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2016 Lectin Analyses of Soybean and Soybean Products Qing Zhao
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COLLEGE OF HUMAN SCIENCES
LECTIN ANALYSES OF SOYBEAN AND SOYBEAN PRODUCTS
By
QING ZHAO
A Thesis submitted to the Department of Nutrition, Food & Exercise Sciences in partial fulfillment of the requirements for the degree of Master of Science
2016 Qing Zhao defended this thesis on June 21, 2016. The members of the supervisory committee were:
Shridhar K. Sathe Professor Directing Thesis
Qinchun Rao Committee Member
John G. Dorsey Committee Member
The Graduate School has verified and approved the above-named committee members, and certifies that the thesis has been approved in accordance with university requirements.
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ACKNOWLEDGMENTS
This thesis becomes a reality with the kind support and help of many individuals. I would like to express my sincere appreciation to all of them.
Foremost, I am very grateful to the mentor, philosopher, and the major professor, Dr. Shridhar K. Sathe, who gave me the opportunity to study and work in his lab in NFES at Florida State University. I am thankful to him for providing ideas and suggestions to make it possible for me to work on this topic that was of great interest to me, for providing the excellent and quality scientific instruments and materials in our lab, for sharing his experience and expertise with me, and making me aware the importance of thinking independently. I would like to thank Dr. Qinchun Rao for serving in my committee, providing me guidance and valuable thinking. I am also thankful for Dr. John G. Dorsey for willing to serve as my committee member and also providing suggestions during my research program at FSU.
I would like to express my gratitude to the Department of Nutrition, Food, and Exercise Sciences for providing the opportunity to pursue my Masters degree. I also want to thank Ms. Tara Hartman, Ms. Ann Smith, Mr. David Parish for helping me during my study here at FSU.
I am sincerely thankful to Changqi Liu (Ben) for taking the time to teach me the basic laboratory techniques and giving the advice for my experiments and thesis writing.
My lab mates, Changqi, Valerie, Sahil, and Sepideh made my life in lab joyful and am thankful for their research support.
Finally, I am extremely thankful to my parents and all family members for their unconditional love, encouragement and financial support. Without your support and love, I could not have come this far.
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TABLE OF CONTENTS
List of Tables ...... v List of Figures ...... vi List of Abbreviations ...... vii Abstract ...... viii
1. INTRODUCTION ...... 1
Background ...... 1 Statement of the Problem ...... 3 Research Hypothesis ...... 4 Specific Aims ...... 4
2. REVIEW OF LITERATURE ...... 6
Food Allergies ...... 6 Soybean ...... 7 Lectins ...... 9 Soy Lectin ...... 10 Food Processing ...... 11
3. MATERIALS AND METHODS ...... 14
Materials ...... 14 Methods ...... 15
4. RESULTS AND DISCUSSION ...... 22
Lectin Purification ...... 22 Competitive ELISA ...... 25 Hemagglutination Assay ...... 40
5. CONCLUSIONS ...... 49
APPENDICES ...... 50
A. ANIMAL CARE AND USE COMMITTEE APPROVAL MEMORANDUM ...... 50 B. HUMAN SUBJECTS COMMITTEE APPRROVAL MEMORANDUM ...... 51 C. INFORMED CONSENT FORMS ...... 52
References ...... 53
Biographical Sketch ...... 65
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LIST OF TABLES
1 Summary of lectin purification by column chromatography ...... 24
2 Intra- and inter-assay variations for the pAb-based soybean lectin competitive ELISA ...... 28
3 Accuracy of constructed competitive ELISA ...... 29
4 Soybean varieties used ...... 30
5 Raw soybean seed lectins analyzed by using the ELISA ...... 31
6 Effects of thermal processing on SBA immunoreactivity as assessed by the competitive ELISA ...... 33
7 Germinated soybean sprouts ...... 36
8 Effects of germination on SBA immunoreactivity as assessed by the competitive ELISA ....38
9 The SBA immunoreactivity of commercial soybean products as assessed by the competitive ELISA ...... 39
10 Pure lectins measured by using the hemagglutination assay ...... 41
11 Raw soybean seed lectins analyzed by using the hemagglutination assay ...... 41
12 Effects of thermal processing on SBA hemagglutination activity of GMO soybean AG5831 as assessed by the hemagglutination assay ...... 43
13 Effects of thermal processing on SBA hemagglutination activity of Non-GMO soybean cobb as assessed by the hemagglutination assay ...... 44
14 Effects of germination on SBA hemagglutination activity as assessed by the hemagglutination assay ...... 46
15 The SBA hemagglutination activity of commercial soybean products as assessed by the hemagglutination assay ...... 47
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LIST OF FIGURES
1 Soybean: acreage planted and value in United States, 1960-2013 (USDA oil crop yearbook, 2014) ...... 8
2 Elution profile for 40% - 80% ammonium sulfate precipitate soybean protein extract off a DEAE DE-53column (2.6 × 30.5 cm)...... 22
3 Elution profile the soybean lectin (DEAE DE-53 pool) off a Sephacryl S200 column (2.6 × 91 cm) ...... 23
4 SDS-PAGE of laboratory purified lectin (1) and commercial lectin (2) ...... 24
5 The schematic representation of the soybean lectin inhibition ELISA procedures ...... 25
6 The logarithmic regression curve of soybean lectin standards ...... 26
7 A sample calculation of LOD for the constructed SBA competitive ELISA ...... 27
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LIST OF ABBREVIATIONS
AP Alkaline Phosphatase Blocking buffer 5% w/v NFDM in TBS-T (pH 7.6) β-ME β-Mercaptoethanol BSA Bovine Serum Albumin BSB Buffer Saline Borate DI water Distilled water ELISA Enzyme Linked Immunosorbent Assay FT Flowthrough GMO Genetically modified Hr Hour HRP Horse Radish Peroxidase HU Hemagglutinating Unit IgE Immunoglobulin E IgG Immunoglobulin G kDa Kilo Dalton mAb Monoclonal Antibody MW Molecular Weight NFDM Nonfat Dried Milk ºC Degree Celsius pAbs Polyclonal Antibodies PBS Phosphate Buffered Saline RT Room Temperature (~22 ºC) SBA Soybean agglutinin SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis TBS-T Tris Buffered Saline with 0.05% (v/v) Tween 20 v/v Volume by Volume w/v Weight by Volume
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ABSTRACT
Soybean allergies are one of top eight types of food allergies, affecting ~ 0.4% children and 0.3% adults in North America. Soybean agglutinin (SBA) protein, also known as soybean lectin, is both an allergen as well as an anti-nutrient that reducing the nutritional value of soybean and soybean products. The objective of this study was to obtain a validated methodology that is precise and accurate in the measurement of SBA while allowing minimally equipped laboratories to effectively conduct the analysis. A competitive ELISA was constructed and optimized by using rabbit anti whole soybean polyclonal antibodies (pAb) as primary Ab. The constructed competitive ELISA was validated to have LOD of 0.063 ng/ml of soybean lectin in all tested samples. The ELISA is reproducible and accurate as CVs of all ELISAs tested were less than 24% and the average recoveries were within 15% of the actual value, which demonstrating accuracy of the assay. However, further investigation is needed to evaluating CV of the assay. The validated ELISA method was able to detect and quantify the SBA in 20 soybean varieties and indicated that the natural variability of SBA is subject to the effects of genotype and environment. Moreover, the ELISA can detect the SBA content in thermal processed soybean, germinated soybean sprouts, and commercial soybean products. The results suggested that processing methods can affect soybean lectin immunoreactivity. Compare to the conventional hemagglutinating assay, ELISA is more sensitive and effective. In conclusion, the constructed pAb-based competitive ELISA provides an efficacious detection and quantification for the soybean lectin.
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CHAPTER 1
INTRODUCTION
Background
According to the Centers for Disease Control and Prevention (2013), more than 50 million Americans have an allergy of some kind and food allergies are estimated to affect 6.53% of children and 9.72% of adults in North America (Patel et al., 2015). Food allergy symptoms and disorders can appear at any age and affect the skin, respiratory tract, gastrointestinal tract and cardiovascular system, which may lead to serious consequences including breathing difficulties, swelling of throat and lips, vomiting and abdominal cramps, and under the worst circumstances death. Most food allergies start in childhood, but people can develop an allergy to foods they have eaten for years with no problems. Sufferers of food allergies are medically required to avoid the consumption of the allergy-causing foods. Eight types of food (milk, egg, peanut, tree nuts, wheat, fish, shellfish, and soybean) account for about 90 percent of food allergies (Sicherer et al., 2006). A foodinduced allergic reaction is usually defined as an immune response to a particular food protein or a group of proteins (Vanga et al., 2015). Food allergies can be classified into three groups based on the mechanism of the allergic response: immunoglobulin E (IgE) mediated (the most common type), non-IgE mediated and mixed IgE and non-IgE-mediated food allergies (NHS Choices, 2014). IgE-mediated hypersensitivity to food is known as type I food allergy. Soybean is an economically important crop, which serves as a source of good-quality protein for animals and humans. According to the USDA, the United States is the world’s largest soybean producer and second largest soybean exporter, contributing 34% of the world’s soybean production and 33% of world’s soybean exports. Soybeans are the second-most-planted field crop in the United States after corn, with 84.8 million acres and $41.8 billion value. The resulting harvest yielded 3.97 billion bushels in 2014 and soybean productivity is continuing to grow (USDA Crop Production 2014 Summary). In addition, soybean is the first dominant genetically modified (GMO) crop grown and traded, which accounts for 75% of the 100 million hectares of soybean planted globally (James, 2011). Soybean seeds contain up to 48% protein and 22% oil,
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which serve as a good and inexpensive source of cooking oil and proteins for human consumption (Derbyshire et al., 1976). Although soybean contains nutritional components such as essential amino acids, vitamin E, essential fatty acids, and isoflavones, soybean allergy is among the top eight types of food allergies, accounting for 0.4% and 0.3% food allergies, respectively, in children and adults in North America (Sicherer et al., 2010). Soybean also contains several anti-nutrients, reducing the nutritional value of unprocessed soybean. These anti-nutrients include lectins, raffinose (a non- digestible carbohydrate), and enzyme inhibitors (Breeze et al, 2015). Soybean lectin is both an allergen as well as an anti-nutrient in soybean. Lectins have long been considered as ‘antinutrients’, mainly due to their negative effects of causing non-pathogenic food poisoning when some legumes were not properly cooked or uncooked before consumption (Van et al., 1998). When consumed in excess by sensitive individuals, lectins can cause three major physiological effects: gastrointestinal damage, type 2 IgG immune responses, and hemagglutination (Pusztai et al., 1983; Gell, 1975; Roitt et al., 1989). Furthermore, many lectins are digestion resistant. They can survive gut passage, bind to gastrointestinal cells, damage the gastric mucosa, and interfere with nutrients absorption, maintaining full biological activity (González et al., 2005; Weaver and Bailey, 1987; Bardocz et al., 1995). The major lectin in soybean seeds, often referred to as soybean agglutinin (SBA), was first characterized by Liener and Pallansch (1952). SBA has a specific affinity towards terminal N-acetyl-Dglucosamine and D-galactose (Schulze et al., 1995). SBA is poorly digestible and has been associated with soybean intolerance and allergy, particularly in human infants and bovine calves (Kilshaw and Sissons, 1979; Heppel et al., 1987). It has been reported that SBA could decrease growth rates in animals such as rats and chickens (Gu et al., 2010; Zang et al., 2006; Douglas et al., 1999). In general, dietary lectins have the ability to survive digestion by the gastrointestinal tract and to bind to the intestinal mucosa, brush border of the gut lumen, or receptors of epithelial cells via specific oligosaccharides or glycopeptides (Oliveira et al., 1989; Pusztai, 1991). As a result of these interactions, a series of harmful reactions are induced, which place soy lectin in antinutritive and/or toxic substances in soybean. Food processing methods play an important role in modification of food allergenicity and anti-nutrient activity, and include moist heat (such as blanching, steaming, boiling, autoclaving
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and microwaving), dry heat (such as dry roasting and frying), and non-thermal processing methods (such as germination, fermentation) (El-Waziry et al., 2007; Sathe et al., 2005). Heat inactivation of SBA helps improve animal nutritional performance and soybean nutritional quality (Qin et al., 1996; Ma and Wang 2010). Due to the toxicity of SBA and the development of new soybean varieties, the interest in monitoring SBA content and activity in food or feed continues.
