMARY AND CONCLUSION 62-64

VII REFERENCES 65-71

1 Research design 15-16

2 Microwave stabilization of rice bran 15-16

3 Probiotic treatment of rice bran 17-18

4 Preparation of biscuit 21-22

5 Preparation of bread 21-22

6 Preparation of muffin 21-22

7 Preparation of chocolate 21-22

8 Preparation of chapati 21-22

9 Percentage of Free Fatty Acid in rice bran on storage 28-29

10 Mean sensory scores of biscuit 32-33

11 Mean sensory scores of bread 36-37

12 Mean sensory scores of muffin 40-41

13 Mean sensory scores of chocolate 42-43

14 Mean sensory scores of chapati 44-45

Plate No. Title Between Pages

(a) Microwave stabilized rice bran

2 Rice bran incorporated biscuit 21-22

3 Rice bran incorporated bread 21-22

4 Rice bran incorporated muffin 21-22

5 Rice bran incorporated chocolate 21-22

6 Rice bran incorporated chapati 21-22

7 Sensory evaluation of products by panel members 23-24

8 Testing of blood glucose in subjects 24-25

Processing of Rice Bran and its Utilization in Food Products 1

2.1 Processing of rice bran

Processing of Rice Bran and its Utilization in Food Products 3

Ramezanzadeh et al. (2000) studied the effect of microwave heat, packaging

Hettiarachchy (2009) conducted a study on yeast fermentation of rice bran

Abdel-Hady (2013) investigated the effect of some thermal processing

2.2 Nutrient composition of rice bran

Processing of Rice Bran and its Utilization in Food Products 5

Rosniyana et al. (2009) studied on nutritional content and storage stability of

The antioxidant and antiproliferative activities of methanolic extracts from

Abdel-Galeel et al. (2012) investigated the effect of milling degree on nutritive

2.3 Development of products and their sensory evaluation

Processing of Rice Bran and its Utilization in Food Products 7

Sairam et al. (2011) studied the physico-chemical characteristics, antioxidant

Premakumari et al. (2012) reported that development of breakfast/ dinner recipes

Processing of Rice Bran and its Utilization in Food Products 9

2.4 Shelf life study of the developed products