Functional Properties of Select Seed Flours and Blackgram (Phaseolus Mungo L.) Storage Globulin Protein Gene Identification Aditya Joshi

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2012 Functional Properties of Select Seed Flours and Blackgram (Phaseolus Mungo L.) Storage Globulin Protein Gene Identification Aditya Joshi

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COLLEGE OF HUMAN SCIENCES

FUNCTIONAL PROPERTIES OF SELECT SEED FLOURS AND

BLACKGRAM (Phaseolus mungo L.) STORAGE GLOBULIN PROTEIN GENE

IDENTIFICATION

ADITYA JOSHI

A Thesis submitted to the Department of Nutrition, Food and Exercise Sciences in partial fulfillment of the requirements of the degree of Master of Science

Degree Awarded: Spring Semester, 2012

Aditya Joshi defended this thesis on March 13, 2012.

The members of the supervisory committee were:

Shridhar K. Sathe Professor Directing Thesis

Yun-Hwa Peggy Hsieh Committee Member

Kenneth H. Roux Committee Member

The Graduate School has verified and approved the above-named committee members, and certifies that the thesis has been approved in accordance with university requirements.

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ACKNOWLEDGEMENT

The Florida State University has provided me great support throughout my duration at Tallahassee campus. I want to thank my mentor and major professor, Dr. Shridhar K. Sathe for diligently supervising my work and also for providing me with continuous guidance and advice. I am thankful to him to train me into an independent researcher. My sincere gratitude is also extended to my thesis committee members, Dr. Kenneth H. Roux and Dr. Yun-Hwa Peggy Hsieh for providing me with valuable advice and input. I want to thank Dr. Kenneth Roux, Dr. Peggy Hsieh, Dr. Bahram Arjmandi and Dr. Jeong-Su Kim for allowing me to use their lab for research purpose. I am also thankful to Dr. Hank Bass and Dr. Kenneth Roux for providing support on blackgram plantation at greenhouse facility and Mission Road Research Facility.

Department of Nutrition, Food, and Exercise Sciences has provided me with this great opportunity to pursue my Master’s degree with financial support and I am really thankful for that. It would not have been possible to complete my degree without department’s support.

I am much obliged to the Core Facility of the Department of Biological Science for their assistance with cloning and gene sequencing facility. A special thanks to Rani Dhanarajan, Andre Irsigler, Cheryl Pye and Brian Washburn.

I am sincerely thankful to Girdhari Sharma, Mengna Su and Shyamali Jayasena for taking the time to teach me most of the basic laboratory techniques during my first semester.

I am truly thankful for all the support, motivation and friendship extended towards me by my colleagues LeAnna Willison, Bhodhana Dole, Pallavi Tripathi, Ruby Tiwari, Julia Katz, Chris Moleno, Sahil Gupta, Changqi Liu, and Ying Zhang. I also want to thank Mukesh Saini, Sanhita Ghosh, and Fatima Wajahat for their continuous support during my stay at Tallahassee. Thank you very much all of you for being there for me always.

Finally, I want to thank all my family members and friends. Masters degree would not have been possible without their endless support and love.

TABLE OF CONTENTS

List of Tables...... v

List of Figures...... vi

List of Abbreviations...... vii

Abstract...... viii

1. INTRODUCTION...... 1

2. LITERATURE REVIEW...... 4

3. MATERIALS AND METHODS...... 11

4. RESULTS AND DISCUSSION...... 20

5. CONCLUSION...... 46

6. APPENDIX...... 47

7. REFERENCE...... 61

8. BIOGRAPHICAL SKETCH...... 69

LIST OF TABLES

Table 1.1 Effect of fermentation on select functional properties of African oil seed flour...... 5

Table 4.1 Proximate composition of full fat flours (as-is basis)...... 21

Table 4.2 Proximate composition of full fat flours (dry weight basis)...... 22

Table 4.3 Proximate composition of defatted seed flours (as-is basis)...... 23

Table 4.4 Proximate composition of defatted seed flours (dry weight basis)...... 24

Table 4.5 Bulk densities of full fat and defatted seed flours...... 25

Table 4.6 Color (L*, a* and b*) readings of full fat and defatted seed flours...... 28

Table 4.7 Average values of chrome and hue angle of full fat and defatted seed flours...... 29

Table 4.8 Oil Holding Capacity of full fat and defatted seed flours...... 31

Table 4.9 Water holding capacity of full fat and defatted seed flours...... 32

Table 4.10 Sequence comparison of BGV with other vicilins...... 40

Table 4.11 Summary of BGV-1 and BGV-2...... 44

LIST OF FIGURES

Figure 3.1 Blackgram pods at various stages of maturity...... 16

Figure 3.2 Levi et al. (1992) method for isolation of total RNA from blackgram...... 17

Figure 4.1 Hue angle explaining various shades in the sample...... 30

Figure 4.2 Least Gelation Concentration of full fat and defatted flours...... 34

Figure 4.3 Total RNA extracted from blackgramseeds...... 36

Figure 4.4 Primary screening of blackgram cDNA library...... 37

Figure 4.5 Tertiary screening showing all positive plaques...... 38

Figure 4.6 Blackgram vicilin DNA Sequence...... 38

Figure 4.7 BGV-3 amino acid sequence...... 39

Figure 4.8 PCR products on 1% agarose gel...... 41

Figure 4.9 Picture representation of band 11, 12 and 2...... 42

Figure 4.10 Agarose gel showing PCR products...... 42

Figure 4.11 Amino acid sequence of BGV-1 and BGV-2...... 43

Figure 4.12 Amino acid comparison between BGV-3 (Vicilin obtained from polyclonal screening), BGV-1 and BGV-2 (Both obtained from PCR screening)...... 44

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LIST OF ABBREVIATIONS

RT: Room temperature BN: Brazil nut LSD: Least significant difference pAB: polyclonal antibodies PCR: Polymerase chain reaction TBS-T: Tris-buffered saline-Tween 20 IPTG: Isopropyl-β-D-thiogalactopyranoside NFDM: Nonfat dried milk LGC: Least gelation concentration WHC: Water holding capacity OHC: Oil holding capacity SG: Specific gravity LB: Luria-Bertani

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ABSTRACT

Plant seed flours are applied in several foods; still an extensive comparative study of seed flour functional properties has not been performed to date. Purpose of the current investigation was to assess functional properties of 16 full fat and defatted seed flours of various seed groups: tree nuts, legumes, oil seeds and cereals.

Tree nut and oil seed full fat flours have lower bulk densities than legume and cereal flours. Defatting caused a decrease in bulk density over a wide range (e.g. 0.49% in millet to 67.20% in Brazil nut). Rice flour was the lightest (L* = 89.62) while pecan flour was the darkest (L* = 53.71). As some of the plant pigments are oil soluble, loss in color, hue and chroma were observed after defatting. Full fat almond flour registered highest oil holding capacity (OHC) of 1.38 g/g however, after defatting macadamia flour registered maximum OHC of 3.11 g/g. Full fat (2.74 g/g) and defatted (3.17 g/g) soybean flour had the highest water holding capacities (WHCs). Water and oil holding capacity of defatted flours were typically greater than their full fat counterparts. Almost all of the defatted flours held greater amount of water and oil than their own weight. In general, defatting improved examined functional properties of all the flours likely due to the increased proportion of protein and carbohydrates when compared to their corresponding full fat counterparts.

Three blackgram vicilin genes (named BGV-3, BGV-1 and BGV-2) were isolated from blackgram cDNA library using rabbit anti-whole blackgram seed proteins polyclonal antibodies and PCR screening methods. BGV-3, BGV-1 and BGV-2 have 445, 452 and 445 amino acids respectively with theoretical pI values 5.54, 5.94 and 5.59 respectively. Signal peptide cleavage site was predicted to be between 25th and 26th amino acid of the derived amino acid sequences of BGV-3, BGV-1 and BGV-2. The estimated molecular weights of mature BGV-3, BGV-1 and BGV-2 were 48.30, 48.77, and 48.29 kDa respectively. Isolated genes exhibited more than 90% sequence similarity with various vicilin genes including, but not limited to, Vigna radiata, Vigna unguiculata, Glycin max, Vigna angularis, Vigna luteola, and Lens culinaris.

CHAPTER 1

INTRODUCTION

Part A: Functional Properties of Select Seed Flours

Globally, plant foods provide 80% of energy and 65% proteins in human food supply (Sathe, 2002). Compared to animal proteins, plant proteins are relatively inexpensive and easy to obtain (Chel-Guerrero et al., 2002) and are therefore especially important sources of food proteins in developing countries. Non-availability and self restrictedness of animal proteins due to religious and cultural practices promote plant protein consumption (Liener, 1962). Plant based diet contains higher percentage of complex carbohydrates to that of fat which is an important factor in reducing the risk of chronic diseases (Tham et al., 1998). There is convincing evidence that vegetarians have lower rates of coronary heart diseases (Fraser, 2009). In 1999, importance of plant food was recognized by the U.S. Food and Drug Administration through a directive that states, “Diets rich in whole grain foods and other plant foods and low in total fat, saturated fat, and cholesterol, may help reduce the risk of heart disease and certain cancers.” Production of plant protein from soy and legumes has low environmental effect than protein production from animal sources (Reijnders & Soret, 2003). Fresh vegetables, cereals, and legumes produce the lowest emissions of greenhouse gases (carbon di-oxide, methane and nitrous oxide) among 22 food items studied by Carlsson-Kanyama & Gonza´lez (2009). Myriads of health benefits; easy availability; wide variety; cultural and religious demands; taste and texture compatibility with other ingredients; relatively inexpensive, and less environmental pollution potential (Carlsson- Kanyama & Gonza´lez, 2009) reinforce the importance of plant foods.

Among diverse plant food groups, edible seeds including cereals, dry beans, oil seeds, legumes, tree nuts, fruit and vegetable seeds, serve as valuable sources of proteins and other nutrients for animals and humans (Sathe et al., 2005). Pulverized seeds, known as flours, serve different and important functions in food manufacturing and processing. Global availability of a variety of seed flours has resulted in preparation of diverse food products to serve diverse needs and demands of contemporary consumers. Emergence of gluten intolerance e.g. celiac disease, 1

demands for replacement for wheat flour. Campbell (1982) has suggested that patients with celiac disease should refrain from wheat, rye, triticale, barley and oats while they can enjoy cereals like corn, rice, buckwheat, millet and sorghum e.g. amaranth flour which is used to make flat breads in some communities (Pagano, 2006) can be utilized by patients with celiac disease. Sorghum has been studied in preparation of breads, parboiled sorghum, sorghum tortillas, snack food, cookies and flat breads (Ciacci et al., 2007). Forty percent of the world sorghum production is used for human consumption in India and Africa while it is mainly used as an animal feed in the western countries (Ciacci et al., 2007). These data suggests sorghum has very huge potential to be utilized as a replacement for wheat. Millet can be a better replacement for bread making as it is nutritious, inexpensive and readily available.

Increasing awareness about legumes and ingredients derived from them is likely to create new and exciting opportunities for food processors and manufacturers. One example of such opportunity is the use of blackgram flour. Modi et al. (2003) found that out of four legume flours studied for buffalo hamburger formulations, roasted blackgram flour had the highest yield, the lowest % shrinkage and the lowest fat absorption. This low fat, high protein, legume flour, although used extensively in South East Asia in numerous food products, remains largely unexplored in the western world. Other legumes like chickpea flour in South East Asia and Turkey; cowpeas in Thailand and many African countries; are used on a routine basis although these are lesser used in the western world. Demand for flours with multitude food applications, has lead to investigation of unconventional seed flours. Bhat et al. (2009) investigated functional properties of lotus seed flours for understanding potential food applications of this unconventional seed flour. Such studies illustrate the interest of food industry in unconventional flours for food product applications with improved properties and reduced cost. Except reported nutritional and health benefits other factors like changes in consumer preferences, increasing demand for variety, change in demographics, rise in food allergy incidences and ongoing research on production and processing technologies etc. are growing interests in use of different flours in developed countries (Boye et al., 2010).

Before exploring use of diverse seed flours in food manufacture and processing, it is important to assess relevant functional properties of the targeted flour in specific food systems. Exploring

functionality of numerous flours in myriad of foods is impractical. However, it is feasible to study comparative assessment of targeted flours under the same defined experimental conditions.

Part B: Blackgram (Phaseolus mungo L.) storage globulin protein gene identification

Blackgram (Phaseolus mungo L. or Vigna mungo L.) is commonly known as urdbean or mash (Souframanien and Gopalakrishna, 2004). It is a highly prized legume in the Indian subcontinent. Blackgram is widely cultivated in the Indian subcontinent and to a lesser extent in Thailand, Australia, and other Asian and South Pacific countries. It is an annual plant with annual production of about 2 million metric tons per year. Blackgram, just as other legumes, is a nitrogen fixing plant and therefore cultivated in combination with cereal crops (i.e. inter cropping) to maintain soil quality. Blackgram is rich in protein and thus used as staple food in combination with cereals such as rice. Blackgram is consumed as whole, dehusked split beans (also known as “dhal”), fermented (alone), fermented in combination with white polished rice or cooked (steamed or fried).

