Effects of Maillard Reaction on the Immunoreactivity of Almond Major Protein in the Food Matrices Guneet Singh Chhabra

Florida State University Libraries

Electronic Theses, Treatises and Dissertations The Graduate School

2013 Effects of Maillard Reaction on the Immunoreactivity of Almond Major Protein in the Food Matrices Guneet Singh Chhabra

Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected]

FLORIDA STATE UNIVERSITY

COLLEGE OF HUMAN SCIENCES

EFFECTS OF MAILLARD REACTION ON THE IMMUNOREACTIVITY OF ALMOND

MAJOR PROTEIN IN THE FOOD MATRICES

GUNEET SINGH CHHABRA

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

Degree Awarded

Fall Semester, 2013

Guneet Singh Chhabra defended this thesis on October 18, 2013. The members of the supervisory committee were:

Shridhar K. Sathe Professor Directing Thesis

Yun-Hwa Peggy Hsieh Committee Member

John G. Dorsey 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.

ACKNOWLEDGMENTS

This master’s thesis is dedicated to my niece, Noor who has come in my life as a blessing of the almighty. Your smile is my remedy. I wish to see you grow all your life and live all your dreams, for that will make my one dream come true, to see you happy. God bless you always. I love you!

The journey that has lead to the completion of this Thesis has been long and challenging and could not have been accomplished without several people. Thank you first to Dr. Sathe, who gave me a chance to come back and supported me to finish my master’s degree. Sir, you literally gave me a new beginning in life and when I needed it the most. I owe my life to my family and friends, whose moral support kept me going when I got discouraged or frustrated. The encouragement I received outside of the academic world overwhelms me.

To perhaps my biggest supporter throughout my life, the almighty, I call him Waheguru, whose blessings carried me through many tough times and kept me going when the end was not insight, thank you. Thank you to my Mom, Harpreet Kaur and Dad, Jasbir Singh, who got me where I am today and always have stood with me in my decisions. To my sister, Gursimran Kaur and brother-in-law, Harpreet Singh for being the best advisors all the time and bringing the angel of my life, Kulpreet Kaur aka Noor, who is most precious to me. To my brother, Mandeep Singh, my best friend for ever, thank you for always being there with all your love.

Thank you so much to my committee members, Dr. Hsieh and Dr. Dorsey, who provided valuable suggestions and guidance in this research and otherwise. I could not have completed this Thesis without the academic and moral support of my lab mates. Coming to lab everyday was so easy when you visited an environment of learning, thinking, and encouragement. I was so lucky to be a part of a lab with so many caring and gifted people that have become dear friends.

iii

They have taught me not only about protein research, but have taught me the value of peer support. Thank you to Changqi Liu “Ben”, who always helped me selflessly and gave me valuable suggestions during this research. There is so much to learn from you and you are a good man my friend. Thank you to Sahil Gupta for being a friend and a colleague and always being there to celebrate good and bad days. I would take this opportunity to thank Ying Zhang for being an inspiration to work hard. Thank you to my colleagues Jasamrit Bakshi and Sepideh

Alasvand. I will cherish all of you.

Finally, thank you again to the person who has had the biggest impact on me through the process of completing my Master’s Thesis, my major professor, Dr. Shridhar Sathe. I cannot thank you enough for everything that you have done for me. Dr. Sathe loves research, and he loves his graduate students. Thank you for giving me the finest education that one could receive in this area of research and for always finding a way to support me financially and giving me the opportunity to teach. Thank you for serving as my major professor for the past three years, for being a mentor and a friend. Most of all, thank you for believing in me, even when I did not. You have instilled in me a confidence that is worth more than I can begin to explain.

TABLE OF CONTENTS List of Tables...... vii

List of Figures...... viii

Abstract...... ix

INTRODUCTION...... 1

Statement of the Problem...... 5

Research Hypothesis...... 5

Limitations of the Study…...... 6

Significance of the Study...... 6

Definitions...... 7

REVIEW OF LITERATURE...... 10

Maillard Reaction Mechanism...... 11

MATERIALS AND METHODS...... 16

Isolation of Almond Major Protein (AMP)...... 21

Protein Extraction...... 22

HunterLab LabScan XE Spectrophotometer...... 22

Water Activity (Aw)…...... 22

Soluble Protein Content...... 23

Immunoassay...... 24

Statistical Analysis...... 28

RESULTS AND DISCUSSION...... 29

Water Activity (Aw) of Food Matrices...... 29

Color of Food Matrices...... 29

Effect of Maillard Reaction and Food Matrix on the Immunoreactivity of Amandin...... 33

Sandwich ELISAs...... 35

Effect of Maillard Reaction on Immunoreactivity of Food Matrices...... 49

Dot Blots...... 50

Western Blots...... 51

Conclusion...... 59

APPENDIX: The Animal Care and Use Committee Approval Memorandum...... 61

REFERENCES...... 64

BIOGRAPHICAL SKETCH...... 74

LIST OF TABLES

2.1 Peptides recognized by murine mAbs...... 11

3.1 List of food matrices for analysis...... 17

3.2 Gradient gel recipe...... 26

4.1 Water activity (Aw at 25 °C) and browning of food matrices containing almonds...... 30

4.2 Sandwich ELISA using 4C10...... 35

4.3 Sandwich ELISA using 4F10...... 40

4.4 Sandwich ELISA using 2A3...... 44

vii

LIST OF FIGURES

4.1 Relationship between the L* and the Aw of the processed food matrices...... 34 4.2 Dot blot for the food matrix samples at room temperature (25 °C)...... 50

4.3Transferred sample proteins (without -me) on nitrocellulose membrane and Ponceau S...... 51 4.4 Transferred sample proteins (with -me) on nitrocellulose membrane and Ponceau S...... 52 4.5 Western blot (without -me) of selected food matrices using 4C10...... 53 4.6 Western blot (without -me) of selected food matrices using 4F10...... 54 4.7 Western blot (without -me) of selected food matrices using 2A3...... 55 4.8 Western blot (with -me) of selected food matrices using 4C10...... 57 4.9 Western blot (with -me) of selected food matrices using 4F10...... 58 4.10 Western blot (with -me) of selected food matrices using 2A3...... 59

viii

ABSTRACT

In the US, among edible tree nut seeds consumed, almonds are ranked number one.

Almonds are considered heart healthy and approved by FDA for qualified health claim. Almonds are consumed as natural raw unprocessed or as variously processed seeds such as blanched, roasted, and fried. Additionally, almond seeds, in various forms (such as chopped, diced, slivered, powdered and others) are used as an ingredient in many foods including baked goods and confectionery items, granola bars, breakfast cereals, and several snack mixes. Inclusion of almonds in these products is valued because almonds provide desirable crunchy texture, sweet mellow flavor, and several micro- and macronutrients. Although safely enjoyed by most, almonds may induce adverse reactions, such as allergic reaction, in sensitive individuals.

Almond allergy has been identified as the third most frequent tree nut allergy in the US and anaphylaxis to almond has been reported. Amandin, a globular storage protein that accounts for

~65% of the extractable proteins is the major allergen in almond seeds. Amandin is a hexameric protein and consists of two trimers. Each trimer is composed of three polypeptides each of which is composed of 40-4β kDa acidic chain (α) linked by a disulfide bond to β0 kDa basic chain ().

Foods, containing almonds, subjected to thermal processing typically experience Maillard reaction. Maillard reaction, chemical reaction between reducing sugars and the amino groups on amino acids/polypeptides/proteins cause non-enzymatic browning of foods. This browning reaction is desirable in many food products due to color and flavor formation. Maillard reaction also may glycate food proteins. As a result of Maillard reaction, destruction of amino groups, glycation, and/or denaturation of proteins may therefore alter amandin immunoreactivity. The

ix current research therefore focused on amandin immunoreactivity in variously processed almonds and almond containing foods.

In the selected commercial and laboratory prepared food matrices, the occurrence of non- enzymatic Maillard browning was objectively assessed by determining color using Hunter L, a, b scale (L* (lightness), a* (green-red), b* (blue-yellow) values). L* values used for different degrees of browning (roasting) were: Light = 53±1, Medium = 48.5±1, Dark = 43±1. The L* values for the tested samples were in the range 31.75-85.28 consistent with Maillard browning or the natural product color (e.g. white chocolate). Immunoassays (ELISA, dot-blot, and Western blot) were used to determine the immunoreactivity. Three murine monoclonal antibodies

(mAbs), 4C10 4F10 and 2A3 were used to probe the desired samples. The mAb 4C10 targeted conformational and 4F10 and 2A3 recognized two independent linear amandin epitopes. The tested food matrices that did not contain almond exhibited no cross-reactivity in the immunoassays indicating their amandin specificity. For sandwich ELISAs, R = protein concentration of sample for 50% of the maximum signal for the corresponding standard curve/ the protein concentration required to register 50% of the maximum signal for the standard curve.

The Fisher’s Least Significant Difference (LSD) at p ≤ 0.05 was calculated for appropriate data.

None of the tested food matrices exhibited increased immunoreactivity. The range of R values for tested food matrices containing almonds were 0.67-15.19 (4C10, LSD = 1.90), 1.00-11.83

(4F10, LSD = 0.86) and 0.77-23.30 (2A3, LSD = 1.34). Results of dot blots and Westerns blots were consistent with the ELISA results. Certain bakery and confectionary samples exhibited significantly decreased immunoreactivity. The observed decrease in immunoreactivity may be due to Maillard reaction, epitope degradation due to loss of amandin disulfide bond(s), loss of protein solubility, amandin thermal aggregation; or a combination thereof. Results of the study

x indicate that the immunoassays using the murine mAbs are specific, sensitive and robust for amandin detection in the tested food matrices and amandin incurred samples.

CHAPTER 1

INTRODUCTION

Among the tree nuts, almonds are popular in many cultures and enjoy a long history of food uses. California Almonds account for about 80% of the global and 100% of the domestic supply. During the 2011/12 crop year California Almonds produced a record 2.02 billion pounds of almonds valued at $4.3 billion (Almond Almanac, 2012, www.almondboard.com). As per

Almond Almanac 2012, among the domestic per capita consumption of competing nuts, almonds have been on the top with 1.81 lb per capita consumption while walnuts, pecans and other tree nuts are under 0.5 lb per capita (www.almondboard.com). Almonds are widely consumed as roasted snacks but in the US they are also used in a variety of forms as ingredients in many foods including granola bars, breakfast cereals, baked goods and confectionery items where they provide texture and flavor plus micro- and macronutrients (Broughton, 1996; Wolf and Sathe,

1998). These value-added products are either produced and packaged in industry or prepared at home (www.agmrc.org). US baking industry accounted for 2.1 per cent of GDP and were about

$311 billion market in total in 2010(ROI report 2011-12, www.americanbakers.org). And the

American confectionery industry ranked top in 2011 with total retail sales of $32.3 billion

(Mediakit, 2013, www.candyindustry.com). According to the breakfast food statistics report

2013 by AIB International, granola bar sales in US in 2012 were worth $1.01 billion

(www.aibonline.org).

Major macronutrients in almonds are oil (54-56%) and protein (16-22%) (Sathe, 1993).

In 2003, FDA proclaimed that consuming 1.5 Oz. almonds as a part of diet low in saturated fat and cholesterol may reduce the risk of heart diseases (www.fda.gov). These heart health claims

1 are mainly due to almonds being a rich source of MUFA (Oleic acid-60.93 g/100g) and PUFA

(Linoleic acid- 29.21 g/100g) (Sathe et al., 2008; Chen et al., 2006; Venkatachalam and Sathe,

2006) and also proteins, dietary fiber, vitamin E, manganese, calcium, etc

(www.almondboard.com).

While almonds may provide several health benefits, it is worth noting that they can also induce adverse reactions in susceptible individuals (Chen et al., 2006). In the US, 10% of the total food allergic patients were reactive to tree nuts (Sicherer et al., 1999). Poltronieiri et al.

(2002) estimated that 10% of allergic individuals will be reactive to two or more nuts and extrapolated this figure to about 500,000 people worldwide (Chen et al., 2006). Amandin, a storage globulin protein which comprises 60-70% of extractable proteins from almonds (Wolf and Sathe, 1998), has been identified as a major almond storage protein and serves as the basis for detecting almonds in food products in nanogram concentrations with rabbit anti-amandin polyclonal antibodies (Sathe et al., 2002; Acosta et al., 1999).

Almonds are often subjected to harsh heat processing conditions prior to or during their incorporation into foods (Venkatachalam et al., 2002). Processing is done to improve flavour, texture, taste, color, preservation, safety, convenience, variety, increase marketability and revenue (Sathe and Sharma, 2009). Processing of foods or its ingredients may either destroy or generate new epitopes known as neoallergens (linear or conformational) as a result of change in protein conformation (Sathe et al., 2005). The formation of neoallergens has been recognized for at least four decades, as evidenced by one of the earlier reports (Spies, 1974) and may partially explain why some people can tolerate unprocessed food or a food ingredient but not the corresponding processed counterpart (Sathe et al., 2005). Almond allergens were reported to be stable to a variety of processing methods including -irradiation alone as well as –irradiation

2 followed by blanching, autoclaving, roasting, frying, and microwave heating (Venkatachalam et al., 2002; Su et al., 2004).

Maillard reaction during food processing may result in formation of neoallergens. The non-enzymatic reaction of amino acids with reducing sugars occurs in the heat treatment during cooking of cakes, biscuits and amylase containing foods or after their long-term storage leading to formation of advanced glycation end-products (AGEs) (Henle, 2005; Hilmeyuk et al., 2009).

Glycation of proteins is a complex series of parallel and sequential reactions collectively called the Maillard reaction (Thorpe and Baynes, 2003; Thornalley, 1999; Ahmed et al., 2005).

Maillard reaction is the foundation of the modern ﬂavor industry. In addition, the Maillard reaction is also called non-enzymatic browning, being one of the main reasons for the occurrence of the non-enzymatic food browning. Thus, the Maillard reaction is of vital importance to food science (Hodge, 1953; Fayle and Gerrard, 2002; Nursten, 2005; Peng et al., 2011).

The early recognition of AGEs occurred in late 1960s when a non-enzymatic glycation process similar to the Maillard reaction (Fayle and Gerrard, 2002; Nursten, 2005) was found in human body by the observation of increased formation of non-enzymatically glycosylated haemoglobins HbAIc (AGE precursors) in diabetic patients (Rahbar et al., 1969; Henle, 2007;

Peng et al., 2011). AGEs (Thorpe and Baynes, 2003) are a group of complex and heterogeneous compounds which are known as brown and fluorescent crosslinking substances such as pentosidine, non-fluorescent crosslinking products such as glyoxal-lysine dimer (GOLD) and methylglyoxal-lysine dimer (MOLD), or non-fluorescent, non-crosslinking adducts such as carboxymethyllysine (CML) and pyrraline (a pyrrole aldehyde) (Rahbar and Figarola, 2002;

Ikeda et al., 1996; Peng et al., 2011). Accumulation of these glycotoxins (Uribarri, 2010) in vivo has been implicated as a major pathogenic process in diabetic complications, including

3 neuropathy, nephropathy, retinopathy and cataract (Ahmed, 2005; Brownlee, 1994) and other health disorders, such as atherosclerosis (Kume et al., 1995; Hilmeyuk et al., 2009; Goldin et al.,

2006), Alzheimer’s disease (Vitek et al., 1994; Munch et al., 1997) and normal aging (Munch et al., 1997; Brownlee, 1995; Peng et al., 2011). There is virtually no information about defined structure-activity relationships documenting the “toxic” effect of individual compounds in physiological concentrations of these uremic toxins (Henle and Miyata, 2003; Vanholder et al.,

2003; Henle, 2007). The receptor for advanced glycation end-products (RAGE) has a well- substantiated role in cell dysfunction and mechanisms of inflammatory diseases (Thornalley,

2007; Goldin et al., 2006).

The fact that the modern diet is a large source of AGEs is now well documented

(Vlassara et al., β004; O’Brien et al., 1989; Goldberg et al., 2004; Uribarri et al., 2010).

Formation of advanced glycation end-products in different food proteins have been studied

(Zhang et al., 2011; Berrens, 1996; Gruber et al., 2004; Hilmenyuk et al., 2009; Oliver et al.,

2005; Suarez et al., 1989). These studies suggest that during processing of foods containing almonds and different reducing sugars either in industrial settings (baked goods) or at home

(meal and/or desserts), glycation of almond major protein (amandin- major allergen) may occur and alter the characteristics of antigenic epitope and resulting immunogenic responses. The extent of the Maillard reaction and the type of reducing sugar are important parameters for the biological properties of food-derived proteins (Maleki et al., 2000; Iwan et al., 2011). Therefore, a study of the food systems containing almonds that are consumed more frequently and in which

Maillard reaction may occur would lead to a practical understanding of AGEs formation and their effects on the immunoreactivity of amandin. A constructive study of various food matrices would be useful in developing optimum conditions for a model system of pure protein i.e.,

4 amandin and various standard sugars. It would also give an insight into the role of various ingredients that are present in a food during the occurrence of Maillard reaction in that food.

