Covalent Reactions Between Flavors and the Model Protein β-Lactoglobulin

A Dissertation

SUBMITTED TO THE FACULTY OF

UNIVERSITY OF MINNESOTA

BY

Vaidhyanathan Anantharamkrishnan

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

Dr. Gary A. Reineccius, Advisor

July 2020

© Vaidhyanathan Anantharamkrishnan 2020 Acknowledgements My PhD journey has been the best time of my life – I enjoyed every moment of it. Without the support of the people around, this would have not been possible.

I would like to express my sincere gratitude to my mentor, teacher, role model, friend, baking instructor, chess opponent, advisor, Prof. Gary Reineccius for his continuous support of my studies and research, for his patience, motivation, emotional and moral encouragement, enthusiasm, confidence he showed on me, and his immense knowledge. His guidance helped me become a better scientist and more importantly a better person. Our talks usually results in us doing something new and interesting, which are outside the box. My first project started with drilling a hole in a newly bought spray drier and ended with milking a cow. I had a simple goal when I came here – To learn everything. He taught me everything I know about flavor, encapsulation, GC, GCMS, how to approach a problem, and so on, and gave me opportunities to apply them to solve issues faced by industry. His passion for research and dedication to this profession has inspired me and I realized that for me to become successful, the easy way is to emulate him.

I would like to acknowledge several contributors to my research. Dr. Baraem Pam Ismail has been supportive of me from the start and taught me everything I know about proteins and food analysis. During the course of the project, we have discussed about the direction of the research, solving problems, college bowl and career. During the time I started, I observed that she was coming to work all days of the week and stayed late. I realized that if a professor is motivated to staying late after accomplishing so much, I have to work harder than that to come closer to that level.

At one point of time, I was going nowhere with this project if I could not find a LCMS that is available and affordable. Dr. Chi Chen welcomed me with open arms – gave access to his high resolution LCMS, guided me through the analysis, insightful suggestions on research. His lab group helped me a lot with sample loading and software - Dan Yao and his PhD student Yuyin Zhou. Yuyin came in during late evenings, weekends to help me load the samples, and importantly find time on the instrument.

i

Dr. Thomas Hoye, Professor, Department of Chemistry, for his help in writing the mechanisms, understand the nuances of carbonyl, Maillard and sulfur chemistry. We had long meetings over the weekends and your enthusiasm and passion towards chemistry has fascinated me. Dr. George Annor has given be many insightful suggestions on the project and been encouraging to me. Dr. Mike Mortenson gave me industrial insights on the research and his expertise in flavor, helped me immensely.

Dr. LeAnn Higgins and Dr. Todd Markowski, Center for Mass Spectrometry and Proteomics taught me everything for the Proteomics part of the project and I am grateful for their openness to teach and collaborate.

Most of my projects involves usage of equipment from pilot plant. Rey Miller, Mitch Maher, Jodi Nelson is greatly thanked for allowing me to use the equipment, their friendship, and for sharing their new creations in cheese, kefir and ice cream. All the Food Science and Nutrition Department faculty and staff is greatly appreciated. I have had several discussion with almost everyone during my term here. Every single time all of them were welcoming and happy to help. Special thanks to Dr. Ted labuza, Dr. Mar Schmidl, Dr. Tonya Schoenfuss, Dr. Dan Gallaher, Dorit Hafner, Nancy Toedt, Sara Cannon, Andrew Howe who were always there to help me or answer any questions.

I needed milk from single cow for isolating single variant protein. Dr. Brian Crooker, Professor, Department of Animal Science helped me to get it and understand the genetic variation in beta lactoglobulin. Jon-Paul Salvador helped me to milk it. Special thanks to the cow 2838 and its stepsister 2708 for their milk!

Jean-Paul Schirle-Keller has been always there for me – friend, mentor, teaching and helping with fixing equipment. I am grateful for his willingness to do all the things he did. To all my lab colleagues over the years, I appreciate their friendship and warmness that made my time in the lab fun. I would like to thank all my friends here whom I met during these years. I am so lucky to have them and this bond would continue.

ii

To my parents, grandmother and family in India for their unconditional love and endless support for supporting me to pursue my dream, a huge Thank you. As a first generation PhD graduate in our family, I hope I have made you all proud.

iii

Dedication To my parents and teachers.

This thesis was written in the middle of a world pandemic. I would also like to dedicate this work to all the health care, essential workers and in the memory of all who suffered from Covid-19.

iv

Abstract Demand for high protein plant and dairy-based diets has been increasing but delivering them has become problematic for the food industry. The flavor issues are due to multifaceted interactions that occur between food proteins and flavoring components. Over 40 years of research has been done on studying temporary interactions between flavor and proteins, but very little work has been done on more permanent interactions – covalent bonding. Covalent bonding takes place between the side chains and terminal amino acids of food proteins and reactive aroma compounds that will change the flavor profile of the product in a permanent manner.

β- lactoglobulin (BLG) was chosen as a model protein for this study as it is well characterized in both amino acid sequence and structure, its molecular weight is suitable for intact protein mass spectrometry and it is a major protein used in food industry. This thesis study developed a methodology using UPLC-ESI/qTOF-MS for monitoring the nature and extent of the covalent reactions based upon the change in molecular weight (Protein + flavor) that occurs after reaction. The cross linking of protein with flavor compounds was evaluated using gel electrophoresis. A proteomics approach using LC and tandem MS after enzymatic digestion was taken to identify the sites of post- translational modification between the flavor compounds and the BLG protein. The UPLC-ESI/qTOF-MS methodology in tandem with proteomics and gel electrophoresis yield a detailed view of flavor/BLG interactions that offered insights on minimizing these undesirable reactions in the future.

A flavoring typically is created from a mixture of volatile chemicals that generally comprise a variety of functional groups. Some of these flavor components when added to a protein matrix form covalent adducts resulting in a change in flavor character or a loss of its potency. The end result of these reactions create an imbalanced flavor, one that is not acceptable to a consumer. This research study analyzed 47 different flavor compounds from 13 different functional groups for their covalent adduct formation with BLG. Aldehydes, sulfur-containing molecules (especially thiols), and functional group- containing furans were found to be the most reactive of the flavor components studied.

v

Thiol-containing compounds reduced disulfide linkages in BLG to result in disulfide interchange and formation of new disulfide linkages with the free cysteine group. Ketones were generally stable, but α-diketones (e.g., diacetyl) were reactive. Some bases (e.g., pyrazines and pyridines) were non-reactive, while the nucleophilic allylamine was reactive. Hydrocarbons, alcohols, acids, esters, lactones, and pyrans did not give observable levels of adduct formation within the time period studied.

Due to the varied environmental conditions present in various food systems, the nature and extent of covalent interactions would likely change. This study investigated the influence of pH, temperature and water activity on the covalent adduct formation between BLG and selected flavor molecules. Covalent adduct formation was slower in acidic pHs. The rate and extent of the reaction increased with increasing pH. The rate of formation of adducts increased with temperature. Higher temperatures (45°C) caused the formation of products that were not observed at lower temperatures (4°C and 25°C). An increase in water activity lead to an increase in formation of adducts for allyl isothiocyanate. There were no observable differences for the effect of water activity on the reaction rate for benzaldehyde, citral and dimethyl disulfide.

Results will help in understanding the conditions at which flavor compounds will covalently bond with a protein and ways to potentially avoid it. Thereby, helping the food industry to develop flavor protein matrices that have a longer shelf life.

vi

Table of Contents

Acknowledgements i

Dedication iv

Abstract v

Table of Contents vii

List of tables xi

List of figures xii

Abbreviations xix

Chapter 1 : INTRODUCTION 1

1.1 Overview 1

1.2 Hypotheses 4

1.3 Objectives 5

1.4 Experimental Approach and Research plan 5

1.5 Novelty 6

1.6 Outcomes 7

1.7 Challenges 8

Chapter 2 : LITERATURE REVIEW 9

2.1 Flavor constituents 9

2.2 Protein – Composition and flavor interactions 11 2.2.1 β -Lactoglobulin - Model protein for study 13

2.3 Protein adducts - Covalent linkages 14

2.4 Mass Spectrometry for measuring protein adducts 15

2.5 Protein: Flavor Interaction 17 2.5.1 Temporary Protein: Flavor interactions 20

vii

2.5.2 Covalent Protein: Flavor reactions 24

2.6 Influence of environmental conditions on protein: flavor interactions 27 2.6.1 pH 27 2.6.2 Temperature 28 2.6.3 Various Proteins and their Concentration 29 2.6.4 Stereochemistry of Functional groups and Concentration 29 2.6.5 Protein functionality and flavor binding 30

Chapter 3 : Method to Characterize and Monitor covalent interaction of Flavor compounds with β-Lactoglobulin using Mass Spectrometry and Proteomics 31

3.1 Preface 31

3.2 Introduction 32

3.3 Materials and Methods 36 3.3.1 Isolation of single variant β-lactoglobulin (BLG) 36 3.3.2 Chemicals: 38 3.3.3 Reacting proteins and flavors 38 3.3.4 UPLC-ESI-MS/QTOF analysis 38 3.3.5 Identification of flavor modified peptides via Proteomics 39 3.3.6 SDS PAGE 42

3.4 Results and Discussion 42 3.4.1 Flavor-protein reactions 42 3.4.2 Flavor compound concentration 44 3.4.3 Intact protein analysis of Protein with no flavor added 44 3.4.4 Reaction of BLG with benzaldehyde 45 3.4.5 Reaction of BLG with Citral 47 3.4.6 Reaction of BLG with allyl isothiocyanate as added flavoring 48 3.4.7 SDS PAGE on BLG and flavor 51 3.4.8 Identifying Post translational modification sites using Proteomics 52

3.5 Conclusions 58

3.6 Supporting Information 59

viii

Chapter 4 : Covalent Adduct Formation Between Flavor Compounds of Various Functional Group Classes and the Model Protein β-Lactoglobulin 67

4.1 Preface 67

4.2 Introduction 68

4.3 Materials and Methods 71 4.3.1 Isolation of single variant β-lactoglobulin (BLG) 71 4.3.2 Chemicals 71 4.3.3 Reaction system: 74 4.3.4 UPLC-ESI-MS/qTOF analysis: 75

4.4 Results and Discussion 76 4.4.1 Protein with no flavor compound added 76 4.4.2 Unreactive Functional Groups 77 4.4.3 Reactive Functional Groups 80

4.5 Conclusion 91

4.6 Supporting Information 93

Chapter 5 : Influence of pH, Temperature, and Water Activity on Covalent Adduct Formation Between Selected Flavor Compounds and the Model Protein β- Lactoglobulin 97

5.1 Preface 97

5.2 Introduction 98

5.3 Materials and Methods 102 5.3.1 Isolation of single variant BLG 102 5.3.2 Chemicals 103 5.3.3 Reaction system 103 5.3.4 UPLC-ESI-MS/qTOF 104

5.4 Results and discussions 105 5.4.1 Protein with no flavor added 105 5.4.2 Variation in pH 106

ix

5.4.3 Variation in Temperature 112 5.4.4 Variation in Water activity 117

5.5 Conclusion 126

5.6 Supporting Information 126

Chapter 6 : Conclusion 142

Chapter 7 : References 144

x

List of tables Table 1-1: Response of the 1,876 internet users aged 18+ who eat plant-based proteins when questioned, “Which attributes are most important when you purchase a product with a plant-based protein?” Source: Light speed/Mintel, Field month: October 2017 2 ... 3 Table 2-1: Examples of aroma compounds in each functional group...... 10 Table 2-2 : One letter, three letter code and the monoisotopic mass of the twenty one amino acids present in protein...... 12 Table 3-1 Methodologies to characterize covalent interaction of protein and flavors ..... 35 Table 3-2: The number and type of reaction observed between the flavor and beta lactoglobulin at six hours...... 51 Table 3-3 : Modified peptides detected by LC-ESI-MS/MS from a tryptic digest of BLG

to which benzaldehyde was added as flavoring at 6 hours after reduction with NaBH4. . 55 Table 3-4 Modified peptides detected by LC-ESI-MS/MS from a tryptic digest of protein to which allyl isothiocyanate was added as flavoring at 6 hours ...... 57 Table 4-1Aroma compounds analyzed for reaction with BLG (reaction at ambient temperature) ...... 72 Table 4-2 Aroma compounds analyzed and their reactivity with BLG (24 h reaction time) ...... 90

xi

List of figures Figure 1-1 Schematic overview of the experimental strategy ...... 6 Figure 2-1 Amino acids (right) and some of the common flavor compounds (left)...... 19 Figure 3-1 : Mechanisms for the formation of Schiff base adduct by benzaldehyde and a

free amine-containing group in a protein and stabilization by NaBH4 reduction ...... 43 Figure 3-2 : Mechanisms for the formation of Schiff base and Michael adduct by citral

and a free amine-containing group in a protein and stabilization by NaBH4 reduction. .. 43 Figure 3-3: Mechanisms for the formation of Michael addition adduct by allyl isothiocyanate and a free amine-containing group in a protein...... 43 Figure 3-4 : Deconvoluted ESI mass spectra of 1% BLG solution in water 6 hrs after putting BLG in solution ...... 45 Figure 3-5: Deconvoluted ESI mass spectra of BLG incubated at room temperature with benzaldehyde at 12ppth for 0h, 10min, 0.5hr, 1h, 6hr and 24 hr...... 46 Figure 3-6: Deconvoluted ESI mass spectra of BLG incubated at room temperature with citral at 12ppth for 0h, 10min, 30min, 1hr, 6hr and 24 hr...... 48 Figure 3-7: Deconvoluted ESI mass spectra of BLG incubated at room temperature with allyl isothiocyanate at 12ppth for 0h, 10mins, 30mins, 1hr, 6hr and 24 hr...... 50 Figure 3-8: SDS-PAGE gel visualization of the BLG profile under reducing and non- reducing conditions with no flavor (named as pure protein), benzaldehyde, citral, and allyl isothiocyanate as flavorings. Lane 1: Molecular weight marker, Lane 2-4: protein with no added flavoring at 0h, 1h, 24 h in non-reducing conditions Lane 5-7: protein with no added flavoring at 0h, 1h, 24 h in reducing conditions. . Lane 8: Molecular weight marker, Lane 9-11: protein with benzaldehyde as flavoring at 0h, 1h, 24 h in non- reducing conditions, Lane 12-14: protein with benzaldehyde as added flavoring at 0h, 1h, 24 h in reducing conditions. Lane 15: Molecular weight marker, Lane 16-18: protein with citral as added flavoring at 0h, 1h, 24 h in non-reducing conditions Lane 19-21: protein with citral as added flavoring at 0h, 1h, 24 h in reducing conditions. Lane 22-24: protein with allyl isothiocyanate as added flavoring at 0h, 1h, 24 h in non-reducing conditions Lane 25-27: protein with allyl isothiocyanate as added flavoring at 0h, 1h, 24 h in reducing conditions...... 52 xii

Figure 3-9 Tandem mass spectra of modified peptides LIVTQTMk (position 17-24, modified m/z = 512.301) through the benzaldehyde- Schiff’s base reaction with the lysine group in BLG protein after reduction with NaBH4...... 57 Figure 3-10 Tandem mass spectra of modified peptide TPEVDDEALEkFDK (position 141-154, modified m/z =578.9367) through the allyl isothiocyanate- Michael addition reaction with the lysine group in BLG protein...... 58 Figure 3-11: Tandem mass spectra of modified peptides (a) GLDIQkVA (position 25-32, modified m/z = 467.275), (b) WENGcAQKk (position 77-86), modified m/z = 664.793), (c) k(+90.05)VLVLDTDYKK (position 107-117, modified m/z = 471.61) (d) WENGEcAQkK (position 77-86, modified m/z = 443.5309), (e) IIAEkTK (position 87- 93, modified m/z = 446.7801), (f) TKIPAVFkIDALNENK (position 92-99, modified m/z = 497.3120), (g) kIDALNENK (position 99-107, modified m/z = 567.8116), (h) VLVLDTDYkK (position 108-117, modified m/z = 642.3671), (i) VLVLDTDYKk (position 108-117, modified m/z = 642.3671), (j) TPEVDDEALEkFDK (position 140- 154, modified m/z = 575.9478), (k) TPEVDDEALEKFDk (position 140-154, modified m/z = 575.9478), (l) ALkALPMHIR (position 155-164, modified m/z = 413.9194) through the benzaldehyde- Schiff’s base reaction with the lysine group in BLG protein after reduction with NaBH4...... 63 Figure 3-12 Tandem mass spectra of modified peptides (a) LIVTQTMk (position 17-24, modified m/z =516.7936) (b) TkIPAVFK (position 92-99, modified m/z =501.7945) (c) VLVLDTDYkK (position 108-117, modified m/z =431.5696) (d) VLVLDTDYKk (position 108-117, modified m/z = 646.8512) (e) TPEVDDEALEKFDk (position 141- 154, modified m/z =578.9365) (f) LkALPMHIR (position 156-164, modified m/z =393.235) through the allyl isothiocyanate- Michael addition reaction with the lysine group in BLG protein...... 65 Figure 3-13 SDS-PAGE gel visualization of the BLG profile under reducing and non- reducing conditions with hexenal as flavoring. Lane 1: Molecular weight marker, Lane 2- 4: protein with hexenal as added flavoring at 0h, 1h, 24 h in non-reducing conditions Lane 5-7: protein with hexenal as added flavoring at 0h, 1h, 24 h in reducing conditions ...... 66

xiii

Figure 4-1 Deconvoluted ESI mass spectrum of BLG (18,276 Da) with no flavor added – i.e., the control spectrum. Adducts at +324 and +648 represent Schiff base lactose adducts of BLG...... 77 Figure 4-2 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with allylamine (57 Da) +BLG (6 h reaction). This ion is likely due to formation of an allylammonium ion adduct of the BLG produced during the ionization (see text)...... 79 Figure 4-3(a) Deconvoluted ESI mass spectrum of diacetyl (86 Da) + BLG. (24 h reaction) (b) Mechanism for the formation of 1:1 addition adducts of diacetyl and the guanidine group in arginine sidechains...... 81 Figure 4-4 Deconvoluted ESI mass spectrum of furfural (78 Da [96 Da– 18 Da (water)]) + BLG. (24 h reaction) ...... 82 Figure 4-5 (a) Deconvoluted ESI mass spectrum of citral (152 Da) + BLG. (6 h reaction)143 (b) Formation of both a Schiff base condensation product as well as a conjugate addition adduct arising from thia-Michael addition reaction...... 83 Figure 4-6 (a) Deconvoluted ESI mass spectrum of citral diethyl acetal (226 Da) + BLG. (24 h reaction time) (b) The net hydrolytic conjugate adduct formation starting from citral diethyl acetal...... 84 Figure 4-7 (a) Deconvoluted ESI mass spectra of propanethiol (76 Da) + BLG (24 h reaction time). (b) Sequential formation of mono- and bis-disulfide adducts with propanethiol. (c) Deconvoluted ESI mass spectra of 2-furfurylmercaptan (114 Da) + BLG (24 h reaction time). (d) Deconvoluted ESI mass spectra of thiophenol (110 Da) + BLG (24 h reaction time)...... 87 Figure 4-8(a) Deconvoluted ESI mass spectra of dimethyl trisulfide (126 Da) + BLG. (24 h reaction time). (b) Rationale for the formation of disulfide and trisulfide adducts via paths indicated by arrows “x” and “y”...... 88 Figure 4-9 (a) Deconvoluted ESI mass spectra of dimethyl trisulfide (126 Da) + BLG. (24 h reaction time). (b) Rationale for the formation of disulfide and trisulfide adducts via paths indicated by arrows “x” and “y”...... 89 Figure 4-10 Deconvoluted ESI mass spectrum of hexanal (100 Da) with BLG. (24 h reaction time). BLG +82 Da and +100 Da represent the Schiff base adduct and the thia-

xiv

(or aza)-Michael addition adduct, respectively. The +164 ion indicates a bis-Schiff base adduct...... 93 Figure 4-11 Deconvoluted ESI mass spectrum of benzaldehyde (106 Da) with BLG. (24 h reaction time). BLG +88 Da represents a mono-Schiff base adduct...... 94 Figure 4-12 Deconvoluted ESI mass spectrum of trans-2-hexenal (98 Da) with BLG. (6 h reaction time). Only noise level spectral data were observed, suggesting that crosslinking had occurred...... 94 Figure 4-13 Deconvoluted ESI mass spectrum of cis-3-hexenal (98 Da) with BLG. (6 h reaction time). Only noise level spectral data were observed, suggesting that crosslinking had occurred...... 94 Figure 4-14 Deconvoluted ESI mass spectrum of trans-trans-2-4-heptadienal (110 Da) with BLG. (6 h reaction time). Only noise level spectral data were observed, suggesting that crosslinking had occurred...... 95 Figure 4-15 Deconvoluted ESI mass spectra of trans-2-hexenal dimethyl acetal (144 Da) with BLG. (24 h reaction time). Only noise level spectral data were observed, suggesting that crosslinking had occurred...... 95 Figure 4-16 Deconvoluted ESI mass spectra of dimethyl disulfide (94 Da) + BLG. (24 h reaction time)...... 96 Figure 5-1 Deconvoluted ESI mass spectrum of BLG with no flavor added – i.e., the control spectrum (24 h storage)...... 106 Figure 5-2 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with benzaldehyde at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 24 h reaction)...... 107 Figure 5-3 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with citral at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 24 h reaction)...... 109 Figure 5-4 Proposed reaction mechanism for the formation of citral-Lys dihydropyridinium adduct...... 109 Figure 5-5 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with allyl isothiocyanate at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 24 h reaction)...... 110

xv

Figure 5-6 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with dimethyl trisulfide at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 24 h reaction)...... 112 Figure 5-7 Deconvoluted ESI mass spectrum of BLG incubated with benzaldehyde at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 24 h reaction)...... 113 Figure 5-8 Deconvoluted ESI mass spectrum of BLG incubated with citral at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 24 h reaction)...... 114 Figure 5-9 Deconvoluted ESI mass spectrum of BLG incubated with allyl isothiocyanate at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 24 h reaction)...... 115 Figure 5-10 Proposed mechanism for adducts and crosslinking between allyl isothiocyanate and the lysine group from the BLG protein (top) which would be reversible to the isothiocyanate in the protein leading to crosslinking...... 115 Figure 5-11 Deconvoluted ESI mass spectrum of BLG incubated with dimethyl trisulfide at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 24 h reaction)...... 117 Figure 5-12 Proposed mechanism for adducts between dimethyl trisulfide and the free cysteine group (top) and , then, with Cys-Cys disulfide residues (bottom) in BLG...... 117 Figure 5-13 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with allyl isothiocyanate at different water activities (a) 0.11 (b) 0.23 (c) 0.33 (d) 0.44 (e) 0.53 (f) 0.64 (g) 0.75 (12 ppth, 48 h reaction)...... 120 Figure 5-14 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with citral at different water activities (a) 0.11 (b) 0.23 (c) 0.33 (d) 0.44 (e) 0.53 (f) 0.64 (g) 0.75 (12 ppth, 48 h reaction)...... 122 Figure 5-15 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with benzaldehyde at different water activities (a) 0.11 (b) 0.23 (c) 0.33 (d) 0.44 (e) 0.53 (f) 0.64 (g) 0.75 (12 ppth, 48 h reaction)...... 124 Figure 5-16 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with dimethyl disulfide at different water activities (a) 0.11 (b) 0.23 (c) 0.33 (d) 0.44 (e) 0.53 (f) 0.64 (g) 0.75 (12 ppth, 48 h reaction)...... 125

xvi

Figure 5-17 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with benzaldehyde at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 1 h reaction)...... 127 Figure 5-18 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with benzaldehyde at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 6 h reaction)...... 127 Figure 5-19 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with citral at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 1 h reaction). .... 128 Figure 5-20 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with citral at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 6 h reaction). .... 129 Figure 5-21 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with allyl isothiocyanate at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 1 h reaction)...... 129 Figure 5-22 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with allyl isothiocyanate at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 6 h reaction)...... 130 Figure 5-23 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with dimethyl trisulfide at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 1 h reaction)...... 131 Figure 5-24 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with dimethyl trisulfide at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 6 h reaction)...... 131 Figure 5-25 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with benzaldehyde at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 1 h reaction)...... 132 Figure 5-26 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with benzaldehyde at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 6 h reaction)...... 133

xvii

Figure 5-27 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with citral at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 1 h reaction)...... 134 Figure 5-28 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with citral at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 6 h reaction)...... 135 Figure 5-29 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with allyl isothiocyanate at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 1 h reaction)...... 135 Figure 5-30 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with allyl isothiocyanate at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 6 h reaction)...... 136 Figure 5-31 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with dimethyl trisulfide at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 1 h reaction)...... 136 Figure 5-32 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with dimethyl trisulfide at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 6 h reaction)...... 137 Figure 5-33 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with allyl isothiocyanate at different water activities (a) 0.11 (b) 0.23 (c) 0.33 (d) 0.44 (e) 0.53 (f) 0.64 (g) 0.75 (12 ppth, 24 h reaction)...... 138 Figure 5-34 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with citral at different water activities (a) 0.11 (b) 0.23 (c) 0.33 (d) 0.44 (e) 0.53 (f) 0.64 (g) 0.75 (12 ppth, 24 h reaction)...... 139 Figure 5-35 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with benzaldehyde at different water activities (a) 0.11 (b) 0.23 (c) 0.33 (d) 0.44 (e) 0.53 (f) 0.64 (g) 0.75 (12 ppth, 24 h reaction)...... 140 Figure 5-36 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with dimethyl disulfide at different water activities (a) 0.11 (b) 0.23 (c) 0.33 (d) 0.44 (e) 0.53 (f) 0.64 (g) 0.75 (12 ppth, 24 h reaction)...... 141

xviii

Abbreviations BLG - Beta lactoglobulin

UPLC/ESI-QTOF MS - Ultra-high-pressure liquid chromatography with electro spray ionization coupled with quadrupole time-of-flight mass spectrometry

ESI/MS - Electro spray ionization mass spectrometry

SDS-PAGE - Sodium dodecyl sulfate polyacrylamide gel electrophoresis ppm – Parts per million ppth – Parts per thousand

aw – Water activity

Single letter abbreviation for amino acids are used. G – glycine, A - alanine, L - leucine,

M – methionine, F – phenyl alanine, W - tryptophan, K – lysine, Q- glutamine, E – glutamic acid, S – serine, P – proline, V – valine, I – isoleucine, C – cysteine, Y – tyrosine, H – histidine, R – arginine, N – asparagine, D – aspartic Acid, T – threonine

xix

Chapter 1 : INTRODUCTION

1.1 Overview The first chapter of this thesis gives a general introduction to the research that has been presented in detail later in this thesis. It outlines the significance, the necessity for conducting this research to the flavor and protein fields, and the objectives and hypotheses. The experimental approach, novelty and potential limitations are also presented. A more traditional literature review is presented in Chapter 2.

Flavor is one of the most important attributes of a food product to be and remain successful in the marketplace. The purpose of adding a flavoring to a food is to impart the flavor of choice, to modify/enhance a flavor that is present, or to mask an undesirable flavor present; ultimately, to enhance the acceptability of the product by the consumer1. Research from Mintel concluded that although consumers are open to plant-based proteins due to the perception of being healthier and kinder to the environment, even if they are more expensive than their traditional alternatives, however, they will not waver when it comes to flavor. As shown in Table 1-1, flavor is the most important attribute influencing consumer acceptance of plant-based proteins that could replace animal based proteins.2 The market demand for healthier foods with increased protein content, reduced fat and sugar, and minimal processing has lead the industry’s and in turn academic research interest towards understanding the basics of how each ingredient of a food matrix interacts and influences flavor release and ultimately perception.

Unfortunately, the process of flavoring foods, especially those high in protein, to gain an acceptable flavor over time is not simple. The delivery, release and overall flavor profile are affected by the physical/chemical properties and amounts of the flavor compounds added to a food matrix.3 The release of aroma, in turn, depends on the interactions that take place between flavor compounds and food. It is generally accepted that food components, e.g., fat,4 carbohydrates,5 or protein,6 interact with flavor compounds. These

1 interactions may make a flavor molecule not volatile and in turn influence flavor perception.

There is a consumer shift towards high protein foods especially plant and dairy protein, and this has posed its own set of flavor problems for food technologists. Protein isolates, used for increasing the protein content of a given food, typically have inherent off flavors associated with the source of the protein or resulting from the processing of the protein to make the isolate. The industry attempts to manage these off flavors by adding high levels of flavoring that may mask the undesirable notes. Unfortunately, this is not a good solution since the added flavoring may not adequately mask the off notes, and during subsequent storage, the added flavoring may react with the protein to become undesirable. Therefore, a robust approach is needed to understand and minimize the interactions that would affect the food during its shelf life.