Blackgram is high in protein with a single dominant protein (known as globulin or phaseolin) which is known to influence the flour functional properties. Despite the importance of blackgram blobulin in determining functionality of blackgram flour, limited investigations have been reported (Susheelamma and Rao, 1974; Susheelamma and Rao, 1978; Susheelamma and Rao, 1979; Susheelamma and Rao, 1989). However until 2006 the importance of the protein in adverse reactions was not known (Kumari et al, 2006). Kumari et al (2006) have reported sensitivity (Type I hypersensitivity) to blackgram in the Indian population. Food hypersensitivity is reported to affect 2% adults and 8% children of the world. Thus it is essential to investigate the blackgram globulin gene for its gene sequence and perform further studies on it. The current study focuses on identification blackgram globulin protein gene.

CHAPTER 2

LITERATURE REVIEW

Functional properties are defined as properties, other than nutritional properties, of food which are essential for using the food source effectively. Abundant research and review articles are available in literature dealing with functional properties of specific flours including wheat flour (Gupta et al., 1992; Weegels et al., 1996), African oil seed bean (Akubor and Chukwu, 1999), lima beans (Granito et al., 2007), horsegram bean (Sreerama et al., 2008), pigeon pea (Tiwari et al., 2008), cashew kernel (Alobo et al., 2009), yellow pea (Aluko et al., 2009), lotus seed (Bhat et al., 2009), adzuki beans (Chau and Cheung, 1998), chickpea (Kaur and Singh, 2006), blackgram seed (Kaur and Singh, 2007), dry beans (Siddiq et al., 2010), rice beans (Chau and Cheung, 1998), pea (Singh et al., 2010). Few research articles focus on protein concentrate, isolates of seed flours e.g cashew (Ogunwulu et al., 2009), peanut (Yu et al., 2007), lima bean (Chel-Guerrero et al., 2002). Functional properties are scrutinized extensively as they manipulate behavior of flour proteins in food systems during processing, storage, preparation and consumption (Boye et al., 2010).

2.1 Functional Properties

Sathe & Salunkhe (1981) evaluated functional properties of Great Northern Bean (Phaseolus vulgaris L.) - flour, albumin, globulin, isolate and concentrate. Great Northern Bean albumins and globulins had similar viscosities at corresponding concentrations. Similar is the case for the bean protein isolates and protein concentrates. Viscosity data suggests that albumins and globulins behave differently together than when separate. The study shows that the bean concentrate has highest water and oil absorption capacities. Sathe and Salunkhe (1981) reported that the bean flour has lower water and oil absorption capacities than soybean and sunflower flour but the bean concentrate has higher water and oil absorption capacities than soybean and sunflower concentrate, soy flour, soy isolate. Further the bean protein concentrate showed 4

highest emulsion capacity (72.6 gram oil per gram sample) while the albumins had highest emulsion stability (5 ml phase separation after 780 hours at 21oC.) and highest foaming capacity (80% volume increase). This study shows that the bean albumins and globulins behave differently when they are separate than together. The bean protein concentrate has better properties than the bean protein isolate which indicates increase in protein concentration does not mean better functional properties. The percentage of protein content and non-protein components manipulate protein functional properties. Least Gelation Concentration (LGC) of the bean protein concentrate (8%) has lowest LGC followed by the bean flour (10%), isolate (12%), albumins (18%) and globulins (20%). These values suggest that other components than proteins play a role in LGC of the bean samples. Presence of fat affects functional properties in cashew flour. Alobo et al. (2009) study focuses on effects of fat component on physiochemical and functional properties of cashew kernel flour. They evaluated functional properties of full fat and defat cashew kernel flour as function of pH and NaCl concentration. Foam capacity, foam stability, nitrogen solubility, emulsion capacity, bulk density, water absorption capacity and oil absorption capacity of the defat flour are better than full fat flour for all pH and NaCl variations. This demonstrates that removal of fat improves functional properties of the cashew kernel flour. Similar study on African oil seed proteins by Akubor & Chukwu (1999) demonstrated the tested functional properties (except emulsion activity) were improved by defatting (Table 1.1).

Table 1.1. Effect of fermentation on select functional properties of African oil seed flour Unfermented flour Fermented flour Property Undefatted Defatted Undefatted Defatted Water Absorption Capacity (%) 88 ± 0.01 195 ± 0.04 120 ± 0.24 225.0 ± 0.35 Oil Absorption Capacity (%) 110 ± 0.20 115 ± 0.1 89.0 ± 0.40 113.0 ± 0.80 Bulk Density (g/cm3) 0.71 ± 0.25 0.62 ± 0.45 0.60 ± 0.31 0.53 ± 0.29 Emulsion Activity (%) 6.0 ± 0.32 5.95 ± 0.49 5.0 ± 0.51 4.0 ±0.09 Emulsion Stability (%) 1.45 ± 0.01 2.38 ± 0.02 1.24 ± 0.09 2.41 ± 0.18 Adopted from Akubor & Chukwu (1999)

It can be seen in Table 1.1 that defatting procedure improved water holding capacity in the unfermented and fermented flour by more than double as compared to respective full fat flours. These studies (Akubor & Chukwu, 1999; Alobo et al., 2009) show a promising improvement in functional properties after defatting and that presence of carbohydrates influences properties like LGC (Sathe & Salunkhe, 1981). Limited studies on this issue demand for similar intervention into other flour types.

Water holding capacity, oil holding capacity, emulsification, foaming, gelation, thickening and flavor binding are essential functional properties for food processing.

2.2 Water Holding Capacity and Oil Holding Capacity

Water holding capacity (WHC) is defined as grams of water that can be retained by gram of sample. It depends on type and amount of protein as well as presence of non protein components in a sample. In non protein components of bean proteins, presence of carbohydrates increases WHC (Sathe, 2002). Electric charges present on proteins determine its interaction with water molecule eventually affecting WHC. Thus pH of the solution affects water holding capacities by manipulating electric charges on protein molecules. WHC values follow same pattern as percentage nitrogen solubility with change in pH of solution. WHC are important for water retaining products like breads, biscuits, puddings, cakes, etc. WHC govern moisture retention in the product which is related to staling of these products. More the water loss more is retrogradation and hence early staling. Thus high WHC values are advantageous for these products to maintain their freshness as they hold water more strongly and efficiently.

Oil Holding Capacity (OHC) is defined as grams of oil that can be retained by gram of sample. Primary mechanism of OHC is physical entrapment of oil by capillary attraction (Chau & Cheung, 1999). Hydrophobic interactions of protein and carbohydrate surfaces with oil play a vital role in determining OHC. OHC is essential in quality of fried products and for retention of flavor components. Chickpea flour, blackgram flour, soy proteins are employed in Asian fried products as they have suitable OHC values (Sathe, 2002). 6

2.3 Emulsification and Foaming Properties

Emulsifying properties and foaming are most important functional properties of proteins. They play critical role in development of traditional and novel foods (Rangel et al., 2003). Proteins act as emulsifying agents by stabilizing oil water interface in products like salad dressings and margarine. Proteins migrate to the oil-water or air-water interface and form an interfacial layer with consequent alteration of the surface properties and stabilization of the dispersion (Rangel et al., 2003). These protein membranes avoid coalescence of droplets and eventually enhancing droplet dispersion in the immiscible phase of emulsions (Aluko et al., 2009). Protein molecular size, pH of medium, protein molecular flexibility, protein surface charge, protein migration rate from bulk phase to interface, degree of denaturation, relative proportions of immiscible phases, ability of the protein to form a “film” or the “skin” at the interface, and the protein solubility in the bulk phase to some extent determine foaming and emulsifying properties (Sathe, 2002). Presence of carbohydrates like starch and fibers may enhance emulsion stability by acting as bulky barriers between the oil droplets, preventing or slowing down the rate of oil droplet coalescence. Therefore, instead of using only proteins or starch, it may be possible to form and stabilize emulsions and foams with suitable combinations (Aluko et al., 2009). Foaming is important in products like idli which is produced from fermented rice and blackgram flour. Blackgram proteins provide desired structure to form the foam translating into desired texture of the product. Dosa is another similar product for which blackgram flour is must to provide foaming properties into the batter.

2.4 Gelation

Pudding, jellies, dessert and meat applications require gelation property from proteins. Least Gelation Concentration (LGC) is an important index of gelling capacity which can be defined as lowest concentration required to form a self-supporting gel (Boye et al., 2010). Among plant proteins heat induced gels are most common. Interactions taking place in protein gel formation include hydrogen bonds, electrostatic interactions, disulfide bonds and hydrophobic interactions. Protein gelation involves several steps that include protein denaturation; formation of protein

strand network(s) mediated through protein-protein and protein-non-protein component interactions, and gel setting (may involve water binding to proteins, protein-protein interactions, and water-water or water-ion binding). The precise mechanism(s) involved in such gel formations depend(s) on several factors, including, the type and source of protein; the protein concentration of the system; the system pH, ionic strength, temperature, the number of different proteins within a system, and presence of non-protein components, as well as the mechanical parameters (shearing, stirring, stretching, etc.) of the system. Several other parameters, such as the type of protein, the fundamental properties of the protein, the operating conditions used to make gel, and the presence or absence of other components in the food system determines type of network formed by proteins (Sathe, 2002).

The review of the literature indicates that the presence of the non-protein components have an essential role in protein functionality. Carbohydrates enhance water holding, gelation, emulsification and foaming properties of proteins by better hydration ability. What happens on removing fat component from whole seed flours? Defatting is an essential step in many protein isolates and concentrates preparations. Defatted flours find applications in several areas as percentage protein in sample increases. Extracted fat can be used for other applications. In case of oil seeds, defatted meal is waste for oil industries which can be economically utilized as protein source. Defatted meal can serve as food ingredient or as filler in food formulations. Limited research articles are available in literature dealing with effect of defatting on protein functionality and physiochemical properties (Alobo et al., 2009; Akubur & Chukwu, 1999).

2.5 Selection of seed flours

Purpose of the current investigation was to compare functional properties of various seed groups under the same set of conditions. Cereals, legumes (beans), oil seeds and tree nuts are 4 the major plant seed groups. Cereals are staple food for the large population of the world. According to FAOSTAT (2012), rice is the most produced commodity in the world (684779898 Metric Tons with total value of ~$178.34 billion). Wheat (~$86.7 billion), soybean (~$57.58 billion) and maize (~$51.16 billion) follow rice in the plant seeds group (FAOSTAT, 2012).World 8

production and worldwide demand for these cereals upholds their importance. Legumes are one of the important protein sources in daily consumption of many people of the world. 23229224 tons of dry beans were produced in the world in year 2010 (FAOSTAT, 2012). Blackgram and chickpea flours are utilized in daily food products of various eastern cultures but it has not been thoroughly exposed to the western communities. Blackgram is one of the major beans used in Asia since the ancient times (Paroda and Thomas, 1987). Beans being inexpensive and a good protein source should be explored for food applications for the new world. Seeds of soybean, peanut, sesame and canola etc. are used worldwide for the oil production purpose. Oil cake, a byproduct of this oil industry, is rich in carbohydrates and protein which can be applied as food ingredient in varied food applications. Functional property determination assists in food product development study e.g. oil absorption capacity gives idea of applicability of that ingredient in food products requiring moisture retention such as cakes. Water holding capacity, color and bulk density are other essential properties which should be studied to understand behavior of the ingredient in food systems.

A. Tree nuts: a. Almond (Prunus dulcis) b. Brazil nut (Bertholletia excels) c. Cashew (Anacardium occidentale) d. Hazel nut (Corylus avellana ) e. Macadamia (Macadamia integrifolia) f. Pecan (Carya illinoinensis) g. Pistachio (Pistachia vera) h. Walnut (Juglans regia) B. Legumes and oil seeds: a. Blackgram (Phaseolus mungo) b. Chickpea (Cicer arietinum) c. Peanut (Arachis hypogaea) d. Soybean (Glycine max) e. Sesame (Sesamum indicum)

C. Cereals: a. Rice (Oryza sativa) b. Wheat (Triticum aestivum) c. Pearl Millet (Pennisetum glaucum)

3.1 Specific Objectives

Part A:

Following objectives were performed for the select seed flours (full fat and defatted flours). A) Estimate proximate composition of the select seed flours: i. Moisture ii. Lipids iii. Proteins iv. Ash v. Carbohydrate

B) Measure physical and functional properties: vi. Water Holding Capacity (WHC) vii. Oil Holding Capacity (OHC) viii. Least Gelation Concentration (LGC) ix. Specific Gravity (SG) x. Color

Part B: Following objectives were performed to isolate major seed storage protein from blackgram: a) Isolate the total RNA b) Construct the blackgram cDNA library c) Isolation of blackgram globulin protein gene from the cDNA library. 10

CHAPTER 3

MATERIALS AND METHODS

Part A: - Functional Properties of select seed flours.

Materials:

All seeds except blackgram and peanuts were obtained from New Leaf Market in Tallahassee, FL. Blackgram and peanuts were obtained from Little India grocery store in Tallahassee, FL, USA.

Flour preparation:

Seeds were ground using an Osterizer grinder at “Grind” setting to particle size of ~20 mesh to obtain full fat flour in single batch. Part of full fat flour was defatted for 8 hrs using a Soxhlet apparatus (Fisher Scientific Co., Orlando, FL) with petroleum ether (boiling point range of 38.2- 54.3 °C) as a solvent (flour-to-solvent ratio of 1:10 w/v). Defatted flours were air-dried in a fume hood, powdered again using a blender (to obtain a homogeneous sample of ~20 mesh). Full fat and defatted flours were stored in screw-capped plastic vials at -20 °C until further use.

