Effects of Vinegar Treatment on Detectivity and Allergenicity of Finfish

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2013 Effects of Vinegar Treatment on Detectability and Allergenicity of Finfish Ye Wang

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COLLEGE OF HUMAN SCIENCES

EFFECTS OF VINEGAR TREATMENT ON DETECTABILITY AND ALLERGENICITY OF

FINFISH

YE WANG

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

Degree Awarded: Summer Semester, 2013

Ye Wang defended this thesis on June 24th, 2013. The members of the supervisory committee were:

Yun-Hwa Peggy Hsieh Professor Directing Thesis

Shridhar Sathe Committee member

Ming Cui 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

I would like to express my sincerest appreciation to my advisor Dr. Yun-Hwa Peggy Hsieh for all the opportunities she afforded throughout the past two years, without her support this work would not have been completed. Her constant inspiration and insightful comments were invaluable to my personal and professional development.

I also would like to thank Dr. Shridhar Sathe and Dr. Ming Cui for being on my thesis committee despite their extremely busy schedule. Additionally, I would like to thank Dr. Jack Ofori, Dr. Yi- Tien Chen, Dr. Yuhong Wang, Behnam Keshavarz, and William Fredericks for their willingness to advise and help me in all areas throughout my course of study. And also thank you to my dear friends Yitong Zhao and Jingjie Xiao for their kindness and help in my life.

Finally, I express my greatest thank to my mother Yonghua Wu for her unconditional love and support. Without her, I would not have had the opportunity to study in the U.S and have this wonderful life.

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TABLE OF CONTENTS

List of Tables ...... vi List of Figures ...... vii Abstract ...... ix

CHAPTER ONE: INTRODUCTION ...... 1

CHAPTER TWO: LITERATURE REVIEW ...... 4 2.1 Fish Allergy ...... 4 2.2 Fish Allergens ...... 5 2.2.1 Fish Parvalbumins ...... 5 2.2.2 Other Fish Allergens ...... 6 2.3 Effects of Processing on Food Proteins ...... 7 2.3.1 Thermal Processing ...... 7 2.3.2 Acid Processing ...... 8 2.4 Detection Methods ...... 9 2.4.1 Diagnosis of Food Allergies ...... 9 2.4.2 Detection of Allergenic Foods ...... 11 CHAPTER THREE: HYPOTHESES AND OBJECTIVES...... 13 3.1 Hypotheses ...... 13 3.2 Objectives ...... 13 CHAPTER FOUR: MATERIALS AND METHODS ...... 14 4.1 Materials ...... 14 4.2 Methods ...... 15 4.2.1 Sample Preparation ...... 15 4.2.2 Protein Extraction ...... 16 4.2.3 Indirect ELISA ...... 16 4.2.4 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blot ...... 17 CHAPTER FIVE: RESULTS AND DISCUSSION ...... 18 5.1 Effects of Different Types of Vinegar on the Detectability of Whiting ...... 18 5.2 Effects of Vinegar Treatment Time on the Detectability of Three Common Species of Finfish ...... 19 5.3 Effects of Vinegar Treatment on Protein Banding Patterns of Finfish ...... 20 5.4 Effects of Vinegar Treatment on Antigenic Protein of MAb 8F5 in Finfish ...... 21 5.5 Effects of Vinegar Induced Chemical Reactions on the Detectability of Finfish ...... 22 5.6 Effects of Vinegar Treatment on the Allergenicity of Finfish ...... 24 5.7 Effects of Vinegar Induced Chemical Reactions on the Allergenicity of Finfish ...... 26 CHAPTER SIX: CONCLUSIONS ...... 29

APPENDIX ...... 30

A. TABLES AND FIGURES ...... 30

REFERENCES ...... 48

BIOGRAPHICAL SKETCH ...... 56

LIST OF TABLES

Table 1. Fish allergens ...... 30

Table 2. Total soluble protein concentration of whiting, cod and red grouper ...... 31

Table 3. Clinical features of patients ...... 31

Table 4. Immunoreactivity of MAb 8F5 against vinegar-treated fish samples determined by iELISA ...... 32

LIST OF FIGURES

Figure 1. Effects of different types of vinegar on the detectability of whiting by MAb 8F5 based- iELISA...... 32

Figure 2. Effects of vinegar treatment on the detectability of whiting, cod and red grouper by MAb 8F5-based iELISA ...... 33

Figure 3. SDS-PAGE profiles of vinegar-treated, water-treated and control samples of whiting, cod and red grouper ...... 34

Figure 4. Antigenic protein banding patterns of vinegar-treated, water-treated and control samples of whiting, cod and red grouper ...... 35

Figure 5. Study of the effects of vinegar induced chemical reactions on the detectability of whiting, cod and red grouper (Group A) ...... 36

Figure 6. Study of the effects of vinegar induced chemical reactions on the detectability of whiting, cod and red grouper (Group B) ...... 37

Figure 7. Study of the effects of vinegar induced chemical reactions on the detectability of whiting, cod and red grouper (Group C) ...... 38

Figure 8. Screen for IgE-binding reactivity of raw and cooked salmon and cod ...... 39

Figure 9. Effects of vinegar treatment on the IgE-immunoreactivity of whiting by human plasma- based iELISA ...... 40

Figure 10. Effects of vinegar treatment on the IgE-immunoreactivity of cod by human plasma- based iELISA ...... 41

Figure 11. Effects of vinegar treatment on the IgE-immunoreactivity of red grouper by human plasma-based iELISA ...... 42

Figure 12. Study of the effects of vinegar induced chemical reactions on the IgE-binding reactivity of whiting, cod and red grouper by human plasma-based iELISA (Group A) ...... 43

Figure 13. Study of the effects of vinegar induced chemical reactions on the IgE-binding reactivity of whiting, cod and red grouper by human plasma-based iELISA (Group B) ...... 44

Figure 14. Study of the effect of vinegar induced chemical reactions on the IgE-binding reactivity of whiting by human plasma-based iELISA (Group C) ...... 45

Figure 15. Study of the effect of vinegar induced chemical reactions on the IgE-binding reactivity of cod by human plasma-based iELISA (Group C) ...... 46

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Figure 16. Study of the effect of vinegar induced chemical reactions on the IgE-binding reactivity of red grouper by human plasma-based iELISA (Group C) ...... 47

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ABSTRACT

Fish provides a valuable source of many essential nutrients. However, it is also one of the “Big Eight” allergenic foods that account for more than 90% of food allergic reactions, so reliable detection of its presence is crucial. The addition of acid ingredients such as vinegar, lemon juice, and tomato sauce has shown to markedly reduce the detectability of fish in an immunoassay using a previously developed fish-specific monoclonal antibody (MAb 8F5), whose antigenic protein is fish tropomyosin-a 36 kDa myofibrilar protein, and acid ingredients may also reduce fish’s allergenicity. This study, therefore, focused on studying the effects of vinegar on the detectability (assay immunoreactivity) and allergenicity of three commonly consumed fish species (whiting, cod, and red grouper).

MAb 8F5 [Immunoglobulin G (IgG)] and human plasma [Immunoglobulin E (IgE)] from three fish allergic patients were individually used to investigate the effects of vinegar on the detectability and allergenicity of each fish sample, using indirect enzyme-linked immunosorbent assay (iELISA). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot were used to reveal changes in the overall and antigenic protein banding patterns in vinegar- treated samples.

The results of iELISA with MAb 8F5 demonstrated that vinegar dramatically reduced the detectability of fish samples (up to 90% of the OD reading) when compared with water-treated and non-treated control samples. SDS-PAGE results showed that the intensity of bands of vinegar- treated samples became lighter than those of controls in all three fish species. The vinegar-treated samples in western blot showed little or no band at 36 kDa, which agreed with the results of the MAb-8F5 based iELISA. Considerable reductions of the OD readings were also apparent in all the fish samples cooked (100 °C) with vinegar for 60 min when tested by IgE-based iELISA. However, there were variations among species and subjects: of the three fish species tested, red grouper was more resistant to vinegar treatment and the subject with a higher IgE concentration in plasma was less affected by vinegar-induced alterations in the fish allergens. Moreover, the chemical reactions that attribute to the vinegar’s effects on antigenic tropomyosin-IgG binding and fish allergen-IgE binding are distinctively different. These results indicate that vinegar treatment of fish decreased

ix the detectability of finfish using MAb 8F5-based iELISA via acidic precipitation of the antigenic protein-tropomyosin, while the decreased allergenicity caused by vinegar was due to the acid denaturation of the allergen.

CHAPTER ONE

INTRODUCTION

Fish, eggs, wheat, tree nuts, peanuts, soy beans, cow’s milk, and crustacean shellfish are the “Big Eight” allergenic foods that account for 90% of allergic reactions to food in humans. Food allergy is an adverse immunological response that is usually mediated by IgE and directed against a specific protein or part of a protein in food. Although most food allergies cause relatively mild symptoms, some can induce severe reactions and may even be life-threatening. In recent years, the number of fish allergy incidences has increased worldwide, probably as a result of the increase in fish consumption over same period.

The current treatment for a food allergy involves strict avoidance of the offending allergen. It is usually suggested that people who are found to be allergic to one kind of fish avoid eating all kinds of fish for the rest of their lives. However, eliminating fish from the daily diet is difficult. Fish and fish ingredients are commonly found in many food products, including some unexpected sources. For example, surimi (processed fish meat) is sometimes used as a substitute for beef or pork (Musmand and others 1996); capsulates often contain fish gelatin; and isinglass, which is mainly composed of fish collagen, is widely used as a fining agent in beer, wines, and champagnes (Taylor and others 2004). Foods like salad dressing, Worcestershire sauce, bouillabaisse, and barbecue sauce, may also be unexpected sources of fish allergens (FAAN website 2012). The unintentional contamination of foods by fish and fish ingredients during manufacturing and household food preparation can also cause reactions for individuals with a fish allergy. To help ensure the safety of individuals with food allergies, the Food Allergen Labeling and Consumer Protection Act (FALCPA) was enacted by Congress in August 2004 and became effective on January 1, 2006 (Public law 108-282). It requires manufacturers to declare on the label of packaged foods that contain an ingredient that is or contains protein from the “Big Eight”. This new regulation has highlighted the need for rapid tests capable of detecting the eight foods or food groups listed in FALCPA as potential sources of allergens in food products. The monoclonal antibody (MAb) 8F5 has therefore been developed by Dr. Peggy Hsieh to detect fish protein in food products (unpublished data). This antibody reacts with protein extracts of 55 common food fish species without cross-reactivity with non-fish animal species and food protein additives. The antigenic

1 component recognized by MAb 8F5 is a heat stable protein with molecular weight of 36 kDa, later identified as tropomyosin (Chen and Hsieh 2012). Preliminary data have indicated that acid ingredients such as vinegar, lemon juice, and tomato sauce could reduce the immunoreactivity, and thus the detectability, of cod in the MAb 8F5-based ELISA (Hou and Hsieh 2012). However, there have been no reports of research into the effects of acid ingredients on the detectability of finfish, which prompted us to initiate this study. MAb 8F5-based ELISA and immunoblot tests were performed in order to determine the effects of vinegar on the immunoreactivity of three common food fish species: whiting (Merlangius merlangus), cod (Gadus morhua), and red grouper (Epinephelus morio). Whiting is the most commonly used inexpensive fish in surimi products, red grouper is a popular fish, in high demand by consumers for its taste and texture, while cod is the most commonly used fish species in allergen related scientific studies. These three species can be easily obtained from a local market and are also habitually eaten with acid ingredients, which led to their selection for this study.