Statement of the Problem
Because of the favorable agronomic characteristics, relatively low price, and high quantity and quality of soybean protein and oil, soybean has become an important and widely used source of food and feed (Liu, 2000). Soybeans have been developed for different types of foods and supplies for humans and the consumption of soybean and its products have been increasing (Traina and Breene, 1994; Albertazzi, 2002; Orf 2013). Because soy lectin is an allergen and an anti-nutrient, detecting and quantifying soy lectin in the diet to support food safety and quality has been of interest. The AOAC (Association of Official Analytical Chemists) International and the American Oil Chemists’ Society (AOCS) have published official methods of analysis for many important analytes present in soybean (AOAC official methods), which are also available from many other international method sources. Nevertheless, there is no official analytical method for SBA even though it is commonly found in soybean seeds. A hemagglutination assay using red blood cells has been used to quantify SBA for several decades. This method, developed in the 1950s, takes advantage of the agglutination properties of lectins (Liener, 1955). When agglutination occurs and is inhibited by mono- or oligosaccharides, it serves as an indication for the carbohydrate specificity to the lectin of interest. The results are defined as the least amount of hemagglutinin that produced positive evidence of agglutination and are presented in hemagglutinating units (HU) (Breeze et al., 2015). Despite simple and rapid procedures in the courses of detection or purification, the results may vary between the sources and conditions, e.g. the origins of cell (human, sheep, rabbits or else), accessibility of the receptor sites (blood type used), cell concentration, cell pretreatments (trypsin or sialidase employments) and temperature or mixing (Wood et al., 2012; Breeze et al., 2015). For many years, many methods have been developed for lectin quantification, such as affinity chromatography and perfusion reversed-phase high performance liquid chromatographic
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(RP-HPLC) (Barca et al., 1991; Anta et al., 2010). A new technique called enzyme linked-lectin assays, or ELLA, is an adapted enzyme-linked-immunosorbent assay (ELISA) using carbohydrates instead of antibodies as detection agents (Mccoy et al., 1984). This method has been explored to measure SBA and a variety of linkage techniques have been applied to immobilize the oligosaccharides on the plates (Hatakeyama et al., 1996; Satoh et al., 1999; Satoh et al., 1998). Additional modifications have been tested, such as the use of immobilized glycoproteins (Rizzi et al., 2003). However, many ELLA techniques have been developed with a focus on determining the properties of the bound carbohydrate residue rather than for the quantification of lectin (Breeze et al., 2015). Immunoassays have enabled rapid qualitative and quantitative detection of soy proteins in complex foods. Thus, they have been used in food industry and research to ensure food safety and quality. Immunohistochemistry, immunoblotting and ELISA technology have facilitated investigation to improve healthful soybean foods and feeds and have permitted investigators to characterize the changes in protein structure during food processing and digestion. Thus, due to the continuing interest to perform SBA analysis globally and the lack of studies on detection and quantification of soy lectin in soybean and soybean products, the objective of this research focued on quantification of soy lectin content in conventional, certified genetically modified, laboratory processed soybean seeds, and commercial soybean products by using soy lectin ELISA and hemagglutination assay.
Research Hypothesis
The experiments outlined here will test the central hypothesis that polyclonal antibody (pAb)-based ELISA can detect trace amounts of soybean lectin and that different processing methods of soybean and soybean products can change the immunoreactivity and hemagglutinating activity of soybean lectin. Understanding the effects of different processing methods on soybean proteins will provide insight into possible processing methods to attenuate or eliminate soybean lectin toxicity.
Specific Aims
1. To develop a competitive ELISA for the specific, sensitive detection of soybean lectin. 2. To purify soybean lectin form raw soybean by using column chromatography.
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3. To determine the effects of select processing methods on immunoreactivity of soybean lectin. 4. To analyze immunoreactivity of soybean lectin of commercial soybean products and raw soybean varieties (GMO and Non-GMO). 5. To assess the effects of a variety of processing methods on hemagglutinating activity of soybean lectin.
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CHAPTER 2
REVIEW OF LITERATURE
Food Allergies
The human immune system may respond to a harmless food as if it were a threat. Exposure to allergens will cause allergic responses in sensitive individuals, which include erythema, pruritus, urticaria, nasal congestion, rhinorrhea, sneezing, wheezing, tongue swelling, nausea, vomiting, diarrhea, hypotension, as well as life threatening anaphylaxis. The term food allergy (hypersensitivity reaction) can be defined as an adverse immune response to foods that, for the vast majority of the population, is part of a healthy diet. Strict avoidance is currently the only solution to prevent food allergy, but accidental exposures are common even if patients attempt to avoid the known allergens. The most clearly understood mechanism for food allergy reaction involves three primary components: food allergens, immunoglobulin E (IgE), and mast cells and basophils. A single food can contain multiple food allergens, the majority of which are proteins. Exposure to allergens can cause a massive production of IgE antibodies which circulate through body, bind to basophils or enter body tissue binding to mast cells. Upon second exposure, allergens cross-link IgEs on mast cells and basophils triggering release of histamine and leukotrienes, which causing allergic reactions (Sampson, 1999a). Symptoms can be mild or severe and are usually seen within minutes of ingesting a food but may not appear for up to 2 hours (Patel et al., 2015). Skin prick testing (SPT), allergen-specific IgE (sIgE), and oral food challenge are the most commonly used methods to diagnose food allergy (Burks et al., 2012). Although 170 foods have been reported as allergenic, only a small number of foods are responsible for a majority of allergy reactions (Patel et al., 2015). The estimated food allergy rates for children and adults, respectively, in North America are listed as follows: milk (2.5%, 0.3%), egg (1.5%, 0.2%), peanut (1%, 0.6%), soybean and wheat (0.4%, 0.3%), tree nuts (0.5%, 0.6%), shellfish (0.1%, 2%), and fish (0.1%, 0.4%) (Sicherer et al., 2010). Allergy to egg, milk, soybean, and wheat tends to occur most commonly during childhood (Savage and Johns, 2015). In general, children are more likely to outgrow a wheat, soybean, milk, and egg allergy, rather than outgrow peanut, tree nut, fish or shellfish allergy (Sampson et al., 2014).
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Soybean
Soybean (Glycine max) is a species of legume which originated in China and was introduced to the United States in 1765 by Samuel Bowen (Hymowitz and Harlan, 1983). Bowen used soybeans to prepare soy sauce and soy noodles for export from Georgia to England. During the 1850’s, large-scale production of soybeans in the United States appeared (Carpenter, 1994; Smith and Circle, 1972). Soybean was used in crop rotation as a method of nitrogen fixation, but did not become an important crop until the 1910’s (Chen et al., 2012). Henry Ford, the founder of Ford Motor Company, was recorded as a great leader of the soybean industry. During 1932- 1933, the Ford Company spent around $1,250,000 on soybean research (Davis, 1941). In the 1960’s, soybean plant acreage was $24.4 million and the value was $1.1 billion based on the USDA Data. Soybean has planted worldwide because of its high protein and oil contents accompanied with a relatively inexpensive price. Soybeans are the second-most-planted field crop in the United States after corn, with 84.8 million acres and a $41.8 billion value (USDA crop production 2014 summary, 2015). According to the USDA, the United States is the world’s largest soybean producer contributing 34% of the world’s production, followed by Brazil (29%), Argentina (19%), China (6%), India (4%), Paraguay (3%), Canada (2%) and others (4%). The United States is the second largest soybean exporter, contributing 33% of the world’s soybean exports (USDA Oilseeds: world market and trade archives, 2015). Soybean production has been increasing over time (Figure 1) (USDA oil crop yearbook, 2014). Two major storage proteins in soybean are glycinin (11S) and β-conglycinin (7S), which account for approximately 70% of total storage proteins in soybean seed (Mujoo et al., 2003). The molecular weight range of soybean storage proteins is from 140 to 300 kDa (Nakai et al., 1996; Wolf, 1970). The main role of soybean major storage proteins (7S and 11S) are to serve as amino nitrogen source for the germinating seeds (Murphy, 2008). The 7S component is a trimer which consists of three subunits without disulfide linkages: α’, α, and β. Their molecular weights are 76, 72, and 53 kDa, respectively (Thanh and Shibasaki, 1978). The subunits associate through strong hydrophobic interactions and hydrogen bonds (Murphy, 2008). Five percent of the 7S weight is carbohydrates of the mannose and glucosamine type, which covalently linked to the peptides of the 7S subunits (Zarkadas et al., 2007). The Glycinin component (11S) is a hexamer, consists of two trimers, each trimer is made up of three monomeric subunits and each
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monomeric subunit is composed of an acidic polypeptide and a basic polypeptide covalently linked by a disulfide bond (Medic et al., 2014). The glycinin component has five major monomeric subunits: A1aB2, A1bB1b, A2B1a, A3B4, and A5A4B3, where A and B stand for acidic and basic polypeptides, respectively (Liu, 1997). The different physicochemical, nutritional and functional properties between soy 11S and 7S have been demonstrated (Prak et al., 2005). These proteins are added to various products such as beverages, bread, and infant formulae (Pszczola, 2005).
Figure 1. Soybean: acreage planted and value in United States, 1960-2013 (USDA oil crop yearbook, 2014). The blue line accounts for the United States soybeans planted. The red line shows the United States soybean value.
Soybeans are used in human foods such as flours, protein isolates and concentrates, and textured fibers. Soymilk, tofu, soy sauce, and miso are the most popular soy foods in the United States and soy proteins have been used for vegetarian meat substitutes (Chen et al., 2012). New soy foods are developed continually (Singh et al., 2000). Soybean contains all essential amino acids and is a good source of lysine. Many studies have been done examing beneficial effects of soy foods on human nutrition and health. The beneficial effects of soybean including lower plasma cholesterol; reduce heart disease risk; prevent cancer, menopausal symptoms, osteoporosis, diabetes, obesity and protect against kidney disease (Taku et al., 2007; Mccue and
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shetty, 2004; Takahashi et al., 2005; Xu and Chang, 2008; Slavin et al., 2009; Zhang et al., 2011). Good-quality soybeans are excellent sources for feeding both animals and humans. They contribute to the nutritional value of many specialty foods and provide texture to many processed foods. Unfortunately there are several components present in soybeans that exert a negative impact on the nutritional quality of soybean and soy-derived foodstuffs. The anti-nutrients may also induce allergic responses in humans and soy allergy is the one of the largest eight types of food allergies (Sathe et al., 2013). These anti-nutrients include raffinose (a non-digestible carbohydrate), enzyme inhibitor, and lectins (Pusztai et al., 1997; Liener, 1994 and 2000).
