Improving the Nutritional Value of Soybean Meal Through Fermentation Using Newly Isolated Bacteria

Improving the nutritional value of soybean meal through fermentation using newly isolated bacteria

Samantha Medeiros

A Thesis presented to The University of Guelph

In partial fulfillment of requirements for the degree of Master of Science In Animal & Poultry Science

Guelph, Ontario, Canada ©Samantha Medeiros, March, 2015

ABSTRACT

IMPROVING THE NUTRITIONAL VALUE OF SOYBEAN MEAL THROUGH FERMENTATION USING NEWLY ISOLATED BACTERIA

Samantha Medeiros Advisor: University of Guelph, 2015 Dr. Julang Li

Soybean meal has limited use in piglet feed due to anti-nutritional factors. Past studies show that using fermented soybean meal can reduce these factors and improve pig growth performance. In this thesis research is reported aimed at using newly isolated strains of bacteria to improve the feeding value of soybean meal through fermentation. Bacteria were isolated from fermented foods and the intestines of grass carp (Ctenpharyngodon idella), screened for enzyme activity, characterized, and then used to ferment soybean meal. The fermented product was analyzed for protein profile, as well as nutrient composition and allergenicity. Results indicate that the bacteria identified as Bacillus amyloliquefaciens decreased large proteins, eliminated allergenic proteins, reduced the oligosaccharide concentration and increased the concentrations of crude protein and amino acids in partly fermented soybean meal. Further studies are needed to investigate the non-starch polysaccharide concentration and the growth performance of piglets fed the fermented soybean meal product.

ACKNOWLEDGEMENTS

First and foremost I would like to thank my advisor Dr. Julang Li. Throughout this journey she has been more than an advisor to me and provided me with guidance and encouragement through the roughest of times. She is a brilliant scientist and it was a privilege to learn under her supervision.

I would also like to thank my advisory committee members, Dr. Kees de Lange and Dr.

Hugh Cai, for all of their advice and insightful comments to this project. I especially would like to thank Dr. Cai for all of his assistance in the identification of the isolates using MALDI-TOF

MS. I would also like to thank our collaborator Dr. Zhang and Jingjing Xie for their contribution of the crude protein, amino acid, and oligosaccharide analyses.

I would also like to acknowledge all of my lab members, especially Dr. Paul Dyce who helped complete the gyrB gene sequencing and the western blot analysis. All of my friends in the department have provided moral support and scientific advice. I was very fortunate to work with such an intelligent and amiable group of people which made my laboratory experience a very pleasant one.

I would also like to thank Ontario Ministry of Agriculture Food and Rural Areas for awarding me their Highly Qualified Personnel Scholarship.

Lastly, I would like to thank my family and my friends for all of their support through these last two years. Being surrounded by such a loving group of people has always made me feel blessed and I could not have come as far as I have without them.

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TABLE OF CONTENTS

ABSTRACT ...... ii

ACKNOWLEDGEMENTS ...... iii

LIST OF FIGURES ...... vii

LIST OF TABLES ...... viii

LIST OF ABBREVIATIONS...... ix

1.0 INTRODUCTION: LITERATURE REVIEW ...... 1

1.1 Challenges faced by piglets ...... 1

1.1.1 Physiological factors ...... 1

1.1.2 Social factors ...... 3

1.1.3 Diet composition ...... 3

1.2 Soybeans ...... 4

1.2.1 Positive factors of soybeans ...... 4

1.2.2 Negative factors of soybeans ...... 7

1.3 Current applications used to improve soybean ...... 19

1.3.1 Processing ...... 19

1.3.2 Biotechnology and selective breeding strategies ...... 20

1.3.3 Addition of enzymes ...... 21

1.3.4 Microbial fermentation ...... 22

1.4 Summary and rationale ...... 26

2.0 HYPOTHESES ...... 27

3.0 OBJECTIVES ...... 27

4.0 MATERIALS AND METHODS ...... 28

4.1 Isolating and screening bacteria with high enzyme activity ...... 28

4.1.1 Primary screening for bacteria with protease activity from fermented food ...... 28

4.1.2 Primary screening for cellulase activity from grass carp source ...... 29

4.1.3 Second round of screening ...... 31

4.2. Identification of bacteria selected ...... 33

4.2.1: MALDI-TOF MS:...... 33

4.2.2: PCR Amplification of partial 16S rRNA gene and gyrB gene: ...... 33

4.3. Soybean meal fermentation...... 36

4.3.1. Preparation of soybean meal and inoculums ...... 36

4.3.2. Liquid-state fermentation ...... 37

4.3.3. Solid-state fermentation ...... 38

4.4 Analysis of soybean meal fermentation products ...... 39

4.4.1 Proximate analysis and amino acid profiling ...... 39

4.4.2 Determination of soluble protein fractions and distribution ...... 39

4.4.3 Determination of oligosaccharide concentrations ...... 40

4.4.4 Detection of Allergenic Proteins...... 41

4.5 Statistical analysis: ...... 43

5.0 RESULTS ...... 43

5.1 Qualitative measurement of enzymes activity...... 43

5.1.1 Primary Screening for Protease Producing Bacteria ...... 43

5.1.2 Primary Screening of Cellulase Producing Bacteria ...... 44

5.1.3 Second round of screening ...... 44

5.2 Identification of selected bacteria ...... 51

5.2.1 Characterization of isolates using MALDI-TOF MS ...... 51

5.2.2 Characterization of isolates using 16S rRNA and gyrB gene sequencing ...... 51

5.3 Liquid-state fermentation ...... 55

5.3.1 Influence of selected bacterium on SBM protein profile ...... 55

5.4 Solid-state fermentation ...... 57

5.4.1 Influence of selected bacterium on SBM protein profile ...... 57

5.4.2 Improved crude protein of fermented soybean meal ...... 58

5.4.3 Amino acid profile of the fermented soybean meal ...... 62

5.4.4 Detection of Allergenic Proteins ...... 65

5.4.5 Oligosaccharide concentration of fermented soybean meal ...... 65

6.0 DISCUSSION ...... 68

REFERENCES: ...... 82

Appendix A ...... 96

LIST OF FIGURES

Figure 1: Simplified figure illustrating the degradation of soy oligosaccharides ...... 17

Figure 2: A simplified diagram illustrating the degradation of cellulose ...... 18

Figure 3: Measuring protease activity using soymilk agar plates ...... 47

Figure 4: Measuring cellulase activity using CMC agar ...... 48

Figure 5: Measuring amylase activity using corn starch agar ...... 49

Figure 6: Gel electrophoresis of PCR products used to identify the bacteria isolates...... 53

Figure 7 : The fermentation effects of using different isolates on the protein profile of SBM in a liquid-state...... 56

Figure 8: The relative changes of total soluble protein of soybean meal when fermented for different lengths and with different bacterial isolates...... 59

Figure 9: Visualizing the changes of total soluble protein profile of soybean meal when fermented for different lengths and with different bacterial isolates with Coomassie Blue gel ... 60

Figure 10: The effects of solid-state fermentation length on crude protein of soybean meal ...... 61

Figure 11: The effect of fermentation length using Isolate-2 on amino acid concentrations ...... 63

Figure 12: Western blot detecting soy allergens in the unfermented and fermented soybean meal products ...... 66

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LIST OF TABLES

Table 1: Summary of the undesirable nutrient components of soybean meal……..………...... 10

Table 2: Summary of the first round of screening……………………………………………....45

Table 3: Summary of the enzyme activity of best performing bacteria isolated from food and grass carp intestine…………………...…………………………………………………………..50

Table 4: Summary of the gene sequencing nucleotide BLAST strain identification of the selected isolates..………………….………………………………………………..…………….54

Table 5: Quantitative representation of the effect of fermentation with Isolate-2 over a 24 and a

48 hour period……………………………………………………………………………………64

Table 6: The oligosaccharide concentration in soybean before and after fermentation with

Isolate-2 for 24 and 48 hours………………………………………………………………….....67

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LIST OF ABBREVIATIONS

AOAC Association of Analytical Communities

ATCC American Type Culture Collections

BHI Brain Heart Infusion bacteria culture media

BLAST Basic local alignment search tool

CFU Colony forming units

CMC Carboxymethyl cellulose

DM Dry matter

EAA Essential amino acids

GIT Gastrointestinal tract gyrB DNA gyrase β subunit

HPLC High-performance liquid chromatography

LAB Lactic acid bacteria

LB Luria Bertani bacteria culture media

MALDI-TOF MS Matrix assisted laser desorption ionization- time of flight mass spectrometry

MRS deMan, Rogosa and Sharpe bacteria culture media

NCBI National

NEAA Non-essential amino acids

NRC National Research Council

NSP Non-starch polysaccharide

P.con Positive control

PCR Polymerase chain reaction

PGA Poly-ɣ-glutamate

PVDF polyvinyl difluoride

RFO Raffinose family of oligosaccharides

SB Sodium borate

SBM Soybean meal

SDS-PAGE sodium –dodecyl sulfate-polyacrylamide gel electrophoresis

SPC Soy protein concentrate

SPI Soy protein isolate

16S rRNA 16S ribosomal ribonucleic acid

1.0 INTRODUCTION: LITERATURE REVIEW

1.1 Challenges faced by piglets

Weaning is the process of separating piglets from the sow’s milk. In nature, piglets are weaned gradually at the age of 10-12 weeks (Lalles et al., 2007); however, in industrial settings piglets are weaned abruptly and much earlier between 21-26 days of age. At this time, the piglet is still undergoing rapid intestinal development. Early weaning is typically associated with a decrease in piglet performance due to decreased feed intake brought on by three main factors: physiological factors, social factors, and change in diet composition. These challenges caused by early weaning decrease as the pig ages, however the losses due to piglet performance during the weaning period remain a major loss to the swine industry.

1.1.1 Physiological factors

When a piglet is first born it consumes colostrum from the sow, a liquid feed. It takes approximately 10-12 weeks post-partum for the piglet’s digestive tract to be able to switch from a liquid feed to a solid feed (Lalles et al., 2007). This abrupt transition when weaning piglets early negatively impacts the piglet because its gastrointestinal tract is still immature and cannot adequately handle solid food yet. There are two intestinal morphological characteristics that have been associated with negative aspects of early weaning: villus atrophy and crypt hyperplasia (Pluske et al., 1997). Villus atrophy observed during early weaning is likely due to an increased rate of cell loss and consequently, an increased crypt-cell production leading to an increased crypt death (Pluske et al., 1997). Consequently, the decreased villi height also has a negative impact on the brush-border enzymes lactase and maltase (Pluske et al., 1997). These

1 changes in gut morphology led to malabsorption of nutrients which increases incidences of diarrhea (Pluske et al., 1997). Early weaning has also been found to effect pancreatic enzyme secretions. The activity of important enzymes responsible for protein digestion, such as: trypsin, chymotrypsin, carboxypeptidase A, and carboxypeptidase B, were found to decrease after early weaning (Hedemann & Jensen, 2004). Without these enzymes, piglets cannot efficiently digest found in soybean meal or successfully hydrolyse the allergenic proteins. The gastric pH of the piglet during weaning also contributes to this decrease in performance. Piglets have a decreased capacity to secrete hydrochloric acid which leads to the gastric pH of 3-4, which is high when compared to that of a mature pig (pH of 1.6-1.7) (Cranwell et al., 1976; Snoeck et al., 2004).

Gastric pH levels that are high negatively affects piglet performance by resulting in an incomplete digestion of protein as well as allowing pathogenic bacteria to survive and colonize the gut (Snoeck et al.,2004). When pathogenic bacteria, such as E.coli, colonize the gut, it leads to the piglet developing the clinical sign of scours.

Apart from of the immature gastrointestinal tract, the piglet also has an immature immune system which contributes to the decrease in piglet performance observed with early weaning.

Piglets acquire immunity when they are first born through the sow’s colostrum but this passive immunity only lasts for a short time (Lalles et al., 2007). At the age of weaning, the piglet has built up a mucosal immune system which is developed enough to activate the immune response

(Lalles et al., 2007). The challenges faced when piglets are fed soybean meal result in damage to the intestine resulting in further malabsorption (Li et al., 1991). During weaning the immune system is still developing which causes the piglet to be more susceptible to bacterial infections

(Kelly and King, 2001). Combined, all of these developmental factors affect piglet performance associated with early weaning.

1.1.2 Social factors

A piglet during weaning also faces a number of non-physiological factors that may be contributing to the negative piglet performance observed during early weaning. During weaning the piglet faces a lot of social challenges such as separation from the sow, mixing litters, and social dominance. When a piglet is separated from its sow, it exhibits separations calls which have been thought to express a need for the sow (Held & Mendl, 2001). It was also observed that the earlier the separation, the more separation calls were exhibited which indicated separation stress (Weary et al., 1999). When piglets from different litters are mixed into one pen, an elevation of cortisol levels is observed (Merlot et al., 2004). These elevated levels of cortisol did not exhibit long-term effects. When piglets are introduced into a new pen a social hierarchy is established. It was observed that dominant piglets showed lower cortisol levels than submissive piglets (Merlot et al., 2004). It was also observed that dominant piglets had a higher weight gain than submissive piglets (Held & Mendl, 2001). All of these factors combined can contribute to the lower feed intake observed during weaning which contributes to a poor growth performance.

1.1.3 Diet composition

Transitioning from sow’s milk which is highly digestible and highly palatable (Klobasa et al., 1987), to a solid-feed which is less palatable presents another challenge. Due to all the anti- nutritional factors associated with plant-based proteins, highly digestible and palatable animal- 3 based proteins are commonly used in piglet diets. These include ingredients like: dried whey, spray dried plasma, poultry by-product meal, and fish meal. In recent times, there has been a move away from using animal-based protein mostly due to the ban in Europe prohibiting any animal-based proteins in animal feed. The rising cost of fish meal is also another reason why researchers are trying to find an alternative protein source for agricultural animals.

1.2 Soybeans

Soybean (Glycine max (L.) Merril) is a legume that is widely used for both extraction of oil and animal feed. Canada produces 4.25 million tons of soybean/year and approximately 90% of that is processed into soybean meal in solvent extraction plants in Ontario (Canadian Institute

Soy 20/20, 2008). More than half of the soybean meal produced in Canada goes to poultry and about a quarter goes to swine operations (Canadian Institute Soy 20/20, 2008). Soybeans carry many advantages such as: high protein level, excellent digestible amino acid profile, as well as their antioxidant properties. Unfortunately, there are also many negative properties of soybeans such as allergenic proteins and other anti-nutritional factors which can limit their use, especially in young animal diets. Soybeans can be processed into several different products such as soybean meal and soybean protein concentrates which eliminates some, but not all, of the anti- nutritional factors, allowing them to be used in animal feeds.

1.2.1 Positive factors of soybeans

There are several factors that contribute to soybean’s positive nutritional properties.

Soybeans are considered to be a high protein source with approximately 40% protein as dry matter (DM) (Saz & Marina, 2007). Soybeans processed into soybean meal can have crude 4 protein values ranging from 44- 48% depending on if hulls are reintroduced into the meal.

Soybeans contain an excellent profile of amino acids, providing most of the essential amino acids (Saz & Marina, 2007). Soybeans are particularly high in lysine and tryptophan, which are the amino acids low in cereal grains (Stein et al., 2008). The digestibility of amino acids can differ depending on heat processing of the soybean flakes. Soybean meal which has been autoclaved at 125 °C contained lower concentrations and reduced digestibility of the amino acids: arginine, lysine, and cysteine (González-Vega et al., 2011). This reduction in digestibility of amino acids was attributed to the Maillard reactions and melanoidins which occur when heating products (González-Vega et al., 2011). Maillard browning reactions decreases the digestibility of amino acids by irreversibly converting available lysine to unavailable lysine complexes (Friedman & Brandon, 2001; González-Vega et al., 2011). Soybeans are also high in arginine which is a great benefit to poultry as they cannot synthesize arginine due to the inactive urea cycle (Stein et al., 2008). However, soybeans are not the perfect protein source as they are deficient in methionine, the first limiting amino acid for poultry. The amino acids found in soybeans are more digestible than amino acids found in other oilseed products (Stein et al.,

2008). The amino acid digestibility and metabolizable energy values can differ depending on if the soybean meal was expelled or solvent extracted. Soybean meal which has been extruded- expelled, rather than extracted, was found to have a greater amino acid digestibility in pigs

(Baker & Stein, 2009). This is likely due to the higher percentage of soybean oil in the extruded- expelled soybean meal. It was also observed that soybean meal had 11-25% higher metabolizable energy content when compared to canola meal, dehulled sunflower meal, cottonseed meal, and peanut meal (Stein et al., 2008). Despite some of the limitations stated above, soybean meal in general is still a great protein source for livestock and poultry feeds.

