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Graduate Theses and Dissertations

12-2019

The Physicochemical Properties of Soy Protein Hydrolysate and The Physicochemical Properties of Soy Protein Hydrolysate and

its Formulation and Stability with Encapsulated Probiotic its Formulation and Stability with Encapsulated Probiotic

John Stricklin Edwards University of Arkansas, Fayetteville

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The Physicochemical Properties of Soy Protein Hydrolysate and its Formulation and Stability

with Encapsulated Probiotic

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Food Science

by

John Stricklin Edwards

Hendrix College

Bachelor of Arts in Biology, 2012

December 2019

University of Arkansas

This thesis is approved for recommendation to the Graduate Council.

Navam S. Hettiarachchy, Ph.D. Thesis Director

Suresh Kumar Thallapuranam, Ph.D. Committee Member

Franck Gael Carbonero, Ph.D. Committee Member

Abstract

Soy protein isolate (SPI) and probiotics can serve as a functional ingredient and beneficial sup-

plement, respectively, to food formulations, but few studies focus on the compatibility of these

two ingredients. The objective of this study was to prepare soy protein isolate (SPI) from defat-

ted soybean flour and identify an optimal hydrolysis protocol to create soy protein hydrolysates

(SPH) with improved solubility and functional properties and achieve an optimum encapsulation

technique for probiotics and to determine the in vitro viability of encapsulated probiotic cells with

SPH in a gastrointestinal environment. The SPI was prepared using an alkaline extraction pro-

cedure for solubility within a beverage-specific pH range. The SPH was prepared from aqueous

extracted soy protein isolate using pepsin and freeze-dried. The physicochemical properties of

the SPH were investigated for solubility, degree of hydrolysis, surface hydrophobicity, and elec-

trophoresis. Alginate was used as an encapsulation agent for L. acidophilus cultures to create

freeze-dried microcapsules. The dried SPH and encapsulated probiotics with alginate in a dry

powder mixture were tested for its gastrointestinal resistance and probiotic viability under in vitro

simulated digestion. The experimental groups were a control of free probiotic cells, encapsu-

lated probiotics only, free probiotics with SPH, and encapsulated probiotics with SPH. Hydroly-

sates from 2, 2.5, and 3 hr. of hydrolysis time achieved a suitable degree of hydrolysis (DH) be-

tween 2.5-5.0%. The 2.5- and 3-hour hydrolysates had higher solubilities of 86 and 88%, re-

spectively, in comparison to SPI with 74% at pH 6.5 (p < .05). Surface hydrophobicity of the hy-

drolysates ranged from 15 to 20 So units, with surface hydrophobicity generally decreasing over

time. Microcapsules measured 1 mm in size using environmental scanning electron microscopy

(ESEM). Probiotic cultures with mean log CFU of 7.74 were obtained before gastrointestinal re-

sistance. Approximately 1-log decrease was observed for all experimental groups after simu-

lated digestion ranged with the lowest value of 6.19 log CFU for free probiotics. No significant

difference was observed among experimental groups for probiotic viability (p = .445). The find-

ings of this research provide an understanding of improved formulation for more sustainable soy

protein hydrolysate and viability of encapsulated probiotics within gastrointestinal environments.

Acknowledgments

Special thanks are extended to the professors and staff within the Department of Food

Science and beyond at the University of Arkansas. I have learned much from them and appreci-

ate their efforts toward greater knowledge. Notable contributors to this research include Dr. Ravi

Gundampati, Dr. Srinivas Jayanthi, Dr. Jiali Li, Dr. Betty Martin, and Dr. Mourad Benamara.

I would like to provide special recognition to Dr. Kumar and Dr. Carbonero for their time,

patience, and advice as committee members.

A very special thanks goes to my advisor, Dr. Hettiarachchy. Her dedication and guid-

ance have been instrumental. I also thank Dr. Horax, and my research group colleagues, partic-

ularly Ali Bisly, for all their teaching, support, and assistance. Many thanks go to Dr. Cameron

Murray, whose advice and friendship continue to be invaluable.

Dedication

This thesis is dedicated to all my family, particularly Cynthia, John, and Jackson. I would

not be where I am today without them. A special dedication goes to Michelle Hastings for her

words of encouragement and exemplifying true bravery.

Table of Contents

Chapter 1: Introduction 1

1.1 Soybean overview 1

1.2 The future of soybean as a genetically engineered crop 2

1.3 Soy protein use and market appeal 3

1.4 Combining protein hydrolysates with probiotics in food products 8

1.5 Hypothesis 10

1.6 Objectives 10

1.7 References 11

Chapter 2: Literature Review 15

2.1 Soybean 15

2.2 Soy protein 18

2.3 Soy protein isolate and hydrolysate use in protein-rich beverages 23

2.4 Probiotics potential health benefits and incorporation into soy protein products 24

2.5 References 28

Chapter 3: Prepare soy protein isolate from non-GE soybean seeds (R08-4004)

and optimize conditions to hydrolyze the protein using pepsin 36

3.1 Introduction 36

3.2 Materials and Methods 37

3.3 Results and Discussion 40

3.4 Conclusion 41

3.5 Tables and Figures 43

3.6 References 45

Chapter 4: Determination of physicochemical properties (degree of hydrolysis, surface

hydrophobicity, and electrophoresis) of soy protein hydrolysates

and probiotic encapsulation viability under simulated gastrointestinal

conditions 49

4.1 Introduction 49

4.2 Materials and Methods 51

4.3 Results and Discussion 57

4.4 Conclusion 63

4.5 Tables and Figures 64

4.6 References 68

Overall Conclusions 72

Appendix: Tables and Figures 73

1

Chapter 1: Introduction

1.1 Soybean overview

Soybeans (Glycine max) are a crucial crop within the agricultural and food processing

industries. Soybeans are cultivated and selected as either two designations: food-grade or

feed-grade. Soybeans are known for their high protein content, with some varieties yielding be-

tween 40-50% protein, and can yield greater amounts of protein per acre than other major vege-

tables or grains, dairy animals, and meat animals (Sharma and others 2014, National Soybean

Research Laboratory 2015, American Soybean Association 2016). Soy protein is also cheaper

for total energy inputs versus energy outputs from the food produced, especially in comparison

to meat proteins (Pimentel 2009). For example, soybeans have an energy input/output ratio of

1:3.7 kcal, while chicken has a ratio of 4:1 kcal, the lowest among meat proteins (Pimentel

2009). Worldwide soybean production has increased 500% within the past 40 years, and the

United States plays a significant role in that production. Greater than 80 million acres of soy-

beans are planted every year in the United States as a major export crop and animal feed

source (National Agricultural Statistics Service 2019). Although not within the top ten of soybean

producing states, Arkansas soybean cultivation and production are increasing, with 3 million

acres planted, a higher acreage than rice (National Agricultural Statistics Service 2019). This

production increases Arkansas’ international presence within the global agricultural and food

markets. At least 50 out of 75 counties in Arkansas have land dedicated to soybean farming,

with a majority concentrated within the eastern Mississippi Delta region of the state. A few soy-

bean-growing counties are found within the Arkansas River Valley and Red River Valley (Na-

tional Soy-bean Research Laboratory 2015, Arkansas Soybean Promotion Board 2014).

Soy is a high protein source and has a great range of benefits to the food sciences. Soy

protein serves as a multifaceted, functional food product, improving qualities such as reduced

fat absorption, enhanced color, and improved texture (Singh and others 2008, National Soybean

Research Laboratory 2015). Soybeans can be processed into textured soy protein (TSP) that

2

provides a healthier alternative to meat, through lower cholesterol and saturated fat while main-

taining texture. Not only does soy protein provide functional physical properties within food sys-

tems, but a high-protein soy food is complemented by beneficial compounds for human health

(i.e. isoflavones) found naturally in soybeans. A diet containing appropriate amounts of soy pro-

tein may reduce the risk of heart disease, reduce breast cancer risk, and contain phytochemi-

cals that may aid in slowing or preventing the progression of several cancers and/or other dis-

eases (Friedman and Brandon 2001; Zhu and others 2005; National Soybean Research Labora-

tory 2015; Rayaprolu and others 2013). The science behind soybean beneficial health qualities

has been communicated and received well by consumers. According to the United Soybean

Board (2015) “Consumer Attitudes about Nutrition” survey, 73% of consumers have adjusted

their diet for health reasons. And 61% of participants believe that soy-based foods can help in

reducing obesity (United Soybean Board 2015). Around 75% of Americans consider soy prod-

ucts as healthy “on an aided basis.” Nearly 40% of consumers are aware of the FDA-approved

health claim stating that “consuming 25 grams of soy protein per day may reduce the risk of cor-

onary heart disease” (United Soybean Board 2015). This growing, positive regard for soy pro-

tein among consumers provides a foundation for greater, improved communication of known

and potential health benefits to these individuals (Mangano and others 2013).

1.2 The future of soybean as a genetically engineered crop

The U.S. Office of Disease Prevention and Health Promotion (ODPHP) has recently

stated that the USDA’s 2015-2020 Dietary Guidelines for Americans describes a healthy diet

that contains “a variety of protein foods, including…soy products” and “oils, including those

from…soybean[s]” (Office of Disease Prevention and Health Promotion 2016). Yet some con-

sumers are concerned about the prevalence of genetically engineered (GE) soybean seeds be-

ing planted (94% of total seeds) and yielding crops entering the food market (United States De-

partment of Agriculture 2015). Even though the National Institute of Health (NIH) and European

Food Safety Authority (EFSA) have approved GE soybean crops as safe, 29% of consumers

3

consider GE soybeans unsafe (The Organic & Non-GMO Report 2007; EFSA 2014; Kareiva

and others 2016). However, there are opportunities for non-GE soybeans to prosper in Arkan-

sas. A study by the Arkansas Soybean Promotion Board (2012) was presented that described

“the potential for Arkansas growers to exploit the market” for non-GE soybeans, ranging from a

variety of food products that would be sold in North America and Asia. This overlap of consumer

and industry interests represents an opportunity for both to benefit. There are myriad possibili-

ties to align agricultural, food industry, and consumer interests by researching and developing

new, non-GE soybean food products, based on food science principles. The goal of this study is

to provide further scientific insights by developing an innovative protein ingredient formulation

that can facilitate the expansion of non-GE soybeans for cultivation. These new soybean culti-

vars should be selected for nutrient density, high-protein content, and increased yields.

1.3 Soy protein use and market appeal

Future developments with soybean protein are expected to be a part of emerging food

protein trends. Recent research and surveys reveal that the maturation of the protein market

has not yet been reached (Health Gauge 2013). The market continues to thrive and grow due to

increased demand for protein, supported by continued science and consumer-health communi-

cation efforts. Protein is viewed as a component to “help maintain overall health” by individuals

of varying age but has great appeal to the “ageing population, people managing their weight,

and growing children” who need it most (Health Gauge 2013). Studies suggest that moderate to

high protein consumption should slow muscle loss and diminish the decrease in protein metabo-

lism that occurs with age (Friedman 1996; Health Gauge 2013). According to research from

Mintel (2013), three-times as many U.S. foods claim to have a high protein content versus other

global products and constitute 19% of new products launched worldwide in 2012. This trend fol-

lows the “‘protein awareness’” that is greater among U.S. consumers than other global consum-

ers (Mintel 2013). High protein snacks cover the greatest portion of the new product market

(20%), followed by meal replacement and other fortified drinks (17%) and yogurt products

4

(15%), from 2012 new product launches (Mintel 2013). Included in this greater awareness of

protein, more consumers are looking for cleaner, healthier, or more ethical, eco-friendly protein

sources. These predominantly include plant proteins, as data shows that products “with both a

high protein and vegan claim” having had a 54% growth (2008-2012) over a five-year period

(Mintel 2013). For the period of 2016-2020, market reports forecasts predict a CAGR of 6.43%

for the global soy food market, representing soybean’s presence as an important, healthy food

protein crop (Business Wire 2016).

High protein products also entice consumers as effective for meal replacement and

providing adequate levels of satiety (Mintel 2013). Packaged Facts (2014) summarized that the

high-protein food product trends include “analogs for meat protein” which would require contin-

ued research in alternative protein sources, like soy protein, and establishing “nutrition-science

based communication…for success with protein-rich and protein-fortified food and beverages.”

This data shows that there is a great demand for protein-fortified beverages from an alternative

protein source like soy protein hydrolysates (Friedlander 2012). An added benefit is the increase

in adaptability of soy products, particularly the use of soy protein in smoothies, energy bars, and

pre-packaged convenience meals, which are noted for serving a market need and may be ex-

panded for future product innovation (Food Business News 2008). Pea protein is another pro-

tein source that is gaining support for use and could be a potential rival to soy protein, particu-

larly for exercise-specific protein (Tulbek and others 2017, Banaszek and others 2019). Collec-

tively, the future of plant protein continues to look promising. The increase in sales of high pro-

tein ingredients, like seeds, nuts, and grains, supports the demand for alternative, non-animal

protein sources, such as soy, that should continue (Granato and others 2010; Foster 2016).