Acid ingredients used as daily food additives may have the potential to decrease the allergenicity of several allergens. For example, in skin prick tests administered to 18 pediatric and 26 adult patients, shrimp soaked in vinegar prior to cooking produced a smaller wheal compared to shrimp prepared conventionally (Perez-Macalalag and others 2007). Armentia and others (2010) reported that the wheal areas of skin prick tests decreased significantly when the test foods (in this case, egg and lentil) were treated with vinegar. This may indicate that adding vinegar during cooking decreases the allergenicity of egg and lentil somewhat. Kim and others (2012) also reported a reduced IgE binding capacity for the major peanuts allergens Ara h 1, Ara h 2, and Ara h 3 after being treated with pH 1.0 acetic acid and commercial vinegar. However, the effect of acid ingredients on the allergenicity of finfish are as yet unknown.

Finfish play an important role in many Americans’ daily diet, especially in coastal areas where fish are more frequently consumed. Eliminating fish from the diet may lead to malnutrition and/or eating disorders, so it is important to find an effective way to reduce the allergenicity of fish and thus benefit those suffering from a fish allergy. Reduced allergenic finfish could be a good candidate for oral immunotherapy for individuals with severe fish allergic reactions. It is also possible that sensitive individuals who take anti-acid medication might face a particularly high

2 risk of fish allergy. In these sensitive individuals, proteins that are normally degradable might act as food allergens in the presence of drugs that hinder peptic digestion (Pali-Schöll and Jensen- Jarolim 2011). If the addition of acidic ingredients reduces the allergenicity of finfish, it might also lower the risk of fish allergy for these patients. According to Thomas and others (2006), IgE- binding methods (e.g., IgE-based ELISA) are useful for investigating the allergenicity of food before and after processing. Hence in this study plasma (IgE) from three fish allergic individuals was used to compare the allergenicity of vinegar -treated and non-treated finfish.

CHAPTER TWO

LITERATURE REVIEW

2.1 Fish Allergy

An allergy is a hypersensitive disorder of the immune system. An allergic reaction is induced by an allergen, which can be any kind of foreign substance, for example pollen, iron, or latex. Food allergens are defined as those food components that induce the production of, and react with, IgE to cause a release of mediators from mast cells and basophiles, resulting in immediate hypersensitive reactions (Taylor and Lehrer 1996). The symptoms of food allergy can vary considerably and include oral allergy syndrome (e.g., itching and angioedema of the lips, mouth and pharynx), skin disorders (e.g., pruritus, angioedema, morbilliform rashes), and gastrointestinal disorders (e.g., nausea, vomiting, gastric retention, intestinal hyper-motility, abdominal pain). At its most dangerous, a food allergen can trigger a life-threatening episode of anaphylaxis.

Food is one of the most common causes of allergy, and fish is one of the leading causes of food allergy. The prevalence of fish allergy varies between countries. A Norwegian study followed 3623 children born in the two main maternity clinics in the capital, Oslo. At one year of age, 1.2% of these children were reported to be allergic to fish but this number increased over time, so that by the age of two, 3.0% of these children were reported to be fish hypersensitive (Eggesbø and others 1999). In Spain, a study of 355 food allergic children with a mean age of 5.4 years found that 30% of these children had fish allergic reactions; another study reported fish to be the third most common cause of food allergy among Spanish children (Crespo and others 1995; Boyano and others 1987). Studies from Italy also showed that fish is one of the leading causes of serious allergic reaction: among 54 episodes of food-induced anaphylaxis in children, 30% were caused by fish (Novembre and others 1998). Asian countries have the highest rate of fish consumption in the world, and as the world’s most populous continent, fish allergy affects a larger population there (Hajab and Selamet 2012). Asian children consume fish for the first time at an earlier age than anywhere else in the world, sometimes as young as seven months. Connett and others (2012) reported that in Southeast Asia, the seafood allergy ratios were 2.29%, 0.26%, and 0.29% for 14- 16 year-old children from the Philippines, Singapore, and Thailand, respectively. Another study

4 conducted in northern Thailand indicated that shrimp, cow’s milk, and fish are the three leading causes of allergy reactions among preschool children in that country (Lao-araya and Trakultivakorn 2012). The frequency of fish allergy in Japan was 10.6% in pediatric patients with a food allergy (Koyama and others 2006). In the U.S, more than one million people (about 0.4% of the total population) are hypersensitive to fish (Munoz-Furlong and others 2004). An even higher fish allergy ratio was found in the U.S. by a nationwide random telephone survey, where 2.3% of 5529 households (14,948 individuals) reported having an allergic reactions to fish (Sicherer and others 2004). The percent of fish allergy among adults in Canada was 0.17%. (Ben- Shoshan and others 2012). Differences in the geographical environment, eating habits, and food processing methods may also be important risk factors for the development of fish allergy in different populations.

2.2 Fish Allergens

2.2.1 Fish Parvalbumins Parvalbumin has been identified to be the major fish allergen. Parvalbumins are a group of intracellular calcium-binding muscle proteins with low molecule weight (10-13 kDa) that promote relaxation in the fast-twitch muscle fibers (Rall 1996). They are divided into two different phylogenic lineages, α and β, according to the composition of their amino acid sequences. β – parvalbumins have been reported to be more allergenic than α-parvalbumins (Roquet and others 1992) and are responsible for the cross-reactivity among different fish species, including salmon, whiff, perch, carp, eel, herring, plaice, catfish, grouper, snapper, pollock, wolffish, halibut, and flounder. It has been found that parvalbumins react with specific IgE in more than 95% of fish- allergic individuals, which strongly supports its identification as the major fish allergen (Griesmeier and others 2010; Chatterjee and others 2006; Poulsen and others 2001; Bugajska- Schretter and others 2000). Gad c1, the cod parvalbumin, is the first and most widely studied fish allergen. It was first identified in Baltic cod (Gadus callarias) and has been well characterized and sequenced. Another parvalbumin, Salmo salar (Sal s l), was found to be the major allergen in the white muscle of Atlantic salmon (Lindstrom and others 1996). Parvalbumins identified as the main allergen in other fish species are listed in Table 1. Parvalbumins share degrees of amino acid homologies ranging from 60 to 80%, which may explain the clinical cross-reactivity of different species of fish in fish-allergic patients.

The quantity of parvalbumin varies in different types of fish muscle. Generally the content of parvalbumin is several times lower in dark muscle than in white muscle, which means the dark muscle is less often implicated in fish allergy than the white muscle. Muscles near the tail have also been found to contain less parvalbumin than the head and middle area of the fish. Tuna is reported to exhibit a significant difference in parvalbumin content between the dorsal and ventral side of their muscles (Coughlin and others 2007). This marked variation in the parvalbumin concentration in different parts of the fish may be explained by the function of parvalbumin. In general, fast twitching white muscles that are responsible for rapid movements contain high levels of parvalbumin, while the dark muscles responsible for continuous swimming contain lower levels of parvalbumin. Fish species such as tuna and swordfish that contain more dark muscle are therefore expected to be less allergenic (Kuehn and others 2010).

Parvalbumins are very stable and have been shown to be resistant to heat, chemical denaturation, and digestive enzymes (Aas and Elsayed 1969, 1971). Unlike most other heat-resistant food allergens that contain linear epitopes, parvalbumins have conformational epitopes that are stabilized by the interaction of metal-binding domains. Although the heat resistance of parvalbumins has been demonstrated, the proteolytic stability of parvalbumins has been questioned by a number of studies. For example, Untersmayer and others (2007) reported that the proteins of cod degrade to small fragments after incubation with simulated gastric fluid for as little as 1 min. Changes to enzyme concentration and pH could therefore have a large effect on the quantity of parvalbumin and this is one of the factors that stimulated our interest in studying the effects of vinegar on the allergenicity of finfish.

2.2.2 Other Fish Allergens Unique IgE-binding bands have been reported at 46 kDa and at 40 kDa in yellow fin tuna (James and others 1997; Yamada and others 1999) and at 25 kDa in swordfish (Kelso and others 1996). Das Dores and others (2002) reported a 41 kDa allergen in addition to the 12 kDa parvalbumin in codfish. This 41 kDa allergen was later identified as aldehyde phosphate dehydrogenase, which is an enzyme located within the cell that is released from the cytosol after cell death, and this release increases in non-frozen fish. Collagen (type I) was found in big eye tuna as a high molecular weight

6 allergen, and has also been shown to exist in Japanese eel, alfonsin, mackerel, and skipjack, indicating that collagen is also a common allergen in several fish species. It was, however, overlooked in early studies (Hamada and others 2001). Wang and others (2011) identified a 28 kDa protein as a new allergen of mackerel (Scomber japonicus) based on the result of IgE- immunoblotting, and this protein was later identified as triosephosphate isomerase (TPI). TPI was also found to be an allergen in non-fish species such as lychee, wheat, latex, archaeopotamobius (Archaeopotamobius sibiriensis), and crangon. A parasite called Anisakis simplex (Moneo and others 2000) has also been proposed as a fish allergen. This parasite is highly resistant to heat and, like other fish allergens, can cause severe allergic reactions including anaphylactic shock.

