The Pennsylvania State University The Graduate School College of Agricultural Sciences REACTIVITY OF EPICATECHIN IN MAILLARD CHEMISTRY A Thesis in Food Science by Vandana M. Totlani © 2006 Vandana M. Totlani Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2006 UMI Number: 3231904 3231904 2006 UMI Microform Copyright All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 by ProQuest Information and Learning Company. The thesis of Vandana M. Totlani was reviewed and approved* by the following: Devin Peterson Assistant Professor of Food Science Thesis Advisor Chair of Committee Robert Beelman Professor of Food Science Donald Thompson Professor of Food Science Koushik Seetharaman Assistant Professor of Food Science A.Daniel Jones Professor of Chemistry, Biochemistry and Molecular biology John Floros Professor Of Food Science Head of the Department of Food Science *Signatures are on file in the Graduate School iii Abstract The Maillard reaction, a carbonyl-amino condensation reaction, is a ubiquitous chemical reaction in nature and a well-documented critical food (flavor, color, nutritional value, toxicity) and biological reaction that involves aging, inflammation, cardiovascular disease, etc. Polyphenolic components (of plant origin) have not been considered as reactants in the thermally catalyzed reactions involved in flavor generation. This study primarily focuses on understanding the mechanisms of how epicatechin (a polyphenolics compound) functions as a reactant in Maillard model systems and in particular with Maillard reaction transient intermediates using GC, GC-MS, LC/MS and NMR analysis for structural confirmation. Preliminary experiments conducted on aqueous Maillard model systems consisting of 4 amino acids plus 2 hexose sugars indicated that the addition of epicatechin or epigallocatechin gallate dramatically altered (reduced) the generation of Maillard-type flavor compounds. The mechanisms of how EC behaves as a reactants in Maillard chemistry were subsequently investigated by labeling studies ( 13 C, 15 N) in an aqueous glucose-glycine model system. Analysis of the volatile flavor compounds was conducted on model consisting of CAMOLA systems (1:1 - 13 C 6 : 12 C 6 )glucose and glycine indicated that the flavor compounds - 2,3-butanedione, acetol, pyrazine, 2- methylpyrazine, 2,5-dimethylpyrazine were essentially derived from intact C 4 , C 3 , C 2 /C 2 , C 2 /C 3 , and C 3 /C 3 sugar fragments, respectively. Analysis of the non-volatiles by LC/MS indicated that epicatechin combined with C 2 , C 3 , and C 4 sugar fragments to form adduct reaction products which were otherwise absent in control a sample with no epicatechin iv added. Glycine was not required under these reaction conditions to catalyze glucose fragmentation, as similar adduct reaction products were obtained when glucose was directly combined with EC. The identity of the individual sugar fragments that were being trapped by epicatechin in aqueous Maillard systems was established by direct comparison of LC/MS peak retention times and ion abundances. The LC/MS chromatograms of epicatechin- sugar fragment adducts generated in the glucose/glycine system were compared with adducts generated via direct combination of epicatechin and well- known sugar carbonyls – glyoxal, acetol, glyceraldehyde, methylglyoxal etc. Based on the comparison of chromatogram of the adduct peaks, the key contributors of the C 2 , C 3 and C 4 epicatechin- sugar fragments were identified as glyoxal, acetol and erythrose (based on their chromatogram responses). To further examine the mechanisms of EC-carbonyl adduct generation, the structural properties of a pure EC-methylglyoxal adduct reaction product was analyzed by NMR. The 1-D ( 1 H and 13 C)and 2-D NMR ( 1 H- 1 H COSY, HMBC, HMQC) analysis were conducted at low temperature (-25°C) to slow down the rapid conformation exchange as indicated by 1 H-NMR analysis of the purified adduct. The 2- D NMR suggested the covalent linkage between the C 1 position of the methylglyoxal and either the C 6 or C 8 position of the epicatechin A-ring by a aromatic substitution reaction. In conclusion, the reduction of flavor generation due to addition of epicatechin in Maillard model systems was ascribed to direct trapping of C 2 , C 3 and C 4 carbonyl compounds in aqueous model systems. Further the identification that EC formed covalent bonds with reactive carbonyl such as glyoxal, methylglyoxal provides a new v mechanism for elucidating the epidemiological evidence supporting a positive health link between dietary intake of polyphenolic flavonoids and the reduction of several age- related chronic diseases. TABLE OF CONTENTS LIST OF FIGURES viii LIST OF TABLES xii Acknowledgements xiii Chapter 1 Literature Review 1 Flavor and Aroma 1 Maillard Reaction 3 Impact of Maillard on food quality 4 Classification of flavor compounds from Maillard reaction 5 Factors influencing Maillard reaction kinetics 7 The influence of pH 8 The influence of temperature-time 9 Water Content 11 Influence of reactant type (sugar and amino