Improving the Solubility of Yellow Mustard Precipitated Protein Isolate in Acidic Aqueous Solutions

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IMPROVING THE SOLUBILITY OF YELLOW MUSTARD PRECIPITATED PROTEIN ISOLATE IN ACIDIC AQUEOUS SOLUTIONS

L. KARINA LORENZO

A thesis submitted in conformity with the requirements for the degree of Master of Applied Science (M.A.Sc.)

Graduate Department of Chemical Engineering and Applied Chemistry University of Toronto

Improving the Solubility of Yellow Mustard Precipitated Protein Isolates

in Acidic Aqueous Solutions

M.A.Sc. Thesis

2008

Laura Karina Lorenzo

Graduate Department of Chemical Engineering and Applied Chemistry

University of Toronto

ABSTRACT

The thesis objective was to investigate methods for improving the solubility of yellow mustard precipitated protein isolate (RTech Laboratories, USA) to allow for its use in protein enhanced acidic beverages along with soluble protein isolate in the pH range of 2 to 4.5. Four treatments were tested: hydrolysis with Alcalase®; cross-linking with transglutaminase; salting in with sodium chloride, sodium tripolyphosphate, and sodium hexametaphosphate; and protective colloid formation with pectin. The effectiveness of each was determined by its ability to improve nitrogen solubility

(Nx6.25, AOCS-Ba11-65). The most effective treatments were hydrolysis and pectin stabilization.

Pectin (1.5 w/v%) improved solubility from 6% to 29% at pH 4. Alcalase increased solubility from

20% to 70% at pH 3 after 2 h of hydrolysis (0.5AU/5g PPI, pH 8.5, 50-55oC) and eliminated the protein’s isoelectric point in the acidic pH range. Investigating the combined use of both treatments to further increase PPI solubility is recommended.

ACKNOWLEDGEMENTS

First and foremost, I would like to thank Professor Diosady, my supervising professor, for his invaluable guidance and support throughout the project. Second, I would like to thank the members of my Master's Oral Committee, Professors Master and Saville, for taking the time to read my dissertation and providing me with valuable feedback. Third, I would like to thank NSERC and the government of Ontario for making this project possible through their financial support. Fourth, I would like to thank the U of T Food Engineering Group (in alphabetical order, Bih-King, Divya, Eilyn, Kadri, Olive, and Veronica) and my summer student, Asyeh, for their assistance. Finally, I would like to thank my family and friends for their support, especially my mother, my sister, and my long-term boyfriend and best friend, Nick.

iii TABLE OF CONTENTS

ABSTRACT ii

ACKNOWLEDGEMENTS iii

LIST OF TABLES vi

LIST OF FIGURES vii

1. INTRODUCTION 1

2. LITERATURE REVIEW 3

2.1 Mustard Seed Overview 3 2.1.1 Mustard Seed Composition 4 2.1.2 Current Uses of Yellow Mustard Products 7

2.2 Mustard Protein Isolates 7 2.2.1 Preparation of Mustard Protein Isolates 7 2.2.2 Mustard Protein Products and Components 11 2.2.3 Mustard Protein Isolate Quality 12 2.2.4 Potential Uses for Mustard Protein Isolates 15

2.3 Protein Solubility 17 2.3.1 Protein Overview 18 2.3.2 Factors Affecting Protein Solubility 20 2.3.3 Methods for Enhancing Protein Solubility 23 2.3.3.1 Protein Modification 23 2.3.3.2 Solution Environment Modification 28

2.4 Project Objectives 32

3. EXPERIMENTAL METHODS 33

3.1 Materials and Equipment 33 3.1.1 Test Materials 33 3.1.2 Reagents and Materials 34 3.1.3 Equipment 35 3.2 Experimental Methods 36 3.2.1 Enzymatic Hydrolysis Treatment 37 3.2.2 Enzymatic Cross-linking Treatment 37 3.2.3 Salt Treatment 38 3.2.4 Stabilizer Treatment 38 3.2.5 Analytical 38

4. RESULTS AND DISCUSSION 40

4.1 PPI Starting Material Analysis 40 4.1.1 Protein Content 40 4.1.2 Solubility 41 4.1.3 Viscosity 44 4.1.4 Organoleptic Qualities 47

4.2 Enhancing PPI Solubility: Protein Modification 51 4.2.1 Enzymatic Hydrolysis 51 4.2.2 Enzymatic Cross-linking 59

4.3 Enhancing PPI Solubility: Solvent Modification 62 4.3.1 Salting In 62 4.3.1.1 Sodium Tripolyphosphate (STPP) 63 4.3.1.2 Sodium Chloride (NaCl) 64 4.3.1.3 Sodium Hexametaphosphate (SHMP) 66 4.3.2 Protective Colloids 67

5. CONCLUSIONS AND RECOMMENDATIONS 69 5.1 Summary 70 5.2 Conclusions 71 5.3 Recommendations 72

6. REFERENCES 74

APPENDICES 82

Appendix A – Detailed Analytical and Experimental Methods 83 Appendix B – Result Tables 104

v LIST OF TABLES

2.1 Essential amino acid content of yellow mustard protein compared to soy and international recommended levels (mg amino acid/ g protein) 13

3.1 Solvents used for experiments 33

4.1 Protein and moisture contents of Rtech PPI, Texas SPI, and soy SPI 40

vi LIST OF FIGURES

2.1 Myrosinase hydrolysis of glucosinolates (adapted from Fahey et al., 2001) 4

2.2 Chemical structure of phytic acid (adapted from Barrientos & Murthy, 1996) 5

2.3 Chemical structure of sinapic acid (left) and p-hydroxybenzoic acid (right) 6

2.4 Flow diagram of the yellow mustard protein isolation process in which only the SPI is filtered (adapted from Xu et al., 2003) 8

2.5 Pressure-driven membrane filtration processes 9

2.6 Schematic diagram of a batch ultrafiltration process (adapted from Tomac, 2003) 10

2.7 Schematic diagram of a batch diafiltration process (adapted from Tomac, 2003) 10

2.8 Protein solubility as a function of pH in 0.2 M NaCl (adapted from Cheftel et al., 1985) 16

2.9 Formation of a peptide bond 18

2.10 A typical protein isoelectric curve 21

2.11 Protease hydrolysis site 23

2.12 Effect of pH on the nitrogen solubility profiles of unmodified and hydrolyzed soy protein isolate (adapted from Walsh et al., 2003) 24

2.13 A typical enzyme reaction progress curve 26

2.14 Transglutaminase cross-linking reaction (adapted from Wartiovaara, 1999) 27

2.15 Protein “salting in” and “salting out” as a function of salt concentration 29

4.1 Unmodified RTech PPI nitrogen solubility as a function of solution pH 42

4.2 Unmodified RTech PPI (represented by crosses) and Texas PPI (represented by circles) nitrogen solubility as a function of solution pH 43

4.3 SDS Polyacrylamide gel electrophoresis of baseline RTech PPI: (a) Protein ladder; (b) PPI protein in solution at PH 3 after NSI Centrifugation; (c) PPI NSI (pH 3) centrifuge cake; original RTech PPI powder 44

4.4 Viscosity (in centipoise) of 1% PPI or SPI enhanced commercial beverages 45

vii 4.5 Viscosity (in centipoise) of various concentrations of PPI enhanced water 46

4.6 Viscosity (in centipoise) of a 1% PPI water solution after several blending sessions 47

4.7 Yellow mustard precipitated protein isolate (PPI) powder 47

4.8 1% aqueous protein solutions in water. From left to right: PPI, MR, SPI, Soy 48

4.9 Sedimentation after 2 hours. From left to right: 1% PPI, MR, SPI, and soy aqueous solutions 48

4.10 Sedimentation after 2 weeks. From left to right: 1% MR, soy, SPI, and PPI aqueous solutions 49

4.11 Left: 1% PPI aqueous solution in repose. Right: 3% PPI aqueous solution after agitation 49

4.12 1% PPI in cranberry juice after agitation 50

4.13 Left to right: cranberry juice, ~0.25 wt% PPI in cranberry juice (after centrifugation/filtration), ~0.25 wt% PPI in cola (after centrifugation/filtration), cola 50

4.14 Solubility and degrees of hydrolysis of Alcalase-treated RTech PPI at an NSI of pH 3 vs. treatment time (treatment constants: 50-55oC, 0.5 AU / 5 g of PPI, pH 8.5). Alpha value used to calculate degrees of hydrolysis: 0.9684 52

4.15 SDS Polyacrylamide gel electrophoresis of baseline and hydrolyzied RTech PPI: (a) protein ladder (1 lane); (b) PPI protein in solution at pH 3 after NSI mixing, decanting, centrifugation, and filtration (2 lanes); (c) Alcalase hydrolyzed PPI (1 h, pH 8.5, 60oC, 0.5 AU / 5 g PPI) in solution at pH 3 after NSI mixing, decanting, centrifugation, and filtration (2 lanes) 55

4.16 Particle size of RTech Alcalase-treated RTech PPI at an NSI of pH 3 vs. treatment time (treatment constants: 50-55oC, 0.5 AU / 5 g of PPI, pH 8.5) 55

4.17 Particle size of the RTech Alcalase-treated RTech PPI at an NSI of pH 3 (treatment constants: 4 h, 50-55oC, 0.5 AU / 5 g of PPI, pH 8.5) 56

4.18 Degrees of hydrolysis of Alcalase-treated RTech PPI at an NSI of pH 3 vs. treatment time (treatment constants: 0.5 AU / 5 g of PPI, pH 8.5). Alpha value used to calculate degrees of hydrolysis: 0.9755 57

4.19 Nitrogen solubility as a function of Alcalse treatment concentration (treatment constants: 1 h, pH 8.5, 50-55oC; NSI pH of 3) 58

4.20 Solubility of Alcalase-treated RTech PPI at an NSI of pH 2.5, 3 and 3.5 (treatment constants: 50-55oC, 0.8 AU / 5 g of PPI, pH 8.5, 1 h) 58

4.21 Solubility of RTech PPI after transglutaminase treatment (55oC, 2.5 hours, pH 6.5) 60

viii 4.22 NSI at pH 3 of RTech PPI after various transglutaminase treatment times (55oC, 10 UE/g protein, pH 6.5) conducted after an Alcalase treatment (5 h, 0.5 AU / 5 g PPI, 55oC, pH 8.5) 61

4.23 NSI at pH 3 of RTech PPI after various transglutaminase treatment concentrations (55oC, pH 6.5, 2.5 h) conducted after an Alcalase treatment (5 h, 0.5 AU / 5 g PPI, 55oC, pH 8.5) 62

4.24 Solubility of STPP-treated RTech PPI (0.04 g of STPP/ 5 g of PPI) 63

4.25 Solubility of NaCl-treated RTech PPI (2.3 g of NaCl/ 5 g of PPI) 64

4.26 SDS Polyacrylamide gel electrophoresis of baseline and NaCl-treated PPI: (a) NaCl-treated RTech PPI (2.3 g of NaCl / 5 g PPI) in solution at pH 3 after NSI mixing, decanting, centrifugation, and filtration (2 lanes) (b) protein ladder (1 lane); (c) PPI protein in solution at pH 3 after NSI mixing, decanting, centrifugation, and filtration (2 lanes) 65

4.27 Theoretical curve for protein “salting in” and “salting out” as a function of salt concentration 65

4.28 Solubility of NaCl-treated RTech PPI at various NaCl concentrations (NSI pH 3) 66

4.29 Solubility of SHMP-treated Texas PPI at pH 2.7 compared to the Texas baseline solubility curve 67

4.30 Solubility of Pectin-treated RTech PPI compared to the unmodified RTech PPI (at an NSI pH of 4) 68

1. INTRODUCTION

The Food Engineering Group at the University of Toronto has, in recent years, developed a process to extract high-grade protein isolates from mustard seeds. The quality of protein obtained from mustard with this process is comparable to that of soy. Therefore, once commercialized, mustard protein has the potential of being used in the food industry as an alternative to soy.

There are several motivations for using mustard protein as an alternative to soy protein. Commercializing mustard protein in Canada would be beneficial from an economic standpoint since Canada is the world’s leading producer and exporter of mustard accounting for over 40% of worldwide mustard production (Agri-Food Canada, 2005; Smith & Jimmerson, 2005). Finding other end uses for mustard seeds would increase the value of Canadian mustard which, in turn, would increase revenue for Canadian mustard farmers. From an ecological standpoint, mustard protein production would be beneficial as it would provide the food industry with a high quality protein source; which, being a plant source, leaves a markedly lower ecological footprint than do animal protein sources. Lastly, from a nutritional standpoint, mustard is a source of high-quality fat-free concentrated protein with a well-balanced essential amino acid composition comparable to that of milk or soy. Moreover, it is a protein source that is free of the allergens typically found in soybeans, thus making it a suitable alternative for those allergic to soy (Diosady et al., 2007).

Research is currently being conducted in our laboratory to gain the necessary knowledge for the use of mustard protein isolates in various commercial applications. One especially interesting application is the use of mustard protein as a nutritional supplement in protein-enhanced beverages, which are recently gaining special attention in the marketplace (CP Kelco, 2006; CP Kelco, 2007; Eckert & Riker, 2005; Lam et al., 2007; Miller, 2005; Sloan, 2007). Mustard protein has a unique solubility profile in the acidic pH range (2 to 4.5): one of the isolates attains a solubility greater than 90% in this pH range whereas other plant protein isolates have much lower solubilities (e.g., the solubility of unmodified soy in this range ranges from 5% to 10% (Walsh et al., 2003)). This makes mustard protein an excellent candidate as a protein extender in acidic protein-enhanced beverages (e.g., fruit juices and carbonated beverages).

In the mustard protein extraction process, two protein isolates are obtained: acid soluble protein isolate (SPI), and precipitated protein isolate (PPI). Of the two, the highest-grade protein product

1 2 is the SPI for its high solubility (>90 wt%) over a broad pH range and high protein content (~90 wt%, dry basis, Nx6.25). PPI is considered to be of a medium grade for its lower solubility (~ 5 to 38 wt%) but high protein content (~90 wt%, dry basis, Nx6.25). Although SPI would ideally be the protein isolate of choice for use in beverages, there is one bottleneck to its use: it is produced in relatively low yields. The yield of PPI ranges from 1.4 to almost 6 times higher than that of the SPI (Diosady et al., 2007). The ideal solution would be to devise a method to improve the solubility of the PPI so that it could be used alongside the SPI in the protein fortification of beverages.

This said, the objective of this thesis project is to investigate methods for improving the solubility of PPI, specifically in the acidic pH range (2 to 4.5). In this report, several solubility enhancing treatments are explored: enzymatic hydrolysis, enzymatic cross-linking, and the addition of solutes and stabilizing agents. Each method is investigated, its strengths and limitations are discussed, and a final treatment recommendation is made.

2. LITERATURE REVIEW

2.1 Mustard Seed Overview

Mustard (Brassica spp.) is a high-protein oilseed that is known for its pungent taste. It is closely related to canola and rapeseed and belongs to the Cruciferae family along with cabbage, broccoli, turnips, and cauliflower. Mustard is believed to have been one of the first crops domesticated by humans, and is now used worldwide as a condiment, spice, oil, and food additive (Agri-Food Canada, 2005; Balke, 2006; Wysocki & Corp, 2002; Oplinger et al., 2000).

The mustard plant is an annual spring crop which grows in cool weather conditions in temperate regions (Agri-Food Canada, 2005; Wysocki & Corp, 2002). In Canada, the majority of the mustard production is concentrated in Manitoba, Alberta, and particularly in Saskatchewan, and it is grown exclusively as a non-GMO crop (Agri-Food Canada, 2005; Canadian Special Crops Association, 2007; Johnston et al., 2002; Wysocki & Corp, 2002). When compared to other oilseeds, mustard is considered less susceptible to pest problems (Wysocki & Corp, 2002; OEC, 2008), and, although the productivity of mustard is lower than that of its close cousins, rapeseed and canola, it is superior to these in its resistance to pod shatter and its tolerance to heat, drought, and mild frosts (Agri-Food Canada, 2005; Oplinger et al., 2000; Wysocki & Corp, 2002; Brown et al., 1999).

There are three main types of mustard: yellow/white mustard (also known as Sinapis alba L., Brassica hirta M., and Brassica alba), oriental/brown mustard (also known as Brassica juncea or Indian mustard), and black mustard (Brassica nigra). Each variety differs in terms of colour, taste, composition, and agronomics (Oplinger et al, 2000; Wysocki & Corp, 2002). This project focuses on improving the solubility of protein from yellow mustard, in particular, as it is by far, the most common mustard variety grown and used in North America (Agri-Food Canada, 2005; Declerq, 2006; Smith & Jimmerson, 2005).

2.1.1 Mustard Seed Composition

Yellow mustard seeds are comprised of: 28-40% protein, 28-36% oil, 20-25% carbohydrates, ~4% minerals, and ~2% fiber on a dry basis (Diosady et al., 2005; George Mateljan Foundation, 2005; Minn-Dak Growers, 2002). These numbers shown are ranges since the composition of yellow

3 4 mustard varies slightly depending on its variety and on the year and region of cultivation (Balke, 2006; Gunasekera et al., 2006). Mustard seeds contain a high content of plant protein; they contain more protein than beans, lentils, and chickpeas, but have a lower protein content than soy (35-52 wt% protein, dry basis) (Esen, 1982; George Mateljan Foundation, 2005; Hammack, 2005). The protein content of beef and chicken muscle meat is roughly double than that of mustard on a dry basis (George Mateljan Foundation, 2005).

Apart from its main constituents, mustard seeds also contain three undesirable compounds: glucosinolates, phenolics, and phytates. These compounds are toxic and/or anti-nutritional and it is imperative that they be removed from the mustard meal before it is to be consumed by humans in large quantities (Diosady et al., 2005). If not removed, these components will also reduce mustard solubility and will limit the use of mustard in foods to a maximum of 1 to 2% (Balke, 2006). Each of these problematic compounds is discussed below.

Glucosinolates

Mustard seeds contain a significant concentration of glucosinolates (> 200 micromol/g of defatted mustard meal (Diosady et al., 2007)); these are bitter-tasting sulfur-containing glycosides that are commonly found in cruciferous plants. The main type of glucosinolate found in mustard is p- hydroxybenzyl glucosinolate (Fabre et al., 1996). Glucosinolates are part of the mustard plant’s natural defense system against predators; when plant tissue is damaged, the glucosinolates are hydrolyzed through the action of an endogenous myrosinase enzyme system. The resulting hydrolysis products are isothiocyanates, nitriles, and thiocyanates (Mithen, 2001).

Figure 2.1 – Myrosinase hydrolysis of glucosinolates (adapted from Fahey et al., 2001)

Of these hydrolysis products, it is the isothiocyanates that are also responsible for the characteristic pungent mustard taste. In high concentrations, isothiocyanates are toxic to humans; they interfere with thyroid function, thereby disrupting growth and reproduction, and cause liver and kidney damage (Diosady et al, 2005; Tookey, 1980; Fahey et al., 2001; Fenwick et al., 1983). While isothiocyanates are toxic, their strong taste prevents their consumption in harmful quantities. When using mustard as a processed food additive (as opposed to a spice), the myrosinase enzyme system is inactivated through heat denaturation to prevent the formation of the glucosinolate hydrolysis products. This ensures that the mustard will not be toxic or overpower the taste of the overall food product. The inactivation results in what is called ‘deheated mustard’ which is bland in flavour, non-toxic, and retains the desirable functional characteristics of conventional mustard (Minn-Dak Growers, 2002; Xu et al., 2003). Unfortunately, over time, residual enzymes or enzymes in the environment cause flavour reversion, which limits the use of deheated mustard.

Phytic Acid

Phytic acid (Figure 2.2; myo-inositol hexaphosphate) comprises 1-3 wt% of most oilseeds and cereals and accounts for 60 to 90% of their total phosphorous content (Gaf & Eton, 1984). This compound is a strong chelating agent that binds to essential polyvalent metal cations in the body such as iron, calcium, zinc and magnesium thus reducing their bioavailability (Erdman, 1979; Liu et al., 1998; Sadeghi et al., 2005). Phytic acid can also bind to proteins and decreases their solubility, thus altering their functionality properties (Erdman, 1979).

Figure 2.2 – Chemical structure of phytic acid at a neutral pH (adapted from Barrientos & Murthy, 1996)

Phenolic Compounds

Phenolic compounds are products of plant metabolism and, as such, are ubiquitous amongst plants and in plant-based food or beverage products (Bravo, 1998; Jobstl et al., 2004). Phenolic compounds are a diverse category of chemicals that have at least one aryl ring which has one hydroxyl group (Bravo, 1998; O’Connell & Fox, 2001). The two main types of phenolic acids present in mustard seeds are: sinapic acid (~68%) and p-hydroxybenzoic acid (~27%), the latter also being the predominant glucosinolate in yellow mustard (Diosady et al., 2007; Kazimierz et al., 1984). These compounds are chelating agents and reduce the bioavailability of minerals and proteins present in the mustard meal thus lowering mustard's nutritional quality (Diosady et al., 2005; Naczk et al., 1998, Xu et al., 2002). These compounds are also responsible for the dark colour and astringent taste present in mustard meal (Rubino et al., 1998; Xu et al., 2002).

Figure 2.3 – Chemical structure of sinapic acid (left) and p-hydroxybenzoic acid (right)

2.1.2 Current Uses of Yellow Mustard Products

Since its first use 4,000 years ago, yellow mustard has been used as a medicine, a condiment, and as a food additive in seed, flour, or oil form (George Mateljan Foundation, 2005). Whole mustard seeds are generally used in pickled products and relishes for their attractive décor, flavor, and texture (Minn-Dak Growers, 2002). Mustard oil is widely used in India and Bangladesh as a spicy cooking oil (Agri-Food Canada, 2005). Ground mustard or mustard flour is added to many processed foods because of its high protein content and flavor enhancement and for the thickening, emulsifying, and water absorption capabilities of its bran. Mustard’s emulsifying properties are largely attributed to the significant amount of mucilage, a heterogeneous group of polysaccharides, present in its bran (Appelqvist, 1971; Balke, 2006; Cui et al., 2001).

Examples of processed foods that contain mustard flour as an emulsifier and texture controlling agent are mayonnaise, salad dressings, soups, and sauces. Mustard flour is also used in meat products as a water binding agent allowing for easier slicing of meat product (Oplinger et al., 2000; Minn-Dak Growers, 2002). In addition, ground yellow mustard is a popular ingredient of dry seasoning and spice blenders, and it is prominently used in North America as ‘prepared mustard’ (also known as ‘hot dog mustard’) consisting of a smooth paste of ground mustard, salt, vinegar, and various spices (Minn-Dak Growers, 2002).

Once protein production from mustard is commercialized, the process will allow for the simultaneous production of mustard bran, oil, and protein from the seeds. The ground hulls or bran can continue to be used as an emulsifying/filling agent in prepared foods; the oil can be used as a vegetable cooking oil, renewable biofuel, lubricant or as a polymer precursor (due to its high erucic acid levels) (Balke, 2006; OEC, 2008); and the protein isolates, as emulsifying agents and non-fat protein extenders for foods and beverage products (Xu et al., 2003).

2.2 Mustard Protein Isolates

2.2.1 Preparation of Mustard Protein Isolates

Mustard protein isolates are obtained by a proprietary process developed by the Food Engineering Group at the University of Toronto (L.L.Diosady, Lei Xu, Bih-King Chen – Production of high- quality protein isolates from defatted meals of Brassica seed, US Patent 6,905,713 (2005)). A flowchart of this process is shown in Figure 2.4. The starting material for this process is hexane- defatted yellow mustard meal. The meal is first mixed with an alkali solution at a pH of 11 (using food-grade NaOH). This causes most of the protein to become solubilized or suspended. At this point, a reducing agent ascorbic acid, 0.05% w/v) is added to the extract solution to reduce the effects of oxidation from the alkaline extraction by partially inhibiting the formation of covalently bonded phenolic-protein complexes. The formation of these complexes is detrimental as it results in darkening the colour of the final product and has an effect on its flavour. The suspended protein is then separated from the residual meal by means of centrifugation. The residual meal is washed, neutralized with acid, and dried resulting in what is called the Meal Residue (MR). MR is a protein- containing flour with high binding capacity which can be sold as a commercial product.

Yellow Mustard Meal

Alkaline Extraction

Supernatant Centrifugation Solids

Ultrafiltration/Diafiltration Washing/Centrifugation

Retentate Drying

Isoelectric Precipitation Meal Residue (MR)

Supernatant Centrifugation Solids

Ultrafiltration/Diafiltration Washing/Centrifugation

Retentate Drying

Drying Precipitated Protein Isolate (PPI)

Soluble Protein Isolate (SPI)

Figure 2.4 – Flow diagram of the yellow mustard protein isolation process (modified from Xu et al., 2003)

The protein-rich supernatant is then heated (to 50-60oC) and NaCl is added to the solution at a level of 0.05M to increase its ionic strength, thereby breaking apart a portion of the ionically bonded phenolic-protein complexes so as to ease the separation of the protein from the phenolics downstream. The supernatant is then subjected to ultrafitration followed by diafiltration to remove the free glucosinolates, phytates, and phenolic compounds from the liquid.

Feed Retentate

Permeate

Figure 2.5 – Operation principle of pressure-driven membrane filtration

Ultrafiltration is a pressure-driven membrane process used to purify streams with large molecules by filtering out smaller sized impurities and to concentrate these streams by reducing their total volume. The membrane processing takes advantage of the large difference in molecular size between the mustard protein and the anti-nutritional compounds, which are small molecules in comparison to the protein molecules. The purified and concentrated stream is called the “retentate”, while the residual stream, the impurity-laden byproduct, is called the “permeate” (Figure 2.5). Diafiltration is commonly used after ultrafiltration to further purify a stream after the ultrafiltration has reached its limit. Diafiltration is essentially identical to ultrafiltration, with the only difference being that the retentate is diluted to keep the retentate volume constant and reduce the concentration of the low molecular weight impurities in the retentate. Figures 2.7 and 2.8 depict the ultrafiltration and diafiltration processes, respectively. In the mustard protein isolation process, the membranes used have a molecular weight cut-off of 5 to 10 kilodaltons.

10 Back Pressure Valve

Retentate

Feed Tank Vo Permeate

Vf Membrane

Peristaltic Pump

Figure 2.6 – Schematic diagram of a batch ultrafiltration process. Vo = Volume of the original solution; Vf = Volume of the final solution (adapted from Tomac, 2003)

Solvent

Vw Back Pressure Valve

Feed Tank V Permeate o Membrane

Peristaltic Pump

Figure 2.7 – Schematic diagram of a batch diafiltration process. Vo = Volume of the original solution; Vw = Total volume of the solvent added (adapted from Tomac, 2003)

The final retentate, now low in anti-nutritional compounds, is passed to a precipitation zone in which a portion of the suspended protein will be precipitated with food grade acid to lower the solution pH to ~ 4.5, the approximate isoelectric point of a large fraction of the yellow mustard protein. The isoelectric point (pI) refers to the pH in which a protein has no net ionic charge (Cheftel et al., 1985). This results in the protein-protein interactions that are stronger than the protein-solvent interactions, thus leading to protein aggregation and precipitation. The precipitated protein is then separated from the soluble fraction by means of centrifugation. The precipitated fraction is washed, neutralized with a food-grade base, and dried. The resulting product is the Precipitated Protein Isolate (PPI), a high- value product that is high in protein content, and has a low solubility near the pI. The soluble protein fraction in the supernatant is neutralized with a base and passed through a second set of membrane filters (ultrafiltration and diafiltration) as a second purification step for the removal of anti-nutritional compounds. The final protein fraction is then dried. This product is the Soluble Protein Isolate (SPI). SPI has a high protein content and is almost completely soluble at all pH ranges.

2.2.2 Mustard Protein Products and Components

The protein extraction process produces three products: precipitated protein isolate (PPI; ~80-90 wt% protein, dry basis), soluble protein isolate (SPI; ~90-99 wt% protein, dry basis), and meal residue (MR; ~20 wt% protein, dry basis) (Diosady et al., 2007). Both the SPI and PPI are bland tasting, fine light coloured powders that are similar in texture to wheat flour. The MR is a coarser powder of slightly darker colour. The composition of the protein products varies slightly depending on the yellow mustard variety, the conditions of growth, and any variability in the process conditions. The yield of the final products relative to each other also varies, but, in general, more PPI is produced than SPI. The ratio of PPI to SPI produced can range from 5.7 to 1.4 (Diosady et al., 2007).

The protein isolates can also be considered to be of a high quality in terms of bioavailability as the glucosinolate and phytate content of the PPI and SPI is either non-detectable by standard methods of analysis or is very low and should not be of any concern (Diosady et al., 2007; Xu et al., 2003). In terms of phenolic acid removal, depending on the process conditions, 10 to 90% of the bound phenolics and 80 to 95% of the free phenolics are removed from the protein isolates. This results in an SPI and PPI with a final concentration of phenolics typically in the range of 0.02 wt%; this concentration is considered to be minimal (Diosady et al., 2007; Xu et al., 2003). The meal residue (MR) is of lower nutritional value than the PPI and SPI due to its lower protein content and higher levels of anti-nutritional compounds, but it is still a useful byproduct that can be sold commercially.

These mustard products have useful functionality. Functional properties include the protein’s fat and water binding properties, gelling, and emulsion stabilizing properties (Diosady et al., 2007). Mustard protein owes its emulsification properties, in particular, to a group of its proteins that are called oleosins (Marnoch, 2004), which are amphipathic structural proteins that are found in the oil bodies of oilseeds (Balke, 2006).

Currently, there is limited information available in the literature on the protein components of these mustard protein isolates (Balke, 2006). The molecular weights of the mustard protein components are known, however. In 2003, Dendukuri & Diosady identified 18 different subunits of alkaline extracted protein by means of SDS Polyacrylamide Gel Electrophoresis, which ranged in size from 6 to 80 kDa. Within this range, Dendukuri & Diosady identified two major bands between 6kDa and 16kDa, three between 20 and 25 kDa, four bands around the 32 kDa mark, and one band at 62 kDa. Proteins smaller than 5-10 kDa in size, are lost in the discarded ultrafiltration permeate during the mustard protein isolation process as the ultrafiltration membrane MW cutoff ranges from ~5-10 kDa.

2.2.3 Mustard Protein Isolate Quality

When the body ingests proteins, it degrades them into their building blocks, the amino acids, and then uses these amino acids to form its own proteins which, in turn, become the building blocks of the human body. Roughly half of these amino acids can be synthesized within the body; the other half cannot be synthesized by the body and must be regularly consumed from external sources (Karp, 2003; Lodish et al., 2003; Whitaker & Tannenbaum, 1977). These are called ‘essential amino acids’. Since the body cannot store proteins or amino acids ingested in surplus, a high quality source of protein must be continuously consumed in adequate amounts in order to ensure the sufficient intake of essential amino acids (Esen, 1982). Doing so is a prerequisite for normal growth and development in children and for the maintenance of protein in adults (Esen, 1982).

Table 2.1 – Essential amino acid content of yellow mustard protein compared to soy and international recommended levels (mg amino acid/ g protein)

Protein Recommendation Yellow Mustard Soy Patterns FAO/W (Bhattach Data Source HO MIT (Sadeg (VanEtten aryya & Tentati (Young & (Miller et (Bell et (Calvins et (Solae, hi et al., et al., Sen, ve Borgonha al., 1962) al., 2000) al., 1972) 2004) 2005) 1976) 2000) Essential (UNU, 2000)

Amino Acids 1996) Isoleucine 38 35 40 37 41 37 38 46 49

Leucine 65 65 79 78 73 59 81 78 82

Lysine 50 50 69 49 59 56 54 64 63

Methionine + 25 25 43 56 ------26 26 Cysteine

Phenylalanine 65 65 84 68 74 47.6 92 88 90 + Tyrosine Threonine 25 25 33 43 46 35 45 39 38

Tryptophan 10 10 -- 16 -- -- 17 14 13

Valine 35 35 57 52 56 43 35 46 50 13

A protein isolate is said to be of a high nutritional quality if it is able to provide all the essential amino acids at or above the FAO/WHO and MIT recommended levels (Balke, 2006; Esen, 1982; Scrimshaw et al., 1996). Table 2.1 shows the essential amino acid composition of yellow mustard seed protein compared to that of the revised FAO/WHO and MIT recommendation patterns and to the essential amino acid composition of soy protein. As can be seen from Table 2.1, yellow mustard protein is able to provide all the essential amino acids in amounts greater than those recommended by the FAO/WHO and MIT standards; it should be deemed a protein source with a well-balanced amino acid profile that is appropriate for protein supplementation in foods. As can also be seen in the table, the quality of yellow mustard protein is essentially equivalent to that of soy protein.

