Isolation,Purification And Biological Activity Of Oyster Protein Peptides

Oyster is the world’s largest marine shellfish,and is cultivated in all coastal provinces of China.Because it is rich in protein and various nutrients,it has high nutritional value and development and utilization prospects.In this paper,oyster protein peptide was prepared from oysters from Dongshan Island in Fujian Province by enzymatic hydrolysis of oysters.The bioactivity and structure-activity relationship of oyster protein peptides were investigated by separation and purification and structural identification.The molecular weight of proteases and the molecular weight of protein peptides were analyzed for oysters.The biological activity of protein peptides provides a reference for the development of oyster protein peptides.The main findings are as follows:1.Using hydrolysis degree as an indicator,the conditions of alkaline protease,neutral protease,trypsin and papain enzymatically hydrolyzed oyster protein were optimized by response surface experiment,and the enzymatic hydrolysis conditions of four proteases for enzymatic hydrolysis of oyster protein were obtained:alkali The enzymatic hydrolysis conditions of the protease are pH 9,temperature 55℃,ratio of material to liquid 2:1 g/mL,enzyme dosage 4000 U/g;neutral protease hydrolysis conditions pH 7.5,temperature 50℃,ratio of material to liquid 2:1 g/mL,enzyme dosage 3000 U/g;trypsin hydrolysis conditions The pH is 8,the temperature is 50℃,the ratio of material to liquid is 2:1 g/mL,and the enzyme dosage is 4000 U/g;the enzymatic hydrolysis conditions of papain are pH 7.5,temperature 60℃,ratio of material to liquid 3:1 g/mL,plus The amount of enzyme is 4000 U/g.Under the above conditions,the hydrolysis degree of oyster protein was 30.96±0.59%,24.43±0.47%,25.07±0.62%and 18.29±0.35%,respectively.2.Hydrolyzed protein peptide products of four proteases were prepared by membrane separation:alkaline protease hydrolysate(OPP-A),neutral protease hydrolysate(OPP-N),trypsin hydrolysate(OPP-T)The papain hydrolysate(OPP-P)was further separated and purified using Sephadex G-15,and four macromolecular peptide components were collected according to the molecular weight(the molecular weight of the components was mainly distributed in 1000 Da-3000 Da)OPP-A1,OPP-N1,OPP-T1,OPP-P1 and four small molecular peptide components(the molecular weight of the components is mainly distributed below 1000 Da)OPP-A2,OPP-N2,OPP-T2,OPP-P2,and The small molecule peptide component was subjected to amino acid sequence analysis to obtain the corresponding peptide sequence,respectively.3.Oyster protein peptide has good antioxidant activity and certain blood pressure lo wering activity,and hypoglycemic activity is not obvious.The effects of molecular weigh t and enzyme species on the antioxidant activity of oyster protein peptides were studied by using three methods of in vitro antioxidant evaluation such as DPPH scavenging,AB TS free radical activity and inhibition of linoleic acid autooxidation.It was found that th e small peptide components were better.Antioxidant activity,the type of enzyme also sig nificantly affects the antioxidant properties of enzymatic hydrolysates:oyster protein pepti de scavenging DPPH·,inhibiting linoleic acid autooxidation and clearing ABTS free radic al activity in order of order:OPP-T2>OPP-P2>OPP-N2>OPP-A2>OPP-T1>OPP-T>OPP-P 1>OPP-P>OPP-A>OPP-N1>OPP-A1>OPP-N,OPP-P2>OPP-T2>OPP-N2>OPP-A2>OPP-P>OPP-P1>OPP-N>OPP-T>OPP-A>OPP-N1>OPP-A1>OPP-T1,OPP-A2>OPP-P2>OPP-A>OP P-P>OPP-N2>OPP-T2>OPP-A1>OPP-N>OPP-P1>OPP-T>OPP-T1>OPP-N1;small peptide c omponent scavenges DPPH free radicals and inhibits linoleic acid The auto-oxidation acti vity was significantly stronger than that of the macromolecular peptide component(p<0.05).Among the various enzymatic hydrolysates,the trypsin hydrolysate scavenged DPPH activity most,the neutral protease hydrolysate