Soybeans have been termed as a functional food. Asian populations with high soy consumption, have been known to have decreased risk of certain cancers (Friedman & Brandon,

2001). Some of the functional properties of the soybean has been attributed to its high levels of specific phenols, which are secondary metabolites produced by plants under normal development or under stressful conditions (Stalikas, 2010). Flavanoids belong to polyphenols and can be classified as anthocyanins, flavanols, flavones, flavonoes, and flavanols (Stalikas, 2010).

Flavonols are more commonly found in vegetables and fruits, whereas isoflavones are found in legumes (Stalikas, 2010). Isoflavones, which are isomeric to flavonoids, have been of interest to the research community. There are twelve types of isoflavones: three free aglycones genistein, daidzein, and glycitein, and their three glucosidic, three acteyl glucosidic, and 3 malonyl glucosidic conjugates (Friedman & Brandon, 2001). Isoflavones, genistein, daidzein, glycitein, biochanin A, and formononectin, are the phytoestrogen molecules found in soybean which have had a particular interest in human health research. These phytoestrogen molecules are able to bind to the estrogen receptor and can act as natural selective estrogen receptor modulators (Fritz et al., 2013). Depending on local estrogen levels, these isoflavones have been found to play either agonistic or antagonistic on the estrogen receptor (Fritz et al., 2013). Isoflavones and their role in certain cancers, especially breast and colon cancers, have been controversial. Some in vitro models suggest that genistein can directly or indirectly interact with the endoplasmic reticulum and promote tumor cell growth in breast tissue (Van Duursen et al., 2011).

Conversely, other studies have reported genestein to have antitumor and anti-angiogenic activity

(Farina et al., 2006). Other effects of soy on human health include inhibition of vascular endothelial growth factor and inhibiting tyrosine kinase, and inducing tumor suppression proteins

(Fritz et al., 2013).

The role of isoflavones in manipulating animal performance has also been investigated.

The concentration of isoflavones in soy can be altered through processing however; soybean meal still contains isoflavones (Kuhn et al., 2004). Because of its estrogen-like properties, soy isoflavones and their effect on carcass characteristic have been studied. The effects of soy isoflavones have been contradictory. Some studies have observed no effect on growth performance, carcass composition, or meat quality of pigs (Kuhn et al., 2004). Other studies indicate isoflavones produce leaner meat in barrows if fed at the level found in conventional soybean meal (Payne et al., 2001). The effects of isoflavones on gilts was also investigated which resulted in no effect on carcass trait (Payne et al., 2001).

1.2.2 Negative factors of soybeans

Despite the apparent benefits of eating soybeans, there are also many negative factors that can impact animal production. Unfortunately, these anti-nutritional factors are part of the legume and are often hard to eliminate. The first anti-nutritional factor to be discussed is the large proteins which may be allergenic to humans and animals. The seed of the soybean is important for storage of nitrogen, sulfur, and carbon (Wilson et al., 2005). Soy seed protein consists of two main fractions, 11S globulin fraction and 7S globulin fraction, which together account for 70 % to 80% of the total protein composition of soybean (Wilson et al., 2005). Soy protein has been listed as one of the “big eight” foods with the most allergens (Wilson et al.,

2005). Two important allergens in soy are glycinin (an 11S globulin fraction) and β-conglycinin

(a 7S globulin fraction). Glycinin is a polypeptide which is composed of multiple acidic (35-40 kDa) (Beardslee et al., 2000) and basic (22 kDa) (Helm et al., 2000) subunits held together by disulfide bonds. There is approximately 40-70 mg of glycinin /g in soybean meal, as indicated in

Table 1 (American Soybean Association, 2004). Glycinin has been well studied in farm animals 7 and has been known to decrease growth performance in piglets (Sun et al., 2008a; Zhao et al.,

2008; Zhao et al., 2010; Li et al., 1990). This decrease in performance can be attributed to the damage to the intestinal morphology, disorder in the immune system, and increased diarrhea

(Sun et al., 2008a; Li et al., 1990; Li et al., 1991; Zhao et al., 2008). Glycinin is a small peptide resistant to heat and most enzymatic digestion. Glycinin is partially insoluble in a piglet’s stomach (Zhao et al., 2008), which presents a further issue to the piglet as it escapes this initial digestion step and can stimulate an immune response when it reaches the small intestine. This was confirmed by the increased levels of immunoglobulins of the mucosal layer of the small intestine (IgA) and immunoglobulins in the serum (IgG) when piglets were fed glycinin (Sun et al., 2008b; Hao et al., 2009). Piglets fed glycinin had increased intestinal mast cells numbers, leading to histamine released (Sun et al., 2008a). Histamine has been known to play a role in increasing intestinal motility and secretion of water which may explain the decrease performance results and increased diarrhea in piglets fed glycinin (Liu et al., 2000; Sun et al., 2008a). It has also been reported that piglets fed increased levels of glycinin experienced villus atrophy and crypt hypertrophy in the jejenum which also indicates intestinal damage (Sun et al., 2008a). The polypeptide glycinin is composed of two subunits; an acidic and a basic subunit. The properties of the different subunits allow glycinin to escape digestion in the duodenum of the small intestine; however evidence suggest that the number of immunoreactive glycinin proteins decreases in the middle of the jejunum (Zhao et al., 2008). However, the digestion of glycinin is inefficient due to its disulfide bonds and hydrophobicity of its subunits (Zhao et al., 2008). The disulfide bonds provide strength and support to the molecular structure. Glycinin’s basic subunits are hydrophobic, and thus can aggregate, hiding cleavage sites that enzymes need to bind in order for the peptide to be digested (Kuipers et al., 2007).

β-conglycinin has also been of great interest. Like glycinin, β-conglycinin is a storage protein consisting of three different subunits: α (72 kDa), α’ (76 kDa) and β (53 kDa) (Doyle et al., 1986). Soybean meal contains approximately 10-40 mg/g of β-conglycinin (American

Soybean Association, 2004; Table 1). Similar to glycinin, β-conglycinin has been known to negatively affect piglet growth performance (Zhao et al., 2010). This decrease in growth performance is likely due to the allergenic effects, direct effect on intestinal cells, and its molecular structure. β-conglycinin can illicit an immune response, increasing IgE levels in the serum and stimulating various cytokines to be released, destroying the integrity of the intestine

(Hao et al., 2009). Apart from its allergenic effects, β-conglycinin has also been found to directly affect the small intestine by altering the cytokine expression, function, and replication in piglet intestinal cells (Chen et al., 2011). Subunits of β-conglycinin can aggregate in the small intestine, hiding enzymatic cleavage sites, leading to its ability to escape digestion (Kuipers et al., 2007). Although both allergenic proteins are mostly resistant to enzymatic digestion, β- conglycinin is found in higher concentrations than glycinin in the lower parts of the small intestine, signifying that β-conglycinin is more resistant to digestion than glycinin (Zhao et al.,

2008). Fortunately, piglets can become desensitized to both glycinin and β-conglycinin as they get older (Wang et al., 2010).

Table 1: Summary of the undesirable nutrient components of soybean meal. 1American

Soybean Association, 2004; 2Choct et al., 2010; 3Bureau et al., 1998.

Category Compound Description

Glycinin - 40-70 mg /g of soybean meal (SBM) 1 - Two subunits: acidic (35-40 kDa) and basic (22 kDa) - Allergenic - Mostly resistant to enzymatic digestion - Decreases digestibility β-conglycinin - 10-40 mg /g of SBM 1 - Three subunits: α (72 kDa), α’ (76 kDa) and β (53 kDa)

- Allergenic - Mostly resistant to enzymatic digestion - Decreases digestibility Trypsin - 4-8 mg/g of SBM 1 Inhibitors - Kunitz trypsin inhibitor (20 kDa) Protein - Bowman-Birk inhibitor (6-12 kDa) - Decreases digestibility of proteins by inhibiting trypsin and chymotrypsin activity - Heat labile Lectins - 50-100 ppm - Binds to the epithelial cells of the intestine causing intestinal damage - Heat labile Raffinose - 1- 1.2 % of SBM 1 - Indigestible due to the 1,6-glycosidic linkage Stachyose - 1.5 - 5 % of SBM 1 Carbohydrates - Indigestible due to the 1,6-glycosidic linkage Soluble NSP - 17% of SBM 2 - Changes viscosity impairing nutrient enzymatic hydrolysis Insoluble NSP - 8% of SBM 2 - Decrease total tract ileal digestibility of dry matter Phytic acid - 0.6 % of SBM 1 - Unavailable source of phosphorus which can chelate Other minerals and contribute to pollution Soyasaponins - 0.87% of SBM3 - Affects intestinal morphology - Affects lipid membranes

Apart from its allergenic aspect, soybeans also contain a high amount of other anti- nutritional factors. These anti-nutritional factors are trypsin inhibitors, lectins, and phytic acid.

Trypsin and chymotrypsin are two of the key enzymes responsible for protein degradation in mammals and birds. They belong to the family of serine proteases and is produced by the pancreas and secreted in the duodenum. Soybeans, similar to other plants, use trypsin inhibitors as a defence mechanism against pests (Jamal et al., 2013). There are two well-known trypsin inhibitors in soybeans: the Kunitz inhibitor and the Bowman-Birk inhibitor. The Kunitz trypsin inhibitor (20 kDa) inhibits only trypsin, whereas the Bowman-Birk inhibitor (6-12 kDa) can also inhibit chymotrypsin (Birk, 1985). Soybeans have approximately 32-123 mg of trypsin inhibitor/ gram of protein while soybean meal only has 4-8 mg/gram (Anderson & Wolf, 1995; American

Soybean Association, 2004). Protease inhibitors bind with proteases which inactivates the enzyme, reducing the animal’s ability to digest proteins. This is evident in rats fed raw soybean which experienced pancreatic hypertrophy and a 60% growth depression (Rackis et al., 1979;

Struthers et al., 1983). Although pigs fed 40% raw soybean flour did not experience a hypertrophic pancreas there was 29% reduction in trypsin activity and an 80% reduction in chymotrypsin activity with an overall 84% growth depression (Struthers et al., 1983).

Fortunately, these trypsin inhibitors are heat labile proteins and can be mostly inactivated with heat treatment (Anderson & Wolf, 1995).

Soybeans also contain glyco-proteins called hemagglutinins, commonly referred to as lectins. Lectins are globulins that contain a carbohydrate-binding domain which allows it to bind to red blood cells of rabbit but not cattle or sheep (Csaky & Fekete, 2004). Lectins are not susceptible to proteolytic digestion in the duodenum which presents the opportunity for lectins to

11 bind to the epithelial cells of the small intestine (Csaky & Fekete, 2004). Soybeans contain 5.7 mg of lectin/g which is greatly reduced to 0.88 mg/g in soybean meal (Canadian National Grain

Institute, 2010). Feeding animals soybeans in which lectins have not been denatured has resulted in decrease growth performance in pigs, poultry, and mice (Palacios et al., 2004; Zhang et al.,

1993; Friedman et al., 1991). In a study which compared the growth performance of chicks and piglets fed modified soybeans, conventional soybeans, and conventional soybean meal, it was observed that chicks and piglets fed lectin-free soybean meal had improved average daily gain and feed conversion ratios compared to that of animals fed raw soybeans (Palacios et al., 2004).

There was also an additive benefit effect when both trypsin inhibitor and lectins were removed from the feed (Palacios et al., 2004). This result may be attributed to lectin’s ability to bind to intestinal mucosa layer which results in an increased secretion of proteins, increasing endogenous nitrogen losses (Palacios et al., 2004). It was also observed that binding to the brush boarder of the intestinal epithelial cells can affect cell viability, the crypt, and the weight of the tissue (Liener, 1994). Lectins do not present a problem if the soybeans are heat treated as in the case of soybean meal. It was reported, however, that lectins can be recovered in soybean meal, which are able to still demonstrate epithelial binding ability, suggesting that a portion of lectins are not destroyed with heat treatment (Maenz, Irish, & Classen, 1999). It is interesting to note that not all types of lectins affect piglet performance in a negative manner. Lectins from kidney beans can actually improve the small intestine development of a piglet and could be potentially used in improving piglet performance during early weaning (Thomosson et al., 2007).

Plants store phosphorus in a form called phytic acid. When phytic acid is bound to a mineral in the seed it is referred to as phytate. Approximately 0.6% of soybean meal consists of

12 phosphorus bound as phytic acid as demonstrated in Table 1 (American Soybean Association,

2010). Phytase can convert phytic acid into an available source of phosphorus but non-ruminant animals do not have phytase and thus require the addition of inorganic phosphorus to be supplemented in diets. This inefficient use of phosphorus has led to issues regarding pollution to the environment. In addition, phytic acid also has the ability to chelate divalent metal ions such as zinc and calcium, decreasing its bioavailability in the gut, which in extreme cases could result in parakeratosis and a depressed growth rate in pigs (Oberleas, Muhrer, & O'dell, 1962).

Carbohydrate concentration is variable by region but is generally around 35% of the soybean (Karr-Lilienthal et al., 2005). These carbohydrates can be categorized as non-structural and structural carbohydrates. Non-structural carbohydrates consists mainly of low molecular weight sugars, polysaccharides (mainly starch), and oligosaccharides. Soy oligosaccharides raffinose and stachyose have also been thought to have a negative impact when fed to animals.

Raffinose and stachyose are structurally very similar and belong to a family called raffinose family of oligosaccharides (RFO) (Figure 1). Raffinose is a tri-saccharide made up of one galactose, glucose, and fructose unit. Stachyose is a tetra-saccharide consisting of two galactose units, one glucose unit, and one fructose unit. Together, raffinose and stachyose make up 5.5-6.2

% of soybean meal (American Soybean Association, 2004; Table 1). RFOs are considered to be anti-nutritional because they are indigestible to non-ruminant animals due to the lack of α- galactosidase enzyme required to digest the raffinose and stachyose. Feeding a non-ruminant animal feed containing RFOs can result in gastrointestinal discomfort and may result growth performance reductions. Piglets fed soy protein isolates, which is soybean meal processed to remove oligosaccharides and other factors, were found to have improved growth rates when

13 compared to piglets fed conventional soybean meal (Sohn et al., 1994). Other studies have reported that stachyose can negatively affect ileal digestibility of dry matter and energy in pigs

(Van Kempen et al., 2006). A decrease in piglet performance and an increase in diarrhea has been observed when stachyose and raffinose levels were increased (Liying et al., 2003). The rate of passage also increased when stachyose levels increased, reducing the amount of time the lower gut flora has to metabolize the oligosaccharides and thus results in higher excretion in the feces (Liying et al., 2003). On the contrary, other studies have found no effect of oligosaccharides on the incidences of diarrhea with only an effect on apparent or true ileal dry matter, nitrogen or amino acid digestibility to a very small degree (Smirick et al., 2002).

Soybeans, as well as other plants, contain steroid glycosides called saponins. There is a class of saponins, called soyasaponin, are found in soybeans and may have an anti-nutritional effect on fish (Bureau et al., 1998). Their anti-nutritional affect may be due to their ability to alter intestine function or their effect on calcium-dependent potassium channels (Bureau et al.,

1998). Unlike other anti-nutritional factors, processing does not eliminate soyasapoinins, unless it has been extracted with alcohol, such as in soy protein concentrate (Bureau et al., 1998). Fish fed soybean meals have been found to suffer from enteritis (Sorensen et al., 2011). It was observed that there are components in purified alcohol extracts of soybeans that can negatively affect the growth performance of both chinook salmon and rainbow trout (Bureau et al., 1998).