The protein quality of mustard is superior to that of most commercially available plant proteins which are usually limited either in lysine or in sulfur containing essential amino acids (methionine and cystine) (Balke, 2006). Wheat protein, for example, contains ~26 mg of lysine per gram of protein (Acquistucci, 1995) while the minimum recommended level is that of 50 mg per gram of protein. Lentils, which are known for their high protein content (29.7 wt%, dry basis), are limiting in their ability to provide sulfur containing amino acids: they contain 21 mg of methionine and cysteine while the recommended minimum is 25 mg per grams of protein (George Mateljan Foundation, 2008). It is because of these essential amino acid deficiencies, and its generally low protein content, that vegetable protein has traditionally not been considered an ideal source of protein for protein isolates.

The bioavailability of the mustard protein largely depends on the amount of anti-nutritional compounds remaining in the protein powder after processing (i.e., polyphenols, phytic acid, and the hydrolysis products of glucosinolates). As is explained in section 2.2.2, only minimal or trace amounts of these compounds are present in mustard protein isolates, hence, their effects should not be of great concern (Xu et al., 2003). Another compound that is detrimental to protein utility is trypsin inhibitor (TI). While many vegetable protein sources contain a significant level of TI (e.g., legumes and soy), its presence in yellow mustard should be of little concern as yellow mustard contains very low levels of TI (ranging from 1.65 to 2.1 TI units per gram) (Bell & Rakow, 1996).

A factor that may undermine the use of a protein isolate is the presence of allergens. Two allergens have been identified in mustard protein, Sin A 1 and Sin A 2 (Menendez-Arias et al., 1988; Mustorp et al., 2007; Palomares et al., 2005); of these, Sin A 1 is known to be a thermostable protein and resistant to degradation by trypsin an other proteolytic enzymes (Rance, 2003). This has resulted in the addition of mustard, along with soy and cow’s milk, to the European Commission list of

15 allergenic foods in 2005 (Food Standards Agency, 2008). Mustard, however, remains unlisted as an allergen in the FAO/WHO Codex Alimentarius Commission (Mustorp et al., 2007). According to the Food Standards Agency in the UK (2008), mustard allergy is rare but it has been reported to cause severe reactions, including anaphylaxis.

In contrast, soy allergies are known to affect a significant percentage of the population (approximately 1-6%) (Bruno et al., 1997; Cordle, 2004; Magnolfi et al., 1996), and soy protein allergy is considered by the Food Standards Agency (2008) to be a common childhood allergy. The symptoms of soy allergy are similar to those of milk allergy (not to be confused with milk intolerance) including rashes, diarrhea, vomiting, stomach cramps, breathing difficulties, and in rare cases, soy allergies can also cause anaphylaxis (Food Standards Agency, 2008).

There is limited literature available on mustard allergies (Rance, 2002). However, it appears as though, in terms of allergen safety, mustard allergies may be considered to be, at worst, equivalent to the use of soy, and, at best, safer for the general population. It is also known that mustard is free of the allergens typically found in soybeans (Diosady et al., 2007), thus making it a suitable alternative for those allergic to soy.

2.2.4 Potential Uses for Mustard Protein Isolates

Commercializing the production of mustard protein isolates would allow these to be used as a protein extender in various food products, and in many cases, as a direct substitute for soy. Mustard may be used as a functional ingredient in protein-containing products (in place of or in combination with other protein isolates such as gluten, casein, whey, and soy). Examples of such products are: processed meats, bakery products, nutritional supplements, infant formulations, protein bars, beverages, frozen confectionary bars, and soups. Mustard protein can also be used in vegetarian meat substitute products (e.g., hamburger patties, wieners, ground round, etc.), and also in non-food protein applications: sizing in paper, adhesives, and adsorbants (Diosady et al., 2007). Additionally, mustard protein isolates can be used as a protein nutritional extender in beverages, and this application is the focus of the present research project.

According to CP Kelco (2006), CP Kelco (2007), Eckert & Riker (2005), Lam et al. (2007), Miller (2005), and Sloan (2007), protein beverages have recently experienced a marked increase in popularity as consumers are purchasing protein-enhanced beverages for their perceived health

16 benefits. Current popular protein drinks include ready-to-drink and powdered milk substitutes, meal replacements, and protein shakes used for body building or recovery (Eckert & Riker, 2005).

Whey protein concentrate 100 Sodium caseinate Soy protein isolate

50 Fish protein concentrate

Nitrogen Solubility (%) 0 2 4 6 8 10 12

Figure 2.8 – Protein solubility as a function of pH in 0.2 M NaCl (adapted from Cheftel et al., 1985)

Most protein drinks available in the market are high pH (>4.5) beverages (Eckert & Riker, 2005). Soy milk, in particular, is generally prepared at a pH of 7 as the solubility of soy begins to decline at a pH 7 to a minimum at pH 4.5 (Figure 2.9) (US FDS, 2007; Lam et al. 2007). According to Diosady et al, (2007) and Eckert & Riker (2005), soy protein isolates have not been typically used in pH <4.5 beverages because, upon protein dispersion, the protein does not dissolve and instead forms a colloidal dispersion, which results in a cloudy beverage. However, while this prevents its use in clear beverages, it does not prevent its use in cloudy fruit juices. Recently, a soy-fortified fruit beverage known as SojaPlus™ has been developed by MinuteMaid® in Spain and has been received with great success. SojaPlus™ is a multi-fruit juice fortified with soy protein hydrolysates (pH ~4, 5.2 g of protein per 250 ml of drink) which recently won the UNESDA (Union of European Beverages Association) 2007 Beverage Innovation award. Examples of other cloudy acidic beverages available for protein enhancement include fruit/vegetable juices such as orange, lemon, tomato, or carrot juices; lemon and orange juice being, according to CP Kelco (2007), the two most popular fruit juice flavours. Thus, if the PPI can not be modified to attain solubility in a clear liquid, another avenue would be to use it in cloudy acidic beverages, in products similar to SojaPlus™.

Whey and SPI, on the other hand, attain a higher solubility at pH levels lower than 4.5, thus enabling their use for clear acidic beverages without the aid of stabilizers (Chefltel et al., 1985, CPKelco, 2006; Davisco, 2007; Diosady et al., 2007). The solubility of whey in a 0.2 M NaCl solution is depicted in Figure 2.8. Examples of clear acidic drinks that may be fortified by whey or SPI include: carbonated or non-carbonated fruit juices (e.g, lemon, apple, cranberry), carbonated soft drinks (e.g., colas and citrus flavoured drinks), carbonated energy drinks, sports drinks (e.g., Powerade™ and Gatorade™) (Diosady et al., 2007).

2.3 Protein Solubility

According to Charalambous & Doxastakis (1989), protein solubility is defined as the fraction of the total protein mass in a sample that is dissolved or enters into colloidal dispersion under specific conditions and does not sediment under moderate centrifugal forces. For a protein to become soluble within a solvent, the protein molecules must become separated from each other, displacing solvent molecules in the process, so as to disperse themselves within the solvent and maximize their interaction with the solvent. Therefore, protein molecules that are soluble are those which have a high degree of interaction with their solvent (e.g., through hydrogen-bond, dipole-dipole, and ionic interactions) (Cheftel et al., 1985); protein solubility is intimately related to protein conformation which determines the distribution of hydrophobic and hydrophilic groups on its surface and its solvation (Wang & Ben-Naim, 1997). A protein’s conformation, and thus its solubility, in water is affected by various factors, the most significant being: pH, ionic strength, and temperature (Buzágh, 1937; Cheftel et al., 1985; Charalambous & Doxastakis, 1989; Fennema, 1982; Golovanov et al., 2004).

Proteins can be categorized based on their solubility and characteristics. For example, albumins are proteins that have very low molecular weights and are soluble over a wide range of pH values (Balke, 2006). An example of an albumin is whey protein which is comprised of ß-lactalbumin and α- lactalbumin (CP Kelco, 2006). Yellow mustard soluble protein isolate is most likely composed mainly of albumins. Other types of proteins are globulins: these are soluble in salt solutions and in weakly acidic or basic solution environments (Balke, 2006). It is possible that the PPI is largely composed of globulins as its solubility is markedly altered with pH and ionic strength (as is shown in the later sections of this report). The major components of soy soluble protein isolate, which displays solubility characteristics close to that of the PPI, are globulins.

2.3.1 Protein Overview

The conformation, or 3-D structure, of a protein is a result of four levels of structural organization: primary, secondary, tertiary, and quaternary. The primary structure of the protein, the basic protein structure, is the linear sequence of its building blocks, the amino acids. This sequence is coded in the DNA and then transcribed into messenger RNA, which forms a template onto which a large polypeptide chain is formed in the ribosomes of a cell. The polypeptide chain is formed by linking together a predetermined sequence of amino acids with peptide bonds formed in a dehydration reaction (Figure 2.9). Once linked into a polypeptide polymer, the individual amino acids are referred to as residues and the linked carbon, nitrogen, and oxygen atoms are referred to as the protein backbone. This linear chain has directionality: the end with the free carboxyl group is called the C- terminus and the end with a free amino group is called the N-terminus. It is believed that this linear sequence of amino acids that is ultimately responsible for the final conformation and, hence, the biological function of the final protein under specified environmental conditions (deMan, 1999; Karp, 2003; Lodish et al., 2003).

Amino Acid 1 Amino Acid 2

Peptide Bond

Figure 2.9 – Formation of a peptide bond

All proteins are formed from 20 base amino acids (Phillips et al., 1994). All amino acids have a similar structure: they contain an α-carbon which is bonded to an amino group (-NH2), a carboxyl group (-COOH), a hydrogen, and a variable side chain (usually represented by R) (Figure 2.9). It is

19 the chemical nature of the amino acid’s side chain or R-group that determines the chemical nature of the amino acid and its interactions with the other amino acid residues in a protein (Phillips et al., 1994).

As the polypeptide chain is produced, the components of the amino acid backbone begin to interact with one another, resulting in hydrogen-bonding between the C=O and N-H groups of the protein backbone. This results in the folding of the amino acid chain forming the protein’s secondary structure. The two predominant secondary structures are the α-helix and the β-sheet. The α-helix structure is characterized by a helical shape with a turn per every 3.6 amino acid residues (Marnoch, 2004). The β-sheet structure is that of pleated sheets plied into a zig-zag shape. The helical structures are stabilized by intramolecular hydrogen bonds while the β-sheet structures are formed by intermolecular hydrogen bonds (Marnoch, 2004). Protein chains that are particularly high in proline, an amino acid with a cyclic structure, will display a disordered secondary structure called the random coil. Proteins do not tend to occur in a complete helical or sheet configuration; only parts of the peptide chains may be helical, with other areas of the chain occurring in a beta or in an unordered configuration (deMan, 1999; Karp, 2003; Lodish et al., 2003; Whitaker & Tannenbaum, 1977).

The tertiary structure of a protein involves the folding of the α-helices, β-sheets and unstructured amino acid chains into a compact unit stabilized by hydrogen bonds, van der Waals forces, disulfide bridges, electrostatic, and hydrophobic interactions (deMan, 1999; Fennema, 1982). Of these, the forces that play the most significant role in maintaining the protein’s tertiary structure are the hydrophobic forces (Fennema, 1982). The folding of a protein into its tertiary structure in an aqueous environment results in most of the polar amino acid residues being located on the surface of the protein and thus being hydrated. The interior of the protein will harbor most of the apolar/hydrophobic side chains and prevent these from being exposed to the aqueous environment (deMan, 1999; Karp, 2003). The final 3-D structure of the protein is one that minimizes the free energy of the compound and it is what makes each type of protein unique (Lodish et al., 2003).

Large proteins (MW > 50,000) may form a quaternary structure, which is the result of several protein subunits interacting and forming a large, complex oligomeric macromolecule (deMan, 1999). These macromolecules may be stabilized by hydrogen bonds, disulfide bridges, and hydrophobic interactions (deMan, 1999; Fennema, 1982). An example of a macromolecule with a quaternary structure is an enzyme, which has several subunits, one or several of which form the active site.

The final conformation or shape, and hence biological function of a protein, is determined by its primary structure as well as the protein’s surrounding environment (e.g., pressure, temperature, pH, ionic strength, solvent type, and presence of other components) (Karp, 2003; Lodish et al., 2003; Phillips et al., 1994). Drastic changes in any of these environmental factors may result in the denaturation of the protein: the unfolding of the protein at the quaternary, tertiary, and in some cases, secondary levels (Lodish et al., 2004). The more severe the denaturation, the more are the structural levels that are affected by unfolding. Changing the 3-D structure of a protein (e.g., through partial denaturation, hydrolysis, or cross-linking), is beneficial in certain cases, such as in the processing of food protein. By intentionally altering the protein’s conformation, its chemical properties, and thus functionality, can be manipulated (e.g., to alter solubility, emulsifying, or foaming characteristics).

2.3.2 Factors Affecting Protein Solubility

Influence of pH

The pH affects the charge and electrostatic balance between protein molecules and between the protein and the solvent. At pH values higher than the isoelectric point (in the basic pH range), proteins carry a net negative surface charge; at pH values lower than the pI (in the acidic pH range), proteins carry a net positive surface charge. This results in an increase in protein solubility as the protein surface charge enables the protein to interact with the neighboring water molecules and protein chains carrying electrical charges of the same sign will repel each other and expand or dissociate, thus promoting dispersibility (Buzágh, 1937; Charalambous & Doxastakis, 1989; Cheftel et al., 1985). Near the isoelectric point, the net surface charge of the protein approaches zero and solubility reaches a minimum (Buzágh, 1937; Charalambous & Doxastakis, 1989). At the pI, the protein’s interaction with the water is minimal and the attractive forces of the proteins dominate resulting in the aggregation and precipitation of the protein molecules. Precipitation is enhanced when the bulk densities of the aggregates differ significantly from the density of the solvent and when the diameter of the aggregates is large (Cheftel et al., 1985; Dixon & Webb, 1979).

)

% (

Protein Solubilit

Figure 2.10 – A typical protein isoelectric curve

The solubility of a protein can be plotted as a function of the pH (as shown in Figure 2.10) usually resulting in a V- or U-shaped curve (Cheftel et al., 1985). The curve’s solubility minimum corresponds to the isoelectric point. Protein solubility is generally greater in the alkaline pH range than the acidic range. The reason for the higher solubility observed at higher pH values is that the number of negatively charged amino acid residues in the alkaline pH range is greater than the number of positively charged amino acid residues in the acidic pH range (Cheftel et al., 1985; Fennema, 1993). The greater solubility that results at basic pH values explains why protein extraction is generally performed in pH ranges of 10 to 12. At pH extremes, however, proteins will denature leading to agglomeration and a marked loss of solubility (CP Kelco, 2004).

According to Diosady (2007), the isoelectric point of PPI occurs between pH 4 to 6, depending on the PPI production conditions and yellow mustard variety used. Mustard protein does not consist of one type of protein but is instead a multi-protein complex comprising an array of different proteins, each with its own pI. For this reason, only a portion of the mustard protein is precipitated in this pH range resulting in the separation of the PPI from the protein fraction that remains soluble at that pH range, the SPI. Yellow mustard protein SPI is unique in that it enjoys an exceptionally high solubility in the acidic pH range of 2 to 5 (90 to 98%) whereas most other protein isolates do not. Examples of other pI points for precipitated protein of other Brassica oilseeds are ~3.5 for canola seeds, ~5 for Chinese rapeseeds, and ~6.5 for Estonian rapeseed (Diosady et al., 2007). According to Cheftel et al. (1985), causing a protein to aggregate and precipitate by reducing the solution pH to the pI, if done at low temperatures, will not cause irreversible unfolding of the protein and should not affect the functionality of the protein thereafter. This research project seeks to enhance the solubility of the PPI

22 in the acidic pH range of 2 to 4.5, which includes its pI range. As is shown in the results section of this thesis, the solubility of unmodified yellow mustard PPI in this range ranges from ~5% at pH 4.5 to ~38% at pH 2.

Influence of Temperature

At constant pH and ionic strength, protein solubility increases with temperature from 0oC to around 50oC. At temperatures higher than 40 or 50oC, the amount of molecular motion experienced by the protein is enough to disrupt its secondary and tertiary structure (Cheftel et al., 1985). This causes the unraveling of the native protein or denaturation (Dixon & Webb, 1979). Denaturation tends to be followed by aggregation causing the protein to lose its solubility and to experience a marked alteration of its functional properties (Diosady et al., 2007; Monarch, 2004), but does not affect the nutritional quality of a protein. According to Cheftel et al. (1985), every subsequent 10oC rise in temperature results in a protein denaturation rate of about 600 times greater. The extent of aggregation is increased if the protein is subjected to the heat treatment at its isoelectric point (Cheftel et al., 1985; Savolainen, 2004).

Influence of Ionic Strength

The solubility of proteins can be altered by the ionic strength of the solvent. Ionic strength, µ, is defined by:

2 µ = (½) (CiZi )

where C is the concentration of charged solutes and Z is valence (Cheftel et al., 1985). The ionic interaction between the protein’s surface, water, and solvated ions of solutes can be increased by means of several treatments: by forming a protective colloid around the protein’s surface with a polyelectrolyte, adding charged solutes to the solution, and by modifying the protein molecule so as to improve its hydrophilicity. These treatments are explained in greater detail in the section below.

2.3.3 Methods for Enhancing Protein Solubility

2.3.3.1 Protein Modification

A protein’s structure can be modified to improve its interaction with the surrounding solvent molecules, thus improving its solubility. This can be done through enzymatic hydrolysis, by cleaving proteins into smaller, highly charged molecules with a greater surface area available for interaction with the solvent. Another strategy for improving solubility through protein modification is to reduce the concentration of exposed hydrophobic groups by cross-linking areas of the protein’s surface together. The latter method, however, will also result in the cross-linking of proteins molecules together which can reduce solubility by increasing the molecular weight of the protein. These two methods are explained in greater detail below.

Enzymatic Hydrolysis

Proteases are a group of enzymes that are capable of hydrolyzing or cleaving peptide bonds (Figure 2.11). These enzymes have been used in the food industry and in research to alter the functionally of food proteins: they have been used to tenderize meat, improve cheese texture, and produce soluble hydrolyzates of animal and vegetable proteins and heat or solvent-denatured proteins (Cheftel et al., 1985; Diosady, 2007; O’Meara & Munro, 1984; Walsh et al., 2003). Although these protein hydrolases generally improve solubility, in special cases, limited specific proteolysis can cause protein coagulation (e.g., as is the case when chymosin, also known as rennin, is used to treat casein to cause it to curdle or when thrombin attacks fibrinogen to produce a blood clot) (Cheftel et al., 1985).

Figure 2.11 – Protease hydrolysis site

According to Cheftel et al. (1985), after subjecting soy protein to a degree of hydrolysis of 3 to 8%, its solubility can be expected to increase from about 10% (in the acidic pH range) to over 50%. Figure

2.12 displays the results from a study conducted by Walsh et al. in 2003 in which Alcalase (a serine protease) was used to hydrolyze a commercial soy soluble protein isolate at hydrolysis levels from 0.5% to 2%. The results are displayed in terms of nitrogen solubility (a measure of protein solubility) versus solution pH. In comparison to the solubility of the control, the solubility of Alcalase-treated soy protein isolates exhibit a significantly greater solubility in the acidic pH range.

100

70 Control 0.5% Hydrolysis 60 1 % Hydrolysis 1.5 % Hydrolysis 50 2 % Hydrolysis

40 30

20 Nitrogen Solubility Index (%)

0 2345678 pH

Figure 2.12 – Effect of pH on the nitrogen solubility profiles of unmodified (control) and hydrolyzed soy protein isolate. Hydrolysis was allowed to proceed to a defined degree of hydrolysis (DH) value of 0.5, 1.0, 1.5, and 2.0 %. Data points are means ± standard deviations of duplicate analyses (adapted from Walsh et al., 2003).

After partial hydrolysis, the solubility profile of a protein is generally improved over the entire pH range, including the isoelectric pH of the multi-protein complex, because large aggregates do not form as the resulting proteins are more hydrophilic and smaller, more solvated polypeptide units (Dixon & Webb, 1979; Cheftel et al., 1985). The particles are more hydrophilic because hydrophilic/charged groups become exposed during hydrolysis a low pH thus improving both interaction with the water and electrostatic repulsion between protein particles (Walsh et al, 2003).

These partial protein hydrolyzates also remain in solution at higher temperatures when compared to the native protein and, as such, are ideal for use in acid protein-enriched beverages that are heat- processed (Cheftel et al., 1985). A drawback of proteolysis is that, when performed extensively, it may result in the release of bitter-tasting hydrophobic peptides (e.g., those with leucine or phenylalanine terminal residues) (Walsh et al., 2003). On the other hand, hydrolyzates are advantageous for consumers with impaired digestive functions (Cheftel et al., 1985; CP Kelco, 2007).

The progress of the hydrolysis can be assessed by calculating the degree of hydrolysis (% DH) by means of a potentiometric titration of the protons released during protein hydrolysis at a constant pH near the 7 to 8 pH range; in this range, each cleaved peptide bond results in one non-ionized α-amino group and one ionized carboxyl group (Adler-Nissen, 1986; Dixon & Webb, 1979; Walsh et al., 2003; Whitaker & Tannenbaum, 1977). The following formula from Cheftel et al. can then be used to calculate the degrees of hydrolysis incurred by a protein:

Where, α is the average dissociation coefficient of alpha-amino groups:

(The average pK of α amino groups varies from 7.7 at 25oC to 6.9 at 60oC)

According to Whitaker & Tannenbaum (1977), the number of peptide bonds per gram of protein can be estimated from the amino acid composition of the protein.

As the protease hydrolyses the protein, a progress curve such as the one shown in Figure 2.13 can be expected. At time zero, the enzyme activity is at its maximum and, gradually, this activity is diminished. There are several causes which may be responsible for this progressive reduction in enzyme activity: the enzyme may undergo inactivation at the pH or temperature of the reaction, the

26 products of the reaction may inhibit the enzyme’s activity, or the degree of substrate saturation of the enzyme may fall over time as the concentration of product increases (Dixon & Webb, 1979).

transformed

Amount of substrate

Time

Figure 2.13 – Typical enzyme reaction progress curve

Based on information obtained from O’Meara and Munro (1984), Alcalase® (Substilisin Carlsberg, E.C. 3.4.21.14) was chosen as the protease of choice for this project as it appears to be the most promising enzyme for improving the solubility of mustard PPI on a commercial-scale. Alcalase is a serine endopeptidase (a serine protease is one that contains serine in its active centre) which cleaves on the carboxyl side of hydrophobic amino acids (Sigma-Aldrich, 2007; Walsh et al., 2003).

It is readily available in bulk quantities and at a low cost in comparison with other enzymes. Moreover, this protease is commonly used in industry to modify the functional properties of an array of protein substrates including soybean protein isolates, dairy protein, chickpea protein, and fish protein (Walsh et al., 2003). When compared to other relatively low-cost commercial enzymes (such as Neutrase 0.5L, bromelain, and papain), Alcalase treatments appear to be the most effective in solubilizing protein (O’Meara & Munro, 1984). In comparison to higher cost enzymes, two enzymes are more effective than Alcalase, namely, Chymotrypsin and Trypsin. However, they are only marginally more effective than is Alcalase. Another advantage that Alcalase has over other enzymes is that its optimum reaction temperature and pH (~50 to 60oC, pH 8.5) are less conducive to microbial

27 growth during reactions compared to other enzymes (e.g. papain or bromelain ~ 40-50oC, pH 7.0), thus, making Alcalase a better enzyme choice in the event of process scale-up.

Enzymatic Cross-linking

Walsh et al. (2003) used enzymatic cross-linking to improve the solubility of hydrolyzed protein isolates. The cross-linking enzyme used in this study was transglutaminase (λ-glutamyltransferase, E.C. 2.3.2.13, referred to as Tgase from this point on). Tgase is an enzyme widely distributed in human tissue which acts to maintain the stability of various biological structures by cross-linking certain protein substrates together (Aeschlimann & Paulsson, 1994). In the food industry, Tgase is commonly used in the processing of dairy, seafood, meat, soy, and bakery products, and has been reported to reduce the bitterness of several types of protein hydrolyzates (including milk and gluten hydrolyzates) (Blinkovsky et al., 2002; Watanabe et al., 1992). Other qualities that are claimed to be improved by Tgase are improvement in mouthfeel, aroma, and crust colour (Blinkovsky et al., 2002). Thus, even if Tgase did not improve the solubility of yellow mustard PPI, its use may still be beneficial (so long as it does not reduce PPI solubility) as it may reduce any bitterness produced by the Alcalase hydrolysis as well as any bitterness present in the unmodified PPI. Figure 2.14 below, shows the cross-linking reaction of transglutaminases (Blinkovsky et al, 2002). Tgase modifies proteins by catalyzing the formation of a covalent bond between a λ-carboxyamide (from glutamine) and lysine and forms an ε-(λ-glutamyl) lysine cross-link resulting in the release of ammonia (NH3) (Walsh et al,. 2003; Wartiovaara, 1999).

Figure 2.14 – Transglutaminase cross-linking reaction (adapted from Wartiovaara, 1999)

Walsh et al. hypothesized that Tgase increases the protein’s solubility by improving its interaction with water by increasing the exposure of the protein’s hydrophillic groups to the solution environment under certain conditions. The fact that it increases solubility at all is surprising, since Tgase is a cross- linking enzyme, which by nature increases the molecular mass of proteins by tethering peptide chains together by means of covalent bonds. This is known to cause a decrease in solubility as it induces aggregation (Cheftel et al., 1985; Dixon & Webb, 1979). Moreover, cross-linking the ε-amino groups of lysine residues usually results in decreased protein solubility (Cheftel et al., 1985), and it results in the linking of two polar amino acids (lysine and glutamine), thus eliminating their ability to interact with the solution environment.

Solubility curves have been obtained by Walsh et al. (2003) for unmodified soybean protein isolates alongside the same protein isolates that are treated solely with Tgase and those that are hydrolyzed with Alcalase as well as being treated with Tgase. These curves represent the protein solubility of the protein isolate (expressed in terms of nitrogen solubility) as the pH is varied. When the soy protein isolated is treated only with Tgase, there is a marked improvement in its solubility at pH 3 while at the remaining pH values the solubility of the protein remains either unchanged or decreases. When combined with hydrolysis, however, it appears that Tgase is capable of further improving the solubility of the Alcalase hydrolysates in the acidic pH range. However, its performance is subject to variations in solution pH, the order in which cross-linking and hydrolysis occurred, and on the degree of hydrolysis, and it appears to be difficult to predict. The effect of Tgase treatment on soy solubility seems to result from a complex interplay of a myriad of factors.

2.3.3.2 Solution Environment Modification

Environment modification refers to the addition of salts or other non-protein solutes that affect the solubility of the protein. These compounds affect the electrostatic interactions between the charged proteins and the ions in the environment, thereby altering protein solubility (Charalambous & Doxastakis, 1989). The following sections describe two potential methods for improving protein solubility through solvent modification, namely, “salting in” and the formation of protective colloids.

Salting In Knowing the ionic strength imparted by an ion in a solution is not enough to predict its effect on the solubility of a protein molecule as different ions have very different effects on the protein

29 conformation and thus its solubility: some ions cause protein precipitation (“salting out”) while others improve the solubility of a protein (“salting in”) (Mizubiti et al., 2000). The theory behind these different effects is still unclear, but there is in existence an empirical series which classifies several ions based on their ability to “salt in” or “salt out” proteins: the Hofmeister series. According to this series, NaCl can be expected to “salt in” a protein; ammonium sulfate on the other hand is well known for its ability to precipitate proteins (Iyer & Przybycien, 1994). Ions that “salt in” protein are thought to do so by interacting with the charged groups on the surface of proteins and decreasing the electrostatic attraction between opposite charges of neighboring protein chains or molecules. Additionally, when these solvated ions interact with the protein, they serve to increase protein solvation as well (Cheftel et al., 1985).

) %

(

Protein Solubilit

Salt Concentration

Figure 2.15 – Protein “salting in” and “salting out” as a function of salt concentration

Ions that do induce “salting in”, however, only do so at certain levels of salt concentration (see Figure 2.15) (Snow, 2007). In general, the concentrations at which neutral salts may increase the solubility of proteins is in the order of 0.5 to 1M. At concentrations greater than 1M, the protein solubility decreases and may result in precipitation. This “salting out” arises from the competition between the protein and salt ions for water molecules for solvation. At high salt concentrations, most of the water molecules will preferentially bind to the salts, leaving the protein without enough water molecules for its own solvation. This causes protein-protein interactions to become stronger than the solvent-protein interactions, which leads to protein aggregation and, in some cases, precipitation. This aggregation is usually reversible as it does not cause extensive unfolding of the protein, especially if the precipitation occurs at low temperatures (Cheftel et al.,1985; Ellinger, 1972).

For this project, three salts were chosen for experimentation on their effects on the PPI solubility: sodium chloride (NaCl), sodium tripolyphosphate (STPP; Na5P3O10), and sodium hexametaphosphate

(SHMP; (NaPO3)6). These were chosen because they have been shown in the literature to improve solubility, are recognized as safe for human consumption within specified limits, and they are commonly used in various food processes. According to Cheftel et al. (1985), Ellinger (1972), Jungwirth (2007), and Pericin et al. (2008), the Na+ ions present in these compounds increases protein solubility and solvation at the isoelectric pH. STPP and SHMP, at a glance, appear to be superior to NaCl as they contain a greater amount of Na+ ions per molecule, however, their polyphosphates have been reported by Ellinger (1972) and Hidalgo et al. (1972) to cause the precipitation of whey and casein proteins at pH values of 3 and 2.5, respectively. STPP and SHMP, however, are common food preservatives and as such are likely to be used in the preservation of acidic beverages where the alternative of hot-filling is not possible or recommended (Eckert & Riker, 2005). Therefore, it would be ideal to test the possible use of STPP and SHMP as potential solubility enhancers as well as preservatives, even if the expected chances of success are low. STPP and SHMP have been reported in the literature to enhance protein dispersion (although generally in the higher pH ranges), specifically meat protein, by improving water absorption, swelling and surface properties (Cheftel et al., 1985; Ellinger, 1972; Hanson, 1974; Knipe, 2004; Savic, 1985). Hence, of the three salts chosen, NaCl is the most promising as a solubility enhancer. SHMP and STPP are less promising due to their polyphosphate component.

One drawback to the use salts in improving solubility is that the addition of these will impart an unpleasant flavour in the final solution which may prevent it from being processed into a commercial beverage. According to Diosady (2006), 0.5 wt% is the threshold concentration below which the added salt will generally not overpower the flavour of the drink. This is also the threshold at which charged ions begin to have a solubility enhancing effect (Figure 2.15). Another drawback to their use is the perceived negative health benefits that consumers associate with chemical preservatives or additives (Eckert & Riker, 2005).

Protective Colloid Formation Another approach to solvent environment modification is to form a protective colloid around the surface of the PPI resulting in a stable protein dispersion. According to Buzágh (1937), a protective colloid occurs when lyophilic particles become adsorbed in a thick layer onto the surface of other

31 particles, the “protected particles”. This results in the “protected particles” acquiring the properties of the lyophilic colloidal solution. The PPI particles can thus be “protected” by mixing these in solution with a lyophilic stabilizing agent such as a gum (Lam et al., 2007). Examples of gums that have been used in the food industry to stabilize protein in low pH ready-to-drink beverages are pectins and alginates (Eckert & Riker, 2005). Of the two, however, pectin is more commonly used (CP Kelco, 2007; Lam et al., 2007). According to Carle (2005), 0.5 to 3 wt% of soy protein can be dispersed in fruit juices with the aid of pectin; without pectin, soy protein tends to precipitate when mixed with fruit juice because of the low pH of the beverage. Using pectin in these solutions results in a homogenous dispersion which remains stable over long periods of time, and which is resistant to heat- treatments. In addition, pectin also contributes positively to beverage flavour, and it can be used to control texture, viscosity, and mouthfeel in the final product (CP Kelco, 2004; Lam et al., 2007). The one main drawback to using pectin is that it will give opacity (Jurlina, 2007; Lam et al., 2007).

Pectin is a polysaccharide that is prepared from the peels of citrus fruits (these usually being lemon and orange) and apples (CP Kelco, 2006a; Lam et al., 2007). Similarly to proteins, its functionality depends on its overall charge and its charge distribution on its chain. The overall charge of the pectin is negative in solutions of pH greater than 3.5. This is the reason pectin may only be used to disperse soy protein or mustard PPI at pH values in between ~3.8 to 4.2. In this pH range, the pectin will be negatively charged and the soy protein or mustard PPI will be very weakly positively charged (as this is very close to the isoelectric point). This results in the attraction of the pectin to the protein, binding, and formation of a protective colloid on the protein’s surface (CP Kelco, 2004; Lam et al., 2007). The negatively charged protein surface will now repel neighbouring protein particles, resulting in a stable dispersion. Conversely, outside of the 3.8 to 4.2 pH range, pectin may enhance protein precipitation. According to Jurlina (2007), pectin is the most widely used stabilizer for protein-enhanced acidic beverages because it is less negatively charged than most other gums; the stronger charge present in these other gums results in the sedimentation of the protein.