Lectins
Lectins are a group of glycoproteins of non-immunological origins which demonstrate carbohydrate binding specificity and cell-agglutinating properties (Wang et al, 2009). In 1888, Peter Hermann Stillmark (Dorpat/Estonia) first described lectin when working on his dissertation on castor bean (Ricinus communis L.) extracts (Allen and Brilliantine, 1969). By the middle of the 20th century, these proteins had become important members of general class of glycan- binding proteins known as “lectins”, adopted from the Latin word “select”. In 1945, William C. Boyd (an American immunochemist) found that some lectins were blood-type specific (Boyd and Shapleigh, 1954). Lectins are prevalently found in plants and animals. The earliest investigated lectins are from plant origins, such as seeds like legumes, cereals, and fruits. Lectin sources that are consumed by humans and animals include fava bean, lentil, and soybean (Rüdiger and Gabius, 2001). Dietary lectins can bind reversibly with free sugars or with sugar residues of polysaccharides, glycoproteins, or glycolipids on erythrocytes or lymphocytes (Goldstein and Poretz, 1986; Goldstein, 1983). Lectins can cause agglutination of red blood cells and function as both allergens and hemagglutinins. The structure of lectins differ depending on the sources. The legume lectins are the best studied and characterized group. Of about 50 plant lectins that are commercially available, the majority originate from the legume (Rüdiger, 1998). Although legume lectins differ profoundly in their carbohydrate binding abilities, many determined complete sequences indicate they are highly homogeneous in structure (Becker et al., 1983; Rouge, 1983). Plant lectins can be subdivided into five groups, according to the monosaccharide
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for which they exhibit the highest affinity: D-mannose/D-glucose, D-galactose/N-acetyl-D- galactosamine, N-acetyl-D-glucosamine, L-fucose, and N-acetylneuraminic acid (Goldstein et al., 1997). The molecular weight of leguminous lectins’ subunits is 30 kDa (Breeze et al, 2015). They may aggregate to the fully active lectin either as dimers or tetramers without further processing (Rüdiger, 1998). Lectins constitute 2-10% of the total seed protein in legumes (Mialonie et al., 1973; Etzler, 1986) and grain products. Lectin activity occurs in 30% of American foods, more so in a whole-grain rich diet (Nachbar, 1980). However, lectins have long been commonly considered as ‘antinutrients’, mainly due to their negative effects of causing non-pathogenic food poisoning when some legumes were not properly cooked or uncooked before consumption (Van et al., 1998). At high intakes, lectins can seriously threaten the lives of consuming animals, including numerous insect pests of crop plants. Based on experiments in which animals fed on diets containing plant lectins, the evident symptoms are: loss of appetite, decreased body weight, and eventually death (Liener et al., 1986; Duranti and Gius, 1997; Lajolo et al., 2002). The mechanisms, by which lectins mediate toxicity and the characteristics which dictate whether a lectin will be deleterious, are not completely understood (Vasconcelos and Oliveira, 2004).
Soy Lectin
The major lectin in soybean seeds often referred to as soybean agglutinin (SBA). SBA is a tetrameric protein consisting of 27 kDa subunits with an estimated molecular weight (MW) of 110 kDa (Sharon and Lis, 1972). Douglas and coworkers (Douglas et al., 1999) indicated that SBA accounted for approximately 15% of the growth depression of raw conventional soybean in chicks. Conversely, Liener (1953) estimated that lectins accounted for approximately 50% of the growth-inhibiting effect of raw soybean meal in rats. In addition, it has been considered that the degree to which some lectins affect metabolism is, in part, dependent on dietary history of the animal and the exact composition of the diet (Grant, 1999). Dietary lectins have the ability to survive digestion by the gastrointestinal tract of consumers and bind to the intestinal mucosa, brush border of the gut lumen, or receptors of epithelial cells via specific oligosaccharides or glycopeptides (Oliveira et al., 1989; Pusztai, 1991). These interactions induce a series of harmful reactions, making soy lectin an antinutritive and/or toxic substance in soybean. Furthermore, lectin binding can cause structural changes in cells that lead to unfavorable outcomes. For
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instance, impairment of brush border continuity and ulceration of villi due to lectin binding may result in depressed growth rate of small animals (Oliveira et al., 1989; Van et al., 1998; Oliveira and Sgarbieri, 1986; Pusztai et al., 1990). Pueppke and coworkers (Pueppke et al., 1978) quantified SBA in growing soybean plants and observed that the lectin level was decreased 12- 15 days after planting. Su et al. (1980) also demonstrated the rapid decline of lectin in the developing plant as well as a poor correlation between nodulation capability with Rhizobium and the apparent content of the major soybean lectin. Gibson et al. (1982) used radioimmunoassay (RIA) to illustrate an additional possible role of SBA in the resistance to pathogens. Vodkin and Raikhel (1986) studied the distribution of soybean lectin in seeds and roots, using immunocytochemistry, immunoblotting, and heterologous sandwich ELISA (two different antibodies). Because, soy lectin is both an allergen and antinutrient in soybean, the interests of soy lectin detection and measurement to ensure food safety and quality are continuing. Recently, F reported a new Enzyme Linked Immunosorbent Assay (ELLA) technique analogous to a sandwich ELISA that can detect lectins (Rizzi et al., 2003). The enzyme linked-lectin assays (ELLA) makes use of the carbohydrate binding specificity of lectins by using polyacrylamide- linked carbohydrates for capture and biotin-labeled polyacrylamide-linked carbohydrates for detection. This assay was applied specifically to SBA by using a polyacrylamide-linked N- acetylgalactosamine (Gal–NAc–PAA) for capture and the biotinylated version (Gal–NAc–PAA– Biotin) for detection (Anta et al., 2010). To eliminate the toxicity of SBA, people usually processed soybean by heat treatments (such as boiling, frying, roasting, and steaming). Many reports indicated that the most efficient way to inactivity lectins is heat treatments (Bajpai, Sharma, & Gupta, 2005; Liener 1994). The beneficial results of such inactivation were manifested by increased weight gain of chick and higher protein efficiency ratio (PER) values for the diet containing the heated lectins (Casaubon- Huguenin et al., 2004; Ahmed, 1986).
Food Processing
Food processing refers to the practices used in the home, restaurant, and food and beverage industries to transform raw plant and animal materials, such as grains, meat and dairy, into products for consumers (Monteiro et al., 2010). Consumers buy processed foods for a
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variety of reasons, which including convenience, variety, and assured consistent quality and safety. During appropriate food processing, food safety, storage ability, digestibility, and sensory properties can be improved. Processing methods can be categorized into home processing (such as peeling, washing, soaking, blending, grinding, milling, pounding, cutting, chopping, rolling, straining, filtration, blanching, cooking, pressure cooking, frying (pan and deep), roasting, toasting, baking, smoking, grilling, extrusion, shredding, dehydration, microwaving, cooling, freezing, canning, bottling, and crystallization), institutional and restaurant processing, and industrial processing (such as spray drying, fluidized bed drying, ultrafiltration, reverse osmosis, c-irradiation, freeze concentration, infra-red heating, high pressure processing, sonication, flash evaporation, electromotive force, chemical sterilization, ozone sterilization, ultrahigh temperature (UHT) short time pasteurization/sterilization) (Sathe and Sharma et al., 2009).
Genetic Modification (GMO) of Soybean
Introduction of transgenic technologies simplifies management appears profitable for seed companies and farmers, as well as promotes efficient industrialization of agriculture, although there is a continuing debate about potential of GMO varieties. Soybean is an important commercial crop grown as an inexpensive source of protein for humans and animals. This has generated significant interest by major agricultural research institutes and biotech industries to enhance the quality of nutrients in soybeans as well as in increase the yield and resistance against various pests (Natarajan, 2010). The commercial cultivation of GMO soybean varieties began in 1996, and they became predominant in the major soybean producing countries. In order to meet increasing global demand of soybean, GMO soybeans have been grown and traded to increase the quality and yield. The largest hectare (ha) crop is soybean, which is planted in 80 million ha among 24 countries, and GMO soybeans occupy 81% of all soybeans grown (Tian et al., 2014). With the expansion of soybean and other crops production, consumers’ concerns are increasing, and they have demanded appropriate information and labeling for foods derived from GMO crops. Labeling systems for GMO foods have been implemented in the European Union (EU), Japan, Australia and other countries (Nikolić et al., 2014). The EU has established the legal basis for the traceability and labeling requirements of genetically modified organisms and GMO derived food and feeds (Regulations
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EC No. 1830/2003). All food and feed products that use GMOs along the production chain need to be labelled, even if no GM content is detectable in the final product (Onori et al., 2013).
Effects of Food Processing on Food Allergens
Foods undergo various processing methods before consumption that may affect conformation of food proteins, their digestion and thereby allergenicity (Sathe and Sharma, 2009). Structural modification of food proteins include unfolding, aggregation, cross-linking between the ingredients and chemical modifications such as oxidation and glycosylation (Lepski and Brockmeyer, 2013). The allergic potential of food proteins may either be reduced or enhanced or sometimes have no effect at all by processing methods (Rahaman, 2016). The food allergen labeling requirements in the US and EU indicate that the presence of any items causing allergy in any form should be mandatorily labeled (Dhakal et al., 2014). Samadi and Yu (2011) have reported that the sensitivity of soybean seeds to moist heating (autoclaving) was much higher than the sensitivity to dry heating in terms of the structure and nutrient profile changes. A few studies have investigated the effect of thermal processing on soybean and soybean protein allergenicity. The IgE-binding capacity of heated (80 ºC or 120 ºC for 60 min) crude soybean and its 7S and 11S protein is significantly reduced (Burks et al., 1991). Gomaa et al. (2013) reported that soy allergen recovery decreased with increasing processing (baking) time and decreasing product size.
13
CHAPTER 3
MATERIALS AND METHODS
Materials
Soybean seeds used in this study were provided by North Florida Research & Education Center in Quincy and Murray State University in Kentucky. Commercial soy products were obtained from local grocery stores. Samples were powdered using a cyclone sample mill (UDY Corporation model 3010-030) or an Osterizer blender (Galaxie model 869-18R) and passed through a 20 mesh sieve for homogeneity. The ground samples were gently flushed with nitrogen gas and stored in airtight containers at -20 °C, until further analysis. Chemicals and reagents were purchased from commercial suppliers: disposable polypropylene columns (5 ml polypropylene column with polyethylene filter disc and top and bottom press-on caps) were from Pierce Inc. (Rockford, IL). Protein G Sepharose 4B Fast Flow beads (2 mg protein G/ml drained medium, average particle size at 90 μm, stored in 20% ethanol). The FARBERWARE CLASSIC SERIES cheese cloth was purchased from Publix. Anti-rabbit IgG (whole molecule) - Alkaline phosphatase (AP) antibody produced in goat (A3687, Lot.105K6071), horseradish peroxidase (HRP) labeled goat anti-rabbit IgG, p- Nitrophenyl Phosphate (Disodium Salt), Ponceau S (P3504, practical grade, 3-Hydroxy-4-(2- sulfo-4-[sulfophenylazo]phenylazo)-2, 7-naphthalenedisulfonic acid sodium salt), luminol (97.0%) and bovine serum albumin [ Sigma Chemical Co. (St. Louis, MO)], electrophoresis and immunoblotting supplies [Hoefer Scientific Co. (San Francisco, CA)] and [Fisher Scientific Co. (Pittsburgh, PA)], BioTek PowerWaveTM 200 Microplate Scanning Spectrophotometer and KC4TM Software [Bio-Tek Instruments, Inc. (Winooski, VT)], pH meter [Corning® Inc. (Lowell, MA)], UltrospecTM 2100 pro UV. Visible Spectrophotometer [GE Healthcare (Piscataway, NJ)], cellulose extraction thimbles (25 mm × 100 mm), filter paper No. 4 and Whatman Chromatography paper 3MM CHR 15 × 17.5 cm [Whatman international Ltd., Maidstone, UK], Protran® nitrocellulose membranes (NC, 0.2 μm, 200 × 3 m) [GE Healthcare (Piscataway, NJ)], X-ray films (BioMax XAR film) [Eastman Kodak Co. (Rochester, NY)], Acrylamide, Chemzymes Ultra PureTM [Polysciences, Inc. (Warrington, PA)], TEMED (N, N, N’ N’-tetramethylenediamhe) and Bis-acrylamide [BioRad, (Hercules, CA)], ninety-six well
14
polyvinyl microtiter ELISA plates [Costar (Corning, NY)]. All other chemicals have been purchased from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific Co. (Pittsburgh, PA) unless otherwise stated and are of reagent or better grade. The rabbit polyclonal antibodies (pAbs) against whole soy protein were produced from a female New Zealand white rabbit (Oryctulagus cuniculus) in 2006 at Florida State University.
