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Characterization and bioevaluation of non-conventional
protein sources for food application
By
Muhammad Sibt-e-Abbas
M.Sc. (Hons.) Food Technology
A thesis submitted in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
IN
FOOD TECHNOLOGY
NATIONAL INSTITUTE OF FOOD SCIENCE & TECHNOLOGY
UNIVERSITY OF AGRICULTURE, FAISALABAD
PAKISTAN
2017
2
3
Dedicated
To
The Holy Prophet
(Peace Be Upon Him)
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i
ACKNOWLEDGEMENT
I feel myself inept to regard the Highness of Almighty Allah, my words have lost their
expressions, knowledge is lacking and lexis scarce to express gratitude in the rightful manner
to the blessings and support of Allah the Almighty who flourished my ambitions and helped
me to attain goals. Perplexed mind feel slur as I seek words of praise for Holy Prophet
Muhammad (P.B.U.H.) for enlightening our lives with the faith in Allah. I present my
humble gratitude from the deep sense of heart to the Holy Prophet Muhammad (Peace Be
Upon Him), that without him the life would have been worthless.
Allah Almighty had been so helpful in His blessings by giving me a prospect to toil under the
esteem supervision of Prof. Dr. Masood Sadiq Butt, Dean, Faculty of Food, Nutrition and
Home Sciences, University of Agriculture, Faisalabad. I have no words to express my
gratitude for his diligent cooperation, scrupulous support and cheering perspective during the
entire degree program. I am indebted to him for consistent encouragement during planning,
execution, and final presentation of this piece of research work. I expand my deepest
appreciation and benedictions to Dr. Moazzam Rafiq Khan Assistant professor, National
Institute of Food Science and Technology, University of Agriculture, Faisalabad for his great
help. I deem it my utmost pleasure in expressing his sympathetic attitude; parental guidance,
scholarly suggestions and criticism indeed are incalculable wealth for me. Abstemious and
resolute appreciation to Dr. Muhammad Shahid, Associate Professor, Department of
chemistry and biochemistry for his compassionate attitude, brotherly advices, valued
suggestions throughout the research project and kind cooperation provided during my
research project. I am also grateful to Dr. Muhammad Tauseef Sultan for his valuable
critical discussions and endorsing support throughout research work and motivating me at
times.
I am thankful to Dr. Mian N. Riaz, Director, Food Protein Research & Development Center
(FPRDC), Texas A&M University, College Station, Texas, USA for hosting me during 6
months research under International Research Support Initiative Program (IRSIP). I am
highly indebted to Higher Education Commission, Pakistan for financial support and assistance during my stay at Texas A&M University, Texas, USA under IRSIP.
I extend my obligations to my amorous Father without his moral support, I wouldn’t have
been at this position today. His endless efforts and best wishes sustained me at all stages of
my life and encouraged me for achieving high ideas of life. My sincere gratitude to my dear
Mother who has always wished to see me glittering high on the skies of success. I am quite
thankful to my wife for the support she gave that helped me to remain steady during the
whole period. My loving daughter Enaab Zahra and cute son Muhammad Shehwar Abbas
are the assets of my life. Extreme love, utmost sincerity and caring behavior of my sweet Brothers and my loving Sister can never be neglected.
Friends are asset of one’s life, my colleagues and friends have really strengthened me a lot
and helped me attain waypoints in the best doable fashion. I am also grateful to my adorable juniors for their valued support throughout the study period.
Muhammad Sibt-e-Abbas
ii
LIST OF ABBREVIATIONS
PEM Protein energy malnutrition
NFE Nitrogen free extract
NSI Nitrogen solubility index
BD Bulk density
WAC Water absorption capacity
OAC Oil absorption capacity
FC Foaming capacity
FS Foaming stability
EC Emulsion capacity
ES Emulsion stability
LGC Least gelation concentration
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
PDCAAS Protein digestibility corrected amino acid score
IVPD In vitro protein digestibility
PI Protein isolates
SPI Sesame protein isolates
FPI Flaxseed protein isolates
CPI Canola protein isolates
PER Protein efficiency ratio
NPR Net protein ratio
RNPR Relative net protein ratio
TD True digestibility
BV Biological value
NPU Net protein utilization
ALT Alanine aminotransferase
AST Aspartate amino transferase
ALP Alkaline phosphatase
SOV Source of variation
Df Degree of freedom
ARM Aromatic amino acid
SAA Sulfur containing amino acid
LAA Limiting amino acid
SGF Straight grade flour
WA Water absorption
DDT Dough development time
DS Dough stability
iii
iv
LIST OF CONTENTS
Acknowledgement i
List of abbreviations ii
Contents iii
List of contents iv
List of tables viii
List of figures x
List of appendices xi
Abstract xii
1. INTRODUCTION 1
2. REVIEW OF LITERATURE 7
2.1. Malnutrition 7
2.2. Oilseeds; nutritional aspects 10
2.2.1. Sesame 10
2.2.2. Flaxseed 12
2.2.3. Canola 14
2.3. Amino acid profile 16
2.4. Functional properties 17
2.5. Gel Electrophoresis (SDS-PAGE) 23
2.6. Bio-evaluation 25
2.7. Composite flours 26
2.8. Bakery products 30
3. MATERIALS AND METHODS 32
3.1. Procurement of raw materials 32
3.2 Preparation of raw material 32
3.3. Proximate composition 32
3.3.1. Moisture content 32
3.3.2. Crude fat 33
3.3.3. Crude protein 33
3.3.4. Crude fiber 33
3.3.5. Ash 33
3.3.6. Nitrogen Free Extract (NFE) 33
3.4. Mineral profile 33
3.5. Defatting of samples 34
3.6. Proximate and mineral analyses of defatted oilseeds 34
3.7. Protein isolates preparation 34
3.8. Protein isolates assays 34
3.8.1. Protein Content 34
3.8.2. Isolate Recovery 34
3.8.3. Protein Yield 36
3.9. Functional properties of defatted oilseed protein isolates 36
3.9.1. Bulk Density 36
3.9.2. Water absorption capacity 36
3.9.3. Oil absorption capacity 36
3.9.4. Foaming properties 36
v
3.9.5. Emulsion properties 36
3.9.6. Nitrogen Solubility Index (NSI) 37
3.9.7. Least Gelation Concentration (LGC) 37
3.10. Gel Electrophoresis (SDS-PAGE) 37
3.11. Amino Acid Profile 37
3.12. Amino Acid Score 38
3.13. PDCAAS Value 38
3.14. In vitro protein digestibility (IVPD) 38
3.15. Bioevaluation 38
3.15.1. Housing of Rats 38
3.15.2. Feed Intake 39
3.15.3. Body Weight Gain 39
3.15.4. Protein Quality Evaluation 39
3.15.5. Safety evaluation 40
3.15.5.1. Serum protein 40
3.15.5.2. Liver and kidney functioning tests 40
3.16. Selection of protein isolates 40
3.17. Development of composite flour 40
3.17.1. Proximate analysis of composite flours 41
3.17.2. Rheological studies 41
3.17.2.1. Farinographic Studies 41
3.17.2.1.1. Water Absorption 41
3.17.2.1.2. Dough Development Time 41
3.17.2.1.3. Dough Stability Time 41
3.17.2.2. Mixographic Studies 41
3.17.2.2.1. Mixing Time 42
3.17.2.2.2. Peak Height Percentage 42
3.17.3. Functional properties of composite flours 42
3.18. Protein enriched muffins preparation 42
3.18.1. Proximate analysis 42
3.18.2. Physical analysis 43
3.18.2.1. Color 43
3.18.2.2. Instrumental texture 43
3.18.2.3. Volume 43
3.18.3. Gross energy 43
3.18.4. Sensory evaluation 43
3.19. Statistical Analysis 44
4. RESULTS AND DISCUSSION 45
4.1. Charaterization of oilseeds 45
4.1.1. Proximate composition 45
4.1.2. Mineral profile of oilseeds 48
4.1.3. Proximate analysis of defatted oilseeds 50
4.1.4. Mineral composition of defatted oilseeds 52
4.2. Protein isolates; recovery, yield and protein content 54
4.3. Functional properties of defatted oilseed protein isolates 56
vi
4.3.1. Bulk density 56
4.3.2. Water and oil absorption capacities 57
4.3.3. Foaming capacity and stability 61
4.3.4. Emulsion capacity and stability 65
4.3.5. Nitrogen solubility index 67
4.3.6. Least Gelation Concentration (LGC) of defatted oilseed protein isolates 70
4.4. SDS-PAGE 72
4.5. Amino acid profile of defatted oilseed protein isolates 74
4.5.1. Amino acid score of defatted oilseed protein isolates 78
4.5.2. Protein digestibility corrected amino acid score (PDCAAS) 78
4.5.3. In vitro protein digestibility (IVPD) 80
4.6. Bioefficacy study 82
4.6.1. Growth study parameters 82
4.6.1.1. Protein efficiency ratio (PER) 82
4.6.1.2. Net protein ratio (NPR) 84
4.6.1.3. Relative net protein ratio (RNPR) 85
4.6.2. Nitrogen balance study 85
4.6.2.1. True Digestibility (TD) 85
4.6.2.2. Biological Value (BV) 87
4.6.2.3. Net protein utilization (NPU) 88
4.7. Safety assessment of defatted oilseed protein isolates 88
4.7.1. Serum protein analysis 88
4.7.2. Renal functioning tests 90
4.7.3. Hepatic functioning tests 92
4.8. Development of composite flours 95
4.8.1. Proximate analysis 95
4.8.2. Rheological characterization of developed composite flours 96
4.8.2.1. Mixographic characteristics of composite flours 96
4.8.2.1.1. Mixing time 96
4.8.2.1.2. Peak height 98
4.8.2.2. Farinographic characteristics of composite flours 98
4.8.2.2.1. Water absorption (WA) 98
4.8.2.2.2. Dough development time (DDT) 100
4.8.2.2.3. Dough stability (DS) 100
4.8.3. Functional properties of composite flours 101
4.8.3.1. Bulk density 101
4.8.3.2. Absorption properties 101
4.8.3.3. Foaming properties 103
4.8.3.4. Emulsion properties 104
4.9. Preparation of protein enriched muffins 106
4.9.1. Proximate analysis 106
4.9.1.1. Moisture content 106
4.9.1.2. Crude protein 107
4.9.1.3. Crude fat 108
4.9.1.4. Crude fiber 108
4.9.1.5. Ash 109
vii
4.9.1.6. Nitrogen free extract (NFE) 109
4.9.2. Gross energy 111
4.9.3. Physical analysis of protein enriched muffins 113
4.9.3.1. Color 113
4.9.3.2. Texture 116
4.9.3.3. Volume 118
4.10. Sensory evaluation of protein enriched muffins 118
4.10.1. Color 120
4.10.2. Flavor 120
4.10.3. Taste 121
4.10.4. Texture 121
4.10.5. Overall acceptability 122
5. SUMMARY 124
RECOMMENDATIONS 130
LITERATURE CITED 131
APPENDICES 162
viii
LIST OF TABLES
Sr.
No. Title Page
3.1 Components of experimental diets 39
3.2 Composite flour treatments 40
4.1 Mean squares for proximate composition of oilseed samples 47
4.2 Proximate composition (%) of oilseed samples 47
4.3 Mean squares for mineral profile of oilseed samples 49
4.4 Mineral profile (mg/100g) of oilseed samples 49
4.5 Mean squares for proximate composition of defatted oilseeds 51
4.6 Proximate composition (%) of defatted oilseeds 51
4.7 Mean squares for mineral profile of defatted oilseeds 53
4.8 Mineral profile (mg/100g) of defatted oilseeds 53
4.9 Mean squares for protein isolates recovery, yield and crude protein 55
4.10 Oilseeds protein isolates recovery, yield and crude protein 55
4.11 Mean squares for bulk density of defatted oilseed protein isolates 58
4.12 Mean squares for absorption properties of defatted oilseed protein isolates 60
4.13 Mean squares for foaming properties of defatted oilseed protein isolates 63
4.14 Mean squares for emulsion properties of defatted oilseed protein isolates 66
4.15 Least gelation concentration of defatted oilseed protein isolates 71
4.16 Mean squares for essential amino acids of defatted oilseed protein isolates 76
4.17 Essential amino acids (g/100g protein) of defatted oilseed protein isolates 76
4.18 Mean squares for non-essential amino acids of defatted oilseed protein isolates 77
4.19 Non-essential amino acids (g/100g protein) of defatted oilseed protein isolates 77
4.20 Amino acids score for defatted oilseed protein isolates 79
4.21 Mean squares for protein digestibility corrected amino acid score (PDCAAS) 81
4.22 Protein digestibility corrected amino acid score (PDCAAS) 81
4.23 Mean squares for In vitro protein digestibility (IVPD) of oilseed protein isolates 83
4.24 In vitro protein digestibility (IVPD) of oilseed protein isolates 83
4.25 Mean squares for PER and NPR of experimental diets 86
4.26 Growth study parameters of test diets 86
ix
4.27 Mean squares for nitrogen balance study parameters of experimental diets 89
4.28 Nitrogen balance study parameters of experimental diets 89
4.29 Mean squares for serum protein analysis of experimental rats 91
4.30 Serum protein profile of experimental rats 91
4.31 Mean squares for renal functioning tests of experimental rats 93
4.32 Renal functioning tests of experimental rats 93
4.33 Mean squares for hepatic functioning tests of experimental rats 94
4.34 Hepatic functioning tests of experimental rats 94
4.35 Mean squares for proximate analysis of composite flour blends 97
4.36 Means for proximate analysis (%) of composite flour blends 97
4.37 Mean squares for mixographic charateristics of composite flours 99
4.38 Means for mixing time and peak height of composite flours 99
4.39 Mean squares for farinographic charateristics of composite flours 102
4.40 Means for farinographic characteristics of composite flours 102
4.41 Mean squares for functional properties of composite flour blends 105
4.42 Functional properties of composite flour blends 105
4.43 Mean squares for proximate analysis of protein enriched muffins 110
4.44 Proximate analysis (%) of protein enriched muffins 110
4.45 Mean squares for gross energy of protein enriched muffins 112
4.46 Means for gross energy (kcal/100g) of protein enriched muffins 112
4.47 Mean squares for crust color of protein enriched muffins 114
4.48 Mean squares for crumb color of protein enriched muffins 114
4.49 Means for crust color of protein enriched muffins 115
4.50 Means for crumb color of protein enriched muffins 115
4.51 Mean squares for texture of protein enriched muffins 117
4.52 Means for texture profile of protein enriched muffins 117
4.53 Mean squares for volume of protein enriched muffins 119
4.54 Means for volume of protein enriched muffins 119
4.55 Mean squares for sensory scores of protein enriched muffins 123
x
LIST OF FIGURES
Sr. No. Title Page
3.1 Systematic flow sheet for oilseed protein isolates recovery 35
4.1 Bulk density of oilseed protein isolates 58
4.2 Water and oil absorption capacity of oilseed protein isolates 60
4.3 Foaming capacity of oilseed protein isolates 64
4.4 Foaming stability of oilseed protein isolates 64
4.5 Emulsion capacity and stability of oilseed protein isolates 66
4.6 Nitrogen solubility (%) of sesame protein isolates (SPI) 68
4.7 Nitrogen solubility (%) of flaxseed protein isolates (FPI) 68
4.8 Nitrogen solubility (%) of canola protein isolates (CPI) 69
4.9 Electrophorogram of oilseed protein isolates 73
4.10 Sensory evaluation of protein enriched muffins 123
xi
LIST OF APPENDICES
Sr.
No. Title Page
1 Vitamin and mineral mixture used in the study 162
2 Sensory Evaluation Performa of Muffins 163
xii
ABSTRACT In present study, characterization and bio-evaluation of protein isolates prepared from different
defatted oilseeds i.e. sesame, flaxseed and canola were carried out to develop protein enriched
muffins. The tested oilseeds were subjected to proximate and mineral analyses prior to protein
isolates preparation using isoelectric precipitation method. The highest protein yield 79.03±2.18%
was assessed in sesame protein isolates (SPI) followed by 78.53±4.02% in canola protein isolates
(CPI) while the lowest yield 74.61±2.93% was recorded in flaxseed protein isolates (FPI).
Moreover, functional properties i.e. bulk density, water & oil absorption capacities, foaming &
emulsifying properties of protein isolates were also determined. Maximum bulk density was
noticed in CPI followed by FPI and SPI. Nonetheless, higher water absorption capacity was
revealed in SPI followed by CPI and FPI. Maximum foaming capacity (FC) was observed in SPI
tracked by FPI while minimum in CPI. Electropherogram through SDS-PAGE revealed that
oilseed protein isolates exhibited protein bands ranging from 15 to 65kDa. Amino acid analysis of
protein isolates indicated that highest lysine content (2.60±0.09 g/100 g) was found in CPI whilst
minimum in SPI (1.48±0.04 g/100 g). Accordingly, amino acid scores were also calculated with
reference to requirement for the pre-school children and the values were recorded as 28.46, 31.15
and 50.00 in SPI, FPI and CPI, respectively. The in vitro protein digestibility indicated highest
value for SPI (87.57±4.41%) whilst lowest for CPI (82.13±2.86%). Moreover, bioevaluation trial
was performed via growth study parameters i.e. protein efficiency ratio (PER), net protein ratio
(NPR) and relative net protein ratio (RNPR). Amongst protein isolates, the highest values were
recorded in SPI followed by CPI and FPI, respectively for these parameters. Likewise, nitrogen
balance study parameters presented maximum values for true digestibility (TD) in SPI trailed by
FPI and CPI. However, biological value (BV) was higher in FPI followed by CPI while lowest in
SPI. Similarly, highest value for net protein utilization (NPU) was noticed in FPI tracked by SPI
and CPI. Moreover, safety assessment of protein isolates was also performed including serum
protein, kidney and liver function tests. All these parameters showed non-significant variations
among the tested protein isolates. On the basis of protein isolates assay, functional properties and
bio-evaluation, one best protein isolate i.e. SPI was selected for the preparation of protein enriched
muffins. Purposely, composite flours with varying levels (5, 10, 15, 20, 25%) of SPI were
prepared and subjected to proximate analysis along with rheological & functional properties. The
treatments were formulated as T0 = 100% Straight grade flour (SGF), T1 = 95% SGF and 5% SPI,
T2 = 90% SGF and 10% SPI, T3 = 85% SGFand 15% SPI, T4 = 80% SGF and 20% SPI and T5 =
75% SGF and 25% SPI. Furthermore, protein enriched muffins were prepared from the composite
blends of SPI. The developed muffins were investigated for chemical composition, gross energy,
color, texture, volume and sensory profile. The gross energy values varied from 422.00±10.24 to
439.59±4.57 kcal/100g for respective treatments. Moreover, the results for crust color of muffins
revealed decreasing trend for L* & b* value, chroma and hue angle in subsequent treatments while
increasing trend was noticed for a* value. Likewise, the texture profile of muffins showed that
firmness increased from 90.42±2.91N for T0 to 115.62±2.62N in T5 with increasing protein level.
However, elasticity decreased from 59.97±2.31% (T0) to 40.38±1.02% (T5). Similarly, for volume
of muffins, the highest value was observed for T0 (145.00±2.69 cm3) followed by T1, T2, T3 and T4,
whilst, the minimum value was noticed for T5 (120.00±3.97). Hedonic response for various
sensory attributes showed acceptance towards protein enriched muffins. The results indicated that
treatment T3 carrying 15% SPI rated better by the sensory judges. Conclusively, the outcomes of
this project explicated that non-conventional protein sources i.e. defatted oilseeds can be
potentially utilized for the extraction of protein isolates with substantial functional and nutritional
properties. It is deduced that the resultant protein isolates are a suitable tool to combat protein
energy malnutrition by their application in food especially bakery products.
1
Chapter 1
INTRODUCTION
Malnutrition is a widely prevalent nutritional disorder in the developing countries that
generally persists due to insufficient intake of nutrients resulting in adverse health effects
(Morgane et al., 2002). According to World Health Organization, malnutrition prevails due
to inadequate supply of nutrients and energy needed for appropriate growth and
maintenance of the body (Batool et al., 2015). It encompasses various health disparities
including under nutrition, over nutrition as well as vitamin & mineral deficiencies that
affect the physical growth and development of human body (Mamiro et al., 2005).
Malnutrition along with other nutritional ailments is the major reason of mortality in
developing as well as under developed economies (Ouedraogo et al., 2008). Some of the
known causes of malnutrition include poor dietary habits, accessability & affordability
issues and family & ethnic reasons (Kouassi, et al., 2010).
Protein energy malnutrition (PEM) is a critical health issue affecting about 200 million
children in developing countries causing death of about 60% infected infants. It is the main
cause of hospitalization of infants. Like malnutrition, the major risk factors associated with
PEM include financial instability, lack of maternal education, large family size, religious &
cultural traditions, improper breast feeding, poor healthcare, prolonged infection and gender
discrimination (Batool et al., 2015).
Besides other deficiencies, protein energy malnutrition has become a serious health concern
particularly in the developing nations where the population is accelerating at an alarming
rate (GOP, 2014). Pakistan, being a developing nation is also facing this menace (Powell,
2007). The national nutritional survey (2011) of Pakistan has depicted higher protein
deficiency in the vulnerable segments due to their poor dietary habits, hence affecting body
formation and function (NNS, 2011). Malnutrition is also linked with higher infant
mortality rate. To tackle the current pathetic situation of protein deficiency, some future
strategies are required. In this regard, nutritional deficiencies can be best dealt via food-
based approaches. The diet diversification or protein supplementation is considered an
appropriate approach for viable control of malnutrition (Müller and Krawinkel, 2005).
2
High quality proteins play an imperative role in maintaining better health of an individual.
Purposely, the proteins obtained from animal sources are of high quality as compared to
plant sources (Salcedo-Chávez et al., 2002). Though, animal proteins exhibit high quality
nevertheless, they are more expensive than vegetable proteins. Owing to high cost and
comparative dearth of food with animal proteins, it has become inevitable to find some new
sources of better quality protein. (Martínez-Flores et al., 2006). Additionally, increasing
cost and insufficient provision of animal proteins have diverted the interest of researchers
towards high protein oilseeds. These non-conventional protein sources also hold interactive
properties with other food components like water and lipids (Enujiugha and Ayodele-Oni,
2003).
Food industry has utilized plant proteins primarily from grains and legumes as potential
ingredients in numerous food products due to their balanced amino acid profile (Horax et
al., 2004). Developed countries have promoted the use of plant based protein in their
routine diet due to associated health claims (Ahmed et al., 2011). Nowadays, protein
supplementation through plant source is also gaining popularity in the developing world
(Khalid et al., 2003). This idea is further strengthened by the fact that animal proteins such
as milk, meat and eggs possess relatively higher cost (Chel-Guerrero et al., 2002).
Additionally, plants being the rich source of fibre, help to manage various bowel diseases
including colon cancer (Sirtori and Lovati, 2001). As a response, there is a dire need to
explore some non-traditional protein sources to combat protein energy malnutrition and
other health related disorders (Iqbal et al., 2006; Nunes et al., 2006; Becker, 2007).
The sesame (Sesamum indicum L.) is an imperative oilseed crop that belongs to the
Pedaliaceae family mostly cultivated in tropical areas. Globally, it is commonly known as
beniseed, gingely, sim sim and til. The height of sesame plant ranges from 50-100 cm and
has been widely grown around the world especially in Africa and Asia since decades.
According to FAOSTAT (2011), Asia and Africa contributed around 62.6% (2,489,518
tons) & 33.1% (1,316,690 tons) sesame to the global production, respectively. The sesame
seed is 2.80 mm long having 1.69 mm width with 0.82 mm thickness. Sesame seed is
mainly used for the extraction of edible oil with an oil extraction up to 47.8-52.2%.
Additionally, it is also used in the preparation of numerous snacks, bakery and
confectionary products (Becker, 2007; Ahmed et al., 2011).
The chemical composition of sesame seed revealed that it contains 25.8-26.9% protein,
2.50-3.90% fiber, 2.00-5.59% ash and 10.10-17.90% carbohydrate (Onsaard, 2012).
3
Sesame seed composition primarily depends on variety, environmental & genetic factors,
climate, ripening stage and time of harvest. It is one of the most ancient crops grown as a
condiment and for edible oil. Owing to high yield and premium quality of oil, sesame is
known as the “queen of the oil seed crops” (Akinoso et al., 2010). The extracted oil is
mainly used for cooking purposes however some proportion can also be used in perfumery,
pharmaceuticals and cosmetic industries.
Sesame oil is also used in the production of insecticides, soaps and paints. It is subjected to
hydrogenation to convert into medium melting fats which are further utilized in
manufacturing vanaspati, margarines and shortenings (Gandhi and Srivastava, 2007). The
fat free meal obtained after oil extraction exhibits a reasonable proportion of high quality
proteins that have the ability to be potentially used as functional ingredient in numerous
food commodities and nutritional supplements. The sesame meal acquired after extraction
of oil is a rich protein source i.e. about 50% and primitively utilized as animal feed (Iqbal et
al., 2006; Nunes et al., 2006; Becker, 2007).
The flaxseed (Linum usitatissimum) belonging to Linaceae family is commonly known as
“Alsi” in Indopak. The annual production of flaxseed has exceeded 3.06 million tons and
Canada leads as the highest flaxseed producer (about 38% of total production) around the
world. Flaxseed is a multipurpose crop mainly cultivated for the production of oil, seed and
textile fiber. It also contains an appreciable amount of high quality proteins and
polyunsaturated fatty acids (Pradhan et al., 2010). Generally, flaxseeds comprised of about
7.7% moisture, 20% protein, 41% fat, 28% fiber and 3.4% ash (Ganorkar and Jain, 2013).
For centuries, flaxseed has been utilized as a source of edible oil around the globe. Flaxseed
fiber also helps in reducing blood cholesterol along with laxation promotion (Payne 2000).
Nevertheless, flaxseed meal is among certain unexplored sources containing high quality
protein for human consumption. It is mainly used for the extraction of oil while the left over
meal is utilized in the production of animal feed. However, the nutritional composition may
vary with location, seed variety and environmental conditions. Since decades, flaxseed has
been successfully incorporated in numerous food products owing to its superior nutritional
and functional attributes. Nowadays, the trend is again shifting towards flax based foods
due to allied nutraceutics and balanced nutritional profile for the improvement of overall
health and wellbeing (Hussain et al., 2012). Flaxseed contains numerous bioactive
components which have fascinated the stakeholders in the field of food production to utilize
them in the development of a variety of functional foods.
4
Earlier, flaxseed was used as a food ingredient only in Asia and Africa (Berglund and
Zollinger, 2002). With the passage of time, its utilization extended around the world mostly
in developed countries. Since 2005, novel foods and other products are being manufactured
using flaxseed or its components. Such products are becoming prevalent in the US market
indicating high growth potential of flax based edibles in functional food industry (Morris,
2007).
Flaxseed contains substantial quantities of numerous essential components. The defatted
meal contains about 35-40% protein that finds its use in the livestock or poultry feed. This
proportion of proteins exhibit higher quantities of lysine, arginine and branched chain
amino acids. The balanced amino acid profile of protein isolated from defatted flaxseed
meal has paved the way for its utilization in value-added food products (Hall et al., 2006).
Flaxseed also contains both soluble and insoluble fibers which play a key role in efficient
digestive system. Soluble fiber in flaxseed exists in the form of mucilage and acts as an
effective cholesterol lowering agent whereas insoluble fiber helps in averting constipation
and the regulation of bowel movements (Jhala and Hall, 2010).
Canola (Brassica napus L.) is a widely cultivated oilseed crop in Canada and nowadays
grown throughout the world including different areas of sub-continent. The extraction rate
of canola oil is about 40% and resultant meal is a rich source of protein. Canola meal
contains about 35-36 g/100 g protein as well as 12 g/100g crude fiber contents along with
some important minerals and vitamins. The protein found in canola meal exhibits balanced
amino acid profile and a higher protein efficiency ratio (PER 3.29) as compared to other
plant based proteins (Knispel and Mclachlan, 2010).
The minerals in canola meal include potassium, calcium, iron, sulphur, phosphorus and
selenium. Some other important components found in canola meal include biotin, choline,
folic acid, thiamin, niacin and riboflavin. Currently, it is being used in livestock feed and
aquaculture industry due to better nutritional profile (Khattab and Arntfield, 2009; Canola
Council of Canada, 2014). The increasing canola oil demand results in the production of
additional meal that needs to be potentially utilized in several food products to enhance
their nutritional quality. Furthermore, the canola protein exhibits better functional
properties as well as superior protein efficiency ratio, biological value and balanced amino
acid ratio (Yoshie-Stark et al., 2008).
5
In the developing economies, inadequate supply and high cost of animal protein has
persuaded the food researchers to use proteins obtained from under-utilized sources i.e.
oilseed meals and legumes (Enujiugha and Ayodele-Oni, 2003). However, proteins isolated
from non-conventional sources must have the ability to properly interact with other food
components (i.e., water & lipids) to assist their incorporation in various food formulations
(Khattab and Arntfield, 2009). Now, the food industries have taken initiative for the
supplementation of protein isolates in numerous food products to fulfill protein
requirements. Purposely, protein isolates are prepared from oilseed meals using isoelectric
precipitation or salt precipitation methods (Iqbal et al., 2006; Nunes et al., 2006; Becker,
2007).
The isoelectric precipitation is considered as efficient technique as compared to salt
precipitation due to lower denaturation rate and higher surface hydrophobicity & foaming
expansion (Tang and Ma, 2009). The eventual use of plant proteins as food ingredients is
mainly dependent on their functional properties and the valuable quality characteristics
imparted to foods. Functionality refers to those attributes of a food ingredient that directly
affect its utilization. Moreover, the quality and acceptance of food products are affected by
the functional properties of specific ingredients (Mahajan and Dua 2002). The functional
attributes of protein isolates are assessed through water and oil absorption capacity,
foaming as well as emulsifying properties (Onsaard et al., 2010). Recently, food industry is
focusing to use cereal based foods as a suitable vehicle for protein supplementation because
of the increasing demand of bakery products (Mishra et al., 2012).
Conventionally, wheat is used as a staple food in Pakistan which is deficient in lysine
(Anjum et al., 2005). Nonetheless, plant proteins are rich in essential as well as non-
essential amino acids providing a balanced protein profile to the foods. Moreover,
numerous food commodities can be supplemented with plant proteins in order to address
the protein deficiency especially in infants and young children.
Product development is not only constrained to formulate innovative food items but also
encompasses the concept of reformulation. Amongst different value added food
commodities, bakery products are an appropriate choice for the supplementation of protein
isolates from non-conventional sources (Bakke and Vickers, 2007). Purposely, food
technologists have successfully striven to develop various composite flour formulations by
supplementing wheat flour with various defatted meals at industrial scale (Junqueira et al.,
2008; Škrbić and Filipčev, 2008).
6
The food products made from composite flours are widely being used in various parts of the
world on commercial scale. These products exhibit additional nutritional and sensory
attributes which help to enhance their acceptability among masses. The protein content of
flour plays an imperative role in the preparation of bakery products (Wieser, 2007).
Moreover, considerable importance has been given to wheat flour fortification with high
quality protein to improve the nutritional and sensory characteristics of the final product.
Hence, plant based protein isolates play imperative role to enhance nutritional and
rheological characteristics of flour, subjected to supplementation process which is further
utilized to address protein energy malnutrition.
Keeping in view the above mentioned facts and severity of protein energy malnutrition in
Pakistan, the present study is aimed to serve the vulnerable segment with high protein diet
by adding protein isolates from different oilseed meals i.e. sesame, flaxseed and canola.
The selection of best protein isolates based on protein yield and functional properties as
well as bioefficacy study is the prime objective of the planned research. Previously, few
research studies have been conducted using oilseed protein isolates i.e. sesame, flaxseed
and canola in preparing certain food products like bread. However, these protein isolates
have not been used in the preparation of muffins in any reported research work. Therefore,
in the product development phase, muffins were prepared by adding selected protein isolate
in varying proportions to enhance protein level in the resultant product. The objectives of
the proposed research are herein;
• Preparation and characterization of protein isolates from sesame, flaxseed and canola
meals
• Selection of best protein isolate on the basis of protein yield, functionality and
bioefficacy assessment
• Supplementation of muffins with the selected protein isolate to evaluate their nutritional
value and hedonic response
7
Chapter 2
REVIEW OF LITERATURE
In developing world, severe food and nutrition insecurity due to rapid increase in
population and rising prices of animal proteins have focused researchers to explore some
new sources of protein. Protein energy malnutrition is being prevalent among the masses
especially infants and young children owing to the scarcity of good quality proteins. In this
perspective, plant proteins are becoming pivotal among dietary protein sources as these
exhibit good quality amino acids especially lysine which is deficient in wheat. Amongst
various plants, oilseeds are non-conventional sources of proteins as these are primarily
utilized for oil extraction purpose. The defatted meal obtained after oil extraction from the
oilseeds contains an appreciable quantity of excellent quality protein. In the current
research investigation, protein isolates were prepared from different oilseed meals. Further,
best selected protein isolates were utilized for the preparation of protein enriched muffins.