Statement of the Problem

Advanced glycation end-products (AGEs) resulting from Maillard reaction of amandin and sugar in selected food matrices may alter allergenicity. The antigenic potential of these glycation products has not been evaluated. A food system based study to determine the effects of glycation on the immunoreactivity of amandin is the main aim of this research. The food matrices selected for the study represent bakery, confectionary, and snack products and several ingredients that may be used in their preparation (Table 3.1). Samples will be powdered into flour (~40 mesh) and their soluble protein will be extracted in 0.1 M Borate Saline Buffer (BSB- pH8.45) (Sathe et al., 2009). The soluble protein content will be determined by Lowry protein assay (Lowry et al. 1951). Glycation of the amandin will be determined by glycoprotein staining of the SDS-PAGE gels. Whole almond flour will be used as a positive control and to prepare the standard curves for ELISAs and other oilseeds and tree nuts (Table 1) will be used as negative controls. Rabbit anti-whole almond polyclonal antibody and murine monoclonal antibodies

4C10, 4F10, and 2A3 against amandin have already been screened, characterized and available in our research laboratory. The same antibodies will be used for evaluation of amandin immunoreactivity. Statistical analysis of data collected from all the experiments will be conducted.

Research Hypothesis

1. Maillard reaction may result in glycation of amandin in bakery and confectionary foods

containing almonds.

2. The possible neoallergen formed might be recognized differently than whole almond or

amandin by the antibodies.

3. Food matrix may interfere with the recognition in addition to Maillard reaction of amandin

and alter the immunoreactivity.

4. Different atopic individual’s sera might recognize the allergen differently and therefore

would show different degree of immunoreactivity on exposure to amandin in the food

matrices.

Limitations of the Study

1. The antigenicity of almonds in the food matrices will be assumed to be independent of the

growing region and season.

2. Antibodies that are raised against almonds and amandin will be used to detect the

neoantigen(s) formed as a result of glycation.

3. The glycation product(s) will not be identified and/or characterized in this study.

4. No model system would be developed for glycation of amandin, but the glycation and its

effects on immunoreactivity will be determined from the selected food matrices using

untreated amandin and whole almonds as control.

Significance of the Study

The effects of Maillard reaction on the immunoreactivity of almond major protein in foods matrices have not been evaluated before. Maillard reaction in foods is important for quality and acceptance by the consumers. A food system based study will represent a realistic situation for Maillard reaction in almond containing foods and therefore, the results would be more applicable in terms of understanding glycation and AGEs. The food matrices may interfere

6 with the immunoreactivity of amandin in addition to alteration of protein by Maillard reaction.

Thus, in order to design better Maillard reaction studies, the results from a food system will be helpful. A model system for glycation of AMP can be developed on the basis of results of food system study to get a further information on the kind of glycation products formed and effect of presence of various ingredients on the type of products formed. Our research lab has all the facilities available to investigate food allergens and we have murine monoclonal antibodies to probe the specific almond allergens and therefore we can conduct this study on food matrices.

Also, this study would serve as a validation of robustness of the monoclonal antibodies based

ELISA assay developed in our lab (Su, 2012). Further, the understanding of immunoreactivity of

AGEs formed in the processed products will help in identifying their connection with diseases such as diabetes, atherosclerosis and Alzheimer’s disease which are more prevalent in both developed and developing world.

Definitions

Advanced Glycation End-Products (AGEs) - Advanced glycation end products (AGEs) are proteins or lipids that become glycated after exposure to reducing sugars.

AMP- Almond major protein also known as prunin or pru du 6, a storage globulin (~65% of total almond proteins).

Anaphylaxis- A type I hypersensitivity reaction to an allergen caused by rapid cross-linking with antigen-specific IgE molecules bound to the surface of tissue mast cells and peripheral blood basophils.

Antibody-Immunoglobulin found in blood, formed by the immune system that recognizes a specific, foreign molecule.

Antigen- Molecule recognized by antibodies.

Cross-reactive antigen- An antigen that binds to antibodies that were raised against different antigens due to similar antigenic determinants (epitopes).

ELISA- Enzyme-Linked Immunosorbent Assay.

Epitope-Specific array of amino acids in a protein that bind to antibodies.

Food allergen- Causative molecule in an offending allergic food. This is a substance that causes an abnormal reaction in some individuals. Type I food allergies are often triggered by food proteins.

Food allergy- An IgE mediated hypersensitive response to a protein or other molecule that is ingested. The clinical response includes urticaria, pruritis, vomiting and diarrhea, abdominal cramps, or in severe cases, systemic anaphylaxis. mAb- Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell, in contrast to polyclonal antibodies (pAbs) which are made from several different immune cells.

Maillard reaction- A non-enzymatic reaction that leads to formation of AGEs.

Monoclonal antibodies- Antibodies raised against a specific antigen, descending from a single cell line.

Polyclonal antibodies- Antibodies raised against a specific antigen, descending from multiple cell lines.

Type-I hypersensitivity- IgE mediated allergy involving an immune release of chemical mediators from mast cells.

Water Activity (Aw) - Water activity is a measure of the energy status of the water in a system, and thus is a far better indicator of perishability than water content.

CHAPTER 2

REVIEW OF LITERATURE

The US National Institute of Allergy and Infectious Diseases (NIAID) defined food allergy as, “adverse health effect arising from a specific immune response that occurs reproducibly on exposure to a given food” (Boyce et al., 2010). Food allergy is an adverse immunological response to a normally tolerated food protein and may be due to IgE mediated or non-IgE mediated mechanisms (Sampson, 1999). Cross-linking of IgE on the surface of mast cells and basophils by the allergen molecule is an obligatory step for manifestation of allergy symptoms. The spectrum of type-I food allergy symptoms may include flushing, urticaria, angioedema, laryngoedema, diarrhea, nausea/vomiting, bronchospasm, or hypotension (Food allergy: a practice parameter, 2006; Sampson, 1999; Chandra and Thompson, 2001). According to The Food Allergy and Anaphylaxis Network (FAAN), about 90% of all food allergies are caused by 8 major foods which include milk, eggs, peanuts, tree nuts, fish, shellfish, soybeans and wheat (www.foodallergy.org). The major cause of anaphylactic reactions treated in emergency rooms in the western countries is due to consumption of food. Sampson (2003) reported that about 30,000 food-induced anaphylactic episodes occur in the United States each year, resulting in 2000 hospitalizations and 150 to 200 deaths. Every 3 min a food allergy reaction sends someone to the emergency department– that is about 200,000 emergency department visits per year, and every 6 min the reaction is one of anaphylaxis. An estimated 1.8 million Americans have an allergy to tree nuts (www.foodallergy.org). Allergic reactions to tree nuts are among the leading causes of fatal and near-fatal reactions to foods. Most individuals diagnosed with an allergy to tree nuts tend to have a life-long allergy (www.foodallergy.org).

Almond allergy has been identified as the 3rd most frequent tree nut allergy in the US (Sicherer

10 et al., 1999; Sicherer et al., 2003) and severe anaphylaxis to almond has been reported (Armentia et al., 2001). Fatalities as a result of consuming tree nuts, including almond, have been documented (Bock et al., 2007).

The allergen in almonds is a major storage globulin (11S legumin) known as amandin, almond major protein, prunin or pru du 6 (Sathe et al., 2002 and Willison et al., 2011). Amandin is hexameric and consists of two trimers of 40-4β kDa acidic chain (α) linked by a disulfide bond to β0 kDa basic chain() (Garcia-Mas et al., 1995 and Sathe et al., 2002). The mAb 4C10 is highly reactive to amandin and recognizes the conformational epitope dependant on the intramolecular disulﬁde bond within the large subunit and/or the intermolecular bond association of the large and small subunits (Willison et al., 2013). 4F10 and 2A3 recognize the sequential

(linear) epitopes only. The whole almond (amandin) peptides recognized by 4C10, 4F10 and

2A3 mAbs in Western blots are listed in Table 2.1.

Table 2.1: Peptides recognized by murine mAbs (Su, 2012). S. No. mAb Peptides recognized Peptides recognized under non-reducing under reducing conditions (kDa) conditions (kDa) 1 4C10 63 63 2 4F10 38,50,55,63 26,28,32,35,38-42,66,72 3 2A3 36,38,50,61 29-36,40 48, 68

Maillard Reaction Mechanism

Hodge (1953) in his integrated review on model systems of sugar-amine browning reactions suggested seven different types of reactions in three different stages of development (Fig.2.2) stated as follows-

1. Initial stage (colorless) -

A. Sugar-amine condensation- It occurs in equimolar ratio. Mechanism involves opening of ring form of sugar, addition of amine group to the carbonyl group followed by elimination of a molecule of water to form N-substituted glycosylamine (Katchalsky and Sharon, 1953).

B. Amadori rearrangement- Isomerisation of N-substituted aldosylamine to 1-amino-1- deoxy-2-ketoses (Kuhn and Weygand, 1937; Kuzin and Polyakova, 1941) in an acid catalyzed reaction. C-2 hydroxyl group of an aldose is essential for occurrence of a significant degree of browning (Beacham and Dull, 1951; Haugaard et al., 1951; Hurd and Kelso, 1948; Wolfrom et al., 1947).

2. Intermediate stage (colorless or yellow with strong absorption in near UV)-

C. Sugar dehydration- In a neutral or acidic system, furfurals are formed. 1, 2-enolization occurs with loss of one mole of water and production of α-enolic- α,-unsaturated aldehyde existing in equilibrium with 3-deoxysone (Taufel and Iwainsky, 1952). Amines are liberated from the Schiff’s base of HMF (hydroxymethyl furfurals) by hydrolysis after sugar dehydration.

Three molecules of water are lost from 1, 2-enolic structure to form Schiff’s base of furfural.

Fraction of unliberated amines is combined in melanoidins (Gottschalk, 1952; Gottschalk and

Partridge, 1950). In presence of amines in basic system, reductones are formed (furfurals without furan ring closure).The dehydro form of reductones is the primary source of browning.

D. Sugar fragmentation- Retroaldolisation catalyzed by anionic form of amines and not the Zwitter ions results in formation of fission products such as triose, pyruvaldehyde, etc.

E. Amino acid degradation/ Strecker degradation- α-amino acids are converted to aldehydes with one carbon less and liberation of carbon-dioxide and transamination for incorporation of amine group into the brown polymer (Schonberg et al., 1948). The aldehydes can condense with themselves, with sugar fragments, with furfurals, etc. to form brown pigments

(Patrick, 1952). Though, it is not a major color producing reaction.

3. Final Stage (highly colored) –

F. Aldol condensation, and

G. Aldehyde-amine polymerization with formation of heterogenous nitrogen compounds-

Formation of heterocyclic nitrogen compounds such as pyrroles, imidazoles, pyridines, pyrazines, aldimines and kitimines.

Maillard browning is important in processed foods for consumer acceptance as well because of the fact that ‘we also eat with our eyes’ (Martins et al., 2000). And therefore understanding Maillard reaction is not only important traditional processing methods such as roasting, baking and cooking but also for development of new technologies such as microwave and high pressure processing (Ames 1998; Shahidi et al., 1998). Griffith and Johnson (1957) reported anti-oxidative components in sugar cookies which provided stability to oxidative rancidity (lipid oxidation). But, Maillard reaction in foods causes loss of nutritive value of proteins including essential amino acids, food quality, decrease in digestibility, etc.

Methylglyoxal and other mutagenic compounds have been found in instant and caffeine-free coffee (Nagao et al., 1979), fried and grilled meat and fish due to formation of heterocyclic amines (Arvidsson et al., 1998).

Nakamura et al., (2008) reported reduction in allergenicity of buckwheat Fag e1 allergen as a result of glycosylation by arabinogalactans and xyloglucans. They suggested shielding of epitope by covalently attached polysaccharide chain and partial denaturation of epitope during

Maillard reaction resulted in neoallergens. Cucu et al., (2012) found that some patients with systemic allergic reaction to hazelnut are at risk when exposed to hazelnut proteins in processed food. These investigators reported a change in IgE binding activity and basophil activation by allergen were observed due to Maillard reaction.

Food industry desires to produce controlled aromas and colors in the processed foods.

Color formation depends mainly on the precursors, thermal processing parameters, pH and ratio of amino nitrogen to reducing sugar (Martins et al., 2000). The extent of browning by Maillard reaction is measured at 420 nm (Martins et al., 2000). At pH> 7, Amadori Rearrangement

Products (ARPs) such as acetic acid, pyruvaldehyde, and other lower sugars are produced in addition to free amino acids. Therefore it is the main pathway for flavour formation (Yaylayan and Huyghues-Despointes, 1996). For instance, the reaction of L-leucine and D-glucose is important in the formation of some volatile compounds for the flavour of cocoa (Hartman, 1983;

Renn and Sathe, 1997).

Influence of process parameters: Temperature, pH, water activity and molar ratios of sugar and amino acids can affect the rate and extent of Maillard browning (Renn and Sathe,

1997).

Temperature- Rate of the reaction is strongly influenced by temperature of reaction system (exponential relationship), and is described by Arrhenius equation:

k= A exp (-Ea/RT), where

A= frequency factor or pre-exponential factor and is the number of collisions in a reaction and the orientation of molecules in that reaction,

Ea= Activation energy, J/mol

R= gas constant (8.3 J/mol/K), and

T= absolute temperature (K)

pH- Rate of the reaction is directly proportional to the pH of reaction system. At high pH, open chain form of sugar and unprotonated form of amino groups are favoured and hence

Maillard reaction.

Water Activity (Aw) - Different components of foods may have different water contents at equilibrium, but have same Aw. And thus relative reaction rates are related to Aw. (Karel and

Buera, 1994). Maillard reaction is favoured in Aw range of 0.3-0.7 (Karel, 1960; Renn and

Sathe, 1997).

CHAPTER 3

MATERIALS AND METHODS

Nonpareil whole almonds were provided by the Almond Board of California (Modesto,

CA). These almonds were grown in Stewart and Jasper Orchards, California. Other tree nuts, seeds, confectionery ingredients, commercial products including cookies, chocolates, granola bars, cereals, spices, dairy products and trail mixes were purchased from the local grocery stores for this study. Required chemicals and reagents were purchased from commercial suppliers:

Anti-mouse IgG (whole molecule) - Alkaline phosphatase (AP) antibody produced in goat

(A3652, Lot. 050M6016, 2.8 mg/ml), horseradish peroxidase (HRP) labelled goat anti-rabbit

IgG, Goat anti-mouse IgG (whole molecule) Peroxidase conjugate antibody develop in goat

(A4416 0.8 mg/ml), p-nitrophenyl phosphate (Disodium Salt), Ponceau S (P3504, practical grade, 3-Hydroxy-4-(2-sulfo-4-[sulfophenylazo]phenylazo)-2,7-naphthalenedisulfonic acid sodium salt), luminol (97.0%) and bovine serum albumin [Sigma Chemical Co. (St. Louis,

MO)], electrophoresis and immunoblotting supplies [Hoefer Scientific Co. (San Francisco, CA)] and [Fisher Scientific Co. (Pittsburgh, PA)], BioTek PowerWaveTM 200 Microplate Scanning

Spectrophotometer and KC4TM Software [Bio-Tek Instruments, Inc. (Winooski, VT)], pH meter

[Corning® Inc. (Lowell, MA)], UltrospecTM 2100 pro UV-Visible Spectrophotometer [GE

Healthcare (Piscataway, NJ)], cellulose extraction thimbles (25 mm × 100 mm), Whatman filter paper No. 4 [Whatman international Ltd., Maidstone, UK], Protran® nitrocellulose membranes

(NC, 0.β μm, β00 × γ m) [Schleicher & Schuell Bioscience, Inc. (Keene, NH)], X-ray films

(BioMax XAR film) [Eastman Kodak Co. (Rochester, NY)], Western Re-ProbeTM for Stripping and Re-Probing western blots [G-Biosciences, (St. Louis, 28 MO)], acrylamide, Chemzymes

Ultra PureTM [Polysciences, Inc. (Warrington, PA)], TEMED (N, N, N’ N’-

16 tetramethylenediamhe) and Bis-acrylamide [BioRad, (Hercules, CA)], ninety-six well polyvinyl microtiter ELISA plates [Costar (Corning, NY)]. All other chemicals were purchased from

Sigma Chemical Co. (St. Louis, MO), Fisher Scientific Co. (Pittsburgh, PA) or VWR (Atlanta,

GA) and unless otherwise stated, and were of reagent or better grade. The rabbit polyclonal antibodies (pAbs) against almond protein were produced in 1999 (Martin Acosta) and the murine monoclonal antibodies (mAbs), 4C10 (145.04 µg/ml), 4F10 (88.24 µg/ml) and 2A3 (28.68

µg/ml) were produced in 1998, 2004, and 2007 in the Biomedical Research Facility [Florida

State University (FSU)] and Hybridoma Core Facility (Department of Biological Science, FSU) by Dr. Mahesh Venkatachalam, Dr. Jason Robotham, Dr. Harshal Kshirsagar, Dr. Girdhari

Sharma, and Shyamali Jayasena under the direction of Profs. Sathe and Roux as well as the assistance from Dr. K. Harper and Mrs. Pushparani Dhanarajan. The cell lines used for fusion in monoclonal antibody production was NS-1(ATCC® TIB-18™, Manassas, VA). And standard medium for cell growth/antibody production included DMEM (GIBCO 11965), 10% FBS

(Sigma), 1X ABAM 100X (GIBCO 15240), 1X Glutamine 100X (GIBCO 25030), and 2% conditioned medium (produced in-house). All mAbs were purified employing affinity column chromatography using Protein G (GE HealthCare Life Sciences, City, State) and systematically titrated for optimum signal prior to their use in the immunoassays by Drs. Mahesh

Venkatachalam and Mengna Su.