Methods
Preparation of Defatted and Full Fat Soybean Flour
Soybeans and commercial soybean products were ground in a cyclone sample mill (UDY Corporation model 3010-030, Fort Collins, CO) or an Osterizer blender (speed setting “grind”; Galaxy model 869-18R, Jaden Consumer Solutions, Boca Raton, FL) to obtain homogeneous flour. Full fat flours were homogenized in the blender and passed through a 20 mesh sieve to obtain uniform flours. The soybean flours were defatted for 8 h using a Soxhlet apparatus (Fisher Scientific Co, Orlando, FL) with petroleum ether (PE, boiling point range of 38.2-54.3ºC, BDH® through VWR Scientific, West Chester, PA) as the extraction solvent (flour to solvent ratio of 1:10 w/v). After overnight air drying in a fume hood, defatted flours were further homogenized in the blender and passed through a US standard 40 mesh sieves [No. 40, 425 μm size, 30.5 cm outside diameter, Fisher Scientific Co. (Pittsburgh, PA)] to obtain uniform flours. The full fat and defatted flours were stored in screw-capped plastic vials at -20ºC until further use.
Protein Extract Preparation
Defatted seed flours and commercial soybean products were extracted (flour to solvent
ratio of 1:10 w/v typically 100 mg/1 mL) with borate saline buffer (BSB, 0.1M H3BO3, 0.025M
Na2B4O7, 0.075M NaCl, pH 8.45). The flour slurries were vortexed (Vortex Genie 2, setting 8, American Scientific Products, McGaw, IL) continuously for 1 hr at RT, followed by centrifugation (13600g, 20 min., RT). Supernatants were collected and residues discarded. Routine protein extractions were performed using BSB as the solvent except where noted otherwise.
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Sample Preparation
GMO Soybean AG5831 and Non-GMO soybean Woodruff 10 grams each were processed under the following processing conditions in duplicate: (A) Dry roasting: Soybean samples were placed in aluminum pans and subjected to roasting in an oven previously heated to the desired temperatures (100, 132, 146, 163°C for 15 and 30 min) and monitored using a thermometer. (B) Frying: Soybean samples were fried in vegetable oil in the pan at 171 °C for 10 min. Fried samples were air-dried at RT (~25°C) in a fume hood until a constant weight was obtained. (C) Microwaving: Soybean samples were processed in a Panasonic Microwave oven at 300, 450, 600, and 750 Watts for 1, 2, and 3 min. (D) Autoclaving: Soybean samples were placed in an autoclave (Barnstead International, Inc., Dubuque, IA, USA) at 121°C, 15 psi for 0, 5, 10, 20, and 30 min. Autoclaved samples were air-dried at RT (~25°C) in a fume hood until a constant weight was obtained. (E) Blanching: Soybean samples were exposed in boiling water at 100°C for 2, 4, 6, 8, and 10 min. The ratio of seeds to water are 1:10 w/v. Samples were patted dry on paper towels and air-dried at RT in a fume hood until a constant weight was obtained. (F) Steaming: Soybean samples were steamed at 100 °C for 5, 10, 20, 40 min. Samples were patted dry on paper towels and air-dried at RT in a fume hood until a constant weight was obtained. (G) Boiling: Soybean samples were boiled for 15, 30, 45, 60, 120 min. Samples were patted dry on paper towels and air-dried at RT in a fume hood until a constant weight was obtained. (H) Germination: 1. Weigh 1000g soybean in a tub, rinse with water several times to get rid of dust. 2. Heat water to ~ 55 °C. 3. Pour the water to the clean beans at 1: 1.6~2 (1Kg +2L water temp. from 55 °C to 45 °C), keep for around 1 hour, change the water once and keep it overnight in the dark at RT. 4. Weigh soybean after soaking. 5. Weigh 200g / container in cheese cloth sprinkle adequate amount of DI water on the top and put in the dark place (open a little).
16
6. Take out samples every day, take scans, pictures and measures of sprout length and grind the sprout, froze and dried. After all the treatments, the samples were extracted with vortexing using (1:10 w/v) BSB for 1h at RT and centrifuged in a tabletop microcentrifuge for 15 min.
Protein Determination
Soluble proteins were determined by the Bradford method (Bradford 1976). Bovine serum albumin (BSA) fraction V (Sigma Chemical Co., St. Louis, MO, USA) were used as the standard protein (0‒600 µg/mL for Bradford method).
Column Chromatography
(A) Anion-Exchange Column Chromatography Column was used to separate and purify soy proteins as described by Sathe et al. (1992). The defatted soy extract was subjected to DEAE DE-53 anion-exchange column chromatography.
The column (2.6 × 30.5 cm) is equilibrated with 50 mM Tris-HCl 1mM NaN3, pH 8.5, and was eluted with 0-250 mM NaCl linear gradient in equilibrium buffer (200 mL each). Fractions (20 min per fraction, column flow rate was 30.5 ml/h) containing the lectin was pooled according to the absorbance at 280 nm and SDS PAGE results. The pooled fractions were dialyzed against distilled water (4 °C, 24 h, six changes) and freeze dried. (B) Gel filtration Sephacryl S200 column Dissolve the freeze dried protein collection in 18 mL sodium phosphate buffer and subjected to the Sephacryl S200 column which was equilibrated and eluted with 150 mM sodium phosphate buffer 1M NaCl, pH 7.5, containing 1mM NaN3. The column flow rate was 18 mL/h. Fractions (20 min per fraction) containing the lectin was pooled according to the absorbance at 280nm and SDS-PAGE results. The pooled fractions were dialyzed against distilled water (4 °C, 24 h, six changes) and freeze dried.
Hemagglutinating Assay
Hemagglutinating assay was used to analyze hemagglutinating activity of the samples as described by Lis and Sharon (1972). A microtiter plate assay was used to determine the Hemagglutinating Activity (HA) of the sample. A commercial (Sigma) and laboratory purified
17 soybean lectin were used as the reference standards. HA activity of the sample extracted in 0.01 M phosphate-buffered saline, pH 6.8, was determined under standard agglutination conditions defined as agglutination of a 4% suspension of fresh human blood (group A, B, AB and O) erythrocytes after 1 h of incubation at 25 °C. Activity was determined from serial dilutions of sample extracts in 0.01 M phosphate-buffered saline, pH 6.8. Hemagglutinating activity was observed visually. A spread fibrous appearance in the wells indicated hemagglutination while appearance of a button like mass indicates no hemagglutination. Note the highest dilution (HD) of the sample showing positive hemagglutination. One hemagglutinating unit (HU) was defined as the least amount of hemagglutinin that produced positive evidence of agglutination. Hemagglutinating activity of the sample was expressed as HU per gram of sample. Hemagglutinating activity (HU/g sample) = HD × dilution factor × volume for extraction (mL) / weight of sample (g)
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Polypeptide profiles of proteins were characterized using SDS-PAGE as described by Fling and Gregerson (1986). Samples were mixed with reducing sample buffer (50 mM Tris- HCl, pH 6.8, 1% SDS, 0.01% bromophenol blue, 30% glycerol, and 2% β-mercaptoethanol) or non-reducing buffer (0.05 M Tris-HCl pH 6.8, 1% SDS w/v, 30% v/v glycerol and 0.01% w/v bromophenol blue) and boiled in water bath for 10 min. The sample solution to sample buffer ratio was 1:1 v/v. And 10-40μg of proteins were loaded into of each lane and electrophoresed in a 4% stacking gel (1.0 cm × 16.5 cm × 1.5 mm) and an 12% acrylamide separating gel (8 cm × 10 cm × 1.5 mm) along with the molecular weight markers ranging from 11 to 170 kDa (Fisher Scientific Co., Orlando, FL). The gels were run at a constant current (20 mA/gel) until the tracking dye reached the gel edge.
Antibody Purification
Antibodies were purified as needed by affinity chromatography using protein G- Sepharose to increase the sensitivity of the ELISA and reduce the background due to non- specific binding. Briefly, antiserum or pAb supernatant was mixed with 0.1 M phosphate buffered saline 0.9% NaCl (PBS, pH 7.2) preswelled protein G matrix and 0.1 M PBS in a 15 ml conical tube at 1:1:1 (v/v/v) ratio. The tube was then incubated for 12 hrs in the cold room on a
18
rocker (60 Hz, Rocker II model 260350, Boekel Scientific, Feasterville, PA). The mixture was transferred to a 5 ml disposable polypropylene column (Pierce Inc., Rockford, IL) containing a bottom filter and allowed to settle. The flowthrough (FT) containing unbound protein(s) was collected. Absorbance using optical density (OD) was read at a wave length of 280 nm using the reference of PBS. The column was then rinsed with additional PBS buffer (three bed volumes a time) and FTs were collected separately until OD280 reached the baseline. Two bed volumes of 0.2 M Glycine sulfate (GS, pH 2.3) [99%, Fisher Scientific Co. (Pittsburgh, PA)] were added to elute Ab from the protein G column and the eluent was collected in 1.5 ml conical micro tubes [Polypropylene, VWR international (West Chester, PA)] containing 0.1ml of 1 M Tris(hydroxymethyl) aminomethane buffer [pH adjusted by HCl, Tris-HCl (pH 8.5)], PBS was then added to wash the column, FT was collected in separate conical micro tubes, the absorbance was read at 280 nm (GS as reference) and the tubes with the OD280 around the peak were combined in a 15 ml conical tube and immediately neutralized with 1 M Tris-HCl (pH 8.5) drop wise. SDS-PAGE was performed to check each fraction to confirm the successful purification of IgG. The IgG fraction was then dialyzed against 10 mM PBS, concentrated to the appropriate
concentration using YM-3 amicon centrifugal filtration device. NaN3 was added to the final concentration of 0.1% (w/v). Aliquots of purified, concentrated Ab were stored at -20 °C.
Western Blot
Following SDS-PAGE, the proteins were transferred onto a 0.22 μm nitrocellulose membrane using a Hoefer TE22 (for 10 × 8 cm) transverse electrophoresis unit at 4ºC for 1 h and 45 min and the transferred polypeptides were visualized by brief staining for 5 min with 0.1% (w/v) Ponceau S stain. Then the nitrocellulose membrane was blocked with 5% w/v nonfat dried milk (NFDM) in Tris-buffered saline containing Tween 20 (TBS-T, 10 mM Tris, 0.9% w/v NaCl, 0.05% v/v Tween 20, pH7.6) for 1 hour at RT. Subsequently the membrane was washed with TBS-T three times for 5 min each. After washing, the membrane was incubated with an appropriate or different dilution of polyclonal antibody (rabbit anti-whole soybean protein extract sera) in TBS-T overnight at 4ºC on a rocker. Following the overnight incubation, the membrane was washed three times with TBS-T for 15 min each and incubated with diluted secondary antibody (1:40000 v/v horseradish peroxidase labeled goat anti-rabbit antibody) in TBS-T for 1 hour at RT on a rocker. The membrane was washed three times with TBS-T for 15
19
min each. The reactive protein bands were detected by incubating the membrane with a mixture of solution A (containing 100 μl of 250 mM luminol, 44 μl of 90 mM p-coumaric acid, 1 ml of 1 M Tris-HCl pH 8.5 and 8.85 ml of DI water) and solution B (6 μl of 30% hydrogen peroxide, 1 ml of 1 M Tris-HCl pH 8.5 and 8.85 ml of DI water) for 5 min and exposed to X-ray film for 60s.
Soybean Antibody (pAbs) Titer
(A). Ab titer: the level of pAb reactivity in various dilutions (Ab: buffer=1:10~1:5000 v/v) with a given amount of soybean protein evaluated by Western blots and ELISA representing the affinity between Ab-Ag. (B). Ag titer: the level of pAb reactivity at a given dilution/titer with various amounts (0.01-10 mg) of soybean protein and soybean lectin evaluated by Western blotting and ELISA representing the affinity between Ab-Ag.
