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Improving Extraction of Allergenic Soy ProteinsFrom Soy ProductsAmma Konadu AmponsahUniversity of Maine

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Recommended CitationAmponsah, Amma Konadu, "Improving Extraction of Allergenic Soy Proteins From Soy Products" (2015). Electronic Theses andDissertations. 2403.http://digitalcommons.library.umaine.edu/etd/2403

IMPROVING EXTRACTION OF ALLERGENIC SOY PROTEINS FROM SOY

PRODUCTS

By

Amma Konadu Amponsah

BSc. Kwame University o f Science and Technology, 2007

MPhil. University o f Ghana, 2010

A THESIS

Submitted in Partial Fulfillment o f the

Requirements for the Degree of

Master o f Science

(in Food Science and Human Nutrition)

The Graduate School

The University o f Maine

December 2015

Advisory Committee:

Balunkeswar Nayak, Assistant Professor, School o f Food and Agriculture, Advisor

Denise Skonberg, Associate Professor, School o f Food and Agriculture

Brian L. Perkins, Laboratory Director & Research Assistant Professor, School o f Food

and Agriculture

IMPROVING EXTRACTION OF ALLERGENIC SOY PROTEINS FROM SOY

PRODUCTS

By Amma Konadu Amponsah

Thesis Advisor: Dr. Balunkeswar Nayak

An Abstract o f the Thesis Presented in Partial Fulfillment o f the Requirements for the

Degree o f Master o f Science (in Food Science and Human Nutrition)

December 2015

With recent increase in the utilization o f soy proteins, there are mounting

concerns o f the escalation o f soy allergies. It is, therefore, important to improve current

methods o f allergen detection to ensure accurate labeling o f foods, produce more reliable

and representative results in studies to create hypoallergenic food and establish allergen

thresholds. The conditions used to extract proteins from foods are important determinants

for appropriate detection and interpretation o f the allergenicity o f food materials. The

extraction of soy proteins for soy allergen detections is conventionally performed with

phosphate buffered saline (PBS) buffer for an extraction period o f at least 2 h at 23 or

4°C. This has been reported to be inefficient due to time consumption and inadequate

protein extraction, resulting in false negative allergen detection and mislabeling o f foods

containing allergenic proteins.

The objectives o f this study were: 1) To improve the extraction o f allergenic

proteins from soy products using different buffers in combination with selected thermal

and non- thermal extraction conditions and 2) To evaluate the efficiency of extraction

methods on the extraction o f allergenic soy proteins from food matrices. With study one,

soy proteins were extracted from raw soy flour, soy protein isolate (SPI) and soy milk

using water bath extraction at 60, 70 and 100°C for 5, 15 and 30 minutes, microwave

assisted extraction (MAE) at 60, 70 and 100°C for 0, 5 and 10 minutes and ultrasound

assisted extraction (UAE) at 4 and 23°C for extraction times of 1, 5 and 10 minutes with

three different buffers namely PBS, Laemmli and urea. Extracts were analyzed for total

proteins, protein molecular weight profile (SDS-PAGE) and antibody-based detection

(ELISA) of soy proteins. Conventional extraction with each of the buffers was used as

controls. Overall, protein recoveries using water bath, MAE and UAE methods were

significantly higher than recoveries from the controls in all soy matrices. Under all

extraction conditions, Laemmli and urea buffer recovered more proteins than PBS.

Electrophoretic analysis of proteins showed bands around 75, 50 and 37 kDa indicating

the presence of soy allergenic proteins β-conglycinin, glycinin and P34, in all samples.

ELISA analyses showed that water bath extraction and MAE as well as the use of urea

reduced the ability of the ELISA kit to detect soy proteins. The use of Laemmli buffer

with conventional extraction and UAE, however, produced similar or better results with

ELISA, compared to conventional extraction with PBS, especially for SPI and soy milk.

These extraction conditions were further studied and their practical use tested on

some commercial samples containing soy as well as samples spiked with known

concentrations of soy flour in study two. The use of Laemmli buffer with conventional

extraction and UAE once again resulted in comparable and in some cases, better

outcomes than conventional extraction with PBS suggesting that these extraction

conditions may be used as alternative or additional extraction methods that could be

employed in the extraction step to improve soy allergen detection.

DEDICATION

I dedicate this manuscript with heartfelt gratitude to my husband, Andrew Jackson and to

our little boy Nathan Jackson.

Ill

ACKNOWLEDGMENTS

My most profound gratitude goes to God Almighty for His grace, protection and

guidance from the beginning to the end of this program.

I would like to acknowledge the financial support of this project through the United

States Department of Agriculture (USDA), National Institute of Food and Agriculture

(NIFA) Hatch No: ME021408 and Maine Agricultural and Forest Experiment Station.

I sincerely thank my advisor, Dr. Balunkeswar Nayak for supervising, guiding and

encouraging me to the successful completion of my study. A special thanks to my

committee members Dr. Denise Skonberg and Dr. Brian Perkins for their support and for

giving me constant access to their labs and equipment throughout my research at the

University of Maine. I also want to thank Dr. Lawrence Leblanc, Dr. Carl Tripp, Dr. Bill

Halteman, Dr. Julie Gosse and her graduate students for making time to train me and

offer assistance which ensured the successful completion of my research. I am most

grateful to Katherine Davis-Dentici and Michael Dougherty for their constant support,

advice and training.

To lab mates and friends, I want to say thanks for your assistance, encouragement

and sense of humor. I also acknowledge the assistance I received from my sister Abena

Amponsah. Lastly I want to thank my wonderful husband, Andrew Jackson. I really

appreciate your support, patience and love over the past two years.

IV

TABLE OF CONTENTS

DEDICATION.............................................................................................................................. iii

ACKNOWLEDGMENTS.......................................................................................................... iv

LIST OF TABLES..................................................................................................................... viii

LIST OF FIGURES..................................................................................................................... ix

CHAPTER 1: INTRODUCTION................................................................................................1

CHAPTER 2: LITERATURE REVIEW................................................................................... 5

2.1 Soybean.................................................................................................................................5

2.2 Soybean Allergy.................................................................................................................. 7

2.3 Allergens in Soybean........................................................................................................10

2.3.1 G lym B d 30K............................................................................................................. 11

2.3.2 Glycinin....................................................................................................................... 13

2.3.3 p-Conglycinin............................................................................................................. 14

2.3.4 G lym B d 28K............................................................................................................. 15

2.3.5 Other Soybean Allergens...........................................................................................17

2.4 Soybean Processing and Allergy Management.............................................................19

2.5 Extraction of Soy Proteins for Allergen Determination.............................................. 22

2.5.1 Conventional Extraction Methods...........................................................................24

2.5.2 High Temperature Extraction with other Buffers..................................................25

v

2.5.3 Ultrasound-Assisted Extraction............................................................................... 28

2.5.4 Microwave-Assisted Extraction.............................................................................. 30

2.6 Soy Allergen Detection..................................................................................................32

2.6.1 Antibody-Based Tests................................................................................................33

2.6.2 Other Soy Detection Methods................................................................................. 35

CHAPTER 3: MATERIALS AND METHODS.................................................................... 38

3.1 Experimental Design........................................................................................................ 38

3.1.1 Sample Preparation.................................................................................................... 39

3.1.2 Water Bath Extraction.............................................................................................. 40

3.1.3 Microwave-Assisted Extraction (MAE).................................................................40

3.1.4 Ultrasound-Assisted Extraction (UAE)..................................................................41

3.1.5 Control samples......................................................................................................... 42

3.1.6 Determination of total protein................................................................................. 42

3.1.7 Electrophoresis...........................................................................................................42

3.1.8 Sandwich Enzyme-linked immunosorbent assay (ELISA).................................43

3.1.9 Statistical analysis...................................................................................................... 44

3.2 Experimental design......................................................................................................... 45

3.2.1 Materials......................................................................................................................45

3.2.2 Preparation of Cookies............................................................................................. 46

vi

CHAPTER 4: RESULTS AND DISCUSSION 48

4.1 Effect o f water bath extraction time at different temperatures on recovery o f

total soy proteins............................................................................................................. 48

4.1.1 Comparison o f conventional and water bath extraction methods on protein

recovery................................................................................................................................ 50

4.2 Effect o f microwave assisted extraction time at different temperatures on

recovery o f total soy proteins.........................................................................................54

4.2.1 Comparison o f conventional extraction and MAE on protein recovery............ 57

4.3 Effect o f ultrasound assisted extraction time at different temperatures on

recovery o f total soy proteins.........................................................................................61

4.3.1 Comparison o f conventional extraction and UAE on protein recovery............ 63

4.4 Comparison o f MAE, UAE, conventional and water bath extraction methods......67

4.5 Characterization o f proteins using SDS-PAGE............................................................ 69

4.6 Effect o f Extraction Conditions on Antibody based detection o f Soy Proteins..... 73

4.7 Effect o f selected extraction conditions on extraction of total proteins from food

matrices............................................................................................................................. 80

4.8 Effect o f selected extraction conditions on protein profiles using SDS-PAGE......82

4.9 Effect o f selected extraction conditions on ELISA analyses o f food matrices.......86

CHAPTER 5: CONCLUSIONS............................................................................................... 90

REFERENCES............................................................................................................................92

BIOGRAPHY OF AUTHOR..................................................................................................106

vii

LIST OF TABLES

Table 3.1. Experimental design of extraction treatments for soy matrices........................38

Table 3.2. Experimental design of extraction treatments for food matrices..................... 45

Table 4.1: MAE Come-up times for samples..........................................................................57

Table 4.2: Protein extracts from soy matrices selected based on high total soluble proteins

for Electrophoresis and ELISA analyses........................................................... 71

Table 4.3: Soy ELISA Analyses of Conventional and Water bath Extraction, MAE

and UAE Extracts..................................................................................................75

Table 4.4: Soy ELISA analyses of commercial samples showing amount of soy proteins

detected (in ppm) using the selected extraction conditions............................ 87

Table 4.5: Soy ELISA analyses of dry mix and cookies spiked with soy flour showing

amount of soy proteins detected (in ppm) using the selected extraction

conditions................................................................................................................87

vm

LIST OF FIGURES

Figure 2.1: Freshly harvested soybeans in pods and dried soybeans 5

Figure 4.1: Quantity of protein obtained from soy flour (a), SPI (b) and soy milk (c)

extracts at different extraction times (5, 15 and 30 minutes) and temperatures

(60, 70 and 100°C) using a water bath............................................................... 49

Figure 4.2: Protein concentrations of soy flour (a), SPI (b) and soy milk (c) extracts

obtained from conventional extraction at 23 °C and water bath extraction at

60, 70 and 100°C and 15 minutes extraction time.............................................51

Figure 4.3: Quantity of protein obtained from soy flour (a), SPI (b) and soy milk (c)

extracts at different holding times (0, 5 and 10 minutes) and temperatures

(60, 70 and 100°C) using microwave-assisted extraction................................55

Figure 4.4: Protein concentrations of soy flour (a), SPI (b) and soy milk (c) extracts

obtained from conventional extraction at 23°C and MAE at 60, 70 and 100°C

and 0 minute holding time....................................................................................59

Figure 4.5: Quantity of protein obtained from soy flour (a), SPI (b) and soy milk (c) at

different extraction times (1,5 and 10 minutes) and temperatures (4 and 23

°C) using ultrasound-assisted extraction............................................................ 62

Figure 4.6: Protein concentrations of soy flour (a), SPI (b) and soy milk (c) extracts

obtained from conventional extraction at 23°C and UAE at 4 and 23°C for 5,

10 and 1 min respectively..................................................................................... 64

IX

Figure 4.7: Protein concentrations of soy flour (a), SPI (b) and soy milk (c) extracts

obtained from conventional and water bath extraction, MAE and UAE.......68

Figure 4.8: Protein profiles of SF (a), SPI (b) & SM (c) extracts from Conventional (B-

D) and water bath extraction (E-G), MAE (H-J) and UAE (K-M) methods

with PBS, Laemmli and urea buffers, respectively, under reduced and non-

reduced conditions, A (molecular weight marker)............................................72

Figure 4.9: Total protein concentrations of commercial samples extracted using

conventional extraction with PBS and Laemmli and UAE with Laemmli.... 81

Figure 4.10: Total protein concentrations of dry mix and cookies spiked with soy

proteins extracts obtained using conventional extraction with PBS and

Laemmli and UAE with Laemmli. DM-dry mix, SFC-soy free cookie, C-

cookie........................................................................................................................ 81

Figure 4.11: Protein profiles of extracts from commercial samples (cheese crackers,

salsa, pancake mix, table crackers and veggie burgers) obtained from

Conventional extraction with PBS (B-F), with Laemmli (G-K) and UAE with

Laemmli (L-P) buffers respectively under reduced conditions, A (molecular

weight marker).........................................................................................................83

Figure 4.12: Protein profiles of extracts from dry mix and cookie samples with 0, 1,5, 10

and 20 mg soy obtained from Conventional extraction with PBS (B-F), with

Laemmli (G-K) and UAE with Laemmli (L-P) buffers, respectively, under

reduced conditions, A (molecular weight marker)................................................83

x

CHAPTER 1: INTRODUCTION

Food allergy is a growing concern worldwide and is considered to be the fourth

most important public health problem by the World Health Organization (WHO) (Kirsch

et al. 2009). It affects the general health status, economy, and legislation of a country

especially if a considerable percentage of its population must deal with dietary

restrictions and food-triggered IgE-mediated allergic reactions ranging from mild to life

threatening (Faeste et al. 2011).

Food allergy is an abnormal immunological, usually, immunoglobulin E (IgE) -

related reaction resulting from exposure to exogenous food macromolecules (typically

proteins) known as allergens or antigens. Exposure can be through ingestion, skin contact

or inhalation ( L’Hocine and Boye 2007, Kirsch et al. 2009). A food allergic reaction is

triggered in two steps. The first step, sensitization, leads to the production of IgE

antibodies specific to one or more proteins in a food. The second step, elicitation, occurs

when a previously sensitized individual is re-exposed to the same food or food proteins

(Gendel 2012). Antibodies, which are allergen- specific IgE molecules reside on the

surfaces of mast cells and basophils and upon binding of the food proteins or allergens to

the antibody during the second exposure, inflammatory mediators such as histamine and

cytokines, induce inflammatory response indicative of an allergic reaction (Wilson et al.

2005, Yang and Mejia 2011). Small regions of allergenic proteins known as epitopes,

composed of 5-7 amino acids or 3-4 sugar residues, upon reaction with an antigen, are

responsible for IgE-mediated allergy (Taylor and Hefle 2001, Yang and Mejia 2011). In

some rare cases, allergic reactions may also occur as a result of cross-reactivity between

similar allergens (Wilson et al. 2005). A study by Wensing et al. (2003) reported cases

1

indicating cross-reactivity between peas and peanuts. In this study, IgE antibodies to pea

vicilin also reacted with peanut vicilin. This reaction has been attributed to homology in

the amino acid sequence found among various allergenic proteins.

Food allergies affect an estimated 3-5% of adults and 8% of children worldwide

(Gendel 2012, Gupta et al. 2011). There is, however no cure for food allergies. It is

therefore important that allergic consumers avoid foods containing ingredients that could

provoke potentially life-threatening reactions (Gendel 2013). The food industry and

public health agencies such as the U.S. Food and Drug Administration (FDA) support

these consumers by ensuring that the labels of packaged foods contain complete and

accurate information about the presence of food allergens through the enactment of the

Food Allergen Labeling and Consumer Protection Act of 2004 (FALCPA). This act

amended the Federal Food Drug and Cosmetic Act to define a “ major food allergen” as

one of eight foods or food groups namely, peanut, milk, egg, soy, tree nuts, fish,

crustacean shellfish, and wheat or ingredients containing proteins from one of these

(Hefle and Taylor 2004, Gendel 2013). Despite awareness of the importance of food

allergy as a public health issue, recalls and adverse reactions linked to undeclared

allergens in foods continue to occur with high frequency (Gendel 2013).

In order to curb this problem and maintain safety, individuals with food allergies

currently rely heavily on accurate labeling and declaration of allergens in foods. There is

also ongoing research to produce hypoallergenic products using processing methods such

as high pressure processing (Li et al. 2012) as well as studies to establish thresholds for

allergens. These tests and studies are all dependent on accurate determination of

allergenic proteins in the food products and such determinations begin with the extraction

2

of these proteins. According to Panda (2012), extraction conditions are an important

determinant for appropriate interpretation of allergenicity.

Conventionally, food proteins are extracted with a buffer, such as phosphate

buffered saline (PBS) at pH 7.4 or Tris/HCl buffer (pH 8.2) prior to analysis by

immunoassay (Koppelman et al. 2004). The extraction is usually done at 4°C or at room

temperature for a period of 2 to 24 hours with or without some form of agitation (Panda

2012). These conditions have, however, been reported to be inefficient in the extraction

of proteins that have undergone modification or denaturation through processing (Poms

et al. 2004, Panda 2012). Few researchers have investigated other extraction processes

and conditions such as the use of extraction temperatures of 60 and 70°C and buffers

other than PBS (Poms et al. 2004, Panda 2012), ultrasound assisted extraction or UAE

(Wang 1978, Jambrak et al. 2009, Karki et al. 2010), microwave heating (Choi et al.

2006), enzymatic and other chemical modifications (Jung et al. 2006) to improve protein

extractability from food matrices such as peanuts, almonds and soybeans.