To support and for better understanding of the study, the literature has been reviewed as
follows:
2.1. Malnutrition
2.2. Oilseeds; nutritional aspects
2.3. Amino acid profile
2.4. Functional properties
2.5. Gel Electrophoresis (SDS-PAGE)
2.6. Bio-evaluation
2.7. Composite flours
2.8. Bakery products
2.1. Malnutrition
Malnutrition exists among the population with inadequate intake of food that is linked with
the socio-economic status (Sereebutra et al., 2006; Padula, et al., 2009). Precisely,
malnutrition is caused by inadequate uptake of nutrients that are crucial for body’s normal
growth and function (Campanozzi et al., 2009). Malnutrition is mostly prevalent among the
infants and children. Resultantly, these children are more vulnerable to various diseases and
infections, which ultimately lead to increased hospitalization and a poor prognosis (Bejon et
al., 2008). Worldwide, approximately one third of the children are affected by malnutrition
8
amongst developing countries. Moreover, it is a serious health problem causing deaths of
about 60% infected children under age five. (Faruque, et al., 2008; Cervantes-Ríos et al.,
2012).
Protein energy malnutrition (PEM) has become a severe threat for a huge segment of
population especially in developing and developed nations. Patients suffering from PEM
are more susceptible to health disorders including hypoglycemia, hypothermia and
electrolyte disturbances. PEM may also result in premature birth, infectious tuberculosis,
mental disorder and vomiting. Moreover, parasitic diseases like measles, diarrhea,
whopping cough and malaria can be caused due to PEM (De-Mutsert et al., 2008). The
severe depletion of protein from the body may result in kwashiorkor and marasmus. The
term “marasmus” is derived from Greek meaning wasting or withering. It refers to the
chronic disorder developing over an extended period of time due to inadequate supply of
energy. Marasmus usually occurs in epidemics as a result of famine and is prevalent in
mostly in Asia, Africa & South America. Patients suffering from long-term infections like
anorexia nervosa and chronic pulmonary disease are more prone to marasmus. Children
suffering from marasmus lack proper growth due to wasting of muscles and lack
subcutaneous fat (Tomlinson et al., 2005).
Kwashiorkor is derived from the Kwa language of Ghana meaning “sickness of weaning.”
In 1933, this term was used by Williams for the first time and it encompasses the
inadequate protein supply with sufficient energy intake. Edema is the distinctive factor
between kwashiorkor and marasmus (Dicko et al., 2006). PEM is mostly prevalent in
children living in areas with limited food supply or affected by famine. It is also
predominant in various countries having rice, corn and beans as main diets (Edhborg et al.,
2000).
PEM may also cause deficiency of various other essential nutrients including vitamins, fatty
acids as well as trace elements that contribute to the growth and maintenance of body
(Black, 2003). The major risk factors associated with PEM include family size, ignorance,
residence, poor maternal education, poverty, religious and cultural food customs,
inadequate breast feeding, lack of quality healthcare, chronic infections, malformations,
child’s gender and incomplete immunization (Nova et al., 2002). Children suffering from
PEM face the deficiency of total proteins which may be reduced up to 50% in severe cases.
Kwashiorkor is the specific condition for the reduction of total serum protein and albumin
which are not reduced in marasmus (Batool et al., 2015).
9
Children with severe PEM also suffer from a lower stroke rate with a thinner heart that
refers to reduced heart muscle mass (Mamoun et al., 2005). The heart is affected by
inappropriate excretion of fluids and sodium from the body by kidneys. Such condition
occurs in kwashiorkor-marasmic or kwashiorkor state. Moreover, Hypoproteinaemia is a
common feature of PEM in which plasma albumin and some glycoprotein fractions are
reduced (Kovesdy et al., 2009). Therefore, PEM may cause enhanced drug toxicity in
children owing to their reactions against drug treatment (Muntner et al., 2005).
PEM is mostly common in children and pregnant women but it may also occur in old
individuals. The statistical data indicate that PEM resulted in hospitalization of about 50%
old people. The reason may be the inability to respond efficiently to the condition of
inappropriate diet along with some other stressful situations. With ageing, qualitative as
well as quantitative changes in concentration of circulating amino acids may occur in
patients suffering from PEM (Keith et al., 2004).
Recently, malnutrition has become the most prominent public health issue in developing
nations. Nutrition and health are interconnected as both are mandatory for a healthy life.
However, there is dire need to utilize nutritional knowledge for the improvement of health.
Though understanding about PEM along with other micronutrient deficiencies is nowadays
common among the masses; nonetheless, these deficiencies still need to be addressed for
complete eradication. As PEM is directly linked to poverty, it can be overcome by effective
programs. Although the ratio of people affected by PEM reduced during 1970s and 1980s,
nonetheless it actually increased in certain areas of Africa. Presently, the total number of
malnourished people continues to increase with rising population (Batool et al., 2015).
Similarly, the total number of children suffering from malnutrition is still increasing at
alarming rate.
Although considerable progress has been made to overcome PEM over the last few
decades, it is still a major public health problem globally. In this context, the United
Nations Food and Agriculture Organization (FAO) indicates that 868 million people are
suffering from PEM worldwide (FAO, 2012). Though two decades earlier, only 130 million
people were suffering from this nutritional problem, but still around 15% of the population
of the developing world has become the victim of this threat. Purposely, one of the
Millennium Development Goals (MDG) was focused to reduce PEM to half by 2015.
Similarly, the World Food Summit Goal (WFSG) of reducing hungry people to half till
2015 has not been achieved (Gómez et al., 2013).
10
2.2. Oilseeds; nutritional aspects
Proteins being integral parts of healthy diet are involved in various physiological functions
thus their adequate intake ensures proper growth and development of the body (Gulati,
2010). High quality protein is an essential component of diet to maintain good health. The
protein from animal source is considered as high quality but relatively more expensive as
compared to vegetable proteins. Due to these hurdles, it has become essential to explore
some new and cost effective sources of protein for the development of protein enriched
quality food products (Salcedo-Chávez et al., 2002). Quality and quantity of proteins
utilized in daily diet can be enhanced by the incorporation of non-conventional protein
sources in numerous diet formulations i.e. supplementation in wheat (Anjum et al., 2005).
In this context, exploring the non-conventional sources rich in quality proteins can play an
imperative role to cope with malnutrition and other health disorders especially in rising
economies (Müller and Krawinkel, 2005; Becker, 2007).
2.2.1. Sesame
Sesame, an oilseed crop belonging to the family Pedaliaceae, is cultivated in many areas of
the world including Eastern Asia Central America and Tropical Africa. Worldwide, its
production is about 3,976,968 tons with Africa (1,316,690 tons) and Asia (2,489,518 tons)
as the major production areas, that constitute about 33.1% & 62.6% of the total world
production, respectively (FAOSTAT, 2011). In sub-continent, sesame seeds are used in
making certain indigenous food products like tahin (sesame butter) and halva. The amount
of oil, protein, carbohydrate and ash in sesame seed are reported as 40-50%, 20-25%, 20-
25% and 5-6%, respectively. Furthermore, sesame seed composition mainly depends on
variety, genetic characteristics, cultivation, environmental factors, ripening stage, climate
and the harvesting time (Onsaard, 2012).
Sesame seeds are anti-aging in nature containing appreciable quantity of vitamins E, A and
B complex. Similarly, sesame exhibits sufficient amount of minerals like calcium, iron,
phosphorus, copper, zinc, potassium and magnesium. Likewise, sesame oil is rich in
linoleic acid that can be utilized in products like shortenings, margarine and vanaspati after
initial processing. This attribute makes sesame a virtually impeccable food and one of the
major sources of good quality edible oil (Bukya and Vijayakumar, 2013). Sesame is also an
imperative source of protein (Onsaard, 2012). The antioxidant lignans (sesamin, sesaminol
and sesamolin) present in sesame oil have shown hypocholesterolemic effects in humans.
11
This phenomenon has created an increasing interest in utilization of sesame oil in numerous
food commodities (Achouri et al., 2012).
Sesame oil is processed using organic solvents or mechanical pressing. The meal left after
oil extraction is a valuable by-product. During sesame oil extraction process, different
forms of defatted meals can be obtained that include whole & dehulled seed meal and
defatted whole & dehulled meal. The sesame meal obtained after oil extraction possesses
various functional properties (Onsaard, 2012). Sesame meal is generally composed of
7.92% moisture, 30.56% protein, 27.83% fat, 6.22% fiber, 28.14% carbohydrates and
5.27% ash. The protein content (41.15-49.58%) of defatted sesame meal is increased after
the extraction of oil (Onsaard et al., 2010).
Sesame seeds contain storage proteins that are composed of albumins (8.6%), globulins
(67.3%), glutelins (6.9%) and prolamines (1.4%) (Zaghloul and Prakash, 2002). Sesame
proteins can be extracted through different methods including isoelectric precipitation (pI)
and alkaline or salt extraction (Cano-Medina et al., 2011). In isoelectric precipitation
method, proteins are extracted at isoelectric point. Generally, the protein molecules exhibit
equal number of positively and negatively charged groups therefore, minimum solubility is
observed at the isoelectric pH.
Previously, Onsaard et al. (2010) used defatted sesame flour to prepare protein concentrates
via isoelectric precipitation using salt and alkali solution. They observed that protein
recovery ranged from 19.5% to 35.3% due to difference in solutions and their pH.
Similarly, the protein contents of sesame protein concentrates (SPC) at pH 9 (82.9%) and
pH 11 (83.3%) were higher as compared to SPC-salt (75.5%). Sesame proteins have been
categorized into various classes based on different criteria including Osborne sequential
extraction and diverse solubility.
Dehulling of sesame seed is essential as the hull exhibits substantial quantity of oxalic acid
(2-3%), that may intricate with calcium thus reduce its bioavailability. Likewise, the
presence of indigestible fiber in hull reduces the protein digestibility. The undesirable
constituents like phytates, fiber, soluble sugar, and oxalates are removed to a large extent
for the production of protein isolates or concentrates using dehulled and defatted seed (Liu
and Chiang, 2008). However, the modification of proteolytic enzyme can be a handy tool to
enhance numerous functional properties and to upsurge the application of protein in
12
numerous food commodities. Partial hydrolysis of proteins results in smaller molecular size
peptides as compared to novel proteins (Bandyopadhyay and Ghosh, 2002).
Sesame protein isolates contain appreciable amounts of sulfur-containing amino acids like
methionine (5.9 g) and cystine (3.7 g) (Onsaard et al., 2010). Most sesame varieties are
deficient in lysine, the first limiting amino acid. However, varieties having darker seed
coats contain lysine contents. Isoleucine is another limiting amino acid as compared to the
reference protein documented by FAO. Tryptophan quantity is also limited in various
proteins but sesame exhibits substantial amount of this amino acid (Ranganayaki et al.,
2012).
Numerous studies have indicated that sesame protein isolates have potential to utilize as
nutritional supplement in various food commodities including beverages and bread (Khalid
et al., 2003; López et al., 2003). Purposely, sesame protein must exhibit exceptional
functional properties in order to incorporate as an ingredient in numerous foods. These
functional properties include solubility, fat absorption capacity, water holding capacity,
foaming and emulsifying properties (Onsaard et al., 2010).
2.2.2. Flaxseed
The flax (Linum usitatissimum) being a member of family Linaceae, is a blue flowering rabi
crop commonly known as “Alsi”. According to Touré and Xueming (2010), the annual
production of flaxseed was 3.06 million tons and Canada is the major producer in the world
(about 38% of total production). The flaxseed grain is oval and flat, with a size of about
2.5×5.0×1.5 mm. It has smooth glossy surface with the color varying from dark brown to
yellow. The texture of flaxseed is crispy and chewy with a pleasant nutty taste.
Furthermore, beyond its ability as an oilseed crop, the proximate composition of flaxseed
makes it an efficient ingredient for utilization in numerous food commodities. In present
era, the trend towards functional food has improved significantly owing to increased health
awareness among the consumers. Therefore, defatted flaxseed can be a novel and high
quality source of protein (Ganorkar and Jain, 2013).
Flaxseed exhibits distinctive compositional characteristics with special reference to high
concentration of oil, proteins and functional components like lignan. Flaxseed oil is rich in
α-linolenic acid (ALA) with antioxidative, anti-inflammatory, hypolipidemic, anti-platelet
and hypotensive properties (Basett et al., 2009). Besides the potential use of flaxseed in
human food, it is also fed to the animals in different areas of the world especially in Europe
13
and Middle East since early times (Oomah and Mazza, 2000; Daun et al., 2003). Apart
from this, flaxseed also finds its use in many other industries including fiber, oil and
chemical industries. Its fiber is used in the production of linen whilst, flaxseed oil finds its
use in the formulation of paints and certain oleo chemicals that are based on linolenic acid
(Daun et al., 2003).
Oomah et al. (2006) indicated that flaxseed contains high quality proteins that are mainly
composed of salt soluble globulin (64-73.4% of total seed protein) and water soluble
albumin (26.6-42% of total seed protein). It possesses sulphur containing and branched
amino acids in well-balanced ratio. Similarly, flaxseed also comprises of high fat contents
along with considerable amount of protein, dietary fiber and potassium. Studies on
proximate composition of flaxseed indicated that it contains 38.76% crude fat, 21.23%
protein, 4.53% moisture, 8.02% crude fiber, 3.47% ash and 23.99% NFE. This composition
may vary with location, environmental conditions and seed variety of the crop. Mineral
profile of flaxseed depicted 826.32 mg/100g of potassium, 430.54 mg/100g magnesium,
240.80 mg/100g calcium, 32.43 mg/100g sodium and 6.10 mg/100g iron (Hussain et al.,
2008). The decrease in protein content of flaxseed with increase in oil content can be
changed by applying appropriate plant breeding methods and can also be affected by
geographical conditions (Daun et al., 2003).
Previous research studies have revealed that the protein content in flaxseed ranged from
10.5-31%. Specifically, Khategaon cultivar in India exhibits 21.9% protein content.
Moreover, the protein content of dehulled and defatted flaxseed may also vary significantly
as it depends upon location and processing of seed. Additionally, dehulling of flaxseed
caused increase in protein level from 19.2-21.8% (Ganorkar and Jain, 2013).
Flaxseed protein contains somewhat high amount of arginine, glutamic acid and aspartic
acid while lysine, cysteine and methionine are considered as limiting amino acids
(Ganorkar and Jain, 2013). Albumin & globulin are the major types of protein in flaxseed
with globulin fraction up to 73.4% and the albumin 26.6% of total protein. During
germination the total amino acid content of the flaxseed increased by 15 times with highest
increase (i.e. 200 times) in leucine and glutamine as compared to the original seed
(Ganorkar and Jain, 2013).
Flaxseed protein has proven effective to lower plasma cholesterol and triglycerides (TAG)
better than casein and soy protein (Bhathena et al., 2002). Another study on the use of
14
flaxseed as a food ingredient indicated that protein in biscuits prepared using composite
flour comprising flaxseed (15%) improved from 6.5-8.52%. Moreover, up to 12% addition
of flaxseed flour revealed that it causes no deleterious effect on the sensory attributes of
cookies (Khouryieh and Aramouni, 2012). Furthermore, as flaxseed is gluten-free, thereby
gluten sensitive people can add flaxseed in their diet (Morris, 2003).
Flaxseed can find its use in numerous diet plans as it can be consumed in either raw or
cooked form. However, heating of flaxseed may cause some physical as well as chemical
changes that ultimately alter the effect of flaxseed in food system. The changes occurring
due to oxidation may cause thermal deformation of vitamins and certain other components.
Likewise, several studies have delineated the lipid lowering effect of flaxseed which may
depend on the dosage. However, the effect of heating (as a food preparation and processing
step) on the lipid lowering characteristic of flaxseed is not evident. Moreover, in numerous
studies, flaxseed has been used in muffins and bread without considering the possible
effects of heating on flaxseed attributes (Khalesi et al., 2011).
Flaxseed (Linseed) exhibits potential health benefits owing to excellent nutritional profile.
Recently, more people are getting awareness about the benefits of flaxseed regarding health
and food applications. Recently, flaxseed is being potentially utilized in baking industry for
incorporation in numerous formulations. Keeping in view the utilization of flaxseed in
foods, the general recommendation for daily intake has been set as 1–3 table spoons for
ground flaxseed and 1 table spoon for flaxseed oil. In recent years, flaxseed has emerged as
an exceptional nutritive and functional ingredient for food commodities (Ganorkar and Jain,
2013).
2.2.3. Canola
Canola (Brassica napus L.) belongs to the mustard family grown in various parts of the
world mainly for oil production and as an ingredient in animal feed. Canola is a summer
crop mostly grown in the temperate and cool areas around the globe. It is also cultivated as
a winter crop in northern Iran. Canola oil exhibits minute quantity of saturated fat as
compared to other vegetable oils (Aminpanah, 2013).
Tan et al. (2011a) explicated that the name “Canola” was first presented in Canada in 1979
particularly representing oil producing rapeseed varieties having reduced amount of erucic
acid (less than 2%) and a lesser quantity of total glucosinolates (30 μmol/g meal). Canola is
an imperative oilseed crop native to Canada which is among the major producers of canola
15
with a total area of 5.24 million ha. In 2006-2007 the yield of canola was approximately
1.72 t/ha and production was recorded as 9.00 million metric tons (Statistics Canada, 2008).
It is mainly utilized for the extraction of oil and the protein-rich meal obtained as a result is
used as livestock feed and in aquaculture industry.
Owing to increasing demand for canola oil around the world, the production of meal will
also escalate due to increased oil extraction. As the oil free meal is rich in protein contents,
therefore, it provides an opportunity for better understanding of canola protein in order to
increase its utilization as a food ingredient for human consumption. This also helps to
upsurge the market demand of meal (Tan et al., 2011b). Furthermore, canola meal has high
PER and BV as compared to soybean which makes it an imperative food ingredient
(Pastuszewska et al., 2000). These characteristics fulfill the nutritional requirement of
young children as well as adults. Canola meal mainly comprises of storage proteins like
napin & cruciferin and a structural protein known as oleosin which is associated with oil
bodies (Ghodsvali et al., 2005). The canola protein isolates prepared from canola meal
possess better functional properties which enables them to become part of food system
(Yoshie-Stark et al., 2008).
Moreover, canola meal (CM) contains high quality protein that can be utilized as ingrdient
in numerous industrial products including bio-polymers, bio-fuels, fertilizers, soil
amendments, adhesives and surfactants (Bonnardeaux, 2007). Canola meal has been
documented as the most extensively traded protein source after soybean. In 2004/2005 the
total production of canola meal was 207 mMT that represents 12.40% of total world meal
protein (Khattab and Arntfield, 2009).
However, resultant canola meal can also be utilized in various food industries for the
manufacturing of protein enriched products. The increasing production rate of canola over
the last 40 years has ranked it as second largest oilseed crop of the world. For animal feed,
canola meal ranked second after soybean meal (USDA, 2010). Canola protein isolates can
also be used as fortificants or supplement in various food products for human consumption
(Uruakpa and Arntfield, 2004; Cumby et al., 2008).
Canola meal contains considerable amount of protein (35-36 g/100g) having balanced
amino acid profile, crude fiber (12 g/100g) along with appreciable quantities of minerals &
vitamins (Pastuszewska et al., 2000). In spite of the fact that canola meal contains high
quality proteins, it has not been widely considered for its functional properties and other
16
nutritional attributes. The functional attributes play imperative role in numerous foods,
enhancing the quality and nutritional status of the commodity (Khattab and Arntfield,
2009).
Canola seeds are initially ground and then subjected to defatting process in Sohxlet
apparatus. Purposely, hexane is used to remove fat from the ground canola seed (Wu and
Muir 2008). Furthermore, the defatted material is placed under fume hood or oven at 40 ◦C
for drying (Ghodsvali et al., 2005). The defatted meal is ground enough to pass through 40
or 60-mesh screen that makes the maximum interaction of defatted meal with chemicals
required for protein isolation (Wu and Muir 2008). The canola protein isolates (CPIs) are
prepared via alkaline extraction using NaOH solution and finally precipated through dilute
acid (Aluko and McIntosh 2001).
2.3. Amino acid profile
Amino acids being important building blocks of protein play imperative role in determining
protein quality. The canola protein isolates (CPI) contain higher amounts of leucine,
glutamine, arginine and glutamic acid while lower quantities of sulfur-containing amino
acids (Aider and Barbana, 2011). Lysine content of CPI mainly depends on the methods of
extraction and ranged from 5.04 to 6.34% that is almost equal to infant’s requirements.
Similarly, CPI contains a considerably higher quantity of threonine (4.49% to 5.30%), in
comparison with both SPI (3.98%) and casein (3.70%) (Wang et al., 2008).
The lysine/arginine ratio determines the atherogenic and cholesterolemic effects of a
protein. This ratio depends on the extraction method for canola protein and varied from 0.7-
0.9 that indicates lower level in comparison with casein (2.2). This suggests that canola
protein isolates have less atherogenic and lipidemic activity as compared to casein.
Moreover, like SPI and casein, CPI contains abundant quantity of glutamine that depends
on the extraction method. CPI comprises 17.27-23.21% glutamine, as compared to casein
(19.00%) and SPI (20.67%). Similarly, CPI exhibit higher histidine content (3.14% to
3.17%) than casein and soy protein. Therefore, in general, CPI is a good source of
glutamine, arginine and histidine (Tan et al., 2011a).
Protein profiles of meals from various canola species were similar in non-reducing
conditions having polypeptides with molecular weights varying from 12-80 kDa. Similarly,
4 major polypeptides were reported to have molecular weights of 16, 18, 30, and 53 kDa
accounting for more than 55% of total polypeptides present in canola meals. Another study
17
conducted by Aluko and McIntosh (2005) indicated that B. juncea meal consists of
polypeptides having molecular weight ranging from 2-80 kDa. Likewise, molecular weight
of CPI ranged from 14 to 59 kDa with 8 major and 6 minor bands (Wu and Muir, 2008).
2.4. Functional properties
Functional properties refer to physical as well as chemical attributes of proteins that affect
their behavior in food system during processing, consumption and storage. Functional
properties of proteins are mainly classified into three categoriess such as (i) attributes
related to structure and rheology like viscosity, thickening & gelation (ii) those associated
with hydration characteristics i.e. water & oil holding capacity and solubility (iii) those
linked to protein surface including foaming & emulsification. Functional properties play
vital role in food formulation and include bulk density, foaming capacity & stability, water
& oil holding capacity, viscosity and gelation. However, some intrinsic factors like protein
sources and their molecular size & structure influence these properties (Tan et al., 2011a).
Proteins exhibit good emulsifying properties therefore, these are important emulsifying
agents to be potentially utilized in various foods. Proteins have tendency to decrease
interfacial tension between oil & water thus facilitating emulsion formation. Moreover,
proteins help to retard coalescence by stabilizing the oil droplets. The formation and
stability of emulsions involve various physicochemical factors which also help to improve
their textural properties (Khattab and Arntfield 2009).
Recently, the emulsifying properties of meals & protein isolates obtained from oilseeds
have been evaluated and certain different terminologies were used for these properties. For
example, emulsifying capacity (EC) and emulsion activity index (EAI) both reflect the
emulsion forming capability of protein. EAI designates the interfacial area available for
coating by the surfactant like proteins. Therefore, it is calculated using the volume of
dispersed phase (Ø), turbidity of emulsion (T) and protein (w/v) of aqueous phase prior to
the formation of emulsion. However, EC can be determined in easier way by measuring the
volume of oil emulsified per gram protein isolate (Yoshie-Stark et al., 2008; Khattab and
Arntfield, 2009). Nonetheless, emulsion stability (ES) is determined by comparing the
initial emulsion volume with that of emulsified layer after 30 min at room temperature i.e.
20◦C (Aluko and McIntosh 2001).
Foaming properties depend upon the formation of foam which is a 2 phase system
comprising air bubbles surrounded by a constant liquid phase. Proteins or surfactants play
18
vital role in the formation and stabilization of foams. Previous research studies evaluated
canola proteins as foaming agent by determining the properties like foaming capacity and
stability. These properties are associated with the binding ability of proteins to the air-water
interface forming foam particles along with protein–protein interaction that helps in the
formation of resilient interfacial membranes in order to stabilize the foam particles
(Sanchez-Vioque et al., 2001).
The gelling behavior of proteins has been illustrated using the term least gelling
concentration (LGC) (Khattab and Arntfield 2009). For the purpose, test tubes containing
solutions are heated to form different gelling concentrations. The test tubes are then
inverted and LGC is determined by observing the concentration at which the gel did not slip
out (Pinterits and Arntfield 2007). Afterwards, various other methods to study the
rheological properties or gel microstructure were also used (Pinterits and Arntfield 2008).
The sesame protein isolates have 2.10 mL/g water holding capacity, 1.50 mL/g oil holding
capacity and 0.71 g/mL bulk density. In a research investigation, sesame protein
concentrate was analyzed for the effect of NaCl concentration and pH on foaming as well
as emulsifying properties along with protein solubility. Results indicated that minimum
protein solubility (2.2%) was observed at pH 4. However, an increase in solubility (6.6 -
13.1%) was noticed as the pH increased from 2 to 10. Likewise, sesame proteins solubility
improved with increasing ionic strength (Onsaard, 2012).
Moreover, 6.2 mL oil/g emulsion capacity of sesame proteins was calculated at 1.0 M salt
concentration. Nonetheless, increase in NaCl concentration enhanced the stability of
emulsion as it ranged from 42-70%. In this study, the most stable foam was produced at 0.5
M NaCl after whipping for 120 min whilst 1.0 M NaCl concentration showed the least
stable foam (Onsaard, 2012). In another research exploration, Khalid et al. (2003) analyzed
sesame proteins for nitrogen solubility and delineated that the maximum nitrogen solubility
was 90% at pH 3 whilst minimum 12% at pH 5. Resultantly, emulsifying and foaming
properties were affected by pH levels as well as salt concentrations.
The sesame proteins also depicted remarkable results for other functional attributes. The
bulk density was reported as 0.71 g/mL, water holding capacity 2.10 mL/g and oil holding
capacity 1.50 mL oil/g. The hydrophilicity/hydrophobicity balance is mainly dependent on
the composition of amino acids, predominantly at protein surface and ultimately affects the
solubility of protein. Therefore, increased protein solubility depends on the factors
19
including amount of hydrophobic residues, higher charge & electrostatic repulsion as well
as ionic hydration near the isoelectric pH (pI). Moure et al. (2006) delineated that
denaturation of proteins affect the solubility owing to changes in the ratio of surface
hydrophilicity/hydrophobicity.
Protein solubility is also affected by the salting-in and salting-out effects that influence
foaming, thickening, emulsification and gelation properties of proteins. In a comparative
study regarding solubility and emulsifying properties of sesame protein isolates and
soybean protein isolates, it was observed that sesame protein isolate exhibited better
emulsifying activity index (EAI) as compared to soybean protein isolate at a specific pH
range (López et al., 2003). Similarly, sesame protein isolates showed about 15 times
increased solubility as compared to soybean protein isolates at pH 2-4, however the
solubility changed in neutral as well as alkaline pH conditions.
In another research study, Kanu et al. (2007) prepared sesame protein isolates by variation
in time, pH and flour to water ratio and concluded that the resultant isolates exhibited least
solubility at pH 4.5-5, which is equivalent to commercial soy protein. According to Onsaard
(2012), isoelectric point (pI) for sesame proteins is between pH 4.4 and 4.8.
Sesame and soy proteins showed an increase in solubility on either side of the isoelectric
region. The pH affects the charge on protein as well as the electrostatic balance between
them. Proteins exhibit positive or negative net charges above or below pI that eventually
improve solubility. Mostly proteins have least solubility at isoelectric pH because
hydrophobic interaction between surfaces is maximum while ionic hydration and
electrostatic repulsion are minimum. The interaction of protein with oil and water play vital
role in food system as it affects the flavor and texture of food (Onsaard, 2012). Moreover,
sesame protein isolates exhibited higher water holding while lower oil holding capacity as
compared to soy protein isolates.
Several research studies have been conducted to elucidate the solubility, hydrophobicity
and structural characteristics of proteins. These properties are important to determine
various functional attributes of protein isolates (Krause et al., 2001). Bulk density is
dependent upon particle size and inter-particle forces. Though, a few studies have indicated
that protein isolates exhibit low bulk density owing to high amount of proteins and low
quantity of carbohydrates (Krause et al., 2002). Earlier, experiments were conducted to
detemine functional properties of three different sesame protein concentrates prepared by
20
two different methods (Onsaard et al., 2010). It was concluded that least protein solubility
was noticed at pH 5. Moreover, the tested protein concentrates showed higher solubility at
pH 3, 8 and 9 in comparison with soy protein.
Additionally, emulsion activity index of prepared sesame proteins was higher while
emulsion stability index was lower as compared to soy proteins. Likewise, sesame proteins
exhibited lower fat absorption capacity, water holding capacity as well as foaming
properties as compared to soy proteins. Furthermore, the viscosity of sesame protein
isolates was affected by temperature and was noted to be higher at 60◦C than at 40oC due to
denaturation of protein at high temperature that increases the viscosity. The sesame protein
isolates with low viscosity are more suitable for the preparation of protein enriched drinks
and beverages. These protein isolates can also be supplemented in infant formulations
(Kanu et al., 2007).
Most proteins have low solubility at isoelectric pH and are deficient in electrostatic
repulsive forces due to which they become poor emulsifiers. This is a vital property of the
protein concentrates which find their use in products like mayonnaise and salad dressings.
In a research study, the maximum value for emulsion stability was observed at pH 8
(88.4%) while minimum at pH 4 (50%). Conclusively, the proteins exhibit more effective
emulsifying properties at a pH range other than their isoelectric point (Martínez-Flores et
al., 2006).
Flaxseed proteins have surface active characteristics and form foam upon whipping.
Generally, flaxseed proteins produce less amount of foam at pH 6 (12%). However, showed
higher foam stability (83.3%) at this pH. Possibly, attractive and repulsive forces in proteins
gain an equlibrium at isoelectric point that helps in the formation of foam and results in
increased foaming stability. The maximum foam capacity (40 & 42%) was observed at pH
2 and 10, respectively (Martínez-Flores et al., 2006).
Aluko and McIntosh (2001) explicated that the emulsion activity index of two canola meals
did not differ significantly. Conversely, momentous variations were observed in foaming
properties of different canola meals. Likewise, low molecular weight and superior
interfacial properties of proteins cause an increase in EAI at the oil-water interface (Aluko
et al., 2005). Similarly, Aluko and McIntosh (2001) illustrated that B. napus meal showed
the ability to form emulsions particularly with lower emulsion stability as compared to B.
21
rapa meal. It occurs due to less effective interaction of proteins interface which helps in the
formation of strong interfacial membrane.
Later, it was observed that the canola proteins have high EAI along with extraordinary
emulsion stability (Khattab and Arntfield, 2009). Regardless of the variations in
emulsifying properties of canola meals, SDS PAGE revealed some resemblance in
polypeptides of 4 different types of seed. However, there may be some potential variation in
structure or conformation of protein (Aluko and McIntosh 2001).
In research studies conducted by Aluko and McIntosh (2001) & Aluko et al. (2005), it was
explained that soybean flour has better emulsifying properties as compared to other oilseed
meals. Nevertheless, research outcomes of Ghodsvali et al. (2005) described that defatted
canola meal exhibit superior emulsifying characteristics. The authors determined the
emulsion forming capacity of proteins and concluded that canola meals (B. napus) exhibit
better emulsifying activity in comparison with commercially available soybean meal.
Likewise, Khattab and Arntfield (2009) recorded comparable results and described that
canola meal (B. napus) has better emulsifying properties than soybean meals. However, the
emulsifying properties of canola meal possibly depend on the extraction and analytical
methods.
Heat treatment causes denaturation of proteins that results in lower emulsion capacity and
stability and thereby reduces nitrogen solubility. Moreover, assessing the variations in
average particle size and distribution is an effective method to determine emulsification
capacity of canola proteins (Agboola et al., 2007). The use of such method to understand
emulsion properties in an efficient manner helps to resolve some of the contradictions in
results.