From the maintained popularity and trend of sales for high protein, particularly soy pro-

teins products, there is a solid foundation for the continued development of food products that

meet consumer trends and benefit their health. There are obstacles when considering a formu-

lation for a new food product, particularly beverages incorporating soy protein. One of the main

5

concerns is that consumers may experience “disutility through changing their diets to increase

the consumption of healthier foods” through fear of taste acceptability, cost, and time (Malla and

others 2015). The most popular beverage proteins for many years have been whey and soy pro-

tein isolate (SPI). Soy protein also has a continued market interest from increased milk protein

prices and supply complications, since it can serve as a complete protein (Rayaprolu and others

2015) and calcium replacement. Soy proteins can replace milk proteins partially or entirely, de-

pending upon the food matrix, while maintaining marketing and consumer appeal at a reduced

cost for the final food product (Tockman 2002; Cosgrove 2005). Although yogurt and smoothie

protein beverages had been popular, many major companies anticipated and expanded bever-

age types that incorporate protein (Cosgrove 2005). However, increasing levels of protein pro-

vides difficulties, including altered flavor/aftertaste, acid/heat stability, clarity, dispersability (for

dry mixes) and solubility (Cosgrove 2005). Especially with acidic beverages, added soy protein

can compromise flavor and result in protein destabilization. Even if the beverage is neutral, high

protein levels can create viscosity issues along with flavor and protein stability compromises

(Cosgrove 2005). Soy protein hydrolysates (SPH), derivatives of SPI, may provide an improved

protein source for beverage purposes.

Even though the majority of U.S. food products contain GE ingredients, the consump-

tion of these products has received negative biotechnology opinions from consumers (Fernan-

dez-Cornejo and Caswell 2006). From analysis of consumer opinions by Consumer Reports

(2014), over 70% of respondents claimed they did not want genetically modified organisms

(GMOs) in their food, and 92% wanted genetically modified ingredients to be labeled. With the

FDA very recently closing its deadline for public commentary submissions on the definition and

use of the term “natural,” there are even more factors to debate regarding GMOs (U.S. Food

and Drug Administration 2015). From the same Consumer Reports survey, nearly 60% believe

that “natural” equates to “no GMOs” (Consumer Reports 2014). These changes are generally

expected with the use of these crops within such a management system, but the extent to which

6

their environmental and economic impacts occur is still of consideration (Watkinson and others

2000; Kamle and Ali 2013; Nicolia and others 2014). For these reasons, producing a food prod-

uct that would contain non-GE soybean varieties would provide environmental sustainability and

crop biodiversity, promote a value-added product, and meet consumer demand.

1.3.1 Soy protein and hydrolysate preparation, characterization, and use for food products

The proposed research will focus on providing more data towards understanding the use

of SPH as an improved beverage ingredient. SPH should improve flavor impact, protein stabil-

ity, and viscosity/texture modifications. Professionals in the industry note that “‘protein ingredi-

ents need to be hydrated properly and…have a homogenization step’” to provide better stability

for shelf-life, flavor, and texture (Cosgrove 2005). Soy protein hydrolysates can even be modi-

fied to provide increased functional and immunological properties, making them usable “as addi-

tives for beverage” and food systems (Chiang and others 1999). For individuals who may have

difficulty digesting intact dietary proteins, protein hydrolysates can provide a solution as smaller,

more easily digestible nutrients.

Protein hydrolysates have been commonly prepared through chemical or enzymatic hy-

drolysis. Enzymatic hydrolysis is preferred over chemical hydrolysis, as its reaction procedures

are less extreme and intensive and provides a more precise, favorable yield of protein hydroly-

sates (Chiang and others 1999). Enzymatic hydrolysis can modify whole proteins into peptides,

providing them with added functional and nutritional benefits (Hartmann and Meisel 2007). Soy

protein can have increased chemical, nutritional, and functional properties through this preferred

enzymatic hydrolysis (Tsumura 2009). These SPH provide improved properties such as solubil-

ity, viscosity, emulsion capacity, and gelation for food product applications.

Properly processing soy protein is important in preparation for hydrolysate creation. The

separation and concentration of soy protein into a protein isolate requires several steps. Soy

flour is defatted, which typically entails a mild heat treatment to minimize the denaturation of the

7

protein (Baker and Rackis 1986). Defatted soy flour is mixed with a large volume of water (typi-

cally 10:1) and the solution is adjusted to pH 8.0-9.0 using sodium hydroxide to solubilize the

protein within the flour. Raising the pH higher than 9.0 can lead to reactions and modification of

amino acids into unwanted derivative compounds (Wang and others 2004). Centrifugation of the

solution allows the supernatant containing the soluble protein to be separated from the centri-

fuged mass of the soy flour. Precipitation of the protein within the supernatant is achieved using

hydrochloric acid to adjust to pH 4.5, which is the isoelectric point of soy protein, rendering it

less soluble in solution (Wang and others 2004). Centrifugation can occur again to now sepa-

rate the supernatant and collect the protein isolate residue, which will be mixed with water and

adjusted to pH 7.0, in preparation for freezing and freeze-drying.

Hydrolysates can be prepared from chemical hydrolysis, enzymatic hydrolysis, or ultrafil-

tration membrane reactors (Chiang and others 2006). This research focused on enzymatic hy-

drolysis for its improved yield and safety, in comparison to chemical hydrolysis, and its ease of

use versus membrane reactors. Determination of the extent of hydrolysis is calculated as de-

gree of hydrolysis, the cleavage of peptide bonds to the total number of available peptide bonds

(Nielsen and others 2001). The preferred method for determining hydrolysis has been the OPA

method as established by Nielsen and others (2001) and was used in this research. Hydrolysis

leads to the alteration of protein structure, potentially causing greater exposure of hydrophobic

groups nestled within the interior protein formation. This exposure of hydrophobic groups is

termed surface hydrophobicity (Nakai 2003). The method for measuring hydrophobic groups in-

volves using fluorescent compounds to bind and yield a signature in response to specific light

wavelength (Nakai 2003). The chemical 1-anilino-8-naphthalene sulfonate (ANS) is a common

fluorescent probe used in detection of surface hydrophobicity and was used in this research (Ju

and others 2001, Paraman and others 2007). Understanding the specific hydrolysis and hydro-

phobicity of hydrolysates can provide useful data towards understanding how the hydrolysates

8

can interact among other food ingredients or what exact qualities their incorporation will bring to

a product.

Protein hydrolysates are smaller peptide sequences or free amino acids that have lower

mass than their origin protein from which they were cleaved (Laemmli 1970). Several methods

exist to determine the relative mass of peptides and are represented in kilodalton (kDa) units.

An effective and reliable technique is polyacrylamide gel electrophoresis containing sodium do-

decyl sulfate (SDS-PAGE). The sodium dodecyl sulfate (SDS) serves as a surfactant that binds

to peptide segments and aids in their size separation during electrophoresis (Reynolds and Tan-

ford 1970). Separating hydrolysate samples and comparing them to their native protein struc-

tures prove useful in providing feasible and fast analysis of hydrolysate peptide composition.

Providing a high-quality protein hydrolysate can ensure its function and compatibility with other

nutraceutical ingredients.

1.4 Combining protein hydrolysates with probiotics in food products

Probiotics and their incorporation into food products that benefit gut health is another

area of food science that has experienced significant sales growth, between 7-8% annually, to

total sales worth billions of dollars in both the U.S. and Europe (Saxelin 2008; Maccaferri 2012;

BCC Research 2014). Probiotics are defined as “live microbial food supplements” which can

confer health benefits through digestion, mostly within the intestinal microbiota (Noland and Ar-

yana 2012; Mitropoulou and others 2013). Using probiotics as a beneficial food ingredient can

dynamically improve the health and wellness of functional foods and appeal among consumers

(Saxelin 2008). Providing a protein-rich dry powder formulation as a food matrix for a probiotic

delivery system does present some challenges. The delivery system, or transport mechanism,

should establish preservation of the bioactive compound without “negatively influenc[ing] the

consumer acceptance of food products” (Sessler and others 2013). The probiotics should be

protected from degradation by chemical, microbial, enzymatic, or even thermal mechanisms.

9

Food ingredients and their intrinsic properties, such as flavor, color, and texture should be pro-

tected from probiotic release and potential interference (Sessler and others 2013; Castro and

others 2013). In respect to probiotics, immobilization-encapsulation technology (IET) has

emerged as a rapidly developing area with useful application for protecting and incorporating

probiotics (Mitropoulou and others 2013). One major application is the function of IET in con-

trolled delivery of microbes within the gut microflora. A benefit to this technology is the gradual,

managed release of probiotics, allowing them to adjust to host microbiota conditions, while at

the same time hindering any side effects of probiotic release into the host’s biome (Mitropoulou

2013). There are a variety of organic compounds that can be utilized for encapsulation of mi-

crobes for life science purposes, from starch-derived cyclodextrins to gelatinous polysaccharide

and colloid capsules (Mourtzinos and others 2008). As Noland and Aryana (2012) state, probiot-

ics, as foreign microorganisms, can face challenges of “low pH [from] bile acids and digestive

enzymes” within human gastrointestinal tracts. Encapsulation provides an effective way to pro-

tect probiotics from these conditions. Although cell immobilization technology has been devel-

oped for some time, the use with probiotics provides limitations. Since probiotics will be incor-

porated as a “live” consumable ingredient, only certain methods are safe for commercial use.

For probiotic incorporation, spray-drying and encapsulation techniques are potential methods for

compatible incorporation with SPH. As these methods are refined and perhaps made more af-

fordable, industrial utilization can increase, providing safe and novel ways to enhance probiotic

incorporation within foods systems.

10

1.5 Hypothesis

The hypothesis for this study was based on the quality of a combined protein hydroly-

sate and probiotic dry mix with acceptable physicochemical properties. The specifics of the hy-

pothesis are:

1. Controlled limited hydrolysis of soy protein would provide hydrolyzed protein that

has increased solubility and decreased bitterness to formulate a quality protein dry

formulation

2. Incorporating an encapsulated probiotic for enhancing the dietary value of the dry

formulation would be effective in improving probiotic viability

1.6 Objectives

This research focused on developing a high-protein dry mix formulation with incorpo-

rated probiotics, using a novel, protein-rich non-GE Arkansas soybean cultivar. Specific

objectives are as follows:

a. Prepared soy protein isolate and hydrolysates using pepsin enzyme

b. Conducted physicochemical evaluation of the soy peptide hydrolysates

c. Characterized microcapsule morphology using environmental scanning electron mi-

croscopy (ESEM)

d. Prepared a dry formulation of hydrolysate and probiotic to determine its gastrointesti-

nal resistance

11

1.7 References

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market in Arkansas. Available from http://www.themiraclebean.com/funded-soybean-re-search. Accessed 2016 January 20.

Baker EC and Rackis JJ. 1986. Nutritional and Toxicological Significance of Enzyme Inhibitors

in Foods. In: Advances in Experimental Medicine and Biology. New York: Plenum Press. p 349-55.

Banaszek A, Townsend JR, Bender D, Vantrease WC, Marshall AC, and Johnson KD. 2019.

The effects of whey vs. pea protein on physical adaptations following 8-weeks of high-inten-sity functional training (HIFT): a pilot study. Sports (Basel) 7(1):12

BCC Research. 2014. The Probiotics Market: Ingredients, Supplements, Foods.

http://www.bccresearch.com/market-research/food-and-beverage/probiotics-market-fod035d.html Accessed 2015 Nov 14

Business Wire. 2016. Available from: http://www.businesswire.com/news/home/20160506005372/en/Global-Soy-Food-Market-2016-2020---Rising. Accessed 2016 February 4.

Castro WF, Cruz AG, Bisinotto MS, Guerreiro LMR, Faria JAF, Bolini HMA, and Cunha RL.

2013. Development of probiotic dairy beverages: rheological properties and application of mathematical models in sensory evaluation. J Dairy Sci 96:16-25.

Chiang WD, Shih CJ, and Chu YH. 1999. Functional properties of soy protein hydrolysate produced from a continuous membrane reactor system. Food Chemistry 65:189-194. Chiang WD, Tsou MJ, Tsai ZY, Tsai TC. 2006. Angiotensin I-converting enzyme inhibitor de-rived from soy protein hydrolysate and produced by using membrane reactor. Food Chemistry 98:725-732 Cosgrove J. 2005. Protein-fortified beverages. Beverage Industry 96(7): 55-57. Available

from:http://www.bevindustry.com/articles/83894-protein-fortified-beverages. Accessed 2016 January 17.

EMSL Analytical, Inc. 2008. Determining shelf life. Available from:

http://shelflifestudy.com/shelf_life.html. Accessed 2016 August 3. European Food Safety Authority. 2014. Scientific opinion on application (EFSA-GMO-NL-2009-

64) for the placing on the market of herbicide-tolerant genetically modified soybean BPS-CV127-9 for food and feed uses, import and processing under regulation (EC) No 1829/2003 from BASF plant science. EFSA Journal 12(1):3505 p 1-30.

Food Business News. 2008. Soy acceptance. Available from:

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Foster A. Foodnavigator. 2016. Top food ingredients for 2016. Available from: http://www.foodnavigator.com/Market-Trends/Food-ingredients-trends-revealed?nocount. Ac-cessed 2016 January 18.