2

Table 1-1: Response of the 1,876 internet users aged 18+ who eat plant-based proteins when questioned, “Which attributes are most important when you purchase a product with a plant-based protein?” Source: Light speed/Mintel, Field month: October 2017 2

Attributes All

Flavor 65%

No artificial ingredients 41%

Protein content 35%

Fiber content 28%

Non-GMO 28%

Organic claim 17%

Brand 16%

Diet friendly (e.g., vegan, vegetarian) 15%

Allergen-free (e.g., dairy, nuts) 10%

Lactose-free 9%

None of the above 8%

A paper published in 19867 concluded that "In practice, this (the trend towards plant proteins (Insert - at that time soy proteins) may prove very troublesome for the food technologist. If the trend toward new foods including more protein extracted from legumes is continued in the future, flavoring problems will probably arise. It is then likely that most of the commercial flavors currently in use will not perform at their best with these new foods since the balance between the flavor components will be disturbed by specific binding to proteins. If new flavors specially intended for these particular applications are to be created, there will be an obvious need for more knowledge in the

3

field of flavor compound-protein interactions. However, it cannot be stressed too strongly that studies should be conducted at sensorially relevant levels of flavor compounds and not just at higher concentrations that are more convenient to analyze." It is ironic that this situation remains the same even after more than 30 years.

There have been four decades of research that deal with temporary interactions of flavor and protein. These interactions are reversible and will reach an equilibrium, therefore, they could be managed by changing the physical conditions of the protein or the proper flavoring formulation. There are few studies on the relatively irreversible interactions that take place. The objective of this study is to measure, understand, and minimize a more permanent bonding: the covalent interaction that takes place between a flavor compound and a protein.

The methodology that has been adopted in this research to measure the covalent interactions uses mass spectrometry. Recent advancements in mass spectrometry have allowed us to analyze large molecules. The methodology of using mass spectrometry has been used in the medical field since the early twenty first century, for finding adducts of lipid degradation products onto a protein. But to our knowledge, this is the first time that this methodology has been applied to study covalent adduct formation between the flavor components and protein. This research paves the way to address the question of how to flavor high protein foods and make them perform well throughout the desired product shelf life by focusing on the covalent interactions.

1.2 Hypotheses 1) The interaction between a flavor and protein is multifaceted. Numerous researchers have studied temporary interactions for four decades. It has been hypothesized but not proven that covalent interactions take place. This study hypothesizes that due to the advancement in mass spectrometry, this analytical technique can be used for measuring the covalent interactions between flavor components and protein. 2) Previous studies have shown that the functional group of a flavor compound has an effect on its temporary interactions. It is hypothesized that the functional group

4

of a flavor molecule will determine its ability to undergo covalent reactions. Understanding how flavor compounds with different functional groups react with proteins can guide the selection and formulation of flavorings that perform well initially and over storage. 3) It is generally accepted, and the studies have shown that the physical conditions

(pH, Temperature (T), water activity (aw)) influence the interaction between flavor compounds and protein. It is hypothesized that the reaction environment

(pH, T, aw) will have an influence on the covalent reactions between flavor compounds and protein.

1.3 Objectives 1) The first objective was to develop a methodology to measure the covalent reactions formed between flavor compounds and proteins. 2) The second objective was to analyze the covalent adduct formation between flavor compounds of various functional group classes and β-Lactoglobulin. 3) The third objective was to study the effect of pH, storage temperature and water activity on the formation of covalent adducts between flavor components and β- Lactoglobulin.

1.4 Experimental Approach and Research plan A schematic overview of the experimental strategy for this research is shown in Figure 1-1. Briefly, a methodology was developed using mass spectrometry to observe and characterize covalent interactions that form between flavor compounds and a model protein, β-lactoglobulin (BLG). BLG was chosen as the model protein as it is the major protein in whey protein which is commonly used in the food industry for protein supplementation. In addition, the molecular weight of BLG is small enough to allow us to use accurate mass intact protein mass spectrometry ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/qTOF MS).

BLG was isolated and purified from a single homozygous cow’s milk to get a single variant of the BLG. Gel electrophoresis was used to determine if covalent crosslinking occurred. For finding the site of adduct formation, a proteomics approach was taken.

5

Changes in functional group of the flavor compound and reaction conditions, e.g., pH, T,

aw, were made, to monitor their effects on the formation of covalent interactions.

Protein Flavor Physical conditions (pH, T, aw)

Change Change

Protein + Flavor

Intact protein MS analysis (UPLC/qTOF MS)

Covalent adduct formation

Yes Proteomics (nanoflow LC/Tandem MS) Gel electrophoresis

Protein + Flavor Enzymatic digestion Protein + flavor

Aggregation of protein over time Site of adduct formation

Yes No

Crosslinking No Crosslinking

Figure 1-1 Schematic overview of the experimental strategy

1.5 Novelty The originality and novelty of the present research to the flavor field are elaborated in this section.

6

 It is generally accepted that there is covalent adduct formation between some flavor compounds, especially aldehydes and protein. However due to a lack of the necessary instrumentation, the formation of covalent bonds was only hypothesized since there was no direct way of measurement. This is the first study that develops a methodology to unambiguously demonstrate the covalent adduct formation between a flavor compound and protein.  A multilevel approach has been taken to characterize the covalent adduct formation as the methodology uses intact protein mass spectrometry to observe adduct formation, gel electrophoresis for monitoring the cross linking, and proteomics to find the site of adduct on the protein  Forty-seven flavor compounds from thirteen different functional groups have been evaluated for their formation of covalent bonds with BLG.  This is also the first study to study the effect of pH, temperature and water activity on covalent adduct formation between flavor compounds and protein.  This study integrates knowledge from other research fields such as medicine, toxicology, and proteomics. It uses instrumentation and techniques (proteomics, accurate mass spectrometer UPLC/qTOF MS) that are not normally used in flavor research.

1.6 Outcomes A problem can be solved only when we can observe, characterize and measure outcomes. Now that covalent bond formation can be measured by the methodology developed from the first objective, solutions can be found to minimize these undesirable reactions. Depending on the final objective of the researcher, the method described can be used at various levels. For example, intact protein mass spectrometry can be used to screen for covalent adduct formation. When the interest is to find the site of adduct formation, proteomics can be adapted. The outcome from the second objective was to find the flavorings that are least reactive with food proteins and explore the opportunity to pair specific flavors with certain proteins. The third objective was about finding a solution to minimize covalent adduct formation by varying physical conditions i.e. pH, temperature and water activity.

7

1.7 Challenges 1) The larger goal for us when we started this research was to apply the results and methodologies to different proteins including plant protein. With the methodology developed, it is best suited for pure/clean protein systems and proteins with a molecular weight of up to 50kDa. Plant proteins are much larger and complex than our model system, however, a proteomics methodology can be explored to analyze covalent adduct formation of flavor components with complex proteins including plant proteins. 2) With the present methodology, accurate quantification of the adducts is not possible. A solution is to conduct a targeted modified m/z run on an LCMS equipped with a quadrupole after finding the sites of adduct using a proteomics approach. 3) When there are multiple variants of a protein, and multiple proteins in a sample, the monitoring of the adduct formation becomes difficult as there is a possibility of multiple adduct formation at multiple sites from multiple proteins.

8

Chapter 2 : LITERATURE REVIEW

Note: Sections of this chapter will be submitted to Food Chemistry journal (Anantharamkrishnan et al. 2020) for publication

2.1 Flavor constituents Flavorings are mixtures of highly reactive chemicals combined in different quantities to give an overall flavor that mimics its natural counterpart. There have been about 11,300 volatile compounds identified8 in nature that could contribute to the aroma of foods. The Flavor and Extract Manufactures Association (FEMA) have approved about 4,878 flavor compounds that are generally recognized as safe (GRAS) under the food additives amendment in its 28th version.

When creating a flavor, a flavorist typically has one main component as a backbone (e.g., Isoamyl acetate for banana) and builds around it by adding other compounds in minute quantities (e.g., ethyl butyrate to provide fruity character) for making a complete, complex flavor. Each chemical has a unique character and contribution to the final aroma and taste of the flavor in the product. These compounds comprise many different functional groups9 and thus potential reactivity (Table 2-1).

9

Table 2-1: Examples of aroma compounds in each functional group.

Functional group Aroma compound Hydrocarbon β - caryophyllene, p-cymene Alcohols (l)-menthol, amyl alcohol, 3-pentanol, methylpentanol, butanol, butanediol Aldehydes vanillin, trans-crotanaldehyde, heptanal, mercaptoacetaldehyde, citral, benzaldehyde Ketones nonenone, methylbutanone, mercaptanone, 1-cyclohexe-3-one Acids hexenoic acid, propionic acid, butyric acid, hexanoic acid, phenylacetic acid Esters iso-amyl acetate, ethyl formate, methyl benzoate, dihydrocarvyl acetate, ethyl lactate, ethyl salicylate Lactones δ-dodecalactone, γ-undecalactone, pantolactone, 4-carbethoxybutyrolactone Bases acetyl pyridine, 2-acetyl-1,4,5,6- tetrahydropyridine, methyl pyrazine, Sulphur containing compounds butyl isothiocyante, diethyldisulfide, dimethyl trisulfide,dimethyl sulfone, Acetals citral diethyl acetal Ethers benzyl methyl ether, bornyl methyl ether Phenols ethyl phenol, 4-vinyl phenol Furans methyl furanthiol, acetylfuran, furaneol, ethyl furanone Epoxides, Pyrans 7-methoxycoumarin, coumarin, epoxylinalool Oxazolines Methyl quinoxaline

10

These flavor compounds are usually added above their sensory threshold to be detected as an aroma compound. The threshold of different chemicals varies widely and sometimes can be at a very small concentration (ppb or ppt). Flavorings are not designed for direct consumption; they have to be diluted to the desired concentration in food systems to impart their intended smell and taste. The sensory character and intensity of a flavoring are strongly influenced by the composition of the food matrix, processing of the food, and the storage environmental conditions.10

• Application of heat during processing would evaporate the aroma compounds and would induce Maillard interactions • Fat in a food matrix would solubilize the liposoluble flavorings which would lead to reduction in perception of those flavor components • Proteins in the food may react with flavorings • Acidic pH may hydrolyze the esters that would catalyze some chemical reactions • Oxygen in the system oxidizes some of the flavorings like terpenes.

These considerations lead to an understanding that the selection of the desirable flavor formulation and appropriate dosage for flavoring a food is complicated. Creation of flavor is still an art, which requires extensive knowledge, experience and practice.11

2.2 Protein – Composition and flavor interactions Proteins are present in all living creatures and play a key role in many biological processes.12 There are twenty-one common amino acids, which are the building blocks of all the proteins. Each amino acid contains a primary amine and carboxylic group present in them with additional functional groups like amine, thiol or sulfhydryl group, or hydroxyl groups. The common amino acids are listed in Table 2-2. The one letter, three letter code is used for the rest of this thesis for representing the amino acids. For proteomics, the monoisotopic mass of the amino acids is used in this study.

11

Table 2-2 : One letter, three letter code and the monoisotopic mass of the twenty one amino acids present in protein.

Amino Acid One letter code Three letter code Monoisotopic mass (Da) Glycine G Gly 75.0320 Alanine A Ala 89.0476 Serine S Ser 105.0425 Proline P Pro 115.0633 Valine V Val 117.0789 Threonine T Thr 119.0582 Cysteine C Cys 121.0197 Leucine L Leu 131.0946 Isoleucine I Ile 131.0946 Asparagine N Asn 114.0429 Aspartic acid D Asp 115.0269 Glutamine Q Gln 128.0586 Lysine K Lys 128.0950 Glutamic acid E Glu 129.0426 Methionine M Met 131.0405 Histidine H His 137.0589 Phenylalanine F Phe 147.0684 Arginine R Arg 156.1011 Tyrosine Y Tyr 163.0633 Tryptophan W Trp 186.0793 Selenocysteine U Sec 168.9642

There is a continuing trend for consumers to increase the amount of protein in their diets, especially plant and dairy proteins. The food industry finds it difficult to flavor high protein products. This is because the problem is multifaceted: there typically is an inherent off

12 flavor present in the protein, and any added flavor compounds, for masking or to impart the required flavor, may react with the protein thereby losing the intended effect. The following sections deals with briefly with off flavors commonly present in protein isolates and then the interaction of flavor compounds with them.

There are many sources of plant-based proteins, e.g., rice, potato, wheat, peas, soy, quinoa, chia, hemp, canola/rapeseed, and other pulses, that are commercially available in the market 13. All of these plants and, diary protein inherently have off flavors present in them. Soy protein is the most popular among all the plant proteins and therefore, it has been extensively studied. The off flavors present in soy proteins are often described as “green”, “beany”, “painty”, “grassy” and/or “bitter” 14,15. They may be due to the source of the raw material, its processing and/or storage. The off notes are commonly attributed to lipoxygenase initiated peroxidation of the unsaturated fatty acids in soybean 16.

The off flavors commonly associated with various whey proteins are grassy, hay, cheesy, and/or astringency notes which are linked to lipid oxidation 17. Bitterness is associated with proteolysis.18 As mentioned earlier, flavorings may be added to the product, sometimes in excess, to compensate for the loss during its storage due to the interaction with proteins. But the common property and the problem faced due to all of the protein sources is its nature to interact with the added flavor compounds, thereby decreasing the effect of flavor, its release and its overall perception in the food matrix.19 The nature of the interactions will also change depending on the mode of delivery of the protein (dry-protein powder, protein bar; liquid-protein drinks, fortified drinks), and its formulation (reduced sugar and/or, fat instead of full fat and/or sugar). All these factors suggest that studying the interactions of proteins and flavor compounds is vital to solve the problems limiting consumer acceptance of high protein food components.

2.2.1 β -Lactoglobulin - Model protein for study β-Lactoglobulin (BLG) constitutes about half of the whey protein in cow’s milk or about 12% of the total protein present in milk.20 It is the most abundant whey protein and is of high nutritional quality. Whey protein is available as byproduct of cheese making. BLG

13

is present in substantial quantities in cow and sheep’s milk and found in lesser abundance in the milk of horses, dogs, and cats. It is absent in milk of humans.21

BLG can be easily and readily isolated from milk. It is a globular protein of the lipocalin family, which has a molecular weight of 18.3 kDa and 162 amino acid residues. BLG is nutritionally valuable as it contains all 20 amino acids. There are two major genetic variants of BLG – A and B. The changes are Asp64Gly and Val118Ala22. This makes the molecular weight of BLG A variant to be 18363 Da and BLG B variant to be 18276 Da. BLG contains 22 Leu, 10 Ile, 15 Lys and 9 Val amino acid residues in the B variant (10 Val in A variant) which makes it one of the richest known food sources of these amino acids. The amino acid sequence of BLG was reported in 197223 and it has been unambiguously determined that there are disulfide bonds between Cys 66-Cys 16024 and Cys 106-Cys 11925,26 with Cys 12127 as the source of free thiol.

The environment of the amino acids present in BLG have been extensively studied and Sawyer28 has reviewed it. The reaction of the free Cys 121 will interfere with the helix structure that destabilizes the dimer29. Disulfide interchange under denaturing conditions leads to aggregation which is an effect that is influenced by the genetic variant30. Lysine methylation, acetylation or succinylation has some antiviral activity.31 For our study in particular, BLG has 15 lysine groups, which is advantageous as the primary flavor reactions are expected to involve the lysine groups. Additionally, the molecular weight (18.3 kDa) of BLG is suitable for our method of analysis: electrospray ionization mass spectrometry (ESI-MS). The fact that BLG is a small food protein, has been well characterized, conformation, contains all amino acids (and been sequenced), and is easily isolated, make BLG a favorite model protein for researchers to try out to new techniques and experiments.

2.3 Protein adducts - Covalent linkages Covalent bonds involve sharing of an electron pair by two atoms resulting in them being a very strong chemical bond. Examples of covalent bonding reactions that are of particular interest in proteins-flavors reactions are carbonyl reactions, e.g., Schiff base and Michael addition, and disulfide linkages32. Most of the reactions of aldehydes,

14

ketones, esters, carboxamides and carboxylic acid derivatives, which are popular functional groups of flavor molecules, directly involve the carbonyl chemistry33. Aldehydes are more reactive than ketones because of the steric effects in the later. The additional substituent increases the steric restrictions for the nucleophilic approach and cause greater steric interaction in the tetrahedral adduct as the hybridization changes from trigonal to tetrahedral. Aldehydes and ketones react with alcohols to form a reversible addition product to form hemiacetals. It is then followed by dehydration and addition of another alcohol molecule leading to the formation of acetal.

In proteins, there are specific groups (e.g.,–NH2, -SS-, -SH) where covalent linkages are most likely to take place with flavor components.9,34 Greater flavor binding has always been observed when covalent binding takes place in combination with the weaker interactions.19 Products of these reactions are salts, amides, esters and aldols35. The most common reaction for forming covalent bonding is between a flavor molecule containing an aldehyde functional group and a lysine group from the protein36. Important compounds in many savory flavors are sulfur containing aroma chemicals. These molecules could interact with free sulfhydryl and disulfide groups on a protein, which would make the flavor moiety non-volatile.37,38 In certain flavor molecules with a higher number of sulfhydryl groups, e.g., diallyl disulfide and diallyl trisulfide, they could react with cysteine and result in disulfide interchange reactions. These reactions are thermodynamically spontaneous and therefore, favorable as the free energy (ΔG = - RTlnK) is negative.39,40

2.4 Mass Spectrometry for measuring protein adducts The advancements in mass spectrometry (MS) have enabled researchers in the detection and characterization of peptides and proteins that covalently bind with small molecules. It has also made it possible the definition of the stoichiometry of the protein/adduct formation and for characterization of the sites of modification. Mass spectrometry when coupled with computational and functional studies helps in explaining the effects of the formation of the adduct formed due to interaction on the functionality of the protein and its relative consequences.41

15

The choice of the mass spectrometry used depends on the type of modification, resolution desired and the available MS. Most of the approaches fall under two categories – intact protein analysis (top-down approach) or analysis of peptides formed by enzymatically digested protein (bottom-up approach)42. Both of these approaches have their own advantages and disadvantages and has been summarized in a review.43

The top-down approach analyzes the intact protein without further sample preparation. The advantages of the top-down approach include the complete sequence coverage, assessment of protein heterogeneity, and identification of posttranslational modifications (PTM). Since proteins are large molecules, its analysis requires the use of appropriate ionization techniques and detector with appropriate resolving power and mass accuracy. Analysis of intact protein analysis became possible only after the invention and development of Matrix assisted laser desorption ionization mass spectrometry (MALDI- MS) and electrospray ionization mass spectrometry (ESI-MS). These techniques made the ionization of large proteins possible without enzymatic digestion. In MALDI, the ions are usually singly charged and therefore the resulting spectra is less complicated than the ESI-MS where the proteins would be multiply charged. A deconvolution step is then needed to convert the ESI-MS to single charged entity for easy interpretation. Fourier transform ion cyclotron resonance (FTICR) mass analyzer, orthogonal acceleration time of flight (oaTOF), quadrupole ion trap, triple quadrupole, hybrid quadrupole/oaTOF (qTOF) are detectors used for this approach with FTICR having the highest resolution, mass accuracy and peak capacity44. The resolving power of MALDI-TOF and ESI-q MS instrument is 600 and 450 for a protein of size 24kDa45, respectively. This would not be sufficient for detecting the posttranslational modification of small molecules and these analyzers have limited opportunities for tandem MS. The development of hybrid ESI qTOF MS46,47 and MALDI qTOF MS48,49 paved the way for protein structural characterization. The hybrid has a better resolution, sensitivity and an extended mass range in comparison to the linear mass analyzer. The mass resolving power of hybrid qTOF mass analyzer is approximately 2300 for a protein of size 24 kDa (trypsinogen) and 10500 for a protein of size 5.7 kDa (insulin) with mass accuracy of 20 ppm in comparison to 100 ppm to the linear mass analyzer.45

16

The identification of PTMs, glycosylation sites, disulfide linkages within and outside the protein source are most often analyzed by bottom-up approach. In this approach, the protein sample would be involved in denaturation, reduction, and alkylation of Cys residues before the enzymatic digestion step and then analyzed using MALDI MS, Orbitrap50 or ESI-MS43. It could also be sent through a LC for separation of peptides before ESI-MS. In Orbitrap MS, ions revolve around a central electrode and oscillate along its axis with a frequency proportional to the square root of m/z. In combination with ESI, it provides good sensitivity, resolution and mass accuracy of about 1-2 ppm.51,52 The front end LC is preferred as the coverage for the sequence is >95% with the usage of a single enzyme whereas a direct MS requires multiple enzymes to obtain similar sequence coverage as there would be ion suppression in complex mixture systems. MALDI MS/MS48 or offline nano spray MS/MS53 or LCMS/MS would be used to perform tandem MS (MS/MS) sequence via collision-induced dissociation (CID), post- source decay or other approaches in the protein to obtain amino acid sequence and identifying the site of PTM54. CID fragmentation of the peptides using QQQ,55 ion trap,56 qTOF57 instruments are done for proteins. In CID fragmentation, there is preferential cleavage of amide bonds of the peptide backbone to generate the b and y ions. The sequence of the amino acid is then identified from the mass difference between a series of b and y ions.58 This makes it more efficient for highly charged ions. The limitation of the bottom-top approach is the need for a series of complicated sample preparation, protein sequence coverage depending on the MS, loss of labile PTM’s and artifactual peptides from enzymatic digestion. However, it currently still is the best approach for identification of the PTM’s in proteomics.

2.5 Protein: Flavor Interaction Food proteins have little to no flavor on their own, but they will influence product flavor by binding, trapping and/or interacting/reacting with added flavor compounds. Due to flavor compound-protein binding, some flavor molecules cannot partition into the gas phase which leads to a decrease in aroma perception and changes the overall flavor balance of the product.59,60 Depending on the nature of the binding, the protein-flavor

17 interactions can also lead to production of off flavors,61 which reduces the shelf life of the food product.

The structure, functional groups, composition, concentration of the protein and the flavor directly influence the interactions between them. Some functional groups,62 common in flavor molecules (e.g., aldehydes, ketones, alcohol, sulfur, esters containing groups) are known to interact with the components of the food matrix, especially proteins14. Globular proteins have a closed structure, which is stabilized by the temporary interactions like ionic, hydrophobic, hydrogen bonding and electrostatic interactions. This makes the amino acids on the protein to have less contact with water, non-polar groups and might reduce the interactions. However, when the same protein is subjected to processing like change in pH,63 heating,64 it could change to open structure or a partially denatured state. This would make the functional groups that were on the interior of the protein to be exposed for interaction.

18

O H2N CHC OH CH O 2 O O O CH2 H N CHC H N CHC H N CHC OH H2N CHC OH 2 OH 2 OH 2 CH2 CH CH CH3 CH2 2 2 NH C O C O SH C NH NH OH O 2 NH2 Citral Hexenal Alanine Arginine Asparagine Aspartic acid Cysteine O O O O O H2N CHC OH H2N CHC OH H N CHC OH H N CHC N S O 2 2 OH C CH2 CH2 CH O H N CHC OH 2 CHCH3 CH2 CH 2 Allyl isothiocyanate Diacetyl 2 CH2 C O C O H N NH CH3 OH NH2 Glutamic acid Glutamine Glysine Histidine Isoleucine O H O S S O O O S H2N CHC OH O H N CHC OH H N CHC OH H N CHC OH 2 CH2 2 2 C OH CH CH CH 2 CH2 2 2 Benzaldehyde Dimethyl trisulfide CHCH CH 3 CH2 2 HN CH S 3 CH2 O CH NH2 3 HO H Leucine Lysine Methionine Phenylalanine Proline O HO O O O O O H2N CHC OH H2N CHC OH O CH H2N CHC OH H2N CHC OH 2 CH2 H2N CHC OH Eugenol Vanillin CH2 CHOH CHCH3 CH CH OH 3 HN 3 OH Serine Threonine Tryptophan Tyrosine Valine

Flavor Compounds Amino acids

Figure 2-1 Amino acids (right) and some of the common flavor compounds (left).

Figure 2-1 shows some of the common flavor compounds that are used in the creation of flavors (e.g., citral used for lemon flavorings, diacetyl for butter flavorings, vanillin for vanilla flavors) on the left and amino acids present in protein on the right. It could be observed that there are potential reactions between the flavor and protein. For example, flavor compounds containing carbonyl group (e.g., benzaldehyde, citral) could react with an amino acid with a free amine group (lysine).

Upon addition of these chemicals (flavor) compounds to high protein food matrices, different types of undesirable reactions will occur which will result in loss of the flavor. The interactions between the flavor and proteins are multifaceted. A compound can be bound in both temporary and a permanent manner. A permanent interaction, i.e. 19 formation of covalent bonds, takes place by the reaction of the functional group of the flavor compound with the N or C terminal and also the amino acid side chains. Also, the proteins may trap flavor compounds in hydrophobic or hydrophilic pockets, and via ionic reactions leading to weaker, temporary but also important interactions. When temporary interactions take place, the heat released is less than 20KJ/mole while it is 40KJ/mole when permanent reactions take place.65

2.5.1 Temporary Protein: Flavor interactions For the past four decades, there have been numerous studies done in an effort to understand and characterize these protein-flavor interactions. The type and rate of the interactions depend on several factors including the functional group of the flavor compounds, amino acid composition of the protein, pH, water activity, storage temperature, protein structure (native or denatured), and also the overall food composition. Studies have been done mainly on temporary interactions, but very little work has been done on a more permanent interaction: i.e. covalent bonding. The temporary interactions are reversible and therefore, by changing the environmental conditions the reaction could be reverted. However, when a covalent bonding takes places between the flavor and protein, the reaction is irreversible.

Soy protein is the most studied plant protein due to its long history of usage. Interactions of alcohols with soy protein was investigated by Chung et al.66 and they concluded that there exists both hydrophobic and to some degree, hydrogen bonding occurring. They also studied the effect of protein denaturation on these interactions. Denaturation by heating might limit the interaction of the proteins with alcohols. The binding constants increased as the carbon chain length increased because the value for hexanol was higher than that of butanol. Some researchers have tried to remove soy protein bound 2-nonanone, the compound responsible for the greeny off note,60 by using β-cyclodextrin. Under the model experimental conditions, it was found that around 94% of the bound 2-nonanone could be stripped off of the protein.67 At equal molar concentration, 2-nonanone would equally partition between the protein and β-cyclodextrin. For this method of removing undesirable aroma compounds to be viable, the β-cyclodextrin had to be present at concentrations many times higher than the soy protein. 20

There are many different types of dry protein isolates on the market, however there has been only one study conducted on a dry protein system (soy protein) and its interaction with flavor compounds at different relative humidity 68. This study studied the effect of flavor compound chemical structure on interaction with proteins. The results showed that the structure greatly determined the binding potential to the soy protein. Nonspecific van der Waals forces were found to be responsible for binding when the flavor compound was nonpolar (hydrocarbons) and as the flavor compound became polar due to functional groups like ketones, aldehydes, esters, or alcohols, there were both nonspecific and specific interactions (hydrogen bonding, dipole forces). Due to steric hindrance, the position of the double bonds in the flavor compound also affected the binding capacity. Interactions were drastically reduced by adsorbed water in the extremely low humidity region and water uptake in 30 to 50% relative humidity range did not make much difference on the binding of polar flavor compounds. Another study also reiterated that aldehydes and ketones undergo both reversible and irreversible interactions with soy protein 69.

A study was conducted by Gkianakis 70 on the interaction of different lactones with soy proteins and concluded that the percentage of binding of gamma lactones (9, 10, or 11 carbons) to the soy protein was almost the same. According to their calculations, soy protein can bind 7 delta- C9 lactone molecules, 6 gamma-C10 lactones and 17 and 13 delta C10 and C11 lactones, respectively.

Pea protein was studied for its interaction with diacetyl and found that a decrease in pH value of the protein solution resulted in a significant decrease in the retention properties of the protein which led to a partial release of the previously bound ligands.7 In addition, the binding constant was low which suggested that the interactions were occurring on the surface of the protein and they were weak and reversible. The effect of denaturation of fababean protein on its interaction with vanillin was studied by Ng, P.K.W. et al.71 They concluded that denaturation increased protein capacity for binding vanillin because the binding sites increased.

β-Lactoglobulin (BLG) has been the most studied among the dairy proteins. BLG was found to have hydrophobic interactions when reacted with ketones: heptanone, octanone and nonanone.72 Furthermore, Reiners, J et al. 72 observed that the interactions were 21

reduced by the modification of the protein structure with urea, i.e. a reduction of disulfide bonds. This study stressed the importance of the native structure of the protein in determining the binding capacity with flavor compounds.