2.3 Effects of Processing on Food Allergens

Generally, in order to improve the quality (e.g., taste, texture, appearance, and preservation time) and safety of food products, processing steps such as preparation, mechanical processes, separation, isolation and purification, thermal processes, biochemical processes, high-pressure treatment, electric field treatment, and irradiation may be involved in the processing of raw material to produce ready-to-eat foods (Thomas and others 2007). These processes can work exclusively or together with other factors to influence the allergenicity of food, and can be generally categorized into two types: thermal and non-thermal processing. Thermal processing methods include boiling, steaming, frying, grilling, roasting, baking, drying, and pasteurization, while germination, fermentation, proteolysis, ultrafiltration, enzymatic tissue disintegration, pulping, peeling, and mashing are classified as non-thermal processing methods (Besler and others 2001; Sathe and others 2005).

2.3.1 Thermal Processing The effects of thermal processing on food proteins, especially food allergens, have been extensively studied. Thermal processing was found to be effective in reducing the IgE-binding reactivity of several well-known allergens. Food allergens such as Mal d 1 apples (Bohle and others 2006), Api g 1 from celery (Jankiewicz and others 1997), and Cor a 1.04 from hazelnuts (Pastorello and others 2002) have all been reported to be heat sensitive. After thermal processing, for example roasting, they may lose some or all of their allergenicity. This reduction in allergenicity can be

7 explained by the alteration of the conformation of heat liable proteins when exposed to heat, and thus the loss of epitopes.

Many other food allergens have been reported to be heat resistant. For instance, Pru p 1 from peaches (Brenna and others 2000), and Ara h 1 from peanuts (Nordlee and others 1981) are all highly resistant to heat. Fish allergens have also been reported to be heat resistant. Parvalbumin- Lep h 1 is the major allergen in whiff, and Griesmeier and others (2010) reported that an extract of cooked whiff samples actually has a higher number of IgE reactive bands and exhibits a higher resistance to pepsinolysis than extracts from raw samples. The exposure of inside epitopes and the formation of aggregates and polymers by heating may explain this increased allergenicity of cooked whiff. Other parvalbumins, for example Gad c 1 in cod, have also been reported to be resistant to heat (Aas and Elsayed 1969). Changes in the food matrix can influence the effect of heat on allergens, and this effect varies with fish species. Chatterjee and others (2006) reported that the allergenicity of boiled and fried extracts of mackerel, pomfret, and hilsa were considerably reduced using IgE based competitive ELISA, while the highest allergenicity of bhetki was found in the fried extract. The increased allergenicity of fried bhetki may be caused by the exposure of new epitopes or the Maillard reaction. Like the major allergen in peanuts, Ara h 1 and Ara h 2 bound higher levels of IgE and were more resistant to heat and digestion after they had been subjected to the Maillard reaction. Roasted peanuts from two different sources bound IgE from patients with a peanut allergy at approximately 90-fold higher levels than raw peanuts from the same source (Maleki and others 2000).

2.3.2 Acid Processing The effects of non-thermal processing on food proteins can vary largely depending on the food type and processing method. Acid processing has been reported to be effective in reducing the allergenicity of several different kinds of foods. Perez-Macalalag and others (2007) examined the effect of vinegar soaking on allergenicity of the major shrimp allergen Sa-II through a skin prick test (SPT), and reported that mean wheal diameters obtained using shrimp extract that had been prepared with preliminary vinegar soaking were significantly smaller than mean wheal diameters obtained using a conventionally prepared extract. Sletten and others (2010) also reported that pickled herring products (rollmops, Bismarck-herring, brathering and jellied herring) which had

8 been prepared in an acetic acid-salt brine showed decreased IgE binding in 3/5 of the sera by using IgE based- competitive ELISA. They also found that other types of processing, such as lye treatment, sugar curing, and salting, may also decrease the allergenicity of fish. A decreased allergenicity of vinegar-treated peanuts was reported by Kim and others (2012). IgE-binding intensities for Ara h 1, Ara h 2, and Ara h 3 were significantly reduced after being treated with pH 1.0 acetic acid or commercial vinegar; the local habit of consuming peanuts with vinegar may thus explain the low prevalence of peanut allergy in Korea. The change in allergenicity can be explained by an alteration in the allergen structures after treatment with vinegar. A partial loss of structure of Lep w 1 in whiff was observed at acidic pH (Griesmeier and others 2010) and Neti and Rehbein (2000) proposed that the proteolytic degradation of parvalbumins by the activity of acidic protease (cathepsin D) may also explain the disappearance of bands in isoelectric focusing (IEF) gel. Several reports have indicated that protease treatment might be the most effective way to destroy the structure of allergens: proteolytic processing has been used to reduce the allergenicity of soy (Yamanishi and others 1996), wheat flour gluten (Watanabe and others 1995), and milk (Thomas and others 2007). However, this must be approached with caution, as incomplete hydrolysis may prevent protease treatments from completely destroying all the epitopes present.

It is possible that acid treatment can alleviate allergic reactions through mechanisms other than changing the content and structure of the allergens. Armentia and others (2010) considered the possibility that vinegar could decrease allergenic response in lentils and eggs by decreasing the gastric pH and thus enhancing the function of digestion and reducing the allergenicity. The importance of pH and its effect on food allergies has also been indicated by a study conducted by Pali-Schöll and Jensen-Jarolim (2011), which suggested that gastric acid levels determine the activation of gastric pepsin and also the release of pancreatic enzymes. When antacid drugs inhibit or neutralize gastric acid, the likelihood of eliciting allergic reactions via the oral route will be dramatically increased.

2.4 Detection Methods

2.4.1 Diagnosis of Food Allergies A diagnosis of a food allergy usually begins with a physical examination and a discussion of the individual’s medical history. These can determine whether the patient has a food-induced allergic 9 disorder and whether an IgE-mediated or non-IgE-mediated mechanism is most likely to be responsible. When IgE-mediated allergic reactions are suspected, laboratory evaluations (e.g., a skin prick test (SPT), radioallergosorbent test (RAST), and/or an enzyme-linked immunosorbent assay (ELISA)) and oral challenges (e.g., a double-blind placebo-control food challenge (DBPCFC)) can be used to pinpoint the specific foods causing the allergic reaction.

SPTs are most frequently used to screen patients with food allergies. Glycerinated food extracts and appropriate positive (histamine) and negative (saline) controls are applied by a pricking technique. Foods producing wheals at least 3mm larger than wheals induced by a negative control are considered positive. The predictive accuracy of a positive SPT response is less than 50% compared with DBPCFC, but the predictive accuracy of a negative SPT response is greater than 95%. Therefore, SPT should be combined with medical history and oral challenges to diagnose patients who are allergic to specific foodstuffs (Sampson 1999). The DBPCFC, however, remains the gold standard for food allergy diagnosis. The food challenge is administered in a fasting state, and the potential allergen is gradually fed to the patient under supervision. It usually begins with a dose that is unlikely to provoke symptoms (25 to 500 mg of lyophilized food), and the level is then doubled every 15 to 60 minutes. Once the patient has tolerated 10 g of lyophilized food blinded in capsules or liquid, clinical reactivity is generally ruled out (Sampson 1999). To confirm the negative result, an open feeding under observation is needed. SPT and food-specific IgE levels are used to assess the risks and benefits of conducting a food challenge. However, in the clinical setting, open oral challenges eliciting no symptoms (negative challenge) or objective symptoms confirming the history can be considered diagnostic (Sampson 1999).

In vitro assays such as ELISA involve the use of allergen-specific IgE antibodies, which are principle components in food-allergic reactions. In the most frequently used allergosorbent-type of assay, after the allergen has been immobilized on a plate, the serum or plasma sample is added and the plate incubated for a pre-determined period. The immobilized allergen will bind to its specific IgE antibody, allowing the bound IgE to be detected with an anti-IgE antibody detection reagent. In the case of ELISA, an enzyme is conjugated to the anti-IgE antibody, but other types of assay such as sandwich ELISA or latex flow may also be used depending on the application. In all the commonly used commercial assay systems utilizing immobilized antigens, a standard curve

10 based on purified IgE calibrators is established and used to convert assay signals to mass units of allergen-specific IgE, given in International Units (IU) per mL of serum or plasma (Asero and others 2007). When interpreting the results of in vitro ELISA, strong positive results are associated with clinical sensitivity, while completely negative results are associated with clinical tolerance (Dorizzi and others 1999). The form of the allergen preparation can have a major effect on the performance of a test for allergen-specific IgE. Moreover, allergen-specific IgE can be applied to determine the allergenicity of a food or a food product. Thomas and others (2006) argued that IgE binding methods are useful for 1) investigating the allergenic potential of proteins; 2) permitting the comparison of foods before and after processing, and 3) improving the management of allergenic risk from foods. IgE obtained from food allergic individuals can also be used to detect allergens in food products. However, the amount of IgE available from sensitized individuals is usually limited and cross-reactivity to more than one allergenic food may be a problem in human serum IgE. Therefore, rather than IgE, allergen-specific IgG raised in animals with high specificity is commonly used in standardized and commercially produced food allergen detection methods (Besler 2001).

2.4.2 Detection of Allergenic Foods In order to fulfill the ever increasing demand to confirm the validity of allergen label statements and assess the risk to food-sensitive consumers, two analytic approaches have become common in recent years. The first involves immunoassays and DNA-based assays such as ELISA and real- time polymerase chain reactions (RT-PCR), both of which are used for large scale and rapid screening. The other approach is more detailed and uses multi-analyte methods such as liquid chromatography–mass spectrometry (LC-MS), which make it possible to carry out full quantification and confirmation of the analytes of interest.

Immunoassay and DNA-based assays are the most developed and frequently used methods for detecting allergenic foods and ingredients. Immunoassay’s high precision, simple handling, high throughput, and good potential for standardization make it a good option for routine food allergen detection (Monaci and Visconti 2010). The DNA-based methods use primers (short stretches of DNA) to facilitate the amplification of DNA originating from the offending food, which is followed by staining the amplified product with a fluorescent dye or using Southern blotting

11 following electrophoresis in an agarose gel to visualize the result. However, using the allergic protein itself is preferred to using markers that indicate the existence of allergic proteins, so immunoassays targeting food allergens such as ELISA and lateral flow devices (dipsticks) have become important tools for detecting food allergens. The first step in developing an immunological detection method for routine food analysis is the identification and purification of target allergens. Polyclonal antibodies for the detection of this specific allergenic protein can then be raised in animals like rabbits, rats, goats, sheep or chickens. Monoclonal antibodies, which are usually IgG and capable of recognizing only one epitope, are better suited for the recognition of a specific antigen. ELISA, which is based on a colorimetric reaction following binding with a specific enzyme-labeled antibody, has been widely used in the detection of allergenic foods and food allergens since its introduction in the 1980s. Commercial ELISA kits against a great variety of food allergens with detection limits ranging from 0.05 to 10 mg kg−1, depending on the allergen and the food matrix, have now been developed (Schubert-Ullrich and others 2009). However, it is important to note that both thermal and non-thermal food processing may lead to changes in target- protein structure, and thus significantly affect the result of immunoassays.