One study showed that Atlantic salmon fed soybean molasses with different subfractions of saponins were found to develop enteritis (Knudsen et al., 2007). The authors attributed the results due to soyasponins alone or in combination with other soybean anti-nutritional factors

(Knudsen et al., 2007). Another study found no negative effects on nutrient digestibility or growth performance of Atlantic salmon when fish meal was supplemented with soyasaponins but

14 did observe a decreased cholesteric effect (Sorensen et al., 2011). It was also found that soysaponins can be hydrolyzed into free aglycones by the intestinal microflora in humans, chicks, rats, and mice (Hu et al., 2004; Gu et al., 2002).

Structural carbohydrates consist of the insoluble and soluble non-starch polysaccharides

(NSP) which together can account for 25% of soybean meal (Choct et al., 2010). Of this 25%,

17% consists of the soluble NSP pectin (Choct et al., 2010). There are three types of pectin polysaccharides commonly present in the soybean meal: rhamnogalacturonans, arabinogalacton, and xylogalacturonon (Choct et al., 2010). Soluble NSP are easily fermentable which increases digesta transit time by forming a gel-like matrix which delays gastric empting (Fabek et al.,

2014; Montagne et al., 2003). In non-ruminants there has been evidence suggesting that soluble

NSP can have an anti-nutritional effect and impact overall animal performance. In poultry, diets containing high amounts of soluble NSP increased the viscosity of the digesta which decreased the nutrient and enzyme interaction in the intestine, negatively affecting the digestibility of other nutrients (Smits & Annison, 1996). The increase in viscosity, as well as the decrease in gastric emptying, allow for other bacteria to repopulate the intestine, negatively disrupting the balance of the intestine (Angkanaporn et al., 1994; Choct et al., 2010). Similar changes were observed in the piglet but were decreased as the piglet aged (Choct et al., 2010). Increasing soluble NSP in the diet of weaning piglets demonstrated an increase in E.coli proliferation and an increased risk of developing swine dysentery (Choct et al., 2010). An explanation for these observations is that the increase in digesta transit time allows the bacteria to proliferate (Kim et al., 2012).

Approximately 8% of soybean meal consists of the insoluble NSP cellulose (Choct et al.,

2010). Cellulose is a high-energy molecule consisting of multiple glucose subunits linked

15 together through a β-1,4 glycosidic linkages (Figure 2). The breakdown of cellulose is complex and requires three different groups of enzymes: cellobiohydrolases, endo-β-1,4-glucanases, and a

β-glucosidase, collectively referred to as the cellulase enzyme complex (Perez et al., 2002).

Ruminant animals have microbes in their rumen that can digest cellulose; however to non- ruminant animals cellulose is virtually indigestible until fermentation in the large intestine.

Insoluble NSP are indigestible and do not increase the viscosity and thus decrease digesta transit time and have been found to increase faecal bulking emptying (Montagne et al., 2003). These properties cause insoluble NSP to have both beneficial and anti-nutritional qualities, depending on if above a minimal level. Microbes in the intestine can utilize insoluble NSP and produce beneficial short chain fatty acids, such as butyrate (Molist et al., 2009). Increases in butyrate concentration are likely due to the longer fermentation time and the more suitable substrate

(Molist et al., 2009). Butyrate has a trophic effect on the colonocytes which have been found to promote both small and large intestine growth (Molist et al., 2009; Montagne et al., 2003).

Young animals utilize insoluble NSP less efficiently than older animals (Montagne et al., 2003) and thus have an anti-nutritional effect on piglets. Feeding piglets diets with increasing percentages of NSP found in soybean hulls was found to negatively affect the daily growth rate and feed conversion compared to that of the control group (Freire et al., 2000).

A) Gal 1

* 6

Gal 1

* 6

Glu 2

Fruc 1

B) Gal 1

* 6

Glu 2

Figure 2 Fruc 1

Figure 1: Simplified figure illustrating the degradation of soy oligosaccharides. A) Soy oligosaccharide stachyose. B) Soy oligosaccharide raffinose. Fruc= fructose; Glu= glucose;

Gal= galactose; *= α-1,6-glycosidic linkage. Figure adapted from previous study (Choct et al.,

2010).

Glu Glu 1 Glu 2 Glu Glu 2 Glu 2 Glu Glu 2 n Cellulose

Glu 3 Glu Glu 3 Glu Glu 3 Glu

Cellobiose Cellobiose Cellobiose

Figure 3 Glu Glu Glu Glu Glu Glu

Multiple glucose molecules

Figure 2: A simplified diagram illustrating the degradation of cellulose. Glu= glucose subunit;

n= number of glucose subunit repeats; 1= cleavage site for cellobiohydrolase; 2= cleavage site

for endo-β-1,4-glucanase; 3= cleavage site for β-glucosidase. Figure was adapted from Deacon,

2005.

1.3 Current applications used to improve soybean

As previously mentioned, there are various negative aspects of soybeans that put restrictions on the quantity of soybean meal that can be used in piglet diets. Fortunately, some of the anti-nutritional factors, such as trypsin inhibitors and lectins are heat-labile and can be reduced during the processing of soybeans into soybean meal. However, some of the anti- nutritional factors of soybeans are heat stable and are still present when young animals are fed soybean meal. There are some current applications being used in the agricultural industry which try to overcome these anti-nutritional factors such as: further processing, biotechnology, breeding strategies, addition of enzymes, and microbial fermentation.

1.3.1 Processing

Processing soybean into soybean meal is one step which reduces the anti-nutritional factors that are sensitive to heat damage. However, there are some negative factors that are not completely eliminated even after the processing into soybean meal. Soybean white flakes can still be further processed into soy protein concentrates (SPC) and soy protein isolates (SPI) which has been found to improve the use of soy as a protein source for young animals. The additional processing involved in creating SPC removes most of the oligosaccharides raffinose, stachyose, and sucrose, effectively increasing the percentage of crude protein to as high as 67%

(Lenehan et al., 2007). There are three main processes that effectively remove the oligosaccharides: extraction with aqueous ethanol, extraction with water at its isoelectric pH

(4.5) and denaturing the protein with moist heat before extraction with water (Lusas & Riaz,

1995). SPC has higher amino acid digestibility than soybean meal which resulted in an improved pig growth performance compared to pigs fed regular soybean meal (Sohn et al.,

1994). It was also noted that different processing techniques, moist-extraction or dry-extraction, can affect pig performance with moist-extracted SPC having a more positive effect than dry- extraction (Friesen et al., 1993). Further processing of soybeans appears to be beneficial in removing the anti-nutritional factors of soybean but additional heating must be properly controlled as excess heat can result in amino acid damage which greatly reduces piglet performance (Friesen et al., 1993). Additional processing may resolve the carbohydrate issue with soybean meal; however, some commercially available SPI demonstrated proteins larger than 37 kDa with less intense bands of the two subunits of β-conglycinin still being present

(Song et al., 2010). Although the additional processing of soybeans into SPI and SPC is beneficial, the high cost of the product limits the use in animal agricultural industries (Opazo et al., 2012).

1.3.2 Biotechnology and selective breeding strategies

Biotechnology can also be used to improve the anti-nutritional aspect of soybeans.

Biotechnology may involve the manipulation of the genome of an animal or plant for the purpose of improving the organism for a particular industry. In Canada, soybeans have already been modified to be resistant to herbicides to try to reduce costs to farmers, this is considered to be first-generation traits (Canadian International Grain Institute, 2010). Manipulation of the soybean genome to reduce anti-nutritional factors or to improve the nutrients for animal and human consumption is considered to be second-generation traits (Canadian International Grain

Institute, 2010). It has been observed that raffinose and stachyose metabolism is not needed for seed germination (Dierkin & Bilyeu, 2009) and Kerr and Sebastian (2000) have developed a strain of soybean that contains high sucrose but a low oligosaccharide content. Similarly, there

20 have been mutations introduced into the phytic acid gene which results in a low phytic acid soybean (Yuan et al., 2007). Biotechnology is a very appealing solution to some of the anti- nutritional factors of soybean meal; however, it is still a controversial solution due to food safety and fears of the public.

Instead of introducing mutations into the genome, plant breeders have been able to identify plants with spontaneous mutations which have been beneficial for industrial uses. Plant breeders use this selective breeding technique to successfully produce soybeans with reduced anti-nutritional factors. There have been mutations identified in the genes responsible for β- conglycinin and glycinin proteins which results in these proteins to be absent in the soybean

(Clarke and Wiseman, 2000). The literature also reveals genetic selection for soybeans with low phytic acid, low trypsin inhibitor, and low lectins which have already been developed and may be a solution to a lot of the issues caused by eating soybean products (Clarke and Wiseman,

2000). The major drawback to selective breeding for positive traits in soybean meal is that it is time consuming. Identifying plants which possess genetic mutations involves screening of multiple plants and using different selection pressures, which are time consuming. Selective breeding may be a strategy to remedy one of the anti-nutritional factors of soybean at a time but it is not a complete solution for all of the negative factors.

1.3.3 Addition of enzymes

Most of the negative factors of soybeans are anti-nutritional because young animals do not possess a source of endogenous enzymes that could digest the factors. Research has shown that adding exogenous enzymes to the soybean can actually reduce the anti-nutritional factors. It 21 has been found that treating soybean meal with proteases produced by Bacillus and Aspergillus species was successful in reducing the allergenic proteins of soybean meal, effectively improving piglet performance (Rooke et al., 1998). Several microbes have the ability to produce phytase which can digest phytic acid, releasing phosphorus which can be used by animals. Several studies have shown that supplementing soybean meal with purified phytase of microbial origin can effectively increase the bioavailability of phosphorus and decrease any chelating effects observed due to phytic acid (Jongbloed et al., 1992). Treating soybean meal with α- galactosidases isolated from Lactobacillus or Aspergillus species also decreased the oligosaccharide stachyose concentrations which improved the nutrient digestion leading to an improved piglet performance (Yoon & Hwang, 2008; Pan et al., 2002). Supplementing β- glucanases and xylases originally from Trichoderma longibrachiatum resulted in a decrease of

NSP, leading to an increase in apparent ileal digestibility of amino acids, decrease in the viscosity of the digesta, which all led to an improvement in overall piglet performance (Yin et al., 2001). The literature suggests that most of the anti-nutritional factors in soybean meal could be reduced using enzyme supplementation. One of the downfalls to using this approach is it is costly to produce and purify all necessary enzymes.

1.3.4 Microbial fermentation

Lastly, microbial fermentation is another way of improving soybeans in order to be fed to humans and animals. Fermentation is one of the oldest techniques widely used in the many cultures. There are many soy products that, when fermented, have improved nutritional value.

Several different microbes can be used to ferment soybeans. Some of the most commonly used microorganism used are: Aspergillus species, Bacillus species, and Lactobacillus species. One

22 of the first studies to experiment with utilizing the fermentation method in soybean meal was

Hong et al., (2004). The authors found that the soybean meal fermented with Aspergillus oryzae for 48 hours was successful in degrading the large allergenic proteins, increasing crude protein content, and improving the non-essential amino acid profile (Hong et al., 2004). One of the short comings of this study was the essential amino acid profile was unchanged which suggested that the mould preferentially uses some amino acids (Hong et al., 2004). Similar to these results, soybeans fermented with A.egypticus has been found to increase the content of free amino acids and antioxidant capacity (Zhang et al., 2007). The effect of feeding soybean meal previously fermented with A.oryzae to animals was also investigated. It was observed that chicks fed the fermented soybean meal had increased villus lengths and deeper crypts (Feng et al., 2007a). It was also observed that the intestinal enzymatic activities were improved in birds fed fermented soybean meal rather than conventional soybean meal, while growth performance was improved but more so starter birds than grower birds (Feng et al., 2007a). Similar to poultry, piglets were also seen to have improved performance when fed fermented soybean meal. Fermented soybean meal at inclusion levels of 10-15% were found to improve feed efficiency, amino acid digestibility, and increase blood urea nitrogen in piglets (Cho et al., 2007). The increase in blood urea nitrogen levels was attributed to the high utility value of the fermented soy protein (Cho et al., 2007). Protein from fermented soybean meal was found to contain more digestible amino acids than conventional soybean meal but had similar standardized ileal digestibility of amino acids when compared to that of enzymatically hydrolyzed soybean meal as well as fish meal

(Cervantes-Pahm & Stein, 2010). The improvement in piglet performance and overall increase in digestibility of fermented soybean meal is due to the elimination multiple types of anti- nutritional factors found in soybeans. Trypsin inhibitor is heat labile and thus is mostly eliminated with heat treating of soybean meal. However residual trypsin inhibitor activity in

23 heat treated soybean meal which was successfully decreased with fermentation (Hong et al.,

2004). Trypsin inhibitor negatively affects protein digestibility in piglets and thus, reducing this increases the digestibility of soybean meal (Kim et al., 2010). Moulds like Aspergillus species are known for secreting a large amount of extracellular enzymes, including proteases, amylases,

α-galactosidases (Cho et al., 2007).

Bacteria have also been used to ferment soybeans with similar beneficial aspects seen with fermented soybean meal using mould. Several Bacillus strains have been used and isolated from different traditional fermented foods. Fermenting soybeans with Bacillus natto has been found to improve antioxidant capacity compared to that of soybeans (Hu et al., 2010) and soybean meal fermented with Bacillus subtilis (Wongputtisin et al., 2007). Soybean meal fermented with Bacillus subtilis has also been found to have a positive effect on piglets. It was observed that piglets fed fermented soybean meal had improved feed intakes, increased villus heights, and improved intestinal enzymatic activity (Feng et al., 2007b). Soymilk fermented with Bacillus natto was found to have reduced IgG reactivity to allergens found in soymilk indicating the bacteria was successful in degrading the allergenic proteins (Yamanishi et al.,

1995). B.subtilis fermented soybean was seen to be superior to that of A.oryzae or R.oryzae fermentation, in terms of reducing allergenic proteins and improving amino acid profiles (Frias et al., 2007). There have also been other beneficial effects of fermenting soybeans using Bacillus as the inoculum. Spores of some species of Bacillus are commonly used as a probiotic in animal feeds with observed improvements in both sow and piglet performance (Kyriakis et al., 1999;

Alexopolilos et al., 2004). Interestingly, growing pigs that were fed a balanced diet with the inclusion of 5% Bacillus subtilis fermented soybean meal were found to have similar average

24 daily intake and average daily gain values as the piglets fed balanced diet with the addition of

100 mg of chlorotetracycline/ kg of feed when challenged with Salmonella Typhimurium; both treatments improved growth performance compared to the piglets in the antibiotic-free control group (Gebru et al., 2010). Some strains of Bacillus licheniformis previously isolated from fermented foods were found to have antimicrobial activity (Kim et al., 2004) which may be a beneficial way of reducing the use of antibiotics in farm animals. Similar to the mould fermentation, the elimination of anti-nutritional factors found in soybean meal can be one of the factors contributing to the beneficial effects on animal performance. The probiotic and increase antioxidant effects of fermented soybean meal with Bacillus species may be another factor contributing to the beneficial effects.

Lactic acid bacteria, such as Lactobacillus and Lactococcus species, are commonly used to ferment milk products such as cheese. Lactobacillus species can secrete α-galactosidase

(Refstie et al., 2005) which is suggests that these bacteria can be used to break down soy oligosaccharides. Fermenting soy white flakes with Lactobacillus brevis for 36 hours resulted in a moderate decrease of raffinose concentrations to 36.7 g/kg from the 41.0 g/kg found in the unfermented white flakes (Refstie et al., 2005). Contents of some essential amino acids, notably arginine, methionine, threonine and valine were increased with fermentation compared to unfermented white flakes (Refstie et al., 2005). The fermentation was also able to reduce trypsin inhibitor levels compared to the unfermented control (Refstie et al., 2005). Lactobacillus plantarum also has the proteolytic activity necessary to degrade the allergenic proteins found in soy flour when fermented in a liquid-state (Frias et al., 2007). It was also found that this method of fermentation resulted in reduced extractable protein compared to the solid-state method of

25 fermentation (Frias et al., 2007). Lactobacillus plantarum with additional supplemented proteases, were able ferment soybean meal and increase the amount of free amino acids and decrease allergenic proteins (Amadou et al., 2011). Similar to fermented soybean meal using

Bacillus species, soybeans fermented with Lactobacillus species were found to have improved antioxidant profiles (Pyo et al., 2005). This increased antioxidant activity was attributed to that bacteria’s ability to convert the soy isoflavones daidzin and genistein into their bioactive components daidzein and genistein (Pyo et al., 2005). Some Lactobacillus species had also been found to be probiotic and which could lead to an additional benefit to piglet performance (De

Angelis et al., 2006). Atlantic salmon fed 40% protein from fermented soy white flakes were found to have improved feed efficiency and less intestinal pathologies when compared to the fish fed unfermented white flakes (Refstie et al., 2005).