The effect of heat treatment on protein-flavor interactions was studied by O’Neal and Kinsella (1988) 64. Their work showed that binding affinity decreased as the number of sites increased when 2-nonanone was added to BLG. Also, the reduction of disulfide bonds and esterification of carboxylic acids group lead to conformational changes in the protein, which also reduced the binding. Heat induced aggregation has also been studied by Hong Y.H et al.73 During heating, the dimer dissociates into monomers leading to a thiol group becoming accessible to the solvent. This leads to aggregates being formed via intermolecular thiol-catalyzed sulfhydryl group interchange and other non-covalent interactions.74,75 There was a change in retention between the aroma compounds and BLG when the pH was changed from 3 to 11.63 For acidic pHs, there was an increase in headspace concentration for limonene and myrcene. The difference was attributed to the flexibility of the protein molecule, i.e. surface that was exposed as the pH changed. Both covalent and non-covalent interactions of benzaldehyde with BLG (BLG used had 10% covalently bound dimers and 15% mono-lactolacted monomers) were studied76 and the study concluded that BLG monomers and benzaldehyde formed complex with a stoichiometry equal to one. This study used a spectrofluorometric technique to measure non-covalently bonded interactions and SDS-PAGE, ESI-MS techniques were used for measuring covalently bound interactions. Alternatively, static headspace technique and HPLC was used to measure the interactions between benzaldehyde and BLG by 77. This study also suggested that hydrocarbons, ketones, esters were reversibly bound to proteins by hydrophobic interactions and hydrogen bonding. Aldehydes tended to form irreversible bonds by covalently bonding with arginine present in the proteins. This group,77 Andriot I et al., also studied the effect of different environmental conditions. Volatile retention (reduced release) was found to be higher at pH 6 than at pH 3. There was a decrease in retention of benzaldehyde in the presence of salt. The presence of ethanol did not affect the rate of interaction when added together with salt. The heat treatment on retention of benzaldehyde had no effect in protein-water solution.

22

A fluorescence quenching technique was used by Dufor et al.,78 to study the interactions between β ionone and related flavor compounds with β lactoglobulin. It was found that β ionone binds with BLG but α ionone, geraniol and limonene did not bind with it. The reason proposed was the conformational constraints that originate from the conjugated double bonds present. They also concluded that β ionone binds near a tryptophan residue. Recent studies by NMR revealed that several amino acids belonging to the side chains that are pointing to the central cavity are affected by the binding of γ-decalactone whereas β ionone affects the amino acids that are present in a groove near the outer surface of the protein.79 This shows the importance of the structure of the aroma compound in the interaction with a protein molecule.

Affinity chromatography was used by Reiners J et al.,72 to determine the binding of esters, pyrazines, and phenolic compounds with proteins. Their work showed that an increase in hydrophobic chain length of an aroma compound increases the affinity for the protein. No interactions were observed between protein and short chain fatty acids or methyl pyrazines. Hydrophobic interactions were involved when binding with esters, which increased with an increase in number of carbon atoms in the acid chain. Contrary to the previous study by Dufor et al., 78, this study suggested strong interactions because of the high binding constants of terpenes. The other class of functional group, furanones, had little or no interaction with the protein.

Competitive binding between flavor molecules and protein was studied by Guichard et al., 80. Their work showed that for the same chemical class, the binding constant increased with increasing chain length as hydrophobic interactions were present. They found a linear relationship between the binding properties and hydrophobicity of flavor compounds for series of ketones, aldehydes, alcohols, lactones and esters. Competitive binding was observed between 2-nonanone and ethyl hexanoate, gamma undecalactone, β ionone and retinol.

BLG was immobilized on a HPLC column and its interaction with flavor molecules was studied by 81. It was established that in the chemical series from unsaturated alcohols, aldehydes, and ketones, the binding constant was fitting an exponential function depending on the chain length of the compound. A decrease in the intensity of fluorescence when 23

hexenal was added to whey proteins and sodium caseinate was attributed to the formation of covalent bonds 82 because the decrease was proportional to the aldehyde concentration and the formation of new fluorophores which exhibited different spectral characteristics depending on the type of aldehyde that was added. In addition, there was a decrease in proportion of the Lys, His residues and the SDS-PAGE showed a higher molecular weight band present.

There are limited studies on the other dairy proteins like α lactalbumin (ALA), bovine serum albumin (BSA) and caseins for their interaction with flavors. ALA binds with ketones and aldehydes but with a poor flavor binding capacity.83 BSA binds with aroma compounds with carbonyl group, which induces conformational change on the protein.84 They also concluded that binding affinity depends on the chain length, functional group and structural state of the protein. The affinity of ketones for BSA increased with increased in chain length84 and addition of one methylene group increased the binding affinity by three fold indicating hydrophobic interaction.

The binding of gamma decalactone to BSA was evaluated using equilibrium dialysis method revealed that BSA has higher affinity to bind with aroma compound at 10°C than 20°C, 30°C while the number of binding sites remained constant85. 2-octanone bound equally by both native and denatured form of BSA but unfolded form of ovalbumin has significant binding affinity to vanillin. Vanillin also does not bind to the denatured form of BSA.86 This suggest there is a non-specific hydrophobic interaction present.

2.5.2 Covalent Protein: Flavor reactions Until now, there have been only observations, i.e. researchers have attributed the reactions to covalent linkages but did not use a method to prove that covalent bonding was taking place.

A study by Kim H et al., proposed that when a saturated aldehyde was added as flavoring to proteins, condensation reactions of alkanals with the primary amino group took place which lead to the formation of non-volatile Schiff base products.6

The other major type of reaction that takes place between carbonyl containing compounds and proteins is Michael addition. During Michael addition, the double bond 24

in the carbon with the carbonyl group will react with the imidazole ring of histidine or lysine containing peptides, which produces non-volatile pyridinium derivatives.87 These reactions could happen multiple times depending on the number of reactive sites on the protein (e.g., number of lysine, histidine). These products lead to the stale and gluey odors in a food system containing casein88 In a soy protein hydrolysate system, hexanal was found to react with free tryptophan to form indole derivatives.89 Acetaldehyde reacted with serum albumin protein to form a Schiff base with the primary amine group, which then proceeded to a cyclic compound by undergoing a condensation reaction with the imidazole ring of histidine.90 A sensory study reported an unpleasant odor was being formed, such as burnt, woody, foul, rotten food, sweat and sulfur compounds, from the reaction products of lysine and 2-furaldehdye.91

Although there are few works done on measuring covalent interaction when flavor reacts with proteins, there are studies reported on the reaction of small molecules with proteins related to human health.

Matrix assisted laser desorption ionization time of flight mass spectra (MALDI-TOF MS) was used to monitor the early and advanced Maillard reaction products, oxidized and cross linked proteins in milk after heat treatments and in infant formulas.92 Hexanal and trans-2-hexenal were added to whey proteins and sodium caseinate solution in water. Reaction resulted in a decrease in the maximum intensity of fluorescence of tryptophanyl residues and the decrease was proportional to the aldehyde concentration. It was also observed that the proportion of free Lys residues decreased with the addition of either aldehyde but the proportion of His decreased only upon the addition of trans-2-hexenal.82 MALDI-TOF MS and liquid chromatography electrospray ionization mass spectrometry (LC –ESI MS) were used for proving that 2(E), 4(E) - decadienal (DDE) covalently bonded to Lys residues of cytochrome c, ribonuclease A and BLG proteins. It was observed that DDE formed Lys Schiff base adducts in ten minutes but the adducts are relatively unstable and could reverse in 24 hrs. However, in BLG, the Lys Schiff adducts increased and further proceeded to form Lys pyridinium adducts and Cys Michael adducts93. In another study, 4-oxo-2-nonenal (ONE), a lipid oxidation product, cross links by conjugate addition of its C=C with the side chain nucleophiles such as sulfhydryl or

25

imidazole groups of the BLG protein to give a ketoaldehyde, which further reacts to form Paal-Knorr condensation with the primary amine from Lys residues. This phenomenon was monitored using SDS-PAGE, MALDI-TOF MS and LC-ESI MS after tryptic and chymotryptic digestion.94 This study also showed that the presence of Glutathione made ONE more reactive in cytochrome c and ribonuclease, but it inhibited the modification by 4-hydroxy-2-nonenal (HNE).

A review article noted that although simple Michael and Schiff base adducts are formed initially, only some of these adducts survive the conditions of proteolysis.95 Some of these adducts that survive are ONE and HNE Michael Cys and His adducts. The reversibly formed Schiff base adducts can be stabilized by reducing with NaBH4 to survive the proteolysis and be detected by LC-ESI MS.95 In a study when HNE was exposed to BLG and human hemoglobin protein, the results concluded that >99% of the adducts were formed via Michael addition and only trace amounts of adducts were formed by Schiff base (using LC-ESI MS, gas chromatography/mass spectrometry, spectroscopic protein carbonyl assays). These results also indicated the availability of the aldehyde adduct for subsequent reaction.96 Another study bolstered the finding about the Michael adduct being the predominant modification mechanism between the HNE in low-density lipoproteins. They also suggested that the Michael adducts could be the reliable marker for atherosclerosis.97

The interaction between allyl isothiocyanate (AITC) with the lysine and phenolic groups of tyrosine residues of the mustard 12 S protein was found to increase with pH, temperature and reaction time and the reaction was complete when the AITC to protein ratio was 100:1.98 AITC reacted slowly with insulin, bovine serum albumin and lysosome protein to cleave the disulfide bonds from the cysteine residues that lead to polymerization.99 It also reacted with the arginine, lysine residues to form thiourea-like adducts. After the reaction, the digestibility with trypsin, chymotrypsin decreased but did not change with pepsin. This was because of the nature of the enzyme – The trypsin and chymotrypsin attack the peptides with basic and aromatic amino acids, but pepsin has a broader range of attack.

26

Benzyl isothiocyanate (BITC), a widely used compound in chemotherapy and cancer chemo preventive action, disturbs the lipid metabolism in rats following the in vivo long- term treatment and in vitro adipocytes.100 This was probably due to the adducts being formed. BITC also decreased the quality of egg proteins when tested by protein quality rat bioassay. BITC reduced the lysine content and its availability and affected significantly the bio utilization of nitrogen and deposition of energy.101

Three methods – fluorescence quenching, equilibrium dialysis, and head space-water equilibrium were compared for characterizing the binding kinetics of covalent binding of AITC with BLG and molecular binding was studied by mass spectrometry. The study concluded that the three methodologies were comparable and reproducible in the presence of high and low ligand concentrations for fluorescence quenching and equilibrium dialysis respectively.102 AITC reacted with the free thiol group in BLG and thereafter its other amino groups. These adducts caused destabilization of secondary and tertiary structure of BLG at pH 7.1, whereas induced molten globule conformation at pH 4.0. Conjugation reduced the heat aggregation of BLG at pH 7.1 but increased it at pH 4.0.Foams produced due to the adducts at pH 4.0 had higher volume in comparison to the foam at pH 7.1.103 Both allicin and diallyl disulfide bonded covalently with BLG but only the later formed non-covalent bond as well. Affinity for allicin was higher in comparison to the diallyl disulfide. In addition, the protein denaturation increased the reaction rate and reduced the number of binding sites for allicin but lead to an increase in non-covalent binding sites for diallyl disulfide.104

2.6 Influence of environmental conditions on protein: flavor interactions

2.6.1 pH Variation in pH would be expected to have a considerable effect on protein and flavor interactions. This is because of the structural changes and the difference in charge on the protein from acidic to basic pH. Previous works have suggested an increasing affinity for flavor compounds from pH 3 to 9 due to the structural flexibility that would give better access to the binding sites but there is a drastic decrease in flavor binding from pH 9 to 27

11.63 This is because of the alkaline denaturation of that protein at pH 11. In addition, another study did an in vivo release test of a mixture of aldehydes and varied the pH values from pH 3 to 9 reported a drastic drop in aldehyde binding between pH 7 to 9 in whey proteins.105 In contrast, another study reported an increase in binding of twenty aroma compounds (except α pinene and alcohols) when moving from pH 3 to 9.106

2.6.2 Temperature Foods will undergo a substantial range in heat treatment depending upon the food and the reason for the heat treatment (prolong shelf life, kill pathogens or provide sterility). Additionally, food products subsequently may be stored under very different temperatures (e.g., ambient, refrigerator or freezer). Due to the wide variation in potential heat treatment and storage temperatures across foods, the effect of temperature on flavor reactions must be understood. There have been several studies done on the effect of temperature on flavor-food interactions however, studies have focused on weak interactions as opposed to covalent reactions.69,70,107

Considering non-covalent flavor interactions, studies found no effect of temperature for the interactions between 2-nonanone and soy proteins (25 and 45 °C), or γ-decalactone with bovine serum albumin (10-30 °C).85 The affinity constant of 2-nonanone with soy protein increased and the number of binding sites between 5 to 25 °C. These effects were proposed to be due to the structural changes in the protein due to changes in temperature.69

A flavoring containing thiol and disulfide functional groups was heated to 100 °C with egg albumin protein and then the concentrations of the unreacted flavor components was determined using a modified Likens-Nickerson apparatus and quantification by gas chromatography-mass spectrometry.38 The results suggested that the occurrence of redox reactions with the protein caused disulfide reduction and formation of its corresponding thiol. In the same study, disulfide reduction of casein was less than egg protein because it had a lower proportion of sulfhydryl groups.

In a similar study with aroma compounds containing disulfide groups, the results indicated that when the protein is denatured, greater losses of the flavor compounds were 28

observed, and some free thiols were produced. Disulfides that contained allyl or furfuryl groups were more reactive than saturated disulfides because of the interchange reactions between protein’s sulfhydryl group and disulfides.108 BLG protein denaturation increased the reaction rate and reduced the number of binding sites for allicin but lead to an increase in non-covalent binding sites for diallyl disulfide.104

2.6.3 Various Proteins and their Concentration Depending on the number of amino acids, types of amino acids, concentration and configuration of the protein, the extent and type of interaction will vary. Amino acids, e.g., lysine, arginine, and cysteine, are more susceptible to covalent interactions and therefore, when a protein has more of these amino acids; it would have higher flavor binding capacities. A report by Reineccius9 noted that the flavor binding capacity decreased in the order: soy protein > gelatin > ovalbumin > casein > corn. Within the different fractions of soy protein, soy glycinin exhibited higher binding capacity than soy beta conglycinin to a series of ketones, alcohols, aldehydes and hexane109. This would be because of the difference in intrinsic molecular structure between the fractions. The increase of protein concentration lead to an increase in total flavor bound.110,111,7,71

2.6.4 Stereochemistry of Functional groups and Concentration The stereochemistry of a protein and a flavor compound plays a major role in determining the extent and the type of interaction that takes place between the two. Most of the work done related to this topic is also only on the temporary interactions. The aldehydes have higher binding affinity to proteins in comparison to ketones.112 But a study concluded that the relative binding strength of acid>lactone>diketone>aldehyde when they compared the adsorption of diacetyl, hexanal, gamma-butyrolactone and butyric acid to soy protein based crackers.113 Diacetyl, which is a di-ketone with two reactive centers, showed a greater adsorption than hexanal (aldehyde). An increase in aliphatic chain length of the flavor molecules increased the binding constants or greater degree of flavor retention when compared within the same functional group for aldehydes107, ketones39,107, alcohols 66and esters114,62. In the case of ketones, moving the carbonyl group to the middle of the chain decreased the binding constant39. The authors

29

attributed it to the steric hindrance of the keto group is limiting the access to the binding sites. Soy proteins had a stronger binding capacity for unsaturated aldehydes in comparison to saturated aldehydes19,115. An increase in flavor concentration lead to an increase in total flavors bound but the percentage of flavors bound remained constant7,71.

2.6.5 Protein functionality and flavor binding Protein-flavor binding could affect the functionality of the protein as well depending upon the amount of flavor added. Protein binding with vanillin was reported to affect the foaming property of different whey proteins.116 Pea protein gels were altered depending upon the functional group of the flavor (aldehyde, ketone), chain length and flavor concentration.117 Proteins are consumed for their biological properties. However, when a high lysine protein is covalently bound to a flavor molecule, it would lose some nutritionally quality. There have been no studies on this but a similar study looking at lactose reactions with proteins draws parallels. During the preparation of whey protein, due to the application of heat, the lactose would bind covalently by Schiff base mechanism with the lysine group. The lactulosyl lysine makes the lysine non- bioavailable. The protein efficiency ratio has been found to be decreased in whey protein concentrate with an average of five lactulosyl lysine residues per protein molecule.118 A detailed study with feeding the pigs with skim milk confirmed the non-bioavailability of lactulosyl lysine. This study also reported a decrease in the digestibility of lysine, phenylalanine, valine, cysteine, aspartic acid, glycine and methionine residues indicating that the lactose adduct hinders the release and utilization of adjacent amino acids. A human study on adolescent male on diets containing different levels of Maillard reaction products showed a decrease in protein digestibility.119

30

Chapter 3 : Method to Characterize and Monitor

covalent interaction of Flavor compounds with β-

Lactoglobulin using Mass Spectrometry and

Proteomics

Note: Sections of this chapter have been published in Journal of Agricultural and Food Chemistry (Anantharamkrishnan et al. 2020) (https://doi.org/10.1021/acs.jafc.9b07978) and are being reproduced here with permission from the editor (Copyright © 2020 American Chemical Society)

3.1 Preface This study develops a method to measure the covalent bonds formed between the side

chains and terminal amino acids of β-Lactoglobulin (BLG) and selected flavor molecules

(benzaldehyde, citral, or allyl isothiocyanate) using electro spray ionization mass spectrometry (ESI/MS) and MS/MS. This technique made it possible to measure increases in molecular weight of BLG as the reaction takes place (BLG+ flavor compound). The observed mass shifts on reaction corresponded to either Schiff’s base or

Michael addition reactions between the chosen flavor compounds and BLG. In the case of citral, SDS-PAGE analysis revealed that these reactions lead to protein cross-linking.

A proteomic approach using tandem MS to identify the sites of post-translational modification between benzaldehyde, allyl isothiocyante and BLG revealed that the lysine groups were the reaction sites. Interestingly, benzaldehyde was found to react with several different lysine groups but never more than one of them per BLG molecule (BLG 31

contains 15 lysine groups/molecule). Furthermore, adducts with benzaldehyde were not observed at two lysine groups. Allyl isothiocyanate was found to react with several sites

on each BLG molecule. The ESI/MS methodology in tandem with proteomics yields a

detailed view of flavor-BLG interactions that may offer insights on minimizing these

undesirable reactions in the future.

Keywords: Flavor, protein, covalent linkage, Schiff base, Michael addition, interactions,

mass spectrometry, proteomics

3.2 Introduction Food flavor is an extremely important determinant of the commercial success of a food

product. The flavor profile and thus, consumer acceptability are highly dependent upon

how the added flavor components interact with the food matrix, primarily carbohydrates

120,121, proteins6, and lipids122. The current market trend towards increased protein in foods

is presenting the industry with a growing flavoring challenge due to flavor-protein interactions.

For the past four decades, there have been a large number of studies done in an effort to understand and characterize how flavoring compounds interact with food components, primarily proteins. The type and rate of the interactions depend on several factors including the functional group of the flavor compounds, amino acid composition of the protein, pH, water activity, storage temperature, protein structure (native or denatured), and also the overall food composition. Weak interactions, e.g., hydrophobic, hydrophilic, and ionic interactions, between flavor compounds and various proteins have been intensely studied but very little work has been done on permanent interactions, i.e. covalent bonding. Some

32

early research has shown that when saturated aldehydes are added as flavorings to a protein

mixture, condensation reactions of alkanals with primary amino groups take place leading

to nonvolatile Schiff’s base formation.6

Carbonyl compounds will also react with proteins via Michael addition. In Michael

addition, the double bond formed will react with the imidazole ring of histidine or lysine

containing proteins and produce non-volatile pyridinium derivatives.87 These flavor reactions with a protein can happen multiple times depending upon the number of reactive sites, e.g., lysine amino acids, present in the protein. Formation of these aldolization compounds was considered to be the cause of stale and gluey odor in a food system containing casein.88 Indole derivatives were found to be formed when hexanal reacts with

the free tryptophan in soy protein hydrolysate.89 When acetaldehyde was reacted with

serum albumin protein, it initially formed a Schiff’s base with the primary amino group,

and then a cyclic compound by undergoing a condensation reaction with the imidazole ring

of histidine.90 Benzaldehyde has been shown to form a Schiff’s base with lysine which on

heating, lead to decarboxylation.

The past work related to covalent bonding between flavor compounds and proteins has

often been observations only with no methods to demonstrate the unequivocal formation

of covalent bonds. Studying the covalent linkages is important because they are irreversible

and permanently negatively affect product flavor and its shelf life.123

While not specifically considering flavor reactions with proteins, some basic studies have

reported on the reaction of small molecules with proteins related to human health. The

covalent interactions of 4-hydroxy-2-nonenal, 2(E),4(E)-decadienal, 4-oxo-2-nonenal

33

(aldehyde products of lipid oxidation) with human hemoglobin protein, BLG, cytochrome

c, and insulin protein have been reported in the literature. 123, 95, 124, 93, 95 This work is of

interest as numerous disease states like cancer, and cardiovascular diseases have been

associated with lipid oxidation-modified proteins.96,97 2(E),4(E)–Decadienal formed lysine:

Schiff’s base adducts, lysine pyridinium adducts and Michael adducts with cysteine in 24

hours with BLG93. Side chain modifying chemistry of 4-oxo-2-nonenal shows that it is a

more reactive protein and cross linking agent than 4-hydroxy-2-nonenal.125 More than 99%

of protein modification occurred via Michael addition and trace amounts of Schiff base

adducts were formed when 4-hydroxy-2-nonenal was reacted with BLG B and measured by electrospray ionization mass spectrometry (ESI MS).96 This study further conducted a

spectrophotometric carbonyl assay and gas chromatography mass spectrometry to confirm

the results.

The methods used to characterize covalent interactions between protein and small

molecules are summarized in Table 3-1. The first four methods in the table assume that the

change in spectral properties is only due to complex or adduct formation – there may be

other options. Also, adducts can also be formed that can lead to no change in fluorescence.

These methods are specific, measuring reactions only at certain amino acids. Mass

spectrometric methods have none of these limitations permitting the measurement of any

adduct that would be formed and identify the site of modification through proteomics.

34

Table 3-1 Methodologies to characterize covalent interaction of protein and flavors

Method What is monitored Theoretically measures References Ellman's test Quantifies free protein Assumes that a decrease 102,126 thiol (–SH) groups in free –SH is the result of a reaction with flavor compound

102 103 o-phthaldialdehyde Measures free protein Loss of free – NH3 is , amine groups (lysine assumed to be result of (OPA) Test side chains) chemical reaction with flavor compound. Spectrophotometer Direct reaction products Formation of reaction 127,128 that give fluorescence. products as a result of E.g.,, monitor the flavor compound (AITC) characteristic absorption –SH reaction. of dithiocarbamate esters (DTC), which are the direct reaction products of AITC with SH-groups of protein. Fluorescence quenching Fluorescence from the The loss of fluorescence 102 three fluorescent indicates covalent emitting amino acids. bonding to amino acids. Flavor adducts formed Any adducts formed after 126 MALDI-TOF MS on intact protein to any protein-flavor reaction. amino acid. (Limited in mass range for high resolution) UPLC/ESI/MS Flavor adducts formed Any adducts formed after 124,93 on intact protein to any protein: flavor reaction. amino acid (Broader mass range with high resolution) Proteomics Adducts to amino acids Post translational 95,93 in a peptide chain after modification on the amino enzymatically digesting acid residues. protein.

35

There have also been studies on the covalent interaction of allyl isothiocyanate (AITC) with BLG, 103 mustard 12S protein,98 soy protein fibers, 129 ovalbumin, 101 legumin, 130 and bovine serum albumin 99 focused on the potential health benefit of AITC. The kinetics of

AITC reaction with BLG was done by using mass spectrometry, fluorescence quenching and equilibrium measurement.102 This work also showed that allyl isothiocyanate was able to cleave the disulfide bond in a protein and bind with the lysine groups present. A study of isothiocyanate reactions with plant proteins131 showed that the reaction with the sulfhydryl groups decreased the activity of the enzyme bromelain. The interaction of AITC with the Mustard 12S protein found that the reaction increased with an increase in pH, temperature, and duration of the interaction within the range studied and the reaction came to completion with AITC to protein ratio of 100:1.98

The purpose of the current study was to adapt previous ESI MS methodology to analyze intact protein: flavor interactions. Further, this method should permit the application of proteomics to determine where on BLG flavor adducts are added. Such a method will subsequently be used to broadly study flavor: protein interactions leading to methodologies to reduce such reactions thereby permitting the development of better-flavored protein enhanced foods.

3.3 Materials and Methods

3.3.1 Isolation of single variant β-lactoglobulin (BLG) BLG was chosen as the model protein for study for several reasons including: it’s conformation is well characterized, amino acid composition/sequence defined, its molecular weight is suitable for ESI MS (18.2kDa), and it is a protein broadly used in the

36 food industry. BLG has 15 lysine groups, which is advantageous as the reaction was expected to take place primarily at the lysine groups. A complicating factor for our work is that commercially available whey protein is a mixture of two genetic variants: the two variants (A/B) changes are Asp64Gly and Val118Ala.22 The molecular weight of BLG A

(18,363Da) and B (18276 Da) differ by 87Da. Most flavor molecules have a molecular weight ranging from ca. 50-180 Da. Therefore, when a flavor compound would be added to a commercial mixed variant BLG and allowed to react, the resultant product MS (BLG

+flavor) would be difficult to monitor by MS, as the adducts would potentially overlap with the genetic variants complicating the measurement of the adducts. Thus, we have chosen to obtain a single variant of BLG by isolating the protein from a homozygous, single cow’s milk. The method used for isolating genetically pure BLG is described below.

Milk from a single cow was obtained from the Dairy Barn on the St Paul campus of

University of Minnesota. The milk was defatted by centrifugation (16,000 rpm for one h at

4°C). The cream layer was removed and the casein was precipitated from the defatted milk by reducing the pH of milk to 4.6 using one molar HCL. The precipitated casein was removed by filtration using a Whatmann No.1 filter paper.

The whey was then passed through a 12 kDa dialysis membrane (Fisher brand dialysis tubing, flat width 45 mm, wall thickness: 20 um, dry cylinder diameter 28.6mm, MWCO:

12000-14000) for removal of small molecular weight molecules e.g., lactose and salts resulting from pH adjustment. The resultant concentrate was freeze-dried. Size exclusion chromatography was used to separate the BLG from other whey proteins. Aliquots of freeze-dried powder (0.3 g) were dissolved in 9.7 ml of 0.05 M phosphate buffer with

0.15M NaCl. Then the sample was added to the head of a HiScale 50/20 size exclusion 37

column packed with SephacrylTM S-200 High resolution (GE Healthcare, Sweden) and

equilibrated with the 0.05M phosphate buffer with 0.15M NaCl. The column was

connected to a LC pump (Shimadzu) and the column effluent was passed through a UV

detector (Shimadzu Corp, Kyoto, Japan), monitoring absorbance at 220nm and 280nm. The

BLG fraction was collected and pooled from multiple runs. The resultant material was

dialyzed using a 12kDa membrane and freeze dried to obtain pure single variant BLG (>90% pure, pH 6.7) that was used in the experiments reported in this work.

3.3.2 Chemicals: All chemicals used were of analytical grade: Benzaldehyde (>99% pure), allyl isothiocyanate (>95% pure) and citral (mixture of isomers) (>96% pure) (Sigma Aldrich,

St. Louis, MO). All the water used in this study was double distilled.

3.3.3 Reacting proteins and flavors A one percent freeze-dried BLG solution was prepared with double distilled water. The flavoring compounds to be studied were added individually to an aliquot of the protein solution at 12ppth (parts per thousand) concentration and vortexed in a closed vessel.

Sample aliquots were taken at different reaction time points and diluted 1:10 with water for MS analysis.

3.3.4 UPLC-ESI-MS/QTOF analysis LC system: A Waters Acquity UPLC coupled to a Waters Synapt G2/Si HDMS

quadrupole orthogonal acceleration time of flight mass spectrometer (UPLC/QTOF/MS)

was used (Waters Corp., Milford, MA USA) in all MS work. A Waters Acquity UPLC

Protein BEH C4 column (2.1 mm x 100 mm column - 1.7 um diameter particles) at 35 °C

38 was used as follows. Mobile phase and gradient : 15 min linear gradient, flow rate of

0.400 mL/min, A: water containing 0.1% formic acid and B: acetonitrile containing 0.1% formic acid: 3% B, 0 min to 3 min; 3% B to 97% B, 3 min to 9 min; 97% B, 9 min to 11 min; 97% B to 3% B, 11 min to 13 min; 3% B 13 min to 15 min. Mass spectra were collected in profile mode over the range m/z 300-2500 every 0.2s during the chromatographic separation.