Sample Preparation- The matrices used for this study are listed in Table 3.1 and include both commercial (superscript ‘c’) and laboratory (superscript ‘l’) prepared products.

Table 3.1: List of food matrices for analysis. S. Food Matrix Name Description No. 1 Almond major protein (AMP)l Isoelectric precipitation (pH= 5.0), nonpareil, defatted

Table 3.1: Continued

S. Food Matrix Name Description No. A. Tree nuts and oil seeds 2 Whole almond Nonpareil, full-fat 3 Roasted almondl Processed, 375 °F, 12 min, full-fat 4 Roasted almondl Processed, 325 °F, 20 min, full-fat 5 Cashew Full-fat 6 Pecan Full-fat 7 Sunflower Full-fat 8 Sesame Full-fat 9 Peanut Unprocessed, #131 (1) 10 Brazil nut Full-fat 11 Hazelnut Full-fat, #80 12 Macadamia Full-fat, #93 13 Pine nut Full-fat, #137 14 Pistachio Full-fat, #138(1) 15 Walnut Full-fat, #180 (1) 16 Soybean Full-fat 17 Lupin Full-fat 18 Coconut Full-fat, # 51 (1) B. Bakery and Confectionery 19 Cookiel Lab made, w/o almond, full-fat 20 Almond cookiel Lab made, 0.5 % almond (w/w), full-fat 21 Almond cookiel Lab made, 1.0 % almond (w/w), full-fat 22 Almond cookiel Lab made, 2.0 % almond (w/w), full-fat 23 Almond cookiel Lab made, 5.0 % almond (w/w), full-fat 24 Shortbread cookiel Commercial, Keebler, Sandies®, w/o almond, full- fat 25 Dark chocolate almond cookiec Commercial, Keebler, Sandies®, w/ almond, full- fat 26 Sponge cakel Lab made, w/o almond, full-fat 27 Almond sponge cakel Lab made, 0.5 % almond (w/w), full-fat 28 Almond sponge cakel Lab made, 1.0 % almond (w/w), full-fat 29 Almond sponge cakel Lab made, 2.0 % almond (w/w), full-fat 30 Almond sponge cakel Lab made, 5.0 % almond (w/w), full-fat 31 White cakel Lab made, w/o almond, full-fat, # 182 (1) 32 Almond white cakel Lab made, w/ almond, full-fat, # 182 (2) 33 Marzipanl Lab made, full-fat 34 Chocolate, darkl Lab made, w/o almond, full-fat 35 Chocolate, dark w/almondl Lab made, 0.5 % almond (w/w) , full-fat 36 Chocolate, dark w/almondl Lab made, 1.0 % almond (w/w) , full-fat 37 Chocolate, dark w/almondl Lab made, 2.0 % almond (w/w) , full-fat

Table 3.1: Continued S. Food Matrix Name Description No. 38 Chocolate, dark w/almondl Lab made, 5.0 % almond (w/w) , full-fat 39 Chocolate, dark w/almondl Lab made, 10.0 % almond (w/w) , full-fat 40 Chocolate, darkc Commercial, Dove, w/o almond, full-fat, # 47 (2) 41 Chocolate, darkc Commercial, Hershey, w/o almond, full-fat, # 47(4) 42 Chocolate, dark w/almondc Commercial, Dove, full-fat, # 47 (6) 43 Chocolate, dark w/almondc Commercial, Hershey, full-fat, # 47 (7) 44 Chocolate, milkl Lab made, w/o almond, full-fat 45 Chocolate, milk w/almondl Lab made, 0.5 % almond (w/w) , full-fat 46 Chocolate, milk w/almondl Lab made, 1.0 % almond (w/w) , full-fat 47 Chocolate, milk w/almondl Lab made, 2.0 % almond (w/w) , full-fat 48 Chocolate, milk w/almondl Lab made, 5.0 % almond (w/w) , full-fat 50 Chocolate, milkc Commercial, Hershey, w/o almond, full-fat, # 47(11) 51 Chocolate, milk w/almondc Commercial, Hershey, full-fat, # 47(13) 52 Chocolate, whitel Lab made, w/o almond, full-fat 53 Chocolate, white w/almondl Lab made, 0.5 % almond (w/w) , full-fat 54 Chocolate, white w/almondl Lab made, 1.0 % almond (w/w) , full-fat 55 Chocolate, white w/almondl Lab made, 2.0 % almond (w/w) , full-fat 56 Chocolate, white w/almondl Lab made, 5.0 % almond (w/w) , full-fat 57 Chocolate, white w/almondl Lab made, 10.0 % almond (w/w), full-fat C. Bars and Brittles 58 Almond cashew barc Commercial, Planters Nut-rition bar, full-fat, # 14 59 Almond barc Commercial, Planters Nut-rition bar, full-fat, # 15 60 Almond brittlel Lab made, full-fat, # 27 (1) 61 Coconut brittlel Lab made, w/o milk, full-fat, # 27 (3) 62 Cashew brittlel Lab made, full-fat, # 27 (4) 63 Granola barl Lab made, full-fat, # 75 (3, 4, 5) 64 Granola oats and honeyc Commercial, Nature Valley, w/o almond, full-fat, # 75 (10) 65 Granola roasted almondc Commercial, Nature Valley, full-fat, # 75 (11) 66 Granola roasted cashewc Commercial, Nature Valley, full-fat, # 75 (12) 67 Granola peanut (br) and almondl Lab made, full-fat, # 75 (7) D. Cereals 68 Cornflakec Commercial, Kellogg’s®, defatted, # 57(1) 69 Cornflake type II Bl Lab made, coarse yellow corn meal, full-fat, # 57(4) 70 Cornflake type III Al Lab made, precooked white corn meal, full-fat, # 57(5) 71 Cornflakel Lab made, w/o almond, full-fat 72 Cornflake w/almondl Lab made, 1% almond (w/w), full-fat 73 Barley Defatted, # 17(1) 74 Basi ragi Defatted, #18

Table 3.1: Continued S. Food Matrix Name Description No. 75 Buckwheat Full-fat, # 31(1) 76 Bulgur wheat Defatted, # 32 77 Cereal, 7 grain Commercial, Publix®, full-fat, #39 78 Oat Commercial, Quaker® oats, full-fat , # 122 (1) 79 Millet Defatted, # 99(1) 80 Quinoa Defatted, # 146 81 Rice Defatted, # 150 (1) 82 Rye Full-fat, # 153 83 Semolina Commercial, Bob’s Red Mill®, full-fat , # 159 84 Sorghum Defatted, # 164 85 All-purpose flour Full-fat , # 181 (3) E. Dairy 86 Non-fat dry milk* Commercial, Saco mix’ n drink® # 98 (2) 87 Whole milk Commercial, Publix®, full-fat, 88 Skimmed milk Commercial, Publix®, 0 % fat F. Colors 89 Color, blue 1 Commercial, Warner Jenkinson Company, Inc., # 54 (1) 90 Color, blue 2 Commercial, Commercial, Warner Jenkinson Company, Inc., # 54 (2) 91 Color, green 3 Commercial, Commercial, Warner Jenkinson Company, Inc., # 54 (3) 92 Color, red 3 Commercial, Commercial, Warner Jenkinson Company, Inc., # 54 (4) 93 Color, red 40 Commercial, Commercial, Warner Jenkinson Company, Inc., # 54 (5) 94 Color, yellow 5 Commercial, Commercial, Warner Jenkinson Company, Inc., # 54 (6) 95 Color, yellow 6 Commercial, Commercial, Warner Jenkinson Company, Inc., # 54 (7) G. Additives and Spices 96 Baking soda Commercial, Arm and Hammer®, full-fat, # 12 97 Brown sugar, dark Defatted, # 29 (1) 98 Sugar Commercial, Domino®, full-fat, # 168 99 Cardamom Defatted, # 34 100 Cinnamon Commercial, McCormick®, full-fat, # 48 (1) 101 Cloves Defatted, # 49 102 Aniseed Defatted, # 6 103 Cumin Defatted, # 61 104 Garlic Defatted, # 73 105 Ginger Defatted, # 74 106 Mustard Defatted # 118 107 Nutmeg Defatted, # 121 (2)

Table 3.1: Continued S. Food Matrix Name Description No. 108 Parsley Defatted, # 128 109 Salt Defatted, # 156 (2) 110 Turmeric Defatted, # 176 111 Vanilla extract Commercial, McCormick®, full-fat, # 179 112 Egg Full-fat H. Fruits 113 Apple Full-fat, # 8 (1) 114 Cherry Defatted, # 4 115 Plum Full-fat I. Others 116 Mix, kheerc Commercial, Shan, w/o almond, # 111 117 Mix, badam kheerc Commercial, Shan, full-fat, full-fat, # 102 118 Trail mixl Lab made, full-fat, # 173 (1) 119 Trail mix, Alaskan almond w/o Commercial, Planters, full-fat, # 173 (4) cashewc 120 Trail mix, Appalachian almond Commercial, Planters, full-fat, # 173 (5) w/o cashewc 121 Trail mix, breakfast almond w/o Commercial, Emerald, full-fat, # 173 (6) cashewc 122 Trail mix, raisin almond cashewc Commercial, Planters, full-fat, # 173 (8) 123 Trail mix, tropical cashew w/o Commercial, Emerald, full-fat, # 173 (9) almondc 124 Trail mix spiked w/almondl Lab made, #173 (1) spiked with 10% almond (w/w)

Isolation of Almond Major Protein (AMP)

AMP was prepared by isoelectric precipitation (pH- 5.0) as described by Wolf and Sathe,

1998. Briefly, 40 mesh defatted almond flour was dissolved in 0.1 M Borate Saline Buffer

(BSB, pH- 8.45) for 1h at RT and then centrifuged at 12,600 x g (4°C) using a J10 rotor in

Beckman model J2-21 centrifuge (California, USA) for 30 min. The pellets of amandin were collected, dissolved in distilled water (1:10 w/v) and pH adjusted to 8.3-8.4 using 1M NaOH and constantly stirred using magnetic stirrer (Sodium azide was added to prevent any contamination).

The reconstituted protein was then stirred for 30 min and transferred to Spectra/Por® 6 dialysis

21 membrane (MWCO- 8000) (Spectrum Labs, Inc., California, USA). Dialysis was carried out for two days and distilled water was changed in between for about 6-8 times. The dialysed amandin was then freeze dried using freeze drier (VirTis bench top k, SP Industries, Warminster, PA), powdered using a pestle and mortar and stored at -20 °C until further use.

Protein Extraction

All the samples were ground to flour (~ 40 mesh) and 100 mg of each sample was extracted in 1.0 ml of 0.1 M Borate Saline Buffer (BSB, pH- 8.45) for 1h at RT with constant mechanical shaking (Vortex Genie 2, VWR Scientific, Atlanta, GA) followed by centrifugation

(16100 g, 10 min, RT) as described by Su, 2012.

HunterLab LabScan XE Spectrophotometer

The method of Sharma et al. (2010) was used. Samples were placed in the glass sample cup (2.5 in. diameter, part 04-7209-00) of the LabScan XE spectrocolorimeter (Hunter

Associates Laboratory, Reston, VA) using 1 inch (diameter) view with 0°/45° geometry and 10° observer. Care was taken to ensure the sample cup bottom was completely covered with the sample. The L*, a*, and b* values were measured with EasyMatch QC software (version 3.90) using the 1 inch sample view port A (25), and the average values of four measurements, two readings for each of the duplicate preparations were reported.

Water Activity (Aw)

The water activity of all the samples was measured at RT using a water activity meter

(Decagon Pawkit, VWR). The hygrometer was calibrated using 6 molal sodium chloride solution

(Aw-0.760) (http://www.aqualab.com/assets/Uploads/Pawkit-Manual.pdf; accessed on August

15, 2013). Samples were placed in sample cup and the sensor port of hygrometer was placed on the top of cup to take a 5 min. reading. All samples were measured in triplicates on the same day.

Soluble Protein Content

Bradford Protein Determination

The method of Bradford (1976) was used to determine the soluble protein content. 10 µL of the standard (0, 50, 100, 200, 300, 400, 500, and 600 µg BSA/mL of solvent) and samples

(containing 50 to 600 µg of protein) were pipetted into a microtiter plate. 200 µL of diluted (5X)

Bradford reagent (0.05% w/v Coomassie Brilliant Blue G-250, 25% v/v ethanol, 50% v/v o- phosphoric acid in distilled water) was added to the wells and plate will be incubated for 5 min at room temperature. Absorbance was read at 595 nm using a PowerWave 200 microplate scanning spectrophotometer (BioTek Instruments, Inc., Vermont). The soluble protein content was determined from the BSA standard curve.

Lowry Protein Assay

The method of Lowry et al. (1951) was used to determine the soluble protein content. 1 mL of the standard (0, 20, 40, 60, 80, 100, 120, 140, 160, 180, and 200 µg BSA per mL of solvent) and samples (containing 20 to 200 µg of protein) were pipetted into small test tubes. 3 mL of Reagent C (100 parts Reagent A with 1 part Reagent B) was added and test tubes were incubated at room temperature for 10 to 60 min without change in final absorbance. Reagent A is

2% w/v Na2CO3, 0.16% w/v potassium tartrate in 0.1 N NaOH. And reagent B is 4% w/v

CuSO4∙5H2O in distilled water. Then, 0.3 mL of diluted phenol reagent (dilute Folin-Ciocalteu’s phenol reagent (2N) 1:1 v/v with distilled water) was added and vortexed immediately. Test tubes were incubated in dark for 45 min at room temperature and the absorbance was recorded at

660 nm using an Ultrospec 2100 pro UV-Vis Spectrophotometer (Amersham, Pharmacia

Biotech, Sweden). The soluble protein content was determined from the standard curve.

Immunoassays

Dot Blot

The method of Venkatachalam et al. (2002) was used. Circles were drawn on a 0.ββ μm nitrocellulose membrane (Protran®, Whatman) using a pencil and a template plate. 1 µL of samples containing 1 mg/mL protein was pipetted within each circle. The labeled membrane was placed on a filter paper and allowed to air dry. The membrane was then blocked with 5% w/v non-fat dry milk (NFDM) in TBST (10 mM Tris, 0.9% w/v NaCl, 0.05% v/v Tween 20, pH 7.6) at room temperature for 1 h. The membrane was washed with 3 changes of TBST for 5 min each at room temperature. Then the membrane was incubated with suitably diluted antibodies (in

TBST) overnight at 4 °C on a rocker. The membrane was again washed with 3 changes of TBST for 15 min each at room temperature and incubated with horseradish peroxidase labeled goat anti-mouse IgG (secondary antibody) for 1 h at room temperature on a rocker and washed with 3 changes of TBST for 15 min each at room temperature. A mixture of freshly prepared solution A

(luminol stock 100 μL, p-coumaric acid stock 44 μL, 1 M Tris-HCl (pH 8.5) 1 mL, distilled water: 8.85 mL) and B (30% v/v H2O2 6 μL, 1 M Tris-HCl (pH 8.5) 1 mL, distilled water: 8.85 mL) (1:1 v/v) was spread evenly to cover the entire membrane and left for 5 min. The membrane was dried and covered with a transparent plastic wrap and exposed to an X-ray film for autoradiographic visualization.