Multi-analyte methods, for example mass spectrometry (MS)-based proteomics methods, are usually used for final confirmation and quantification of the presence of an allergen in different foodstuffs. In mass spectrometry-based proteomics methods, complete protein sequencing is both time consuming and difficult to achieve. Specific peptides are used to confirm the presence of target allergens in food samples. Therefore, prior to submitting samples for MS analysis to identify intact proteins, pre-fractionation techniques including proteolytic digestion (typically by trypsin) or advanced liquid chromatography (LC) are performed to separate the peptides. The extract can then be subjected to MS or tandem-MS (MS2) to obtain a complete sequence of the peptides of interest. To identify these peptides, spectra are scanned against protein-sequence databases using search algorithms (Monaci and Visconti 2009). These methods are very sensitive and have been used to detect the peanut allergen- Ara h 1 in chocolate, with a detection limit as low as 2 ppm (Shefcheck and others 2006). However, for samples with a high protein content, the results of MS- based proteomics methods may affected by the levels of other proteins in solution and a false- negative result would be likely if a high content of other proteins masks the peptide signals from the allergenic protein.

CHAPTER THREE

HYPOTHESES AND OBJECTIVES

3.1 Hypotheses

1. If vinegar can influence the detectability of fish proteins, the results of anti-fish monoclonal antibody (MAb 8F5)-based iELISA and immunoblot against vinegar-treated fish samples will be different compared with water-treated and non-treated control samples.

2. If vinegar can affect the allergenicity of fish allergens, the results of iELISA using human plasma (IgE) against vinegar-treated fish samples will be different compared with water-treated and non- treated control samples.

3. If the effects of vinegar on both the detectability and allergenicity of fish are due to the same chemical reaction, which may consist of acid denaturation, acid proteolysis or acidic precipitation of antigenic/allergenic protein, their resulting patterns should be similar.

3.2 Objectives

The overall goal of this research was to investigate the effects of vinegar treatment on detectability by IgG-based immunoassay, and allergenicity by IgE-based immunoassay of whiting, cod and red grouper.

The specific objectives were to: 1) study the effect of vinegar on the detectability of cooked fish using anti-fish monoclonal antibody (MAb 8F5)-based iELISA and Western blot; 2) identify the effect of vinegar on the allergenicity of cooked fish using human plasma (IgE)-based iELISA; and 3) study the chemical action that attribute to the effects of vinegar on fish muscle proteins and fish allergens.

CHAPTER FOUR

MATERIALS AND METHODS

4.1 Materials

White vinegar (Heinz), and fresh whiting, cod, and red grouper were purchased from the Publix supermarket in Tallahassee, Florida. The fish samples were stored at -20 oC in a freezer until use. MAb 8F5 was previously developed in our laboratory. Hydrogen peroxide, horseradish peroxidase conjugated goat anti-mouse IgG (Fc specific), ABTS (2,2′-azino-bis 3-ethylbenzthiazoline- 6- sulfonic acid), and β-mercaptoethanol were purchased from Sigma-Aldrich Co., St. Louis, MO. Bromophenol blue sodium salt was purchased from Allied Chemical Corporation, New York.

Sodium chloride (NaCl), sodium phosphate dibasic anhydrous (Na2HPO4), sodium phosphate monobasic anhydrous (NaH2PO4), bovine serum albumin (BSA), sodium bicarbonate (NaHCO3), sodium carbonate (Na2CO3), citric acid monohydrate, sodium dodecyl sulfate, Tween 20 and all other chemicals, reagents, filters (Whatman No. 1 and No. 4 paper), and 96 well polystyrene microplates (Costar 9018) were purchased from Fisher Scientific, Fair Lawn, NJ. 0.5 M Tris-HCl buffer (pH 6.8), 1.5 M tris-HCl (pH 8.8), TEMED (N,N,N,N′-tetra-methyl ethylenediamine), Precision Plus Protein Kaleidoscope Standards, 30% acrylamide/ bis solution, Tris buffer saline (TBS), Tris/glycine buffer, 10 ×Tris/glycine/SDS buffer, supported nitrocellulose membrane (0.2 μm), and thick blot paper were purchased from Bio-Rad Laboratories Inc., Hercules, CA. All solutions were prepared using distilled deionized pure water (DDI water) from the NANO pure Diamond ultrapure water system (Barnstead International, Dubuque, IA). All chemicals and reagents were analytical grade. Plasma samples from three fish allergic patients (Table 3) were purchased from International Plasma Lab, WA. Horseradish peroxidase conjugated mouse anti- human IgE (ε chain specific) secondary antibody was purchased from Southern Biotech (Birmingham, Al), and alkaline phosphatase conjugated mouse anti-human IgE secondary antibody was purchased from Thermo Scientific (Pittsburgh, PA).

4.2 Methods

4.2.1 Sample Preparation Fish samples were prepared differently to satisfy the research requirements of the various experimental objectives.

In order to study the effects of vinegar treatment and treatment time on the detectability and allergenicity of finfish, 4 g portions of half thawed frozen fish muscle from each species were weighed into beakers. Vinegar or water was added to the samples in the ratio of 1:2 w/v, and the samples soaked for <1 min, 15 min, 30 min, and 60 min. A blank control received no processing. After treatment, the liquid was drained and the samples rinsed with D.I water and patted dry on a clean paper towel. The beakers containing muscle samples were then covered with aluminum foil, sealed with adhesive tape, and cooked for 5 min in a boiling water bath.

To study the chemical reactions that caused by vinegar treatment on the detectability and allergenicity of finfish, three groups of samples were prepared in different ways. In Group A, 4 g portions of partially-thawed frozen fish muscle were weighed into beakers. Vinegar or water was added to the samples in the ratio of 1:2 w/v, and the samples soaked for <1 min, 15 min, 30 min, and 60 min. A blank control received no processing. After treatment, the liquid was drained and the samples rinsed with D.I water and patted dry on a clean paper towel. The beakers containing muscle samples were then covered with aluminum foil, sealed with adhesive tape, and cooked (100°C) for 5 min in a boiling water bath. In Group B, 4 g portions of half-thawed frozen fish muscle were weighed into beakers. After cooking (100°C at 5 min), vinegar or water was added to the samples in the ratio of 1:2 w/v and soaked for <1 min, 15 min, 30 min, and 60 min. The liquid was drained and the samples rinsed with D.I water and patted dry on a clean paper towel. Control samples were cooked (100°C at 5 min) with no treatment. In Group C, 4 g portions of half thawed frozen fish muscle were weighed into beakers. Vinegar or water in the ratio of 1:2 w/v was added to the samples and the beakers covered with aluminum foil, sealed with adhesive tape, and immediately cooked (100°C) for 5, 15, 30, and 60 min. Control samples were cooked for 5, 15, 30, 60 min with no treatment.

4.2.2 Protein Extraction All prepared samples were mashed into small pieces using a glass rod and scissors after cooling to room temperature. Extraction buffer (0.15 M NaCl) in the ratio 1:5 w/v was then used to extract the soluble proteins. Each sample was homogenized at 1000 rpm (Model Ultra-turrax T25 basic, IKA) for 2 min followed by 1 hour incubation at 4°C. After incubation, samples were centrifuged (Model 5810R, Eppendorf) at 3220×g at 4°C for 30 minutes. Supernatants obtained after centrifugation were filtered through Whatman No.4 filtration paper and stored at -20 °C for later use.

4.2.3 Indirect ELISA Extracts of the fish samples were tested using iELISA to examine any changes in their immunoreactivities. As described earlier, MAb 8F5 supernatant and human plasma from fish allergic individuals were utilized as the source of primary antibodies, while horseradish peroxidase-conjugated goat anti-mouse IgG-Fc specific antibody (1:3000) and horseradish peroxidase conjugated mouse anti-human IgE (ε chain specific) antibody (1:1000) were used as the secondary antibodies for the studies of detectability and allergenicity, respectively. The ELISA procedure proceeded as follows: 2 μg/100μL of sample protein extract diluted in 0.06 M carbonate buffer (pH 9.6) was coated onto each well of a 96-well polystyrene microplate and incubated at 37°C for 1 hour. The plate was then washed three times with PBST (0.05% v/v Tween-20 in 10 mM PBS, pH 7.2) and incubated with 200 μL/well blocking solution (1% BSA in 10 mM PBS) at 37°C for 1 hour, followed by another washing step. Properly diluted sample protein extracts in 0.06 M carbonate buffer (pH 9.6) were coated (2 μg/100 μL per well) onto the wells of a 96-well polystyrene microplate and incubated at 37° C for 1h. After washing three times with PBST, 100 μL of the secondary antibody was added to each well and the plate was incubated at 37°C for 1 hour. After incubation, the plate was washed 5 times and then 100 μl/well ABTS substrate solution was added to the wells, color was developed at room temperature for 20 min, then 100 μl/well of 0.2 M citric acid was added to stop the reaction. The absorbance was read at 415 nm (Model MQX200R, BioTek).

4.2.4 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blot Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were used to reveal any changes in the antigenic component. SDS-PAGE was performed to resolve the soluble proteins in different sample extracts according to the method of Laemmli (1970), with modifications. Briefly, soluble proteins in fixed volumes (15 μl per lane) from the sample extracts were loaded on 5% stacking gel (pH 6.8) and separated on 12% polyacrylamide separating gel (pH 8.8). The gel was subjected to electrophoresis at 200 V for 45 min using a Mini-Protein 3 electrophoresis cell (Model 161- 3301, Bio-Rad) connected to a power supply (Model 3000, Bio- Rad).