1.4 Summary and rationale

Soybean meal remains to be one of the most widely used protein sources in animal feeds.

Its high content of crude protein, excellent amino acid profile, and isoflavones are just some of the benefits of using soybeans. Unfortunately soybean’s unprocessed form contains several allergenic proteins and other anti-nutritional factors. Industry pressures have adopted the early weaning practice for piglets. Feeding the desired amount of soybean protein to the early weaned piglet becomes a challenge due to its allergens, other anti-nutritional factors and the underdeveloped gut of the piglet. Microbial fermentation of soybean meal is a means for decreasing these undesirable factors, improving the amino acid profile, increasing antioxidant capacity and potentially providing a protective benefit (probiotic) for the piglet depending on the bacterial strain used. Identifying new food-derived bacteria that are capable of effective

26 fermentation would offer opportunity for more cost effective fermentation of soybean meal, and thus be beneficial to the animal production industry.

2.0 HYPOTHESES

Isolating bacteria derived from fermented food and intestine of the Ctenopharyngodon idella

(grass carp) should yield bacteria that demonstrate high protease, cellulase, and amylase activity.

Using bacteria with high enzymatic activity should improve the nutritional value of soybean meal largely through the reduction of allergenic proteins and other anti-nutritional factors typically found in soybean meal.

3.0 OBJECTIVES

1. Screen bacteria from various fermented food sources and grass carp to isolate the bacteria

with the highest protease, amylase, and cellulase activity.

2. Characterize the potentially useful bacteria

3. Determine the efficiency of fermenting soybean meal using isolated bacteria, by

characterizing the soluble protein content, protein profile, crude protein and amino acid

concentrations, detect soy allergens, and raffinose and stachyose concentrations in partly

fermented soybean meal.

4.0 MATERIALS AND METHODS

4.1 Isolating and screening bacteria with high enzyme activity

4.1.1 Primary screening for bacteria with protease activity from fermented food

One of the main ways of improving the nutrient value of soybean meal is decreasing the high-molecular weight and allergenic proteins. To successfully accomplish this, various bacteria from different fermented foods were screened. Five types of fermented bean curds and fermented bean products were obtained from local Chinese supermarkets for bacteria isolation.

Approximately 1 cm3 of each source was cut using a clean scalpel and put into a 1.5 mL

Eppendorf tube previously filled with 500 µL of sterile water. The mixture inside the tube was mixed using a vortex. Serial dilutions were performed by taking 10 µL of the solution and re- suspending it in 990 µL of sterile water to obtain a dilution of 10-2. Subsequently, 10 µL of the

10-2 diluted sample was re-suspended in 990 µL of sterile water, obtaining a dilution of 10-4.

This was repeated to obtain 10-6 and 10-8 dilutions. Serial dilutions were performed to obtain approximately 100 colonies of bacteria per culture plate to allow isolation of clearly defined individual colonies. Diluted samples were screened for ability to grow on soymilk agar using methods previously described with modifications (Amoa-Awua et al., 2006). In brief, soy milk agar was made by mixing together 1 gram of soy milk powder (Bulk Barn, Canada) and 2.5 grams of BactoAgar (Becton, Dickinson & Company, U.S.A) with 100 mL of water and autoclaved for 25 minutes at 121°C. 10 mL of this soymilk agar was poured into petri dishes and left to cool. 100 µL of the 10-4 and 10-6 diluted of each of the five sources was pipetted onto the soymilk agar plates and spread using sterile glass beads, aseptically using a microbiology cabinet. Plates were inverted and incubated for 24 hours in 37 °C. Cultures that were able to grow on this minimal media and were able to demonstrate a clear zone around its colony was

28 selected for the second round of screening. A glycerol stock of each selected bacteria was created and stored at – 80 °C in a cryopreservation vial. To do this, a liquid culture for each of the selected bacteria was generated by inoculating the colony into 1 mL of previously prepared sterile Bacto Brain Heart Infusion (BHI) (Becton, Dickinson & Company, U.S.A) culture media according to manufacturer’s protocol. Each of the liquid cultures were incubated for 24 hours at

37 °C. Glycerol stocks were prepared by adding 250 µL of 50% glycerol and 250 µL of bacteria liquid culture into a cryopreservation vial. Bacteria from this liquid culture was also streaked onto a previously prepared BHI agar plate and incubated for 24 hours at 37 °C. BHI agar plates were prepared following manufacturing recommended procedures. These plates were used for phenotypic characterization of the bacteria using MALDI-TOF MS.

4.1.2 Primary screening for cellulase activity from grass carp source

In order to reduce the amount of non-starch polysaccharides in soybean meal, bacteria which secreted cellulase, in particular β-1,4-glucanase, were screened. In order to achieve this we chose the herbivorous fish, the grass carp (Ctenopharyngodon idella) as the source for selecting cellulolytic bacteria. Two sets of intestines from freshly killed grass carp were obtained from a local Chinese supermarket and kept on ice until processed. Bacteria were isolated according to the methods previously described (Li et al., 2009). In brief, the intestines of each of the two fish were stretched out and the proximal and distal ends of the intestine were identified. The intestine was sectioned by introducing two cuts using a scalpel at approximately

1/3 and 2/3 length from the proximal end of the intestine. The most proximal third of the intestine was considered to be the “beginning” of the intestine, the second third the “middle” and the most distal third the “end”. Contents from each of the three sections were emptied into a 15

29 mL sterile conical tube and mixed using a vortex. One mL of the content was diluted in 9 mL of sterile deionized water and vortexed to create a more homogenous solution. Serial dilutions of each section’s contents of both fish intestines were performed as described above. 100 µL of liquid from each of the serial diluted solutions (10-2 , 10-4, 10-6, and 10-8) were plated on previously prepared Luria Bertani (LB), BHI, and DeMan, Rogosa and Sharpe (MRS) (Oxoid,

England) agar plates and spread using sterile glass beads. BHI and MRS agar plates were prepared according to manufacturer’s procedures with the addition of 1% (w/v) BactoAgar

(Becton, Dickinson, & Company, U.S.A) and autoclaved. LB agar plates were prepared by adding 1.0 gram of Tryptone Powder (Bio Basics Canada INC, Canada), 0.5 grams of Yeast

Extract (Bio Basics Canada INC, Canada), 1.0 grams of NaCl (Fisher Scientific, U.S.A) to 80 mL of deionized water stirred on a magnetic plate with a magnetic stir bar. The pH of the solution was adjusted to 7.5 using NaOH (Fisher Scientific, U.S.A), 1.5 grams of BactoAgar was added, and more deionized water was added to reach 100 mL before being autoclaved for 25 minutes at 121 °C. These plates were inverted and incubated at 37 °C overnight. Any colonies present on the plates were blotted onto previously prepared soymilk agar and carboxymethyl cellulose (CMC) (Acros Organics, U.S.A) plates using sterile filter paper, to test if the colony can grow using soybean nutrients and has cellulose degrading activity. CMC agar plates were prepared according to previously described procedures with slight modifications (Sazci et al.,

1986). CMC agar plates were prepared by adding 1.0 grams of CMC and 1.5 grams of

BactoAgar to 100 mL of deionized water and autoclaved for 25 minutes at 121 °C. Soymilk and

CMC agar plates were inverted and incubated at 37 °C for 72 hours. CMC agar plates were stained by flooding the agar plates with 1.0 % (w/v) Congo Red Staining Solution for 15 minutes; Congo Red solution was prepared by adding 0.1 gram of Congo Red Dye (Fisher

Science Education, U.S.A) to 10 mL of deionized water and mixed thoroughly until dye was

30 dissolved. Stained agar plates were de-stained with 1M NaCl (Fisher Scientific, U.S.A) by first by discarding any excess stain into a waste container and flooding the plates with 1M NaCl with gentle agitation for 15 minutes (Ruijssenaars & Hartmans, 2001 ). Colonies that exhibited an orange halo after being de-stained were matched up to the colonies that grew on the soymilk agar. Colonies that exhibited an orange halo and were able to grow on the soymilk agar were selected for the second round of screening.

4.1.3 Second round of screening

The first round of screening was intended to identify isolates, from each of the five fermented food sources and the grass carp, which demonstrated high protease and cellulase activity, respectively. One of the factors required to create an optimal fermentation is a shorter length of fermentation. In order to achieve this, the bacteria selected must grow and secret large concentration of extracellular enzymes in a short period of time. The second round of screening sought out to rank the protease, cellulase, and amylase activity of the isolates. To identify the bacteria which demonstrate the highest protease, amylase, and cellulase activity, we needed to first extract the extracellular enzymes of each bacterium. Crude enzymes of the bacteria were extracted according to methods previously described (Chantawannakul et al., 2002). First, a single colony from each of the selected bacteria was obtained from the previously streaked BHI agar. The colony was inoculated into 1 mL of BHI culture media and incubated for 18 hours at

37 °C. A commercially available source of B. subtilis ATCC 6633 was used as a positive control due to its previous observation for production of protease activity (Dias et al., 2008), amylase activity (Mitrica & Granum, 1979) and cellulase activity (Mawadza et al., 2000). After the 18 hours of incubation, each of the liquid cultures were emptied into a clean 1.5 mL Eppendorf tube

31 and centrifuged at 10,000 rpm for 15 minutes at 4 oC. Centrifuging the liquid culture allows the enzymes to be separated from the bacteria pellet so that only secreted enzyme containing supernatant can be harvested. Agars for screening for protease activity, using 1.0% (w/v) soymilk agar, and cellulase activity, using 1.0% (w/v) CMC agar, were prepared in similar methods as described in the first round of screening. Identification of amylase activity was conducted by using 1.0% corn starch agar plates as previously described (Amoa-Awua et al.,

2006). To prepare starch agar plates, 1 gram of corn starch (Bulk Barn, Canada) and 1.5 grams of BactoAgar was added to 100 mL of deionized water and autoclaved for 25 minutes at 121 °C.

To screen for each of the three enzymes, 15 mL of their respectful nutrient agars was poured onto a petri dish (90 mm x 15 mm) and allowed to settle on a flat surface. Once the agars were solidified, a 7 mm hole was punctured into the middle of the plate using the end of a sterile P-

100 pipette tip. Subsequently, 20 µL of the crude enzymes extract was pipetted into the 7 mm hole for each of the three nutrient agar plates and, after five minutes, were inverted and incubated at 37 °C for 72 hours. An additional plate, for each of the three select agars, was filled with 20

µL of just blank culture media to be used as a negative control. For protease activity no destaining was required and clearance zone measurements were obtained from measuring the length from the edge of the clearance zone to the edge of the bacterial growth. For cellulase activity, plates were stained with 1.0% (w/v) Congo Red Staining solution and destained using

1M NaCl, as detailed in first round screening. Clearance zone measurements were taken from the edge of the punctured hole to the outer edge of the orange halo. To detect amylase activity, corn starch agar plates were stained with Gram’s Iodine Stain as previously described (Amoa-

Awua et al., 2006). Amylase clearance zone measurements were obtained by measuring the length from the edge of the punctured hole to the edge of the clearance zone. Each observation was replicated three times using fresh inoculums each time.

4.2. Identification of bacteria selected

4.2.1: MALDI-TOF MS:

All the bacteria that were selected for the second round of screening were also identified.

Each isolate was streaked from the glycerol stock onto a previously prepared BHI agar plate, inverted, and incubated for 18 hours at 37 °C. Colonies on agar plates were submitted to the

Animal Health Laboratory to be identified using a matrix-assisted laser desorption ionization- time of flight mass spectrometry (MALDI-TOF MS, Bruker, Canada). Briefly, a fresh cultured bacterial colony was spotted onto the target plate (Bruker) and left to air dry. The sample was overlayed with 1 µL matrix solution mixed with an organic solvent solution made of 50% acetonitrile (Sigma Aldrich, Canada) and 2.5% trifluoroacetic acid (AnalaR Normapur through

VWR, Canada). The target plate was air dried then placed onto the MALDI-TOF instrument

(Bruker) for analysis. Peptide mass fingerprint spectra was read from the target plate and scored using MALDI-TOF MS software and database (MALDI Biotyper 3.0, Bruker). The software automatically identified an isolate to species level if the score was 2.0 to 3.0, or to genus level if the score was 1.7 to 1.999, and no reliable identification is generated if the score was less than

1.7.

4.2.2: PCR Amplification of partial 16S rRNA gene and gyrB gene:

The next step required for identification of the bacteria used in the second round of screening is sequence analysis of two genes: partial 16S ribosomal RNA gene and gyrB gene.

Genomic DNA of each isolate was extracted from the bacterial cells using the PureLink

Genomic DNA Mini Kit (Invitrogen, U.S.A) according to manufacturers’ instructions. The 16S rRNA gene in the DNA was amplified using PCR previously described (Cai et al., 2003). In

33 brief, primers BSF8/20 (5’-AGAGTTTGATCCTGGCTCAG-3’) and BSR534/18 (5’-

ATTACCGCGGCTGCTGGC-3’) were used to amplify the 16S rRNA gene with the expected product size of around 500 bp. For the three genes the PCR reaction had 2 µl of extracted DNA as a template, 3 U/100 µL of Taq Polymerase (Invitrogen, U.S.A), 0.1 mM dNTPs, 0.2 µM of each primer, and 1X PCR Buffer for a total of 25 µL per reaction. For the 16S rRNA gene, PCR was performed using the PCR thermo cycler under the following conditions: 95 °C for 3 min; 35 cycles of amplification with template denaturing at 95 °C for 20 sec, primer annealing at 55 °C for 30 sec, and primer extension at 72 °C for 90 sec; with a final extension at 72 °C for 5 min.

After the reaction was complete, the concentration of the PCR products was measured using a

BioPhotometer Plus spectrometer (Eppendorf, U.S.A) to ensure that the minimum concentration of DNA needed for sequencing was obtained.

In order to further identify the strain of the isolates the DNA gyrase subunit B (gyrB) gene was sequenced. The gyrB gene is very fast evolving gene, necessary for DNA replication, and is distributed universally among bacteria species (Yamamoto & Harayama, 1995), thus making it a perfect candidate gene for bacteria identification. To sequence the gyrB gene, two sets of primers were ordered from Integrated DNA Technology (Iowa, U.S.A). The first set of primers was:

UP-1: 5’-GAAGTCATCATGACCGTTCTGCA(TC)GC(TCAG)GG(TCAG)GG(TCAG)

AA(AG)TT(TC) GAGAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA- 3’

UP-2r: 5′-AGCAGGGTACGGATGTGCGAGCC(AG)TC(TCAG)AC(AG)TC(TCAG)GC(AG)

TC(TCAG)GTCATAGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-

3’ and the second set of primers were UP1S : 5’-GAAGTCATCATGACCGTTCTGCA-3’ and 34

UP2SR – 5’-AGCAGGGTACGGATGTGCGAGCC-3’ according to previous study (Yamamoto

& Harayama, 1995). The UP-1 and UP-2r primers were designed by reverse transcribing the gyrB protein of three different types of bacteria (Yamamoto & Harayama, 1995). The first twenty-three nucleotides of primers UP-1 and UP-2r are not degenerate and therefore may not bind to the complementary strand on the target gene (Yamamoto & Harayama, 1995). This allows the 5’ regions of both primers to be used as a tag sequence for sequencing (Yamamoto &

Harayama, 1995) which is what UP1S and UP2SR primers are for. The PCR mix contained 1.0

µM of each UP-1 and UP-2r primers, 1.25 mM MgCl2, 0.2 mM dNTP, 1.5 U Taq Polymerase,

1X PCR Buffer and 1.0 µL of template DNA. The PCR was performed using the PCR thermo cycler under the following conditions: 94 °C for 3 min; 35 cycles of amplification with template denaturing at 94 °C for 1 min, primer annealing at 48 °C for 1 min, and primer extension at 72°C for 3 mins. After the reaction was complete, the concentration of the PCR products was measured using a BioPhotometer Plus spectrometer (Eppendorf, U.S.A) to ensure that the minimum concentration of DNA needed for sequencing was obtained.