Enzyme-Linked Immunosorbent Assay (Sandwich ELISA)

The method of Venkatachalam et al. (2002) was used. A 96-well “U” bottom microtiter plate (Serocluster ™, Costar; Corning, NY) was coated with 50 µL/well of rabbit anti whole almond 99R20 pAb (polyclonal antibody) (604 ng/well, 1:5000 dilution) in coating buffer (48.5 mM Citric acid, 103 mM Disodium phosphate (Na2HPO4) pH-5.0) and incubated at 38 °C for 2 h. 200 µL/well of 5% w/v of NFDM in TBST (10 mM Tris, 0.9% w/v sodium chloride, 0.05% v/v Tween 20, pH 7.6) was added to block the pAb and incubated for 1 h at 38 °C. Unblocked

NFDM was dumped and the plate was then washed for 3 times with TBST. 90 µL/well of sample protein of suitable concentration based on Bradford protein assay (Antigen) in 1% w/v of NFDM in TBST were added to the plate and incubated for 2 h at 38 °C. Leftover sample in plate was dumped and washed with TBST for 3 times. 50 µL/well of mAb 4C10 (1:1200)/4F10 (1:500)/

2A3 (1:100) (primary antibody) in 1% w/v of NFDM in TBST was loaded in the plate and incubated for 1 h at 38 °C. The unbound was again dumped followed by 3 times washing with

TBST. 50 µL/well of alkaline phosphatase-labeled goat anti-mouse IgG pAb (28 ng/well)

(1:5000) in 1% w/v NFDM in TBST was loaded in the plate and incubated for 1 h at 38 °C. The unbound was again dumped followed by 3 times washing with TBST. 50 µL/well of substrate p- nitrophenyl phosphate (pNPP) in substrate buffer (0.0049% w/v magnesium chloride, 0.096% diethanolamine, pH 9.8) was loaded in the plate and incubated for 30 min at 38 °C (covered with aluminum foil to avoid light). 50µL/well of 3 M NaOH was added to stop the color reaction. The absorbance of microtiter plate was read using PowerWave 200 microplate scanning spectrophotometer (BioTek Instruments, Inc., Vermont) at 405 nm.

For all sandwich ELISAs, a 4X serial dilution of whole almond was prepared from 8000-

0.488 ng/ml in each plate and the samples were diluted to ~125 ng/ml based on the soluble

25 protein values estimated by Bradford assay. The selected concentration of samples was used to avoid low intensity signals due to presence of other components in the food matrices and also to compare directly with 125 ng/ml signal value from whole almond standard curve.

SDS-PAGE

The method of Fling and Gregerson (1986) was used. The separating gels were made by mixing 8% and 25% acrylamide solutions (15 mL each) using a gradient maker and peristaltic pump. For monomer gels, 12% resolving gel solution was filled in the gel cassette. 4% stacking gel was prepared on top of the solidified resolving gel. The recipe for making gels is listed in

Table 3.2. Sample proteins were mixed with suitable volumes of sample buffer (50 mM Tris-

HCl, 1% w/v SDS, 0.01% w/v bromophenol blue, γ0% v/v glycerol, and/or β% - mecaptoethanol, pH 6.8) to make final concentration of 0.50mg/ml of each sample and heated for 10 min in a boiling water bath. 40 µl of protein samples were loaded on the gels along with standard molecular weight marker. The gels were run in running buffer (50 mM Tris-HCl, 190 mM glycine, 0.1% w/v SDS, pH 8.7-8.8) at a constant current (8-12 mA/gel) until the tracking dye reached the gel edge.

Table 3.2: Gradient gel recipe 8% resolving gel 25% resolving gel 4% stacking gel

Lower/upper stock (mL) 7.5 (lower) 7.5 (lower) 2.5 (upper)

Acrylamide stock (mL) 2.4 7.5 0.8

Distilled water (mL) 5.1 0 6.64

APS (μL) 50 50 50

TEMED (μL) 10 10 10

 Acrylamide stock: 50% w/v acrylamide, 1.35% w/v bis-acrylamide

 Lower stock: 1.5 M Tris-HCl, 0.2% w/v SDS, pH 8.8

 Upper stock: 0.5 M Tris-HCl, 0.4% w/v SDS, pH 6.8

 APS: 10% w/v ammonium persulfate

Western Blot

The method of Venkatachalam et al. (2002) was used. Following SDS-PAGE, proteins were transferred onto a 0.ββ μm nitrocellulose membrane in Towbin buffer (β5 mM Tris, 19β mM glycine, 20% v/v methanol, 0.1% w/v SDS, pH 8.4; small gel: 1300 mL, big gel: 5 L) at 1

A/tank for 4 h. Stacking order used were black frame / sponge/ blotting paper / gel / membrane / blotting paper / sponge / white frame. The transferred polypeptides were visualized by Ponceau S staining (0.1% w/v for 5 min) and the membrane was scanned and blocked with 5% w/v NFDM in TBST at room temperature for 1 h. Then the membrane was washed with 3 changes of TBST for 5 min each at room temperature and incubated with suitably diluted antibodies (in TBST) overnight at 4 °C on a rocker. In each well, 20 µg of sample protein was loaded and the 4C10,

4F10 and 2A3 were used in 1:2000 dilutions in TBST buffer during all incubations. The membrane was again washed with 3 changes of TBST for 15 min each at room temperature and incubated with horseradish peroxidase labeled goat anti-mouse IgG (1:10000 in TBST) for 1 h at room temperature on a rocker. The membrane was washed with 3 changes of TBST for 15 min each at room temperature. Equal volumes of freshly prepared solution A (luminol stock 100 μL, p-coumaric acid stock 44 μL, 1 M Tris-HCl (pH 8.5) 1 mL, distilled water: 8.85 mL) and B (30% v/v H2O2 6 μL, 1 M Tris-HCl (pH 8.5) 1 mL, distilled water: 8.85 mL) (1:1 v/v) were mixed and spread evenly to cover the entire membrane and left for 5 min. The membrane was dried and

27 covered with a transparent plastic wrap, and exposed to an X-ray film for autoradiographic visualization. The exposed film was developed by immersing in a developer (1:5 in water) (GBX developer, Carestream®, Kodak®, VWR, Atlanta, GA) followed by rinsing in water and fixed in a fixer solution (1:5 in water) (GBX fixer, Carestream®, Kodak®, VWR, Atlanta, GA), rinsed again in water and air dried.

Statistical Analysis

Appropriate data was statistically analysed (SPSS 21.0 Inc., Chicago, IL; 2012). Data were reported as the mean ± SEM. All experiments were carried out at least in duplicates.

Fisher’s least significant difference test as described by Ott et al. (1977) were used to determine statistical significance, and results were considered to be significant if p ≤ 0.05.

CHAPTER 4

RESULTS AND DISCUSSION

Water Activity (Aw) of Food Matrices

All the processed food matrices including the dry roasted almonds, bakery and confectionary products, and bars and brittles containing whole almond flour were examined physically for the occurrence of Maillard reaction by measuring the water activity and color

(Hunter L, a, b scale). The Maillard reactions depend strongly on the water activity (Aw) and take place at Aw as low as 0.3 and upto 0.8 with maximum rate of reaction at Aw between 0.5-

0.6 (Fig 4.1) (Labuza and Baiser, 1992). The Aw ranges from 0.37-0.80 (n=3) for the tested products in the present study (Table 4.1) and therefore is in the range of optimum Aw for

Maillard reaction to occur. Although the sugar used to prepare these food matrices was sucrose, but at temperatures above 70 °C, hydrolysis of sucrose into equal moles of glucose and fructose occurs even at pH 7 during typical processing times (Davies and Labuza, 1997).

Color of Food Matrices

The Hunter L, a, b scale (Opponent-color scales) gives measurements of color in units of approximate visual uniformity throughout the color solid. L* measures lightness and varies from

100 for perfect white to zero for black. The chromaticity dimensions, a measures redness when positive, gray when zero, and greenness when negative while b measures yellowness when positive, gray when zero, and blueness when negative. Color in Maillard reaction occurs due to the formation of high molecular weight (>12, 000 Daltons) polymeric compounds also known as melanoidins (Davies and Labuza, 1997). In the present study, the L* values (Table 4.1) for the tested food matrix flours (n=4) was compared to the roast color value scale, Light=53±1,

Medium=48.5±1, Dark=43±1 to determine the lightness and darkness of the product (McDaniel et al., 2012). The cookies, cakes, almond brittles, granola with roasted almond and white chocolates had L* values above 53.00 and were light roasted, granola peanuts and almond, and granola control were near 48.00 and thus medium roasted color while milk and dark chocolates were in the dark roast color (L* < 43.00) due to the polyphenols and not the processing. And a* and b* values (Table 4.1) exhibit more redness and yellowness in the products indicating

Maillard browning except in white chocolates due to the natural color which had green color on a* scale. The L*, a*, b* values for the Maillard product containing almonds in this study were similar to those reported by Ajila et al. (2008) in soft dough biscuits using LabScan XE.

Therefore the browning of the selected food matrices can be attributed to Maillard reaction.

Figure 4.2 exhibits the occurrence of Maillard reaction in the selected processed food matrices.

Based on the pattern of the relationship between the L* values and the Aw, we can see that at

Aw between 0.5-0.6 (maximum rate of Maillard reaction), the L* value is in the dark range (<

43). And for the Aw near 0.3 and 0.8, where the rate of reaction decreases, the L* value is in the light range (> 53). The observed relationship is in agreement with the theoretical concept of occurrence of Maillard reaction and Aw. Therefore, the browning of the selected food matrices can be attributed to Maillard reaction.

Table 4.1: Water activity (Aw at 25 °C) and browning of food matrices containing almonds.Superscripts denote significant difference from control (whole almond): a =water activity, xyz= L*, a* and b* values. Food Matrix Name Aw L* a* b*

Mean ± S.D Mean ± S.D Mean ± S.D Mean ±S.D

Whole Almond 0.51 ± 0.01 66.65 ± 0.43 5.66 ± 0.10 22.57 ±0.04

Bakery and Confectionery

Cookiel, w/o almondaz 0.37 ± 0.02 70.80 ± 0.18 5.26 ± 0.13 30.56 ±0.21

Table 4.1: Continued Food Matrix Name Aw L* a* b*

Mean ± S.D Mean ± S.D Mean ± S.D Mean ± S.D

Cookiel, w/o almondaz 0.37 ± 0.02 70.80 ± 0.18 5.26 ± 0.13 30.56 ± 0.21

Almond cookiel (0.5 %)az 0.37± 0.01 69.56 ± 0.09 5.61 ± 0.04 29.42 ± 0.08

Almond cookiel (1.0 %)ayz 0.37 ± 0.02 62.13 ± 0.07 9.75 ± 0.03 33.05 ± 0.06

Almond cookiel (2.0 %)az 0.39 ± 0.01 69.62 ± 0.04 6.28 ± 0.03 30.55 ± 0.02

Almond cookiel (5.0 %)az 0.39 ± 0.02 66.58 ± 0.05 7.82 ± 0.02 31.23 ± 0.03

Shortbread cookiec, w/o 0.39 ± 0.01 67.75 ± 0.05 7.18 ± 0.00 29.45 ± 0.05 almondaz

Dark chocolate almond 0.43 ± 0.02 41.39 ± 0.13 9.63 ± 0.01 20.37 ± 0.02 cookiecaxy

Sponge cakelayz 0.70 ± 0.02 74.19 ± 0.24 1.77 ± 0.30 26.06 ± 0.88

Almond sponge cakel (0.5 0.76 ± 0.02 74.36 ± 0.15 1.52 ± 0.63 28.31 ± 1.45

%)ayz

Almond sponge cakel (1.0 0.79 ± 0.01 73.23 ± 0.05 0.91 ± 0.07 27.64 ± 0.13

%)ayz

Almond sponge cakel (2.0 0.79 ± 0.01 73.60 ± 0.06 1.51 ± 0.06 28.06 ± 0.14

%)ayz

Almond sponge cakel (5.0 0.75 ± 0.03 75.63 ± 0.11 3.17 ± 0.02 26.71 ± 0.04

%)a

White cakel, w/o almondax 0.43 ± 0.01 83.30 ± 0.09 3.14 ± 0.09 19.08 ± 0.17

Almond white cakela 0.45 ± 0.01 75.91 ± 0.12 4.92 ± 0.02 23.71 ± 0.21

Chocolate darkl w/oalmondaxz 0.54 ± 0.01 32.88 ± 1.25 6.87 ± 0.38 11.02 ± 0.79

Table 4.1: Continued Food Matrix Name Aw L* a* b*

Mean ± S.D Mean ± S.D Mean ± S.D Mean ± S.D

Chocolate, dark w/almondl 0.56 ± 0.00 31.99 ± 2.75 8.66 ± 0.87 12.56 ± 2.13

(0.5 %)axyz

Chocolate, dark w/almondl 0.62 ± 0.01 34.49 ± 0.03 8.85 ± 0.01 13.09 ± 0.01

(1.0 %)axyz

Chocolate, dark w/almondl 0.58 ± 0.02 32.55 ± 2.00 8.59 ± 0.83 12.25 ± 1.75

(2.0 %)axz

Chocolate, dark w/almondl 0.57 ± 0.01 32.53 ± 2.17 8.71 ± 0.79 11.77 ± 1.94

(5.0 %)axyz

Chocolate, dark 0.58 ± 0.01 31.75 ± 1.48 8.62 ± 0.83 11.53 ± 1.56 w/almondl(10.0 %)axz

Chocolate, milkl, w/o 0.50 ± 0.01 39.24 ± 2.44 8.95 ± 0.62 14.50 ± 2.00 almondaxyz

Chocolate, milk w/almondl 0.52 ± 0.00 38.86 ± 1.49 9.76 ± 0.41 15.00 ± 1.06

(0.5 %)xyz

Chocolate, milk w/almondl 0.51 ± 0.01 42.01 ± 1.47 9.77 ± 0.10 15.80 ± 0.84

(1.0 %)xyz

Chocolate, milk w/almondl 0.52 ± 0.02 40.90 ± 0.95 9.79 ± 0.27 15.62 ± 0.61

(2.0 %)xyz

Chocolate, milk w/almondl 0.53 ± 0.01 39.77 ± 1.78 9.91 ± 0.42 15.03 ± 1.25

(5.0 %)axyz

Table 4.1: Continued Food Matrix Name Aw L* a* b*

Mean ± S.D Mean ± S.D Mean ± S.D Mean ± S.D

Chocolate, milk w/almondl 0.54 ± 0.01 41.14 ± 1.66 10.40 ± 0.30 16.78 ± 0.99

(10.0 %)axyz

Chocolate, whitel, w/o 0.48 ± 0.01 85.06 ± 0.16 -2.10 ± 0.03 22.89 ± 0.25 almondaxy

Chocolate, white w/almondl 0.48 ± 0.01 85.28 ± 0.90 -2.02 ± 0.08 17.62 ± 0.31

(0.5 %)axyz

Chocolate, white w/almondl 0.48 ± 0.01 82.13 ± 1.10 -2.29 ± 0.06 14.49 ± 0.19

(1.0 %)axyz

Chocolate, white w/almondl 0.48 ± 0.00 84.06 ± 0.31 -1.95 ± 0.01 16.90 ± 0.12

(2.0 %)axyz

Chocolate, white w/almondl 0.49 ± 0.01 85.26 ± 0.33 -1.20 ± 0.14 17.50 ± 0.68

(5.0 %)axyz

Chocolate, white w/almond 0.50 ± 0.01 82.01 ± 1.76 -0.97 ± 0.06 16.54 ± 1.05

(10.0%)xyz

Bars and Brittles

Almond cashew barcaxyz 0.57 ± 0.02 54.49 ± 0.08 9.26 ± 0.04 30.69 ± 0.08

Almond barcxyz 0.52 ± 0.01 53.11 ± 0.11 9.71 ± 0.03 28.37 ± 0.07

Almond brittlelaz 0.41 ± 0.01 73.81 ± 0.06 3.22 ± 0.05 16.10 ± 0.07

Granola barlaxyz 0.48 ± 0.01 48.60 ± 0.19 12.17 ± 0.11 31.88 ± 0.20

Granola oats and honeycaz 0.49 ± 0.00 76.88 ± 0.16 4.67 ± 0.05 17.79 ± 0.12

Granola roasted almondcaxyz 0.56 ± 0.02 82.05 ± 0.04 2.49 ± 0.01 12.78 ± 0.03

Table 4.1: Continued Food Matrix Name Aw L* a* b*

Mean ± S.D Mean ± S.D Mean ± S.D Mean ± S.D

Granola peanut (br) and 0.45 ± 0.02 49.15 ± 0.04 9.30 ± 0.03 23.99 ± 0.07 almondlaxy

LSD (p≤0.05) 0.020 11.68 3.00 4.45

Fig 4.1: Relationship between the L* and the Aw of the processed food matrices. Some of the outliers’ circled red and green are due to the natural product color viz. white chocolate and dark chocolate. y = 551.7x2 - 644.7x + 240.23; R² = 0.2002.