The Western blot assay was carried out according to the method of Towbin and others (1979) with modifications. After separation of the proteins through SDS-PAGE, protein bands were transferred electrophoretically (1 hour at 300mA) from the gel to nitrocellulose membranes using a MiniTrans- Blot unit (Bio-Rad). After completion of the transfer, the membrane was washed with TBST (20 mM Tris, 500 mM NaCl, 0.05% Tween-20, pH 7.5), and then blocked with 1% BSA in TBS for 1 hour. After another washing step, the membrane was incubated with the primary antibody for 1 hour. The membrane was washed with TBST to remove excess antibody and then incubated for 1 hour at room temperature with the secondary antibody (goat anti-mouse IgG- alkaline phosphatase conjugate diluted 1:3000 in antibody buffer). After washing, the membrane was incubated with 5-bromo-4-chloro-3-indolyl phosphate/p-nitroblue tetrazolium chloride (BCIP/NBT) in 0.1 M Tris buffer (pH 9.5) for about 3 minutes to develop the color. D.I water was applied to stop color development. The appearance of a dark purplish band indicated the antibody- binding site. Prestained broad range protein standards (Precision Plus Protein Kaleidoscope Standards, Bio-Rad, 161-0375) were used as molecular weight markers for both the SDS-PAGE and Western blot.

CHAPTER FIVE

RESULTS AND DISCUSSION

5.1 Effects of Different Types of Vinegar on the Detectability of Whiting

A preliminary study tested the effects of vinegar, lemon juice and tomato sauce on the immunoreactivity of cod (Hou and Hsieh 2012). The results showed that among these three acid ingredients, vinegar has the strongest capacity to reduce immunoreactivity and thus the detectability of cod by 8F5-based ELISA. Therefore, in this study vinegar was selected as the acid ingredient used in all the subsequent experiments to study the effect of acid on the detectability of fish samples. In order to ascertain whether there was any variation between different types of vinegar, MAb 8F5-based iELISA tests were performed on whiting samples treated by five different types of vinegar (white, red wine, garlic wine, rice, and apple vinegar). Raw muscle of whiting was soaked with vinegar in the ratio of 1:2 m/v for 30 min, and then cooked (100C) for 5 min. The results are shown in Figure 1.

The OD reading decreased dramatically in all the vinegar-treated samples compared with the water-treated and non-treated control samples. The sample treated with rice vinegar showed the lowest OD reading (0.45±0.02), while the OD readings of the water-treated and non-treated control samples were 1.93±0.10 and 1.78±0.00, respectively. There was no considerable difference amongst the OD readings of the samples treated by different types of vinegar, indicating that these vinegar products all had a similar effect on reducing the detectability of whiting. The pH of these vinegars ranged from 2.5 to 3.0. It is plausible that the acid environment caused by adding vinegar leads to the change in the detectability of whiting. Therefore, the similarity in pH values may explain the similar effects of the different types of vinegar. Considering that white vinegar is the most widely consumed vinegar in the U.S., fish samples in the following studies were all treated with white vinegar.

5.2 Effects of Vinegar Treatment Time on the Detectability of Three Common Species of Finfish

As described earlier, MAb 8F5 is a previously developed fish-specific monoclonal antibody for fish protein detection. To study the effects of vinegar treatment as well as the treatment time on the detectability of different fish species (whiting, cod and red grouper), the total soluble protein concentration of fish samples was determined first. For fish samples treated by vinegar for different time lengths, less than 1 min treatment was indicated to be most effective in decreasing the total soluble protein concentration (Table 2). Comparing to water-treated control, decreases of 88.9%, 28.0%, 59.3% in the protein concentrations were observed in vinegar treated (<1 min) whiting, cod and red grouper sample, respectively, indicating that the amount of antigenic fish muscle protein present may also have decreased. However, the prolonged vinegar treatment time did not further intense the decrease trend of crude protein concentrations. In opposite, the total soluble protein concentrations of vinegar treated samples increased with the increase of treatment time.

The extracts of vinegar treated and control fish samples were examined using the MAb 8F5-based iELISA. The results are shown in Figure 2. In general, vinegar treatment reduced the immunoreactivity in the fish samples from all three fish species dramatically (up to 90% of the OD reading). As shown in Figure 2, the detectability of the vinegar-treated samples decreased markedly compared with the water-treated and non-treated control samples. After treatment with vinegar for 60 min, decreases of 81.3%, 76.3%, and 71.3% in the OD readings were observed in the whiting, cod and red grouper, respectively. These decrease in the OD readings indicated a lower detectability of the antigenic protein, either due to the decreased amount of the antigen present or the destruction of the epitope structure of the target antigen. As previously reported (Chen and Hsieh 2012), the antigenic protein of MAb 8F5 is fish tropomyosin, a 36 kDa myofibrilar protein. Therefore, vinegar treatment induced changes in tropomyosin, either by altering its conformation or by lowering its solubility.

Tropomyosin is an essential protein in muscle contraction, both in invertebrates and vertebrates. Fish tropomyosin is not allergenic, whereas shrimp tropomyosin is the major allergen in shellfish and is responsible for the cross-reactivity between crustaceans (Zhang and others 2006) and mollusks (Taylor 2008), as well as insects and mites (Witteman and others 2009; Ree and others

1996). Several MAbs against tropomyosin have been documented (Jeounga and others 1997; Lu and others 2007; Werner and others 2007), but there have been no reports of the effects of acid ingredients or pH value on the detectability of shellfish using immunochemical assays. However, in a study to detect mustard in salad dressing, Lee and others (2009) found acidic salad dressing matrices markedly reduced the detectability of mustard by ELISA. When vinegar was spiked with mustard flour at pH 3, 3.5, and 4, the detectability of mustard decreased considerably, with the lowest detectability at pH 3. The authors considered that this reduced detectability was probably due to the acid precipitation of mustard proteins, which rendered them insoluble and non- extractable.

As shown in Figure 2, the overall immunoreactivity of vinegar-treated whiting, cod, and red grouper samples was generally much lower than their water-treated and non-treated counterparts. However, the vinegar treatment time that produced the greatest change in detectability varied among the three fish species. For whiting, the peak decrease in the OD reading appeared in the sample treated with vinegar for 15 min (a 90% reduction), whereas whiting samples treated with vinegar for 30 min or longer showed slight increases in immunoreactivity. Compared with the whiting sample treated with vinegar for 15 min, which showed a negative iELISA result (OD reading <0.2), the iELISA result was positive (OD reading= 0.35±0.03) after 60 min treatment. For cod and red grouper, the OD reading decreased 97% for both samples treated for less than 1 min and an increase in the OD reading can also be seen in the cod and red grouper samples treated with vinegar for longer times. Long vinegar treatments may expose hidden epitopes, making them more accessible for antibody binding or the formation of new epitopes, which would both yield increased immunoreactivity and could explain the increasing trend observed among vinegar- treated samples.

5.3 Effects of Vinegar Treatment on Protein Banding Patterns of Finfish

SDS-PAGE was performed to reveal any changes in the protein banding pattern under treatment with vinegar. In general, the intensity of the bands of the vinegar-treated samples became lighter than that of the corresponding controls (Figure 3). In the vinegar-treated (for less than 1 min) cod and red grouper samples, there was no visible band below 150 kDa and in vinegar-treated whiting

20 there were several barely visible bands below 36 kDa. Electrophoretic separation of the fish extracts showed strongly stained single bands at 36 kDa in water-treated and non-treated control samples of whiting, cod and red grouper corresponding to fish tropomyosin. Compared to the control samples, the intensity of the 36 kDa bands in vinegar-treated samples was dramatically decreased. The fading of the bands indicates a decreased protein content due to the vinegar treatment. Sletten and others (2009) also reported this reduction in the intensity of the protein bands in acetic acid-salt brined herring products. Although the overall protein band intensity in vinegar-treated samples decreased, Figure 3 shows several novel bands appearing between 37 and 100 kDa in the vinegar-treated cod and red grouper samples. These minor novel bands may be caused by the acid hydrolyzation of larger proteins. Additional protein bands common to whiting and cod were seen at 10, 12, 17, 19, 24, 36, and 150 kDa, while bands at 12, 36, and 150 kDa were seen for red grouper. Interesting variations between species were noticed; the banding pattern for the control samples of cod were more similar to that for whiting, while that of the vinegar-treated samples of cod more closely resembled the banding pattern for vinegar-treated red grouper. A major band at 10-13 kDa in these fish samples also faded in vinegar-treated samples when compared with the control samples. This protein is likely to be fish parvalbumin based on its molecular weight (Elsayed 1971).

5.4 Effects of Vinegar Treatment on Antigenic Protein of MAb 8F5 in Finfish

Western blot using MAb 8F5 was performed to reveal any changes in the levels of antigenic protein under the treatment with vinegar. As shown in Figure 4, the monoclonal anti-fish tropomyosin antibody, MAb 8F5, recognized the 36 kDa tropomyosin bands in all the fish samples. The 72 and 108 kDa bands were recognized by MAb 8F5 in the whiting samples, but not in either the cod or red grouper samples, and so may be the dimeric and trimeric forms of whiting tropomyosin. As with the SDS-PAGE results, vinegar-treated samples of the three fish species in Western blot showed little or no band at 36 kDa, which is also consistent with the decreased iELISA OD reading in the vinegar-treated fish samples. Furthermore, the changes in the antigenic protein banding pattern of all three fish species once again agreed with the iELISA results. For whiting, the sample treated by vinegar for 15 min, which showed the lightest band at 36 kDa, also showed the lowest OD reading in iELISA. In cod and red grouper, there were no visible bands at all at 36 kDa for samples treated with vinegar for less than 1 min, and the iELISA results of these two samples were 21 negative (OD reading <0.2). This trend of increased iELISA readings in the samples that received longer vinegar treatment times supports the findings in Western blot. The combined results of SDS-PAGE and Western blot provide further evidence that vinegar treatment decreases the IgG- binding activity by either lowering solubility, and thus the amount of antigenic protein in the extract, or destroying its epitope. In addition, the exposure of hidden epitopes that slightly increased the IgG-binding could explain the enhanced intensity of bands of the lengthier vinegar- treated fish samples.