Pedroche et al. (2004) focused on acid-precipitated protein isolates for emulsifying
properties and concluded that these properties are affected by extraction pH. They
explicated that proteins of B. carinata isolated at various alkaline pH levels exhibited poor
emulsifying properties as compared to meal. Resultantly, the emulsifying properties
declined with an increase in extraction pH (10-12). Moreover, defatting process greatly
affect the emulsifying and other properties of proteins (Vioque et al., 2000).
The protein isolates (B. napus) prepared using ultra filtration process have higher EC as
compared to soy, whole egg (Gao et al., 2001) and some other plant proteins like mung
bean, lupin, pea and sesame (El-Adawy 2000; El-Adawy et al., 2001; Gao et al., 2001;
22
Khalid et al., 2003). Thus, ultra filtered protein isolates have substantial and improved
emulsifying properties than the alkali-extracted isolates.
Moreover, the interaction of polysaccharides with canola protein isolates (CPI) proved
effective to enhance emulsifying properties. Likewise, Uruakpa and Arntfield (2005)
observed a momentous improvement in the emulsifying properties of CPI by adding κ-
carrageenan or guar gum. The addition of polysaccharides and their interaction with CPI
require different pH. Modification of protein through hydrolysis is an alternative technique
to improve solubility along with emulsifying properties of proteins. Furthermore, the
foaming properties of Brassica napus meal are considerably superior in comparison with B.
rapa meal and soybean flour (Khattab and Arntfield (2009). Both studies indicated that
foaming capacity of canola meal was comparatively higher than soybean meal irrespective
of the method of analysis.
Numerous research studies have indicated that the foaming properties of meals were
relatively superior as compared to protein isolates prepared through acid or calcium
precipitation. Moreover, the protein isolates preparation process consistently reduced the
foaming capacity of different brasicca meals (Aluko et al., 2005). Likewise, lower foaming
stability of meals was noticed as compared to protein isolates. The reason might be the
protein denaturation occurring at high pH during the protein isolates preparation procedure.
Protein isolates having high foaming capacity (FC) may not exhibit high foaming stability
(FS). Likewise, Aluko and McIntosh (2001) documented maximum foaming capacity but
least stability for B. napus protein isolates prepared at pH 7. Comparatively, protein isolates
prepared through acid-precipitation showed more stable and superior foaming properties as
compared to those isolated via calcium-precipitation (Aluko and McIntosh 2001). However,
foaming properties of CPI prepared through acid or calcium precipitation were also better
in comparison with SPI.
A few research studies indicated that rapeseed flours, isolates and concentrates exhibit
inferior gelation properties. Researchers have determined that hydrogen and ionic bonds are
not the key factors for crosslinking in the gel though some disulfide bondings are involved.
Recently, it is revealed that gel forming ability of canola meal was comparatively better
than soybean meal. Similarly, least gelation concentration (LGC) of canola meal is quite
higher as compared to soybean meal which indicates its poor gelation characteristics
(Khattab and Arntfield, 2009).
23
The gelling properties of proteins are mostly dependent on their molecular size. Moreover,
large molecular size proteins play vital role in the formation of more extensive 3
dimensional cross-linking networks. These networks provide improved gelling properties.
Furthermore, the formation of polymers having high molecular weight is dependent upon
the modification of protein structure by the treatment of transglutaminase (TG) which helps
in polypeptides cross-linking. The treatment of canola proteins with TG made them
sustainable gelling agents. In a previous research investigation, considerably higher
emulsion capacity was noticed for protein isolates obtained from canola meal (~515.6 g
oil/g protein) as compared to flaxseed protein isolates (~498.9 g oil/g protein). Likewise,
emulsion stability index measured for oilseed protein isolates ranged between ~10.5–15.5
min (Karaca et al., 2011).
2.5. Gel Electrophoresis (SDS-PAGE)
Proteins can be separated on molecular weight basis by using Sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) technique. It refers to the movement of
protein molecules in an electrical current forming polypeptide chain in the presence of SDS
detergent (Wang et al., 2007). The SDS-PAGE technique gives information regarding
molecular size along with intermolecular disulfides bonds of proteins. The proteins as well
as their fractions are presented on electrogram and characterized as fingerprints (Klomklao
et al., 2010).
Numerous research studies have been done to purify 11S globulin, the main storage protein
in sesame, by using gel filtration chromatography. Purposely, the samples are treated with
reducing agents for SDS-PAGE analysis. The results expounded that 11S globulin exhibited
acidic (34.5 and 30.5 kDa) as well as basic polypeptides (19.2 kDa) (Orruño and Morgan,
2007). Moreover, SDS-PAGE analysis of 11S globulin with non-reducing conditions
indicated that it consists of four protein bands i.e. 36.0, 41.0, 46.6 & 51.8 kDa. The protein
was isolated under homogeneous conditions via ion-exchange chromatography using
DEAE-Sephacel along with native PAGE. Finally, immunoblotting was done for the
identification of protein through polyclonal globulin antibodies. Several scientists have
purified the sesame 2S albumin by the use of gel filtration chromatography (Orruño and
Morgan, 2007).
SDS-PAGE has been used to characterize and purify 7S globulin protein. This
characterization may be carried out either with or without reducing agent. Moreover,
24
analysis with reducing agents showed that 7S globulin comprised of 8 polypeptides. The
molecular weight of these polypeptides ranged from 12.4-65.5 kDa. Additionally, SDS-
PAGE was used in the absence of reducing agent to explicate the strength of bonds between
polypeptides present in 7S globulin. The research study on sesame proteins elucidated that
the 7S globulin lack disulfide bonds that hold the polypeptides together (Orruño and
Morgan, 2007).
Earlier, a study was conducted to explore the SDS-PAGE pattern for flaxseed protein
isolates and concluded that isolates consist of similar fractions like sesame proteins.
However, a slight variation exists in the ratio of polypeptides. Moreover, the flaxseed
protein isolates differ in a legumin-like 11S seed globulin which does not exist in sesame
proteins. Conclusively, four groups of 11S subunits were identified (36, 46, 50 and 55 kDa).
Each subunit consists of a pair of disulfide-linked α- and β-chains. Furthermore, the
molecular masses of the α- and β-chains were calculated as 38-34 & 25 kDa and 19-21 kDa,
respectively. Moreover, very faint bands of 7S globulin subunits (54, 36 and 21 kDa) were
observed along with a minor quantity of low molecular mass components (7–10 kDa).
These subunits were free from interchain disulfide bonds (Krause et al., 2002).
In a previous research investigation, canola protein isolates were analyzed by SDS-PAGE.
The analysis was carried out in the presence and absence of β-mercaptoethanol (ME).
Resultantly, canola protein isolate showed 8 major and 6 minor bands. The molecular
weight of major bands varied from 14-59 kDa (Wu and Muir, 2008). In another study
conducted by Aluko and McIntosh (2001), 4 major bands of canola proteins with molecular
weights of 16, 18, 30 and 53 kDa were detected. However, the bands of 14 and 59 kDa
were not reported in this study. The band having a molecular weight of 14 kDa is similar to
the most abundant band separated by Sephacryl S-300 chromatography. The purity of this
band (91.4%) was calculated using SDS-PAGE. This band indicated 2S albumin (napin),
which accounts for 25.3% of canola protein. However, the presence of β-mercaptoethanol
(ME) caused dissociation of 2S albumin and the mono-napin band into small polypeptide
chains. These results were in agreement with the conclusions of previous studies that napins
consist of a small as well as large polypeptide chain linked via disulfide bridges. However,
small polypeptide chain was not identified as it ran through the gel. Therefore, a gel with a
lower molecular weight limit will be required to detect this small polypeptide (Wu and
Muir, 2008).
25
Previously, the polypeptide composition of cruciferin was determined. The molecular
weight ranged from 230 to 300 kDa owing to the analytical method and isolation process.
Cruciferin has been proved to be an oligometric protein consisting of 6 subunits, each
comprising of 2 polypeptide chains. The SDS-PAGE analysis of cruciferin determined 10
polypeptide bands. However, presence of any proteinous contaminants in cruciferin may
alter the results. The results showed two major and 1 minor bands with molecular weights
of 50 & 29.5 KDa and 44 KDa, respectively (Wu and Muir, 2008).
2.6. Bio-evaluation
Processing techniques affect the extraction yield and quality of proteins. The bioavailability
of proteins also depends on processing technology. According to Ekanayake et al., 2000,
bioavailability of protein is evaluated by growth as well as nitrogen balance studies.
Numerous foods are analyzed through in vivo assay to assess their ability for growth and
usual metabolic activity. The protein efficiency elucidates the quality of protein destined for
human consumption particularly growing infants and children (Gropper et al., 2005).
The nutritional potential of protein isolates plays vital role in growth and maintenance of
body especially in infants and young children. In various research studies, rat bioassay has
been used for efficacy purpose. Bio-evaluation of protein encompasses the parameters like
protein efficiency ratio (PER), net protein ratio (NPR) and relative net protein ratio (RNPR)
whereas nitrogen balance study comprises of biological value (BV), net protein utilization
(NPU) and true digestibility (TD) (Seena et al., 2006). Accordingly, food consumed by
experimental animals differs with protein level in the diet and the amount needed for
physiological and metabolic functions (White et al., 2000).
Protein efficiency ratio (PER) below 1.5 indicates low quality protein whereas PER
between 1.5 and 2.0 shows an intermediate protein quality nevertheless, PER above 2.0
means good quality protein (Amza et al., 2013). Earlier, Alajaji and El-Adawy, (2006)
expounded that PER is significantly enhanced by cooking. According to Yasothai (2014),
protein efficiency ratio of sesame meal is 1.35. However, the partially defatted flaxseed
flour showed 1.87 protein efficiency ratio, 84.6% true digestibility, 64.6% biological value
and 54.65% net protein utilization (Hussain et al., 2012). The protein efficiency ratio (PER)
of canola protein isolates has been reported as 2.64 (Tan et al., 2011b).
Rangel et al. (2004) elucidated that diet containing cow pea protein isolates showed NPR
0.7 and digestibility of 87%. Later, Ingbian and Adegoke (2007) explained that soybean
26
blended food has PER 1.6-2.19 as compared to 0.2-1.5 for groundnut enriched products.
Similarly, African yam bean protein isolates have higher true digestibility (Francis et al.,
2009). Previously, Bhagya et al. (2006) recorded an increase in PER, NPR, protein
retention efficiency (PRE), TD, BV and NPU in cooked Canavalia cathartica.
Similarly, Vadivel et al. (2008) evaluated protein quality of black cumin through PER and
NPU. The results showed that Turkish seeds have NPU 63.1% whereas Syrian type has
54.6%. Furthermore, values for NDPE were documented as 5.6 & 5.3% for Turkish and
Syrian samples, correspondingly. Nonetheless, Syrian sample exhibited PER of 1.9.
The true protein digestibility for navy bean and cowpea have been recorded as 62.6-78.2%
& 73.7-87.5%, respectively (Jackson, 2009). Later, Vadivel et al. (2010) supplemented diet
with sword beans and noticed the values for PER 2.66, TD 73.35%, BV 70.51% and NPU
56.48%. However, the utilized protein (UP) was recorded as 39.16%. Moreover, yam bean
presented higher PER, NPR and RNPR as compared to non-germinated sample (Francis et
al., 2009).
2.7. Composite flours
In the developing nations, due to high cost and inadequate access to animal proteins, there
is dire need for the utilization of plant proteins which are more economical. The plant
protein sources include defatted oilseeds and legumes that contain excellent quality proteins
(Enujiugha and Ayodele-Oni, 2003). For the incorporation of non-conventional proteins in
the food formulations, one must have considerable knowledge about their functional and
rheological characteristics. Amongst value added food products, baked items offer an
excellent opportunity to incorporate food ingredients such as proteins from legumes, grains
or other non-conventional food sources (Bakke and Vickers, 2007).
Composite flour refers to the combination of flour with protein, starch and some other
ingredients totally or partially replacing wheat flour in bakery and other food products.
Previous research studies have indicated that the bakery products made by composite flour
exhibited better quality. Wheat is deficient in lysine and some other essential amino acids.
Therefore, supplementing wheat flour with low-cost, non-conventional sources like defatted
oilseed meals and legumes is a suitable strategy to improve nutritional quality of wheat
products (Dhingra and Jood, 2001).
Bakery products vary in nutritional profile owing to adding various nutritionally rich
ingredients. Therefore, the utilization of composite flour in bakery products has been
27
potentially increased which encouraged scientists to study the functional as well as
physicochemical attributes of resultant blends. These studies elucidated the vitality and role
of various ingredients used to produce composite flour (Sudha et al. 2007). Numerous
studies regarding product technology as well as consumer acceptance have revealed that
wheat is an indispensable constituent of composite flours. Furthermore, wheat gluten plays
an imperative role in determining the percentage of wheat flour needed to attain some
specific effect in composite blends. It also depends on the nature of the intended food
product. Thus, the quality of bakery products manufactured using composite flour must be
similar to the products made from wheat flour (Mepba et al., 2007).
Composite flour technology involves mixing of different flours including legumes and
cereals with wheat flour. Keeping in view the cost effectiveness, this technology has paved
the way for utilizing locally produced materials in the manufacturing of high quality food
products. It has been documented that cereals i.e. rice, wheat, barley, sorghum and maize
fulfill about 68% food requirements around the world (Olaoye et al., 2006; Švec and
Hrušková, 2010). Nonetheless, wheat being a dietary staple in Pakistan is utilized by two
third of population (Rehman et al., 2007).
In recent years, numerous organizations including Food and Agriculture Organization
(FAO) have supported the development of composite flour blends by partially replacing
wheat flour with various protein sources. This will certainly help to handle the protein
energy malnutrition especially in developing countries. Similarly, Abdel-Kader (2000)
explicated that composite flour technology has become a suitable choice to develop low
cost dietary substitutes. Furthermore, this technology has potentially utilized indigenous
crops thus providing additional health benefits to the native community (Anjum et al.,
2005).
Later, Ade-Omowaye et al. (2008) elucidated that recent development in nutritional
guidelines around the world demands the utilization of composite flours especially in
baking industry. The world population is dependent on cereals to fulfill overall calorific
requirements (Shittu et al., 2009). In Pakistan, wheat is the major source of protein and
energy providing >60% of the total daily protein and calories however, it is deficient in
lysine. Similarly, lysine and tryptophan are the limiting amino acids in most cereal based
diets (Onwulata and Konstance, 2006).
28
Dietary interventions are required to improve the nutritional status of diets of vulnerable
groups in order to combat protein energy malnutrition. Purposely, exploitation of composite
flour in the manufacturing of numerous food commodities is an appropriate option to
overcome this peril. Moreover, the protein as well as amino acids content of various foods
can be improved through composite flours (Hall and Johnson, 2004). Previously, cowpea
and plantain blends were prepared for the production of nutritionally balanced food for
infants as well as adults. These blends have excessive quantity of protein along with
improved nutritional profile. Furthermore, these blends are low cost as compared to animal
proteins. Therefore, the use of these blends has stimulated the exploration of other protein
rich plant sources (Akubor, 2003; Arshad et al., 2007).
Numerous composite blends have been prepared by substituting wheat flour with flaxseed
& sunflower meal, raw & defatted soy, lupin flour and wheat germ (Škrbić and Filipčev,
2008). Furthermore, biscuits and bread with composite flours made by replacing wheat
flour with sorghum (10 and 20%) showed acceptable quality (Elkhalifa and El-Tinay,
2002). Likewise, chickpea and wheat composite blends were utilized to prepare layer cakes
(Gómez et al., 2008).
Previous research studies indicated that composite blends of defatted soy with cereal
increased the quality and quantity of protein in final product. A composite blend consisting
of wheat and soy exhibits high quality protein owing to high lysine content in soy.
However, wheat is deficient in lysine thus making it a limiting amino (Jiang et al., 2008). In
another research investigation, replacing wheat flour with soybean isolates (5.6%) and
defatted soybean (11.1%) enhanced the nutritional and sensory attributes of end product.
Similarly, some studies elucidated that fortified flours contain more than 35% protein and a
much higher quantity of lysine in comparison with pure wheat flour. Addition of 15% full
fat as well as defatted soy and barley in wheat flour improved protein and certain other
nutrients in the resultant breads (Dhingra and Jood, 2001). Moreover, amalgamation of
protein in composite flours potentially enhanced the quality and acceptability of cookies
(Singh and Mohamed, 2007).
Composite blends were prepared by adding sesame peels flour in wheat flour. The cookies
produced from these composite flours showed acceptable sensory characteristics up to 30%
replacement of sesame peels flour (Zouari et al., 2016). Likewise, sesame and millet
composite flours produced biscuits with enhanced nutritional, baking and hedonic
characteristics (Alobo, 2001). Similarly, cookies containing wheat and germinated sesame
29
flour exhibited better nutritional quality due to increased protein and lysine content
(Olagunju and Ifesan, 2013).
Successful supplementation of nutritional components obtained from nonconventional
sources in food formulations needs necessary information about their functional and
rheological characteristics. Purposely, bakery products supplemented with essential
nutrients play an imperative role in fulfilling nutritional needs as these are utilized at
massive scale (Bakke and Vickers, 2007). Therefore, the supplementation of wheat flour
with nonconventional protein sources i.e. oilseed meals can improve the nutritional worth
of bakery items.
The rheological characteristics of composite blends affect the process efficiency and quality
of product. Addition of protein isolates in wheat flour will alter protein percentage of
composite flour that will eventually affect the mixing time as well as the hydration
requirements of flour to form gluten matrix. The rheological as well as mechanical
properties of dough affect the product quality. Furthermore, the information about rheology
of dough helps in controlling the online baking process conditions (Torbica et al., 2010).
Moreover, some alteration in rheological characteristics may occur due to factors like
structure of materials, ingredients interaction and proteins arrangement (Švec and
Hrušková, 2010). These factors affect the final product quality by influencing dough
rheology during the manufacturing process. Likewise, the textural characteristics and final
volume of bakery products are also influenced by these factors (Hadnađev et al., 2011).
The quality of final product is based on the interaction of ingredients and handling behavior
of dough. Purposely, numerous techniques have been employed to determine the
rheological characteristics of dough. These techniques include mixograph, extensograph
and farinograph (Pasha et al., 2011). The addition of other flours in wheat alters the dough
rheology and increases quality of end product. Additionally, assessment of the
characteristics of composite flour blends is essential to define the suitability of
nonconventional sources for their utilization in bakery products (Hadnađev et al., 2011).
Similarly, Jia et al. (2011) explicated that mixograph is used to estimate the mixing as well
as hydration requirements of flour to form gluten matrix.
Previously, Baixauli et al. (2008) expounded that the rheology of batter is also affected by
numerous factors like nature & quantity of ingredients, mixing and beating time along with
baking conditions. Moreover, flour, water, fat, salt and sugar are the key ingredients.
30
Furhtermore, Saha et al. (2011) have revealed that millet and wheat (60:40) composite
blends resulted in high quality bakery products.
Protein structure and ability to form network also affect the rheology of dough.
Furthermore, the functional properties like water absorption capacity of dough are also
affected by the quality as well as quantity of protein (Rosell et al., 2007). Moreover,
nonconventional ingredients also affect the viscoelastic properties of wheat flour dough.
Moreover, the protein network is also vital for the textural characteristics of final product
(Amjad et al., 2010).
The supplementation of rice flour with soy & pea proteins resulted in altered mechanical
characteristics. The rice, soy & pea protein isolates composite blends showed considerably
higher water absorption capacity (Marco and Rosell, 2008). The supplementation of protein
isolates also affects the textural properties of final product. Earlier explorations have
indicated that the dough rheology and product attributes are significantly affected by the
addition of soybean protein isolates (Wanyo et al., 2009).
The farinographic studies of wheat and flaxseed blends (5, 10, 15 & 20%) indicated a
significant increase in water absorption, mixing tolerance index and dough development
time while a reduction in dough stability with rise in flaxseed flour addition (Koca and
Anil, 2007). Various researches have been carried out on composite flours in which wheat
flour is substituted with raw or defatted wheat germ, soy or defatted soy flour, sunflower
meal and flaxseed (Junqueira et al., 2008; Škrbić and Filipčev, 2008).
2.8. Bakery products
Amongst bakery products, muffin is a prominent breakfast or afternoon snack food i.e.
sweet in taste and exhibit soft texture. The baked items have high consumer demand
especially due to better quality and textural attributes (Sanz et al., 2009). Muffins are
conventionally prepared using wheat flour, sugar, eggs, milk and oil/fat. Muffins being
sweet and high-energy bakery products are extremely liked by the consumers owing to their
excellent taste and textural attributes (Matos et al., 2014). Additionally, muffins exhibit
porous structure with high volume, therefore, considered as soft product with spongy
texture.
In this context, Chetana et al. (2010) elaborated the nutritional attributes of muffins
prepared from wheat flour and raw & roasted flaxseed powder. Moreover, the decrease in
31
volume of muffins is attributed to the gradual addition of flaxseed powders. Furthermore,
the texture is significantly affected by flaxseed powders and the muffins became softer.
Conclusively, adding flaxseed in muffins proved beneficial as it helped in improving their
nutritional quality and overall acceptability. Therefore, flaxseed can be a potential
ingredient in the preparation of protein enriched muffins.
In another research exploration, ground flaxseed was incorporated into bakery products like
bread & muffins and their nutritional & sensory attributes were analyzed. It was concluded
that muffins with 50% flaxseed obtained better overall acceptability scores. Additionally,
supplementation of proteins in bakery products i.e. muffins play a key role in improving
their functional properties (Matos et al., 2014). Further, proteins also result in greater height
and volume of muffin owing to swelling and denaturation at elevated temperature,
consequently helps in better gas retention through structural support (Ziobro et al., 2013).
Previously, the sesame and canola protein isolates have not been reported to be utilized in
the preparation of muffins. Therefore, the present project was focused on their potential
utilization in bakery products.
In the nut shell, utilization of oilseed protein isolates can be a handy tool for the
development of novel food formulations. Moreover, oilseed protein isolates will play an
imperative role in improving the protein content and nutritional status of numerous food
commodities. The current study regarding wheat flour supplementation with defatted
oilseed protein isolates for the preparation of protein enriched muffins will help in
addressing the menace of protein energy malnutrition.
32
Chapter 3
MATERIALS AND METHODS
The current study was carried out in the Postgraduate Research Laboratory, National
Institute of Food Science and Technology (NIFSAT), University of Agriculture, Faisalabad
(UAF). However, characterization of protein isolates was conducted at Food Protein
Research & Development Center (FPRDC), Texas A&M University, TX, USA. In this
context, protein isolates were extracted using defatted sesame, flaxseed and canola meals.
The resultant protein isolates were used in developing protein enriched muffins. The detail
of protocols and procedures used is mentioned herein:
3.1. Procurement of raw materials
Oilseeds i.e. sesame (TS-5), flaxseed (Chandni) and canola (Faisal canola) were procured
from Ayub Agriculture Research Institute (AARI), Faisalabad. Wheat flour and other
required ingredients were purchased from local market, Faisalabad. The chemicals &
standards were bought from Merck (Merck KGaA, Darmstadt, Germany) and Sigma-
Aldrich (Sigma-Aldrich Tokyo, Japan). The Sprague Dawley rats needed for bioevaluation
trial were kept in the Animal Room of NIFSAT, UAF.
3.2. Preparation of raw material
The seeds of sesame, flax and canola were initially cleaned and then ground to fine powder
for further analyses.
3.3. Proximate composition
The selected oilseed materials were subjected to proximate analysis i.e. moisture, crude
protein, crude fat, crude fiber, ash and nitrogen free extract (NFE) following the respective
methods (AACC, 2000; AOAC, 2006).
3.3.1. Moisture content
To estimate moisture contents, samples were dried in Air Forced Draft Oven (Model: DO-
1-30/02, PCSIR, Pakistan) at 105±5 °C till constant weight according to the AOAC (2006)
Method No. 925.10.
33
3.3.2. Crude fat
It was estimated via Soxhtec System (Model: H-2 1045 Extraction Unit, Hoganas, Sweden)
using hexane as solvent as mentioned in AOAC (2006) Method No. 920.85.
3.3.3. Crude protein
The nitrogen content determination was carried out through digestion of sample with conc.
sulfuric acid and digestion mixture while heating till light greenish color using Kjeltech
Apparatus (Model: D-40599, Behr Labor Technik, Gmbh-Germany). Afterwards, dilution
and distillation was done using 40% NaOH solution (10 mL) in distillation apparatus. 4%
boric acid (H3BO3) solution was used to collect the liberated ammonia in the presence of
methyl red indicator. Lastly, titration was done using 0.1N sulphuric acid (H2SO4) to attain
golden brown color. For crude protein (%) determination, N2 was multiplied with 6.25
according to AOAC (2006) Method No. 920.87.
3.3.4. Crude fiber
To deteremine crude fiber, defatted samples were subjected to digestion using 1.25%
H2SO4 followed by 1.25% NaOH using Labconco Fibertech (Labconco Corporation
Kansas, USA). The resultant residues were filtered after washing prior to ignition in Muffle
Furnace according to AACC (2000) Method No. 32-10.
3.3.5. Ash
To determine ash content, the samples were first subjected to charring followed by
incineration using a Muffle Furnace (MF-1/02, PCSIR, Pakistan). Incineration was done at
550±50 °C and was contined till grayish white residues appear (AOAC, 2006; Method No.
923.03).
3.3.6. Nitrogen free extract (NFE)
NFE was calculated using the formula;
NFE % = 100 – (crude fat% + crude protein% + crude fiber% + ash%)
3.4. Mineral profile
The oilseeds were subjected to mineral profiling using AOAC (2006) methods. The
samples were wet digested prior to estimation of calcium, zinc, and iron (Method 968.08,
991.11 and 985.01) using Atomic Absorption Spectrophotometer (Varian AA240,
Australia). However, the other minerals like sodium and potassium (Method 968.08,
34
968.08) were estimated through Flame Photometer-410 (Sherwood Scientific Ltd.,
Cambridge).
3.5. Defatting of samples
The conventional solvent (hexane) method was employed to extract oil from the selected
samples using soxtec system (Model: H-2 1045 Extraction Unit, Hoganas, Sweden)
(AOAC, 2006). Resulting defatted oilseeds were dried and stored for further analyses.
3.6. Proximate and mineral analyses of defatted oilseeds
The defatted oilseeds obtained after oil extraction were assessed for moisture, crude fat,
crude protein, ash, crude fiber and nitrogen free extract (NFE). Moreover, the minerals like
sodium (Na), calcium (Ca), potassium (K), zinc (Zn) and iron (Fe) were also estimated
following the described procedures.
3.7. Protein isolates preparation
For the preparation of protein isolates (Fig. 1), defatted oilseeds were disolved in distilled
water (1/10) with pH 9.5. Furthermore, centrifugation was carried out at 4000 rpm for 20
min to separate the supernatant. Afterwards, the pH of collected supernatant was set at 4.5
following re-centrifugation, neutralization and freeze drying (Makri et al., 2005).
3.8. Protein isolates assays
3.8.1. Protein content
The protein isolates were analyzed for crude protein content by Kjeltech Apparatus
following the respective protocols (AACC, 2000). The protein percentage was estimated as:
% N2×6.25.
3.8.2. Isolate Recovery
Isolates recovery was calculated as;
Weight of protein isolates /100 g of respective sample
(Wang et al., 1999).
35
Defatted oilseeds + water (1:10)
` pH adjustment (9.5)
Centrifugation (4000 rpm, 20 min)
Supernatant
pH adjustment(4.5)
Centrifugation (4000 rpm, 20 min)
Precipitate (protein isolates)
Neutralization
Freeze drying
Storage
Figure 3.1: Systematic flow sheet for oilseed protein isolates recovery
36
3.8.3. Protein yield
Following expression was used to calculate protein isolates yield (Wang et al., 1999).
Yield (%) =Weight (g) of protein isolates
Weight (g) of defatted meal ×
Protein content of protein isolates (%)
Protein content (%) of defatted meal× 100
3.9. Functional properties of defatted oilseed protein isolates
3.9.1. Bulk density
To determine bulk density, 10 g protein isolates sample was poured into 100 mL graduated
cylinder that was tapped several times on laboratory bench till the sample completely
settled. The bulk density was described as g/cm3 (Siddiq et al., 2010).
3.9.2. Water absorption capacity
For the estimation of water absorption capacity (WAC), 3 g of sample was mixed in 25 mL
distilled water and the solution was stirred prior to centrifugation at 3000×g for 25 min. The
resultant supernatant was reweighed after decanting and removal of excess moisture. WAC
was calculated as:
Water absorbed (g)/g sample (Kaur and Singh (2007)
3.9.3. Oil absorption capacity
Oil absorption capacity (OAC) was determined by adding 0.5 g of respective protein isolate
in 6mL of corn oil. The dispersion was stirred for 1 min to dissolve the sample in oil. After
keeping for 30 min, the dispersions were centrifuged at 3000 × g for 25 min. After
removing the separated oil, the tubes were inverted for 25 min to drain the oil and then
reweighed. OAC was calculated as:
Oil absorbed (g)/ g sample (Kaur and Singh, 2007).
3.9.4. Foaming properties
These were measured by mixing 1g protein isolate in 50 mL of distilled water and
transferred to a graduated cylinder (250 mL). Foaming capacity was estimated as foam
volume after air current incorporation (15 min). Foaming stability was determined by
observing the cylinder after 60 min (Siddiq et al., 2010).
3.9.5. Emulsion properties
Emulsifying properties were determined by mixing 0.5 g protein isolate in 3 mL distilled
water. Afterwards, 3 mL oil was added and vigorously shaken (5 min) prior to
37
centrifugation (2000 × g for 30 min). Emulsifying activity was estimated as ratio of the
emulsified layer height to that of liquid layer after 30 min centrifugation (mL/100 mL).
Moreover, the emulsifying stability was determined by heating (80oC) the emulsion in
water bath (WNB-29, Memmerts, Germany). The samples were then centrifuged at 3000 ×
g. The emulsifying stability was calculated using following expression (Siddiq et al., 2010).
Volume of emulsifying layer ×100
Heated slurry
3.9.6. Nitrogen Solubility Index (NSI)
NSI was estimated by forming protein solutions using deionized water and adjusting pH
between 2 to 12 (0.01N HCL or NaOH solutions). Initially, samples were agitated at 120
rpm for 30 min (30ºC) and then centrifuged (2000 × g). The supernatant was collected to
measure nitrogen solubility index (%) (Shand et al., 2007).
3.9.7. Least Gelation Concentration (LGC)
The LGC of respective protein isolates was determined by following the method described
by Siddiq et al. (2010). Initially, 2 to 20% (w/v) suspensions were made and then heated for
1 hr in distilled water prior to immediate cooling. Water bath was used to heat the test tubes
carrying dispersions. These test tubes were then cooled under cold running water. The LGC
was estimated as concentration of sample when it did not slip from the inverted test tube
and described as complete (+), partial (±) or no (−) gelation as.
3.10. Gel Electrophoresis (SDS-PAGE)
Initially, 250 µL sample buffer was used to solubilize the protein isolate samples. In order
to perform the electrophoresis on Bio-Rad Mini Protean 3 System (Bio-Rad Laboratories,
Hercules, CA, USA), 12.5% and 4% stacking and separating gels were used, respectively.
Purposely, samples’ loading was done @ 10 µL/lane. A constant voltage (60 V) was
supplied for 2.5 hr to run the loaded gels till the front dye moved far down the gel.
Coomassie Brilliant Blue (CBB) was used to stain the gels while methanol water mixture
was used for destaining purpose (Tang and Sun, 2011).
3.11. Amino Acid Profile
The amino acid profiling was done at the University of veterinary and animal sciences
(UVAS) Lahore, Pattoki campus. Purposely, calculated volume of the prepared supernatant
was injected in Bio Chrom 30+ Amino Acid Analyzer (Adeyeye and Afolabi, 2004).
38
However, for tryptophan, samples were hydrolyzed in the presence of Ba(OH)2, isolated
through gel filtration and colorimetrically analyzed.
3.12. Amino Acid Score
Amino acid score was determined by following the amino acid requirement for
preschoolers (WHO/FAO/UNU 2007; Gurumoorthi et al. 2008).
3.13. PDCAAS Value
Protein digestibility corrected amino acid score (PDCAAS) was calculated via true
digestibility of the respective protein isolates and the lowest amino acid score by following
expression (Kannan et al., 2001).