Friedlander J. 2012. Soy versus dairy: what’s the footprint of milk? The Conversation. Available from: http://theconversation.com/soy-versus-dairy-whats-the-footprint-of-milk-8498. Accessed 2016 April 12. Friedman M. 1996. Review: Nutritional value of proteins from different food sources. J Agric Food Chem 44:6-29. Friedman M, Brandon DL. 2001. Nutritional and health benefits of soy proteins. J Agri & Food Chem 49(3):1069-1086 Granato D, Branco GF, Nazzaro F, Cruz AG, and Faria JAF. 2010. Functional foods and

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Hartmann R and Meisel H. 2007. Food-derived peptides with biological activity: from research to

food applications. Curr Op Biotech 18:163-169. Health Gauge. 2013. Growing demand for protein. Available from: http://www.healthgauge.com/read/growing-demand-for-protein/. Accessed 2016 January 17. Ju ZY, Hettiarachchy NS, and Rath N. 2001. Extraction, denaturation and hydrophobic properties of rice proteins. J Food Sci 66(2):229-232. Kareiva P, Glenna L, and Gallo MA. 2016. New report on GE crops avoids simple answers-and

that’s the point, study members say. The Conversation. Available from: https://theconversa-tion.com/new-report-on-ge-crops-avoids-simple-answers-and-thats-the-point-study-mem-bers-say-59289. Accessed 2016 June 13.

Laemmli UK.1970. Clevage of structural proteins during the assembly of the head of bacterio-

phage T4. Nature. 227:680-685 Maccaferri S, Klinder A, Brigidi P, Cavina P, and Costabelle A. 2012. Potential probiotic

Kluyveromyces marxianus B0399 modulates the immune response in Caco-2 cells and pe-ripheral blood mononuclear cells and impacts the human gut microbiota in an in vitro colonic model system. J App Envr Micro. 2012:956-964

Malla S, Hobbs JE, and Sogah EK. 2015. Estimating the potential benefits of new health claims in Canada: The case of soluble fiber and soy protein. Can J Ag Econ 00:1-25. Mangano KM, Hutchins-Wiese HL, Kenny AM, Walsh SJ, Abourizk RH, Bruno RS, Lipcius R, Fall P, Kleppinger A, Kenyon-Pesce L, Prestwood KM, and Kerstetter JE. 2013. Soy proteins

and isoflavones reduce interleukin-6 but not serum lipids in older women: a randomized con-trolled trial. Nutr Research 33:1026-33.

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Mintel. 2013. U.S. Consumers Have a Healthy Appetite for High Protein Food. The U.S. Leads the Way in Global Launches of High Protein Products. Available from: http://www.mintel.com/press-centre/food-and-drink/us-consumers-have-a-healthy-appetite-for-high-protein-food-the-us-leads-the-way-in-global-launches-of-high-protein-products. Ac-cessed 2016 January 18.

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15

Chapter 2: Literature Review

2.1 Soybean

2.1.1 Soybean seed composition

The soybean seed is classified as a legume plant species. Sharing horticultural traits

with other legume crops, soy has a higher value for its combination of valuable nutrients, phyto-

chemicals, and other bioactive compounds that can yield health benefits. Soybeans contain sig-

nificant quantities of the three major macronutrients: protein (38%), carbohydrates (30%), lipids

(18%), with the remainder yielding moisture (water content), ash, and hull (14% combined) in

total composition (American Soybean Association 2016). However, the specific composition of

each soybean seed can differ due to cultivar genetic variety, geographical (soil, climate, and en-

vironmental) conditions, and cultivation methods (Berk 1992). From these varying conditions,

some soybean breeders have developed cultivars that have improved characteristics for yield,

in terms of increased protein content (Berk 1992; Anderson and Wolf 1995, Sharma and others

2014)

Figure 1: General composition of mature soybean

(adapted from American Soybean Association)

Soybean oil is low in saturated lipids, higher in unsaturated essential fatty acids, and

serves as a rich source of vitamin E (Hunter and Cahoon 2007). Polyunsaturated fatty acids

compose between 61-63% of the total lipid content within the seed (United Soybean Board

2015c; Reinwald and others 2010). Soybeans serve as a source of omega-6 and omega-3 fatty

acids, particularly linolenic acid (7-8% of total lipid content). Soybean oils high unsaturated fatty

16

acid content classifies it as a semi-drying oil that is vulnerable and prone to lipid oxidation and

deterioration. Improved methods and technologies used in the oil extracting and refining pro-

cess have reduced issues with oil degradation, allowing soybean oil to presently serve as a

quality, versatile, and economical edible oil (Berk 1992). Soybean has lower germination re-

quirements than corn and, as a legume, has nitrogen-fixation capabilities, due to naturally oc-

curring specialized bacteria facilitated by its root nodules (Coulter and others 2010).

The major antinutritional factor within soybean seeds is phytic acid. Phytic acid can bind

to positively charged molecules, such as proteins and cationic minerals found in food, attributed

to their strong negative charge within a wide range of pH values. Removal of phytic acid can be

achieved through proper processing of soy protein isolate to reduce its impact on dietary inter-

actions (Hurrell and others 1992; Zhou and Erdman 1995).

2.1.2 Soybean production

Soybeans are a crucial crop within the agricultural and food processing industries. It

ranks second behind corn as the most planted crop (89.2 million acres in 2018) within the U.S.

and accounts for 90% of U.S. oilseed production (United States Department of Agriculture 2016,

National Agriculture Statistics Service 2019). From 1996 to 2013, soybean food product sales

have gradually increased from $1 billion to $4.5 billion, an average of more than $200 million

per year. Beverages containing soy (excluding soymilk) increased by 12.5% compounded an-

nual growth rate (CAGR) within a two-year period (2011-2013) to $210.5 million alone (United

Soybean Board 2015a). These products are projected to increase in sales, due to increased

consumer demand for value-added, high protein food products. The growth and significance of

soybean food products, particularly its acceptance as a healthy and functional source of protein,

vitamins, and minerals, provide new opportunities for developing innovative food products that

incorporate soybean components, particularly soy protein.

17

Soybeans are harvested for numerous purposes. Most soybeans are processed for oil

extraction and feed meal. Soybean oil is the major vegetable oil within the United States. Soy-

bean meal is primarily used to feed livestock within the meat industry (Food and Agriculture Or-

ganization of the United Nations 2006; Pelletier and Tyedmers 2010). A small remainder of soy-

beans are used for products benefiting human consumption, like tofu, soy flour, soymilk, soy

protein derivatives, and functional ingredients for other food products, such as lecithin for sur-

factant and emulsifying properties. Soybean seed varieties are numerous as well, providing

unique sets of traits that contribute to efficient growth for varied climates, yield, and protein qual-

ity for a specific food product. Soybeans thus provide a valuable source of nutrition domestically

and internationally. Soy products can be incorporated across food cultures, due to nutrient feasi-

bility, wide acceptability, and economic affordability (Carlsson-Kanyama and Gonzalez 2009).

Soybeans and soy products can serve as an affordable and nutritious source of food to reduce

chronic malnutrition within several regions of the world. Developing countries dealing with hun-

ger and nutrient deficiency can use soy products to improve infantile health and assist in creat-

ing healthier, stronger communities (National Soybean Research Laboratory 2016).

Soybeans were one of the first crops to become genetically engineered (GE) to resist

herbicide application, becoming commercially available in the 1990s. Because of their general

acceptance within the agricultural community, GE soybeans are the predominant variety used

throughout food production. For these reasons, producing a food product that would contain

non-GE soybean varieties with enhanced nutritional qualities could be more beneficial to envi-

ronmental sustainability, product marketing, and consumer appeal.

For this research, use of a conventional (non-GE) cultivar (R08-4004, Natural Soybean

and Grain Alliance, Fayetteville, AR) will be used. This soybean is a newer (2013-14) large-

seeded variety, containing a yellow hilum with a mid-MG 5 maturity, and well suited for tofu and

soymilk products (Arkansas Soybean Promotion Board 2014).

18

2.2 Soy protein

Soybean has a higher protein content (35-50%) than other legumes, providing quality

protein and low levels of saturated fat per recommended serving (Messina 1999; United Soy-

bean Board 2015b). Soy protein lacks adequate methionine content but is sufficient in cysteine

for sulfur-containing amino acid content (Friedman and Brandon 2001). The four globulin frac-

tions that compose soy protein, based on sedimentation rate, are 2S (22%), 7S (36%), 11S

(31%), and 15S (11%), comprising total protein content (Shewry and others 1995; Barać and

others 2004). For food applications, soy protein can be processed into differing levels of con-

centration, from lower concentration soy flour to much higher levels in soy protein concentrate

and isolate.

2.2.1 Soy protein isolate preparation

Before the protein extraction, whole soybeans are milled and separated from the natu-

rally occurring soybean oils to get defatted soy flour (Scheide and Brand 1987). Soy protein ex-

traction falls into two general categories: the aqueous extraction method and the non-aqueous

methods (Scheide & Brand, 1987). The non-aqueous methods separate the protein component

from the non-protein component using organic solvents (Scheide and Brand 1987). However,

organic solvents usually cause undesirable effects to protein; for example, they can cause seri-

ous alteration of the protein resulting in denatured protein, which is less palatable and has poor

functionalities such as poor heat gel ability, water binding, and heat coagulation compared to

undenatured protein (Scheide and Brand 1987). In contrast, aqueous extraction techniques gen-

erally result in palatable undenatured protein with good heat gel ability, water binding, and heat

coagulation properties (Scheide and Brand 1987). The principle of aqueous extraction is taking

advantage of the insolubility in water at the iso-electric point (pI) of the glycinin proteins found in

soy (Scheide and Brand 1987). At its pI (pH about 4.0 to 4.8), the protein is insoluble and pre-

cipitated while a large portion of soy flour remains in aqueous solution; and the protein-rich pre-

cipitate can be separated from the supernatant, yielding a high-quality protein concentrate

19

(Scheide and Brand 1987). Wolf (1983) described SPI process using defatted soy flour, which

had oil extracted using hexane, as starting material. Protein was extracted by solubilizing the

defatted meal in water at 60ºC with the meal solvent ratio of 1:10 and pH 8-11. The insoluble

fiber was removed from the solution by centrifuging. The solution was adjusted pH to 4.2- 4.5,

so that the protein would be precipitated. The protein curd was separated from soluble sugars

by centrifuging. Water-wash and centrifugation had been applied several times before the

washed-protein was neutralized to pH 6.8 and spray-dried. According to Russin and others

(2007), the particle size of soy flour could affect the yield of SPI. Particularly, the smaller the

particle size was, the higher the recovery of protein could be achieved. The pH range between

7.5 and 9.0 is most commonly preferred to extremely high pH during the protein extraction be-

cause the excessively high pH can stimulate the protein-carbohydrate interaction that results in

protein loss as well as in formation of dark pigments in solution (Berk 1992).

2.2.2 Enzymatic soy protein hydrolysates

a. Definition

Protein hydrolysates can be prepared through chemical or enzymatic hydrolysis. Enzy-

matic hydrolysis uses enzymes to catalyze protein cleavage, based on affinity to specific amino

acid bonding sites. Enzymatic hydrolysis is preferred over chemical hydrolysis, as the reaction

procedure is less extreme and intensive and provides a more precise, favorable yield of protein

hydrolysate products to reactants (Geisenhoff 2009).

b. Specific enzymes used

The enzymes, or proteases, commonly used for protein enzymatic hydrolysis are al-

calase, trypsin, chymotrypsin, and pepsin. Protease specificity affects the size, amount, and

composition of free amino acids and peptide sequences, influencing the bioactivity and antioxi-

dant nature of resulting hydrolysates (Sarmadi and Ismail 2010). Pepsin is an acidic protease

that cleaves multiple peptide sites, mimicking digestion within the human stomach. Pepsin also

serves to provide bioactive soy protein hydrolysates containing a low DH value, complementary

20

to the intent of this research (Chen and others 1995). The table below provides information of

various types of proteases, their sources, and catalytic specificities.

Source Type of protease Common names

pH range

Preferential specificity

Ox, pig

Aspartic Pepsin 1-4 Aromatic-COOH and -NH2, Leu-, Asp-, Glu-COOH

Serine Trypsin 7-9 Lys-, Arg-COOH

Serine Chymotrypsin 8-9 Phe-, Tyr-, Trp-COOH

Papaya fruit Cysteine Papain (pure) 5-7 Lys-, Arg-, Phe-X-COOH

Fig latex Cysteine Ficin 5-8 Phe-, Tyr-COOH

Pineapple stem Cysteine Bromelain 5-8 Lys-, Arg-, Phe-, Tyr-COOH

Bacillus licheni-formis

Serine Alcalase 6-10 Broad specificity, mainly hydro-phobic –COOH

Bacillus amyloliq-uefaciens

Serine Substilisin 6-10 Broad specificity, mainly hydro-phobic –COOH

(B. subtilis) Metalloprotease Neutrase 6-8 Leu-, Phe-NH2, and other

Table 1: List of proteases commonly used for soy protein hydrolysates

(Adapted and modified from Adler-Nissen 1986)

c. Physicochemical properties and functional properties of soy protein hydrolysates

A protein hydrolysate is a mixture of shorter oligopeptide chains (between 2-20 amino

acids) and free amino acids. Research conducted by Nielsen and others (2001) supports the

use of the o-phthalaldehyde (OPA) method as a more accurate and safer method to determine

degree of hydrolysis (DH), especially with food protein hydrolysates. The DH value indicates the

fraction of peptide bonds that have been cleaved from the starter protein and serves as an im-

portant physicochemical property towards functionality within a food matrix.