To verify the correct PCR product sizes, the PCR products of both gene reactions were subjected to agarose gel electrophoresis in separate gels. A 1% agarose gel for electrophoresis was prepared by mixing 0.7 grams of agarose powder (Invitrogen, U.S.A) with 70 mL of 1X sodium borate (SB) buffer and boiling the mixture for approximately 2 minutes in the microwave. Once the agarose was cooled, 10 µL of RedSafe was added in order to visualize the

DNA under UV light. The solution was then slowly added to the plate of the gel-casting tray and a sample comb with 10 wells was added to create wells in the finished gel. Once the gel was completely solidified, the sample comb was removed and the gel was placed in the

35 electrophoresis equipment filled with 1X sodium borate (SB) buffer. Each of the wells were loaded with 9 µL of PCR product, mixed with 1 µL of 10X loading buffer, and the gel electrophoresis was set to run for 35 minutes at 100 mV. The DNA on the gel was visualized using UV illumination for verification of the expected product size. Once the correct product size was observed, the remaining PCR products were sent for sequencing at a DNA Sequencing

Facility (CBS, University of Guelph, Canada) by using an ABI Prism 3100 Automated

Sequencer. For the 16S rRNA gene sequencing the same primers used for the PCR reaction were used for sequencing. For the gyrB gene sequencing primers UP1S and UP2SR were used for sequencing. Bacterial strains were identified by using the sequencing results from both genes and comparing the nucleotide sequences in the GeneBank database

(http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the Basic Local Alignment Search Tool (BLAST) search algorithm (Altschul et al.,1990).

4.3. Soybean meal fermentation

4.3.1. Preparation of soybean meal and inoculums

After the isolates with high enzyme activities were identified, their ability to ferment soybean meal was investigated. The first step in preparing for the fermentation was carefully heat treating the soybean meal to reduce the number of bacteria present in the stock soybean meal. Sterilizing soybean meal in the autoclave has been found to be detrimental to lysine

(González-Vega et al., 2011) and would be costly and impractical to do in the feed industry. To prevent any damage to lysine and other amino acids, the soybean meal used was gently heated according to previous methods which showed no negative effects to lysine (González-Vega et al., 2011). This was performed by spreading approximately 50 grams of 48 % crude protein

36 soybean meal (Grand Valley Fortifiers, Canada) on an aluminum tray and heated in the oven- dryer for 30 minutes at 125 °C (González-Vega et al., 2011). Treated soybean meal was removed from the oven, covered in aluminum foil, and stored in a previously autoclaved glass jar until needed.

Bacteria inoculums of Isolate-2, Isolate-4, Isolate-7, Isolate-8, F-8, F-9, and F-11

(selected based on enzyme activity measure described above) were prepared fresh for each fermentation. Each isolate was streaked onto a previously prepared BHI agar plate from each of their glycerol stocks. Bacteria on the agar plates were grown for 18 hours in 37 °C. Each inoculum was prepared by inoculating a single colony from each isolate’s agar plate, obtained using a sterile inoculating loop, into a 50 mL sterile flask containing 10 mL of BHI culture media. Liquid cultures were incubated for 18 hours at 37 °C and a colony forming unit (CFU) measurement was taken by diluting the inoculum by a serial dilution and plating 20 µL of the dilutions on BHI agar plates. To obtain the CFU/mL the number of colonies on the plate was multiplied by the dilution number and divided by 0.02 mL. The liquid cultures incubated under these conditions obtained a culture of 108 CFU/mL.

4.3.2. Liquid-state fermentation

The first fermentation investigated was a high-moisture fermentation further referred to as liquid-state fermentation. The entire preparation of the fermentation was performed under sterile conditions. In the liquid-state fermentation only the bacteria isolated from food sources

(Isolate-2, Isolate-4, Isolate-7, and Isolate-8) were used. The fermentation process was initiated by weighing 2 grams of the previously treated soybean meal into a sterile 50 mL flask. 37

Subsequently, 10 mL of sterilized deionized water was added and the mixture was set aside for an hour to allow the soybean meal to absorb the water. After the hour, 1 mL of each bacteria liquid culture was inoculated into the soybean meal mixture. There were three replicates per treatment. This mixture was secured into a shaker incubator (New Brunswick, U.S.A) set at 37

°C, 350 rpm, for 48 hours. There was an additional soybean meal fermentation set-up that used the same soybean meal as the treatments with the exception of only being inoculated with blank culture media. This was considered to be the negative control and was directly stored in the -80

°C freezer once the water was added to the soybean meal, representing the soybean meal pattern completely unfermented. Samples of the fermented soybean meal were taken at 24 and 48 hours and stored at -80 °C. All of the samples of each treatment group were were lyophilized with a freeze dryer (Labconco, U.S.A) for 24 hours and ground into a fine powder, using a mortar and pestle, and used for further analysis.

4.3.3. Solid-state fermentation

The second fermentation condition was low-moisture fermentation, further referred to as solid-state. The solid state fermentation was initiated by weighing 2 grams of previously heated soybean meal into a sterile 50 mL flask. There were seven treatment groups and two controls for each fermentation. Each treatment and control groups were replicated three times. The treatment groups consisted of inoculum prepared from isolated with high enzymatic activity

(Isolate-2, Isolate-4, Isolate-7, Isolate-8, F-8, F-9, and F-11). The negative control contained blank culture media instead of inoculums and was directly stored in -80 °C. The treatment groups were set up by mixing 2 mL of each inoculum (108 CFU/mL) with 2 mL of sterile deionized water. This was done in order to evenly distribute the inoculum. This diluted liquid

38 culture was inoculated into the 2 grams of soybean meal for each of the treatment groups. This mixture was set to ferment in an incubator at 42 °C for 48 hours without any agitation. Samples were collected at 24 and 48 hours and stored at -80 °C until lyophilized. Samples were lyophilized using a freeze dryer (Labconco, U.S.A) for 24 hours and then ground into a fine powder using a mortar and pestle. Only lyophilized, finely ground unfermented and fermented products were used for further analysis.

4.4 Analysis of soybean meal fermentation products

4.4.1 Proximate analysis and amino acid profiling

Finely ground lyophilized solid-state unfermented and fermented samples were sent to

Dr. Zhang’s group at the Institute of Animal Husbandry, China Academy of Agricultural

Science (Beijing, China) to determine dry matter (AOAC official method 934.01), crude protein

(AOAC official method 954.01), and amino acid concentrations (AOAC official method 994.12).

4.4.2 Determination of soluble protein fractions and distribution

Total soluble protein of the unfermented and fermented soybean meal was isolated as previous described with slight modifications (Hong et al., 2004). First, 0.05 grams of lyophilized finely ground fermented product was homogenized using an ultrasonic cell disruptor

(Misonix, U.S.A) with 1 mL of deionized water for 30 seconds on ice. The mixture was then centrifuged at 10,000 rpm for 15 minutes at 4 °C. The supernatants of each product were transferred into a clean 1.5 mL Eppendorf tube and used for total soluble protein quantification using the DC Assay Kit (Bio-Rad Laboratories, U.S.A).

Samples of soluble protein(supernatants) were analyzed by sodium –dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) using an 11% tris gel which used a 1X Tris running buffer (0.12 M Tris, 0.96 M Glycine, and 0.5% SDS). The total volume loaded per well was 20 µL which consisted of 15 µL of the crude protein sample diluted with 5 µL of 5X loading buffer (5% SDS, 20% glycerol, 0.05% β-Mercaptoehtanol, and 0.001% Bromophenol) and boiled for 7 minutes at 90 °C prior to being loaded and subsequently subjected to electrophoresis for 100 minutes using 100 V. Ten µL of Unstained Protein Ladder (Bio-Rad,

U.S.A) was used as a marker for molecular mass from 10-250 kDa. Following electrophoresis, the gel was stained using a Coomassie blue staining solution which consists of 0.5% Coomassie

Brilliant Blue G-250, 50% methanol, and 10 % acetic acid for 5 minutes. The gel was de-stained using solution containing 40% methanol and 10% acetic acid, for two hours replacing the destaining solution every 20 minutes and left to continue de-staining overnight in Milli Q water.

An image of the gel separating the proteins of the liquid-state fermentations was captured using white light conversion screen on the GelDoc (Syngene, U.S.A). An image of the gel separating the proteins from the solid-state fermentation was taken using white light conversion screen on the ChemiDoc XRS+ (Bio-Rad Laboratories, U.S.A) and analyzed using the Image Lab software.

4.4.3 Determination of oligosaccharide concentrations

Finely ground lyophilized unfermented and fermented samples were sent to Dr. Zhang’s group at the Institute of Animal Husbandry, China Academy of Agricultural Science (Beijing,

China) to determine concentrations of raffinose and stachyose. Contents of stachyose and

40 raffinose were determined using high-performance liquid chromatography (HPLC) (Shimadzu

LC-15C, Japan) equipped with refractive index detector (Shimadzu RID-10A, Japan). Briefly, stachyose and raffinose were first extracted using ethanol. A total amount of 2.00 g of soybean meal samples were mixed with 50 ml of 70% (v/v) ethanol, microwaved to boiling and then cooled down to room temperature. The liquid was removed and transferred to EP tube. The extraction procedure was repeated and the remaining was rinsed with 70% (v/v) ethanol. All liquid was collected in the same EP tube for each sample. The collected liquid was centrifuged at 5000 r/min for 10 min and the supernatant was concentrated to a volume less than 10 ml by rotary evaporation and finally filled with deionized water to the final volume of 25 ml. The solution was then centrifuged at 4000 rpm for 10 min to obtain the supernatant, which was filtrated with 0.22 μm filter before injected into the HPLC system. A volume of 20 μl of each sample was injected into the HPLC system with 70% acetonitrile (v/v = 7:3) as the mobile phase.

The temperature was set at 35 °C, and the flow rate was 1 ml/min. Standards of stachyose (Dr.

Ehrenstorfer, China) and raffinose (Dr. Ehrenstorfer, China) were used.

4.4.4 Detection of Allergenic Proteins.

A Western blot was performed to detect the allergenic proteins of the unfermented and fermented soybean meal. The total soluble protein of the unfermented and fermented soybean meal samples of 50 µg were loaded into an 11% polyacrylamide gel similar to the procedures described above. Once the electrophoresis step was completed, the proteins were transferred to an Immobolin-P (Millipore, Billerica, USA) polyvinyl difluoride (PVDF) transfer membrane using an electro-blotting transfer tank (Biorad, U.S.A). To do this, the PVDF membrane was immersed in methanol and then washed in deionized water for two minutes. Subsequently, the

SDS-PAGE and the membrane were equilibrated with chilled 1X transfer buffer (25 mM Tris,

192 mM glycine, and 20% methanol) in order to remove buffer salts, detergents, and to allow methanol-dependent shrinking. A transfer cassette was assembled in the following order: fiber pad, filter paper, SDS-PAG, PVDF membrane, filter paper, fiber pad. The assembled cassette was placed in the transfer tank and the transfer was run at 100 V for 100 minutes. Once the proteins have been successfully transferred onto the membrane, the cassette was disassembled and the membrane washed for 8 minutes in 1X TBS. The membrane was then blocked using blocking buffer comprised of 5% skim milk, 0.1% Tween 20, and 1X TBS for an hour. The blocked membrane was subsequently washed for 30 minutes using washing buffer (0.1% Tween and TBS) and replacing the buffer every ten minutes on an orbital shaker. After the wash, the membrane was incubated in primary antibody in 4 °C overnight on an orbital shaker. The primary antibody was prepared following a published protocol (Song et al., 2010). Briefly, plasma was collected from eight piglets (8-week old) that were previously exposed to feed containing soybean meal. Piglets develop an immune response to soybean allergens by 21 days of exposure, and thus should have pre-formed antibodies that should react to the two allergenic proteins glycinin and β-conglycinin. The primary antibody used was comprised of 20 µL of pooled pig serum and 10 mL of blocking buffer for a dilution ratio of 1:500. The membrane was washed again for 30 minutes using washing buffer replacing the buffer every ten minutes on an orbital shaker. Following the wash, the membrane was incubated with secondary antibody (1:

100,000, rabbit anti-pig horseradish peroxidase-linked IgG; Abcam) for 1 hour at room temperature. The membrane was washed again for 30 minutes using washing buffer and replacing the solution every ten minutes. Proteins were detected by using Clarity Western

Enhanced Chemiluminescence (ECL) Substrate (Bio-Rad Laboratories, U.S.A) according to manufacturer’s instructions. In brief, equal parts of solution A and solution B were mixed and

42 poured onto the membrane and incubated for 1 minute. The membrane was developed by using the ChemiDoc XRS+ (Bio-Rad Laboratories, U.S.A) and analyzed using the Image Lab software.

4.5 Statistical analysis:

All statistical analysis was performed using PRISM GraphPad Prism Version 5.03 (San

Diego, CA). The screening data was analyzed using one-way ANOVA with a Dunnet post-test to determine if any treatment groups were significantly different from the positive control. For the screening data, observations were made in triplicate and simultaneously. The statistical analysis of the unfermented and fermented samples was performed using one-way ANOVA with a Tukey post-test; observations were made in triplicate, with one observation obtained daily.

The assumption of no day effect was believed to be true. Data was expressed as mean ± standard error of the mean and considered to differ significant by p< 0.05.

5.0 RESULTS

5.1 Qualitative measurement of enzymes activity.

5.1.1 Primary Screening for Protease Producing Bacteria

The first round of screening identified one colony which demonstrated the largest clearance zone from each of the five sources (Table 1). One source had two colonies which demonstrated similar clearance zones and were both selected for further screening. Of the six colonies selected, two of the bacteria demonstrated an additional, different bacterial colony growth on the agar plate (Isolate-3 and Isolate-6). These two colonies as well as the original six, were selected for the second round of screening. 43

5.1.2 Primary Screening of Cellulase Producing Bacteria

Of all the bacteria screened, only thirty colonies were able to grow on both soymilk agar and on CMC agar. Of these 30 bacteria, only 20 colonies demonstrated an orange halo when stained with congo red dye. Of these 20 colonies only three isolates demonstrated high cellulase activity, i.e. formed distinguishably larger halos than the other colonies. These three colonies (F-

8, F-9, and F-11) were selected for the second round of screening to be ranked with the other food-derived bacteria isolates.

5.1.3 Second round of screening

Figure 3A shows the representative images of proteinase positive (right image), and negative (left image), respectively. The results of the secondary screening for protease activity is shown in Figure 3. The radius of each clearance zones, reflecting protease activity, were measured. Of the eight isolates from food sources, only six demonstrated protease activity on the soymilk agar plates (Figure 3B). From the fermented food source, the isolate which had the largest clearance zone, and thus the most protease activity, was Isolate-4 (Figure 3B). The three bacteria isolated from the grass carp demonstrated protease activity. Among them, F-11 had the largest clearance zone (Figure 3B). Protease activity of Isolate-4 and F-11 were significantly higher than that of B.subtilis ATCC6633, the positive control (Figure 3).

Table 2: Summary of first round of screening. Five fermented food sources were used to isolate bacteria which demonstrated high protease activity on soymilk agar plates.

Source Primary Secondary Screening ID/ Glycerol Screening ID Stock ID

Black Fermented Beans A-1 Isolate-1

Red Fermented Beans B-1 Isolate-2

Red Fermented Beans B-2 Isolate-3

Yellow Fermented Beans C-1 Isolate-4

Soft Fermented Beans D-1 Isolate-5

Soft Fermented Beans D-2 Isolate-6

Japanese Natto E-1 Isolate-7

Japanese Natto E-2 Isolate-8

For the cellulase screening, the positive control plates demonstrated an orange halo, indicating cellulase activity, as expected (Figure 4 A, right image). All of the negative control plates did not have orange halos (Figure 4 A, left image). Of the eight bacteria isolated from food sources, only three isolates (Isolate-2, Isolate-7 and Isolate-8) were able to demonstrate cellulase activity (Figure 4 B). All three of the bacteria isolated from the fish tested positive for cellulase activity. Of all the isolates tested, F-8 had a significantly different level of cellulase activity compared to the positive control (p< 0.05; Figure 4B).

