Elucidation of pro-oxidant mechanisms of the bioactive polyphenol, (-)-epigallocatechin gallate, in food emulsions
Open Access
- Author:
- Zhou, Lisa
- Graduate Program:
- Food Science
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 07, 2012
- Committee Members:
- Ryan John Elias, Dissertation Advisor/Co-Advisor
Joshua D Lambert, Committee Member
John Coupland, Committee Member
Ming Tien, Committee Member - Keywords:
- Food oil-in-water emulsions
polyphenols
(−)-epigallocatechin-3-gallate
EGCG
hydrogen peroxide
protein oxidation - Abstract:
- Polyphenols are widely regarded as antioxidants, due in large part to their free radical scavenging activities and their ability to disrupt radical chain propagation. Besides the anticipated antioxidant activity in foods, polyphenols are also attractive ingredients for their potential health benefits after consumption. However, recent studies have demonstrated that the oxidation of some phenolic compounds result in the generation of hydrogen peroxide (H2O2), a precursor for the highly reactive hydroxyl radical (•OH), which can potentially compromise the oxidative stability of foods and beverages. Due to conflicting results regarding phenolic effects on lipid oxidation in the literature, I surveyed the effects of pH (2-7) and phenolic concentration (0-500 M) on the generation of lipid oxidation markers in flaxseed emulsions to determine conditions for future oxidation studies. (-)-Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, was used as the model polyphenol in all oxidation studies. A low pH (2-4) resulted in pro-oxidant activity, while higher pH values (5-7) resulted in a net antioxidant activity. Higher EGCG concentrations (100, 500 M) also showed the largest changes in lipid oxidation markers compared to the no EGCG controls after 96 h. Disrupting redox cycling by metal chelation may be a viable solution for controlling metal-catalyzed phenolic oxidation, since redox cycling of transition metals is essential for the metal to exert a catalytic function. I added the iron chelators, EDTA and 2,2- bipyridine (BPY) to hexadecane emulsions containing EGCG (400 M) and Fe3+ (25 M) at pH 3 and pH 7 to determine their effects on EGCG oxidation and the resulting ROS produced in the absence of an oxidatively labile lipid. I then repeated the studies in flaxseed emulsions to determine the effect on lipid oxidation. At neutral pH, EDTA accelerated EGCG oxidation and hydroxyl radical (•OH) formation. BPY treatment slightly slowed EGCG oxidation compared to EGCG-only samples, though both treatments showed H2O2 accumulation and very slow •OH radical generation. Even with rapid •OH radical formation in EDTA treatments, all EGCG-containing samples showed antioxidant activity at neutral pH. Conversely, at acidic pH, EDTA strongly inhibited EGCG oxidation and •OH radical formation resulting in strong antioxidant activity in preventing flaxseed emulsion oxidation, while EGCG readily oxidized in BPY and EGCG-only treatments, leading to rapid •OH radical generation, and increased lipid oxidation in flaxseed emulsions. Though EDTA showed promising results by preventing phenolic and lipid oxidation at acidic pH, EDTA may cause problems at neutral pH by accelerating phenolic loss even if no increased lipid oxidation occurs. Proteins may be another viable solution in inhibiting increased lipid oxidation by scavenging the H2O2 generated from metal-catalyzed phenolic oxidation, prior to •OH radical formation. We show that casein (CAS) and whey protein isolate (WPI) readily scavenge H2O2 in emulsions. However, differences in peroxide scavenging activity were dependent on more than total methionine (Met) and cysteine (Cys) content; Met and Cys being the two amino acids capable of directly reacting with peroxides. A major factor for the variable peroxide scavenging activity observed in proteins may be due to differences in Met and Cys accessibility. To further examine this, I effectively blocked Met and Cys residues in proteins by reacting them with peroxides and collecting the oxidized proteins. CAS and -lactoglobulin (BLG) were pre-treated with the peroxides, t-butyl hydroperoxide (TBHP) or H2O2. TBHP is a bulky peroxide whose accessibility to buried Met and Cys residues is sterically hindered; H2O2, on the other hand, has been shown to be able to access buried residues. Using the prepared proteins, I proceeded to examine the proteins’ ability to scavenge exogenously added H2O2 and improve EGCG stability. In Tween-stabilized hexadecane emulsions at neutral pH, CAS treatments showed decreasing peroxide scavenging activity and EGCG stability as peroxide pre-treatments decreased solvent accessible Met concentration. Similar to WPI, BLG displayed much weaker peroxide scavenging activity and lower EGCG stability compared to CAS, since the Met residues were not readily accessible to the ROS present. However, in SDS-stabilized emulsions, BLG readily scavenged H2O2 and showed increased EGCG stability compared to CAS. As SDS denatured BLG, buried Cys residues became accessible to oxidation. When I repeated experiments in flaxseed emulsions at pH 3, BLG was capable of preventing increased lipid oxidation from the addition of high (400 M) and low (100 M) EGCG concentrations in Tween-stabilized flaxseed emulsions. The addition of BLG-CTRL-1% or BLG-HOOH-1% maintained lipid oxidation markers in EGCG treatments well below the control. However, the addition of BLG-HOOH-0.2% with EGCG (400 M) was unable to keep lipid oxidation markers in EGCG treatments below the control for the full duration of the 8-day study. This suggests that the presence of solvent accessible Met residues is important to BLG’s protective effect. At the low EGCG concentration (100 M), both BLG-0.2% treatments showed higher lipid oxidation markers by the end of the study, signifying phenolic concentration also impacts the effects on lipid oxidation. Overall, I showed that EGCG readily oxidized to generate ROS in emulsions with a low pH being a strong promoter of increased lipid oxidation due to metal-catalyzed phenolic oxidation. Though all my studies were performed using the polyphenol EGCG, many of the mechanistic investigation, such as the effects of metal chelators on oxidation, may be extrapolated to other phenolics that undergo metal-catalyzed oxidation. Further work is necessary to elucidate the impact of the different reactions resulting from metal-catalyzed phenolic oxidation, such as •OH radical formation and Fe reduction, on inducing lipid oxidation. However, controlling metal redox cycling with chelators holds promise as a potential strategy for mitigating the pro-oxidant activity of phenolics in foods. The quenching of EGCG-generated H2O2 by solvent accessible Met and Cys residues in proteins is also a viable strategy for stabilizing lipids in foods. Though uncontrolled metal-catalyzed phenolic oxidation may yield many problems, my research shows that phenolics may be safely incorporated into food emulsions without negative consequences on lipid stability by controlling key phenolic pro-oxidant mechanisms of the polyphenol, such as transition metal reduction and •OH radical formation.