PDCAAS (%) = True Digestibility × lowest amino acid score
3.14. In vitro protein digestibility (IVPD)
IVPD (%) of protein isolates was determined using the procedure outlined by Aboubacar
(2001). For the purpose, protein isolates samples (200 mg) were weighed into Erlenmeyer
flasks and mixed with 35 mL of porcine pepsin solution (1.5 g of pepsin/L in 0.1M
KH2PO4, pH 2.0). Samples were digested for 2 hr at 37°C in a shaking water bath.
Digestion was stopped by adding 2 mL of 2N NaOH. Samples were centrifuged (4900 × g,
4°C) for 20 min, and the supernatant was discarded. The residues were washed and
centrifuged twice with 20 mL of buffer (0.1M KH2PO4, pH 7.0). Undigested nitrogen (N)
was determined with a Technicon nitrogen analyzer. Digestibility was calculated as:
% Digestibility = (N in sample – undigested N)/N in sample × 100.
3.15. Bioevaluation
Bioevaluation of oilseed protein isolates was conducted by feeding respective diets to
different groups of Sprague Dawley rats. The control diets consisted of soy, casein and no
protein diet (Table 3.1). Commercial soy protein isolate and casein had protein content
93.48% & 96.35%, respectively. The diets were made iso-nitrogenous by maintaining the
protein content at 10% level. Likewise, vitamins and minerals mixtures were added. The
nitrogen free mixture comprised of sucrose, cellulose and corn starch.
3.15.1. Housing of Rats
For bioevaluation study, thirty male Sprague Dawley rats were housed in the Animal Room
of NIFSAT, UAF. Initially, rats were divided into six groups, five in each. The rats were
given respective diets for 10 days. The temperature (23±2ºC) and humidity (50±5%) were
39
maintained throughout the experimental period. The spilled diet and feces were collected on
daily basis. At the termination of trial, overnight fasted rats were decapitated and their
bodies were allowed to dry till constant weight by placing in a hot air oven (105 °C). The
dried carcass was grinded and its nitrogen content was estimated (AACC, 2000).
Table 3.1. Components of experimental diets
Diet constituents (g) SPI FPI CPI Soy
diet Casein diet No protein diet
SPI 11.09 0.0 0.0 0.0 0.0 0.0
FPI 0.0 11.58 0.0 0.0 0.0 0.0
CPI 0.0 0.0 11.14 0.0 0.0 0.0
Soy Protein 0.0 0.0 0.0 10.69 0.0 0.0
Casein 0.0 0.0 0.0 0.0 10.37 0.0
Corn oil 5.0 5.0 5.0 5.0 5.0 5.0
Mineral mixture 5.0 5.0 5.0 5.0 5.0 5.0
Vitamin mixture 1.0 1.0 1.0 1.0 1.0 1.0
N-free mixture 77.91 77.42 77.86 78.31 78.63 89.00
Total diet weight (g) 100 100 100 100 100 100
* All diets contain 10% protein except “no protein diet”
SPI – Sesame protein isolates
FPI – Flaxseed protein isolates
CPI– Canola protein isolates
3.15.2. Feed Intake
Feed intake of experimental rats was calculated on daily basis by eliminating spilled diet
from the total diet consumed during the entire experimental period (Wolf and Weidbrode,
2003).
3.15.3. Body Weight Gain
Gain in body weight of each group was calculated for growth index with regard to
respective diet (Seena et al., 2006).
3.15.4. Protein Quality Evaluation
Net feed intake and body weight gain were employed to determine growth study parameters
like protein efficiency ratio (PER), net protein ratio (NPR) and relative net protein ratio
(RNPR). The spilled diet, feces, urinary outputs and dried rat bodies were analyzed for
nitrogen content to estimate nitrogen balance study parameters such as true digestibility
40
(TD), biological value (BV) and net protein utilization (NPU) (Ingbian and Adegoke,
2007).
3.15.5. Safety evaluation
Safety assessment was conducted via serum protein as well as kidney & liver function tests.
3.15.5.1. Serum protein
Serum total protein, albumin, globulin and albumin/globulin ratio were estimated by
following the procedures described by Al-Gaby (1998).
3.15.5.2. Liver and kidney functioning tests
The sera of rats were subjected to liver function tests via enzymatic evaluation i.e. alanine
aminotransferse (ALT), aspartate amino transferase (AST) and alkaline phosphatase (ALP)
(Basuny (2009). Moreover, for kidney functioning tests, GLDH-method was used to
analyze the serum urea while creatinine was estimated by following Jaffe-method using
commercial kits (Jacobs et al., 1996; Thomas et al., 1998).
3.16. Selection of protein isolates
On the basis of overall yield, functional properties and bioevaluation trial, one best protein
isolate sample i.e. sesame protein isolate was selected for further utilization in protein
enriched muffins preparation.
3.17. Development of composite flour
Straight grade wheat flour was replaced with sesame protein isolates to prepare different
blends of composite flour as mentioned in Table 3.2.
Table 3.2: Composite flour treatments
Treatments Wheat flour (%) Sesame protein isolates
(%)
T0 100 0
T1 95 5
T2 90 10
T3 85 15
T4 80 20
T5 75 25
41
3.17.1. Proximate analysis of composite flours
The composite flours were subjected to analysis for moisture, crude protein, crude fat,
crude fiber, ash and nitrogen free extract (NFE) (AACC, 2000).
3.17.2. Rheological studies
The flour samples were tested for their rheological characteristics using Brabender
Farinograph and Mixograph according to their standard procedures outlined by AACC
(2000).
3.17.2.1. Farinographic studies
Brabender Farinograph was used to prepare the farinograms of treatment flours by
following the instructions provided in AACC (2000) Method No. 54-21. Farinograph
equipped with a bowl of 50 g capacity was used and constant flour weight method was
employed. The parameters given in AACC (2000) were interpreted from each farinogram
according to the instructions as detailed below.
3.17.2.1.1. Water Absorption
Water absorption is the percentage of water required to reach the center of curve on 500
Brabender Unit (BU) line at maximum consistency of the dough. In this context, the water
absorption (%) was visualy observed from microburette.
3.17.2.1.2. Dough Development Time
This is the time required for the curve to reach its full development or maximum
consistency before indicating the weakening of dough. High peak values depict strong
wheat having extended mixing time.
3.17.2.1.3. Dough Stability Time
It refers to the time difference between intersection and departure point of the top of curve
with 500 B.U. line. Better tolerance would indicate the flour stability against mixing
3.17.2.2. Mixographic Studies
Mixograph instrument was used to prepare mixograms of various flour samples. Mixograph
equipped with 10 g bowl capacity was used. 60% water was added to each sample and run
through the mixograph (AACC, 2000; Method No. 54-40A). The following parameters
were interpreted from mixograms.
42
3.17.2.2.1. Mixing Time
It refers to maximum development time of dough. This is estimated by crossing of two lines
through the centers of both sides of curve. The distance from the starting point of the curve
to the intersection of two lines is the optimum mixing line. This time may be known as peak
time.
3.17.2.2.2. Peak Height Percentage
It refers to the maximum peak height (%) of Mixogram.
3.17.3. Functional properties of composite flours
Functional properties i.e. bulk density, foaming capacity & stability water & oil absorption
capacities and emulsion activity & stability of composite flours were determined by
following the respective procedures described earlier.
3.18. Protein enriched muffins preparation
Composite flours were used to prepare muffins (Table 2) as outlined by Shearer and Davies
(2005) with some modifications. The resultant muffin samples were evaluated for
physicochemical analyses, nutritional value and sensory response.
Initially, sugar and oil were mixed in mixing pan and shake well for 6 minutes. Then egg,
flour and baking powder was added and shaken well till grains of sugar become completely
ground. Liquid milk was added in pan and thoroughly mixed for 2 minutes till viscous
batter formed.
Butter paper was placed in muffin pans and then batter was placed. Each pan filled with 1/2
to 2/3 of batter. Afterwards, muffin pans were placed in a baking tray. Baking tray was
placed in the oven at 175oC for 15-20 minutes. Finally, the muffins were cooled at room
temperature.
3.18.1. Proximate analysis
The prepared muffins were subjected to proximate analysis by following the respective
methods outlined by AACC (2000).
43
3.18.2. Physical analysis
3.18.2.1. Color
The crust and crumb color of muffins was estimated as L*, a* & b* values; L* value
determining lightness, a* value estimating redness and b* value yellowness using
HunterLab miniScan XE Plus colorimeter (Model 45/0-L, HAL, USA) following the
method described by Goswami et al. (2015). Chroma (C) and hue angle (ho) were
calculated by the following forlumae:
C = [a*2 + b*2]1/2
ho = tan-1(b*/a*)
3.18.2.2. Instrumental texture
Texture of protein enriched muffins was determined using a TA.XT2i Texture Analyzer
(Texture Technologies Corp., Scarsdale, NY/Stable Micro Systems, Godalming, Surrey,
UK) according to the American Institute of Baking’s (AIB, Manhattan, KS) Standard
Procedure for Muffin Firmness and Elasticity (AACC method No. 74-09) as described by
Shearer and Davies (2005). It is an automatic equipment having software attached that
gives measurements of firmness and elasticity of muffins to bend or snap.
3.18.2.3. Volume
Muffin volume was determined via rapeseed displacement method (AACC, 2000).
Purposely, the muffin was placed in rapeseeds container filled and the volume of rapeseeds
displaced by the muffin was noted. Each treatment was measured in triplicate and the
average value was recorded according to Keskin et al. (2004).
3.18.3. Gross energy
The gross energy or calorific value (CV) of muffins was determined through Oxygen Bomb
Calorimeter (C-2000, IKA WERKE) (AOAC, 2006). For the purpose, 0.5 g sample was
placed in calorimeter bucket to allow high pressure internal burning. The generated
combustion energy was calculated by the instrument.
3.18.4. Sensory evaluation
The muffins were assessed for color, flavor, taste, texture and overall acceptability by
consumer panel using 9-point hedonic scale (Meilgaard et al., 2007). Purposely, 9 panelists
(25 to 45 years age) were given training to perform sensory evaluation through simple
orientation. Panelists were selected based solely on interest, time availability, willingness
44
and lack of allergies to food ingredients used in the muffins under study. The individual
rating i.e. liked extremely-9 to disliked extremely-1 was used. The sensory evaluation was
performed in Sensory Evaluation Laboratory at NIFSAT. During sensory session, four
muffins from each treatment were served to the panelists. Serving order was determined by
following the permutation principle. Purposely, the muffins made from different flour
blends were labeled with three digit random codes. The panel was directed to assign scores
to different sensory attributes according to their opinion.
3.19. Statistical Analysis
The collected data was statistically analyzed using Statistical Package (Costat-2003, Co-
Hort, v 6.1.). Accordingly, level of significance was estimated by analysis of variance
(ANOVA) using completely randomized design (CRD) as defined by Steel et al., (1997).
45
Chapter 4
RESULTS AND DISCUSSION
Plant based foods are one of the key contributors of protein in human diet. Despite their
importance as major sources of oil, the defatted oilseeds contain appreciable amount of
quality proteins. These proteins play imperative role in growth, repair and maintenance of
human body. Purposely, the current research study was designed to isolate protein from
different defatted oilseed meals i.e. sesame, flaxseed and canola. Furthermore, the resultant
isolates were analyzed for yield as well as functional properties i.e. bulk density, water &
oil absorption, emulsifying & foaming properties, gelation and amino acid profile. The
amino acid score was also determined with reference to requisite profile for pre-schoolers.
Moreover, bio-evaluation of these isolates was carried out using growth parameters i.e.
protein efficiency ratio, net protein ratio & relative net protein ratio and nitrogen balance
study including true digestibility, biological value & net protein utilization using Sprague
Dawley rats. Afterwards, based on overall yield, functional attributes and bio-evaluation,
one best protein isolate i.e. sesame protein isolate (SPI) was selected for the preparation and
characterization of composite flour blends. Subsequently, protein enriched muffins were
prepared using flour blends and analyzed for various physicochemical & sensorial
attributes. The findings for the above mentioned parameters are explained herein:
4.1. Characterization of oilseeds
Raw and defatted oilseeds (sesame, flaxseed and canola) were investigated for proximate
and mineral profile to determine their quality and nutritional significance.
4.1.1. Proximate composition
The oilseeds were assessed for moisture, crude protein, crude fat, crude fiber, ash content
and nitrogen free extract (NFE). Mean squares indicated momentous differences among the
oilseed samples (Table 4.1).
The means indicated moisture content ranging from 4.53±0.37 to 6.32±0.10% in sample
oilseeds. The maximum crude protein content was observed in sesame (22.41±0.55%)
followed by flaxseed (21.62±0.38%) and canola (19.93±0.56%). Moreover, crude fat
differed significantly among sesame (41.29±1.24%), canola (39.70±1.35%) and flaxseed
(34.99±1.42%). Crude fiber ranged from 3.42±0.13 to 7.55±0.29% for oilseed samples
46
while ash content varied from 3.05±0.11 to 5.44±0.19%. Likewise, NFE exhibited
significant differences with values ranging from 21.74±0.50 to 27.97±1.22% (Table 4.2).
The instant outcomes are in consensus with previous research studies, though, minor
differences exist owing to difference in varieties or the environmental conditions.
Previously, Ogungbenle and Onoge (2014) revealed the presence of moisture, crude
protein, crude fat, crude fiber and ash in sesame as 11.69, 33.91, 40.97, 5.63 and 4.04
g/100g, respectively. Likewise, Bukya and Vijayakumar (2013) explicated moisture,
protein, fat, fiber and ash in sesame as 5.30, 18.30, 43.30, 2.90 and 5.20%, correspondingly.
Similar results were illustrated by Makinde and Akinoso (2013), they stated moisture
content ranging from 4.18 to 5.41%, protein 21.94-23.64%, fat 45.63-46.09%, fiber 4.70-
7.15 and ash 6.16-7.34% for different sesame varieties.
Present findings for flaxseed are also in conformity with the results of Amin and Thakur
(2014), they expounded that moisture, crude protein, crude fat, crude fiber and ash were
6.89, 28.86, 33.43, 5.61 and 3.80%, correspondingly. The instant findings are also closely
associated with the verdicts of Ganorkar and Jain (2013), they recorded 7.7% moisture in
flaxseed, 20% crude protein, 41% fat, 28% fiber and 3.4% ash. Likewise, Herchi et al.
(2015) explained that moisture, protein, fat, fiber and ash were 5.22, 22.65, 35.10, 30.00
and 2.90%, respectively in flaxseed.
The results of current investigation for proximate composition of canola are also in concord
with the findings of Fernández et al. (2014), documented 7.1% moisture, 18.7% protein and
44.2% fat. Similar results were elucidated by Klassen et al. (2011) for moisture, crude
protein, crude fat and ash as 3.37, 22.30, 44.10 and 4.32%, respectively. The current
outcomes are also in accordance with the findings of Arif et al. (2012). The authors
deduced crude protein, crude fat and ash for canola as 23.33, 43.87 and 6.93%,
correspondingly. Likewise, Anwar et al. (2015) presented values for moisture and crude
fiber as 6.0% and 10.0%, respectively in canola seeds.
However, differences in proximate composition of oilseeds as sesame, flaxseed and canola
might be due to differences in genotypes, environmental conditions as well as analytical
techniques. Previously, Najib and Al-Khateeb (2004) worked on oilseeds and delineated
that oilseeds offer better quality proteins.
47
Table 4.1. Mean squares for proximate composition of oilseed samples
SOV df Moisture Crude
protein
Crude
fat
Crude
fiber Ash NFE
Samples 2 3.298** 6.406** 42.981** 17.452** 5.687** 39.611**
Error 9 0.061 0.251 1.801 0.082 0.035 0.766
Total 11
P value <0.05
** Highly significant NFE=Nitrogen free extract
Table 4.2. Proximate composition (%) of oilseed samples
Oilseeds Moisture Crude
protein Crude fat
Crude
fiber Ash NFE
Sesame 4.53±0.37c 22.41±0.55a 41.29±1.24a 3.42±0.13c 4.27±0.24b 24.08±0.75b
Flaxseed 6.32±0.10a 21.62±0.38b 34.99±1.42c 6.05±0.38b 3.05±0.11c 27.97±1.22a
Canola 5.64±0.19b 19.93±0.56c 39.70±1.35b 7.55±0.29a 5.44±0.19a 21.74±0.50c
Means with similar letters in a column are significantly alike
48
Conclusively, tested oilseeds i.e. sesame, flaxseed and canola possess good nutritional
profile with respect to protein, fat and fiber. Furthermore, these oilseeds are accessible and
exhibit quality protein that can be replaced with the conventional animal protein. It is
evident from the present investigation that oilseeds are nutritionally favorable in terms of
protein availability. Therefore, these can play a pivotal role to alleviate PEM especially in
the developing nations.
4.1.2. Mineral profile of oilseeds
The mean squares (Table 4.3) for mineral profile showed momentous variations among the
oilseeds. The results (Table 4.4) indicated that sodium was maximum in canola
651.70±21.44 mg/100g followed by sesame 76.30±6.26 mg/100g, while minimum was
observed in flaxseed 30.36±0.50 mg/100g. Likewise, the results for potassium were in
subsequent manner for canola (1048.50±29.51 mg/100g), flaxseed (824.12±14.32 mg/100g)
and sesame (549.91±13.40 mg/100g). Similarly, for calcium the results were
1226.05±41.82, 1146.25±34.48 and 195.09±7.94 mg/100g for canola, sesame and flaxseed,
respectively. Iron was in higher concentration in canola (22.51±0.87 mg/100g) as compared
to sesame (9.45±0.36 mg/100g) and flaxseed (4.15±0.26 mg/100g). However, zinc was
higher in sesame 5.62±0.31 mg/100g followed by flaxseed 3.37±0.12 mg/100g and canola
2.78±0.10 mg/100g.
Earlier, Obiajunwa et al. (2005) delineated calcium as key mineral in sesame seed.
Nevertheless, the differences in mineral profile may be due to variations in environmental
conditions and genotypes. Likewise, Ogungbenle and Onoge (2014) described that sesame
seeds contain 87.21 mg/100g Na, 96.33 mg/100g K, 61.37 mg/100g Ca, 7.29 mg/100g Fe
and 19.29 mg/100g Zn. However, Özcan et al. (2013) observed that sesame seed contains
122.50 mg/100g of sodium and 851.35 mg/100g potassium. Similarly, Zebib et al. (2015)
explicated that calcium ranged from 1172.08-1225.71 mg/100g in sesame, whilst minimum
ranges were documented for iron (10.2-10.75 mg/100g) and zinc (4.23 - 4.45 mg/100g).
Another researchers group explained that K and Ca were prevailing in flaxseed whilst, Na,
Fe and Zn were comparatively in lower concentration (Katare et al. 2012). Later,
Bernacchia et al. (2014) delineated that flaxseed contain 831 mg/100g K, 236 mg/100g Ca,
27 mg/100g Na, 5.0 mg/100g Fe and 4.0 mg/100g Zn. Similarly, Karande (2014) reported
potassium, calcium, iron and zinc as 813, 255, 5.73 and 4.34 mg/100g, respectively in
flaxseed.
49
Table 4.3. Mean squares for mineral profile of oilseed samples
SOV df Sodium Potassium Calcium Iron Zinc
Samples 2 479508.992** 249423.192** 1315972.875** 356.884** 8.970**
Error 9 166.334 418.474 1000.130 0.319 0.040
Total 11
P value <0.05
** Highly significant
Table 4.4. Mineral profile (mg/100g) of oilseed samples
Oilseeds Sodium Potassium Calcium Iron Zinc
Sesame 76.30±6.26b 549.91±13.40c 1146.25±34.48b 9.45±0.36b 5.62±0.31a
Flaxseed 30.36±0.50c 824.12±14.32b 195.09±7.94c 4.15±0.26c 3.37±0.12b
Canola 651.70±21.44a 1048.50±29.51a 1226.05±41.82a 22.51±0.87a 2.78±0.10c
Means with same letters in a column are significantly alike
50
Previously, Hussain et al. (2008) explicated the presence of Na, K, Ca, Fe and Zn in
flaxseed as 32.43, 826.32, 6.10 and 4.51 mg/100g, correspondingly. According to Acikgoz
and Deveci (2011), canola exhibited essential minerals like potassium, calcium, iron and
zinc as 3.06, 2.65, 23.96 and 2.95 mg/100g, respectively. Likewise, Kandil and Gad (2012)
documented that canola contains 0.74 g/100g Na, 2.05 g/100g K, 1.59 g/100g Ca, 24.77
mg/100g Fe and 3.00 mg/100g Zn. Varietal differences, environmental aspects and
genotypic changes may be the reason for variations in mineral profile explicated in the
current findings (Iqbal et al., 2011).
4.1.3. Proximate analysis of defatted oilseeds
Mean squares for proximate composition (Table 4.5) showed significant differences among
defatted oilseed samples. The means (Table 4.6) showed that moisture varied from
7.34±0.60 to 9.37±0.15% in defatted oilseeds. Maximum crude protein content was
observed in sesame 40.90±1.00% followed by flaxseed 36.57±0.64% and canola
34.88±0.98%. Crude fat was found as 3.97±0.12% in sesame, 2.48±0.09% in canola whilst
1.91±0.08% in flaxseed. Moreover, significant differences were observed for crude fiber as
12.81±0.50%, 11.85±0.74% and 7.82±0.30% for canola, flaxseed and sesame, respectively.
The ash content varied from 5.30±0.18 to 7.49±0.42% while NFE presented significant
differences in the range of 32.48±1.01 to 35.09±0.81%.
Current results of defatted sesame are in agreement with the findings of Hassan (2013),
they delineated the ranges for moisture, protein, fat, fiber and ash as 1.72- 2.76, 45-55,
2.20-3.58, 22.49-24.48 and 5.11-6.94%, respectively. Likewise, Jimoh and Aroyehun
(2011) elucidated the composition of defatted sesame as 10.68% moisture, 38.73% protein,
12.75% fat, 5.78% fiber and 9.48% ash. The instant outcomes are also supported by the
findings of Essa et al. (2015), they stated moisture content, protein, fiber and ash for
defatted sesame as 8.79%, 51.05%, 18.26% & 6.05%, respectively.
The present findings for defatted flaxseed are also in accordance with the outcomes of
Gupta and Shivhare (2012). The researchers noticed moisture, crude protein, fat, fiber and
ash as 7.83, 38.16, 0.94, 15.14 and 5.86%, correspondingly. Likewise, Bhise and Kaur
(2013) delineated 2.61% moisture, 38.24% crude protein, 2.71% fat and 12.24% fiber.
Moreover, Mueller et al. (2010) observed 1.67% fat 43.30% protein and 6.40% ash in
linseed meal whilst, Eastwood et al. (2009) observed 8.40% moisture in defatted flaxseed.
51
Table 4.5. Mean squares for proximate composition of defatted oilseeds
SOV df Moisture Crude
protein Crude fat
Crude
fiber Ash NFE
Samples 2 4.142** 38.651** 4.526** 28.052** 4.826** 8.779**
Error 9 0.153 0.787 0.009 0.295 0.087 1.334
Total 11
P value <0.05
** Highly significant NFE=Nitrogen free extract
Table 4.6. Proximate composition (%) of defatted oilseeds
Oilseeds Moisture Crude
protein Crude fat
Crude
fiber Ash NFE
Sesame 7.34±0.60c 40.90±1.00a 3.97±0.12a 7.82±0.30c 7.49±0.42a 32.48±1.01b
Flaxseed 9.37±0.15a 36.57±0.64b 1.91±0.08c 11.85±0.74b 5.30±0.18c 35.00±1.52a
Canola 8.26±0.27b 34.88±0.98c 2.48±0.09b 12.81±0.50a 6.48±0.23b 35.09±0.81a
Means having same letters in a column are significantly alike
52
The present results for defatted canola are in concordance with the findings of Aider and
Barbana (2011), who found 12.30% moisture, 32.10% protein, 4.40% fat and 8.20% ash.
The findings of Khattab and Arntfield (2009) also supported these results. They elucidated
11.35 moisture, 36.13 crude protein, 2.77 crude fat, 11.54 crude fiber and 6.26g/100g ash
content. Likewise, Li et al. (2012) expounded 0.89% crude fat, 49.26% crude protein and
8.62% crude fiber while Tan et al. (2011a) illustrated 10.24% moisture and 5.34% ash in
defatted canola. The variations in proximate composition might be due to difference in
genotypes and analytical methods.
4.1.4. Mineral composition of defatted oilseeds
The mean squares (Table 4.7) showed that defatted oilseeds exhibit significant differences
regarding mineral profile. The mean values (Table 4.8) noted for sodium were
776.73±17.89, 133.88±4.18 and 52.69±2.29 mg/100g for canola, sesame and flaxseed,
respectively. Likewise, maximum potassium content was observed in flaxseed
1430.14±42.54 mg/100g followed by canola 1249.65±51.69 mg/100g and sesame
964.89±30.52 mg/100g.
Similarly maximum calcium was noticed in sesame 2011.25±61.07 mg/100g, tracked by
canola 1461.27±78.99 mg/100g and flaxseed 338.55±12.28 mg/100g. Nonetheless, highest
concentration of iron was depicted in canola (26.83±1.47 mg/100g) while minimum was
observed in flaxseed (7.21±0.34 mg/100g). Nevertheless, zinc was higher in sesame
(9.86±0.59 mg/100g) whereas lower in flaxseed (5.85±0.15 mg/100g) and canola
(3.31±0.04 mg/100g).
Earlier, Essa et al. (2015) observed calcium as the major mineral in defatted sesame seed.
They illustrated 0.31% Na, 0.53% K, 1.91% Ca, 0.13% Fe and 0.03% Zn. Similarly,
Ogungbenle and Onoge (2014) explicated that defatted sesame seeds contain 59.88
mg/100g Na, 63.42 mg/100g Ca, 7.26 mg/100g Fe and 17.29 mg/100g Zn. Nonetheless,
Hassan (2013) delineated 0.29-0.35 g/100g Na, 0.51-0.77 g/100g K and 1.40-2.00 g/100g
Ca in defatted sesame. Previously, Hussain et al. (2008) elucidated that potassium was
dominant in defatted flaxseed whilst, sodium, iron and zinc were in lower concentrations.
They described the results as 58.16 mg/100g Na, 1369.31 mg/100g K, 398.21 mg/100g Ca,
10.96 mg/100g Fe and 7.86 mg/100g Zn. Likewise, Khan et al. (2010) explained that
flaxseed contains Na 0.05%, K 1.41%, Ca 0.39%, Fe 50.56 ppm and Zn 13.55 ppm.
According to Khajali and Slominski (2012), defatted canola contains essential minerals like
53
Table 4.7. Mean squares for mineral profile of defatted oilseeds
SOV df Sodium Potassium Calcium Iron Zinc
Samples 2 629392.738** 220089.531** 2907265.833** 385.076** 43.556**
Error 9 114.286 1804.158 3373.221 1.138 0.125
Total 11
P value <0.05
** Highly significant
Table 4.8. Mineral profile (mg/100g) of defatted oilseeds
Oilseeds Sodium Potassium Calcium Iron Zinc
Sesame 133.88±4.18b 964.89±30.52c 2011.25±61.07a 16.59±1.07b 9.86±0.59a
Flaxseed 52.69±2.29c 1430.14±42.54a 338.55±12.28c 7.21±0.34c 5.85±0.15b
Canola 776.73±17.89a 1249.65±51.69b 1461.27±78.99b 26.83±1.47a 3.31±0.04c
Means having different letters in a column vary significantly
54
sodium, potassium and calcium as 0.08, 1.17 and 0.67%, respectively. Similarly, Kubik et
al. (2012) explicated that defatted canola exhibits 0.13% Na, 1.32% K and 0.80% Ca.
4.2. Protein isolates; recovery, yield and protein content
Oilseeds, mainly utilized for oil extraction purpose are also amongst the vital sources of
high quality proteins that can be extracted by isoelectric precipitation with substantial yield.
Purposely, protein isolates of the selected oilseeds were evaluated for their recovery,
protein content and yield. Mean squares regarding isolates recovery, crude protein &
protein yield elucidated momentous difference amongst the isolates (Table 4.9).
The results indiacted that maximum recovery (36.86±1.22 g/100g) was noticed in sesame
protein isolates (SPI) followed by 31.59±0.98 g/100g in flaxseed protein isolates (FPI).
However, the lowest protein isolate recovery (30.52±1.20 g/100g) was observed in canola
protein isolates (CPI). Likewise, maximum crude protein 76.14±2.00% was recorded in SPI
trailed by FPI 73.37±3.13% and CPI 69.75±2.78%. Similarly, the highest protein yield was
recorded for SPI 79.03±2.18% whilst, 78.53±4.02% for CPI. Nonetheless, the lowest yield
was observed in FPI (74.61±2.93%) (Table 4.10).
Previously, Gandhi and Srivastava (2007) obtained 29.20% recovery for SPI. Similarly,
Kanu et al. (2007) delineated 81% recovery for SPI. Later, Ribeiro et al. (2013) recorded
70% recovery for FPI. However, Kaushik et al. (2016) explicated 12.10-20.29% FPI
recovery. The current findings for canola protein isolates (CPI) are in consensus with the
outcomes of Tan et al. (2011b), explicated 73.77% and 71.49% recovery for canola protein
isolates extracted using Osborne and direct alkaline extraction method, respectively.
In present study, maximum crude protein was noted in SPI. Similarly, Essa et al. (2015),
documented 92.43% crude protein in SPI. Earlier, Biswas et al. (2010) explicated 91.50%
crude protein in sesame protein isolates obtained from defatted sesame meal. Likewise,
Onsaard et al. (2010) determined crude protein of sesame protein concentrates ranging from
75.50 to 83.30%. Later, Kuhn et al. (2014) elucidated 68.53% crude protein in FPI.
Previously, Silva et al. (2013) observed 71.80% crude protein in flaxseed protein isolates.
The current findings for crude protein content in canola protein isolates are in agreement
with the previous literature. Moreover, Karaca et al. (2011) prepared canola protein isolates
using isoelectric precipitation & salt extraction and depicted crude protein as 75.31% &
93.10% for both methods, respectively. Likewise, Aider and Barbana (2011) explicated
90% crude protein in canola protein isolates. Similarly, it was observed that extracted
55
Table 4.9. Mean squares for protein isolates recovery, yield and crude protein
SOV df
Protein isolate
recovery
(g/100g defatted
oilseed)
Protein yield
(% defatted oilseed
protein)
Crude protein
Samples 2 46.076** 23.427** 41.065**
Error 9 1.300 9.821 7.182
Total 11
P value <0.05
** Highly significant
Table 4.10. Oilseeds protein isolates recovery, yield and crude protein
Protein isolate
Protein isolate
recovery
(g/100g defatted
oilseed)
Protein yield
(% defatted oilseed
protein)
Crude protein
(%)
SPI 36.86±1.22a 79.03±2.18a 76.14±2.00a
FPI 31.59±0.98b 74.61±2.93c 73.37±3.13b
CPI 30.52±1.20c 78.53±4.02b 69.75±2.78c
Means with similar letter in a column are essentially alike
SPI= Sesame protein isolates
FPI= Flaxseed protein isolates
CPI= Canola protein isolates
56
protein isolates from rapeseed exhibited 91.20% crude protein. Protein yield of CPI is lesser
than FPI and SPI due to protein-protein interactions (Haar et al., 2014).
Previously, Das et al. (2009) recorded 65.3% yield for sesame protein isolates. The current
results regarding the yield of FPI are supported by the outcomes of Ho et al. (2007), they
expounded 66.8% yield. Likewise, Gutiérrez et al. (2010) observed 53.15% yield for FPI.
The findings of the present study regarding CPI yield were in concord with the results of
Akbari and Wu (2015), who depicted 56.20% yield. However, Tan et al. (2011) noticed
higher yield (71.30%) for canola protein isolates.
Conclusively, oilseed protein isolates with appreciable yield can play significant role in the
development of protein enriched products. These have ability to be potentially utilized in
novel food commoditied. Additionally, protein isolation from non-conventional sources can
help to fulfill the protein requirements of an appreciable part of population.
4.3. Functional properties of defatted oilseed protein isolates
These are the intrinsic characteristics affecting product behavior during different processing
phases. These properties are mainly dependent upon the interaction of protein with three
imperative components i.e. water, oil and gas. Proteins interact with water to impart
solubility, viscosity and gelling characteristics to the end product. Likewise, interaction
with oil and gas indicates foaming and emulsifying properties. Furthermore, the functional
behavior of proteins in a food system also depends upon shape, size, amino acid
composition, structure, sequence and interaction with other food constituents. Moreover, it
is also dependent on the external environmental factors like temperature, pH and salt
concentration (Aremu et al., 2007). Moreover, protein functionality is also influenced by
isolation method and purification conditions.