Figure 5A shows representative amylase positive (right), and negative (left) images. Of all the bacteria isolated from food sources, only three demonstrated amylase activity (Figure 5

B). All of the three bacteria isolated from the fish also tested positive for amylase activity but only Isolate-2 and Isolate-8 demonstrated significantly (p<0.05) higher amylase activity than the positive control. A summary of the activities of the three enzymes from bacteria isolated from food and fish are shown in Table 3.

A) Negative Control F-11 Clearance Zone

8 * * 6

2 Clearance Zone (mm) Zone Clearance 0

F-8 F-9 F-11 P.con Isolate-1Isolate-2Isolate-3Isolate-4Isolate-5Isolate-6Isolate-7Isolate-8

Figure 4 Figure 3: Measuring protease activity using soymilk agar plates. Bacteria Isolate-1 to Isolate-8 were isolated from fermented food sources and bacteria F-8 to F-11 were isolated from the intestines of a grass carp. Agar plates were incubated for 72 hours to observe clearance zones.

B.subtilis ATCC 6633 was used a positive control and blank culture media was used as a negative control. (A) Representative image of clearance zones. (B) Measurement of clearance zones. Error bars represent the SEM of the mean of three independent observations. *= p<0.05 compared to positive control.

A) Negative Control F-11 Clearance Zone

20 * 15

5 Clearance Zone (mm) Zone Clearance 0

F-8 F-9 F-11 P.Con Isolate-1Isolate-2Isolate-3Isolate-4Isolate-5Isolate-6Isolate-7Isolate-8

Figure 5 Figure 4: Measuring cellulase activity using CMC agar. Bacteria Isolate-1 to Isolate-8 were isolated from fermented food sources and F-8 to F-11 from the intestines of a grass carp. CMC agar plates were incubated for 72 hours and stained with Congo Red and de-stained using NaCl to observe clearance zones. B.subtilis ATCC 6633 was used a positive control and culture media was used as a negative control. (A) Representative images of clearance zones.(B) Measurement of clearance zones. Error bars represent the SEM of the mean of three independent observations.

*= p<0.05 compared to positive control.

A) Negative Control Isolate-2 Clearance Zone

* 15 * 10

5 Clearance Zone (mm) Zone Clearance 0

F-8 F-9 F-11 P.con Isolate-1Isolate-2Isolate-3Isolate-4Isolate-5Isolate-6Isolate-7Isolate-8

Figure 6 Figure 5: Measuring amylase activity using corn starch agar. Bacteria Isolate-1 to Isolate-8 were isolated from fermented food sources and F-8 to F-11 were from the intestines of a grass carp.

Starch agar plates were incubated for 72 hours and stained with Gram’s Iodine stain to observe clearance zones. B.subtilis ATCC 6633 was used a positive control and culture media was used as a negative control. (A) Representative image of clearance zones. (B) Measurement of clearance zones. Error bars represent the SEM of the mean of three independent observations.

*= p<0.05 compared to positive control.

Table 3: Summary of the enzyme activity of best performing bacteria isolated from food and grass carp intestine. +++= significantly different higher than the Bacillus subtilis ATCC6633 control (p>0.05).

Source Genus Enzyme Activity Isolate MALDI-TOF MS Results Protease Cellulase Amylase

Isolate -1 Fermented Food Bacillus cereus ++ - -

Isolate -2 Fermented Food No ID but Bacillus gram + + +++ positive

Isolate -3 Fermented Food Enterococcus faecium - - -

Isolate -4 Fermented Food No ID but Bacillus gram +++ - ++ positive

Isolate -5 Fermented Food Bacillus cereus ++ - -

Isolate -6 Fermented Food Lysinibacilllus fusiformis - - -

Isolate-7 Fermented Food Bacillus subtilis ++ ++ ++

Isolate-8 Fermented Food Bacillus subtilis ++ ++ +++

F-8 Grass Carp No ID but Bacillus gram ++ +++ ++ positive

F-9 Grass Carp No ID but Bacillus gram ++ ++ ++ positive

F-11 Grass Carp No ID but Bacillus gram +++ ++ +++ positive

ATCC Commercially Bacillus subtilis ++ ++ ++ 6633 available (ATCC)

5.2 Identification of selected bacteria

5.2.1 Characterization of isolates using MALDI-TOF MS

The group of bacteria that were identified for second round screening, were characterized using matrix assisted laser desorption ionization- time of flight mass spectrometry (MALDI-TOF

MS). Table 3 summarizes all of the MALDI-TOF MS results. Of the eight bacteria isolated from food, only five were identified to species level. Isolate-1 and Isolate-5 were identified as

Bacillus cereus. Isolate-3 was identified as Enterococcus faecium, Isolate-6 was Lysinibacilllus fusiformis and Isolate-7 and Isolate-8 were Bacillus subtilis. Isolate-2 and Isolate-4 and the bacteria isolated from fish intestine were unable to be identified using MALDI-TOF MS but gram staining identified the bacteria as Bacillus gram positive.

5.2.2 Characterization of isolates using 16S rRNA and gyrB gene sequencing

Figure 6 is a gel image showing the PCR product amplified with the expected size. The

BLAST results revealed that Isolate-2, Isolate-4, and F-9 were of a Bacillus amyloliquefaciens strain, summarize in Table 4. The partial 16S rNA gene sequence of Isolate-2 had a 99% identity with B.amyloliquefaciens strain IHB B 3373 (GeneBank accession # KF475869.1) and a

98% identity with B.amyloliquefaciens subsp. plantarum str. FZB42 (GeneBank accession #

NC_009725.1) for its gyrB gene sequence. The partial 16S rNA gene sequence of Isolate-4 had a

99% identity with B.amyloliquefaciens strain BAB-68 16S (GeneBank accession # KC250107.1) and a 95% identity with B.amyloliquefaciens subsp. plantarum str. FZB42 (GeneBank accession

# NC_009725.1) for its gyrB gene sequence. Isolate-9 had a 99% identity with

B.amyloliquefaciens strain IHB B 3373 (GeneBank accession # KF475869.1) and a 98% identity with B.amyloliquefaciens subsp. plantarum str. FZB42 (GeneBank accession # NC_009725.1)

51 for its partial 16S rRNA and gyrB gene sequences, respectively. F-11’s gene sequences had a

99% identity to B.amyloliquefaciens supsp. plantarum strain UCMB5033 (GeneBank Accession

# HG328253.1) and a 97% identity to B.amyloliquefaciens subsp. plantarum str. FZB42

(GeneBank Accession # NC_009725.1) to partial 16S rRNA and gyrB sequences, respectively.

Isolate-7, Isolate-8, and F-8 were all of a Bacillus subtilis strain, summarized in Table 4. Isolate-

7’s gene sequences had a 99% identity to B.subtilis strain GLMP6 (GeneBank Accession #

KF364495.1) and a 98% identity to B.subtilis subsp. subtilis str. 168 (GeneBank Accession #

NC_000964.3) to partial 16S rRNA and gyrB sequences, respectively. Isolate-8’s gene sequences had a 99% identity to B.subtilis strain YLB-P1 (GeneBank Accession # KF220577.1) and a 97% identity to B.subtilis subsp. subtilis str. 168 (GeneBank Accession # NC_000964.3) to partial 16S rRNA and gyrB sequences, respectively. F-8’s gene sequences had a 99% identity to

B.subtilis strain IHB B 4270(GeneBank Accession # KF475879.1) and a 97% identity to

B.subtilis subsp. subtilis str. 168 (GeneBank Accession # NC_000964.3) to partial 16S rRNA and gyrB sequences, respectively.

A) 1 2 3 4 5 6 7

500

Figure 7 1 2 3 4 5 6 7

1650 1000

Figure 6: Gel electrophoresis of PCR products used to identify the bacteria isolates. (A) The partial sequence of 16S rRNA gene product. PCR products were electrophoresed on a 1.0% agarose gel and visualized under UV light. Low molecular weight DNA ladder (New England

Labs, U.K) was used to confirm expected size of product (500 kp). (B) The gyrB gene product.

1 kb DNA Plus Ladder (Life Technologies, U.S.A) was used to confirm expected size of product

(1,200 bp).1=Isolate-2, 2= Isolate-4, 3= Isolate-7; 4= Isolate-8; 5= F-8, 6= F-9, and 7= F-11

nucleotide isolates. sequencing the selected of gene identification BLAST strain

Summary of the Summary of

: 4 Table

5.3 Liquid-state fermentation

5.3.1 Influence of selected bacterium on SBM protein profile

Figure 7B is a representative image of the results. It was observed that the abundance of high molecular weight proteins had decreased, and the abundance of low molecular weight proteins increased, with fermentation. The large allergenic subunits of β-conglycinin (72 kDa,

76 kDa and 53 kDa) were no longer visible in the gel, suggesting the allergenic proteins have been reduced. Of all the isolates, Isolate-2 was determined to be the best isolate in terms of reducing high molecular weight proteins, increasing low molecular weight proteins, and reducing the allergenic proteins.

A) 300

200

100

0 to Unfermented Soybean Meal (%) Unfermented Natural Isolate-2 Isolate-4 Isolate-7 Isolate-8 Soluble Protein Concentration Relative Treatments B) 1 2 3 4 5 6 7

180 β-Conglycinin 115 α’ subunit 82 α subunit 64 β subunit 49 Glycinin acidic subunit basic subunit 37

Figure 7: The fermentation effects of using different isolates on the protein profile of SBM in a liquid-state. (A) The total soluble protein concentration in the supernatant was quantified using

DC Assay and then expressed as a percent relative to the unfermented control . Error bars represent SEM of three independent observations. *= p<0.05 compared to unfermented control.

(B) Coomassie Blue staining representing the protein profile. Samples of 20 µL of each sample were loaded into an 11% SDS-PAGE set at 100 V for 100 mins. The gel was stained with

Coomassie Blue and visualized using white light conversion screen.

1= Unfermented soybean meal, 2=Natural; 3= Isolate-1, 4= Isolate-4;5= Isolate-2; 6=Isolate-7; and 7=Isolate-8 56

5.4 Solid-state fermentation

5.4.1 Influence of selected bacterium on SBM protein profile

After obtaining the encouraging result from liquid-state fermentation, we next examined the feasibility of the more industry-applicable solid-state fermentation of SBM. Soybean meal was fermented at a liquid: substrate ratio of 2:1 at 42 °C for 48 hours, with sampling at 24 and 48 hours. Total soluble protein concentrations were increased in five fermentations, compared to the unfermented SBM control, as shown in Figure 8. No effect of fermentation time on total soluble protein levels (Figure 9) of the same isolate were observed except for Isolate-2 and

Isolate-4. The total soluble protein of soybean meal fermented with Isolate-2 appears to lower at

48 hours as compared to 24 hours. The opposite effect is observed with soybean meal fermented with Isolate-4 which was increased at 48 hours when compared to 24 hours. In addition, fermentation with the selected isolates was able to decrease the high molecular weight proteins while increasing the low molecular weight proteins (Figure 9). This was different among bacteria isolated from food rather than from the intestine of the grass carp. In general, bacteria isolated from food effectively reduced most proteins to approximately less than 25 kDa (Figure

9A). Most of the bacteria isolated from food demonstrated maximal SBM peptide degradation at

24 hours of fermentation. Bacteria isolated from the intestine of the grass carp were able to degrade the proteins to less than 35 kDa (Figure 9B). This is 10 kDa higher than that fermented by bacteria isolated from the food sources. Similar to the bacteria isolated from fermented food, no real improvement in peptide profile was observed after the 24 hour fermentation. The fermentations from both sources were also successful in reducing the allergenic proteins of β- conglycinin (72 kDa, 76 kDa and 53 kDa). Of all the bacteria used for fermentation, Isolate-2 was the isolate that degraded most of the high molecular proteins and increase most of the low

57 weight proteins at the 24 hour time point, and thus was selected and further tested to investigate changes in crude protein and amino acid profiles.

5.4.2 Improved crude protein of fermented soybean meal

Once the most efficient isolate was identified, its ability to improve soybean meal nutrient composition was examined. The best isolate to improve the peptide profile visualized using Coomassie Blue gel was Isolate-2, a bacterium isolated from food source. The lyophilized ground fermented product of Isolate-2 and the unfermented product were tested for crude protein and amino acid profile changes. It was determined that fermentation with Isolate-2 significantly increased crude protein concentrations from 48.1 ± 0.5% to 53.3 ± 0.67% (24 hours) and 55.9±

0.9% (48 hours), respectively (Figure 10; Table 5), although the difference is not significant between 24 hours and 48 hours.

Figure 8

400

* 300 * * *

200

100 Unfermented Soybean Meal (%) Soluble Protein Concentration Relative to 0

F-8 (24 hr) F-8 (48 hr) F-9 (24 hr) F-9 (48 hr) F-11 (24 hr) F-11 (48 hr) Unfermented Isolate-2 (24 Isolate-2hr) (48 Isolate-4hr) (24 Isolate-4hr) (48 Isolate-7hr) (24 Isolate-7hr) (48 Isolate-8hr) (24 Isolate-8hr) (48 hr) Treatments

Figure 8: The relative changes of total soluble protein of soybean meal when fermented for different lengths and with different bacterial isolates. The total soluble protein concentration in the supernatant was quantified using DC Assay and then expressed as a percent relative to the unfermented control. Error bars represent the SEM of the means data of three independent observations. *= significant difference compared to the unfermented soybean meal control (p <

0.05).

Figure 9 A) 1 2-a 2-b 3-a 3-b 4-a 4-b 5-a 5-b 245 β-Conglycinin

α’ subunit 75 α subunit 63 β subunit 48 Glycinin 35 acidic subunit 25

basic subunit 20 17

B) 1 6-a 6-b 7-a 7-b 8-a 8-b

245 β -Conglycinin α’ subunit 75 α subunit 63 β subunit 48 35 Glycinin acidic subunit 25 basic subunit 20 17 11

Figure 9: Visualizing the changes of total soluble protein of soybean meal when fermented in a solid-state for different lengths and with different bacterial isolates with Coomassie Blue gel.

(A) Bacteria isolated from fermented food sources. (B) Bacteria isolated from grass carp.

Coomassie Blue staining representing the peptide profile. 20 µL of each sample was loaded into an 11% SDS-PAGE set at 100 V for 100 mins. The gels were stained with Coomassie Blue and visualized using white light conversion screen. 1= Unfermented soybean meal, 2= Isolate-8, 3=

Isolate-7, 4=Isolate-2, 5= Isolate-4, 6= F8, 7= F9, and 8=F11 where “a”= 24 hours and “b”= 48 hours.

60 b b a

Crude Protein Concentration (% Dry Matter) 0 Unfermented Isolate-2 (24 hr) Isolate-2 (48 hr) Treatments Figure 10

Figure 10: The effects of solid-state fermentation length on crude protein of soybean meal.

Crude protein was measured using Ketuo Kjeldalh nitrogen analyzer (KDY-9820) of lyophilized fermented and unfermented soybean meal products. Fermentation using Isolate-2 illustrated a significantly higher crude protein level than the unfermented control. Error bars represent the

SEM of the means data of three independent observations. Columns that do not share the same letter are significantly different (p <0.05).

5.4.3 Amino acid profile of the fermented soybean meal

As shown in Figure 11, at 24 hours, both the total essential (Figure 11 A) and total non- essential (Figure 11 B) amino acids concentrations were significantly increased by fermentation using Isolate-2, when compared to unfermented control. By 48 hours of fermentation, the essential amino acid (EAA) profile decreased to the same level of the control whereas the non- essential amino acids remained unchanged. Table 5 depicts the change detected in all the amino acids when fermented with Isolate-2 for 24 and 48 hours, respectively. With the exception of arginine, all the essential amino acids analysed show a numerical increase at 24 hour of fermentation when compared to the unfermented control, with the changes in valine and phenylalanine being the only amino acids that increased statically significantly (p<0.05). At 48 hours of fermentation, the concentration of phenylalanine and histidine were significantly higher, but concentrations of arginine were significantly lower than the unfermented control. Of the eight non-essential amino acids analysed, levels of aspartic acid, glutamic acid, cysteine, tyrosine, and proline were found to be significantly from than the unfermented control. Levels of aspartic acid, glycine, and proline significantly increased at 24 hours of fermentation, but declined back to the same level of unfermented control at 48 hours. Levels of glutamic acid were significantly higher than the unfermented control at 24 and 48 hours of fermentation.