Mass Spectrometry: MS parameters: positive electrospray ionization mode, capillary, 0.5 kV; sampling cone, 35.0 V; extraction cone, 4.0 V; desolvation gas flow, 800 L/h; source temperature, 100 °C; desolvation temperature, 350 °C; cone gas flow, 40 L/h; trap CE, off. Lockspray (on-the-fly mass calibration) configuration consisted of infusion of a

0.5 ug/mL solution of leucine-enkephalin and acquisition of one mass spectrum (0.2s scan, m/z 300-2500) every 10s. Three lockspray m/z measurements of protonated

(positive ionization mode) leucine-enkephalin were averaged and used to apply a mass correction to measured m/z values during the course of the analysis. MaxEnt probability software from Waters was used for the deconvoluting the spectra from m/z to m. The programmed mass ranges for MaxEnt is 10000: 20000 for a resolution of 0.1Da/channel.

Uniform Gaussian model was used with width at half height 0.33Da and minimum left and right intensity ratios at 33% and the iteration was programmed for auto convergence.

3.3.5 Identification of flavor modified peptides via Proteomics One percent protein sample (1 ml) was allowed to react with a flavor compound (12 ppth) at room temperature for six hrs. NaBH4 (7.6 mg) was then added to the sample to reduce the reaction products (stabilize the adducts for MS analysis).

39

A Bradford assay was performed on the reacted protein samples to determine the protein content. This involved transferring 20 ug volume of each sample into a 1.5 ml tube and adding 4x sample volumes of denaturing buffer (7M urea, 0.4M triethylammonium bicarbonate, pH 8.5, 20% acetonitrile). The samples were sonicated at 30% amplitude for

5 sec with a Branson Digital Sonifier 250 (Branson Ultrasonics, Danbury, CT). methyl methanethiosulfonate MMTS (200 mM) was added to a final concentration of 8 mM to alkylate any reduced cysteine residues. The sample was then incubated at room temperature for 30 mins. The samples were subsequently diluted 5-fold to bring the urea concentration below 2 M. Trypsin was prepared at 0.05 ug/ul with water/10 mM CaCl2 and 10 ul was added to each sample. The samples were incubated at 37 °C overnight. The samples were then frozen at -80 °C for 30 min, and then dried in a speed-vac (concentrating the sample by evaporating the solvent in vacuum). The samples were desalted using a solid phase extraction C18 desalting cartridge and the resulting sample was used for MS analysis.

LC-MS analysis: Aliquots of ~0.05 ug of total peptide were dissolved in load solvent

(98:2:0.01, water: acetonitrile: formic acid; B Solvent, 98:2:0.01) and loaded directly onto a 14 cm x 100 um internal diameter fused silica pulled-tip (New Objective) capillary column packed in-house with Magic C18AQ resin (5 um, 200 Å pore size; Michrom Bio

Resources) with load solvent at a flow rate of 1.1 ul/min using an Eksigent 1D+ LC

nanoflow system and a MicroAS auto sampler. Peptides were eluted using a gradient of 5

– 30% B Solvent (A Solvent, 98:2:0.01, water: acetonitrile: formic acid; B Solvent,

98:2:0.01, acetonitrile: water: formic acid) over 65 at 330 nl/min with an Orbitrap Velos

(ThermoFisher Scientific) mass spectrometer in data dependent acquisition mode

40

targeting the top 6 most intense peaks with higher energy collision-induced dissociation

(HCD) collision and Orbitrap MS2 detection.

Detailed Orbitrap Velos parameters: REF: PMID 23148228 with slight variations in the acquisition parameters: lock mass was not used, MS1 scan range 380 – 1800 m/z; maximum MS1 injection time 150 ms (milliseconds); maximum MS1 injection time 200 ms; dynamic exclusion mass width +/- 15 ppm, list size 200 and duration 30 s.

Peaks® Studio [PMID: 14558135] 8.5 (Bioinformatics Solutions, Inc, Waterloo, ON CA)

was used for interpretation of tandem MS (mass spectra) and protein inference. Search

parameters for the post translational modifications were: bovine (taxonomy ID 9913)

protein sequence database from UniProt (http://www.uniprot.org/) downloaded April

30th, 2019 concatenated with the common lab contaminant database from http://www.thegpm.org/crap/ minus bovine proteins minus bovine proteins; precursor

mass error tolerance 50.0 ppm; fragment mass error tolerance 0.1 Da; precursor mass

search type monoisotopic; trypsin enzyme specificity with 1 missed cleave site and

specific digest mode; variable modifications methionine oxidation and di-oxidation, pyroglutamic acid, protein N-terminal acetylation, deamidation of N and Q, custom modifications Lactosylation with mass 324.1056 Da, benzschiffNaBH4Red with mass

90.0470 Da, benz2schiffNaBH4Red with mass 180.0939 Da, benz3schiffNaBH4Red with mass 270.1409 Da; maximum variable modifications per peptide 2; false discovery rate calculation On; spectra merge off; no charge state correction; spectral filter quality >0.65.

41

3.3.6 SDS PAGE SDS-PAGE was performed on the original protein and protein with adducts. The samples were run in both reducing and non-reducing conditions. The method used was that of 132,133.

Briefly, for non reducing conditions, an aliquot (100 ul) of the sample at each time point

(0, 6, and 24 h) was taken and mixed 1:1 (v/v) with the Laemmli buffer and boiled for five min. Samples (5 ul) and Precision PlusTM molecular weight standard (10 ul) were loaded

into the wells of a Criterion 8-16% TGX tris-glycine acrylamide gel. The gel was run at

approximately 150 V for one hour. The gel was stained with Imperial Protein Coosmassie

blue stain for one hr and then destained with water. The staining and de-staining process

was repeated three times and then the gel was destained with water overnight. A molecular

Imager Gel Doc XR system (Bio-Rad Laboratories) was used to scan the gels.

3.4 Results and Discussion

3.4.1 Flavor-protein reactions The flavor molecules used in this study, benzaldehyde, citral, and allyl isothiocyante, were

chosen for different reasons. First, they are commonly used flavor molecules

(benzaldehyde for cherry/almond flavoring, citral for lemon flavoring and allyl

isothiocyanate for garlic flavorings and other savory notes). Second, due to their structure

they can undergo only certain types of reactions. Benzaldehyde has its alpha carbon

involved in the aromatic ring and thus cannot undergo Michael addition but only Schiff’s

base reaction (Figure 3-1). Citral being a linear structure and an alpha carbon not involved on the aromatic ring, it can undergo both Schiff’s base and Michael addition (Figure 3-2).

Allyl isothiocyanate can undergo only Michael addition and not Schiff’s base reaction

(Figure 3-3). 42

H O H N H N H Protein Protein H NaBH4 + H2N Protein

Benzaldehyde (106 u) Reduced Schiff base adduct (+90 u) Schiff base adduct (+88 u)

Figure 3-1 : Mechanisms for the formation of Schiff base adduct by benzaldehyde and a

free amine-containing group in a protein and stabilization by NaBH4 reduction

NaBH4 O HO NH NH Protein Protein

Michael adduct (+ 152 u) Reduced Michael adduct (+ 152 u)

+ O H2N Protein Citral ( 152 u)

NaBH4 Protein Protein N N H

Schiff base adduct (+134 u) Reduced Schiff base adduct (+134 u)

Figure 3-2 : Mechanisms for the formation of Schiff base and Michael adduct by citral and a free amine-containing group in a protein and stabilization by NaBH4 reduction.

H H N N N S + C Protein C H2N Protein S Allyl isothiocyanate (99 u) Micheal adduct (+ 99 u)

Figure 3-3: Mechanisms for the formation of Michael addition adduct by allyl

isothiocyanate and a free amine-containing group in a protein.

43

Due to the nature of both the aldehyde and the amino acid it reacts with, the reaction product may hydrolyze, which makes it necessary to reduce the sample to its corresponding stable secondary amine or alcohol before LC MS analysis. Sodium borohydride (NaBH4) can be used at the end of the reaction to stabilize this reaction product.134, 135, 136, 123 In alkaline conditions, the addition of sodium borohydride reagent leads to the reduction of both the amino acid residue and the aldehydes reacted which would essentially stop the reaction. The advantage of reduction is that the resulting modified peptides are resistant to acid hydrolysis (HCl 6 N at 110 °C) conditions for 24 h.136, 135 In our study, this was done only for the samples that contained aldehyde groups (benzaldehyde) and that were used for identifying the post-translational modifications by enzyme digestion by proteomics. This was because for intact protein analysis, no further sample preparation steps such as enzymatic digestion or hydrolysis were involved after the reaction.

3.4.2 Flavor compound concentration The flavor industry typically uses flavoring components in final products at levels in the low ppm range. We used a much higher level (12 ppth) in this study for two reasons.

First, the task of measuring reactions is much easier/more accurate when the reaction rate/extent is higher. It would be difficult to measure reactions in the ppm range but easy at ppth levels. The same reaction would occur at lower concentration but at a much slower rate. Second, there are 15 lysine groups present in the BLG molecule. We wanted to add the flavor at a level that would potentially allow ALL lysines to react if possible.

3.4.3 Intact protein analysis of Protein with no flavor added The deconvoluted spectra of BLG with no flavor added (Figure 3-4) shows a peak of molecular mass of 18,276 Da as the major peak which is the mass of unreacted BLG B 44 variant. There was no α-lactalbumin (ALA) peak indicating the isolation procedure for

BLG was successful. However, there were major peaks with mass of 18,600 and 18,924

Da. These peaks (18,600 = BLG+ Lactose, 18,924 = BLG+ 2 Lactoses) are assumed to be

Maillard reaction adducts of lactose (molecular weight 324Da) with the main protein peak

(18,276 Da). While the sample was not heated during the isolation process in order to avoid these reactions, the Maillard reaction appeared to occur at ambient temperatures. Previous studies have reporting lactose adducts but their samples had been heated.137, 92, 138

Figure 3-4 : Deconvoluted ESI mass spectra of 1% BLG solution in water 6 hrs after putting BLG in solution

3.4.4 Reaction of BLG with benzaldehyde Upon the addition of flavor compounds to the BLG solution, new masses were observed which were not present in the BLG with no flavor added. The sample with added benzaldehyde had an adduct peak with a delta shift of 88 Da [106 (Molecular weight of benzaldehyde) – 18 (molecular weight of water lost during Schiff’s base) = 88 Da]. This new mass corresponds to the molecular weight of the Schiff’s base adduct of the BLG and benzaldehyde. The reaction pathway was shown in (Figure 3-1(a)). The new adducts were 45

present in the BLG that had lactose adducts as well. Samples were prepared at increasing

reaction times (0, 10 min, 30 min, 1 h, 6 h, 24 h) (Figure 3-5) and the deconvoluted spectra show the intensity of the adduct peaks increased and the intensity of the unreacted BLG peaks decreasing.

Figure 3-5: Deconvoluted ESI mass spectra of BLG incubated at room temperature with benzaldehyde at 12ppth for 0h, 10min, 0.5hr, 1h, 6hr and 24 hr. 46

3.4.5 Reaction of BLG with Citral The deconvoluted spectra of BLG with added citral shows adduct peaks of mass shifts

138Da and 152Da corresponding to the Schiff’s base and Michael addition reaction, respectively (Figure 3-6). The same new masses were observed on the BLG peak with the lactose adduct as well. As time of reaction increased, the native BLG peak and the native

BLG lactose adduct peak both decreased in intensity. At 24 hours (Figure 3-6(f)), peaks of both the protein and the adduct were not detected. This is due to the crosslinking that takes place between the BLG molecules when reacted with an alpha hydroxyaldehyde (e.g., citral)

– the mass of the cross-linked protein was above the useful mass range of the MS instrument.

47

Figure 3-6: Deconvoluted ESI mass spectra of BLG incubated at room temperature with citral at 12ppth for 0h, 10min, 30min, 1hr, 6hr and 24 hr.

3.4.6 Reaction of BLG with allyl isothiocyanate as added flavoring The deconvoluted spectra of the protein solution with added allyl isothiocyanate (AITC) shows that there were multiple Michael adducts formed corresponding to a mass shift of 48

99Da consecutively (Figure 3-7). Similar peaks were observed for native BLG with lactose adducts as well. This was different from what was observed from citral and benzaldehyde because only one adduct peak was detected. Also, in Figure 3-7 (e) and (f) it can be observed that all of the protein (18276 Da) has at least one allyl isothiocyanate adduct, i.e. the 18276 Da peak is at the noise level, the protein + 2 AITC being the peak with highest intensity at six hours and the protein + 3 AITC adducts being the peak with highest intensity at 24 hours. This also indicates the reaction continues to proceed and probably until all the lysine groups have adducts or the isothiocyanate is depleted. There have been studies102 reporting the reaction of AITC with cysteine to form a disulfide linkage but we have not observed this reaction at the reaction time and reaction conditions used in the current study. Our reaction time was 6 hours compared to 24 hours for the cited study. Zhu et al.94 have postulated that the free cysteine group Cys 121 is deeply buried in the protein, making it difficult to access and react with a flavor molecule. With an increase in reaction time, AITC could have been able to access the cysteine group and potentially form a disulfide linkage.

49

Figure 3-7: Deconvoluted ESI mass spectra of BLG incubated at room temperature with allyl isothiocyanate at 12ppth for 0h, 10mins, 30mins, 1hr, 6hr and 24 hr.

The type of reaction, number of adducts and the mass shifts that were observed for the reaction of the tested flavor molecules with BLG are summarized in Table 3-2.

50

Table 3-2: The number and type of reaction observed between the flavor and beta lactoglobulin at six hours.

Protein - Beta lactoglobulin Observed Type of Mass shift Incubation Flavor number of reaction (amu) time (h) bound molecules No Flavor 2 Schiff's base +324 0 Benzaldehyde 1 Schiff's base +88 6 Schiff's base, Citral 2 Michael addition +134, +152 6

Allyl isothiocyanate 4 Michael addition +99 6

3.4.7 SDS PAGE on BLG and flavor Results from the SDS PAGE (Figure 3-8) shows that for some of the flavor compounds that could undergo both Michael addition and Schiff’s base reactions, e.g., citral, the protein started to aggregate or form higher molecular weight compounds, which were not present in the pure protein sample. The same phenomena was not observed for benzaldehyde or AITC. This explains the reason that there was no native protein mass detected after 24 hours in the protein: citral sample in the LC MS analysis (Figure 3-6).

From the Figure 3-13, it can be observed that hexenal crosslinks the protein and aggregates after 24 h in both reducing and non-reducing conditions. As the SDS-PAGE was done in both reducing and non-reducing conditions, the sample result helped us to understand if the nature of the adduct was of sulfur crosslinking. Previous studies using SDS-PAGE have shown that 4-oxo-2-nonenal will cross link glutathione and carnosine to BLG protein.94

Cross linking of BLG with different aldehydes, e.g., acrolein, 2(E),4(E)-decadienal, 4- hydroxy-2-nonenal, 4-oxo-2-nonenal, and malondialdehyde, has been demonstrated by

SDS–PAGE.95 51

Figure 3-8: SDS-PAGE gel visualization of the BLG profile under reducing and non- reducing conditions with no flavor (named as pure protein), benzaldehyde, citral, and allyl isothiocyanate as flavorings. Lane 1: Molecular weight marker, Lane 2-4: protein with no added flavoring at 0h, 1h, 24 h in non-reducing conditions Lane 5-7: protein with no added flavoring at 0h, 1h, 24 h in reducing conditions. . Lane 8: Molecular weight marker, Lane 9-11: protein with benzaldehyde as flavoring at 0h, 1h, 24 h in non- reducing conditions, Lane 12-14: protein with benzaldehyde as added flavoring at 0h, 1h, 24 h in reducing conditions. Lane 15: Molecular weight marker, Lane 16-18: protein with citral as added flavoring at 0h, 1h, 24 h in non-reducing conditions Lane 19-21: protein with citral as added flavoring at 0h, 1h, 24 h in reducing conditions. Lane 22-24: protein with allyl isothiocyanate as added flavoring at 0h, 1h, 24 h in non-reducing conditions Lane 25-27: protein with allyl isothiocyanate as added flavoring at 0h, 1h, 24 h in reducing conditions.

3.4.8 Identifying Post translational modification sites using Proteomics The site of the flavor adduct on the protein was identified using proteomics. The post- translational modification (PTM) for the BLG with benzaldehyde as flavoring showed a mass shift of 90 Da (106 [molecular weight of Benzaldehyde] – 18 [molecular weight of water that is lost during Schiff’s base reaction] + 2 Da [addition of two hydrogen while reducing with NaBH4] = 90Da) at the lysine groups. The following sequence of the BLG was identified by the PEAKS software for analysis and used: 52

MKCLLLALALTCGAQALIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDAQSA

PLRVYVEELKPTPEGDLEILLQKWENGECAQKKIIAEKTKIPAVFKIDALNENKVL

VLDTDYKKYLLFCMENSAEPEQSLACQCLVRTPEVDDEALEKFDKALKALPMHI

RLSFNPTQLEEQCHI

The MKCLLLAL TCGAQA indicates that the protein has been obtained from cow.21

The following post translational modifications were programmed into the software : single benzaldehyde mass shift (90.05 Da) after reduction; two benzaldehyde mass shift

(180.09 Da) after reduction; three benzaldehyde mass shift (270.14 Da) after reduction on the K, R, C, H groups, lactosylation on the K (324.11 Da) because lactose adducts were observed to be present in the intact protein mass spectrometric analysis; oxidation

(+15.99 Da) on the M group; deamidation in the N, Q group, beta-methylthiolation on the

C group as they were added in during sample preparation and acetylation on the N term of the residues.

The results showed that the single benzaldehyde mass shift of 90.05 Da being present on the BLG sample on the lysine groups preferentially. With reference to the protein sequence shown above, the post translational modifications were observed on the lysine groups present in positions 24, 30, 85, 86, 91, 93, 99, 107, 116, 117, 151, 154, and 157. There were no modifications observed on the lysine groups present in positions 63 and 76. Table 3-3 shows all the modified peptides that have occurred due to benzaldehyde reactions. The criteria for confirming a modified peptide is the positive b or y ion for the PTM, consecutive positive five amino acid identifications after/before/along with the PTM, error lesser than 5ppm and identification of all the major spectra above the noise level. The

MS/MS spectra for the modified lysine site at 24 is shown in Figure 3-9. The 219.15 (Figure 53

3-11 Supplementary information) is the immonium ion of the lysine group with the

modification present. From the peptide sequence k(+90.05)VLVLDTDYKK (position

107-117, modified m/z = 471.61) (Figure 3-11 Supporting information), it could be

observed that even when multiple lysine are present on the sequence, there is only one

confirmatory modification that has taken place.

The post-translational modification (PTM) for the protein with allyl isothiocyanate as

flavoring showed a mass shift of 99 Da (molecular weight of allyl isothiocyanate) at the

lysine groups. The results showed that the single allyl isothiocyanate mass shift of 99.01

Da being present on the protein sample on the lysine groups preferentially. The reference

protein sequence that was used for identification is the same as the one used for finding

modification with benzaldehyde. The posttranslational modification that were observed on

the lysine groups are present in the position 24, 93, 116, 117, 151, 154, 157. The

modifications could not be confirmed on the other lysine groups present in the protein. The

Table 3-4 shows all the modified peptides that has taken place on the protein. The peptides

were confirmed by using the same criteria as that of the benzaldehyde.

The MS/MS spectra for the modified lysine site at 151 is shown in Figure 3-10 . From the

peptide sequence TPEVDDEALEkFDK (position 141-154, modified m/z =578.9367)

(Figure 3-10) and TPEVDDEALEKFDk (position 141-154, modified m/z =578.9365)

(Supporting information Figure 3-12 (e)) it could be observed that even when multiple lysine are present on the sequence, there is only one confirmatory modification that has taken place at one time.

54

Table 3-3 : Modified peptides detected by LC-ESI-MS/MS from a tryptic digest of BLG to which benzaldehyde was added as flavoring at 6 hours after reduction with NaBH4.

Mass Positio Modifie Modifie Assignme Peptides without n d m/z d Mass nt PTM

Reduced 932.536 1022.583 LIVTQTMK(+90.05) 17-24 512.301 Lys Schiff 5 5 base

Reduced 842.486 GLDIQK(+90.05)VA 25-32 467.275 932.5331 Lys Schiff 2 base

Reduced WENGEC(+45.99)AQK(+90. 1191.53 77-86 443.531 1327.569 Lys Schiff 05)K 4 base

Reduced WENGEC(+45.99)AQKK(+9 1191.53 77-86 664.793 1327.569 Lys Schiff 0.05) 4 base

Reduced

IIAEK(+90.05)TK 87-93 446.78 801.496 891.543 Lys Schiff

base

Reduced 902.558 TK(+90.05)IPAVFK 92-99 497.312 992.6059 Lys Schiff 9 base

55

Reduced 1043.56 K(+90.05)IDALNENK 99-107 567.812 1133.608 Lys Schiff 1 base

Reduced 107- 1320.76 1410.812 K(+90.05)VLVLDTDYKK 471.61 Lys Schiff 117 5 3 base

Reduced 108- 1282.717 VLVLDTDYK(+90.05)K 642.367 1192.67 Lys Schiff 117 3 base

Reduced 108- 1282.717 VLVLDTDYKK(+90.05) 642.367 1192.67 Lys Schiff 117 3 base

Reduced TPEVDDEALEK(+90.05)FD 140- 1634.76 1724.814 575.948 Lys Schiff K 154 8 5 base

Reduced TPEVDDEALEKFDK(+90.05 140- 1634.76 1724.814 575.947 Lys Schiff ) 154 8 5 base

Reduced 155- 1148.68 1238.732 ALK(+90.05)ALPMHIR 413.919 Lys Schiff 164 5 2 base

56

Table 3-4 Modified peptides detected by LC-ESI-MS/MS from a tryptic digest of protein to which allyl isothiocyanate was added as flavoring at 6 hours

Mass Modifie Modifie Assignmen Peptides Position without d m/z d Mass t PTM Lys 516.793 1031.550 LIVTQTMK(+99.01) 17-24 932.54 Michael 6 8 addition Lys 501.794 1001.573 TK(+99.01)IPAVFK 92-99 902.56 Michael 5 2 addition Lys 431.569 1291.684 VLVLDTDYK(+99.01)K 108-117 1192.67 Michael 6 6 addition Lys 646.851 1291.684 VLVLDTDYKK(+99.01) 108-117 1192.67 Michael 2 6 addition Lys 578.936 1733.781 TPEVDDEALEK(+99.01)FDK 141-154 1635.74 Michael 7 7 addition Lys 578.936 1733.781 TPEVDDEALEKFDK(+99.01) 141-154 1634.77 Michael 5 7 addition Lys 1176.662 LK(+99.01)ALPMHIR 156-164 393.235 1077.65 Michael 4 addition

Figure 3-9 Tandem mass spectra of modified peptides LIVTQTMk (position 17-24,

modified m/z = 512.301) through the benzaldehyde- Schiff’s base reaction with the lysine

group in BLG protein after reduction with NaBH4.

57

Figure 3-10 Tandem mass spectra of modified peptide TPEVDDEALEkFDK (position

141-154, modified m/z =578.9367) through the allyl isothiocyanate- Michael addition

reaction with the lysine group in BLG protein.

3.5 Conclusions In summary, this chapter reports on a methodology for detecting and monitoring covalent

interactions between flavor compounds and BLG. UPLC/ESI-QTOF MS was used to monitor the mass shift that occurs when a covalent protein: flavor molecule bond is formed.

The primary reaction mechanisms that were observed were Schiff’s base and/or Michael addition involving lysine. While there has been some previous work published that demonstrates other amino acid: flavor compound reactions, only lysine reactions were observed in this study. This may be due to the reaction conditions (e.g., pH, time, temperature, etc.) or sample environment used in this study.

As one would expect based on the reaction mechanisms (Schiff’s base or Michael addition), benzaldehyde underwent only Schiff’s base reaction, citral both Schiff’s base and Michael addition, and AITC only Michael addition. SDS-PAGE data showed that the Michael addition reaction proceeds to cross-linking and subsequent BLG aggregation. Proteomics show that there is no specificity for the benzaldehyde molecule to preferentially choose a site in the protein. However, only one modification takes place on the protein at a time 58

even though there are 15 lysine groups and an abundance of flavor compound. Proteomics data also confirmed the modification sites on BLG due to allyl isothiocyanate. Multiple adducts were found in the intact mass spectra but couldn’t be confirmed by proteomics.

This study provides an analytical methodology to further study the reactivity of other flavor compounds, the influence of sample environment and reaction conditions, and potentially how flavor compound reactions occur in a simple, but real protein system.

3.6 Supporting Information Tandem mass spectra of other peptides with the posttranslational modifications are

provided.

a)

b)

59 c)

d)

e)

f)

60

g)

h)

i)

61

j)

k)

l )

62

Figure 3-11: Tandem mass spectra of modified peptides (a) GLDIQkVA (position 25-32, modified m/z = 467.275), (b) WENGcAQKk (position 77-86), modified m/z = 664.793), (c) k(+90.05)VLVLDTDYKK (position 107-117, modified m/z = 471.61) (d) WENGEcAQkK (position 77-86, modified m/z = 443.5309), (e) IIAEkTK (position 87- 93, modified m/z = 446.7801), (f) TKIPAVFkIDALNENK (position 92-99, modified m/z = 497.3120), (g) kIDALNENK (position 99-107, modified m/z = 567.8116), (h) VLVLDTDYkK (position 108-117, modified m/z = 642.3671), (i) VLVLDTDYKk (position 108-117, modified m/z = 642.3671), (j) TPEVDDEALEkFDK (position 140- 154, modified m/z = 575.9478), (k) TPEVDDEALEKFDk (position 140-154, modified m/z = 575.9478), (l) ALkALPMHIR (position 155-164, modified m/z = 413.9194) through the benzaldehyde- Schiff’s base reaction with the lysine group in BLG protein after reduction with NaBH4.

(a)

(b)

63

(c)

(d)

64

(e)

(f)

Figure 3-12 Tandem mass spectra of modified peptides (a) LIVTQTMk (position 17-24, modified m/z =516.7936) (b) TkIPAVFK (position 92-99, modified m/z =501.7945) (c)

VLVLDTDYkK (position 108-117, modified m/z =431.5696) (d) VLVLDTDYKk

(position 108-117, modified m/z = 646.8512) (e) TPEVDDEALEKFDk (position 141-

154, modified m/z =578.9365) (f) LkALPMHIR (position 156-164, modified m/z

=393.235) through the allyl isothiocyanate- Michael addition reaction with the lysine group in BLG protein.

65

Figure 3-13 SDS-PAGE gel visualization of the BLG profile under reducing and non- reducing conditions with hexenal as flavoring. Lane 1: Molecular weight marker, Lane 2-

4: protein with hexenal as added flavoring at 0h, 1h, 24 h in non-reducing conditions

Lane 5-7: protein with hexenal as added flavoring at 0h, 1h, 24 h in reducing conditions

66

Chapter 4 : Covalent Adduct Formation Between

Flavor Compounds of Various Functional Group

Classes and the Model Protein β-Lactoglobulin

Note: Sections of this chapter have been published in Journal of Agricultural and Food Chemistry (Anantharamkrishnan et al. 2020) (https://doi.org/10.1021/acs.jafc.0c01925) and are being reproduced here with permission from the editor (Copyright © 2020 American Chemical Society)

4.1 Preface The formation of covalent bonds between forty-seven flavor compounds belonging to

thirteen different classes of functional groups and β-lactoglobulin (BLG) has been

evaluated using electrospray ionization protein mass spectrometry. Covalent bond

formation was determined by the appearance of ions in the mass spectra corresponding to

BLG + flavor molecule(s). The observed processes for covalent bond formation were

Schiff base, Michael addition and disulfide linkages. Some reactions resulted in protein

cross-linking. Aldehydes, sulfur-containing molecules (especially thiols), and functional group-containing furans were the most reactive flavor components. The thiol-containing compounds cleaved one or both electrophilic disulfide linkages in BLG to form disulfide linkages and the sulfides formed covalent bonds with the free cysteine group. Ketones were generally stable but α-diketones (e.g., diacetyl) were reactive. Some bases (e.g., pyrazines and pyridines) were unreactive while the nucleophilic allylamine was reactive.

Hydrocarbons, alcohols, acids, esters, lactones, and pyrans did not give observable levels

67

of adduct formation within the period studied. The formation of covalent bonding (flavor-

protein) is potentially responsible for the loss of flavor, limiting the shelf life of many

foods.

Keywords: protein, flavor, covalent interaction, functional group, mass spectrometry,

UPLC-TOF-MS

4.2 Introduction Flavorings consist of complex mixtures of reasonably small and volatile organic chemicals that bind to major food components, such as carbohydrates, fats, and proteins, as well as to other minor components. Understanding the nature of the interactions that

occur between flavorings and food components is important, as it is only possible to

smell or taste flavorants that are released in the oral cavity during consumption. Flavor

compounds that are bound to food components in a fashion where they will not be

released during consumption will not contribute to food flavor perception.