The stability of a pectin-protein solution may be evaluated by determining the particle size distribution. The lower the d(0.5) (the size at which 50% of the sample is smaller than that cutoff), the greater the tendency to stability. As an example, a d(0.5) of 12.7 corresponds to that of an unstable drink, while a drink with a d(0.5) of 1.2 is considered to be stable. According to CP Kelco (2004), commercial soy-fruit juice drinks may also be prepared with soy protein hydrolysates, however, the ideal conditions of pectin use in this case may be different than the ideal conditions specified for non- hydrolyzed protein (since the protein surface charge characteristics are different).

2.4 Project Objectives

The objective of this project was to investigate methods for maximizing the solubility of yellow mustard precipitated protein isolate (PPI) in aqueous solutions in the pH range of 2 to 4.5. In this report, several solubility enhancing treatments are described: enzymatic hydrolysis, enzymatic cross- linking, and the addition of solutes for “salting in” and of stabilizing agents to form a protective colloid. Each of these treatments was tested on sample PPI obtained from a pilot-scale test in the RTech Laboartories (MN, USA). Each of these treatments was tested with the following agents:

• Enzymatic hydrolysis: Alcalase® • Enzymatic cross-linking: transglutaminase • Salting in: sodium chloride, sodium tripolyphosphate, and sodium hexametaphosphate • Protective colloid formation: pectin

Based on information obtained from the literature, it was hypothesized that the hydrolysis treatment would result in a moderate increase in solubility (of ~40% or greater) even at low levels of hydrolysis (3 to 8% degrees of hydrolysis). This method was also expected to improve solubility within the entire pH range of interest (2 to 4.5) and including the isoeletric pH of the multi-protein complex. The effect of Tgase treatment on the PPI solubility was difficult to predict as its effect appears to result from a complex interplay of a myriad of factors including the order in which cross-linking and hydrolysis takes place, the pH of the solution, and the type of protein subjected to treatment. The salting in treatments were expected to improve solubility mainly near the isoelectric pH, and their effect was expected to be diminished as the pH deviates from the pI. Of the three salts proposed for testing, NaCl appeared to be the most promising. The expected chances of success in using STPP and SHMP were low; however, they were tested nonetheless to determine their effects on protein solubility as they are common preservatives which could be proposed for use in a low-pH soft drink. The results of the last method, the use of pectin to form protective colloids, were difficult to predict but appeared to have the potential of achieving almost complete dissolution after the optimization of several parameters: solution pH, protein concentration, and pectin concentration. The effectiveness of each of these treatments was determined in terms of their ability to improve protein solubility (measured in terms of nitrogen solubility) compared to the baseline solubility curve of PPI.

3. EXPERIMENTAL METHODS

3.1 Materials and Equipment

3.1.1 Test Materials

Protein Products • Yellow Mustard PPI, SPI, and MR: produced in a pilot-scale test in the RTech laboratories (St. Paul, MN, USA). In this test, the centrifugation step did not result in a clear supernatant. The centrifugation force was not set high enough and, as a result, the meal residue was not completely separated from the SPI and PPI. Thus, it was expected that the resulting RTech PPI and SPI powders would have a lower protein content and slightly altered functional properties. • Yellow Mustard PPI and SPI: produced in a pilot-scale test at the Food Protein Research and Development Centre at the Texas A&M University (College Station, Texas, USA). • Soy Protein Isolate (Supro 500E): Solae™, St. Louis, MO, USA

Solvents The chart below lists the solvents used for the experiments.

Table 3.1 – Solvents used for experiments

Solvent Source Distilled water Wallberg building tap

Cola Pepsi, bottled

Carbonated Water San Benedetto, bottled

Orange Juice (100% pure from concentrate) Tropicana, boxed Cranberry Juice (Cranberry Cocktail) President’s Choice, boxed Apple Juice Minute Maid, boxed

Peach-Flavoured Carbonated Water Life Brand, bottled

33 34

Enzymes • Alcalase® (E.C. 3.4.21.14; Subtilisin Carlsberg (also known as Subtilisin A); a serine protease from Bacillus licheniformis; enzyme activity is equal to 2.67 Anson Units per gram of preparation; Product No. P4860): Sigma-Aldrich™, St. Louis, MO, USA • Transglutaminase (E.C. 2.3.2.13; Activa-TI powder contains calcium independent transglutaminase and maltodextrin; the powder contains 100 units of enzyme activity per gram of powder): Ajinomoto Food Ingredients LLC, Chicago, IL, USA.

Salts/Stabilizing Agents

• Sodium Hexametaphosphate ((NaPO3)6, analytical grade): BDH Chemicals Ltd., Poole, England • Sodium Chloride (NaCl, analytical grade): EMD™, Gibbstown, NJ, USA

• Sodium Tripolyphosphate (STPP; Na5P3O10, analytical grade): Sigma-Aldrich™, St. Louis, MO, USA • Pectin (GENU® Pectin Type YM-100-L; sample contains pectin standardized with sucrose): CP Kelco, San Diego, CA, USA

3.1.2 Reagents and Materials

• Nitrogen Solublity Index (NSI) and Solubility Enhancing Treatments o AntiFoam B Emulsion (Cat. #R0664436-70; contains octamethylcyclotetrasiloxane and hydrogenated silica gel): EMD™, Gibbstown, NJ, USA o Liquid nitrogen: U of T Medstore house brand, Toronto, Canada o Phosphoric acid (analytical grade): Caledon Laboratories Ltd., Georgetown, ON, Canada o Sodium hydroxide (N/5, analytical grade): Fisher Scientific, Nepean, ON, Canada

• Kjeldahl Method

o Boric acid (H3BO3, powder, analytical grade): BDH Chemicals Ltd., Poole, England o Glass wool (low in lead): BDH Chemicals Ltd., Poole, England

o Kelmate® MT-37 Kjedahl digestion mixture (3.5g K2SO4/0.175g HgO per tablet, Product No. KX0015A-1): EMD™, Gibbstown, NJ, USA o N-Point Indicator (Product No. NX0847/3; contains methanol, water, bromocresol green, and methyl red): EMD™, Gibbstown, NJ, USA

o Sodium hydroxide (50 w/w%, analytical grade): VWR™, West Chester, PA, USA o Sodium thiosulfate (analytical grade): Fisher Scientific, Fair Lawn, NJ, USA o Stock solutions (preparation instructions available in Appendix A)

Boric acid 4 w/v% (H3BO3) . Sodium thiosulfate 8 w/v% (Na2S2O3 5H2O) Sodium hydroxide 32 w/w% (NaOH) o Sulfuric acid (0.1000 N, analytical grade): VWR™, West Chester, PA, USA o Sulfuric acid (concentrated, 95-98%): EMD™, Gibbstown, NJ, USA

• SDS Polyacrylamide Gel Electrophoresis Analytical grade, VWR®, West Chester, PA, USA o Acrylamide o Ammonium persulfate o Beta-mercaptoethanol o Bis o Bromophenol blue o Butanol o Coomassie Brilliant Blue R-250 o Glacial acetic acid o Glycerol o Glycine o Methanol o Sodium dodecyl sulfate o TEMED o Tris base

• Particle Sizing o Nanopure water: Wallberg fourth floor laboratory tap, U of T, Toronto, Canada

3.1.3 Equipment

• Nitrogen Solublity Index (NSI) and Solubility Enhancing Treatments o Centrifuge (Beckman Coulter™ - Avanti™ Centrifuge J-20 XP): Beckman Instruments, Fullerton, CA, USA o Emulsifier (Model L2R): Silverson Machines Ltd., Waterside, England o Freeze dryer (Freezeone 12 Plus, Catalog No. 7960044): Labconco, Kansas City, MO, USA o pH meter (Model 8000): VWR Scientific, West Chester, PA, USA o Mechanical stirrer (Type RZR 50): Caframo, Wiarton, ON, Canada

• Kjeldahl Method o Kjeldahl digestor (Model No. 425): Buchi, Switzerland o Kjeldahl digestion tubes (300mL), holder, manifold and clamps: Buchi, Switzerland o Kjeldahl distillation unit (Model No. K-350): Buchi, Switzerland

• SDS Polyacrylamide Gel Electrophoresis o Electrophoresis station: Bio-Rad Laboratories, Hercules, CA, USA

• Particle Sizing o Beckman Coulter Counter Multisizer 3 3.51: Beckman Instruments, Fullerton, CA, USA

• Viscosity Measurements o Blender (Cat #702): Waring Products Corp, New York, NY, USA o Cannon-Fenske Routine Viscometer: Cannon Instrument Company, PN, USA

• General o Convection oven (Blue M- Constant Temperature Cabinet Model No. 0V-490A-2): Electric Company, Blue Island, IL, USA o Microscale (Model No. 2001 MP2): Sartorius, Germany o Refrigerator (1-4oC) / Freezer (-18oC) o Balance (Mettler PC 4400-DeltaRange®): Mettler Instruments A.G., Zurich, Switzerland

3.2 Experimental Methods

The project consisted of creating a solubility curve for unmodified PPI and comparing this baseline curve to the resulting solubility curves obtained from PPI treated with enzymatic hydrolysis, enzymatic cross-linking, and the addition of charged solutes and stabilizing agents. The solubility of the PPI, both unmodified and modified, was determined by measuring its Nitrogen Solubility Index (NSI; Standard AOCS Official Method Ba 11-65; detailed procedure available in Appendix A1). The standard NSI was used, as opposed to a modified version, to ensure that the results could be compared to the results from other studies that have followed the standard NSI procedure. The following sections describe each of the solubility enhancement treatments used in this project.

3.2.1 Enzymatic Hydrolysis Treatment

The enzyme chosen for the enzymatic hydrolysis treatment for this project was Alcalase (E.C. 232- 752-2). The PPI was treated with this enzyme at various concentrations (0.1 to 0.3 g of Alcalase / 5 g of PPI) and temperatures (50oC and 60oC) and for varying lengths of time (0.5 h to 24 h). The hydrolysis pH was chosen as 8.5, which according to Novo Industri A/S (1978) is deemed to be the optimum pH for Alcalase hydrolysis. Since enzymes are denatured by strong alkalis and acids, the addition of sodium hydroxide and phosphoric acid (for pH control) was done by carefully running a diluted base or acid down the side of the beaker into the solution and mixing immediately with a mechanical stirrer. The formation of froth was also avoided during the experiment by keeping the mechanical stirring speed low and the blades of the stirrer deeply immersed in the solution as froth can also result in enzyme denaturation and, hence, inactivation (Dixon & Webb, 1977). After an enzymatic treatment, enzymes are usually deactivated by means of heat typically in the temperature range of 70 to 100oC, at which point the enzyme’s active site becomes denatured (Dixon & Webb, 1977; Walsh et al., 2003). However, denaturation by means of heat at these temperatures may cause a significant decrease in protein solubility (Cheftel et al., 1985; deMan, 1999). Since enzymes are active only over a very limited pH range, it was decided to instead deactivate the Alcalase protease by dropping the solution pH to 3.5 and cooling the solution in the refrigerator immediately after treatment completion; at this pH and temperature the enzyme is outside of the range of activity (Blinskovsky et al., 2002; Dixon & Webb, 1977; Sigma-Aldrich, 2007). It is important to note that the enzyme at this pH may not have been destroyed and may simply be temporarily inactive (Dixon & Webb, 1977); care was taken to not subject the PPI solution to a pH over 5 again during the NSI measurement as the enzyme may return to activity and confound the final results. After the enzyme deactivation step, the solution was flash frozen with liquid nitrogen and lyophilized over a period of two to three days. Lyophilization was chosen as the drying method to minimize protein denaturation. The dry, treated protein was then manually pulverized with a mortar and pestle and its Nitrogen Solubility Index was determined. Appendix A6 contains a detailed procedure for the enzymatic hydrolysis treatment.

3.2.2 Enzymatic Cross-linking Treatment

The yellow mustard PPI was cross-linked with calcium-independent transglutaminase (E.C. 2.3.2.13). The procedure used for the enzymatic hydrolysis experiment was also used for the cross-linking experiment; the only difference being the optimal reaction pH. According to Ajinomoto Food

Ingredients LLC (2007), the optimum reaction temperature for transglutaminase is between 45 to 55oC and the optimal pH is between 6 and 7 (55oC and pH 6.5 were chosen for the experiment). The enzyme concentration in the PPI solution and the reaction time were varied. The enzyme, which was made available in powdered form, was pre-mixed in warm distilled water to ensure that it was well- mixed prior to its addition to the PPI solution. After treatment, the enzyme was deactivated by decreasing pH and temperature, as was done in Alcalase treatments. According to Ajinomoto Food Ingredients LLC (2007), at these pH and temperature conditions, the action of the enzyme is minimal. After the enzyme deactivation step, the solution was flash frozen with liquid nitrogen and lyophilized over a period of two to three days. The dry treated protein was then manually pulverized with a mortar and pestle and its Nitrogen Solubility Index was determined.

3.2.3 Salt Treatment

The salt treatment consisted of adding a set amount of a salt into the PPI solution at the beginning of the Nitrogen Solubility Index determination procedure (Appendix A1). The salts used in this project were: sodium hexametaphosphate, sodium chloride, and sodium tripolyphosphate.

3.2.4 Stabilizer Treatment

The stabilizing agent used for this project was pectin. The procedure used was virtually the same as the procedure described in subsection 3.2.3. The one difference, however, was that the pectin was first mixed with 200 ml of distilled water, heated to 70oC, mixed with a blender, and then added to the PPI powder (as was recommended by CP Kelco (2004) and Lam et al., (2007)). The NSI was then determined according to standard procedure. While the NSI pH could be altered when used with salts to determine the solubility of the salt-treated PPI under various pH values, CP Kelco (2007) advised that the pectin only be used within the pH range of 3.8 to 4.2; outside of this pH range, the pectin could be expected to precipitate the protein.

3.2.5 Analytical

• Nitrogen Solubility Index (NSI) Determination: AOCS Ba11-65 with minor modifications (detailed procedure presented in Appendix A1) • Kjeldahl Procedure: AOCS Ba4d-90 (detailed procedure presented in Appendix A2)

• SDS Gel Electrophoresis: Detailed procedure presented in Appendix A3 • Particle Size Analysis: Detailed procedure presented in Appendix A4 • Moisture Content: Detailed procedure presented in Appendix A5

4. RESULTS AND DISCUSSION

4.1 PPI Starting Material Analysis

4.1.1 Protein Content

Table 4.1 shows the protein content of Rtech and Texas yellow mustard PPI samples used in this project compared with the values obtained for soy protein isolate (Supro 500E, Solae™). The protein content was determined ‘as is’ via the standard Kjeldahl method (AOCS Official Method Ba 4d-90) using the nitrogen-protein conversion factor of 6.25, the accepted standard. The protein content value for the RTech PPI is a result of six experiments; Texas PPI, of four experiments; and soy, of one. Each experiment consisted of a blank and three replicate samples. The uncertainty values shown are standard deviation values. As can be seen in the table, the standard deviation of the soy protein isolate is notably lower than that for the other samples. The main reason for this was the significantly larger sample mass used for the soy Kjeldahl measurement (~1 g) compared to the amount used for the yellow mustard protein powders (~0.1 g). Later on in this study, the sample masses for all protein weight percent determination tests were increased to several grams which resulted in standard deviations of less than 0.15 in most cases.

Table 4.1 – Protein and moisture contents of Rtech PPI, Texas SPI, and soy protein isolate

Protein Content Moisture Content Protein Content Sample (wt %, As Is) (wt %) (wt %, Dry Basis)

RTech PPI 69.1 ± 2.3 5.7 ± 0.0 73.3 ± 2.4

Texas PPI 79.2 ± 2.9 7.0 ± 0.0 85.2 ± 3.1

Soy Protein Isolate 84.6 ± 0.1 6.4 ± 0.0 90.5 ± 0.4

The amount of moisture present in the protein samples was measured by drying (in triplicate) 10 g of each powder at 110oC for 24 hours, dividing the difference in mass by the original powder weight, and multiplying by 100%.

40 41

After the moisture content was determined, the ‘dry basis’ protein concentration of the powders was calculated by using the following equation:

The uncertainty for the ‘as is’ protein content was calculated by uncertainty propagation using the ‘weakest-link uncertainty rule’. Detailed result tables for the above results are found in Appendices B1 through B6.

The results displayed in Table 4.1, are in close agreement with the unpublished values obtained by Chen in 2007 (Rtech PPI moisture content of 5.7 wt %; Texas PPI ‘as is’ protein content of 78.2 wt%) and Tomac in 2003 (Texas PPI ‘dry basis’ protein content of 84.7 wt%). It is evident in Table 4.1, that the protein content of the RTech and Texas PPI powders are different. The principal cause for this difference is most likely the different processing conditions for each. According to Chen (2007), during RTech PPI pilot-test production, high enough centrifuge speeds were not achieved during the alkaline protein extraction step. This suggests that the PPI produced in the RTech PPI test is a blend of PPI and meal residue (MR), resulting in a lower protein content. According to Diosady et al. (2007), under more standard processing conditions, such as those attained during Texas PPI production, the protein content of the PPI powder generally is higher than 80 wt% on a ‘dry basis’. In this project, RTech PPI was used as the starting material for the majority of the experiments not because of its quality but because it was available for research in the greatest quantity.

4.1.2 Solubility

The baseline solubility curve for the unmodified PPI in water was determined so that it could be used as a point of comparison, or control, for the subsequent solubility enhancement treatments. Figure 4.1 below shows the protein solubility of the RTech PPI represented by means of the standard nitrogen solubility index.

A 5 g sample of PPI powder was mixed with 250 ml of distilled water (125 rpm, 120 min, 30oC, at a constant chosen pH) and was then decanted, centrifuged, and filtered. The final solution contains protein deemed to be soluble according to Charalambous’ definition (1989) of protein solubility. The concentration of the protein for each prepared solution was then determined in triplicate by the standard Kjeldahl method (Appendix B7). Each point on the graph represents an average of three replicates; for each set of triplicates, one blank (distilled water) was also tested. As with all experimental results from this point on, the error bars in the graph represent the standard deviation. In some graphs, however, the error bars are difficult to discern as some of the standard deviation values are quite low.

100

80 70 60 50 (%) 40 30 20 10 Nitrogen Solubility Index Solubility Nitrogen 0 12345678 pH

Figure 4.1 – Unmodified RTech PPI nitrogen solubility as a function of solution pH

The RTech PPI isoelectric curve has a U-shape in agreement with the literature. According to Cheftel et al. (1985), a protein isoelectric curve will be U or V-shaped with the solubility reaching a minimum at the pI. In the case of the RTech PPI curve, the minimum appears to be in between pH 4 and 4.5, which is only slightly lower than the precipitation pH of 4.5 reported in Diosady et al.’s 2007 patented protein isolation process description. This slight difference could have been caused by a slight change in processing conditions during the pilot plant test.

The baseline solubility curve of another PPI sample, Texas PPI, was also determined, using a slightly modified version of the NSI index. This modified NSI differs from the standard version in mixing time (30 min), temperature (25oC), PPI-to-solvent ratio, centrifugation (6,000 rpm x 20 min),

43 filtration after centrifugation (vacuum filtration with Hardened Ashless No. 541 Filter paper). As with the previous curve, each point represents the mean of three sample replicates. Detailed results are available in Appendix B8.

100

90 80 70 60 50 40

Nitrogen Solubility Index (%) Index Solubility Nitrogen 10

0 12345678 pH

Figure 4.2 – Unmodified RTech PPI (represented by crosses) and Texas PPI (represented by circles) nitrogen solubility as a function of solution pH

Even with the different NSI procedures, the solubility curves for both PPI samples appear to be consistent in shape and only slightly different in terms of pI values (the pI value of Texas PPI appears to be in between 4.5 and 5). Whether this difference in pI values is due to the difference in the powders or to the NSI protocol cannot be said conclusively. Another solubility curve was prepared from RTech PPI using the altered NSI method to determine whether the slight difference in curve shift was because of a difference in the PPI powders or the NSI procedure, however, unfortunately, this solubility curve was discarded: not enough sample was used for the Kjeldahl analysis of the final solutions and the results were unreliable.

Yellow mustard PPI enjoys, at most, only a marginally higher solubility than commercial soy soluble protein isolates do in the acidic pH range. The nitrogen solubility of the soy soluble protein isolate used in Walsh et al.’s 2003 tests ranged from ~5% at pH 4 to ~10% at pH 3. While the soy protein isolate tested by Walsh et al. (2003) displays low solubility, other soy protein isolates that are subject

44 to different processing conditions or which contain stabilizing agents such as lecithin are able to attain a higher solubility. The NSI at pH 3 of a soy protein isolate sample from Solae™ (Supro 500E) was measured to be 20.2 ± 0.2% (in duplicate, results available in Appendix B9). In contrast, the average solubility of the PPI powders ranges from ~10% at pH 4 to an average of ~25% at pH 3. The solubility of yellow mustard SPI at this pH is much higher ranging from 90 to 98% (Diosady et al., 2007).

An SDS Polyacrylamide Gel Electrophoresis was prepared with the NSI solutions (at pH 3) of RTech PPI to determine if the soluble particles had a different molecular weight than did the PPI particles that were centrifuged out during the NSI procedure. The gel below shows that the PPI that is deemed as soluble during the NSI, has a lower molecular weight than does the original protein powder or the centrifuge cake. This is in accordance with the literature which states that the lower the molecular weight, the greater the solubility of a protein.

a b c d a

Figure 4.3 – SDS Polyacrylamide gel electrophoresis of baseline RTech PPI: (a) protein ladder (1 lane); (b) PPI protein in solution at pH 3 after centrifugation (2 lanes); (c) PPI (pH 3) centrifuge cake (2 lanes); (d) original RTech PPI powder

4.1.3 Viscosity

According to Cheftel et al. (1985) and CP Kelco (2004) three of the most important factors determining the use of a protein product in a beverage are its solubility at different pHs, its resulting viscosity, and its appearance/mouthfeel. The major aim of this project was to investigate the protein

45 solubility and to devise methods for improving it, however the baseline PPI solution viscosity and the appearance and mouthfeel were also characterized.

To characterize the solution viscosity of protein-enhanced acidic beverages, the viscosity of the 1% SPI and PPI mixed into various commercial beverages was measured to determine if the addition of the protein powder would change the final beverage viscosity and, hence, affect its acceptance as a protein enhanced beverage. To determine the final solution viscosity 1% SPI and PPI solutions were prepared by blending the powder into each of the chosen commercial beverages for 30 seconds (on the “low-speed blend” setting of a Waring blender); the control solutions (with no PPI or SPI addition) were also blended for 30 seconds so as to keep SPI or PPI addition as the sole changing variable in the experiment. The viscosity was then measured in centipoises using a Routine Cannon- Fenske viscometer. The results of the viscosity of 1% PPI and SPI enhanced carbonated water, water, apple juice, cola, cranberry juice, orange juice and flavoured carbonated water are displayed in Figure 4.4. Each bar in the graph represents a mean of 25 to 30 viscosity measurements, all of which took place at room temperature. The viscosity of 1% MR enhanced beverages could not be measured as the MR particles were large enough to result in the clogging of the viscometer. The results for this experiment are found in Appendices B10 through to B16.

Figure 4.4 – Viscosity (in centipoises) of 1% PPI or SPI enhanced commercial beverages

Figure 4.4 shows that the addition of SPI or PPI does not to result in large changes in solution viscosity. In the case of orange juice, however, a small increase in viscosity is observed. Although these measurements were made with a viscometer that has an ideal measuring range of 3 to 15 cP, the accuracy of these results should not be expected to be severely compromised. To determine the expected accuracy of the measurements with this instrument, the viscosity of water was measured prior to beginning the experiment: 0.967 ± 0.01 cP (the result of 28 measurements). This value is comparable to the value available in the literature for the viscosity of pure water at room temperature: 0.89 cP. Although the measurements conducted were out of the ideal range of the instrument, they may be regarded as only slightly imprecise and are useful in terms of comparison between solutions.

As expected, as the concentration of protein was increased, the viscosity of the solutions increased as well. Figure 4.5 depicts the viscosity of water enhanced with PPI at various weight percentages. Up to a protein weight concentration of 3% (the approximate protein content of milk), the viscosity can be expected to range between the viscosity of water to that of orange juice.

3.5

3.0

2.5

2.0

1.5 Viscosity (cP) 1.0

0.5 0.0 012345

wt % PPI

Figure 4.5 – Viscosity (in centipoises) of various concentrations of PPI enhanced water

Figure 4.6 depicts the viscosity of a 1% PPI solution after several 30-second blending sessions. Increasing the amount of blending increases the solution viscosity slightly, which is to be expected as each 30-second blending session increases mixing and this, in turn, improves the protein’s interaction with the solution environment.

3.0

2.5

2.0 y = 0.0368x + 0.973 R2 = 0.8763

1.5

Viscosity (cP)

1.0

0.5 0123456 Number of Times Blended

Figure 4.6 – Viscosity (in centipoises) of a 1% PPI water solution after several blending sessions

4.1.4 Organoleptic Qualities

Yellow mustard precipitated protein isolate (Figure 4.7) is a light brown powder that is bland in taste. Its odour is faint and is similar to that of wheat flour. According to a soy protein expert, the taste of the mustard PPI is bland enough to be incorporated into commercial products (Nummelin, 2007).

Figure 4.7 – Yellow mustard precipitated protein isolate (PPI) powder

Figure 4.8 – 1% aqueous protein solutions in water. From left to right: PPI, MR, SPI, soy protein isolate

Figure 4.8 shows 1% PPI, MR, and SPI mustard solutions and a 1% soy protein isolate solution. The yellow mustard protein products are darker in colour than the bone white soy solution. While its darker colour may present itself as a hindrance to commercial acceptability (as the desired colour for protein isolates is white), its colour may also be beneficial for use in brown coloured beverages such as chocolate-flavoured beverages. Research is being conducted in the U of T Food Laboratory on methods for lightening the colour of the protein products.

Figure 4.9 – Sedimentation after 2 hours. From left to right: 1% PPI, MR, SPI, and soy aqueous solutions

Figures 4.9 and 4.10 depict the protein solutions from Figure 4.8 after two hours and two weeks of quiescence, respectively. Interestingly, the PPI, SPI, and soy solutions experience sedimentation, while the MR solution does not sediment even after one week of storage. This is to be expected as the SPI, PPI, and soy protein isolates are powders high in protein whereas the MR contains a smaller amount of protein and a high quantity of mucilage, a well-known emulsifying agent.

Figure 4.10 – Sedimentation after 2 weeks. From left to right: 1% MR, soy, SPI, and PPI aqueous solutions

Another general observation made is that the more PPI or SPI is added to a solution or beverage, the greater the amount of foam produced upon agitation. After agitation, a layer of foam of up to 1 cm was observed on the surface of the protein enhanced solution. This is expected as protein is a bio- surfactant. After quiescence, the foam disappears completely. The protein powder’s ability to form foam could be seen as an advantage for certain food products, but a hindrance for other applications. In any case, foam formation can easily be avoided by using an antifoaming agent.

Figure 4.11 – Left: 1% PPI aqueous solution in repose. Right: 3% PPI aqueous solution after agitation

Upon addition of the SPI and PPI to commercial beverages, the general observation was that the powder resulted in an opaque unstable dispersion. Some of the dispersions had a rather pleasant physical appearance such as the 1% PPI solution in cranberry juice. After agitation, the solution resembles that of a strawberry milkshake (Figure 4.12).

Figure 4.12 – 1% PPI in cranberry juice after agitation

All these solutions also exhibited a grainy mouthfeel. According to CP Kelco (2006), proteins have a sandy mouthfeel when the protein is suspended and not dissolved. This is in accordance with the solubility results obtained from Figure 4.2 as 70% to 90% of the PPI added to these solutions is not dissolved and is easily removed upon centrifugation. According to Nummelin (2007), a grainy mouthfeel can be improved by reducing the particle size of the protein powder. The mesh size of a typical commercial soy protein powder is considerably smaller than that of mustard RTech PPI or SPI (Nummelin, 2007). Reducing the powder mesh size will increase the surface area of the protein powder exposed to the solution thus improving solubility, but this may also result in a decreased wettability.

Figure 4.13 – Left to right: cranberry juice, ~0.25 wt% PPI in cranberry juice (after centrifugation/filtration), ~0.25 wt% PPI in cola (after centrifugation/filtration), cola

Once the insoluble protein is separated from the dissolved protein (by means of centrifugation and filtration), the resulting solutions range in appearance from very clear to cloudy (but not opaque) and have a significantly less perceptible graininess in the mouthfeel. It would be expected that greater protein concentration in these solutions would result in cloudier final solutions. Figure 4.13 shows ~0.25 wt % PPI in cola and cranberry juice. These protein enhanced solutions and control solution differ slightly in colour, smell, taste, and mouthfeel. A possible explanation for the difference in colour between the protein-enhanced solutions and the control solutions could be that the protein binds to colour pigments in the drink during the mixing step leading to a portion of the pigments being separated from the drink along with the insoluble protein during centrifugation. This would also explain why the centrifuge cake is coloured pink when protein enhanced cranberry juice is prepared and dark brown in the case of protein enhanced cola. According to a soy protein expert, the difference in these organoleptic qualities is minimal enough to be masked (Nummelin, 2007). The solutions in Figure 4.13 were stored in the refrigerator to test their stability. The soluble protein remained in solution for over one week, and all solutions remained unspoiled for over a month. Not surprisingly, the protein-enhanced solutions spoiled before the control solutions showed signs of spoilage.

4.2 Enhancing PPI Solubility: Protein Modification

4.2.1 Enzymatic Hydrolysis

Alcalase® was chosen as the enzyme of choice for the enzymatic hydrolysis experiments for reasons explained in the literature review (section 2.3.3.1). According to the manufacturer, the optimum temperature and pH for Alcalase are 50-60oC and 8.5. For these experiments, a reaction temperature range of 50-55oC was chosen as per O’Meara & Munro’s recommendations (1984) who attained the highest solubilities at this temperature range. A higher reaction temperature will lead to a higher initial reaction rate but will also result in faster enzyme deactivation. According to Blinkovsky et al. (2002), the concentration of endopeptidases conventionally used in industry ranges from 0.001 to 0.08 Anson Units (AU) per g of system protein (this range is equivalent to 0.004 AU to 0.3 AU per 5 g of PPI). The majority of the experiments in this section were conducted at an enzyme concentration of 0.2 g of enzyme per 5 g of PPI sample (0.5 AU per 5 g of PPI) as it represents the maximum amount of Alcalase that would be acceptable for commercial use (slightly above the maximum amount

52 commonly used). The NSI pH for the first experiment was chosen to be 3 as most acidic drinks are in the pH range between 2.5 and 3.5 (USFDA, 2007), and pH 3 lies in the middle of this range.

The goal of the first experiment conducted was to establish the reaction time necessary to attain the maximum solubility via Alcalase hydrolysis. Each point in the curve represents one experiment consisting of three replicates and one blank. The results are displayed in Figure 4.14 (the result table is presented in Appendix B24).

100 90

70 Degrees of Hydrolysis - Exp. 1 60 Degrees of Hydrolysis - Exp. 2

50 Degress of Hydrolysis - Exp. 3 (%) Nitrogen Solubility 40 30

20 10

0 0 5 10 15 20 Alcalase Treatment Time (hours)

Figure 4.14 – Nitrogen solubility (pH 3) and degrees of hydrolysis of Alcalase-treated RTech PPI vs. treatment time (treatment constants: 50-55oC, 0.5 AU / 5 g of PPI, pH 8.5). Alpha value used to calculate degrees of hydrolysis: 0.9684.

As can be seen in the graph, through Alcalase hydrolysis, PPI was solubility was increased from 20% (the PPI control; no treatment) to a maximum of 75% (a solubility increase of 55%). After 5 hours of reaction, the solubility of the PPI can be considered to have nearly attained the maximum solubility; after 5 hours, solubility does continue to increase but only at a very low rate. Therefore, 5 hours was chosen as the ideal Alcalase reaction time that will maximize PPI solubilities under the specified Alcalase concentration and reaction temperature. Should this process be scaled-up however, choosing a reaction time of 2 hours may be more appropriate as the solubility is close to the maximum at this point (this is the point at which the slope of solubility enhancement curve begins to markedly

53 decline). For the transglutaminase experiments shown in the next section, however, an Alcalase pre- treatment time of 5 hours was used.

In terms of appearance, the PPI solution became notably darker as the reaction time was increased (the colour changed from a medium tan colour to a medium brown). The increased solubility results were coupled with visual observations that indicated that the solubility was drastically improved: the Alcalase-treated PPI, when mixed with distilled water, resulted in a solution that was much more viscous, could no longer be decanted, and less solid cake was produced by centrifugation. The final solutions were slightly cloudy and they remained stable in the refrigerator for over a week.

The progress of the latter hydrolysis reaction was determined by calculating the degrees of hydrolysis (%DH) incurred by the PPI protein. This was done by means of a potentiometric titration of the protons released during protein hydrolysis at the constant reaction pH of 8.5. The formula below (from Cheftel et al., 1985) was used to calculate the degrees of hydrolysis incurred by the PPI protein:

Where α is the average dissociation coefficient of alpha-amino groups:

According to Cheftel et al., the average pK of α amino groups varies from 7.7 at 25oC to 6.9 at 60oC. Assuming that the pK value follows a linear relationship from 25oC to 60oC, the pK value for this experiment (conducted at ~55oC) was calculated to be 7.0. This results in an alpha value of 0.97.