Levels of cysteine and tyrosine increased with fermentation to a significantly different level than the control at 48 hours. The total amino acid concentration in soybean meal was significantly increased by fermentation at 24 hour, but not at 48 hours (Table 5).

A)igure 11

b a a 20

10 Total Essential Amino TotalAcids Essential Amino

Concentrations (% Dry Matter) (% Dry Concentrations 5

0 Unfermented Isolate-2 (24 hr) Isolate-2 (48 hr) Treatments

B) 30 b b a

10 Total Non-Essential Total

Amino Acids Concentrations (% Concentrations Dry Matter) Acids Amino 0 Unfermented Isolate-2 (24 hr) Isolate-2 (48 hr) Treatment

Figure 11: The effect of fermentation length using Isolate-2 on changes of amino acid concentrations. (A) EAA profile. (B) NEAA profile. Error bars represent the SEM of the means data of three independent observations. Columns that do not share the same letter are significantly different (p <0.05).

Table 5: Quantitative representation of the effect of fermentation with Isolate-2 over a 24 and a

48 hour period. Data represents the means± SEM of three independent observations. Numbers in the same rows that do not have a common letter are significantly different (p <0.05).

* With the exception of tryptophan, asparagine, and glutamine.

Treatment Type of Amino Acid Unfermented Isolate-2 (24 hours) Isolate-2 (48 hours) Amino Acid (% DM) Threonine 1.77 ab ± 0.06 1.83 a ± 0.03 1.59 ab ± 0.05 Valine 1.99 a ± 0.06 2.29 b ± 0.07 2.26 ab ± 0.07 Methionine 0.47 a ± 0.01 0.50 a ± 0.03 0.52 a ± 0.01 Essential a a a Isoleucine 1.87 ± 0.09 2.09 ± 0.07 1.90 ± 0.05 a a a Leucine 3.41 ± 0.13 3.75 ± 0.05 3.37 ± 0.09 a b b Phenylalanine 2.19 ± 0.07 2.57 ± 0.03 2.63 ± 0.12 Lysine 2.80 a ± 0.10 3.24 a ± 0.04 3.25 a ± 0.19 Histidine 1.18 a ± 0.04 1.41 ab ± 0.03 1.47 b ± 0.09 Arginine 3.17 a ± 0.11 3.10 a ± 0.10 2.72 b ± 0.00 Aspartic acid 4.95 a ± 0.17 5.49 b ± 0.03 5.11 a ± 0.06 Non- Serine 2.25 a ± 0.08 2.24 a ± 0.05 1.96 a ± 0.06 Essential Glutamic acid 8.36 a ± 0.28 10.42 b ± 0.20 11.28 b ± 0.66 a b a Glycine 1.92 ± 0.07 2.12 ± 0.15 1.94 ± 0.01 a a a Alanine 1.93 ± 0.07 2.03 ± 0.04 1.92 ± 0.03 a ab b Cysteine 0.57 ± 0.01 0.64 ± 0.01 0.67 ± 0.03 Tyrosine 1.75 a ± 0.04 1.99 ab ± 0.02 2.04 b ± 0.10 Proline 2.22 a ± 0.09 2.53 b ± 0.02 2.31 ab ± 0.05

Total* of AA (%DM) 42.80 a ± 1.38 48.26 b ± 0.17 46.93 a ± 1.07

Crude protein (%DM) 48.05 a ± 0.47 53.26 b ± 0.69 55.89 b ± 0.94

Dry matter (%DM) 95.22 a ± 0.66 94.93 a ± 0.17 94.12 a ± 0.20

5.4.4 Detection of Allergenic Proteins

The results of the SDS-PAGE suggest that the subunits of the soy allergens β-conglycinin

(76, 72, and 53 kDa) and glycinin (35 and 22 kDa) were decreased or eliminated by fermentation with the different isolates. To validate that the allergens in fermented soybean meal did decrease, a western blot was performed. The results of the western blot indicated that antibodies found within the piglet plasma were able to bind to three distinct proteins found in the unfermented soybean meal product (Figure 12). These three distinct soy proteins were found approximately at: 75 kDa, 40-50 kDa, and 30-40 kDa. At 24 hours soybean meal fermented with

Isolate-2 or F-9 showed no reactivity to the piglet antibodies. However, piglet antibodies reacted to a peptide of 37 kDa at 24 hours with Isolate-4, F-8, or F-11 (Figure 12A). By 48 hour of fermentation, no allergen reactive band was detected (Figure 12B).

5.4.5 Oligosaccharide concentration of fermented soybean meal

Two major problematic soy oligosaccharides for piglets are raffinose and stachyose. As shown in Table 6, the concentration of stachyose and raffinose in the lyophilized unfermented soybean meal were 41.73 mg/g and 9.88 mg/g, respectively. Starting from 24 hours, fermentation with Isolate-2 reduced the amount of both oligosaccharides in SBM to non- detectable level (Table 6).

Figure 12

A) 1 2 3 4 5 6 7

β-Conglycinin α’ subunit 75

β subunit 50

Glycinin 37 acidic subunit

B) 1 2 3 4 5 6 7

β -Conglycinin 75 α’ subunit

β subunit 50 Glycinin acidic subunit 37

Figure 12: Western blot detecting soy allergens in the unfermented and fermented soybean meal products. ( A) Soybean meal fermented with various isolates for 24 hours. (B) Soybean meal fermented with various isolates for 48 hours. 50 µg of protein was loaded for each well and incubated with piglet serum (primary antibody) followed by rabbit anti-piglet IgG (secondary antibody). 1= Precision Plus Protein Dual Colour Standards (Biorad, U.S.A) ; 2= Unfermented soybean; 3= Isolate-2 product; 4= Isolate-4 product; 5= F-8 product; 6= F-9 product; 7= F-11 product.

Table 6: The oligosaccharide concentration in soybean before and after fermentation with

Isolate-2 for 24 and 48 hours.

Treatment Raffinose (mg/g) Stachyose (mg/g)

Unfermented Soybean Meal 9.88 41.73

Isolate-2 (24 hours) Not detectable Not detectable

Isolate-2 (48 hours) Not detectable Not detectable

6.0 DISCUSSION

Fermenting soybean meal with bacteria isolated from fermented food sources, or from the intestine of grass carp, was found to be successful in improving the nutritional value of soybean meal. The eight candidate bacteria selected for fermentation of soybean meal were able to improve the nutrient value of soybean meal through the reduction of allergenic and other high molecular weight proteins, and an increase in low-weight proteins. Of these eight candidate fermentations, Isolate-2 was considered to be the best fermented product and further analysis indicated that it also improved the amino acid profile, and decreased the concentration of soy oligosaccharides stachyose and raffinose. Six of the eight bacteria isolated from traditional fermented food sources, successfully demonstrated protease activity. The two colonies (Isolate-3 and Isolate-5) that did not demonstrate protease activity were considered to be contaminating

Isolate-2 and Isolate-4 and thus, were also selected for second round screening. Traditional

Chinese fermented soy foods undergo natural spontaneous fermentation (Han et al., 2001), which suggests that the bacteria isolated from these sources break down soybean components to utilizable nutrients. Another benefit to using bacteria derived from fermented foods and intestine is the safety of the bacteria. Bacteria isolated from fermented foods are considered to be safe as they are consumed by humans and may contain other beneficial properties such as being probiotic or having antimicrobial activity (Alexopolilos et al., 2004; Kim et al., 2004). Bacteria isolated from fish intestines may also have probiotic potential. Fish fed probiotic B.subtilis, previously isolated from the intestines of a carp fish, were found to have significantly higher lengths and weights and were also found to have significantly higher survival rates when challenged with Aeromonas hydrophila compared to that of the fish not fed the probiotic (Ghost et al., 2008). Since this study did not investigate the antimicrobial or probiotic qualities of our

68 isolates, we cannot conclude that our isolates are antimicrobial or probiotic. Further studies should be performed to determine if any of our isolates share any of these probiotic properties.

The bacteria isolated from this study were identified using common molecular tools such as MALDI-TOF MS and genetic sequencing. The MALDI- TOF MS results of this study indicated all the bacteria are of the Bacillus species. This is consistent with other studies that isolated bacteria from other traditionally fermented foods such as Dawadawa (Amoa-Awua et al., 2006), Meju (Cho et al., 2003), Cheonggukjang (Baek et al., 2009), and Douchi (Peng et al.,

2003). These bacteria can survive on legumes because they have the ability to secrete extracellular enzymes- such as proteases (Cho et al., 2003). MALDI-TOF MS uses bacterial protein to identify the bacteria and matches the peaks from the mass spectrometry to find its closest match in the database. However, there are some limitations of the assay. First, in order for the unknown isolate to have a match, the suspect species would have to be previously characterized and the profile saved in the database (Hotta et al., 2013). Secondly, the database must be accurate or else identification will not be reliable (Hotta et al., 2013). Lastly, there might be some issues with post-translational modification that could alter the peaks which could incorrectly identify the bacteria (Hotta et al., 2013). It is thus important to confirm the MALDI-

TOF MS identification by other methods, such as 16S rRNA gene sequencing used in this study.

The results of the partial 16S rRNA gene sequencing revealed the Isolate-2, Isolate-4, and F-9 were Bacillus amyloloquefaciens; and Isolate-7, Isolate-8, F-8, and F-11 were Bacillus subtilis.

Although partial 16S RNA gene sequencing can identify many bacterial species, it is more reliable to sequence the complete 16S rRNA gene, and some other housekeeping gene such as the gyrB gene. The gyrB gene is responsible for producing the β subunit protein of a DNA

69 gyrase and is one of the genes essential in DNA replication (Wang et al., 2007). This gene is distributed universally among species and has a faster rate of molecular evolution than the 16S rRNA gene, thus it has been used to identify different strains of bacteria of the same species

(Wang et al., 2007). Using the gyrB sequence results, we were able to further identify Isolate-2,

Isolate-4, F-9 and F-11 as Bacillus amyloliquefaciens; and Isolate-7, Isolate-8, and F-8 as

Bacillus subtilis. It is interesting to note that the same strain of bacteria were found in both fermented food and fish intestines, but yet have different enzyme efficiencies. The source from which bacteria is isolated is very important and can determine which enzymes the bacteria will secrete to survive. Isolate-2 was one of the eight bacteria we isolated from traditional fermented food. Given the source, we expected the bacteria to demonstrate high protease activity. This is important because the bacteria will need to break down the plant’s high molecular weight proteins in order to better utilize them for their growth. Grass carp is known to be a herbivore but can utilize both animal and plant sources for nutrients (Zhou et al., 2013). The fish itself does not produce cellulase but have been known to have microbes in the intestine that can secrete various enzymes, including cellulase (Li et al., 2009; Zhoa et al.,2013). It was also found that supplying an exogenous source of cellulase can alter the microbe population in the intestine which can increase cellulase production ( Zhoa et al.,2013). Given this source we expected the bacteria we isolated (F-8, F-9, and F-11) to demonstrate the ability to break down cellulose in order to survive. There has been a study which has successfully isolated cellulase producing bacteria from the intestine of the grass carp (Li et al., 2009) and thus, we expect NSP concentrations to also decrease in our fermentation. The change in the NSP concentration of soybean meal after fermentation with Isolate-2 will be investigated in future experiments.

Of the entire Bacillus genus, Bacillus subtilis is the most commonly used in soybean fermentations. Previous studies have observed an improvement on the total soluble protein profiles, elimination of allergenic proteins, and a reduction of anti-nutritional factors when soybean has been fermented with various strains Bacillus subtilis (Wongputtisin et al., 2014;

Ying et al; 2009; Frias et al., 2007). These effects were due the bacteria’s ability to produce extracellular enzymes which can digest the large proteins into smaller proteins and peptides

(Frias et al., 2007). This species of bacteria has also been found to increase the antioxidant capacity of soybeans by (Wongputtisin et al., 2007). Many strains of Bacillus subtilis have been found to have antimicrobial and probiotic effects (Kyriakis et al., 1999; Alexopolilos et al.,

2004; Guo et al., 2006).

Bacillus amyloliquefaciens has been less investigated in fermentation of soybean compared to Bacillus subtilis. To our knowledge, only one study has investigated Bacillus amyloliquefaciens in soybean fermentation (Chistyakov et al., 2015). This study was performed using the spores of the bacteria to ferment the whole soybean as a target dietary supplement for use in poultry feeds (Chistyakov et al., 2015). The results of the study indicate that the birds fed the treatment had an 8% higher growth rate and an 8.8% higher feed efficiency when compared to the control birds (Chistyakov et al., 2015). This species of bacteria has been also found to have found to have anti-bacteria activity against a strain of E.coli (Chi et al., 2014) and

Vibrionaceae species (Xu et al., 2014) that were both found to be resistant to multiple types of antibiotics. This anti-microbial activity was attributed to the lipopeptides produced by the bacteria which destroyed the membrane and whole cells of the pathogens, acting like porins, causing the cells to rupture (Chi et al., 2014; Xu et al., 2014). The species has also been found

71 to have anti-fungal properties (Song et al., 2013) and produce phytase (Shim & Oh, 2012).

Again, it is important to keep in mind the source of which bacteria is isolated in order to determine the safety of the bacteria for consumption. Bacteria of the same species can exhibit different characteristics depending on where it is isolated from, in order to adapt to its environment. Isolate-2, the bacteria isolated from fermented food and chosen for further fermentation of soybean meal, has been identified as Bacillus amyloliquefaciens. A strain of

Bacillus amyloliquefaciens isolated from soil was found to have beneficial traits such as being a probiotic strain (Islam et al., 2011), but other strains isolated from other sources such as the inside of a moisture damaged building may secrete substances that can be toxic to both humans and animals (Mikkola et al., 2004).

Preliminary data from the liquid state fermentation shows that the majority of the large soybean proteins were successfully cleaved to less than 37 kDa, using the newly isolated bacteria with the exception of Isolate-1. The protein concentration of all the fermented products was improved compared to the unfermented control. Soy flour fermented by Lactobacillus plantarum in a liquid state was found to decrease most of its proteins to less than 15 kDa (Frias et al., 2008). The particle size of the substrate was found to have an influence on these results, as the same bacteria fermented in a liquid state, using cracked soybean as the substrate, was found to degrade proteins to less than 40 kDa (Frias et al., 2008). The reduction of particle size increases the surface area which provides the bacteria with more products to utilize (Frias et al.,

2008). Fermenting soybean in a liquid-state solubilizes the soy proteins which make it easier for the bacteria to access and thus, hydrolyze using extracellular enzymes (Frias et al., 2008).

Although this study and other studies provide evidence that there are benefits to using liquid-state fermentation, the solid-state application is more desirable to the agricultural industry setting due to less costly equipment and lower moisture levels (Singhania et al., 2009; Opazo et al., 2012). Lower moisture content of fermented soybean meal reduces the amount of drying necessary to eliminate the risk of mould growth (Opazo et al., 2012), and the cost of transportation. To compensate for fermentations with higher moisture ratios, a previous study has oven-dried their fermentation product to a moisture level of 10% (Chen et al., 2010). This extra processing step further increases the cost and the risk of Maillard reactions, negatively affecting lysine, which effectively decreases the nutrient bioavailability of the feed (Friedman &

Brandon, 2001; Gonzalez-Vega et al., 2011).