These extraction methods and conditions have been reported to be more effective

than conventional extraction of proteins for allergen detection, but despite the advantages

of these extraction methods, there is very little published data on their use to improve the

extraction of soy proteins (which is the focus of this study), especially for the purpose of

allergen detection. There is also no report on the use of these extraction methods in

combination with buffers (other than PBS) to improve the extraction process for better

detection of allergenic soy proteins.

3

The objectives of this study were to:

1) Improve the extraction of allergenic proteins from soy products using different

buffers in combination with selected thermal and non- thermal extraction

conditions and

2) Evaluate the efficiency of extraction methods on the extraction of allergenic soy

proteins from food matrices.

4

CHAPTER 2: LITERATURE REVIEW

2.1 Soybean

Soybean (Glycine max) is an economically important crop native to China and

Southeast Asia. It is popularly known as the miracle crop due to its many uses, health

benefits and high nutritional value. Mature soybean seeds contain about 18-25% of fat

and 38-50% of protein (Müller et al. 1998, Cucu et al. 2012, Panda 2012). They also

contain approximately 31% carbohydrates, 5% mineral and 12% moisture (L'Hocine and

Boye 2007). For the purposes of human nutrition, soybean protein contains adequate

amounts of the essential amino acids, histidine, isoleucine, leucine, phenylalanine,

tyrosine, threonine, tryptophan and valine. It is, however, deficient in the sulfur

containing amino acids methionine and lysin.

The Unites States of America (US) is the largest producer of soybeans,

worldwide. About 38% of the world’s soybean crop is grown in the US, followed by

Brazil (25%), Argentina (19%), China (7%), India (3%), Canada (2%), and Paraguay

(2%) (Singh et al. 2008).

Figure 2.1: Freshly harvested soybeans in pods and dried soybeans(http://www.anaphylaxis.ca/images/Allergens_Soy.jpg&imgrefurl=http://www.anaphylaxis.ca/en/anaphylaxislOl/allergens.html).

5

The use of soybean has generated a lot of interest due to evidence suggesting that

diets containing soybean have beneficial effects and because of its nutritional and

functional properties, it is consumed worldwide. In Asian countries soybean is processed

into various fermented and non-fermented food stuffs such as soy sauce, miso, natto,

yogurts, kinako, protein crisp, desserts, soy milk, tofu, aburage and yuba. In western

countries relatively unprocessed soybeans are used mainly in soy milk, tofu, soy sprouts

and edamame (Hammond and Jez 2011, Panda 2012). In addition, soybean proteins in the

form of concentrates or isolates are largely used, as ingredients in formulated foods, meat

and poultry products, bakery and pastry products, dairy products, edible spreads, frozen

desserts, ice cream and cheese analogues (L’Hocine and Boye 2007, Cucu et al. 2012).

They are also used in the production of infant formula and are generally introduced into

the diet early in life particularly for infants with an intolerance or allergy to cow milk.

Soybean based products are also used as the primary source of protein for individuals

with disorders such as lactose intolerance and severe gastroenteritis in infants (Businco

et al. 1992, Panda 2012). Aside from its usefulness in the food industry, soybean is also

of great value as an ingredient in a variety of pharmaceutical and industrial applications

(Endres 2001, Verma et al. 2013).

Several health benefits have been associated with the consumption of soybean. It

has been reported to lower plasma cholesterol (Anthony et al. 1996), prevent cancer

(Kennedy 1998), provide protection against bowel and kidney disease (Friedman and

Brandon 2001), improve bone mineral density (Kreijkamp-Kaspers et al. 2004), and

reduce the risk of cardiovascular diseases (L’Hocine and Boye 2007, Cucu et al. 2012).

These health benefits have been attributed to the presence of isoflavones, saponins,

6

proteins, and peptides in soybean (Friedman and Brandon 2001, Michelfelder 2009). The

benefits of soybean have resulted in increased production and consumption of soy

products especially in western countries. For instance, in a survey by Michelfelder

(2009), approximately 33% of Americans recalled consuming soybean products at least

once a month.

Despite the importance of soybean in human nutrition and welfare, it is also an

important source of food-related allergies. It ranks among the 'big eight’ that are the most

significant foods known to cause more than 90% of food allergies (Pedersen 1988). Many

studies have been performed on peanut allergies because of the generally held belief that

peanut allergy is more severe and is sometimes life threatening (Sicherer et al. 2000).

However, with the increasing usage of soybean proteins in the food and other industries,

there are mounting concerns related to the escalation of allergies to soybeans.

2.2 Soybean Allergy

Minimal attention has been given to soy allergy relative to peanut allergy because

soy allergy has been reported to affect about 0.2-0.4 % of the population (Becker et al.

2004, FDA 2006, Savage et al. 2010) as compared to the 0.6% and 0.8% of adults and

children, respectively, affected by peanut allergies (Sicherer et al. 2003, Sampson 2004,

FDA 2006). Soybean consumption has, however increased over the years. Soybean and

its derivatives have become ubiquitous in many vegetarian and meat-based food products

as well as in the diets of infants with cow’s milk allergy (CMA). It has thus become

difficult to avoid soybean (Wilson et al. 2005) and underscores the need to take soybean

allergy as seriously as peanut allergy.

7

A study by Sicherer et al. (2000) showed that the prevalence and incidence of soy allergy

in the general population is unknown and is likely to be dependent on local dietary habit

and exposure. Errahali et al. (2002) reported that 6% of sensitive children are affected

with soy allergy while Ito et al. (2011) also reported that the prevalence of soy allergy is

considered to be higher in Asian countries than Europe and US and it has been reported

to be the fifth most common food allergen causing anaphylaxis in Japan.

According to Panda (2012), there have been several inconsistent reports on the

prevalence of soybean allergy. For instance, a study by Bruno et al. (1997) showed that

soy allergy is not common in children with atopic disorders and is also rare in children

fed soy early in life. In another study by Magnolfi et al. (1996) in Italy, out of 131 soy

skin prick positive children, only 6% showed positive oral challenge with soybean,

representing 1.1% of 704 atopic children used in the study (Magnolfi et al. 1996, Panda

2012). Zeiger et al. (1999) also found that 14% of IgE mediated cow’s milk allergic

children also have soy allergy, based on double blind placebo controlled food challenge

(DBPCFC), open challenge, and history of anaphylactic reaction to soy. Children have

been shown to outgrow their allergy later in life. It has been shown that the median age at

which children outgrow their allergies is around 10 years. The rate of soy allergy

resolution depends on soy specific IgE levels although children with higher soy specific

IgE levels tend to have persistent soy allergy (Savage et al. 2010).

Soy allergy can also be as a result of cross-reactivity with peanut or birch pollen

and this usually occurs later in the lives of individuals (Savage et al. 2010). Several

soybean allergens have been identified, with some such as the glycinin being homologs

of peanut allergens. Several studies have been conducted with reference to soy allergy

8

due to cross-reactivity. A 3-year study by Foucard and Malmheden-Yman (1999)

conducted in Sweden showed that soybean allergies may be underestimated. Life-

threatening reactions that occurred in four peanut-sensitive individuals were attributed to

the possible consumption of soybean. In one study, IgE from serum of individuals

allergic to both peanut and soybean bound to several proteins were present in soy and

peanut seed extracts with an apparent molecular weight of 50-60 kD (likely to be beta-

conglycinin) (Herian et al. 1990, Xiang 2003). In another study by Mittag et al. (2004)

soybean allergy was shown to be prevalent in adult patients allergic to birch pollen, and

the allergy was due to cross-reactivity or shared IgE binding of the soybean allergen Gly

m 4 with the birch pollen allergen Bet v 1. Unlike mild oropharyngeal symptoms in

patients with pollen related food allergy, patients with marked allergies to birch pollen

showed systemic reactions in DBPCFC to soybean (Mittag et al. 2004). Other studies

have also shown that the soybean allergen Gly m Bd 30K shares approximately 70%

sequence homology with peanut’s main allergen (Ara h 1) and 50% to 70% with the

immunodominant cow’s milk allergen (2-S1-casein) (Wilson et al. 2005). Due to this

homology and close botanical relationship, Immunoglobulin E antibodies to peanut

proteins can also react with soybean proteins (Xiang 2003). This may explain a study in

Sweden that reported three anaphylactic deaths in patients ages 9 to 17 after consumption

of meat products fortified with 2.2% to 7% soy protein; these patients had a previously

known allergy, to peanuts but not to soybeans (Wilson et al. 2005).

Clinical manifestations of soy allergy range from severe enterocolitis to atopic

eczema and immediate IgE-mediated systemic multi-system reactions (Sicherer et al.,

2000). Life threatening reactions to soy have been reported to be quite rare and much

9

lower in prevalence than severe reactions to peanut and other allergenic foods (Cantani

and Lucenti 1997). A study by Rolinck-Werninghaus et al. (2012) showed that in an oral

food challenge, most of the severe reactions to soy in soy sensitized children occurred at

doses greater than 1.1 gram compared to milk and egg allergic reactions which occurred

at 3 mg. Becker et al. (2004) have reported the threshold levels required for eliciting

allergenic response to soy vary from 0.0013 to 500 mg. A few cases of anaphylactic

reactions, including food dependent exercise induced anaphylaxis, have been reported♦

due to consumption of soybean (Foucard and Malmheden-Yman 1999, Taramarcaz et al.

2001, Adachi et al. 2009), making the study of soy allergy important.

2.3 Allergens in Soybean

Through the use of techniques such as acid precipitation and ultracentrifugation,

different fractions of soybean proteins have been isolated and their relation to soy

allergies studied. The common soy protein fractions isolated are the 2S, 7S and 1 IS

fractions (Appu and Narasinga 1977). According to studies by Burks Jr et al. (1988) and

Sicherer et al. (2000), the 2S fraction includes a-conglycinin which is composed of 18.2

and 32.6 kD proteins, the Kunitz and Bowman Birk trypsin inhibitors, albumins and

cytochrome C. The 7S fraction is mainly composed (about 50%) of p-conglycinin, a

trimer of 150-170 kD. The 1 IS fraction is composed almost entirely of glycinins, which

are 12mer units of 320-360 kD. The allergenicity documented for these fractions has been

different in several studies. One early study demonstrated that the 2S fraction contained

the most potent allergens (Shibasaki et al. 1980). A later study, however, indicated that

the 7S or 1 IS fractions were more allergenic than the 2S fraction (Burks Jr et al. 1988),

10

while yet another study indicated that the 7S fraction displayed stronger allergenicity

(Ogawa et al., 1991).

In other studies, at least 16 soybean proteins with molecular weights ranging from

14 kDa to 70 kDa have been shown to bind IgE from sera of patients with atopic

dermatitis (Ogawa et al. 1991, Panda 2012). Some of these proteins such as the Gly m

Bd 30K, glycinin (Gly m 6), P-conglycinin (Gly m 5), Gly m Bd 28K and some others

have been thoroughly studied and described as major soybean allergens, and are

discussed below.

2.3.1 Gly m Bd 30K

Gly m Bd 30K, a cysteine protease from soybean, also known as soybean

vacuolar protein P34 is a major soybean allergen and according to Ogawa et al. (1991)

probably the most frequent and strongly recognized allergen from soybean in the

Japanese population. P34 was first identified as a 34 kD oil body associated protein

(Herman 1987) but further studies indicated that it is a vacuolar protein and the oil body

association was an artifact arising during cellular lysis of the fractionation procedure used

to prepare oil bodies (Kalinski et al. 1992). It has since been described as a monomeric

insoluble glycoprotein (glycosylated in vivo at Asn 170) consisting of 258 amino acid

residues contained by disulfide linkages in the 7S globulin fraction of soybean storage

protein (Wilson et al. 2005). It is a relatively minor seed constituent comprising less than

1% of total seed protein (Helm et al. 1998) and may play a role in protein folding (Bando

et al. 1996, Hosoyama et al. 1996, Helm et al. 2000). Gly m Bd 30K is a post-

translationally processed form of a 46 to 47 kD precursor protein, whose N-terminal 122

11

amino acids residues are partially removed during soybean seed development. It can be

further processed to a 32 kD protein during the fourth through sixth days of seed

germination when an additional 10 amino acids are removed from the N-terminus. Gly m

Bd 30K also exhibits tertiary conformation consistent with the thiol proteinase of the

papain superfamily (Herman et al. 1990, Kalinski et al. 1990, Ogawa et al. 1995, Yaklich

et al. 1999, Ogawa et al. 2000).

The allergenicity of Gly m Bd 30K has been discussed in many studies. For

instance, it has been reported that IgE binding assays using immunoglobulins from

soybean sensitive individuals that 65% of the total allergenic response was accounted for

by Gly m Bd 30K (Ogawa et al. 1995, Helm et al. 2000, Herman 2003). According to

Tsuji et al. (1995) Gly m Bd 30K is very stable and its IgE binding ability is insensitive

to trypsin, chymotrypsin or heat treatment. It is therefore able to survive most types of

food processing, hence making its concentration significant in several soybean products,

ranging from 0.14 - 5.54 mg/g, fresh weight (Tsuji et al. 1995). In some fermented

soybean products such as miso, shoyu and natto, however, Gly m Bd 30K has been

reported to be undetectable, an indication that this allergen may be destroyed through

fermentation. Babiker et al. (2000) also compared the allergenicity of native and

recombinant Gly m Bd 30K and concluded that the recombinant Gly m Bd 30K protein

expressed in Escherichia coli displayed IgE binding similar to the native allergen, an

indication that both recombinant and native proteins contained similar IgE binding

epitopes. These results also indicated that the glycan moiety of the native protein was

unlikely to be a significant component for IgE binding or elicitation in allergic

12

individuals (Babiker et al. 2000). P34 has been shown to have a tendency to bind to

lipids, which is likely significant in its role as an allergen (Mills et al. 2004).

2.3.2 Glycinin

Glycinin or Gly m 6 is a major storage protein present in the 1 IS fraction of

soybean seeds. It constitutes about 20% of the seed dry weight (Nielsen et al. 1989) and

has been implicated as a major allergen in soybean (Holzhäuser et al. 2009, Ito et al.

2011). It is a hexameric protein with a molecular mass of 300-380 kDa and composed of

five subunits ( Adachi et al. 2003, Maruyama et al. 2003). The subunit composition of

soybean glycinin has been reported to vary by cultivar (Mori et al. 1981, Panda 2012).

Each glycinin subunit contains at least one acidic chain of approximately 34 to 45 kDa,

and one basic chain of approximately 19 to 22 kDa. These chains are generated by post-

translational removal of a signal peptide, cleavage of the proglycinin monomer, and

linkage of the acidic and basic subunits through disulfide bonds (Burks Jr. et al. 1988,

Adachi et al. 2001). Five major kinds of subunits namely, AlaBlb, A2Bla, AlbB2,

A3B4, and A5A4B3 have been identified in glycinin (Adachi et al. 2003). Each of these

subunits is composed of an acidic chain (Ala, Alb, A2, A3, A4, A5) of 37 to 42 kDa and

isoelectric point (pi) of 4.2 to 4.8 and a basic chain (Bla, Bib, B2, B3, B4) of 17 to 20

kDa and pi of 8 to 8.5 (where A, stands for the acidic chain and B, represents the basic

chain) linked by a single disulfide bond and are not glycosylated (Petruccelli and Anon

1995, Maruyama et al. 2003).

Studies exploring the contribution of these chains to the allergenicity of glycinin

have been inconsistent. Pedersen and Djurtoft (1989), Djurtoft et al. (1991) and Zeece et

al. (1999) have all indicated that the acidic glycinin chain is mainly responsible for IgE

13

binding with sera from soy allergic patients. Helm et al. (2000) and Panda (2012) in a

later study, however, also reported IgE binding to the basic chain of glycinin. Irrespective

of which chain is responsible, glycinin has been implicated in a number of soy allergies.

Eleven linear epitopes have been identified in glycinin by Beeil epitope mapping among

which, four are immuno-dominant (Helm et al. 2000). The IgE binding epitopes of the

acidic chain of glycinin were shown to be identical to that of major peanut allergen Ara h

3 in other studies (Beardslee et al. 2000, Xiang 2003, Panda 2012). In a study by

Holzhäuser et al. (2009) 36% of study subjects with positive DBPCFC and with history

of anaphylaxis to soy had specific IgE binding to glycinin. In another study, 58% of

children with severe allergic reactions to soybean also exhibited IgE binding to glycinin

(Ito et al. 2011, Panda 2012).

2.3.3 p-Conglycinin

Soybean P-conglycinin or Gly m 5 together with glycinin form the two dominant

storage proteins in soybean seeds. These two proteins account for approximately 80% of

the storage proteins in soybean seeds (Nielsen et al. 1989). P-conglycinin is a trimeric

protein of approximately 150-200 kDa. It is composed of different combinations of three

subunits namely a, a ’ and P which are about 67 kDa, 71 kDa and 50 kDa, respectively.

Each subunit is processed by co- and post-translational modifications during

biosynthesis. For example, all subunits of p -conglycinin are glycosylated by addition of

a polymannose or complex glycan and the a and a ’ subunits are processed at N-terminal

regions (Utsumi 1992, Maruyama et al. 2003). The a and a' subunits each have two

glycosylated sites, while the P subunit has only one. According to Maruyama et al.