Enzyme Linked Immunosorbent Assay (ELISA)
Competitive inhibition ELISA: column purified soybean lectin was used to coat the plate wells. To test for soybean lectin, a sample extract was pre-incubated with rabbit anti-soybean pAbs. A quantitative determination of the amount of soybean lectin present in a test sample was provided by the degree to which the targeted soybean lectin inhibits the binding of the pAbs to the lectin used for coating the plate. Comparison to a standard curve based on inhibition by known quantities of the soybean lectin facilitated quantification. Specifically, the plate 1 was coated with 50 μl of 10 µg/ml BSB soluble soybean lectin flour proteins in the coating buffer
(pH 5, 48.5 mM citric acid, 103 mM Na2HPO4) for 1 hr at 37 °C and then blocked with 200 μl of blocking buffer (5% NFDM in TBST) for 1 hr at 37 °C. To perform the assay, a soybean lectin standard and the test samples were serially diluted into a predetermined amount of pAb (diluted 1:50 v/v in 1% NFDM in TBS-T, 100 µl each well), in a separate microtiter plate. Standard protein (unprocessed soybean lectin extract) of 0.1 mg/ml were added in the first well, and serially diluted 10 times into the subsequent wells. After 1 hr incubation at 37 °C, 50 μl aliquots/well was transferred to the soybean lectin coated plate and incubated for 1 hr at 37 °C. This preincubation step, in which the pAb and inhibitor was allowed to react prior to exposure to soybean lectins coated on the plate, can significantly increase sensitivity. The plates were
20
subsequently incubated with 1:5,000 v/v alkaline phosphatase-labeled goat anti-rabbit IgG in 1% NFDM in TBS-T for 1 h at 37°C. Between each step, the plate was washed 3 times with TBST. The strength of the signal was assessed by color development, following the addition of 50 μl/well of p-nitrophenyl phosphate (pNPP) in substrate buffer followed by 30 min of incubation in dark. To stop color development, 50 μl of 3 M NaOH was added to each well and the absorbance was read at 405 nm. A logarithmic regression curve was generated by Excel software. The concentration of soybean lectin in test sample was indirectly proportional to the color intensity of the test sample. On the same plate standards, negative control, and blanks were included using freshly prepared soybean lectin; 0.1% BSA; and TBST, no-coating of soybean protein, no primary Ab, no enzyme-labeled Ab conjugate, or no substrate. Standard curve was also prepared in each plate using freshly prepared soybean lectin assayed in duplicate.
Data Analysis and Statistical Procedures
All statistical analyses were performed using SPSS statistical software (version 19; Chicago, IL). All experiments were carried out at least in duplicate, and data were expressed as mean ± standard deviation. One-way ANOVA and Fisher’s least significant difference test as described by Ott et al. (1977) was used to determine statistical significance, and results were compared for significance at p ≤ 0.05. Statictical significance was determined using Fisher’s LSD at p ≤ 0.05.
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CHAPTER 4
RESULTS AND DISCUSSION
Lectin Purification
Anion-Exchange Column Chromatography
The method of column chromatography for purification of soybean lectin was described by Sathe et al. (1992). Soybean protein extracted in 50 mM Tris-HCl buffer was precipitated by ammonium sulfate. The 40% - 80% ammonium sulfate fraction was dialysed, freeze dried, and re-dissloved in 50 mM Tris-HCl buffer and subjected to DEAE DE-53 column. The column volume and flow rate were 161.85 mL and 30.83 mL/h separately. Fractions (20 min/fraction) rich in lectin (tubes 88 – 96 in Figure 2) were pooled according to the absorbance at 280nm and the SDS-PAGE results. Pooled fractions were dialyzed against distilled water (4 °C, 24 h, six changes) and freeze dried. The column profile for the ammonium sulfate precipitate soybean protein off DEAE DE-53 is given in Figure 2.
Figure 2. Elution profile for 40% - 80% ammonium sulfate precipitate soybean protein extract off a DEAE DE-53column (2.6 × 30.5 cm). Column was equilibrated with 50 mM Tris-HCl 1mM NaN3, pH 8.5. Fractions containing lectins (tubes 88 – 96) were pooled. Inset: SDS-PAGE (reducing conditions) analysis of fractions (20 µL was loaded on each lane) eluted off the
22
column indicated by number on the top of the gel lane. L = protein loaded on to the column. S = protein marker, molecular mass of each standard is indicated in the left margin of the inset.
Gel Filtration: Sephacryl S200 Column
Freeze dried protein collection off the DEAE DE-53 column was dissolved in 18 mL sodium phosphate buffer and subjected to a 2.6 × 91 cm Sephacryl S200 column which was equilibrated and eluted with 150 mM sodium phosphate buffer, pH 7.5, containing 1mM NaN3. The column flow rate was 18 mL/h. Fractions (20 min per fraction) containing the lectin was pooled according to the absorbance at 280nm and SDS-PAGE results. This fraction was soybean lectin and resulted in a single peak when further purified by S200 column. The pooled fractions were dialyzed against distilled water (4 °C, 24 h, six changes) and freeze dried for the future use. The column profile for the soybean lectin (DEAE DE-53 pool) off a Sephacryl S200 column is given in Figure 3.
Figure 3. Elution profile the soybean lectin (DEAE DE-53 pool) off a Sephacryl S200 column (2.6 × 91 cm). Column was equilibrated with 150 mM sodium phosphate buffer, pH 7.5, containing 0.02% NaN3.Tubes 42 – 46 were pooled to yield soybean lectin. Inset: SDS-PAGE (reducing conditions) analysis of fractions (10 µL was loaded on each lane) eluted off the column indicated by number on the top of the gel lane. L = protein loaded on to the column. S = protein marker, molecular mass of each standard is indicated in the left margin of the inset.
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As evident from Figure 2, lectin account for a very small fraction of the total soybean protein loaded on the column. After S200 column, only 28.4 mg of lectin was purified from 40 g of defatted soybean flour. Table 1 summarizes typical yield data of soybean lectin.
Table 1. Summary of lectin purification by column chromatography a. Purification step Total protein (mg) Original soybean protein extract 17, 590 Extract loaded on DEAE DE-53 6000 lectin off DEAE DE-53 33.5 Collected fractions loaded on S200 33.5 lectin off S200 28.4 a Data are for a typical preparation starting with 40 g of defatted soybean flour and using the 40% - 80% ammonium sulfate precipitated proteins.
Figure 4. SDS-PAGE of laboratory purified lectin (1) and commercial lectin (2). S = protein marker, molecular mass of each standard is indicated in the left margin of the inset.
The lectin purification results indicated that the method provided by Sathe et al. (1992) is repeatable, reproducible, and reliable. Furthermore, purity of the laboratory purified soybean lectin (~ 90%) is higher than commercial soybean lectin (~ 70%) purchased from Sigma (Figure 4).
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Competitive ELISA
ELISA Construction
Figure 5. The schematic representation of the soybean lectin inhibition ELISA procedures.
A competitive assay was done to demonstrate the feasibility of this pAb-based 96-well plate immunoassay. Figure 5 is the schematic representation of the inhibition ELISA Procedures. Protein extracts were normalized to the same concentration (80000 ng/ml) in BSB buffer to compensate for the loss of protein solubility due to processing treatments before the ELISA. The optimal dilutions for the soybean pAb is 1:50 (v/v). Immunoreactivity was measured by using the logarithmic equation of the standard curve obtained by using soybean lectin extract as an inhibitor on each plate. The higher the concentration of the soybean lectin was, the fewer antibodies bound to captured antigen. Signals were reported by substrate buffer reacting to the enzyme labeled on secondary antibody that bound to the primary antibody. Concentration of soybean lectin standard curve were designed to be 1×105, 1×104, 1×103, 1×102, 10, 1, 0.1, and 0.01 ng/mL, each in two replicates. As shown in
25
Figure 6, the absorbance read at 405nm decreased proportionally to the increasing concentration of the soybean lectin. The logarithmic dose response correlation was very high (R2=0.992).This result demonstrated a stable detection capability of this pAb-based 96-well filtration plate immunoassay.
Figure 6. The logarithmic regression curve of soybean lectin standards. The standard curve was repeated two times within a plate. Relative standard deviation (Standard deviation/Average×100, RSD)of results in each concentration were 1.1%, 2.1%, 4.3%, 1.0%, 2.7%, 2.6%, 8.7%, 4.9%, respectively from right to left.The higher the concertration of soybean lectin, the lower absorbance in average.
ELISA Validation
Once the competitive ELISA was constructed and optimized, the assay performance should be evaluated for its validation. Validation provides LOD, reproducibility, and accuracy of the assay by statistical analysis on data collected from required tests over a period of time.
Limit of Detection (LOD): LOD is defined as the minimum concentration of an analyte that can be reliably distinguished from background in the assay. The LOD was calculated using the formula LOD = M - 3σ, where M is the mean absorbance of the blank (negative control) and σ is the standard deviation of the blank (mean).
26
In this assay, as little as 0.063 ng/ml (6.3×10-5 ppm) of soybean lectin can be reliably distinguished from background (sample buffer). The LOD (0.063 ng/ml) for soluble soybean lectin suggested the assay is sensitive. A sample calculation is given in Figure 7.
Figure 7. A sample calculation of LOD for the constructed SBA competitive ELISA
Reproducibility: Reproducibility, also called precision, defined as the ability of the assay to duplicate results in repeat determinations. It was determined by the coefficient variation (%CV) between replicate determinations in the same assay (intraassay variability) and in different assays (interassay variability). Variation of at least four replicates of each sample was measured. At least three different samples within the plate for intraassay variability and in different plates for interassay variability were measured. Percent coefficient variations (%CV) were calculated for the intraassay or interassay variability by using the formula CV (%) = (σ/X) × 100% where σ is the standard deviation and X is the mean of the replicate determinations. The intra- and inter-assay variability of the SBA competitive ELISA was < 24% CV, demonstrating that the assay is reproducible. Two replicates of each sample carried out within a plate and at least six replicates of each sample carried out in different plates or on different days, were measured for the coefficient of variation (CV). Table 2 shows the calculations for both
27 intra- and interassay variability. Note that the samples for inter assays were not the same samples because such low concentration samples are not stable in storage; therefore, samples were always prepared fresh from stock using the same procedure to achieve the same concentration for testing. Variability may have been introduced during the sample preparation.
Table 2. Intra- and inter-assay variations for the pAb-based soybean lectin competitive ELISA Intra Assay Concentration Measured (ng/ml) Mean SD CV (%) (ng/ml) 100 100.20 87.59 93.89 8.92 9.50 10 12.52 9.50 11.01 2.13 19.38 1 1.53 1.11 1.32 0.30 22.53 Inter Assay Measured (ng/ml) Mean SD CV (%) 100.20 87.59 100 111.51 88.67 95.63 11.89 12.43
80.55 105.28
7.19 11.41
10 12.52 9.50 10.37 2.06 19.84
12.26 9.35
1.53 1.11
1 0.95 0.84 1.22 0.29 23.90
1.45 1.43
The CVs of intra- and inter-assay ranged from 9.5% to 22.5% and from 12.4% to 23.9%, respectively (Table 2). Due to some of the CVs of intra- and inter-assay were higher than 15%, the futher investigation is needed to evaluating the CV of the assay.
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Accuracy: Accuracy describes the closeness of mean test results obtained by the assay to the true value (concentration) of the analyte. Multiple dilutions of purified soybean lectin were evaluated, the percent recovery was determined using the following formula: recovery (%) = (measured concentration / predicted concentration) × 100%. Three samples, each were tested 6 times using the constructed competitive ELISA. The recoveries were calculated using the formula above and results were shown in Table 3. The average recoveries for the relative difference of each dilution relative to the average value were within 15%, which suggesting accuracy of the assay.