During the hydrolysis reaction, each cleaved peptide bond results in one nonionized α-amino group and one ionized carboxyl group. Therefore, each mol of NaOH required to maintain the solution pH at a constant of 8.5 is equivalent to one mol of hydrolyzed peptide bonds. Figure 4.14 displays the moles of NaOH required to maintain solution pH at 8.5. The cumulative moles of NaOH required were determined by measuring the cumulative NaOH volume used during the reaction.

According to Dixon & Webb (1977), the moles of peptide bonds per gram of protein can be estimated from the amino acid composition of the protein. The amino acid profile of a 0.525 mg sample of RTech PPI was sent for an amino acid analysis to the Toronto Hospital for Sick Children HSC Advanced Protein Technology Centre. The information from this amino acid profile was used to estimate the moles of peptides per gram of RTech PPI: 0.00612 (See Appendix B26 for analysis). This number was estimated by assuming that the number of amino acid residues present in the protein is equivalent to the number of peptide bonds in the protein. Of course, this is only an approximation, which in fact is an overestimation as the terminal amino acids are counted into the final number. A more accurate number could be estimated if the respective molecular weights and concentrations of the protein fractions present in the sample were known.

The resulting reaction progression curve, in terms of degrees of hydrolysis (%DH), is shown in Figure 4.14. The figure indicates that the RTech PPI incurred a maximum of ~40% degrees of hydrolysis during the treatment. The result tables are available in Appendices B27 through B29. The shape of this reaction progression curve is as expected, according to the literature (see Figure 2.13). At time zero, the enzyme velocity is at its maximum and, gradually, this velocity is diminished. This is may be caused by several factors, one of them being enzyme inactivation at the pH or temperature of the reaction.

The solubility curve and the degrees of hydrolysis curves are similar in shape, but after 2 hours of reaction, the solubility curve appears to have a flatter slope than the degrees of hydrolysis curve. After 2 hours, the PPI solubility increased to 70% at a DH% of 30%; after 5 hours, the solubility is 73% at a DH% of 35%; and at 16 hours, the solubility has reached 76% whereas the DH% has jumped to 40%. After two hours, the rate of increase in solubility is approximately 2 times lower than the rate of increase in degrees of hydrolysis. It is therefore not worthwhile to continue hydrolyzing the protein after 5 hours.

An SDS Polyacrylamide Gel Electrophoresis was prepared using aqueous solutions (at pH 3) of RTech PPI treated with Alcalase (1 h, 0.5 AU/ 5 g of PPI, 60oC, pH 8.5) and of the non-hydrolyzed RTech PPI. The gel confirms that the endopeptidase cleaves the PPI protein into particles smaller than those present in the unmodified PPI.

a b c

Figure 4.15 – SDS Polyacrylamide gel electrophoresis of baseline and hydrolyzed RTech PPI: (a) protein ladder (1 lane); (b) PPI protein in solution at pH 3 after mixing, decanting, centrifugation, and filtration (2 lanes); (c) Alcalase hydrolyzed PPI (1 h, pH 8.5, 60oC, 0.5 AU / 5 g PPI) in solution at pH 3 after mixing, decanting, centrifugation, and filtration (2 lanes)

8.000 8 7.000 7

6.000 6

5.0005 44 hh ofof AlcalaseAlcalase TreatmentTreatment 88 hh ofof AlcalaseAlcalase TreatmentTreatment 4.000 4 1616 hh ofof AlcalaseAlcalase TreatmentTreatment

% of Particles 3.000 % of Particles % of 3

2.0002

1.0001

0.0000 6 6 7 8 9

6 7 9 111418222835435411 12 13 15 17 20 22 25 28 32 37 41 47 53 60 ParticleParticle Diameter Diameter (in micrometers)micrometers)

Figure 4.16 – Particle size of RTech Alcalase-treated RTech PPI at pH 3 vs. treatment time (treatment constants: 50-55oC, 0.5 AU / 5 g of PPI, pH 8.5).

A particle size analysis was also conducted (detailed results are available in Appendix B35). The particle sizes of the soluble PPI hydrolyzates at 4, 8, and 16 hours are very similar as can be seen in Figure 4.16. This is in accordance with the results shown in Figure 4.14 in which these same

56 hydrolyzates are shown to have similar solubilities. The difference in the particle sizes between the soluble hydrolyzates (centrifuged during the NSI) and insoluble hydrolyzates (the resulting NSI centrifuge cake) was also measured (Figure 4.17). As expected, the size of the particles of the PPI in the centrifuge cake was larger than the size of the particles remaining in solution after centrifugation. This is in accordance with the literature which states that the lower its particle size, the greater the solubility of a protein.

8.0008.000 8.000

7.000 7.0007.000

6.0006.0006.000

5.0005.0005.000 CentrifugedCentrifugedCentrifuged

DecantedDecantedDecanted 4.000 4.0004.000 CentrifugeCentrifugeCentrifuge Cake Cake Cake

% of Particles % of % of Particles % of 3.0003.000 % of Particles % of 3.000

2.0002.000 2.000

1.0001.000 1.000

0.0000.000 6 6 7 8

0.000 6 6 7 8 10 11 13 15 17 19 22 25 29 33 38 43 50 57

10 11 13 15 17 19 22 25 29 33 38 43 50 57 6 6 7 8 10 ParticleParticle11 13 Diameter Diameter15 17 (in19 (in micrometers) 22 micrometers)25 29 33 38 43 50 57 Particle Diameter (in micrometers)

Figure 4.17 – Particle size of the RTech Alcalase-treated RTech PPI at pH 3 (treatment constants: 4 h, 50-55oC, 0.5 AU / 5 g of PPI, pH 8.5).

The results in Figure 4.14 indicate the maximum solubility that could be attained by means of Alcalase hydrolysis in a practical setting; the Alcalase concentration was slightly above the industry maximum, the temperature used was the optimum for longer reaction times, and the effects of the Alcalase treatment were measured over a span of 16 hours of reaction time. Due to the high cost of enzymes, however, it would be beneficial to optimize the reaction conditions for this Alcalase treatment so as to minimize operational costs in the event of process scale-up. In essence, now that the practical maximum is known, it is now necessary to determine how to maximize solubility while minimizing reaction time and Alcalase concentration. In theory, this could be done by increasing the reaction temperature from 50-55oC to 60oC. Although the higher temperature will deactivate the

57 enzyme more rapidly, it was hypothesized to possibly allow for a faster initial reaction velocity which may result in a high solubility with a lower reaction time and Alcalase concentration.

One preliminary test was conducted to determine the effect on final solubility of treating the protein with Alcalase at 60oC instead of 50-55oC as was previously done. The treatment constants were: 0.5 AU/ 5 g of PPI, pH 8.5, 1 hour. The NSI was conducted at a pH of 3. Detailed results are available in Appendix B22. The final solubility of the PPI subjected to the 60oC treatment was: 61.6 ± 0.3 (% nitrogen solubility), while that of the PPI that underwent the 50-55oC treatment was 56.8 ± 0.1 (% nitrogen solubility). These data suggest that the reaction is proceeding more quickly as the temperature is increased. However, the reaction rate appears to be the same at both temperatures as is shown in Figure 4.18 (results in Appendices B27, 28, 29, 31). Thus, it appears that there is either no difference or a very slight increase in hydrolysis when the temperature is increased to 60oC. It is also possible that the increased solubility was caused by a higher mixing temperature rather than by improved hydrolysis.

100 90 80 70 60 deg C 60 50-55 deg C Exp 1

50 50-55 deg C Exp 2 40 50-55 deg C Exp 3

(%) Hydrolysis Degrees of 10

0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Alcalase Treatment Time (hours)

Figure 4.18 – Degrees of hydrolysis of Alcalase-treated RTech PPI at a pH 3 vs. treatment time (treatment constants: 0.5 AU / 5 g of PPI, pH 8.5). Alpha value used to calculate degrees of hydrolysis: 0.9755.

Another experiment was conducted to determine the effects of decreasing the concentration of Alcalase from 0.5 and 0.8 AU/ 5 g of PPI to the maximum level used in industry (0.3 AU / 5 g of PPI). The treatment constants for this experiment were 50-55oC, pH of 8.5, 1 h, and an NSI pH of 3.

Detailed results are available in Appendix B23. As expected, the solubility increased gradually as enzyme concentration was increased. The difference, however, between the solubility attained with the Alcalase concentrations used in these experiments and that that would be attained at levels typical to industry is very small.

100

80 70

20 Nitrogen Solubility Index (%) Index Solubility Nitrogen

0 0.0 0.3 0.5 0.8 Alcalase Concentration (AU / 5 g PPI) Figure 4.19 – Nitrogen solubility as a function of Alcalase treatment concentration (treatment constants: 1 h, pH 8.5, 50-55oC; solution pH 3)

100

90 Alcalase-Treated RTech PPI 80 Baseline RTech PPI

70 60

50 40

30 20

2.5 3.0 3.5 NSI pH

Figure 4.20 – Solubility of Alcalase-treated RTech PPI at a solution pH of 2.5, 3 and 3.5 (treatment constants: 50-55oC, 0.8 AU / 5 g of PPI, pH 8.5, 1 h)

In one last experiment, the solubility of Alcalase-treated RTech PPI versus NSI pH was prepared at a pH range of 2.5 to 3.5 (Figure 4.20; results available in Appendix B24). The pH of 3.5 was not exceeded as there would be a chance of the enzyme becoming reactivated at a pH greater than 4 as was explained in section 3.2.1 (although, should reactivation occur, the enzyme activity would be very low at a pH lower than 6). The reaction time was chosen to be 1 hour.

As expected, hydrolysis appears to have eliminated the isoelectric area from the PPI solubility curve; all samples attain similar solubility values within the pH range of 2.5 to 3.5, while the solubility curve of the baseline RTech PPI has a negative slope in this same pH range. Higher solubility results than those attained in this experiment can be expected by increasing the reaction time to over one hour, however one hour was chosen for this final NSI curve as it would be more adequate for use in industry. The Alcalase hydrolysis treatment proved to be successful in increasing PPI nitrogen solubility from 35% to 65% at pH 2 and from 15% to almost 70% at pH 3.5 near the isoelectric point. As will be seen in the later sections of this report, Alcalase hydrolysis is one of the most effective solubility enhancing treatments investigated in this project.

4.2.2 Enzymatic Cross-linking

Transglutaminase (Tgase) was used in an attempt to improve the solubility of the PPI via enzymatic cross-linking. The Tgase used for these experiments was Ca2+ independent Tgase as calcium ions have been reported in the literature to induce protein precipitation (Ellinger, 1972). The main reason for testing the use of Tgase was that it appeared to have the potential to further increase solubility after Alcalase hydrolysis (albeit slightly) while improving the bitterness typically present in protein powders. The first experiment was conducted to determine what levels of Tgase would be appropriate for use. According to the manufacturer (Ajinomoto Food Ingredients LLC), common commercial levels of Tgase use range from 0.1 to 5 U of enzyme per gram of system protein, and in some cases, 10 U of enzyme per gram of system protein are required. In Walsh et al.’s study, a level of 0.04 UE/g of system protein was used. All these concentrations are represented in Figure 4.21. Ajinomoto states that Tgase’s optimum temperature and pH are 45 to 55oC and 6 to 7, respectively. Therefore, a pH of 6.5 and a temperature of 55oC were chosen. The temperature chosen was the highest temperature possible within the optimum range; the reason for this being to minimize microbial growth should this treatment prove effective for use on a commercial scale. The treatment time was set to 2.5 hours, the reaction time used in Walsh et al.’s study. The solubility of the treated

60 protein powder was measured by means of an NSI at pH 3. The results are presented on a logarithmic scale in Figure 4.21 (the result table is available in Appendix B38).

100 90

80 70

60 50

40 30 20

Nitrogen (%) Solubility Index 10 0 0 0.001 0.1 1 10

Transglutaminase Concentration (UE / g system protein)

Figure 4.21 – Solubility of RTech PPI after transglutaminase treatment (55oC, 2.5 hours, pH 6.5)

The results indicate a decrease in solubility upon Tgase-treatment. The higher the Tgase concentration, the lower the resulting PPI solubility will be up to a decrease of almost 10%. These results are different than what was obtained by Walsh et al. who obtained a solubility increase of 50% upon treating a soy protein isolate with Tgase under similar conditions. This decrease in solubility, however, is not surprising since cross-linking does increase protein MW which, according to the literature, decreases solubility, and, according to Cheftel et al. (1985), cross-linking the ε-amino groups of lysine residues tends to result in decreased protein solubility. In terms of appearance, the Tgase-treated PPI was easier to pulverize, resulting in a finer powder (which may improve mouthfeel upon dispersion) and, consistently with the literature, was lighter in colour than Alcalase-treated PPI and baseline PPI.

To determine if Tgase had an effect on the solubility of Alcalase-hydrolyzed PPI, two more experiments were conducted. PPI that had been lyophilized after being hydrolyzed with Alcalase (5 h, 0.5 AU / 5 g PPI, 50-55oC, 8.5) was treated with Tgase over several treatment times and concentrations. Again, the resulting solubilities of each treated powder were determined by an NSI at pH 3.

100

) 90 80

40 30

Nitrogen Solubility Index (% Index Solubility Nitrogen 10 0 0 2 4 6 8 10 12 14 16 18 Transglutaminase Treatment Time (hours)

Figure 4.22 – NSI (pH 3) of RTech PPI after various transglutaminase treatment times (55oC, 10 UE/g protein, pH 6.5) conducted after an Alcalase treatment (5 h, 0.5 AU / 5 g PPI, 55oC, pH 8.5)

The amount of Tgase used for this first experiment (Figure 4.22; result table in Appendix B36) was 10 UE/g of system protein, which represents the maximum concentration that would be used in an industrial setting. This experiment also indicated a loss in solubility. The solubility of the control hovered around ~75% while the solubility of Tgase-treated PPI was 20 to 25% lower. Interestingly, the Tgase decreases the solubility of the PPI to a maximum level after as little as 30 minutes of treatment time. After that time, it appears as though additional Tgase treatment has no effect. This is most likely due to the inactivation of the enzyme which may be caused by several factors: the enzyme may have undergone inactivation at the pH or temperature of the reaction (as the experiment was conducted at the highest possible optimum temperature, 55oC, and known Tgase deactivation occurs at 60oC). Alternatively, the degree of substrate saturation of the enzyme may have fallen over time as the concentration of product increased.

The last experiment, shown in Figure 4.23 (results available in Appendix B37), consisted of treating Alcalase hydrolyzed PPI with Tgase over a span of several concentrations. Each bar represents the average of three replicates. Again, the solubility of Tgase-treated PPI was markedly lower than the solubility of the control.

100

Nitrogen Solubility Index (%) Index Solubility Nitrogen 20

0 0 0.001 0.01 0.1 1 10 Transglutaminase Concentration (UE / g system protein)

Figure 4.23 – NSI at pH 3 of RTech PPI after various transglutaminase treatment concentrations (55oC, pH 6.5, 2.5 h) conducted after an Alcalase treatment (5 h, 0.5 AU / 5 g PPI, 55oC, pH 8.5)

In conclusion, it is clear from the results that Tgase is not an effective method for improving the solubility of the PPI at this pH, as it decreases the solubility of both unhydrolyzed and hydrolyzed PPI over a broad range of concentrations. The final solubility of Tgase and Alcalase treated PPI is, however, higher than that of the baseline PPI. Therefore, if Tgase cross-linking were to be used as a method for improving protein powder taste and colour, its use even at the expense of maximizing PPI solubility may be justified.

4.3 Enhancing PPI Solubility: Solvent Modification

4.3.1 Salting In

The protein solution environment was modified by adding sodium tripolyphosphate (STPP), NaCl, and sodium hexametaphosphate (SHMP) to the PPI suspension during the dissolution phase of the NSI procedure. A 2 hour mixing period of the PPI with the aforementioned salts was used. After

63 mixing, the solutions were decanted, centrifuged, and filtered followed by immediate Kjeldahl protein concentration analysis. The NSI values were determined in the pH range of 2 to 5 as this was the pH range of interest.

4.3.1.1 Sodium Tripolyphosphate (STPP)

Sodium tripolyphosphate (STPP) was added to the PPI solution at a concentration level of 0.0016 w/v% (0.04 g of STPP/ 5 g of PPI in 250 ml of distilled water). This would result in a STPP concentration in the PPI suspension of well below ~0.5 w/v%, which is the taste threshold (Diosady, 2007). The results of the experiment are depicted in Figure 4.24 and are shown in comparison to the solubility results obtained with the baseline RTech PPI powder (results available in Appendix B39).

100

70 STPP Treated RTech PPI 60 Baseline RTech PPI 50

Nitrogen Solubility Index (%) Index Solubility Nitrogen 20 10 0 012345678 pH

Figure 4.24 – Solubility of STPP-treated RTech PPI (0.04 g of STPP/ 5 g of PPI)

It appears as though the STPP had no effect on the RTech PPI solubility near its isoelectric point, and it decreased the solubility as the pH deviated from that of the isoelectric point (as the PPI protein becomes progressively more positively charged) and had no affect again at pH 2. One explanation could be that the negatively charged STPP molecule binds to the positively charged amino acid moieties on the PPI’s protein surface thus transforming these moieties into negatively charged regions in a protein that is predominately positively charged. This could cause increased protein-protein interactions thus decreasing solubility, as would be expected of polyphosphates in the pH ranges of

2.5 to 3, according to the literature (Ellinger, 1972). At pH 2, perhaps the number of positively charged amino acid moieties on the protein’s surface is large enough for the binding of the STPP to have no effect. Another explanation is that the STPP simply has no effect on the RTech PPI solubility. This explanation would be plausible if it is assumed that the point corresponding to pH 2.5 deviated from the expected due to an experimental error, however, an additional experiment was conducted at pH 2.5 and resulted in the same effect. In either case, whether the STPP has no effect on protein solubility or whether it decreases solubility, it is clear that it certainly does not enhance PPI solubility, at least not at the levels used in this experiment. The use of STPP as a solubility enhancing agent for the purposes of the project was deemed to be ineffective. In addition, its use as a preservative in the final drink may not be advisable at pH values of 2.5 and 3.

4.3.1.2 Sodium Chloride

As expected based on the literature, NaCl improves PPI solubility near the isoelectric point and has no effect as the pH is lowered. The maximum increase in solubility for PPI was displayed at the isoelectric point with an increase of 25%. The amount of NaCl used in this experiment was 2.3 g of salt per 5 g of PPI resulting in a salt concentration of 0.92 w/v% in the 250 ml NSI PPI solution. Although this amount of salt is over the 0.5% taste threshold, it was the concentration recommended by Altemueller & Guevara (2002): their patent claims that ~0.7 g of NaCl/g of plant protein is the optimal amount of NaCl to be used to improve solubility.

100

80 70

60 NaCl-Treated RTech PPI 50 Baseline RTech PPI 40

Nitrogen Solubility Index (%) 20 10

0 012345678 pH Figure 4.25 – Solubility of NaCl-treated RTech PPI (2.3 g of NaCl/ 5 g of PPI)

An SDS Polyacrylamide Gel Electrophoresis was prepared with the NSI solutions (at pH 3) of RTech PPI treated with NaCl and that of the baseline RTech PPI. In accordance with the literature, the salt does not modify protein size.

a b c

Figure 4.26 – SDS Polyacrylamide gel electrophoresis of baseline and NaCl-treated PPI: (a) NaCl-treated RTech PPI (2.3 g of NaCl / 5 g PPI) in solution at pH 3 after mixing, decanting, centrifugation, and filtration (2 lanes) (b) protein ladder (1 lane); (c) PPI protein in solution at pH 3 after mixing, decanting, centrifugation, and filtration (2 lanes)

) % (

~30%

Protein Solubilit

? 0.92

NaCl Concentration (w/v%)

Figure 4.27 – Theoretical curve for protein “salting in” and “salting out” as a function of salt concentration

The amount of NaCl used for the experiment (Figure 4.25) was effective in improving solubility near the isoelectric point but was above the flavour threshold. Therefore, another experiment was conducted to determine the minimum NaCl concentration required before its effects on PPI solubility were inconsequential (see Figure 4.27). The NaCl concentrations used for this experiment were 10%, 25%, 50% and 70% of the amount used in the first experiment, corresponding to 0.2, 0.6, 1.15 and 1.6

66 g of NaCl per 5 g of PPI protein, respectively. These amounts translate to: ~0.1, 0.2, 0.5 and 0.6 w/v%, respectively.

100

70 NaCl-Treated RTech PPI 60 Baseline RTech PPI 50

Nitrogen Solubility Index (%) Index Solubility Nitrogen 20

0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 NaCl Concentration (w/v%)

Figure 4.28 – Solubility of NaCl-treated RTech PPI at various NaCl concentrations (pH 3)

The results from this experiment (displayed in Figure 4.28, Appendix B41), follows the theoretical curve shown in Figure 4.27. At low NaCl concentrations (up to ~0.5 w/v%, the taste threshold), the salt addition appears to have no effect on the PPI solubility. At a point in between ~ 0.5 and 0.66 w/v%, the effect of NaCl increases. At 0.66 and 0.92 w/v%, the effects of the NaCl enhances the solubility of the PPI by about 10%. The test shows that NaCl could not be used to effectively improve solubility without degrading the product taste.

4.3.1.3 Sodium Hexametaphosphate (SHMP)

One trial run was performed with sodium hexametaphosphate to test its effectiveness. The pH chosen was pH 2.7 and Texas PPI was used. A sample of ~0.35 g of SHMP was added to the solution (0.14w/v%, 0.0006M). Figure 4.29 shows the results of two SHMP experiments, each conducted in triplicate and with one blank, compared to the baseline solubility curve of Texas PPI. The SHMP reduced the solubility at this pH by ~7%. This is consistent with the literature as polyphosphates have been reported to reduce solubility in the acidic pH range (Ellinger, 1972). Using sodium

67 hexametaphosphate for solubility enhancing purposes was thus deemed ineffective at this pH. If possible, the use of sodium hexametaphosphate should be avoided as an antimicrobial agent in the final beverage, as it decreases protein solubility even at low levels.

100 90

60 SHMP-Treated Texas PPI 50 Baseline Texas PPI

(%) Index Solubility Nitrogen 20 10

0 01234567 pH

Figure 4.29 – Solubility of SHMP-treated Texas PPI at pH 2.7 compared to the Texas baseline solubility curve

4.3.2 Protective Colloids

The stabilizer chosen for this treatment was pectin as it was recommended in the literature and has been used on several occasions to stabilize protein in commercial juice beverages. As recommended by CP Kelco, pectin was used at pH 4 at a solution concentration of 1.5 w/v%, which has been deemed to be optimal. This amounted to 3.75 g of pectin per 5 g of PPI powder. While the NSI pH could be altered, when used for salting in to determine the solubility of the salt-treated PPI under various pH values, CP Kelco (2007) advised that the pectin only be used within the pH range of 3.8 to 4.2; outside of this pH range, the pectin could be expected to precipitate the protein.

The resulting PPI-pectin solution had a different appearance than the typical solution observed during the other treatments: a very homogenous dispersion with a thicker consistency similar to that of soy

68 milk. After centrifugation, a moderate amount of solid cake was observed, and the supernatant was very cloudy, translucent, and its colour resembled that of toasted almonds. The solution was easily separated from the cake, but was too viscous to be filtered. Figure 4.30 displays the resulting solubility of the pectin-treated RTech PPI compared to the solubility of the unmodified RTech PPI. The unmodified PPI value is the average of two experiments, each performed in triplicate, and the pectin-PPI value is the average of one experiment performed in triplicate. The error bars have been included although they are not perceptible in the graph. Detailed results are found in Appendix B43.

100

80 70 Pectin-Treated RTech PPI

Baseline RTech PPI 60 50

40 30

10 0

4.0 NSI pH

Figure 4.30 – Solubility of pectin-treated RTech PPI compared to the unmodified RTech PPI (at a pH of 4)

The results indicate that this treatment is effective in improving PPI nitrogen solubility by ~25% (to 29%). Although expressed in terms of nitrogen solubility, it is important to note that pectin does not improve solubility according to some definitions; instead forms a stable dispersion of the protein in water. The pectin-protein system was stored in the refrigerator and remained in a stable colloidal dispersion for over one week (see Figure 4.31).

The major disadvantages of this treatment are that the final solution is cloudy/opaque and must be prepared at a pH of 4. However, as was observed in this project, all other treatments also resulted in cloudy solutions as the concentration of soluble protein increased. This treatment does, however, improve texture and may help reduce the grainy mouthfeel that was observed upon addition of the PPI into fruit juices.

5. CONCLUSIONS AND RECOMMENDATIONS

5.1 Summary

The objective of this project was to investigate methods for maximizing the solubility of yellow mustard precipitated protein isolate (PPI) in aqueous solutions in the pH range of 2 to 4.5. Several solubility enhancing treatments were explored: enzymatic hydrolysis, enzymatic cross-linking, and the addition of solutes and stabilizing agents. Each method was investigated, and its strengths and limitations were discussed. The following is a summary of the treatments investigated and the results obtained.

Enzymatic Hydrolysis • Enzyme used: Alcalase (relatively inexpensive compared to other enzymes, widely used in food industry, one of the most effective in solublizing protein, reaction conditions are less conducive to microbial growth compared to those of other enzymes) • Maximum N solubility attained: ~70% (at an NSI pH of 3) at 0.5 AU/ 5 g PPI, 5 h, pH 8.5, 50-55oC • 90% of this maximum is attained after 2 hours of treatment • After 5 hours, enzyme continues to hydrolyze protein, however, rate of solubility increase is very low, and after 4 hours, the particle size does not decrease with increased hydrolysis • Improved RTech PPI solubility evenly across the acidic pH range of 2.5 to 3.5 (isoelectric point disappeared) by 30% at pH 2.5 to 55% at pH 3.5.

Enzymatic Cross-linking • Enzyme used: transglutaminase (widely used in the food industry to improve bitterness, texture and colour; reported by Walsh et al. (2003) to improve soy protein solubility at pH 3) • Decreased the solubility of unmodified PPI (by ~5%) over a span of concentrations (0.001 - 10 UE/g system protein) (reaction conditions: 55oC, 2.5 h, pH 6.5; NSI pH of 3) • Decreased solubility of hydrolyzed PPI (by ~15-25%) over a span of concentrations (0.001 - 10 UE/g system protein) and within 0.5 h of reaction time (reaction conditions: 55oC, pH 6.5; NSI pH of 3)

69 70

Sodium Tripolyphosphate • STPP chosen because it is widely used in the food industry as a preservative in protein containing foods and has been reported to improve protein dispersion although not in the acidic pH range • 0.04 g of STPP / 5 g of PPI was used; 0.0016 w/v% in solution which is well below the taste threshold of 0.5% • Reduced solubility at pH 2.5 by ~15%, at pH 3 by 5%, virtually no effect at other acidic pH values

Sodium Chloride • NaCl chosen because it is widely used in the food industry, it is inexpensive, and has been widely reported to improve protein solubility in acidic pH ranges • 2.3 g of NaCl / 5 g of PPI was used; 0.92 w/v% in solution; recommended by literature but above taste threshold of 0.5% • Increased solubility by 25% at the isoelectric point, at the pH deviated from the isoelectric point, solubility enhancement was subdued • NaCl had no effect on PPI N solubility at or below the taste threshold (at an NSI pH of 3) • Addition of NaCl did not alter protein molecular weight

Sodium Hexametaphosphate • SHMP chosen because it is widely used in the food industry as a preservative in protein containing foods and acidic liquids and has been reported to improve protein dispersion although not in the acidic pH range • 0.35 g of SHMP / 5 g of PPI was used; 0.14 w/v%; well below the taste threshold of 0.5% • SHMP decreased solubility by ~7% at a pH of 2.7

Pectin • Pectin was chosen as the preferred stabilizer as it has been reported in the literature as being successfully used to prepare fruit juice-soy enriched acidic beverages • Increased the solubility of PPI from ~5% to ~29% at an NSI pH of 4 and 1.5 w/v% of pectin

General Observations All NSI prepared protein-enhanced solutions were clear and displayed a yellow hue at low protein concentrations and were brown and cloudy at high protein concentrations. All solutions had the

71 apparent viscosity of water. Pectin-protein solutions were an exception, however. These solutions were viscous and completely opaque, even at a lower protein concentration.

5.2 Conclusions

The solubility of isoelectrically precipitated mustard protein (PPI) could be significantly increased by treatments acceptable to the food industry.

• The most promising treatments were Alcalase hydrolysis and pectin stabilization. Alcalase hydrolysis of the PPI results in a final solubility of 70% at pH ranges of 2.5 to 3.5 while pectin results in a final solubility of 29% at a pH of 4. The combined use of both of these treatments may result in a further increase in solubility. The reasons for believing so are the solubility results attained for the individual treatments and the fact that the combined use of both of these treatments has been reportedly used in the preparation of commercial ready-to- drink acidic protein-beverages.

• PPI can be hydrolyzed during the protein isolate production process, after separation of PPI and SPI. The hydrolyzed and now soluble protein would then be mixed with the SPI and the insoluble hydrolyzed protein would remain as PPI. This would increase the yield of SPI.

• The use of Alcalase hydrolysis alone in the preparation of the PPI for a protein-enhanced beverage will not result in a product as competitive as SPI or whey which are over 90% soluble. PPI will be 70% soluble, which is a very large improvement from the solubility of the original PPI, but will still result in the discarding of 30% of the insoluble protein when preparing a protein-enhanced drink.

• The remaining treatments were either not promising or ineffective:

o The NaCl treatment is only effective at the isoelectric point and at levels above the taste threshold. Therefore, it cannot be utilized in the final composition of a commercial drink. It cannot be used to increase the yield of the SPI during the protein isolation process either. The addition of an amount greater than 0.5 w/v% to the mustard protein solution will result in improved solubility only prior to the ultrafiltration/diafiltration process step. At this point the NaCl will be filtered out as

an impurity thus reverting solubility back to its original value. In addition, NaCl cannot be used after the filtration step as it would then be dried along with the protein, thus imparting a taste on the protein isolates, which would lower their quality.

o Transglutaminase may be used to improve bitterness, colour, texture; however, this will be done at the expense of final PPI solubility at a solution pH of 3

o The use of STPP as a preservative in the final drink should be used with caution in the preparation of beverages with a pH near 2.5 and 3 since STPP may decrease solubility at these pH values even at low concentration levels

o The use of SHMP as a preservative in the final drink should be avoided as it decreases solubility even at low levels of use at a solution pH of 2.7

5.3 Recommendations

To build upon the knowledge gained in this project, further investigation should be conducted on the following:

• On the use of enzymatic hydrolysis in combination with pectin

• On the use of hydrolysis during protein isolate production: o Determine the solubility of Alcalase-treated PPI over a span of enzyme concentrations o Perform a cost-benefit analysis to determine the ideal Alcalase concentration to be used in the process (i.e., a higher concentration will increase production costs but will improve solubility and thus SPI yield which has a higher market value than PPI)

• On the use of pectin alone to improve PPI dispersibility o Other methods should be used to further determine the effectiveness of pectin as the NSI is not an adequate means of determining the stability of a colloidal dispersion (the rpm of mixing recommended by the NSI standard procedure is too low, pectin- protein solutions require the use of a homogenizer). One method that is used in

industry to determine the stability of pectin colloidal dispersions is particle size analysis.

• On the taste, odour, viscosity, and mouthfeel of pectin-protein and pectin-hydrolyzed protein and hydrolyzed-protein

• On the use of Tgase at other NSI pH values to determine if it results in improved solubility at pH values other than 3.

• On the use of Tgase to improve the colour, texture, and taste of PPI not destined for beverage enhancement (for use in protein-containing products such as processed meats, bakery products, and meat substitutes)

• On the effect of reducing PPI particle size (by dry milling) on PPI solubility and mouthfeel

• On the effects of common food preservation techniques on final solubility (e.g., chemical preservation, hot filling, etc.)

The knowledge gained from this project, provides the groundwork for improving the solubility of yellow mustard PPI in acidic aqueous solutions. Devising an optimized method for increasing PPI solubility will allow for its use alongside SPI in the production of low pH protein enhanced beverages. The commercialization of PPI and SPI as soluble protein isolates will provide the beverage industry with a non-fat, high quality plant protein source. The presence of this protein source will be beneficial as it will provide the food industry with an acceptable alternative to soy protein; yellow mustard protein is comparable to soy protein in quality but is free of the allergens typically found in soy. Lastly, large-scale commercialization of yellow mustard protein will increase the value of mustard seeds, which would benefit the farming industry in Canada, currently the world’s leading exporter and producer of mustard.

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Young, V.R. & Borgonha, S. (2000). Nitrogen and amino acid requirements: the Massachusetts Institute of Technology amino acid requirement pattern. The Journal of Nutrition, 130 (7):1841S-9S.