With the consumer trend towards increased protein in foods, the interactions between

flavor components and proteins have become an issue of growing importance.2 These

interactions may be through either adsorption involving weak interactions (e.g., ionic,

hydrophilic, or Van der Waals) or through formation of stronger and more permanent

engagements (i.e., covalent bond formation). Most weak interactions come to an

equilibrium state quickly. Both the on and the off rate of binding can be expected to be

relatively rapid, and the latter is directly relevant to release and perception of the

flavorant. Accordingly, flavorant adsorption to a protein can be readily managed in flavor

formulation. However, covalent adducts are not likely to release the flavorant during

68

consumption, so the extent to which they have occurred following addition of flavoring to

a food source will influence the perception of the consumer. It is reasonable to posit that

these covalent bond forming reactions will proceed until the flavoring is essentially

exhausted or until essentially all the reactive sites on the protein are occupied. Covalent

reactions relate directly to the issue of shelf life. While there has been more than four

decades of research done to gain an understanding of the weak interactions between flavor compounds and proteins, very little research has been devoted to understanding the covalent reactions between a flavor compound and a protein.

To understand how covalent reactions influence perception, some background on

flavorings may be useful. A formulated flavoring typically is composed of 10 to 20

different aromatic flavor molecules (essential oils may contribute 100+ components).

These flavor components generally contain numerous different functional groups. The

functional group is often what imparts the characteristic aroma of a chemical. Each flavor

component imparts a unique character and contribution to the final aroma of the product.

Covalent chemical reactions between a flavorant component will typically result in a

reduction or complete loss of that component, thereby altering the characteristic of the

overall flavor (e.g., loss of citral would result in the loss of the lemon character, or loss of

hexenal would result in a loss of the green, fresh character). Therefore, understanding the

interaction of different functional groups with food components, primarily protein, is

important for the flavorist, flavor application scientist, and food technologist.139

Although there are numerous functional groups potentially used in creating a given

flavoring, researchers have studied only a few of these classes of compounds, and then

the focus has largely been on the nature of their weak interactions.62,140,141,66,19,69 Of these 69 functional groups, aldehydes have been a favorite for study because of their broad usage and importance in many of the fruit flavors.112 Studies of other functional groups (e.g., ketones, alcohols, esters, acids, pyrazines, and hydrocarbons) are more limited. Aroma compounds having, e.g., aldehyde, enal, iso thiocyanate, disulfide, or thiol functional groups could, in principle, readily form covalent interactions with proteins by forming, e.g., Schiff base adducts, Michael addition adducts, or disulfide linkages in addition to their weak interactions.65,89,35 These covalent interactions are particularly important to understand as these bonds are relatively stable. Once formed, they can be expected to be cleaved only slowly, making them difficult to be compensated for in formulation.

The aim of the present study was to evaluate by mass spectrometry the ability of aroma compounds having a broad array of different functional groups to react with the model protein β-lactoglobulin (BLG). BLG was chosen for this study as it is well characterized in both amino acid composition/sequence and structure, its molecular weight is suitable for ESI MS, and it is a major protein used in the food industry. Interactions of BLG with several of the flavorants we have examined here have been studied,142 although the nature of those “interactions” (covalent vs. non-covalent binding) was not determined. BLG has two major variants (A and B) in nature. The differences in the variants are at Asp64Gly and Val118Ala.22 The molecular weight of the variants differ by 87 Da. The molecular weight of flavor molecules typically ranges from about 50-180 Da and, therefore, when

BLG and flavor molecules react to form covalent adducts, the adduct (BLG+ flavor) may overlap in mass with the second variant. This could complicate monitoring of adduct formation with flavor compounds by mass spectrometry. Thus, single variant BLG was isolated from a single homozygous cow’s milk.

70

Here we describe observations of covalent bond formation between representative

flavorants containing aldehyde, enal, α-diketone, thiol, di- and trisulfide functionality with BLG using intact protein mass spectrometry. Non-covalent adducts are not expected to be identified by mass spectrometry (i.e., to alter the molecular weight of the BLG).

4.3 Materials and Methods

4.3.1 Isolation of single variant β-lactoglobulin (BLG) The procedure for isolation of B variant of BLG was adapted from a previous

publication143 and is briefly described below. Milk was defatted by removing the cream

layer that formed on centrifugation. By reducing the pH to 4.6, casein was precipitated

and then removed by filtration. Small molecular weight sugars and salts were removed

from the resultant whey by dialysis using a 12 kDa membrane. The resulting whey protein concentrate was then freeze-dried. A Hi Scale 50/20 size exclusion column (50

mm ID, 200 mm L) packed with high-resolution SephacrylTM S-200 connected to a LC

pump (Shimadzu) and UV detector (Shimadzu Corp, Kyoto, Japan) was used to separate

the BLG from the other whey proteins. The collected BLG fraction was again dialyzed

using a 12 kDa membrane to remove salts used during size exclusion and then freeze- dried. The resultant pure single BLG variant, >90% pure, pH 6.7 was used for the experiments reported in this article.

4.3.2 Chemicals All chemicals used in this study were of analytical grade and purchased from Sigma

Aldrich, St. Louis, MO (see Table 4-1for aroma compounds studied, reason for including

71 a compound in this study, and compound molecular weight). The water used in the study was doubly distilled.

Table 4-1Aroma compounds analyzed for reaction with BLG (reaction at ambient temperature)

Functional Group Molecular of the flavor Compound Reason for study weight (Da) compound added pure protein No Flavor control 18,276 (BLG) Hydrocarbon p-cymene aromatic 134 (l)-menthol terpene alcohol 156 double bond to alpha geraniol 154 Alcohols carbon 2-pentanol secondary alcohol 88 2,3-butanediol dihydroxy 90 diacetyl diketone 86 2-heptanone carbonyl with six carbons 114 Ketones 2-nonenone carbonyl with nine carbons 140 cyclotene cyclic, diketone 112 butyric acid fatty acid 88 2-hydroxybenzoic Acids phenolic acid 138 acid m-toluic acid aromatic acid 136 methyl free amine group 151 anthranilate Esters iso-amyl acetate ester 130 ethyl lactate lactate 118 methyl salicylate phenolic group 152

72

delta lactone (six- δ-dodecalactone 198 membered ring) gamma lactone (five- Lactone γ-butyrolactone 86 membered ring) gamma lactone (five- γ-decalactone 170 membered ring) 3-acetyl pyridine pyridine 121 allylamine amine 57 Amine Bases 2,5-dimethyl pyrazine 108 pyrazine 4-methyl-5-vinyl- thiazole 125 thiazole dimethyl sulfide sulfide 62 dimethyl disulfide disulfide 94 dimethyl trisulfide 126 trisulfide Sulfur-containing dimethyl sulfone sulfone 94 propanethiol aliphatic thiol 76 thiophenol aromatic thiol 110 allyl isothiocyanate 99 isothiocyanate 2-methyl thiophene 98 thiophene trans-2-hexenal- acetal 144 dimethyl acetal Acetal citral diethyl acetal 226 acetal 2,6- dimethylphenol 122 Phenols dimethylphenol 4-vinylphenol vinylphenol 120

73

allyl chain-substituted eugenol 206 guaiacol furfuryl furan substituted with a 114 mercaptan thiomethyl group Furans 2,5-dimethylfuran carrying furaneol additional oxo 128 and hydroxy groups Pyranone maltol a 4-pyranone 126 vanillin phenolic aldehyde 152 hexanal saturated aldehyde 100

trans-2-hexenal conjugated enal 98

cis-3-hexenal nonconjugated enal 98

furfural furanyl aldehyde 96 Aldehydes trans,trans-2,4- conjugated dienal 110 heptadienal citral conjugated enal 152

benzaldehyde aromatic aldehyde 106

4.3.3 Reaction system: A one weight-percent freeze-dried BLG solution was prepared in doubly distilled water.

Flavor compounds were added individually to the protein solutions at 12 ppth (parts per thousand by weight) concentration, vortexed in a closed vessel, and then analyzed at storage intervals ranging from 10 min to 24 h and, on occasion, extending to 48 h

(storage at ambient temperature). Samples were diluted 1:10 with water for analysis by

MS. The molar ratio of flavor compound to BLG ranged from ca. 50–250. BLG contains

15 Lys, 1 Cys, 2 Cys-disulfides (from 4 additional Cys), and 3 Arg residues. The concentration of flavor used in this study is much higher than that used in the industry.

74

The reason for this is the ease and accuracy of measuring the adducts when the reaction occurs in greater numbers than what would be found when adding the model flavor compounds at typical industry usage levels i.e. lower parts per million levels.143

4.3.4 UPLC-ESI-MS/qTOF analysis: The method for mass spectrometry is adapted from a previous publication.143

LC system: A Waters Acquity UPLC coupled to a Waters Synapt G2/Si HDMS quadrupole orthogonal acceleration time of flight mass spectrometer (UPLC/qTOF/MS)

(Waters Corp., Milford, MA, USA) was used for analyzing the protein and flavor interaction. A Waters Acquity BEH C4 column (2.1 mm x 100 mm column – 1.7 um diameter particles) at 35 °C was programmed as follows: 15 min linear gradient separation at a flow rate of 0.400 mL/min using A: water containing 0.1% formic acid and B: acetonitrile containing 0.1% formic acid: 3% B, 0 min to 3 min; 3% B to 97% B, 3 min to 9 min; 97% B, 9 min to 11 min; 97% B to 3% B, 11 min to 13 min; 3% B 13 min to 15 min. Mass spectra were collected in profile mode over the range m/z 300-2500 every 0.2 s during the chromatographic separation.

Mass spectrometry: MS parameters in positive electrospray ionization mode were as follows: capillary, 0.5 kV; sampling cone, 35.0 V; extraction cone, 4.0 V; desolvation gas flow, 800 L/h; source temperature, 100 °C; desolvation temperature, 350 °C; cone gas flow, 40 L/h; trap CE, off. Lockspray (on-the-fly mass calibration) configuration consisted of infusion of a 0.5 ug/mL solution of leucine-enkephalin and acquisition of one mass spectrum (0.2 s scan, m/z 300-2500) every 10 s. Three lockspray m/z measurements of protonated (positive ionization mode) leucine-enkephalin

75

were averaged and used to apply a mass correction to measured m/z values during the

course of the analysis. MaxEnt probability software from Waters was used for

deconvoluting the spectra from m/z to m. The programmed mass ranges for MaxEnt was

10000:50000 for a resolution of 0.1 Da/channel. Uniform Gaussian model was used with

width at half height 0.33 Da. The iterations were programmed for auto convergence to

obtain the deconvoluted spectra. The results of the deconvoluted spectra reported are

accurate to ± 1 Da.

4.4 Results and Discussion

4.4.1 Protein with no flavor compound added The deconvoluted mass spectra of the BLG solution with no flavor added shows that the

major peak is of molecular weight 18,276 Da, which corresponds to the molecular weight

of the B variant of BLG (Figure 4-1). There were two additional major peaks: one of

molecular weight 18,600 Da (BLG + lactose) and the other of 18,924 Da (BLG + 2

lactose), which correspond to the molecular weights of Schiff base lactose adducts [342

Da (molecular weight of lactose) – 18 (water molecule) = 324 Da] onto the protein.

Adducts form at relatively low temperatures, and only the advanced compounds require

more heat. Because Maillard reaction products typically require elevated temperature and because our sample of BLG was not heated at any stage (e.g., the milk was not pasteurized), we presume that these lactose adducts were present in the raw milk precursor from which the BLG was isolated. There are previous studies that show that lactose adducts of BLG are formed at lysine groups.137,92,138

76

Figure 4-1 Deconvoluted ESI mass spectrum of BLG (18,276 Da) with no flavor added – i.e., the control spectrum. Adducts at +324 and +648 represent Schiff base lactose adducts of BLG.

4.4.2 Unreactive Functional Groups

Hydrocarbons

Hydrocarbons do not have any reactive functional group and, therefore, they were not expected to covalently bond with BLG. As expected, the hydrocarbon tested, p-cymene, did not show any adduct formation with BLG.

Alcohols

None of the alcohols tested [i.e., (l)-menthol (cyclic), geraniol (primary alcohol, unsaturation in the chain), 2-pentanol (secondary alcohol), and 2,3-butanediol (two hydroxy groups)] were observed to covalently react with BLG within the 48 h observation period. One might imagine the reaction of these alcohols with aspartic or glutamic acid to form an ester linkage, but this was not observed. Alcohols have been reported to form non-covalent interactions with protein.66,19

77

Phenols, furaneol, and maltol

Perhaps not surprisingly, phenols (2, 6-dimethyl phenol, eugenol, and 4-vinylphenol),

furaneol, or maltol (a 2-hydroxy-4-pyrone derivative) did not react with BLG.

Acids

In the timeframe studied, there were no covalent reactions observed for the acids studied

– butyric acid, 2-hydroxybenzoic acid, or m-toluic acid. Although one might have

anticipated some formation of esters from the reaction of these acids with serine and

threonine or of amides with lysine, no such adducts were observed. Previous work has

shown that butyric acid shows strong hydrogen bonding and ionic interactions with a soy

protein-containing soda cracker, but there was no mention of the formation of covalent

bonding via ester formation.113

Esters and lactones

Esters and lactones (cyclic esters) are relatively unreactive; thus, no reaction was expected. Esters (methyl anthranilate, iso-amyl acetate, ethyl lactate, methyl salicylate) did not form any detectable covalent bonds with BLG under our test conditions. Again, no covalently bonded adducts were observed between lactones (δ-dodecalactone, γ-

butyrolactone and γ-decalactone) and BLG.

Ketones

Molecules with a single keto group (2-heptanone, 2-nonenone, cyclotene) were not observed to covalently react with BLG, irrespective of the carbon chain length or their

78

linear or cyclic structure. However, later discussion will elaborate on the unique

reactivity of diacetyl (2, 3-butanedione).

Amine bases

Three different types of amines were analyzed – pyrazines, pyridines, and the primary

aliphatic amine, allylamine (prop-2-en-1-amine). 2, 5-Dimethylpyrazine and 3- acetylpyridine did not react to form covalent bonds with BLG. However, allylamine, surprisingly, showed formation of a simple, 1:1 addition complex (Figure 4-2) with BLG.

We speculate that this is a result of ion-pairing of allylammonium ions instead of protons with the BLG in the ESI mass measurement. Allylamine is the most basic of the nitrogen compounds (heteroaromatics and anilines) that were studied and, therefore, the most likely to form a BLG•ammonium complex in the ionization chamber.

Figure 4-2 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with allylamine (57 Da) +BLG (6 h reaction). This ion is likely due to formation of an allylammonium ion adduct of the BLG produced during the ionization (see text).

79

4.4.3 Reactive Functional Groups

α-Diketone

Diacetyl (2,3-butandione, 86 Da) contains more reactive (electrophilic) carbonyl groups

than the simple (and unreactive) monoketones because of the inductive effect of the

adjacent carbonyl. Diacetyl was observed to react with BLG to form a covalent 1:1

addition adduct of 86 higher Da (Figure 4-3 (a)). Importantly, this is not a condensation

product, which would be 68 mass units greater than the protein [i.e., 86 Da minus loss of

18 Da (the mass of water)].

Diacetyl, a characterizing compound in butter flavorings, has been associated with

causing obliterative bronchiolitis in microwave popcorn plant workers.144 Because of this,

there have been numerous studies on its reactivity with proteins and further, determining

the sites of interaction. Previous studies have suggested that diacetyl reacts with arginine

and/or lysine residues in a protein, proceeding further to crosslinking.145,146,147 However, the reactions of simple primary amines with diacetyl readily give Schiff bases (i.e., condensation products) and not simple addition adducts (i.e., hemiaminals). In contrast, the guanidine group in arginine residues is known to form 1:1 adducts, giving the dinitrogen alicyclic compound as shown in Figure 4-3(b) and we suggest that similar adducts of BLG are giving rise to the observed +86 Da derivatives.

80

a

b Me OH Me OH O NH OH O Me Me Protein HN Me N NH Me 2 HN NH NH H O ProteinN ProteinNH . diacetyl (86 Da) arginine residue adduct (BLG + 86 Da)

Figure 4-3(a) Deconvoluted ESI mass spectrum of diacetyl (86 Da) + BLG. (24 h reaction) (b) Mechanism for the formation of 1:1 addition adducts of diacetyl and the guanidine group in arginine sidechains.

Aldehydes

Aldehydes are well known to react with proteins through Schiff base formation. Hexanal

showed multiple adducts, presumably engaging multiple lysines. Both benzaldehyde and

furfural (Figure 4-4), less electrophilic aldehydes than hexanal, were observed to give

only a single Schiff base imine. The reason for the lack of further reactivity compared

with that seen for hexanal is unclear. Finally, we saw no evidence for reaction between

BLG and vanillin within 48 h, even though there are reports148,40,149,150 suggesting that

slow engagement of vanillin occurs with a number of proteins.

81

Figure 4-4 Deconvoluted ESI mass spectrum of furfural (78 Da [96 Da– 18 Da (water)])

+ BLG. (24 h reaction)

Enals and Enal-Acetals

Several additional alkene-aldehydes capable of either condensation reaction (Schiff base

formation) or conjugate addition of, most likely, the free cysteine thiol (thia-Michael

reaction) were studied. These were trans-2-hexenal, cis-3-hexenal, trans, trans-2, 4- heptadienal, and citral. All of these enals gave adducts with BLG. trans-2-Hexenal, cis-3- hexenal, trans,trans-2,4-heptadienal all reacted quickly as judged by the loss of protein signal in the mass spectrum of each, even of aliquots measured at only 10 min after mixing. This is consistent with the known ability of these reactive enals to cross-link whey proteins.82 Citral did so as well, but much more slowly, consistent with the more

hindered nature of the β-carbon of the aldehyde (or its imine). Both Schiff base formation

(134 Da) and a thia-Michael adduct (+ 152 Da) (Figure 4-5b), were observed for citral

(Figure 4-5a), although the mass of the isomeric aza-Michael adduct in which a lysine

amino group is the nucleophile that has undergone the conjugate addition143 would also

be + 152 Da.

82

a

b CHO BLG CHO N SH H2N S Me Me BLG Me H2N SH BLG

Me Me Me Me Me Me

BLG Schiff base thia-Michael adduct citral (152 Da) protein (+ 134 Da) (+ 152 Da)

Figure 4-5 (a) Deconvoluted ESI mass spectrum of citral (152 Da) + BLG. (6 h reaction)143 (b) Formation of both a Schiff base condensation product as well as a

conjugate addition adduct arising from thia-Michael addition reaction.

Citral diethyl acetal and trans-2-hexenal dimethyl acetal, somewhat surprisingly, were also observed to form covalent linkages with BLG. Acetals are stable under basic conditions but will hydrolyze under acidic conditions to yield the carbonyl compound and ethanol.151 The BLG also underwent crosslinking to form aggregates as it did when

reacted with the corresponding aldehyde, as suggested with the near-complete loss of the

83

BLG signal in the LC-MS experiment. Although both the Schiff base and a hetera-

Michael adduct were observed for citral (Figure 4-5), only the latter (i.e., +152 corresponding to hetera-Michael adduct) was observed using the citral diethyl acetal

(Figure 4-6(a)). A possible rationale for this is shown in Figure 4-6 (b).

a

OEt b CHO CH(OEt)2 -EtOH -EtOH S(NH) Me Me MeS(NH) HS NH 2 BLG BLG BLG Me Me Me Me Me Me the citral citral diethyl acetal BLG Michael adduct (226 Da) protein (+ 152 Da)

Figure 4-6 (a) Deconvoluted ESI mass spectrum of citral diethyl acetal (226 Da) + BLG.

(24 h reaction time) (b) The net hydrolytic conjugate adduct formation starting from citral diethyl acetal.

84

Sulfur-containing aroma compounds

Different sulfur-containing functional groups were tested for their reactivity with BLG.

None of the 4-methyl-5-vinylthiazole (1% in ethanol), 2-methylthiophene and dimethyl

sulfone formed adducts with BLG. In contrast, simple thiols readily combined with BLG,

giving 1:1 (and 2:1) addition compounds.

Many flavor compounds contain thiol groups. We studied propanethiol, furan-2- ylmethanethiol , and thiophenol and all three compounds formed covalent adducts with

BLG. The thiol group is a reactive (and soft) nucleophile capable of cleaving one or both of the (soft) electrophilic disulfide linkages in BLG. The mass spectrum of the reaction of

BLG with propanethiol (n-PrSH) (76 Da) is shown in Figure 4-7a. Both mono- (+76 Da) and bis-adducts (+152 Da) of BLG were observed (Figure 4-7b). Moreover, nearly none of the 2:1 (+152 Da) adduct was present at 6 h, but it can clearly be seen in the 24 h

(Figure 4-7a).

Furan-2-ylmethanethiol (114 Da), the most important aroma compound in roasted coffee aroma, also reacted readily with BLG to form covalent linkages (Figure 4-7(c)). In close analogy to the reactions with n-PrSH (Figure 4-7b), both 1:1 and 2:1 adducts were observed. Finally, thiophenol (PhSH) also formed a covalent linkage with BLG as demonstrated by the appearance of a MS peak at mass 18,388 (+110 Da) (Figure 4-7d).

In this instance, however, no appreciable formation of a 2:1 adduct (i.e., +220 Da) was observed even at the 48 h reaction time. Perhaps subtle differences in the steric access of the bulkier PhSH to the second disulfide linkage in BLG (cf. Figure 4-7b graphic) is responsible for this observation.

85

a

b

n-PrS n-PrS S n-PrSH S S n-PrS S SH SH n-PrSH S S S

S S SH

BLG BLG BLG + 76 Da + 152 Da protein

c

86

d

Figure 4-7 (a) Deconvoluted ESI mass spectra of propanethiol (76 Da) + BLG (24 h

reaction time). (b) Sequential formation of mono- and bis-disulfide adducts with propanethiol. (c) Deconvoluted ESI mass spectra of 2-furfurylmercaptan (114 Da) + BLG

(24 h reaction time). (d) Deconvoluted ESI mass spectra of thiophenol (110 Da) + BLG

(24 h reaction time).

Dimethyl sulfide (MeSMe) is not electrophilic, consequently, it did not react with the protein. However, both of the soft electrophiles dimethyl disulfide (MeSSMe) and dimethyl trisulfide (MeSSSMe) were observed to form covalent bonds with BLG. The disulfide formed an adduct 46 Da greater than BLG, corresponding to the net addition of

CH2S. This is consistent with formation of BLG-CysSSMe by reaction of the BLG-CysH with MeSSMe. The trisulfide homolog gave a more complex pattern of adducts, as seen in Figure 4-8a. Both BLG-CysSSMe (+46 Da) and BLG-CysSSSMe (+78 Da) were observed, formed by the competitive paths “x” and “y”, respectively, shown in Figure

4-8b. In the reaction time studied, the extent of adduct formation for dimethyl disulfide was less than that for dimethyl trisulfide. Dimethyl disulfide is less electrophilic and is

expected to engage the BLG-CysSH more slowly.

a 87

b x x –MeSSH S S S Protein Me S Me HS Protein Me S

dimethyl trisulfide cysteine residue disulfide (+ 46 Da) (126 Da) y y –MeSH S S S S Me S Me HS Protein Me S Protein

dimethyl trisulfide cysteine residue trisulfide (+ 78 Da) (126 Da)

Figure 4-8(a) Deconvoluted ESI mass spectra of dimethyl trisulfide (126 Da) + BLG. (24 h reaction time). (b) Rationale for the formation of disulfide and trisulfide adducts via paths indicated by arrows “x” and “y”.

Isothiocyanate

We have previously shown that allyl isothiocyanate (CH2=CHCH2N=C=S) (99 Da) readily forms covalent linkages through the addition of varying numbers of the (15)

143 lysine NH2 groups to the isothiocyanate. This reactivity is also supported by other studies.102,103 A representative spectrum of this reaction is shown in Figure 4-9.

88

Figure 4-9 (a) Deconvoluted ESI mass spectra of dimethyl trisulfide (126 Da) + BLG. (24 h reaction time). (b) Rationale for the formation of disulfide and trisulfide adducts via paths indicated by arrows “x” and “y”.

A summary of all compounds and their reactivity is presented in Table 4-2. One should note that the reaction time allowed in this study was quite short, a maximum of 48 h.

However, compounds with less reactive functional groups might still engage over longer periods to impact their contribution to flavor. We plan additional studies to probe this question.

89

Table 4-2 Aroma compounds analyzed and their reactivity with BLG (24 h reaction time)

Covalent Adduct Functional Group Compound Formation?

No Flavor pure protein no Hydrocarbon p-cymene no (l)-menthol no geraniol no Alcohols 2-pentanol no 2,3-butanediol no 2,6-dimethyl phenol no Phenols eugenol no 4-vinyl phenol no Enol furaneol no Pyranone maltol no butyric acid no Acids 2-hydroxybenzoic acid no m-toluic acid no methyl anthranilate no iso-amyl acetate no Esters ethyl lactate no methyl salicylate no δ dodecalactone no Lactone γ butyrolactone no γ decalactone no 2-heptanone no Ketones 2-nonenone no cyclotene no 2,5 dimethyl pyrazine no Bases 3-acetyl pyridine no allylamine no α-Diketone diacetyl yes vanillin no hexanal yes Aldehydes benzaldehyde yes furfural yes

90

trans-2-hexenal yes cis-3-hexenal yes Aldehydes (Enals) trans,trans-2,4-heptadienal yes citral yes citral diethyl acetal yes Enal-Acetal trans-2-hexenal dimethyl yes acetal 4-methyl-5-vinyl-thiazole, no 1% 2-methyl thiophene no dimethyl sulfone no Sulfur-containing propanethiol yes compounds thiophenol yes 2-furfuryl mercaptan yes dimethyl sulfide no dimethyl disulfide no dimethyl trisulfide yes Isothiocyanate allyl isothiocyanate yes

4.5 Conclusion This work clearly demonstrates that many aroma compounds that play a key role (e.g.,

benzaldehyde in a cherry flavor and eugenol in clove flavor) or supporting roles (e.g.,

trans-2-hexenal contributing a green/unripe fruity note and dimethyl trisulfide to achieve

savory, sulfury notes in cooked meat-like flavor) in food flavorings, covalently react with

BLG. The formation of these bonds would likely result in a temporal change of flavor characteristics, which could occur either slowly (impacting shelf life issues) or rapidly

(affecting, e.g., the initial flavoring of food products). The primary reactions that occur are Schiff base formation, conjugate (hetera-Michael) addition reactions, or thiol and di-

/tri-sulfide exchange reactions. The observed reactions involve carbonyl- or sulfur- containing aroma compounds. This last class of reactions not only influences the flavor

91 properties of the food, but also could affect protein stability by virtue of cleavage or scrambling of disulfides present in the native protein.

The reactivity we have identified using BLG is not unique to that model. Other proteins, including plant-based (e.g., pea or soy) proteins, will present the same basic amino acids, and the nature of their fundamental reactivity and covalent adduct formation can be expected to mirror what we established for BLG. Also, the rate and extent of the reactions will change according to the processing conditions (such as freezing of proteins in ice cream like foods, retorting in the case of savory flavors, extrusion or spray drying in meat analogs or protein powder, and, of course, cooking) experienced prior to consumption. Additional work using a subset of the studied flavorants is ongoing using longer reaction times, increased temperatures, and a range of pH and water activities. We anticipate that results addressing the rates of various reactions will arise from these studies to complement the qualitative information about reactivity that we report here.

It is interesting that 20 years ago one of the senior authors of this paper raised the question of product shelf life. Is the principal contributor to the deterioration of flavor quality that eventually ends a product’s life a result of the formation of off flavors (e.g., through oxidation), as is often thought to be the case, or, instead, the loss of desirable flavor due to processes that consume some flavorants such as the reactions described here?152

92

4.6 Supporting Information The deconvoluted ESI MS spectra are shown here for experiments mentioned in the manuscript where reactions were occurring but for which the spectra were not shown in the text itself.

Figure 4-10 Deconvoluted ESI mass spectrum of hexanal (100 Da) with BLG. (24 h reaction time). BLG +82 Da and +100 Da represent the Schiff base adduct and the thia-

(or aza)-Michael addition adduct, respectively. The +164 ion indicates a bis-Schiff base adduct.

93

Figure 4-11 Deconvoluted ESI mass spectrum of benzaldehyde (106 Da) with BLG. (24 h

reaction time). BLG +88 Da represents a mono-Schiff base adduct.

Figure 4-12 Deconvoluted ESI mass spectrum of trans-2-hexenal (98 Da) with BLG. (6 h

reaction time). Only noise level spectral data were observed, suggesting that crosslinking

had occurred.

Figure 4-13 Deconvoluted ESI mass spectrum of cis-3-hexenal (98 Da) with BLG. (6 h reaction time). Only noise level spectral data were observed, suggesting that crosslinking had occurred.

94

Figure 4-14 Deconvoluted ESI mass spectrum of trans-trans-2-4-heptadienal (110 Da) with BLG. (6 h reaction time). Only noise level spectral data were observed, suggesting that crosslinking had occurred.

Figure 4-15 Deconvoluted ESI mass spectra of trans-2-hexenal dimethyl acetal (144 Da) with BLG. (24 h reaction time). Only noise level spectral data were observed, suggesting that crosslinking had occurred.