Effect of Maillard Reaction and Food Matrix on the Immunoreactivity of Amandin

The selected food matrices were categorised in nine groups based on their types (Table

3.1). The list included a variety of foods to determine the specifity, sensitivity and robustness of the mAb based sandwich ELISA (Su, 2012) (for cross-reactivity with non-almond matrices) and the effect of type of food matrices, processing and Maillard reaction on immunoreactivity of almond major protein. Three murine mAbs 4C10, 4F10 and 2A3, specific for amandin detection, which can sensitively detect both native and denatured amandin were used for detecting amandin

34 immunoreactivity of full fat almond containing food matrices. The mAb 4C10 has been reported to recognise conformational epitope on amandin while 4F10 and 2A3 can recognise independent linear epitopes on amandin (Su, 2012). Therefore, the effect of processing of food matrices and

Maillard reaction as a result of protein and sugar interaction on the epitope structure and stability could be assessed using these three mAbs. Heat denaturation typically decreases protein solubility (Sathe et al., 1989) therefore all protein extracts were suitably diluted to contain

1mg/ml prior to all the assays. The soluble protein concentration of the sample protein extracts as determined by Bradford protein assay were used for immunoassay data collection.

Sandwich ELISAs

From Tables 4.2, 4.3 and 4.4, we can see that all three murine mAbs did not cross-react with the samples # 5-18, 61-64, 66, 68-71, 73-115. These samples did not contain almonds and did include tree nuts and oil seeds, tree nuts incorporating foods, and cereals, several food additives, spices, and fruits. The results of these assays indicated that the mAbs correctly detected the presence of almonds in tested samples (i.e., no false positives or negatives). Similar results for no cross-reactivity of these mAbs were reported by Su (2012). Immunoreactivity was calculated as, R = (protein concentration of sample for 50% of the maximum signal for the corresponding standard curve)/ (protein concentration required to register 50% of the maximum signal for the standard curve).

Table 4.2: Sandwich ELISA using 4C10. Immunoreactivity (R) of selected food matrices compared with whole almond by sandwich ELISA using whole almond 99R20 pAb (604 ng/well) as capture antibody and 4C10 (6.05 ng/well) as detection antibody. (N.D- Not detected). * denotes that the mean R of sample is significantly lower than unprocessed almond (control). S. Food Matrix Name R 1 R 2 R 3 R No. Mean ± SEM

1 Almond major protein (AMP)l 0.98 0.96 0.92 0.95 ± 0.02

Table 4.2: Continued

S. Food Matrix Name R 1 R 2 R 3 R No. Mean ± SEM

A. Tree nuts and oil seeds 2 Whole almond 1.00 1.00 1.00 1.00 ± 0.00 3 Roasted almondl* 2.62 2.84 3.54 3.00 ± 0.28 4 Roasted almondl 0.70 0.66 0.67 0.67 ± 0.01 5 Cashew N.D N.D N.D N.D 6 Pecan N.D N.D N.D N.D 7 Sunflower N.D N.D N.D N.D 8 Sesame N.D N.D N.D N.D 9 Peanut N.D N.D N.D N.D 10 Brazil nut N.D N.D N.D N.D 11 Hazelnut N.D N.D N.D N.D 12 Macadamia N.D N.D N.D N.D 13 Pine nut N.D N.D N.D N.D 14 Pistachio N.D N.D N.D N.D 15 Walnut N.D N.D N.D N.D 16 Soybean N.D N.D N.D N.D 17 Lupin N.D N.D N.D N.D 18 Coconut N.D N.D N.D N.D B. Bakery and Confectionery 19 Cookiel N.D N.D N.D N.D 20 Almond cookiel* (0.5 %) 3.52 4.94 4.31 4.26 ± 0.41 21 Almond cookiel (1.0 %) 1.81 1.06 1.24 1.37 ± 0.23 22 Almond cookiel (2.0 %) 1.27 1.31 1.26 1.28 ± 0.02 23 Almond cookiel (5.0 %) 0.76 0.79 0.78 0.77 ± 0.01 24 Shortbread cookiec N.D N.D N.D N.D 25 Dark chocolate almond cookiec* 2.23 1.80 1.90 1.98 ± 0.13 26 Sponge cakel N.D N.D N.D N.D 27 Almond sponge cakel* (0.5 %)* 5.77 6.38 6.14 6.10 ± 0.18 28 Almond sponge cakel* (1.0 %) 3.24 3.45 3.59 3.43 ± 0.10 29 Almond sponge cakel (2.0 %) 2.43 2.82 2.65 2.63 ± 0.11 30 Almond sponge cakel (5.0 %) 2.12 2.16 2.15 2.14 ± 0.01 31 White cakel N.D N.D N.D N.D 32 Almond white cakel 2.57 2.62 2.67 2.62 ± 0.03 33 Marzipanl 2.37 2.42 2.46 2.42 ± 0.03 34 Chocolate, darkl N.D N.D N.D N.D 35 Chocolate, dark w/almondl*(0.5 %) 9.49 10.78 9.88 10.05 ± 0.38

Table 4.2: Continued

S. Food Matrix Name R 1 R 2 R 3 R No. Mean ± SEM

36 Chocolate, dark w/almondl*( 1.0 %) 9.09 9.73 10.78 9.87 ± 0.49 37 Chocolate, dark w/almondl*(2.0 %) 7.41 6.62 7.52 7.18 ± 0.28 38 Chocolate, dark w/almondl*(5.0 %) 4.14 5.22 5.41 4.92 ± 0.4 39 Chocolate, dark w/almondl* (10.0 %) 5.12 4.15 4.41 4.56 ± 0.29 40 Chocolate, darkc, Dove N.D N.D N.D N.D 41 Chocolate, darkc, Hershey N.D N.D N.D N.D 42 Chocolate, dark w/almondc, Dove 1.49 1.42 1.44 1.45 ± 0.02 43 Chocolate, dark w/almondc, Hershey 1.41 1.43 1.40 1.42 ± 0.01 44 Chocolate, milkl N.D N.D N.D N.D 45 Chocolate, milk 5.55 5.65 5.73 5.64 ± 0.05 45 w/almondl*(0.5 %) 46 Chocolate, milk w/almondl*(1.0 %) 5.71 5.46 5.35 5.51 ± 0.11 47 Chocolate, milk w/almondl*(2.0 %) 6.61 6.00 5.48 6.03 ± 0.33 48 Chocolate, milk w/almondl*(5.0 %) 4.73 4.42 4.52 4.56 ± 0.09 49 Chocolate, milk w/almondl* (10.0 %) 3.62 3.75 3.79 3.72 ± 0.05 50 Chocolate, milkc , Hershey N.D N.D N.D N.D 51 Chocolate, milk w/almondc, Hershey 1.46 1.60 1.51 1.53 ± 0.04 52 Chocolate, whitel N.D N.D N.D N.D 53 Chocolate, white w/almondl*(0.5 %) 3.57 3.60 3.58 3.58 ± 0.01 54 Chocolate, white w/almondl*(1.0 %) 3.89 3.80 3.63 3.77 ± 0.08 55 Chocolate, white w/almondl*( 2.0 %) 2.99 3.03 2.94 2.99 ± 0.03 56 Chocolate, white w/almondl(5.0 %) 2.60 2.62 2.48 2.57 ± 0.04 57 Chocolate, white w/almondl(10.0 %) 2.64 2.58 2.35 2.52 ± 0.09 C. Bars and Brittles 58 Almond cashew barc 1.80 1.54 2.02 1.79 ± 0.14 59 Almond barc 1.55 1.46 1.59 1.54 ± 0.04

Table 4.2: Continued

S. Food Matrix Name R 1 R 2 R 3 R No. Mean ± SEM

60 Almond brittlel 1.54 1.48 1.43 1.48 ± 0.03 61 Coconut brittlel N.D N.D N.D N.D 62 Cashew brittlel N.D N.D N.D N.D 63 Granola barl N.D N.D N.D N.D 64 Granola oats and honeyc N.D N.D N.D N.D 65 Granola roasted almondc 1.63 1.71 1.82 1.72 ± 0.05 66 Granola roasted cashewc N.D N.D N.D N.D 67 Granola peanut (br) almondl 1.66 1.53 1.58 1.59 ± 0.04 D. Cereals 68 Cornflakec N.D N.D N.D N.D 69 Cornflake type II Bl N.D N.D N.D N.D 70 Cornflake type III Al N.D N.D N.D N.D 71 Cornflake l N.D N.D N.D N.D 72 Cornflake w/almondl*(1%) 3.75 3.99 3.64 3.80 ± 0.10 73 Barley N.D N.D N.D N.D 74 Basi ragi N.D N.D N.D N.D 75 Buckwheat N.D N.D N.D N.D 76 Bulgur wheat N.D N.D N.D N.D 77 Cereal, 7 grain N.D N.D N.D N.D 78 Oat N.D N.D N.D N.D 79 Millet N.D N.D N.D N.D 80 Quinoa N.D N.D N.D N.D 81 Rice N.D N.D N.D N.D 82 Rye N.D N.D N.D N.D 83 Semolina N.D N.D N.D N.D 84 Sorghum N.D N.D N.D N.D 85 All-purpose flour N.D N.D N.D N.D E. Dairy 86 Non-fat dry milk* N.D N.D N.D N.D 87 Whole milk N.D N.D N.D N.D 88 Skimmed milk N.D N.D N.D N.D F. Colors 89 Color, blue 1 N.D N.D N.D N.D 90 Color, blue 2 N.D N.D N.D N.D 91 Color, green 3 N.D N.D N.D N.D 92 Color, red 3 N.D N.D N.D N.D 93 Color, red 40 N.D N.D N.D N.D 94 Color, yellow 5 N.D N.D N.D N.D 95 Color, yellow 6 N.D N.D N.D N.D G. Additives and Spices 96 Baking soda N.D N.D N.D N.D 97 Brown sugar, dark N.D N.D N.D N.D

Table 4.2: Continued

S. Food Matrix Name R 1 R 2 R 3 R No. Mean ± SEM

98 Sugar N.D N.D N.D N.D 99 Cardamom N.D N.D N.D N.D 100 Cinnamon N.D N.D N.D N.D 101 Cloves N.D N.D N.D N.D 102 Aniseed N.D N.D N.D N.D 103 Cumin N.D N.D N.D N.D 104 Garlic N.D N.D N.D N.D 105 Ginger N.D N.D N.D N.D 106 Mustard N.D N.D N.D N.D 107 Nutmeg N.D N.D N.D N.D 108 Parsley N.D N.D N.D N.D 109 Salt N.D N.D N.D N.D 110 Turmeric N.D N.D N.D N.D 111 Vanilla extract N.D N.D N.D N.D 112 Egg N.D N.D N.D N.D H. Fruits 113 Apple N.D N.D N.D N.D 114 Cherry N.D N.D N.D N.D 115 Plum N.D N.D N.D N.D I. Others 116 Mix, kheerc N.D N.D N.D N.D 117 Mix, badam kheerc 1.46 1.35 1.37 1.39 ± 0.04 118 Trail mixl 2.06 1.62 1.96 1.88 ± 0.13 119 Trail mix, Alaskan almond w/o cashewc 2.26 1.76 2.29 2.10 ± 0.17 120 Trail mix, Appalachian almond w/o cashewc 1.43 1.58 1.83 1.62 ± 0.12 121 Trail mix, breakfast almond w/o cashewc 1.48 1.41 1.33 1.41 ± 0.04 122 Trail mix, raisin almond cashewc* 15.66 22.62 7.27 15.19 ± 4.44 123 Trail mix, tropical cashew w/o almondc N.D N.D N.D N.D 124 Trail mix spiked 1.50 1.60 1.39 1.50 ± 0.06 w/almondc(10%) LSD (n=3) = 1.90, Least significant difference. Differences between two means exceeding LSD value are significant (p ≤ 0.05) (p<0.05).

Table 4.3: Sandwich ELISA using 4F10. Immunoreactivity (Ra)of selected food matrices compared with whole almond by sandwich ELISA using whole almond 99R20 pAb (604 ng/ml) as capture antibody and 4F10 (8.824 ng/ml) as detection antibody. (N.D- Not detected). * denotes that the mean R of sample is significantly lower than unprocessed almond (control).

S. Food Matrix Name R 1 R 2 R 3 R No. Mean ± SEM

1 Almond major protein (AMP)l* 1.94 1.98 2.00 1.97 ± 0.02 A. Tree nuts and oil seeds 2 Whole almond 1.00 1.00 1.00 1.00 ± 0.00 3 Roasted almondl* 4.85 4.07 4.20 4.37 ± 0.24 4 Roasted almondl* 2.25 2.25 2.20 2.23 ± 0.02 5 Cashew N.D N.D N.D N.D 6 Pecan N.D N.D N.D N.D 7 Sunflower N.D N.D N.D N.D 8 Sesame N.D N.D N.D N.D 9 Peanut N.D N.D N.D N.D 10 Brazil nut N.D N.D N.D N.D 11 Hazelnut N.D N.D N.D N.D 12 Macadamia N.D N.D N.D N.D 13 Pine nut N.D N.D N.D N.D 14 Pistachio N.D N.D N.D N.D 15 Walnut N.D N.D N.D N.D 16 Soybean N.D N.D N.D N.D 17 Lupin N.D N.D N.D N.D 18 Coconut N.D N.D N.D N.D B. Bakery and Confectionery 19 Cookiel N.D N.D N.D N.D 20 Almond cookiel* (0.5 %) 4.72 5.50 3.51 4.58 ± 0.58 21 Almond cookiel* (1.0 %) 2.31 2.98 2.54 2.61 ± 0.20 22 Almond cookiel* (2.0 %) 2.26 2.17 2.08 2.17 ± 0.05 23 Almond cookiel* (5.0 %) 2.18 1.93 2.05 2.05 ± 0.07 24 Shortbread cookiec N.D N.D N.D N.D 25 Dark chocolate almond cookiec* 2.78 2.64 3.05 2.82 ± 0.12 26 Sponge cakel N.D N.D N.D N.D 27 Almond sponge cakel* (0.5 %) 2.49 2.48 2.59 2.52 ± 0.03 28 Almond sponge cakel* (1.0 %) 2.10 1.95 1.77 1.94 ± 0.10 29 Almond sponge cakel (2.0 %) 1.62 1.71 1.86 1.73 ± 0.07 30 Almond sponge cakel (5.0 %) 1.50 1.49 1.49 1.49 ± 0.00 31 White cakel N.D N.D N.D N.D 32 Almond white cakel* 1.87 1.84 1.91 1.87 ± 0.02 33 Marzipanl 1.59 1.65 1.75 1.66 ± 0.05

Table 4.3: Continued

S. Food Matrix Name R 1 R 2 R 3 R No. Mean ± SEM

34 Chocolate, darkl N.D N.D N.D N.D 35 Chocolate, dark w/almondl*(0.5 %) 10.88 13.01 11.59 11.83 ± 0.62 36 Chocolate, dark w/almondl*(1.0 %) 7.62 5.93 7.73 7.09 ± 0.58 37 Chocolate, dark w/almondl*(2.0 %) 3.95 3.60 2.06 3.20 ± 0.58 38 Chocolate, dark w/almondl*(5.0 %) 2.48 2.41 2.50 2.47 ± 0.03 39 Chocolate, dark w/almondl* (10.0 %) 2.00 2.17 2.35 2.17 ± 0.1 40 Chocolate, darkc, Dove N.D N.D N.D N.D 41 Chocolate, darkc, Hershey N.D N.D N.D N.D 42 Chocolate, dark w/almondc*, Dove 5.86 5.69 4.82 5.46 ± 0.32 43 Chocolate, dark w/almondc*, Hershey 4.61 4.62 4.74 4.66 ± 0.04 44 Chocolate, milkl N.D N.D N.D N.D 45 Chocolate, milk w/almondl*(0.5 %) 5.61 6.18 6.07 5.96 ± 0.17 46 Chocolate, milk w/almondl$(1.0 %) 5.38 4.88 5.47 5.24 ± 0.18 47 Chocolate, milk w/almondl$(2.0 %) 5.17 4.39 5.83 5.13 ± 0.42 48 Chocolate, milk w/almondl*(5.0 %) 4.90 4.88 4.70 4.83 ± 0.06 49 Chocolate, milk w/almondl* (10.0 %) 4.46 4.66 5.30 4.81 ± 0.25 50 Chocolate, milkc , Hershey N.D N.D N.D N.D 51 Chocolate, milk w/almondc*, Hershey 4.97 6.92 6.75 6.21 ± 0.63 52 Chocolate, whitel N.D N.D N.D N.D 53 Chocolate, white w/almondl*(0.5 %) 3.32 3.42 3.69 3.48 ± 0.11 54 Chocolate, white w/almondl*(1.0 %) 3.99 4.05 4.30 4.11 ± 0.10 55 Chocolate, white l* w/almond (2.0 %) 3.48 3.69 3.64 3.60 ± 0.06 56 Chocolate, white w/almondl*(5.0 %) 3.61 3.60 3.09 3.43 ± 0.17 57 Chocolate, white w/almondl*(10.0 %) 3.35 3.60 3.05 3.33 ± 0.16