Young, L.S., G.A. Taylor, & A.T. Bonkowski. (1986). Use of soy protein products in injected and absorbed whole muscle meats. ACS Symposium Series, 312: 90-98.

APPENDICES

82 83

APPENDIX A

Detailed Analytical and Experimental Methods

A1 – Determination of the Nitrogen Solubility Index

(AOCS Official Method Ba 11-65 – with minor modifications)

1. Weigh 5 g of the sample into a 500 ml beaker. Record mass.

2. Measure 200 ml of distilled water at 30oC.

3. Add a small portion of the water at a time and disperse it thoroughly with a stirring rod. Stir in the remainder of the water, using the last of it to wash off the stirring rod.

4. Stir the mixture at 125 rpm with a mechanical stirrer for 120 min at 30oC in the water bath. Record the original pH of the solution. At this point the pH is changed to the desired pH using sodium hydroxide or phosphoric acid. This pH is maintained throughout the two hours of mixing time.

5. Transfer the mixture to a 250 ml volumetric flask by carefully washing out the contents of the beaker into the flask. Add two drops of Antifoam B, dilute to the mark with distilled water and mix the contents of the flask thoroughly.

6. Leave solution standing for 15 minutes. Then, decant off ~80 ml of the supernatant into two 50 ml Falcon tubes.

7. Centrifuge for 10 minutes at 1,500 rpm and decant supernatant through a funnel containing a plug of glass fiber. Collect the clear filtrate into a 100 ml beaker.

8. Record the appearance of the solution.

9. Weigh out 20 g of this solution into 3 Kjeldahl tubes (use 20 g of distilled water for the blank) and determine the nitrogen content of the liquids using the standard Kjeldahl procedure (AOCS Official Method Ba 4d-90)

A2 – Kjeldahl Standard Operating Procedure

(AOCS Official Method Ba 4d-90 – with minor modifications)

A2.1 Kjedahl Digestion

1. Preheat the Digestion unit (Büchi 425) to setting 4.

2. Place Büchi tubes (one per sample + one blank) on the tube rack and label accordingly.

3. If sample is solid: Weigh out ~0.1g of each sample onto nitrogen free paper (previously folded in each direction to form a “boat”). Record the exact measured weight. Pull the corners of the paper together and tightly twist these ends of the paper to form a bag which holds the sample. Drop one “bag” into each sample tube. For the blank, drop a (sample-free) crumpled nitrogen free paper into the blank Büchi tube.

If sample is liquid: Weigh out ~20 g of each sample directly into the Büchi digestion tubes and ~20 g of distilled water into the blank digestion tube. Since the digestion tubes will not stand upright on the scale, it is recommended to place the digestion tube into a beaker which can then be placed stably onto the scale.

4. Add 4 Kjedahl Kelmate® Tablets to each tube (including the blank) in the fumehood. Slide each tablet down the side of the tilted tube to prevent a shock upon landing.

5. Add 25 mL of concentrated H2SO4 to each tube (including the blank) in the fumehood.

6. Place a glass manifold on the tubes (make sure that the glass fiber end is on the same side as the blank). Clamp the cover on tightly with 4 clamp wires.

7. Place the tube rack with the tubes near the Digestion unit (Büchi 425). Connect the glass manifold with the suction tube and turn on the tap water. This will ensure that the sulfuric acid vapours do not exit the glass manifold.

8. Place the connected tubes onto the Digestion unit (Büchi 425).

9. Heat the tubes on setting 4 for 20 minutes. Raise the setting to 6 for 10 minutes or until the foam subsides and the air in the tube starts to show mist. Then, turn the setting up to 10 (full power) and digest for 35 minutes ensuring that the walls of the glass are clean and that the solution is colourless for at least 5 to 10 minutes before removing from heat. Turn off the digester when done.

10. Remove the tubes from the digester and place in a rack with the suction continuing until the solution is cool (~30 minutes). Once the solution is sufficiently cool, the suction tube can be removed and the rack moved into the fumehood to finish cooling.

11. As the tubes cool, a white precipitate will appear within the clear liquid. At this point, the tubes may be left to cool overnight and the distillation may be performed the next day. Alternatively, the tubes may be left to cool (for approximately 30 min) and the distillation part of the experiment may be performed on the same day.

A2.2 Kjedahl Distillation

1. Remove the glass manifold (be careful not to spill any condensed acid remaining within the manifold). Rinse the manifold with water and leave it in the sink to air dry.

2. Swirl the samples and blank to mix the clear liquid with the white precipitate. Then, add 50 mL of distilled water to each tube. Swirl the samples once again until all the white precipitate has dissolved leaving you with a clear liquid. Then, add 25 mL of sodium thiosulfate (8%

Na2S2O3·5H2O) to each tube. The samples and blank will now have a dark brown colour and a nebulous texture. Swirl the tubes and cover. Wait until the tubes are cool before distilling the samples. It is advisable to swirl the sample tubes every 3-5 minutes to increase the extent of the reaction in the tubes.

3. Check the Distillation Unit to make sure that the distilled water compartment is 3/4 full and that the 32 wt/wt% NaOH compartment is at least 1/3 full. If not, refill it before beginning the distillation.

4. Label one Erlenmeyer flask per sample and blank. Add 60 mL of 4 wt/v% boric acid to and three drops of red methyl indicator to each Erlenmeyer flask.

5. Turn on the tap water (the tube connected to the Kjedahl distillation unit) and press the green switch on the right side of the distillation unit. The machine will now warm up and beep when it is ready for use.

6. After hearing the beep, open the glass compartment, lift the metal restraint, and remove the tube. Pour out the water from this the tube and refill it half-way with distilled water. Remove the Erlenmeyer flask from the distillation unit, remove water, and refill it with fresh distilled water.

7. Set the distillation time to 2 minutes by pressing the time button and press start. The unit will make a loud water hammer noise, this is normal. When the machine is finished, it will beep twice. After the second beep, remove the water tube (which will now be very hot) with the tongs.

8. Replace the water tube with your blank tube. Replace the water Erlenmeyer flask with one of the boric acid + phenolphthalein Erlenmeyer flasks. Ensure that the tip of the plastic tube is fully submerged in the boric acid solution. This may be done by putting paper towels under the Erlenmeyer flask to increase the height of the solution.

9. Add 90 mL of NaOH reagent by pressing the reagent button until the total solution hits the 180 mL mark or until the sample solution stops bubbling (which means that it has reached its neutralization point). Add this reagent manually and slowly to minimize the formation and escape of H2S from the unit.

10. Press the time button and increase the distillation time to 5 minutes.

11. Place a paper towel in the bottom slide-out compartment of the distillation unit and under the Kjedahl tube to trap any spills during distillation or during sample changing.

12. Press the start button.

13. When the instrument is finished, it will beep twice. At this time, open the cover and use tongs to remove the sample tube from the distillation unit. Put this tube back onto the rack.

14. Place a new sample in the tube and a new Erlenmeyer flask into the unit. The Erlenmeyer flask liquid should be green for all samples with the exception of the blank. Cover this flask with Parafilm® until titration is begun.

15. Press the reagent button twice to manually add the reagent to the sample. If the button is pressed only once, the distillation unit will add the reagent automatically. It is preferable to add the reagent manually so as to be able to add it slowly.

16. Press time (it should say 5 minutes) and press start.

17. Repeat the procedure for the remaining samples.

18. Once finished running all the samples through the distillation unit, fill the storage tube (1/2 way) and the storage Erlenmeyer flask (1/2 way) with fresh distilled water. Run the instrument with the water for five minutes to clean the apparatus. No reagent should be added for this cleaning step.

19. Repeat the cleaning procedure if necessary. When finished, replace the used water with fresh distilled water, turn off the unit by pressing the green button on the side of the apparatus, and turn off the tap water.

20. Sign the logbook.

A2.3 Titration

1. Pump the 0.1 N sulfuric acid slowly until it is set to 0 ml.

2. Titrate the sample until the pink endpoint is reached. During the titration, the sample will transition from green to clear to pink. The endpoint is the same shade of pink as the blank.

3. Record the volume of sulfuric acid to reach the endpoint.

A2.4 Calculating the Concentration of Protein in the Sample

1. First, determine the % crude N in the sample by means of the following formula:

[H SO ml used for sample − H SO ml used for blank]× 0.1N ×1.4 wt % of N in sample = 2 4 2 4 weight of sample (g)

Sample Calculation: Assuming, - The blank required no titration (sample was already a pink shade after distillation)

Volume of H2SO4 used for the blank = 0 ml

- 2.6 mL of H2SO4 were required to titrate sample 1 to the same shade of pink seen in the blank

Volume of H2SO4 used for sample 1 = 2.6 ml

- Sample 1 consisted of 0.0572g of canola seeds

Substituting into the equation: [2.6 mL − 0 mL]× 0.1N ×1.4 wt % of N in sample 1 = ∴wt % of N in sample 1 = 6.36 % 0.0572 g

2. Next, the wt % of protein in the sample can be determined from the N wt% by means of the following formula:

Wt % of protein in sample = (wt % of N in sample) x (6.25)

Sample Calculation: If the wt % of N for sample 1 = 6.36 %, then the wt % of protein in sample 1 is:

Wt % of protein in sample = 6.36 % x (6.25) = 39. 77%

A2.5 Calculating the PPI Concentration in the Solution

In order to determine the PPI concentration of a solution, the protein concentration of this solution must first be determined by means of the standard Kjedahl method. Once protein concentration is determined, this value can be converted to PPI concentration by means of the following ratio: 1 g of PPI = 0.7922 g of protein

This ratio was determined for this project by means of four Kjedahl experiments (each in triplicate).

Sample calculation: Find the weight percent of PPI in a solution that has a 0.67 wt% of protein:

PPI wt% = 0.846

A2.7 Calculating the Solubility of PPI

Once the PPI concentration of a solution is determined, the PPI solubility can be easily calculated. This is done by first determining, by means of calculation, the total mass of dissolved PPI in the final solution and then dividing this value by the mass of PPI initially mixed into the solvent which will have been recorded.

To find the mass of the dissolved PPI:

All the components of this equation will be known with the exception of “mass of PPI in final solution”. Once this term is found, the PPI % solubility can be calculated by means of the following equation:

Sample Calculation:

Find the PPI % solubility of a PPI-enhanced solution to which 10.01g of PPI and 93.01 g of solvent were added. After centrifugation, after the PPI cake was removed, the PPI wt% in the solution was found to be 0.846 wt%.

First find the mass of PPI dissolved in the final solution:

0.846 wt% = (mass of PPI in final solution) x 100 % (93.01 g + mass of PPI in final solution)

0.7869 + 0.00846 x (mass of PPI in final solution) = (mass of PPI in final solution)

mass of PPI in final solution = 0.7936 g of PPI

Now that the mass of PPI in the final solution is known, the PPI % solubility can be found:

PPI solubility (%) = 0.7936 g of PPI in final solution x 100 % 10.01 g of PPI in initial solution

PPI solubility (%) = 7.93 %

A2.8 Preparing Kjeldahl Reagent Solutions

32% (w/w) NaOH

It is preferable to prepare this solution from 50 wt/wt% liquid NaOH as opposed to preparing it from NaOH pellets. The reason for this is that preparation time will be much shorter for the former option.

To determine the ratios of NaOH and distilled water needed for the final solution, use the following equation:

Calculation Steps:

1. Determine what mass of the 50 wt/wt% NaOH solution is necessary to prepare the desired mass of 32 wt/wt% NaOH.

Example: To prepare a 1,500 g solution of 32 wt/wt% NaOH, 480 g of NaOH are needed.

∴mass of NaOH = 480 g

This mass of NaOH is obtained from the 50 wt/wt% NaOH solution. To determine what total mass of this 50 wt/wt% solution is necessary,

∴total mass of 50 wt/wt% solution = 960 g

2. To determine the mass of distilled water that must be added to the 960 g of the 50 wt/wt% NaOH solution,

Total mass of final solution = mass of NaOH + mass of distilled water

Substituting in the previously calculated values, 1,500 g = 480 g + mass of distilled water ∴mass of distilled water = 1020 g

3. Calculate the volume of this solution to determine what beaker size to use:

Density of 32 wt/wt% NaOH = 1.349 g/mL

Density = mass / volume

Volume of the final solution = (1,500 g)/(1.349 g/mL) = 1111.9 mL

Therefore, a 2 L beaker may be used for the preparation of this solution.

Solution Preparation Steps: 1. Weigh 960 g of 50 wt/wt% NaOH solution into a 2 L beaker 2. Place beaker in the fume hood onto a stir plate 3. Add a spin bar into the NaOH solution 4. Weigh 1020 g of distilled water onto a separate beaker

5. Add the water slowly to the NaOH solution 6. Allow the solution to mix in the fume hood until it no longer appears cloudy

4% (w/v) Boric Acid

Generally, 2 L of this solution are prepared at a time. This volume should last for 6 to 8 Kjedahl distillations. The boric acid used to prepare this solution was in powder form. However, it is recommended to use the flake form of boric acid in the future since this form is more soluble than the powder form. Using the flake form will decrease preparation time. The preparation procedure is as follows:

1. Weigh 80 g of boric acid into a 2 L beaker 2. Fill the beaker with distilled water up to the 1,800 mL mark 3. Add a large spin bar into the beaker, place the beaker on a stir plate and mix the solution until clear (~3 hours) 4. Cover the beaker with aluminum foil to prevent the contamination of the solution 5. Once the boric acid is completely dissolved, transfer the solution to a 2 L volumetric flask and add distilled water up to the mark

8% (w/v) Sodium Thiosulfate

Generally, 2 L of this solution are prepared at a time. This volume should last for approximately 13 to 15 Kjedahl distillations. The preparation procedure is as follows:

1. Weigh 160 g of sodium thiosulfate into a 2 L volumetric flask 2. Fill the flask up to the mark (while filling with distilled water, swirl the flask to dissolve the sodium thiosulfate)

A2.9 Monthly Kjeldahl Unit Checks

These tests are performed to check that the distillation unit is functioning properly. These tests consist of a nitrogen analysis using an ammonia salt. It is recommended that one standard test be performed per month.

Substance used: Ammonium dihydrogen phosphate, MW: 115.03, N content: 12.17 %

Procedure:

1. Weigh 450 mg of ammonium dihydrogen phosphate into Kjeldahl tubes 1, 2, & 3, and record weights. Leave blank empty.

2. Add 20 ml of distilled water to ALL tubes

3. Prepare 4 beakers with 60 ml of boric acid in each (with 3 drops of indicator)

4. During distillation, add 20 ml of NaOH reagent

5. Set the distillation time to 4 minutes

6. Titrate with 0.1 N sulfuric acid – consumption will be approx. 7 to 10 ml

7. Calculate the % N:

a. Acceptable recovery rate for the standard: 99.0 to 102.0%

b. Acceptable RSD standard: less than 1%

A3 – SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)

1. Gather the starting materials: a. Plastic cover slip bags b. Gelling apparatus c. Glass plates d. Two separator plastics (thin, long, white) e. Gel comb

2. Clean the glass plates with water and ethanol and wipe dry with Kimwipes 3. Put the two plates together and place the two separators between these on either ends 4. Place the plates and separators into a plastic slip bag. Be careful not to move the plates. 5. Place plastic slip bag into the gelling apparatus and tighten the knobs

6. Prepare “resolving-gel solution”: Material 10% Gel 12% Gel 15% Gel Distilled Water 3.25 ml 2.7 ml 1.92 ml

4x Resolving 2 ml 2 ml 2 ml Buffer 30% Acrylamide 2.7 ml 3.2 ml 4 ml 10% APS 80 µl 80 µl 80 µl TEMED 3 µl 3 µl 3 µl

Notes: o Add TEMED immediately before pouring the gel o APS last only one week once prepared o The higher the gel %, the smaller the gel pore size and the slower the sample will run. Use a higher gel % for proteins of lower molecular weight.

7. Quickly mix the solution and pour in between plates with a dropper. The final height should be ~2 cm below the lower ledge. 8. Add butanol on top to remove bubbles and to smoothen the surface 9. Leave the gel for ~30 minutes to allow for polymerization

10. Prepare the “stacking gel”: Material Volume Distilled Water 4.6 ml 4 x Stacking Gel Buffer 2.0 ml 30 % Acrylamide 1.3 ml 10 % APS 48 µl TEMED* 5 µl

Note: Add TEMED immediately before pouring the gel

11. Before adding the TEMED, pour out the butanol (into a waste bottle) and dry the inner sides of the plates with a Kimwipe 12. Add the stacking gel on top of the resolving gel with a dropper and place the comb in between the two plates, into the liquid gel, being careful not to form bubbles 13. Wait for the stacking gel to polymerize (~30 min) 14. While waiting for the stacking gel to polymerize, prepare the samples. Place 300 µl of each sample followed by 100 µl of loading buffer into Eppendorf tubes. Label the sample tubes accordingly and place in boiling water for 5 minutes. 15. Once the gel has been polymerized, remove plates from the plastic sleeve and place into the electric current apparatus ensuring that the lower lip of the plates faces inwards. Secure the plates in place (tightening the four knobs). 16. Prepare non-native buffer: 1 part native buffer concentrate mixed with 3 parts distilled water 17. Pour the non-native buffer into the instrument chamber until the sliver filament is covered 18. Load the samples into the gel wells using a superfine micropipette tip and freeze the remaining samples for later use. Use the following loading volumes: a. Ladders: 7 µl ladder + 8 µl loading buffer (total 15 µl) b. Samples: 15 µl 19. Place cover on instrument, press the switch on the lower left hand side of the instrument, and press the (on/off) grey button. The Amps will remain constant at 9 and the volts will vary between 100 and 150 V. The display should be on ‘actual’ and the mode on “V”. 20. Turn off the instrument when the leading edge nears the bottom of the gel 21. Remove plates from instrument and gently remove gel from plates 22. Stain the gel in the Coomassie blue staining solution overnight

23. Destain the gel in 50% methanol/10% acetic acid/40% water for 1 hour or until the gel is pale blue in colour 24. Remove the gel from the destain solution and place in a 10% methanol/10% acetic acid/80% distilled water solution for a minimum of one hour (it may be left in this solution up to one week) 25. The final gel may be left in solution in the refrigerator (for a maximum duration of one week) or may be dried using a low temperature oven

A3.1 Preparing SDS-PAGE Solutions

4x Resolving Gel Buffer 20 ml 1.5 M Tris-HCl, pH 8.8 1.6 ml 10% SDS 18.4 ml distilled water

30% Acrylamide Solution 29.2 g acrylamide 0.8 g bis 100 ml distilled water

10% Ammonium persulfate (APS) 1 g ammonium persulfate Dilute to 10 ml with distilled water (shelf-life: 1 week)

4x Stacking Gel Buffer 20 ml 0.5 M Tris-HCl, pH 6.8 1.6 ml 10% SDS 18.4 ml distilled water

Non-Native Electrophoresis Buffer Concentrate 12 g Tris base 57.6 g glycine 4 g SDS 1 litre distilled water (no need to adjust the pH)

4x Sample Loading Buffer 10 ml 4x stacking gel buffer 18 ml 10% SDS 2 ml beta-mercaptoethanol 20 ml glycerol 0.01% w/v bromophenol blue

50/10 Fixing/Destaining Solution 50% vol/vol methanol 10% vol/vol glacial acetic acid

10/10 Destaining Solution 10% vol/vol methanol 10% vol/vol glacial acetic acid

Coomasie Blue Staining Solution 1 g Coomassie Brilliant Blue R-250 500 ml fixing/destaining solution

1.5 M Tris-HCL, pH 8.8 18.15 g Tris base 50 ml distilled water Adjust pH to 8.8 with 1 N HCl (~24.3 ml) Dilute to 100 ml with distilled water

0.5 M Tris-HCL, pH 6.8 3 g Tris base 40 ml distilled water Adjust pH to 6.8 with 1 N HCl Dilute to 50 ml with distilled water

10% SDS 10 g SDS Dilute to 100 ml with distilled water

100

A4 – Beckman Coulter Multisizer 3 3.5 Particle Size Analysis

The instrument will return a graph plotting Number (%) on the y-axis and Particle Diameter (µm) on the x-axis. The procedure for using this equipment is as follows:

1. Turn on the nanopure water system (press the red power button and wait for 10 minutes) 2. Turn on the computer, enter password, and click on the Multisizer program 3. Prepare the electrolyte solution: 9.8 g NaCl / 1 litre of nanopure water (use the large Erlenmeyer flask) 4. Clean probe and mixing blade with electrolyte solution (use squeeze bottle with blue top) 5. Fill the one third of the Coulter beaker with electrolyte solution 6. Put beaker onto stand inside instrument 7. Press lever under the stand to raise the stand until the beaker liquid level is high enough to activate the stirrer 8. Set stirrer at velocity 8 9. Unblock aperture using the Multisizer program (system button drawdown menu) 10. Click on flush 11. Prepare sample: 90 ml electrolyte + 10 ml sample 12. Load sample into beaker 13. Click on start

101

A5 – Determining Moisture Content

Moisture content of the PPI samples was measured by the following procedure:

1. Weigh, in triplicate, ~10 g of sample into a beaker (record exact weight) 2. Record exact sample weight for each replicate 3. Record total weight for each replicate 4. Dry samples in an oven at 110oC for 24 hours 5. After drying, record total final mass 6. Determine the mean moisture content in sample in w/w%:

Moisture Weight (g) = Final Sample Mass (g) – Initial Sample Mass (g)

Moisture % = Moisture Weight (g) / Initial Sample Mass (g) x 100%

102

A6 – Enzymatic Hydrolysis Treatment – Detailed Procedure

The procedure used to hydrolyze the PPI is as follows:

1. Heat 150 ml of distilled water to 50oC

2. Once heated, measure 150 ml of it into a volumetric flask

3. Weigh 10 g of PPI, record exact weight, and place it in a dry 200 ml beaker

4. Slowly, pour half of the water from the volumetric flask into the beaker containing PPI sample, making sure to wash the sides of the beaker and leaving ~50 ml of the water aside.

5. Stir the mixture with a glass rod

6. Using a portion of the remaining distilled water, wash the rod and the sides of the beaker so that no PPI remains on the wall or the rod

7. Place the beaker with the solution on the hot plate under the mechanical stirrer and set the stirrer rpm to 125

8. Place a thermometer and a pH meter probe (calibrated for basic pH values) into the solution, being careful not to let either touch the sides of the beaker or the blade

9. Heat the solution to in between 50 and 55o C (or alternate temperature) and set the pH to 8.5 using NaOH (wait for the temperature and pH to stabilize)

10. Weigh 1 g (or alternate amount) of Alcalase liquid on a weigh boat and dilute to 10 ml in a volumetric flask and mix

11. Add 1 ml (or alternate amount) of this mixture to the PPI solution

12. Set the timer to two hours (or alternate time)

103

13. Maintain the pH at 8.5 and record the volume of 0.5 N NaOH used (for the %DH calculation).

14. After the two hours, reduce the pH to 3.5 and transfer the solution to the refrigerator to stop enzyme activity

15. Flash-freeze the solution with liquid nitrogen and freeze dry

16. After PPI is freeze dried, collect the powder, grind it with a mortar and pestle, and determine the solubility of the now treated PPI by means of NSI (AOCS Official Method Ba-11-65)

104

APPENDIX B

Result Tables

105 B1

Experiment Overall RSTDV: Overall STDV (%): Overall (%): Mean – RtechPPI–Protein Content(a 6 5 4 3 2 1 # 06/07/2007 06/15/2007 06/14/2007 06/13/2007 06/13/2007 06/06/2007 Date

Sample ln .00 .0 0.00 0.00 Blank 0.0000 0.00 0.00 Blank 0.0000 0.00 0.00 Blank 0.0000 0.00 0.00 Blank 0.0000 0.00 0.00 Blank 0.0000 0.00 0.00 Blank 0.0000 .02 .1 110.50 7.91 3 0.1002 .01 .9 10.65 7.69 10.24 2 0.1011 7.42 1 0.1014 10.98 8.01 11.17 2 0.1021 8.05 1 0.1009 11.03 7.90 11.21 2 0.1002 8.20 1 0.1024 11.38 8.92 10.65 2 0.1097 8.29 1 0.1090 .01 .1 11.36 8.21 11.03 2 0.1011 8.24 1 0.1018 11.04 8.12 10.93 2 0.1030 7.98 1 0.1022 .03 .1 11.32 8.11 3 0.1003 10.96 7.94 3 0.1014 10.61 7.65 3 0.1009 11.28 8.09 3 0.1004 11.29 8.18 3 0.1014 69.12 3.27 2.26 Mass (g) s is;Nx6.25)DeterminedbyKj Titration Volume (ml) Sample % N in in % N eldahl Standard Procedure % Protein in Sample 69.07 66.55 64.03 68.63 69.81 68.94 70.06 71.15 66.55 71.83 70.83 68.99 68.32 70.75 68.35 66.34 70.50 73.41 0.00 0.00 0.00 0.00 0.00 0.00 Mean 93 .1 0.88 69.36 0.61 11 .0 0.85 71.14 0.60 0.55 68.55 0.38 2.13 65.64 1.40 1.36 69.65 0.95 4.97 70.37 3.50 STDV RSTDV 106 B2 No sodiumadded (thiswasdoneaspartofatest) *Discarded: thiosulfate was Experiment #

Overall RSTDV: Overall STDV (%): Overall (%): Mean – Texas PPI–Protein Content(a 4 3 2 1

19/01/2006 18/01/2006 15/01/2006 10/01/2006 Date

Sample ln 000 NA N/A 0.00 N/A* 0.20 0.0000 0.0000 Blank 2 Blank 1 ln .00 .2 0.00 0.22 Blank 0.0000 0.00 0.00 Blank 0.0000 0.00 0.32 Blank 0.0000 .97 .0 12.07 12.01 8.30 2 0.0937 8.68 1 0.0986 12.30 8.62 2 0.0958 12.48 12.38 8.06 2 0.0904 9.32 1 0.1054 12.93 13.12 9.70 2 0.1016 11.52 1 0.1195 .92 .0 12.44 8.50 3 0.0932 12.70 10.06 3 0.1087 12.78 9.85 3 0.1079 13.55 11.40 3 0.1145 79.22 3.65 2.89 Mass (g) s is;Nx6.25)DeterminedbyKj Titration Volume (ml) Sample % N in in % N % Protein in eldahl Standard Procedure Sample 75.45 75.08 76.91 79.39 78.73 80.78 82.01 77.74 79.37 81.28 84.67 0.00 0.00 0.00 0.00 N/A Mean 60 .4 1.89 76.09 1.44 2.23 78.14 1.74 1.66 79.80 1.32 2.41 82.49 1.99 STDV RSTDV 107 B3 Experiment # Overall RSTDV: Overall STDV (%): Overall (%): Mean

– Soy Protein Isolate –Protein Content (asis Procedure 1

02/14/2008 Date

Sample ln .00 .0 0.00 0.40 Blank 0.0000 .97 72 13.56 13.52 97.20 96.98 2 0.9997 1 1.0004 .03 72 13.55 97.22 3 1.0003 84.63 Mass(g) 0.16 0.14 Change inVolume(mL) ; Nx6.25)Determinedby % Sample N in % inSample Protein 84.73 84.47 84.69 0.00 KjeldahlStandard Mean 46 0.140.16 84.63 STDV RSTDV 108 B4 Experiment

Overall Mean: % PROTEIN DRY BASIS Overall RSTDV: Overall STDV: Overall Mean: % MOISTURE Overall RSTDV: Overall STDV: Overall Mean: % PROTEINAS IS – RtechPPI–Protein Content(dryba 1 #

13/06/2007 Date

69.12 73.35 3.27 2.26 0.71 0.04 5.77 Sample 004 051 9.4531 0.5815 3 10.0346 007 050 9.4370 0.5800 2 10.0170 9.4316 0.5724 1 10.0040

Initial PPIMass (g) sis; Nx6.25)andMoisture Content Moisture Mass (g) Final PPI Mass Final PPIMass (g) Moisture 5.7949 5.7902 5.7217 (%) Mean 5.77

STDV 0.04

RSTDV 0.71

109 B5

Experiment # Overall Mean: % PROTEIN DRY BASIS Overall RSTDV: Overall STDV: Overall Mean: % MOISTURE Overall RSTDV: Overall STDV: Overall Mean: % PROTEINAS IS – Texas PPI –Protein Content (dryba 1

01/10/2006 Date

79.22 85.20 0.32 0.02 7.02 3.65 2.89 Sample .77 .85 9.0862 0.6865 2 9.7727 9.6753 0.7280 1 10.4033 055 074 9.8210 0.7440 3 10.5650 Initial PPIMass (g) sis; Nx6.25)andMoisture Content Moisture Mass (g) Final PPI Mass Final PPIMass (g) Moisture 7.0247 6.9978 7.0421 (%) Mean .200 0.32 7.02 0.02 STDV RSTDV

110 B6 Experiment #

Overall Mean: % PROTEIN DRY BASIS Overall RSTDV: Overall STDV: Overall Mean: % MOISTURE Overall RSTDV: Overall STDV: Overall Mean: % PROTEINAS IS

– Soy Protein Isolate –Protein Content (dry 1

02/14/2008 Date

84.63 90.47 0.03 6.46 0.16 0.14 0.40 Sample 004 063 9.3610 0.6432 3 10.0042 002 066 9.3567 0.6462 9.3247 2 10.0029 0.6461 1 9.9708 Initial Soy Mass (g) Moisture Mass basis;Nx6.25)andMoisture Content (g) Final Soy Mass Final Soy Mass (g) Moisture 6.4293 6.4601 6.4799 (%) Mean .600 0.40 6.46 0.03 STDV RSTDV 111 B7 –RTech PPI –Nitrogen Solub pH

.51.6 .0 5.65 0.60 5.45 10.66 0.42 0.04 5.48 9.40 0.13 0.02 5.99 14.31 0.37 0.05 6.06 14.36 0.25 0.05 6.49 21.51 0.20 0.05 7.01 23.45

Solubility Solubility Mean N (%) STDEV RSTDEV 22/06/2007 04/07/2007 09/07/2007 11/07/2007 10/07/2007 10/07/2007 Date lity Index(%) vs.Solution pH

Sample ln 00 00 000 .02055 .17 0.00 5.0022 0.2147 0.5452 0.000 0.00 Blank 20.00 0.00 5.0062 0.2149 0.5456 0.000 0.00 Blank 20.01 ln 19 01 000 .06055 .17 0.00 5.0016 0.2147 0.5451 0.000 0.13 Blank 21.92 0.00 5.0015 0.2147 0.5451 0.000 0.00 Blank 20.00 0.00 5.0004 0.2146 0.5450 0.000 0.00 Blank 20.00 0.00 5.0010 0.2147 0.5451 0.000 0.08 Blank 20.00 3 20.32 3.42 0.023 5.0016 0.5451 10.56 0.2147 3 20.01 3.00 0.020 5.0015 0.5451 9.35 0.2147 3 20.00 4.39 0.031 5.0004 0.5450 14.32 0.2146 3 20.00 4.50 0.031 5.0010 0.5451 14.41 0.2147 3 20.00 6.58 0.046 5.0022 0.5452 21.45 0.2147 3 20.00 12.61 2 20.00 7.19 0.050 5.0062 1 20.00 7.21 0.050 0.5456 5.0062 23.42 0.2149 0.5456 23.49 0.2149 2 25.20 4.50 0.024 5.0016 1 22.82 3.67 0.022 0.5451 5.0016 11.31 0.2147 0.5451 10.12 0.2147 2 20.00 3.02 0.020 5.0015 1 20.01 3.02 0.020 0.5451 9.42 5.0015 0.2147 0.5451 9.42 0.2147 2 20.00 4.38 0.031 5.0004 1 20.00 4.39 0.031 0.5450 5.0004 14.28 0.2146 0.5450 14.32 0.2146 2 20.00 4.48 0.031 5.0010 1 20.01 4.47 0.031 0.5451 5.0010 14.35 0.2147 0.5451 14.31 0.2147 2 20.00 6.61 0.046 5.0022 1 20.01 6.61 0.046 0.5452 5.0022 21.55 0.2147 0.5452 21.54 0.2147 Mass Liq. (g)

Volume Titrant (ml) brwn soln Final N mixed w Conc. Discrd: dstllte (%)

.02055 024 N/A 0.2149 5.0062 0.5456 Initial Mass PPI (g) Initial mass (g) N Initial N Conc. (%) Solubility Solubility (%) N 112 B7 –(continued)RTech PPI–Nitrogen pH

.298 00 0.93 0.09 5.02 9.83 .959 01 2.71 0.16 3.99 5.95 0.97 0.07 4.01 6.71 0.68 0.05 4.48 6.83 0.67 0.07 5.00 10.37 1.70 0.16 5.00 9.50

Solubility Solubility Mean N (%) STDEV RSTDEV 22/06/2007 21/06/2007 28/06/2007 25/06/2007 20/06/2007 09/07/2007 Date