Keeping in view the functional behavior, proteins play an imperative role in the
manufacturing process by contributing solubility, oil absorption, emulsification, foaming
and gelling characteristics. Owing to physicochemical changes in processing conditions,
proteins interact with other ingredients of food, thus imparting diversified functional
characteristics. Therefore, proteins isolated from plant sources can be effectively used in a
food system depending on the suitability of their functional attributes with the final product.
4.3.1. Bulk density
Mean squares showed significant variations among various defatted oilseed protein isolates
(Table 4.11). The maximum value was recorded for CPI 0.57±0.04 g/cm3 trailed by SPI
57
(0.53±0.02 g/cm3) and flaxseed protein isolates 0.45±0.03 g/cm3 (Fig. 4.1). Bulk density is
dependent on particle size, inter particle forces along with contact points strength. These
factors ultimately play vital role in defining the packaging behavior of the end product
(Oladele and Aina, 2007). In flaxseed protein isolates, the variations in textural porosity
may cause lower bulk density. However, in sesame protein isolates particle size refinement
helped appropriate settling of isolate, therefore improved bulk density.
The present results for bulk density of oilseed protein isolates are in accordance with
previous literature. Earlier Onsaard (2012) demarcated 0.71 g/mL bulk density for sesame
protein isolates. In another research trial, Taha and Ibrahim (2002) hydrolyzed sesame meal
with papain and bromelain enzymes. They observed bulk density for sesame hydrolysates
ranging from 0.25 to 0.78 g/cm. Contrarily, Escamilla-Silva et al. (2003) delineated 420
kg/m3 bulk density for sesame protein concentrates.
The results regarding bulk density of FPI are in concordance with the outcomes of Singer et
al. (2011), they elucidated 0.4 g/mL bulk density for protein product prepared using
defatted flaxseed. Recently, Khan and Saini (2016) expounded 0.66 & 0.48 g/cm3 bulk
density for flaxseed and roasted flaxseed flour, respectively.
In another research investigation, Singh et al. (2014) documented a linear decrease in bulk
density 702.24-582.37 kg/m3 of flaxseed with increase in moisture content 4.62-18.39%.
Moreover, Mailer (2004) delineated 565 kg/m3 bulk density for canola meal. In contrast to
this, Swick and Wu (2016) working on canola meal observed 61.68 kg/100L bulk density.
A powdered food product requires high bulk density as it assists in packaging by allowing
more weight in less volume (Asma et al., 2006).
4.3.2. Water and oil absorption capacities
These properties indicate the amphiphilic nature of protein isolates. The mean squares
regarding water and oil absorption capacity revealed momentous differences amongst the
tested oilseed protein isolates (Table 4.12). Results indicated that SPI showed highest water
absorption capacity (WAC) 2.12±0.08 tracked by CPI & FPI i.e. 1.47±0.06 & 1.43±0.03
mL/g, correspondingly. Likewise, highest OAC was revealed by SPI 3.11±0.12 mL/g
trailed by FPI 2.77±0.18 mL/g and CPI 1.14±0.07 mL/g (Fig. 4.2).
The WAC and OAC reflect the tendency of protein to bind water and oil molecules,
respectively. The conformational attributes of protein and their interfacial tension affect the
58
Table 4.11: Mean squares for bulk density of defatted oilseed protein isolates
SOV df Bulk density
Oilseed protein isolates 3 0.048**
Error 8 0.001
Total 11
P value <0.05
** Highly significant
Figure 4.1: Bulk density of oilseed protein isolates
0.53
0.45
0.57
0.75
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
SPI FPI CPI Casein
g/c
m3
Oilseed Protein Isolates
Bulk Density
Bulk Density
59
WAC that is also affected by the temperature and pH. Moreover, oil absorption capacity
helps in flavor preservation, improves mouthfeel as well as emulsion characteristics of the
food commodities (Martínez-Flores et al., 2006).
The instant results for absorption properties of oilseed protein isolates are comparable with
previous literature. In this context, Onsaard (2012) observed 2.10 mL/g water holding
capacity while Essa et al. (2015) noted 3.07 g/g oil holding capacity for sesame protein
isolates. Likewise, Demirhan and Özbek (2013) delineated 2.67 g/g water holding capacity
for SPI. Previously, sesame protein concentrates were prepared by using two different
solutions i.e. salt and alkali solution through isoelectric precipitation (Onsaard et al., 2010).
This srudy revealed water and oil holding capacity ranging from 1.98-3.53 & 1.19-2.69 g/g,
correspondingly.
Similarly, the present results for water and oil/fat absorption capacity of FPI are in
agreement with the outcomes of Martínez-Flores et al. (2006), i.e. 2.70 g/g water holding
capacity and 1.18 g/g oil absorption capacity. Previously, Krause et al. (2002) observed that
water binding capacity for FPI ranged from 3.7 to 9.8 g/g. Likewise, Teh et al. (2014)
presented results for WAC & OAC of FPI as 4.2 & 6.5 mL/g, respectively.
The current findings regarding WAC and OAC of canola PI are in concord with the
outcomes of Haar et al. (2014). They noted 1.6 mL/g water binding capacity and 1.3 mL/g
oil binding capacity for rapeseed protein isolates. In contrast to this, Teh et al. (2014)
delineated WAC and OAC of canola PI as 7.8 & 7.0 mL/g. Later, Gerzhova et al. (2015)
expounded 1.30 & 1.11 g/g water and fat absorption capacity for CPI in respective manner.
The WAC of SPI might be due to polar amino acids at protein-water interface. However,
conformational changes in protein may result in lower WAC in canola protein isolates. The
oil binding characteristics of protein isolates depict their efficiency to contact with oil
molecules. In present research, SPI developed strong oil binding as compared to FPI and
CPI; might be owing to the existence of more non-polar side chains that bind with hydro-
carbon chains leading to improved oil absorption. However, decreased oil absorption is
possibly attributable to the occurrence of hydrophilic groups on the protein molecules.
Moreover, the technique used for recovery of protein can also affect water holding capacity.
It is obvious from previous research studies that proteins recovered using isoelectric pre-
cipitation revealed higher water binding capacity as compared to those obtained by
ultrafiltration.
60
Table 4.12: Mean squares for absorption properties of defatted oilseed protein isolates
SOV Df Water absorption Oil absorption
Oilseed protein isolates 3 2. 565** 2.508**
Error 8 0.004 0.014
Total 11
P value <0.05
** Highly significant
Figure 4.2: Water and oil absorption capacity of oilseed protein isolates
2.12
3.11
1.43
2.77
1.47
1.14
3.41
1.72
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
Water absorption capacity Oil absorption capacity
mL
/g
Absorption properties
SPI
FPI
CPI
Casein
61
Protein solubility is dependent on balance in hydrophobic behavior of the protein molecule.
Water absorption causes swelling of molecules depending on protein, fiber and starch
concentration. Owing to its high swelling power in water, starch contributes to higher
swelling ability when combined with proteins and fiber. Therefore, oilseed protein isolates
have momentous role in swelling and water absorption properties (Torruco-Uco and
Betancur-Ancona, 2007). The fat absorption mechanism includes physical entrapment of
oil. Therefore, OAC can be influenced by various factors like, particle size, moisture
content and microstructure. Furthermore, different protein composition & quantity of non-
polar amino acids along with conformational changes and starch-protein-lipid binding may
cause variations in oil retention attributes of oilseed proteins (Lazou and Krokida, 2010).
The proteins impart form, viscosity and thickness to the foods like bakery products,
custards and soups by absorbing water while keeping the proteins intact (Seena and
Shridhar, 2005). Bakery products handling require improved WAC. The flours exhibiting
higher OAC can be potentially utilized in manufacturing doughnuts and pancakes as OHC
is a crucial trait in these commodities (Akubor, 2003).
4.3.3. Foaming capacity and stability
These properties play imperative role in the determination of functional characteristics of
proteins. Moreover, higher water solubility, flexibility and the ability of protein to become
part of cohesive film at the air-water interface help in the formation of better foam (Cano-
Medina et al., 2011). The foaming capacity represents relative increase in the volume of
protein solution by the incorporation of air.
Nonetheless, foam stability indicates the ability of food molecules to keep air bubbles. It is
estimated either by the reduction or separation of foam volume from food over a short time
period (Boye et al., 2010).
The mean squares for FC and FS depicted momentous differences amongst various oilseed
protein isolates (Table 4.13). In this context, sesame protein isolates (SPI) showed highest
foaming capacity 18.51±0.60 mL followed by flaxseed protein isolates (FPI) i.e.,
14.13±0.52 mL, however, lowest FC was depicted by canola protein isolates (CPI)
12.29±0.53 mL (Fig. 4.3). Likewise, maximum foaming stability was noticed in SPI
46.98±0.90 min and minimum in CPI 35.46±1.19 min while FPI indicated foaming stability
as 39.87±1.43 min (Fig. 4.4).
62
In general, foams are required to develop better texture, appearance and consistency in
foods (Akubor, 2003). Moreover, foaming properties indicate the whipping ability of
protein isolates. Liquid or semi liquid material is whipped to entrap air and produce foam in
the form of bubbles. The resultant foam reduces the interfacial tension and enhances
stickiness of liquid resulting in the formation of strong film. Furthermore, the FC is
dependent on dispersion of protein at air-water interface caused by the unfolding of
structure while, foaming stability indicates the formation of thick cohesive layer around the
air bubble (Mepba et al., 2007).
The highest foaming capacity was noticed in SPI due to upsurge in foam hydration and
stable molecular layer formation at water & air interface. Nonetheless, CPI showed low FC
as the disulfide bonds are denatured resulting in decreased flexibility. Previously,
Alamanou and Doxastakis (1997) explained that the protein isolation process also affects
the degree of denaturation. In a research investigation, Demirhan and Özbek (2013)
revealed that sesame cake protein hydrolysates exhibit 45.2% FC and 31.5 mL FS.
Similarly, Onsaard et al., 2010 expounded FC and FS of sesame protein concentrates as
58% and 14 min, respectively. These results differ with the present findings owing to
different methods of protein isolates preparation. Contrarily, Ogungbenle and Onoge (2014)
noticed 6.53% foaming capacity and 3.25% foaming stability in sesame protein
concentrates. Previously, Boye et al. (2010) explicated that recovery methods do not affect
the foaming characteristics of protein isolates. Moreover, protein isolates have capability to
yield foam having improved solubility as it requires maximum net charge that eventually
modifies the adsorption characteristics (Hassan, et al., 2010).
Previously, Hussain et al. (2008), reported 17.40 mL FC and 9.00 mL FS in partially
defatted flaxseed flour. Likewise, Martínez-Flores et al. (2006) revealed 12% FC and
83.3% FS in flaxseed protein concentrates. In contrast to this, Rabetafika et al. (2011)
delineated 80% FC and 22 min FS in flaxseed protein isolates. Gerzhova et al. (2015)
obtained similar results for the foaming properties of canola protein isolates. They observed
57.83% foaming capacity and 18% foaming stability for CPI. Similarly, Moure et al. (2006)
explicated 56% FC in commercial canola protein isolates. Flexible proteins exhibit good
foaming ability due to reduced surface tension (Khuwijitjaru et al., 2007; Jitngarmkusol et
al., 2008).
63
Table 4.13: Mean squares for foaming properties of defatted oilseed protein isolates
SOV df Foaming capacity Foaming stability
Oilseed PI samples 4 41.022** 3926.370**
Error 10 0.362 1.950
Total 14
P value <0.05
** Highly significant
64
Figure 4.3: Foaming capacity of oilseed protein isolates
Figure 4.4: Foaming stability of oilseed protein isolates
18.51
14.13
12.29
19.15
21.17
0
5
10
15
20
25
FC
mL
Foaming Capacity
SPI
FPI
CPI
Casein
Egg White
46.98
39.8735.46
77.54
122.02
0
20
40
60
80
100
120
140
FS
mim
Foaming Stability
SPI
FPI
CPI
Casein
Egg White
65
4.3.4. Emulsion capacity and stability
Protein exhibits better tendency to form emulsions by facilitating their formation and
improving the stability. Moreover, proteins from plant sources help in the production of
required physicochemical attributes in various emulsions. The emulsifying ability of protein
is attributed to its hydrophobic as well as hydrophilic structure. Furthermore, protein
reduces the oil-water interfacial tension and its electrostatic repulsion mechanism assists in
the stabilization of oil droplets, thus facilitating the emulsion formation (Brewer et al.,
2016). The mean squares regarding emulsion properties of different oilseed protein isolates
illustrated significant variations in emulsion capacity and stability (Table 4.14).
The maximum emulsifying capacity (EC) was recorded in SPI 81.36±2.19% followed by
FPI 73.24±2.50% whereas, minimum in CPI 65.40±3.13% (Fig. 4.5). Emulsion stability
refers to the tendency of isolate to create resistance against emulsion breakdown. The
results for emulsion stability (ES) revealed higher stability in SPI 78.69±1.08% while,
lower in FPI 75.08±3.22% and CPI 71.97±2.50% (Fig. 4.5). The lowest emulsifying
capacity of CPI might be due to fewer hydrophobic residues on protein surface. Resultantly,
the oil droplets diffused in continuous aqueous phase. Protein denaturation may enhance the
emulsifying properties owing to increased elasticity and hydrophobic surface (Raymundo et
al., 1998). Furthermore, the emulsion properties are influenced by molar mass,
conformational stability hydrophobicity and some physicochemical factors like pH,
temperature & ionic strength (Lam and Nickerson, 2013).
Heat treatment increases emulsification and surface activity of proteins due to formation of
hydrophobic units thus facilitating protein interface with non-polar solvents. The protein-
lipids interaction is expedient to promote the emulsion stability that results in reduction of
conformational stability and hydrophilic attributes of proteins. The emulsion capacity and
stability are affected by numerous physical factors including cream formation, aggregation
and coalescence. These factors play a fundamental role in the separation phase. Consistency
against coalescence is the most common prerequisite of emulsion during storage
(Tolstoguzov, 1997).
The instant findings are in contrast with the outcomes of Ogungbenle and Onoge (2014),
estimated 27.43% EC and 30.50% ES for sesame protein isolates (SPI) that might be due to
difference in pH as the emulsion properties are significantly affected by changes in pH.
Likewise, Essa et al. (2015) extracted proteins from sesame meal and studied emulsifying
66
Table 4.14: Mean squares for emulsion properties of defatted oilseed protein isolates
SOV df Emulsion capacity Emulsion stability
Oilseed PI samples 3 299.819** 1197.10**
Error 8 7.037 5.390
Total 11
P value <0.05
** Highly significant
Figure 4.5: Emulsion capacity and stability of oilseed protein isolates
81.3678.69
73.24 75.08
65.40
71.97
88.50
32.38
0
10
20
30
40
50
60
70
80
90
100
Emulsion Capacity Emulsion Stability
Perc
en
tag
e (
%)
Emulsion properties
SPI
FPI
CPI
Casein
67
properties of the resultant isolates at different pH levels. They delineated 37% EC and
43.24% ES at pH 6. In another research investigation, Khalid et al. (2003) found 70.00%
emulsion activity (EA) and 70.02% emulsion stability (ES) for sesame seed proteins.
The results of the present research regarding emulsion properties of flaxseed protein
isolates (FPI) are supported by the findings of Rabetafika et al. (2011), reported 63 & 59%
EC and 81 & 70% ES at pH 4 & 9, respectively. Previously, Martínez-Flores et al. (2006)
expounded 84.8% EC at pH 6 whilst 88.4% ES at pH 8 for flaxseed proteins. Similarly,
Singer et al. (2011) estimated 95.3% emulsion capacity for flaxseed protein product while,
Kaushik et al. (2016) explicated 86% emulsion stability for flaxseed protein isolates.
Earlier, Stone et al. (2014) delineated 63.34% EC and 76.00% ES for CPI. Likewise, Teh et
al. (2014) documented 50% emulsion activity and 100% emulsion stability for CPI. Later,
Moure et al. (2006) described 41.6% emulsion activity index and 70 % emulsion stability
for commercial canola proteins.
The emulsion properties (EC & ES) are the momentous attributes of food proteins that play
imperative role in the stabilization of food system. Moreover, the oil absorption ability of
proteins is enhanced by hydrophobicity and the surface area. The proteins have tendency to
bind with fat depending on numerous factors including size of protein molecules,
flexibility, degree of denaturation and non-polar sides (Ulloa et al., 2011). Previous
research investigations have proven that protein rich materials exhibit better emulsion
attributes hence can be potentially utilized in various food products like mayonnaise, cake
batter and salad dressings (Akubor, 2003).
4.3.5. Nitrogen solubility index
NSI of defatted oilseed protein isolates was pH dependent as shown in Fig. 4.6 to 4.8. The
lowest NSI 7.32-23.43% was observed at pH 4.0 owing to isoelectric region. Furthermore,
an increasing trend for solubility was noted on either side of pH i.e. acidic and basic.
Moreover, a noticeable rise in nitrogen solubility was detected till pH 8.0 where it showed
an index of 34.46 to 54.31%. A progressive increase was noticed up to pH 12.0, where
nitrogen solubility index ranged from 62.51 to 82.56%.
Solubility is mainly dependent on physicochemical attributes of protein affecting functional
properties like foaming, gelling and emulsification capacity. The present results for the
nitrogen solubility index of oilseed protein isolates are supported by the outcomes of
previous research studies. Earlier, Bandyopadhyay and Ghosh (2002) delineated 55.97%
68
Figure 4.6: Nitrogen solubility (%) of sesame protein isolates (SPI)
Figure 4.7: Nitrogen solubility (%) of flaxseed protein isolates (FPI)
70.14
23.43
28.32
54.31
71.23
82.56
0
10
20
30
40
50
60
70
80
90
2 4 6 8 10 12
N.
So
lub
ilit
y (
%)
pH
SPI
40.42
12.0513.84
50.25
58.72
65.01
0
10
20
30
40
50
60
70
2 4 6 8 10 12
N. S
olu
bil
ity
(%
)
pH
FPI
69
Figure 4.8: Nitrogen solubility (%) of canola protein isolates (CPI)
52.03
7.3210.14
34.46
58.12
62.51
0
10
20
30
40
50
60
70
2 4 6 8 10 12
N.
Solu
bil
ity (%
)
pH
CPI
70
protein solubility for sesame protein isolates at pH 7. Similarly, Karaca et al. (2011)
observed 40% nitrogen solubility index (NSI) for flaxseed protein isolates. Whilst,
Gerzhova et al. (2015) explicated that nitrogen solubility index for canola protein isolates
ranged from 8.09-56.82% at various pH levels. It was also noticed that alkali caused
disaggregation and dissociation of proteins that generally helps to improve protein
solubility (Hojilla-Evangelista et al., 2009). Previous research investigation has proven that
nitrogen solubility index (NSI) determines protein solubility primarily caused by protein
dispersion in solvent. NSI has also become a vital functional property of protein isolates
and hydrolysates (Thiansilakul et al, 2007).
Earlier, Tomotake et al. (2002) expounded that net negative charge on protein is increased
at higher pH values resulting in the dissociation of its aggregates. However, the carboxyl
and amino groups are protonated as -COOH and -NH, respectively at lower pH value that
generally results in positive charge. Moreover, the amino groups disassociate into -NH2 and
-H+ with increase in pH causing the protein to be negatively charged due to -COO- group.
Nevertheless, a gradual rise in pH causes a few carboxyl groups to dissociate into -COO-
and -H+ (Nicole et al., 2010).
Solubility of protein isolates is influenced by processing conditions. Previous studies have
indicated highest protein solubility at low acidic and high basic pH values. Nevertheless,
lowest solubility was noticed at pH values near isoelectric point. Nitrogen solubility of
protein isolates and concentrates can be increased by hydrolysis and physicochemical
modifications (Boye et al., 2010).
4.3.6. Least Gelation Concentration (LGC) of defatted oilseed protein isolates
The gelation ability is typically stated as least gelation concentration. LGC refers to the
qualitative attribute that determines least concentration of protein required to form gel.
Furthermore, this gel must not slide along the inverted test tube walls owing to the
formation of self-supporting network (Rai et al., 2014). Gel formation of oilseed protein
isolates occurs at a temperature higher than protein denaturation.
The results (Table 4.15) indicated that SPI exhibited high least gelation concentration 16%
followed by FPI 15% and CPI 14%. Gelation ability was observed from 12 to 14%
concentration of protein isolates, whilst, a stable and strong gel was detected from 16%
concentration to onward. Furthermore, lower concentration solution of protein isolates
showed higher liquid phase. Soy protein revealed a sticky tendency at 12% concentration;
71
Table 4.15: Least gelation concentration of defatted oilseed protein isolates
Concentration
(%) SPI FPI CPI Soy protein
2 (−) (−) (−) (−)
4 (−) (−) (−) (−)
6 (−) (−) (−) (−)
8 (−) (−) (−) (−)
10 (−) (−) (±) (−)
12 (±) (±) (±) (±)
14 (±) (±) (+) (±)
16 (+) (+) (+) (+)
18 (+) (+) (+) (+)
20 (+) (+) (+) (+)
LGC 16 15 14 16
Gelation levels: (−) no, (±) partial, (+) complete gel
72
however, a stable gel was noticed at 16%. Moreover, protein denaturation and gel strength
caused lesser LGC for canola protein isolates. The gelling ability of proteins is dependent
on the concentration and pH balance of cation & anion. Therefore, at neutral pH, viscid gel
was not formed below 16% concentration. Least gelation concentration relies on certain
characteristics like viscosity, elasticity and plasticity. The gel forming ability of protein
gives structural matrix that helps in water binding. The variations in gelling ability of
different protein isolates were due to the differences in their protein, lipid and carbohydrate
contents. Moreover, LGC plays imperative role in food system by contributing towards
texture and rheology of end product (Nicole et al., 2010).
Previously, Fekria et al. (2012) explicated 6.0% least gelation concentration for defatted
sesame seeds. Likewise, Singer et al. (2011) elucidated 11% gelation for flaxseed.
However, for canola protein isolates 14.9-15.7% LGC was indicated by Nithiyanantham et
al. (2013). Later, He et al. (2014) observed 15% LGC for rapeseed proteins. Earlier, Cheng
et al. (2009) explained that protein-protein interaction of isolates at isoelectric point affects
the gelation ability due to lack of net charge on the surface of protein molecules.
Conclusively, the outcomes of current study indicated that oilseed protein isolates are rich
in quality protein and exhibit remarkable functional characteristics that can be explored in
the food systems. Further, these protein isolates have ability to be potentially utilized in
various food formulations depending upon the required characteristics and function. The
protein isolates can be incorporated into bakery products. Nevertheless, their possible
effectiveness depends on functional properties that ultimately affect sensory attributes of
the food.
4.4. SDS-PAGE
The proteins of resultant isolates (SPI, FPI and CPI) were characterized for their molecular
weight using Sodium dodecyl sulfate polyacrylamide gel electrophoresis. The
electropherogram for sesame, flaxseed & canola protein isolates and the reference standard
is illustrated in figure 4.9. The respective isolates were recorded ranging from 15-65kDa.
The electropherogram also presented numerous fractions having low molecular weights.
Moreover, the SPI included several polypeptide bands ranging from 15 to 45 kDa while,
FPI bands lied between 25 to 48 kDa. Furthermore, the CPI bands ranged from 16-65 kDa
with fewer bands than other tested protein isolates.
73
Figure 4.9: Electropherogram of oilseed protein isolates. Sesame protein isolates (SPI),
Flaxseed protein isolates (FPI), Canola protein isolates (CPI), standard (Std)
74
Nonetheless, a trivial difference in the movement was observed in the electrophoretic bands
for SPI, FPI & CPI. Certain variations may be attributed to structural as well as
compositional changes in protein along with their interaction with salt. The present findings
are in conformity with the outcomes of Achouri and Boye (2013), they delineated
molecular weight between 10-40 kDa for sesame proteins. Likewise, Chatterjee et al.
(2015) explicated that molecular weight of proteins in sesame protein concentrates and
hydrolysates ranged from 20-40 & 14-26 kDa, respectively. Earlier, Achouri et al. (2012)
observed molecular weight of sesame proteins ranging from 10-50 kDa. Previously, Chung
et al. (2005) studied SDS-PAGE for major fractions of flaxseed proteins and observed three
major bands with molecular weights 20, 23 & 31 kDa. Likewise, Krause et al. (2002)
demarcated the molecular weight of flaxseed proteins in the range of 19-38 kDa. According
to the findings of Pinterits and Arntfield (2008), canola protein isolates exhibited proteins
with molecular weight ranging from 22-31 kDa. Likewise, Pinterits and Arntfield (2007)
treated canola protein isolates with different levels of trypsin and observed same results for
molecular weight of canola proteins via SDS-PAGE. Later, Tan et al. (2011b) delineated
that canola protein isolates exhibit 8 major bands in the range of 14-59 kDa.
4.5. Amino acid profile of defatted oilseed protein isolates
Mean squares in Table 4.16 and 4.18 explicated significant differences for essential and
non-essential amino acids of oilseed protein isolates. These isolates exhibit better amino
acids profile as protein quality mainly depends on essential amino acids. The maximum
lysine content was recorded in CPI as 2.60±0.09 g/100g followed by FPI 1.62±0.07 g/100g
whilst, SPI showed lowest value as 1.48±0.04 g/100g (Table 4.17).
Data regarding essential amino acids of sesame protein isolates (SPI) showed values for
aromatic amino acids (phenylalanine+tyrosine) 3.36±0.11, leucine 4.39±0.11, sulfur
containing amino acids (methionine+cysteine) 1.59±0.05 and valine 4.95±0.19 g/100g,
respectively. Likewise for FPI, the maximum values were observed for leucine (4.37±0.22
g/100g) and aromatic amino acids (3.30±0.90 g/100g) while, minimum values 1.67±0.04 &
1.96±0.03 g/100g were noted for histidine and tryptophan, respectively. Moreover, CPI
exhibited maximum value for leucine 4.30±0.23 g/100g followed by aromatic amino acids
2.89±0.15 g/100g, valine 2.69±0.05 g/100g and threonine 2.38±0.08 g/100g (Table 4.17).
Means pertaining to non-essential amino acids of oilseed protein isolates are given in Table
4.19. Highest value for alanine (3.30±0.08 g/100g) was observed in FPI whilst, lowest
75
(2.66±0.11 g/100g) in CPI. Glutamic acid was varying from 9.70±0.33 to 12.70±0.76
g/100g in the tested protein isolates whilst, serine ranged from 2.51±0.11 to 3.17±0.05
g/100g. The highest value for arginine was noted in FPI (7.49±0.18 g/100g) whilst, the
lowest was observed in CPI (3.91±0.06 g/100g). Moreover, SPI and FPI exhibited higher
aspartic acid levels as 5.87±0.29 and 5.86±0.08 g/100g in contrast to CPI 0.51±0.02 g/100g.
Furthermore, glycine ranged from 2.82±0.13 to 3.09±0.12 g/100g in different oilseed
protein isolates.
The present results regarding amino acid composition of SPI are in consensus with the
outcomes of Biswas et al. (2010), they noted histidine (2.40 g/100g), isoleucine (3.30
g/100g), leucine (7.70 g/100g), lysine (2.10 g/100g), threonine (3.40 g/100g) and valine
(5.40 g/100g). Similarly, for non-essential amino acids the values were recorded for alanine
2.30, arginine 9.50, aspartic acid 7.80, glutamic acid 19.50, glycine 8.40 and serine 6.50
g/100g.
The current outcomes of amino acids for FPI are in consensus with Kaushik et al. (2016).
They observed highest value for valine (55.30 mg/g) while lowest for methionine
(18.60mg/g). The values were also noticed for histidine 21.80, isoleucine 45.40, leucine
54.90, lysine 27.50, phenylalanine 53.10 and threonine 33.90 mg/g. For non-essential
amino acids the values ranged from 37.70 mg/g for proline to 108.0 mg/g for arginine. The
values were also observed for for alanine 43.60, aspartic acid 101.80, glutamic acid 185.10,
glycine 48.20 and serine 47.0 mg/g.
The present results for amino acid profile of CPI are supported by the outcomes of
Fleddermann et al. (2013), explicated histidine as 2.53 g/100g, isoleucine 3.33 g/100g,
leucine 6.96 g/100g, lysine 4.78 g/100g, threonine 4.37 g/100g, methionine 1.57 g/100g and
valine 4.12 g/100g. Likewise, non-essential amino acids ranged from 4.15 g/100g (serine)
to 19.10 g/100g (glutamic acid). The values were noted for alanine (4.23 g/100g), arginine
(6.79 g/100g), aspartic acid (8.34 g/100g), glycine (4.92 g/100g) and proline (5.58 g/100g).
Keeping in view the aforementioned amino acid profile, oilseed protein isolates can be
potentially utilized in numerous food preparations. These proteins and their amino acids are
essential components of food in order to provide better growth and maintenance to the
body.
Deficiency of these essential amino acids in diet prevents normal growth and metabolic
activities (Bosch et al., 2006). Furthermore, essential amino acids cannot be synthesized by
76
Table 4.16: Mean squares for essential amino acids of defatted oilseed protein isolates
SOV df ARM Histidine Isoleucine Leucine Lysine
Oilseed
PI 2 0.263** 0.098** 0.043* 0.009ns 1.492**
Error 9 0.014 0.002 0.006 0.038 0.005
Total 11
SOV df Threonine SAA Tryptophan Valine
Oilseed
PI 2 0.013ns 0.431** 1.472** 0.054*
Error 9 0.012 0.002 0.001 0.012
Total 11
P value <0.05
** = Highly significant; * = Significant; ns= Non-significant
PI = Protein isolates
Table 4.17: Essential amino acids (g/100g protein) of defatted oilseed protein isolates
Amino acid SPI FPI CPI
ARM* 3.36±0.11a 3.30±0.90a 2.89±0.15b
Histidine 1.65±0.05a 1.67±0.04a 1.39±0.05b
Isoleucine 2.31±0.08a 2.12±0.05b 2.29±0.09a
Leucine 4.39±0.11 4.37±0.22 4.30±0.23
Lysine 1.48±0.04c 1.62±0.07b 2.60±0.09a
Threonine 2.41±0.16 2.49±0.08 2.38±0.08
SAA** 1.59±0.05b 2.00±0.06a 1.36±0.04c
Tryptophan 0.98±0.01b 1.96±0.03a 0.85±0.02b
Valine 2.83±0.11a 2.60±0.15b 2.69±0.05b
Means having similar letter in a row do not differ significantly
* Aromatic amino acid (Phenyl alanine + Tyrosine)
** Sulfur containing amino acid (Methionine + Cysteine)
SPI= Sesame protein isolates FPI=Flaxseed protein isolates
CPI=Canola protein isolates
77
Table 4.18: Mean squares for non-essential amino acids of defatted oilseed protein
isolates
SOV df Alanine Arginine Aspartic
acid
Glutamic
acid Glycine Serine
Oilseed
PI 2 0.505** 16.793** 38.238** 11.328** 0.849* 0.474**
Error 9 0.006 0.037 0.030 0.232 0.018 0.011
Total 11
P value <0.05
Table 4.19: Non-essential amino acids (g/100g protein) of defatted oilseed protein
isolates
Amino acid SPI FPI CPI
Alanine 3.23±0.03a 3.30±0.08a 2.66±0.11b
Arginine 7.43±0.28a 7.49±0.18a 3.91±0.06b
Aspartic acid 5.87±0.29a 5.86±0.08a 0.51±0.02b
Glutamic acid 12.52±0.07a 12.70±0.76a 9.70±0.33b
Glycine 3.05±0.15a 3.09±0.12a 2.82±0.13b
Serine 3.01±0.13a 3.17±0.05a 2.51±0.11b
Means with similar letter in a row are momentously alike
SPI= Sesame protein isolates x
FPI=Flaxseed protein isolates CPI=Canola protein isolates
78
the body therefore, these are mandatory to be supplied through diet (Sinclair, 2005).
Oilseed protein isolates can be obtained with high protein contents, lack of impurities and
exhibiting appropriate sensory attributes. Previous research studies have indicated that
essential amino acids derived from oilseeds can play imperative role as quality protein
source in food system, imparting improved functional properties (Olaofe et al., 1994).
4.5.1. Amino acid score of defatted oilseed protein isolates
Amino acid score of defatted oilseed protein isolates was associated with the reference
pattern required for pre-school children. The respective amino acid score have been given
in Table 4.20. The sesame protein isolates (SPI) revealed relatively better essential amino
acid score as compared to FPI and CPI. Oilseed protein isolates exhibited good quality
proteins, ensuring the provision of required amount of essential amino acids for pre-
schoolers (WHO/FAO/UNU, 2007).