Table 4.3: Continued

S. Food Matrix Name R 1 R 2 R 3 R No. Mean ± SEM

C. Bars and Brittles 58 Almond cashew barc* 4.81 6.22 5.22 5.41 ± 0.42 59 Almond barc* 4.81 4.79 5.57 5.06 ± 0.26 60 Almond brittlel* 5.52 5.20 5.94 5.55 ± 0.21 61 Coconut brittlel N.D N.D N.D N.D 62 Cashew brittlel N.D N.D N.D N.D 63 Granola barl N.D N.D N.D N.D 64 Granola oats and honeyc N.D N.D N.D N.D 65 Granola roasted almondc* 4.86 5.04 6.72 5.54 ± 0.59 66 Granola roasted cashewc N.D N.D N.D N.D 67 Granola peanut (br) and almondl* 5.76 5.37 5.06 5.4 ± 0.2 D. Cereals 68 Cornflakec N.D N.D N.D N.D 69 Cornflake type II Bl N.D N.D N.D N.D 70 Cornflake type III Al N.D N.D N.D N.D 71 Cornflake l N.D N.D N.D N.D 72 Cornflake w/almondl*(1%) 5.41 4.52 3.93 4.62 ± 0.43 73 Barley N.D N.D N.D N.D 74 Basi ragi N.D N.D N.D N.D 75 Buckwheat N.D N.D N.D N.D 76 Bulgur wheat N.D N.D N.D N.D 77 Cereal, 7 grain N.D N.D N.D N.D 78 Oat N.D N.D N.D N.D 79 Millet N.D N.D N.D N.D 80 Quinoa N.D N.D N.D N.D 81 Rice N.D N.D N.D N.D 82 Rye N.D N.D N.D N.D 83 Semolina N.D N.D N.D N.D 84 Sorghum N.D N.D N.D N.D 85 All-purpose flour N.D N.D N.D N.D E. Dairy 86 Non-fat dry milk N.D N.D N.D N.D 87 Whole milk N.D N.D N.D N.D 88 Skimmed milk N.D N.D N.D N.D F. Colors 89 Color, blue 1 N.D N.D N.D N.D 90 Color, blue 2 N.D N.D N.D N.D 91 Color, green 3 N.D N.D N.D N.D 92 Color, red 3 N.D N.D N.D N.D 93 Color, red 40 N.D N.D N.D N.D 94 Color, yellow 5 N.D N.D N.D N.D

Table 4.3: Continued

S. Food Matrix Name R 1 R 2 R 3 R No. Mean ± SEM

95 Color, yellow 6 N.D N.D N.D N.D G. Additives and Spices 96 Baking soda N.D N.D N.D N.D 97 Brown sugar, dark N.D N.D N.D N.D 98 Sugar N.D N.D N.D N.D 99 Cardamom N.D N.D N.D N.D 100 Cinnamon N.D N.D N.D N.D 101 Cloves N.D N.D N.D N.D 102 Aniseed N.D N.D N.D N.D 103 Cumin N.D N.D N.D N.D 104 Garlic N.D N.D N.D N.D 105 Ginger N.D N.D N.D N.D 106 Mustard N.D N.D N.D N.D 107 Nutmeg N.D N.D N.D N.D 108 Parsley N.D N.D N.D N.D 109 Salt N.D N.D N.D N.D 110 Turmeric N.D N.D N.D N.D 111 Vanilla extract N.D N.D N.D N.D 112 Egg N.D N.D N.D N.D H. Fruits 113 Apple N.D N.D N.D N.D 114 Cherry N.D N.D N.D N.D 115 Plum N.D N.D N.D N.D I. Others 116 Mix, kheerc N.D N.D N.D N.D 117 Mix, badam kheerc* 6.02 6.43 6.27 6.24 ± 0.12 118 Trail mixl* 6.68 5.16 5.48 5.77 ± 0.46 119 Trail mix, Alaskan almond w/o cashewc* 6.19 6.75 5.39 6.11 ± 0.39 120 Trail mix, Appalachian almond w/o cashewc* 6.13 5.94 6.22 6.10 ± 0.08 121 Trail mix, breakfast almond w/o cashewc* 5.50 6.55 5.32 5.79 ± 0.38 122 Trail mix, raisin almond cashewc* 7.64 8.20 9.83 8.55 ± 0.66 123 Trail mix, tropical cashew w/o almondc N.D N.D N.D N.D 124 Trail mix spiked w/almondc*(10%) 5.41 5.35 5.06 5.27 ± 0.11 LSD (n=3) = 0.86, Least significant difference. Differences between two means exceeding LSD value are significant (p ≤ 0.05) (p<0.05).

Table 4.4: Sandwich ELISA using 2A3. Immunoreactivity (Ra)of selected food matrices compared with whole almond by sandwich ELISA using whole almond 99R20 pAb (604 ng/ml) as capture antibody and 2A3 (14.34 ng/ml) as detection antibody. (N.D- Not detected). * denotes that the mean R of sample is significantly lower than unprocessed almond (control). S. Food Matrix Name R 1 R 2 R 3 R No. Mean ± SEM

1 Almond major protein (AMP)l* 3.76 4.01 4.33 4.03 ± 0.17 A. Tree nuts and oil seeds 2 Whole almond 1.00 1.00 1.00 1.00 ± 0.00 3 Roasted almondl* 8.12 9.11 7.85 8.36 ± 0.38 4 Roasted almondl* 4.24 4.05 3.84 4.04 ± 0.12 5 Cashew N.D N.D N.D N.D 6 Pecan N.D N.D N.D N.D 7 Sunflower N.D N.D N.D N.D 8 Sesame N.D N.D N.D N.D 9 Peanut N.D N.D N.D N.D 10 Brazil nut N.D N.D N.D N.D 11 Hazelnut N.D N.D N.D N.D 12 Macadamia N.D N.D N.D N.D 13 Pine nut N.D N.D N.D N.D 14 Pistachio N.D N.D N.D N.D 15 Walnut N.D N.D N.D N.D 16 Soybean N.D N.D N.D N.D 17 Lupin N.D N.D N.D N.D 18 Coconut N.D N.D N.D N.D B. Bakery and Confectionery 19 Cookiel N.D N.D N.D N.D 20 Almond cookiel* (0.5 %) 7.76 7.60 6.99 7.45 ± 0.23 21 Almond cookiel* (1.0 %) 5.70 5.61 5.12 5.47 ± 0.18 22 Almond cookiel* (2.0 %) 4.14 4.06 4.29 4.17 ± 0.07 23 Almond cookie* (5.0 %) 3.59 3.77 3.78 3.71 ± 0.06 24 Shortbread cookiec N.D N.D N.D N.D 25 Dark chocolate almond cookiec* 4.71 4.79 4.69 4.73 ± 0.03 26 Sponge cakel N.D N.D N.D N.D 27 Almond sponge cakel* (0.5 %) 9.49 9.40 8.85 9.24 ± 0.20 28 Almond sponge cakel* (1.0 %) 6.47 5.78 6.06 6.10 ± 0.20 29 Almond sponge cakel* (2.0 %) 4.78 4.96 4.39 4.71 ± 0.17 30 Almond sponge cakel* (5.0 %) 4.01 3.96 3.94 3.97 ± 0.02

Table 4.4: Continued

S. Food Matrix Name R 1 R 2 R 3 R No. Mean ± SEM

31 White cakel N.D N.D N.D N.D 32 Almond white cakel* 4.59 5.01 4.77 4.79 ± 0.12 33 Marzipanl* 6.71 7.04 6.27 6.67 ± 0.22 34 Chocolate, darkl N.D N.D N.D N.D 35 Chocolate, dark w/almondl* (0.5 %) 25.87 20.59 23.46 23.30 ± 1.53 36 Chocolate, dark w/almondl*(1.0 %) 14.69 16.18 13.45 14.77 ± 0.79 37 Chocolate, dark w/almondl* (2.0 %) 10.40 11.82 12.20 11.47 ± 0.55 38 Chocolate, dark w/almondl*(5.0 %) 7.57 7.04 7.66 7.42 ± 0.19 39 Chocolate, dark w/almondl* (10.0 %) 6.77 6.77 7.45 7.00 ± 0.23 40 Chocolate, darkc, Dove N.D N.D N.D N.D 41 Chocolate, darkc, Hershey N.D N.D N.D N.D 42 Chocolate, dark w/almondc*, Dove 12.02 8.26 8.79 9.69 ± 1.18 43 Chocolate, dark w/almondc*, Hershey 8.69 11.76 9.23 9.90 ± 0.94 44 Chocolate, milkl N.D N.D N.D N.D 45 Chocolate, milk w/almondl( 0.5 %) 1.46 1.72 2.38 1.86 ± 0.27 46 Chocolate, milk w/almondl(1.0 %) 1.22 1.97 1.24 1.47 ± 0.25 47 Chocolate, milk w/almondl ( 2.0 %) 1.33 2.06 1.37 1.59 ± 0.24 48 Chocolate, milk w/almondl (5.0 %) 1.16 1.63 1.93 1.57 ± 0.22 49 Chocolate, milk w/almondl (10.0 %) 1.39 1.62 1.37 1.46 ± 0.08 50 Chocolate, milkc , Hershey N.D N.D N.D N.D 51 Chocolate, milk w/almondc*, Hershey 11.51 10.68 11.12 11.10 ± 0.24 52 Chocolate, whitel N.D N.D N.D N.D 53 Chocolate, white w/almondl (0.5 %) 0.98 0.67 0.95 0.67 ± 0.95 54 Chocolate, white w/almondl (1.0 %) 1.20 1.28 1.07 1.28 ± 1.07 55 Chocolate, white w/almondl (2.0 %) 0.91 0.84 0.87 0.84 ± 0.87

Table 4.4: Continued

S. Food Matrix Name R 1 R 2 R 3 R No. Mean ± SEM

56 Chocolate, white w/almondl (5.0 %) 0.99 0.73 0.76 0.73 ± 0.76 57 Chocolate, white w/almondl (10.0 %) 0.80 0.77 0.73 0.77 ± 0.73 C. Bars and Brittles 58 Almond cashew barc* 7.60 7.26 7.33 7.40 ± 0.18 59 Almond barc* 6.96 9.18 8.17 8.10 ± 1.11 60 Almond brittlel* 11.84 7.90 8.60 9.45 ± 2.10 61 Coconut brittlel N.D N.D N.D N.D 62 Cashew brittlel N.D N.D N.D N.D 63 Granola barl N.D N.D N.D N.D 64 Granola oats and honeyc N.D N.D N.D N.D 65 Granola roasted almondc* 8.98 8.13 8.51 8.54 ± 0.25 66 Granola roasted cashewc N.D N.D N.D N.D 67 Granola peanut (br) and almondl* 10.15 8.65 8.34 9.05 ± 0.56 D. Cereals 68 Cornflakec N.D N.D N.D N.D 69 Cornflake type II Bl N.D N.D N.D N.D 70 Cornflake type III Al N.D N.D N.D N.D 71 Cornflake l N.D N.D N.D N.D 72 Cornflake w/almondl*(1%) 8.43 7.82 10.09 8.78 ± 0.68 73 Barley N.D N.D N.D N.D 74 Basi ragi N.D N.D N.D N.D 75 Buckwheat N.D N.D N.D N.D 76 Bulgur wheat N.D N.D N.D N.D 77 Cereal, 7 grain N.D N.D N.D N.D 78 Oat N.D N.D N.D N.D 79 Millet N.D N.D N.D N.D 80 Quinoa N.D N.D N.D N.D 81 Rice N.D N.D N.D N.D 82 Rye N.D N.D N.D N.D 83 Semolina N.D N.D N.D N.D 84 Sorghum N.D N.D N.D N.D 85 All-purpose flour N.D N.D N.D N.D E. Dairy 86 Non-fat dry milk* N.D N.D N.D N.D 87 Whole milk N.D N.D N.D N.D 88 Skimmed milk N.D N.D N.D N.D F. Colors 89 Color, blue 1 N.D N.D N.D N.D

Table 4.4: Continued

S. Food Matrix Name R 1 R 2 R 3 R No. Mean ± SEM

90 Color, blue 2 N.D N.D N.D N.D 91 Color, green 3 N.D N.D N.D N.D 92 Color, red 3 N.D N.D N.D N.D 93 Color, red 40 N.D N.D N.D N.D 94 Color, yellow 5 N.D N.D N.D N.D 95 Color, yellow 6 N.D N.D N.D N.D G. Additives and Spices 96 Baking soda N.D N.D N.D N.D 97 Brown sugar, dark N.D N.D N.D N.D 98 Sugar N.D N.D N.D N.D 99 Cardamom N.D N.D N.D N.D 100 Cinnamon N.D N.D N.D N.D 101 Cloves N.D N.D N.D N.D 102 Aniseed N.D N.D N.D N.D 103 Cumin N.D N.D N.D N.D 104 Garlic N.D N.D N.D N.D 105 Ginger N.D N.D N.D N.D 106 Mustard N.D N.D N.D N.D 107 Nutmeg N.D N.D N.D N.D 108 Parsley N.D N.D N.D N.D 109 Salt N.D N.D N.D N.D 110 Turmeric N.D N.D N.D N.D 111 Vanilla extract N.D N.D N.D N.D 112 Egg N.D N.D N.D N.D H. Fruits 113 Apple N.D N.D N.D N.D 114 Cherry N.D N.D N.D N.D 115 Plum N.D N.D N.D N.D I. Others 116 Mix, kheerc N.D N.D N.D N.D 117 Mix, badam kheerc* 8.05 7.71 7.60 7.79 ± 0.14 118 Trail mixl* 9.50 10.09 7.02 8.87 ± 0.94 119 Trail mix, Alaskan almond w/o cashewc* 7.30 7.93 8.60 7.94 ± 0.38 120 Trail mix, Appalachian almond w/o cashewc* 8.01 6.54 7.08 7.21 ± 0.43 121 Trail mix, breakfast almond w/o cashewc* 6.62 6.49 6.99 6.70 ± 0.15 122 Trail mix, raisin almond cashewc* 10.55 10.28 9.67 10.17 ± 0.26 123 Trail mix, tropical cashew w/o almondc N.D N.D N.D N.D

Table 4.4: Continued

S. Food Matrix Name R 1 R 2 R 3 R No. Mean ± SEM

124 Trail mix spiked w/almondc*(10%) 6.59 6.67 6.87 6.71 ± 0.08 LSD (n=3) = 1.34, Least significant difference. Differences between two means exceeding LSD value are significant (p ≤ 0.05). Rb = (protein concentration of sample for 50% of the maximum signal for the corresponding standard curve)/ (protein concentration required to register 50% of the maximum signal for the standard curve).

The sandwich ELISAs using whole almond pAb 99R20 as the capture antibody and

4C10, 4F10 and 2A3 as the detection antibody were able to detect almond in both commercial and lab-made food matrices as seen in table 4.2, 4.3 and 4.4. The assay did not give any false positives for the matched food matrices without almond or any false negatives for food matrices containing almond. This indicates that the food matrix did not interfere with amandin detection and the assays are robust. The R value for the lab-made samples spiked with 0.5, 1, 2 and 5%

(and 10% for chocolates) whole almond flour increased with increase in the level of spiking. The almond content of commercial samples is not known to us; however, absence of almond in the corresponding controls of their matched pairs confirmed that the assay is applicable to the commercial samples. Dark chocolates including dark chocolate cookies (sample #25) decrease the immunoreactivity of amandin possibly due to interaction of polyphenols present in the food matrix with the proteins, although the possible mechanism for such effect is not known. The phenolic compounds may also interfere in the ELISA although the experience in our laboratory

(Zhang 2012) suggests that such interference by phenolics is not likely. Similar effects on reduced immunoreactivity due to polyphenol-protein interaction were observed in trail mix containing raisin almond cashew (sample #122) while the other trail mixes did not show a low

I.R for all three antibodies. Laboratory prepared marzipan with blanched almonds which is a high sugar containing food matrix showed a decrease in immunoreactivity. And the dry roasted

48 whole almonds showed an increase in the immunoreactivity, but the increase was not significant for 4C10. Both of these results are consistent with the findings reported Venkatachalam et al.

(2002) which states that thermal processing does not affect the stability of almond allergen, although wet heating decreases the immunoreactivity while dry heating increases the immunoreactivity.

Effect of Maillard Reaction on Immunoreactivity of Food Matrices

In addition to thermal processing, aggregation of protein and/or loss of solubility of protein, Maillard reaction between sugar and almond proteins in a food matrix can significantly reduce immunoreactivity in presence of sugars. Studies reported by, (i) Zhang (2012) for reduction of almond allergenicity in presence of 20% sugar (specially corn syrup), (ii) Gruber et al. (2004) for reduction of allergenicity of Pru av 1, cherry major allergen in presence of glucose and ribose, (iii) Su (2012) for a significant decrease in immunoreactivity of roasted almond flour with sugar (i.e. importance of Maillard reaction in immunoreactivity) and thus the importance of

HunterLab LabScan XE spectrophotometer to determine L*, a*, b* values of processed samples to describe the extent of Maillard reaction, (iv) Nakamura et al. (2007) for a drastic reduction of the reactivity of buckwheat Fag e 1 with polysaccharides against human sera of buckwheat- allergy subjects due to the Maillard-type glycosylation, and (v) Iwan et al. (2011) for a decrease in IgE/IgG binding properties as a result of glycation of hazelnut allergen Cor a 11 with glucose at 60 and 145 °C, support the results found in the present study. Therefore, occurrence of

Maillard reaction as confirmed by the water-activity and color tests in the selected food matrices containing almonds may be responsible for a decrease in the immunoreactivity of the food matrices.