All of the following analyses were performed at least in duplicate.

A) Proximate composition: a. Moisture: Moisture (AOAC Official Method 925.40). An accurately weighed sample (~1 g) was placed in an aluminum pan and then dried in a previously heated vacuum oven (Barnstead lab- Line, Melrose Park, IL; model 3608-5; 95-100 °C, 20-25 in. of Hg) to a constant weight. b. Lipid: (AOAC Official Method 948.22). A known weight of the sample (~15 g/thimble) was defatted in a Soxhlet apparatus using petroleum ether (boiling point range 38.2-54.3 °C) as the solvent (flour-to-solvent ratio of 1:10 w/v) for 8 h. Defatted samples were dried overnight (~10- 12 h) in a fume hood to remove residual traces of petroleum ether and the samples were weighed

to calculate lipid content. Difference in weight before and after extraction were taken as fat in the sample. Lipid (%) = (Initial wt of full fat flour (g) – Final wt of defatted flour (g)) x 100 Initial wt (as is) of full fat flour (g)

c. Protein: Protein (AOAC Official Method 950.48). The micro-Kjeldahl method were used to determine total proteins. Briefly, 0.1 g of sample was placed in a micro-Kjeldahl flask. A catalyst

(mixture of 0.42 g of CuSO4 + 9.0 g of K2SO4), a few glass beads (to prevent sample bumping), and 15 mL of concentrated H2SO4 (36 N) was added to each sample. Sample digestion was done at 410 °C for 30-75 min (until a clear green solution was obtained, which ensured complete oxidation of all organic matter). The digest was diluted with 50 mL of distilled water, and the micro-Kjeldahl flask was attached to the distillation unit. After the addition of 45 mL of 15 N NaOH, sample distillation was commenced and released ammonia was collected into a 0.1N boric acid solution containing the indicators methylene blue and methyl red. Borate anion

(proportional to the amount of nitrogen) was titrated with standardized 0.1 N H2SO4. H2SO4 was standardized against 0.1 N Na2CO3 solution. A reagent blank was run simultaneously.

%N = (mL of H2SO4 for sample - mL of H2SO4 for blank) x 0.136 x 1.4007 Wt of sample (g)

Sample nitrogen content was calculated using the formula Protein (%) = (total N (%) x appropriate factor for sample). The conversion factors used were 5.18 for almond, 5.46 for peanut, and 5.3 for Brazil nut, cashew, hazel nut, macadamia, pecan, pistachio and walnut. Conversion factors for wheat, soybean, and millet were 5.59, 5.69, 5.60 respectively (Tkachuk, 1969). Conversation factor 5.6 was used for remaining flours.

d. Ash: (AOAC Official Method 923.03). Accurately weighed sample (~0.1 g) was placed in a ceramic crucible (previously heated and cooled until constant weight was obtained) and subjected to ashing in a muffle furnace maintained at 550 °C until a constant final weight for ash was achieved. Ash percentage was calculated ‘as is basis’ and dry weight basis.

e. Carbohydrate: Total carbohydrates was determined by difference (%) Carbohydrates = [100-(%) Moisture - (%) Fats – (%) Proteins – (%) Ash].

B) Bulk density:

Bulk density was measured as method of Kaur and Singh (2007). Flour samples were gently filled in 10 ml graduated cylinder (with least count 0.5ml). The bottom of the cylinder was gently tapped 5 times until there was no further diminution of the sample level after filling to the 10 ml mark. Bulk density was calculated as mass of sample per unit volume of sample (kg/m3).

C) Color:

Color of each sample was measured with LabScan XE spectrophotometer (Hunter Associates Lab., Reston, Virginia, U.S.A.). LabScan XE spectrophotometer was standardized using white and black plates provided by the manufacturer. A thick layer of sample (~50g) was filled in the 2.5inch diameter sample cup and gently tapped to remove all voids. Color was measured using 1 inch diameter view with 0o/45o geometry and 10o observer. L*, a* and b* values of the sample were recorded using EasyMatch QC software. Chroma and hue angle were calculated as per formulas stated below.

D) Least Gelation Concentration:

Least Gelation Concentration (LGC) was determined by the method of Sathe & Salunkhe (1981). For each type of flour, dispersions of 2, 4,6,8,10,12,14,16,18 and 20% (w/v) were prepared in 5ml distilled water in 20ml pyrex test tubes. These test tubes were heated in a boiling water bath (100oC) for 1 hour followed by rapid cooling under running cold tap water (25oC). The tubes were further cooled at 4oC for 2 hours before LGC determination. The LGC is the minimum concentration at which sample from inverted tube doesn’t fall or slip from wall of the tube.

E) Water Holding Capacity (WHC) and Oil Holding Capacity (OHC):

Water Holding Capacity (WHC) and Oil Holding Capacity (OHC) were determined by method of Sze-Tao and Sathe (2000). Sample (0.1g) was weighed and vortex (Fisher Brand, Pittsburgh, PA) mixed for 30 s at RT with 1ml water/vegetable oil. These flour suspensions were allowed to stand for 30 minutes before centrifugation (13600g, 10 min, RT) (Eppendof 5415D, Brinkmann Instruments, Inc., Westbury, NY). The excess water/oil was drained for 1 minute by keeping microcentrifuge tube at 45 degree angle. The tube was weighed again and weight of supernatant was obtained by difference of weights before and after draining. Water-holding capacity was expressed as gram of water held per gram of flour sample. Oil-holding capacity was expressed as gram of oil held per gram of flour sample. The procedure is repeated three times for each sample.

F) Statistical Analysis

The results are expressed as means ± standard deviation. The data were subjected to one-way analysis of variance (ANOVA) using SPSS (Version 15.0) statistic program for Windows (SPSS Inc. Chicago, IL) and Fisher’s Least Significant Difference (LSD) at p=0.05 was calculated as described by Ott (1977).

Part B: - Identification and extraction of blackgram (Phaseolus mungo) protein gene.

Materials

Dry blackgram seeds (Phaseolus mungo L.) were obtained from local grocery store (Little India, Tallahassee, FL, USA) and planted in FSU Biology greenhouse and The Florida State University Mission Road Research Facility experimental plots (on 2nd week of June, 2009). Isopropyl-β-D- 1-thiogalactopyronoside (IPTG), dithiotheritol, thiourea, 2-mercaptoethanol, sodium ethylidenediaminetetraacetic acid was obtained from Fisher Scientific Co (Pittsburgh, PA). Lithium dodecylsulfate, polyvinylpyrrolidone, aurintricarboxylic acid and diethylpolycarbonate were obtained from Sigma Chemical Co. (St. Louis, MO). Tween-20 was obtained from BDH chemicals (West Chester, PA) and Tris (ultrapure) was obtained from MP Biochemicals (Solon, OH). 10mM IPTG solution was passed through 0.25um filter. SM buffer, LB media was autoclaved for 15 minutes at 15psig. Whenever required materials/chemicals were sterilized using 0.2um filters or autoclave at 15 psig for 15-20 minutes.

Isolation of total RNA and blackgram cDNA library construction

Blackgram pods, grown in the greenhouse as well as a field (Florida State University, Mission Road Research Facility, Tallahassee, FL), at different degree of maturity (Figure 3.1) were picked on several different days during August to September 2009. The harvested pods were immediately stored at -80oC until further use.

Figure 3.1. Blackgram pods at various stages of maturity.

Pods were manually opened with care and seeds at various stages of development were used to isolate the total RNA as described by Levi et al. (1992). Approximately 0.4 g tissue was manually ground with a mortar and pestle, and mixed with 5 ml homogenization buffer (200 mM Tris HCl, pH 8.5, 1.5% lithium dodecylsulfate, 300 mM lithium chloride, 10 mM sodium EDTA, 1.5% sodium deoxycholate, 1.5% Nonidet P-40, and 2% soluble and insoluble polyvinylpyrrolidone each, 0.5mM thiourea, 1 mM aurintricarboxylic acid, 10 mM dithiothreitol, and 75 mM 2-mercaptoethanol). The steps for isolation are described in figure 3.2. All steps, unless indicated, were carried out at 4°C.

Total RNA was dissolved in 0.1% (v/v) diethyl pyrocarbonate (DEPC) treated water and stored at -80°C until further use. The mRNA was separated from the total RNA using PolyATtract kit (Promega, Madison, WI) according to the manufacturer’s protocol. The synthesis of cDNA library was performed with the ZAP-cDNA Gigapack III Gold cloning kit (Stratagene Inc, Cedar Creek, TX) according to the manufacturer’s instruction. The double stranded cDNA was cloned directionally into the lambda Uni-ZAP XR expression vector, packaged in vitro, and transfected in E.coli XL1-Blue MRF’ strain to determine the titer of the cDNA library and for amplification of the cDNA library (See Appendix for complete description of protocol). 16

Ground tissue (0.4 g) + 5 ml Homogenization buffer Mix well (manually) Add 5 ml chloroform, shake for 5 min, add 5 ml chloroform Mix gently (manually) Centrifuge at 2,500 g for 15 min

Upper aqueous phase (transferred to new tube) + 5 ml chloroform Mix for 5 min (Vortex at low speed) Centrifuge at 2,500g for 15 min, 4oC

Upper aqueous phase (transferred to new tube) + 0.5 ml 3M NaCl + 10 ml ethanol Mix well Incubate at -20°C for 1 hr

Centrifuge at 4,000g for 15 min, 4oC

Dissolve pellet in 0.5 ml TE buffer (50 mM Tris-HCl, 10 mM sodium EDTA, pH 8.0)

Centrifuge at 12000g for 10 min, 4oC

Supernatant (transferred to new tube) + 50 µl 3M NaCl + 275 µl isopropanol

Incubate at -20°C for 1 hr

Centrifuge at 14,000g for 10 min, 4oC

Wash pellet with 400 µl 70% ethanol

Dissolve pellet in 300 µl TE buffer + 100 µl 8M lithium chloride

Incubate overnight at refrigeration temperature (~4°C)

Centrifuge at 14,000g for 10 min, 4oC

Dissolve pellet in 300 µl TE buffer + 450 µl 5M potassium acetate

Incubate for 5 hr on ice

Centrifuge at 14,000g for 10 min

Dissolve pellet in 300 µl TE buffer + 30 µl 3M NaCl + 660 µl ethanol

Incubate at -20°C for 1 hr

Centrifuge at 14,000g for 10 min, 4oC

Wash pellet with 400 µl 70% ethanol and Centrifuge at 14,000g for 10 min

Dry the pellet by keeping slant at 45 degree and dissolve in 300 µl DEPC treated water

Clarify at 12,000g for 5 min and transfer the total RNA (supernatant) to new tube

Figure 3.2. Levi et al. (1992) method for isolation of total RNA from blackgram.

Production of rabbit polyclonal antibody (pAb)

Two New Zealand white female rabbits were immunized each with whole blackgram protein extract (500g) in 0.5 ml RIBI adjuvant as described by Acosta et al. (1999). Three booster doses were administered in RIBI adjuvant each at 4 week intervals. Each rabbit was subsequently bled and the serum was collected and stored at -20oC until further use. Pre immune serum was collected to serve as control when determining the antibody titer.

cDNA library screening using blackgram pAB cDNA library was screened using rabbit polyclonal antibody as per procedure of Sambrook and Russell (2001). Luria-Bertani (LB) agar plates (150 mm and 90 mm) were prepared in house from autoclaved LB media and stored at 4oC until further use. cDNA library was diluted suitably to plate 0.5 x 104 to 5 x 104 plaques on 150 mm LB agar plates. Each plate was incubated for 3.5 hours at 42oC. 10 mM IPTG soaked labeled nitrocellulose membranes were overlaid on the agar plate and plates are further incubated at 37oC for 4 hours. Membranes were marked on the plate using needle (to trace back position of membrane/plaques on the plate) and carefully transferred to Tris-buffered saline (TBS-T; 10 mM Tris, 0.9% w/v NaCl, 0.05% v/v Tween 20, pH 7.6). Plates were stored in 4oC till results from membrane were obtained. Membranes were carefully washed using TBS-T buffer and blocked using 5% NFDM. Positive plaques were screened using Western blot method using rabbit anti-blackgram polyclonal antibodies. Positive plaques were traced back on the plate (by matching needle marks on the membrane and on plate), picked using sterile pipette tips and transferred to 1ml SM buffer. The positive plaques were further screened in similar manner till all the plaques were positive on the plate. Positive insert was obtained from Uni-Zap XR vector according to the manufacturer’s protocol. The insert was cloned into pCR 2.1-TOPO (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and plasmid purified using QIAprep spin plasmid miniprep kit (Qiagen Inc., Valencia, CA) for sequencing purpose.

cDNA library screening using PCR

Specific primers were designed using conserved sequence of Vigna species. Known sequences of Vigna radiata and Vigna unguiculata were used to determine “GAGAACAACCAGAGGAACTTCC” forward primer (BgVf) and “GGAAGTTCCTCTGGTTGTTCTC” reverse primer (BgVf-r). BgVf, BgVf-r, M13 forward and M13 reverse primers were used in combinations to PCR amplify target genes. Amplified PCR product was purified, sequenced and new primers were designed until complete sequence (containing start codon “AUG” and stop codon) was obtained. Sequence was confirmed by designing new primers from non-coding region and using high fidelity PCR to avoid any mutations in PCR. Two identified sequences (BGV-1) and (BGV-2) were amplified using PCR and cloned into pCR 2.1-TOPO (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and plasmid purified using QIAprep spin plasmid miniprep kit (Qiagen Inc., Valencia, CA) for sequencing purpose.