Lysine was found as limiting amino acid in oilseed protein isolates i.e. SPI, FPI and CPI.
The protein score of the oilseed protein isolates was noted as 28.46, 31.15 and 50.00 for
SPI, FPI and CPI, correspondingly (Table 4.20). In the present study, several essential
amino acids in oilseed protein isolates explicated good quality protein that can be
recommended for human utilization (Seena et al., 2006; FAO, 2012).
Nutritional proficiency of proteins is estimated by their ability to fulfill human amino acid
requirements. Amino acid score clearly indicates the existence of different essential amino
acids in the samples as compared to reference pattern. In a recent research investigation,
amino acid profile of different oilseeds like soybean, rapeseed and canola were studied to
assess their nutritional performance (Hojilla‑Evangelista et al., 2015). They delineated that
the tested oilseeds were rich in lysine, leucine and proline that can satisfy the human needs
for essential amino acids. It was also described earlier that oilseeds, legumes, cereals and
their products exhibit appreciable quantities of quality protein comprising of adequate
essential amino acids. Moreover, heat treatment can lower the quality of protein by
affecting lysine content during processing (McKevith, 2004).
4.5.2. Protein digestibility corrected amino acid score (PDCAAS)
PDCAAS is estimated by the ratio between first limiting amino acid in sample protein and
the respective amino acid in the reference pattern (Rutherfurd et al., 2015). The present
results indicated significant variations among different protein isolates (Tables 4.21)
depicting variation in amino acid content and digestibility of protein isolate samples.
79
Table 4.20. Amino acids score for defatted oilseed protein isolates
Amino acid SPI FPI CPI
ARM* 73.04 71.74 62.83
Histidine 91.67 92.78 77.22
Isoleucine 74.52 68.39 73.87
Leucine 69.68 69.37 68.25
Lysine 28.46 31.15 50.00
SAA** 63.60 80.00 54.40
Threonine 89.26 92.22 88.15
Tryptophan 140.00 280.00 121.43
Valine 69.02 63.41 65.61
Protein Score 28.46 31.15 50.00
L.A.A.*** Lys Lys Lys
* Aromatic Amino acid (Phenylalanine + Tyrosine)
** Sulfur containing amino acid (Methionine + Cysteine)
*** Limiting amino acid
SPI= Sesame protein isolates
FPI= Flaxseed protein isolates
CPI= Canola protein isolate
80
The PDCAAS results are illustrated in Table 4.22 that revealed maximum value in CPI
35.17±1.31% followed by FPI 22.58±0.66% and SPI 21.98±1.22%.
The PDCAAS is a method to assess quality of protein requiring description of limiting
amino acid and the true digestibility. Moreover, PDCAAS method for protein quality
determination is a way to assess the ability of proteins to fulfill human requirements of
essential amino acids (Aider and Barbana, 2011). Previously, PDCAAS value of 61% was
revealed for extruded flaxseed meal (Giacomino et al., 2013). Likewise, Sadeghi et al.
(2006) noted PDCAAS values for rapeseed and mustard protein isolates as 83 and 79%,
respectively. Earlier, Hoffman and Falvo (2004) documented that animal proteins exhibit
relatively better quality as compared to plants due to rich amino acid profile ranging from
92 to 100%.
PDCAAS is a simple method of protein quality determination and advantageous owing to
its ease and direct association with human protein requirements. The reference pattern
depicts minimum quantity of amino acids required for proper growth and maintenance of
body tissues. The PDCAAS is calculated by using three different protein quality assessment
parameters i.e. profile of essential amino acids, their digestibility as well as ability to fulfill
children’s requirements (Betancur-Ancona et al., 2008). According to authenticated
technique, PDCAAS values i.e. 1.00 or 100% indicate that protein provides adequate
quantity of essential amino acids for children as well as adults (Hughes et al., 2011).
The PDCAAS is extensively used and approved method for protein quality evaluation of
plant based foods especially infant formulations. Previously, World Health Organization
(WHO) adopted an alternate method to estimate protein quality in comparison with
PDCAAS. This method is used to evaluate amino acid scores for 2 to 5 year old children.
Conclusively, PDCAAS imparted relatively good protein with improved digestibility
(Messina, 1999). The differences in methods of PDCAAS determination have expounded
that reference amino acid score affects the PDCAAS value for a product. The accuracy of
PDCAAS is recommended by WHO (2001) to determine protein quality in numerous
commodities (Rutherfurd et al., 2015).
4.5.3. In vitro protein digestibility (IVPD)
The IVPD is a key factor in determining the availability of amino acids. Therefore, it plays
an imperative role in nutritional quality assessment of food proteins.
81
Table 4.21: Mean squares for protein digestibility corrected amino acid score
(PDCAAS)
SOV df PDCAAS
Oilseed PI 2 221.987**
Error 9 1.214
Total 11
P value <0.05
** Highly significant
Table 4.22. Protein digestibility corrected amino acid score (PDCAAS)
Oilseed protein isolates PDCAAS (%)
SPI 21.98±1.22c
FPI 22.58±0.66b
CPI 35.17±1.31a
Means with different letter in a column are not momentously alike
SPI= Sesame protein isolates
FPI= Flaxseed protein isolates
CPI= Canola protein isolates
82
The mean squares for in vitro protein digestibility revealed significant variations among
different defatted oilseed protein isolates (Table 4.23). Means indicated that the highest in
vitro protein digestibility was recorded for SPI (87.57±4.41%) followed by FPI
(85.41±2.04%) and CPI (82.13±2.86%). However, for soy the IVPD was observed as
91.35±3.12% whilst, 95.42±2.68% for casein (Table 4.24). Soy and casein are considered
as the reference proteins. Increase in IVPD mainly depends on the elimination of anti-
nutritional factors as well as denaturation of protein during cooking or its exposure to
enzymatic action.
The instant findings are in consensus with Monroy-Torres et al. (2008), they demonstrated
92.20% in vitro protein digestibility for soybean flour. Likewise, Asma et al. (2006)
illustrated that IVPD of weaning blends containing numerous legumes and oilseeds (sesame
& groundnut) varied from 84.6 to 92.0%. Similarly, Kenawi (2003) explicated IVPD of
three chickpea based breakfast foods ranged from 73.22 to 83.76%. Earlier, Chavan et al.
(2001) found that the in vitro protein digestibility of beach pea protein isolates varied from
80.6 to 82.6% whereas, for flaxseed the corresponding value was documented as 90%.
Likewise, Lόpez et al. (2003) explicated 94.5% protein digestibility for sesame protein
isolates. Furthermore, Milán-Carrillo et al. (2007) found the IVPD value for extruded
chickpea flour as 82.6%.
4.6. Bioefficacy study
The protein quality of defatted oilseed protein isolates was assessed through bioefficacy
trial. In this context, Sprague Dawley rats were used as test animals and soy & casein as
reference diets. The growth study parameters comprised of protein efficiency ratio (PER),
net protein ratio (NPR) & relative net protein ratio (RNPR). Whilst, nitrogen balance study
parameters consist of true digestibility (TD), biological value (BV) & net protein utilization
(NPU).
4.6.1. Growth study parameters
The mean squares regarding PER, NPR and RNPR showed significant variation among the
tested diets based on SPI, FPI and CPI accompanied by casein and soy protein as presented
in tables 4.25 and 4.26.
4.6.1.1. Protein efficiency ratio (PER)
PER is the ratio of weight gained by rat over the protein consumed during trial period
(Becker, 2007). The present results revealed that the highest PER was observed for SPI diet
83
Table 4.23. Mean squares for In vitro protein digestibility (IVPD) of oilseed protein
isolates
SOV df IVPD
Oilseed PI 4 133.647**
Error 20 9.748
Total 24
P value <0.05
** Highly significant
Table 4.24: In vitro protein digestibility (IVPD) of oilseed protein isolates
Oilseed protein isolates IVPD (%)
SPI 87.57±4.41c
FPI 85.41±2.04d
CPI 82.13±2.86e
Soy 91.35±3.12b
Casein 95.42±2.68a
Means with different letter in a column have significant difference
84
(2.14±0.10) trailed by CPI (2.09±0.06) while, minimum PER was recorded for the FPI diet
(1.98±0.07). Nonetheless, reference diets exhibited PER values as 2.74±0.12 (casein) and
2.52±0.09 (soy) (Table 4.26).
Protein play imperative role in growth, maintenance as well as provision of energy to the
body. Recently, FAO/WHO has recommended the use of biological indices in nutritional
studies to assess the quality and nutritional potential of proteins. Furthermore, biological
assessment of protein isolates is important to evaluate their nutritional quality that further
illuminates their potential food applications. Therefore, growth parameters study is a
promising tool to estimate nutritional attributes of test proteins (Gigliotti et al., 2008). The
PER is dependent upon the amount of essential amino acids as well as the capability of
body to digest them.
The results of current exploration regarding protein efficiency ratio of SPI based diet are in
harmony with the outcomes of Jimoh and Aroyehun (2011). They delineated that the PER
exhibited a decreasing trend (1.71-1.40) in fish diets with increasing levels of defatted
sesame seed meal. Likewise, Yasothai (2014) documented PER 1.22 for sesame seed and
1.35 for sesame meal in diets fed to rats. Furthermore, 2.60 PER was recorded for lupinus
species (Pastor-Cavada et al., 2009).
Moreover, the present results for PER of FPI based diet are in consensus with the outcomes
of Rabetafika et al. (2011), described that flaxseed proteins have higher PER as compared
to soy proteins. In another research investigation, Hussain et al. (2012) recorded 1.87
protein efficiency ratio for diet having 16% partially defatted flaxseed flour. Previously,
Khattab and Zeitoun (2007) described that defatted flaxseed meal indicated 2.21 PER.
The instant outcomes regarding protein efficiency ratio of CPI are in agreement with the
results presented by Zhou and Yue (2010), they explicated that the PER ranged from 1.83
to 2.33 for different canola meal based diets. Later, Tan et al. (2011b) revealed that canola
protein isolates (CPI) exhibited PER of 2.64. Likewise, Wanasundara et al. (2016) also
reported the similar protein efficiency ratio (2.64) for rapeseed flour. Earlier, Sadeghi et al.
(2006) expounded that the computed PER (2.57) of mustard protein isolate was higher as
compared to meal (2.35).
4.6.1.2. Net protein ratio (NPR)
The results of the present study regarding net protein ratio (NPR) revealed that SPI based
diet exhibited highest NPR (5.03±0.26) followed by CPI (4.98±0.11) and FPI (4.67±0.13).
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However, the reference protein diets i.e. soy & casein exhibited NPR values 5.52±0.13 and
5.93±0.19, respectively (Table 4.26). Net protein ratio (NPR) is NPR is an efficient
biological method to assess the utilization of protein by the experimental rats (Sousa et al.
2011).
Likewise, Frías et al., (2011) documented that NPR for raw and extruded pea varied from
2.9 to 2.5. One of their peers, Sousa et al. (2011) reported NPR for diets containing almond,
cashew nut and peanut as 3.53, 3.17 & 2.91, correspondingly. In another research
investigation, 3.04 NPR was noticed for complementary diet containing germinated
cowpea, maize and sesame seeds (Ikujenlola and Fashakin 2005). Likewise, current
outcomes are also in corroboration with the findings of Giacomino et al. (2013), noticed
NPR of extruded flaxseed meal as 3.22.
4.6.1.3. Relative net protein ratio (RNPR)
The RNPR was calculated via reference protein (casein as 100). RNPR is a ratio of NPR
values of oilseed protein isolates and the standard protein. The means for RNPR of test
diets ranged from 78.75 to 84.82 (Table 4.26). The SPI based group revealed appreciable
nitrogen balance that indicated greater nitrogen intake as compared to fecal and urinary
excretion.
Earlier, Giacomino et al. (2013) observed RNPR (58.6) for extruded flaxseed meal. In
another research exploration, RNPR (64) in pea protein concentrates was noted (Mitchell et
al., 1989). Later, Frías et al. (2011) worked on nutritional quality of pea and delineated
RNPR (59.6) for raw pea, whilst, RNPR values for extruded pea ranged from 50.6 to 54.2.
4.6.2. Nitrogen balance study
It includes true digestibility (TD), biological value (BV) and net protein utilization (NPU).
The mean squares for these parameters showed momentous variation among diets
containing defatted oilseed protein isolates (Table 4.27).
4.6.2.1. True Digestibility (TD)
TD is assessed by determining the amount of nitrogen in feed, feces as well as dried body
of rats (Abdel-Aziz et al., 1997). The present results indicated that highest TD was
observed for SPI based diet 77.23±3.20 followed by FPI and CPI as 72.47±2.27 &
70.34±2.10%, respectively (Table 4.28). Moreover, the reference diets containing soy and
casein reflected TD as 90.05±2.87 and 91.34±4.39, correspondingly. The maximum TD for
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Table 4.25: Mean squares for PER and NPR of experimental diets
SOV df PER NPR
Diets 4 0.511** 1.227**
Error 20 0.008 0.030
Total 24
P value <0.05 ** Highly significant PER= Protein efficiency ratio
NPR= Net protein ratio
Table 4.26: Growth study parameters of test diets
Diets PER NPR RNPR*
SPI 2.14±0.10c 5.03±0.26c 84.82
FPI 1.98±0.07e 4.67±0.13d 78.75
CPI 2.09±0.06d 4.98±0.11c 83.98
Soy 2.52±0.09b 5.52±0.13b 93.09
Casein 2.74±0.12a 5.93±0.19a 100
Means with dfferent letters in a column have significant difference
RNPR= Relative net protein ratio
* Calculated from standard protein casein as 100.
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SPI indicated exceptional digestibility as well as absorption of protein in the body in
comparison with other protein isolates. Moreover, protein digestibility depends on the
quantity of limiting as well as essential amino acids in the tested diets. Previously, Mepba
and Achinewhu (2003) elaborated that animal protein exhibit higher digestibility (90-99%)
as compared to plant protein (70-90%). The results revealed lower true digestibility for the
tested diets as compared to casein nonetheless, it was higher than certain other cereal
proteins.
In a research study, Giacomino et al. (2013) elucidated that extruded flaxseed meal
exhibited 73% TD. Moreover, Fleddermann et al. (2013) documented true digestibility for
canola protein isolates as 93.3%. Similar results were expounded by Wanasundara et al.
(2016), reported 95% true digestibility for canola protein isolates. Previously, Rangel et al.
(2004) documented 87% TD for cowpea protein isolates. One of the researchers group
expounded 78.42% true digestibility for chickpea based diet (Tavano et al., 2008).
4.6.2.2. Biological value (BV)
Biological value serves as an indicator of protein quality and reflects the absorbance of
protein from food that ultimately plays its role in body growth. Means regarding BV
indicated highest value for FPI (69.35±3.47%), whilst the diets containing CPI and SPI
exhibited 67.66±2.59 and 63.94±2.50% BV, respectively (Table 4.28). Nevertheless, the
BV for soy and casein based diets were 91.55±2.46 and 92.72±3.10%, correspondingly.
Higher biological value noted in FPI as compared to other protein isolates indicates
improved profile, digestibility and bioavailability of amino acids. According to Al-Gaby
(1998), these factors are associated with digestion and absorption that consequently
influence the quantity of nitrogen lost during digestion. A group of researchers assessed
defatted wheat germ supplemented wheat flour in comparison with casein and stated
gradual enhancement in protein quality parameters of the experimental subjects (Arshad et
al., 2007).
Similarly, Khattab and Zeitoun (2007) reported the BV of defatted sesame meal as 67.73%.
Moreover, the biological value for defatted flaxseed meal was noted as 76.35%. Later,
Hussain et al. (2012) documented 64.60% BV for unleavened flat bread diet comprising of
16% partially defatted flaxseed flour. Moreover, Giacomino et al. (2013) indicated 80.0%
BV for extruded flaxseed meal. Earlier, Sadeghi et al. (2006) corroborated biological value
of 87% for mustard protein isolates while BV for defatted meal was reported as 86%. In the
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nutshell, oilseed protein isolates exhibit better quality proteins with balanced amino acid
profile that can play an imperative role in improving the growth of test animals.
4.6.2.3. Net protein utilization (NPU)
Mean values regarding NPU indicated that highest NPU (50.26±2.44%) was observed for
FPI based diet, while 49.38±1.58 & 47.59±1.85% for SPI and CPI, respectively. Moreover,
soy and casein based diets performed better than other diets with NPU values 82.44±2.07
and 84.69±3.47%, correspondingly (Table 4.28).
Net protein utilization is comparable to net protein ratio as it indicates nitrogen retention in
the body. It is the estimation of tested protein retained in the body i.e. denoted as %
nitrogen absorbed. Moreover, it reflects the effect of test diets on experimental subjects.
The instant results for NPU are in corroboration with the findings of Alobo (2001), they
documented that sesame seeds exhibit 54% net protein utilization. Likewise, Hussain et al.
(2012) expounded that diets containing partially defatted flaxseed flour showed 54.65%
NPU. One of their peers, Giacomino et al. (2013) observed 58.4% NPU for extruded
flaxseed meal.
Moreover, Elango et al. (2009) explicated that net protein utilization values for soy proteins
ranged from 71 to 78%. In the present study, FPI based diet showed lowest NPU value
owing to reduced absorption and increased excretion of consumed nitrogen in the body.
Moreover, higher NPU value designates the existence of high quantity of essential amino
acids.
4.7. Safety assessment of defatted oilseed protein isolates
Safety assessment of protein isolates is important for their potential utilization as food
ingredients. Purposely, the experimental rats were fed on test diets containing defatted
oilseed protein isolates and later subjected to serum protein analysis and kidney & liver
function tests.
4.7.1. Serum protein analysis
Serum protein analysis includes the determination of serum total protein, albumin, globulin
and A/G ratio. Mean squares indicated that the experimental diets have non-significant
effect on serum protein profile during the experimental trial (Table 4.29).
The result values regarding serum total protein were ranging from 6.45±0.27 to 6.86±0.67
g/dL. These results clearly indicated that highest protein was observed in group fed on SPI
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Table 4.27: Mean squares for nitrogen balance study parameters of experimental diets
SOV df TD BV NPU
Diets 4 483.515** 968.769** 1791.820**
Error 20 9.446 8.131 5.630
Total 24
P value <0.05 ** Highly significant TD= True digestibility
BV= Biological value
NPU= Net protein utilization
Table 4.28: Nitrogen balance study parameters of experimental diets
Diets TD (%) BV (%) NPU (%)
SPI 77.23±3.20c 63.94±2.50d 49.38±1.58cd
FPI 72.47±2.27cd 69.35±3.47c 50.26±2.44c
CPI 70.34±2.10d 67.66±2.59cd 47.59±1.85d
Soy 90.05±2.87b 91.55±2.46b 82.44±2.07b
Casein 91.34±4.39a 92.72±3.10a 84.69±3.47a
Means with different letter in a column have significant difference
SPI= Sesame protein isolates FPI=Flaxseed protein isolates
CPI= Canola protein isolates
90
based diet 6.79±0.32 g/dL trailed by CPI & FPI as 6.66±0.51 and 6.45±0.27, respectively.
However, for reference diets, i.e. soy and casein the values were 6.78±0.38 and 6.86±0.67,
correspondingly (Table 4.30). Serum albumin concentration was in the range of 3.08±0.35
to 3.16±0.11 g/dL for different experimental groups. Likewise, the globulin was observed
to be varying from 2.91±0.13 to 3.04±0.13 g/dL. Furthermore, A/G ratio ranged from
1.04±0.09 to 1.07±0.08 for the experimental diets (Table 4.30).
The current outcomes are supported by the findings of Azab (2014), who explicated serum
total proteins as 6.86 g/dL in male albino mice treated with sesame oil. Moreover, the
serum albumin was recorded as 4.30 g/dL. Likewise, Matusiewicz et al. (2015) reported
albumins and globulins as 3.67 and 27.17 g/dL, respectively, in rats fed with diet containing
genetically modified flaxseeds. Moreover, the value for total protein was observed as 58.83
g/dL.
In another research trial, Munish et al. (2011) studied the hepatoprotective effect of
ethanolic extract of sesame seeds in rats suffering from liver damage. They inferred that the
serum albumin increased from 4.00 to 4.18 g/dL with increasing concentration of sesame
extract. Similarly, serum total protein increased from 6.95 to 7.03 g/dL. Likewise, Njidda
and Isidahomen (2011) reported that the albumin level increased from 2.2 to 4.2 g/dL with
varying levels of sesame seed meal in rabbit diets. The values for serum globulin were
observed in the range of 1.3 to 2.0 g/dL whilst, the total protein varied from 5.5 to 6.1 g/dL
in various diets.
According to Saleh and Amer (2009), the values for serum albumin, globulin and total
protein for control diet were 3.42, 3.25 and 6.13 g/dL, respectively. They also studied the
effect of sesame seed addition in the diet of lambs and elucidated an increase in these traits
in experimental diets as compared to control. The values reported for albumin, globulin and
total protein were 3.69, 4.08 and 7.77 g/dL, respectively, for diet containing 4% sesame
seeds. Likewise, for diet having 8% sesame seeds the values for these traits were recorded
as 3.78, 4.19 and 7.97 g/dL, correspondingly.
4.7.2. Renal functioning tests
Mean squares for renal functioning parameters revealed non-momentous variation in rats
fed on different experimental diets (Table 4.31). The means for urea content indicated that
it ranged from 14.85±0.70 to 15.35±0.62 mg/dL while, creatinine varied from 0.30±0.02 to
0.33±0.01 mg/dL (Table 4.32). The production of urea in the body is related to the amino
91
Table 4.29: Mean squares for serum protein analysis of experimental rats
SOV df Total
protein Albumin Globulin A/G ratio
Diets 4 0.128ns 0.005ns 0.014ns 6.422ns
Error 20 0.204 0.095 0.041 0.010
Total 24
ns=Non-significant
Table 4.30: Serum protein profile of experimental rats
Diets Total protein
(g/dL) Albumin (g/dL) Globulin (g/dL) A/G ratio
SPI 6.79±0.32 3.12±0.25 3.01±0.25 1.04±0.09
FPI 6.45±0.27 3.08±0.35 2.95±0.18 1.05±0.07
CPI 6.66±0.51 3.10±0.49 2.91±0.13 1.07±0.08
Soy 6.78±0.38 3.15±0.19 3.01±0.28 1.05±0.08
Casein 6.86±0.67 3.16±0.11 3.04±0.13 1.04±0.15
SPI=Sesame protein isolates FPI=Flaxseed protein isolates
CPI=Canola protein isolates
92
acid deamination. The protein metabolism results in the production of some waste in the
form of urea, and uric acid that is expelled out via kidneys however, a significant increase
in these traits reflects malfunctioning of kidneys (Elmonem, 2014). An increase in urea
level may occur due to diet, dehydration and antidiuretic drugs while, creatinine is more
related to kidney (Zari and Al-Attar, 2011). Later, Abdou et al. (2012) treated female rats
with sesame oil and deduced plasma urea and creatinine as 26.90 and 0.84 mg/dL,
correspondingly. Earlier, Barakat and Mahmoud (2011) studied the renal protective effect
of a mixture of seeds (flaxseed, purslane, pumpkin) on hypercholesterolemic rats and
reported values for urea and creatinine in control group as 24.05 and 1.01 mg/dL,
respectively. Furthermore, the group fed with flaxseed/pumpkin mixture showed increased
levels of urea (29.97 mg/dL) and creatinine (1.19 mg/dL) as compared to control.
In another scientific exploration, it was explicated that urea nitrogen level decreased from
46.603 to 38.109 mg/dL with increasing level of flaxseed in the diet of rats suffering from
liver damage. Moreover, the creatinine level also decreased from 1.376 to 0.972 mg/dL (El-
Bahy et al., 2011). Likewise, Mohamed et al. (2012) studied the benefits of defatted
flaxseed supplemented bread in normal and type 2 diabetic subjects. They observed
creatinine levels in normal and diabetic subjects fed with supplemented bread as 0.791
mg/dL and 0.86 mg/dL, correspondingly.
According to Njidda and Isidahomen (2011), the blood urea level of weanling rabbits
increased from 2.5 to 5.8 mmol/L with increasing levels of sesame seed meal. Furthermore,
the creatinine level varied from 44.0 to 59.0 mmol/L for diets with different levels of
sesame seed meal. Likewise, Saleh and Amer (2009) elucidated the values for urea and
creatinine in control diet as 26.4 & 0.94 mg/dL, respectively. They further explicated that
the urea and creatinine levels decreased with increasing levels of sesame seeds in the
experimental diet of lambs.
4.7.3 Hepatic functioning tests
Mean squares indicated non-significant difference among various diets containing defatted
oilseed protein isolates (Table 4.33). The means revealed that the enzymes activities i.e.
ALT, AST & ALP varied as 38.92±2.90 to 40.10±2.64, 76.48±9.92 to 78.45±5.65 and
144.66±10.90 to 146.22±6.91 U/L, respectively (Table 4.34). The rats fed on protein
isolates based experimental diets exhibited comparable results for liver enzymes with
control (Table 4.34). The activity of these enzymes determines the hepatic functioning.
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Table 4.31: Mean squares for renal functioning tests of experimental rats
SOV df Urea Creatinine
Diets 4 0.259ns 6.451ns
Error 20 0.624 3.991
Total 24
ns = Non-Significant
Table 4.32: Renal functioning tests of experimental rats
Diets Urea (mg/dL) Creatinine (mg/dL)
SPI 15.25±0.78 0.32±0.02
FPI 14.98±1.02 0.30±0.02
CPI 14.87±0.78 0.31±0.02
Soy 15.35±0.62 0.33±0.01
Casein 14.85±0.70 0.32±0.03
SPI= Sesame protein isolates FPI=Flaxseed protein isolates
CPI= Canola protein isolates
94
Table 4.33: Mean squares for hepatic functioning tests of experimental rats
SOV df ALT AST ALP
Diets 4 1.691ns 2.829ns 2.856ns
Error 20 10.249 39.240 89.954
Total 24
ns =Non-Significant
Table 4.34: Hepatic functioning tests of experimental rats
Diets ALT (U/L) AST (U/L) ALP (U/L)
SPI 40.08±2.69 78.14±4.64 146.12±7.98
FPI 38.92±2.90 76.48±9.92 144.66±10.90
CPI 39.36±2.35 77.57±5.74 146.22±6.91
Soy 38.95±4.81 77.56±3.38 144.75±13.75
Casein 40.10±2.64 78.45±5.65 145.84±5.52
SPI= Sesame protein isolates FPI=Flaxseed protein isolates CPI= Canola protein isolates
95
In a research trial, Rezaeipour et al. (2016) evaluated liver functioning of Japanese quails
by providing sesame meal based diets. They revealed that the values for ALT ranged from
24.03 to 25.94 IU/L whilst, the values for AST and ALP varied from 247.3 to 274.7 and
1278 to 1360 IU/L, respectively. In another experimental trial, Saleh and Amer (2009)
assessed the effect of sesame seeds supplementation on the growth of lambs and reported
ALT (18.1 to 18.7 IU/L), AST (33.9 to 35.6 IU/L) & ALP (43.5 to 45.7 IU/L) for the
experimental diets.
Likewise, El-Bahy et al. (2011) observed the effect of flaxseed on the liver enzymes of rats
suffering from liver damage and illustrated that the AST and ALT reduced with increase in
flaxseed in the experimental diets. The values for these traits decreased from 186.137 to
175.535 and 92.892 to 83.971 U/L in respective manner. Likewise, Abdou et al. (2012)
observed the activities of ALT and AST in female rats treated with sesame oil as 21.2 and
31.5 U/L, respectively. Similarly, Azab (2014) documented ALT (27.4 U/L), AST (48.61
U/L) and ALP (46.01 U/L) for male albino mice treated with sesame oil.
4.8. Development of composite flours
In order to estimate the effectiveness of incorporating resultant protein isolates in food
preparations, these were added with wheat flour at varying levels and assessed for chemical
as well as rheological properties.
4.8.1. Proximate analysis
The mean squares for proximate composition indicated that the treatments were
significantly different (Table 4.35). The highest moisture was found in T0 (11.27±0.55%)
tracked by T1 (10.95±0.90%), T2 (10.49±0.45%), T3 (10.44±0.44%) and T4 (9.88±0.10%)
while, lowest was recorded in T5 (9.76±0.64%). Moreover, maximum protein content
(31.14±1.46%) was noted in T5 (75% SGF and 25% SPI) whilst, minimum (10.05±0.27%)
was observed in T0 (control). Nonetheless, other treatments presented values as14.31±0.46
(T1), 18.45±0.90 (T2), 22.71±0.24 (T3) and 26.62±0.99% (T4). The crude fat content
declined from 1.28±0.04 to 1.01±0.03% in T0 to T5. Similarly, crude fiber content also
showed decreasing trend with values ranging from 0.52±0.02 to 0.43±0.02% in
corresponding treatments. The mean values for ash were 0.68±0.04 (T0), 0.67±0.02 (T1),
0.66±0.01 (T2), 0.64±0.01 (T3), 0.63±0.02 (T4) and 0.62±0.02% (T5). Moreover, results for
NFE were noted as 76.20±2.84 (T0), 72.33±3.54 (T1), 68.74±2.51 (T2), 64.62±2.58 (T3),
61.35±0.93 (T4) and 57.03±1.37% (T5) (Table 4.36).
96
Previously, Chetana et al. (2010) found 11.50% moisture, 10.70% crude protein, 6.50%
fiber and 0.48% ash in wheat flour. They also documented proximate composition of raw &
roasted flaxseed powder and explicated moisture as 7.10 & 2.40% in respective manner.
Similarly, the ash content varied between 2.8 and 3.06%, protein 20.3 to 21.8% and fat 44.7
to 46.4%. In a previous research study conducted by Farooq et al. (2001), straight grade
flour was evaluated and ranges were recoded as moisture (12.05-12.57%), protein (10-
12.03%), fat (1.25-1.40%), fiber (0.30-0.40%), ash (0.50-0.60%) and NFE (85.68-87.77%).
Later, Mridula et al. (2007) elucidated proximate composition of sorghum, wheat &
defatted soy flours and documented that wheat contains 10.47% protein, 0.42% fiber,
1.41% fat and 0.73% ash. Similarly, sorghum and defatted soy flours exhibited 9.67 &
56.3% protein, 3.18 & 3.78% fiber, 1.11 & 1.09% fat and 1.41 & 6.42% ash, respectively.
Likewise, Mohamed et al. (2006) obtained 12% protein in bread flour. Recently, David et
al. (2015) expounded proximate composition of soft wheat and revealed crude protein
(10.23%), crude fiber (0.51%), moisture (3.33%), ash (1.00%), crude fat (1.33%) and
carbohydrate (83.60%).
4.8.2. Rheological characterization of developed composite flours
The developed composite flours were subjected to rheological evaluation including
mixographic and farinographic studies in order to determine the behavior of dough during
processing.
4.8.2.1. Mixographic characteristics of composite flours
The structure and interaction of protein molecules affect the dough rheology. Purposely, the
composite flour blends were analyzed for mixing time and peak height using mixograph.
Moreover, the mixograph elucidates physical attributes of dough by estimating dough
resistance during mixing. It gives idea about the mixing requirements of flour.The mean
squares showed significant differences amongst various treatments (Table 4.37).
4.8.2.1.1. Mixing time
It refers to the time needed for complete development of dough. The present results were
recorded as 3.14±0.25 (T0), 3.06±0.11 (T1), 2.73±0.02 (T2), 2.58±0.04 (T3), 2.39±0.09 (T4)
& 2.19±0.08 min (T5) (Table 4.38). The results clearly depicted decrease in mixing time by
the addition of SPI in straight grade flour. In current study, straight grade flour showed
maximum mixing time i.e. 3.28 min and is related with peak height. Moreover, strong
wheat exhibits maximum peak strength along with extended mixing time. The momentous
97
Table 4.35: Mean squares for proximate analysis of composite flour blends
SOV df Moisture Crude protein Crude fat Crude fiber Ash NFE
Treatments 5 1.033* 184.347** 0.030* 0.004* 0.002* 151.564**
Error 12 0.320 0.712 0.002 0.0004 0.0004 6.047
Total 17
P value <0.05 ** = Highly significant; * =Significant
Table 4.36: Means for proximate analysis (%) of composite flour blends
Treatments Moisture Crude protein Crude fat Crude fiber Ash NFE
T0 11.27±0.55a 10.05±0.27f 1.28±0.04a 0.52±0.02a 0.68±0.04a 76.20±2.84a
T1 10.95±0.90ab 14.31±0.46e 1.24±0.05a 0.50±0.02ab 0.67±0.02ab 72.33±3.54b
T2 10.49±0.45b 18.45±0.90d 1.16±0.03b 0.49±0.01b 0.66±0.01b 68.74±2.51c
T3 10.44±0.44b 22.71±0.24c 1.13±0.04bc 0.46±0.02bc 0.64±0.01bc 64.62±2.58d
T4 9.88±0.10c 26.62±0.99b 1.07±0.04c 0.45±0.03c 0.63±0.02c 61.35±0.93e
T5 9.76±0.64c 31.14±1.46a 1.01±0.03d 0.43±0.02d 0.62±0.02d 57.03±1.37f
Means with similar letter in a column do not differ significantly
T0 = 100% Straight grade flour
T1 = 95%SGF and 5% SPI
T2 = 90% SGF and 10% SPI
T3 = 85% SGFand 15% SPI
T4 = 80% SGF and 20% SPI
T5 = 75% SGF and 25% SPI
98
difference in mixing time can be attributed to variations in protein quantity. In general,
flour blends comprising high protein indicated less mixing time.
