Study On The Effect Of Hemoglobin-induced Fatty Acid Oxidation On The Silver Carp Myofibrillar Protein Oxidation And Antioxidant Effect Of Rosmarinic Acid

Freshwater fish have become the main raw materials for the production of frozen surimi in China because of rich resource and low price.One major obstacle in using freshwater fish is that they are highly susceptible to oxidation because of the relatively high content of polyunsaturated fatty acids.There is no bleeding process during freshwater fish surimi production.The hemoglobin left in the fish mince after washing could induce lipid and protein oxidation,which is one of the important reasons for the deterioration of fish fillet and surimi-based products during their processing and storage conditions.There is a lack of in-depth understanding of the effects of hemoglobin and its induced lipid oxidation on silver carp surimi.Based on the understanding of the interaction between hemoglobin and fatty acid in this study,the changes of silver carp myofibrillar protein properties and the antioxidant effect of rosmarinic acid were investigated under the hemoglobin-induced fatty acid oxidative stress.1.Binding interaction between hemoglobin and fatty acids ? Oleic Acid(OA),?-Linolenic Acid(ALA)and Docosahexaenoic acid(DHA)was selected to study the binding interaction between fatty acid and hemoglobin(Hb).UV/Visible spectra showed the complex formation between Hb and fatty acids with increased in absorbance value.Increased fluorescence intensity indicated tertiary structural changes after binding between Hb and fatty acid.The negative values of ?G? for binding of HB to fatty acid in the fluorescence studies indicates the process to be spontaneous.Thermodynamic parameters of the Hb-fatty acids interaction in the complex reflected dominance of hydrophobic interactions.The binding ability increased with increase in the unsaturation level,and the binding constants were 7.30,8.89,9.46 for OA,ALA and DHA respectively.The binding interaction resulted in the changes of the secondary structure from ?-helix to ?-sheet and random coil.Docosahexaenoic acid showed a more binding affinity to hemoglobin which was indicated by thermodynamic parameters.2.Effect of Hemoglobin forms on Myofibrillar protein oxidation.Different forms of hemoglobin and fatty acids affected the silver carp myofibrillar protein oxidation during storage at 4 ?C.Deoxyhemoglobin(Deoxy Hb),methemoglobin(Met Hb)and ferrylhemoglobin(Ferryl Hb)in the presence of fatty acids significantly increased the lipid and protein oxidation(P < 0.05)as indicated by higher thiobarbituric acid reactive substances(TBARS)and carbonyl content,respectively.Moreover,Met Hb and Ferryl Hb significantly reduced the total sulfhydryl content of MP(P < 0.05).Fluorescence experiments proved the quenching of myofibrillar protein intrinsic fluorescence under oxidative stress,resulted the change in tertiary structure of MP.All forms of hemoglobin accelerated the protein oxidation throughout storage time,but Met Hb and Ferryl Hb were found to be more dominant to enhance lipid and protein oxidation.The results also suggested that the degree of unsaturation of fatty acids played a significant role.?-linolenic acid(ALA)and docosahexaenoic acid(DHA)promoted lipid and protein oxidation more rapidly than oleic acid(OA).Their oxidation rate reaction was dependent on the carbon chain length as well as double bond number.3.Influence of rosmarinic acid on MP functional properties under oxidative stress.The present study showed the dose-dependent retardation effect of rosmarinic acid(RA)on the oxidized myofibrillar protein.The incorporation of low(10 ?M/g protein)and middle(75 ?M/g protein)dose of rosmarinic acid significantly suppressed lipid oxidation,protein carbonyl formation and prevented the loss of total thiol(-SH)and free amine content(P < 0.05).However,high(250 ?M/g protein)concentration couldn't prevent the carbonyl formation,loss of total-SH and free amine content in oxidized MP.Analysis of tryptophan fluorescence indicated that RA induced the unfolding of MP structure in a dose-dependent manner.Polymers were formed along with marked attenuation of myosin heavy chain in oxidized MP with 250 ?M/g RA.Oxidized MP without rosmarinic acid had a higher gelling property as compared to the dose-dependent addition of rosmarinic acid.The high concentration(250 ?M/g protein)of rosmarinic acid couldn't improve the gelling properties of oxidized MP.Moreover,SDS-PAGE and microstructure results showed the interaction between oxidized MP and rosmarinic acid possibly by adduct formation and aggregation of MP with increasing concentration of rosmarinic acid.Hence,the dose of phenolic compounds played a significant role in MP functional and structural properties.