Table 3. Accuracy of constructed competitive ELISA Predicted Measured Recovery Mean recovery SEM Sample (ng/ml) (ng/ml) (%) (%) (%) 1 1072.61 107.26 2 1111.60 111.16 3 1476.10 147.61 A 1000 110.64 10.39 4 1302.67 130.27 5 875.06 87.51 6 800.33 80.03 1 100.20 100.20 2 111.51 111.51 3 80.55 80.55 B 100 95.63 4.85 4 87.59 87.59 5 88.67 88.67 6 105.28 105.28 1 7.19 71.91 2 12.52 125.20 3 12.26 122.56 C 10 103.71 8.40 4 11.41 114.09 5 9.50 95.02 6 9.35 93.48
The validation experiments confirm that the constructed competitive ELISA can accurately and precisely detect SBA.
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Immunoreactivity Comparison of Unprocessed Soybean Seed Lectins
Due to the worldwide demand and development of soybean plantation, there is a wide range of soybean varieties. With soybean varieties dramatically increased by transgenic technologies (Nararajan et al., 2010), a discussion of the relationship between traditional seeds and GMO seeds for the safty of food uses are important. In this study, we examined the lectin immunoreactivity of the 20 soybean varieties (GMO and Non-GMO) using a competitive ELISA constructed in our laboratory. Table 5 contains the results of immunoreactivity of soybean lectins in raw soybean seeds. The 20 different soybean seed varieties used were shown in Table 4. Of all analyzed soybean seeds samples, the AGS 5911 LL GMO soybean has shown the least immunoreactivity of lectin and the AGS 75R27 RR GMO soybean shows the highest immunoreactivity of lectin. As the data shown in Table 5, like other soybean components (Mujoo et al., 2003), the SBA content is subjected to the effects of genotype and environment, which shows the variability among soybean varieties. The results indicate the SBA content and immunoreactivity are different among soybean population, but whether it is because of the genetic modification or the difference of soybean varieties is still remaining to be discussed. Based on the results presented, the ELISA can be used to detect and quantify the SBA regardless of the tested soybean genotype and variety. However, further work is needed to assess applicability of the assay to detect and quantify lectin in several additional soybean varieties.
Table 4. Soybean varieties used Soybean varieties Released year Yield (bu/acre) Non-GMO Cobb 1977 56.2 ± 6.7 Non-GMO GSG 483 NAa NA Non-GMO GSG 510 NA NA Non-GMO Hinson Long Juvenile 2001 NA Non-GMO Hutcheson 1987 NA Non-GMO Jake 2006 NA Non-GMO Stoddard 2006 ~43 Non-GMO Woodruff 2008 37.6 ± 4.1 GMO 95Y70 2009 51.1 ± 5.2
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Table 4 – continued. Soybean varieties Released year Yield (bu/acre) GMO AG5831 2011 48.5 ± 1.9 GMO AG6931 2011 42.5 ± 8.0 GMO AG7733 2012 46.7 ± 7.0 GMO AG7934 2013 46.6 ± 3.8 GMO AGS 568 RR 2006 38.9 ± 4.7 GMO AGS 5911 LL 2011 38.9 ± 8.8 GMO AGS 75R27 RR 2013 35.5 ± 1.8 GMO AGS 787 RR 2012 37.8 ± 6.4 GMO AGS 828 RR NA 35.3 ± 7.3 GMO NK S78-G6 NA 41.4 ± 4.5 a NA means not applicable
Table 5. Raw soybean seed lectins analyzed by using the ELISAa Varieties Lectin Conc. (ng/100ng) Non-GMO Cobb 1.93±0.22 Non-GMO GSG 483 1.83±0.32 Non-GMO GSG 510 2.25±0.34 Non-GMO Hinson Long Juvenile 2.66±0.06 Non-GMO Hutcheson 2.06±0.33 Non-GMO Jake 4.88±0.30 Non-GMO Stoddard 4.29±0.53 Non-GMO Woodruff 2.67±0.11 GMO 95Y70 3.28±0.29 GMO 95Y71 3.75±0.18 GMO AG5831 2.13±0.16 GMO AG6931 4.30±0.25 GMO AG7733 2.57±0.21 GMO AG7934 2.66±0.55 GMO AGS 568 RR 2.36±0.12
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Table 5 – continued. Varieties Lectin Conc. (ng/100ng) GMO AGS 5911 LL 1.64±0.40 GMO AGS 75R27 RR 6.18±0.80 GMO AGS 787 RR 2.57±0.16 GMO AGS 828 RR 1.66±0.16 GMO NK S78-G6 2.04±0.11 LSDb 0.54 a Data are expressed as ng lectin/100ng soybean soluble protein on immunoreactivity, mean ± standard deviation (STD). b LSD = Fisher’s Least Significant Difference, the differences between means exceeding this value are significant (p ≤ 0.05).
Effects of Processing on Soybean Lectin Immunoreactivity
Raw soybeans contain about 2% lectin (Medic et al., 2014). When lectin concentration is above 7 g/kg of certain food, it is harmful for digestibility and is unfit for consumption (Pusztai 1993, Pusztai et al. 1998). To improve the food safety and nutritional quality of soybean or soybean products, inactivation or removal of the anti-nutrients are important. Soybeans are often processed by many different processing methods before being used as human food and animal feed. The effects of processing methods (microwaving, dry roasting, boiling, blanching, steaming, autoclaving, frying, and germination) on the immunoreactivity of SBA was therefore assessed. Protein extracts of all the samples were normalized to 8000 ng/ml in BSB to compensate for the loss of protein solubility due to processing treatments before using the protein extracts for the ELISA examination.
Thermal Processing. Lectin activity was used to quantify in selected unprocessed and processed GMO and non-GMO soybean seeds. The results shown in Table 6 indicate that the immunoreactivities of SBAs were significantly reduced to undetectable amounts by the thermal processing methods. But the inactivation of the SBA may not always be complete. The processing method and time are the important factors to affect the lectin activity. We found the boiling, autoclaving, and frying are the most effective methods to lower the immunoreactivity of SBA to the undetectable levels. A similar inactivation of lectin has also been found for processed soybean (Machado et al, 2008), kidney bean (Shimelis and Rakshit, 2007), and fava bean (Luo
32
and Xie, 2012) proteins. Microwaving at 300W for 1 and 2 min does not seem to decrease the SBA immunoreactivity. For the dry rosting and microwaving methods, the immunoreactivity of SBAs are reduced but remain detectable by ELISA. These results are in an agreement with the study that soybean lectins can be resistant to inactivation by dry heat (de Muelenaere, 1964). Interestingly, microwaving at 300W for 1 min and blanching at 100 ºC for 2, 4, 6, and 8 min resulted in an increase of SBA immunoreactivity. Moreover, among the two selected soybean varieties, exhibited difference in the immunoreactivity. Processing may modify or destruct epitopes on allergens resulting in the increase, reduction, and/or elimination of immunoreactivity (Sathe et al., 2005). Maenz et al (1998) indicated that the quaternary and tertiary structure of soybean lectins would be destroyed during soybean processing. The increase of SBA immunoreactivity may be affect by (1) unfolding of protein molecule, exposure of barred epitopes, and (2) influence of interactions (i.e. Maillard reaction) with non-lectin food ingredients. Maillard reaction affects the allergenicity of proteins depending on the combination of proteins and reducing sugars (Nakamura et al., 2005). Many studies have been reported that the food (peanut, wheat, cow milk, shrimp) allergenicity can be enhanced by heat processing (Blanc et al., 2011; Pasini et al., 2001; Bu et al., 2009; kamath et al., 2013).
Table 6. Effects of thermal processing on SBA immunoreactivity as assessed by the competitive ELISAa Soy AG5831 GMO Soy Cobb Non-GMO Conditions LSDb (ng/100ng) (ng/100ng) Unprocessed seeds 2.1 ± 0.3 1.9 ± 0.3 0.50 Microwaving 300W 1min 4.7 ± 0.8 3.2 ± 0.4 1.14 Microwaving 300W 2min 1.5 ± 0.3 0.8 ± 0.2 0.41 Microwaving 300W 3min 0.9 ± 0.2 0.3 ± 0.1 0.24 Microwaving 500W 1min 0.8 ± 0.1 1.3 ± 0.3 0.33 Microwaving 500W 2min 0.1 ± 0 0.1 ± 0 0.05 Microwaving 500W 3min 0.1 ± 0 NDc 0.03 Microwaving 600W 1min 0.3 ± 0.1 0.8 ± 0.1 0.19 Microwaving 600W 2min ND 0.1 ± 0 0.02 Microwaving 600W 3min ND ND 0.01
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Table 6 – continued. Soy AG5831 GMO Soy Cobb Non-GMO Conditions LSDb (ng/100ng) (ng/100ng) Microwaving 800W 1min ND 0.1 ± 0 0.02 Microwaving 800W 2min ND ND 0.001 Microwaving 800W 3min ND ND 0.01 Roasting 100°C 15min 0.8 ± 0.2 2 ± 0.6 0.76 Roasting 100°C 30min 0.8 ± 0.1 1.8 ± 0.6 0.70 Roasting 132°C 15min 1 ± 0.3 1 ± 0.2 0.39 Roasting 132°C 30min 0.8 ± 0.1 0.5 ± 0.2 0.27 Roasting 146°C 15min 0.4 ± 0.1 0.7 ± 0.2 0.28 Roasting 146°C 30min 0.2 ± 0 0.3 ± 0.1 0.08 Roasting 163°C 15min 0.1 ± 0 0.1 ± 0 0.01 Roasting 163°C 30min ND 0.1 ± 0 0.03 Boiling 15min 0.1 ± 0 ND 0.02 Boiling 30min ND ND 0.002 Boiling 45min ND ND 0.001 Boiling 60min ND ND 0.001 Boiling 120min ND ND 0.001 Blanching 2min 3.1 ± 0.5 1.8 ± 0.4 0.77 Blanching 4min 2.7 ± 0.2 1.6 ± 0.4 0.54 Blanching 6min 2.8 ± 0.6 2 ± 0.4 0.84 Blanching 8min 4.5 ± 1.2 2.7 ± 0.4 1.51 Blanching 10min 1.9 ± 0.1 0.9 ± 0.2 0.28 Steaming 5min 0.3 ± 0.1 0.7 ± 0.1 0.16 Steaming 10min 0.2 ± 0 0.3 ± 0 0.06 Steaming 20min 0.1 ± 0 ND 0.03 Steaming 40min ND ND 0.002 Autoclaving 5min ND ND 0.00 Autoclaving 10min ND ND 0.00
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Table 6 – continued. Soy AG5831 GMO Soy Cobb Non-GMO Conditions LSDb (ng/100ng) (ng/100ng) Autoclaving 20min ND ND 0.00 Autoclaving 30min ND ND 0.00 Frying 10min ND ND 0.01 LSD 0.4 0.3 a Data are expressed as ng lectin/100ng soybean soluble protein on immunoreactivity, mean ± standard deviation (STD). b LSD = Fisher’s Least Significant Difference, the differences between means exceeding this value are significant (p ≤ 0.05). c ND: signal cannot be detected.
Non-thermal Processing: Germination. Soybean seeds were germinated in the laboratory (Table 7) and the values of SBA immunoreactivity were analysed by the competitive ELISA. Values of SBA concentrations are given in ng lectin / 100ng soybean soluble protein. During the germination process, levels of lectin immunoreactivity was significantly (P ≤ 0.05) reduced after three days (Table 8). The decreasing of SBA content during seeds germination is in agreement with the study reported by Chen and Pan (1977). However, Palmer et al. (1973) demonstrated that germination did not remove the toxicity of kidney beans. Interestingly, our result shows a slight increase of SBA expression at the first day and 9th day of germination. And this slight increase of SBA expression is also reported by Rizzi et al. (2003). They indicated that the increase of SBA value could be the result of an increase of vegetative tissues (SVL) expression and the new soybean lectin from SVL might be recognised by the same antibody used in the ELISA. Overall the results demonstrate that the immunreactivity of the SBA could be modulated during germination.