One of the researchers group noticed mixing time of 6 min for patent grade wheat flour
(Qammar et al., 2010). Later, Ahmad et al. (2013) noted 3.5 min mixing time for straight
grade wheat flour. Previously, Gómez et al. (2011) explained that the mixing time ranged
from 4.57 to 6.80 min for two Argentinean commercial wheat flours.
4.8.2.1.2. Peak height
It refers to the highest point on mixogram that reflects maximum development of dough.
This point is recognized as peak height. In current study, different treatments of composite
flour blends revealed a decreasing trend for peak height. The maximum peak height
57.14±3.72% was observed for T0 (control) followed by T1 (55.23±2.26%), T2
(53.42±2.76%), T3 (49.32±2.19%), T4 (46.13±1.30%). However, T5 showed the minimum
value 42.37±1.55% (75% SGF and 25% SPI) (Table 4.38).
In present research investigation, straight grade flour (SGF) exhibited maximum peak
height in comparison with other composite blends supplemented with SPI. In this context,
Lei et al. (2008) indicated a significant decrease in peak height during storage owing to
presence of proteolytic enzymes. Previously, Anjum and Walker (2000) delineated that
Pakistani wheat variety Barani-83 exhibited increased peak height due to higher gluten
contents. One of the researchers groups explicated that straight grade wheat flour showed
62% peak height (Ahmad et al., 2013).
4.8.2.2. Farinographic characteristics of composite flours
These characteristics of composite flours containing varying levels of SPI were assessed via
farinograph. The mean squares for these parameters depicted significant variations among
different treatments (Table 4.39).
4.8.2.2.1. Water absorption (WA)
The data pertaining to water absorption (Table 4.40) in current study indicated highest
value (73.36±3.47%) for T5 followed by T4 (69.62±3.80%), T3 (66.27±2.18%), T2
(63.22±3.60%) and T1 (59.23±2.58%) whilst the lowest value was recorded for T0
(56.39±2.41%).
In farinographic study, water absorption refers to the water quantity needed to achieve
maximum consistency of dough. The current results revealed that flour supplemented with
99
Table 4.37: Mean squares for mixographic characteristics of composite flours
SOV df Mixing time Peak height
Treatments 5 0.413** 96.812**
Error 12 0.015 5.902
Total 17
P value <0.05 ** Highly significant
Table 4.38: Means for mixing time and peak height of composite flours
Treatments Mixing time (min) Peak height (%)
T0 3.14±0.25a 57.14±3.72a
T1 3.06±0.11ab 55.23±2.26b
T2 2.73±0.02b 53.42±2.76c
T3 2.58±0.04bc 49.32±2.19d
T4 2.39±0.09c 46.13±1.30e
T5 2.19±0.08d 42.37±1.55f
Means havingg similar letters in a column are statistically alike
100
25% SPI exhibited better water absorption compared to other treatments. The existing
outcomes are in concord with the results of Ahmad et al. (2013), stated 57.6% water
absorption for straight grade wheat flour dough. Previously, Asghar et al. (2012) found
61.2% water absorption in commercial wheat flour. Moreover, the flour having less water
absorption yields dough with low moisture that ultimately results in dry, stiff and poor
sensory quality baked products (Farooq et al., 2001).
In a research trial, Pasha et al. (2011) assessed mungbean based composite flour and
recorded a substantial increase in water absorption capacity. Later, Rajiv et al. (2012)
studied the effect of addition of roasted and ground flaxseed on the farinographic properties
of wheat flour. They delineated water absorption ranging from 59.5 to 62.0% for different
levels of flaxseed supplementation in wheat flour blends. Previously, Chetana et al. (2010)
evaluated wheat flour blends having raw & roasted flaxseed powder and deduced that water
absorption increased from 52.5 to 55.5% with progressive addition flaxseed powders.
4.8.2.2.2. Dough development time (DDT)
In present study, maximum DDT (7.09±0.23 min) was observed for T5 (75% SGF and 25%
SPI) while, the lowest for T0 (control) i.e. 4.53±0.08 min. Moreover, T1 (95% SGF and 5%
SPI), T2 (90% SGF and 10% SPI), T3 (85% SGF and 15% SPI) and T4 (80% SGF and 20%
SPI) revealed DDT as 5.63±0.14, 6.43±0.17, 6.71±0.22 & 6.98±0.18 min, respectively
(Table 4.40).
The time (minutes) required by dough to attain maximum consistency after adding water
before the weakening process initiates is referred as dough development time. In a research
study, Ahmad et al. (2013) described straight grade wheat flour dough development time of
4.50 min. Previously, Chetana et al. (2010) delineated gradual reduction (5 to 3.7 min) in
dough development time by adding raw and roasted flaxseed powder. The instant outcomes
are also in conformity with the findings presented by Rajiv et al. (2012). The authors
noticed increase in dough development time from 3.0 to 6.5 min by adding roasted and
ground flaxseed.
4.8.2.2.3. Dough stability (DS)
In current investigation, the means indicated significant decrease in dough stability owing
to protein supplementation in flour blends. The values were noted as 4.98±0.19 (T0),
4.79±0.23 (T1), 4.61±0.10 (T2), 4.18±0.25 (T3), 3.85±0.13 (T4) & 3.56±0.22 min (T5)
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(Table 4.40). The observed decrease in DS may be due to differences in quality as well as
quantity of supplemented protein (Shahzadi et al., 2005).
Dough stability is the difference between departure and arrival time during dough formation
that indicates its strength. Previously, Chetana et al. (2010) elucidated that dough stability
decreases significantly (8.6 to 2.1 min) by the addition of raw and roasted flaxseed powder.
In a research exploration, Ribotta et al. (2005) observed that soy protein isolate exhibits
opposite effect on dough stability. Later, Rajiv et al. (2012) also documented significant
reduction (6.8 to 4.0 min) in dough stability owing to the incorporation of roasted and
ground flaxseed. The variations in farinographic attributes might be due to differences in
protein quantity of non-conventional protein sources utilized at different levels.
4.8.3. Functional properties of composite flours
4.8.3.1. Bulk density
The mean squares (Table 4.41) for bulk density indicated non-significant variations among
various treatments of supplemented flour blends. The bulk density varied from 0.64±0.03
(T0) to 0.67±0.01 g/cm3 (T5) as mentioned in Table 4.42. The current results for bulk
density of different flour blends are in agreement with the outcomes of Bukya and
Vijayakumar (2013), documented bulk density for sesame cake and defatted sesame flour
as 0.67 & 0.70 g/mL, respectively.
Likewise, Zouari et al. (2016) elucidated 0.83g/cm3 bulk density for sesame peels flour
whilst, wheat/sesame peels flour blends bulk density in the range of 0.725 to 1.05 g/cm3.
Similarly, Azeez et al. (2015) elaborated that the bulk density of unripe plantain and
defatted sesame flour blends in the range of 0.69 to 0.75 g/mL.
In another research investigation, Khan and Saini (2016) explicated that unroasted flaxseed
showed 0.47 g/cm3 bulk density, while, 0.48 g/cm3 was observed for roasted flaxseed flour.
Earlier, Hussain et al. (2008) explicated that bulk density of flaxseed flours subjected to
defatting and roasting treatments varied from 0.77 to 0.83 g/cm3.
Moreover, Bhise et al. (2013) worked on texturized defatted flaxseed meal and reported a
range of 0.189 to 0.359 g/mL for bulk density. Previously, Mepba et al. (2007) observed
that the bulk density of wheat/plantain composite flours ranged from 0.57 to 0.63 g/cm3.
4.8.3.2. Absorption properties
The mean squares regarding water and oil absorption capacity (Table 4.41) revealed
102
Table 4.39: Mean squares for farinographic characteristics of composite flours
SOV df WA DDT DS
Treatments 5 121.736** 2.902** 0.935**
Error 12 9.442 0.032 0.038
Total 17
P value <0.05
** Highly significant WA = Water absorption
DDT = Dough development time
DS = Dough stability
Table 4.40: Means for farinographic characteristics of composite flours
Treatments WA (%) DDT (min) DS (min)
T0 56.39±2.41f 4.53±0.08e 4.98±0.19a
T1 59.23±2.58e 5.63±0.14d 4.79±0.23b
T2 63.22±3.60d 6.43±0.17c 4.61±0.10bc
T3 66.27±2.18c 6.71±0.22bc 4.18±0.25c
T4 69.62±3.80b 6.98±0.18b 3.85±0.13d
T5 73.36±3.47a 7.09±0.23a 3.56±0.22e
Means showing similar letter in a column do not differ significantly
T0 = 100% Straight grade flour T1 = 95% SGF and 5% SPI
T2 = 90% SGF and 10% SPI
T3 = 85% SGFand 15% SPI
T4 = 80% SGF and 20% SPI
T5 = 75% SGF and 25% SPI
103
momentous variations among tested flour treatments. Results revealed water absorption
capacity (WAC) for T0, T1, T2, T3, T4 and T5 as 0.70±0.04, 0.83±0.03, 0.95±0.03, 1.08±0.06,
1.20±0.05 & 1.32±0.07 mL/g, correspondingly. Likewise, for OAC the results were
1.85±0.06, 1.91±0.01, 1.97±0.11, 2.04±0.05, 2.10±0.07 and 2.17±0.05 mL/g for respective
treatments (Table 4.42).
Previously, Doxastakis et al. (2002) revealed 57.2% water absorption for wheat-soya flour
blends (95% wheat flour + 5% soya flour). Recently, Azeez et al. (2015) explicated water
absorption index ranging from 224.74 to 242.57% for unripe plantain and defatted sesame
flour blends. Later, Bukya and Vijayakumar (2013) reported water holding capacity for
defatted sesame flour (DSF). Additionally, fat absorption capacity for DSF was observed as
181.61%. In another research trial, Bhise et al. (2013) worked on the production of
texturized defatted flaxseed meal and delineated that the water absorption index of protein
flour was ranging from 1.35 to 4.85 g/g. They also observed a range of 71.07 to 91.48% for
fat absorption capacity of protein flour.
In previous decade, one of the scientists groups worked on wheat/plantain composite flours
and inferred 65 to 284% water absorption capacity while, 110 to 130% oil absorption
capacity. Furthermore, 130% water absorption was noted for soy flour (Mepba et al., 2007).
In another research investigation, Baljeet et al. (2010) demarcated 151% WAC and
169.97% OAC for refined wheat flour. Earlier, Mansour et al. (1999) observed an increase
in water absorption by adding pumpkin and canola proteins to wheat flour.
Later, Oyeyinka et al. (2014) delineated the functional properties of wheat-cowpea flours
and revealed 0.76 g/g water absorption capacity (WAC) and 0.70 g/g oil absorption
capacity (OAC) for wheat flour. Furthermore, the results for WAC and OAC of wheat-
cowpea flours were determined as 2.06 and 1.20 g/g, respectively. Likewise, Akubor (2003)
documented 75% WAC and 196% OAC for wheat flour.
4.8.3.3. Foaming properties
The mean squares for foaming capacity (FC) illustrated non-significant difference whilst
the results for foaming stability (FS) showed momentous variations among various
treatments (Table 4.41). In this perspective, the results for foaming capacity were
28.72±0.98 mL, 28.30±1.64 mL, 27.89±0.46 mL, 27.47±1.62 mL, 27.05±0.66 mL and
26.63±0.41 mL for the treatments T0, T1, T2, T3, T4 & T5, respectively. Nonetheless, foaming
stability gradually decreased from 60.35±1.91 min for T0 to 51.10±1.96 min for T5 (Table
104
4.42).
Previously, Baljeet et al. (2010) explicated foaming capacity and foaming stability of
refined wheat flour as 12.42 & 96.48%, respectively. Likewise, Bhise et al. (2013) worked
on the production of texturized defatted flaxseed meal and observed the FC of protein flour
in the range of 9.23 to 17.81%. In another research trial, Zouari et al. (2016) observed
13.19% foaming capacity for wheat flour. Furthermore, the foaming capacity increased
from 13.68 to 15.99% with gradual substitution of wheat flour with sesame peels flour.
In another research experiment, Aremu et al. (2007) delineated the functional properties of
various groundnut & cowpea flours and revealed that the foaming capacity ranged from 7.9
to 28.1% whilst, the foaming stability varied from 98.1 to 89.1%. Likewise, Radha et al.
(2007) prepared protein hydrolysate using mixture of oilseed flours (sesame, soybean,
peanut). They observed the foaming capacity and stability of protein hydrolysate as 122%
& 3 mL, correspondingly.
Later, Khan and Saini (2016) expounded the functional properties of wheat and flaxseed
flour. They revealed 30.40% foaming capacity and 56.05% foaming stability for wheat
flour. Similarly, the foaming capacity and stability for unroasted flaxseed flour was noted
as 9.23% & 54.43%, respectively. Nevertheless, the foaming capacity and stability for
roasted flaxseed flour were observed as 7.82 & 48.60%, correspondingly.
4.8.3.4. Emulsion properties
The mean squares for emulsifying properties of different treatments of flour blends
demonstrated significant difference in emulsion capacity and stability (Table 4.41). The
emulsifying capacity (EC) of different treatments was observed as T0 (15.27±0.66%), T1
(18.06±0.33%), T2 (20.86±0.47%), T3 (23.66±0.83%), T4 (26.46±1.01%) and T5
(29.26±0.65%). The results for emulsion stability (ES) revealed the values as 17.84±0.57
(T0), 20.64±0.21 (T1), 23.45±1.09 (T2), 26.25±0.77 (T3), 29.06±0.70 (T4) & 31.86±1.07%
(T5) (Table 4.42).
The present results are supported by the outcomes of Mepba et al. (2007), documented that
the emulsion capacity of wheat and soy flour ranged from 10.1 to 25.6% whilst, the result
for sunflower flour was observed as 95.1%. Furthermore, the emulsion capacity of wheat
plantain flour varied from 3.5 to 12.8% and the emulsion stability ranged from 2.4 to
18.6%. Similarly, Zouari et al. (2016) revealed 42.77% emulsion capacity for wheat flour
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Table 4.41: Mean squares for functional properties of composite flour blends
SOV df Bulk
density WAC OAC FC FS EC ES
Treatments (T) 5 0.0004ns 0.163** 0.043** 1.830ns 35.937** 82.232** 82.635**
Error 12 0.0005 0.002 0.004 1.180 3.313 0.486 0.630
Total 17
P value <0.05 ** Highly significant ns Non-Significant
Table 4.42: Functional properties of composite flour blends
Treat-
ments
BD
(g/cm3) WAC (mL/g) OAC (mL/g) FC (mL) FS (Min) EC (%) ES (%)
T0 0.64±0.03 0.70±0.04f 1.85±0.06d 28.72±0.98 60.35±1.91a 15.27±0.66f 17.84±0.57f
T1 0.65±0.02 0.83±0.03e 1.91±0.01cd 28.30±1.64 58.50±2.76b 18.06±0.33e 20.64±0.21e
T2 0.66±0.02 0.95±0.03d 1.97±0.11c 27.89±0.46 56.65±1.06c 20.86±0.47d 23.45±1.09d
T3 0.66±0.04 1.08±0.06c 2.04±0.05b 27.47±1.62 54.80±1.49d 23.66±0.83c 26.25±0.77c
T4 0.67±0.01 1.20±0.05b 2.10±0.07ab 27.05±0.66 52.95±1.20e 26.46±1.01b 29.06±0.70b
T5 0.67±0.01 1.32±0.07a 2.17±0.05a 26.63±0.41 51.10±1.96f 29.26±0.65a 31.86±1.07a
Means with same letter in a column are not significantly different
BD = Bulk density
WAC = Water absorption capacity
OAC = Oil absorption capacity FC = Foaming capacity , FS = Foaming stability
EC = Emulsion capacity, ES = Emulsion stability
106
whereas the wheat-sesame peels flour blends showed emulsion capacity ranging from
44.09 to 52.98%.
Previously, Baljeet et al. (2010) reported 0.43% emulsion activity for refined wheat flour.
Furthermore, Eltayeb et al. (2011) delineated that the emulsion capacity of Bambara flour
ranged from 89 to 134 mL/g with varying pH levels while, the emulsion stability varied
from 15 to 75%. In another research study, El-Adawy et al. (2001) expounded that the
emulsification capacity of bitter and sweet lupin protein isolates ranged from 164.0 to
210.2 mL/g.
4.9. Preparation of protein enriched muffins
The nutritional as well as functional attributes along with bioevaluation study of the
protein isolates (SPI) have enlightened their importance as potential ingredient for the
preparation of protein enriched muffins. Purposely, wheat flour was replaced with
selected oilseed protein isolates in different combinations @ 5, 10 15, 20 and 25% and the
resultant blends were utilized for muffins preparation (Table 3.2). Moreover, muffins
were evaluated for proximate composition, physicochemical and sensory characteristics
to assess the appropriateness of SPI. The pertinent results are discussed herein;
4.9.1. Proximate analysis
The mean squares elucidated momentous effect of treatments on crude protein and
NFE whereas, non-significant effect was observed for moisture, crude fat, crude fiber
and ash content of muffins (Table 4.43).
4.9.1.1. Moisture content
The moisture content is among the most common parameters used for the analysis of food
product as lower moisture content indicates better storage stability. The means regarding
moisture content of different treatments (Table 4.44) ranged from 11.09±0.48% (T5) to
11.25±0.30% (T4) reflecting non-momentous rise in moisture with increasing quantity of
protein in muffins. Means were 11.20±0.46, 11.21±0.41, 11.23±0.48 and 11.24±0.22%
for T0, T1, T2 and T3, correspondingly.
The upsurge in moisture might be due to increased quantity of polar amino acids (Mohsen
et al. 2009). According to Chetana et al. (2010), moisture content of muffins increased
from 17.90 to 23.10% by the addition of raw and roasted flaxseed powder. This retention
of moisture might be due to the presence of fiber content during baking. The results of
107
present exploration are also in agreement with the outcomes of Srivastava et al. (2012),
they elucidated that muffins prepared by adding fenugreek seed husk has increased
moisture content 21.62% as compared to the control muffins (20.39%).
Contrarily, Goswami et al. (2015) corroborated that muffins prepared by the addition of
barnyard millet flour exhibited lower moisture content 22.40% than the control muffins
25.61%. Moreover, Shearer and Davies (2005) explicated that moisture content of
muffins differed non-momentously by adding flaxseed meal in wheat flour. Similarly,
Rajiv et al. (2011) delineated that moisture content of muffins decreased by adding finger
millet flour in wheat flour.
4.9.1.2. Crude protein
The present results elucidated that the crude protein in muffins significantly increased
with increasing levels of SPI (Table 4.44). Maximum protein content (12.81±0.17%) was
noticed for T5 trailed by T4 (11.68±0.50%), T3 (10.55±0.24%), T2 (9.42±0.46%), and T1
(8.29±0.30%) while, the minimum value was shown by T0 (7.16±0.36%).
The current outcomes are in accordance with the verdicts of Chetana et al. (2010),
expounded significant increase in protein content from 8.5% to 12.4% by adding raw
flaxseed powder. Similar rise in protein content (11.9%) was noticed by the incorporation
of roasted flaxseed powder. Later, Srivastava et al. (2012) delineated that muffins
prepared by adding fenugreek seed husk exhibited higher protein content (7.97%) in
comparison with control (7.26%).
Likewise, Jisha et al. (2010) described that the protein content of muffins prepared from
cassava based composite flours varied from 4.50 to 5.58%. Similarly, Jisha and Padmaja
(2011) reported momentous increase in protein content (7.96 to 14.36%) of muffins
developed from composite flours supplemented with whey protein concentrate. In another
research exploration, Rosa et al. (2006) delineated 9.7 & 9.8% protein content in muffins
with mesquite pod flour toasted at 60 and 70oC, respectively. However, Goswami et al.
(2015) reported 6.05% protein in muffins prepared from barnyard millet.
The recent results are also in accord with the findings of Lipilina and Ganji (2009),
revealed an increasing trend for protein content (6.4 to 8.4%) of muffins made by
substituting wheat flour with ground flaxseed. Later, Jauharah et al. (2014) observed a
significant increase in protein content of muffins developed with partial replacement of
wheat flour with young corn powder that ranged from 6.73 to 7.93%. Likewise, Younas et
108
al. (2015) assessed the protein content of apple pomace enriched muffins varying from
6.38 to 10.13%.
4.9.1.3. Crude fat
The results of current study explicated non-momentous variations in fat contents of
various treatments. The recorded values were 29.29±1.12 (T0), 29.27±0.29 (T1),
29.25±1.30 (T2), 29.23±0.90 (T3), 29.21±1.13 (T4) & 29.20±0.68 (T5) (Table 4.44).
The fat content of food is an important parameter to evaluate its physical and sensory
attributes i.e. texture, flavor, appearance and mouthfeel. In a previous research study,
Chetana et al. (2010) indicated 29.6% fat content of muffins. Nonetheless, Goswami et
al. (2015) delineated 16.86% fat content in control muffins while 16.92% in barnyard
millet flour based muffins. In another research exploration, Srivastava et al. (2012)
observed 29% fat content in muffins made by supplementing wheat flour with fenugreek
seed husk. Likewise, Jisha et al. (2010) expounded that the fat content of muffins
prepared from cassava based composite flours ranged from 7.15 to 9.95%. Moreover,
Jisha and Padmaja (2011) stated that the fat content of muffins prepared from cassava
flour mixes incorporated with whey protein varied from 7.25 to 12.80%.
Previously, Rosa et al. (2006) reported 11.2% fat content in muffins containing mesquite
pod flour. Similarly, Yaseen et al. (2012) found that fat content of muffins varied from
26.7-27.4% by adding date syrup in wheat bran. Likewise, Jauharah et al. (2014)
corroborated that the fat content of muffins decreased from 13.43 to 12.12% by the
supplementation of young corn powder in wheat flour. Similarly, Younas et al. (2015)
elucidated that the fat content of muffins reduced from 21.92 to 19.59% by the
enrichment of wheat flour with apple pomace.
4.9.1.4. Crude fiber
In present study, muffins prepared by adding SPI showed non-momentous variation in
fiber. The results indicated that the fiber content was in the range of 0.65±0.01 to
0.69±0.04% (Table 4.44).
Previously, Chetana et al. (2010) documented that total dietary fiber increased (3.1 to
18.3%) in muffins by the addition of raw and roasted flaxseed powder. Later, Srivastava
et al. (2012) observed 29% total dietary fiber in muffins made by the incorporation of
fenugreek seed husk in wheat flour. Likewise, Rosa et al. (2006) documented 7.8% crude
109
fiber for mesquite pod flour muffins. Similarly, Gambuś et al. (2004) & Frank and Sarah
(2006), reported that fiber content of muffins and cookies increases by adding 3-5% flax.
Likewise, Lipilina and Ganji (2009) observed increase in fiber content (1.09-4.80%) by
the substitution of wheat flour with ground flaxseed. Moreover, Goswami et al. (2015)
recorded 2.09% fiber content in muffins prepared from barnyard millet flour. Likewise,
Yaseen et al. (2012) expounded that fiber content of muffins increased from 1.3 to 5.6%
by the addition of wheat bran along with date syrup. Likewise, Younas et al. (2015)
described increase in fiber content from 0.75 to 3.13% by gradually adding apple pomace
in wheat flour.
4.9.1.5. Ash
The instant results revealed non-significant variations in ash content. Mean values for ash
content varied from 0.90±0.05% (T5) to 0.93±0.04% (T0) as presented in Table 4.44.
Earlier, Chetana et al. (2010) found that ash content of muffins prepared from composite
flours supplemented with raw and roasted flaxseed powder increased from 0.8 to 1.3%.
Likewise, Srivastava et al. (2012) noticed 1.16% ash content in wheat flour-fenugreek
seed husk muffins. In another research investigation, Mohsen et al. (2009) explicated that
supplementation of protein isolates from soy decreases ash contents. Previously, Rosa et
al. (2006) observed 8.8% ash content of muffins with mesquite pod flour.
Recently, Sudha et al. (2015) delineated 0.84% ash content in muffins with 25% dried
mango pulp fiber waste. In another research investigation, Jauharah et al. (2014) revealed
that the ash content of muffins prepared by partial replacement of wheat flour with young
corn powder increased from 1.22 to 1.56%. Recently, Younas et al. (2015) reported ash
content (0.93-1.69%) in apple pomace enriched muffins.
4.9.1.6. Nitrogen free extract (NFE)
The instant results revealed significant variation in NFE of muffins prepared from
different treatments of composite flour blends (Table 4.43). The highest NFE value
50.75±0.44% was obtained for T0 (control) followed by T1 49.63±2.26%, T2
48.49±1.64%, T3 47.37±1.90% and T4 46.24±0.81% whereas, minimum in T5
45.35±2.61% (Table 4.44).
In a previous study, Yaseen et al. (2012) explained that the NFE of muffins made by
adding varying concentrations (30, 40, and 50%) of date syrup in wheat bran decreased
110
Table 4.43: Mean squares for proximate analysis of protein enriched muffins
SOV df Moisture Crude
protein
Crude
fat
Crude
fiber Ash NFE
Treatments 5 0.010ns 13.402** 0.004ns 0.001ns 0.000ns 12.582**
Error 12 0.162 0.127 0.930 0.000 0.002 3.171
Total 17
P value <0.05
** Highly significant; ns Non-Significant
Table 4.44: Proximate analysis (%) of protein enriched muffins
Treatments Moisture Crude
protein Crude fat
Crude
fibre Ash NFE
T0 11.20±0.46 7.16±0.36f 29.29±1.12 0.67±0.02 0.93±0.04 50.75±0.44a
T1 11.21±0.41 8.29±0.30e 29.27±0.29 0.67±0.01 0.93±0.05 49.63±2.26b
T2 11.23±0.48 9.42±0.46d 29.25±1.30 0.68±0.02 0.93±0.03 48.49±1.64c
T3 11.24±0.22 10.55±0.24c 29.23±0.90 0.69±0.02 0.93±0.07 47.37±1.90d
T4 11.25±0.30 11.68±0.50b 29.21±1.13 0.69±0.04 0.93±0.02 46.24±0.81e
T5 11.09±0.48 12.81±0.17a 29.20±0.68 0.65±0.01 0.90±0.05 45.35±2.61f
Means carrying similar letters in a column are significantly alike
T0 = 100% Straight grade flour
T1 = 95% SGF and 5% SPI
T2 = 90% SGF and 10% SPI
T3 = 85% SGFand 15% SPI
T4 = 80% SGF and 20% SPI
T5 = 75% SGF and 25% SPI
111
from 23.24 to 20.97%. Previously, decrease in NFE content was noticed by addition of
soy proteins (Mohsen et al., 2009). Previously, Borrelli et al. (2003) observed 45-50%
NFE content in muffins.
In nutshell, muffins supplemented with SPI exhibit better nutritional attributes owing to
the presence of high quantity of proteins. The utilization of protein enriched muffins may
be helpful for both children and adults to acquire recommended daily requirement of
protein. Therefore, the muffins prepared by the addition of sesame protein isolates have
ability to cope with the menace of PEM especially in under developed countries.
4.9.2. Gross energy
Mean squares indicated non-momentous differences among treatments (Table 4.45). The
lowest gross energy 422.00±10.24 kcal/100g was recorded in T0 (control) whilst the
highest value was reported in T5 439.59±4.57 kcal/100g. Nevertheless, the means were
recorded as 424.73±10.15 (T1), 428.44±4.87 (T2), 431.68±19.98 (T3) and 436.13±2.18
kcal/100g (T4) (Table 4.46).
The instant findings are in accord with the outcomes of Lipilina and Ganji (2009), they
found that the energy values varied from 1481 to 1509 kcal for ground flaxseed
incorporated muffins. Likewise, Jisha et al. (2010) explicated that energy content of
muffins prepared from cassava based composite flours increased from 1421 to 1569
kJ/100g. Similarly, Jisha and Padmaja (2011) reported that muffins containing whey
protein-incorporated cassava flour mixes exhibited energy content ranging from 1392 to
1695 kJ/100g. In another research investigation, Younas et al. (2015) delineated the
caloric value for apple pomace enriched muffins ranging from 4520.0 to 4633.0 cal/g.
Similarly, Jauharah et al. (2014) expounded the energy range for muffins incorporated
with young corn powder as 406.48 to 514.83 kcal/100g. Comparable results were inferred
by Mrabet et al. (2016), they documented energy values varying from 447.22 to 465.32
kcal/100g for muffins having different levels of date fiber concentrates. However, Yaseen
et al. (2012) concluded that calorific value of date bran muffins decreased with an
increase in the quantity of date syrup.
112
Table 4.45: Mean squares for gross energy of protein enriched muffins
SOV df Gross energy
Treatments 5 135.256ns
Error 12 109.426
Total 17
ns Non-Significant
Table 4.46: Means for gross energy (kcal/100g) of protein enriched muffins
Treatments Gross Energy
T0 422.00±10.24
T1 424.73±10.15
T2 428.44±4.87
T3 431.68±19.98
T4 436.13±2.18
T5 439.59±4.57
Means with same letters in a column are significantly alike
T0 = 100% Straight grade flour
T1 = 95% SGF and 5% SPI
T2 = 90% SGF and 10% SPI
T3 = 85% SGFand 15% SPI
T4 = 80% SGF and 20% SPI
T5 = 75% SGF and 25% SPI
113
4.9.3. Physical analysis of protein enriched muffins
4.9.3.1. Color
The mean squares pertaining to crust and crumb color of protein enriched muffins
revealed significant variations among different treatments as shown in Tables 4.47 &
4.48. The means for lightness (L*), color (a*, b* value), chroma (C) & hue angle (ho) of
the crust and crumb are presented in Tables 4.49 & 4.50, respectively. The L* values for
crust decreased gradually among the treatments with highest value exhibited by T0
44.52±2.49 followed by T1 41.26±0.74, T2 38.50±0.47, T3 35.98±1.40 and T4 32.84±0.29,
whilst, the lowest value was observed for T5 30.12±1.88. Similar trend was noticed for L*
values of crumb that declined from 30.73±1.01 (T0) to 17.88±0.92 (T5). This decrease in
lightness can be attributed to the dull color of SPI. Additionally, decrease in crumb
lightness might also be due to increase in fiber content of muffins.
The redness (a* value) of crust also increased significantly from 15.03±0.81 in T0
(control) to 18.48±0.72 in T5. Moreover, a* values were also recorded for T1
(15.73±0.61), T2 (16.19±0.37), T3 (16.68±0.56) and T4 (17.20±0.45). Similar results were
observed for redness (a* value) of crumb with values ranging from 11.02±0.45 (T0) to
8.45±0.37 (T5). Nevertheless, a significant decrease in yellowness (b* value) of crust and
crumb was also observed. The b* values for crust varied from 21.23±0.61 in control
muffins (T0) to 12.79±0.06 in T5 (75% SGF and 25% SPI). Likewise, the b* values for
crumb of muffins were noted as T0 (17.22±0.15), T1 (15.80±0.91), T2 (13.36±0.08), T3
(11.86±0.38), T4 (9.98±0.50) and T5 (7.78±0.15).
This decrease in redness (a* value) and yellowness (b* value) of muffins may be
attributed to color of SPI. Moreover, the chroma and hue angle of muffins also decreased
significantly by increasing the level of SPI. The results indicated that chroma for crust
decreased from 26.01±0.43 (T0) to 22.48±0.17 (T5) whereas for crumb, the values were
20.44±0.48 (T0), 20.29±0.44 (T1), 17.42±0.23 (T2), 15.94±0.26 (T3), 13.56±0.16 (T4) and
11.48±0.17 (T5). Likewise, for hue angle of crust, the values exhibited by various
treatments were T0 (54.70±1.31), T1 (51.55±0.94), T2 (48.62±0.80), T3 (45.34±0.61), T4
(41.10±0.29) and T5 (34.69±0.33). Furthermore, the values for hue angle of crumb ranged
from 57.39±1.32 (T0) to 42.63±0.24 (T5). These changes in color did not affect the
sensory acceptability of muffins enriched with SPI.