Nucleotide sequencing and analysis of cDNA

The plasmid cDNAs were sequenced from both directions on an ABI 3100 Genetic Analyzer (Foster City, CA) by using capillary electrophoresis and Version 2 Big Dye Terminators, as described by the manufacturer. Similarity searches for deduced nucleotide and amino acid sequences were performed by using BLAST program accessible at the website of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/BLAST/). Signal peptide cleavage site was predicted by using SignalP 3.0 server (www.cbs.dtu.dk/services/SignalP/). The multiple sequence alignment of the nucleotide and amino acid sequences were performed using the ClustalW program accessible at the website of European Bioinformatics Institute (www.ebi.ac.uk/clustalw/).

CHAPTER 4

RESULTS AND DISCUSSION

Part A: Functional Properties of Select Seed Flours

Proximate Composition:

Protein content (as-is basis) in tree nut full fat flours 12.53% to 22.45%, legumes 13.41% to 19.89%, oil seeds 24.21% to 29.05% and cereals 6.78% to 15.44%. Protein content on dry weight basis is reported in Table 4.2 (full fat flours) and Table 4.4 (defatted flours). Tree nuts and oil seeds have higher percentage of protein content. Higher protein content with higher fat content than other seed groups (42.12% to 66.71%) makes tree nuts a high calorie food. Legumes have low fat content (1.67% to 3.59%) with good amount proteins and carbohydrates which makes them regular protein source for vast population. Cereals are high in carbohydrates (71.45% to 78.21%) with low in fat and proteins making them staple food for a large population of the world.

Table 4.1. Proximate composition of full fat floursa (as-is basis) Full fat flours Sample Moisture (%) Protein (%) Lipid (%) Ash (%) Carbohydrate (%) Almond 3.40 ± 0.01 22.45 ± 0.31 45.07 ± 0.63 2.14 ± 0.07 26.85 ± 0.57 Brazil Nut 3.42 ± 0.08 13.43 ± 0.11 64.66 ± 0.41 3.04 ± 0.06 15.34 ± 0.92 Cashew 4.72 ±0.019 18.63 ± 0.65 42.12 ± 0.60 2.14 ± 0.07 32.73 ± 0.44 Hazel 3.08 ± 0.03 14.09 ± 0.95 61.85 ± 0.62 1.20 ± 0.07 19.65 ± 0.22 Macadamia 1.48 ± 0.08 12.53 ± 0.58 66.71 ± 0.41 1.25 ± 0.07 18.11 ± 0.85 Pecan 1.84 ± 0.05 15.32 ± 0.68 62.78 ± 0.15 1.15 ± 0.07 18.87 ± 0.62 Pistachio 3.25 ± 0.03 18.08 ± 0.19 44.56 ± 0.38 1.05 ± 0.21 33.03 ± 0.08 Walnut 1.27 ± 0.36 18.62 ± 0.06 65.56 ± 0.40 1.65 ± 0.07 12.70 ± 0.08 Blackgram 9.17 ± 0.05 19.89 ± 0.59 1.67 ± 0.23 1.15 ± 0.21 68.24 ± 0.69 Chickpea 8.24 ± 0.26 13.41 ± 0.05 3.59 ± 0.26 1.50 ± 0.14 73.07 ± 0.10 Peanut 4.79 ± 0.16 24.21 ± 0.08 40.66 ± 0.25 2.35 ± 0.20 28.21 ± 0.33 Soybean 7.71 ± 0.04 29.05 ± 0.69 4.57 ± 0.26 2.39 ± 0.01 56.41 ± 0.62 Sesame 3.93 ± 0.05 13.46 ± 0.05 49.26 ± 0.28 3.34 ± 0.22 29.97 ± 0.34 Wheat 12.84 ± 0.07 15.44 ± 0.42 1.15 ± 0.15 1.80 ± 0.13 78.21 ± 0.71 Rice 8.94 ± 0.02 6.78 ± 0.62 2.30 ± 0.31 1.05 ± 0.06 71.45 ± 0.90 Millet 8.73 ± 0.07 12.63 ± 0.69 3.63 ± 0.35 1.85 ± 0.07 72.92 ± 0.59 LSDb 0.93 1.08 0.28 0.51 1.22 a All values are expressed in gram per 100 gram (as-is basis), and the data reported is mean ± standard deviation (n=2) b Differences between means within the same column exceeding the LSD value are significant(p=0.05).

As expected, protein and carbohydrates content is higher in defatted flours as compared to their full fat counterparts. Increase in protein content of defatted flours is directly proportional to original fat content of the full fat flours. All the defatted flours had more than 50% carbohydrates (dwb) and 14% (dwb) proteins (except rice is 7.22% protein).

Table 4.2. Proximate composition of full fat seed floursa (dry weight basis) Sample Moisture (%) Protein (%) Lipid (%) Ash (%) Carbohydrates (%) Almond 3.52 ± 0.03 23.25 ± 0.34 46.75 ± 0.87 2.21 ± 0.07 27.79 ± 0.60 Brazil Nut 3.60 ± 0.19 13.91 ± 0.14 67.05 ± 0.71 3.14 ± 0.07 15.89 ± 0.92 Cashew 4.95 ± 0.05 19.55 ± 0.69 43.85 ± 0.17 2.25 ± 0.07 34.35 ± 0.44 Hazel 3.16 ± 0.08 14.54 ± 0.97 63.96 ± 0.87 1.24 ± 0.14 20.27 ± 0.24 Macadamia 1.53 ± 0.20 12.72 ± 0.56 67.63 ± 0.4 1.26 ± 0.07 18.39 ± 0.90 Pecan 1.85 ± 0.11 15.6 ± 0.71 64.02 ± 0.04 1.17 ± 0.06 19.21 ± 0.61 Pistachio 3.34 ± 0.05 18.69 ± 0.21 46.1 ± 0.52 1.08 ± 0.21 34.13 ± 0.10 Walnut 1.55 ± 0.63 18.91 ± 0.05 66.52 ± 0.14 1.68 ± 0.08 12.89 ± 0.00 Blackgram 10.07 ± 0.13 21.9 ± 0.67 1.73 ± 0.24 1.27 ± 0.23 75.11 ± 0.68 Chickpea 9.15 ± 0.62 14.63 ± 0.14 3.98 ± 0.35 1.63 ± 0.14 79.75 ± 0.35 Peanut 4.96 ± 0.40 25.41 ± 0.18 42.53 ± 0.26 2.46 ± 0.21 29.60 ± 0.24 Soybean 8.39 ± 0.07 31.48 ± 0.76 4.80 ± 0.13 2.59 ± 0.01 61.14 ± 0.63 Sesame 4.11 ± 0.13 14.01 ± 0.08 51.31 ± 0.47 3.48 ± 0.23 31.20 ± 0.31 Wheat 14.8 ± 0.14 7.79 ± 0.72 1.18 ± 0.00 1.20 ± 0.07 89.83 ± 0.65 Rice 9.81 ± 0.03 16.95 ± 0.46 2.62 ± 0.41 1.98 ± 0.14 78.45 ± 1.01 Millet 9.61 ± 0.17 13.84 ± 0.74 4.2 ± 0.05 2.03 ± 0.07 79.92 ± 0.77 LSD 0.93 1.08 0.28 0.51 1.22 a All values are expressed in gram per 100 gram (dry weight basis), and the data reported is mean ± standard deviation (n=2) b Differences between means within the same column exceeding the LSD value are significant (p=0.05).

Table 4.3. Proximate composition of defatted seed floursa (as-is basis) Defatted Flours Sample Moisture (%) Protein (%) Ash (%) Carbohydrate (%) Almond 5.81 ± 0.0484 30.03 ± 0.15 6.13 ± 0.04 58.04 ± 0.09 Brazil Nut 5.72 ± 0.16 34.19 ± 0.12 6.83 ± 0.23 53.40 ± 0.45 Cashew 6.41 ± 0.11 31.48 ± 0.04 3.69 ± 0.15 58.41 ± 0.23 Hazel 5.14 ± 0.04 35.18 ± 0.09 2.24 ± 0.06 57.41 ± 0.02 Macadamia 8.55 ± 0.08 22.17 ± 1.00 3.04 ± 0.22 66.32 ± 0.70 Pecan 5.12 ± 0.16 20.39 ± 0.09 4.54 ± 0.06 69.85 ± 0.28 Pistachio 5.68 ± 0.15 29.97 ± 0.74 2.05 ± 0.08 62.51 ± 0.98 Walnut 9.37 ± 0.16 34.84 ± 0.05 4.79 ± 0.15 51.14 ± 0.00 Blackgram 11.39 ± 0.01 20.71 ± 0.13 1.54 ± 0.07 66.38 ± 0.06 Chickpea 10.50 ± 0.03 14.79 ± 0.06 2.20 ± 0.13 72.49 ± 0.23 Peanut 8.19 ± 0.05 37.07 ± 0.24 3.65 ± 0.07 51.11 ± 0.06 Soybean 10.33 ± 0.04 32.07 ± 0.87 3.00 ± 0.14 54.60 ± 1.06 Sesame 7.18 ± 0.19 34.23 ± 0.14 5.74 ± 0.06 52.85 ± 0.13 Wheat 12.55 ± 0.00 18.28 ± 0.04 1.35 ± 0.07 78.89 ± 0.85 Rice 9.22 ± 0.05 7.22 ± 0.78 1.95 ± 0.07 70.56 ± 0.12 Millet 10.37 ± 0.21 15.39 ± 0.03 2.20 ± 0.0 72.04 ± 0.44

LSDb 0.93 0.25 0.40 1.06

a All values are expressed in gram per 100 gram (as-is basis), and the data reported is mean ± standard deviation (n=2) b Differences between means within the same column exceeding the LSD value are significant(p=0.05).

Table 4.4. Proximate composition of defatted seed floursa (dry weight basis) Sample Moisture (%) Protein (%) Ash (%) Carbohydrates (%) Almond 6.17 ± 0.13 31.88 ± 0.13 6.5 ± 0.04 61.61 ± 0.17 Brazil Nut 5.9 ± 0.12 36.21 ± 0.17 7.23 ± 0.25 56.55 ± 0.42 Cashew 6.86 ± 0.21 33.64 ± 0.02 3.95 ± 0.17 62.41 ± 0.19 Hazel 5.45 ± 0.06 37.1 ± 0.07 2.36 ± 0.06 60.53 ± 0.01 Macadamia 9.26 ± 0.11 24.22 ± 1.07 3.32 ± 0.24 72.46 ± 0.84 Pecan 5.51 ± 0.34 21.51 ± 0.02 4.79 ± 0.08 73.70 ± 0.05 Pistachio 5.87 ± 0.18 31.66 ± 0.84 2.17 ± 0.09 66.17 ± 0.93 Walnut 10.16 ± 0.23 38.38 ± 0.03 5.28 ± 0.15 56.34 ± 0.12 Blackgram 12.84 ± 0 23.36 ± 0.15 1.74 ± 0.08 74.90 ± 0.07 Chickpea 11.77 ± 0.05 16.53 ± 0.07 2.45 ± 0.15 81.02 ± 0.22 Peanut 8.89 ± 0.13 40.37 ± 0.21 3.97 ± 0.08 55.65 ± 0.13 Soybean 11.51 ± 0.08 35.76 ± 0.99 3.35 ± 0.15 60.89 ± 1.14 Sesame 7.74 ± 0.39 36.88 ± 0.02 6.19 ± 0.05 56.93 ± 0.06 Wheat 14.35 ± 0 8.25 ± 0.88 1.54 ± 0.09 90.21 ± 0.97 Rice 10.15 ± 0.11 20.14 ± 0.03 2.15 ± 0.08 77.72 ± 0.05 Millet 11.57 ± 0.58 17.17 ± 0.06 2.45 ± 0.01 80.37 ± 0.07 LSD 0.40 0.93 0.25 1.06 a All values are expressed in gram per 100 gram (dry weight basis), and the data reported is mean ± standard deviation (n=2) b Differences between means within the same column exceeding the LSD value are significant (p=0.05).

Bulk Density:

Blackgram and rice exhibited highest bulk densities. Brazil nut and walnut have highest bulk densities within tree nut flours, followed by pecan, cashew, pecan, macadamia, almond, pistachio, and hazel nut. Higher packing of particles in Brazil nut and walnut flours as compared to other tree nuts might be reason for difference in bulk density. On comparison of lipid content with bulk densities, it can be seen that there is inverse relation between fat content and bulk densities of full fat flours. More the fat content lower is the bulk density. Blackgram and rice with 1.67% and 1.15% lipid content respectively have highest bulk densities. Hazel nut had lowest bulk density closely followed by pistachio, almond, macadamia and peanut.