14

(2001), the biological function of glycosylation seems to be linked to the prevention of

protein aggregation during assembly in the endoplasmic reticulum and it apparently does

not affect the structure and thermal stability of the protein.

The allergenic property of soybean ß-conglycinin has been studied extensively.

Most of these studies have shown and concluded that all three subunits bind IgE to some

extent. In one study the a subunit of ß-conglycinin was recognized by sera from 16 out of

69 soybean allergic patients tested, though this observation was not made for the a' and ß

subunits (Ogawa et al. 1995, 1991). Contrary to this, a study by Krishnan et al. (2009)

using IgE immunoblot analysis with soybean allergic patient sera showed that the a' and

ß subunits of ß-conglycinin could also be recognized by IgE and are therefore likely

allergens. Another study by Holzhäuser et al. (2009) also showed all three subunits of ß-

conglycinin were bound by IgE from subjects who were allergic to soybean as

demonstrated by DBPCFC (Panda 2012). Other ß-conglycinin related studies include one

by Adachi et al. (2009) which identified ß-conglycinin as the soybean allergen

responsible for food dependent exercise- induced anaphylaxis induced during the

consumption of tofu. Another study by Ito et al. (2011) indicated that 67% of children

with severe allergic reactions to soybean showed IgE reactivity against ß-conglycinin

(Panda 2012).

2.3.4 Gly m Bd 28K

Soybean allergen Gly m Bd 28K is a minor soybean glycoprotein component

found in the 7S fraction of soybean extracts that has been shown in some studies to be

another important soybean allergen. It has a molecular mass of 26 to 28 kDa and a pi of

15

6.1 (Ogawa et al. 2000, Tsuji et al. 2001). This protein is reported to be quite stable

during processing of soybean meals. A study by Bando et al., (1998) showed that it was

present in noticeable quantities of 0.28 to 14.4 pg/g (fresh weight) in soybean protein

isolate (SPI) and tofu. It was, however, less stable than Gly m Bd 30K during the

preparation of some processed soybean products, such as meatballs and fried chicken to

which SPI has been added. It was also destroyed in fermented soybean products as has

been observed for Gly m Bd 30K (Bando et al. 1998).

The allergenic property of Gly m Bd 28K has been studied by Ogawa et al.

(1991). The authors reported that out of 69 soybean allergic patients tested, 23%

produced antibodies (IgE) that recognized this protein (Ogawa et al. 1991). The glycan

moiety of Gly m Bd 28K has been described by Tsuji et al. (1997) as being similar to that

of other glycoprotein allergens such as bromelain, horseradish peroxidase, ascorbate

oxidase and Gly m Bd 30K and has also been reported to play an important role in

allergic reactions. A study by Hiemori et al. (2000) showed that the glycan at Asn 20 of

Gly m Bd 28K could bind to IgE. Another study by Hiemori et al. (2004) also reported

that a C-terminal peptide fragment of Gly m Bd 28 K, which is a 23 kDa glycoprotein,

can bind IgE from soybean sensitive patients, primarily due to the glycan moiety (Xiang

2003, Panda 2012)

16

2.3.5 Other Soybean Allergens

Other soybean allergens of importance include soybean profilin (Gly m 3), Gly m

4, Gly m 1, soybean albumin and some aeroallergens.

Soybean profilin (Gly m 3) is a 14 kDa soybean protein that has been isolated and

identified by PCR based c-DNA cloning. It has been shown to bind IgE from 69% of

soybean sensitive patient sera (Rihs et al. 1999, Panda 2012). The IgE binding properties

of this protein is, however, only observed the full length profilin and not profilin

fragments. Cross-reactivity of soybean recombinant Gly m 3 (rGly m 3) with the birch

pollen profilin allergen Bet v 2, has also been shown (Mittag et al. 2004, Rihs et al.

1999). However, soybean profilin has not been demonstrated to elicit clinical food allergy

(Panda 2012).

Gly m 4 is another important soybean allergen. It was first described as a stress-

induced protein in soybean, also known as SAM 22 (starvation associated message 22)

(Crowell et al. 1992). It is a 16 kDa protein, which is homologous to a number of other

plant proteins and has been associated with some allergies caused through cross­

reactivity. It has been reported to show a 53% sequence identity with the major birch

pollen aeroallergen, Bet v 1; 58% with the major hazelnut food allergen Cor a 1.0401;

53% with the major apple food allergen Mai d 1; and 54% with the major cherry food

allergen Pru a v (Crowell et al. 1992, Kleine-Tebbe et al. 2002). A study by Kleine-Tebbe

et al. (2002) showed that recombinant Gly m 4 (rSAM 22) caused the production of high

IgE levels in sera from patients with severe oropharyngeal and anaphylactic reactions to a

soy-containing nutritional supplement drink. A study by Mittag et al. (2004) found that

71% of patients allergic to birch pollen with high titers of Bet v 1-specific IgE were

17

sensitized to Gly m 4, an indication of cross-reactivity between Gly m 4 and Bet v 1

(Mittag et al. 2004, Panda 2012). Another study also reported that soybean-dependent

pollen-food cross-reaction in children due to ingestion of soy milk was likely due to the

presence of high concentrations of Gly m 4 in moderately processed soy milk (Kosma et

al. 2011).

Soybean albumins have also been identified as allergens but a study by Lin et al.

(2006) has indicated that the two isolated 2S albumins of soybean (AL 1 and AL 3) are

minor allergens at best, based on IgE binding of the sera of 23 soybean allergic patients

(Panda 2012)

Soybean lecithin proteins have also been implicated in soy allergies. Several cases

of allergic responses specifically induced by soybean lecithin have been reported (Fine

1991, Lavaud et al. 1994, Renaud et al. 1996). At least five IgE binding proteins have

been identified in soybean lecithin (P7, PI2, P57, P39 and the soybean Kunitz type

trypsin inhibitor (SKTI) or P20 (Xiang et al. 2008). Of these, SKTI has been reported as a

minor allergen, even though in rare circumstances, it could cause severe allergic reactions

(Burks et al. 1994, Xiang 2003). It has also been identified as an aeroallergen from

soybean flour and has been shown to be recognized by sera from 68% of bakers suffering

from workplace-related respiratory symptoms and sensitized to soybean (Baur et al.

1996, Quirce et al. 2002).

Several soybean hull allergens such as Gly m 1, a component of the 7S fraction

and Gly m 2 have also been identified as aeroallergens in soybean. These were reportedly

responsible for very common airway allergies around the shipping port in Barcelona,

Spain (Codina et al. 1999, Panda 2012).

18

2.4 Soybean Processing and Allergy Management

The utilization of soybean and its products has increased worldwide within the

last two decades. According to Hou and Chang (2004) approximately 60% of processed

foods are estimated to contain ingredients derived from soybean. They are added to foods

in forms such as unprocessed soy flour which contains approximately 40-50% protein,

soy protein concentrate (SPC) which contains approximately 70% protein and soy

protein isolate (SPI), the most refined form which is about 90% protein (Lusas and Riaz

1995, Friedman and Brandon 2001, Singh et al. 2008). Soybean proteins are used in

various baked products to provide specific nutritional and functional properties such as

improved texture, moisture, fat retention and emulsification (Panda 2012). Soy flour for

instance, may be incorporated at up to 12% with wheat flour for bread and also up to a

level of 5-20% in cookies to improve the protein quantity and quality (Riaz 1999, Singh

et al. 2008). Soybean proteins are also used in sausages, meatballs, pizza toppings, soups,

sauces, confections, imitation nut meats, coffee creamer, ice cream, low fat spreads,

chocolate products and yogurts (Lusas and Riaz 1995, Singh et al. 2008, Panda 2012).

Different methods have been employed in the processing of soybeans for the

purposes of destroying anti-nutritional factors as well as producing hypoallergenic foods.

Thermal processing such as the application of moist or dry heat and non-thermal

processing methods such as fermentation, germination, enzyme hydrolysis and ultra­

filtration have all been used in soybean processing (Thomas et al. 2007).

These processing methods may alter the allergenicity of the food product and these

changes could be due to inactivation or destruction of epitope structures, formation of

new epitopes, or improved access of previously hidden epitopes. These alterations in

19

allergenicity may, however, differ from one process to the other and from product to

product (Paschke and Besler 2002). Current knowledge of the impact of food processing

on allergen structure indicates that it is difficult to predict how different allergens respond

to various food processing treatments. Some allergens such as soy (3-conglycinin are

labile to common food processing technologies while others (glycinin) are relatively

stable. Several studies have indicated a wide variation in the allergenicity of processed

food products (Panda 2012).

In the manufacture of soy milk, for instance, the use of temperatures above 90°C

has been shown to increase the dispersion stability of soy milk protein and emulsions.

The increased stability has been attributed to the denaturation of glycinin and p-

conglycinin present in soymilk and subsequent formation of soluble aggregates due to

disulfide bonding (Shimoyamada et al. 2008, Panda 2012). The denaturation of these

major allergenic proteins may also result in a reduction in the allergenic properties of soy

milk. In a study by Franck et al. (2002), commercially available soy flour, soy milk,

hydrolyzed and unhydrolyzed infant formula milk products and texturized soy protein

products were tested for serum IgE binding using sera from nine soy allergic individuals.

The infant formula soy milk did not show IgE binding, but IgE binding was observed for

the other soy milk with all nine soy allergic sera. Texturized soy protein also showed IgE

binding, with seven out of nine sera tested (Franck et al. 2002). In an earlier study by

Burks et al. (1992), soy flour extract, purified 1 IS globulin and 7S globulin fractions of

soybean were heat treated at 37°C and 56°C for 1 hour and 100°C for 5, 20 and 60

minutes but no differences in IgE or IgG binding compared to control samples were

observed using a pool of soybean allergic patient sera (Burks et al. 1992). A study by

20

Herian et al. (1990) also showed enhanced IgE binding of roasted soybeans to a 20 kDa

band with sera from two out of six patients. A study by Panda (2012) also indicated that

the majority of thermal treatment conditions utilized in making soybean products will not

affect their allergenicity and hydrolysis of soybean proteins by different enzymes, doesI

not make them less allergenic compared to the untreated proteins and may increase their

allergenicity. These variations in the effects of processing on soy allergenicity are a clear

indication of a need for further studies. Processing conditions that may make epitopes

less available to antibody receptors would be valuable in the mitigation of food allergies

(Wilson et al. 2005). Understanding the impact of food processing and food structure on

allergenic potential, however, is crucial in managing allergen risks in the food chain.

Shibasaki et al. (1980) investigated the effect of heat processing on the

allergenicity soybean glycinin, P-conglycinin and 2S globulin at 80°C, 100°C and

autoclaved at 120°C for 30 minutes prior to testing IgE binding by radioallergosorbent

test (RAST) and RAST inhibition using five soy allergic sera. A reduction in IgE binding

to glycinin and [3 - conglycinin was observed, indicating a possible reduction in

allergenicity. A more recent study by Li et al. (2012) also showed that high hydrostatic

pressure (HHP) processing considerably reduced the allergenicity of soy protein isolate

(SPI) for infant formula. At 300 MPa and 15 minutes, the allergenicity decreased by

48.6% compared to native SPI. The group is also exploring HHP assisted enzymatic

methods to further decrease allergenicity of SPI (Li et al. 2012).

According to Wilson et al. (2008) soybean allergy, like many food allergies can

be managed by avoidance or the consumption of low allergen-content foods and medical

treatment. Avoidance is the most practiced due to the enactment of FALCPA into the

21

United States law. Avoidance is still very difficult especially when studies by Gendel

(2013) have indicated that the enactment of FALCPA has not prevented recalls due to

undeclared food allergens. In addition, the consumer has little or no control over food

ingredients used in food services such as restaurants, causing sensitive individuals to

avoid foods that might otherwise be safe for their consumption. There are also a number

of strategies for medical treatment of food allergies being developed, including new

drugs and immunotherapy. These treatments are promising, however, when or if they will

be available remains uncertain (Joseph et al. 2006). With respect to the consumption of

low allergen-content foods, there is research to eventually ensure the possibility of

producing hypoallergenic foods for sensitive individuals.

2.5 Extraction of Soy Proteins for Allergen Determination

In order to maintain safety, individuals with food allergies rely heavily on

accurate labeling and declaration of allergens in foods they consume. Accurate labeling

as well as studies on the effect of processing on allergenicity are both dependent on the

correct determination of allergenic proteins. Such analyses begin with the extraction of

these proteins. According to Panda (2012), the conditions used to extract proteins from

both raw and processed foods is an important determinant for appropriate interpretation

of the allergenicity of a given food material. The extraction step is therefore very

essential but it has also been described to be the hardest to do efficiently (Panda 2012).

Most studies simply use water-soluble extraction methods followed by antibody detection

and this has resulted in some studies reporting reduced allergenicity of various foods

when there may have been no control for possible poor extraction of allergenic proteins.

22

The inefficient extraction of proteins from processed food products can occur due to

matrix effects including reduced solubility of denatured proteins, cross-linking through

glycation and binding of hydrophobic proteins to the food matrix (Panda 2012). Tan et al.

(2 0 1 1 ) have also reported that protein yield and properties are influenced by the

extraction process and by other extraction factors such as pH, salts concentration, and the

ionic strength of the medium of extraction. According to Poms et al. (2004) it is often

necessary to optimize sample extraction in order to ensure that an analytical result

represents the true impact of a process on allergenic activity.

A wide variety of extraction and fractionation tools for proteins and peptides are

available based on their physicochemical and structural characteristics, such as solubility,

hydrophobicity, molecular weight, isoelectric point, and so on. Generally, different

technologies based on cell disruption, solubilization or precipitation, and enrichment

systems are needed to obtain the protein fraction of interest. Removal of interfering

compounds (mainly lipids, nucleic acids, phenolic compounds, carbohydrates, proteolytic

and oxidative enzymes, and pigments) is crucial and these procedures need to be

optimized to minimize protein modifications and proteolysis, as well as to be compatible

with subsequent analysis (Martinez-maqueda et al. 2013). Different extraction conditions,

processes such as ultrasound assisted extraction, microwave heating (Choi et al. 2006),

enzymatic modifications (Jung et al. 2006) and chemical modifications have been

investigated to improve protein extractability from soy substrates.

23

2.5.1 Conventional Extraction Methods

Traditionally, soy proteins and other food proteins are extracted with buffers such

as phosphate buffered saline (PBS) ( pH 7.4) or Tris/HCl buffer (pH 8.2) especially when

they are to be analyzed by immunoassays (Koppelman et al. 2004, Rudolf et al. 2012).

The extraction is usually done at 4°C or at room temperature (18-30°C) for a period of 2

to 24 hours, with or without some form of agitation (Panda 2012). These conditions are

widely used in protein extraction but they have been reported to be inefficient, especially

in the extraction of proteins that have undergone some form of modification through

processing (Panda 2012). The addition of chaotropic agents (urea or thiourea), reducing

agents (beta-mercaptoethanol) or detergents such as sodium dodecyl sulphate (SDS) to

the extraction buffers has also been explored and reported to increase the solubility and

hence improve extraction (Pedreschi et al. 2010, Schmitt et al. 2010). Other studies have,

however, explained that the effect of SDS in improving the extraction process is

dependent on the buffer in question (Rudolf et al. 2012, Panda 2012).

In a study aimed at improving peanut protein extraction using different extraction

buffers, the addition of SDS or urea to the buffers resulted in the doubling of protein

concentrations in most samples (Rudolf et al. 2012). In another study, however, reducing

and non-reducing formulations of Laemmli buffer (with or without beta-

mercaptoethanol) showed that extraction was more efficient in the absence of the

reducing agent. The addition of salts has also been explored. Rudolf et al. (2012) reported

that the addition of NaCl to low- and neutral pH buffers increased the extractability of

peanut proteins, but solubility was reduced in combination with high-pH buffers such as

carbonate and borate buffer, an indication that the effectiveness of these additives may be

24

buffer-dependent. A study by Poms et al. (2004) also demonstrated that the extraction

efficiency of allergenic proteins varied with the use of different extraction buffers.

The disadvantages of conventional methods and the limitations in the use of

additives with extraction buffers have resulted in the exploration of other methods to

improve the extraction of proteins. Researchers have and are still investigating extraction

at temperatures higher than the conventional ambient and 4°C, the use of harsh extraction

buffers, microwave and ultrasound-assisted extractions.

2.5.2 High Temperature Extraction with other Buffers

Most of the in-vitro methods utilized to assess the presence of food allergens are

based on the assumption that most or all of the proteins to be detected are water-soluble

and extracted into aqueous buffer. Heat treatment of foods containing allergens has,

however, been said to cause denaturation and unfolding of proteins leading to surface

exposure of hydrophobic and sulfhydryl groups located in the interior of the molecule.

This can result in irreversible protein aggregation and formation of covalent complexes

resulting in decreased protein solubility and extractability (Renkema et al. 2000, Li et al.

2012, Panda 2012). According to Schmitt et al. (2010), inter and intra molecular covalent

cross- linking among proteins can also occur during heat processing due to chemical

modification of proteins by the Maillard reaction, resulting in further reduction in

solubility. Thermally-induced unfolding and aggregation of P-conglycinin and glycinin

has been shown to occur at temperatures above 75°C and 60°C, respectively (Mills et al.