Solublity Index(%)vs.SolutionpH Sample ln 00 00 000 .08 .44 .18 0.00 0.2148 0.5454 0.00 5.0038 0.000 0.2146 0.00 0.5450 5.0005 20.00 Blank 0.000 0.30 10.31 Blank ln .0 .8 .0 .08055 .17 0.00 0.00 5.0028 0.000 0.5453 0.2147 0.18 Blank 9.40 0.2147 0.00 0.5451 5.0006 0.2147 5.0013 0.5450 0.000 0.00 0.00 Blank 20.01 0.000 0.2150 0.11 0.5460 20.05 5.0092 Blank 0.000 0.00 9.72 Blank 3 10.13 1.52 0.021 5.0005 0.5450 9.79 0.2146 3 9.50 1.02 0.012 5.0028 0.5453 5.76 0.2147 3 20.02 2.22 0.014 5.0006 0.5450 6.65 0.2147 3 20.02 2.22 0.015 5.0013 0.5451 6.87 0.2147 4 10.00 1.60 0.022 5.0092 0.5460 10.42 0.2150 3 20.02 N/A 1 20.00 2.95 0.021 5.0038 0.5454 9.61 0.2148 2 10.28 1.54 0.021 5.0005 1 8.14 1.24 0.021 0.5450 9.77 5.0005 0.2146 0.5450 9.94 0.2146 2 9.49 1.06 0.013 1 9.28 5.0028 1.04 0.013 5.0028 0.5453 6.05 0.2147 0.5453 6.04 2 20.02 0.2147 2.26 0.015 5.0006 1 20.01 2.24 0.014 0.5450 6.78 5.0006 0.2147 0.5450 6.71 0.2147 2 20.00 2.19 0.015 5.0013 1 20.01 2.21 0.015 0.5451 6.78 5.0013 0.2147 0.5451 6.84 0.2147 3 8.98 1.48 0.023 2 9.28 5.0092 1.58 0.024 1 9.59 0.5460 5.0092 1.52 0.022 10.73 0.2150 0.5460 5.0092 11.09 0.2150 0.5460 10.32 0.2150 2 20.00 2.88 0.020 5.0038 0.5454 9.39 0.2148 Mass Liq (g) Volume Titrant (mL) Final N pink soln Conc Discrd: (%) .08055 024 N/A 0.2148 5.0038 0.5454 Initial Mass PPI (g) Initial mass (g) N Conc (%) Initial N Solubility Solubility (%) N 113 B7 –(continued)RTech PPI–Nitrogen pH

. 48 02 0.81 0.28 2.5 34.84 1.36 0.18 3.5 13.22 80 06 1.73 0.66 2 38.05 3.12 0.69 3 22.12 0.11 0.02 3 20.48

Solubility Solubility Mean N (%) STDEV RSTDEV 21/06/2007 21/06/2007 04/07/2007 29/06/2007 04/07/2007 Date

Solublity Index(%)vs.SolutionpH Sample ln 00 00 000 .02 .44 .18 0.00 0.2148 0.5454 5.0042 0.000 0.00 20.01 Blank ln .3 .9 .0 499 054 024 0.00 0.2146 0.5449 4.9996 0.00 0.000 0.2147 0.29 0.5452 0.00 9.63 5.0019 Blank 0.000 0.2146 0.00 0.5450 5.0004 20.01 Blank 0.000 0.00 0.00 0.2146 9.46 0.5450 5.0004 Blank 0.000 0.00 20.00 Blank 2 20.01 4.02 0.028 5.0042 1 20.02 4.10 0.029 0.5454 5.0042 13.09 0.2148 0.5454 13.35 0.2148 2 9.23 5.78 0.083 1 10.12 4.9996 6.12 0.081 4.9996 0.5449 0.5449 38.80 0.2146 37.58 0.2146 2 20.01 11.04 0.075 1 20.01 5.0019 11.02 0.075 0.5452 5.0019 35.03 0.2147 0.5452 34.97 0.2147 2 9.78 3.39 0.049 1 9.98 5.0004 3.31 0.046 0.5450 5.0004 22.61 0.2146 0.5450 21.63 0.2146 2 20.02 6.28 0.044 5.0004 1 20.02 6.29 0.044 0.5450 5.0004 20.46 0.2146 0.5450 20.49 0.2146 3 10.57 6.41 0.081 4.9996 0.5449 37.77 0.2146 3 9.39 4.11 3 20.01 0.00 3 13.85 7.62 0.074 5.0019 0.5452 34.51 0.2147 3 20.00 0.00 Mass Liq (g)

Volume Titrant (mL) Final N Pink, like overshot Discard: Discard: Discard: Conc Outlier blank (%) .04055 024 N/A 0.2146 5.0004 0.5450 0.00 0.2146 5.0004 0.5450 0.00 0.2148 5.0042 0.5454 Initial Mass PPI (g) Initial mass (g) N Conc (%) Initial N Solubility Solubility (%) N B8 – Texas PPI –Nitrogen Solub

5.06 4.58 4.09 3.38 3.03 2.00 114 pH

Solubility Solubility Average 23.69 0.32 1.36 23.69 0.32 1.87 32.02 0.60 3.03 37.10 1.12 4.14 0.06 1.38 3.22 0.15 4.72 9.60 0.07 0.75 (%) N STDEV RSTDEV 15/01/2006 20/01/2006 20/01/2006 25/01/2006 31/01/2006 23/01/2006 Date

lity Index(%) vs.Solution pH Sample Blank 15.35 0.26 0.000 134. 0.000 0.26 Blank 15.35 ln 65 00 000 88. 0.000 0.00 Blank 16.50 95. 0.000 0.00 Blank 19.08 102. 0.000 0.00 Blank 15.50 99. 0.000 0.00 Blank 10.07 110. 0.000 0.40 Blank 15.37 2 13.95 33.40 0.333 1 14.91 134.10 33.80 0.315 134.10 1 15.98 6.10 0.053 88.20 2 18.11 4.98 0.038 1 18.56 95.37 5.42 0.041 95.37 2 15.82 12.12 1 15.97 0.107 12.40 102.85 0.109 102.85 2 10.26 19.92 1 10.80 0.272 99.57 20.98 0.272 99.57 2 15.66 38.22 0.338 1 16.10 110.62 39.70 0.342 110.62 3 10.24 20.36 0.278 99.57 3 16.63 39.54 0.330 110.62 3 14.92 35.58 0.331 134.10 2 15.89 5.98 0.053 88.20 3 19.83 5.28 0.037 95.37 3 15.78 12.10 0.107 102.85 Mass Liq (g) Volume Titrant (mL) Final N Conc (%) Mass(g) Water Dist 01.1128 121 0.00 1.2919 20 10.01 1.2688 0.00 1.2095 37 10.06 1.2751 0.00 1.1568 57 10.00 1.2675 01.0 .65 .76 0.00 0.8796 1.2675 10 10.00 51.0 .65 .22 0.00 1.1232 1.2675 85 10.00 0.00 1.0508 1.2675 62 10.00 Initial Mass 00 .65 .76 37.81 0.8796 10.00 1.2675 35.80 0.8796 10.00 1.2675 00 .68 .99 4.14 1.2919 10.01 1.2688 3.18 1.2095 3.38 10.06 1.2751 1.2095 10.06 1.2751 9.55 1.1232 9.68 10.00 1.2675 1.1232 10.00 1.2675 23.50 1.1568 10.00 1.2675 23.51 1.1568 10.00 1.2675 32.18 1.0508 10.00 1.2675 32.52 1.0508 10.00 1.2675 00 .65 .58 24.06 1.1568 10.00 1.2675 31.36 1.0508 10.00 1.2675 37.68 0.8796 10.00 1.2675 00 .68 .99 4.08 1.2919 10.01 1.2688 3.08 1.2095 10.06 1.2751 9.56 1.1232 10.00 1.2675 PPI (g) mass (g) Initial N Conc (%) Initial N Solubility Solubility (%) N B9 –SoyProtein Isolate (Supro 5.95 5.50

pH 115 3 20.18 0.22 3 20.18 1.07 15/02/2008

Average N Solubility Solubility 7.85 0.06 0.77 6.56 0.09 (%) STDEV RSTDEV 1.38

31/01/2006 25/01/2006 Date 500E, Solae™)–Nitrogen Sol

Sample ln 64 00 000 92. 0.000 0.00 Blank 16.41 92. 0.000 0.00 Blank 18.03 Blank 20.02 0.32 0.000 5.0015 0.000 0.00 0.6772 0.32 0.2667 Blank 20.02 3 18.95 10.90 0.081 92.61 2 16.37 11.50 1 15.73 0.098 92.08 10.94 0.097 92.08 2 17.97 10.62 1 17.86 0.083 92.61 10.46 0.082 92.61 3 15.82 6.12 0.054 88.20 3 15.84 10.96 0.097 92.08 3 20.01 2 20.02 7.96 0.053 1 20.03 8.08 5.0015 0.054 0.6772 5.0015 0.6772 0.2667 20.03 0.2667 20.34 Liq Mass (g) cloudy pink Volume Titrant Discard: (mL) ublity Index(%)atpH3 Final N Conc N/A 5.0015 0.6772 N/A 0.2667 (%) 81.1128 122 0.00 1.2428 08 10.01 1.2688 0.00 1.2464 61 10.10 1.2802 Initial PPI Mass (g) 01 .82 .44 6.46 1.2464 10.10 1.2802 00 .68 .48 7.91 1.2428 7.83 10.01 1.2688 1.2428 10.01 1.2688 6.64 1.2464 6.58 10.10 1.2802 1.2464 10.10 1.2802 4.19 1.2919 10.01 1.2688 00 .68 .48 7.79 1.2428 10.01 1.2688 mass (g) Initial N Initial N Conc (%) N Solubility (%) 116 B10 –Rtech SPI andPPIinCola – Viscosity Measurements (incP) STDEV 0.009 Mean 1.226

Control .2 .1 1.225 1.247 1.212 1.235 1.203 1.219 1.227 1.212 1.206 1.226 1.203 1.221 1.230 1.213 1.226 1.224 1.220 1.196 1.220 1.216 1.225 1.229 1.216 1.245 1.216 1.220 1.234 1.230 1.217 1.223 1.212 1.221 1.211 Measurement 1.218 1.219 1.220 1.215 1.194 1.217 1.197 1.211 1.206 1.206 .2 .1 1.211 1.232 1.237 1.240 1.225 1.241 1.232 1.244 1.244 1.228 1.229 1.214

1.223 1.225 1.214 1.239 1.228

STDEV 0.008 Mean 1.212 1% SPI 1% SPI .2 1.208 1.217 1.242 1.222 1.226 1.215 1.205 1.218 1.220 1.214 1.201 1.208 STDEV 0.018 Mean 1.217 1% PPI 1% PPI 1.202 1.195 1.196 1.189 1.210 1.193 1.214 1.208 1.216 1.214 1.227 1.219

s Prevented byClogging 1% MR 117 B11 –RtechSPIandPPIin Apple Juic

SDTEV 0.012 Mean 1.259 1.285 1.245 1.274 1.287 1.253 1.269 1.281 1.268 1.259 1.280 1.268 1.246 1.259 1.291 1.266 1.259 1.302 1.277 1.256 1.322 1.260 1.223 1.254 1.306 1.227 1.252 1.301 1.229 1.247 1.306 1.248 1.231 1.253 1.279 1.245 1.252 1.291 1.243 1.248 1.298 1.256 1.203 1.252 1.278 1.206 1.245 1.281 1.206 1.236 1.276 1.271 1.190 1.272 1.240 1.188 1.274 1.239 1.189 1.275 1.238 Control 1.272 1.261 1.253 1.250 1.247 1.273 SDTEV 0.023 Mean 1.280 1.268 1.281 1.295 1.274 1.266 1.240 1% SPI 1% SPI e – Viscosity Measurements (in cP) 1.279 1.299 1.302 1.286 1.275 1.239 SDTEV 0.026 Mean 1.227 1.231 1.236 1.220 1.224 1.202 1.192 1% PPI 1% PPI 1.249 1.256 1.225 1.236 1.204 1.190

Measurements Prevented by Clogging 1% MR

118 B12 –RtechSPIandPPIinFlavoured Carbonat

STDEV 0.011 Control -Blending Mean 1.055 1.073 1.079 1.178 1.065 1.196 1.066 1.200 1.064 1.067 1.176 1.061 1.183 1.049 1.187 1.068 1.211 1.066 1.171 1.108 1.183 1.059 1.175 0.068 1.190 1.058 1.181 1.113 1.127 1.063 1.146 1.178 1.057 1.175 1.146 1.127 1.189 1.057 1.189 1.117 1.192 1.048 1.199 1.146 1.111 1.052 1.115 1.167 1.048 1.175 1.091 1.134 1.214 1.046 1.179 1.099 1.188 1.041 1.187 1.110 1.053 1.154 1.049 1.187 1.088 1.178 1.045 1.191 1.091 1.191 1.043 1.194 1.098 1.048 1.152 1.044 1.198 1.074 1.154 1.040 1.201 1.081 1.158 1.032 1.205 1.090 1.071 1.060 1.063 1.056 1.047 1.048 1.041 Control -No Blending STDEV 0.011 Mean 1.185 1.171 1.174 1.172 1.186 1.191

1.174 1.184 1.178 1.189 1.199 STDEV 0.018 Mean 1.176 1.155 1.154 1.172 1.166 1.160 1.163 1.147 1% SPI 1% SPI ed Water– Viscosity cP) Measurements(in 1.182 1.175 1.189 1.181 1.187 1.171 1.152 STDEV 0.197 Mean 1.069 1.105 1.104 1.103 1.094 1.091 1.086 1.077 1% PPI 1% PPI 1.128 1.116 0.852 1.121 1.098 1.091 1.080

Measurements Prevented by Clogging 1% MR 119 B13 –Rtech SPI andPPIinCarbonated

Control -Blending 0.871 1.255 0.870 0.889 1.074 1.255 0.869 0.890 1.087 1.236 0.866 0.896 1.090 0.869 1.211 0.869 0.886 1.053 1.205 0.867 0.887 1.070 1.195 0.868 0.892 1.072 0.870 1.164 0.873 0.888 1.035 1.166 0.869 0.888 1.041 1.157 0.890 0.892 1.051 0.895 1.132 0.867 0.891 1.020 1.111 0.899 0.890 1.021 1.111 0.863 0.894 1.024 0.871 1.050 0.869 0.891 0.990 1.054 0.865 0.894 1.001 1.049 0.866 0.894 1.008 0.870 1.016 0.869 0.910 0.959 1.026 0.886 0.912 0.965 1.018 0.889 0.906 0.978 0.869 0.868 0.876 0.881 0.868 0.878 Control -No Blending 0.892 0.887 0.886 0.888 0.897 0.908 0.892 0.888 0.888 0.891 0.894 0.909

Water – Viscosity Measurements (in cP) 1.259 1.215 1.172 1.130 1.053 1.008 1% SPI 1% SPI 1.251 1.207 1.165 1.121 1.051 1.017 1.067 1.048 1.029 1.006 0.979 0.954 1% PPI 1% PPI 1.079 1.061 1.039 1.018 0.995 0.964

Measurements Prevented by Clogging 1% MR 120 B13 –(Continued)RtechSPIand

STDEV 0.009 Control -Blending Mean 0.874 0.873 0.874 0.871 0.873 0.872 0.881 0.890 0.868 0.873 0.878

Control -No Blending STDEV 0.008 Mean 0.894 PPI inCarbonated Water – Viscosity Measurements (incP)

STDEV 0.084 Mean 1.135 1% SPI 1% SPI STDEV 0.052 Mean 1.048 1.113 1.129 1.137 1.091 1.100 1.112 1.113 1.107 1% PPI 1% PPI 1.121 1.104

1% MR 121 B14 –Rtech SPI and PPI in Water – Viscosity Measurements (incP)

Control -No Blending STDEV 0.010 Mean 0.967 .6 .2 0.923 0.963 1.023 0.938 0.967 1.025 0.940 0.968 1.030 0.980 0.946 0.978 1.034 0.945 0.979 1.044 0.959 0.979 1.062 0.975 0.931 0.975 1.046 N/A 0.975 1.059 0.957 0.967 1.067 0.999 1.059 0.969 1.042 1.031 0.965 1.056 1.029 0.973 1.065 0.957 0.944 0.957 1.034 0.933 0.957 1.047 0.951 0.959 1.057 0.957 0.930 0.955 1.033 0.940 0.955 1.045 0.950 0.956 1.053 0.962 0.932 0.962 1.016 0.943 0.961 1.021 0.951 0.960 1.030 0.967 0.979 0.973 0.976 0.958 0.956 0.961 0.966 STDEV 0.015 Mean 1.039 1.033 1.041 1.036 1.030 1.026 1.017 1.018 1% SPI 1% SPI 1.043 1.053 1.050 1.042 1.039 1.021 1.024

STDEV 0.050 Mean 0.957 0.941 0.925 1.138 0.943 0.925 0.926 0.920

1% PPI 1% PPI 0.948 0.938 1.064 0.943 0.936 0.938 0.930 Measurements Prevented by Clogging 1% MR

122 B15 –RtechSPIinOrangeJuice

STDEV 0.115 Control -Blending Mean 2.236 2.357 2.368 2.144 2.735 2.231 2.106 2.885 2.164 2.107 2.664 2.224 2.347 2.116 2.518 2.241 2.102 2.528 2.368 2.093 2.813 2.351 2.282 2.109 2.537 2.344 2.105 2.418 2.195 2.109 2.655 2.113 2.100 2.272 2.470 2.051 2.220 2.468 2.048 2.226 2.426 2.280 2.295 2.293 2.078 Control -No Blending STDEV 0.060 Mean 2.155 2.163 2.238 2.145 2.222 2.130 2.137 2.117 2.235 – Viscosity Measurements (incP) STDEV 0.157 Mean 2.599 2.697 2.836 2.476 2.450 1% SPI 1% SPI

2.745 2.674 2.521 2.454

Prevented by Clogging Measurements 1% PPI 1% PPI

Prevented by Clogging Measurements 1% MR 123 B16 –RtechPPIandSPIinCranberryJu

Control -Blending STDEV Mean 1.222 1.212 1.213 1.223 1.203 1.204 1.201 1.201 1.201 1.205 1.193 1.177 1.197 1.205 1.212 1.194 1.197 1.191 1.190 1.186 1.201 1.198 1.194 1.188 0.011 1.200 1.217 1.202 1.194 1.202 1.191 1.195 Control -No Blending STDEV Mean 1.363 1.370 1.395 1.369 1.362 1.360 1.357 1.364 1.353 1.351 1.351 1.350 1.352 1.362 1.356 1.351 1.368 1.369 1.375 1.380 1.370 1.370 1.374 1.380 0.011 1.365 1.374 1.361 1.351 1.355 1.373 1.374 STDEV Mean 1.369 1.380 1.384 1.406 1.342 1.355 1.367 1.374 1.458 1.457 1.464 1.478 1.431 1.441 1.445 1.457 1.390 1.407 1.402 1.481 1.407 1.421 1.427 1.439 ice – Viscosity Measurements (incP) 1% SPI 1% SPI 1.385 1.360 0.040 1.416 1.464 1.443 1.420 1.424 STDEV Mean 1.262 1.269 1.278 1.313 1.251 1.265 1.268 1.292 1.287 1.298 1.308 1.335 1.280 1.288 1.302 1.324 1.280 1.284 1.306 1.339 1.282 1.287 1.305 1.326 1% PPI 1% PPI 1.280 1.269 1.307 1.299 1.302 1.300 0.023 1.293

Measurements Prevented by Clogging 1% MR 124 B17 – Viscosity of1%PPI vs. Number ofBlendingSessions

STDEV Mean .9 .7 11 1.131 1.148 1.1 0.996 1.071 1.091 0.987 1.065 1.127 1.146 1.088 0.971 1.058 1.121 1.134 1.076 0.978 1.076 1.13 1.122 0.011 0.011 0.012 0.011 0.007 0.009 1.092 0.983 1.074 1.129 1.140 1 2 3 1 2 4 1.088 1.088 1.137 1.084 1.107 1.136 1% PPI–Number ofBlending Sessions TE .0 .0 0.054 0.096 1.393 3.149 0.027 0.005 1.002 1.845 STDEV 0.007 Mean 0.955

oto Wtr %PI %PI %PI 4% PPI 3% PPI 2% PPI 1% PPI Control (Water) 0.9549 1.001 1.361 0.9474 1.002 1.82 1.321 0.9612 1.009 3.09 1.302 1.825 1.872 3.183 3.302 B19 – Viscosity vs.PPIConcentration .3 185 1.885 3.109 1.868 1.85 1.433 1.821 3.063 1.445 1.419 1.423 1.821 1.386 0.997 .4 1.449 5 B18 – Viscosity of4%PPIat0hand24 TE .9 0.054 STDEV 0.096 3.013 Mean 3.149 3.109 2.995 3.063 2.987 3.090 2.980 3.183 2.983 3.302 3.121 0h 24h 0h 125 B22 –NSIatpH3vs. Alcalase Treatment Temperat B20 –Distilled Water pHMeasurements

Treatment Temp. 50-55 ( o 60 C) 28-Jun-07 6.38 20-Jun-07 5.95 27-Jul-07 5.46 11-Jul-07 5.97 04-Jul-07 5.99 04-Jul-07 6.01 STDEV 0.29 Mean 5.96 Date Solubility Solubility Mean N 61.57 56.82 (%) STDEV 0.25 0.12 pH

RSTDEV 0.41 0.20

Sample Blank Blank 3 2 1 3 2 1 Liq Mass 20.02 20.03 20.01 20.01 20.02 20.01 20.00 20.01 (g) Volume B21 –pHof2%RTech Titrant 19.18 19.18 19.30 16.88 16.86 16.80 (mL) 0.32 0.24 20-Jun-07 5.63 25-Jun-07 5.40 22-Jun-07 5.82 22-Jun-07 5.63 21-Jun-07 6.04 21-Jun-07 6.02 21-Jun-07 5.62 20-Jun-07 5.62 ure (RTech PPI,1h,pH 8.5,0.5 AU/ 5gPPI) 20-Apr-07 5.54 19-Apr-07 5.64 18-Apr-07 5.64 26-Apr-07 5.63 13-Apr-07 5.79 26-Apr-07 5.61 18-Apr-07 5.60 18-Apr-07 5.58 Date Final N 0.132 0.132 0.133 0.000 0.116 0.116 0.116 0.000 Conc (%) pH Mass (g) 5.0007 5.0007 5.0007 5.0007 4.7591 4.7591 4.7591 4.7591 Initial PPI 1Sp0 5.98 5.98 21-Sep-07 6.01 21-Sep-07 6.00 20-Sep-07 6.00 20-Sep-07 20-Sep-07 29-Jun-07 5.84 28-Jun-07 6.03 28-Jun-07 5.82 27-Jun-07 5.99 27-Jun-07 5.77 27-Jul-07 5.49 11-Jul-07 5.67 04-Jul-07 5.96 04-Jul-07 6.04 04-Jul-07 5.85 mass (g) Initial N PPI Aqueous Solutions STDEV 0.5450 0.5187 0.5187 0.5187 0.5187 0.5450 0.5450 0.5450 Mean Conc (%) Initial N 0.19 5.78 0.2147 0.2045 0.2045 0.2045 0.2045 0.2147 0.2147 0.2147 N Solub. 56.91 56.87 56.69 61.44 61.41 61.86 0.00 0.00 (%) 126 B23 –NSIatpH 3vs. Alcalase Alcalase AU / 5 g AU/5

Conc. PPI 0.8 0.5 0.3 0.0 Average N Solub 62.78 56.82 55.26 20.48 (%) STDEV 0.34 0.12 0.18 0.02 RSTDEV 0.54 0.20 0.33 0.11 Treatment(RTech Concentration Sample Blank Blank Blank Blank 1 3 2 1 3 2 1 3 2 1 3 2 Liq Mass 20.03 20.03 20.01 20.02 20.02 20.00 20.03 20.02 20.03 20.02 20.02 20.01 20.00 20.01 20.02 20.02 (g) Volume Titrant 17.00 19.18 19.38 19.28 16.88 16.86 16.80 17.00 16.90 (mL) 0.00 0.00 6.28 6.29 0.00 0.00 0.24 Conc (%) Final N 0.119 0.000 0.000 0.044 0.044 0.000 0.134 0.136 0.135 0.000 0.116 0.116 0.116 0.000 0.119 0.118 PPI,1h,pH8.5,50-55 Initial PPI Mass (g) 5.0016 5.0016 5.0016 5.0016 4.7591 4.7591 4.7591 4.7591 5.0014 5.0014 5.0014 5.0014 5.0004 5.0004 5.0004 5.0004 mass (g) Initial N 0.5451 0.5451 0.5451 0.5451 0.5187 0.5187 0.5187 0.5187 0.5451 0.5451 0.5451 0.5451 0.5450 0.5450 0.5450 0.5450 Conc (%) Initial N 0.2147 0.2147 0.2147 0.2147 0.2045 0.2045 0.2045 0.2045 0.2147 0.2147 0.2147 0.2147 0.2146 0.2146 0.2146 0.2146 o C) N Solub 62.44 63.12 62.77 56.91 56.87 56.69 55.37 55.05 55.35 20.46 20.49 0.00 0.00 0.00 0.00 0.00 (%) 127 B24 –NSIatpH3vs. Alcalase Treatm Treatment

Time 16.0 0.0 8.0 4.0 2.0 0.5 (h) Solubility Solubility Mean N 72.40 69.29 52.99 20.48 76.44 74.54 (%) STDEV 0.21 0.21 0.10 0.02 0.90 0.08 RSTDEV 0.18 1.18 0.10 0.29 0.31 0.11 Sample Blank Blank Blank Blank Blank Blank 2 1 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 3 ent Time (RTech PPI,pH8.5,50-55 Mass (g) 20.01 20.01 20.00 20.01 20.02 20.02 20.00 20.02 20.03 20.04 20.02 20.03 20.01 20.01 20.01 20.04 20.03 20.02 20.03 20.00 20.00 20.01 20.02 20.02 Liq Volume Titrant 16.40 16.34 23.24 23.40 23.80 22.88 22.90 22.86 20.20 20.08 20.16 21.36 21.44 21.32 16.38 0.12 0.00 6.28 6.29 0.00 0.00 0.00 0.00 0.12 (ml) Final N 0.114 0.113 0.000 0.000 0.044 0.044 0.000 Conc 0.163 0.164 0.166 0.000 0.160 0.160 0.160 0.000 0.141 0.140 0.141 0.000 0.149 0.149 0.148 0.000 0.114 (%) Mass (g) 5.0019 5.0019 5.0019 5.0015 5.0015 5.0015 5.0015 4.5222 4.5222 4.5222 4.5222 5.0012 5.0012 5.0012 5.0012 4.9986 4.9986 4.9986 4.4680 5.0004 5.0004 5.0004 5.0004 5.0019 Initial PPI o mass (g) Initial N C, 0.5 AU / 5gPPI) 0.5452 0.5452 0.5452 0.5451 0.5451 0.5451 0.5451 0.4929 0.4929 0.4929 0.4929 0.5451 0.5451 0.5451 0.5451 0.5448 0.5448 0.5448 0.4870 0.5450 0.5450 0.5450 0.5450 0.5452 Conc. (%) Initial N 0.2147 0.2147 0.2147 0.2147 0.2147 0.2147 0.2147 0.1945 0.1945 0.1945 0.1945 0.2147 0.2147 0.2147 0.2147 0.2146 0.2146 0.2146 0.1922 0.2146 0.2146 0.2146 0.2146 0.2147 Solub. 76.18 77.44 74.49 74.63 74.50 72.56 72.17 72.49 69.26 69.52 69.09 52.99 53.08 52.89 20.46 20.49 75.69 0.00 0.00 0.00 0.00 0.00 0.00 0.00 (%) N 128 B25 –SolubilityCurveof Alcalase -Treat

NSI pH 3.0 62.78 0.34 0.54 2.5 63.87 0.04 0.06 3.5 67.92 0.51 0.75 Solubility Solubility Average (%) N STDEV RSTDEV Sample ln 2.1 .0 .0 501 .41024 0.00 5.00110.54510.2147 0.000 0.00 20.01 Blank ln 2.3 .0 .0 502 .42024 0.00 5.00200.54520.2147 0.000 0.00 20.03 Blank 0.00 5.00160.54510.2147 0.000 0.00 20.02 Blank 20.0019.600.137 5.0011 2 0.5451 20.0219.600.137 5.0011 1 0.2147 63.91 0.5451 0.2147 63.85 20.0320.880.146 5.0020 2 0.5452 20.0320.700.145 5.0020 1 0.2147 67.97 0.5452 0.2147 67.38 20.0219.380.136 5.0016 2 0.5451 20.0319.280.135 5.0016 1 0.2147 63.12 0.5451 0.2147 62.77 20.0319.180.134 5.0016 3 0.5451 0.2147 62.44 20.0219.600.137 5.0011 3 0.5451 0.2147 63.85 20.0221.00 0.147 5.0020 3 0.5452 0.2147 68.40 Liq Mass ed PPI(RTech PPI,1h,pH8.5,50-55 (g) Volume Titrant (ml) Final N Conc (%) Mass (g) Initial PPI mass (g) Initial N o C, 0.8 AU /5gPPI) Initial N Conc (%) Solubility Solubility (%) N 129

B26 – Moles of Amino Acids per g of RTech PPI

Amino Acid AA picomoles* AA moles AA MW AA mass

Asp 159453.936 1.59454E-07 133.1 2.12233E-05 Glu 248315.450 2.48315E-07 146.1 3.62789E-05

Ser 121768.103 1.21768E-07 147.1 1.79121E-05

Gly 228888.808 2.28889E-07 75.1 1.71895E-05 His 61461.122 6.14611E-08 155.2 9.53877E-06 Arg 115344.067 1.15344E-07 174.2 2.00929E-05 Thr 109694.264 1.09694E-07 119.1 1.30646E-05

Ala 157137.061 1.57137E-07 89.1 1.40009E-05

Pro 147752.723 1.47753E-07 115.1 1.70063E-05 Tyr 69451.562 6.94516E-08 181.2 1.25846E-05 Val 143877.572 1.43878E-07 117.1 1.68481E-05 Met 37074.830 3.70748E-08 149.2 5.53156E-06 Cys 8964.546 8.96455E-09 121.1 1.08561E-06

Ile 116950.463 1.1695E-07 131.2 1.53439E-05 Leu 205540.653 2.05541E-07 131.2 2.69669E-05 Phe 113905.610 1.13906E-07 165.2 1.88172E-05 Lys 173005.159 1.73005E-07 146.2 2.52934E-05 Nleu 3125.000 3.125E-09 131.2 4.10E-07

RTech PPI Sample Mass* = 0.525 mg

RTech PPI protein wt%: 69.12

Mass of protein in sample = 0.0003623 g

Moles of AA per g of RTech PPI protein: 0.00612

130

B27 – % Degrees of Hydrolysis vs. Alcalase Treatment Time Experiment 1 (Alcalase Treatment: 50-55oC, 0.5 AU / 5 g of RTech PPI, pH 8.5, variable time)

Note: Aliquots were removed and flash frozen at: 0.5 h, 4 h, 8 h, 16 h

Cumulative Moles of Mass % Deg. Time Time Moles of Mass Volume of NaOH Protein of (h) (min) NaOH PPI (g) NaOH (ml) (sectioned) (g) Hydrolysis 0.00 0.00 0.00 0.0000 0.0000 20 13.824 0.00 0.06 3.45 22.72 0.0045 0.0045 20 13.824 5.55 0.06 3.87 24.82 0.0050 0.0050 20 13.824 6.06 0.08 4.87 27.00 0.0054 0.0054 20 13.824 6.59 0.09 5.42 28.44 0.0057 0.0057 20 13.824 6.94 0.10 6.07 30.12 0.0060 0.0060 20 13.824 7.35 0.13 7.82 35.68 0.0071 0.0071 20 13.824 8.71 0.15 8.87 38.82 0.0078 0.0078 20 13.824 9.48 0.16 9.80 41.12 0.0082 0.0082 20 13.824 10.04 0.17 10.35 42.12 0.0084 0.0084 20 13.824 10.28 0.20 11.80 44.92 0.0090 0.0090 20 13.824 10.97 0.21 12.77 46.84 0.0094 0.0094 20 13.824 11.43 0.23 13.57 47.92 0.0096 0.0096 20 13.824 11.70 0.24 14.37 49.26 0.0099 0.0099 20 13.824 12.02 0.27 16.17 51.88 0.0104 0.0104 20 13.824 12.66 0.30 17.70 53.58 0.0107 0.0107 20 13.824 13.08 0.32 19.38 55.80 0.0112 0.0112 20 13.824 13.62 0.35 20.82 57.36 0.0115 0.0115 20 13.824 14.00 0.38 22.90 59.92 0.0120 0.0120 20 13.824 14.63 0.41 24.67 61.94 0.0124 0.0124 20 13.824 15.12 0.43 25.97 63.38 0.0127 0.0127 20 13.824 15.47 0.44 26.68 63.78 0.0128 0.0128 20 13.824 15.57 0.46 27.77 65.00 0.0130 0.0130 20 13.824 15.87 0.48 28.65 65.84 0.0132 0.0132 20 13.824 16.07 0.49 29.10 66.24 0.0132 0.0132 20 13.824 16.17 0.60 36.03 70.00 0.0140 0.0008 15 10.368 17.39 0.62 36.97 70.60 0.0141 0.0009 15 10.368 17.59 0.63 38.00 71.28 0.0143 0.0010 15 10.368 17.81 0.69 41.42 73.34 0.0147 0.0014 15 10.368 18.48 0.71 42.70 74.06 0.0148 0.0016 15 10.368 18.72 0.74 44.15 74.82 0.0150 0.0017 15 10.368 18.96 0.76 45.78 75.68 0.0151 0.0019 15 10.368 19.24 0.79 47.23 76.54 0.0153 0.0021 15 10.368 19.52 0.81 48.62 77.42 0.0155 0.0022 15 10.368 19.81 0.83 49.82 78.10 0.0156 0.0024 15 10.368 20.03 0.85 50.77 78.80 0.0158 0.0025 15 10.368 20.26 0.87 52.22 79.48 0.0159 0.0026 15 10.368 20.48 0.92 55.25 80.78 0.0162 0.0029 15 10.368 20.90 0.94 56.62 81.30 0.0163 0.0030 15 10.368 21.07