Another indicator of hydrolysate quality is surface hydrophobicity. Hydrophobicity deter-

mines polarity, or the degree of interaction with water. The greater the hydrophobicity, the less

likely the protein hydrolysate surface will bind and dissolve within an aqueous medium, disrupt-

ing its hydrophobic and hydrophilic balance (Scarsi and others 1999). Hydrophobicity also gives

information on the hydrophobic and hydrophilic nature of the peptides that provides information

for its use in suitable products for functionality. Beyond functional properties, hydrophobicity can

contribute to digestive stability and in vivo transport and efficacy in proteins (Xie and others

21

2015). With protein hydrolysates, achieving the proper degree of hydrolysis is important to hy-

drophobicity, as excessive hydrolysis can lead to a reduction of total hydrophobicity in peptides

(Cho and others 2004; Xie and others 2015).

An appropriate level of enzymatic hydrolysis is needed in some food proteins to prevent

a reduction of these beneficial effects in food products (Kayashita and others 1997). The same

precautions may need to be practiced with soy proteins, especially for limiting bitterness (Adler-

Nissen 1984). For individuals who may have difficulty digesting intact dietary proteins, protein

hydrolysates can provide a solution. Di- and tripeptides are absorbed more intact than larger

peptides, even after luminal and brush-border enzymatic digestion (Manninen 2009). Thus,

smaller peptides are generally absorbed faster than intact proteins. Due to this more favorable

rapid absorption, protein hydrolysates have been observed as being more effective in stimulat-

ing skeletal muscle protein synthesis than intact proteins (Kim and Egan 2008). Soy protein hy-

drolysate have been measured with enhanced solubility (70-75%) across a wide pH range from

2.0 to 9.0 (Utsumi and others 1997; Chiang and others 1999).

Analyzing the yielded hydrolysates of enzyme catalysis from protein isolate is readily

performed using sodium dodecyl sulfate-polyacyrlamide gel electrophoresis (SDS-PAGE). The

technique was refined by Laemmli during the 1970s and modifications and improvements have

been incorporated over time, while the remaining principles of his initial research, including us-

ing the namesake Laemmli buffer (Pederson 2008). The use of SDS is important as a surfactant

(within the buffer solutions) that can bind to protein segments in quantifiable portions relative to

protein size. The ratio is understood to be 1.4 gram of SDS bound to 1 gram of protein (Smith

1984). The SDS envelopes the proteins and provide an overall anionic charge to the structure

that facilitates their migrations through the gel (Smith 1984). Numerous protein samples can be

run in a gel (up to ten), with each sample isolated within a specific gel lane. A protein ladder,

22

containing a collection of known proteins standards of varying sizes, and a sample of the spe-

cific native protein that corresponds to the respective hydrolysates are typically included with hy-

drolysate samples for comparison.

Polyacrylamide gels serve as the matrix which sieve the protein segments along the

electrical gradient created by the voltage introduced by the gel-containing apparatus. Smaller

protein structures migrate faster through the gel than larger proteins. Under the most commonly

used method, discontinuous SDS-PAGE, two gels are created. A stacking gel of 4-5% poly-

acrylamide concentration is prepared separately from a 10-20% resolving gel. The stacking gel

has a lower concentration of acrylamide compounds and contains larger gel matrix pores to al-

low for collection of the various protein compounds, while the resolving gel has formed smaller

pores from the increase in acrylamide particles that provide for greater separation and resolu-

tions of protein structures by size as they migrate through the gel matrix (Schägger 2006). A dif-

ference between the pH of the Tris-glycine-chloride buffers in the stacking (pH 6.8) and resolv-

ing (pH 8.8) gels creates the stacking effect that occurs with the protein samples, as they transi-

tion between the two gel layers (Hachmann and Amshey 2005). After the protein samples mi-

grate along the gel towards the end of the gel, the electrical current is halted, and the gel can be

removed for staining and de-staining. Several staining compounds exist, with Coomassie blue

being the most common and easiest to use, to silver staining, which is very sensitive and details

a greater number of compounds within a gel. Other types of fluorescents stains can be used

that can potentially provide higher discrimination of protein compounds. These are more com-

mon in immunological detection techniques, like the western blot (Schägger 2006). After a brief

period of staining, a de-staining step removes the stain at a greater proportion from non-protein-

aceous compounds within the gel, providing visual representation of the samples, in corre-

spondence with their molecular weight.

23

2.3 Soy protein isolate and hydrolysate use in protein-rich beverages

Protein is an important macronutrient that has increased use in product development.

From 2009-2013, new food products containing high protein claims increased 45%, with meal

replacement products showing a 96% increase in high protein claims (Bloom 2014). Sports bev-

erages also doubled in their use of high protein claims, providing expanded use to functional

soluble protein. Beverages that are higher in protein or perceived as specific “protein drinks” are

50% more likely to be selected for consumption over nutritional (39%) and sports drinks (36%)

for their energy content. The view of protein as an ideal energy source for physical activity and

exercise enhances the preference for high-protein beverages (Bloom 2014).

Soy food products continue to be featured and favored by consumers (Food Business

News 2008). Nutritional shakes and meal replacement beverages have served as popular food

items for a variety of consumers and can be prepared using soy. Common beverage uses for

soy protein isolate include dairy-type replacement beverages (beverage powders, infant for-

mula, and soymilk), bottled fruit drinks, and weight-management/fitness shakes (Soyfoods As-

sociation of North America 2004). Weight-management/fitness beverages are typically dry pow-

der protein or meal replacement shakes, and soy protein can serve as a suitable ingredient in

these products. Soy-based beverages and diets are observed as being beneficial to weight loss.

The significant levels of dietary fiber within a soy-based diet increase satiety and aids in de-

creasing fat mass (Liao and others 2007). Soy protein also stimulates a quick rise and digestion

of amino acids, which can aid in maintaining muscle mass. Even for consumers who are not the

most athletic or physically active, protein beverages can serve as a convenient source of nutri-

tional supplementation within their daily diet.

Many factors are considered for developing and implementing a protein source for bev-

erage applications. Tsumura and others (2005) state that enzymatically-derived hydrolysates

achieve minimal solubility at pH 4.5, similar to soy protein isolates. However, these hydrolysates

achieve greater solubility at other pH levels than unhydrolyzed soy protein (Cho and others

24

2008). A suitable protein for dry blended beverages (DBB) or ready-to-drink (RTD) products re-

quires attributes including quality solubility, high dispersibility, and controlled viscosity (Paulsen

2009). Another attribute to consider is powder density, as the density of the soy protein can im-

mensely alter a high-protein DBB at levels above 25% protein (Paulsen 2009).

If properly incorporated with a probiotic, soy protein can serve as an effective component

for a health-oriented high protein probiotic beverage (Nguyen and others 2016b). These are

some of the properties that will be the focus of this research, and their efficacy should be im-

proved through the development of highly functional soy protein hydrolysates.

2.4 Probiotics potential health benefits and incorporation into soy protein products

Probiotic-incorporation is another area of food science that has experienced significant

growth, between 7-8% annually with billion-dollar sales, in both the U.S. and Europe (Saxelin

2008; Maccaferri 2012; BCC Research 2014). Probiotics are defined as “live microbial food sup-

plements” which can confer health benefits through digestion, mostly within the intestinal micro-

biota (Annan and others 2008; Farnworth and others 2007; Mitropoulou and others 2013). Using

probiotics as a beneficial food ingredient can provide microbiota supplementation to functional

foods. Lactic acid bacteria, mainly Lactobacilli and Bifidobacterium species, are the most com-

mon microorganisms used as potential probiotics (Farnworth and others 2007; Annan and oth-

ers 2008; Champagne and others 2009). Probiotic cell concentrations should be maintained be-

tween 108-1010 colony forming units (CFU) ml-1 in the gut to provide the highest potential surviv-

ability and health effects (Heidebach and others 2010). Food products are digested in the gut

between 2-4 hr, so the probiotics would have to maintain viable concentrations in the human

gastrointestinal (GI), particularly as they pass through the stomach, during that time (Annan and

others 2008). Studies have suggested that soy beverage provides a suitable, if not better, probi-

otic growth environment than milk (Champagne and others 2009). Farnworth and others (2007)

observed greater acidification and more extensive lactobacilli growth rates in soy beverages

versus milk beverages, suggesting that soy beverages provide a more favorable acidic growth

25

environment. However, milk appears to have a better buffering capacity than soy beverages,

providing a more stable progression for varied probiotic growth. But probiotics also grow more

extensively in soy beverages, due to the greater availability of free amino acids (Farnworth and

others 2007). Bifidobacteria also grow well in soy substrates because of their utilization of raffi-

nose and stachyose saccharides found in soy (Farnworth and others 2007). But their sensitivity

to a variety of acidic environments (both food and human) make them precarious to use (Annan

and others 2008). Lactic acid bacteria provide a more natural resistance to acidic environment

but are vulnerable to high-acid conditions. To provide greater protection to food-bound probiot-

ics, encapsulation technologies can be utilized. More importantly, understanding the mecha-

nisms between food composition and the interrelated human gut microbiota are thought to be of

more importance than ever before. Therefore, more microbiological work is needed or requires

expansion into the field of food science to provide more data and insights on the potential use of

probiotics within the diet.

2.4.1 Probiotic encapsulation and formulation

For probiotic incorporation in a beverage, spray-drying and microencapsulation tech-

niques are potential methods for compatible incorporation within a soy protein substrate (Farn-

worth and others 2007). Spray-drying, for probiotics, is the “atomization of an aqueous or oily

suspension of probiotics and carrier material into a drying gas” which leads to an evaporation of

water (Gbassi and Vandamme 2012). There are many advantages to spray-drying, including the

improved preservation and viability of probiotic cells in storage. However, there are complica-

tions with the procedure that can affect probiotic quality. Proper adjustments must be made to

the parameters of the procedure, as high temperatures and length of exposure can limit the

number of probiotic cells that are viable and their activity in gastrointestinal environments

(Gbassi and Vandamme 2012). Selection of the proper coating agents (polysaccharides and

proteins) is also important, to reduce stickiness in the mixture suspension or excess moisture in

26

the drying chamber (Hernández-Carranza and others 2014). Coating agent options include solu-

ble starches, pectins, alginates, gums, and food proteins, or even a combination of two or more

of these options. Additional non-digestible oligosaccharides may complement the beverage mix-

ture as prebiotics. Hernández-Carranza and others (2014) observed a notable increase in

spray-drying product yield by using maltodextrin as the primary coating agent, with fruit and veg-

etable extracts serving as prebiotic sources. For microencapsulation technologies, there are a

variety of options, classified by final product size. Capsules, or macrocapsules, are encapsu-

lated products greater than 5,000 μm, microcapsules are scaled between 0.1 and 5,000 μm,

and nanocapsules are sized below 0.1 μm (Hernández-Carranza and others 2014). Some stud-

ies recommend microcapsules with a diameter below 100 μm as the best particle delivery sys-

tem that minimally affects texture within a food matrix (Annan and others 2008). The two main

methods used for encapsulating particles are extrusion and emulsification. Extrusion is a sim-

pler and easier process to emulsification, with both providing a retention of a high number of

probiotic cells (Gbassi and Vandamme 2012).

Adapted from Gbassi and Vandamme (2012)

27

Between spray-drying and microencapsulation methods, spray-drying is a more cost-ef-

fective, one-step process in comparison to encapsulation, but the protection from degradation

provided by encapsulation methods is generally greater and more extensive (Hernández-Car-

ranza and others 2014). However, an improved microencapsulation method will be researched

for the purpose of this thesis.

Gastrointestinal resistance or protection is also important to the efficacy of consumed

probiotics, and many researchers use simulated gastrointestinal digestion in vitro in their meth-

ods. Although in vivo models can provide more accurate digestion data, in vitro digestion simu-

lations are cheaper, faster and easier to perform, and more ethical (Passannanti and others

2017). Simulated models can control primary digestion factors such as pH, concentration of di-

gestive enzymes, and, in more dynamic digestion systems, the flow of food and chemical com-

positions (Passannanti and others 2017). The proper encapsulation techniques and beverage

formulation/environment must be established to ensure the probiotic viability. The methods and

coatings used for encapsulating probiotics should maintain cell survival through food pro-

cessing, storage before consumption, and consumption from the gastrointestinal tract to the co-

lon (Hernández-Carranza and others 2014). Although research has provided a way to determine

the causes of bitterness/off-taste and digestibility for SPH and probiotics each, a greater focus

on the compatibility and digestion of both products together has not been provided.

28

2.5 References

Aaslyng MD, Elmore JS, and Mottram DS. 1998. Comparison of the aroma characteristics of acid-hydrolyzed and enzyme-hydrolyzed vegetable proteins produced from soy. J Agri Food Chem. 46:5225-31.