2001, Mills et al. 2003). It is therefore possible that most of the reduction of IgE binding

25

previously reported following heat treatment of soybean ingredients is simply due to

protein aggregation and insolubility. Soybean products that are heat- treated may thus not

have easily extractable and detectable proteins and would still contain insoluble allergens

that could cause allergic reactions upon ingestion by sensitive individuals (Panda 2012).

Many investigators have reported that thermal processing has become an important factor

in allergen quantitation, affecting detection of allergenic food residues at low levels

(Mills et al. 2009, Nowak-Wegrzyn and Fiocchi 2009, Scaravelli et al. 2009). Another

concern is the possible generation of novel allergens due to thermal processing of foods

(Thomas et al. 2007).

Despite these setbacks, the use of high temperatures in protein extraction has been

investigated and reported to improve the extraction of allergenic proteins. While some

investigators have used the disadvantages mentioned earlier as a basis to disagree with

the exploration of extraction at high temperatures, others have justified this with the

explanation that the use of other buffers (and pH) instead of the conventional PBS (pH

7.4 with low salt concentration) which has been described as ‘very mild' by Panda (2012)

with elevated temperature may increase the solubility of modified proteins (Poms et al.

2004, Albillos et al. 2011, Panda 2012).

In a study by Poms et al. (2004) to extract proteins (allergens) from dry and oil

roasted of peanuts, they employed the use of a variety of buffers at different pH levels

and performed the extraction at 4°C overnight and 60°C for 20 minutes. Their results

showed that for peanut protein extraction efficiency, the most significant factor appeared

to be the pH of the selected extraction buffer. According to their study, the best yields

were obtained with buffers in the range of pH 8-11 with urea buffer (pH 8.7) showing

26

relatively high yields for processed peanut protein. Elevated temperatures (60°C for 20

min) in this case however, did not increase the yield of extractable protein compared to

extraction at 4°C overnight, contrary to expectation (Poms et al. 2004). This study,

however, did demonstrate that the extraction efficiency of the allergenic proteins varied

with the use of different extraction buffers.

In another study by Albillos et al. (2011), the extraction of proteins from almonds

was optimized using different buffers and elevated temperatures. Almonds were roasted

at 230, 260 and 400 °C for 10 minutes, ground to meals, defatted and extracted with

water, PBS and PBST (PBS with tween 20) for 30 minutes at 25, 40, 60 and 70 °C. In this

study, the temperature of the extraction buffer was shown to be important, with higher

protein yields at 60 and 70 °C. Use of a higher temperature in combination with

sonication for 5-10 minutes, increased the amount of protein extracted with PBST

(Albillos et al. 2011). The presence of the detergent, tween 20, in the PBS may have also

played an important role in improving the solubility of the almond protein. Schmitt et al.

(2 0 1 0 ) reported that the use of detergents and reducing agents maximize protein

extraction from insoluble peanut pellets.

In a more recent study by Panda (2012), soybean samples heat treated under

different conditions were evaluated for IgE binding by extracting the proteins using a

variety of extraction buffers for testing by immunoblotting to maximize protein

solubilization for a more complete evaluation of potential allergenicity compared to

extraction in regular PBS. Results from this study showed that Laemmli buffer (described

as a ‘harsh’ buffer) had higher overall protein solubilization, especially in heat processed

samples. It was also observed that some proteins were extracted more efficiently by

27

NaCl- containing buffer especially proteins from unheated soybean samples. This study

showed that the differential solubility of soybean proteins in the different extraction

buffers resulted in varying IgE binding patterns. The study concluded that it is essential

to evaluate proteins extracted with different extraction buffers and not just a single buffer

while making any interpretation about allergenicity. This is because the choice of

extraction buffer could affect interpretation of results from in vitro assays that utilize

soluble antigens (Panda 2012).

2.5.3 Ultrasound-Assisted Extraction

Ultrasound-Assisted Extraction (UAE) is a method of extraction that has been

reported to accelerate heat and mass transfer. Ultrasounds have frequencies of 20 kHz to

10 MHz. This frequency is above human hearing but below microwaves frequencies. As

a sound wave passes through an elastic medium, it induces a longitudinal displacement of

particles. The source of the sound wave acts as a piston on the surface of the medium

(Mason 1990). Ultrasound waves, after interaction with subjected plant material, alter its

physical and chemical properties and their cavitational effect facilitates the release of

extractable compounds and enhances the mass transport by disrupting the plant cell walls.

UAE is a clean method that avoids the use of large quantities of solvent and reduces

extraction time (Lopez-Avila et al. 1996, Luque-Garci 'a and De Castro, 2003). UAE has

been utilized in the extraction of natural products that typically needed hours or days to

reach completion with conventional methods. Using ultrasound, full extractions can be

completed in minutes with high reproducibility. Several classes of food components such

as aromas, pigments, antioxidants, and other organic and mineral compounds have been

28

extracted and analyzed efficiently from animal tissues, food and plant materials (Khanal

et al. 2007, Karki et al. 2010). Ultrasound has been used to enhance oil extraction from

soybeans (Li et al. 2004); active xylan and heteroxylan extraction from corn cobs and

corn hulls (Ebringerova et al. 1998), and glucose release from corn slurry from dry-grind

ethanol plants (Khanal et al. 2007).

For the extraction of proteins, ultrasonic extraction has proven efficient in

increasing protein homogenization and solubility in soybean meal (Wang 1978; Karki et

al. 2010). Ultrasound induces cavitation, which causes proteins to undergo physical

disruption and/or chemical transformation. It heats by specific absorption of acoustic

energy, dynamic agitation and shear stresses and turbulence (Floros and Liang 1994). A

study by Jambrak et al. (2009) reported that the solubility of soy proteins increased after

ultrasound treatment and this was attributed to unfolding and to the breaking of peptide

bonds by hydrolysis. Karki et al. (2010) also conducted a study that focused on the use of

high-power ultrasound prior to soy protein extraction to simultaneously enhance protein

and sugar release in the extract. Defatted soy flakes dispersed in water were sonicated for

durations of 15, 30, 60 and 120 seconds. The particle size of the soy flakes decreased

nearly 10-fold following ultrasonic treatment at high amplitudes (84 pmpp) resulting in a

high increase in total sugar released (50%) and protein yield (46%) after 120 seconds of

sonication when compared with non-sonicated samples (control).

In another study by Albillos et al. (2011) the extraction of proteins from almonds was

optimized using different buffers and elevated temperatures after which the extracts were

sonicated in a temperature-controlled water bath and aliquots were taken after 0, 1, 3, 5

and 10 minutes of sonication. They reported that ultrasonic treatment improved protein

29

extraction from almonds, especially those roasted at 260 and 400°C. These studies show

that there is great potential in the use of UAE for improving the extraction of proteins for

the determination of allergens in foods such as soy.

2.5.4 Microwave-Assisted Extraction

Microwave is a form of electromagnetic energy and is generally regarded as

occupying the frequency from 300MHz to 300GHz, with corresponding wavelengths

ranging from 1mm to 1m (Gupta and Wong, 2007). On the electromagnetic spectrum,

microwaves region lies between the infrared and radio waves region. Unlike X-rays and

gamma rays, microwaves are nonionizing radiations and do not break chemical bonds or

cause molecular changes in a compound by removal of electrons (Gupta and Wong,

2007).

Microwave assisted extraction (MAE) was first reported by Ganzler et al. (1986).

They reported applications using microwave radiation to enhance extraction of organic

compounds from solid matrices such as soils, seeds, food, and feeds as a novel sample

preparation method for chromatography analysis (Ganzler et al. 1986, Hu 2011). It has

since attracted growing interest in scientific research due to the efficient use of

microwave energy. Application of microwave in some heating processes have been

reported to have advantageous thermal effects such as quick heating, thawing, selective

energy dissipation, same-direction heat and mass transfer (Rosenberg and Bogl 1987,

Choi et al. 2006). The thermal effects of microwave energy result in the disruption of

biological tissues, making microwave energy very important in the efficient extraction of

useful components in plant tissues (Choi et al. 2006). According to Camel (2000), MAE

30

allows for rapid extraction of biological components, with extraction efficiency similar or

superior to that of conventional techniques. In MAE, heating occurs in a targeted and

selective way with practically no heat being lost to the environment. The apparatus can

thus significantly diminish the extraction time (Huie 2002) and reduce solvent

consumption (Jain et al. 2009). When using MAE, factors such as solvent type and

volume, microwave power, extraction time, temperature and matrix characteristics must

be taken into consideration to ensure efficient extraction (Mandal et al. 2007, Chan et al.

2011, Salomon et al. 2014).

MAE has been used in many studies and for the extraction of compounds from

various biological systems. Onuska and Terry (1993) used microwave to extract

organochlorine pesticides from sediment samples. They reported nearly 100% recovery

for most compounds and no degradation as a result of exposure to microwave energy

(Onuska and Terry 1993). In a study by Pan and Tangratanavalee (2003), it was also

reported that MAE improved the efficiency of the extraction of polyphenols and caffeine

from green tea leaves. In another study by Hu (2011), MAE was used for the extraction

of saikosaponins from Radix Bupleuri. This study showed that for the extraction of

saikosaponins a, c, and d using MAE, power level of 300 to 500 W and extraction time of

5 min at 75 °C were favorable extraction conditions. The study also showed that

compared with conventional extraction methods, MAE can significantly reduce the

extraction time, resulting in better extraction efficiency (Hu 2011). In a very recent study

by Salomon et al. (2014), mangiferin, a bioactive metabolite having potent antioxidant

and pharmacological properties was extracted from Mangifera indica (mango) leaves

using MAE. With a microwave power of 900W, a maximum mangiferin yield of 63.22%

31

was obtained. This yield was not statistically different from that achieved using

conventional methods but MAE extraction was achieved within 5 minutes, which was

reported to be shorter. The study therefore concluded that MAE provides a very good and

consistent extraction method of a bioactive metabolite from natural plants (Salomon et al.

2014).

Microwave energy and MAE have also been used on soy products. In a study by

Ashida et al. (1998), microwave heating of a soya slurry was applied for the preparation

of soya milk and tofu. The study reported that a higher protein concentration in soya

milk was obtained by this method than by conventional methods of heating such as the

use of boiling water. The study also reported that microwave oven heating of soya slurry

was effective for protein extraction (Ashida et al. 1998). Choi et al. (2006) also compared

the extraction of soluble protein from soybeans using MAE and a conventional shaking

water bath extraction system. The study reported that the yield of soluble protein

increased with time within a range of 30 minutes. It also reported that scanning electron

microscopy showed the destruction of the microstructure of soybean cells, which

increased the extraction of soluble soy protein (Choi et al. 2006). Based on these positive

results, the use of MAE could be further investigated in improving the extraction of soy

proteins for soy allergen detection.

2.6 Soy Allergen Detection

The safest way for individuals with food allergies to remain healthy is to

completely avoid the foods in question. This will only be possible if products containing

these allergy-causing ingredients are accurately labeled. It is therefore critical that

32

efficient methods of soy allergen detection are available to ensure accurate labeling and

hence, ensuring the safety of individuals with soy allergies. The detection of allergens in

foods is, however, very challenging because food allergens are typically present in trace

amounts and are mostly masked by the food matrix (Poms et al. 2004).

Many different methods have been developed for the detection of soy and other

allergenic proteins. These methods are mostly antibody or DNA-based and are used to

detect proteins extracted from the sample. Other detection methods involve the detection

of components besides proteins and are useful when considering the insolubility of

proteins (Koppelman and Hefle 2006). These methods are discussed in detail below.

2.6.1 Antibody-Based Tests

Antibody-based tests are the most commonly used detection method for soy

proteins. Enzyme-linked immunosorbent assay (ELISA) is the commonly used detection

method in this category and many ELISA kits specific for soy allergens have been

developed (Brandon et al. 2004, Koppelman et al. 2004, Pedersen et al. 2008, Sakai et al.

2010). It is a powerful analysis tool used for the detection of total or specific soybean

proteins (Koppelman and Hefle 2006). Antibodies are raised against native soybean

proteins, one single protein such as Gly m Bd 30 K (P34), ß- conglycinin, glycinin,

Kunitz Trypsin Inhibitor, or denatured/renatured soybean proteins (Cucu et al. 2012).

Ravestein and Driedonks (1986) raised antibodies against SDS-denatured acidic

polypeptide of glycinin. They constructed an ELISA with a detection limit of 0.5% (5000

ppm) with these antibodies which were successfully used in an immunoblotting

experiment. Plumb et al. (1994) also raised antibodies against native glycinin. They

33

reported that heating glycinin at pH 7.6 caused its immunoreactivity to decrease by about

50%. The immunoreactivity of the glycinin, however, increased when heated above 92°C

at pH 7.6 (Plumb et al. 1994, Koppelman and Hefle 2006). Plumb et al. (1995) also

raised antibodies against P- conglycinin and reported that the immunoreactivity of this

protein was increased with heat, reaching a maximum at the denaturation temperature of

65°C.

Several other methods have raised antibodies against the whole soy protein

fraction. Hitchcock et al. (1981) raised antibodies against soy protein which had been

renatured from hot urea solution by removing or diluting the denaturant. ELISA

developed from this method was able to recognize heat-treated and native soy protein

fractions. Based on the method of Hitchcock et al. (1981), commercial soy protein assay

kits were developed (Hitchcock et al. 1981; Koppelman and Hefle 2006). There are now

many commercially available ELISA kits to detect soy allergens based on various

studies. ELISA kits are manufactured by companies like Tepnel, Elisa Sytems, Congen,

Neogen and Romer laboratories (Koppelman and Hefle 2006). These kits have been

useful in the rapid detection of allergens in the food industry but they all have some

limitations.

Platteau et al. (2011) have reported that the detection of soybean proteins by

commercial ELISA kits is severely affected by food processing methods. Cucu et al.

(2012) compared ELIS As using antibodies against modified soybean proteins and against

soybean KTI. The applicability of these ELISAs was evaluated to detect the presence of

soybean proteins in cookies. They reported that both matrix interferences and the baking

process affected analytical recovery even though the recoveries were found to be much

34

higher using soybean-ELISA than KTI-ELISA. The low recovery obtained using the

KTI-ELISA was attributed to the inability of the antibodies used to recognize the

modified proteins. They thus concluded that using antibodies developed towards

allergens modified through food processing is a better approach to be used in food

allergen detection (Cucu et al. 2012). In a more recent study, Gomaa and Boye (2013)

investigated the effects of baking time, temperature profile and cookie dimensions and

weight on the detection of four allergens including soy simultaneously spiked in a non­

wheat flour cookie using ELISA. They reported that allergen recovery generally

decreased as baking time increased and cookie size was decreased and that no recoveries

were obtained for soy in some of the thermally processed samples. Other detection

methods have therefore been explored even though ELISA still remains the most widely

used.

2.6.2 Other Soy Detection Methods

Due to the fact that ELISAs rely on the extraction of soluble proteins, a method

that does not require soluble proteins has been developed for use in the detection of soy

proteins. This is referred to as the indirect protein analysis and it is based on non-protein

markers associated with soy. Examples of markers that have been used for assay

development include insoluble polysaccharides, oligosaccharides, protein-bound sugars,

free amino acids, free peptides, phytate, saponins, sterols, and metals. Microscopic

examinations of calcium oxalate crystals and plant cell components have also been

reported to indicate the presence of soy (Hitchcock et al. 1981). The detection level of

this method is, however, too high for quantification of traces of soy.

35

The use of Polymerase Chain Reaction (PCR) and soy DNA has been investigated

as another soy allergen detection method. In a PCR test, DNA is extracted from the food

matrix and subsequently tested for a specific DNA sequence. For the detection of soy, the

lectin gene is usually investigated (Gatti and Ferretti, 2010). Work by Espineira et al.

(2010) described the development and validation of two PCR methods, end-point and

real-time PCR, for the detection of soy protein in a wide range of foodstuffs. They

described these techniques as reliable and sensitive, allowing detection of trace amounts

of soybean in processed products. In the study, 35 meat, fish and bakery processed

products, which could potentially contain soy but was not declared on the label, were

tested for the presence of soy DNA using the proposed PCR methods. The study

concluded that the methodologies will be valuable in issues regarding the presence of soy

protein in processed products, especially in verifying labeling and security regulations to

protect consumers’ rights (Espineira et al. 2010).

Electrophoresis, which has the advantage of being easy to perform, has also been

used for analyzing soy proteins (Koppelman et al. 2004). It is a separation technique

based on the movement of charged protein molecules within a fluid medium under the

influence of an electric field. Protein molecules are separated based on their molecular

weight. The molecular weights of many soy allergenic proteins have been determined

through the use of electrophoresis.

A homogeneous aggregation immunoassay involving the use of gold

nanoparticles (AuNPs) and light scattering detection has also been described by Sanchez-

Martinez et al. (2009) for soy protein determination in food samples. According to the

study, AuNPs act as enhancers of the precipitate that appears when the antigen-antibody

36

complex is formed. The AuNPs-antibody conjugate was synthesized by physical

adsorption of polyclonal anti-soy protein antibodies onto the surface of commercial

AuNPs with a nominal diameter of 20 nm. The direct assay is based on the reaction of the

conjugate with soy protein, which reaches the equilibrium in about 10 minutes, and the

measurement of the light scattering intensity at 530 nm, which is proportional to the

analyte concentration. The results obtained with this method were reported to be similar

to those obtained using a commercial ELISA kit, but the assay time was significantly

shorter and the detection limit was about 10 times lower (Sanchez-Martinez et al. 2009).