95

Figure 4-16 Deconvoluted ESI mass spectra of dimethyl disulfide (94 Da) + BLG. (24 h reaction time).

96

Chapter 5 : Influence of pH, Temperature, and

Water Activity on Covalent Adduct Formation

Between Selected Flavor Compounds and the

Model Protein β-Lactoglobulin

Note: Sections of this chapter will be submitted for publication in Journal of Agricultural and Food Chemistry (Anantharamkrishnan et al. 2020)

5.1 Preface Covalent adduct formation between a flavor compound and protein leads to a decrease in shelf life because of the diminished consumer acceptability. The delivery of protein can be through multiple systems – powders, low pH’s and refrigerated protein beverage. Due to the varied environmental conditions, the nature and extent of covalent interactions would change. This study investigates the influence of pH, temperature and water activity using liquid chromatography/electrospray ionization mass spectrometry. At acidic pH 3, the covalent adducts were formed more slowly for citral, AITC, and DMTS in

comparison to basic pH’s. There were no adduct observed for benzaldehyde at pH 3 due

to the carbonyl chemistry involved during the covalent adduct formation. Increase in

formation of adduct were observed as the pH was increased to pH 7 and pH 8. The rate of

formation of adducts increased with increase in temperature for the flavor compounds

that were studied. Higher temperatures (45 °C) led to the formation of products that were

not observed at lower temperatures (4 °C and 20 °C). Increase in water activity led to an

97

increase in formation of adducts for AITC. There were no observable difference for the

effect of change in water activity for adduct formation with benzaldehyde, citral, and

dimethyl disulfide in the period studied.

5.2 Introduction While proteins may contribute an undesirable flavor to a product due to off-flavor

compounds inherent in the starting material, proteins themselves, do not provide any

flavor to the finished food product. However, chemical reactions between a protein and

added flavor constituents can cause the overall flavor profile of a food to become

imbalanced and unpleasant. The reactions between a protein and a flavoring are

multifaceted. There can be both weak interactions like ionic bonding and hydrogen

bonding and strong interactions such as covalent bonding. Flavorings are ideally

formulated to adjust for the weak interactions to provide a high quality flavor to meet

consumer expectations. This is possible in the case of weak flavor-protein interactions

but not so in the case of covalent interactions. The weak flavor-protein interactions will

relatively quickly come to some pre-packaging equilibrium that is then stable over the

subsequent shelf life of the product. Covalent reactions typically occur more slowly but

continue throughout shelf life ultimately resulting in key parts of the flavoring being

consumed through formation of strong chemical bonds with the protein. The type and extent of chemical reactions depend on the intrinsic characteristics of both the flavoring and the protein and how these reactions are influenced by environmental parameters such as pH, temperature, and water activity. For over forty years, there has been a great deal of research focused on studying the interactions between flavor components and proteins;

however, in most cases, the combined effect of both weak interactions and covalent bond

98

formation has been measured. The techniques for measuring interaction have seldom

been able to distinguish between the types of reactions that have occurred and have only measured an overall effect. A summary of previous work reporting on the effect of the reaction environment (e.g., pH, water activity and temperature) on flavor: protein reactions follows.

The effect of sample pH on the overall reaction of flavor components (e.g., through the loss of free volatiles from both weak and covalent bonding) with proteins has been studied by several authors 153,60,154,69. An increase in temporary interactions was observed

with increasing pH between pH 3 and 9 for methyl ketones, esters, and terpenes.63,85 This

was attributed to the flexibility of the protein, which allows better access to hydrophobic

binding sites. A severe decrease was observed at pH 11 and the reason for it would be

due to the alkaline denaturation of the protein.155,156 Similar results were found by

another group in which the interaction of benzaldehyde with β-lactoglobulin (BLG) was

higher at pH 6 than pH 3.110 In an in-vivo release test using a mixture of aldehydes and whey protein, a drastic drop in aldehyde binding was observed between pH 7 to 9 when the pH values were varied between pH 3-9.157. In contrast, for BLG an increase in pH

from 3-9 resulted in an increase in binding of twenty flavor compounds (except for α-

pinene and alcohols).106 In a study of reactions between a mixture of disulfides and

ovalbumin, there were significant losses observed when the pH of the solution was

increased to 8.0 in comparison to its native pH 6.7.108

Similar to pH, there have been several studies done on the effect of temperature on

protein-flavor interactions, primarily focused on weak interactions.69,70,107 The binding of

flavor compounds with bovine serum albumin or soy protein did not vary for the 99

temperature range between 25–45 °C.158 Similar results were found when the interaction

between 2-nonanone and soy proteins did not change between 25 and 45 °C, and γ-

decalactone interaction with bovine serum albumin protein was independent of

temperature between 10-30 °C.85 The affinity constant of 2-nonanone with soy protein

increased between 5 to 25 °C and the number of binding sites halved at 5 °C in

comparison to 25 °C. This was proposed to be due to the structural changes of the protein

due to changes in temperature.69 One study reported that increasing the temperature to

76 °C did not affect the binding constant of benzaldehyde with β-lactoglobulin.110 In

contrast, another study reported that an increase in temperature leads to an increase in

binding.159 This was proposed to be caused by an unfolding of the protein that would

expose the hydrophobic sites that were buried before, leading to an increase in binding

constant.66,71 In an opposite case, an increase in temperature would cause heat aggregation of the protein, which would modify the structure of the protein, leading to a decrease in association constant and increase in the number of binding sites.160 This was also demonstrated with 2-nonanone as a flavor compound in its reactions with β- lactoglobulin.64 The binding of trans-2-nonenal, 1-nonanal, 2-nonanone with whey

protein isolate was evaluated after treating the matrix with heat and high pressure.115 The authors concluded that covalent interactions were enhanced upon heat denaturation but there was a decrease in hydrophobic interactions. A commercial savory flavoring that contained thiol and disulfide flavorings was heated to 100 °C with egg albumin protein and then the concentration of the flavor components was analyzed.38 The results

suggested that the occurrence of redox reactions with the protein caused the disulfide

reduction and formation of its corresponding thiol. In the same study, when casein

100

protein was used, there was a small amount of disulfide reduction because it has a low

proportion of sulfhydryl groups. In a similar study with aroma compounds from disulfide

groups, the results show that when the protein is denatured, greater losses of the flavor

compounds were observed, and some free thiols were also produced. Disulfides that

contained allyl or furfuryl groups were more reactive than saturated disulfides. This

would be because of the interchange reactions between a protein’s sulfhydryl group and

disulfides.108

The delivery of high protein system can be through many systems including protein

powder, crackers, protein bars, meat analog patties, and beverages. There is a difference

in water activity between these systems, which would be influencing the reaction

between flavor and protein because the mobility of the system increases with increase in

161 water activity (aw). Most of the studies on flavor: protein interactions have been done

with aqueous systems162,163 There is only one group that studied the influence of storage

relative humidity on the interaction between aroma compounds and proteins.164 Soy protein was used as the protein and inverse gas chromatography was used to measure interaction.165 The study concluded that polar aroma compounds (esters, ketones,

aldehydes, and alcohols) exhibited both specific and nonspecific interactions and their

binding was greatly influenced at low relative humidity (close to 0%). There was no

significant effect between the 30 to 50% relative humidity region for polar compounds,

except for alcohols, which further increased.

From all the discussion above, it is obvious that the extrinsic factors like pH, temperature,

and water activity have a significant impact on protein-flavor interactions. Understanding

how these interactions take place would help in optimizing the processing and storage 101

conditions of the high protein products and potentially control their release during

consumption. This whole process becomes very complex when covalent interactions also

take place. Surprisingly, there is no literature that has been reported dealing with the

influence of pH, temperature, and water activity on the covalent interaction with flavor

components. However, there are studies on the influence of extrinsic factors on the

velocity of Maillard reaction in foods from which parallels can be drawn for interpreting

the results of this study.166,167,168 The objective of this study is to fill that void in

understanding, which would be valuable for the food industry in developing protein

products with acceptable flavors over shelf life. This study uses BLG as the model

protein and benzaldehyde, citral, allyl isothiocyanate, and dimethyl trisulfide as the flavor

components for evaluating the effect of pH and temperature and benzaldehyde, citral,

allyl isothiocyanate, and dimethyl disulfide for studying the effect of water activity.

5.3 Materials and Methods

5.3.1 Isolation of single variant BLG The procedure for the isolation of BLG was adapted from a previous publication143 with

some modifications. Milk was obtained from a single homozygous cow from the Dairy

Barn on the St. Paul campus of the University of Minnesota. The milk was defatted by

centrifugation (16000 rpm for 1h at 4°C). The fat layer was removed, and the casein was

precipitated from the defatted milk by reducing the pH to 4.6 using 1 M HCl. The

precipitated casein was removed using Whatman No.1 filter paper. After adjusting the pH

of the whey to 7.0, it was passed through a 10 kDa size exclusion membrane for

removing small molecular weight compounds such as salts and lactose. The pH of the resultant concentrate was adjusted to 3.8. The solution was heated and maintained at 102

45°C for 30 minutes. The precipitated α lactalbumin, bovine serum albumin, and

immunoglobulin were removed by centrifugation (16000 rpm for 1 h at 4 °C). The

supernatant was passed through a 10 kDa size exclusion membrane to remove the salts

used during pH adjustment and the concentrate was freeze dried to yield a protein

fraction comprising 95% β-lactoglobulin.

5.3.2 Chemicals All chemicals [benzaldehyde, >99.5% pure, citral (mix of cis and trans), >96% pure, allyl

isothiocyanate, >95% pure, dimethyl trisulfide, >98% pure, dimethyl disulfide, >99%

pure, HCl ACS reagent, 37%] used were of analytical grade and purchased from Sigma

Aldrich (St. Louis, MO, USA). All the water used in this study was doubly distilled.

5.3.3 Reaction system A one-weight percent freeze dried aqueous BLG solution was prepared for studying pH

and temperature effects on reactions. For studying the effect of pH, the BLG solution was

adjusted with 0.5 M HCl (to pH 3) or 0.5 M NaOH (to pH 7, or 8) to its desired pH. pH

was measured using VWR SympHony pH meter. The above pH values were chosen as

they are within the range of what one would observe in food matrices. For variation in

reaction temperature, the BLG solution (pH 7) was equilibrated to three different

temperatures: 4°C, ambient (ca. 20 °C), and 45°C. Flavoring compounds were added individually to an aliquot of the protein solution at 12 parts per thousand (ppth) concentration and vortexed. Sample aliquots were taken at different reaction time points

(1 h, 6 h, and 24 h) and diluted 1:10 with water for MS analysis.

103

For studying the effect of water activity, freeze dried BLG was equilibrated for six weeks in a desiccator saturated with the respective salts: LiCl for 0.11, CH3CO2K for 0.23,

MgCl2.6H2O for 0.33, K2CO3 for 0.44, Mg(NO3)2 for 0.53, NaNO3 for 0.64 and NaCl for

0.75. After equilibration, the flavoring compound (benzaldehyde, citral, allyl isothiocyanate, dimethyl disulfide) to be studied was added individually to an aliquot of the protein at each water activity at 12 parts per thousand concentration and mixed thoroughly. The sample vials were then closed tightly and stored for the desired time and temperature. Powder samples were taken at different reaction time points (24 h and 48 h) and mixed with water to make a 0.1% solution for MS analysis.

5.3.4 UPLC-ESI-MS/qTOF The method for mass spectrometry was adapted from a previous publication.143

LC system: A Waters Acquity UPLC coupled to a Waters Synapt G2/Si HDMS quadrupole orthogonal acceleration time of flight mass spectrometer (UPLC/qTOF/MS)

(Waters Corp., Milford, MA, USA) was used for analyzing the protein and flavor interaction. A Waters Acquity BEH C4 column (2.1 mm x 100 mm column – 1.7 um diameter particles) at 35 °C was programmed as follows: 15 min linear gradient separation at a flow rate of 0.400 mL/min using A: water containing 0.1% formic acid and B: acetonitrile containing 0.1% formic acid: 3% B, 0 min to 3 min; 3% B to 97% B, 3 min to 9 min; 97% B, 9 min to 11 min; 97% B to 3% B, 11 min to 13 min; 3% B 13 min to 15 min. Mass spectra were collected in profile mode over the range m/z 300-2500 every 0.2 s during the chromatographic separation.

104

Mass spectrometry: MS parameters in positive electrospray ionization mode were as

follows: capillary, 0.5 kV; sampling cone, 35.0 V; extraction cone, 4.0 V; desolvation gas

flow, 800 L/h; source temperature, 100 °C; desolvation temperature, 350 °C; cone gas

flow, 40 L/h; trap CE, off. Lockspray (on-the-fly mass calibration) configuration

consisted of infusion of a 0.5 ug/mL solution of leucine-enkephalin and acquisition of

one mass spectrum (0.2 s scan, m/z 300-2500) every 10 s. Three

lockspray m/z measurements of protonated (positive ionization mode) leucine-enkephalin

were averaged and used to apply a mass correction to measured m/z values during the

analysis. MaxEnt probability software from Waters was used for the deconvoluting the spectra from m/z to m. The programmed mass ranges for MaxEnt was 10000:50000 Da for a resolution of 0.1 Da/channel. The uniform Gaussian model was used with a width at half height 0.33 Da. The iterations were programmed for auto convergence to obtain the deconvoluted spectra. The results of the deconvoluted spectra reported are accurate to ± one Da.

5.4 Results and discussions

5.4.1 Protein with no flavor added The deconvoluted spectra of the BLG solution with no flavor added (Figure 5-1) show

that the highest peak is of molecular weight 18,362 Da. This corresponds to the A variant

of BLG found in nature.22 The other small peaks are oxidation products of the protein.

The peak at 18,686 Da corresponds to the lactose adduct (BLG + 324 Da).

105

Figure 5-1 Deconvoluted ESI mass spectrum of BLG with no flavor added – i.e., the

control spectrum (24 h storage).

5.4.2 Variation in pH

Benzaldehyde

Benzaldehyde has been shown to form covalent adducts with BLG by Schiff base

formation.143 At low pH, the formation of this covalent adduct does not take place

because the lysine amines are substantially protonated (Figure 5-2(a)). However, at neutral and basic pH, Schiff base formation is much faster and the covalent adducts are observed (Figure 5-2). The relative intensity of the adduct peak in 24 h is less in comparison to our previous publication.143 This could be because another variant (A) of

the protein is used for this study whose reactivity and native structure is different than the

B variant that was used in the previous study.

106

Figure 5-2 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with

benzaldehyde at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 24 h reaction).

Citral

In the case of citral as an added flavoring, there were no adducts observed at the lowest

pH (Figure 5-3(a)). The mechanism for adduct formation is like benzaldehyde, Schiff

base formation, but citral can also undergo aza-Michael addition reaction. Both mechanisms to form these two adducts require the participation of free base primary amines. In neutral and basic conditions, covalent adducts were observed (Figure 5-3(b) and (c)). The +152 adduct corresponds to the aza-Michael addition adduct. The +268 adduct could be two Schiff base adducts that have been formed with multiple lysine groups present or the pyridinium adduct that is formed with a single lysine group and two citral molecules (Figure 5-4). Various lysine pyridinium adducts involving two or more alkenal molecules have been shown in previous studies using MS and/or NMR

107

spectroscopy and model protein/peptides.93,169,87,170,171,172 The reaction mechanism is

shown in Figure 5-4. The +402 and +554 adducts would correspond to the additional

Schiff base and Michael adducts at the other reactive sites (e.g., lysine, and arginine). The intensities of the unreacted protein and adduct peaks were reduced at the basic pH (Figure

5-3(c)) compared to the other two pHs. This would be due to the crosslinking of the protein by citral, thereby making the mass of the cross-linked molecule above the detection limits of the MS. This is consistent with the previous studies where alkenals were shown to form crosslinking between the proteins.82 Multiple adducts were present

suing the B variant of the protein, whereas the reaction with variant A showed only one

adduct.143 This could again be due to the change in the variant and native structure of the

protein.

108

Figure 5-3 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with

citral at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 24 h reaction).

- O H2O + R NH N 2 R

Citral Protein + O

+ H H N R

NH + R O O

H

N R N R OH H

N R

Pyridinium ion (Py+) 286

Figure 5-4 Proposed reaction mechanism for the formation of citral-Lys dihydropyridinium adduct.

Ally isothiocyanate

Allyl isothiocyanate (AITC) undergoes addition reactions with the lysine groups present

in BLG. In acidic conditions, the adducts form but at a slower rate than in comparison to

neutral and basic conditions (Figure 5-5). Multiple covalent adducts were observed between AITC and BLG. As time progressed (Supporting Information Figure 5-21 and 109

Figure 5-22) the intensity of the protein peak reduced and ultimately disappeared as is seen in Figure 5-5(c). In addition, the major peak in Figure 5-5(b) is 18,560 Da and in

Figure 5-5(c) is 18,659 Da. The graphs follow suit when the corresponding time points

(1h and 6h) provided in the Supporting Information was compared with the 24h point.

This suggests that the reaction is progressing and would theoretically continue until all the flavor molecules have reacted or all the sites of the protein are bonded with adducts.

In Figure 5-5(b) and (c), dehydration reactions are observed (18,362 (BLG) – 18 (water molecule) = 18344 Da) and correspondingly, adducts of this dehydrated compound were also observed (18,443, 18,452, 18,641 Da).

Figure 5-5 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with allyl isothiocyanate at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 24 h reaction).

110

Dimethyl trisulfide

Dimethyl trisulfide (DMTS) forms covalent adducts with cysteine groups. BLG has five

cysteine groups present, four of which are engaged in disulfide bonds with each other. In the previous article, we have shown the mechanisms for formation of +46 and +78 Da adducts.173 Once the free cysteine group is reacted, the disulfide bond present in the Cys-

Cys would be cleaved and the adduct with the DMTS would be formed (Figure 5-12). In

acidic conditions, there were no appreciable adducts formed with BLG (Figure 5-6(a)). In

neutral conditions, the two adducts +46 and +78 Da, as explained above, could be

observed (Figure 5-6(b)). At pH 8, multiple adducts with an increment of +32

(corresponding to the mass of a sulfur atom) could be observed. This suggests the adducts

are formed after cleaving the Cys-Cys disulfide linkage. The proposed sequential

mechanism is shown in Figure 5-12.

111

Figure 5-6 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with

dimethyl trisulfide at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 24 h

reaction).

In summary, for the types of covalent adduct formation studied (i.e., Schiff base, Michael

addition, disulfide exchange reaction), the rate of covalent adduct formation increases at higher pH. At a basic pH, multiple adducts that were not observed in the lower pH values were observed suggesting that the structure of the protein could be changing and reactive sites that were stable at acidic pH are exposed in basic pH values.

5.4.3 Variation in Temperature

Benzaldehyde

At refrigeration temperature (4 °C), the +88 Da Schiff adduct could be observed but to a

lesser extent compared to the sample stored at room temperature (20 °C) and 45 °C

(Figure 5-7). There was no appreciable difference between the adducts formed for the

sample incubated at 20 °C and 45 °C. (Figure 5-7(b) and (c)) in the reaction time period

studied. The result is consistent with the previous studies where no difference in

reactivity was observed for samples stored at 25 °C and 45 °C.158

112

Figure 5-7 Deconvoluted ESI mass spectrum of BLG incubated with benzaldehyde at

different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 24 h reaction).

Citral

In the case of citral, there is an increase in intensity of the adduct peak (mass 18,450 Da)

for samples incubated at 4 °C and 20 °C (Figure 5-8(a) and (b)). The reason for observing

the peaks at a noise level for the sample stored at 45 °C could be because of crosslinking.

The progression of the signal to noise level can be observed from Supporting Information

Figure 5-27(c) and Figure 5-28(c). In one hour, the adducts were observable (Supporting

Information Figure 5-27(c)), and the peak intensity decreased after six hours (Supporting

Information Figure 5-28(c)), finally leading to the noise level in Figure 5-8(c). The enals are known to crosslink proteins.143,82,174

113

Figure 5-8 Deconvoluted ESI mass spectrum of BLG incubated with citral at different

temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 24 h reaction).

Allyl isothiocyanate

An increase in temperature lead to an increase in the rate of formation of an adduct

between AITC and BLG. The major peak in Figure 5-9(a) is 18,362 Da whereas it is

18,461 in Figure 5-9(b). The deconvoluted MS spectrum of the sample incubated at

45 °C (Figure 5-9(c)) shows peaks in the noise level. The increase in temperature appears

to have induced cross linking resulting in the molecular weight of the adducts being

above the mass range of the ESI/MS measurement. The mechanism for this would be because the addition reaction between allyl isothiocyanate and BLG is partially reversible. When the reaction reverses, the isothiocyanate group could be formed with the protein instead of the allyl group - (b) product in (Figure 5-10). This would further react to induce cross linking or form higher molecular weight compounds. The progression of 114 the reaction over different time points can be seen from the Supporting Information

Figure 5-29(c) and Figure 5-30(c).

Figure 5-9 Deconvoluted ESI mass spectrum of BLG incubated with allyl isothiocyanate at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 24 h reaction).

H H N N N S + H N Protein C 2 C Protein S Allyl isothiocyanate (99u) BLG Protein Addition product

H H N N NH + N S C Protein 2 Protein C S (b) Addition product (a)

Figure 5-10 Proposed mechanism for adducts and crosslinking between allyl isothiocyanate and the lysine group from the BLG protein (top) which would be reversible to the isothiocyanate in the protein leading to crosslinking.

115

Dimethyl trisulfide

There was only a small amount of adduct formation for DMTS with BLG for the sample incubated at 4 °C (Figure 5-11 (a)). The was a small spike at +46 Da adduct but not yet significant. Figure 5-11(b) shows the presence of +46 Da adduct at 24 h when the sample was incubated at 20 °C. For the sample incubated at 45 °C, multiple adducts were being observed. The major unreacted protein peak has completely disappeared and the adducts observed correspond to a reaction with the cysteine groups present in BLG. At 45 °C, the

Cys-Cys disulfide bond would be cleaved giving rise to an adduct with one of the sulfur atoms from the disulfide linkage. The mechanism is shown in Figure 5-12. It can be a mix and match of the combination between the +46 and +78 Da ions, which is formed in the intermediate steps in addition to the reaction product formed with the free Cys group in BLG.

116

Figure 5-11 Deconvoluted ESI mass spectrum of BLG incubated with dimethyl trisulfide at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 24 h reaction).

S S S Me-S-S-S-Me + S S S S S S S S S S S SH S S Me S Me

BLG BLG BLG + 46 Da protein + 78 Da

Me Me (S)1,2 (S)1,2 S S S Me-SH/ Me-S-SH Me (S) S SH SH 1,2 Me-SH / Me-S-SH S S S S S S S S (S)1,2 SH (S)1,2 (S)1,2 Me Me Me

BLG BLG + 46/78 Da BLG + 46/78 Da + 46/78 Da +46/78 Da + 46/78 Da +46/78 Da

Figure 5-12 Proposed mechanism for adducts between dimethyl trisulfide and the free cysteine group (top) and , then, with Cys-Cys disulfide residues (bottom) in BLG.

5.4.4 Variation in Water activity Water activity is considered to be an important factor controlling several chemical reactions in foods.175 In general, one would expect an increase in water activity (to some point, some studies have shown it to be 0.75) 176,177 would increase reactant solubility and mobility and thereby, affect the rate of chemical reactions, especially the Maillard

117

reaction. The reaction rate would decrease with the further increase in aw, as the moisture

content would dilute the concentration of reactants.

Lactose adducts (18,686 Da, BLG +324 Da) can be observed in (Figure 5-13 a and b) at

0.11 aw and they generally increased in intensity with increases in storage water activity.

Previous studies have shown lysine-lactose adduct formation increases with aw to a

maximum around 0.6-0.7.176,177

Lactose adducts were not found in the temperature and pH studies (previous sections) since they were done shortly after protein isolation and the samples were maintained at low aw (0.18) until use. Therefore, there were no lactose adducts observed in the MS. The

water activity part of this study was conducted several months after protein extraction,

during which time, lactose adducts were formed and thus, are observed in all of the mass

spectra.178,179

Allyl isothiocyanate

An increase in water activity led to an increase in rate of formation of adducts between

AITC and BLG. This would be due to the increase in mobility of the flavor molecule to

reach the reactive amino acid moiety with increase in water activity.175 The reaction

progression can be observed in Figure 5-13(a) – (g). At 0.11 water activity, the major

peak is 18,362 Da, which is the molecular weight of BLG, and +99 Da adducts

correspond to the adducts with AITC. The reduction in intensity of the native protein

peak (18,362 Da) and increase in the intensity of adduct peaks and formation of new

adducts can be seen with increase in water activity. At water activity 0.75 Figure 5-13(g),

up to six adducts can be observed with the major peak being 18,659 Da. For the same

118

water activity 0.75, the major peak in Figure 5-33(g) is 18461 Da (24h time point) and the major peak in Figure 5-13(g) is 18,659 Da (48h time point), which suggest that the reaction is progressing over time. For lower water activities, the rate of adduct formation is slower and therefore, no significant change is observed between time points 24 h and

48 h (Figure 5-33 (a)-(f)).

119

Figure 5-13 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with allyl isothiocyanate at different water activities (a) 0.11 (b) 0.23 (c) 0.33 (d) 0.44 (e)

0.53 (f) 0.64 (g) 0.75 (12 ppth, 48 h reaction).

120

Citral

From Figure 5-14, it can be observed that citral forms covalent adducts with BLG at all

water activities. However, there was no observable difference between the samples at

different water activities. This could be because of the shorter reaction period studied –

24h (Figure 5-34) and 48h (Figure 5-14). As time progresses, there could be larger differences between the samples.

121

Figure 5-14 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with citral at different water activities (a) 0.11 (b) 0.23 (c) 0.33 (d) 0.44 (e) 0.53 (f) 0.64

(g) 0.75 (12 ppth, 48 h reaction).

122

Benzaldehyde and dimethyl disulfide

For the other two flavor compounds (benzaldehyde and dimethyl disulfide) that were

tested, there were no adduct peaks formed for water activities between 0.11 and 0.64 in

48 h (Figure 5-15 a – f) and (Figure 5-16 a-f). However, both of these compounds have

been shown to form covalent adducts with BLG in aqueous solution.173 There is a peak with relatively low intensity, corresponding to a molecular weight of 18,450 Da and

18,409 Da for benzaldehyde and dimethyl disulfide, respectively at 0.75 water activity

(Figure 5-15 (h) and Figure 5-16 (h)). There is no observable increase in the intensity of the adducts between 24 h and 48 h (Figure 5-15 h and Figure 5-35h). Benzaldehyde undergoes Schiff’s base reaction. Previous studies have shown that this reaction is not favorable at lower water activity as the mobility of the reactant is slow.161 Our data

suggest there is a water activity effect but this effect is not obvious at short reaction times

(48 h). The difference might become more apparent at longer time points.180

123

Figure 5-15 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with benzaldehyde at different water activities (a) 0.11 (b) 0.23 (c) 0.33 (d) 0.44 (e) 0.53

(f) 0.64 (g) 0.75 (12 ppth, 48 h reaction).

124

Figure 5-16 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with dimethyl disulfide at different water activities (a) 0.11 (b) 0.23 (c) 0.33 (d) 0.44 (e)

0.53 (f) 0.64 (g) 0.75 (12 ppth, 48 h reaction).

125

5.5 Conclusion Results from this study have demonstrated that the covalent binding of flavor molecules

with BLG varies with pH, temperature and water activity. The rate (and, therefore extent)

of covalent bond formation increased with pH (from pH 3 to 8), temperature (5 to 20°C),

and, somewhat, water activity (0.11-0.75). The results also show that the effect of these

variables on the rate and extent of the reaction depends on the reactive group (functional

group) on the flavor compound, which, in turn, dictates the nature of the adduct-forming

reaction. Further investigations on the effect of the environmental conditions on reaction rates and extent are needed over longer reaction times to gain further understanding.

5.6 Supporting Information Deconvoluted ESI mass spectra of BLG+ flavor compound at time points not shown in

this main chapter for variations in pH (1 h and 6 h), temperature (1 h and 6 h) and aw (24

h) are presented.

126

Figure 5-17 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with benzaldehyde at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 1 h reaction).

Figure 5-18 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with benzaldehyde at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 6 h reaction).

127

Figure 5-19 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with citral at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 1 h reaction).

128

Figure 5-20 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with citral at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 6 h reaction).

Figure 5-21 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with allyl isothiocyanate at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 1 h reaction).

129

Figure 5-22 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with allyl isothiocyanate at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 6 h reaction).

130

Figure 5-23 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with dimethyl trisulfide at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 1 h reaction).

Figure 5-24 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with dimethyl trisulfide at different pH values (a) pH 3 (b) pH 7 (c) pH 8 (12 ppth, 6 h reaction).

131

Figure 5-25 Deconvoluted ESI mass spectrum of BLG incubated at room temperature

with benzaldehyde at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 1 h reaction).