Dot Blots

The dot blots probed with all three mAbs were also consistent with the ELISAs in detecting only the food matrices containing almond (Fig 4.3) and did not show any cross- reactivity or a false positive signal, thereby showing the specificity and sensitivity (due to detection of low concentration of almonds in food matrices) of the selected murine based mAbs in almond detection.

Figure 4.2: Dot blot for the food matrix samples at room temperature. Protein loaded on each dot was 1 mg/ml. Detection antibodies for respective blots: 4C10 (290.08 ng/ml), 4F10 (176.48 ng/ml) and 2A3 (286.8 ng/ml). Exposure time was 90 seconds.

Western Blots

Based on the results of both of ELISAs and dot blots, 69 samples were selected from the food matrices for western blotting. Essentially, most of the non cross-reactive samples were eliminated from this assay except some tree nuts and oilseeds and the members of Rosaceae family. The samples selected were # 1-10, 19-60, 62, 64-68, 72, and 113-124. Western blotting was done on the food matrices protein extracts in both non-reducing (without -ME) (Fig 4.4) and reducing conditions (with -ME) (Fig 4.5).

Fig 4.3: Transferred sample proteins (without -me) on nitrocellulose membrane and Ponceau S. Standard: EZ-Run Pre-stained Protein Ladder. Protein loaded in each lane was 20 µg. Lane numbers refer to the sample numbers from Table 3.1.

Fig 4.4: Transferred sample proteins (with -me) on nitrocellulose membrane and Ponceau S. Standard: EZ-Run Pre-stained Protein Ladder. Protein loaded in each lane was 20 µg. Lane numbers refer to the sample numbers from Table 3.1.

Non-reducing samples that were probed with 4C10 (Fig. 4.6) recognised ~63 kDa band in all the samples containing almond and did not show any cross-reactivity with the food matrices without almonds. The intensity of signal recognised in laboratory made confectionary samples increased with an increase in the spiking level and is similar to the pattern observed in sandwich ELISA results for these samples. For matrices containing dark chocolate, the signal intensity was low compared to other food matrices with similar spiking levels. Sample 122, which contained raisins did not show any signal in the western blot, though ELISAs were able to detect a low signal indicating a low

52 immunoreactivity in the matrix. Similar results were obtained in the western blots probed with the linear epitope recognising 4F10 (Fig 4.7) and 2A3 (Fig 4.8) for the non-reduced sample proteins.

For 4F10, ~63, 55 and 38 kDa bands and for 2A3 ~61, 50 and 38 kDa bands were observed in the western blots. No cross-reactivity was observed with these mAbs as well. From these results we can verify that linear epitope and conformational epitopes overlap (~63 kDa) and due to processing some of the disulfide bond between the acidic and basic chains in amandin are broken and are recognized by 4F10 and 2A3 mAbs. The results indicate that conformational epitopes still exist in the processed food matrices and the amandin is thermally stable and can be detected accurately without any hindrance from other ingredients in a food matrix. Similar results for stability of amandin in thermally processed samples were reported by Roux et al. (2001),

Venakatchalam et al. (2002), Tiwari et al. (2010) and Willison et al. (2013).

Fig 4.5: Western blot (without -me) of selected food matrices using 4C10. Standard: EZ-Run Pre- stained Protein Ladder. Protein loaded in each lane was 20 µg. Lane numbers refer to the sample numbers from Table 3.1. Detection antibody 4C10 (72.53 ng/ml). Exposure time 90 seconds.

Fig 4.5: Continued.

Fig 4.6: Western blot (without -me) of selected food matrices using 4F10. Standard: EZ-Run Pre-stained Protein Ladder. Protein loaded in each lane was 20 µg. Lane numbers refer to the sample numbers from Table 3.1. Detection antibody 4F10 (44.12 ng/ml). Exposure time 90 seconds.

Fig 4.6: Continued.

Fig 4.7: Western blot (without -me) of selected food matrices using 2A3. Standard: EZ-Run Pre-stained Protein Ladder. Protein loaded in each lane was 20 µg. Lane numbers refer to the sample numbers from Table 3.1. Detection antibody 2A3 (14.34 ng/ml). Exposure time 90 seconds.

Fig 4.7: Continued.

As expected, after adding β% w/v -mercaptoethanol to the sample protein extracts, most of the disulfide bonds between the acidic and basic chain were broken which resulted in degradation of the conformational epitopes on amandin. Therefore, 4C10 did not give any positive signal for the loaded food matrix proteins in the western blots and could detect only whole almond that too with a very weak signal as seen in Fig. 4.9. The mAb 4F10 recognized

~40 kDa band for all the food matrices containing almond and with increase in the almond spiking level from 1 to 10% in the food matrices, other bands ~ 66, 33 and 28 and 72 kDa were recognised in a pattern (Fig 4.10). Additionally, for the almond spiked cookies (sample 19-23) and trail mixes (sample 118-121 and 124) other bands ~20 and 17 kDa (for trail mixes only) are also detected by 4F10. Similarly, 2A3 detected ~40 kDa bands for all the sample proteins containing almonds, and with increase in the almond spiking level other bands, ~68, 38, 24, 17 kDa were also observed (Fig 4.11). Similar results for decrease in immunoreactivity of processed

56 whole almond seeds were reported by Su (2012). Therefore, all the three immunoassays are in agreement with detection of amandin, indicating that 4C10, 4F10 and 2A3 are excellent murine mAbs that display a high affinity and specificity for targeted almond detection and development of reproducible and robust immunoassays. Also, possible glycation of epitope of amandin due to

Maillard reaction and thermal aggregation of amandin resulting in loss of amandin solubility may result in decreased immunoreactivity of amandin.

Fig 4.8: Western blot (with -me) of selected food matrices using 4C10. Standard: EZ-Run Pre-stained Protein Ladder. Protein loaded in each lane was 20 µg. Lane numbers refer to the sample numbers from Table 3.1. Detection antibody 4C10 (72.53 ng/ml). Exposure time 90 seconds.

Fig 4.9: Western blot (with -me) of selected food matrices using 4F10. Standard: EZ-Run Pre-stained Protein Ladder. Protein loaded in each lane was 20 µg. Lane numbers refer to the sample numbers from Table 3.1. Detection antibody 4F10 (44.12 ng/ml). Exposure time 90 seconds.

Fig 4.10: Western blot (with -me) of selected food matrices using 2A3. Standard: EZ-Run Pre-stained Protein Ladder. Protein loaded in each lane was 20 µg. Lane numbers refer to the sample numbers from Table 3.1. Detection antibody 2A3 (14.34 ng/ml). Exposure time 90 seconds.

Conclusion

Immunoreactivity of processed food matrices containing almonds depends on the stability of the targeted allergen to maintain its native form. Detection of allergen is affected by the processing methods (dry or moist heat as shown by Venkatachalam et al., 2002, Zhang, 2012),

59 thermal aggregation and loss of protein solubility (Sathe et al., 1989), and/or sugar which is present in many ingredients naturally and added to the food preparations as well. Glycation as a result of Maillard reaction can either mask the epitope responsible for allergenicity and thereby decreasing the immunoreactivity, or prevent refolding of the allergen to its original and native configuration to form neoallergen(s) which might have a decreased or increased immunoreactivity. Although, in the present study, we found that the immunoreactivity of the food matrices decreased compared to unprocessed almond and since amandin is thermally stable, we can assess that may be no neoallergens were formed in the food matrices. Therefore, the reduction in allergenicity can be due to a) glycation of the epitope on amandin as a result of Maillard reaction, b) loss of disulfide bond between the acidic and basic polypeptides of native amandin due to thermal processing, c) thermal aggregation of amandin, d) loss of amandin solubility or a combination thereof. The results of this study also indicate that immunoassays developed by murine based mAbs are specific, sensitive and robust for amandin detection in the tested food matrices and amandin incurred samples.

APPENDIX

THE ANIMAL CARE AND USE COMMITTEE APPROVAL MEMORANDUM

REFERENCES

Acosta, M. R.; Roux, K. H.; Teuber, S. S.; Sathe, S. K. Production and characterization of rabbit polyclonal antibodies to almond (Prunus dulcis L.) major storage protein. J. Agric. Food Chem. 1999, 47, 4053-4059.

Ahmed, N. Advanced glycation endproducts-role in pathology of diabetic complications. Diabetes Res. Clin. Pract. 2005, 67, 3-21.

Ahmed, N.; Mirshekar-Syahkal, B.; Kennish, L.; Karachalias, N.; Babaei-Jadidi, R.; Thornalley, P.J. Assay of advanced glycation endproducts in selected beverages and food by liquid chromatography with tandem mass spectrometric detection. Mol. Nutr. Food Res. 2005, 49, 691 - 699.

Ahrens, S.; Venkatachalam, M.; Mistry, A.M.; Lapsley, K.; Sathe, S.K. Almond (Prunus Dulcis L.) protein quality. Plant Foods Hum. Nutr. 2005, 60, 123-128.

Ajila, C.M.; Leelavathi, K.; Prasada Rao, U.J.S. Improvement of dietary ﬁber content and antioxidant properties in soft dough biscuits with the incorporation of mango peel powder. J. Cereal Sci. 2008, 48, 319-26.

Ames, J.M. Applications of the Maillard reaction in the food industry. Food Chem. 1998, 62, 431-439.

Armentia, A.; Barber, D.; Lombardero, M.; Santos, J.M.M.; Gil, F.J.M.; Pena, M.L.A.; Callejo, A.; Salcedo, G.; Sanchez-Monge, R. Anaphylaxis associated with antiphospholipid syndrome. Annals Allergy Asthma Immunol. 2001, 87, 54-59.

Arvidsson, P.; Van Boekel, M.A.J.S.; Skog, K.; Jagerstad, M. Formation of mutagenic maillard reaction products. The Maillard Reaction in Foods and Medicine. 1998, (O’Brien, J.; Nursten, H.E.; Crabbe, M.J.C.; Ames, J.M., eds), Royal Society of Chemistry, Cambridge, UK. 219-224.

Beacham, H. H.; DULL, M. F.Some observations on the browning reaction a,b,c. J. of Food Sci. 1951, 16, 439-445.

Berrens, L. Neoallergens in heated pecan nut: Products of maillard-type degradation? Allergy 1996, 51, 277-278.

Bock, S.A.; Munoz-Furlong, A.; Sampson, H.A. Further fatalities caused by anaphylactic reactions to food, 2001-2006. J. Allergy Clin. Immunol. 2007, 119, 1016-1018.

Boyce, J.A.; Assa’ad, A.; Burks, A.W.; Jones, S.M.; Sampson, H.A.; Wood, R.A.; Plot, M.; Cooper, S.F.; Fenton, M.J. Guidelines for the diagnosis and management of food allergy in the United States: Report of the NIAID-sponsored Expert Panel. J. Allergy Clin. Immunol. 2010, 126, S1-S58.

Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 1976, 72, 248- 254.

Broughton, C. Almonds enhance food value. Cereal Foods World 1996, 41, 460-462.

Brownlee, M. Glycation and diabetic complications. Diabetes. 1994, 43, 836-841.

Brownlee, M. Advanced protein glycosylation in diabetes and aging. Annu. Rev. Med. 1995, 46, 223-234.

Chandra, R.K., and Thompson, K. The management and prevention of food anaphylaxis. Nutrition Research 2001;22, 89-110.

Chen, C-Y.; Lapsley, K.; Blumberg, J. A nutrition and health perspective on almonds. J. Sci. Food Agric. 2006, 86: 2245-2250.

Cucu, T.; Meulenaer, B.D.; Bridts, C.; Devreese, B.; Ebo, D. Impact of thermal processing and the Maillard reaction on the basophil activation of hazelnut allergic patients. Food Chem. Toxicology 2012, 50, 1722–1728.

Davies, C. G. A.; Labuza, T. P. The Maillard reaction: application to confectionery products. Confectionery science. Pennsylvania: Penn State Univ. Press. 1997, 35-66.

Fayle, S.E.; Gerrard, J.A. The Maillard Reaction, ed. P. S. Belton, Royal Society of Chemistry, Cambridge, 2002, ch. 1, 1–6.

Fling, S. P.; Gregerson, D. S. Peptide and protein molecular weight determination by electrophoresis using a high-molarity Tris buffer system without urea. Anal. Biochem. 1986, 155, 8γ‒88.

Food allergy: a practice parameter. Ann. Allergy Asthma Immunol.2006, 96, S1-S68.

Garcia-Mas, J.; Messeguer, R.; Arus, P.; Puigdomenech, P. Molecular characterization of cDNAs corresponding to genes expressed during almond (Prunus amygdalus Batsch) seed development. Plant Mol. Bio. 27, 1995, 205–10.

Goldberg, T.; Cai, W.; Peppa, M.; Dardaine, V.; Baliga, B.S.; Uribarri, J.; Vlassara, H. Advanced glycoxidation end products in commonly consumed foods. J. Am. Diet Assoc. 2004, 104, 1287-1291.

Goldin, A.; Beckman, J.A.; Schmidt, A.M.; Creager, M.A. Advanced Glycation End Products: Sparking the development of diabetic vascular injury. Circulation 2006, 114, 597-605.

Gottschalk, A. Some biochemically relevant properties of N-substituted fructosamines derived from amino-acids and N-arylglucosylamines. Biochem. J. 1952, 52, 455-60.

Gottschalk, A.; Partridge, S. M. Interaction between simple sugars and amino-acids. Ibid. 1950, 165, 684-685.

Griffith, T.; Johnson, J.A. Relation of the browning reaction to storage of sugar cookies. Cereal Chem.1957, 34, 159–169.

Gruber, P.; Vieths, S.; Wangorsch, A.; Nerkamp, J.; Hofmann, T. Maillard reaction and enzymatic browning effect the allergenicity of Pru av1, the major allergen from cherry (Prunus avium). J. Agric. Food Chem. 2004, 52, 4002-4007.

Hartman, M. Formation of volatile compounds from the reaction of leucine and D-glucose in propylene glycol. Perfum.Flavor. 1983, 8, 81-86.

Haugaard, G.; Tumerman, L.; Silvestri, H. A study on the reaction of aldoses and amino acids. J. Am. Chem. Soc.1951, 73: 4594-4600.

Henle, T.; Miyata, T. Advanced glycation end products in uremia. Adv. Ren. Repl. Ther. 2003, 10, 321 - 331.

Henle, T. Dietary advanced glycation end products – a risk to human health? A call for an interdisciplinary debate. Mol. Nutr. Food Res. 2007, 51, 1075-1078.

Henle, T. Protein-bound advanced glycation endproducts (AGEs) as bioactive amino acid derivatives in foods. Amino Acids 2005, 29, 313-322.

Hilmenyuk, T.; Bellinghausen, I.; Heydenreich, B.; Ilchmann, A.; Toda, M. Grabbe, S.; Saloga, J. Effects of glycation of the model food allergen ovalbumin on antigen uptake and presentation by human dendritic cells. Immunology 2009, 129, 437–445.

Hodge, J.E. Chemistry of browning reactions in model systems. Agric. and Food Chem. 1953, 15, 928-943. Hurd, C. D.; Kelso, C. D. Hydroxylation of 2, 3-dihydropyran and the application of desoxyaldopentoses in the browning reaction1. J. Am. Chem. Soc. 1948, 70, 1484- 1486.

Iwan, M.; Vissers, Y.M.; Fiedorowicz, E.; Kostyra, H.; Kostyra, E.; Savelkoul, H.F.J.; Wichers, H.J. Impact of Maillard reaction on immunoreactivity and allergenicity of the hazelnut allergen Cor a 11. J.Agric. Food Chem. 2011, 59, 7163–7171.

Kamil, A.; Chen, C-Y. O.; Health benefits of almonds beyond cholesterol reduction. J. Agric. Food Chem. β01β, 60, 6694−670β.

Karel, M. Some effects of water and oxygen on rates of reactions of food components. Ph.D. Thesis, Massachussets Institute of Technology, 1960.

Karel, M.; Buera, M.P. Glass transition and its potential effects on kinetics of condensation reactions and in particular on non-enzymatic browning. Special Publications of the Royal Society of Chemistry. 1994, 151, 164-169.

Katchalsky, A.; Sharon, N. Kenetics of aldose-amino acid interaction. Biochem. Et Biophys. Acta. 1953, 10, 290-301.

Kuhn, R.; Weygand, F. The Amadori rearrangement. Ibid. 1937, 70, 769-772.