The use of Fourier transform infrared (FTIR) and X-ray diffraction spectroscopy

using aqueous and AOT reverse micelle extracted soy proteins has also been used to

determine the conformation of the 7S and 1 IS globulins. This method could be further

explored in the detection of soy allergens (Chen et al. 2011).

Irrespective of the method of detection being used, it is important that where a

protein extraction step is involved, it is done efficiently, thus the aim of this study to

improve the extraction of allergenic proteins from soy products.

37

CHAPTER 3: MATERIALS AND METHODS

OBJECTIVE 1: IMPROVING THE EXTRACTION OF ALLERGENIC PROTEINS

FROM SOY PRODUCTS USING DIFFERENT BUFFERS WITH SELECTED

THERMAL AND NON- THERMAL EXTRACTION CONDITIONS

3.1 Experimental Design

This study consisted of four sets of extraction treatments each with two replicates.

The extraction treatments consisted of conventional extraction (control method), water

bath extraction, microwave-assisted extraction (MAE) and ultrasound-assisted extraction

(UAE). Each extraction treatment was performed on three soy matrices (soy flour, soy

protein isolate and soy milk) using three extraction buffers (phosphate buffered saline,

Laemmli and urea buffer). The water bath and MAE had a 3x3x3 full factorial design

while UAE had a 2x3x3 full factorial design for each soy matrix. The treatment variables

for each of the extraction treatments are outlined in table 3.1.

Table 3.1. Experimental design of extraction treatments for soy matricesE xtraction m ethod E xtraction tem perature E xtraction tim e(s) E xtraction

buffer

Conventional room temperature (23 °C) 2 hours PBS, Laemmli, urea

Water bath 60, 70, 100°C 5, 15, 30 minutes PBS, Laemmli, urea

Microwave-assisted 60, 70, 100°C 0, 5, 10 minutes PBS, Laemmli, urea

Ultrasound-assisted 4 °C, room temperature(23 °C)

1,5, 10 minutes PBS, Laemmli, urea

38

3.1.1 Sample Preparation

The soy matrices used in this study were soy flour (SF), soy protein isolate (SPI)

and soy milk (SM) with protein contents of 35.3, 87.0, and 3.6%, respectively. Protein

content of each sample was determined by nitrogen combustion analysis at 1050°C using

the LECO FP-2000 Protein Analyzer. Approximately 99.5% pure

ethylenediaminetetraacetic acid or EDTA (Sigma-Aldrich, St. Louis, MO) was used as

the nitrogen standard, and a conversion factor of 6.25 was used to determine crude

protein content of the soy matrices.

All soy matrices were obtained from a local organic grocery store. SPI and soy

milk were used in the form they were obtained but for soy flour, dried organic soybeans

were obtained, ground and passed through a 60-mesh sieve. All soy samples were

defatted using 1:3 sample to solvent ratio with hexane (Fisher Scientific Pittsburgh, PA)

as the solvent at room temperature. Each soy sample was thoroughly mixed with the

hexane using an orbital shaker (DS-500, VWR, Scientific Chicago, IL) at 70 rpm for 1

hour in a fume chamber. Hexane was decanted and the process repeated two more times.

Defatted samples were then dried in the fume chamber overnight before use.

Extraction buffers used were 0.01M phosphate buffered saline (PBS) at pH 7.4,

2X Laemmli at pH 6.8 and 6M urea at pH 8.7 (Fisher Scientific Pittsburgh, PA). A 1L

preparation of 0.01M PBS contained 137 mM sodium chloride (NaCl), 2.7 mM

potassium chloride (KC1), lOmM anhydrous sodium phosphate dibasic (Na2HP04), 1.8

mM potassium phosphate monobasic (KH2PO4). A 1L preparation of Laemmli buffer

was composed of 100 mM Tris HC1, 4% SDS and 10% glycerol. All buffer components

39

were obtained from Fisher Scientific, Pittsburgh, PA. The buffers used were selected

based on literature and some preliminary studies.

3.1.2 Water Bath Extraction

Extraction was carried out with the selected buffers using a shaking water bath

(Model SW22, Julabo U.S.A., Inc., Allentown, Pa., U.S.A.). One gram of each sample

was weighed into 50 mL centrifuge tubes and 19 ml of each buffer (PBS-7.4, Laemmli-

6.8 and urea-8.7) was added. The mixture was well mixed by gently shaking the tube to

prepare a uniform dispersion. The tubes were then placed securely in the water bath. The

water bath was set at 60, 70 and 100°C and to shake at a constant speed of 200 rpm for 5,

15 and 30 minutes. Extraction was performed in two replications (n=2) for each soy

matrix and for each water bath extraction treatment. After the extraction process at each

temperature for the required period of time, samples were cooled and centrifuged at 7800

rpm for 20 minutes at room temperature. The supernatants (protein extracts) obtained

after centrifugation were then collected and stored at -20°C for soy protein analyses.

3.1.3 Microwave-Assisted Extraction (MAE)

MAE was achieved with a microwave accelerated reaction system (Model:

MARS 5, CEM Corporation, Matthews, NC, USA) at a standard frequency of 2450 MHz.

The instrument is equipped with a carousel of 14 teflon vessels in a closed-system.

Temperature was controlled by a sensor inserted in a control vessel and extraction was

performed at 600W. One gram of each sample was weighed into the Teflon reaction

vessel and 19 mL of buffer (PBS-7.4, Laemmli-6.8 and urea-8.7) added. The mixture was

well mixed to prepare a uniform dispersion. The vessel was then placed in the carousel.

40

Extraction of soy proteins was carried out at temperatures of 60, 70 and 100°C and

holding times of 0, 5 and 10 minutes. Come-up times were also monitored and excluded

from the holding time. Extraction was performed in two replications (n=2) for each soy

matrix and for each MAE treatment. After the extraction process, vessels were cooled.

Cooled samples were transferred into 50 mL centrifuge tubes and centrifuged at 7800

rpm at room temperature for 20 minutes. The supernatants (protein extracts) obtained

after centrifugation were then collected and stored at -20°C for soy protein analyses.

3.1,4 Ultrasound-Assisted Extraction (UAE)

The use of UAE in soy protein extraction was investigated using low temperatures

(room temperature and 4°C) or non- thermal conditions in order to minimize the effect of

heat on soy proteins. UAE has been used to achieve efficient extraction at low

temperatures. Extraction was carried out with an ultrasonic dismembrator (Model 300,

60Hz, 300W, Artek System Corporation) equipped with a 3/8 inch intermediate probe.

One gram of each sample was weighed into a 50 ml beaker and 19 mL of buffer (PBS-

7.4, Laemmli-6.8 and urea-8.7) were added. The sample was well mixed by gently

stirring the mixture with a glass stirrer to prepare a uniform dispersion. The probe was

then inserted about one inch into the mixture. Extraction of soy protein was performed at

room temperature (23°C) and 4°C for 1, 5 and 10 minutes. Extraction was performed in

two replications (n=2) for each soy matrix and for each UAE treatment. The UAE treated

samples obtained were transferred into 50 mL centrifuge tubes and centrifuged at 7800

rpm at room temperature for 20 minutes. The supernatants (protein extracts) obtained

after centrifugation were then collected and stored at -20°C for soy protein analyses.

41

3.1.5 Control samples

Control samples were prepared with each of the three buffers in the same way as

described above, but extraction was achieved by allowing samples to settle without

agitation (conventional extraction) at room temperature for 2 hours.

3.1.6 Determination of total protein

Total soluble proteins in the extracts obtained from the soy matrices under each of

the extraction processes described above was quantified following the procedure of Smith

et al. (1985) using bicinchoninic acid (BCA) assay. A commercial BCA assay kit was

purchased from Pierce Biotechnology Inc., (Rockford, IL) and used for total protein

quantitation according to manufacturer’s instructions. Bovine serum albumin (BSA),

provided with the kit, was used as the protein standard. Soy flour and SPI samples were

diluted 16-fold while soymilk samples were diluted 2-fold. Each of the replicates

obtained for each of the extraction treatments were analyzed in triplicate (n=6 ) and mean

values reported. Extracts of soy samples from each extraction treatment that recorded

significantly high total soluble proteins were selected in addition to all extracts obtained

from conventional extraction for electrophoresis and enzyme-linked immunosorbent

assay (ELISA) analyses.

3.1.7 Electrophoresis

The protein profiles of control and treated soybean extracts were determined by

separating the proteins by SDS-PAGE using The Mini-PROTEAN® Tetra cell (BioRad,

Hercules, CA, USA, Cat # 165-8004). Samples were run under reducing conditions by

diluting the high concentration extracts to a protein content of 7.5pg per 15 pi in 2X

42

Laemmli SDS sample buffer (BioRad, Hercules, CA, USA, Cat # 161-0737) containing

the reducing agent 5% 2-mercaptoethanol (Sigma, St Louis, MO, USA, Cat # M3148).

Samples were heated at 100°C for 5 min after dilution in the 2X Laemmli and 5% 2-

mercaptoethanol mixture.

Samples were also run under non-reducing conditions by diluting to a protein

content of 7.5pg per 15 pi in 2X Laemmli SDS sample buffer (BioRad, Hercules, CA,

USA, Cat # 161-0737) without mercaptoethanol and without heating prior to loading in

the gel. Samples were loaded in wells of a 12% tris glycine gel (Bio-Rad, Hercules, CA,

USA, Cat #456-1046). Protein sizes were estimated based on migration of protein

standards with predetermined molecular weights from 10 to 250 kDa. A 10 pi sample of

Precision Plus protein standards (BioRad, Hercules, CA, USA, Cat # 161-0374) was

loaded in the same gel. Electrophoresis was carried out at a constant 170 V for

approximately 1 hour.

The proteins separated in the gels were fixed in a solution of 7% acetic acid and 40%

methanol in water and then stained with GelCode Blue stain reagent (Pierce

Biotechnology Inc., Rockford, IL, Cat # 24592) for at least 1 hour. After staining, the

gels were de-stained with distilled water for at least 1 hour until the background was clear

of blue dye.

3.1.8 Sandwich Enzyme-linked immunosorbent assay (ELISA)

The quantification of soy protein in the extracts was determined with Neogen's

Veratox for Soy Allergen (Neogen Corporation, Lansing, MI, Cat #8410) following

manufacturer’s instructions. Five milliliters of each of the sample extracts was transferred

into a 250 mL extraction bottle and one level scoop of extraction additive, provided with

43

the kit, added. To this mixture, 125 mL of extraction solution (60°C, lOmM PBS) was

added. Further extraction was carried out in a water bath set at 60°C at a shaking speed of

150 rpm for 15 minutes. The new extracts were centrifuged and allowed to cool to room

temperature. 100 pL of controls (containing 0, 2.5, 5, 10, and 25 ppm soy) provided with

the kit and sample extracts (appropriately diluted) were transferred into antibody-coated

wells, thoroughly mixed for 20 seconds and incubated for 10 minutes at room

temperature. Contents were emptied after incubation and washed 5 times with the wash

buffer provided with the kit. After thoroughly removing all wash buffer, 100 pL of

conjugate was transferred into the wells, thoroughly mixed for 20 seconds and incubated

for 10 minutes at room temperature. Wells were then washed again. Substrate solution

(lOOpL) was transferred into the wells, thoroughly mixed for 20 seconds and incubated

for 10 minutes at room temperature for color development. Color development was

stopped by adding 100 pL of Red Stop solution and thoroughly mixed for 20 seconds.

Absorbance of the samples was obtained at a wavelength of 650 nm in a plate reader.

Results were interpreted using Neogen's log/ Logit software. The final results are usually

reported as ppm soy proteins but this has been converted to mg/mL soy proteins present

100 pL of extract in this study for easy presentation of results.

3.1.9 Statistical analysis

Results obtained for total proteins and ELISA were statistically analyzed by

analysis of variance (ANOVA) with the Statistical Package for Social Scientists (SPSS)

for Windows, version 19.0 (SPSS Inc., Chicago, IL). Differences between means were

determined by Tukey’s multiple comparison test procedure at 5% significance level

(p< 0.05).

44

OBJECTIVE 2: DETERMINATION OF THE EFFICIENCY OF EXTRACTION

METHODS ON THE EXTRACTION OF ALLERGENIC SOY PROTEINS FROM

FOOD MATRICES.

3.2 Experimental design

For this study, soluble proteins were extracted from five commercial samples and 10 in-

house prepared samples using the extraction treatment from study (objective) 2 that was

most compatible with ELISA analysis. The treatment variables for each of the selected

extraction treatments are outlined in table 3.2.

Table 3.2. Experimental design of extraction treatments for food matrices

E xtraction m ethod E xtraction tem perature E xtraction tim e(s) E xtractionbuffer

Conventional room temperature (23 °C) 2 hours PBS, LaemmliUltrasound-assisted room temperature (23 °C) 10 minutes Laemmli

The in-house prepared samples (dry flour mix and model cookies) consisted of

five treatments with two replicate batches. The treatments consisted of the control (dry

flour mix or cookie with no soy proteins) and dry flour mix samples spiked with 1,5, 10

and 20 mg of soy proteins per 450 g dry flour mix.

3.2.1 Materials

Five commercial food products, baked cheese crackers, salsa, pancake mix,

gluten-free table crackers and veggie burgers were purchased from a local grocery store.

These food samples were selected based on the fact that they were labeled with “may

45

contain traces of soy” (baked cheese crackers and salsa), “may contain soy” (pancake

mix) or “contains soy” (gluten-free table crackers and veggie burgers).

Dry flour mix and model cookies were also prepared in-house using the

experimental design above. The dry flour mix was prepared using a non-wheat gluten

composite flour of buckwheat flour (180 g), rice flour (90 g), sorghum flour (90 g), and

tapioca starch (90 g). The composite flour was mixed with sugar (180 g), salt (1.3 g),

sodium bicarbonate (2.23 g), baking soda (1.8 g) and different quantities of soy flour.

Other ingredients used were sunflower oil and water. All ingredients were obtained from

a local organic grocery store.

3.2.2 Preparation of Cookies

Soy-free dry flour mix was prepared in-house according to the method by Gomaa

and Boye (2013) with slight modifications. All the materials mentioned above, with the

exception of the soy flour, were thoroughly mixed together using an electric mixer (Stand

mixer, KitchenAid Appliances, St. Joseph, MI USA). Samples of the dry mix were then

collected for further analyses. The rest of the dry mix was divided into four parts, each

weighing 450 g. Soy flour was added to each dry mix and thoroughly mixed for

approximately 2 hours to obtain the spiked dry mix containing 1,5, 10 and 20 mg of soy

proteins per 450 g of flour mix. This was performed in two replications. Samples of each

spiked dry mix were taken for further analyses.

Soy allergen-free and cookies spiked with soy proteins were prepared from the

soy-free dry flour mix and the spiked dry mix by adding 300 g of the flour mix to 60 g

sunflower oil and lOOg water to obtain a dough. Cookies weighing 43 g and measuring

46

76 mm in diameter were prepared from the dough and baked for 15 minutes in an oven

preheated to 177°C (350.6°F). The baked cookies were cooled to room temperature and

subsequently ground into fine powders for further analyses.

Soluble proteins were extracted from the soy-free flour mix and cookies in

addition to spiked flour mix and cookies using the extraction treatments described in the

experimental design (Conventional extraction with PBS and Laemmli buffer and UAE

with Laemmli buffer). After the extraction process, samples were transferred into 50 mL

centrifuge tubes and centrifuged at 7800 rpm at room temperature for 20 minutes. The

supernatants (protein extracts) obtained after centrifugation were then collected and

analyzed for total proteins by BCA, protein profile using SDS-PAGE and soy

allergenicity using ELISA as described in objective 1. Statistical analysis was also done

as described in objective 1.

47

CHAPTER 4: RESULTS AND DISCUSSION

OBJECTIVE 1: IMPROVING THE EXTRACTION OF ALLERGENIC PROTEINS

FROM SOY PRODUCTS USING DIFFERENT BUFFERS WITH SELECTED

THERMAL AND NON- THERMAL EXTRACTION CONDITIONS

4.1 Effect of water bath extraction time at different temperatures on recovery of

total soy proteins

Results on the effect of extraction time at 60, 70 and 100°C using water bath

extraction (thermal) are presented in Figure 4.1a-c. There was, overall, a slight increase

in total protein concentration with time at each of the three temperatures for all the soy

matrices. This increase in protein concentration with time was, however, not statistically

significant (p > 0.05) for each extraction temperature and for all extracts with the

exception of SPI extracted with urea at 100°C for 15 and 30 minutes. The protein

concentrations of these extracts were significantly higher (p < 0.05) than the other SPI

extracts at 60 and 70°C.

Various investigators have used different extraction times for the extraction of

proteins from numerous food matrices using water bath extraction and this served as a

guide in the selection of times used in this study. In a study by Poms et al. (2004),

extraction of proteins from roasted peanuts for allergen detection was performed with

different buffers including PBS and urea either at 60°C for 20 minutes or at ambient

temperature or 4°C overnight.

48

Figure 4.1: Quantity of protein obtained from soy flour (a), SPI (b) and soy milk (c) extracts at different extraction times (5, 15 and 30 minutes) and temperatures (60, 70 and 100°C) using a water bath. Each value is the mean ± standard deviation (n = 6). All treatments were analyzed by ANOVA. Significant differences were analyzed by Tukey’s HSD post-hoc test.