132

Figure 5-26 Deconvoluted ESI mass spectrum of BLG incubated at room temperature

with benzaldehyde at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 6 h reaction).

133

Figure 5-27 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with citral at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 1 h reaction).

134

Figure 5-28 Deconvoluted ESI mass spectrum of BLG incubated at room temperature

with citral at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 6 h reaction).

Figure 5-29 Deconvoluted ESI mass

spectrum of BLG incubated at room temperature with allyl isothiocyanate at different

temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 1 h reaction).

135

Figure 5-30 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with allyl isothiocyanate at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 6 h reaction).

Figure 5-31 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with dimethyl trisulfide at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 1 h reaction).

136

Figure 5-32 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with dimethyl trisulfide at different temperatures (a) 4 °C (b) 20 °C (c) 45 °C (12 ppth, 6 h reaction).

137

Figure 5-33 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with allyl isothiocyanate at different water activities (a) 0.11 (b) 0.23 (c) 0.33 (d) 0.44 (e)

0.53 (f) 0.64 (g) 0.75 (12 ppth, 24 h reaction).

138

Figure 5-34 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with citral at different water activities (a) 0.11 (b) 0.23 (c) 0.33 (d) 0.44 (e) 0.53 (f) 0.64

(g) 0.75 (12 ppth, 24 h reaction).

139

Figure 5-35 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with benzaldehyde at different water activities (a) 0.11 (b) 0.23 (c) 0.33 (d) 0.44 (e) 0.53

(f) 0.64 (g) 0.75 (12 ppth, 24 h reaction).

140

Figure 5-36 Deconvoluted ESI mass spectrum of BLG incubated at room temperature with dimethyl disulfide at different water activities (a) 0.11 (b) 0.23 (c) 0.33 (d) 0.44 (e)

0.53 (f) 0.64 (g) 0.75 (12 ppth, 24 h reaction).

141

Chapter 6 : Conclusion

The inability to measure the covalent adduct formation between the flavor compounds and a protein has meant that scientific understanding has lagged behind the observations of flavor scientists for many decades – namely, that flavorings are highly reactive when added to high protein products.

This thesis starts with developing an analytical methodology to observe the nature and extent of chemical reaction that occurs in a flavor-protein system. Using UPLC/qTOF MS, intact protein analysis was performed on the flavor-protein system, screening for the occurrence of the reaction with a chosen flavor compound and model protein (BLG). The observance of new masses in the mass spectrum corresponding to BLG+ flavor molecule was the indicator for covalent adduct formation. SDS-PAGE was performed on samples that underwent chemical reaction to check for crosslinking of the protein due to the reaction with a given flavor compound. Then using proteomics, the site of modification on the protein was identified. A mechanism of the reaction for the observed new peaks from the mas spectra corresponding to the covalent adducts was proposed.

Using the UPLC/qTOF MS methodology developed from the first study, the formation of covalent bonds between 47 different flavor compounds belonging to 13 different functional groups and BLG was evaluated. The different mechanisms through which BLG and flavor reacted were Schiff base formation, Michael addition, and di-poly- sulfide linkages. Aldehydes, sulfur containing molecules, furans were the most reactive functional groups. The cleavage of one or both the electrophilic disulfide linkages in BLG was observed with selected flavor compounds containing thiol groups and the sulfides reacted with the free cysteine group. Ketones were stable but α diketones were very reactive. Bases, for example pyridines, and pyrazines, were not reactive. Hydrocarbons, alcohols, acids, esters, lactones, pyrans were not found to be reactive within the time period studied. (Future studies should involve longer time frames.)

The influence of pH, temperature, and water activity was studied using UPLC/qTOF MS. Covalent adduct formation increased with an increase in pH. There were no observable 142

adducts for benzaldehyde at pH 3 while substantial activity occurred at pHs 7 and 8. The formation of adducts increased with an increase in temperature for all of the flavor compounds studied. There was formation of new products at higher temperatures (45 °C) that were not observed at lower temperatures (4 °C and 25 °C). These trends are expected because of the rate of a chemical reaction decreases at lower temperature. An increase in water activity increased the formation of adducts for AITC because there was more mobility in the system. There were no observable differences in covalent adduct formation for the other flavor compounds that were studied.

In conclusion, the results that have been obtained using BLG and some the flavor compounds studied are not expected to unique to this protein model. Certainly other proteins, including plant based (e.g., soy, pea etc.) proteins would have the same basic amino acid groups and therefore, the nature of fundamental reactivity and covalent adduct formation can be expected to mirror, at least somewhat, the reactivity of the BLG model. The rate and extent of the reactions will change according to the nativity of the protein, number of the reactive amino acids present, processing conditions (heating, freezing, retorting, extrusion, spray drying etc.), but none of these conditions would change the fundamental nature of the reaction itself. Thus, this study fills an important void in the literature regarding the chemical reactions that occur in flavor-protein matrices, which unquestionably present a serious shelf life issue.

143

Chapter 7 : References

(1) Reineccius, G. The Flavor Industry. In Source Book of Flavors; Springer US: Boston, MA, 1995; pp 1–23. https://doi.org/10.1007/978-1-4615-7889-5_1.

(2) Roberts Jr, B. Plant-Based Proteins - US - January 2018 - Market Research Report; 2018.

(3) Bakker, J.; Langley, K. R.; Elmore, J. S.; Brown, W.; Martin, A.; Salvador., D. Release of Flavour from Food Matrices and Effect on Flavour Perception. In Aroma: Perception, formation, evaluation; Rothe, M., Kruse, H. P., Eds.; Deutsche Institut fur Ernahrungsforschung: Potsdam-Rehbrucke, 1995; pp 191–207.

(4) Plug, H.; Haring, P. The Role of Ingredient-Flavour Interactions in the Development of Fat-Free Foods. Trends in Food Science and Technology. 1993, pp 150–152. https://doi.org/10.1016/0924-2244(93)90035-9.

(5) Rutschmann, M. A.; Solms, J. Formation of Inclusion Complexes of Starch in Ternary Model Systems with Decanal, Menthone, and 1-Naphthol. Leb. und - Technologie 1990, 23 (5), 457–464.

(6) Kim, H.; Min, D. B. Interaction of Flavor Compounds with Protein. In Flavor Chemistry of Lipid Foods; Min, D. B., Smouse, T. H., Eds.; American Chemical Society: Washington, DC, 1988; pp 404–420.

(7) Dumont, J. P.; Land, D. G. Binding of Diacetyl by Pea Proteins. J. Agric. Food Chem. 1986, 34 (6), 1041–1045. https://doi.org/10.1021/jf00072a028.

(8) VCF - Volatile Compounds in Food https://www.vcf- online.nl/VcfCompounds.cfm (accessed Dec 26, 2019).

(9) Reineccius, G.; Heath, H. B. Flavor Chemistry and Technology.; Taylor & Francis, 2006.

(10) Ziegler, E.; Ziegler, H. Flavourings : Production, Composition, Applications, Regulations; Wiley-VCH, 1998. 144

(11) Wang, K. Evaluation of Protein-Flavour Binding on Flavour Delivery and Protein Thermal-Gelation Properties in Regards to Selected Plant Proteins. 2015.

(12) Phillips, G. O.; Williams, P. A. Introduction to Food Proteins. In Handbook of Food Proteins; Phillips, G. O., Williams, P. A., Eds.; Woodhead Publishing: Philadelphia, 2011; pp 1–12.

(13) Wang, K.; Arntfield, S. D. Effect of Protein-Flavour Binding on Flavour Delivery and Protein Functional Properties: A Special Emphasis on Plant-Based Proteins. Flavour Fragr. J. 2017, 32 (2), 92–101. https://doi.org/10.1002/ffj.3365.

(14) Wilson, L. A. Flavor-Binding and the Removal of Flavors from Soy Protein. In World Soybean Research Conference III: Proceedings; R. Shibles, Ed.; Westview Press: Boulder, CO, 1985; pp 158–165.

(15) Rackis, J. J.; Sessa, D. J.; Honig, D. H. Flavor Problems of Vegetable Food Proteins. JAOCS, J. Am. Oil Chem. Soc. 1979, 56, 262–271.

(16) MacLeod, G.; Ames, J. Soy Flavor and Its Improvement. Crit. Rev. Food Sci. Nutr. 1988, 27 (4), 219–400.

(17) Hill, L. M.; Hammond, E. G.; Carlin, A. F.; Seals, R. G. Organoleptic Evaluation of the Effectiveness of Antioxidants in Milk Fat. J. Dairy Sci. 1969, 52 (12), 1917–1921. https://doi.org/10.3168/jds.S0022-0302(69)86874-8.

(18) Harwalkar, V. R.; Cholette, H.; McKellar, R. C.; Emmons, D. B.; Rosenberger, W. S.; Nelson, R. E.; Whitney, R. M. Relation Between Proteolysis and Astringent Off-Flavor in Milk. J. Dairy Sci. 1993, 76 (9), 2521–2527. https://doi.org/10.3168/jds.S0022-0302(93)77587-6.

(19) Gremli, H. A. Interaction of Flavor Compounds with Soy Protein. J. Am. Oil Chem. Soc. 1974, 51 (1), 95–97. https://doi.org/10.1007/BF02542100.

(20) Phillips, G. O.; Williams, P. A. Chemistry of the Major Whey Proteins. In Handbook of Food Proteins ; Woodhead Publishing, 2011.

(21) Sawyer, L. B -Lactoglobulin. In Advanced Dairy Chemistry: Volume 1A: Proteins: 145

Basic Aspects, 4th Edition; Paul L.H. McSweeney, Fox, P. F., Eds.; Springer: Cork, Ireland, 2013; pp 211–259. https://doi.org/10.1007/978-1-4614-4714-6_2.

(22) Bewley, M. C.; Baker, E. N.; Qin, B. Y.; Jameson, G. B.; Sawyer, L. Bovine Beta- Lactoglobulin and Its Variants: A Three-Dimensional Structural Perspective. 1997, 100–109.

(23) Braunitzer, G.; Chen, R.; Schrank, B.; Stangl, A. Automatische Sequenzanalyse Eines Proteins ( b -Lactoglobulin AB). Hoppe-Seyler’s Z Physiol. Chem. 1972, 353, 832–834.

(24) Préaux, G.; Lontie, R. Revised Number of the Free Cysteine Residues and of the Disulphide Bridges in the Sequence of b -Lactoglobulins A and B. Arch. Int. Physiol. Biochim . 1972, No. 80, 980–981.

(25) McKenzie, H. A.; Shaw, D. C. Alternative Positions for the Sulphydryl Group in b -Lactoglobulin: The Signi Fi Cance for Sulphydryl Location in Other Proteins. Nat. New Biol. 1972, 238, 147–149.

(26) Papiz, M. Z.; Sawyer, L.; Eliopoulos, E. E.; North, A. C. T.; Findlay, J.B.C., Sivaprasadarao, R.; Jones, T. A.; Newcomer, M. E.; Kraulis, P. J. The Structure of b -Lactoglobulin and Its Similarity to Plasma Retinolbinding Protein. Nature 1986, 324, 383–385.

(27) Phelan, P.; Malthouse, J. P. G. 13C-n.m.r. of the Cyanylated β-Lactoglobulins: Evidence That Cys-121 Provides the Thiol Group of β-Lactoglobulins A and B. Biochem. J. 1994, 302 (2), 511–516. https://doi.org/10.1042/bj3020511.

(28) Sawyer, L. β-Lactoglobulin. In Advanced Dairy Chemistry—1 Proteins; Springer US, 2003; pp 319–386. https://doi.org/10.1007/978-1-4419-8602-3_7.

(29) Sakai, K.; Sakurai, K.; Sakai, M.; Hoshino, M.; Y., G. Conformation and Stability of Thiol-Modi Fi Ed Bovine b -Lactoglobulin. Prot. Sci . 2000, 9, 1719–1729.

(30) Chamani, J. Comparison of the Conformational Stability of the Non-Native a - Helical Intermediate of Thiol-Modi Fi Ed b -Lactoglobulin upon Interaction with

146

Sodium n-Alkyl Sulfates at Two Different PH. J. Colloid Interf. Sci . 2006, 299, 636–646.

(31) Sitohy, M.; Billaudel, S.; Haertle, T.; Chobert, J.-M. Antiviral Activity of Esteri Fi Ed a -Lactalbumin and b -Lactoglobulin against Herpes Simplex Virus Type 1. Comparison with the Effect of Acyclovir and L-Polylysines. J. Agric. Food Chem. 2007, 55, 10214–10220.

(32) Bhagavan, N. V.; Ha, C.-E. Three-Dimensional Structure of Proteins. In Essentials of Medical Biochemistry; Elsevier, 2011; pp 29–38. https://doi.org/10.1016/b978- 0-12-095461-2.00004-7.

(33) Carey, F. A.; Sundberg, R. J. Addition, Condensation and Substitution Reactions of Carbonyl Compounds. In Advanced Organic Chemistry; Springer US, 2007; pp 629–711. https://doi.org/10.1007/978-0-387-44899-2_7.

(34) Van Ruth, S. M.; Roozen, J. P. Delivery of Flavours from Food Matrices. In Food Flavour Technology: Second Edition; Wiley-Blackwell: Oxford, UK, 2010; pp 190–206. https://doi.org/10.1002/9781444317770.ch7.

(35) Mills, O. E.; Solms, J. Interaction of Selected Flavour Compounds with Whey Proteins. Leb. Wiss. Technol. 1984, 17, 331–335.

(36) Preininger, M. Interactions of Flavor Components in Foods; 2006; pp 477–542. https://doi.org/10.1201/9781420028133.ch14.

(37) Mottram, D. S.; Nobrega, I. C. C. Interaction between Sulfur-Containing Flavor Compounds and Proteins in Foods. ACS Symp.Ser. 2000, 763 (Flavor Release), 274–281.

(38) Mottram, D. S.; Dodson, A.; Szauman-Szumski, C. Interaction of Thiol and Disulfide Flavor Compounds with Food Components. J. Agric. Food Chem. 1996, 44 (8), 2349–2351. https://doi.org/10.1021/jf960170o.

(39) Damodaran, S.; Kinsella, J. E. Interaction of Carbonyls with Soy Protein: Conformational Effects. J. Agric. Food Chem. 1981, 29 (6), 1253–1257.

147

https://doi.org/10.1021/jf00108a038.

(40) Li, Z.; Grun, I. U. U.; Fernando, L. N. N. Interaction of Vanillin with Soy and Dairy Proteins in Aqueous Model Systems: A Thermodynamic Study. J. Food Sci. 2000, 65 (6), 997–1001. https://doi.org/10.1111/j.1365-2621.2000.tb09406.x.

(41) Carini, M.; Orioli, M. Mass Spectrometric Strategies for Identification and Characterization of Carbonylated Peptides and Proteins. Biomarkers Antioxid. Def. Oxidative Damage Princ. Pract. Appl. 2010, 173–197. https://doi.org/10.1002/9780813814438.ch11.

(42) Reid, G. E.; McLuckey, S. A. ‘Top down’ Protein Characterization via Tandem Mass Spectrometry. J. Mass Spectrom. 2002, 37 (7), 663–675. https://doi.org/10.1002/jms.346.

(43) Srebalus Barnes, C. A.; Lim, A. Applications of Mass Spectrometry for the Structural Characterization of Recombinant Protein Pharmaceuticals. Mass Spectrom. Rev. 2007, 26 (3), 370–388. https://doi.org/10.1002/mas.20129.

(44) Bogdanov, B.; Smith, R. D. Proteomics by FTICR Mass Spectrometry: Top down and Bottom Up. Mass Spectrom. Rev. 2005, 24 (2), 168–200. https://doi.org/10.1002/mas.20015.

(45) Rouse, J. C.; McClellan, J. E.; Patel, H. K.; Jankowski, M. A.; Porter, T. J. Top- down Characterization of Protein Pharmaceuticals by Liquid Chromatography/Mass Spectrometry: Application to Recombinant Factor IX Comparability- a Case Study. Methods Mol. Biol. 2005, 308, 435–460. https://doi.org/10.1385/1-59259-922-2:435.

(46) Morris, H. R.; Paxton, T.; Dell, A.; Langhorne, J.; Berg, M.; Bordoli, R. S.; Hoyes, J.; Bateman, R. H. High Sensitivity Collisionally-Activated Decomposition Tandem Mass Spectrometry on a Novel Quadrupole/Orthogonal-Acceleration Time-of-Flight Mass Spectrometer. Rapid Commun. Mass Spectrom. 1996, 10 (8), 889–896. https://doi.org/10.1002/(SICI)1097-0231(19960610)10:8<889::AID- RCM615>3.0.CO;2-F.

148

(47) Shevchenko, A.; Chernushevich, I.; Ens, W.; Standing, K. G.; Thomson, B.; Wilm, M.; Mann, M. Rapid “de Novo” Peptide Sequencing by a Combination of Nanoelectrospray, Isotopic Labeling and a Quadrupole/Time-of-Flight Mass Spectrometer. Rapid Commun. Mass Spectrom. 1997, 11 (9), 1015–1024. https://doi.org/10.1002/(SICI)1097-0231(19970615)11:9<1015::AID- RCM958>3.0.CO;2-H.

(48) Loboda, A. V.; Krutchinsky, A. N.; Bromirski, M.; Ens, W.; Standing, K. G. A Tandem Quadrupole/Time-of-Flight Mass Spectrometer with a Matrix- Assisted Laser Desorption/Ionization Source: Design and Performance. Rapid Commun. Mass Spectrom. 2000, 14 (12), 1047–1057. https://doi.org/10.1002/1097- 0231(20000630)14:12<1047::AID-RCM990>3.0.CO;2-E.

(49) Loboda, A. V.; Ackloo, S.; Chernushevich, I. V. A High-Performance Matrix- Assisted Laser Desorption/Ionization Orthogonal Time-of-Flight Mass Spectrometer with Collisional Cooling. Rapid Commun. Mass Spectrom. 2003, 17 (22), 2508–2516. https://doi.org/10.1002/rcm.1241.

(50) Makarov, A. Electrostatic Axially Harmonic Orbital Trapping: A High- Performance Technique of Mass Analysis. Anal. Chem. 2000, 72 (6), 1156–1162. https://doi.org/10.1021/ac991131p.

(51) Hardman, M.; Makarov, A. A. Interfacing the Orbitrap Mass Analyzer to an Electrospray Ion Source. Anal. Chem. 2003, 75 (7), 1699–1705. https://doi.org/10.1021/ac0258047.

(52) Yates, J. R.; Cociorva, D.; Liao, L.; Zabrouskov, V. Performance of a Linear Ion Trap-Orbitrap Hybrid for Peptide Analysis. Anal. Chem. 2006, 78 (2), 493–500. https://doi.org/10.1021/ac0514624.

(53) Wilm, M.; Mann, M. Analytical Properties of the Nanoelectrospray Ion Source. Anal. Chem. 1996, 68 (1), 1–8. https://doi.org/10.1021/ac9509519.

(54) Biemann, K.; Scoble, H. A. Characterization by Tandem Mass Spectrometry of Structural Modifications in Proteins. Science (80-. ). 1987, 237 (4818), 992–998.

149

https://doi.org/10.1126/science.3303336.

(55) Derf A. Lewis; Andrew W. Guzzetta; Hancock, W. S. Characterization of Humanized Anti-TAC, an Antibody Directed Against the Interleukin 2 Receptor, Using Electrospray Ionization Mass Spectrometry by Direct Infusion, LC/MS, and MS/MS; UTC, 1994; Vol. 66.

(56) Hirayama, K.; Yuji, R.; Yamada, N.; Kato, K.; Arata, Y.; Shimada, I. Complete and Rapid Peptide and Glycopeptide Mapping of Mouse Monoclonal Antibody by LC/MS/MS Using Ion Trap Mass Spectrometry. Anal. Chem. 1998, 70 (13), 2718– 2725. https://doi.org/10.1021/ac9712153.

(57) Zhang, W.; Marzilli, L. A.; Rouse, J. C.; Czupryn, M. J. Complete Disulfide Bond Assignment of a Recombinant Immunoglobulin G4 Monoclonal Antibody. Anal. Biochem. 2002, 311 (1), 1–9. https://doi.org/10.1016/s0003-2697(02)00394-9.

(58) Biemann, K. Contributions of Mass Spectrometry to Peptide and Protein Structure. Biol. Mass Spectrom. 1988, 16 (1–12), 99–111. https://doi.org/10.1002/bms.1200160119.

(59) Guichard, E. Interactions between Flavor Compounds and Food Ingredients and Their Influence on Flavor Perception. Food Rev. Int. 2002, 18 (1), 49–70. https://doi.org/10.1081/FRI-120003417.

(60) O’Neill, T. E.; Kinsella, J. E. Flavor Protein Interactions: Characteristics of 2- Nonanone Binding to Isolated Soy Protein Fractions. J. Food Sci. 1987, 52 (1), 98–101. https://doi.org/10.1111/j.1365-2621.1987.tb13980.x.

(61) O’Neill, T. E. Flavor Binding by Food Proteins: An Overview; UTC, 2020; Vol. 14.

(62) Pelletier, E.; Sostmann, K.; Guichard, E. Measurement of Interactions between Beta-Lactoglobulin and Flavor Compounds (Esters, Acids, and Pyrazines) by Affinity and Exclusion Size Chromatography. J. Agric. Food Chem. 1998, 46 (97), 1506–1509.

150

(63) Jouenne, E.; Crouzet, J. Effect of PH on Retention of Aroma Compounds by Beta- Lactoglobulin. J. Agric. Food Chem. 2000, 48 (4), 1273–1277. https://doi.org/jf990215w [pii].

(64) O’NEILL, T.; KINSELLA, J. E. E. Effect of Heat Treatment and Modification on Conformation and Flavor Binding by Beta-Lactoglobulin. J. Food Sci. 1988, 53 (3), 906–909. https://doi.org/10.1111/j.1365-2621.1988.tb08982.x.

(65) Maier, H. G.; Hartmann, R. U. The Adsorption of Volatile Aroma Constituents by Foods. Z. Leb. Unters.-Forsch 1977, 163, 251–254.

(66) Chung, S.; Villota, R. Binding of Alcohols by Soy Protein in Aqueous Solutions. J. Food Sci. 1989, 54 (6), 1604–1606. https://doi.org/10.1111/j.1365- 2621.1989.tb05170.x.

(67) Arora, A.; Damodaran, S. Competitive Binding of Off-Flavor Compounds with Soy Protein and Beta-Cyclodextrin in a Ternary System: A Model Study. JAOCS, J. Am. Oil Chem. Soc. 2010, 87 (6), 673–679. https://doi.org/10.1007/s11746-009- 1535-8.

(68) Cadwallader, Ke. R. Effect of Flavor Compound Chemical Structure and Environmental Relative Humidity on the Binding of Volatil(2).Pdf. 2006, 15–17.

(69) Damodaran, S.; Kinsella, J. E. Interaction of Carbonyls with Soy Protein: Thermodynamic Effects. J. Agric. Food Chem. 1981, 29 (6), 1249–1253. https://doi.org/10.1021/jf00108a037.

(70) Gkionakis, G. A.; David Anthony Taylor, K.; Ahmad, J.; Heliopoulos, G. The Binding of the Flavour of Lactones by Soya Protein, Amino Acids and Casein. Int. J. Food Sci. Technol. 2007, 42 (2), 165–174. https://doi.org/10.1111/j.1365- 2621.2006.01184.x.

(71) Ng, P. K. W.; Hoehn, E.; Bushuk, W. Binding of Vanillin by Fababean Proteins. J. Food Sci. 1989, 54 (1), 105–107. https://doi.org/10.1111/j.1365- 2621.1989.tb08578.x.

151

(72) Reiners, J.; Nicklaus, S.; Guichard, E. Interactions between β-Lactoglobulin and Flavour Compounds of Different Chemical Classes. Impact of the Protein on the Odour Perception of Vanillin and Eugenol. 2000, 80, 347–360.

(73) Hong, Y. H.; Creamer, L. K. Changed Protein Structures of Bovine β- Lactoglobulin B and α-Lactalbumin as a Consequence of Heat Treatment - ScienceDirect. Int. Dairy J. 2002, 12 (4), 345–359. https://doi.org/10.1016/S0958- 6946(02)00030-4.

(74) Hoffmann, M. A. M.; Van Mil, P. J. J. M. Heat-Induced Aggregation of β- Lactoglobulin: Role of the Free Thiol Group and Disulfide Bonds. J. Agric. Food Chem. 1997, 45 (8), 2942–2948. https://doi.org/10.1021/jf960789q.

(75) Prabakaran, S.; Damodaran, S. Thermal Unfolding of β-Lactoglobulin: Characterization of Initial Unfolding Events Responsible for Heat-Induced Aggregation. J. Agric. Food Chem. 1997, 45 (11), 4303–4308. https://doi.org/10.1021/jf970269a.

(76) Relkin, P.; Molle, D.; Marin, I. Non-Covalent Binding of Benzaldehyde to Beta- Lactoglobulin: Characterisation by Spectrofluorimetry and Electrospray Ionisation Mass-Spectrometry. J. Dairy Res. 2001, 68 (1), 151–155. https://doi.org/Doi 10.1017/S0022029900004684.

(77) Andriot, I.; Marin, I.; Feron, G.; Relkin, P.; Guichard, É. Binding of Benzaldehyde by β-Lactoglobulin, by Static Headspace and High Performance Liquid Chromatography in Different Physico-Chemical Conditions. Le Lait, INRA Ed. 1999, 79 (6), 577–586. https://doi.org/10.1051/lait:1999647.

(78) Dufour, E.; Haertle, T. Binding Affinities of B-Ionone and Related Flavor Compounds to b-Lactoglobulin: Effects of Chemical Modifications. J. Agric. Food Chem. 1990, 38 (8), 1691–1695. https://doi.org/10.1021/jf00098a014.

(79) Lübke, M.; Guichard, E.; Tromelin, A.; Le Quéré, J. L.; O ’keefe, S. F.; Resurreccion, A. P.; Wilson, L. A.; Murphy, P. A.; Damodaran, S.; Kinsella, J. E.; et al. Nuclear Magnetic Resonance Spectroscopic Study of β-Lactoglobulin

152

Interactions with Two Flavor Compounds, γ-Decalactone and β-Ionone. J. Agric. Food Chem. 1998, 54 (4), 1506–1509. https://doi.org/10.1021/jf801810b.

(80) Guichard, E.; Langourieux, S. Interactions between Beta-Lactoglobulin and Flavour Compounds. Food Chem. 2000, 71 (3), 301–308. https://doi.org/10.1016/S0308-8146(00)00181-3.

(81) Sostmann, K.; Guichard, E. Immobilized Alpha-Lactoglobulin on a HPLC- Column: A Rapid Way to Determine Protein-Flavour Interactions. Food Chem. 1998, 62 (4), 509–513. https://doi.org/10.1016/S0308-8146(97)00182-9.

(82) Meynier, A.; Rampon, V.; Dalgalarrondo, M.; Genot, C. Hexanal and T-2-Hexenal Form Covalent Bonds with Whey Proteins and Sodium Caseinate in Aqueous Solution. Int. Dairy J. 2004, 14 (8), 681–690. https://doi.org/10.1016/j.idairyj.2004.01.003.

(83) Kay L. Franzen* and John E. Kinsella. Parameters Affecting the Binding of Volatile Flavor Compounds in Model Food Systems. I. Proteins. J. Agric. Chem. 1974, 22 (4), 675–678.

(84) Damodaran, S.; Kinsella, J. E. Flavor Protein Interactions. Binding of Carbonyls to Bovine Serum Albumin: Thermodynamic and Conformational Effects. J. Agric. Food Chem. 1980, 28 (3), 571–578. https://doi.org/10.1017/CBO9781107415324.004.

(85) Druaux, C.; Lubbers, S.; Charpentier, C.; Voilley, A. Effects of Physico-Chemical Parameters of a Model Wine on the Binding of γ-Decalactone on Bovine Serum Albumin. Food Chem. 1995, 53 (2), 203–207. https://doi.org/10.1016/0308- 8146(95)90789-A.

(86) Burova, T. V.; Grinberg, N. V.; Grinberg, V. Y.; Tolstoguzov, V. B. Binding of Odorants to Individual Proteins and Their Mixtures. Effects of Protein Denaturation and Association. A Plasticized Globule State. Colloids Surfaces A Physicochem. Eng. Asp. 2003, 213 (2–3), 235–244. https://doi.org/10.1016/S0927- 7757(02)00516-2.

153

(87) Baker, A.; Žídek, L.; Wiesler, D.; Chmelík, J.; Pagel, M.; Novotny, M. V. Reaction of N-Acetylglycyllysine Methyl Ester with 2-Alkenals: An Alternative Model for Covalent Modification of Proteins. Chem. Res. Toxicol. 1998, 11 (7), 730–740. https://doi.org/10.1021/tx970167e.