acids) 13 Mechanisms of Maillard reaction 15 Hodge Scheme 15 Stage One: 16 Stage two 18 Degradation reaction of Amadori Product 18 Strecker Degradation 21 Stage three 23 Free Radical (Alternate Browning Pathway) 24 Maillard Model Systems 30 Labeling Studies 30 Nutritional Aspects 32 The Maillard reaction and Aging 33 Flavonoids 36 Reactivity of Phenols with Carbonyl Groups 42 Polyphenol and Maillard Chemistry 43 Hypothesis and Objectives 46 References 47 Chapter 2 Influence of Epicatechin and Epigallocatechin gallate on the Generation of Maillard-type Aroma Compounds 54 Introduction 54 Materials and Methods 56 Results and Discussion 58 Conclusion 59 References 67 vii Chapter 3 Reactivity of Epicatechin in Aqueous Glucose-Glycine Systems: Quenching of C 2 , C 3 and C 4 Sugar Fragments 68 Abstract 68 Introduction 70 Materials and Methods 73 Results and Discussion 77 References 85 Chapter 4 Epicatechin-Carbonyl Trapping Reactions in Aqueous Maillard Systems: Identification and Structural Elucidation 88 Abstract 88 Introduction 89 Materials and Methods 91 Results and Discussion 97 References 116 Chapter 5 Suggested Future Work 119 Appendix A Abbreviations 121 Appendix B 123 Optimization of the condition for synthesis of methylglyoxal-epicatechin adduct for NMR analysis 123 Methods 123 Results 126 viii LIST OF FIGURES Figure 1-2: Structures of the functional group of compounds formed by the interaction of sugar and amino acid degradation during Maillard reaction 6 Figure 1-3: The Hodge Scheme from reproduced from source (8) 16 Figure 1-4: Formation of Amadori Rearrangement Product reproduced from source (42) 19 Figure 1-5: Enolization of the Amadori Compound reproduced from source(19) 21 Figure 1-6: The Strecker Degradation reaction between Glycine and Glyoxal adapted from source (47) 23 Figure 1-7: Alternate pathway for free radical formation in Maillard reaction adapted from source (54) 27 Figure 1-8: Pathways of Melanoidin Formation reproduced from source (56) 28 Figure 1-9: Formation of Carboxymethyllysine (CML) in vitro reproduced from source (77) 36 Figure 1-10: a)Base Flavan Nucleus b) Structure of Epicatechin 38 Figure 1-11: Structure of Flavonoids reproduced from source (80) 39 Figure 1-12: Antioxidant Activity reproduced from source (82) 40 Figure 1-13: Dimer of catechin-epicatechin in presence of acetaldehyde at acidic pH reproduced from source(88) 42 Figure 1-14: Epigallocatechin gallate-ascorbic acid condensation product reproduced from source (89) 43 Figure 2-15: Relative difference in the aroma compounds generated in a model Maillard reaction systems with and without epicatechin at pH 6.0, 125 o C for 30 minutes; TIC Peak Area of Epicatechin (EC) Treatment/TIC Peak Area of Control * 100; peak area adjusted by internal standard (dodecane) 60 Figure 2-16: Relative difference in the aroma compounds generated in a model Maillard reaction systems with and without epicatechin at pH 7.0, 125 o C for 30 minutes; TIC Peak Area of Epicatechin (EC) Treatment/TIC Peak Area of Control * 100; peak area adjusted by internal standard (dodecane) 61 ix Figure 2-17: Relative difference in the aroma compounds generated in a model Maillard reaction systems with and without epicatechin at pH 8.2, 125 o C for 30 minutes; TIC Peak Area of Epicatechin (EC) Treatment/TIC Peak Area of Control * 100; peak area adjusted by internal standard (dodecane) 62 Figure 2-18: Relative difference in the aroma compounds generated in a model Maillard reaction systems with and without epicatechin at pH 7.0, 125 o C for 15 and 30 minutes; TIC Peak Area of Epicatechin (EC) Treatment/TIC Peak Area of Control * 100; peak area adjusted by internal standard (dodecane) 63 Figure 2-19: Relative difference in the aroma compounds generated in a model Maillard reaction systems with and without epicatechin at pH 7.0, 125 o C and 150 o C for 30 minutes; TIC Peak Area of Epicatechin (EC) Treatment/TIC Peak Area of Control * 100; peak area adjusted by internal standard (dodecane) 64 Figure 2-20: Relative difference in the aroma compounds generated in a model Maillard reaction systems with and without epicatechin at pH 7.0, 100 o C for 60 minutes; TIC Peak Area of Epicatechin (EC) Treatment/TIC Peak Area of Control*100; peak area adjusted by internal standard (dodecane) 65 Figure 2-21: Relative difference in the aroma compounds generated in a model Maillard reaction systems with and without epicatechin or epigallocatechin gallate at pH 7.0, 125 o C for 30 minutes; TIC Peak Area of Phenolic Antioxidant Treatment/TIC Peak Area of Control * 100; peak area adjusted by internal standard (dodecane) 66 Figure 3-1: Theoretical isotopomeric distribution in pyrazine in CAMOLA studies if formed from intact two-carbon sugar fragments 79 Figure 3-2: Measured Isotopomers of Select LC/MS Analytes from Model B, D, and E 82 Figure 3-3: Proposed Composition of LC/MS Analytes [M-H] - from Model B 83 Figure 3-4: Chromatogram of LC/MS analytes (m/z 343, 347, 359, 361,371,633) from Model B 85 Figure 4-1: Chromatogram of analyte M.W. 348 (m/z 347 [M-1] - ) generated from models: (A) Glucose (Glu) + Glycine (Gly) + Epicatechin (EC); (B) Glyoxal (GO) + EC; (C) Erythrose (ERY) + EC. All reactions were conducted at pH 7, 125°C for 30 minutes 99 Figure 4-2: Chromatogram of analyte - M.W. 362 (m/z 361 [M-1] - ) generated from models: (A) Glucose (Glu) + Glycine (Gly) + Epicatechin (EC); (B) Methyl glyoxal (MGO) + EC; (C) Acetol (ACT) + EC; (D) Dihydroxy [...]