49

Gomaa et al. (2012) also obtained protein extracts from casein, egg powder and soy

protein concentrates for allergen detection by mixing each sample with PBS (pH 7.4) and

shaking the mixture for 15 minutes in a stirring water bath at 60°C.

In this study, 15 minutes of extraction was found to be most suitable at the

temperatures used for all three soy matrices (Figure 4.1a-c). It was long enough to ensure

adequate extraction with minimal alteration of the integrity of the proteins. Based on this

an extraction time of 15 minutes was selected for further comparison at the different

temperatures and among the different buffers for soy flour, soy milk and soy protein

isolate.

4.1.1 Comparison of conventional and water bath extraction methods on protein

recovery

Figure 4.2a-c show the quantity of total proteins recovered from the soy matrices

using conventional extraction at room temperature for 2 hours and water bath extraction

at 60, 70 and 100°C for 15 minutes.

Under conventional extraction, the soy matrices displayed similar trends in the

concentration of proteins recovered in terms of the extraction buffer used. Laemmli and

urea buffers had extracts with significantly higher protein concentration (p< 0.05)

compared to PBS. For Laemmli buffer under conventional extraction, protein

concentrations of 15.07, 17.25 and 1.00 mg/mL were obtained from soy flour, SPI and

soy milk, respectively. These concentrations correspond to approximately 77, 40 and

56%, respectively, of the total protein contents of 1 gram of the products.

50

20

PBS-7.4Laemmli-6.8Urea-8.7

Conventional

bX e

£

WBE 60”C WBE 70°C

Extraction Temperature

e-J-

iHWBE 100°C PBS-7.4

53 Laemmli-6.8 ES Urea-8.7

c Extraction TemperatureLaemmli-6.8Urea-8.7

Figure 4.2: Protein concentrations of soy flour (a), SPI (b) and soy milk (c) extracts obtained from conventional extraction at 23°C and water bath extraction at 60, 70 and 100°C and 15 minutes extraction time. Each value is the mean ± standard deviation (n = 6 ). Bars for treatments not followed by the same letter are significantly different (p < 0.05). All treatments were analyzed by ANOVA (Tukey’s HSD post-hoc).

51

The protein recovery from urea buffer under the same conditions was significantly lower

(p< 0.05) except in SPI, where a protein concentration of 20.33 mg/ml corresponding to

47% of the total protein content of SPI was obtained. The lowest recovery under

conventional conditions was obtained with the use of PBS buffer, where protein

concentrations of 6.12 (31%), 6.03 (14%) and 0.34 mg/mL (19%) were obtained from

soy flour, SPI and soy milk, respectively.

Some observations made with the use of different extraction buffers under

conventional extraction in this study correspond to a study by Panda (2012). This

investigator evaluated soybean samples treated under different heating conditions for the

detection of IgE binding by extracting the proteins using a variety of extraction buffers,

including Laemmli and PBS, at room temperature for 2 hours (conventional extraction).

Panda (2012) reported that Laemmli buffer (described as a ‘harsh’ buffer in their study)

had higher overall protein solubilization properties, especially in heat processed samples,

compared to PBS. The effect of Laemmli buffer in protein extraction may be attributed to

the fact that it contains about 4% sodium dodecyl sulfate (SDS), which has denaturing

properties.

Comparing the effect of conventional and water bath extraction on total protein

recovery from the soy matrices, there was an overall increase in protein yields at elevated

temperatures, especially at 100°C for most samples. The effect of the buffers at higher

temperatures was similar to that in conventional extraction as shown in Figure 4.2 a-c.

With water bath extraction, PBS once again recorded significantly lower (p< 0.05)

protein recovery compared to Laemmli and urea under all three temperatures and for all

soy matrices.

52

For this study, comparison of the other extraction conditions was made mostly to

extraction with PBS under conventional extraction since that this is the most commonly

used method for the extraction of proteins. For soy flour, the total protein recovered in 15

minutes using PBS at elevated temperatures was not significantly different (p>0.05) from

that obtained under conventional methods, an indication that the use of elevated

temperatures could considerably lower extraction time and produce comparable results.

This is probably why most ELISA kits employ the use of 15 minutes extraction at 60°C

with PBS for the extraction step during allergen detection. It may, however, be necessary

to consider the use of other buffers since PBS recovered the least protein compared to

Laemmli and urea buffers under all extraction conditions and in all soy matrices. With

water bath extraction, the highest protein recovery for soy flour and SPI (17.9 mg/mL and

29.8 mg/mL corresponding to about 92% and 6 8 %, respectively, of the total protein

content in 1 g of sample) was obtained with urea buffer at 100°C. For soy milk, Laemmli

buffer at 100°C resulted in the best protein recovery of 1.04 mg/mL, representing 58% of

the total protein content in 1 g of soy milk.

Results obtained in this study correspond to the findings of some investigators. In

a study by Poms et al. (2004) with peanuts, the investigators reported that for peanut

protein extraction efficiency, the most significant factor appeared to be the pH of the

extraction buffer and that the best protein yields were obtained with buffers in the range

of pH 8-11. In their study, urea buffer at pH 8.7 provided relatively high yields for

processed peanut protein. The higher recovery of proteins in soy flour and SPI using urea

could be attributed to higher pH of urea buffer as shown by Poms et al. (2004). In another

study, Rudolf et al. (2012) tested the capacity of 30 different buffers to extract proteins

53

from mildly and strongly roasted peanut samples at 60°C for 15 minutes in a water bath.

From their results most of the tested buffers showed good extraction capacity for putative

Ara h lfrom mildly roasted peanuts but protein extraction from dark-roasted samples

required denaturing additives such as 6M urea. The denaturing effect of 6M urea may

have also contributed to the increased protein recovery in soy flour and SPI in the present

study. In the study of Rudolf et al. (2012), however, overall best results were achieved

using neutral phosphate buffers. The property of Laemmli buffer having high overall

protein solubilization properties especially in heat processed samples as described by

Panda (2012) may have contributed to it being a better extraction buffer for soy milk

compared to PBS and urea.

4.2 Effect of microwave assisted extraction time at different temperatures on

recovery of total soy proteins

For MAE, efficient extraction of soluble proteins was achieved in all soybean

matrices within the come-up time (Figure 4.3a-c), that is, the time it took for the

equipment to heat up samples to the required temperature of 60, 70 or 100°C. Samples

were immediately removed, once the temperature was reached (0 minute holding time).

Come-up times varied among samples, buffers and extraction temperatures as shown in

table 4.1. Generally, the come-up time at 60 and 70°C for all samples was under 2

minutes and under 4 minutes at 100°C. The longest come-up time was 3 minutes, 47

seconds for the extraction of proteins from SPI with PBS at 100°C and this was a shorter

extraction time than both conventional and water bath extraction times.

54

Figure 4.3: Quantity o f protein obtained from soy flour (a), SPI (b) and soy milk (c) extracts at different holding times (0, 5 and 10 minutes) and temperatures (60, 70 and 100°C) using microwave-assisted extraction. Each value is the mean ± standard deviation (n = 6). All treatments were analyzed by ANOVA. Significant differences were analyzed by Tukey’s HSD post-hoc test.

55

The extraction times that resulted in adequate yields in this study were shorter than those

used by other investigators. Choi et al. (2006) investigated the efficacy of MAE in the

extraction of soluble proteins from six Korean cultivars of soybeans compared with

conventional extraction with a shaking water bath. In their study, the microwave

extraction process was performed with 10 mL of distilled water and 1 g of ground

soybean at 80°C for 10 minutes. Salomon et al. (2014) also extracted mangiferin, a

bioactive metabolite with antioxidant properties from mango leaves using a microwave

power of 900 W and extraction time of 5 minutes. The differences in extraction times

may be attributed to type of sample, microwave and solvent as well as microwave power

and capacity being used.

56

Table 4.1: MAE Come-up times for samples

Sample Extraction Buffer Extraction Temperature Come-up Time (mins)

Soy flour PBS 60 1:0070 1:27100 3:04

Laemmli 60 1:0070 1:25100 3:00

Urea 60 1:1270 1:42100 3:20

SPI PBS 60 1:2670 1:30100 3:47

Laemmli 60 1:0570 1:20100 3:00

Urea 60 1:1970 1:30100 3:20

Soy milk PBS 60 1:2570 1:50100 3:08

Laemmli 60 1:1570 1:52100 3:04

Urea 60 1:1970 1:28100 3:20

n = 2

4.2.1 Comparison of conventional extraction and MAE on protein recovery

Upon selection of the best extraction time under MAE (0 minute holding time),

protein recoveries at the different MAE temperatures of 60, 70 and 100°C using the three

buffers were compared in order to select the best extraction condition for each of the soy

matrices. Protein recovered from MAE samples were also compared to recoveries from

samples extracted using the conventional method. These results are shown in

figure 4.4a-c.

57

With MAE, there was an overall significant (p < 0.05) increase in protein

recovery with increasing MAE temperature for all soy matrices. In soy flour, however,

the amount of proteins recovered using PBS buffer was reduced significantly (p < 0.05)

from 10.40 mg/ml (53% of the protein content) to 7.53 mg/ml (39%) as the MAE

temperature increased from 60 to 100°C. The reverse occurred with protein extracts from

SPI and soy milk using PBS; Protein recovery increased with increasing MAE

temperature. Irrespective of whether protein recovery decreased or increased with

temperature, recovery was still significantly higher (p < 0.05) than that of conventional

extraction in all soy matrices. PBS still resulted in the lowest protein recovery compared

to Laemmli and urea buffer.

Laemmli buffer exhibited a less predictable effect when used with MAE. For soy

flour, protein yields at all MAE temperatures were not significantly different (p>0.05)

with the use of Laemmli. Laemmli also produced significantly higher (p < 0.05) protein

yields under conventional conditions compared to MAE (Fig. 4.4a). The reverse occurred

in soy milk where protein recovery at 70 and 100°C with Laemmli using MAE were

significantly higher (1.22 mg/ml corresponding to 68% of the total protein content of soy

milk) than conventional extraction (Fig. 4.4c). MAE with Laemmli for SPI produced

extracts with protein concentrations which did not differ significantly (p>0.05) with

conventional extraction and with temperature increase.

58

PBS-7.4Laemmli-6.8Urea-8.7

Conventional MAE 60°C MAE 70°C MAE 100°C

Extraction Conditions■ PBS-7.4

Laemmli-6.8 Q Urea-8.7

■ PBS-7.4 @ Laemmli-6.8 □ Urea-8.7

Figure 4.4: Protein concentrations of soy flour (a), SPI (b) and soy milk (c) extracts obtained from conventional extraction at 23°C and MAE at 60, 70 and 100°C and 0 minute holding time. Each value is the mean ± standard deviation (n = 6). Bars for treatments not followed by the same letter are significantly different (p < 0.05). All treatments were analyzed by ANOVA (Tukey’s HSD post-hoc).

59

MAE with urea showed a trend of increased or similar protein recovery compared

with conventional extraction. For soy flour, the highest recovery of 16.61 mg/ml (85%)

was achieved at 100°C. This was not significantly different (p>0.05) from recovery at

70°C but significantly higher (p< 0.05) than conventional extraction. For SPI, the highest

recovery of 26.18 mg/ml (60%) was also achieved at 100°C, also significantly higher

(p< 0.05) than conventional extraction. For soy milk, a recovery of approximately 0.84

mg/ml (47%) was achieved at all MAE temperatures and this was significantly higher

(p< 0.05) than the recovery from the extracts under conventional extraction.

Microwave assisted extraction has been reported to be a very advantageous

extraction process because microwave energy provides thermal effects such as quick

heating, thawing, selective energy dissipation, same-direction heat and mass transfer

(Rosenberg and Bogl 1987, Choi et al. 2006). In this study, MAE was proven to be a

quick and efficient extraction method. In most instances, MAE recovered more protein

than both UAE and conventional extraction. Choi et al. (2006) made a similar

observation in their study. They compared the extraction of soluble protein from

soybeans using MAE and a conventional shaking water bath extraction system. The study

reported that the yield of soluble protein increased with time within a range of 30

minutes. The authors also reported that scanning electron microscopy showed the

destruction of the microstructure of soybean cells, which increased the extraction of

soluble soy protein (Choi et al. 2006). In an earlier study by Ashida et al. (1998),

microwave heating of soya slurry was applied for the preparation of soya milk and tofu.

The study reported that a higher protein concentration in soya milk was obtained by this

method than by conventional methods of heating such as the use of boiling water. The

60

study also reported that microwave oven heating of soya slurry was effective for protein

extraction and this has been shown in this study. Based on the results obtained using

MAE, the extracts with the highest protein recovery for each extraction buffer and for

each soy matrix were selected together with all conventional extraction samples for

further analyses.

4.3 Effect of ultrasound assisted extraction time at different temperatures on

recovery of total soy proteins

For UAE, extraction time varied among the soy matrices. For soy flour, 5 minutes

was selected as the best extraction time. This was because for the three buffers and at

both extraction temperatures, extraction for 5 minutes resulted in protein yields higher

than extraction for 1 minute and were comparable to yields at 10 minutes extraction as

shown in Figure 4.5a. For SPI, 10 minutes was selected as the best extraction time

(Figure 4.5b) for similar reasons as mentioned for soy flour. Additionally, a study by Hu

et al. (2013) reported SPI dispersed in water and ultrasonicated for 15 and 30 minutes

improved protein solubility. For soy milk, 1 minute extraction was selected since it

resulted in higher or comparable protein yields for all three buffers and for both

extraction temperatures (Figure 4.5c).

6 1

Figure 4.5: Quantity o f protein obtained from soy flour (a), SPI (b) and soy milk (c) at different extraction times (1 ,5 and 10 minutes) and temperatures (4 and 23 °C) using ultrasound-assisted extraction. Each value is the mean ± standard deviation (n = 6). All treatments were analyzed by ANOVA. Significant differences were analyzed by Tukey’s HSD post-hoc test.

62

4.3.1 Comparison of conventional extraction and UAE on protein recovery

As was done with water bath extraction and MAE, samples obtained at the best

extraction time using UAE (5 ,10 and 1 minute for soy flour, SPI and soy milk,

respectively) were compared based on extraction temperature and buffer. These samples

were also compared to conventional extraction samples and the results are presented in

Figure 4.6 a-c.

Using UAE, protein recoveries obtained with the use of the different buffers

varied with the different soy matrices. In soy flour, the highest recoveries of 12.78 and

14.25 mg/ml at 4 and 23°C (room temperature), respectively, were achieved with urea

buffer (Fig. 4.6a). These correspond to about 66 and 73%, respectively, of the total

protein content of 1 g of the soy flour and were significantly higher (p < 0.05) than the

use of urea in conventional extraction. The recoveries from Laemmli buffer at both UAE

temperatures (11.58 and 12.17 mg/ml) were significantly lower (p<0.05) than those from

urea buffer and from conventional extraction with Laemmli. PBS buffer, again recorded

the lowest protein recovery of 9.11 (47%) and 10.36 mg/ml (53%), respectively, at both

UAE temperatures. With the use of PBS and UAE, however, protein recovery was

significantly higher (p< 0.05) than in conventional extraction with PBS.

With the use of UAE on SPI, a slightly different trend was observed (Fig. 4.6b).

The highest recovery was achieved with urea buffer at 23°C (21.55 mg/mL). This was

about 50% of 1 g of the SPI protein content and it was not significantly different (p>0.05)

from recovery obtained with urea at 4°C. The highest recovery was also approximately

3% more protein recovered compared to urea with conventional extraction.

63

Figure 4.6: Protein concentrations of soy flour (a), SPI (b) and soy milk (c) extracts obtained from conventional extraction at 23°C and UAE at 4 and 23°C for 5, 10 and 1 min respectively. Each value is the mean ± standard deviation (n = 6). Bars for treatments not followed by the same letter are significantly different (p < 0.05). All treatments were analyzed by ANOVA (Tukey’s HSD post-hoc).

64

Protein recoveries obtained with Laemmli were slightly lower (19.37 mg/ml at 23°C) and

not significantly different (p>0.05) with reference to the extraction temperature. PBS

recorded the lowest recoveries of 6.36 (15%) at 4°C and 6.97 (16%) at 23°C, which was

slightly higher, but not significantly different (p>0.05) from conventional extraction with

PBS.

With soy milk, Laemmli buffer was observed to produce the highest recovery at

both UAE temperatures (Fig. 4.6c). Protein concentrations of 0.98 and 1.02 mg/ml

corresponding to 54 and 57% were recorded for extraction at 4 and 23°C, respectively.

These recoveries for Laemmli were comparable to that of conventional extraction. Urea

and PBS buffers recorded significantly lower recoveries (p < 0.05). The protein yield of

urea at 4°C was significantly higher (p < 0.05) than its yield under conventional

extraction. PBS recorded the lowest of 0.55 (31%) and 0.59 (33%) at 4 and 23°C,

respectively. These recoveries were, however, significantly higher (p < 0.05) than that of

conventional extraction.

Ultrasound assisted extraction (UAE), is based on mechanical waves at a

frequency above the threshold of human hearing (>16 kHz) and has been reported to be a

beneficial extraction method, especially where the use of moderate temperature ranges is

required (Lopez-Avila et al. 1996; Hu et al. 2013). For the extraction of proteins,

ultrasonic extraction has been shown by investigators like Wang (1978) and Karki et al.