Table 4.5. Bulk Densities of full fat and defatted seed flours Sr. Bulk Densitya (g cm-3) Sample No. Full Fat Defatted 1 Almond 0.512 ± 0.016 0.331 ± 0.002 2 Brazil Nut 0.713 ± 0.003 0.234 ± 0.002 3 Cashew 0.536 ± 0.005 0.432 ± 0.002 4 Hazel Nut 0.466 ± 0.008 0.220 ± 0.006 5 Macadamia 0.515 ± 0.033 0.202 ± 0.005 6 Pecan 0.544 ± 0.029 0.179 ± 0.001 7 Pistachio 0.510 ± 0.014 0.289 ± 0.001 8 Walnut 0.701 ± 0.021 0.257 ± 0.001 9 Blackgram 0.930 ± 0.004 0.918 ± 0.011 10 Chickpea 0.776 ± 0.005 0.762 ± 0.006 11 Peanut 0.542 ± 0.024 0.343 ± 0.001 12 Soybean 0.629 ± 0.005 0.527 ± 0.009 13 Sesame 0.656 ± 0.025 0.315 ± 0.004 14 Rice 0.927 ± 0.005 0.913 ± 0.010 15 Wheat 0.796 ± 0.003 0.766 ± 0.006 16 Millet 0.872 ± 0.008 0.867 ± 0.008 LSDb 0.0003 0.0002

a All values are mean ± standard deviation (n=3) b Differences between means within the same column exceeding the LSD value are significant (p=0.05)

Yellow tigernut flour (32% lipid) and Brown tigernut (35% lipid) flour have bulk density of 0.62g cm-3 and 0.55 g cm-3 respectively (Oladele and Aina, 2007). Padilla et al. (1996) reported soybean bulk density of 0.5598 g cm-3 which is close to one reported in this study. Flour bulk densities have been studied previously including fluted pumpkin (0.2 g/ml) (Giami and Bekebain, 1992), yam flour (0.75 g/cm3) (Hsu et al., 2003), adzuki beans (0.62 g/ml), rice bean (0.59 g/ml) and Indian bean (0.64g/ml) (Chang and Cheung, 1998).

Bulk density is important for packaging, transport and warehousing purposes. More the bulk density of the product less is the space taken per unit weight, which further translates into money saving. Particle size plays an important role in the bulk density determination as indicated by Dench et al. (1981). Smaller particles increase bulk density by compact packing. Fat content in sample affects particles friction during grinding. Cereals create higher number of smaller particles during grinding due to reduced fat content. Lipids present in tree nuts or oil seeds provide cushion, avoiding friction between starch/protein particles during grinding process. Thus it is hard to produce fine tree nut flours and primary reason why tree nut meals are sold in the market today. Tree nut meals are typically expensive and difficult to obtain.

Defatting decreased bulk densities of all seed flours (0.5% for millet to 67.2% for Brazil nut). Percentage change in bulk densities have reported in the Table 4.5. Alobo et al. (2009) reported increase in densities after defatting of cashew flours (0.71 g cm-3 full fat and 0.78 g cm-3 defat). Ogunwulu et al. (2009) reported 0.48 g cm-3 bulk density of defatted cashew nut flour. Difference in bulk densities of Alobo et al. (2009) and the current study can be due to difference in the initial particle size and method of defatting. Alobo et al. (2009) had used hexane as solvent with 2 hours refluxing as compared to petroleum ether with 8 hours refluxing in the current study. Akpata and Akubor (1999) reported bulk densities of full fat and defat dehulled orange seed flours 0.51 g cm-3 and 0.40 g cm-3 respectively showing decrease in bulk densities after defatting. Future studies should be conducted to understand effect of fat on packing of the particles and effect of friction on packing during milling process.

Color:

L*(lightness), a*(red-green) and b*(yellow-blue) values of full fat and defat seed flours are reported in the Table 4.6. Full fat macadamia and cashew have highest L* values indicating lightest tree nut flours while pecan and walnut are darkest tree nut flours with low L* values (53.71 and 59.4 respectively). High tannin content in pecan and walnut (Venkatachalam and Sathe, 2006) may be the reason for darker shade. Xu et al. (2007) have reported significant negative co-relation between total phenolic content and L* value of the flour samples. Polished rice lacks color pigments which is evident by highest L* = 89.62. Positive a* values denotes red color of the sample. Full fat flours exhibited redness which was lost on defatting in few samples. Almond, Brazil nut, hazel nut, pecan and walnut had significant redness as compared to other tree nut flours. The tree nut seeds mentioned above were ground with seed coat which may have provided redness to the flour samples. Similar is the case with peanut, chickpea, soybean samples. For future it will be interesting to understand color difference between flours made with and without seed coat.

b* reading describe yellow-blueness of samples. Pistachio showed maximum yellow tone with b* value of 41.96. Pistachio contains chlorophyll a, chlorophyll b and lutein pigments in concentration of 38.68 mg/kg, 32.83mg/kg and 25.77mg/kg respectively (Bellomo et al., 2009). Combination of chlorophyll a (green pigment); chlorophyll b and lutein (both are yellow) produce pistachio’s color yellow-green which is evident from above values.

Colors of flours play essential role in the new product development process. Granato and Ellendersen (2009) have determined color as quality parameter for cookies prepared from almond and peanut (gluten-free products). These gluten-free cookies received great acceptance due to their color and texture (Granato and Ellendersen, 2009).

Table 4.6. Color (L*, a* and b*) readings of full fat and defatted seed floursa. Sr Full Fat Defatted Sample No L* a* b* L* a* b* 1 Almond 69.4 ± 0.88 5.31 ± 0.24 20.31 ± 0.29 87.01 ± 0.56 1.89 ± 0.09 10.78 ± 0.18 2 Brazil Nut 60.38 ± 0.67 3.50 ± 0.13 16.52 ± 0.15 88.39 ± 0.05 0.74 ± 0.01 9.22 ± 0.10 3 Cashew 75.36 ± 0.34 1.71 ± 0.13 25.07 ± 0.48 93.86 ± 0.24 -0.36 ± 0.01 6.58 ± 0.13 4 Hazel Nut 68.06 ± 0.27 4.70 ± 0.05 20.84 ± 0.08 84.84 ± 0.24 2.00 ± 0.04 10.68 ± 0.14 5 Macadamia 76.43 ± 0.07 0.70 ± 0.03 25.84 ± 0.03 90.64 ± 0.16 -0.25 ± 0.03 12.85 ± 0.09 6 Pecan 53.71 ± 0.59 6.42 ± 0.08 21.94 ± 0.24 86.03 ± 0.18 1.01 ± 0.03 9.84 ± 0.03 7 Pistachio 66.44 ± 0.11 0.98 ± 0.03 41.96 ± 0.26 90.02 ± 0.14 -1.67 ± 0.04 14.57 ± 0.15 8 Walnut 59.4 ± 0.40 4.70 ± 0.09 25.97 ± 0.30 82.47 ± 0.12 1.09 ± 0.04 15.73 ± 0.14 9 Blackgram 76.45 ± 0.27 0.11 ± 0.02 10.00 ± 0.15 74.45 ± 0.59 -0.01 ± 0.03 9.11 ± 0.09 10 Chickpea 86.38 ± 0.04 2.96 ± 0.06 19.87 ± 0.25 88.11 ±0.21 1.46 ± 0.05 14.71 ± 0.10 11 Peanut 71.32 ± 0.31 4.54 ± 0.04 19.63 ± 0.28 88.79 ± 0.04 0.96 ± 0.04 9.01 ± 0.06 12 Soybean 71.4 ±0.20 2.14 ± 0.10 22.51 ± 0.44 89.51 ± 0.21 0.17 ± 0.06 14.69 ± 0.10 13 Sesame 85.14 ± 0.38 1.37 ± 0.06 24.62 ± 0.12 91.72 ± 0.38 -0.21 ± 0.06 8.68 ± 0.24 14 Rice 89.62 ±0.30 0.05 ± 0.08 10.55 ± 0.19 90.04 ± 0.32 0.06 ± 0.08 9.13 ± 0.20 15 Wheat 83.78 ± 0.19 2.80 ± 0.14 14.75 ± 0.21 85.81 ± 0.14 2.27 ± 0.08 11.75 ± 0.11 16 Millet 84.67 ± 0.12 2.53 ± 0.10 25.98 ± 0.34 88.59 ± 0.20 1.73 ± 0.25 15.95 ± 1.58 LSDb 0.65 0.17 0.44 0.47 0.13 0.69 a All values are mean ± standard deviation (n=3). b Differences between means within the same column exceeding the LSD value are significant (p=0.05).

Defatting improved flour appearance as a result of removal of some coloring pigments. Chlorophyll, xanthophyll and carotenoids are fat soluble pigments. Fat soluble color pigments (chlorophyll, xanthophyll, and carotenoids) were extracted by Soxhlet extraction process. Similar trend is observed with a* values where few flours show faint green hue in them (negative values are close to zero). Mostly all a* values are close to zero since most red-green pigments in seeds are fat soluble. Yellow (positive b* values) color had been reduced in defatted flours as compared to full fat flours. Typically flours had exhibited yellow color tone. In food products involving emulsions or fat phase, all the color components from flour will be dissolved in lipid layer. In such products it will be important that lipid phase is evenly distributed throughout the product to get an even color.

Chroma and hue

Chroma is color saturation value of a sample. It is evident that defatting decreased color saturation for all flours (except blackgram). Flours lose their characteristic colors and become dull on defatting. Sensory analysis should be done to understand color preference of consumers for full fat and defatted flours. Full fat pistachio flour has highest chroma value due to higher content of chlorophyll and xanthophyll. Other tree nuts lack pigmentation the flesh of the seed. Xu et al. (2006) reported color, hue and chroma of various legumes. Hue and chroma of soybean is -1.5 radians and 17.3 respectively while that of chickpea is -1.5 radians and 19.7 respectively which is in comparison with current investigation.

Table 4.7. Chroma and hue angle of full fat and defatted seed floursa Full Fat Defatted Seed Flour Chroma Hue Angle (o) Chroma Hue Angle (o) Almond 21.00 ± 0.34 75.35 ± 0.46 10.95 ± 0.19 80.04 ± 0.27 Brazil Nut 16.88 ±0.15 78.04 ± 0.42 9.25 ± 0.10 85.41 ± 0.08 Cashew 25.13 ± 0.49 86.11 ± 0.23 6.59 ± 0.13 -86.87 ± 0.15 Hazel Nut 21.36 ± 0.09 77.29 ± 0.10 10.86 ± 0.14 79.41 ± 0.14 Macadamia 25.85 ± 0.03 88.44 ± 0.06 12.86 ± 0.09 -88.87 ± 0.12 Pecan 22.86 ± 0.25 73.68 ± 0.06 9.89 ± 0.03 84.12 ± 0.18 Pistachio 41.97 ± 0.26 88.66 ± 0.05 14.67 ± 0.15 -83.46 ± 0.11 Walnut 26.39 ± 0.30 79.75 ± 0.20 15.77 ± 0.14 86.03 ± 0.12 Blackgram 10.00 ± 0.15 89.35 ± 0.10 9.11 ± 0.09 -89.84 ± 0.05 Chickpea 20.09 ± 0.25 81.52 ± 0.06 14.78 ± 0.10 84.34 ± 0.16 Peanut 20.14 ± 0.28 76.98 ± 0.11 9.06 ± 0.06 83.94 ± 0.28 Soybean 22.61 ± 0.45 84.57 ± 0.14 14.69 ± 0.10 89.32 ± 0.24 Sesame 24.66 ± 0.12 86.82 ± 0.12 8.69 ± 0.24 -88.59 ± 0.39 Rice 10.55 ± 0.19 89.52 ± 0.08 9.13 ± 0.19 89.34 ± 0.24 Wheat 15.01 ± 0.23 79.24 ± 0.37 11.97 ± 0.12 79.07 ± 0.28 Millet 26.10 ± 0.35 84.45 ± 0.18 16.05 ± 1.60 83.82 ± 0.28 LSD 0.45 0.36 1.06 0.34 a All values are mean ± standard deviation (n=3). b Differences between means within the same column exceeding the LSD value are significant (p=0.05).

Hue of cookies made with combinations of wheat, chickpea and fonio flours has been reported by McWatters et al. (2003). Hue angle close to 75 is brown while that close to 90 is yellow. Most of the full fat flours had brown to yellow hue except rice which showed red. Defatting did not change hue angle for most of the samples. Defatted cashew, macadamia, pistachio and sesame revealed negative hue angle values indicating blue hue. Defatted blackgram flour showed hue angle -29 which indicates pink hue. Hue angle values with different shades is exhibited in figure 4.1.

Figure 4.1. Hue angle explaining various shades in the sample (Adopted from www.wikipedia.com)

Oil Holding Capacity: Almond closely followed by hazel nut had the highest oil holding capacities in full fat flour samples while pecan closely followed by macadamia had highest oil holding in defatted flours. Among other full fat flours blackgram showed the lowest oil holding while millet and blackgram showed lowest oil holding in defatted flours. Tree nuts, peanut and sesame full fat flours held more oil than their own weight. Remaining full fat flours demonstrated oil holding less than 1. Plant proteins typically have oil holding capacity ~5g oil/g sample (Sathe, 2002), which is true for most of the samples studied. Defatting demonstrated improved oil holding capacity. Defatting increased percentage protein in the sample (refer Table 4.1.) which translates to more oil holding capacity per unit weight of defatted flours. Oil holding of proteins depends on surface properties like surface area, hydrphobicity, electrical charge distribution etc. Defatting exhibited as low as 14% (millet) to 181% (macadamia) increase in oil holing capacity.