5.5 Effects of Vinegar Induced Chemical Reactions on the Detectability of Finfish

Three hypotheses were proposed to explain the changes of immunoreactivity of vinegar-treated fish samples with MAb 8F5. The reduction of immunoreactivity may be caused by 1) proteolysis of the antigenic protein by acidic proteases in raw fish muscle, which is activated by vinegar; 2) acid denaturation/hydrolysis of the protein; or 3) the precipitation of antigenic protein at pH around the pI value of tropomyosin. In order to verify these hypotheses, samples were treated by vinegar in three different ways according to the detailed sample preparation protocols described in Chapter Four. In Group A, fish samples were treated with vinegar first, and then cooked (100oC, 5 min). The iELISA results for Group A (Figure 5) were identical with the data recorded previously: vinegar treatment dramatically reduced the immunoreactivity in the fish samples of all three fish species. In Group B, fish samples were cooked to inactivate the proteases in the fish muscle (if such proteases were present), then the samples were treated with vinegar for different lengths of time (<1, 15, 30, and 60 min). The results showed that the detectability of all the vinegar-treated fish samples in Group B also decreased (Figure 6), which implies that the heat inactivation of proteases had no effect on the action of vinegar on the detectability of these three fish species. Therefore Hypothesis 1, that the decreased detectability of finfish could be attributed to the acid activated intrinsic acidic protease, was rejected.

Group C sought to verify Hypothesis 2, that acid denaturation contributes to the decreased immunoreactivity of finfish. Samples in Group C were cooked with vinegar for gradually increasing lengths of time (5, 15, 30, and 60 min). The results, shown in Figure 7, revealed that the iELISA readings of vinegar-treated whiting and cod samples decreased with increasing treatment time, whereas the OD reading of vinegar-treated red grouper remained fairly stable. The

22 trend of decreased detectability for the fish samples with prolonged vinegar treatment time could have therefore supported Hypothesis 2, but since the OD readings of the control samples without vinegar treatment also decreased, and to a similar extent, the dramatically reduced detectability in vinegar-treated finfish is not likely to be primarily induced by the effect of acid denaturation so Hypothesis 2 was also rejected.

Subsequently, the pH of fish extracts was determined using a pH meter in an attempt to verify Hypothesis 3. The pHs of the extracts of the vinegar-treated samples were all around 4.4, while those of the water-treated and non-treated samples were around 7.1. As the pI value of tropomyosin is 4.6 (Hamoir 1951), which is very close to the pH of vinegar-treated samples, it is reasonable to attribute the decreased immunoreactivity of vinegar-treated finfish to the acid precipitation of the antigenic protein of MAb 8F5 at a pH near its pI, which renders the protein insoluble and non- extractable. The fact that the total soluble protein concentration of the vinegar-treated finfish samples was lower than that of the control samples also suggests that the decreased immunoreactivity was indeed mainly caused by the precipitation of antigenic protein, thus Hypothesis 3 was supported.

In this study, a number of fish species-specific pattern changes were observed in the results. In Group B, the patterns for vinegar-treated cod and red grouper samples were different to those in Group A, although both showed dramatically lowered iELISA readings. As shown in Figures 5 and 6, a longer vinegar treatment time was required to reach the lowest reading in Group B compared to that in Group A. Hatae and others (1990) reported that fish species with a firm texture had thin muscle fibers with considerable heat-coagulating material between them, while species with a soft texture had thick muscle fibers with little heat-coagulating material. Since Group B samples were cooked first, this heat-coagulating material may have obstructed the rapid penetration of vinegar into the tissue, therefore lengthening the time needed for vinegar to react on the cooked muscle compared to the raw muscle. For the Group C samples, a slight decrease in immunoreactivity was observed in both whiting and cod. This finding indicates that although tropomyosin is thermal-stable, prolonged cooking (60 min) can still denature the protein to a small extent. Furthermore, as reported by Huang and Ochiai (2005) in their study of thermal stability of tropomyosin in different fish species, the thermal stability of fish tropomyosins may be species-

23 specific. They found clear differences in thermal stability among fish tropomyosins of six fish species, even though the identity of their amino acid sequences was more than 93.3%. This may explain our finding that unlike the other two fish species, the immunoreactivity of red grouper remained quite stable during the 60 min cooking.

In conclusion, based on the results of this study, factors such as fish species, the timing of the vinegar addition and the vinegar treatment time all had an effect on the detectability of vinegar- treated finfish. These effect factors and their corresponding iELISA results are summarized in Table 4. Generally, if the finfish was cooked together with vinegar, the longer the treatment time, the lower the immunoreactivity. Whether the finfish was treated with vinegar before or after cooking affected the treatment time that induced the most significant decreased assay signal, depending on the species of finfish.

5.6 Effects of Vinegar Treatment on the Allergenicity of Finfish

The investigation of altered immunoreactivity thus the detectability in the vinegar-treated finfish using MAb 8F5-based immunoassays revealed that the total soluble protein concentrations (Table 2) of vinegar-treated samples decreased as a result of the treatment, indicating that the amount of fish allergens present may also have decreased. Based on the SDS-PAGE gel results, the size of the protein band at 10-13 kDa found in all three fish species tested corresponded to the amount of the major fish allergen, parvalbumin, present. The intensity of this band became lighter in vinegar- treated samples compared with the corresponding control samples (Figure 3). This finding may provide evidence for a decrease in the amount of fish allergen in vinegar-treated fish samples, supporting the findings of studies that have reported that vinegar treatment decreased the allergenicity of shrimp, herring and peanuts (Perez-Macalalag and others 2007; Sletten and others 2009; Kim and otthers 2012); there have been no previous reports of the effects of vinegar on allergenicity in finfish. For the current study, the effects of vinegar treatment on the allergenicity of whiting, cod and red grouper were studied using human plasma (IgE) of three individuals (subject no. 21290, 18440, and 17427) with a confirmed fish allergy (PlasmaLab International, Everett, WA). Initially, the IgE-binding reactivity of extracts of raw and cooked salmon and cod were verified by iELISA. Serological cross-reactivity (OD reading ≥0.2) to different fish samples was observed in all three individuals. IgE from subject no. 21290 demonstrated the strongest IgE- 24 binding reactivity, while the IgE-binding reactivity of plasma from subject no. 18440, 17427 are relatively weaker (Figure 8). The IgE-binding reactivity of these three individuals corresponded to the information on their specific IgE concentrations (kU/I) provided by the commercial source (Table 3). The individuals with higher plasma IgE concentrations are known to exhibit stronger IgE-binding reactivity and Sampson (2001) reported that the quantification of food-specific IgE is a useful test for diagnosing symptomatic allergies to eggs, milk, peanuts, and fish, and could eliminate the need to perform double-blind, placebo-controlled food challenges. Therefore, it is appropriate to use a test measuring alterations in IgE-binding reactivity to predict changes in an individual’s allergenicity to finfish. All three individuals showed a positive reaction to both raw and cooked fish samples. Fish allergens were proven to be thermally stable by comparing the results of iELISA of raw and cooked fish samples.

IgE-based iELISA was performed in order to study the effects of vinegar treatment and treatment time on the overall allergenicity of whiting, cod and red grouper. A considerable reduction in the iELISA readings for two of the three plasma samples was observed in vinegar-treated fish samples of whiting and cod (Figures 9-10). However, vinegar appeared to have little effect on lowering the OD reading of red grouper (Figure 11). Furthermore, variations were found when comparing the results of the IgE-iELISA among different individuals. A decrease in OD reading of up to 69.2% and 83.9% for plasma from subjects no. 18440 and 17427, respectively, indicated that vinegar- treated whiting and cod exhibited reduced IgE-binding when compared with water-treated controls. A decreased reactivity for vinegar-treated whiting and cod samples for plasma from subject no. 21290 was also observed, but the effect was mild. A similar finding of individual variations has also been reported in other studies examining the allergenicity of various fish products using human IgE (Sletten and others 2009; Chatterjee and others 2006).

Interestingly, differences between the water-treated and non-treated samples were observed in the whiting samples (Figure 9). In general, water-treated whiting samples showed higher OD readings than non-treated ones, which indicates that the presence of water during cooking (100 °C) may slightly increase the IgE-binding reactivity of whiting. However, this effect was not observed in either the cod or the red grouper samples. In contrast to our finding, Sletten and others (2009) reported that increased IgE-binding by dried cod was observed in all 12 sera in their study, as

25 revealed by the decreased IC50 values in competitive ELISA. Studies in peanuts have also indicated that the involvement of water during cooking reduces IgE-binding reactivity; Mondonlet and others (2005) found that the IgE-binding reactivity of whole peanut protein extracts prepared from boiled peanuts was half that of the extracts prepared from raw and roasted peanuts, confirming the results reported in an earlier study by Beyer and others (2001). However, the effect of dry or wet heat treatment on the IgE-binding reactivity, which could be used to access the allergenicity of finfish is unclear, and needs further study.

A similar pattern in the results for IgE-based iELISA (Figures 9 and 10) and MAb 8F5-based iELISA (Figure 2) was noticeable. Whiting (vinegar-treated for 15 min) and cod (vinegar-treated for less than 1 min) samples showed the lowest reactivity in both the IgE-based iELISA and MAb 8F5-based iELISA. The increased OD reading with prolonged vinegar treatment time, which may be caused by either the exposure of hidden epitopes or the formation of new epitopes, was also observed in IgE-based iELISA for both whiting and cod.

5.7 Effects of Vinegar Induced Chemical Reactions on the Allergenicity of Finfish

Does this similarity in reactivity changes between the IgG and IgE based iELISA results indicate that vinegar affects both the detectability and allergenicity of finfish through the same chemical action? As discussed earlier, vinegar decreases the detectability of finfish mainly by precipitating the antigenic protein, tropomyosin, of the MAb 8F5 at a pH around 4.4, which is close to its pI value (pI=4.6). The pI value of the major allergen, parvalbumin, varies for different species. The α-parvalbumins have a pI of 5.0 or higher, while the more allergenic β-parvalbumins contain more acidic amino acids, resulting in a pI value of 4.5 or lower (Goodman and Pechére 1977). Depending on the pI range, the decreased allergenicity of finfish could also be induced by the precipitation of the major fish allergen, parvalbumin. However, previous studies have suggested that other chemical reactions may also contribute to the reduction of allergenicity. A study by Fink and others (1994) on the acid denaturation of frog parvalbumin reported that frog parvalbumin unfolds in solutions with pH 3-4. Since the IgE-binding epitopes on parvalbumin are located on a unique conformational site in the tertiary structure of the parvalbumin (Perez-Gordo and others 2011; Van and others 2005), acid induced unfolding may destroy the epitopes, thus decreasing the IgE-

26 binding reactivity of parvalbumin. Therefore, the decrease of allergenicity in vinegar-treated samples may also be caused by acid denaturation and the following study of different vinegar treatments on the allergenicity of finfish was therefore conducted to verify these hypotheses. Fish samples of whiting, cod and red grouper were treated with vinegar in three ways following the detailed sample preparation protocol described in Chapter Four. Briefly, samples in Group A were treated for 15 min, and then cooked (100 C 5 min); samples in Group B were cooked (100 C 5 min) then treated with vinegar for 15 min; and samples in Group C were cooked with vinegar for different lengths of time (5, 15, 30, and 60 min). Proteins were extracted using 0.15 M NaCl.