Our solid-state fermentation was also successful in improving soybean meal protein content. Interestingly, the bacteria isolated from fermented soy foods cleaved most of the soy protein to 27 kDa and under, while fermentation with bacteria isolated from the intestines of the grass carp, resulted in majority of the soybean protein being degraded to 35 kDa and less. It is possible that different species and subspecies bacteria may secrete distinct arrays of proteinases that target different subsets of soybean proteins. The protein distribution patterns are comparable between 24 and 48 hours of fermentation using Isolate-2 and F-6, respectively. This suggests that 24 hours is sufficient to achieve maximal protein degradation. Similar peptide profiles have also been previously observed. Cracked soybeans fermented with A.oryzae for 48 hours resulted in majority of soybean protein that were 50 kDa or smaller (Frias et al., 2008). In the same study, cracked soybean fermented with B.subtilis for 48 hours was observed to decrease soybean proteins to less than 20 kDa, which appears to be more of an improvement than using A.oryzae

(Frias et al., 2008). Soybean meal fermented with A.oryzae for 48 hours was found to have

13.4% of its proteins concentrated at 20-60 kDa, while the rest of the proteins were less than 20 kDa (Hong et al., 2004). The reduction of protein sizes is an important aspect to increase the digestibility of soybean protein as small peptides can be more easily hydrolyzed to free amino acids that can be absorbed in the intestine (Silk et al., 1979).

The results of the piglet immunoreactivity of the unfermented soybean meal indicated three distinct soy proteins at: 75 kDa, 40-50 kDa, and 30-40 kDa. These bands would correspond to the α’ (76 kDa) and β (53 kDa) subunits of β-conglycinin and the acidic (35-40 kDa) subunit of glycinin. The α(72 kDa) subunit of β-conglycinin and the basic (22 kDa) subunit of glycinin were not visible on the western blot. The western blot as well as the SDS-

PAGE indicated that Isolate-2 was successful at reducing and completely eliminating all subunits of both allergens. Allergenic proteins found in soybean meal activate the immune system leading to an inflammation of the intestine, releasing chemical such as histamine, which increases the incidences of diarrhea (Hao et al., 2009; Zhao et al., 2010). Additionally, they have been known to impede digestion (Cervantes-Pahm & Stein, 2010). β-conglycinin has also been found to directly affect piglet intestinal cells in vitro by decreasing cell growth, damaging the cytoskeleton, and stimulating apoptosis of the intestinal cells (Chen et al., 2010). Thus, removal of these allergenic soybean proteins is beneficial for both digestion and animal health.

Soybean meal manufactures use a quality control measurement called protein dispersibility index which should be around 40% (Soy 20/20, 2008). This indicates that at least

40% of the protein found in soybean meal should be recovered when suspended in water. The 74 allergenic proteins focussed in this paper are β-conglycinin and glycinin and each of its subunits has different degrees of solubility (Zheng et al., 2009). The solubility of the subunits can vary depending on pH or temperature (Zheng et al., 2009). The subunits of β-conglycinin were found to have 50% solubility in water at pH equal to or greater than 6 (Zheng et al., 2009). The subunits of glycinin were found to have a solubility of approximately 100% at pH greater than

6.5 at 20 °C at varying ionic strengths (Lakemond et al., 2000). This thesis only investigated the changes in the soluble protein when fermented with our isolates and did not measure the reduction of the insoluble allergenic proteins. Although it was not measured, it cannot be concluded that the insoluble portion of the allergenic proteins were not also reduced, and thus, future studies should investigate the changes in the insoluble protein or total protein when soybean meal is fermented with Isolate-2.

The solid-state fermentation with Isolate-2 significantly increased the crude protein by

5.21 % and 7.84% in 24 hour and 48 hours, respectively. This level of increase is within the range of crude protein increased using A.oryzae. Soybean meal fermented with A. oryzae has been reported to have crude protein increased ranging from 1.95 % (Chen et al., 2007) to 10%

(Hong et al., 2004). The increase in crude protein can be a result of two processes. Firstly, the increase crude protein observed can be due in part to the decrease in carbohydrates (Hong et al.,

2004). Some bacteria can break down cellulose, polysaccharides, and oligosaccharides and utilize its sugar subunits for their metabolism and respiration processes (Opazo et al., 2012). As crude protein is a percentage of dry matter, any reduction in dry matter would lead to an increase in percentage of crude protein. Decrease in dry matter content could also be due to the required gaseous (carbon dioxide) losses during fermentation. Our finding that Isolate-2 has cellulase activity is consistent with this possibility. Another aspect of fermentation that would cause an increase in crude protein, is the production of proteins by the bacteria. Many Bacillus species, 75 including Bacillus amyloliquefaciens, have been found to produce a polymer of glutamic acid called poly-ɣ-glutamate (PGA) (Feng et al., 2014). Our finding of glutamic acid level increase in the fermented soybean meal may be a result of PGA production. PGA is biodegradable, edible, and is safe for humans (Shih & Van, 2001). It can be found in many traditional fermented foods such as the Japanese fermented food natto (Kada et al., 2012), has high antioxidant activity, as well as play a role in the immune response (Lee et al., 2014; Lee et al.,

2011). It interacts with toll-like receptors to decrease pathological symptoms caused by airway asthma (Lee et al., 2011), as well as reduce inflammation caused by angiogenesis and mucosal inflammation observed in irritated bowel disorder (Sung et al., 2013). It would be interesting to further investigate if PGA concentration is indeed increased in soybean meal during fermentation.

The amino acid profile was also improved in the solid-state fermentation with Isolate-2.

Of the 17 amino acids analyzed, levels of valine, phenylalanine, aspartic acid, glutamic acid, glycine, and proline were significantly higher than the unfermented soybean meal at 24 hours of fermentation. At 48 hours, levels of histidine, cysteine, and tyrosine increased from a numerical change (24 hours) to significance; however the levels of valine, aspartic acid, glycine and proline decline and were no longer statistically significantly different than the unfermented control. The increase of selective amino acids was also observed in other studies as well. Hong et al. (2004) observed a significant increase in glycine, glutamic acid, and aspartic acid when soybean meal was fermented with A. oryzae. Another study using A.oryzae observed similar results with levels of amino acids threonine and glutamine only significantly increasing when Lb. casei was added

(Chen et al., 2007). On the other hand, using cracked soybean fermented with A.oryzae reported

76 significant increases in all amino acids except arginine and lysine (Frias et al., 2008). Similarly, cracked soybeans fermented with B.subtilis increased aspartic acid, glutamic acid, serine, alanine, proline, valine, methionine, cysteine, isoleucine, phenylalanine, tyrosine and lysine levels after 48 hours (Frias et al., 2008). This increase in amino acids may be in part due to the protein constituents of bacterial mass and microbial metabolism (Chen et al., 2010). The results of the current study also showed that levels of arginine decrease at 48 hours of fermentation.

Our result on the increase of total essential amino acid in 24 but not 48 hours (which has an increase in nonessential amino acid only) of fermentation indicates that bacteria at different phases of the fermentation may utilize and synthesize a different array of amino acids. This data again suggests that the 24 hour time point is not only more cost effective, but also desirable for the fermentation when compared to the 48 hours. The length of the fermentation is an important factor to consider at the production scale. In an agricultural production facility, like any other production facility, the manufacturing time of the product may at times be a limiting factor. The time to manufacture a product may dictate the required fermentation capacity and how quickly the product can be sold, which influences the profit of the product. The faster a product can be made the more potential revenue could be had. The length of the fermentation can be greatly affected by choice of inoculum. The fermentation of soybean into traditional soy sauce can be greatly reduced from 7 days to 40 hours by using selected strains of A.oryzae which secrete high amounts of extracellular enzymes (Valyasevi & Rolle, 2002). Other published soybean fermentation systems have reported fermentation lengths of 40 hours (Chen et al., 2010), 48 hours (Hong et al., 2004, Frias et al., 2008), 72 hours (Lee et al., 2010) or 10 days (Opazo et al

2012). Our fermentation with the newly isolate bacteria has shortened the required length to only 24 hours in order to obtain the desired protein and amino acid profile.

Moisture level is another important factor when designing a fermentation system. Too little moisture will not allow the bacteria to grow and survive, yet a moisture level too high may result in undesirable mould growth. Another advantage to our solid-state system is our ratio of

2:1 (weight of liquid: weight of substrate). Previous studies have reported ratios such as

2.4:1(Opazo et al., 2012), 3.5:1 (Refstie et al., 2005), and 10:1 (Rodrigues et al., 2010) which are all (slightly) higher than our ratio. By optimizing fermentation conditions to have a low moisture level, we decrease the risk of mould growing and also make it more efficient for the fermented soybean meal to be inoculated animal feed. Many animal feeds, including swine and poultry feed, are processed into pellets. The pelleting process itself is very delicate and if moisture level of the ingredients is too high the pellets will not be strong enough to withstand the transport to the farm. Our solid-state fermented soybean meal product contains a lower moisture level than other previous studies, and thus, requires less drying time. Decreasing any additional heating required for processing also decreases the risk of further heat damage to the proteins.

The results of this study demonstrate that the protein profile, in terms of decreasing allergenic proteins, decreasing high molecular weight proteins, increasing low molecular weight proteins, and increasing total amino acids, can be improved by fermenting soybean meal using our Isolate-2, we were also able to observe a decrease in the oligosaccharides. Our results indicate that raffinose and stachyose levels decreased to non-detectable levels in soybean meal after fermentation with Isolate-2. The oligosaccharides in soy are indigestible to piglets and have been found to cause a decrease in piglet performances (Sohn et al., 1994). Increased levels of oligosaccharides have been found to negatively affect nutrient and enzyme interaction, as well as decrease the digestibility of dry matter in the ileum (Smits & Annison, 1996; Van Kempen et al., 2006). Studies have reported a decrease in soy oligosaccharides, stachyose and raffinose, 78 and NSPs when soybean meal was fermented with bacteria (Refstie et al.,2005; Opazo et al.,

2012) although a much longer term (10 days) fermentation was required. The reduction of these oligosaccharides could have been due to hydrolysis of the α-1,6-galactose linkage by the enzyme

α-1,6-galactosidase. Some strains of Bacillus have been found to secrete extracellular α-1,6- galactose (Lee & Cho, 2012) which may explain the reduction of oligosaccharides observed.

This study investigates the fermentation of soybean meal using single bacterium. Other studies have investigated the benefits of adding more than one microbe to soybean meal fermentation. It was observed that when a combination of B.subtilis, Lb. fermentum, and

Saccharomyces cerevisae were used to ferment soybean meal with wheat bran, it could reduce the growth of Enterobacteriaceae in vivo (Ying et al., 2009). The authors attributed this reduction of pathogenic bacteria to the antimicrobial proteins produced by their strain of

B.subtilis and to the secondary metabolites produced by the lactic acid bacteria which inhibits spoilage (Ying et al., 2009). Viable Lactobacillus cells and Bacillus spores were found after fermentation and have led the authors to conclude that this new method of fermentation could be an alternative to using antibiotics in the animal industry (Ying et al., 2009). Another study investigated the effects of three newly isolated strains of cellulolytic bacteria isolated from various sources on soybean meal fermentation (Opazo et al., 2012). The soybean meal was fermented with 1×109 cells of inoculum (a mixture of bacteria from

Streptomyces, Cohnella and Cellulosimicrobium genuses) /g of substrate for 10 days in 37 °C under 2.4:1 (weight of water: weight of substrate) with constant agitation. The fermentation reduced stachyose levels to 0.44 grams/ 100 grams of soybean meal and raffinose levels to 0.48 grams/100 grams of soybean meal compared to that of that of the non-inoculated control groups

79 which were 3.52 grams and 2.11 grams, respectively (Opazo et al., 2012). NSP concentrations were also reduced to 12.17 grams/ 100 grams of soybean meal compared to the 16.01 grams/100 grams of soybean meal found in the non-inoculated group (Opazo et al., 2012). There have also been studies exploring the possibility of using a two-step fermentation to observe the dual benefits of using A.oryzae and Lb.casei in soybean meal fermentation (Chen et al., 2010). The two-step fermentation, total fermentation time of 40 hours at 30 °C (first-step), and 37 °C

(second-step), successfully decreased soy oligosaccharides to non-detectable levels, increasing essential amino acids by 9.5%, and decreasing most allergenic proteins, compared to that of the unfermented control (Chen et al., 2010). Three-step fermentation of soybeans using Lactococcus lactis, A.orzyae, and B.subtilis was found to have a higher total amino acid concentration than when fermented with B.subtilis alone (Lee et al. 2007). This improvement in fermentation is due to the mixture of enzymes secreted by each type bacteria (Lee et al., 2007). Some microbes, such as A.oryzae, prefer select amino acids, which may result in only certain amino acids being increased (Hong et al., 2004). Using a combination of bacteria in soybean meal fermentation may result in the increase in different amino acids, resulting in a larger increase of total amino acids (Lee et al., 2007). Including a lactic acid bacteria (LAB) in soybean fermentation may add additional benefits such as increasing amino acid concentration and decreasing the pH of the fermented product (Lee et al., 2007). In traditional fermentations, many LABs are used as a starter culture to inhibit the growth of any pathogenic microbes (Leroy& De Vuyst, 2004).

Using LAB in soybean fermentation may also be beneficial to early-weaned pigs. Feeding piglet metabolites of LAB have demonstrated to reduce incidences of diarrhoea, decreased faecal pH and pathogenic bacteria, and increase short chain fatty acids (Thu et al., 2011). It would be of interest to further investigate if combination of other microbes with Isolate-2 can further improve fermentation of soybean meal.

In conclusion, this in vitro solid-state fermentation using newly isolated, food derived bacteria, resulted in an improved protein profile, an increase in crude protein concentration, an increase in total amino acid concentration, and a decrease in raffinose and stachyose. This fermentation system achieved effective fermentation after 24 hours and a relatively low moisture level. A follow-up study should investigate if further optimization of fermentation conditions results in further improvements in nutritional value of fermented soybean meal product and to compare these to commercial or other experimental fermented products. Decreasing the fermentation length would allow decrease of facilities running cost and product turnover time.

Also, the low moisture level of our system reduces drying needs. The improvement in all of these aspects could potentially lead to a plant protein source that is less expensive to produce, highly digestible, and allergenic-free for piglets and other animal species, in order to reduce some of the negative performance observed during early weaning due to feeding soybean meal. Further studies should investigate the NSP profiles, as well as the antioxidant profile of the fermented soybean meal product. Isolate-2 should be further investigated to determine if there are any toxic effects to mammalian cells and if there is an advantage of combining different a LAB with

Isolate-2 to ferment soybean meal.

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Appendix A

Type of Amino Amino Acid Treatment Acid (% DM) Unfermented P.con Isolate-2 Isolate-4 Isolate-8 Isolate-7 Essential Lysine 3.52 3.89 3.91 3.68 3.82 4.1 Threonine 2.22 1.9 2.24 1.72 1.97 1.87 Methionine 0.84 0.91 0.94 0.85 0.93 0.93 Isoleucine 2.46 2.41 2.84 2.83 2.56 2.48 Leucine 4.64 4.82 5.41 5.09 4.98 5.24 Histidine 1.37 1.11 1.23 1.23 1.36 1.54 Valine 2.6 2.73 3.4 3.16 2.75 2.9 Arginine 3.94 3.18 3.03 2.59 3.11 3.06 Tryptophan 0.68 0.76 0.8 0.74 0.76 0.82 Phenylalanine 2.78 3.15 3.33 3.21 3.19 3.31 Serine 2.79 2.34 2.36 1.86 2.33 2.22 Glutamic acid 9.14 9.03 9.52 9.03 8.9 9.08 Non- Essential Proline 2.46 2.56 3.08 2.47 2.85 2.69 Glycine 2.32 2.19 2.47 2.13 2.26 2.15 Alanine 2.4 2.38 2.57 2.24 2.26 2.25 Cysteine 0.76 0.81 0.82 0.79 0.78 0.77 Tyrosine 1.44 1.62 1.19 1.55 1.14 1.56 Aspartic acid 6.24 5.93 6.14 5.53 6.15 6.16

Total* AA (% DM): 52.59 51.71 55.28 50.69 52.1 53.13 Total EAA (% DM): 25.05 24.86 27.13 25.1 25.43 26.25 Total* NEAA (% DM): 27.55 26.86 28.15 25.6 26.67 26.88

Table 2 Quantitative preliminary data showing the effect of fermentation on each amino acid when fermented with different isolates under high-moisture conditions. Bolded numbers represents increase value compared to the unfermented control.

*With the exception of glutamine and asparagine.