Table 4.8. Oil Holding Capacity of full fat and defatted seed floursa. Sr. OHC Seed Flour No. Full Fat Defatted 1 Almond 1.386 ± 0.037 2.188 ± 0.026 2 Brazil Nut 0.993 ± 0.115 2.170 ± 0.077 3 Cashew 0.909 ± 0.039 2.270 ± 0.062 4 Hazel Nut 1.350 ± 0.025 1.530 ± 0.036 5 Macadamia 1.106 ± 0.061 3.113 ± 0.243 6 Pecan 1.229 ± 0.151 3.151 ± 0.051 7 Pistachio 1.012 ± 0.028 2.121 ± 0.048 8 Walnut 1.176 ± 0.238 1.987 ± 0.011 9 Blackgram 0.582 ± 0.740 0.913 ± 0.072 10 Chickpea 0.858 ± 0.005 1.064 ± 0.045 11 Peanut 0.935 ± 0.018 1.989 ± 0.007 12 Soybean 1.137 ± 0.051 1.442 ± 0.058 13 Sesame 1.121 ± 0.018 2.159 ± 0.015 14 Rice 0.654 ± 0.004 0.959 ± 0.032 15 Wheat 0.955 ± 0.063 1.146 ± 0.079 16 Millet 0.787 ± 0.002 0.899 ± 0.063 LSD 0.17 0.22 a All values are (as-is basis) mean ± standard deviation (n=3) b Differences between means within the same column exceeding the LSD value are significant (p=0.05).

Oil absorption is essential for food industry standpoint. Various rice flours were applied as partial replacement for wheat flour in donuts to reduce oil uptake (Shih et al., 2006). Study showed that by replacing wheat (control) with 50% gelatinized rice flour oil uptake of donut decreased from ~26% to ~12% (oil uptake on dry basis minus original donut oil content 3.47%). Further studies with flours such as millets, chickpea and blackgram should be undertaken to find potential application with lowered oil uptake. Dough batters used in deep frying of food products like onion rings can be replaced by millet, chickpea or rice flours to reduce fat content. Chickpea, blackgram, rice and similar flours can be partially or completely replaced for wheat flour in oil fried products to reduce oil uptake and increase protein content, making a healthy and nutritious product. Flours such as chickpea and blackgram will not only provide reduced oil absorption but also increased protein content providing added health benefits. Flours with high oil absorption capacity (e.g. almond) may be suitable for food products that require high oil holding capacity (e.g. cakes) with ability to retain moisture as such flours are suitable emulsion formations. 31

Water Holding Capacity:

Table 4.9. Water holding capacity of full fat and defatted seed flours. Sr WHC Seed Flour No. Full Fat Defatted 1 Almond 1.438 ± 0.031 2.075 ± 0.046 2 Brazil Nut 0.673 ± 0.005 1.846 ± 0.019 3 Cashew 0.919 ± 0.009 1.986 ± 0.010 4 Hazel Nut 1.082 ± 0.019 1.831 ± 0.028 5 Macadamia 0.647 ± 0.065 1.448 ± 0.021 6 Pecan 1.167 ± 0.133 2.293 ± 0.155 7 Pistachio 1.403 ± 0.017 1.964 ± 0.018 8 Walnut 1.238 ± 0.025 2.221 ± 0.041 9 Blackgram 2.478 ± 0.029 2.354 ± 0.042 10 Chickpea 2.106 ± 0.038 2.274 ± 0.072 11 Peanut 1.067 ± 0.159 1.864 ± 0.004 12 Soybean 2.740 ± 0.009 3.169 ± 0.018 13 Sesame 1.527 ± 0.044 2.550 ± 0.015 14 Rice 1.094 ± 0.028 1.346 ± 0.004 15 Wheat 1.688 ± 0.097 1.746 ± 0.004 16 Millet 1.285 ± 0.069 1.330 ± 0.004 LSD 0.19 0.07 a All values are (as-is basis) mean ± standard deviation (n=3) b Differences between means within the same column exceeding the LSD value are significant (p=0.05).

Fats avoid wetting of flour which is evident by Water holding capacity (WHC, g water per g sample) of full fat tree nut and oil seed flours. WHC of full fat tree nut and oil seed flours is lower than defatted flours, legumes and cereals. Soybean flour (defatted and full fat) had the highest WHC followed by legumes. Macadamia full fat flour holds the least amount of water. Amount of lipid in the sample is not directly proportional to WHC e.g. hazel nut with almost same lipid content as Brazil nut had greater WHC. This indicates that non-fat components play essential role in WHC. Almond had comparable WHC to that of wheat flour which possibly explains replacement of almond flour to wheat flour in bread making process. WHC of various flours have reported before including, cashew flour (full fat flour 2.4g/g and defatted 3.2g/g) (Alobo et al., 2009), pulse protein concentrate (0.6 – 2.7 g/g) (Boye et al., 2010), broadbean

(flour 3.04g/g; isolate 2.55g/g) (Vioque et al., 2012), soy protein isolate (4.4g/g), blackgram protein concentrate (5.9g/g) (Abbey and Ibeh, 1987), Chickpea (1.28g/g) (Han and Khan, 1990) , pinto bean flour (1.53g/g) (Han and Khan, 1990), green mung bean flour (2.1 g/g) (Dzudie and Hardy, 1996), Great northernbean (1.77g/g) (Deshpande et al., 1982), defatted peanut flour, (~2.3 g/g) (Wu et al,. 2009), peanut flour (1.67 g/g) (Yu et al., 2007). A comprehensive list of bean WHC has been compiled in Sathe (2002). WHC of each oat protein is studied in detail by (Mohammed et al., 2009). Reported WHC of chickpea and blackgram are lower than that reported here.

WHC is essential in food products like bread, cookies where water is essential in staling or crust formation during processing. For example soy can be added in gluten-free breads (rice, corn, almond flour bread etc.) to increase water holding capacity helping improved crumb softening and retarded staling (Belitz and Grosch 1987). Isanga and Zhang (2009) exhibited increased WHC of peanut milk yogurt than cow milk yogurt. WHC is affected by certain intrinsic factors like amino acid composition, protein conformation and surface polarity/hydrophobicity (Barbut, 1999). Processing showed to have less effect on WHC of variety of the pulse samples (Boye et al., 2010). Wu et al. (2009) reported strong acid or alkali extraction reduced WHC while alcohol leaching increased WHC of peanut protein isolates. According to authors acid or alkali might have denatured proteins to expose more hydrophobic groups while alcohol’s effect is complex through diverse mechanisms. Effect of processing on peanut flours was studied by Yu et al. (2007). Roasting peanut flour decreased WHC from 1.67 g/g to 1.00 g/g while fermentation increased WHC from 1.67 g/g to 2.25 g/g. Heat denaturation of proteins during roasting to expose hydrophobic areas explains reduced WHC. Fermentation creates soluble oligopeptides by proteolysis increasing WHC. Further research should be done with group of flours at same setting for understanding the effect of heat and other food processing methods on water holding capacity.

Least Gelation Concentration:

Figure 4.2. Least Gelation Concentration of full fat and defatted flours.

Cashew full fat flour formed gel at least flour concentration (8%) (LGC, % (w/v)) while pecan, macadamia, rice and millet all formed get at 18% (w/v). LGCs for several seed proteins are comparable to reported in the current study. Some examples of reported LGCs include Great Northern bean protein concentrate (8%) and isolate (12%) (Leggett, 2008), lupin protein concentrate (8%) (Sathe et al., 1982), winged bean protein concentrate (14%) (Sathe et al., 1982), pumpkin (8%) (Lazos, 1992), quinoa (16%) (Ogungbenle, 2003), adzuki bean flour (12%) (Chau and Cheung, 1998), rice bean flour (13%) (Chau and Cheung, 1998), soybean (17%) (Chau and Cheung, 1998), chickpea flour (8-12%) (Kaur and Singh , 2005), cowpea (16%) (Abbey and Ibeh, 1988), cowpea protein isolate (6%) (Ragab et al., 2004), African locust bean (4-18%) (Lawal et al., 2005), jack bean proteins (4-12%) (Lawal, 2009), sunflower proteins (10%) (Gonzalez-Perez and Vereijken, 2007), and canola (14.9-15.7%), soybean (9.7-11%), and 34

flaxseed (8.5-9.7%) meals (Khattab and Arntfield, 2009). Non protein components are important in gelation process. Yusuf (2003) reported LGC for almond protein concentrate (89.95% protein, 3.15% carbohydrates) to be 25% compared to LGC of 14% for almond flour (30.13% protein, 47.70% fat, and 9.63% carbohydrates). Mulvihihill and Kinsella (1987) reported that gel strength is improved with increased protein concentrations.

Except cereal and legume flours all flours had decrease in LGC after defatting. This is not in agreement with Akpata and Akubor (1999) who reported that defatting increased LGC due to denaturation in orange seed proteins. Proteins in orange oil seeds might have denatured due to sun drying and air oven drying applied after defatting process. In the current investigation, flours were not heated during or after defatting which may explain the difference. Alobo et al. (2009) reported LGCs of cashew full fat (14%) and defat (10%) which is slightly higher than reported in this study. Difference in raw material, mesh size and method of defatting may have led to variation in LGC values. Alobo et al. (2009) used smaller mesh size and defatting was done using n-hexane for 2 hours as compared to 8 hours with petroleum ether in the current study. It can be concluded that there is no direct relation in defatting and LGC as in some studies LGC increased after defatting while in other LGC decreased after defatting. LGC is important in food products requiring thickening and gelling such as sauces and puddings.

Part B: Isolation of Blackgram Vicilin gene.

Total RNA extraction:

Extraction of total RNA using methods of TRIzol (Invitrogen, Carlsbad, CA), Ding et al. (2008), Ince and Karaka (2009) from blackgram was unsuccessful. Extraction method reported by Levi et al. (1992) for pecan was successful on blackgram. Mung bean (Vigna mungo) and blackgram belong to same family still method successful with mung bean was not successful with blackgram. Further research should be done to understand difficulty in total RNA extraction from blackgram.

Figure 4.3. Total RNA extracted from blackgram seeds. S – DNA standard marker. I – Negative control. II- Blackgram total RNA.

Polyclonal antibody screening

Primary screening of the blackgram cDNA library resulted in few positive plaques as shown in figure 4.4. Selected positive plaques were plated again to undergo secondary screening.

Figure 4.4. Primary screening of blackgram cDNA library showing positive plaques circled and numbered 1 to 10.

Plaques with strong signal were selected and screened in similar manner until all plaques gave a positive signal. Figure 4.6 demonstrates tertiary screening where all the plaques were positive confirming presence of a desirable insert. Two plaques were selected at end of tertiary screening to undergo pBluescript phagemid extraction from uni-Zap XR vector. The separated phagemid were cloned into TA vector for DNA sequencing.

Figure 4.5. Tertiary screening of a plaque showing all positive plaques.

Results of both inserts were same indicating presence of similar inserts. The gene sequence is indicated in Figure 4.6.

Figure 4.6. Blackgram vicilin (BGV-3) DNA Sequence.

ProtParam tool was utilized to analyze BGV-3 amino acid sequence (http://web.expasy.org/protparam/ accessed on Feb 15 2012). BGV-3 contains 445 amino acids with theoretical molecular weight of 51020.3 Da and theoretical of pI 5.58. BGV-3 contains signal peptide with 25 amino acids suggesting 420 amino acids in mature blackgram vicilin with theoretical molecular weight of ~48.30 kDa. Chavan and Djurtoft (1982) reported 3 subunits of blackgram vicilin (64.5kDa, 55 kDa and 50 kDa). BGV theoretical weight is close to 50kDa subunit mentioned by Chavan and Djurtoft (1982). Amino acid sequence of BGV showed absence of amino acid cysteine indicating absence of di sulfide linkage. Globulins are major proteins in blackgram (81%, Padhye and Salunkhe, 1977; 63%, Mahajan et al. (1988)). Mahajan et al. (1988) further reported that legumin to vicilin ratio is 4:1 in globulins of blackgram. Mung beans (Vigna mungo) consists of three storage proteins basic 7S, vicilin type (8S) and legumin type (11S) at 3.4%, 89% and 7.6% level (Tecson-Mendoza et al., 2001).

MMRARIPLLLLLGILFLASLSVSFG*IREQHESQDVSVSRGKNNPFYFNSDRWFRTL FRNQFGHLRVLQRFDQRSNQLQNLENYRVVEFQSKPNTLLLPHHADADFLLVVLNGR AVLTLVNPDGRDSYILEQGHAQKIPAGTIFFLVNPDDNENLRIIKLAVPVNNPHRFQ DFFLSSTEAQQSYLQGFSKNILEASFDSDIKEINRVLFGEEGQQQQQGQESQQEGVI VELKREQIRELTKHAKSSSKKSLSSEDEPFNLRNQKPIYSNKFGEFYEITPKKNPQL RDLDVFLSYVDIKEGSLLLPHYNSKAIVILVINEGKANIELVGLKEEQQQQQQQDER LEVQRYRAEVSEHDVFVIPAGHPVAIDATSNLNFFAFGINAENNQRNFLAGEKDNVI SEIPTEVLDLAFPAPGEKVEKLIEKQSRSHFVDAQPEEQQNRGHSTE- Figure 4.7. BGV-3 amino acid sequence. * shows location of signal peptide.

BGV-3’s similarity with other genes was compared using BLAST (Basic Local Alignment Search Tool) tool on National Center for Biotechnology Information website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). BGV-3 demonstrated more than 90% coverage and more than 57% identity with various seed storage proteins, some of which are reported in Table 4.10. The sequence is similar to vicilin proteins from mung bean, cowpeas, adzuki beans, lentil, soybean and peas etc.

