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 BY 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 © Copyright by Laura Karina Lorenzo (2008) 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. ii 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 iv 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 ix 1.