Unlike the decrease in OD readings observed in the IgG-based iELISA of vinegar-treated samples in both Groups A and B (Figures 6 and 7), the decreased OD readings were seen only in the IgE- based iELISA of vinegar-treated samples in Group A, and not those in Group B. As shown in Figures 12 and 13, the vinegar treatment before cooking (100 °C, 5min) decreased the OD reading of whiting and cod considerably, whereas implementing the vinegar treatment after cooking had no effect on the OD reading. As mentioned earlier, the decrease in the OD reading indicates a reduction in the IgE-binding reactivity, and thus the allergenicity of the allergens. This demonstrates that vinegar treatment after cooking does not decrease the allergenicity of whiting and cod. These findings suggest that the chemical reactions that cause the decrease in detectability and allergenicity are different. The reduction in the allergenicity of finfish as a result of the vinegar treatment may not be due to the precipitation of fish allergens in an acidic pH environment.

The pattern of results for the Group C samples suggests that rather than allergen precipitation, acid denaturation may be the chemical action that responsible for the decrease in the allergenicity of finfish. These results clearly show that 60 min cooking of fish muscle of whiting, cod or red grouper with vinegar can dramatically decrease their IgE-binding reactivity. Compared with control samples, an average decrease of 68.9%, 62.0%, and 60.7% of OD reading was observed in the whiting, cod and red grouper samples, respectively, when tested by IgE from three individuals. Especially for subjects 18440 and 17427, the iELISA result for whiting and red grouper was considerably reduced (OD reading <0.2) after the 60 min vinegar treatment, which indicates a considerable reduction in allergenicity. Moreover, prolonged vinegar cooking time with vinegar intensified this decreased allergenicity in all three fish species. Comparing the OD reading of the

27 samples cooked with vinegar for 5 min and those for samples cooked with vinegar for 60 min, a clear decrease is visible (Figures 13-15), indicating that the effect of vinegar on the IgE-binding reactivity of finfish may be due to acid denaturation -- the combination of heat and acid.

It is noticeable that red grouper is more resistant to acid treatment than either whiting or cod. Vinegar treatment, either before or after cooking, had no effect on the IgE-binding reactivity of red grouper. In Group B, vinegar-treated red grouper samples even showed higher IgE-binding reactivity than control samples when tested by IgE from all three individuals. It is possible that the 15 min vinegar treatment exposed hidden epitopes in the red grouper samples but was unable to destroy them. However, in Group C, an average decrease of 61.5% in the OD reading was observed between vinegar-treated (for 60 min) and water-treated (for 60 min) red grouper samples. Looking at the Group C results (Figures 14-16), iELISA using IgE from subject no. 21290 showed 46.1%, 37.1%, 31.0% decreases in the OD reading for whiting, cod and red grouper samples, respectively, treated with vinegar for 60 min. This decrease was not observed in samples treated with vinegar before (Group A) or after cooking (Group B). Therefore, unlike the effects of vinegar on the detectability of finfish tested by MAb 8F5-based iELISA, it is difficult to determine which treatment method and treatment time produce the most significant decrease in detectability for different fish species. Overall, lengthy cooking of fish muscle, together with vinegar treatment, is the most effective way to reduce the allergenicity of finfish for all fish allergic individuals, despite the existence of specie and individual variations.

CHAPTER SIX

CONCLUSIONS

Changes in immunoreactivity, manifested in terms of the detectability using IgG-based iELISA, and allergenicity, measured using IgE-based iELISA, as a result of treating whiting, cod and red grouper samples with vinegar were investigated in this study. The results indicate that vinegar treatment can decrease both the detectability and allergenicity of whiting, cod and red grouper. However, the chemical action by which vinegar affects the binding properties between antigenic tropomyosin-IgG binding and fish allergen-IgE binding are distinctively different. Vinegar treatment decreased the detectability of finfish to MAb 8F5 by acidic precipitation of the antigenic protein-tropomyosin, while the allergenicity was reduced mainly by acid denaturation of the allergenic protein. Therefore, it is not appropriate to use changes in the IgG-binding activity under the effect of vinegar to predict changes in IgE-binding reactivity, although they do share some common patterns of immunoreactivity changes. In MAb 8F5-based iELISA, whiting, cod and red grouper treated for 15 min, <1 min and <1 min, respectively showed negative results (OD reading< 0.2), indicating the most dramatically decreased detectability. To address the difficulty of detecting fish in acidic pH environments, the production of antibody targeting acid-resistant fish proteins could be a solution. Moreover, although cooking with vinegar for 60 min decreased the overall allergenicity of whiting, cod, and red grouper, the variations among individual subjects and different fish species clearly demonstrates the unpredictable nature of food-allergic responses. The clinical significance of these findings remains to be established. Although the efficiency of vinegar treatment in avoiding or alleviating clinical fish allergic reactions is still not clear, vinegar-treated fish products with decreased allergenicity can be potentially used in oral desensitization therapy for IgE mediated fish allergic individuals, and it could also be used to lower the risk of fish allergy in those patients taking anti-acid medications.

APPENDIX A

TABLES AND FIGURES

Table 1. Fish allergens Origin Allergen M.W Protein Reference (kDa) Classification Baltic cod Gad c 1 12.3 β-parvalbumin Elsayed and Bennich 1975 (Gadus callarias) Atlantic salmon Sal s 1 11.9 β-parvalbumin Lindstrom and others 1996 (Salmo salar) Atlantic cod Gad m 1 11.5 β-parvalbumin Das Dores and others 2002 (G. morhua) Whiff Lep w 1 12 β-parvalbumin Griesmeier and others 2010 (Lepidorhombus whiffiagonis) Mackerel Sco j 1 11 β-parvalbumin Hamada and others 2003 (Scomber japonis) Mackerel Sco a 1 11 β-parvalbumin Hamada and others 2003 (S. australasicus) Mackerel Sco s 1 11 β-parvalbumin Hamada and others 2003 (S. scombrus) Pacific Pilchard Sar sa 13.1 β-parvalbumin Beale and others 2009 (Sardinops sagax) 1.0101 Alaska Pollock The c 1 11.5 β-parvalbumin Van Do and others 2005 (Theragra chalcogramma) Cod 41 aldehyde phosphate Das Dores and others 2002 (Gadus morhua) dehydrogenase Bigeye tuna 120 collagen type I Hamada and others 2001 (Thunnus obesus) Swordfish 25 Kelso and others 1996 (Xiphias gladius) Mackerel 28 triosephosphate Wang and others 2010 (Scomber japonicus) isomerase Anisakis Ani s 1 21 Moneo and others 2000 (Anisakis simplex)

Table 2. Total soluble protein concentration of whiting, cod and red grouper Soluble Protein Concentration (mg/ml) Fish Vinegar treated Water treated Non treated Whiting(< 1 min) 0.06 0.54 0.58 Whiting(15 min) 0.15 0.60 0.53 Whiting(30 min) 0.12 0.57 0.47 Whiting(60 min) 0.17 0.66 0.43 Cod(< 1 min) 0.36 0.50 0.59 Cod(15 min) 0.42 0.50 0.54 Cod(30 min) 0.45 0.57 0.63 Cod(60 min) 0.55 0.52 0.58 Red grouper(< 1 min) 0.77 1.89 2.64 Red grouper(15 min) 1.25 1.99 3.14 Red grouper(30 min) 1.81 1.96 2.86 Red grouper(60 min) 1.95 1.80 3.19

Table 3. Clinical features of patients. (Source: Plasmalab International, Everett, WA) No. Age Sex Total IgE kU/I Specific IgE, kU/I (ImmunoCAP) C T S 21290 13 M 2432 84.4 40.4 85.0 18440 32 F 4105 35.8 - 29.7 17427 42 M 398 42.1 - 39.2 C= cod; T= tuna; S= salmon

Table 4. Immunoreactivity of MAb 8F5 against vinegar-treated fish samples determined by iELISA. Samples in group A, B and C were treated with vinegar before, after, and during cooking, respectively. Absorbance readings at 415 nm: <0.19—“-”, 0.20-0.49—“+”, 0.50- 1.49—“++”, >1.50—“+++”. Treatment Group A Group B Group C time(min) W C R W C R W C R <1 +++ - - +++ +++ +++ 5 + ++ - 15 + + + + - + + ++ - 30 - + + ++ - - + + - 60 - + + ++ + - - + - W= Whiting, C= Cod, R= Red grouper.

Figure 1. Effects of different types of vinegar on the immunoreactivity of whiting by MAb 8F5 based-iELISA. Raw whiting was treated with different types of vinegar for 30 min, after cooking (100 °C, 5 min) the fish protein was extracted with 0.15 M NaCl. Plate was coated with samples at the concentration of 2μg/100 μL. MAb 8F5 supernatant diluted 1:3 was used in the assay. Results were expressed as A415  SD, n =2.

Figure 2. Effect of vinegar treatment on the immunoreactivity of whiting, cod and red grouper by MAb 8F5-based iELISA. Raw whiting, cod and red grouper was treated with vinegar for different time lengths, after cooking (100 °C, 5 min) the fish protein was extracted with 0.15 M NaCl. Plate was coated with samples at the concentration of 2μg/100 μL. MAb 8F5 supernatant diluted 1:3 was used in the assay. Results were expressed as A415  SD, n =2.

(a) whiting

(b) cod

(c) red grouper Figure 3. SDS-PAGE profiles of vinegar-treated, water-treated and control samples of whiting, cod and red grouper. Fish extracts of whiting, cod and red grouper diluted 1:1, 1:1, 1:3 v/v, respectively, with sample buffer were used in the assay. Each lane was loaded with 15 μl of diluted sample extract.

(a) whiting

(b) cod

(c) red grouper Figure 4. Antigenic protein banding patterns of vinegar-treated, water-treated and control samples of whiting, cod and red grouper. MAb 8F5 supernatant (1:3) was used as the primary antibody, anti-mouse IgG-AP conjugated antibody (1:1000) was used as the secondary antibody.