114
Table 4.47: Mean squares for crust color of protein enriched muffins
SOV df L* a* b* Chroma Hue Angle
Treatments
(T) 5 85.397** 4.363** 29.232** 5.798** 159.510**
Error 12 0.395 0.171 0.142 0.268 0.636
Total 17
P value <0.05
** Highly significant
Table 4.48: Mean squares for crumb color of protein enriched muffins
SOV df L* a* b* Chroma Hue Angle
Treatments
(T) 5 63.847** 6.996** 37.680** 38.925** 71.337**
Error 12 0.289 0.025 0.055 0.100 0.635
Total 17
P value <0.05
** Highly significant
115
Table 4.49: Means for crust color of protein enriched muffins
Treatments L* a* b* Chroma Hue Angle
T0 44.52±2.49a 15.03±0.81d 21.23±0.61a 26.01±0.43a 54.70±1.31a
T1 41.26±0.74b 15.73±0.61cd 19.82±0.57b 25.30±0.32b 51.55±0.94b
T2 38.50±0.47c 16.19±0.37c 18.38±0.98c 24.50±0.69c 48.62±0.80c
T3 35.98±1.40d 16.68±0.56bc 16.88±0.96d 23.72±0.63d 45.34±0.61d
T4 32.84±0.29e 17.20±0.45b 15.00±0.60e 22.82±0.65e 41.10±0.29e
T5 30.12±1.88f 18.48±0.72a 12.79±0.06f 22.48±0.17e 34.69±0.33f
Means with different letters in a column are not significantly alike
T0 = 100% Straight grade flour
T1 = 95% SGF and 5% SPI
T2 = 90% SGF and 10% SPI T3 = 85% SGFand 15% SPI
T4 = 80% SGF and 20% SPI
T5 = 75% SGF and 25% SPI
Table 4.50: Means for crumb color of protein enriched muffins
Treatments L* a* b* Chroma Hue Angle
T0 30.73±1.01a 11.02±0.45b 17.22±0.15a 20.44±0.48a 57.39±1.32a
T1 27.21±0.76b 12.72±0.50a 15.80±0.91b 20.29±0.44a 51.16±0.89b
T2 25.45±1.01c 11.18±0.07ab 13.36±0.08c 17.42±0.23b 50.09±0.81c
T3 22.94±0.28d 10.66±0.32c 11.86±0.38d 15.94±0.26c 48.06±0.67d
T4 20.80±0.53e 9.17±0.49d 9.98±0.50e 13.56±0.16d 47.43±0.33e
T5 17.88±0.92f 8.45±0.37e 7.78±0.15f 11.48±0.17e 42.63±0.24f
Means having same letters in a column are significantly identical
T0 = 100% Straight grade flour
T1 = 95% SGF and 5% SPI
T2 = 90% SGF and 10% SPI
T3 = 85% SGFand 15% SPI
T4 = 80% SGF and 20% SPI
T5 = 75% SGF and 25% SPI
116
Previously, Shearer and Davies (2005) explicated that L* value for flaxseed meal
supplemented muffins decreased from 52.04 to 50.53. Similarly, a* and b* values
differed from 6.28-6.74 and 15.94-15.84, respectively. In another research exploration,
Goswami et al. (2015) evaluated the crumb color parameters of barnyard millet flour
based muffins and inferred a decreasing trend in lightness (L* value) with values ranging
from 63.51 to 72.33. However, an increasing trend was noticed for a* value (2.11 to
4.72). Likewise, the b* values decreased from 30.56 to 27.57 with increasing levels of
barnyard millet flour in wheat flour. For chroma (C), the values declined from 30.63 to
27.97, whilst hue angle for crumb of muffins reduced from 86.06 to 80.29. Likewise,
Srivastava et al. (2012) assessed crumb color of muffins prepared by the addition of
fenugreek seed husk in wheat flour at various levels. The L* values showed a decreasing
trend from 68.00 to 57.30 with increasing levels of fenugreek seed husk. Similarly for
redness (a* value), the values increased from 2.08 to 3.60 while, a similar increasing
trend was noticed for b* value that ranged from 17.63 to 23.00.
4.9.3.2. Texture
The protein enriched muffins were evaluated for different parameters of texture including
firmness and elasticity. The means squares (Table 4.51) regarding these traits showed
significant variation among various treatments. The means for firmness indicated a
momentous increase with increasing level of SPI among the treatments as depicted in
Table 54. The highest value was revealed by T5 (115.62±2.62 N) while the lowest was
noticed in T0 (90.42±2.91 N). The other treatments exhibited firmness as 95.58±2.25 N
(T1), 99.65±3.59 N (T2), 105.17±2.22 N (T3) and 110.42±6.17 N (T4). Increase in
firmness of muffins might be attributed to less availability of water and higher quantity of
proteins. Contrarily, elasticity showed a decreasing trend among the treatments with the
highest value 59.97±2.31% for T0 followed by T1 (55.94±2.82%), T2 (51.49±2.27%), T3
(47.68±2.30%) and T4 (44.10±1.30%) whilst, the lowest value was observed for T5
(40.38±1.02%) as depicted in Table 4.52.
The instant outcomes are in concordance with the findings of Shearer and Davies (2005),
they elucidated that maximum force for flaxseed meal supplemented muffins increased
from 396.9 to 594.9 g with increasing levels of flaxseed meal. This force depicts the
firmness of muffins. However, the elasticity of muffins showed a decreasing trend with
values varying from 60.2 to 55.4%. Likewise, Jauharah et al. (2014) revealed increasing
trend for the hardness of muffins incorporated with young corn powder.
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Table 4.51: Mean squares for texture of protein enriched muffins
SOV df Firmness Elasticity
Treatments (T) 5 266.053** 161.709**
Error 12 6.848 0.800
Total 17
P value <0.05 ** Highly significant
Table 4.52: Means for texture profile of protein enriched muffins
Treatments Firmness (N) Elasticity (%)
T0 90.42±2.91f 59.97±2.31a
T1 95.58±2.25e 55.94±2.82b
T2 99.65±3.59d 51.49±2.27c
T3 105.17±2.22c 47.68±2.30d
T4 110.42±6.17b 44.10±1.30e
T5 115.62±2.62a 40.38±1.02f
Means having same letters in a column are momentously alike T0 = 100% Straight grade flour
T1 = 95% SGF and 5% SPI
T2 = 90% SGF and 10% SPI
T3 = 85% SGFand 15% SPI
T4 = 80% SGF and 20% SPI
T5 = 75% SGF and 25% SPI
118
Contrarily, Rajiv et al. (2011) delineated a decrease in textural characteristics including
cohesiveness, springiness and chewiness of the muffins with increasing levels of finger
millet flour in wheat flour blends. Likewise, Goswami et al. (2015) stated a progressive
decrease in hardness from 90.63 to 52.36 N and other textural attributes like springiness,
cohesiveness and chewiness of muffins prepared from wheat flour supplemented with
barnyard millet flour.
4.9.3.3. Volume
The mean squares regarding volume of muffins indicated momentous variation among
various treatments (Table 4.53). Means elucidated a gradual decreasing trend for volume
of muffins among the treatments. The maximum volume was exhibited by T0 145.00±2.69
cm3 followed by T1 140.00±2.17 cm3, T2 135.00±0.23 cm3, T3 130.00±6.98 cm3 and T4
125.00±8.82 cm3 whilst lowest value was noticed for T5 120.00±3.97 cm3 (Table 4.54).
Earlier, Chetana et al. (2010) expounded a momentous decrease in volume of muffins
with increasing levels of raw and roasted flaxseed powder. The volume decreased from
150 cc in control to 120 cc in muffins having 40% roasted flaxseed powder. Recently,
Shevkani et al. (2015) delineated that the volume of gluten-free rice muffins
supplemented with cowpea protein isolates ranged from 40.9-54.7 mL. This designated
that the volume of muffins was momentously affected by the addition of protein isolates.
Likewise, Srivastava et al. (2012) explicated that the volume of muffins prepared by the
addition of fenugreek seed husk in wheat flour varied from 70 to 140 cc. In another
research exploration, Nasar-Abbas and Jayasena (2012) explained that the volume of
muffins is reduced by the incorporation of high dietary fiber sources. Similarly, Mrabet et
al. (2016) reported momentous decrease in volume of muffins having different levels of
date fiber concentrates with values ranging from 97.56 to 104.83 mL.
4.10. Sensory evaluation of protein enriched muffins
Sensory evaluation was performed by panel of judges to assess the treatment effect on
color, flavor, texture, taste and overall acceptability. It plays an imperative role for the
estimation of quality attributes, consumer’s response and acceptability. The mean squares
indicated that the treatments momentously affected the sensory characteristics of protein
enriched muffins (Table 4.55).
119
Table 4.53: Mean squares for volume of protein enriched muffins
SOV df Volume
Treatments (T) 5 262.500**
Error 12 25.703
Total 17
P value <0.05 ** Highly significant
Table 4.54: Means for volume of protein enriched muffins
Treatments Volume (cm3)
T0 145.00±2.69a
T1 140.00±2.17b
T2 135.00±0.23c
T3 130.00±6.98d
T4 125.00±8.82e
T5 120.00±3.97f
Means having same letters in a column are momentously alike T0 = 100% Straight grade flour
T1 = 95% SGF and 5% SPI
T2 = 90% SGF and 10% SPI
T3 = 85% SGFand 15% SPI
T4 = 80% SGF and 20% SPI
T5 = 75% SGF and 25% SPI
120
4.10.1. Color
Color is amongst the required attributes for baking performance and the product’s
demand & acceptability. It indicates appropriateness of raw materials as well as necessary
information regarding quality of the end product with respect to its formulation. The
results for sensory response elucidated that color scores gradually decreased by the
addition of sesame protein isolates (SPI).
The maximum color score 7.50±0.33 was recorded for T0 trailed by T3 (7.30±0.27), T2
(7.25±0.16), T1 (7.20±0.15) and T4 (6.97±0.11) while, minimum score was observed for
T5 (6.59±0.30) as depicted in Fig. 4.10. The present results elucidated that muffins made
by adding 20 and 25% protein isolates were least suitable. A steady decline in color score
of muffins was noticed as the quantity of protein isolates increased. This color
differencemight be attributed to color and quantity of protein isolates (Mridula et al.,
2007). Moreover, the color difference may be attributed to moisture absorption resulting
in oxidation.
The instant findings are in accordance with the outcomes of Chetana et al. (2010), they
described decreasing trend for color of muffins prepared by the incorporation of wheat
flour with raw and roasted flaxseed powder. Contrarily, Lipilina and Ganji (2009)
observed an increasing trend for color of muffins made by the substituting wheat flour
with ground flaxseed. Likewise, Rahman et al. (2015) delineated significant variations in
color of wheat grass incorporated muffins.
In another research investigation, Nasar-Abbas and Jayasena (2012) depicted similar
scores for the color of control muffins and those prepared by 50% incorporation of lupin
flour. However, the other treatments (10, 20, 30, 40% lupin flour) varied significantly.
Similarly, Ramcharitar et al. (2005) expounded significant differences in color scores for
control (6.61) and flax muffins (4.97).
4.10.2. Flavor
Flavor is an imperative characteristic to determine the likeness or dislikeness of a product .
It is the combination of smell, taste as well as texture of the end product. The present
results for flavor indicated that the maximum score was attained by T0 7.38±0.28 tracked
by T3 (6.46±0.23), T2 (7.18±0.11), T1 (7.14±0.25) and T4 (6.95±0.41) whilst, minimum by
T5 (6.69±0.31) as presented in Fig. 4.10.
121
In a previous research study, Srivastava et al. (2012) noted a significant decrease in flavor
scores of muffins supplemented with fenugreek seed husk. Moreover, Jia et al. (2011)
explained that the bakery products undergo staling process very quickly that converts rich
flavor and aroma of freshly baked product into undesirable off flavor. Recently, Goswami
et al. (2015) delineated a declining trend in the flavor score of muffins prepared by the
replacement of refined wheat flour with barnyard millet flour.
In another study, Mrabet et al. (2016) observed significant differences in flavor scores of
muffins with varying levels of date fiber concentrates. The oxidation process taking place
due to moisture content results in the development of off flavor (Sharif et al., 2009).
Later, Nasar-Abbas and jayasena (2012) explicated a decreasing trend in flavor scores of
muffins with progressive substitution of lupin flour. Similarly, Martínez-Cervera et al.
(2012) expounded decrease in flavor score of muffins made with increasing levels of a
polydextrose/sucralose mixture (PD-SC) as sucrose replacer.
4.10.3. Taste
It is perceived via taste buds and affected by numerous factors. The sensory results of
muffins indicated that maximum taste score was given to T0 (7.36±0.37) tracked by T3
(7.20±0.20), T2 (7.16±0.16), T1 (7.14±0.24), T4 (6.80±0.32) and T5 (6.53±0.27) (Fig.
4.10). The results clearly depicted that muffins with 15% protein isolates were more liked
by judges panel whereas increased quantity of SPI imparted adverse effect.
Earlier, Chetana et al. (2010) documented decreasing trend for taste scores of muffins
having raw and roasted flaxseed flour. Later, Goswami et al. (2015) also delineated
gradual decrease in the taste of barnyard millet flour muffins. In another research
exploration, Yaseen et al. (2012) studied date bran muffins and noticed significant
variation for taste scores. Likewise, Nasar-Abbas and Jayasena (2012) observed a
significant effect of substitution of lupin flour on the taste score of muffins.
4.10.4. Texture
The mean squares revealed that the treatments momentously affected the texture of
muffins as depicted in Table 4.55. T0 exhibited the highest scores 7.43±0.16 while T5
achieved minimum scores 6.77±0.37. Moreover, other treatments were scored as T1
(7.17±0.21), T2 (7.21±0.13), T3 (7.28±0.36) and T4 (7.00±0.50) (Fig. 4.10).
The texture is an imperative quality characteristic of muffins that is related to the
freshness of product. Moreover, texture is considered as an indicator of food quality as
122
well as safety. Later, Sudha et al. (2015) described a declining effect of mango pulp fiber
waste addition on muffins texture scores. Similarly, Rajiv et al. (2011) elucidated that
texture scores for muffins decreased significantly with increasing levels of finger millet
flour addition in wheat flour with. Later, Chetana et al. (2010) observed momentous
decrease in texture score of muffins prepared using various levels of raw and roasted
flaxseed powder. Similarly, Yaseen et al. (2012) explicated significant variations in
texture scores for date bran muffins. Likewise, Goswami et al. (2015) expounded that the
sensory scores for texture decreased significantly in barnyard millet flour muffins.
4.10.5. Overall acceptability
The mean squares for overall acceptability showed significant variation among different
treatments (Table 4.55). The results revealed that maximum scores were given to T0
(7.37±0.25) whilst, the minimum for T5 (6.55±0.29). Nonetheless, the scores were
assigned to T1 (7.10±0.33), T2 (7.16±0.29), T3 (7.25±0.23) and T4 (6.73±0.26) (Fig. 4.10).
The present results for overall acceptability of protein enriched muffins showed harmony
with the outcomes of Sudha et al. (2015). The authors described that acceptability score
of muffins decreased by increasing levels of mango pulp fiber waste. Likewise, Rajiv et
al. (2011) observed decrease in overall quality scores of muffins with gradual addition of
finger millet flour. Previously, Sanz et al. (2009) observed a significant decrease in
overall acceptance of different resistant starch containing muffins.
The current outcomes are also in conformity with the verdicts of Rahman et al. (2015),
indicated a momentous effect of wheat grass powder on the overall acceptability of
muffins. Likewise, Ramcharitar et al. (2005) delineated that flax muffins showed less
overall acceptability in comparison with control muffins. In another research study
conducted by Batool et al. (2013), the overall acceptability scores of baked products
revealed a decreasing trend with respect to storage time.
It is deduced from the current exploration that adding sesame protein isolates (SPI) up to
15% in wheat flour imparts no adverse effect on the baking characteristics of composite
flours. Furthermore, the protein enriched muffins prepared using composite flours exhibit
better sensory attributes. Conclusively, these protein enriched muffins possess the
tendency to provide sufficient quantity of proteins needed by the body to perform its
functions. Therefore these muffins can be potentially utilized as a dietary intervention to
cope with protein energy malnutrition.
123
Table 4.55: Mean squares for sensory scores of protein enriched muffins
SOV df Color Flavor Taste Texture Overall
acceptability
Treatments 5 0.506* 0.584* 0.473* 0.268* 0.509*
Error 24 0.270 0.375 0.351 0.479 0.371
Total 29
* Significant
Figure 4.10: Sensory evaluation of protein enriched muffins
T0 = 100% Straight grade flour
T1 = 95% SGF and 5% SPI T2 = 90% SGF and 10% SPI
T3 = 85% SGFand 15% SPI
T4 = 80% SGF and 20% SPI
T5 = 75% SGF and 25% SPI
5.80
6.00
6.20
6.40
6.60
6.80
7.00
7.20
7.40
7.60Color
Flavor
TasteTexture
Overall acceptabilityT0
T1
T2
T3
T4
T5
124
Chapter 5
SUMMARY
Protein energy malnutrition has become a major nutritional dilemma in under developed
countries owing to increasing population as well as limited resources. In the developing
countries like Pakistan, people mostly rely on wheat to fulfill their energy requirements.
Nevertheless, wheat is deficient in lysine and certain other amino acids that paves the way
to explore some non-conventional sources of protein. In this perspective, defatted
oilseeds, i.e. sesame, flaxseed and canola exhibit high quality proteins that can cope with
the existing deficiency. Purposely, oilseed protein isolates were prepared through
isoelectric precipitation method. Moreover, these isolates were examined for recovery &
yield along with functional properties, bioevaluation and safety assessment. On the basis
of these analyses, one best protein isolate was selected for the preparation of protein
enriched muffins.
The proximate analysis of sesame, flaxseed and canola indicated that the crude protein,
ranged from 19.93±0.56 to 22.41±0.55. Furthermore, these oilseeds contained
considerable quantity of minerals like potassium and calcium with values ranging from
549.91±13.40 to 1048.50±29.51 mg/100g and 195.09±7.94 to 1226.05±41.82 mg/100g,
correspondingly. Nevertheless, iron, zinc and sodium were in small quantities. The
chemical composition of defatted oilseeds revealed protein varying from 34.88±0.98 to
40.90±1.00. Moreover, the mineral profile of defatted oilseeds indicated higher values for
potassium and calcium while lower for sodium, iron & zinc.
Mean square for crude protein content, recovery and yield of isolates explicated
significant variation among the tested oilseeds. Means for crude protein content revealed
highest value for sesame protein isolates (SPI) 76.14±2.00% as compared to flaxseed and
canola protein isolates. Likewise, highest protein isolate recovery was also noted for SPI
(36.86±1.22%) tracked by FPI (31.59±0.98%) while the lowest was observed in CPI
(30.52±1.20%). The protein yield also showed significant variations among the tested
isolates. The means indicated that maximum protein yield 79.03±2.18% was observed for
SPI.
125
The functional properties i.e. bulk density as well as water & oil absorption capacity
revealed momentous difference among different samples. The results indicated maximum
bulk density for CPI 0.57±0.04 g/cm3 whilst, the minimum was observed in FPI
0.45±0.03 g/cm3. Moreover, the highest value for water absorption capacity was noted in
SPI 2.12±0.08 mL/g. Likewise, SPI depicted the highest oil absorption capacity
(3.11±0.12 mL/g) as compared to FPI and CPI.
The foaming properties showed significant variations among the tested samples. The
maximum FC was revealed by SPI (18.51±0.60 mL) followed by FPI and CPI. Likewise,
the maximum foaming stability was noticed in SPI (46.98±0.90 min) trailed by FPI
whereas, the minimum was observed in CPI. Furthermore, the emulsion properties also
presented significant variations among different protein isolates. The maximum emulsion
capacity was recorded for SPI (81.36± 2.19%) while minimum was noted in CPI
(65.40±3.13%). Likewise, maximum emulsion stability was recorded for SPI 78.69±
1.08% followed by FPI and CPI. Moreover, minimum nitrogen solubility of SPI 23.43%
was noted at pH 4.0. However, from pH 8.0 to 12 the solubility of SPI ranged from 54.31-
82.56%. Similar trend was observed for FPI and CPI regarding nitrogen solubility.
The least gelation concentration (LGC) refers to the measure of gelling ability. The LGC
results indicated that SPI exhibited higher concentration (16%) followed by FPI (15%)
and CPI (14%). The electropherogram of SDS-PAGE revealed that the tested proteins
were found ranging from 15 to 65kDa. The SPI contained several polypeptide bands
ranging from 15 to 45 kDa whilst, FPI showed bands between 25 & 48 kDa and CPI
bands ranged from 16 to 65 kDa.
The CPI indicated high amount of lysine i.e. 2.60±0.09 g/100g followed by FPI and SPI.
The SPI depicted highest values for leucine (4.39±0.11 g/100g) and valine (2.83±0.11
g/100g) as compared to FPI and CPI. Likewise, for aromatic amino acids (phenylalanine
+ tyrosine), the highest values were recorded in SPI i.e. 3.36±0.11 g/100g while for sulfur
containing amino acids (methionine + cysteine), the maximum value was recorded for
FPI (2.00±0.06 g/100g). Moreover, the highest values for isoleucine 2.31±0.08 g/100g
and tryptophan 1.96±0.03 g/100g were observed in SPI & FPI, correspondingly.
Furthermore, FPI indicated highest value for histidine and threonine as 1.67±0.04 &
2.49±0.08 g/100g, correspondingly. Likewise, significant variation was observed for non-
essential amino acids in the tested protein isolates. The results revealed that maximum
values for alanine (3.30±0.08 g/100g), arginine (7.49±0.18 g/100 g), glutamic acid
126
(12.70±0.76 g/100 g), glycine (3.09±0.12 g/100 g) and serine (3.17±0.05 g/100 g) were
exhibited by FPI. However, maximum value for aspartic acid (5.87±0.29 g/100 g) was
recorded in SPI.
The amino acid scores revealed momentous variations among the tested oilseed protein
isolates. The results indicated lysine as the limiting amino acid for SPI, FPI and CPI. The
protein scores were recorded as 28.46, 31.15 & 50.00 for SPI, FPI and CPI, respectively.
Moreover, the results for protein digestibility corrected amino acid score (PDCAAS)
revealed highest value as 35.17±1.31% in CPI trailed by FPI whilst, the lowest value was
observed in SPI. Furthermore, significant variations were noticed among the protein
isolates regarding in vitro protein digestibility (IVPD). The maximum value for this trait
was noted in SPI (87.57±4.41%) followed by FPI while the minimum was recorded in
CPI.
The oilseed protein isolates were subjected to bioevaluation using Sprague Dawley rats.
Purposely, growth study parameters like protein efficiency ratio (PER), net protein ratio
(NPR) & relative net protein ratio (RNPR) and nitrogen balance study parameters i.e. true
digestibility (TD), net protein utilization (NPU) & biological value (BV) were measured.
Resultantly, the highest PER was recorded for SPI (2.14±0.10), followed by CPI
(2.09±0.06) however, the lowest value was noticed for FPI (1.98±0.07). The net protein
ratio (NPR) refers to the utilization of dietary protein. The highest NPR was revealed by
SPI 5.03±0.26 followed by CPI and FPI. Moreover, the results for relative net protein
ratio (RNPR) indicated that the values ranged from 78.75 to 84.82 for respective diets.
The results revealed momentous differences in the tested diets. The highest value for true
digestibility (TD) was observed for SPI (77.23±3.20%) trailed by FPI and CPI. However,
the results for biological value (BV) showed that FPI exhibited maximum value i.e.
69.35±3.47% whilst, CPI and SPI based diets indicated BV as 67.66±2.59 and
63.94±2.50%, respectively. Likewise, the highest value for net protein utilization (NPU)
was observed in FPI 50.26±2.44% followed by SPI and CPI.
Furthermore, serum biochemical parameters were determined to assess the safety of
respective diets. The results for serum total protein, albumin, globulin & A/G ratio
showed non-significant effect. The serum total protein varied from 6.45±0.27 to
6.79±0.32 g/dL. Likewise, serum albumin concentration was observed in the range of
3.08±0.35 to 3.12±0.25 g/dL for tested groups. Moreover, the values for globulin were
127
ranging from 2.91±0.13 to 3.01±0.25 g/dL. Similarly, the A/G ratio indicated values
varying from 1.04±0.09 to 1.07±0.08. Furthermore, kidney and liver function tests
revealed non-significant variations among the diets containing oilseed protein isolates.
After determining the functional attributes, overall yield and bioassessment study, SPI
was selected as the best protein isolate to prepare protein enriched muffins. Purposely,
composite flours with varying levels of SPI i.e. T1 (5%), T2 (10%), T3 (15%), T4 (20%) &
T5 (25%) were prepared and evaluated for proximate composition indicating 10.05±0.27
to 31.14±1.46% protein among different treatments.
The composite flours were further analyzed for rheological characteristics i.e. mixograph
and farinograph studies. The results for mixograph revealed highest value for mixing time
was noticed in T0 (3.14±0.25 min) followed by T1, T2, T3, T4 and T5. Likewise, the
maximum peak height was recorded in T0 (control) 57.14±3.72% while, minimum in T5
42.37±1.55%. Moreover, the results for farinographic characteristics revealed maximum
water absorption for T5 73.36±3.47% trailed by T4, T3, T2, T1 and T0. Likewise, the
highest dough development time was noted for T5 (7.09±0.23 min) however, the lowest
time was recorded for T0 i.e. 4.53±0.08 min. Furthermore, the dough stability of different
blends varied from 3.56±0.22 to 4.98±0.19 min.
The functional properties of composite flours were also determined and the results
indicated non-momentous variation in bulk density (BD) with values varying from
0.64±0.03 to 0.67±0.01 g/cm3 for different treatments. However, significant differences
were observed among treatments regarding absorption properties i.e. water and oil
absorption capacity. The results revealed that the maximum WAC was exhibited by T5
1.32±0.07 mL/g followed by T4, T3, T2, T1 and T0. Likewise, maximum oil absorption
capacity (OAC) was recorded in T5 (2.17±0.05 mL/g) while minimum was observed for
T0 (1.85±0.06 mL/g).
The results pertaining to foaming properties revealed that foaming capacity (FC) of
different composite flours was affected non-significantly with means ranging from
26.63±0.41 to 28.72±0.98 mL. Nonetheless, foaming stability (FS) showed momentous
variations among the treatments. The maximum value was recorded in T0 (60.35±1.91
min) while minimum foaming stability was noticed in T5 (51.10±1.96 min). Moreover,
the emulsion properties indicated significant variations among the treatments. The
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emulsion capacity (EC) and stability (ES) ranged from 15.27±0.66 to 29.26±0.65% and
17.84±0.57 to 31.86±1.07%, respectively.
The protein enriched muffins were prepared and analyzed for compositional profile. The
results for crude protein revealed significant variations among different treatments. The
highest value for crude protein was recorded for T5 (12.81±0.17%) tracked by T4, T3, T2
and T1 whilst, minimum was observed for T0 (7.16±0.36%). However, moisture, crude
fiber, crude fat, ash and nitrogen free extract (NFE) exhibited non-momentous variation
among different treatments. Moreover, the lowest gross energy was observed in T0
422.00±10.24 kcal/100g that increased non-significantly among the treatments T1, T2, T3
T4 & T5, respectively.
The crust and crumb color of protein enriched muffins indicated significant variations.
The results revealed that the L* value for crust varied from 30.12±1.88 (T5) to 44.52±2.49
(T0), a* value from 15.03±0.81 (T0) to 18.48±0.72 (T5), b* value 12.79±0.06 (T5) to
21.23±0.61 (T0), chroma 22.48±0.17 (T5) to 26.01±0.43 (T0) and hue angle 34.69±0.33
(T5) to 54.70±1.31 (T0). Likewise, L* value for crumb ranged from 17.88±0.92 (T5) to
30.73±1.01 (T0) whilst, a* value from 8.45±0.37 (T5) to 12.72±0.50 (T1). However, the
b* value, chroma & hue angle were observed ranging from 7.78±0.15 (T5) to 17.22±0.15
(T0), 11.48±0.17 (T5) to 20.44±0.48 (T0) and 42.63±0.24 (T5) to 57.39±1.32 (T0),
correspondingly.
The muffins texture i.e. firmness & elasticity revealed momentous variations among
treatments. The results indicated that maximum firmness was recorded in T5 (115.62±2.62
N) followed by T4, T3, T2 and T1 whereas, minimum was noticed for T0 (90.42±2.91 N).
However, maximum elasticity was observed in T0 59.97±2.31% while T5 exhibited
minimum elasticity 40.38±1.02%. Moreover, significant variations were noted among the
treatments regarding volume of protein enriched muffins. The maximum volume was
observed in T0 as 145.00±2.69 cm3 nonetheless, minimum value was shown by T5
120.00±3.97 cm3.
Sensory evaluation plays a significant role in assessing quality of final product keeping in
view the consumer requirements and preferences. Present results showed that highest
color score was exhibited by T0 (7.50±0.33) followed by other treatments. Likewise, for
flavor, T0 received maximum score i.e. 7.38±0.28 while minimum was attained by T5
6.46±0.23. Likewise, maximum taste score was obtained by T0 (7.36±0.37) followed by
129
T3, T2, T1, T4 and T5. The texture of muffins indicated highest score for T0 7.43±0.16
while minimum for T5 6.77±0.37. Likewise, the maximum overall acceptability score was
assigned to T0 7.37±0.25 whereas, minimum was recorded for T5 (6.55±0.29).
Conclusively, sesame protein isolates (SPI) exhibited better nutritional, functional and
biological attributes. Furthermore, it was evident that addition of SPI up to 15% was
relatively acceptable. The present study deduced that supplementation of muffins with
oilseed protein isolates can be a handy tool to cope with the protein deficiency among the
masses. Findings of the present investigation are helpful for nutritionists and dietitians to
understand the functional and nutritional role of oilseed protein isolates. Moreover, the
development of protein isolates enriched products could be a way forward to alleviate
protein energy malnutrition amongst the vulnerable segments. These scientific
contributions of the present research project can play vital role in creating nutritional
awareness among the masses.
130
RECOMMENDATIONS
Agro industrial byproducts like oilseed meals should be encouraged in dietary
modules to attain food and nutritional security in developing economies
The protein isolates from non-conventional sources should be used for the production
of protein enriched bakery products
Defatted oilseed protein isolates are valuable source of quality protein hence their
utilization should be promoted among the masses
Other valuable sources of quality protein should be identified in future explorations to
tackle the dilemma of protein energy malnutrition
Commercial exploitation of protein isolates based on advanced extraction procedures
with improved quality should be introduced for product development and value
addition
Concept of composite blends ought to be promoted by adding protein isolates in
wheat flour
Mass awareness and community based educational tools should be used to create
intellectual harmonization among allied stake holders
Nutritionists and dietitians should practice the dietary induction of protein enriched
products in meal plans particularly in the malnourished segments
131
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Appendix-I
Vitamin and mineral mixture used in the study
Vitamins Weight
(g) Minerals `
Weight
(g)
Thiamine hydrochloride 0.060 Calcium citrate 308.2
p-aminobenzoic acid 12.000 H2HPO4 218.7
Choline chloride 12.000 Ca(H2PO4).2H2O 112.8
Nicotine acid 4.000 HCL 124.7
Inositol 4.000 NaCl 77.0
Calcium pentothenate 1.200 CaCO3 68.5
Pyridoxine hydrochloride 0.040 MgCO3.
Mg(OH)2.3H2O 35.1
Riboflavin 0.200 MgSO4 anhydrous 38.3
Biotin 0.040 Ferric ammonium
citrate
91.41
16.7
Folic acid 0.040 CuSO4.5H2O 5.98
Cyanocobalamin 0.001 NaF 0.76
Maize starch 966.419 Mn(SO4)2. 12H2O 1.07
KAl(SO4)2. 12H2O 0.54
KI 0.24
1000.00 100.00
163
Appendix-II
Sensory Evaluation Performa of Muffins
Name of Judge --------------------------------------------
Age --------------------------------------------
Date --------------------------------------------
Hedonic Scale
9 Like extremely
8 Like 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
No. Parameter Score obtained
T0 T1 T2 T3 T4 T5
1 Color
2 Flavor
3 Taste
4 Texture
5 Overall
Acceptability