131

0.98 58.88 82.08 0.0164 0.0032 15 10.368 21.33 1.01 60.68 82.72 0.0165 0.0033 15 10.368 21.53 1.04 62.63 83.42 0.0167 0.0034 15 10.368 21.76 1.07 64.33 83.90 0.0168 0.0035 15 10.368 21.92 1.11 66.83 84.66 0.0169 0.0037 15 10.368 22.17 1.15 68.90 85.24 0.0170 0.0038 15 10.368 22.35 1.19 71.32 85.88 0.0172 0.0039 15 10.368 22.56 1.23 73.72 86.48 0.0173 0.0040 15 10.368 22.76 1.26 75.43 87.04 0.0174 0.0042 15 10.368 22.94 1.28 76.55 88.08 0.0176 0.0044 15 10.368 23.28 1.32 78.98 88.52 0.0177 0.0045 15 10.368 23.42 1.34 80.57 88.98 0.0178 0.0045 15 10.368 23.57 1.41 84.85 89.64 0.0179 0.0047 15 10.368 23.79 1.45 86.83 89.94 0.0180 0.0047 15 10.368 23.88 1.49 89.20 90.70 0.0181 0.0049 15 10.368 24.13 1.51 90.58 91.30 0.0183 0.0050 15 10.368 24.33 1.54 92.30 91.88 0.0184 0.0051 15 10.368 24.52 1.58 94.72 92.36 0.0185 0.0052 15 10.368 24.67 1.60 96.28 92.58 0.0185 0.0053 15 10.368 24.74 1.64 98.23 92.70 0.0185 0.0053 15 10.368 24.78 1.70 102.15 93.40 0.0187 0.0054 15 10.368 25.01 1.71 102.82 93.70 0.0187 0.0055 15 10.368 25.11 1.74 104.63 94.12 0.0188 0.0056 15 10.368 25.24 1.76 105.42 94.80 0.0190 0.0057 15 10.368 25.47 1.78 106.55 95.16 0.0190 0.0058 15 10.368 25.58 1.81 108.73 95.50 0.0191 0.0059 15 10.368 25.69 1.83 109.60 95.62 0.0191 0.0059 15 10.368 25.73 1.89 113.53 96.12 0.0192 0.0060 15 10.368 25.90 1.94 116.38 96.56 0.0193 0.0061 15 10.368 26.04 1.97 117.95 96.82 0.0194 0.0061 15 10.368 26.12 1.99 119.23 97.02 0.0194 0.0062 15 10.368 26.19 2.05 122.95 97.56 0.0195 0.0063 15 10.368 26.36 2.11 126.38 98.14 0.0196 0.0064 15 10.368 26.55 2.19 131.43 98.92 0.0198 0.0065 15 10.368 26.81 2.31 138.37 100.04 0.0200 0.0068 15 10.368 27.17 2.38 142.97 100.82 0.0202 0.0069 15 10.368 27.43 2.51 150.37 101.92 0.0204 0.0071 15 10.368 27.78 2.62 157.33 102.70 0.0205 0.0073 15 10.368 28.04 2.67 160.10 103.06 0.0206 0.0074 15 10.368 28.15 2.75 164.87 103.50 0.0207 0.0075 15 10.368 28.30 2.84 170.18 104.36 0.0209 0.0076 15 10.368 28.58 2.98 178.67 105.54 0.0211 0.0079 15 10.368 28.96 3.06 183.60 106.10 0.0212 0.0080 15 10.368 29.14 3.09 185.67 106.64 0.0213 0.0081 15 10.368 29.32 3.15 189.00 107.50 0.0215 0.0083 15 10.368 29.60 3.33 200.05 107.98 0.0216 0.0083 15 10.368 29.76 3.38 202.78 108.52 0.0217 0.0085 15 10.368 29.93 3.56 213.72 109.30 0.0219 0.0086 15 10.368 30.19 3.84 230.10 110.96 0.0222 0.0089 15 10.368 30.73

132

3.86 231.30 111.08 0.0222 0.0090 15 10.368 30.76 3.91 234.83 111.36 0.0223 0.0090 15 10.368 30.86 4.27 256.07 112.80 0.0226 0.0003 10 6.912 31.56 4.31 258.65 113.10 0.0226 0.0003 10 6.912 31.71 4.42 265.15 113.50 0.0227 0.0004 10 6.912 31.90 4.51 270.77 113.84 0.0228 0.0005 10 6.912 32.07 4.58 274.72 114.26 0.0229 0.0006 10 6.912 32.27 4.78 286.98 114.86 0.0230 0.0007 10 6.912 32.56 4.82 289.13 115.16 0.0230 0.0008 10 6.912 32.71 4.99 299.23 115.62 0.0231 0.0009 10 6.912 32.94 5.13 307.83 116.20 0.0232 0.0010 10 6.912 33.22 5.33 320.00 116.78 0.0234 0.0011 10 6.912 33.50 5.37 322.37 117.16 0.0234 0.0012 10 6.912 33.69 6.15 369.02 118.72 0.0237 0.0015 10 6.912 34.45 6.42 385.25 119.20 0.0238 0.0016 10 6.912 34.68 6.58 394.73 119.42 0.0239 0.0016 10 6.912 34.79 6.81 408.80 120.06 0.0240 0.0017 10 6.912 35.10 7.04 422.55 120.36 0.0241 0.0018 10 6.912 35.25 7.32 439.28 121.40 0.0243 0.0020 10 6.912 35.76 7.62 457.48 122.02 0.0244 0.0021 10 6.912 36.06 7.72 463.12 122.36 0.0245 0.0022 10 6.912 36.23 8.30 497.97 122.68 0.0245 0.0001 5 3.456 36.54 11.04 662.32 125.46 0.0251 0.0006 5 3.456 39.25 12.94 776.30 126.10 0.0252 0.0007 5 3.456 39.88 13.61 816.88 126.40 0.0253 0.0008 5 3.456 40.17 14.23 853.90 126.66 0.0253 0.0009 5 3.456 40.43 14.86 891.88 127.70 0.0255 0.0011 5 3.456 41.44

133

0.030

0.025

0.020

0.015

0.010

0.005

Cumulative Moles of NaOH 0.000 0246810121416 Alcalase Treatment Time (hours)

100 90

80 70 60

50 40 30

20 Degrees of Hydrolysis (%) Hydrolysis Degrees of 10 0 0 2 4 6 8 10 12 14 16

Alcalase Treatment Time (hours)

134

B28 – % Degrees of Hydrolysis versus Alcalase Treatment Time Experiment 2 o (Alcalase Treatment: 50-55 C, 0.5 AU / 5 g of RTech PPI, pH 8.5, variable time)

Cumulative Mass Mass Time Moles of % Degrees of Time (h) Volume of PPI Protein (min) NaOH Hydrolysis NaOH (ml) (g) (g) 0.00 0.00 0.00 0.0000 5 3.46 0.00 0.03 1.50 4.22 0.0008 5 3.46 4.12 0.05 2.70 5.56 0.0011 5 3.46 5.43 0.06 3.67 6.34 0.0013 5 3.46 6.19 0.09 5.15 7.46 0.0015 5 3.46 7.28 0.11 6.40 8.56 0.0017 5 3.46 8.36 0.13 7.52 9.48 0.0019 5 3.46 9.26 0.15 8.82 10.70 0.0021 5 3.46 10.45 0.19 11.35 12.04 0.0024 5 3.46 11.75 0.22 13.40 12.78 0.0026 5 3.46 12.48 0.26 15.38 13.38 0.0027 5 3.46 13.06 0.29 17.25 13.86 0.0028 5 3.46 13.53 0.31 18.78 14.18 0.0028 5 3.46 13.84 0.36 21.55 15.10 0.0030 5 3.46 14.74 0.46 27.42 16.64 0.0033 5 3.46 16.25 0.53 31.92 17.22 0.0034 5 3.46 16.81 0.61 36.57 18.06 0.0036 5 3.46 17.63 0.66 39.53 18.68 0.0037 5 3.46 18.24 0.79 47.38 19.44 0.0039 5 3.46 18.98 0.86 51.48 20.18 0.0040 5 3.46 19.70 0.98 58.97 20.98 0.0042 5 3.46 20.48 1.07 63.98 21.62 0.0043 5 3.46 21.11 1.22 73.32 22.30 0.0045 5 3.46 21.77 1.53 91.87 23.88 0.0048 5 3.46 23.31 2.21 132.85 26.82 0.0054 5 3.46 26.18 2.75 164.97 28.08 0.0056 5 3.46 27.41 3.08 184.58 29.02 0.0058 5 3.46 28.33 3.55 212.95 29.78 0.0060 5 3.46 29.07 4.16 249.88 30.42 0.0061 5 3.46 29.70 4.51 270.63 31.36 0.0063 5 3.46 30.62 4.69 281.27 32.18 0.0064 5 3.46 31.42 5.13 307.88 32.64 0.0065 5 3.46 31.87 5.34 320.32 33.06 0.0066 5 3.46 32.28 6.71 402.47 34.62 0.0069 5 3.46 33.80

135

0.030

0.025

0.020

0.015

0.010

0.005 Cumulative Moles of NaOH of Moles Cumulative

0.000 0 2 4 6 8 10 12 14 16 Alcalase Treatment Time (hours)

100 90 80 70 60

50 40 30 20

Degrees of Hydrolysis (%) Hydrolysis of Degrees 10 0 0246810121416 Alcalase Treatment Time (hours)

136 B29 – % Degrees of Hydrolysis versus Alcalase Treatment Time Experiment 3 o (Alcalase Treatment: 50-55 C, 0.5 AU / 5 g of RTech PPI, pH 8.5, variable time)

Note: Aliquots were removed and flash frozen at: 0.5 h, 1 h, 2 h

Cumulative Moles of Mass Time Moles of Mass % Deg. of Time (h Volume of NaOH Protein (min) NaOH PPI (g) Hydrolysis NaOH (ml) sectioned (g) 0.00 0.00 0.00 0.0000 0.0000 20 13.824 0.00 0.03 2.00 14.60 0.0029 0.0029 20 13.824 2.84 0.07 4.33 24.34 0.0049 0.0049 20 13.824 4.73 0.11 6.72 32.36 0.0065 0.0065 20 13.824 6.29 0.26 15.52 49.02 0.0098 0.0098 20 13.824 9.52 0.34 20.12 56.56 0.0113 0.0015 15 10.368 11.98 0.36 21.63 58.80 0.0118 0.0020 15 10.368 12.06 0.39 23.23 62.30 0.0125 0.0027 15 10.368 13.85 0.41 24.75 64.60 0.0129 0.0031 15 10.368 14.59 0.44 26.23 66.26 0.0133 0.0034 15 10.368 13.99 0.45 27.17 67.20 0.0134 0.0036 15 10.368 14.23 0.50 30.00 70.48 0.0141 0.0043 15 10.368 15.08 0.63 37.82 71.14 0.0142 0.0044 15 10.368 15.25 0.68 40.73 73.12 0.0146 0.0048 15 10.368 15.77 0.73 44.00 74.70 0.0149 0.0051 15 10.368 16.18 0.78 46.60 75.54 0.0151 0.0053 15 10.368 16.39 0.81 48.62 76.56 0.0153 0.0055 15 10.368 16.66 0.89 53.13 78.42 0.0157 0.0059 15 10.368 17.14 0.91 54.43 79.00 0.0158 0.0060 15 10.368 17.29 0.93 56.00 79.46 0.0159 0.0061 15 10.368 17.41 0.96 57.47 79.90 0.0160 0.0062 15 10.368 17.52 0.98 58.83 80.20 0.0160 0.0062 15 10.368 17.60 1.11 66.47 80.90 0.0162 0.0001 10 6.912 17.94 1.15 69.25 81.88 0.0164 0.0003 10 6.912 18.42 1.24 74.27 84.26 0.0169 0.0008 10 6.912 19.58 1.57 94.47 87.40 0.0175 0.0014 10 6.912 21.12 1.63 97.87 88.38 0.0177 0.0016 10 6.912 21.59 1.85 110.75 92.20 0.0184 0.0024 10 6.912 23.46 1.88 112.78 92.38 0.0185 0.0024 10 6.912 23.55 1.92 115.47 92.54 0.0185 0.0025 10 6.912 23.62 1.99 119.53 93.10 0.0186 0.0026 10 6.912 23.90 2.17 130.38 93.30 0.0187 0.0000 5 3.456 24.09 2.33 139.77 93.80 0.0188 0.0001 5 3.456 24.58 2.36 141.53 94.04 0.0188 0.0002 5 3.456 24.82 2.71 162.83 95.72 0.0191 0.0005 5 3.456 26.46

137

0.030

0.025

0.020

0.015

0.010 0.005

Cumulative Moles of NaOH NaOH of Moles Cumulative 0.000 0246810121416 Alcalase TreatmentTime (hours)

100 90 80 70

50 40 30 20

Degrees of Hydrolysis (%) Hydrolysis of Degrees 10

0 0 2 4 6 8 10121416 Alcalase Treatment Time (hours)

138

B30 – % Degrees of Hydrolysis versus Alcalase Treatment Time Experiment 4 (Alcalase Treatment: 60oC, 0.5 AU / 5 g of RTech PPI, pH 8.5, variable time)

Cumulative % Degrees Time Moles of Mass Mass Time (min) NaOH Volume of (h) NaOH PPI (g) Protein (g) (ml) Hydrolysis 0.00 0.00 0.00 0.0000 5.0007 3.46 0.00 0.02 1.13 2.92 0.0006 5.0007 3.46 2.83 0.03 2.03 5.62 0.0011 5.0007 3.46 5.45 0.05 2.93 7.38 0.0015 5.0007 3.46 7.15 0.06 3.47 8.16 0.0016 5.0007 3.46 7.91 0.08 4.50 9.48 0.0019 5.0007 3.46 9.19 0.09 5.63 10.54 0.0021 5.0007 3.46 10.22 0.12 6.98 11.36 0.0023 5.0007 3.46 11.01 0.14 8.22 12.06 0.0024 5.0007 3.46 11.69 0.15 8.93 12.44 0.0025 5.0007 3.46 12.06 0.16 9.60 12.90 0.0026 5.0007 3.46 12.50 0.18 11.08 13.84 0.0028 5.0007 3.46 13.41 0.23 13.90 15.30 0.0031 5.0007 3.46 14.83 0.29 17.45 16.38 0.0033 5.0007 3.46 15.88 0.33 20.03 17.14 0.0034 5.0007 3.46 16.61 0.37 22.08 17.74 0.0035 5.0007 3.46 17.19 0.40 23.88 18.30 0.0037 5.0007 3.46 17.74 0.49 29.50 19.18 0.0038 5.0007 3.46 18.59 0.54 32.43 19.74 0.0039 5.0007 3.46 19.13 0.68 40.60 20.24 0.0040 5.0007 3.46 19.62 0.72 43.48 20.86 0.0042 5.0007 3.46 20.22 0.84 50.60 21.48 0.0043 5.0007 3.46 20.82 1.00 60.00 22.20 0.0044 5.0007 3.46 21.52

100

80 70 60

50 40 30 20

Degrees of Hydrolysis (%) Hydrolysis of Degrees 10 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Alcalase Treatment Time (hours)

139

B31 – % Degrees of Hydrolysis versus Alcalase Treatment Time Experiment 5 (Alcalase Treatment: 50-55oC, 0.8 AU / 5 g of RTech PPI, pH 8.5, 1 hour)

Cumulative % Degrees Time Moles of Mass PPI Mass Time (min) NaOH of (h) NaOH (g) Protein (g) Volume(ml) Hydrolysis 0.00 0.00 0.00 0.0000 15.0015 10.37 0.00 0.08 5.00 22.68 0.0045 15.0015 10.37 7.32 1.00 60.00 64.74 0.0129 15.0015 10.37 20.89

B32 – % Degrees of Hydrolysis versus Alcalase Treatment Time Experiment 6 o (Alcalase Treatment: 50-55 C, 0.3 AU / 5 g of RTech PPI, pH 8.5, 1 hour)

Cumulative % Degrees Time Moles of Mass PPI Mass Time (min) NaOH Volume of (h) NaOH (g) Protein (g) (ml) Hydrolysis 0.00 0.00 0.86 0.0002 15.0021 10.37 0.28 0.03 2.00 10.32 0.0021 15.0021 10.37 3.33 0.05 3.00 12.8 0.0026 15.0021 10.37 4.13 0.22 13.40 28.6 0.0057 15.0021 10.37 9.23 0.48 28.60 34.16 0.0068 15.0021 10.37 11.02 0.72 43.17 39.5 0.0079 15.0021 10.37 12.74 0.84 50.50 42.32 0.0085 15.0021 10.37 13.65 1.00 60.00 44.8 0.0090 15.0021 10.37 14.45

140

B33 – % Degrees of Hydrolysis vs. Alcalase Treatment Time (Alcalase Treatment for Tgase Experiment Shown in Figure 4.22) o (Alcalase Treatment: 50-55 C, 0.5 AU / 5 g of RTech PPI, pH 8.5, 5 h)

Cumulative Time NaOH Volume Moles of Mass Mass % Deg. of (h) Time (min) (ml) NaOH PPI (g) Protein (g) Hydrolysis 0.00 0.00 0.00 0.0000 35.0080 24.20 0.00 0.22 13.43 67.96 0.0136 35.0080 24.20 9.48 0.25 15.23 70.90 0.0142 35.0080 24.20 9.89 0.28 16.80 73.20 0.0146 35.0080 24.20 10.21 0.31 18.80 76.20 0.0152 35.0080 24.20 10.63 0.35 20.80 78.60 0.0157 35.0080 24.20 10.96 0.36 21.85 79.54 0.0159 35.0080 24.20 11.09 0.39 23.50 81.26 0.0163 35.0080 24.20 11.33 0.43 25.65 83.88 0.0168 35.0080 24.20 11.70 0.44 26.47 84.64 0.0169 35.0080 24.20 11.80 0.47 28.45 86.70 0.0173 35.0080 24.20 12.09 0.50 29.77 87.86 0.0176 35.0080 24.20 12.25 0.51 30.58 88.62 0.0177 35.0080 24.20 12.36 0.53 32.08 89.44 0.0179 35.0080 24.20 12.47 0.56 33.80 91.58 0.0183 35.0080 24.20 12.77 0.59 35.53 93.58 0.0187 35.0080 24.20 13.05 0.64 38.27 96.32 0.0193 35.0080 24.20 13.43 0.67 39.98 97.98 0.0196 35.0080 24.20 13.66 0.71 42.63 100.78 0.0202 35.0080 24.20 14.05 0.75 45.20 103.32 0.0207 35.0080 24.20 14.41 0.79 47.62 105.70 0.0211 35.0080 24.20 14.74 0.84 50.28 108.32 0.0217 35.0080 24.20 15.11 0.86 51.65 109.98 0.0220 35.0080 24.20 15.34 0.90 54.25 112.50 0.0225 35.0080 24.20 15.69 0.93 56.08 114.52 0.0229 35.0080 24.20 15.97 0.98 58.93 117.78 0.0236 35.0080 24.20 16.43 1.04 62.48 121.08 0.0242 35.0080 24.20 16.89 1.08 65.07 122.82 0.0246 35.0080 24.20 17.13 1.12 67.18 124.42 0.0249 35.0080 24.20 17.35 1.20 72.15 128.02 0.0256 35.0080 24.20 17.85 1.24 74.57 128.56 0.0257 35.0080 24.20 17.93 1.27 76.45 131.14 0.0262 35.0080 24.20 18.29 1.31 78.85 132.90 0.0266 35.0080 24.20 18.53 1.36 81.47 135.00 0.0270 35.0080 24.20 18.83 1.42 85.42 137.88 0.0276 35.0080 24.20 19.23 1.53 91.95 141.92 0.0284 35.0080 24.20 19.79 1.68 100.83 147.78 0.0296 35.0080 24.20 20.61 1.78 106.57 151.94 0.0304 35.0080 24.20 21.19 1.85 110.87 154.46 0.0309 35.0080 24.20 21.54 1.89 113.30 156.16 0.0312 35.0080 24.20 21.78 1.94 116.18 157.70 0.0315 35.0080 24.20 21.99

141

2.07 124.48 160.90 0.0322 35.0080 24.20 22.44 2.15 129.20 162.86 0.0326 35.0080 24.20 22.71 2.23 134.05 164.80 0.0330 35.0080 24.20 22.98 2.32 139.02 167.40 0.0335 35.0080 24.20 23.35 2.37 141.92 169.06 0.0338 35.0080 24.20 23.58 2.52 151.02 173.40 0.0347 35.0080 24.20 24.18 2.57 153.93 173.86 0.0348 35.0080 24.20 24.25 2.62 157.22 174.70 0.0349 35.0080 24.20 24.36 2.69 161.13 175.42 0.0351 35.0080 24.20 24.46 2.78 166.73 177.00 0.0354 35.0080 24.20 24.68 2.81 168.52 178.08 0.0356 35.0080 24.20 24.84 2.84 170.53 179.02 0.0358 35.0080 24.20 24.97 2.87 172.23 179.80 0.0360 35.0080 24.20 25.08 2.89 173.55 180.32 0.0361 35.0080 24.20 25.15 2.92 175.47 181.10 0.0362 35.0080 24.20 25.26 2.98 178.57 181.94 0.0364 35.0080 24.20 25.37 3.01 180.77 182.56 0.0365 35.0080 24.20 25.46 3.06 183.35 183.22 0.0366 35.0080 24.20 25.55 3.09 185.37 183.80 0.0368 35.0080 24.20 25.63 3.16 189.35 184.96 0.0370 35.0080 24.20 25.79 3.21 192.48 186.28 0.0373 35.0080 24.20 25.98 3.25 194.95 187.38 0.0375 35.0080 24.20 26.13 3.30 198.20 189.06 0.0378 35.0080 24.20 26.37 3.42 205.18 191.00 0.0382 35.0080 24.20 26.64 3.59 215.57 193.10 0.0386 35.0080 24.20 26.93 3.70 221.90 194.72 0.0389 35.0080 24.20 27.16 3.75 225.17 195.96 0.0392 35.0080 24.20 27.33 3.85 230.75 198.16 0.0396 35.0080 24.20 27.64 4.07 244.22 200.34 0.0401 35.0080 24.20 27.94 4.23 253.68 202.24 0.0404 35.0080 24.20 28.20 4.42 265.08 205.76 0.0412 35.0080 24.20 28.70 4.47 268.00 206.28 0.0413 35.0080 24.20 28.77 4.54 272.63 206.90 0.0414 35.0080 24.20 28.85 4.68 280.55 207.84 0.0416 35.0080 24.20 28.99 4.75 285.20 207.92 0.0416 35.0080 24.20 29.00 4.77 286.22 208.10 0.0416 35.0080 24.20 29.02 4.80 287.72 208.56 0.0417 35.0080 24.20 29.09 4.83 289.65 208.78 0.0418 35.0080 24.20 29.12 4.86 291.72 209.80 0.0420 35.0080 24.20 29.26 4.89 293.38 210.48 0.0421 35.0080 24.20 29.35 4.91 294.32 210.76 0.0422 35.0080 24.20 29.39 4.93 295.55 211.16 0.0422 35.0080 24.20 29.45 4.96 297.37 211.52 0.0423 35.0080 24.20 29.50

142

100

90 80 70 60

40 30 20

Degrees of Hydrolysis (%) Hydrolysis of Degrees 10 0 0123456 Alcalase Treatment Time (hours)

0.045 0.040 0.035 0.030 0.025 0.020 0.015 0.010 0.005 Cumulative Moles of NaOH 0.000 0123456 Alcalase Treatment Time (hours)

143

B34 – % Degrees of Hydrolysis versus Alcalase Treatment Time (Alcalase Treatment for Tgase Experiment Shown in Figure 4.23) (Alcalase Treatment: 50-55oC, 0.5 AU / 5 g of RTech PPI, pH 8.5, 5 h)

Cumulative Time Time Moles of Mass PPI Mass % Degrees of NaOH Volume (h) (min) NaOH (g) Protein (g) Hydrolysis (ml) 0.00 0.00 0.00 0.0000 35.0041 24.19 0.00 0.33 19.95 76.42 0.0153 35.0041 24.19 10.66 0.36 21.38 79.48 0.0159 35.0041 24.19 11.09 0.39 23.37 83.80 0.0168 35.0041 24.19 11.69 0.42 25.05 86.62 0.0173 35.0041 24.19 12.08 0.44 26.42 88.22 0.0176 35.0041 24.19 12.30 0.46 27.67 90.22 0.0180 35.0041 24.19 12.58 0.49 29.10 91.92 0.0184 35.0041 24.19 12.82 0.50 30.25 93.32 0.0187 35.0041 24.19 13.02 0.54 32.20 96.08 0.0192 35.0041 24.19 13.40 0.56 33.53 97.82 0.0196 35.0041 24.19 13.64 0.62 37.28 103.30 0.0207 35.0041 24.19 14.41 0.65 38.97 105.40 0.0211 35.0041 24.19 14.70 0.69 41.37 107.66 0.0215 35.0041 24.19 15.02 0.71 42.53 108.92 0.0218 35.0041 24.19 15.19 0.75 45.13 111.50 0.0223 35.0041 24.19 15.55 0.80 48.07 113.76 0.0228 35.0041 24.19 15.87 0.86 51.68 117.18 0.0234 35.0041 24.19 16.34 0.90 54.10 119.70 0.0239 35.0041 24.19 16.70 0.95 57.07 122.06 0.0244 35.0041 24.19 17.02 0.98 58.72 123.56 0.0247 35.0041 24.19 17.23 1.01 60.68 124.72 0.0249 35.0041 24.19 17.40 1.12 67.20 128.88 0.0258 35.0041 24.19 17.98 1.15 69.23 130.72 0.0261 35.0041 24.19 18.23 1.21 72.65 133.38 0.0267 35.0041 24.19 18.60 1.25 75.20 135.38 0.0271 35.0041 24.19 18.88 1.30 78.03 137.06 0.0274 35.0041 24.19 19.12 1.32 79.22 137.62 0.0275 35.0041 24.19 19.19 1.35 81.07 138.48 0.0277 35.0041 24.19 19.31 1.39 83.40 139.78 0.0280 35.0041 24.19 19.50 1.43 85.90 141.00 0.0282 35.0041 24.19 19.67 1.48 89.00 143.04 0.0286 35.0041 24.19 19.95 1.53 91.60 144.70 0.0289 35.0041 24.19 20.18 1.58 94.90 146.70 0.0293 35.0041 24.19 20.46 1.74 104.10 151.16 0.0302 35.0041 24.19 21.08 1.87 112.20 154.26 0.0309 35.0041 24.19 21.52 2.03 121.78 157.82 0.0316 35.0041 24.19 22.01 2.16 129.58 163.26 0.0327 35.0041 24.19 22.77 2.29 137.32 165.28 0.0331 35.0041 24.19 23.05

144

2.35 140.80 166.54 0.0333 35.0041 24.19 23.23 2.42 145.18 168.10 0.0336 35.0041 24.19 23.45 2.52 150.92 169.64 0.0339 35.0041 24.19 23.66 2.64 158.42 171.42 0.0343 35.0041 24.19 23.91 2.80 167.88 174.00 0.0348 35.0041 24.19 24.27 2.92 175.25 177.46 0.0355 35.0041 24.19 24.75 2.98 178.95 179.20 0.0358 35.0041 24.19 24.99 3.19 191.57 182.08 0.0364 35.0041 24.19 25.40 3.27 196.15 183.70 0.0367 35.0041 24.19 25.62 3.49 209.18 187.32 0.0375 35.0041 24.19 26.13 3.64 218.38 189.02 0.0378 35.0041 24.19 26.36 3.90 234.02 192.30 0.0385 35.0041 24.19 26.82 4.11 246.77 195.20 0.0390 35.0041 24.19 27.23 4.41 264.50 198.62 0.0397 35.0041 24.19 27.70 4.87 291.97 202.86 0.0406 35.0041 24.19 28.29 4.93 296.08 203.40 0.0407 35.0041 24.19 28.37

100 90 80 70 60 50 40 30 20 10

Degrees of Hydrolysis (%) Hydrolysis of Degrees 0 0123456

Alcalase Treatment Time (hours)

0.045 0.040

0.035 0.030 0.025 0.020 0.015

0.010 0.005 Cumulative Moles of NaOH 0.000 0123456 Alcalase Treatment Time (hours)

145

B35 – Particle Size Analysis for Alcalase-Treated RTech PPI (Alcalase Treatment: 50-55oC, 0.5 AU / 5 g of RTech PPI, pH 8.5, variable time)

Particle 8h 8 h 16 h 16 h 16 h 4 h 4 h 4 h 8 h Diameter decant centrif cake decant centrif cake decant centif cake (µm) 5.632 0.548 0.569 0.756 0.541 1.036 0.665 0.450 0.717 0.910 5.696 4.790 5.498 5.815 4.159 6.736 5.207 3.893 6.093 6.317 5.761 4.574 5.213 5.926 3.883 4.145 4.675 3.774 6.810 4.863 5.827 4.265 4.834 5.229 4.023 4.145 4.205 3.325 5.376 4.190 5.893 4.266 5.403 5.165 3.791 4.145 3.725 3.375 4.659 3.809 5.96 3.931 4.550 4.886 3.698 4.663 3.504 3.366 5.018 3.382 6.028 3.945 4.455 4.510 3.760 1.554 3.467 3.075 4.301 3.192 6.097 3.685 5.118 4.540 3.459 4.663 3.195 3.013 2.867 2.951 6.167 3.702 3.981 4.275 3.227 4.145 3.113 3.028 5.018 2.921 6.237 3.472 3.128 3.909 3.381 1.554 2.967 2.827 2.151 2.698 6.308 3.288 3.318 3.742 3.113 2.073 2.862 2.876 3.584 2.434 6.38 2.879 2.749 3.241 2.726 4.145 2.490 2.421 1.434 2.210 6.453 3.117 3.886 3.544 2.994 2.591 2.660 2.737 2.867 2.355 6.526 2.917 3.223 3.240 2.680 2.591 2.626 2.561 0.717 2.326 6.601 2.637 2.938 3.086 2.673 1.036 2.513 2.440 3.943 2.237 6.676 2.561 2.654 3.031 2.646 2.073 2.449 2.432 1.792 2.209 6.752 2.521 2.844 2.744 2.542 1.036 2.365 2.326 0.717 2.130 6.829 2.287 2.180 2.600 2.420 1.554 2.282 2.093 1.792 2.005 6.907 2.099 2.275 2.463 2.309 2.591 2.147 2.120 1.792 2.040 6.986 2.208 1.991 2.268 2.271 2.591 2.164 2.005 1.434 2.049 7.065 1.979 1.611 2.098 2.140 2.073 2.008 1.887 2.151 1.816 7.146 1.892 1.896 1.983 2.015 1.036 1.958 1.870 1.792 1.897 7.227 1.824 1.611 1.777 2.043 1.554 1.941 1.905 1.792 1.855 7.31 1.722 1.801 1.668 1.809 1.554 1.844 1.830 1.075 1.776 7.393 1.637 0.948 1.524 1.869 0.518 1.825 1.682 1.434 1.710 7.477 1.543 1.232 1.473 1.637 2.073 1.691 1.621 1.075 1.713 7.563 1.507 1.043 1.267 1.574 0.518 1.665 1.536 1.792 1.611 7.649 1.436 1.232 1.249 1.500 1.554 1.625 1.497 0.358 1.577 7.736 1.353 1.232 1.089 1.590 0.518 1.542 1.494 0.717 1.597 7.824 1.193 2.085 1.007 1.393 1.554 1.438 1.389 0.717 1.481 7.913 1.224 1.517 0.882 1.252 1.036 1.388 1.325 1.434 1.495 8.004 1.082 0.758 0.833 1.210 1.554 1.323 1.295 0.717 1.394 8.095 1.018 0.948 0.715 1.176 1.554 1.312 1.311 0.000 1.319 8.187 0.952 1.517 0.684 1.071 0.518 1.235 1.209 0.717 1.331 8.281 0.914 0.664 0.622 1.016 0.518 1.142 1.107 0.717 1.209 8.375 0.798 0.948 0.589 1.016 1.554 1.082 1.058 0.358 1.151 8.47 0.810 0.569 0.500 0.917 0.518 0.994 1.061 0.358 1.144 8.567 0.790 0.569 0.388 0.930 1.554 1.004 0.976 0.000 1.104 8.665 0.783 0.664 0.393 0.814 0.518 0.913 0.964 0.358 1.022 8.763 0.711 0.569 0.348 0.785 1.036 0.869 0.922 0.717 0.967 8.863 0.662 0.569 0.330 0.729 0.000 0.808 0.868 0.717 0.916 8.964 0.605 0.190 0.322 0.710 0.518 0.730 0.813 0.717 0.891 9.067 0.551 0.379 0.272 0.644 1.554 0.719 0.789 1.075 0.756