Kume, S.; Takeya, M.; Mori, T.; Araki, N.; Suzuki, H.; Horiuchi, S.; Kodama, T.; Miyauchi, Y.; Takahashi, K. Immunohistochemical and ultrastructural detection of advanced glycation end products in atherosclerotic lesions of human aorta with a novel specific monoclonal antibody. Am. J. Pathol. 1995, 147, 654–667.

Kuzin, A. M.; Polyakova, O. I. Formation and properties of addition com-pounds of amino acids and sugars. Biokhimiya. 1941, 6, 113-121.

Labuza, T. P. and Baiser, W. M. The kinetics of non-enzymatic browning. In H. G. Schwartzberg & R. W. Hartel (Eds.), Phys. chem. foods. 1992, 595-649). New York: Marcel Dekker.

Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with Folin phenol reagent. J. Biol. Chem. 1951, 193, 265–275.

Maleki, S. J.; Chung, S. Y.; Champagne, E. T.; Raufman, J. P. The effects of roasting on the allergenic properties of peanut proteins. J. Allergy Clin. Immunol. 2000, 106, 763– 768.

Martins, S. I.; Jongen, W. M.; Van Boekel, M. A. A review of Maillard reaction in food and implications to kinetic modelling. Trends Food Sci Tech. 2000, 11, 364-373.

McDaniel, K.A.; White, B.L.; Dean, L.L.; Sanders, T.H.; Davis, J.P. Compositional and mechanical properties of peanuts roasted to equivalent colors using different time/temperature conditions. J. Food Sci. 2012, 77, C1292-98.

Munch, G.; Thome, J.; Foley, P.; Schinzel, R.; Riederer, P. Advanced glycation endproducts in ageing and Alzheimer’s disease. Brain Res. Rev. 1997, βγ, 1γ4–143.

Nagao, M.; Takahashi, Y.; Yamanaka, H.; Sugimura, T. Mutagens in coffee and tea Mutat. Res. 1979, 68, 101–106.

Nakamura, S.; Suzuki, Y. Ishikawa, E.; Yakushi, T.; Jing, H.; Miyamoto, T.; Hashizume, K. Reduction of in vitro allergenicity of buckwheat Fag e 1 through the Maillard-type glycosylation with polysaccharides. Food Chem. 2008, 109, 538–545.

Nursten, H.E. The Maillard Reaction: Chemistry, Biochemistry, and Implications, ed. H. Nursten, Royal Society of Chemistry, Cambridge, 2005. ch. 1, 1–4.

O’Brien, J.; Morrissey, P.A. Nutritional and toxicological aspects of the Maillard browning reaction in foods. Crit. Rev. Food Sci. Nutr. 1989, 28, 211-248.

Oliver, C.M.; Melton, L.D.; Stanley, R.A. Glycation of caseinate by fructose and fructo- oligosaccharides during controlled heat treatment in the ‘dry’ state. J. Sci. Food Agric. 2006, 86, 722-731.

Patrick Jr, T. M. The Reaction of Aldehydes with Aldimines. J. Am. Chem. Soc. 1952, 74, 2984-2986.

Peng, X.; Ma, J.; Chen, F.; Wang, M. Naturally occurring inhibitors against the formation of advanced glycation end-products. Food Funct. 2011, 2, 289-301.

Poltronieri, P.; Cappello, M.S.; Dohmae, N.; Conti, A.; Fortunato, D.; Pastorello, E.A. Identification and characterisation of the IgE-binding proteins 2S albumin and conglutin γ in almond (Prunus dulcis) seeds. Int. Arch. Allergy Immunol. 2002,128, 97-104.

Rahbar, S.; Blumenfeld, O.; Ranney, H. M. Studies of an unusual hemoglobin in patients with diabetes mellitus. Biochem. Biophys. Res. Commun. 1969, 36, 838 - 843.

Renn, P.T.; Sathe, S.K. Effect of pH, temperature, and reactant molar ratio on L-leucine and D-glucose maillard browning reaction in an aqueous system. J. Agric. Food Chem. 1997, 45, 3782-3787.

Roux, K.H.; Teuber, S.S.; Robotham, J.M.; Sathe, S.K. Detection and stability of the major almond allergen in foods. J. Agric. Food Chem. 2001, 49, β1γ1−β1γ6.

Sampson, H.A. Food Allergy. Part 1: immunopathogenesis and clinical disorders. J. Allergy Clin. Immunol. 1999, 103, 717-728.

Sampson, H.A. Anaphylaxis and emergency treatment. Pediatrics 2003, 111, 1601-1608.

Sathe, S. K. Isolation and characterization of the protein that co-purifies with soy bean (Glycine max L.) glycinin. J. Food Biochem. 1991, 15, 33-49.

Sathe, S.K. Solubilization, electrophoretic characterization and in vitro digestibility of almond (Prunus-Amygdalus) proteins. J. Food Biochem. 1993, 16, 249-264.

Sathe, S.K.; Mason, A.C.; Weaver, C.M. Thermal aggregation of soybean (Glycime max L.) sulfur-rich protein. J. Food Sci. 1989, 54:319-42. Sathe, S.K.; Sharma, G.M. Effects of food processing on food allergens. Mol. Nutr. Food Res. 2009, 53, 970 - 978.

Sathe, S.K.; Seeram, N.P.; Kshirsagar, H.H.; Lapsley, K.A. Fatty acid composition of California grown almonds. J. Food Sci. 2008, 73, C607-C614.

Sathe S.K.; Wolf, W.J.; Roux, K.H.; Teuber, S.S.; Venkatachalam, M.; Sze-Tao, K.W.C. Biochemical characterization of amandin, the major storage protein in almond (Prunus Dulcis L.). J. Agric. Food Chem. 2002, 50, 4333-4341.

Sathe, S.K.; Venkatachalam, M.; Sharma, G.M.; Kshirsagar, H.H.; Teuber, S.S.; Roux, K.H. Solubilization and electrophoretic characterization of select edible nut seed proteins. J. Agric. Food Chem. 2009, 57, 7846-7856.

Schönberg, A.; Moubasher, R.; Mostafa, A. Degradation of α-amino-acids to aldehydes and ketones by interaction with carbonyl compounds. J. Chem. Soc. 1948, 176-182.

Shahidi, F.; Ho, C-T.; Van Chuyen, N. Process induced chemical changes in food. Adv. Exp. Med. Bio. 1998, 434 (eds.).

Sharma, G. M.; Su, M.; Joshi, A. U.; Roux, K. H.; Sathe, S. K. Functional properties of select edible oilseed proteins. J. Agric. Food Chem. 2010, 58, 5457-5464.

Sicherer, S.H.; Munoz-Furlong, A.; Burks, A.W.; Sampson, H.A. Prevalence of peanut and tree nut allergy in the US determined by a random digit dial telephone survey. J. Allergy Clin. Immunol. 1999, 103, 559–562.

Sicherer, S.H.; Munoz-Furlong, A.; Sampson, H.A. Prevalence of peanut and tree nut allergy in the United States determined by means of a random digit dial telephone survey: 5- year follow-up study. J. Allergy Clin. Immunol. 2003, 112, 1203–1207.

Spies, J.R. Allergens. J.Agric. Food Chem. 1974, 22, 30-36.

Su, M.; Venkatachalam, M.; Teuber, S.S.; Roux, K.H.; Sathe, S.K. Impact of γ -irradiation and thermal processing on the antigenicity of almond, cashew nut and walnut proteins. J. Sci. Food Agric. 2004, 84, 1119–1125.

Su, M. Development of a specific, sensitive and robust enzyme-linked immunosorbent assay (ELISA) to detect trace amounts of almond (Prunus dulcis L.). Ph.D. Thesis, The Florida State University, 2012.

Suarez, G.; Rajaram, R.; Oronsky, A.L.; Gawinowicz, M.A. Non-enzymatic glycation of bovine serum albumin by fructose (Fructation). J. Bio. Chem. 1989, 264, 3674-3679.

Taufel, K.; Iwainsky, H. Analytical and chromatographic studies of Maillard's reaction. Biochem. Z. 1952, 323: 299-308.

Tiwari, R.S.; Venkatachalam, M.; Sharma, G.M.; Su, M.; Roux, K.H.; Sathe, S.K. Effect of food matrix on amandin, almond (Prunus dulcis L.) major protein, immunorecognition and recovery. LWT - Food Sci. Tech. 2010, 43, 675–83.

Thornalley, P. J. Clinical significance of glycation. Clin. Lab. 1999, 45, 263 - 273.

Thornalley, P.J. Dietary AGEs and ALEs and risk to human health by their interaction with the receptor for advanced glycation end products (RAGE) – an introduction. Mol. Nutr. Food Res. 2007, 51, 1107 - 1110.

Uribarri, J.; Woodruff, S.; Goodman, S.; Cai, W.; Chen, X.; Pyzik, R.; Yong, A.; Striker, G.E.; Vlassara, H. Advanced glycation end products in foods and a practical guide to their reduction in the diet. J Am Diet Assoc. 2010, 110, 911-916.

Vanholder, R.; De Smet, R.; Glorieux, G.; Argiles, A. Review on uremic toxins: Classification, concentration, and intraindividual variability. Kidney Int. 2003, 63, 1934 - 1943.

Venkatachalam, M.; Sathe, S.K. Chemical composition of selected edible nut seeds. J. Agric. Food Chem. 2006, 54, 4705-4714.

Venkatachalam, M.; Teuber, S.S.; Rox, K.H.; Sathe, S.K. Effects of roasting, blanching, autoclaving, and microwave heating on antigenicity of almond (Prunus Dulcis L.) proteins. J. Agric. Food Chem. 2002, 50, 3544-3548.

Vissers, Y.M.; Iwan, M.; Adel-Patient, K.; Sthal Skov, P.; Rigby, N.M.; Johnson, P.E.; Mandrup Muller, P.; Przybylski-Nicaise, L.; Schaap, M.; Ruinemans-Koerts, J.; Jansen, A.P.H.; Mills, E.N.C.; Savelkoul, H.F.J.; Wichers, H.J. Effect of roasting on the allergenicity of major peanut allergens Ara h 1 and Ara h 2/6: the necessity of degranulation assays. Clin. Expt. Allergy 2011, 41, 1631-1642.

Vitek, M.P.; Bhattacharya, K.; Glendening, M. Stopa, E.; Vlassara, H.; Bucala, R.; Manogue, K.; Cerami, A. Advanced glycation end products contribute to amyloidosis in Alzheimer disease. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 4766-4770.

Vlassara, H.; Uribarri, J. Glycoxidation and diabetic complications: Modern lessons and a warning? Rev. Endocrin. Metab. Disord. 2004, 5, 181-188.

Vojdani, A. Detection of IgE, IgG, IgA and IgM antibodies against raw and processed food antigens. Nutr. Metab. 2009, 6, 22.

Westermeier R. 1993. Electrophoresis in practice. A guide to theory and practice. Method 8: SDS polyacrylamide electrophoresis, p 182, VCH Verlagsgesellschaft mbH, D-6940 Weinheim.

Willison, L.N.; Tripathi, P.; Sharma, G.; Teuber, S.S.; Sathe, S.K.; Roux, K.H. Cloning, expression and patient IgE reactivity of recombinant Pru du 6, an 11S globulin from almond. Int. Arch. Allergy and Immunol. 2011, 156, 267–81.

Willison, L.N.; Zhang, Q.; Su, M.; Teuber, S.S.; Sathe, S.K.; Roux, K.H. Conformational epitope mapping of Pru du 6, a major allergen from almond nut. Mol. Immunol. 2013, 55, 253–263.

Wolf, W.J.; Sathe, S.K. Ultracentrifugal and polyacrylamide gel electrophoretic studies of extractability and stability of almond meal proteins. J. Sci. Food Agric. 1998, 78, 511-521.

Wolfrom, M. L.; Cavalieri, L. F.; Cavalieri, D. K. Chemical interactions of amino compounds and sugars. II. 1 Methylation Experiments2, 3. J. Am. Chem. Soc. 1947, 69, 2411-2413. http://www.agmrc.org/commodities__products/nuts/almond-profile/ (accessed on July 30, 2013). https://www.aibonline.org/resources/statistics/breakfast_products.html (accessed on July 30, 2013). www.almondboard.com (accessed on Feb 05, 2013). www.americanbakers.org/industry-data/ (accessed on July 30, 2013). www.candyindustry.com/ext/resources/issues/February/CIN_mediakit_2013_FebLR.pdf (accessed on July 30, 2013). www.fda.gov; Docket No. 02P-0505 (accessed on Feb 05, 2013). www.foodallergy.org (accessed on Feb 05, 2013).

Yaylayan, V.A.; Huyghues-Despointes, A. Retroaldol and redox reactions of amadori compounds: mechanistic studies with various labelled D-[13C] glucose. J. Agric. Food Chem. 1996, 44, 672–681.

Zhang, G.; Huang, G.; Xiao, L.; Mitchell, A.E. Determination of advanced glycation endproducts by LC-MS/MS in raw and roasted almonds (Prunus Dulcis). J. Agric. Food Chem. 2011, 59, 12037–12046.

Zhang, Y. Effects of select phenolic compounds, sugars and thermal processing on immunoreactivity of almond (Prunus dulcis) proteins. Master’s thesis of Florida State University. 2012.

BIOGRAPHICAL SKETCH

Guneet Singh Chhabra

Education

 Florida State University- M.S. Food and Nutrition Science, Spring 2011 to present, GPA: 3.87 on the scale of 0-4.0.  IMS Engineering College/ Gautam Buddh Technical University- B.Tech (Hons.) Biotechnology, August 2006 to July 2010, GA: 78% on the scale of 0-100%.  New State Academy (CBSE) - Class XII, May 2005 to April 2006, GA: 84.2% on the scale of 0-100%.  Guru Harkrishan Public School (CBSE) – Class X, May 2003 to April 2004, GA: 78% on the scale of 0-100%.

Professional Experience

 Researcher with Dr. Shridhar K. Sathe at Department of Nutrition, Food and Exercise Science, Florida State University from September 2013 to present.  Graduate Research Assistant on a USDA grant with Dr. Shridhar K. Sathe at Florida State University from August 2012 to August 2013. And management of resources in the research laboratory and food science teaching laboratory.  Set-up undergraduate food science teaching lab worth $90000 at Florida State University with Dr. Shridhar K. Sathe in Spring 2011.  Trainee in Genetic Engineering training program at IMS Engineering College co- sponsored by Medox- life sciences and G-Biosciences in Summer 2009.  Trainee in Microbiology and R&D Division at Central Indian Pharmacopoeia Laboratory, Ghaziabad, Uttar Pradesh in Summer 2008.

Projects

 Effects of Maillard Reaction on the Immunoreactivity of Almond Major Protein in the Food Matrices Containing Almonds (On-going; includes samples of processed food matrices prepared in our laboratory, commercial samples and ingredients to check interference, for analysis).  Chhabra, G.S., Gupta, S., Bakshi, J.S., Liu, C., Sathe, S.K. 2013. Functional Properties of Selected Dry Bean Flours. IFT Abstract No. 255-14.  Bakshi, J. S., Chhabra, G. S., Gupta, S., Liu, C., Sathe, S. K. 2013. Physical Properties of Selected Dry Bean Seeds. IFT Abstract No. 031-25.  Liu, C., Gupta, S., Bakshi, J. S., Chhabra, G. S., Sathe, S. K. 2013. Hemagglutinating activity, trypsin inhibitor activity, and tannin content in selected dry bean flours. IFT Abstract No. 255-189.

 Gupta, S., Liu, C., Bakshi, J. S., Chhabra, G. S., Sathe, S. K. 2013. Proximate Composition of Commercially Important Dry Bean Flours. IFT Abstract No. 255-23.

Honors

 Florida State University Congress of Graduate Students Conference Support grant in Summer 2013.  Betty M. Watts Memorial Scholarship for Food Science in Spring 2013.  Interdisciplinary waiver for three credit hours from The Graduate School, Florida State University in Spring 2013.  Judged bakery product competition at North Florida Fair 2012, Tallahassee.  Head Co-ordinator of annual fest “Burgeon β010” at the Department of Biotechnology in IMS Engineering College, Ghaziabad.  Vice-President of GEMS (Genetic Engineering and Molecular Scientists Club) 2009-10 at IMS Engineering College, Ghaziabad.

Skills

 Food chemistry, food safety/quality, protein chemistry, food processing, microbiology, biotechnology, immunology, and statistics.  Analytical techniques: Texture Analyser TA.XT plus, HunterLab LabScan XE, Soxhlet Apparatus, Micro-Kjeldahl, Gel-Electrophoresis, Spectrophotometer Ultrospec 2100 pro and Powerwave 200, VirTis bench-top freeze drier.  Immunological Techniques: ELISA, Dot-blot, Western blotting.  Computer: MS Office, SPSS.

Memberships

 Institute of Food Technologists (IFT),  American Oil Chemists’ Society, and  Glenn Society, FSU.