Table 4.10. Sequence comparison of BGV-3 with other vicilins. Source Accission No. % Coverage % Identity

Vigna radiata ABW23574.1 98% 88%

Vigna angularis BAF56571.1 99% 83%

Vigna luteola AAZ06661.1 98% 81%

Vigna unguiculata CAP19902.1 96% 84%

Glycin max BAE44299.1 96% 68%

Lens culinaris CAD87731.1 93% 57%

Len c 1 (accession number CAD87731.1) is reported a major allergen from lentil seeds (Lopez- Torrejon et al., 2003). Len c1 exhibited 93% coverage and 57% identity with BGV-3 suggesting BGV-3 can be a potential allergen. Kumari et al. (2006) studied blackgram extract for hypersensitivity I reactions using serum from patients sensitive to blackgram. They reported that raw blackgram extract showed 14 reactive polypeptides. 47kDa polypeptide which is closer in weight to BGV-3 also showed the reactivity. Processing (roasting and boiling) did not affect immunoreactivity of this band (Kumari et al., 2006). BGV-3 can be one of immune reactive protein. Further research should be done to understand immunoreactivity of BGV-3.

cDNA library Screening using PCR:

Primers were designed using conserved sequence from Vigna radiata, Glycin max and other vicilins. Primers BgV-f - “GAGAACAACCAGAGGAACTTC C” and its complement BgVf-r - “GGAAGTTCCTCTGGTTGTTCTC” were designed by comparing 38 sequences from NCBI database. M13 forward, M13 reverse with designed primers were used to perform PCR using cDNA library as template (figure 4.11).

Figure 4.8. PCR products on 1% agarose gel. Lane 1- Marker; Lane -2; Primers BgVf-r and M13 reverse; Lane 3- Primers BgVf and M13 forward. Blackgram cDNA library was used as template

Comparing location of conserved region in V. radiata it was estimated that conserved region in blackgram will be located towards 3’ side of the gene (Refer figure 4.10). Two distinct bands observed in lane 2 (11 and 12) are made up of nucleotide between conserved region and 3’ end of vicilin gene. Two distinct bands showed possibility of isomers of blackgram vicilin gene. More primers were designed from the gene sequence obtained from bands 11, 12 and 2.

Figure 4.9. Schematic representation of band 11, 12 and 2.

It was assumed that 11 and 12 are two different 3’ ends of blackgram vicilin isomers with same (band 2) 5’ side. Forward primer F1 (designed from band 2) produced 2 different PCR products with reverse primer 11R2 (designed from band 11) and 12R2 (designed from band 12) as seen in figure 4.11.

Figure 4.10. 1% Agarose gel showing PCR products. M – Marker; Lane 1- Primers F1 and 11R2; Lane 2 – Primers F1 and 12R2. Blackgram cDNA library was used as template.

It can be seen that as assumed PCR product from lane 1 (BGV-1) contains more base pairs than that from lane 2 (BGV-2). These PCR products were sequenced and new primers were designed 42

from non-coding region of both sequences. Hi fidelity PCR was performed to find complete sequence of BGV-1 and BGV-2. Complete amino acid sequence of BGV-1 and BGV-2 is shown in figure 4.11.

>BGV-1 MMRARIPLLLLLGILFLASLSVSFG*IREQHESQDVSVSRGKNNPFYFNSDRWFRTLFRN QFGHLRVLQRFDQRSNQLRNLENYRVVEFQSKPDTLLLPHHADADFLLVVLNGRAVLT LVNPDGRDSYILEQGHAQKIPAGTIFFLVNPDDNENLRIIKLAVPVNNPHRFQDFFLSSTE AQQSYLQGFSKNILEASFDSDIKEINRVLFGEEGQQQKGEESQQEGVIVELKREQIRELTK HAKSSSKKSLSSEDEPFNLRNQKPVYSNRFGRLHEITPEKNPQLRDLDVFLSYVDIKEGG LLIPSYNSKATVILVVNEGEANIELVGLKQQQQQQQQSWEVQRYSAELSEDDVFVIPAA YPVAINATSNLNFLAFGINAKNNQRNFLAGEKDNVISEIPNQVLEVAFPGSGEKVVKLIN KQSLSYFVDAQSQQKEKQSKGRKDPLSSILDTLH

>BGV-2 MMRARIPLLLLLGILFLASLSVSFG*IREQHEGQDVSVSRGKNNPFYFNSDRWFRTLFRN QFGHLRVLQRFDQRSNQLQNLENYRVVEFQSKPNTLLLPHHADADFLLVVLNGRAVLT LVNPDGRDSYILEQGHAQKIPAGTIFFLVNPDDNENLRIIKLAVPVNNPHRFQDFFLSSTE AQQSYLQGFSKNILEASFDSDIKEINRVLFGEEGQQQQQGQESQQEGVIVELKREQIRELT KHAKSSSKKSLSSEDEPFNLRNQKPIYSNKFGEFYEITPKKNPQLRDLDVFLSYVDIKEGS LLLPHYNSKAIVILVINEGKANIELVGLKEEQQRQQQQDERLEVQRYRAEVSEHDVFVIP AGHPVAIDATSNLNFFAFGINAENNQRNFLAGEKDNVISEIPTEVLDLAFPAPGEKVEKLI EKQSRSHFVDAQPEEQQNRGHSTE Figure 4.11. Amino acid sequence of BGV-1 and BGV-2. * indicates location of signal peptide.

BGV-1 and BGV-2 both have 25 amino acid signal peptide (indicated by * in figure 4.11). Properties of BGV-1 and BGV-2 are summarized in Table 4.11.

Table 4.11. Summary of BGV-1 and BGV-2. Mol Wt Sr. # of # of amino Signal Peptide Name (excluding pI No. nucleotides acids cleavage site signal peptide) 1 BGV-1 1356 452 25th and 26th 48.77 kDa 5.94 2 BGV-2 1335 445 25th and 26th 48.29 kDa 5.50

BGV-1 and BGV-2 showed more than 90% sequence identity with the vicilin genes of mung beans and more that 60% identity with vicilins of other beans such as adzuki beans, cowpeas etc. Figure 4.11 shows that BGV and BGV-2 are almost same except difference in an amino acid at the location 32. Glycine in BGV-2 is replaced by a serine at BGV producing change in theoretical pI and total molecular weight of the protein.

BGV2 MMRARIPLLLLLGILFLASLSVSFGIREQHEGQDVSVSRGKNNPFYFNSDRWFRTLFRNQ 60 BGV3 MMRARIPLLLLLGILFLASLSVSFGIREQHESQDVSVSRGKNNPFYFNSDRWFRTLFRNQ 60 BGV1 MMRARIPLLLLLGILFLASLSVSFGIREQHESQDVSVSRGKNNPFYFNSDRWFRTLFRNQ 60 *******************************.****************************

BGV2 FGHLRVLQRFDQRSNQLQNLENYRVVEFQSKPNTLLLPHHADADFLLVVLNGRAVLTLVN 120 BGV3 FGHLRVLQRFDQRSNQLQNLENYRVVEFQSKPNTLLLPHHADADFLLVVLNGRAVLTLVN 120 BGV1 FGHLRVLQRFDQRSNQLRNLENYRVVEFQSKPDTLLLPHHADADFLLVVLNGRAVLTLVN 120 *****************:**************:***************************

BGV2 PDGRDSYILEQGHAQKIPAGTIFFLVNPDDNENLRIIKLAVPVNNPHRFQDFFLSSTEAQ 180 BGV3 PDGRDSYILEQGHAQKIPAGTIFFLVNPDDNENLRIIKLAVPVNNPHRFQDFFLSSTEAQ 180 BGV1 PDGRDSYILEQGHAQKIPAGTIFFLVNPDDNENLRIIKLAVPVNNPHRFQDFFLSSTEAQ 180 ************************************************************

BGV2 QSYLQGFSKNILEASFDSDIKEINRVLFGEEGQQQQQGQESQQEGVIVELKREQIRELTK 240 BGV3 QSYLQGFSKNILEASFDSDIKEINRVLFGEEGQQQQQGQESQQEGVIVELKREQIRELTK 240 BGV1 QSYLQGFSKNILEASFDSDIKEINRVLFGEEGQQQK-GEESQQEGVIVELKREQIRELTK 239 ***********************************: *:*********************

BGV2 HAKSSSKKSLSSEDEPFNLRNQKPIYSNKFGEFYEITPKKNPQLRDLDVFLSYVDIKEGS 300 BGV3 HAKSSSKKSLSSEDEPFNLRNQKPIYSNKFGEFYEITPKKNPQLRDLDVFLSYVDIKEGS 300 BGV1 HAKSSSKKSLSSEDEPFNLRNQKPVYSNRFGRLHEITPEKNPQLRDLDVFLSYVDIKEGG 299 ************************:***:**.::****:********************.

BGV2 LLLPHYNSKAIVILVINEGKANIELVGLKEEQQRQQQQDERLEVQRYRAEVSEHDVFVIP 360 BGV3 LLLPHYNSKAIVILVINEGKANIELVGLKEEQQQQQQQDERLEVQRYRAEVSEHDVFVIP 360 BGV1 LLIPSYNSKATVILVVNEGEANIELVGLKQQQQQQQQS---WEVQRYSAELSEDDVFVIP 356 **:* ***** ****:***:*********::**:***. ***** **:**.******

BGV2 AGHPVAIDATSNLNFFAFGINAENNQRNFLAGEKDNVISEIPTEVLDLAFPAPGEKVEKL 420 BGV3 AGHPVAIDATSNLNFFAFGINAENNQRNFLAGEKDNVISEIPTEVLDLAFPAPGEKVEKL 420 BGV1 AAYPVAINATSNLNFLAFGINAKNNQRNFLAGEKDNVISEIPNQVLEVAFPGSGEKVVKL 416 *.:****:*******:******:*******************.:**::***..**** **

BGV2 IEKQSRSHFVDAQPE--EQQNRGHSTE------445 BGV3 IEKQSRSHFVDAQPE--EQQNRGHSTE------445 BGV1 INKQSLSYFVDAQSQQKEKQSKGRKDPLSSILDTLH 452

*:*** *:*****.: *:*.:*:.

Figure 4.12. Amino acid comparison between BGV-3 (Vicilin obtained from polyclonal screening), BGV-1 and BGV-2 (Both obtained from PCR screening).

BGV-1 and BGV-2 exhibited similar coverage with other seed proteins as that of BGV. It is evident that BGV-1 and BGV-2 can also be potential allergens in blackgram. Further detailed investigations should be performed to confirm allergenic properties of BGV, BGV-1 and BGV-2 polypeptides.

CHAPTER 6

CONCLUSION

Seed flour functional properties

Tree nuts are high caloric foods with 12.72% (macadamia) - 23.25% (almond) proteins and 43.85% (cashew) - 67.05% (Brazil nut) fat content (dwb). Legumes are good source of proteins (14.63% - 21.9%) with low fat content (1.73% - 3.98%) while cereals are good source of carbohydrates (78.45% to 89.78%) (all % values are on dwb). Defatting increased protein and carbohydrate content of all flours. Full fat flours with higher fat content have lower bulk densities e.g. tree nuts and oil seeds have the lower bulk densities than legumes and cereals. Rice flour is the whitest while pecan is the darkest flour. Defatting extracted pigments from the flours resulting loss in color and improved overall appearance. Color loss of flours should be considered during application of defatted flours in food products.

Defatting increased water and oil holding capacities of all flour samples. Soybean flour exhibited maximum water holding capacity (full fat and defat). Full fat almond had maximum oil holding capacity while defatted macadamia flour had the maximum oil absorption capacity. Defatted flours (except rice) held more water and oil than their own weight. These flours can be used successfully in food preparations (breads, cookies and cakes etc.) requiring water and/or oil holding properties. Cashew full fat flour gel at lowest concentration (8% w/v). LGC improved in some defatted flours as compared to their full fat counterpart but not for all.

Blackgram vicilin gene isolation

Genes encoding blackgram vicilin were isolated using rabbit anti whole blackgram polyclonal antibody screening and PCR screening method. Polyclonal antibody screening of blackgram cDNA library led to identification of genes, named BGV3. BGV3 codes for 445 amino acids with more than 57% identity with other vicilin like genes of mung beans, soybean, lentils and cowpea etc. Primers designed using conserved region of Vigna vicilin led to identification of two genes (BGV-1 and BGV-2). BGV-1 and BGV-2 code for 452 and 445 amino acids respectively. Both genes exhibited more than 55% identification with vicilin like 46

genes from other plants as mentioned above. BGV-3, BGV-1 and BGV-2 showed 93% similarity of len c 1, an identified allergen from lentil. Further research should be carried out to study BGV-3, BGV-1 and BGV-2 as potential blackgram allergen.

