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Lipid co-oxidation of proteins: Reaction pathways, products, and consequences in foods Faculty PI: K.M. Schaich Students: Y. Dong W.I. Wan Zunair Lipid oxidation is not always what it seems Lipids oxidize and produce free radicals, hydroperoxides, aldehydes, and epoxides that are measured to follow the process and determine extent of reaction. BUT… • Each of these intermediates and products also react with proteins to broadcast damage while also eliminating some footprints of lipid oxidation. Lipid oxidation thus can appear to be low when it is actually quite rapid and evidence of oxidation is clearly visible in browning and texture changes. Hypothesis and Position Statement All intermediates and products of lipid oxidation react with and damage proteins, but they do so differentially, each with specific consequences. By intercepting lipid radical chain reactions and removing reactive lipid products, protein co-oxidations may make lipid oxidation seem to slow while transferring oxidation potential to different molecular sites and change observable system properties. Some consequences of protein oxidation have been misattributed to lipid oxidation, particularly flavors. Thus, co-oxidations must be accounted for when analyzing extent of lipid oxidation in any food system Limiting co-oxidation of proteins (and other biomolecules) must be included in all strategies for stabilizing food quality. Project Goals Determine the absolute and sequential modifications in protein properties and chemistry induced by four classes of lipid oxidation products -- free radicals, hydroperoxides, epoxides, and carbonyl (mostly aldehyde) secondary products Differentiate the types of reactions and patterns of damage induced by each oxidant Show where such reactions occur in some specific food applications Experimental Approach 1. Model systems (lyophilized and fluid emulsions oxidizing Me Linoleate EPR – free radicals 13C NMR – struct and funct grp MLOOH + proteins PAGE – xlkg and fragmentn. ML aldehydes ML epoxides Ab rxn (protein oxidation) Protease digestion LC-MS -- ID modified amino acids 2. Simultaneously monitor changes in oxidizing lipids to determine how the footprint of lipid oxidation changes with protein co- oxidation. 3. Coordinate model system studies with analyses of protein and lipid oxidation in representative foods (e.g. dairy, meats, grains/bread products). Studies with tortilla chips Baked Fried Thermal damage Thermal + lipid oxidation Studies with tortilla chips Extraction a. Non-reducing 1.5% SDS, 0.0125 M Borate, pH = 10 b. Reducing Above + 2% 2-mercaptoethanol Chemical Protein solubility (Bradford) analyses SH/S-S Protein fragmentation and crosslinking PAGE 12.5% and 15% resolving gel, 4% stacking gel Ab rxns Identification of proteins with C=O products Observations Lipids reduce protein solubility S-S doesn’t alter solubility in baked chips Extractable Protein 0.6 Other 0.5 S-S 0.4 ug/ul 0.3 0.2 0.1 0 baked, baked, non- fried, fried, non- reducing reducing reducing reducing Solubility reduced by both S-S and other mechanisms in fried chips. Lipids alter dye-binding amino acids Reducing Non-Reducing Fried Baked Fried Baked −L +L −L +L St −L +L −L +L R vs NR suggest more S-S crosslinking than was shown in Major loss Bradford assay of dye binding to proteins in fried chips Proteins extracted w/ − L: lipid removed + L: lipid present CBB binding sites (His, Arg, Lys, Trp) are major targets for lipid radical attack Form stable radicals with oxidizing lipids Georgiou et al, Anal. Bioanal. Chem. 2008 Silver staining of proteins Heat alone ↑ S-S crosslinking, Lipids crosslink by other mechanisms Reducing Non-Reducing Polymeric Fried Baked Fried Baked fractions, −L +L −L +L −L +L −L +L Lost HIGH mw probably with fractions bound lipid potentially bound to lipids β-MCE eliminates S-S High, Mod and crosslinking, but low mol wt glutelin fractions fractions still missing in missing fried chips Zeins Antibodies reveal extensive oxidation (R-CHO) in both baked and fried chips Reducing Non-Reducing C F C B St F C B C Heat alone generates Glutelins aldehydes in all protein fractions. Other mechanisms must be active in loss Zeins A of dye binding and B 25 kd specific fractions. RSP-1 C 15 kd D C: control F: Fried B: Baked Further characterization of protein modifications • EPR for free radicals • PAGE variations for further differentiation – 2D, selective staining for lipid-protein and glycoprotein adducts • Analysis of specific lipid oxidation products • Dye binding to detect lipid adducts to proteins • Fluorescence analysis for Schiff bases (RCHO and epoxide adducts) • Immobilized enzyme hydrolysis to amino acids, LC-MS analysis of modified amino acids • Storage studies – following protein changes over time, 40 °C • Coordinate with model system studies Peanut butter Studies of Products in Controlled Release Packaging with Antioxidants Quercetin + tocopherols EVOH EVA EVA Control 40 °C 60 °C Are these changes from lipid oxidation -- OR -- from protein oxidation? Processed Cheese Spread Studies of Products in Controlled Release Packaging with Antioxidants No N2 N2 N2 + tocopherols 40 °C Low viscosity Increased Normal consistency Gritty cohesiveness and flavor Off-flavors Less gritty Smooth Are these changes from lipid oxidation -- OR -- from protein or starch oxidation? Lipid co-oxidations → extensive degradation of food quality • Texture -- both ↑and ↓ viscosity, so both crosslinking and fragmentation both ↑and ↓ cohesion grittiness • Color changes – browning and pigment bleaching • Flavor changes – definitely not all classical lipox • Loss of essential amino acids These damage processes must be elucidated to develop more effective stabilization strategies in formulation, processing, and packaging. Research plans now reverse initial expectations 1. Study protein degradation in these real foods, identify key protein modifications responsible for quality changes over time. 2. Supplement with studies of maize, milk, and peanut proteins in model systems with actively oxidizing lipids (free radicals) isolated LOOH lipid aldehydes lipid epoxides to determine damage kinetics and products associated with different lipid species. 3. Use model system results to elucidate co-oxidation pathways active in foods. Thank you for your attention… Any questions?
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