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Chapter 3: Preparation of soy protein isolate from non-GE soybean seeds (R08-4004) and

optimization of conditions to hydrolyze the protein using pepsin

3.1 Introduction

Soy protein, particularly soy protein isolate (SPI), is consumed in $4.5 billion worth of

food sales by U.S. consumers, and its growth is on the rise through the expansion of functional

beverages containing soy or emphasizing high protein (Katahdin Ventures 2014). This ingredi-

ent is commercially incorporated into bottled fruit drinks, energy bars, soups and sauces, meat-

alternative products, baked goods, breakfast cereals, and fitness protein powder (Soyfoods As-

sociation of North America 2004). However, the conversion of SPI into soy protein hydrolysates

(SPH) is not as widely utilized in food products but has potential for soy-based products, particu-

larly beverages.

For SPI production, the aqueous method is preferred for better protein functionality, pro-

tein palatability, and structure, due to less modification from organic solvents (Scheide and

Brand 1987). Soy protein isolate solubility is considerable (>90%) at alkaline pH. Soy protein

solubility diminishes near its isoelectric point of pH 4.5, and lower pH levels require extreme

temperatures and longer incubation times to improve the solubility (Ortiz and Wagner 2002). En-

zymatic hydrolysis can serve as a better method for improving soy protein solubility in hydroly-

sate form at lower pH levels (Deeslie and Cheryan 1988; Hettiarachchy and Kalapathy 1997).

Modification of soy protein isolate in this manner can improve the use of the protein within bev-

erages. Enzymatic hydrolysis is also preferred to acid hydrolysis for its benefits of minimal unde-

sirable by-products via side reactions, safer yields, and more consistent product through con-

trolled, specified reactions (Campbell and others 1996; Sun 2011). Pepsin is a common natural

enzyme used for hydrolysis. Pepsin serves as an acidic peptidase. Obtaining the optimal enzy-

matic conditions is crucial to preparing the most functional hydrolysates within a specified food

environment.

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3.2 Materials and Methods

3.2.1 Materials

Arkansas-grown non-GE soybeans (R08-4004 Kirksey lineage cultivar) was obtained

from the National Soybean and Grain Alliance (Fayetteville, AR). This variety was used for the

preparation of soy protein isolate and hydrolysates (Nguyen and others 2016b). A coffee grinder

(Model IDS77, Sunbeam Products, Inc., Cleveland, OH, USA) and larger industrial grinder

(Model A20, Tekmar Company, Cincinnati, OH, USA) were used to grind the soybeans into

flour. A RapidStill (Labconco, Kanasas City, MO, USA) system was used for Kjeldahl protein di-

gestion and distillation procedures (Edwards and others 2019). Standard-grade hexane, sulfuric

acid, food-grade pepsin (Sigma-Aldrich Corp., St. Louis, MO), and other reagents and chemi-

cals were purchased from VWR (Radnor, PA) and Sigma-Aldrich Corp. (St. Louis, MO) and

used to prepare analytical grade solutions specific to the experiments conducted.

3.2.2 Preparation of soy protein isolate

a. Whole soybean milling and defatting

One kilogram of soybeans was ground into flour using a larger industrial grinder

(Tekmar) initially, with the coffee grinder (Sunbeam) being used later to more finely grind the

flour for finer sieving. The lipid content of the flour was removed using hexane (1:2 flour-to-hex-

ane ratio) and continued twice more to remove residual oil. The residual hexane in the defatted

flour was evaporated for 24 hours under a hood. The hexane-free soy flour was ground and

passed through 40-mesh (425 μm) again, followed by 60-mesh (250 μm) sieving, to obtain fine

particle flour for protein isolate preparation. After 60-mesh sieving, a final hexane defatting wash

(using a 1:4 flour/hexane ratio) was performed to remove any remaining lipids (Edwards and

others 2019).

i. Soy protein isolate preparation

The defatted soy flour was mixed with deionized water to form a 10% colloidal solution

(10 g/100 mL) and adjusted to pH 9.0 from, to provide optimal protein solubility. The dispersion

38

was stirred for 2 hours and centrifuged at 10,000 x g for 20 minutes at 20 °C (Model J2-21,

Beckman-Coulter, Fullerton, CA, USA). The supernatant was collected, and the residue was re-

extracted once more to yield more soluble protein (Edwards and others 2019). The combined

supernatants were adjusted to isoelectric pH 4.5 to precipitate the proteins and centrifuged at

10,000 x g for 20 minutes at 20 °C. The protein precipitate was washed twice to remove other

soluble compounds with deionized water at pH 4.5, with each wash followed by centrifugation at

10,000 x g for 20 minutes at 20 °C. The washed SPI was adjusted to pH 7.0 and freeze-dried

(VirTis Genesis 25LE, SP Scientific, Warminster, PA, USA) and stored at 5 °C. The protein con-

tent was determined using the Kjeldahl method (Edwards and others 2019).

b. Protein content determination

i. Kjeldahl protein digestion and distillation

Protein content was determined using a micro-Kjeldahl method (AACC Method 46-11.02

“Crude Protein – Improved Kjeldahl Method, Copper Catalyst Modification”): approximately 0.25

g of SPI sample was digested at 410ºC in digestion tubes with the addition of the copper cata-

lyst tablet and 5 mL of 10N sulfuric acid for 90 min. Sample in Kjeldahl digestion flask was

cooled to ambient temperature in a fume hood. The digest was quantitatively transferred to 25-

mL volumetric flasks and diluted. Samples were then distilled into boric acid. The protein con-

tent of sample was measured from 0.025 N HCl titration of the sample into the distillate solution,

based on the equations below:

% 𝑁𝑖𝑡𝑟𝑜𝑔𝑒𝑛 = (𝑉 0.025 𝑁 𝐻𝐶𝑙 𝑥 0.025 𝑁 𝐻𝐶𝑙 𝑥 14(𝑛𝑖𝑡𝑟𝑜𝑔𝑒𝑛 𝑎𝑡𝑜𝑚𝑖𝑐 𝑚𝑎𝑠𝑠)

(𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑚𝑔 𝑥 𝑟𝑎𝑡𝑖𝑜 𝑜𝑓𝑠𝑎𝑚𝑝𝑙𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑡𝑜 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑑𝑖𝑙𝑢𝑡𝑒𝑑 𝑣𝑜𝑙𝑢𝑚𝑒)

% 𝑃𝑟𝑜𝑡𝑒𝑖𝑛 = 6.25 𝑠𝑜𝑦 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡/𝑓𝑎𝑐𝑡𝑜𝑟 𝑥 % 𝑒𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡𝑎𝑙 𝑛𝑖𝑡𝑟𝑜𝑔𝑒𝑛

Based on the average nitrogen content of protein to be 16% (1/0.16 - 6.25)

39

3.2.3 Soy protein isolate (SPI) hydrolysis optimization

a. SPI solubility testing

SPI was solubilized at varying pHs. The pH of the SPI in water was adjusted along the

range of 2.0-10.0 at 1.0 pH increments in ascending order, and centrifuged at 16,000 x g for 20

min at 25 °C using a centrifuge (Model 5415 C, Eppendorf, Hamburg, Germany) to collect the

supernatant within solubilized protein sample aliquots. The protein content in the supernatant

samples was determined by the Biuret method (Onishi and Barr 1978) and expressed as a per-

centage of soluble protein to total protein.

b. Soy protein enzymatic hydrolysis using pepsin

To obtain a degree of hydrolysis (DH) ranging between 2-10%, or limited hydrolysis, var-

ying durations (1-3 hr.) were tested for the enzyme while maintaining other hydrolysis condi-

tions, such as temperature (37 °C) and enzyme to protein substrate ratio.

A weighted sample of 3.0 g SPI was dissolved in DI water to make a 10% SPI solution.

The protein hydrocolloidal solution was adjusted to 2.0 pH for pepsin. After a 30 min. pre-warm-

ing period in an incubator water bath, pepsin (0.5% w/w) was added to the protein hydrocolloidal

solution and incubated for periods from 60-180 min in a 37 °C water bath with a stirrer platform

(Grant Model OLS200, Cambridge, England) at a speed of 140 rpm. A 10% SPI solution served

as a control (no pepsin/incubation used). The hydrolysates were pipetted in 10 mL samples, in-

activated for enzymatic activity by adjusting the pH to 9.0 for pepsin, centrifuged, and evaluated

for the degree of hydrolysis and surface hydrophobicity (Rayaprolu and others 2013; Nguyen

and others 2016a).

40

3.2.4 Statistical analysis

The reported data were expressed as means of triplicate observations ± standard deri-

vation. Data were subjected to one-way analysis of variance (ANOVA) using JMP Pro 14.0 soft-

ware (SAS Institute, Cary, NC, USA).

3.3 Results and Discussion

3.3.1 Kjeldahl digestion for protein determination

The results of the soy protein isolate (SPI) samples tested in this study are summarized

in Table 1. Three trials of SPI Kjeldahl digestions were performed in triplicate to verify con-

sistency of protein content. Assessment of significant differences among treatments were deter-

mined by LSD. No significant differences were found between the SPI preparations to be used

for hydrolysis. A high standard deviation with SPI Trial 2 (±4.1) was noted, but Trial 2 also dis-

played the highest protein composition at 88.4 %, the highest among the SPI samples. The re-

sults indicate that all the SPI trial samples contain a very high protein content, with average con-

tent being approximately 87% among the prepared samples.

3.3.2 Effect of pH on extracted SPI solubility

Figure 1 demonstrates the effects of a pH gradient from 2.0 to 10.0 on the solubility of

SPI. Significant differences were observed for SPI extracted at pH 7.0 or greater. The SPI from

pH 2.0 was the only acidic pH of significance that displayed nearly 90% solubility. Solubility was

lowest at pH 4.0 and 5.0, around the natural isoelectric value of soy protein (Malhotra and Cou-

pland 2004, Hefnawy and Ramadan 2011, Kong and others 2017). This represents the increas-

ing solubility relationship that is obtained with a greater degree of pH change (higher acidic or

alkaline pH levels) from the isoelectric pH of a protein (Malhotra and Couplan 2004). This is typi-

cal due to the greater polarity of charge that is created at extreme pH, allowing for improved sol-

ubility and bonding.

41

3.3.3 Effect of optimized enzymatic hydrolysis on protein solubility

Using pepsin enzyme, the enzymatic hydrolysis of SPI was optimized. Hydrolysis pH

(2.0) and temperature (37 °C) were kept constant to maintain the ideal hydrolysis conditions for

pepsin, while hydrolysis time varied for treatments. Hydrolysis was performed for 1, 1.5, 2, 2.5,

3, and 20 hr. to establish a hydrolysis gradient range. As DH increased, the solubility increased,

as is expected from the degradation of the protein secondary structure and cleavage of small

peptides from the larger SPI protein structure (Bao and others 2017). Longer incubation time re-

sulted in higher soluble protein content, and soy protein hydrolysates (SPH) with satisfactory

solubility were obtained from 2 hr., 2.5 hr., and 3 hr. treatments (Figure 2). It was determined

that 2.5 hr. and 3 hr. hydrolysis times provided the best overall SPH solubility, averaging above

85% within the target beverage pH range (Edwards and others 2019). These were also favored

for their inclusion with the pepsin hydrolysis time range of 1-3 hr. (Edwards and others 2019).

This method of pepsin hydrolysis also improved SPH solubility to higher levels than alcalase

from previous research (Nguyen and others 2016a).

The results indicate that the protein solubility between the prepared SPI and hydrolyzed

SPI from pepsin differed significantly for protein solubility (Figure 2). Hydrolysis increases solu-

bility through the alteration of the secondary protein structure of native SPI and formation of

smaller peptides derived from the native protein by enzymatic catalysis (Adler-Nissen 1976). As

the pH increased, the differences in solubility were less significant, especially at the pH level ap-

proached closer to optimal soy solubility at pH 9.0 (Berk 1992, Wang and others 2004). Extrapo-

lating this information for further experimentation, longer incubation time can result in higher sol-

uble protein. However, it is important to gauge how longer hydrolysis incubation times impact

physicochemical properties that can impact functionality of SPH (Edwards and others 2019).

3.4 Conclusion

This study displays the potential protein hydrolysates with improved solubility

42

and higher protein quality can be obtained from this non-GE soy cultivar. The established hy-

drolysis conditions provide SPH with high soluble protein, even at varying pH levels. Optimal hy-

drolysis conditions are: 150 min. incubation using 0.5 wt% (2 U/mg) pepsin at 37 ºC tempera-

ture (Edwards and others 2019). SPH from non-GE soybeans are a protein-rich nutraceutical

that can be incorporated in food products, especially beverages. Further studies can include pu-

rification of SPH for enhanced activity, alternative combination of hydrolysis methods, and prod-

uct application for potential commercialization (Edwards and others 2019).

43

3.5 Tables and Figures

Table 1: Protein content of SPI by Kjeldahl method (g/100g)

SPI Trials Protein Content1

Trial 1 86.1 ± 1.39a

Trial 2 88.4 ± 4.10a

Trial 3 85.8 ± 1.46a

1 Values are presented as means ± standard deviation in triplicate; those not connected

with the same letters in each column are significantly different (p < .05)

Figure 1. Percent solubility of soy protein isolate (SPI) along pH range 2.0-10.0 (results are in triplicate ±SD, p < .05)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0

Solu

bili

ty (

%)

pH

SPI Solubility Curve

44

Figure 2. Percent soluble protein at pH 6.5, 7.5, and 8.5 for various pepsin-hydrolyzed hydrolysate intervals. Values are presented as means ± standard deviation in triplicate; values not connected with the same letters are significantly different (p < .05)

45

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Berk Z. 1992. Isolated soybean protein. In: Berk Z, editor. Technology of production of edible flours and protein products from soybeans. Rome: Food and Agriculture Organization of the United Nations.

Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248-254

Burgain J, Gaiani C, Linder M, and Scher J. 2011. Encapsulation of probiotic living cells: from laboratory-scale to industrial applications.

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Champagne CP and Fustier P. 2007. Microencapsulation for the improved delivery of bioactive compounds into foods. Current Opinion in Biotechnology 18:184-190.

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Cook MT, Tzortzis G, Charalampopoulos D, and Khutory VV. 2011. Production and evaluation of dry alginate-chitosan microcapsules as an enteric delivery vehicle for probiotic bacteria. Bi-omacromolecules 12:2834-2840.

Conforto E, Joguet N, Buisson P, Vendeville JE, Chaigneau C, and Maugard T. 2015. An opti-mized methodology to analyze biopolymer capsules by environmental scanning electron microscopy. Materials Science and Engineering C 47:357-366

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Edwards JS, Hettiarachchy NS, Kumar TKS, Carbonero F, Martin EM, and Benamara M. 2019. Physicochemical properties of soy protein hydrolysate and its formulation and stability with encapsulated probiotic under in vitro gastrointestinal environment. Manuscript submitted for publication.

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Gueimonde M and Sánchez B. 2012. Enhancing probiotic stability in industrial processes. Micro-bial Ecology in Health & Disease 23:18562

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Hefnawy HTM and Ramadan MF. 2011. Physicochemical characteristics of soy protein isolate and fenugreek gum dispersed systems. J Food Sci Technol. 48(3):371-377.

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Jung S, Rickert DA, Deak NA, Aldin ED, Recknor J, Johnson LA, and Murphy PA. 2003. Compar-ison of Kjeldahl and Dumas methods for determining protein contents of soybean products. J American Oil Chem Sci 80(12):1169-1173.

Katahdin Ventures. 2014. Soyfoods in U.S. retail 2014. Available from: http://www.soy-foods.org/soy-products/sales-and-trends. Accessed 2016 August 19.

Kong X, Jia C, Zhang C, Hua Y, and Chen Y. 2017. Characteristics of soy protein isolate/gum arabic-stabilized oil-in-water emulsions: influence of different preparation routes and pH. RSC Adv. 7:31875-31885

Llano DG, Herraiz T, and Polo MC. 2004. Chapter 6: Peptides. In: Handbook of food analysis: Physical characterization and nutrient analysis (Revised ed). CRC Press, Boca Raton, FL,

p 125-148.

Malhotra A and Coupland JN. 2004. The effect of surfactants on the solubility, zeta potential, and viscosity of soy protein isolates. Food Hydrocolloids 18(1):101-108.

Manninen AH. 2009. Protein hydrolysates in sports nutrition. Nutr & Metabol 6:38.

Meinlschmidt P, Sussmann D, Schweiggert-Weisz U, and Eisner P. 2016. Enzymatic treatment of soy protein isolates: effects on the potential allergenicity, technofunctionality, and sensory properties. Food Science and Nutrition 2016 4(1):11-23.

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Nguyen Q, Hettiarachchy N, Rayaprolu S, Jayanthi S, Thallapuranam S, and Chen P. 2016a. Physicochemical properties and ACE-I inhibitory activity of protein hydrolysates from a non-genetically modified soy cultivar. J Am Oil Chem Soc 93:595-606.

Nguyen Q, Hettiarachchy N, Rayaprolu S, Seo HS, Horax R, Chen P, and Kumar TKS. 2016b. Protein-rich beverage development using non-GM soybean (R08-4004) and evaluated for sensory acceptance and shelf-life. J Food Sci Technol (Online).

Nielsen PM, Petersen D, Dambmann C. 2001. Improved method for determining food protein degree of hydrolysis. J Food Chem and Tox 66:642–6.

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Tsumura K, Saito T, Kugimiya W, and Inouye K. 2004. Selective proteolysis of the glycinin and β- conglycinin fractions in a soy protein isolate by pepsin and papain with controlled pH and temperature. Journal of Food Science 60(6):C363-C367

Wang H, Johnson LA, and Wang T. 2004. Preparation of soy protein concentrate and isolate from extruded-expelled soybean meals. J AOCS 81(7):713-717. Wu W, Hettiarachchy N, and Qi M. 1998. Hydrophobicity, solubility, and emulsifying properties of

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49

Chapter 4: Determination of physicochemical properties (degree of hydrolysis, surface

hydrophobicity, and electrophoresis) of soy protein hydrolysates and probiotic encapsu-

lation viability under simulated gastrointestinal conditions

4.1 Introduction

Soy protein chemical, nutritional, and functional properties can be enhanced through en-

zymatic hydrolysis. These soy protein hydrolysates (SPH) provide improved properties such as

solubility, foaming properties, and emulsion capacity for food product applications (Ortiz and

Wagner 2002). Important functional properties that were the focus of this research through en-

zymatic hydrolysis include solubility, degree of hydrolysis (DH) functionality, and surface hydro-

phobicity (S0). DH essentially is the percentage of cleaved peptides from original starter peptide

chains (Manninen 2009). Most cleaved peptides are smaller size protein fragments including di-

and tripeptides, resulting in faster absorption in the bloodstream and providing a nutritional ad-

vantage to functional foods (Manninen 2009). Limited hydrolysis is critical to obtaining an appro-

priate DH level for functional purposes. However, a high DH can lead to intense bitterness and

alteration of protein bioactivity (Klompong and others 2007). Another structural feature, S0 as-

sists the functional properties, gastrointestinal stability, and in vivo efficiency, in some cases, of

peptides (Xie and others 2015). Protease treatment has been analyzed for hydrophobicity,

which can be potentially responsible for bitterness with SPH. Evidence has been provided for

peptide fractions below 1,000 Da being much less bitter than higher mass fractions (Moure and

others 2005). Through properly modified enzymatic hydrolysis methods, a balance of functional-

ity and reduced bitterness could be achieved for food soy protein hydrolysates. To better indi-

cate bitterness levels, the measure of S0 can be obtained from the slopes of the regression cor-

relation between peptide concentration and fluorescence intensity. Wu and others (1998) ob-

tained significant hydrophobicity values of soy protein peptides, and rapeseed protein hydroly-

sate values have been obtained by He and others (2013), with both using ANS detection meth-

ods. ANS can bind to accessible hydrophobic regions of peptides and increase in fluorescence

50

at excitation and emission wavelengths of 390 and 470 nm, respectively. Understanding the hy-

drophobic properties of peptides can assist in understanding their behavior in a beverage envi-

ronment and indicate denaturation during storage and shelf-life testing (Tang and others 2002).

Information on these properties can also be useful in understanding the dynamics between SPH

and probiotic integration for improved functionality, particularly with degradation and shelf-life.

Using SDS-PAGE provides a complimentary quantitative value to the previous methods by

providing the approximate hydrolysate weight in kilodaltons (kDa). Understanding the composi-

tion of hydrolysate sizes within each treatment group gives insight into how a higher number of

smaller hydrolysates corresponds to higher DH. In addition, SDS-PAGE can identify which pro-

tein subunits likely served as the source for the hydrolysates from the native SPI protein.

Probiotics are another area of food science that is important to research and better un-

derstand for food applications. The three major considerations for probiotic use and function are

their strain variety, their size for encapsulation, and the viable population number that can be

established and maintained within a food product (Burgain and others 2011). Probiotics are lim-

ited to food products that do not undergo extensive heat treatment, as the thermal processing

can kill the bacteria and lower viable population values (Burgain and others 2011, Gueimonde

and Sanchez 2012). A dry powder mixture provides a suitable food environment for probiotics.

The packaging of a soy protein powder formulation in an aseptic package provides a dry, sterile

environment for probiotic storage and facilitates probiotic growth upon reconstitution into a bev-

erage and consumption. Of the microencapsulation technologies (ME) available for preparing

probiotics for dry storage, polysaccharides, modified starch, and protein capsules provide the

best protection and stability for probiotics within acidic environments, from beverage to digestive

acids (Annan and others 2008, Champagne and Fustier 2007, Heidbach and others 2010, Huq

and others 2013). Lactobacillus and Bifidobacteria species serve as suitable probiotics (Ruiz

and others 2011). Lactobacillus strains will be selected for encapsulation, due to their improved

51

resistance to lower pH levels and their durability within microcapsules (Burgain and others

2011).

The objectives of this study were to: 1) determine the solubility, degree of hydrolysis

(DH), surface hydrophobicity (SH) of the prepared soy protein hydrolysates and conduct electro-

phoresis of the protein and hydrolysates size and 2) encapsulate the probiotics for incorporation

in a dry mix with the selected protein hydrolysates for simulated gastrointestinal resistance.

4.2 Materials and Methods:

4.2.1 Materials

Selected Lactobacillus species (HOWARU Dophilus 100 gm Std Probiotic Sample, Dan-

isco, USA) were used in this study. Alginate (Grinsted, Danisco, USA) was used as the encap-

sulating agent. Other reagents and chemicals were purchased from VWR (Radnor, PA) and

Sigma Chemical Co. (St. Louis, MO) and used to prepare analytical grade solutions specific to

the experiments conducted (Edwards and others 2019).

4.2.2 Protein content determination in the enzyme catalyzed hydrolysates

The Bradford assay, based on methods and technical aspects by Sigma-Aldrich (Tech-

nical Bulletin, Catalog Number B6916, St. Louis, MO, USA), was used to determine the concen-

tration of proteins in solution in preparation for SDS-PAGE.

The Bradford assay principles are based on the creation of a complex between

the dye in the Bradford reagent, Brilliant blue G, and the proteins available in solution. The com-

plex forms and results in a shift in the absorption maximum of the dye between 465-595 nm.

The absorption is proportional to the protein present (Bradford 1976). The Bradford assay is

suitable for samples being used in SDS-PAGE, because the reagent is compatible with reducing

agents.

The Micro Cuvette 2 mL Assay Protocol was used (Sigma-Aldrich). Protein standards

from bovine serum albumin (BSA) were prepared, ranging from 25 μg/mL to 1.25 μg/mL in

52

phosphate buffer saline (PBS), resulting in a total of 7 standards, plus a PBS blank. The Brad-

ford reagent was gently mixed at room temperature. 1 mL of each protein standard was added

to 1 mL of reagent. The samples incubated at room temperature for 5 min. The standards and

experimental samples were transferred to cuvettes and measured using a spectrophotometer

set at 595 nm. The net absorbance vs. the protein concentration was plotted for the standards.

From the standard curve, the protein concentration of the experimental samples was deter-

mined from the net absorbance.

4.2.3 Determination of degree of hydrolysis

Nielsen and others (2001) method was used to determine the DH. This method is based

on a reaction between primary amino groups of the hydrolyzed bonds and o-phthaldialdehyde

(OPA) in the presence of dithiothreitol (DTT) forming a compound that can absorb energy at a

wavelength of 340 nm. Serine was selected as the standard, since a serine reaction can provide

the closest response to the average amino acid OPA reaction. The serine solution was made by

dissolving 50 mg of serine in 500 mL of DI water. OPA reagent were prepared as follows: 100

mg of sodium-dodecyl sulfate and 3.81 g of disodium tetraborate decahydrate (DTD) were dis-

solved in 60 mL of water; 80 mg of o-phthaldialdehyde was weighted and dissolved in 2 mL of

ethanol; both solutions were mixed, added with 88 mg of (DTT) and stirred to form a clear solu-

tion; deionized water was added to make a total volume of 100 mL. All the soy protein hydroly-

sates from pepsin digestion were diluted to provide a suitable protein hydrolysate concentration

for spectrophotometric readings.

Four hundred µL of the diluted protein hydrolysate solutions were placed in test tubes

with 3 mL of OPA reagent, and mixed. Spectrometer absorbance at 340 nm (Shimadzu Model

UV-1601, Kyoto, Japan) was taken exactly 2 minutes after addition of OPA reagent. Blank and

standard were prepared using 400 µL of DI water and serine solution, respectively. DH was cal-

culated using the following equations (Nielsen 2001):

53

where V is the sample volume (in liters; 0.1 L); X is sample weight (in grams; 0.125 g); P

is soluble protein content (percentagewise; 90%) of the sample, and Serine–NH2 is in

meq Serine-NH2/g protein.

where and values prescribed for soy protein are standards of 0.342 and 0.97, re-

spectively, and h is the number of hydrolyzed bonds (Nielsen 2001).

where htot equals 7.8 for the number of peptide bonds per protein equivalent (Li and oth-

ers 2012). Hydrolysates with limited hydrolysis (DH 2-10%) were selected for obtaining

the advantages of hydrolysis without the disadvantages of extensive hydrolysis (Klom-

pong and others 2007, Manninen 2009) and subjected to further investigation for solubil-

ity and surface hydrophobicity.