APPENDIX A

ANIMAL CARE AND USE COMMITTEE APPROVAL MEMORANDUM

APPENDIX B

Table B.1. Full fat flour proximate composition raw data Sample Lipid Protein Ash Moisture Carbohydrates Almond 44.56 22.68 2.09 3.42 27.25 45.77 22.23 2.18 3.38 26.44 Brazil nut 64.32 13.35 2.99 3.35 15.99 65.12 13.51 3.08 3.6 14.69 Cashew 41.65 19.09 2.09 4.75 32.42 41.91 18.17 2.19 4.69 33.04 Hazel nut 62.56 13.42 1.1 3.12 19.80 61.44 14.76 1.3 3.01 19.49 Macadamia 66.24 12.12 1.29 1.64 18.71 66.98 12.94 1.2 1.37 17.51 Pecan 62.78 15.8 1.1 1.89 18.43 62.93 14.84 1.19 1.74 19.30 Pistachio 44.24 18.22 1.19 3.27 33.08 44.98 17.95 0.9 3.2 32.97 Walnut 65.89 18.67 1.6 1.09 12.75 65.12 18.58 1.7 1.96 12.64 Blackgram 1.42 19.48 1.3 9.07 68.73 1.72 20.31 1.0 9.22 67.75 Chickpea 3.41 13.44 1.4 8.75 73.00 3.89 13.37 1.59 8.01 73.14 Peanut 40.45 24.15 2.49 4.47 28.44 40.59 24.26 2.2 4.98 27.97 Soybean 4.34 29.53 2.38 7.78 55.97 4.51 28.56 2.39 7.7 56.84 Sesame 49.56 13.5 3.18 4.03 29.73 49.01 13.42 3.5 3.86 30.21 Rice 1.03 6.35 1.09 12.82 78.71 1.11 7.22 1 12.97 77.70 Wheat 2.65 15.74 1.89 8.91 70.81 2.12 15.14 1.71 8.95 72.08 Millet 3.86 12.14 1.8 8.87 73.33 3.81 13.12 1.9 8.67 72.50

Table B.2. Defatted flour proximate composition raw data Sample Protein Ash Moisture Carbohydrates Almond 30.14 6.16 5.73 57.97 29.92 6.09 5.89 58.10 Brazil Nut 34.28 6.99 5.65 53.08 34.11 6.67 5.5 53.72 Cashew 31.51 3.59 6.29 58.61 31.45 3.8 6.55 58.20 Hazel nut 35.12 2.28 5.21 57.39 35.25 2.2 5.13 57.42 Macadamia 21.46 3.19 8.54 66.81 22.88 2.89 8.41 65.82 Pecan 20.33 4.58 5.44 69.65 20.45 4.5 5.01 70.04 Pistachio 29.38 1.99 5.43 63.20 30.43 2.1 5.66 61.81 Walnut 34.81 4.69 9.36 51.14 34.87 4.9 9.09 51.14 Blackgram 20.61 1.59 11.38 66.42 20.8 1.49 11.38 66.33 Chickpeas 14.75 2.1 10.5 72.65 14.83 2.29 10.56 72.32 Peanut 36.91 3.7 8.24 51.15 37.24 3.6 8.09 51.07 Soybean 31.46 2.91 10.28 55.35 32.68 3.1 10.37 53.85 Sesame 34.33 5.79 6.94 52.94 34.13 5.7 7.42 52.75 Rice 6.67 1.29 12.55 79.49 7.76 1.4 12.55 78.29 Wheat 18.25 2 9.28 70.47 18.31 1.9 9.15 70.64 Millet 15.41 2.2 10.04 72.35 15.37 2.2 10.7 71.73

Table B.3. Full fat flour raw color data ID L* a* b* FF Almond 1 70.39 5.04 19.98 FF Almond 2 68.71 5.51 20.46 FF Almond 3 69.11 5.38 20.5 FF Brazil Nut 1 59.61 3.65 16.54 FF Brazil Nut 2 60.74 3.41 16.36 FF Brazil Nut 3 60.79 3.44 16.65 FF Cashew 1 74.97 1.86 25.62 FF Cashew 2 75.62 1.61 24.77 FF Cashew 3 75.48 1.65 24.82 FF Hazel nut 1 68.37 4.67 20.75 FF Hazel Nut 2 67.85 4.76 20.91 FF Hazel nut 3 67.97 4.67 20.85 FF Macadamia 1 76.37 0.67 25.81 FF Macadamia 2 76.5 0.72 25.86 FF Macadamia 3 76.42 0.72 25.86 FF Pecan 1 54.12 6.5 22.22 FF Pecan 2 53.04 6.42 21.83 FF Pecan 3 53.97 6.35 21.77 FF Pistachio 1 66.51 0.98 42.12 FF Pistachio 2 66.49 0.95 42.09 FF Pistachio 3 66.31 1.01 41.66 FF Walnut 1 59.19 4.76 26.3 FF Walnut 2 59.15 4.74 25.71 FF Walnut 3 59.87 4.59 25.91 FF Blackgram 1 76.42 0.13 9.83 FF Blackgram 2 76.74 0.11 10.1 FF Blackgram 3 76.2 0.1 10.06 FF Chickpea 1 86.38 3.03 20.15 FF Chickpea 2 86.42 2.93 19.68 FF Chickpea 3 86.35 2.93 19.78 FF Peanut 1 71.64 4.57 19.94 FF Peanut 2 71.29 4.49 19.42 FF Peanut 3 71.03 4.55 19.52 FF Sesame 1 71.21 2.25 23.01 FF Sesame 2 71.37 2.11 22.35 FF Sesame 3 71.61 2.06 22.18 FF Soy 1 85.45 1.35 24.62 FF Soy 2 85.24 1.32 24.5 FF Soy 3 84.72 1.43 24.74 FF Rice 1 89.96 0.04 10.36 FF Rice 2 89.42 0.08 10.73 FF Rice 3 89.48 0.1 10.56 FF Wheat 1 83.56 2.96 14.97 FF Wheat 2 83.89 2.7 14.56 FF Wheat 3 83.89 2.75 14.71 FF Millet 1 84.74 2.56 25.8 FF Millet 2 84.53 2.61 26.37 FF Millet 3 84.74 2.41 25.76

Table B.4. Defatted flour raw color data ID L* a* b* DF Almond 1 86.37 1.99 10.98 DF Almond 2 87.27 1.86 10.73 DF Almond 3 87.39 1.83 10.63 DF Brazil nut 1 88.33 0.74 9.34 DF Brazil Nut 2 88.42 0.73 9.16 DF Brazil Nut 3 88.42 0.75 9.17 DF Cashew 1 93.64 -0.37 6.44 DF Cashew 2 93.82 -0.35 6.69 DF Cashew 2 94.12 -0.36 6.62 DF Hazel Nut 1 84.88 2 10.57 DF Hazel Nut 2 84.58 2.03 10.83 DF Hazel Nut 3 85.06 1.96 10.63 DF Macadamia 1 90.8 -0.28 12.77 DF Macadamia 2 90.65 -0.25 12.84 DF Macadamia 3 90.49 -0.23 12.95 DF Pecan 1 86.21 0.98 9.86 DF Pecan 2 86.04 1.02 9.8 DF Pecan 3 85.85 1.04 9.85 DF Pistachio 1 90.13 -1.7 14.55 DF Pistachio 2 89.87 -1.68 14.73 DF Pistachio 3 90.08 -1.63 14.44 DF Walnut 1 82.59 1.06 15.61 DF Walnut 2 82.35 1.14 15.88 DF Walnut 3 82.47 1.08 15.71 DF Blackgram 1 75.08 0.03 9.21 DF Blackgram 2 74.35 -0.03 9.03 DF Blackgram 3 73.92 -0.02 9.1 DF Chickpea 1 88.31 1.41 14.63 DF Chickpea 2 88.11 1.45 14.67 DF Chickpea 3 87.9 1.51 14.82 DF Peanut 1 88.81 1 8.95 DF Peanut 2 88.82 0.92 9.01 DF Peanut 3 88.75 0.95 9.07 DF Soy 1 89.29 0.24 14.69 DF Soy 2 89.7 0.12 14.59 DF Soy 3 89.53 0.16 14.78 DF Sesame 1 91.8 -0.24 8.82 DF Sesame 2 91.31 -0.15 8.82 DF Sesame 3 92.06 -0.25 8.41 DF Rice 1 90.34 -0.02 9.13 DF Rice 2 90.08 0.13 8.94 DF Rice 3 89.71 0.08 9.33 DF Wheat 1 85.72 2.35 11.82 DF Wheat 2 85.97 2.2 11.62 DF Wheat 3 85.73 2.26 11.81 DF Millet 1 88.46 2.01 17.72 DF Millet 2 88.48 1.67 15.48 DF Millet 3 88.82 1.52 14.66

Table B.5. Oil Holding Capacity raw data Full Fat Full fat flour OHC Defatted flour OHC Flour (g/g) (g/g) 1.3240 2.2380 Almond 1.4510 2.1700 1.3840 2.1550 1.1690 2.0160 Brazil 1.0330 2.2380 Nut 0.7780 2.2560 0.9490 2.1450 Cashew 0.8309 2.3270 0.9470 2.3370 1.3080 1.5960 Hazel Nut 1.3930 1.5220 1.3500 1.4720 1.1050 3.1830 Macadamia 1.2130 3.4930 1.0010 2.6620 1.5010 3.2440 Pecan 0.9800 3.1400 1.2070 3.0680 1.0660 2.1910 Pistachio 0.9960 2.1440 0.9739 2.0270 1.2040 2.0070 Walnut 1.1960 1.9700 1.1290 1.9840 0.43795 0.8620 Blackgram 0.68395 1.0560 0.62295 0.8220 0.86795 1.1480 Chickpea 0.85595 1.0510 0.84995 0.9929 0.9530 2.0020 Peanut 0.9530 1.9860 0.8980 1.9790 1.0460 1.3410 Soybean 1.2220 1.5430 1.1440 1.4430 1.1480 2.1710 Sesame 1.1280 2.1770 1.0860 2.1280 0.6620 0.9579 Rice 0.6519 1.0140 0.6490 0.9050 0.8549 1.2490 Wheat 0.9400 1.1990 1.0700 0.9910 0.7910 0.9310 Millet 0.7830 0.9880 0.7860 0.7779

Table B.6. Water holding capacity raw data Flour Full fat flour Defatted flour Name WHC (g/g) WHC (g/g) 1.4070 2.0454 Almond 1.5000 2.1274 1.4070 2.0524 0.6650 1.8398 Brazil 0.6730 1.8673 Nut 0.6810 1.8314 0.9310 1.9835 Cashew 0.9250 1.9973 0.9010 1.9780 1.0510 1.8592 Hazel Nut 1.0770 1.8315 1.1170 1.8024 0.6480 1.4598 Macadamia 0.5340 1.4603 0.7600 1.4243 1.4240 2.4312 Pecan 0.9800 2.1249 1.0970 2.3214 1.3910 1.9634 Pistachio 1.4360 1.9821 1.3820 1.9453 1.1880 2.2424 Walnut 1.2590 2.2466 1.2670 2.1732 2.435 2.3482 Blackgram 2.534 2.3987 2.465 2.3153 2.041 2.3572 Chickpea 2.174 2.2289 2.102 2.2348 0.9680 1.8653 Peanut 1.3780 1.8662 0.8560 1.8589 2.758 3.1586 Soybean 2.733 3.18988 2.728 3.1598 1.484 2.5672 Sesame 1.614 2.5392 1.483 2.5425 1.146 1.341 Rice 1.086 1.3471 1.05 1.3489 1.708 1.7498 Wheat 1.845 1.7462 1.51 1.7423 1.188 1.3258 Millet 1.417 1.3287 1.249 1.3341

Table B.7. Least gelation concentration values raw data Sr. LGC Flour Name No. Full Fat Defat 14 8 1 Almond 14 8 Brazil 14 10 2 Nut 14 10 8 6 3 Cashew 8 6 14 14 4 Hazel Nut 14 14 18 14 5 Macadamia 18 14 18 14 6 Pecan 18 14 10 12 7 Pistachio 10 12 18 14 8 Walnut 18 14 12 12 9 Blackgram 12 12 12 12 10 Chickpea 12 12 16 10 11 Peanut 16 10 16 14 12 Soybean 16 14 16 12 13 Sesame 16 12 18 18 14 Rice 18 18 14 14 15 Wheat 14 14 18 18 16 Millet 18 18

APPENDIX C

PROTOCOL 1 – SCREENING cDNA LIBRARY

PROTOCOL 2 - QIAprep Spin Miniprep Kit (Qiagen Inc.)

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BIOGRAPHICAL SKETCH

EDUCATION:

Florida State University Tallahassee, Florida Master of Science (M.S.) in Food Science and Nutrition Current GPA – 3.81

University Institute of Chemical Technology (Formerly -UDCT) Mumbai, India Bachelor of Technology (B.Tech.) in Food Engineering and Technology Percentage – 72.27% (Ist Class with Distinction)

EXPERIENCE: Campbell Soup Company (Camden, New Jersey) Process Engineer R&D Co-op

Florida State University (Tallahassee, Florida) Teaching and Research Assistant, Food Science Lab

Cadbury Plc (Pune, India) Quality Assurance and Production Summer Intern

Frolic Foods (Mumbai, India) Product Development Intern

Coca-Cola Company (Mumbai, India) Quality Assurance Summer Intern

PUBLICATION: Sharma, G. M.; Su, M.; Joshi, A. U.; Roux, K. H.; Sathe, S. K. Functional Properties of Select Edible Oilseed Proteins. Journal of Agricultural and Food Chemistry, 2010, 58, 5457-5464.

ACTIVITIES AND HONORS: Betty M Watts Memorial Funds – Florida State University- April 2011 2011 IFT Quality Assurance Division Travel Grant Student Member, Institute of Food Technologists (IFT). Student member of Florida Association of Food Protection.