146

9.17 0.552 0.284 0.245 0.618 1.036 0.681 0.715 0.000 0.761 9.275 0.526 0.190 0.250 0.547 0.000 0.631 0.732 0.358 0.715 9.38 0.480 0.190 0.212 0.531 0.000 0.586 0.703 0.000 0.692 9.487 0.443 0.000 0.174 0.409 0.000 0.527 0.670 0.358 0.589 9.595 0.406 0.284 0.168 0.459 0.518 0.471 0.603 0.717 0.591 9.705 0.388 0.190 0.155 0.446 1.036 0.452 0.625 0.358 0.536 9.816 0.376 0.095 0.137 0.416 0.518 0.428 0.564 0.000 0.523 9.927 0.388 0.190 0.124 0.384 0.000 0.422 0.562 0.358 0.498 10.04 0.362 0.569 0.125 0.345 0.518 0.378 0.478 0.000 0.443 10.16 0.304 0.190 0.098 0.313 0.518 0.349 0.492 0.000 0.389 10.27 0.266 0.095 0.095 0.298 0.000 0.324 0.503 0.000 0.386 10.39 0.280 0.284 0.092 0.310 0.000 0.322 0.454 0.000 0.344 10.51 0.466 0.095 0.125 0.401 1.554 0.426 0.726 1.075 0.515 10.63 0.241 0.000 0.072 0.278 0.000 0.240 0.353 0.358 0.264 10.75 0.224 0.000 0.078 0.261 0.000 0.218 0.373 0.358 0.248 10.87 0.201 0.095 0.054 0.209 1.036 0.213 0.356 0.000 0.256 10.99 0.192 0.095 0.062 0.189 0.000 0.208 0.347 0.000 0.224 11.12 0.149 0.095 0.053 0.194 0.000 0.189 0.349 0.000 0.210 11.25 0.163 0.000 0.048 0.170 0.000 0.173 0.348 0.717 0.198 11.37 0.167 0.190 0.054 0.168 0.000 0.170 0.256 0.000 0.172 11.5 0.172 0.095 0.048 0.190 0.000 0.144 0.254 0.000 0.176 11.64 0.116 0.000 0.037 0.130 0.000 0.139 0.284 0.358 0.158 11.77 0.159 0.095 0.039 0.142 0.518 0.139 0.244 0.000 0.154 11.9 0.118 0.095 0.030 0.141 0.518 0.122 0.227 0.358 0.139 12.04 0.124 0.000 0.032 0.144 0.000 0.106 0.221 0.000 0.122 12.17 0.134 0.095 0.019 0.130 0.518 0.113 0.224 0.000 0.105 12.31 0.105 0.095 0.017 0.122 0.000 0.103 0.200 0.358 0.109 12.45 0.103 0.190 0.025 0.097 0.000 0.095 0.190 0.000 0.117 12.6 0.110 0.190 0.031 0.107 0.000 0.096 0.174 0.000 0.092 12.74 0.085 0.000 0.019 0.097 0.518 0.093 0.160 0.358 0.091 12.88 0.100 0.000 0.018 0.093 0.518 0.079 0.167 0.000 0.089 13.03 0.082 0.095 0.023 0.085 0.000 0.067 0.147 0.358 0.078 13.18 0.088 0.000 0.021 0.079 1.036 0.063 0.142 0.358 0.086 13.33 0.072 0.000 0.023 0.071 0.000 0.051 0.125 0.000 0.069 13.48 0.075 0.000 0.023 0.075 0.000 0.060 0.145 0.358 0.066 13.64 0.057 0.000 0.009 0.059 0.000 0.060 0.128 0.358 0.061 13.79 0.075 0.000 0.011 0.063 0.000 0.059 0.141 0.000 0.059 13.95 0.070 0.000 0.012 0.066 0.000 0.046 0.097 1.075 0.050 14.11 0.043 0.000 0.014 0.055 0.000 0.037 0.107 0.000 0.051 14.27 0.071 0.095 0.010 0.049 0.518 0.048 0.093 0.358 0.037 14.43 0.039 0.000 0.012 0.051 0.000 0.041 0.101 0.358 0.042 14.6 0.037 0.000 0.016 0.046 0.518 0.032 0.110 0.717 0.034 14.76 0.050 0.000 0.011 0.048 0.000 0.026 0.108 0.000 0.035 14.93 0.038 0.000 0.016 0.052 0.518 0.027 0.096 0.000 0.033 15.1 0.041 0.000 0.003 0.041 0.518 0.027 0.075 0.358 0.031 15.27 0.034 0.000 0.012 0.030 0.518 0.029 0.093 0.000 0.027 15.45 0.024 0.000 0.003 0.026 0.000 0.022 0.064 0.358 0.033 15.62 0.034 0.000 0.008 0.026 0.000 0.033 0.067 0.358 0.026 15.8 0.028 0.095 0.004 0.029 0.000 0.022 0.065 0.000 0.023

147

15.98 0.042 0.000 0.009 0.031 0.518 0.015 0.057 0.358 0.020 16.16 0.029 0.000 0.002 0.027 0.518 0.022 0.044 0.717 0.021 16.35 0.028 0.000 0.003 0.027 0.000 0.018 0.077 0.000 0.014 16.54 0.022 0.000 0.001 0.036 0.000 0.016 0.057 0.358 0.022 16.72 0.008 0.000 0.000 0.019 0.000 0.010 0.026 0.358 0.007 16.91 0.007 0.000 0.000 0.004 0.000 0.002 0.010 0.000 0.004 17.11 0.020 0.000 0.002 0.018 0.000 0.018 0.039 0.000 0.014 17.3 0.021 0.000 0.003 0.029 0.518 0.013 0.055 0.000 0.020 17.5 0.025 0.000 0.004 0.029 0.000 0.011 0.041 0.358 0.012 17.7 0.016 0.000 0.004 0.022 0.000 0.008 0.031 0.000 0.015 17.9 0.013 0.095 0.001 0.031 0.000 0.008 0.037 0.000 0.011 18.1 0.012 0.095 0.002 0.019 0.000 0.013 0.035 0.358 0.011 18.31 0.020 0.000 0.002 0.010 0.000 0.008 0.025 0.000 0.009 18.52 0.007 0.000 0.000 0.016 0.000 0.010 0.029 0.000 0.015 18.73 0.012 0.000 0.003 0.016 0.000 0.005 0.030 0.000 0.013 18.94 0.007 0.000 0.001 0.018 0.000 0.010 0.023 0.000 0.008 19.16 0.018 0.000 0.000 0.011 0.000 0.007 0.031 0.000 0.006 19.38 0.012 0.000 0.000 0.008 0.000 0.005 0.026 0.358 0.007 19.6 0.009 0.000 0.001 0.015 0.000 0.009 0.023 0.000 0.007 19.82 0.012 0.000 0.000 0.018 0.000 0.006 0.022 0.000 0.004 20.05 0.005 0.000 0.003 0.012 0.000 0.006 0.018 0.000 0.006 20.28 0.005 0.000 0.000 0.016 0.518 0.002 0.027 0.000 0.004 20.51 0.014 0.000 0.003 0.010 0.000 0.002 0.018 0.000 0.005 20.74 0.009 0.000 0.000 0.012 0.000 0.004 0.017 0.358 0.004 20.98 0.004 0.000 0.000 0.011 0.000 0.005 0.019 0.000 0.003 21.22 0.003 0.000 0.000 0.007 0.000 0.003 0.014 0.000 0.004 21.46 0.008 0.000 0.001 0.010 0.000 0.004 0.019 0.000 0.004 21.71 0.004 0.000 0.001 0.008 0.000 0.000 0.011 0.358 0.002 21.95 0.003 0.000 0.000 0.004 0.000 0.002 0.019 0.000 0.003 22.2 0.003 0.000 0.000 0.008 0.000 0.002 0.006 0.000 0.003 22.46 0.007 0.000 0.000 0.000 0.000 0.007 0.009 0.000 0.003 22.71 0.000 0.000 0.000 0.008 0.000 0.002 0.005 0.000 0.003 22.97 0.004 0.000 0.001 0.007 0.000 0.001 0.012 0.358 0.000 23.23 0.005 0.000 0.000 0.001 0.000 0.003 0.004 0.000 0.000 23.5 0.005 0.000 0.000 0.003 0.000 0.002 0.006 0.000 0.000 23.77 0.007 0.000 0.000 0.003 0.000 0.001 0.004 0.000 0.001 24.04 0.005 0.000 0.001 0.005 0.000 0.002 0.006 0.358 0.003 24.31 0.004 0.095 0.000 0.001 0.518 0.002 0.011 0.000 0.000 24.59 0.004 0.000 0.000 0.000 0.000 0.001 0.006 0.358 0.001 24.87 0.001 0.000 0.000 0.000 0.000 0.002 0.003 0.000 0.001 25.15 0.003 0.000 0.000 0.004 0.000 0.001 0.007 0.000 0.002 25.44 0.001 0.000 0.000 0.007 0.000 0.001 0.007 0.000 0.001 25.73 0.000 0.000 0.000 0.003 0.000 0.002 0.004 0.000 0.000 26.02 0.003 0.000 0.000 0.004 0.000 0.001 0.001 0.000 0.001 26.32 0.003 0.000 0.000 0.001 0.000 0.002 0.004 0.000 0.001 26.62 0.003 0.095 0.000 0.001 0.000 0.001 0.003 0.358 0.001 26.92 0.003 0.000 0.000 0.001 0.000 0.000 0.003 0.000 0.000 27.23 0.000 0.000 0.000 0.000 0.000 0.001 0.003 0.000 0.000 27.54 0.001 0.000 0.000 0.000 0.000 0.001 0.001 0.000 0.000

148

27.85 0.003 0.000 0.000 0.004 0.000 0.000 0.001 0.000 0.000 28.17 0.000 0.000 0.000 0.001 0.000 0.001 0.001 0.000 0.000 28.49 0.001 0.000 0.000 0.003 0.000 0.001 0.000 0.000 0.000 28.82 0.000 0.000 0.000 0.000 0.000 0.000 0.001 0.000 0.001 29.15 0.000 0.000 0.000 0.003 0.000 0.001 0.000 0.000 0.000 29.48 0.001 0.000 0.001 0.003 0.000 0.000 0.002 0.000 0.000 29.82 0.000 0.000 0.000 0.000 0.000 0.000 0.001 0.000 0.000 30.16 0.000 0.000 0.000 0.001 0.000 0.000 0.002 0.000 0.000 30.5 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 30.85 0.000 0.000 0.000 0.001 0.000 0.001 0.000 0.000 0.000 31.2 0.000 0.000 0.000 0.001 0.000 0.000 0.002 0.000 0.000 31.55 0.000 0.000 0.000 0.001 0.000 0.000 0.000 0.000 0.000 31.91 0.000 0.000 0.000 0.000 0.000 0.000 0.001 0.000 0.000 32.28 0.000 0.000 0.000 0.001 0.000 0.000 0.000 0.000 0.000 32.65 0.000 0.000 0.000 0.001 0.000 0.000 0.000 0.000 0.000 33.02 0.001 0.000 0.000 0.000 0.000 0.000 0.001 0.000 0.000 33.39 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 33.78 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 34.16 0.000 0.000 0.000 0.001 0.000 0.001 0.000 0.000 0.000 34.55 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 34.94 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 35.34 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 35.75 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 36.15 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 36.57 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 36.98 0.000 0.000 0.001 0.000 0.000 0.000 0.000 0.000 0.000 37.4 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 37.83 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 38.26 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 38.7 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 39.14 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 39.59 0.000 0.000 0.000 0.000 0.000 0.001 0.000 0.000 0.000 40.04 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 40.49 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 40.95 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 41.42 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 41.89 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 42.37 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 42.85 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 43.34 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 43.84 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 44.34 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 44.84 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 45.35 0.000 0.000 0.000 0.000 0.000 0.000 0.001 0.000 0.000 45.87 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 46.39 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 46.92 0.000 0.000 0.000 0.000 0.000 0.001 0.000 0.000 0.000 47.46 0.001 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 48 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

149

48.55 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 49.1 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 49.66 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 50.23 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 50.8 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 51.38 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 51.96 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 52.56 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 53.16 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 53.76 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 54.37 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 54.99 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 55.62 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 56.26 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 56.9 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 57.55 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 58.2 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 58.87 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 59.54 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 60.22 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 60.9 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 61.6 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 62.3 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 63.01 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 63.73 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 64.45 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

150 B36 –NSIatpH 3vs. Tgase Treatment Alcalase Treatm

Treatment Time (h) 16.0 0.5 0.0 8.0 2.0 1.0 (Tgase Treatment:50-55 Solubility Solubility Average 50.92 52.48 53.58 51.79 50.74 75.50 (%) N STDEV 0.09 0.17 0.16 0.04 0.12 1.10 ent: 5h,0.2gE/ 5gPPI, pH 8.5,50-55 RSTDEV 0.08 0.24 2.06 0.17 0.34 0.21 o Sample C, pH6.5,10EUnits/gprotein; Blank Blank Blank Blank Blank Blank 3 20.02 13.08 0.090 4.1006 0.4469 0.1766 50.91 3 2 1 2 20.03 13.08 1 0.090 4.1006 3 0.4469 2 1 0.1766 50.88 3 2 1 3 2 1 3 2 1 Time on Alcalase-Treated RTech PPI Liq Mass 20.03 20.00 20.02 20.02 20.03 20.02 20.03 20.01 20.01 20.02 20.01 20.05 20.01 20.07 20.01 20.02 20.01 20.01 20.02 20.04 20.02 20.01 (g) Volume Titrant 23.42 23.38 23.32 13.10 13.58 13.58 13.52 16.34 16.50 16.98 16.14 16.20 16.18 15.72 15.84 15.76 (mL) 0.20 0.22 0.26 0.16 0.28 0.20 Conc (%) Final N 0.162 0.162 0.162 0.000 0.090 0.000 0.093 0.093 0.093 0.000 0.113 0.114 0.118 0.000 0.111 0.111 0.111 0.000 0.109 0.109 0.109 0.000 o C) PPI Mass 4.1006 4.1148 4.1148 4.1148 4.1148 5.0001 5.0001 5.0001 5.0001 5.0013 5.0013 5.0013 5.0013 4.9986 4.9986 4.9986 4.9986 5.0010 5.0010 5.0010 5.0010 4.1006 Initial (g) mass (g) Initial N 0.4469 0.4485 0.4485 0.4485 0.4485 0.5450 0.5450 0.5450 0.5450 0.5451 0.5451 0.5451 0.5451 0.5448 0.5448 0.5448 0.5448 0.5451 0.5451 0.5451 0.5451 0.4469 0.1766 0.1772 0.1772 0.1772 0.1772 0.2146 0.2146 0.2146 0.2146 0.2147 0.2147 0.2147 0.2147 0.2146 0.2146 0.2146 0.2146 0.2147 0.2147 0.2147 0.2147 0.1766 Initial Conc (%) N Solub. 52.53 52.58 52.34 52.74 53.16 54.83 51.69 51.86 51.82 50.58 50.92 50.71 75.60 75.59 75.32 50.96 0.00 0.00 0.00 0.00 0.00 0.00 (%) N 151 Alcalase B37 –NSIatpH 3vs. Tgase Treatment Concentrationon Alcalase-Treated RTech PPI

protein) system Tgase (UE/g 0.001 0.000 Conc 0.01 0.1 10 1

Solubility Solubility Average 49.34 52.84 54.09 51.41 54.24 68.35 (%) N (Tgase Treatment: 50-55 STDEV 0.09 0.09 0.21 0.04 0.05 0.10 Treatment: 5h,0.5 AU / RSTDEV 0.19 0.17 0.40 0.08 0.09 0.15 Sample Blank Blank Blank Blank Blank Blank 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 Liq Mass 20.03 20.03 20.03 20.00 20.00 20.03 20.02 20.02 20.06 20.03 20.02 20.04 20.01 20.01 20.00 20.03 20.00 20.03 20.01 20.03 20.04 20.01 20.01 20.02 (g) 5 gPPI, pH8.5,50-55 o C, pH6.5,2.5h; Volume Titrant 21.34 21.28 21.32 15.50 15.48 15.46 16.60 16.52 16.54 16.74 16.80 16.66 12.74 12.74 12.74 16.66 16.66 16.66 (mL) 0.32 0.32 0.32 0.14 0.24 0.00 Final N 0.147 0.147 0.147 0.000 Conc 0.106 0.106 0.106 0.000 0.114 0.113 0.113 0.000 0.116 0.117 0.116 0.000 0.088 0.087 0.087 0.000 0.116 0.117 0.117 0.000 (%) Mass (g) 5.0008 5.0008 5.0008 5.0008 5.0011 5.0011 5.0011 5.0011 3.9463 3.9463 3.9463 3.9463 5.0036 5.0036 5.0036 5.0036 5.0013 5.0013 5.0013 5.0013 5.0008 5.0008 5.0008 5.0008 Initial PPI o C) mass (g) Initial N 0.5450 0.5450 0.5450 0.5450 0.5451 0.5451 0.5451 0.5451 0.4301 0.4301 0.4301 0.4301 0.5454 0.5454 0.5454 0.5454 0.5451 0.5451 0.5451 0.5451 0.5450 0.5450 0.5450 0.5450 Conc (%) Initial N 0.2147 0.2147 0.2147 0.2147 0.2147 0.2147 0.2147 0.2147 0.1701 0.1701 0.1701 0.1701 0.2148 0.2148 0.2148 0.2148 0.2147 0.2147 0.2147 0.2147 0.2147 0.2147 0.2147 0.2147 Solub. 52.93 52.75 52.84 54.10 54.30 53.87 51.44 51.36 51.41 54.19 54.27 54.27 68.44 68.24 68.37 49.50 49.36 49.32 0.00 0.00 0.00 0.00 0.00 0.00 (%) N 152 B38 –NSIatpH 3vs. Tgase Treatmen

protein) system Tgase Conc. (UE/g 0.001 0.001 0.1 10 1 0 Solubility Solubility Mean N 12.42 13.71 15.33 20.48 9.55 9.58 (%) STDEV 0.58 0.37 0.05 0.20 4.68 0.02 RSTDEV 48.84 6.09 2.95 0.34 1.28 0.11 Sample Blank Blank Blank Blank Blank Blank 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 3 2 1 t Concentration(RTech PPI, 50-55 Liq Mass 20.02 20.01 20.02 20.02 20.00 20.04 20.01 20.01 20.05 20.03 20.02 20.02 20.01 16.49 20.02 20.03 20.02 20.02 20.05 20.01 20.01 20.03 20.01 20.02 (g)

Volume Titrant (mL) 0.16 6.28 6.29 0.00 2.76 3.10 3.02 0.00 4.20 4.04 3.98 0.26 3.48 4.22 4.20 0.00 4.50 4.50 4.40 0.20 3.24 4.46 1.60 N/A Final N 0.000 0.044 0.044 0.000 Conc 0.019 0.022 0.021 0.000 0.028 0.026 0.026 0.000 0.030 0.030 0.029 0.000 0.030 0.030 0.029 0.000 0.022 0.030 0.010 N/A (%) Mass (g) 5.0504 5.0000 5.0000 5.0000 5.0000 5.0085 5.0085 5.0085 5.0085 4.5238 4.5238 4.5238 4.5238 5.0010 5.0010 5.0010 5.0010 5.0004 5.0004 5.0004 5.0004 5.0504 5.0504 5.0504 Initial PPI o C, pH6.5,2.5h) mass (g) Initial N 0.5505 0.5450 0.5450 0.5450 0.5450 0.5459 0.5459 0.5459 0.5459 0.4931 0.4931 0.4931 0.4931 0.5451 0.5451 0.5451 0.5451 0.5450 0.5450 0.5450 0.5450 0.5505 0.5505 0.5505 Conc (%) Initial N 0.2167 0.2146 0.2146 0.2146 0.2146 0.2150 0.2150 0.2150 0.2150 0.1946 0.1946 0.1946 0.1946 0.2147 0.2147 0.2147 0.2147 0.2146 0.2146 0.2146 0.2146 0.2167 0.2167 0.2167 Solub. 12.83 12.32 12.12 13.74 13.73 13.65 15.46 15.43 15.10 10.03 14.01 20.46 20.49 10.01 0.00 0.00 0.00 0.00 4.69 0.00 0.00 8.90 9.75 N/A (%) N 153

B39 –NSIvs.pHfor STPP-Treated RTech PPI

2.00 2.50 2.50 3.00 3.50 4.00 pH Solubility Solubility Average 78 .0 0.26 37.89 0.10 8.54 21.44 1.83 2.33 23.16 0.54 0.85 15.92 0.14 5.29 16.65 0.88 7.73 1.11 14.41 (%) N STDEV RSTDEV Sample Blank 20.01 0.00 Blank 0.000 Blank 20.00 5.0016 0.00 Blank 20.01 0.5451 0.000 0.00 Blank 20.01 0.2147 5.0011 0.000 0.00 Blank 20.00 0.00 0.5451 5.0016 0.000 0.00 0.2147 0.5451 5.0006 0.000 0.00 0.2147 0.5450 5.0009 0.00 0.2147 0.5451 0.00 0.2147 0.00 3 3 3 3 3 3 2 20.01 1 20.03 2.70 2.02 0.019 0.014 5.0009 5.0009 0.5451 0.5451 0.2147 0.2147 8.80 6.58 20.02 11.660.082 5.0016 0.5451 20.02 11.600.081 5.0016 0.2147 37.98 0.5451 0.2147 37.78 2 1 2 1 2 20.02 1 20.00 7.30 2 20.01 7.02 1 20.01 0.051 4.92 0.049 4.84 2 20.02 1 20.01 0.034 5.0011 0.034 4.92 5.0011 5.30 5.0016 0.034 0.5451 5.0016 0.037 0.5451 0.5451 5.0006 0.2147 0.5451 5.0006 0.2147 0.2147 0.5450 23.78 0.2147 0.5450 22.89 16.03 0.2147 15.77 0.2147 16.03 17.28 STPP (g) 0.0403 0.0415 0.0405 0.0399 0.0406 0.0400 Mass Total

20.03 7.18 0.050 20.03 7.18 5.0003 0.5450 0.2146 23.38 20.02 0.00 0.000 20.02 0.00 5.0003 0.5450 0.2146 0.00 20.03 11.64 0.081 5.0016 0.5451 0.2147 37.89 20.02 7.00 0.049 20.02 7.00 5.0011 0.5451 0.2147 22.80 0.034 20.01 4.90 5.0016 0.5451 0.2147 15.97 20.01 N/A 0.017 20.02 2.40 5.0009 0.5451 0.2147 7.82 Mass 20.02 6.06 0.042 20.02 6.06 5.0003 0.046 20.00 6.50 0.5450 5.0003 0.2146 0.5450 19.74 0.2146 21.20 Liq (g)

Volume Titrant (ml) Final N Discard: Conc Cloudy Pink (%) Mass (g) .06055 .17 N/A 5.0006 0.5450 0.2147 Initial PPI Initial N mass (g) Initial N Conc (%) Solub. (%) N 154 B40 –NSIvs.pHfor NaCl-Treated RTech PPI

. 24 27 8.42 2.73 2.5 32.43 1.63 0.52 2.5 32.01 15.51 5.05 3.5 32.57 2.30 0.66 3.5 28.68 pH 89 .8 0.62 0.18 4 28.97 01 .2 0.41 0.12 3 30.11 Solubility Solubility Average (%) N STDEV RSTDEV Sample ln 00 0.00 Blank 20.00 0.000 0.00 Blank 20.00 5.0046 0.000 0.00 0.5455 Blank 20.01 5.0016 0.000 0.00 0.2148 0.5451 Blank 20.00 5.0030 0.000 0.75 0.00 0.2147 0.5453 Blank 20.02 5.0033 0.000 0.00 0.00 0.2148 0.5453 Blank 20.01 5.0051 0.000 0.00 0.2148 0.5455 5.0086 0.00 0.2148 0.5459 0.00 0.2150 0.00 3 3 3 3 3 3 00 8.90 8.84 2 20.00 1 20.00 0.062 0.062 5.0086 5.0086 0.5459 0.5459 0.2150 0.2150 28.98 28.78 00 9.43 10.92 2 20.00 1 20.00 0.066 9.83 0.076 9.65 2 20.00 1 20.00 5.0046 0.069 9.28 0.068 5.0046 9.22 2 20.00 1 20.00 5.0016 0.065 0.5455 9.09 5.0016 0.065 0.5455 9.11 2 20.00 1 20.01 5.0030 0.5451 0.064 9.50 0.2148 5.0030 0.5451 0.064 9.78 0.2148 2 20.00 1 20.01 5.0033 0.5453 0.061 0.2147 5.0033 0.5453 0.063 30.73 0.2147 35.58 5.0051 0.5453 0.2148 5.0051 0.5453 32.05 0.2148 31.46 0.5455 0.2148 0.5455 30.25 0.2148 30.05 0.2148 29.63 0.2148 29.68 28.51 29.41 NaCl (g) 2.3007 2.3012 2.3041 2.3014 2.3006 2.3000 Mass Total

Mass 00 8.95 0.063 5.0086 0.5459 20.00 0.2150 29.14 00 9.210.064 5.0030 0.5453 20.00 0.2148 30.02 00 9.510.067 5.0046 0.5455 20.00 0.2148 30.99 9.970.070 5.0016 0.5451 20.00 0.2147 32.51 9.060.082 5.0033 0.5453 15.38 0.2148 38.40 9.38 0.060 5.0051 0.5455 20.00 0.2148 28.12 Liq (g) Volume Titrant (ml) Conc (%) Final N Initial PPI Mass (g) mass (g) Initial N Initial N Conc (%) Solub. (%) N 155 B40 –(Continued)NSIvs.pHfor NaCl-Treated RTech PPI

2.2 63 .6 4.29 1.56 2 36.32 3.33 1.32 2 39.48

38.20 4.82 12.63 ln 00 0.00 Blank 20.01 0.000 0.00 Blank 20.00 5.0028 0.000 0.00 0.5453 Blank 20.01 5.0034 0.000 0.2147 0.5453 5.0073 0.00 0.2148 0.5458 0.00 0.2149 0.00 3 3 00 11.08 1 20.00 11.94 0.078 12.58 2 20.00 1 20.01 0.084 12.10 5.0028 0.088 10.10 2 20.01 1 20.00 5.0034 0.085 5.0034 0.5453 0.071 5.0073 0.5453 5.0073 0.5453 0.2147 0.5458 0.2148 0.5458 0.2148 36.12 0.2149 38.92 0.2149 40.98 39.39 32.89 00 11.65 2 20.00 0.082 5.0028 0.5453 0.2147 37.98 3 2.3032 2.3045 2.3076

20.01 11.83 0.083 5.0034 11.830.083 0.5453 0.2148 20.01 38.54 20.00 10.70 0.075 5.0028 10.700.075 0.5453 0.2147 20.00 34.88 20.01 13.00 0.091 5.0073 13.000.091 0.5458 0.2149 20.01 42.32 156 B41 –NSIatpH 3for NaCl-Treated RTech PPI

Mass

NaCl 0.23 0.57 1.15 0.46 1.65 2.30 2.30 0.92 (g) (w/v%) (w/v%) Conc. NaCl 0.09 0.23 0.66 0.92

Average Solub. 89 .2 1.69 18.99 0.32 5.46 22.16 1.21 0.93 35.19 0.33 3.93 33.79 1.33 20 .2 2.37 22.02 0.52 0.41 30.11 0.12 (%) N STDEV RSTDEV Sample Blank 20.01 0.00 0.000 5.0030 0.5453 0.00 0.2148 Blank 20.00 0.00 Blank 20.00 Blank 0.00 0.000 Blank 20.01 0.000 5.0048 0.00 Blank 20.01 5.0052 0.5455 0.00 0.000 0.00 0.5455 0.2148 0.000 5.002 0.00 0.2148 5.0054 0.5452 0.00 0.5455 0.2147 0.00 0.2149 3 3 3 3 2 20.00 1 20.00 9.28 9.22 0.065 0.065 5.0030 5.0030 0.5453 0.5453 30.25 0.2148 30.05 0.2148 2 20.00 1 20.00 2 20.00 5.88 1 20.00 5.89 35.50 0.5452 10.89 0.0765.002 20.00 0.2147 35.21 0.5452 10.80 0.0765.002 20.00 2 0.2147 6.69 1 0.041 6.50 0.041 2 1 0.047 0.046 5.0048 5.0048 2 20.00 1 20.00 5.0052 5.0052 0.5455 0.5455 10.60 9.90 0.5455 19.16 0.5455 0.2148 19.19 0.074 0.2148 0.069 21.80 0.2148 21.18 0.2148 5.0054 5.0054 0.5455 0.5455 34.54 32.25 0.2149 0.2149 3 3 Mass NaCl 0.2326 0.5757 1.6539 2.3016 1.1501 2.3012 Total (g)

at Various NaClSolution Concentrations 20.02 5.72 0.040 5.0048 18.62 0.5455 0.2148 18.07 6.52 0.051 5.0052 23.51 0.5455 0.2148 34.85 0.5452 10.690.0755.002 20.00 0.2147 20.00 10.61 0.074 34.57 5.0054 0.5455 0.2149 20.00 6.58 0.046 5.0004 20.00 6.78 21.46 0.047 0.5450 5.0004 0.2146 22.11 0.5450 0.2146 Mass 20.01 6.90 0.048 5.0004 22.49 0.5450 0.2146 20.01 0.00 0.000 5.0004 0.00 0.5450 0.2146 20.00 9.21 0.064 5.0030 30.02 0.5453 0.2148 Liq (g) Volume Titrant (ml) Conc. Final (%) N Initial Mass PPI (g) N mass Initial (g) Conc. Initial (%) N Solub. (%) N 157 B42 –NSIatpH 2.7for SHMP-Treated Texas PPI

B43 –NSIatpH 4.0for Pectin-Treated RTechPPI

2.65 2.69 pH .02.9 .9 1.02 0.29 4.00 28.89 pH

Average Solub. Average 54 .2 1.64 25.45 0.42 0.93 25.45 0.24 Solub. (%) N (%) N STDEV STDEV RSTDEV RSTDEV Sample Sample Blank 10.60 0.00 124.54 0. Blank 10.15 0.00 123.50 0. Blank 20.02 0.00 0.000 0.5531 5.0013 0.00 0.2178 2 10.80 1 10.60 19.16 19.00 124.54 124.54 0.248 0.251 2 11.70 1 10.47 20.26 17.82 123.50 123.50 0.242 0.238 3 3 3 2 20.03 1 20.03 8.88 8.78 0.062 0.061 0.5531 5.0013 0.5531 5.0013 28.91 28.59 0.2147 0.2147 Pectin 0.3513 0.3645 SHMP 3.7526 Mass Mass Total Total (g) (g) 00 .6 .6 .03053 .1729.17 0.5531 0.063 0.2147 20.03 8.96 5.0013 Mass Mass 10.19 17.92 123.50 10.19 17.92 0.246 124.54 10.70 19.34 0.253 Liq. Liq (g) (g) Titration Volume Volume Titrant (ml) (ml) Final N Solvent Conc Mass (%) (g) Initial Mass Conc. Final PPI (g) (%) 0 05 .39095 0.00 0.9856 000 10.50 1.3309 0 00 .74092 0.00 0.9521 000 10.03 1.2714 N Initial N mass Initial 05 .39095 25.20 0.9856 25.46 10.50 1.3309 0.9856 10.50 1.3309 00 .74092 25.46 0.9521 25.03 10.03 1.2714 0.9521 10.03 1.2714 00 .74092 25.86 0.9521 10.03 1.2714 25.67 0.9856 10.50 1.3309 Mass (g) PPI (g) Initial Conc Initial Mass (%) N (g) N Solubility Solubility Conc. Initial (%) N (%) N Solub. (%) N