4.2.4 Determination of surface hydrophobicity

Surface hydrophobicity was determined using a modified hydrophobic fluorescence

probes method (Paraman and others 2007). The 1.0% SPH solution was prepared by dissolving

the proteins in 0.01 M phosphate buffer. Protein hydrolysates were diluted with 0.01 M phos-

phate buffer at concentrations spanning 0.001-0.01%. Ten microliters of 1-anilino-8-naphthalene

sulfonate (ANS) solution (8 mM in 0.01 M phosphate buffer) were added to the protein solutions

immediately before readings. Fluorescence intensity of ANS-protein conjugates was measured

at 390 nm of excitation and 470 nm of emission, using a spectrofluorophotometer (Shimadzu

Model RF-1501). Absorbance readings were taken for each sample to provide the approximate

𝑆𝑒𝑟𝑖𝑛𝑒 − 𝑁𝐻2 = 𝑆𝑎𝑚𝑝𝑙𝑒 𝑂𝐷 −𝐵𝑙𝑎𝑛𝑘 𝑂𝐷

𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑂𝐷 −𝐵𝑙𝑎𝑛𝑘 𝑂𝐷 𝑥

(0.9516 𝑥 𝑉 𝑥 100)

(𝑋 𝑥 𝑃);

ℎ = (𝑆𝑒𝑟𝑖𝑛𝑒 −𝑁𝐻2)− 𝛽

𝛼;

𝐷𝐻 =ℎ

ℎ𝑡𝑜𝑡 𝑥 100; where htot for soy protein is 7.8, which is the total number

54

fluorescence absorbance. The surface hydrophobicities, expressed as a slope of fluorescence

intensity compared with protein concentration (ppm), were calculated by linear regression and

established as a protein hydrophobicity index (Nguyen and others 2016a).

4.2.5 SDS-PAGE protein hydrolysate molecular size determination

SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) was

performed per the method of Laemmli (1970). Protein samples (2 microgram/microliter) were

prepared in a reducing buffer containing 0.5 M Tris-HCl (pH 6.8), 10% (w/v) SDS, 10% glycerol,

5% 2-mercaptoethanol, and 0.5% (w/v) bromophenol blue. Protein samples of 10 microliters

were used for the gel. SDS-PAGE was carried out on a slab gel using SDS-Tris-Glycine discon-

tinuous buffer system. Stacking gel was 5%, and the resolving gel was from an 18.0 to 22.0%

value (Edwards and others 2019). The gels ran for 50 min. and followed by a staining and de-

staining wash. Stain solution was prepared using 400 mL ethanol, 100 mL acetic acid, 500 mL

of de-ionized water, and 1 g of Coomassie Brilliant Blue. De-staining solution was prepared

without Coomassie, using the same volume of all other chemicals used in the staining solution.

The approximate molecular weights of proteins were determined using dual-range molecular

weight standards (BioRad). Molecular sizes of the protein standards range from 6.5 to 200 kDa

(mysosin 200 kDa, β-galactosidase 116.25 kDa, phosphorylase B 97.4 kDa, serumalbumin 66.2

kDa, ovalbumin 45 kDa, carbonic anhydrase 31 kDa, trypsin inhibitor 21.5 kDa, lysozyme 14.4

kDa, and aprotinin 6.5 kDa).

4.2.6 Probiotic encapsulation

Lactobacilli were grown in MRS medium (Sigma, St. Louis, MO, USA) at 37 °C for 14-16

hr anaerobically using the Gas Pak Plus System (BBL-71040 anaerobic system envelopes with

palladium catalyst, Becton Dickinson, Cockeysville, MD, USA). Resistant starch (RS) in the form

of a prebiotic fiber blend was purchased from Gut Garden LLC (Chicago, IL, USA). Standard

vegetable (canola) oil was obtained from a local store (Edwards and others 2019).

55

Alginate was tested for their effectiveness as encapsulating agents. These hydrocolloids

are stable at acidic environments and are of food-grade quality, making them appealing for food

products (Burgain and others 2011). A method for encapsulating probiotics in calcium alginate

beads was adopted, with modifications, from Cook and others (2011). All glassware and solu-

tions used in the protocol were sterilized at 121 °C for 15 min. A 1.0% alginate mixture was pre-

pared and combined in a 9:1 ratio with probiotic cell slurry diluted in PBS (Cook and others

2011). The mixture was immersed into a 62.5 mmol solution of calcium chloride, containing

Tween 80 (0.025%), and 0.025 g/mL of soy lecithin (emulsifier). The mixture stood for 30 min for

the calcium-alginate microcapsules to develop and harden (Edwards and others 2019). The mi-

crocapsules were collected by low speed centrifugation (3000 x g, 15 min, 4 °C), washed once

with PBS, and stored at refrigeration temperatures (4-7 °C). Physical assessment of the micro-

capsules was determined via light and environmental scanning electron (ESEM) microscopy

(Edwards and others 2019).

Probiotic microcapsules were freeze-dried using a commercial freeze dryer (VirTis Gen-

esis 25LE, SP Scientific, Warminster, PA, USA). The samples were initially frozen at −10 °C

outside of the freeze dryer at atmospheric pressure. The following freeze-drying process was

carried out under these conditions for 1-2 days.

4.2.7 Probiotic characterization and viability

Encapsulated probiotics beads were measured for size based on methods from

previous research (Cook and others 2011, Heidebach and others 2009, Conforto and others

2015). Light microscopy (Olympus, Shinjuku, Tokyo, Japan) accompanied with a measuring

scale attachment was used to measure the approximate size of the encapsulated beads and

provide imaging. More exact measurements and physical assessments were obtained with envi-

ronmental scanning electron microscopy (ESEM), using a Phillips XL30 Series ESEM micro-

scope (Institute for Nanoscience and Engineering, Fayetteville, Arkansas).

56

Encapsulated probiotics and non-encapsulated probiotics were subjected to gas-

trointestinal simulations to determine the stability of the microcapsules and assess the

viability of probiotics during the digestion process. To determine the survival and record

changes of probiotics cells in soy protein hydrolysate powders, four treatment groups

were used: one group of only capsules containing probiotics, one group with free probi-

otic cells in SPH powder, and another with SPH powder and encapsulated probiotics.

One group of five grams of free cells was subjected to the same gastrointestinal condi-

tions as other groups as a control. Five milliliter aliquots were drawn from each group at

30, 60, 90, and 120 min. during the simulated digestion processes (Edwards and others

2019).

Simulated gastric juice (SGJ) was prepared from sodium chloride (0.2 g) and hy-

drochloric acid (0.7 mL) being added to deionized water (90 mL) in a 100 mL volumetric

flask and stirred for 30 minutes. The final volume was made up to 100 mL with deionized

water and transferred into a beaker. The pH was adjusted to 2.0 using 6 M sodium hy-

droxide. Purified pepsin enzyme from Sigma-Aldrich Corp. (St. Louis, MO) (0.32 g) was

added and stirred. The solution temperature was maintained at 37 °C. Ten grams of

freeze-dried probiotic-loaded capsules and hydrolysate were dissolved in the simulated

gastric juice and allowed to incubate at 37 °C with constant agitation. After 120 min., the

pH was adjusted to 7.5 to inactivate the enzyme. After ten minutes, the pH was adjusted

to 4.5 for centrifugation. The reaction mixture was centrifuged at 3000xg for 20 min. to

collect peptide hydrolysates and probiotics from the supernatant. The resistant hydroly-

sate and probiotic substances were then treated with simulated intestinal juice (Edwards

and others 2019).

Simulated intestinal juice (SIJ) was prepared from the addition of potassium

phosphate monobasic (0.68 g) and sodium hydroxide 0.2 N (7.7 mL) to deionized water

(90 mL) in a 100 mL volumetric flask, followed by stirring for 30 minutes. Final volume

57

was made up to 100 mL and transferred into a titration flask. The pH of the solution was

adjusted to 8.0, and the mixture was maintained at 37 °C. Pancreatin (Sigma-Aldrich

Corp.) at a final concentration of 0.1% was added and stirred. The simulated gastric

juice treated hydrolysate and probiotic slurry was dissolved in the simulated intestinal

juice and allowed to incubate at 37 °C with constant shaking. After 120 min., enzyme

was inactivated by adjusting the pH to 4.5. The reaction mixture was centrifuged at

3000xg for 20 min. to obtain the slurry mix of hydrolysate and probiotics from the super-

natant. The mixture was stored at 4 °C. The number of probiotics with and without gas-

trointestinal treatment were counted and viability determined (Edwards and others

2019).

4.3 Results and Discussion

4.3.1 Degree of Hydrolysis of the Hydrolysates

During the incubation time (0, 30, 60, 90, 120, 150, 180, and 1200 min), the degree of

hydrolysis increased gradually over the initial 180 min period and had risen to a very high per-

centage by 1200 minutes. This observed progression is comparable to other studies that

demonstrate the reaction process of soy protein treated with pepsin enzyme (Peña-Ramos and

Xiong 2002, Meinlschmidt and others 2016). Higher rates of hydrolysis were observed from 60-

90 min. and 150-180 min. period of digestion (Figure 1). As seen in Figure 1, the hydrolysis rate

between 90 and 150 min of incubation was lower and not significantly different (p < .05) from

hydrolysis during the initial 180 minutes (Edwards and others 2019). However, degree of hydrol-

ysis was much higher between 120-180 min versus 0-120 min, with significant difference be-

tween 120 min. (2.4%) and 180 min. (4.2%). The consistent hydrolysis rate but lower DH, com-

pared to other broad-cleaving enzymes, could be due to the specificity of pepsin enzyme at Leu-

, Asp-, Glu-, and aromatic amino acid bond sites within protein structures, inhibition of active

sites, or slower initial degradation of a more compact protein structure (Nguyen and others

58

2016a, Edwards and others 2019). However, within the later time period of hydrolysis, more

bonds have been cleaved and exposed more surface area of the proteins that can be more eas-

ily catalyzed by the enzyme, resulting in a greater degree of hydrolysis. This is exemplified by

the DH at 1200 min (Figure 1), showing that incubation time is a significant variable for achiev-

ing degree of hydrolysis (Edwards and others 2019). However, lengthy exposure of SPI to pep-

sin (more than 6 hours) can result in intense catalysis of the protein into oligopeptides, and

smaller size peptides which were examined by previous researchers (Peña-Ramos and Xiong

2002).

Exposure of the protein structure to the enzyme must be controlled for the optimal dura-

tion to achieve ideal DH for functional purposes, typically between 2-10% DH. For these rea-

sons, hydrolysates from 120, 150, and 180 min. displayed the most ideal DH values (Edwards

and others 2019). In addition, greater hydrolysis can potentially result in a larger portion of hy-

drophobic amino acids, which would increase surface hydrophobicity (Llano and others 2004).

4.3.2 Surface Hydrophobicity (S0) of Hydrolyzed SPI

From the hydrolysis treatment of SPI, 120, 150, and 180 min. hydrolyzed samples were

the focus for determining surface hydrophobicity, as these three hydrolysis times yielded the

best DH among treatments (Figure 1). Non-hydrolyzed SPI produced a S0 of 6.5 at neutral pH

(Figure 2). Hydrolysis led to an overall increase, but at a decreasing trend, in surface hydropho-

bicity. Hydrolysis of 120 min. produced the greatest S0 of 19.4, while 150 min. showed a signifi-

cant decrease of S0 to 16.6 afterward. 180 min. of hydrolysis increased to hydrophobicity of

17.3, but it was not significantly different to other hydrolysis treatments. Protease digestion by

pepsin cleaves both aromatic and negatively charged amino acids, providing a blend of expo-

sure groups. Over the hydrolysis incubation, it is likely that smaller protein fragments with less

hydrophobicity were produced, or the confluence of thermal treatment and acidic conditions of

hydrolysis yielded more hydrophilic species (Ortiz and Wagner 2002). The greater exposure of

non-polar and hydrophobic amino acids during hydrolysis, which causes an increase in surface

59

hydrophobicity, through an increased interaction with the aqueous medium of the sample solu-

tion, may be measured as lower through a greater proportion of hydrophilic species revealed

over time (Nielsen and Olsen 2002, Chen and others 2011). The exposure of greater numbers

of hydrophobic amino acids can correlate to the rise of bitterness, especially in comparison to

the native SPI (FitzGerald and O’Cuinn 2006). The decrease in hydrophobicity with hydrolysis

time can be ideal, as this could lead to less bitterness among the peptides while improving in

solubility. However, perhaps there was inconsistency in the absorption of the ANS compound

within the hydrophobic proteins resulting in a lower hydrophobicity at 150 min. of hydrolysis. Ali

and others (1999) provided insight into how ANS can induce folding within proteins as a result of

electrostatic interactions. These mechanisms were considered by other researchers to poten-

tially be responsible for higher ANS fluorescence that is not indicative of true hydrophobic inter-

actions alone (Qadeer and others 2012). In addition, ANS is bound better among partially un-

folded protein structures than native protein or extensively denatured proteins (Qadeer and oth-

ers 2012). Perhaps this explains the progression of reduced hydrophobicity with hydrolysis incu-

bation, since the proteins become more denatured from their SPI structure.

4.3.3 Molecular Size of Soy Protein Hydrolysates (SPH)

Figure 3 displays the distribution of protein fractions and approximate molecular sizes

among soy protein isolate (SPI) and pepsin-hydrolyzed SPH samples within an SDS-PAGE gel.

SPI (Lane 2) was compared to hydrolyzed soy protein isolate samples from 1 hr. (Lane 3), 2 hr.