(2010) to be efficient in increasing protein homogenization and solubility in soybean

meal. This was the general observation made in this study and studies have attributed the

increase in protein solubility to the fact that ultrasound induces cavitation, which causes

proteins to undergo physical disruption and/or chemical transformation. It heats by

65

specific absorption of acoustic energy, dynamic agitation and shear stresses and

turbulence (Floros and Liang 1994).

A study by Jambrak et al. (2009) reported that the solubility of soy proteins is

increased after ultrasound treatment and this was attributed to the unfolding and breaking

of peptide bonds by hydrolysis. Karki et al. (2010) also conducted a study that focused on

the use of high-power ultrasound prior to soy protein extraction from soy flakes to

simultaneously enhance protein and sugar release in the extract. In their study the particle

size of the soy flakes decreased nearly 10-fold following ultrasonic treatment at high

amplitude of 84 pmpp resulting in a high increase in total sugar released (50%) and

protein yield (46%) after 120 seconds of sonication when compared with non-sonicated

samples (control). In another study by Albillos et al. (2011) proteins from almonds were

extracted using different buffers, after which the extracts were sonicated in a

temperature-controlled water bath and aliquots were taken after 0, 1, 3, 5 and 10 minutes

of sonication. They reported that ultrasonic treatment improved protein extraction from

the almonds, especially those roasted at 260 and 400°C. These results obtained by these

investigators correspond to the general trends observed in this study especially with the

use of PBS buffer on all soy matrices. This study also showed that UAE has varying and

less predictable effects when used in combination with buffers other than PBS and on

different food matrices. For instance, with Laemmli buffer, protein recovery decreased

significantly (p< 0.05) compared to the control in soy flour but for soy milk, recovery

was not significantly different from the control (p>0.05). The use of urea with UAE on

soy flour resulted in increased recovery, but recovery from SPI and soymilk were not

significantly different (p>0.05) from the control. This may be an indication that different

6 6

food matrices may require different extraction conditions to achieve efficient extraction

for allergen detection purposes.

4.4 Comparison of MAE, UAE, conventional and water bath extraction methods

Under each extraction method, water bath, MAE and UAE, and taking each of the

buffers into consideration, the extracts with the highest protein concentrations,

(significantly) together with the controls (conventional extraction), were selected for

further analyses. For soy flour, with water bath extraction, extracts obtained at 60°C were

selected for PBS and extracts obtained at 100°C were selected for Laemmli and urea.

With MAE, extracts of PBS and Laemmli at 60°C and urea at 70°C were selected.

Samples extracted at room temperature for all three buffers were selected from UAE for

further analyses. For SPI, the highest recovery for all buffers was achieved at 100°C with

water bath and MAE and at 23°C with UAE. These samples were thus, selected for

subsequent analyses together with all extracts from conventional extraction. For soy

milk, all samples obtained from conventional extraction, water bath at 100°C for PBS and

Laemmli and 60°C for urea, MAE using PBS at 100°C, urea at 60°C, Laemmli buffer at

70°C and UAE with PBS and Laemmli at room temperature and urea at 4°C were

selected for further analyses.

Figure 4.7 a-c show a comparison of all four extraction methods based on the

selected samples. For soy flour, water bath extraction (100°C) using urea buffer resulted

in the highest protein yield of 17.9 mg/mL. Water bath extraction (in combination with

urea buffer) was the overall best extraction method for SPI. For soy milk, MAE (in

combination with Laemmli buffer) was the overall best extraction method. Overall,

67

irrespective of the extraction method, higher protein concentrations were obtained with

the use of Laemmli and urea buffers and not the traditional PBS.

Figure 4.7: Protein concentrations of soy flour (a), SPI (b) and soy milk (c) extracts obtained from conventional and water bath extraction, MAE and UAE.Each value is the mean ± standard deviation (n = 6). Bars for treatments not followed by the same letter are significantly different (p < 0.05). All treatments were analyzed by ANOVA (Tukey’s HSD post-hoc).

68

4.5 Characterization of proteins using SDS-PAGE

For the comparison of protein profiles, 7.5 pg per 15uL of protein in each of the

extracts (from each soy matrix) selected based on high soluble protein concentration

(table 4.2) were loaded onto a gel. Due to the possible denaturation effects of the

different extraction conditions used, extracts were treated under reduced (addition of p-

mercaptoethanol plus heating) and non-reduced (no P-mercaptoethanol and no heating)

conditions before loading into gels. Protein profiles for each of the soy matrices are

shown in Figure 4.8a-c.

Bands observed in soy flour were more numerous under all extraction conditions

compared to those found in SPI and soymilk and this is due to the fact that the flour was

the least processed soy matrix. The application of heat and processing chemicals duration

the manufacture of SPI and soy flour may have contributed to the reduced number of

protein bands observed in these samples. This confirms the effects of processing methods

on soy proteins and the possible effect this might have on allergen detection. The

conditions under which extracts were prepared before loading into the gel was observed

to impact the types and intensities of bands present in each of the matrices. Overall, more

bands were observed in extracts prepared under reduced conditions, compared to extracts

prepared under non reduced conditions. There were, however, bands observed between

100 and 150 kDa for extracts prepared under non reducing conditions for all soy products

that were either very faint or not present at all in samples prepared under reduced

conditions. There were also bands around the 50 kDa marker that were more intense in

extracts prepared under non reduced conditions for all soy products compared to those

prepared under reduced conditions. This may correspond to the P- subunits of P-

69

conglycinin, a major soy allergenic protein that has been reported by Krishnan et al.

(2009) to have a molecular weight of 52kDa.

70

Table 4.2: Protein extracts from soy matrices selected based on high total soluble proteins for Electrophoresis and ELISA analysesSoy Product Extraction Buffer Extraction Method Extraction Conditions

Soy flour PBS Conventional 23°C for 2h(7.4) Water bath 60°C for 15 mins

MAE 60°C for 0 minUAE 23°C for 5 mins

Laemmli Conventional 23°C for 2h(6.8) Water bath 100°C for 15 mins

MAE 60°C for 0 minUAE 23°C for 5 mins

Urea Conventional 23 °C for 2h(8.7) Water bath 100°C for 15 mins

MAE 70°C for 0 minUAE 23°C for 5 mins

SPI PBS (7.4) Conventional 23 °C for 2hWater bath 100°C for 15 mins

MAE 100°C for 0 minUAE 23°C for 10 mins

Laemmli (6.8) Conventional 23°C for 2hWater bath 100°C for 15 mins

MAE 100°C for 0 minUAE 23°C for 10 mins

Urea (8.7) Conventional 23 °C for 2hWater bath 100°C for 15 mins

MAE 100°C for 0 minUAE 23°C for 10 mins

Soy milk PBS (7.4) Conventional 23°C for 2hWater bath 100°C for 15 mins

MAE 100°C for OminUAE 23 °C for 1 min

Laemmli (6.8) Conventional 23°C for 2hWater bath 100°C for 15 mins

MAE 70°C for 0 minUAE 23 °C for 1 min

Urea (8.7) Conventional 23 °C for 2hWater bath 60°C for 15 mins

MAE 60°C for OminUAE 4°C for 1 min

71

Red uced N on -re d u ce d

a)A B C D E F G H I J K L M A B C D E F G H I J K L M

b)A B C D E F G H I J K L M A B C D E F G H I J K L M

c)A B C D E F G H I J K L M A B C D E F G H I J K L M

F igu re 4.8: Protein profiles of SF (a), SPI (b) & SM (c) extracts from Conventional (B-D) and water bath extraction (E-G), MAE (H-J) and UAE (K-M) methods with PBS, Laemmli and urea buffers, respectively, under reduced and non-reduced conditions, A (molecular weight marker). Bands around 75 and 50 kDa correspond to (3-conglycinin and bands around 37kDa correspond to glycinin (33 kDa) and the 34 kDa P34 allergenic soy protein.

72

Aside from this band, the other major bands of interest were more visible under reduced

conditions.

In all soy matrices and for all samples, bands were observed around the 75 kDa

region under reduced conditions. These may correspond to the 70 kDa a ’ and 72 kDa a-

subunits of p-conglycinin. Protein bands with these molecular weights have also been

observed and identified by Krishnan et al. (2009) as the a ’ (70 kDa) and a- (72 kDa)

subunits of p-conglycinin. The other major bands observed under reduced conditions

were around the 37 kDa band. These probably correspond to the 34 and 33 kDa P34

allergenic protein and the A3 chain of glycinin, respectively, as has been reported by

Yang et al. (2014). These have also been reported as major soy allergens. Soy glycinin

and P-conglycinin, also referred to as the 1 IS and 7S globulins, respectively, are the two

major storage protein components in soybean. They account for approximately 70% of

total storage proteins in soybean seed (Chen et al. 2013). The presence of these proteins

in all three soy matrices is an indication that they are very resistant to processing as well

as extraction conditions. Each of the three buffers and extraction methods employed in

this study was capable of extracting these allergenic proteins.

4.6 Effect of Extraction Conditions on Antibody based detection of Soy Proteins

In the detection of soy proteins, antibody-based tests are mostly used, especially

in the food industry because they are rapid and their protocols are relatively easy to

follow. Enzyme-linked immunosorbent assay (ELISA) is the commonly used detection

method in this category and ELISA kits specific for soy allergen detection have been

developed (Brandon et al. 2004, Koppelman et al. 2004, Pedersen et al. 2008, Sakai et al.

73

2010). It is a powerful analysis tool used for the detection of total or specific soybean

proteins (Koppelman and Hefle 2006). Antibodies specific to native soybean proteins,

one single protein such as Gly m Bd 30 K (P34), (3- conglycinin, glycinin or Kunitz

Trypsin Inhibitor, or denatured/ renatured soybean proteins are raised for the detection of

these proteins (Cucu et al. 2012).

The effect of the different extraction conditions on the detection of soy proteins in

the extracts from the different soy matrices was determined using Neogen’s Veratox Soy

kit according to manufacturer’s instructions. This kit detects total soy proteins in the

sample of interest. The results obtained are recorded in table 4.3 for each of the extracts

obtained from each of the soy matrices. Different concentrations of total soy proteins

were detected in each of the extracts obtained from each soy matrix. Overall, the highest

levels of total soy protein detected were from soy flour extracts obtained with all

extraction methods and all buffers with the exception of urea used with water bath

extraction and MAE. This observation was because the soy flour used in this study was

not cooked and had undergone minimal processing. Native proteins have been reported to

show strong detection in these antibody based detection kits due to strong binding of the

proteins (since they have been subjected to little or no protein modification) to the

antibodies used in these kits (Cucu et al. 2012).

Comparing the performances of the different extraction methods, for soy flour the

strongest detection of soy proteins (28.99 mg/ml) was observed under conventional

methods with PBS buffer. This extraction method also had the overall highest detection

of soy proteins and as mentioned earlier, this is because the soy flour used was uncooked

and the proteins extracted were minimally modified.

74

Table 4.3: Soy ELISA Analyses of Conventional and Water bath Extraction, MAEand UAE ExtractsSoy Extraction Extraction Extraction Detected SoyProduct Buffer Method Conditions Proteins (mg/ml)Soy PBS Conventional 23°C for 2h 28.99 ± 2.59 aFlour (7.4) Water bath 60°C for 15 mins 14.19 ±0.45 c

MAE 60°C for 0 min 19.51 ± 0.35 bUAE 23°C for 5 mins 14.07 ± 0.07 c

Laemmli Conventional 23°C for 2h 14.40 ± 1.16 c(6.8) Water bath 100°C for 15 mins 9.63 ± 0.20 d

MAE 60°C for 0 min 9.51 ±0.21 dUAE 23°C for 5 mins 9.27 ± 0.14 d

Urea Conventional 23°C for 2h 7.76 ± 0.84 d(8.7) Water bath 100°C for 15 mins 0.09 ± 0.02 f

MAE 70°C for 0 min 1.56 ±0.21 eUAE 23°C for 5 mins 14.33 ± 0.20 c

SPI PBS (7.4) Conventional 23°C for 2h 2.39 ± 0.03 aWater bath 100°C for 15 mins 0.56 ± 0.07 f

MAE 100°C for 0 min 0.88 ± 0.03 bUAE 23 °C for 10 mins 1.55 ±0.03 ce

Laemmli (6.8) Conventional 23 °C for 2h 1.53 ± 0.02 eWater bath 100°C for 15 mins 1.14 ±0.02 bf

MAE 100°C for 0 min 1.98 ±0.05 dUAE 23°C for 10 mins 6.52 ± 0.18 g

Urea (8.7) Conventional 23°C for 2h 1.97 ± 0.24 dWater bath 100°C for 15 mins 0.52 ± 0.03 f

MAE 100°C for 0 min 1.91 ±0.02 cdUAE 23°C for 10 mins 2.75 ± 0.02 a

Soy PBS (7.4) Conventional 23°C for 2h 0.24 ± 0.01 aMilk Water bath 100°C for 15 mins 0.06 ± 0.01 f

MAE 100°C for Omin 0.16 ±0.01 deUAE 23 °C for 1 min 0.17 ± 0.04 cde

Laemmli (6.8) Conventional 23°C for 2h 0.22 ± 0.02 abWater bath 100°C for 15 mins 0.02 ±0.001 e

MAE 70°C for 0 min 0.19 ±0.01 bedUAE 23 °C for 1 min 0.19 ±0.03 be

Urea (8.7) Conventional 23°C for 2h 0.18 ± 0.02 cdeWater bath 60°C for 15 mins 0.02 ± 0.001 e

MAE 60°C for Omin 0.15 ±0.02 deUAE 4°C for 1 min 0.19 ±0.02 bed

75

Laemmli and urea buffer used in conventional extraction on the other hand, resulted in

significantly lower (p< 0.05) soy protein detection of 14.40 and 7.76 mg/ml, respectively.

This was likely because these two buffers modified the soy proteins hence, altering to

some extent the immunological binding ability of the proteins extracted. A similar

observation was made in a study by Rudolf et al. (2012) where a lateral flow device

(LFD) which is also an antibody-based detection method was developed for the detection

of peanut protein. In their study the capacity of 30 different buffers to extract proteins

from mildly and strongly roasted peanut samples as well as their influence on the LFD

were investigated and it was reported that most extraction buffers showed inhibiting

effects on LFD performance compared with 0.1M PBS. Urea caused denaturation of

antibodies, inhibiting antibody-antigen binding in their study. This may also be the

reason PBS is the preferred buffer used in the extraction step of the ELISA kit protocol.

The buffers in combination with water bath extraction, MAE and UAE also

affected the detection of proteins extracted using these methods and extraction conditions

(table 4.3). With soy flour, extracts obtained with the use of PBS and MAE (60°C)

resulted in strong detection of soy proteins (19.51 mg/mL), although this was

significantly lower (p< 0.05) than that of PBS. The least soy protein detection was

observed in extracts obtained from water bath extraction in combination with urea buffer

(1.56 mg/mL).

For SPI, the extract from UAE with Laemmli recorded the strongest detection of

soy proteins (6.52 mg/mL). This was very interesting as this value was significantly

higher (p< 0.05) than the soy proteins detected in PBS extracts under conventional

extraction (2.39 mg/mL). Another interesting observation was the fact that the quantity of

76

soy proteins detected in extracts from UAE with urea (2.75 mg/mL) was not significantly

different (p>0.05) from those from conventional extraction with PBS. Water bath

extraction (100°C) with PBS and urea showed the weakest soy protein detection of 0.56

and 0.52 mg/mL, respectively, for SPI.

For Soy milk, the strongest detection of soy proteins was observed in extracts

obtained from conventional extraction with PBS (0.24 mg/mL). The amount of soy

proteins detected was, however, not significantly different (p>0.05) from the amount of

soy proteins detected in the soy milk extracts obtained from conventional extraction with

Laemmli buffer. The lowest detection of 0.02 mg/ml was observed in extracts obtained

from water bath extraction with Laemmli (100°C) and urea buffer (60°C) and were not

significantly different (p>0.05).

These results suggest that for products like SPI and soy milk containing soy

proteins that have undergone some level of modification due to processing, UAE as an

extraction method and Laemmli as an extraction buffer may be good extraction

conditions that could be used to improve extraction of proteins for the detection of soy

allergens using antibody based detection methods. In a study by Panda (2012) where

different extraction buffers including PBS and Laemmli were used to extract proteins

from soy flour processed with different heat treatments for allergen detection, it was

reported that Laemmli buffer- extracted samples showed higher IgE binding by

immunoblot compared to the other extraction buffers especially at the 75, 50 and 35 kDa

bands.

77

Extraction based on heating techniques as was used in water bath extraction and

MAE may improve the extraction of soy proteins but reduce the ability of these proteins

to be detected by immunological methods. Heat treatment has been reported to alter

proteins, hence the low level of detection of soy proteins in extracts from these methods.

Gomaa and Boye (2013) investigated the effects of baking time, temperature profile,

cookie dimensions and weight on the detection of four allergens, including soy in a non­

wheat flour cookie using ELISA. They reported that allergen recovery decreased as

baking time increased and cookie size was decreased and that no recoveries were

obtained for soy in some of the thermally processed samples. Cucu et al. (2012) also

reported in their study to evaluate the applicability of different soy ELISAs to detect the

presence of soybean proteins in cookies, that both matrix interferences and the baking

process affected analytical recovery and detection of the proteins. This further explains

the poor detections made in the heat-based water bath and MAE extracts. Additionally,

even though these kits have been useful in the rapid detection of allergens in the food

industry, they have limitations. Platteau et al. (2011) have reported that the detection of

soybean proteins by commercial kits is severely affected by food processing reactions.