Figure 5. Study of the effects of vinegar induced chemical reactions on the immunoreactivity of whiting, cod and red grouper (Group A). The samples in Group A were treated with vinegar, water or left untreated for <1, 15, 30, 60 min separately and then cooked (100 °C) for 5 min. Plate was coated with samples at the concentration of 2μg/100 μL. MAb 8F5 supernatant diluted 1:3 was used in the assay. Results were expressed as A415  SD, n =2.

Figure 6. Study of the effects of vinegar induced chemical reactions on the immunoreactivity of whiting, cod and red grouper (Group B). The samples in Group B were cooked for 5 min then treated with vinegar, water or left untreated for <1, 15, 30, 60 min separately. Plate was coated with samples at the concentration of 2μg/100 μL. MAb 8F5 supernatant diluted 1:3 was used in the assay. Results were expressed as A415  SD, n =2.

Figure 7. Study of the effects of vinegar induced chemical reactions on the immunoreactivity of whiting, cod and red grouper (Group C). The samples in Group C were cooked (100 °C) with vinegar, water or left untreated for 5, 15, 30, 60 min separately. Plate was coated with samples at the concentration of 2μg/100 μL. MAb 8F5 supernatant diluted 1:3 was used in the assay. Results were expressed as A415  SD, n =2.

Figure 8. Screen for IgE-binding reactivity of raw and cooked salmon and cod. The fish proteins in raw and cooked (100 °C, 5 min) salmon and cod were extracted with 0.15 M NaCl. Plate was coated with samples at the concentration of 2μg/100 μL. Human plasma from three individuals diluted 1:2 v/v were used in the assay. Results were expressed as A415  SD, n =2.

Figure 9. Effect of vinegar treatment on the IgE-immunoreactivity of whiting by human plasma-based iELISA. Raw whiting was treated with vinegar, water or left untreated for different time lengths before cooking (100 °C, 5 min). The fish proteins were extracted with 0.15 M NaCl. Plate was coated with samples at the concentration of 2μg/100 μL. Human plasma from three individuals diluted 1:4, 1:2, and 1:2 v/v, respectively were used in the assay. Results were expressed as A415  SD, n =2.

Figure 10. Effect of vinegar treatment on the IgE-immunoreactivity of cod by human plasma-based iELISA. Raw cod was treated with vinegar, water or left untreated for different time lengths before cooking (100 °C, 5 min). The fish proteins were extracted with 0.15 M NaCl. Plate was coated with samples at the concentration of 2μg/100 μL. Human plasma from three individuals diluted 1:4, 1:2, and 1:2 v/v, respectively were used in the assay. Results were expressed as A415  SD, n =2.

Figure 11. Effect of vinegar treatment on the IgE-immunoreactivity of red grouper by human plasma-based iELISA. Raw red grouper was treated with vinegar, water or left untreated for different time lengths before cooking (100 °C, 5 min). The fish proteins were extracted with 0.15 M NaCl. Plate was coated with samples at the concentration of 2μg/100 μL. Human plasma from three individuals diluted 1:4, 1:2, and 1:2 v/v, respectively were used in the assay. Results were expressed as A415  SD, n =2.

Figure 12. Study of the effects of vinegar induced chemical reactions on the IgE-binding reactivity of whiting, cod and red grouper by human plasma-based iELISA (Group A). The samples in Group A were treated with vinegar, water or left untreated for 15 min and then cooked (100 °C) for 5 min, the fish proteins were extracted with 0.15 M NaCl. Plate was coated with samples at a concentration of 2μg/100 μL. Human plasma from three individuals diluted 1:8, 1:4, and 1:4 v/v, respectively were used in the assay. Results were expressed as A415  SD, n =2.

Figure 13. Study of the effects of vinegar induced chemical reactions on the IgE-binding reactivity of whiting, cod and red grouper by human plasma-based iELISA (Group B). The samples in Group B were cooked (100 °C) for 5 min then treated by vinegar, water or left untreated for 15 min, the fish proteins were extracted with 0.15 M NaCl. Plate was coated with samples at a concentration of 2μg/100 μL. Human plasma from three individuals diluted 1:4, 1:2, and 1:2 v/v, respectively were used in the assay. Results were expressed as A415  SD, n =2.

Figure 14. Study of the effect of vinegar induced chemical reactions on the IgE-binding reactivity of whiting by human plasma-based iELISA (Group C). The samples in Group C were cooked (100 °C) with vinegar, water or left untreated for 5, 15, 30, 60 min, the fish proteins were extracted with 0.15 M NaCl. Plate was coated with samples at a concentration of 2μg/100 μL. Human plasma from three individuals diluted 1:4, 1:2, and 1:2 v/v, respectively were used in the assay. Results were expressed as A415  SD, n =2.

Figure 15. Study of the effect of vinegar induced chemical reactions on the IgE-binding reactivity of cod by human plasma-based iELISA (Group C). The samples in Group C were cooked (100 °C) with vinegar, water or left untreated for 5, 15, 30, 60 min separately, the fish proteins were extracted with 0.15 M NaCl. Plate was coated with samples at a concentration of 2μg/100 μL. Human plasma from three individuals diluted 1:4, 1:2, and 1:2 v/v, respectively were used in the assay. Results were expressed as A415  SD, n =2.

Figure 16. Study of the effect of vinegar induced chemical reactions on the IgE-binding reactivity of red grouper by human plasma-based iELISA (Group C). The samples in Group C were cooked (100 °C) with vinegar water or left untreated for 5, 15, 30, 60 min separately, the fish proteins were extracted with 0.15 M NaCl. Plate was coated with samples at a concentration of 2μg/100 μL. Human plasma from three individuals diluted 1:4, 1:2, and 1:2 v/v respectively were used in the assay. Results were expressed as A415  SD, n =2.

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BIOGRAPHICAL SKETCH

EDUCATION

The Florida State University Tallahassee, FL Master of Science Major: Nutrition and Food Science August 2011- present Vinegar on Detectability

Thesis: The Effects of and Allergenicity of Finfish. Zhejiang Gongshang University Hangzhou, China Bachelor of Science in Food Science Major: Food Safety and Quality August 2006- June 2010

Thesis: Screening of Biological Preservative Lactic Acid Bacteria and Characterization of Bateriocin. RESEARCH EXPERIENCE

The Florida State University Tallahassee, FL January 2012- present  Effects of acid ingredients on immunoreactivity and allergenicity of finfish.  Food allergens and fish proteins.  Determination of protein concentration, Enzyme-linked immunosorbent assay (ELISA), SDS-PAGE and Western blot.  Developing research, include paperwork writing, research planning, time arrangement and budget.

Zhejiang Gongshang University Hangzhou, China June 2009- November 2009  Screening of biological preservative lactic acid bacteria and characterization of the bateriocin.  Lactic acid bacteria, bacteriocin and their potential to be biological preservative.  Screening bacteriocin-producing lactic acid bacteria, basic microbiology characterization skills and 16S rDNA identification technique.

Zhejiang Gongshang University Hangzhou, China January 2009- May 2009  Efficiency of microwave-assisted acid hydrolysis to gas chromatography for monosaccharide composition analysis of polysaccharide.  Internal standard method, t-test, gas chromatography.  Efficiency of microwave assisted-acid hydrolysis for sample preparation of different test methods.

INTERNSHIP EXPERIENCE

Administration of Quality Supervision, Inspection and Quarantine in Zhejiang, Research Assistant Hangzhou, China Nov. 2009- May 2010

 Determination of contents of illegal food additives (e.g. melamine) in animal products using high- performance liquid chromatography (HPLC).  Hepatitis E virus infection in swine in Zhejiang province using reverse transcription polymerase chain reaction (RT-PCR).  Quality management and techniques for illegal food additives in China.  Record management and classification.

PROFESSIONAL EXPERIENCE

Training Experience  HACCP Certificate Registered, International HACCP Alliance August 2012 Experience: training of HACCP background knowledge (seven principles HACCP, physical, chemical, biological hazards and hazard control), HACCP plans writing and implement HACCP in food processing, distribution and preparation environment.

 Hazardous Waste Awareness Refresher Training, The Florida State University May 2012 Experience: training of hazardous waste determination and definitions, hazardous waste generator status, accumulation limits, container management, spill control etc.

Teaching Experience  Instructor of English Reading course, Nasi Education January- June 2011 Responsibilities: Instruct knowledge about how to improve English reading ability and skills; Develop lectures; Make PPT and video tutorials to trains students,; Prepare and graded exams.

Computer Skills and Certificates  MS Office (Word, Excel, PowerPoint), SPSS, Origin 7.0, Auto CAD  International HACCP Alliance HACCP Certification (USA, 2012)  Food Quality Inspector Certification (China2010)  National Compute Level II certification (China, 2008)

Leadership Activities  Investigation of Hygienic Production Conditions in Small Workshops, China July 2007 Responsibilities: event planning and organization, communication with local government food safety department and media, etc. The team won the award of Provincial Excellent Team of Summer Social Practice  Popularizing Food and Drug Safety Knowledge in Impecunious County, China July 2008 . Responsibilities: event planning and organization  Student Union in College of Food and Biology Engineering, Zhejiang Gongshang University-Minister of Secretariat May 2007- May 2008 Responsibilities: organization and communication between different departments of College Student Union, document management, assets management.

PUBLICATION

 Junli Zhu, Ye Wang, Jianrong Li. Screening of biological preservative Lactic acid bacteria and characterization of the bacteriocin[J], China Chewing, 2010, 5: 42-45

AWARDS AND HORNORS

 Research Assistantship, Florida State University, 2013  Florida-China linkage scholarship, Florida, 2012-2013  7th Annual Experience Asia Festival, Tallahassee- Finger language performer, 2011  Excellent Undergraduate Thesis Paper, Zhejiang Gongshang University, 2010  Social Practice Special Scholarship, Zhejiang Gongshang University, 2009  College Comprehensive Scholarship, Zhejiang Gongshang University, 2007, 2008  Provincial Excellent Team of Summer Social Practice award, Zhejiang, China, 2007  Gongshang University, 2007  Outstanding Member in Summer Internship, Zhejiang Gongshang University, 2007 Excellent Student Award, Zhejiang

AFFILIATION

 Student Membership of Institute of Food Technologists