(Lane 4), and 3 hr. (Lane 5) of hydrolysis, using a reducing buffer. The reducing buffer con-

tained 2-mercaptoethanol, a compound that reduces disulfide bonds between protein groups,

allowing for easier separation of the protein groups (National Center for Biotechnology Infor-

mation 2019).

The increase in hydrolysis incubation resulted in an increase in lower-weight peptides,

with the β-conglycinin fractions showing more resistance to enzymatic hydrolysis than glycinin

fractions (Tsumura and others 2004). Noticeable disappearance of bands at 37 kDa and 21 kDa

60

were observed, especially with 3 hr. (Lane 5) of hydrolysis. This result indicates that pepsin has

a greater effect on hydrolyzing glycinin proteins into smaller fractions, with some approximately

6.5 kDa or less (Figure 3). However, these results differ from the research by Peña-Ramos and

Xiong (2002) but are supported by other research from Meinlschmidt and others (2016). The dif-

ference in results indicate the variations in concentration of SPI in solution during hydrolysis can

have an impact on protein hydrolysis. The lower concentration of SPI seems to allow the en-

zyme kinetics of pepsin to have a greater impact on the larger protein subunits found within the

β-conglycinin range (Peña-Ramos and Xiong 2002). In concurrence with this research, the

longer hydrolysis incubation with pepsin resulted in a greater concentration of lower molecular

weight peptides (under 10 kda), particularly as the proteins in the glycinin structure were de-

graded (Meinlschmidt and others 2016). The concentration of these lower weight peptides was

too high to discriminate within the electrophoresis gels used. For future experiments, utilizing

lower molecular size standards within tricine gels or mass spectrometry can more precisely de-

termine the size of the soy protein hydrolyzed peptides.

4.3.4 Determination of Capsule Characteristics

The vacuum mode to which the capsules were exposed to and the absence of metal

deposition and heating from metal sputtering procedure were chosen to prevent damage to the

capsules, based on previous literature (Conforto and others 2015). Samples were exposed to a

30 kV-electron beam mode using a GSE detector between 0.8-1.0 Torr of water vapor pressure

in ESEM for at least 5 min. In comparison, freeze-dried alginate capsules appeared smaller,

more compressed, and rougher in texture. Freeze-drying did not appear to impact the structural

integrity of the microcapsules at significant levels. The compression could have been greater

and the volume lower if metallization had been used with the capsules (Conforto and others

2015).

4.3.5 Encapsulated Probiotic Viability within Gastrointestinal Simulation

61

The growth of L. acidophilus was measured by absorbance at wavelength of 600 nm

(OD600) over a span of 30 hours. Max absorbance occurred at 24 hr. (OD600 = 1.910 ± 0.055)

and began to level at the 30-hr. interval, indicating stationary phase of growth (Figure 4). This is

supported by the typical culturing procedures used by other researchers, with Lee and others

(2004) specifying 28 hr. of growth as late log-phase (Iyer and Kailasapathy 2005, Sabikhi and

others 2010). Bacterial cultures during log phase were desired for encapsulation, to capitalize

on their growth potential to improve viability. Based on the growth pattern from Figure 4, L. aci-

dophilus cultures were grown for 16-18 (OD600 = 1.610 ± 0.207) hours to ensure that many via-

ble cells were encapsulated as opposed to late-stationary or less viable cells. L. acidophilus

cultures after 18 hr. incubation achieved average growth of 9.10 (± 0.12) log CFU/mL on MRS

plates and used for encapsulation.

When probiotic cultures were exposed to simulated gastric juice (SGJ, pH = 2.0), OD600

patterns decreased significantly after 30 min. for free probiotics, while encapsulated probiotics

did not deviate significantly (Figure 5). Free probiotics and encapsulated probiotics did not vary

significantly from average OD600 values from all time points. Free probiotics and encapsulated

probiotics paired with SPH both increased in OD600 values over incubation, with all four time

point values being significant (Figure 5). Encapsulated probiotics with SPH samples had higher

overall OD600 values and differed significantly from the free probiotics with SPH samples.

After exposure to simulated intestinal juice (SIJ, pH = 8.0), OD600 patterns differed from

SGJ absorbance patterns. The microcapsules with probiotics were observed to be intact in SGJ

solution after incubation but began dissolving after 30 min. of incubation in SIJ solution. Free

probiotic OD600 values decreased over time, with 120 min. (OD600 = 0.003 ± 0.002) significantly

different from all other time points (Figure 6). Free probiotics also had the lowest log CFU of

6.19 after 120 min from an initial log CFU of 6.93 (Table 1). Encapsulated probiotics had an in-

verse pattern to free probiotics. Values increased over time for encapsulated probiotics, with 0

62

min. (OD600 = 0.004 ± 0.002) exposure to SIJ being the lowest absorbance (Figure 6). Encapsu-

lated probiotics had a low log-difference 0.30 between 0 min. and 120 min. of incubation (Table

1). Free probiotics and encapsulated probiotics paired with SPH varied in their incubation val-

ues. Free probiotics with SPH increased in value over incubation time, with 90- (OD600 = 0.849 ±

0.048) and 120-min. (OD600 = 0.877 ± 0.047) time intervals being significantly higher than other

values (Figure 6). These higher values also corresponded to higher log CFU values of 6.35 and

6.26, respectively (Table 1). Encapsulated probiotics with SPH had values which remained stag-

nant over incubation. Values did not differ significantly across all time points, with 90 min. sam-

ples providing the highest value ((OD600 = 0.584 ± 0.050) during incubation (Figure 6). Log CFU

of 6.55 was highest for SPH encapsulated probiotics among all groups at 120 min. incubation

(Table 1). The SPH may provide a protective and nutritional advantage to probiotics under intes-

tinal conditions. More studies on the contributions of soy protein to probiotics would provide bet-

ter understanding.

Absorbance values between free and encapsulated SPH groups became significantly

different after 90 min. of incubation, with free probiotic SPH group values being higher on aver-

age (Figure 6). SPH probiotic groups displayed higher absorbance values naturally from the hy-

drolysates present in the sample, compared to samples without SPH. However, the increase in

values for both SGJ and SIJ treated free probiotic with SPH groups may be due to the increased

ability of the enzymes used within each solution to catalyze the hydrolysate, resulting in higher

values over time. This pattern was also demonstrated with the SPH and encapsulated probiotic

group in SGJ but not in SIJ. In comparison, log CFU values were significant for time, with initial

log values greater than all other time values. However, even though free probiotics had the low-

est log value among all groups, no significant differences were found for improved probiotic cell

viability between experimental groups, based on least square means from two-way ANOVA (p =

.445).

63

4.4 Conclusion

Encapsulation has been shown to provide protection for probiotic bacteria during in vitro

gastrointestinal conditions in the literature. Overall microcapsule integrity was observed to be

stable during acidic gastric conditions. Log values for samples from this research oscillated

more over time compared to samples from other researchers. This research did differ from other

research in its use of experimental groups containing protein hydrolysates and probiotics and

utilizing digestive enzymes. There is evidence that a rise in microencapsulated L. acidophilus is

possible at colonic pH. Perhaps slight increases in simulated intestinal conditions demonstrate

the growth of the probiotics that were resistant and viable to gastric conditions. This research

determined that encapsulation with alginate was not significant in improving L. acidophilus sur-

vival under digestion conditions. Studies that focus on improving microcapsule carrying capacity

may be essential for obtaining higher log values during the end of digestion. Other researchers

have determined that encapsulated probiotics experienced improved resistance to acidic condi-

tions, conditions near the initial gastric environment in humans. Their research also compared

alginate capsules to alginate capsules coated with chitosan, which showed significant improve-

ment to alginate alone. Further research on expanding upon encapsulation agent combinations

and composition can provide new insights on how to improve protection for probiotics via micro-

encapsulation.

64

4.5 Tables and Figures

Figure 1: Degree of hydrolysis (%) of SPI treated with pepsin (0.5% w/w) at various incubation times. Points on the curve are presented as means ± standard deviation in triplicate; those not connected with same letters are significantly different (p < .05)

Figure 2. Surface hydrophobicities of 2 hr. (SPH 2, SD = 1.1), 2.5 hr. (SPH 2.5, SD = 1.1), 3 hr. (SPH 3, SD = 1.2), and SPI samples (SD = 1.6). Results are presented in triplicate (p < .05).

a

b

cbc

0

5

10

15

20

25

SPI SPH 2 SPH 2.5 SPH 3

Surf

ace

Hyd

rop

ho

bic

ity

(So)

65

Figure 3. Electrophoretograms of soy protein isolate (Lane 2) and and hydrolyzed soy protein isolate after 1, 2, and 3 hr. incubation (Lane 3-5, respectively) using reducing buffers. The highlighted areas mark the beta-conglycinin (top) and glycinin (bottom) pro-tein structures. Molecular sizes of the protein standards ranged from 200 to 6.5 kDa (mysosin 200 kDa, β-galactosidase 116.25 kDa, phosphorylase B 97.4 kDa, serumalbu-min 66.2 kDa, ovalbumin 45 kDa, carbonic anhydrase 31 kDa, trypsin inhibitor 21.5 kDa, lysozyme 14.4 kDa, and aprotinin 6.5 kDa).

Figure 4. Time course of L. acidophilus growth measured by absorbance (mean ± SD, n = 3) at 600 nm (OD600).

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25 30 35

Ab

sorb

ance

(6

00

nm

)

Time (hr.)

66

Table 1. Survival of free and microencapsulated L. acidophilus groups after incubation in simulated intestinal juice (pH 8.0) at 37 °C

Viabilitya (log cfu/mL)

Sample 0 min. 30 min. 60 min. 90 min. 120 min.

Free probiotics 6.93 ± 0.28 6.10 ± 0.10 6.33 ± 0.03 6.15 ± 0.09 6.19 ± 0.10

Encapsulated probiotics 6.80 ± 0.10 6.66 ± 0.06 6.26 ± 0.14 6.26 ± 0.13 6.50 ± 0.25

SPH + free probiotics 7.10 ± 0.24 6.33 ± 0.20 6.11 ± 0.08 6.35 ± 0.05 6.26 ± 0.14

SPH + encapsulated probiotics 7.18 ± 0.29 6.36 ± 0.18 6.17 ± 0.09 6.10 ± 0.10 6.55 ± 0.14

a. Values represented as means ±SD in triplicate for all groups

Figure 5. Survival of free, encapsulated, and SPH-paired probiotics in simulated gastric juice. The error bars represent mean ± SD of three replicates for all treatments ● = SPH with free cells; ◊ = encapsulated cells only; ∆ = free cells; □ = SPH with encapsulated cells.

67

Figure 6. Survival of free, encapsulated, and SPH-paired probiotics in simulated intesti-nal juice. The error bars represent mean ± SD of three replicates for all treatments ● = SPH with free cells; ◊ = encapsulated cells only; ∆ = free cells; □ = SPH with encapsu-lated cells

68

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Overall Conclusions

This thesis displays how potential protein hydrolysates with improved solubility

and higher protein quality can be obtained from this non-GE soy cultivar. The established hy-

drolysis conditions provide soy protein hydrolysates (SPH) with highly soluble protein, even at

varying pH levels. Optimal hydrolysis conditions used 150 min. incubation with 0.5 wt% (2

U/mg) pepsin at 37 ºC temperature. The SPH from non-GE soybeans are a protein-rich

nutraceutical that can be incorporated in food products, especially beverages. Regarding probi-

otic protection, encapsulation has been shown to provide protection as equally as free probiotics

contained within an SPH formulation under simulated digestion conditions.

Further studies can include purification of SPH for enhanced activity, alternative combi-

nation of hydrolysis methods, and product application for potential commercialization. Other re-

search has shown that encapsulation can provide improved protection for bacteria during in vitro

gastrointestinal conditions. Some experiments in the literature focused on using layered micro-

capsules with more than one encapsulating agent. These differences likely provided improved

differences between encapsulated and free probiotics. Further research on expanding upon a

combination of encapsulating agents, comparison between different probiotic strains, and more

dynamic digestion simulations can provide new insights on how to improve protection for probi-

otics via microencapsulation.

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Appendix Tables and Figures

Figure 1. Protein concentration of soy protein isolate and hydrolysates compared to bovine serum albumin (BSA) standard

Figure 2. Absorbance of soy protein isolate per protein concentration under spectro-fluorophotometry (390 nm excitation; 470 nm of emission, n = 3)

0

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ance

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Figure 3. Absorbance of soy protein hydrolysate (2 hr hydrolysis) per protein concentra-tion under spectrofluorophotometry (390 nm excitation; 470 nm of emission, n = 3)

Figure 4. Absorbance of soy protein hydrolysate (2.5 hr hydrolysis) per protein concen-tration under spectrofluorophotometry (390 nm excitation; 470 nm of emission, n = 3)

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Figure 5. Absorbance of soy protein hydrolysate (3 hr. hydrolysis) per protein concentration under spectrofluorophotometry (390 nm excitation; 470 nm of emission, n = 3)

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a. b.

c. d.

a. b.

c. d.

Figure 6. An alginate capsule observed dry without any preparation using environmental scanning electron mi-croscopy (ESEM). Microscope utilized GSE in low vac-uum mode.

Figure 7. Freeze-dried alginate capsules observed using environmental scanning electron microscopy (ESEM). Microscope utilized SE detector under 9.2e-5 mBar.