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Table 7. Germinated soybean sproutsa Germinated soybean seed Time (days) Length of sprout (mm)
Day 0 0 ± 0
Day 1 6.9 ± 3.2
Day 2 15.2 ± 5.8
Day 3 28.4 ± 6.0
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Table 7 – continued. Germinated soybean seed Time (days) Length of sprout (mm)
Day 4 39.0 ± 8.8
Day 5 48.5 ± 9.1
Day 6 66.4 ± 11.0
Day 7 78.8 ± 13.0
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Table 7 – continued. Germinated soybean seed Time (days) Length of sprout (mm)
Day 8 84.8 ± 11.6
Day 9 87.7 ± 12.7
Day 10 89.2 ± 11.7
a Data are expressed as length of sport (mm), mean ± standard deviation (STD).
Table 8. Effects of germination on SBA immunoreactivity as assessed by the competitive ELISAa Time (days) Lectin (ng/100ng) Day 0 2.3 ± 0.4 Day 1 2.7 ± 0.5 Day 2 2.2 ± 0.4
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Table 8 – continued. Time (days) Lectin (ng/100ng) Day 3 1.4 ± 0.3 Day 4 1.2 ± 0.2 Day 5 0.7 ± 0.1 Day 6 0.6 ± 0.1 Day 7 0.5 ± 0.1 Day 8 0.4 ± 0.1 Day 9 0.9 ± 0.1 Day 10 0.2 ± 0 LSDb 0.4 a Data are expressed as ng lectin/100ng soybean soluble protein on immunoreactivity, mean ± standard deviation (STD). b LSD = Fisher’s Least Significant Difference, the differences between means exceeding this value are significant (p ≤ 0.05).
Commercial Soybean Products. Commercial soybean products purchased from local grocery stores were analysed by the competitive ELISA and the data are shown in Table 9. Values of SBA concentrations are given in mg lectin / g products soluble protein. Very low levels of SBA immunoreactivity were found in these commercial soybean products. Even the highest value of SBA in these products was 0.342 mg/g, which is the soy nuts. These commercial products are not only processed by a signal processing method, most of them are processed by the combination of different processing methods (i.e. boiling, roasting, autoclaving, pasteurization etc.) and other food ingredients are added. Due to the complex food processing and ingredients adding, commercial soybean products presented very low levels of SBA immunoreactivity. But due to the sensitivity of our ELISA, we still can detect the trace amount of SBA in the products.
Table 9. The SBA immunoreactivity of commercial soybean products as assessed by the competitive ELISAa Commercial samples Lectin (mg/g) Edamame 0.010 Tempeh 0.008 Soy meat 0.275
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Table 9 – continued. Commercial samples Lectin (mg/g) Soy cheese 0.073 Soy nuts 0.342 Miso 0.006 Silken Tofu 0.015 Water packed tofu 0.001 Baked tofu 0.018 Dried soy slices 0.003 Soy dried curd 0.007 Soy milk sweetened 0.014 Soy milk unsweetened 0.041 Soybean oil 0.001 Soybean sauce 0.003 Tang jiang preserved soybean 0.095 LSDb 0.02 a Data are expressed as mg / g soluble protein on immunoreactivity, mean ± standard deviation (STD). b LSD = Fisher’s Least Significant Difference, the differences between means exceeding this value are significant (p ≤ 0.05).
Hemagglutination Assay
Hemagglutination assay is a classic way to detect lectin, which is the aggregation of red blood cell (RBC). This method was used to analyse hemagglutinating activity of the samples as described by Lis and Sharon (1972). Hemagglutination activity is assessed by visual discrimination of the settling pattern. Soybean and soybean products protein extracts were assayed in hemagglutination assays at 1:2 serial dilutions. A commercial and laboratory purified SBA was used to provide the reference standard (Table 10). In the study, we are not only investigated the hemagglutination activity of SBA, but also examined the variations of four different human blood groups (group A, B, AB, O).
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Table 10. Pure lectins measured by using the hemagglutination assaya Blood Group Blood Group Blood Group Blood Group Lectin Standard A B AB O Commercial lectin 6788 ± 367 6788 ± 368 6788 ± 368 6788 ± 367 Purified lectin 7259 ± 449 7259 ± 449 7259 ± 449 7259 ± 449 a Data are expressed as HU/g lectin, mean ± standard deviation (STD).
Hemagglutination Activity Comparison of Unprocessed Soybean Seed Lectins
The same samples that analysed by the ELISA were also analysed in the hemagglutination assays. The humagglutination activities of the SBA in soybean and soybean products indicated very low levels of hemagglutination unit (HU) (Table 11). And there was no significant difference of SBA hemagglutination activity between soybean varieties. The effect of blood group variations on agglutination also showed that there was no significant difference among human blood group A, B, AB, and O (Table 11). The similar results have been reported by Patel and Willis (1992).
Table 11. Raw soybean seed lectins analyzed by using the hemagglutination assaya Blood Group Blood Group Blood Group Blood Group Soybean Varieties A B AB O Non-GMO Cobb 4.998 ± 0.003 4.996 ± 0.005 4.996 ± 0.005 4.998 ± 0.003 Non-GMO GSG 483 4.996 ± 0.002 4.999 ± 0.004 4.999 ± 0.004 4.996 ± 0.002 Non-GMO GSG 510 4.991 ± 0.004 4.992 ± 0.002 4.992 ± 0.002 4.991 ± 0.004 Non-GMO Hinson 4.992 ± 0.004 4.993 ± 0.003 4.993 ± 0.003 4.992 ± 0.004 Long Juvenile Non-GMO Hutcheson 4.991 ± 0.004 4.992 ± 0.006 4.992 ± 0.006 4.991 ± 0.004 Non-GMO Jake 4.989 ± 0.004 4.989 ± 0.001 4.989 ± 0.001 4.989 ± 0.004 Non-GMO Stoddard 4.993 ± 0.004 4.992 ± 0.004 4.992 ± 0.004 4.993 ± 0.004 Non-GMO Woodruff 4.995 ± 0.003 4.998 ± 0.006 4.998 ± 0.006 4.995 ± 0.003 GMO 95Y70 4.994 ± 0.005 4.993 ± 0.003 4.993 ± 0.003 4.994 ± 0.005 GMO 95Y71 4.992 ± 0.004 4.992 ± 0.007 4.992 ± 0.007 4.992 ± 0.004
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Table 11 – continued. Blood Group Blood Group Blood Group Blood Group Soybean Varieties A B AB O GMO AG5831 4.998 ± 0.003 4.997 ± 0.001 4.997 ± 0.001 4.998 ± 0.003 GMO AG6931 4.992 ± 0.003 4.996 ± 0.003 4.996 ± 0.003 4.992 ± 0.003 GMO AG7733 4.991 ± 0.005 4.991 ± 0.006 4.991 ± 0.006 4.991 ± 0.005 GMO AG7934 4.989 ± 0.003 4.991 ± 0.001 4.991 ± 0.001 4.989 ± 0.003 GMO AGS 568 RR 4.993 ± 0.005 4.995 ± 0.004 4.995 ± 0.004 4.993 ± 0.005 GMO AGS 5911 LL 4.995 ± 0.004 4.998 ± 0.004 4.998 ± 0.004 4.995 ± 0.004 GMO AGS 75R27 RR 9.998 ± 0.010 9.997 ± 0.007 9.997 ± 0.007 9.998 ± 0.010 GMO AGS 787 RR 4.995 ± 0.003 4.996 ± 0.003 4.996 ± 0.003 4.995 ± 0.003 GMO AGS 828 RR 4.996 ± 0.003 4.995 ± 0.002 4.995 ± 0.002 4.996 ± 0.003 GMO NK S78-G6 4.996 ± 0.003 4.997 ± 0.002 4.997 ± 0.002 4.996 ± 0.003 LSDb 0.007 0.007 0.007 0.007 a Data are expressed as HU/g sample, mean ± standard deviation (STD). b LSD = Fisher’s Least Significant Difference, the differences between means exceeding this value are significant (p ≤ 0.05).
Effects of Processing on Soybean Lectin Hemagglutination Activity
Thermal Processing. A comparison of the hemagglutination activity of thermally processed and unprocessed soybean seed protein extracts and of two different soybean varieties were evaluated. Similar to the ELISA results, hemagglutination activities of thermal processed soybean were reduced (Table 12, Table 13). Moreover, most of soybean samples become undetectable by using the hemagglutination assay after thermal processing. Microwaving at 300W for 1 min, roasting at 100°C for 15min and 30min, and blanching at 100 ºC for 2, 4, 6, and 8 min showed a positive signal of hemagglutination activity. In addition, no difference of hemagglutination activities was found between the two selected soybean varieties (Table 12, Table 13). Many studies have been demonstrated that lectin is a heat-sensitive protein and heat treatments could reduce or completely eliminate its hemagglutination activity (Habiba, 2000; Martin-Cabrejas et al., 2009; Shimelis & Rakshit, 2007). However, compare to our ELISA results, thermally processed soybean that lack the hemagglutination activity could retain the
42 immunoreactivity. Maenz et al (1999) also reported that processing of the soybean does not completely denature all the lectin and a significant quantity of lectin remain a capacity to bind glycoprotein.
Table 12. Effects of thermal processing on SBA hemagglutination activity of GMO soybean AG5831 as assessed by the hemagglutination assay a Conditions Blood Group Blood Group Blood Group Blood Group (Soy AG5831 GMO) A B AB O Unprocessed seeds 4.998 ± 0.003 4.997 ± 0.001 4.997 ± 0.001 4.998 ± 0.003 Microwaving 300W 1min 4.993 ± 0.004 4.995 ± 0.003 4.995 ± 0.003 4.993 ± 0.004 Microwaving 300W 2min NDc ND ND ND Microwaving 300W 3min ND ND ND ND Microwaving 500W 1min ND ND ND ND Microwaving 500W 2min ND ND ND ND Microwaving 500W 3min ND ND ND ND Microwaving 600W 1min ND ND ND ND Microwaving 600W 2min ND ND ND ND Microwaving 600W 3min ND ND ND ND Microwaving 800W 1min ND ND ND ND Microwaving 800W 2min ND ND ND ND Microwaving 800W 3min ND ND ND ND Roasting 100°C 15min 4.989 ± 0.001 4.992 ± 0.001 4.992 ± 0.001 4.989 ± 0.001 Roasting 100°C 30min 4.992 ± 0.002 4.993 ± 0.003 4.993 ± 0.003 4.992 ± 0.002 Roasting 132°C 15min ND ND ND ND Roasting 132°C 30min ND ND ND ND Roasting 146°C 15min ND ND ND ND Roasting 146°C 30min ND ND ND ND Roasting 163°C 15min ND ND ND ND Roasting 163°C 30min ND ND ND ND Boiling 15min ND ND ND ND
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Table 12 – continued. Conditions Blood Group Blood Group Blood Group Blood Group (Soy AG5831 GMO) A B AB O Boiling 30min ND ND ND ND Boiling 45min ND ND ND ND Boiling 60min ND ND ND ND Boiling 120min ND ND ND ND Blanching 2min 4.993 ± 0.004 4.994 ± 0.003 4.994 ± 0.003 4.993 ± 0.004 Blanching 4min 4.993 ± 0.003 4.995 ± 0.003 4.995 ± 0.003 4.993 ± 0.003 Blanching 6min 4.989 ± 0.003 4.990 ± 0.002 4.990 ± 0.002 4.989 ± 0.003 Blanching 8min 4.987 ± 0.002 4.988 ± 0.002 4.988 ± 0.002 4.987 ± 0.002 Blanching 10min ND ND ND ND Steaming 5min ND ND ND ND Steaming 10min ND ND ND ND Steaming 20min ND ND ND ND Steaming 40min ND ND ND ND Autoclaving 5min ND ND ND ND Autoclaving 10min ND ND ND ND Autoclaving 20min ND ND ND ND Autoclaving 30min ND ND ND ND Frying 10min ND ND ND ND LSDb 0.002 0.002 0.002 0.002 a Data are expressed as HU/g sample, mean ± standard deviation (STD). b LSD = Fisher’s Least Significant Difference, the differences between means exceeding this value are significant (p ≤ 0.05). c ND: signal cannot be detected.
