Analysis Of Black Bean Antioxidant Peptide Inhibiting Oil Oxidation

Oil oxidation leads to deterioration of the quality of edible oil,loss of essential fatty acids and formation of harmful substances,which seriously affect the nutrition and safety of oil food.Adding fat-soluble antioxidants is the most direct and effective way to control the oxidation of fats and oily foods.Currently,TBHQ,BHT and other chemical synthesis antioxidants most commonly used in industry have certain safety risks,while lipophilic natural polyphenols can promote oxidation in addition to limited types.Antioxidant peptides can scavenge free radicals and inhibit lipid oxidation,but the mechanism of anti-lipid oxidation is still unclear.Therefore,black bean antioxidant peptides as the research object,through the construction of fingerprint,explored the relationship between structure and antioxidant function,and clarified the mechanism of black bean antioxidant peptides inhibiting lipid oxidation through the change law of edible oil system and simulated oil system.In order to offer the application basis for the substitution of synthetic antioxidants,and to provide a scientific basis and theoretical reference for the application in complex oil system.The main results are as follows:Firstly,the protein hydrolysates of black bean were prepared,and the lipid-soluble peptides were extracted by ultrasonic-assisted solvent method,their antioxidant activities and structural characteristics were evaluated.Response surface analysis showed that the optimal hydrolysis conditions of bromelain were as follows: hydrolysis time 4.2 h,p H 7.0,enzyme concentration 1.8%,temperature 55°C,and the inhibition rates of primary and secondary oxidation products of lipid-soluble peptides were 7.75 g/L and 5.12 g/L,respectively.The structure of the lipid-soluble peptide changed significantly,mainly as follows: the CMC value of the extracted lipid-soluble protein was smaller,and the ultraviolet absorption spectrum showed a strong absorption peak near 280 nm;he increase of fluorescence absorption peak indicated that the extracted peptide had stronger hydrophobicity.Compared with the hydrolysates,resulting in the formation of new conformations with high β-type which may be beneficial to antioxidant capacity.Secondly,HPLC fingerprint of lipid-soluble black bean antioxidant peptideswas constructed to identify the activity.Total 30 common peaks were analyzed in the characteristic fingerprint.The similarity analysis of 11 groups of hydrolysates was carried out,and the similarity among each group was between 0.961 and 0.999.Grey correlation analysis was used to collect 7 components of main characteristic peaks,and their antioxidant activities were measured,among which characteristic peaks 1 and 8 showed the strongest antioxidant activity.The inhibition rates of primary oxidation products were 55.34±0.57,41.15±3.34and30.22±7.38,29.82±2.00,respectively.After the analysis of LC-MS/MS,there were 57 and 44 peptides identified from characteristic peaks 1 and 8 with molecular weights ranging from258.65 Da to 1443.70 Da.Ten peptides with low molecular weight were selected,including MFVPHY,ILF,LPSVL,RIRLL,YPFVVN,PAGTTYYVV,PFLLG,FPPPKVI,FPPPHVI and FPAI.The inhibition rate of primary products of the 10 peptides was from 7.17% to32.09%,and the inhibition rate of the secondary products ranged from 11.24% to 41.15%.Then sunflower seed oil was selected as the representative of edible oils to explore the effect of antioxidant peptides on the oxidation stability of sunflower seed oil.The results showed that both ILF and LPSVL could effectively reduce the acid value and peroxide value of sunflower oil.The ESR results showed that the antioxidant peptides with different sequences can reduce the signal intensity of alkyl radicals and alkane peroxyl radicals in sunflower oil,which has a good effect on inhibiting the generation of free radicals in sunflower oil.Among the heated volatile products,LPSVL showed a better effect on the production of(E,E)-2,4-decadienal,while for hexanal,(Z)-2-heptenal and(E)-2-octenal,ILF showedmore obvious inhibition effect.Under transmission electron microscope,most oil droplets with black bean antioxidant peptides still maintained round structure,which significantly delayed the oxidation process compared with fresh sunflower oil.Raman results showed that the molecular conformation of ILF and LPSVL did not change during the heating and oxidation process.Finally,a simulated oil system represented by methyl linoleate was constructed to analyze the oxidation mechanism of black bean antioxidant peptides.The oxidation products in the oxidation system were analyzed qualitatively and quantitatively by NMR.It was found that ILF and LPSVL could effectively inhibit the formation of oxidation products such as N-alkaldehydes and primary alcohols.ESR was used to detect the change of the total number of spins during the oxidation of methyl linoleate.The inhibition effect of ILF and LPSVL samples on free radicals was greater than that of the samples without peptide,indicating that ILF had a certain antioxidant effect,and the antioxidant effect of ILF was better.It was found that the contents of ILF and LPSVL decreased in the process of the heating and oxidation.The comparison of mass spectrograms at 0 h and 15 h showed that ILF lost isoleucine during heating,while LPSVL lost leucine and proline.