... reactants include free amino acids, protein, ammonia, nucleic acids and peptides Although the type of amino acids can influence reaction kinetics, basic amino acid are known to be more reactive and tend to brown faster than the acidic amino acids in the following order: lysine> β-alanine > α-alanine > glutamic acid (9) The incorporation of various hetero-atoms (sulfur, nitrogen, oxygen) of the amino acid... pKa of glycine is 9 which means at neutral pH the concentration of the unprotonated amino group of glycine (effective reactant concentration) is less than 1%(16) However, in contrast to the reduced reactivity of amino group at lower pH, the reactivity of the carbonyl moiety under acidic conditions increases due to the interaction of protons with the oxygen molecule of the carbonyl group which increases... analogs, which include mono and dicarbonyl compounds formed mainly via retroaldolization of sugars are known to react with amino acids more rapidly than sugar itself (9) and are key intermediates of the Maillard reaction In addition of the influence of sugars on Maillard reaction kinetics, the amino acid component can have a dramatic impact on the flavor character of food systems(41) Typical food amine-type... divided into three stages: 1 The reaction pathways involving the sugar-amine condensation (initiation) reaction including the Amadori rearrangement, are included in stage one These reaction mechanisms are well understood; no browning (color formation) occurs during this stage 2 The second stage involves numerous reactions occurring simultaneously and therefore is highly complex involving the degradation of. .. generation of lipid oxidation products (i.e decadienal, etc) are important direct contributors to the flavor profiles of many thermally processed foods (2) Indirectly, lipid-derived carbonyl compounds can further combine with a amino group (from protein, peptides or amino acid) and participate in Maillard chemistry (3) The industrial definition of caramelization is a degradation reaction of sugars when... optimization of the reaction to produce the desired results is a challenging task Normally, studies evaluating the influence of these parameters are conducted on simple model systems comprising of amino acid(s) and sugar(s) as it is easier to manipulate and interpret the mechanisms of MRPs formation 8 The literature presented in the following section is a discussion of the influence of reaction conditions... is difficult to establish in the complex systems (35) 13 Influence of reactant type (sugar and amino acids) Carbonyl compounds are important precursors of the non-enzymatic browning reaction These can include reducing sugars, vitamin C, and low molecular weight compounds (aldehyde, ketones) formed via lipid oxidation or during the intermediates of Maillard reaction For reducing sugars, the acyclic form... 125°C for 30 minutes 106 Figure 4-6: 1H spectra for (A) Epicatechin at 23°C, (B) Epicatechinmethylglyoxal adduct Peak D at 23°C; (C) Epicatechin- methylglyoxal adduct peak D at -25°C in CD3OD In figure b and c, the two peaks at 1.9ppm are from acetic acid/ammonium acetate (residual from LC mobile phase buffer) 111 Figure 4-7: 2-D Plots of HMQC for (A) epicatechin and (B) epicatechinmethylglyoxal... temperatures are associated with the increased reactivity of the precursors (22) In a study conducted by Lea and Hannan (23) the loss of amino-nitrogen in a casein-glucose mix was reported to increase by 40,000-fold over the temperature range of 0° to 80°C The Q10 coefficient (x-fold increase in the reaction rate per 10oC), ranges from 2 to 8 for generation of the Maillard reaction products, which is... important for the complexity of the flavor perceived by Maillard- type reactions A key flavor pathway of the Maillard reaction, the Strecker degradation, involves the transformation of the amino acid side chain into an aldehyde reaction product which contains one less carbon atom; by this reaction methionine generates methional, sulfur containing flavor compound with a low threshold of detection Strecker aldehydes . from Maillard reaction 5 Factors influencing Maillard reaction kinetics 7 The influence of pH 8 The influence of temperature-time 9 Water Content 11 Influence of reactant type (sugar and amino. Beelman Professor of Food Science Donald Thompson Professor of Food Science Koushik Seetharaman Assistant Professor of Food Science A.Daniel Jones Professor of Chemistry, Biochemistry. confirmation. Preliminary experiments conducted on aqueous Maillard model systems consisting of 4 amino acids plus 2 hexose sugars indicated that the addition of epicatechin or epigallocatechin gallate