(88) Pokorný, J.; Luan, N.-T.; Kondratenko, S. S.; Janíček, G. Changes of Sensory Value by Interaction of Alkanals with Amino Acids and Proteins. Food / Nahrung 1976, 20 (3), 267–272. https://doi.org/10.1002/food.19760200306.

(89) Arai, S.; Noguchi, M.; Yamashita, M.; Kato, H.; Fujimaki, M. Studies on Flavor Components in Soybean. Agric. Biol. Chem. 1970, 34 (10), 1569–1573. https://doi.org/10.1080/00021369.1970.10859810.

(90) Zavodnik, I. B.; Stepuro, I. I.; Ostrovskiǐ, I. M. Various Enzymatic and Nonenzymatic Pathways of Acetaldehyde Transformation. Ukr. Biokhim. Zh. 1988, 60 (4), 51–57.

(91) Pokorný, J.; Karnet, J.; Davídek, J. Sensory Profile of a Fernet Appetizer. Food / Nahrung 1981, 25 (6), 553–559. https://doi.org/10.1002/food.19810250607.

(92) Fenaille, F.; Parisod, V.; Visani, P.; Populaire, S.; Tabet, J. C.; Guy, P. A. Modifications of Milk Constituents during Processing: A Preliminary Benchmarking Study. Int. Dairy J. 2006, 16 (7), 728–739. https://doi.org/10.1016/j.idairyj.2005.08.003.

(93) Zhu, X.; Tang, X.; Zhang, J.; Tochtrop, G. P.; Anderson, V. E.; Sayre, L. M. Mass Spectrometric Evidence for the Existence of Distinct Modifications of Different Proteins by 2(E),4(E)-Decadiena. Chem. Res. Toxicol. 2010, 23 (3), 467–473. https://doi.org/10.1021/tx900379a.

(94) Zhu, X.; Gallogly, M. M.; Mieyal, J. J.; Anderson, V. E.; Sayre, L. M. Covalent Cross-Linking of Glutathione and Carnosine to Proteins by 4-Oxo-2-Nonenal. Chem. Res. Toxicol. 2009, 22 (6), 1050–1059. https://doi.org/10.1021/tx9000144.

(95) Sayre, L. M.; Lin, D.; Yuan, Q.; Zhu, X.; Tang, X. Protein Adducts Generated from Products of Lipid Oxidation: Focus on HNE and ONE. Drug Metab. Rev. 154

2006, 38 (4), 651–675. https://doi.org/10.1080/03602530600959508.

(96) Bruenner, B. A. B. A.; Jones, D. A.; German, J. B.; German, B. J.; German, J. B. Direct Characterization of Protein Adducts of the Lipid Peroxidation Product 4- Hydroxy-2-Nonenal Using Electrospray Mass Spectrometry. Chem. Res. Toxicol. 1995, 8 (4), 552–559. https://doi.org/10.1021/tx00046a009.

(97) Uchida, K.; Toyokuni, S.; Nishikawa, K.; Kawakishi, S.; Oda, H.; Hiai, H.; Stadtman1, E. R. Michael Addition-Type 4-Hydroxy-2-Nonenal Adducts in Modified Low-Density Lipoproteins: Markers for Atherosclerosis. Biochemistry 1994, 33 (41), 12487–12494. https://doi.org/10.1021/bi00207a016.

(98) Kishore, N. V; Murthy, K.; Rao, N. M. S. Interaction of Allyl Isothiocyanate with Mustard 12S Protein. 1988, 34, 448–452. https://doi.org/https://doi.org/10.1021/jf00069a017.

(99) Kawakishi, S.; Kaneko, T. Interaction of Proteins with Allyl Isothiocyanate. J. Agric. Food Chem. 1987, 35 (1), 85–88. https://doi.org/10.1021/jf00073a020.

(100) Okulicz, M.; Hertig, I. Benzyl Isothiocyanate Disturbs Lipid Metabolism in Rats in a Way Independent of Its Thyroid Impact Following in Vivo Long-Term Treatment and in Vitro Adipocytes Studies. J. Physiol. Biochem. 2013, 69 (1), 75– 84. https://doi.org/10.1007/s13105-012-0189-4.

(101) Hernández-Triana, M.; Kroll, J.; Proll, J.; Noack, J.; Petzke, K. J. Benzyl- Isothiocyanate (BITC) Decreases Quality of Egg White Proteins in Rats. J. Nutr. Biochem. 1996, 7 (6), 322–326. https://doi.org/10.1016/0955-2863(96)00033-2.

(102) Keppler, J. K.; Koudelka, T.; Palani, K.; Stuhldreier, M. C.; Temps, F.; Tholey, A.; Schwarz, K. Characterization of the Covalent Binding of Allyl Isothiocyanate to β -Lactoglobulin by Fluorescence Quenching, Equilibrium Measurement, and Mass Spectrometry. J. Biomol. Struct. Dyn. 2014, 32 (7), 1103–1117. https://doi.org/10.1080/07391102.2013.809605.

(103) Rade-Kukic, K.; Schmitt, C.; Rawel, H. M. Formation of Conjugates between β- Lactoglobulin and Allyl Isothiocyanate: Effect on Protein Heat Aggregation, 155

Foaming and Emulsifying Properties. Food Hydrocoll. 2011, 25 (4), 694–706. https://doi.org/10.1016/j.foodhyd.2010.08.018.

(104) Wilde, S. C.; Keppler, J. K.; Palani, K.; Schwarz, K. β-Lactoglobulin as Nanotransporter - Part I: Binding of Organosulfur Compounds. Food Chem. 2016, 197, 1015–1021. https://doi.org/10.1016/j.foodchem.2015.11.010.

(105) Weel, K. G. C.; Boelrijk, A. E. M.; Alting, A. C.; Van Mil, P. J. J. M.; Burger, J. J.; Gruppen, H.; Voragen, A. G. J.; Smit, G. Flavor Release and Perception of Flavored Whey Protein Gels: Perception Is Determined by Texture Rather than by Release. J. Agric. Food Chem. 2002, 50 (18), 5149–5155. https://doi.org/10.1021/jf0202786.

(106) Van Ruth, S. M.; Villeneuve, E. Influence of Beta-Lactoglobulin, PH and Presence of Other Aroma Compounds on the Air/Liquid Partition Coefficients of 20 Aroma Compounds Varying in Functional Group and Chain Length. Food Chem. 2002, 79 (2), 157–164. https://doi.org/10.1016/S0308-8146(02)00124-3.

(107) O’Keefe, S. F.; Resurreccion, A. P.; Wilson, L. A.; Murphy, P. A. Temperature Effect on Binding of Volatile Flavor Compounds to Soy Protein in Aqueous Model Systems. J. Food Sci. 1991, 56 (3), 802–806. https://doi.org/10.1111/j.1365- 2621.1991.tb05386.x.

(108) Adams, R. L.; Mottram, D. S.; Parker, J. K.; Brown, H. M. Flavor - Protein Binding: Disulfide Interchange Reactions between Ovalbumin- and Volatile Disulfides. J. Agric. Food Chem. 2001, 49 (9), 4333–4336. https://doi.org/10.1021/jf0100797.

(109) O ’keefe, S. F.; Wilson, L. A.; Resurreccion, A. P.; Murphy, P. A. Determination of the Binding of Hexanal to Soy Glycinin and 8-Conglycinin in an Aqueous Model System Using a Headspace Technique. J. Agric. Food Chem. 1991, 39 (6), 1022–1028. https://doi.org/10.1021/jf00006a003.

(110) Andriot, I.; Marin, I.; Feron, G.; Relkin, P.; Guichard, E. Binding of Binzaldehyde by B-Lactoglobulin, by Static Headspace and High Performance Liquid

156

Chromatography in Different Physico-Chemical Conditions. Lait 1999, 79, 577– 586.

(111) Landy, P.; Druaux, C.; Voilley, A. Retention of Aroma Compounds by Proteins in Aqueous Solution. Food Chem. 1995, 54 (4), 387–392. https://doi.org/10.1016/0308-8146(95)00069-U.

(112) Heng, L.; Van Koningsveld, G. A.; Gruppen, H.; Van Boekel, M. A. J. S.; Vincken, J. P.; Roozen, J. P.; Voragen, A. G. J. Protein-Flavour Interactions in Relation to Development of Novel Protein Foods. Trends in Food Science and Technology. Elsevier Ltd 2004, pp 217–224. https://doi.org/10.1016/j.tifs.2003.09.018.

(113) Zhou, Q.; Lee, S.-Y.; Cadwallader, K. R. Inverse Gas Chromatographic Evaluation of the Influence of Soy Protein on the Binding of Selected Butter Flavor Compounds in a Wheat Soda Cracker System. J. Agric. Food Chem. 2006, 54, 5516–5520. https://doi.org/10.1021/jf060538+.

(114) Fares, K.; Landy, P.; Guilard, R.; Voilley, A. Physicochemical Interactions Between Aroma Compounds and Milk Proteins: Effect of Water and Protein Modification. J. Dairy Sci. 1998, 81 (1), 82–91. https://doi.org/10.3168/jds.S0022- 0302(98)75554-7.

(115) Kühn, J.; Considine, T.; Singh, H. Binding of Flavor Compounds and Whey Protein Isolate as Affected by Heat and High Pressure Treatments. J. Agric. Food Chem. 2008, 56 (21), 10218–10224. https://doi.org/10.1021/jf801810b.

(116) Dickinson, E.; Miller, R.; Relkin, P.; Vermersh, J. Binding Properties of Vanillin to Whey Proteins: Effect on Protein Conformational Stability and Foaming Properties. In Food Colloids - Fundamentals of Formulation; Dickinson, E., Miller, R., Eds.; Royal Society of Chemistry: Cambridge, 2001; pp 282–292.

(117) Wang, K.; Arntfield, S. D. Interaction of Selected Volatile Flavour Compounds and Salt-Extracted Pea Proteins: Effect on Protein Structure and Thermal-Induced Gelation Properties. Food Hydrocoll. 2015, 51, 383–394.

157

https://doi.org/10.1016/j.foodhyd.2015.05.044.

(118) Higgs, K.; Boland, M. J. Changes in Milk Proteins during Storage of Dry Powders. In Milk Proteins; Elsevier, 2014; pp 343–357. https://doi.org/10.1016/b978-0-12- 405171-3.00011-8.

(119) Gilani, G. S.; Xiao, C. W.; Cockell, K. A. Impact of Antinutritional Factors in Food Proteins on the Digestibility of Protein and the Bioavailability of Amino Acids and on Protein Quality. Br. J. Nutr. 2012, 108 (SUPPL. 2). https://doi.org/10.1017/S0007114512002371.

(120) Versic, R. J. Flavor Encapsulation: An Overview. In Flavor Encapsulation; Reineccius, G. A., Risch, S. J., Eds.; American Chemical Society: Washington, DC, 1988; pp 1–11.

(121) Voilley, A.; Beghin, V.; Charpentier, C.; Peyron, D. Interactions between Aroma Substances and Macromolecules in a Model Wine. Leb. Technol. 1981, No. 24, 469–472.

(122) Plug, H.; Haring, P. The Influence of Flavour-Ingredient Interactions on Flavour Perception. Food Qual. Prefer. 1994, 5 (1–2), 95–102. https://doi.org/10.1016/0950-3293(94)90013-2.

(123) Fenaille, F.; Guy, P. A.; Tabet, J. C. Study of Protein Modification by 4-Hydroxy- 2-Nonenal and Other Short Chain Aldehydes Analyzed by Electrospray Ionization Tandem Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2003, 14 (3), 215–226. https://doi.org/10.1016/S1044-0305(02)00911-X.

(124) Tang, X.; Sayre, L. M.; Tochtrop, G. P. A Mass Spectrometric Analysis of 4- Hydroxy-2-(E)-Nonenal Modification of Cytochrome C. J. Mass Spectrom. 2011, 46 (3), 290–297. https://doi.org/10.1002/jms.1890.

(125) Doorn, J. A.; Petersen, D. R. Covalent Modification of Amino Acid Nucleophiles by the Lipid Peroxidation Products 4-Hydroxy-2-Nonenal and 4-Oxo-2-Nonenal. Chem. Res. Toxicol. 2002, 15 (11), 1445–1450. https://doi.org/10.1021/tx025590o.

158

(126) Kehoe, J. J.; Brodkorb, A.; Mollé, D.; Yokoyama, E.; Famelart, M. H.; Bouhallab, S.; Morris, E. R.; Croguennec, T. Determination of Exposed Sulfhydryl Groups in Heated β-Lactoglobulin a Using IAEDANS and Mass Spectrometry. J. Agric. Food Chem. 2007, 55 (17), 7107–7113. https://doi.org/10.1021/jf070397r.

(127) Dinkova-Kostova, A. T.; Holtzclaw, W. D.; Cole, R. N.; Itoh, K.; Wakabayashi, N.; Katoh, Y.; Yamamoto, M.; Talalay, P. Direct Evidence That Sulfhydryl Groups of Keap1 Are the Sensors Regulating Induction of Phase 2 Enzymes That Protect against Carcinogens and Oxidants. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (18), 11908–11913. https://doi.org/10.1073/pnas.172398899.

(128) Blagrove, R. J.; Gruen, L. C. Spectrophotometric Determination of Thiourea. Mikrochim. Acta 1971, 59 (4), 639–643. https://doi.org/10.1007/BF01235070.

(129) Vega-Lugo, A. C.; Lim, L. T. Controlled Release of Allyl Isothiocyanate Using Soy Protein and Poly(Lactic Acid) Electrospun Fibers. Food Res. Int. 2009, 42 (8), 933–940. https://doi.org/10.1016/j.foodres.2009.05.005.

(130) Rawel, H. M.; Kroll, J.; Schröder, I. In Vitro Enzymatic Digestion of Benzyl-and Phenylisothiocyanate-Derivatized Food Proteins. Am. Chem. Soc. 1998, 46 (12), 5103–5109. https://doi.org/10.1021/jf980244r.

(131) Rawel, H. M.; Kroll, J.; Schröder, I. Reactions of Isothiocyanates with Food Proteins: Influence on Enzyme Activity and Tryptical Degradation. Nahrung - Food 1998, 42 (3–4), 197–199. https://doi.org/10.1002/(sici)1521- 3803(199808)42:03/04<197::aid-food197>3.0.co;2-b.

(132) Laemmli, U. K. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature 1970, 227 (5259), 680–685. https://doi.org/10.1038/227680a0.

(133) Boyle, C.; Hansen, L.; Hinnenkamp, C.; Ismail, B. P. Emerging Camelina Protein: Extraction, Modification, and Structural/Functional Characterization. JAOCS, J. Am. Oil Chem. Soc. 2018, 95 (8), 1049–1062. https://doi.org/10.1002/aocs.12045.

(134) Bolgar, M. S.; Yang, C. Y.; Gaskell, S. J. First Direct Evidence for Lipid/Protein 159

Conjugation in Oxidized Human Low Density Lipoprotein. J. Biol. Chem. 1996, 271 (45), 27999–28001. https://doi.org/10.1074/jbc.271.45.27999.

(135) Requena, J. R.; Fu, M. X.; Ahmed, M. U.; Jenkins, A. J.; Lyons, T. J.; Baynes, J. W.; Thorpe, S. R. Quantification of Malondialdehyde and 4-Hydroxynonenal Adducts to Lysine Residues in Native and Oxidized Human Low-Density Lipoprotein. Biochem. J. 1997, 322 (1), 317–325. https://doi.org/10.1042/bj3220317.

(136) Uchida, K.; Stadtman, E. R. Modification of Histidine Residues in Proteins by Reaction with 4- Hydroxynonenal. Proc. Natl. Acad. Sci. U. S. A. 1992, 89 (10), 4544–4548. https://doi.org/10.1073/pnas.89.10.4544.

(137) Meltretter, J.; Becker, C. M.; Pischetsrieder, M. Identification and Site-Specific Relative Quantification of β-Lactoglobulin Modifications in Heated Milk and Dairy Products. J. Agric. Food Chem. 2008, 56 (13), 5165–5171. https://doi.org/10.1021/jf800571j.

(138) Siciliano, R. A.; Mazzeo, M. F.; Arena, S.; Renzone, G.; Scaloni, A. Mass Spectrometry for the Analysis of Protein Lactosylation in Milk Products. Food Res. Int. 2013, 54 (1), 988–1000. https://doi.org/10.1016/j.foodres.2012.10.044.

(139) Matheis, G. Applications. In Flavourings : Production, Composition, Applications, Regulations; Ziegler, E., Ziegler, H., Eds.; Wiley-VCH: Weinheim, 1998; pp 385– 509.

(140) Frapin, D.; Dufour, E.; Haertle, T. Probing the Fatty Acid Binding Site of β- Lactoglobulins. J. Protein Chem. 1993, 12 (4), 443–449. https://doi.org/10.1007/BF01025044.

(141) Luebke, M.; Guichard, E.; Le-Quere, J. L.; Lübke, M.; Guichard, E.; Le Quéré, J. L. Infrared Spectroscopic Study of β-Lactoglobulin Interactions with Flavor Compounds. In Flavor Release ACS Symposium Series; Roberts, D. D., Taylor, A. J., Eds.; American Chemical Society (ACS): Washnigton, DC, 2000; Vol. 763, pp 282–292. https://doi.org/10.1021/bk-2000-0763.ch023.

160

(142) Boudaud, N.; Dumont, J. P. Interaction between Flavor Components and β- Lactoglobulin. In Flavor-Food Interactions; McGorrin, R. J., Leland, J. V., Eds.; American Chemical Society (ACS): Washington, DC, 1996; Vol. 633, pp 90–97. https://doi.org/10.1021/bk-1996-0633.ch008.

(143) Anantharamkrishnan, V.; Reineccius, G. A. Method To Characterize and Monitor Covalent Interactions of Flavor Compounds with β-Lactoglobulin Using Mass Spectrometry and Proteomics. J. Agric. Food Chem. 2020. https://doi.org/10.1021/acs.jafc.9b07978.

(144) Mathews, J. M.; Watson, S. L.; Snyder, R. W.; Burgess, J. P.; Morgan, D. L. Reaction of the Butter Flavorant Diacetyl (2,3-Butanedione) with N-α- Acetylarginine: A Model for Epitope Formation with Pulmonary Proteins in the Etiology of Obliterative Bronchiolitis. J. Agric. Food Chem. 2010, 58 (24), 12761– 12768. https://doi.org/10.1021/jf103251w.

(145) Yankeelov, J. A.; Mitchell, C. D.; Crawford, T. H. A Simple Trimerization of 2,3- Butanedione Yielding a Selective Reagent for the Modification of Arginine in Proteins. J. Am. Chem. Soc 1968, 90 (6), 1664–1666. https://doi.org/10.1021/ja01008a056.

(146) Yankeelov, J. A. Modification of Arginine in Proteins by Oligomers of 2, 3- Butanedione. Biochemistry 1970, 9 (12), 2433–2439. https://doi.org/10.1021/bi00814a007.

(147) Riordan, J. F. Functional Arginyl Residues in Carboxypeptidase A. Modification with Butanedione. Biochemistry 1973, 12 (20), 3915–3923. https://doi.org/10.1021/bi00744a020.

(148) O’Neill, T. E. Flavor Binding by Food Proteins: An Overview. In Flavor-Food Interactions, ACS Symposium Series; McGorrin, R. J., Leland, J. V., Eds.; American Chemical Society: WashingtonDC, 1996; Vol. 633, pp 59–74. https://doi.org/10.1021/bk-1996-0633.ch006.

(149) McNeill, V. L.; Schmidt, K. A. Vanillin Interaction with Milk Protein Isolates in

161

Sweetened Drinks. J. Food Sci. 1993, 58 (5), 1142–1144. https://doi.org/10.1111/j.1365-2621.1993.tb06133.x.

(150) Ng, P. K. W.; Hoehn, E.; Bushuk, W. Sensory Evaluation of Binding of Vanillin by Fababean Proteins. J. Food Sci. 1989, 54 (2), 324–325. https://doi.org/10.1111/j.1365-2621.1989.tb03072.x.

(151) Sandler, S. R.; Karo, W. Acetals and Ketals. In Sourcebook of Advanced Organic Laboratory Preparations; Sandler, S. R., Karo, W., Eds.; Elsevier: San Diego, 1992; Vol. 3, pp 250–257. https://doi.org/10.1016/B978-0-08-092553-0.50038-8.

(152) Chen, M.; Reineccius, G. A. The Stability of Flavor Compounds during Storage in the Presence of Glucose and Protein. In Frontiers of Flavour Science; Schieberle, P., Engel, K. H., Eds.; Deutsche Forschungsanstalt fur Lebensmittelchemie: Garching, 2000; Vol. 1, pp 334–340.

(153) Tromelin, A.; Andriot, I.; Guichard, E. Protein-Flavour Interactions. In Flavour in food; Voilley, A., Etievant, P., Eds.; Woodhead Publishing Limited.: Cambridge, 2002; pp 172–207.

(154) Dickinson, E. Interfacial, Emulsifying and Foaming Properties of Milk Proteins. In Advanced Dairy Chemistry—1 Proteins; Springer US, 2003; pp 1229–1260. https://doi.org/10.1007/978-1-4419-8602-3_33.

(155) Cayot, P.; Lorient, D. Protéines “Majeures”Du Lactosérum. In Structure and technical properties of milk proteins.; Technique et Documentation Lavoisier: Paris, 1998; pp 37–50.

(156) Jouenne, E.; Crouzet, J. Determination of Apparent Binding Constants for Aroma Compounds with β-Lactoglobulin by Dynamic Coupled Column Liquid Chromatography. J. Agric. Food Chem. 2000, 48 (11), 5396–5400. https://doi.org/10.1021/jf000423k.

(157) Weel, K. G. C.; Boelrijk, A. E. M.; Burger, J. J.; Claassen, N. E.; Gruppen, H.; Voragen, A. G. J.; Smit, G. Effect of Whey Protein on the in Vivo Release of Aldehydes. J. Agric. Food Chem. 2003, 51 (16), 4746–4752. 162

https://doi.org/10.1021/jf034188s.

(158) Beyeler, M.; Solms, J. Interactions of Flavor Model Compounds with Soy Protein and Bovine Serum Albumin. Leb. Technol. 1974, 7 (4), 217.

(159) Hansen, A. P.; Booker, D. C. Flavor Interaction with Casein and Whey Protein. ACS Symp. Ser. 1996, 633, 75–89. https://doi.org/10.1021/bk-1996-0633.ch007.

(160) Kühn, J.; Considine, T.; Singh, H. Interactions of Milk Proteins and Volatile Flavor Compounds: Implications in the Development of Protein Foods. J. Food Sci. 2006, 71 (5), R72–R82. https://doi.org/10.1111/j.1750-3841.2006.00051.x.

(161) Wong, C. W.; Wijayanti, H. B.; Bhandari, B. R. Maillard Reaction in Limited Moisture and Low Water Activity Environment. In Food Engineering Series; Springer, 2015; pp 41–63. https://doi.org/10.1007/978-1-4939-2578-0_4.

(162) Aspelund, T. G.; Wilson, L. A. Adsorption of Off-Flavor Compounds onto Soy Protein: A Thermodynamic Study. J. Agric. Food Chem. 1983, 31 (3), 539–545. https://doi.org/10.1021/jf00117a019.

(163) Alan Crowther, Lester A. Wilson, C. E. G. Effects of Processing on Adsorption of off Flavors onto Soy Protein. J. Food Process Eng. 1981, 4 (1980), 99–115.

(164) Zhou, Q.; Cadwallader, K. R. Effect of Flavor Compound Chemical Structure and Environmental Relative Humidity on the Binding of Volatile Flavor Compounds to Dehydrated Soy Protein Isolates. J. Agric. Food Chem. 2006, 54, 1838–1843. https://doi.org/10.1021/jf052269d.

(165) Zhou, Q. X.; Cadwallader, K. R. Inverse Gas Chromatographic Method for Measurement of Interactions between Soy Protein Isolate and Selected Flavor Compounds under Controlled Relative Humidity. J. Agric. Food Chem. 2004, 52 (20), 6271–6277. https://doi.org/Doi 10.1021/Jf049273u.

(166) FOX, M.; LONCIN, M.; WEISS, M. INVESTIGATIONS INTO THE INFLUENCE OF WATER ACTIVITY, PH AND HEAT TREATMENT ON THE VELOCITY OF THE MAILLARD REACTION IN FOODS. J. Food Qual. 1983,

163

6 (2), 103–118. https://doi.org/10.1111/j.1745-4557.1983.tb00760.x.

(167) T.P., L.; M., S. The Nonenzymatic Browning Reaction as Affected by Water in Foods [Shelf Life of Food Products]. In 2nd International Symposium on Properties of Water; Osaka, 1978.

(168) WARMBIER, H. C.; SCHNICKELS, R. A.; LABUZA, T. P. NONENZYMATIC BROWNING KINETICS IN AN INTERMEDIATE MOISTURE MODEL SYSTEM: EFFECT OF GLUCOSE TO LYSINE RATIO. J. Food Sci. 1976, 41 (5), 981–983. https://doi.org/10.1111/j.1365-2621.1976.tb14372.x.

(169) Žídek, L.; Doležel, P.; Chmelík, J.; Baker, A. G.; Novotny, M. Modification of Horse Heart Cytochrome c with Trans-2-Hexenal. Chem. Res. Toxicol. 1997, 10 (6), 702–710. https://doi.org/10.1021/tx960118m.

(170) Baker, A. G.; Wiesler, D.; Novotny, M. V. Tandem Mass Spectrometry of Model Peptides Modified with Trans-2-Hexenal, a Product of Lipid Peroxidation. J. Am. Soc. Mass Spectrom. 1999, 10 (7), 613–624. https://doi.org/10.1016/S1044- 0305(99)00029-X.

(171) Pournamdari, M.; Saadi, A.; Ellis, E.; Andrew, R.; Walker, B.; Watson, D. G. Development of a Derivatisation Method for the Analysis of Aldehyde Modified Amino Acid Residues in Proteins by Fourier Transform Mass Spectrometry. Anal. Chim. Acta 2009, 633 (2), 216–222. https://doi.org/10.1016/j.aca.2008.11.070.

(172) Ichihashi, K.; Osawa, T.; Toyokuni, S.; Uchida, K. Endogenous Formation of Protein Adducts with Carcinogenic Aldehydes: Implications for Oxidative Stress. J. Biol. Chem. 2001, 276 (26), 23903–23913. https://doi.org/10.1074/jbc.M101947200.

(173) Anantharamkrishnan, V.; Hoye, T.; Reineccius, G. Covalent Adduct Formation Between Flavor Compounds of Various Functional Group Classes and the Model Protein β-Lactoglobulin. J. Agric. Food Chem. 2020, 68 (23), 6395–6402. https://doi.org/10.1021/acs.jafc.0c01925.

(174) Miller, A. G.; Meade, S. J.; Gerrard, J. A. New Insights into Protein Crosslinking 164

via the Maillard Reaction: Structural Requirements, the Effect on Enzyme Function, and Predicted Efficacy of Crosslinking Inhibitors as Anti-Ageing Therapeutics. Bioorganic Med. Chem. 2003, 11 (6), 843–852. https://doi.org/10.1016/S0968-0896(02)00565-5.

(175) Wijewickreme, A. N.; Kitts, D. D. Influence of Reaction Conditions on the Oxidative Behavior of Model Maillard Reaction Products. J. Agric. Food Chem. 1997, 45 (12), 4571–4576. https://doi.org/10.1021/jf970040v.

(176) LENGES, J. Influence of the Activity of Water on the Spoilage of Foodstuffs. Int. J. Food Sci. Technol. 1968, 3 (2), 131–142. https://doi.org/10.1111/j.1365- 2621.1968.tb01448.x.

(177) JOKINEN, J. E.; RElNECCIUS, G. A.; THOMPSON, D. R. LOSSES IN AVAILABLE LYSINE DURING THERMAL PROCESSING OF SOY PROTEIN MODEL SYSTEMS. J. Food Sci. 2008, 41 (4), 816–819. https://doi.org/10.1111/j.1365-2621.1976.tb00730_41_4.x.

(178) Pereyra Gonzales, A. S.; Naranjo, G. B.; Leiva, G. E.; Malec, L. S. Maillard Reaction Kinetics in Milk Powder: Effect of Water Activity at Mild Temperatures. Int. Dairy J. 2010, 20 (1), 40–45. https://doi.org/10.1016/j.idairyj.2009.07.007.

(179) Malec, L. S.; Pereyra Gonzales, A. S.; Naranjo, G. B.; Vigo, M. S. Influence of Water Activity and Storage Temperature on Lysine Availability of a Milk like System. Food Res. Int. 2002, 35 (9), 849–853. https://doi.org/10.1016/S0963- 9969(02)00088-1.

(180) Reineccius, G. Flavoring Materials. In Flavor Chemistry and Technology, Second Edition; Reineccius, G., Ed.; CRC Press: Boca Raton, 2005; Vol. 1, pp 203–256.

165