UAE, even though performed at room temperature resulted in weak soy protein

detection in soy flour and can be explained by because the mechanism of soluble protein

release modifying the proteins during the extraction process. UAE was however, the best

extraction method in terms of detection with ELISA for SPI. It is also important to note

that the different soy matrices produced varying detection results under different

extraction conditions; while PBS with conventional extraction gave the best results for

soy flour and soy milk, UAE with Laemmli was the best condition for SPI. This is an

78

indication that no single extraction method may be the best for all samples, something

that should be taken into consideration in allergen detection. Panda (2012) also

mentioned in their study that it is essential to evaluate proteins extracted with different

extraction buffers and not just a single buffer while making any interpretation on

allergenicity.

Based on the compatibility of SPI and soy milk extracts from UAE and

conventional extraction using Laemmli buffer with the soy ELISA detection method,

these extraction conditions in addition to conventional extraction with PBS (control) were

selected as the best extraction conditions and used for the second study.

79

OBJECTIVE 2: EVALUATION OF THE EFFICIENCY OF EXTRACTION

METHODS ON THE EXTRACTION OF ALLERGENIC SOY PROTEINS FROM

FOOD MATRICES.

4.7 Effect of selected extraction conditions on extraction of total proteins from food

matrices

In this study proteins were extracted from commercial samples (baked cheese

crackers, salsa, pancake mix, gluten-free table crackers and veggie burgers) and in-house

prepared dry flour mix and model cookies (spiked with 0, 1,5, 10, and 20 mg soy

proteins) using UAE and conventional extraction with Laemmli and conventional

extraction with PBS as the control extraction method. Results are presented in figures 4.9

and 4.10.

For the commercial samples, conventional extraction with PBS resulted in the

least protein recovery compared to UAE and conventional extraction with Laemmli

buffer. With the exception of veggie burger, UAE and conventional extraction with

Laemmli recovered comparable protein yields from all commercial samples. For veggie

burger, which was labeled to contain soy proteins, conventional extraction with Laemmli

recovered the most proteins. UAE with Laemmli and conventional extraction with PBS

recovered comparable protein yields, which were significantly lower (p<0.05) than

conventional extraction with Laemmli buffer.

80

Figure 4.9: Total protein concentrations of commercial samples extracted using conventional extraction with PBS and Laemmli and UAE with Laemmli.Each value is the mean ± standard deviation (n = 6). Treatments within each sample were analyzed by ANOVA. Significant differences were analyzed by Tukey’s HSD post-hoc test.

Figure 4.10: Total protein concentrations of dry mix and cookies spiked with soy proteins extracts obtained using conventional extraction with PBS and Laemmli and UAE with Laemmli. DM-dry mix, SFC-soy free cookie, C-cookie.Each value is the mean ± standard deviation (n = 6). All treatments were analyzed by ANOVA. Significant differences were analyzed by Tukey’s HSD post-hoc test.

81

A similar observation was seen in the in-house dry flour mixes and cookies. For all

samples in this set, conventional extraction with PBS recovered the least amount of

proteins. For all dry mix samples and the soy-free cookies (0 mg soy protein), UAE and

conventional extraction with Laemmli buffer recovered protein yields that were not

significantly different (p >0.05) but significantly higher (p< 0.05) than conventional

extraction with PBS. For all cookie samples containing soy proteins, UAE with Laemmli

recovered the highest protein yields compared to the other two extraction conditions.

As mentioned earlier, the presence of the detergent SDS in Laemmli buffer may

have resulted in its ability to recover high protein yields compared to PBS. Panda (2012)

has also reported that Laemmli buffer is more efficient in the recovery of proteins from

processed food matrices compared to PBS. Since the use of UAE with Laemmli also

resulted in comparable protein recoveries for almost all samples and higher recoveries for

all cookie samples with soy, UAE may be a more time efficient alternative to

conventional extraction, especially for the extraction of soy proteins.

4.8 Effect of selected extraction conditions on protein profiles using SDS-PAGE

In order to determine if the proteins extracted from the various food samples

contained allergenic soy proteins, gel electrophoresis under reduced conditions was

performed for all samples. The protein profiles are shown in figures 4.11 and 4.12 for

commercial and in-house samples, respectively.

82

C o m m e rcia l sam p le s

A B C D E F G H I J K L M N O P250kDa ----- „

Figure 4.11: Protein profiles of extracts from commercial samples (cheese crackers, salsa, pancake mix, table crackers and veggie burgers) obtained from Conventional extraction with PBS (B-F), with Laemmli (G-K) and UAE with Laemmli (L-P) buffers respectively under reduced conditions, A (molecular weight marker).Bands around 75 and 50 kDa correspond to (3-conglycininn and bands around 37kDa correspond to glycinin (33 kDa) and the 34 kDa P34 allergenic soy protein.

Dry Mix Cookies

A B C D E F G H I J K L M N O P A B C D E F G H I J K L M N O P

Figure 4.12: Protein profiles of extracts from dry mix and cookie samples with 0, 1,5, 10 and 20 mg soy obtained from Conventional extraction with PBS (B-F), with Laemmli (G- K) and UAE with Laemmli (L-P) buffers, respectively, under reduced conditions, A (molecular weight marker).Bands around 75 and 50 kDa correspond to (3-conglycininn and bands around 37kDa correspond to glycinin (33 kDa) and the 34 kDa P34 allergenic soy protein.

83

For commercial samples no protein bands were seen in salsa (lanes C, H, M) and table

crackers (lanes E, J, O) under all three extraction conditions (figure 4.11). This may have

been due to the fact that the salsa was labeled ‘may contain traces of soy’ and thus if any

soy protein was present at all, may not have been enough to be captured in the analysis.

The table crackers on the other hand, contained soy flour and soy lecithin, but all three

extraction conditions may have denatured the proteins or may not have extracted enough

soy proteins to be seen after electrophoresis. For the other commercial samples, cheese

crackers (lanes B, G, L), pancake mix (lanes D, I, N) and veggie burgers (lanes F, K, P),

bands were seen around the 75 and 50 kDa marker. These bands were very faint in

conventional extraction with PBS extracts (B-F) but more visible in conventional with

Laemmli (G-K) and UAE with Laemmli (L-P) extracts. These proteins have been

discussed earlier and are reported to correspond to the 70kDa a ’ and 72kDa a- subunits

of [3-conglycinin and the 52kDa P- subunits of P-conglycinin, which has been reported by

Krishnan et al. (2009) as a major soy allergenic protein. The presence of this protein in

these samples may be an indication that they contain soy. This was expected for the

veggie burger since it contains soy protein, soy sauce and soy lecithin.

For the dry-mix and cookie samples prepared in-house bands were seen at the 75,

50 and 37 kDa markers for most of the samples containing soy (figure 4.12). As was

expected, these bands were not present in soy-free dry mix and cookie samples. The

number of bands observed in the samples with soy varied with the different extraction

conditions.

For the dry-mix samples, extracts obtained from conventional extraction with

PBS showed visible bands at 75, 50 and 37 kDa markers (which is reported to correspond

84

to the 34 and 33 kDa P34 allergenic protein and the A3 chain of glycinin, respectively)

for samples containing 1,5, 10 and 20 mg soy (lanes C-F). This was not the case for

conventional extraction and UAE with Laemmli. For extracts obtained from conventional

extraction with Laemmli, bands at the 75 and 50 kDa markers were observed only in

samples containing 5, 10 and 20 mg soy (lanes I-K). For extracts from UAE with

Laemmli, bands at the 75 kDa marker were faint in 1 and 5 mg soy dry mix samples, but

clearly visible in 10 and 20 mg soy samples. The 50 kDa bands were only seen clearly in

the 20 mg soy dry mix sample. These results are an indication that despite the fact that

conventional extraction with PBS recovered the least amount of total proteins in all

samples, it was effective in extracting the allergenic soy proteins in the dry mix samples.

For the cookie samples, faint bands were seen at the 75 kDa marker for all

extracts containing soy from conventional extraction with PBS (lanes B-F), while the

same bands were seen only in the 10 and 20 mg soy cookie samples for conventional

extraction (lanes G-K) and UAE with Laemmli (lanes L-P). For all three extraction

conditions and for all cookie samples, bands were seen at the 37 kDa marker. There were,

however, no bands seen at the 50 kDa marker and this may imply that the processing

conditions (baking at 350°F) used in the preparation of the cookies modified these

proteins. Cucu et al. (2012) made similar observations after incubating soy protein extract

with glucose and sunflower oil at 70 °C in order to mimic major protein changes that

occur during the interaction of proteins with lipids and reducing sugars. They reported

that the bands representing the 1 IS glycinin and the 7S P-conglycinin lost intensities and

attributed this to a combined effect of incubation with glucose, as well as with sunflower

85

oil. Since the cookie samples contained sugar and sun flower oil, this may have

contributed to the absence of the 50 kDa band in the samples.

4.9 Effect of selected extraction conditions on ELISA analyses of food matrices

Extracts from commercial samples, as well as dry mixes and cookies prepared in-

house, were analyzed to detect the effect of the extraction conditions on the detection of

allergenic soy proteins present in each sample using anti-body based detection method

(ELISA). Results are reported in table 4.4 for commercial samples and table 4.5 for dry

mix and cookie samples.

For the commercial cheese crackers, salsa and pancake mix, the ELISA kit

detected comparable levels of soy proteins in extracts from all three extraction

conditions. The soy protein concentrations detected from these samples were not

significantly different (p>0.05). For table crackers and veggie burgers, which definitely

contained soy, conventional extraction with Laemmli extracts produced the highest

quantity of soy proteins detected (79.93 and 113.43 ppm for table crackers and veggie

burgers, respectively). Conventional extraction with PBS resulted in the least soy protein

detection for table crackers and comparable soy protein detection with UAE and Laemmli

for veggie burgers.

8 6

Table 4.4: Soy ELISA analyses of commercial samples showing amount of soy proteins detected (in ppm) using the selected extraction conditions

Commercial Sample

Cheese cracker SalsaPancake mixTable cracker Veggie burger

Conventional with PBS11.60 ±5.73 A 5.28 ±0.32 A

11.03 ±4.00 A 3.00 ± 0.64 C

34.38 ± 8.52 B

Conventional with Laemmli

8.23 ± 2.93 A 7.58 ±0.39 A

17.03 ±5.98 A 79.93 ±7.18 A

113.43 ±4.63 A

UAE with Laemmli5.30 ± 1.13 A4.10 ±0.35 A9.45 ± 3.32 A

12.95 ± 4.74 B 49.63 ± 8.66 B

Values above represent the mean of triplicate batches ± standard deviations (n =3). Values within columns not sharing a letter are significantly (p< 0.05) different. All treatments were analyzed by ANOVA. Significant differences were analyzed by Tukey’s HSD post-hoc test.

Table 4.5: Soy ELISA analyses of dry mix and cookies spiked with soy flour showing amount of soy proteins detected (in ppm) using the selected extraction conditions

Sample Conventional with PBS

Conventional with Laemmli

UAE (10mins+23°C) with Laemmli

Dry Mix (no soy) <LOD <LOD <LODDry Mix+lmg soy 153.50 ± 13.44 B 76.80 ±7.35 C 222.20 ± 4.53 ADry Mix+5mg soy 1051.71 ± 11.02 B 1646.00 ±19.80 A 535.70 ± 46.24 CDry Mix+lOmg soy 1645.50 ±34.65 A 1828.00 ±83.44 A 1234.60 ± 57.42 BDry Mix+20mg soy 2676.00 ±299.8 IB 3434.50 ±289.21 A 2560.00 ± 124.45 BSoy Free Cookies <LOD <LOD <LODCookies+lmg soy 2.53 ±0.15 A 2.63 ± 0.25 A 5.54 ±0.29 ACookies+5mg soy 10.38 ± 0.88 B 32.05 ± 4.03 A 40.37 ± 2.38 ACookies+lOmg soy 41.15 ± 1.63 B 69.65 ± 3.04 A 89.91 ±5.43 ACookies+20mg soy 556.90 ± 26.45 A 188.40 ±9.05 B 131.60 ± 5.09 BValues above represent the mean of triplicates ± standard deviations (n= 3). Values within columns not sharing a letter are significantly (p< 0.05) different. All treatments were analyzed by ANOVA. Significant differences were analyzed by Tukey’s HSD post-hoc test.

87

Since ELISA is an anti-body based method of detecting allergenic soy proteins, it

is important that during the extraction process, the integrity of the proteins is not

compromised as this may affect detection of allergenic soy proteins. Efficient extraction

of the proteins is also important since processing of foods may reduce the ability of the

ELISA kit to detect any allergenic proteins that may be present. The high values recorded

in conventional extraction with Laemmli samples for table crackers and veggie burgers

may be an indication that this extraction method maintains soy protein integrity making it

easy for anti-body based detection. The comparable results obtained for cheese crackers,

salsa and pancake mix, which may have contained relatively smaller or trace quantities of

soy proteins could also be an indication that the use of UAE with Laemmli may be a

faster alternative to conventional extraction with PBS.

For the dry mix and cookie samples prepared in-house, no soy proteins were

detected in the samples not spiked with soy flour (table 4.5). This was expected since the

samples had no soy in them. Overall, higher quantities of soy proteins were detected in

the dry mix samples incurred with soy flour compared to their corresponding cookie

samples. This was due to the fact that these samples had not been cooked and thus

contained little or no modified proteins, which are more difficult to detect with ELISA.

The proteins in the cookies on the other hand, had undergone some chemical and physical

modification during processing and baking reducing the ability of ELISA to detect

allergenic proteins present in these samples.

Comparing the effect of the three extraction conditions on ELISA output,

conventional extraction with Laemmli produced the overall best results for dry mix

samples (table 4.4). UAE with Laemmli produced the best results for dry mix containing

8 8

1 mg soy protein. For the cookie samples, conventional extraction and UAE with

Laemmli produced comparable and overall, best ELISA results. Conventional extraction

with PBS produced the best results in cookies with 20 mg soy protein. These results

suggest that again, for the purposes of allergen detection, the use of UAE and the use of

Laemmli buffer may be an alternative to conventional extraction. The results also suggest

that no one extraction method may be the best for different food matrices.

89

CHAPTER 5: CONCLUSIONS

This study set out to investigate extraction methods that could be used as

improved, more efficient and less time consuming alternatives to conventional extraction

used in the extraction of soy proteins for allergen detection. Water bath extraction, MAE

and UAE, were compared to conventional extraction. These extraction methods were

found to increase protein recovery from the soy matrices used. The allergen detection

method used in the study was, however, unable to efficiently detect the presence of

allergenic soy proteins from extracts obtained using water bath extraction and MAE,

possibly due to the use of high temperatures in these extraction methods. It might,

therefore, be a better option to use these extraction methods and conditions with allergen

detection methods that are not antibody- based such as liquid chromatography mass

spectrophotometry (LC-MS). The use of other extraction buffers besides the traditional

PBS in combination with these extraction methods was also investigated and it was found

that the use of urea also contributed to decreasing soy allergen detection with ELISA.

The use of Laemmli buffer with conventional extraction and UAE proved to be

comparable or better extraction methods compared with conventional extraction with

PBS for processed soy samples. These extraction conditions were also compatible with

ELISA and produced comparable or better results with reference to conventional

extraction conditions.

These extraction conditions were further studied and their practical use tested on

some commercial samples containing soy as well as some samples spiked with known

concentrations of soy flour. The use of Laemmli buffer with conventional extraction and

UAE once again proved to be comparable and in some cases better than conventional

90

extraction with PBS, suggesting that these extraction conditions may be better

alternatives or additional extraction methods that could be employed in the extraction

step in soy allergen detection.

Another consistent finding in this study was the fact that different food matrices

showed best results (whether it was for the recovery of total proteins or detection by

ELISA) under different extraction conditions suggesting that no single extraction method

is the best for all samples. It is therefore important that further research be conducted to

know which extraction conditions work best for a particular food product before the

selection of an extraction method. The need for such research makes this study very

important as two additional extraction conditions have been shown to produce similar or

better results than conventional extraction. Further study of these extraction methods,

especially UAE and the use of Laemmli buffer instead of PBS, could be helpful in

improving the extraction step employed by ELISA manufacturers. This could save

allergen detection time and more importantly, produce more accurate results that could

help curb the problem of false negatives in allergen detection.

91

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BIOGRAPHY OF AUTHOR

Amma K. Amponsah was bom in Mamponteng, a suburb in the Ashanti region of

Ghana, West Africa. She was raised in Accra, the capital city o f Ghana and graduated

from Wesley Girls’ High School in 2002. She then attended the Kwame Nkrumah

University o f Science and Technology, Kumasi, Ghana and graduated in 2007 with a

Bachelor o f Science degree in Biological Sciences. In 2008, she pursued a Masters

program in Food Science at the University o f Ghana and graduated in 2010 after which

she was employed as a Research Assistant in the same university. In July 2013, Amma

enrolled in the graduate program in the School o f Food and Agriculture at the University

of Maine.

She is a candidate for the Master o f Science degree in Food Science and Human

Nutrition from the University o f Maine in December 2015.

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