Table 13. Effects of thermal processing on SBA hemagglutination activity of Non-GMO soybean cobb as assessed by the hemagglutination assay a Conditions (Soy Cobb Blood Group Blood Group Blood Group Blood Group Non-GMO) A B AB O Unprocessed seeds 4.998 ± 0.003 4.996 ± 0.005 4.996 ± 0.005 4.998 ± 0.003 Microwaving 300W 1min 4.997 ± 0.002 4.998 ± 0.004 4.998 ± 0.004 4.997 ± 0.002
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Table 13 – continued. Conditions (Soy Cobb Blood Group Blood Group Blood Group Blood Group Non-GMO) A B AB O Microwaving 300W 2min NDc ND ND ND Microwaving 300W 3min ND ND ND ND Microwaving 500W 1min ND ND ND ND Microwaving 500W 2min ND ND ND ND Microwaving 500W 3min ND ND ND ND Microwaving 600W 1min ND ND ND ND Microwaving 600W 2min ND ND ND ND Microwaving 600W 3min ND ND ND ND Microwaving 800W 1min ND ND ND ND Microwaving 800W 2min ND ND ND ND Microwaving 800W 3min ND ND ND ND Roasting 100°C 15min 4.994 ± 0.003 4.997 ± 0.003 4.997 ± 0.003 4.994 ± 0.003 Roasting 100°C 30min 4.989 ± 0.003 4.991 ± 0.003 4.991 ± 0.003 4.989 ± 0.003 Roasting 132°C 15min ND ND ND ND Roasting 132°C 30min ND ND ND ND Roasting 146°C 15min ND ND ND ND Roasting 146°C 30min ND ND ND ND Roasting 163°C 15min ND ND ND ND Roasting 163°C 30min ND ND ND ND Boiling 15min ND ND ND ND Boiling 30min ND ND ND ND Boiling 45min ND ND ND ND Boiling 60min ND ND ND ND Boiling 120min ND ND ND ND Blanching 2min 4.989 ± 0.003 4.994 ± 0.003 4.994 ± 0.003 4.989 ± 0.003 Blanching 4min 4.991 ± 0.003 4.992 ± 0.003 4.992 ± 0.003 4.991 ± 0.003 Blanching 6min 4.989 ± 0.003 4.991 ± 0.003 4.991 ± 0.003 4.989 ± 0.003
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Table 13 – continued. Conditions (Soy Cobb Blood Group Blood Group Blood Group Blood Group Non-GMO) A B AB O Blanching 8min 4.990 ± 0.004 4.991 ± 0.006 4.991 ± 0.006 4.990 ± 0.004 Blanching 10min ND ND ND ND Steaming 5min ND ND ND ND Steaming 10min ND ND ND ND Steaming 20min ND ND ND ND Steaming 40min ND ND ND ND Autoclaving 5min ND ND ND ND Autoclaving 10min ND ND ND ND Autoclaving 20min ND ND ND ND Autoclaving 30min ND ND ND ND Frying 10min ND ND ND ND LSDb 0.003 0.003 0.003 0.003 a Data are expressed as HU/g sample, mean ± standard deviation (STD). b LSD = Fisher’s Least Significant Difference, the differences between means exceeding this value are significant (p ≤ 0.05). c ND: signal cannot be detected.
Non-thermal Processing: Germination. Same germinated soybean seeds that used in the ELISA were also used in the hemagglutination assays. After 4 days of germination, the agglutination activity dropped down significantly to the undetectable levels in hemagglutination assay (Table 14). During the first 4 days of germination, SBA hemagglutination activities of 4 human blood groups remained unchanged. Chen and Pan (1977) observed the hemagglutinating activity of eight varieties of peas and beans seeds were reduced during 4-day germination.
Table 14. Effects of germination on SBA hemagglutination activity as assessed by the hemagglutination assay a Time (days) Blood Group A Blood Group B Blood Group AB Blood Group O Day 0 4.999 ± 0.005 5.001 ± 0.003 5.001 ± 0.003 4.999 ± 0.005 Day 1 4.988 ± 0.004 4.988 ± 0.003 4.988 ± 0.003 4.988 ± 0.004 Day 2 4.987 ± 0.002 4.988 ± 0.003 4.988 ± 0.003 4.987 ± 0.002
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Table 14 – continued. Time (days) Blood Group A Blood Group B Blood Group AB Blood Group O Day 3 4.987 ± 0.003 4.989 ± 0.003 4.989 ± 0.003 4.987 ± 0.003 Day 4 NDc ND ND ND Day 5 ND ND ND ND Day 6 ND ND ND ND Day 7 ND ND ND ND Day 8 ND ND ND ND Day 9 ND ND ND ND Day 10 ND ND ND ND LSDb 0.003 0.003 0.003 0.003 a Data are expressed as HU/g sample, mean ± standard deviation (STD). b LSD = Fisher’s Least Significant Difference, the differences between means exceeding this value are significant (p ≤ 0.05). c ND: signal cannot be detected.
Commercial Soybean Products. Same commercial soybean products were analysed by the hemagglutination assay and the data are shown in Table 15. Values of SBA concentrations are given in HU / g products soluble protein. Due to the complex processing methods and ingredients adding, most of commercial soybean products showed undetectable of hemagglutination activity. However, soy meat shows the positive signal of agglutination activity at 9.987 ± 0.009 HU/g. For the result of soy meat product, other food ingredients may affect the activity. Actually, some studies reported that other factors, like the presence of agglutinating substances of non-lectin nature, can determine misinterpretation of the agglutination results (Carratu et al., 1995; Whitmore, 1992).
Table 15. The SBA hemagglutination activity of commercial soybean products as assessed by the hemagglutination assaya Blood Group Blood Group Blood Group Blood Group Commercial samples A B AB O Edamame NDc ND ND ND Tempeh ND ND ND ND Soy meat 9.987 ± 0.009 10.002 ± 0.002 10.002 ± 0.002 9.987 ± 0.009
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Table 15 – continued. Blood Group Blood Group Blood Group Blood Group Commercial samples A B AB O Soy cheese ND ND ND ND Soy nuts ND ND ND ND Miso ND ND ND ND Silken Tofu ND ND ND ND Water packed tofu 1 ND ND ND ND Backed tofu ND ND ND ND Dried soy slices ND ND ND ND Soy dried curd ND ND ND ND Soy milk sweetened ND ND ND ND Soy milk unsweetened ND ND ND ND Soybean sauce ND ND ND ND Tang jiang preserved ND ND ND ND soybean LSDb 0.001 0.001 0.001 0.001 a Data are expressed as HU/g sample, mean ± standard deviation (STD). b LSD = Fisher’s Least Significant Difference, the differences between means exceeding this value are significant (p = 0.05). c ND: signal cannot be detected.
For the hemagglutination assay, the variation between the origin, preparation of red blood cells, temperature and the laboratories conducting the analyses can be significant (Wang et al 2009). As the data shown in both ELISA and hemagglutination assay, the lectin content has been underestimated in the hemagglutination assay. Our data demonstrate that the ELISA assay is more sensitive and effective than hemagglutinating assay for soy lectin detection and it was able to readily detect and quantify SBA in all tested soybean samples.
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CHAPTER 5
CONCLUSIONS
The lectins in soybean were purified using column chromatography. The lectin purification method provided by Sathe et al. (1992) is repeatable, reproducible, and reliable. Purity of laboratory purified soybean lectin is higher than commercial soybean lectin. The ELISA was able to readily detect and quantify soybean lectin in all tested genotypes, marketing varieties harvested from different locations/years. The ELISA was also able to detect and quantitatively soybean lectin in samples following different food processing procedures, such as autoclaving, boiling, steaming, blanching, frying, microwaving, dry roasting, and germination. However, processing methods can affect soybean lectin immunoreactivity detectable by the ELISA. Detectable amounts of soybean lectin were found in soy-based edible products. Compared to the standard hemagglutination assay (HU/g sample), ELISA is more useful and quantitative for soybean lectin detection. The lectin content has been underestimated in the hemagglutination assay. Recovery and accuracy experiments suggest that the ELISA can detect 0.063 ng/ml of soybean lectin in all tested samples. The average recoveries of the competitive ELISA were < 15% of the actual value, demonstrating that the assay is reproducible. However, further investigation is needed to re-evaluating CV of the assay. Overall, the ELISA is useful in detecting trace amounts of soybean lectin, an allergen and anti-nutrient in soybean seeds.
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APPENDIX A
ANIMAL CARE AND USE COMMITTEE APPROVAL MEMORANDUM
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APPENDIX B
HUMAN SUBJECTS COMMITTEE APPRROVAL MEMORANDUM
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APPENDIX C
INFORMED CONSENT FORMS
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BIOGRAPHICAL SKETCH
EDUCATION
2014 – Present M.S., Food Science, Department of Nutrition, Food and Exercise Sciences,
Florida State University, Tallahassee, FL, USA.
2009 – 2013 B.S., Chemistry, College of Chemistry and Chemical Engineering, Nanjing
University of Technology (NJUT), Nanjing, China.
RESEARCH & WORK EXPERIENCES
2015. 06 – 2015. 08 Graduate Research Assistant, Department of Nutrition, Food and Exercise
Sciences, Florida State University, Tallahassee, FL, USA
Research: a. Antibody purification
b. Protein purification and characterization
c. Protein immunoreactivity and immunoassay
2013 Visiting Student, School of civil and environment engineering, Brook
Byers Institute for Sustainable Systems, Georgia Institute of Technology,
Atlanta, GA, USA.
Finished and completed the undergraduate graduation thesis at Georgia
Tech.
Project: Effects and Toxicity of Nano-CeO2 toward Three Kinds of
Protozoa.
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2013 – 2014 Assistant Engineer, Center for Environmental Analysis and Measurement
Research, Chinese Research Academy of Environmental Sciences, Beijing,
China.
Project: Measurement of heavy metal ions in soils and vegetables using
ICP-MS and Chromatograph.
2011 – 2012 Dr. Zongping Shao’s research lab of fuel cell materials, Nanjing
University of Technology, Nanjing, China.
Research: La-doped BaFeO3-d PerovSkite as A Cobalt-free Oxygen
Reduction Electrode for Solid Oxide Fuel Cells with Oxygen-ion
Conducting Electrolyte.
AWARDS AND HONORS
Florida State University Betty M. Watts Memorial Fund for Food Science, 2015
Excellent Graduation Thesis and Graduate student of NJUT, 2013
First class Scholarship of NJUT, 2013
Feiyang Scholarship by Pujing Chemical Industry Limited Co., 2012
Second Class Scholarship of the NJUT, 2012
Third Class Scholarship of the NJUT , 2011
Excellent Student Leader in the School of Chemistry and Chemical Engineering, NJUT, 2010
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PUBLICATION
Feifei Dong, Dengjie Chen, Yubo Chen, Qing Zhao, and Zongping Shao, La-doped BaFeO3-d PerovSkite as A Cobalt-free Oxygen Reduction Electrode for Solid Oxide Fuel Cells with Oxygen-ion Conducting Electrolyte. Journal of Materials Chemistry, 2012, 22, 15071–15079.
ABSTRACT
Zaffran, V.D.; Liu, C.; Gupta, S.; Zhao, Q.; Sathe, S.K. 2015. Pecan (Carya illinoinensis) detection using a monoclonal antibody-based direct sandwich enzyme-linked immunosorbent assay. Pacifichem 2015: Honolulu, HI, USA.
Gupta, S.; Liu, C.; Zaffran, V.D.; Zhao, Q.; Sathe, S.K. 2015. Hazelnut (Corylus avellana) detection using a monoclonal antibody-based direct sandwich enzyme-linked immunosorbent assay. Pacifichem 2015: Honolulu, HI, USA.
SERVICE
North Florida Fair Bakery Judge, Florida, 2015
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