scavenged DPPH activity was the weakest;papain hydrolysate inhibited Linoleic acid has the strongest ability to auto-oxidize(p<0.05);the small peptide component has relative Good ABTS radical scavenging activity,va rious kinds of hydrolyzate,the alkaline protease and papain activity stronger product.The oyster protein peptide sample has hypotensive activity,and the macromolecular peptide c omponent activity is relatively stronger.At the sample concentration of 10 mg/mL,the A CE inhibitor inhibition rates of OPP-A1 and OPP-P1 are 56.38%and 49.62%,respectively.For other components.The hypoglycemic activity of oyster protein peptide was not o bvious.Only OPP-A and OPP-A2 showed lower hypoglycemic activity when the sample concentration reached 10 mg/mL,and the α-glucosidase inhibition rate was only about 23%.4.Oyster protein peptide has protective effect on H2O2 oxidative damage of LO2 cells.Both molecular weight and enzyme species affect the antioxidant activity of protein peptide cells.Oyster protein peptide can significantly reduce the amount of MDA production in LO2 cells with H2O2 oxidative damage.Except for OPP-A1 and OPP-N1,the MDA production of LO2 cells in each purified sample treatment group was significantly lower than that of Vc treatment group;the same enzyme water product In the middle,the MDL activity of the small molecular peptide component is stronger than that of the macromolecular component,and the difference in activity between the enzymatic hydrolyzed products of different enzymes is not significant.The oyster protein peptide significantly reduced the LDH activity in the supernatant of LO2 cells with H2O2 oxidative damage(p<0.05).The enzyme activity level of each sample treatment group was close to that of Vc group,and the activity difference between oyster protein peptide samples was not significant.OPP-T,OPP-P,OPP-N1,OPP-T1,OPP-P1,OPP-A2,OPP-N2,and OPP-T2 can significantly increase the level of GSH-Px activity in LO2 cells with H2O2 oxidative damage(p<0.05),except for papain,the activity of small peptide components of other three hydrolysates is stronger than that of macromolecules;OPP-T,OPP-P1,OPP-A2 activities are equivalent to Vc(p>0.05),OPP-T2 activity was stronger than VC(p<0.05),and the enzymatic hydrolysate of trypsin showed better GSH-Px activity than other enzymes.OPP-T,OPP-A1,OPP-T1,OPP-P1,OPP-A2,OPP-N2,OPP-T2,OPP-P2 can significantly increase cell CAT activity(p<0.05),of which OPP-A2,OPP-T2,OPP-P2 activity was comparable to Vc(p>0.05),OPP-N2 activity was stronger than Vc(p<0.05),small molecule components were relatively more active,and trypsin hydrolysate products showed significant CAT enzyme.Live enhances activity.Oyster protein peptide can significantly reduce SOD activity in LO2 cells with H2O2 oxidative damage(p<0.05).OPP-T and OPP-A1 activities are equivalent to Vc,OPP-N,OPP-P,OPP-T1,OPP-P1.The activities of OPP-T2 and OPP-P2 were stronger than those of Vc(p<0.05),indicating that papain and trypsin hydrolysate had stronger activity in increasing SOD activity,while molecular weight had no significant effect on increasing SOD activity.5.Oyster protein peptide has lipid-lowering activity.Oyster protein peptide can reduce the TC content in LO2 high-fat cells,and there is a certain dose-effect relationship of TC activity.When the sample concentration is 0.5mg/mL,the TC content of the cells in each sample treatment group and the control group are extremely significant.(p<0.01),OPP-P,OPP-P1,OPP-P2 decreased TC activity was significantly lower than other protein peptide samples,indicating that papain hydrolysate product TC activity is relatively weak.When the concentration of oyster protein peptide was 0.1 mg/mL,only OPP-T2 and OPP-P2 could significantly reduce the TG content in LO2 high-fat cells(p<0.05).When the concentration increased to 0.5 mg/mL,OPP-A2,OPP-N2,OPP-P2 decreased TG activity to a significant level(p<0.05),OPP-T2 reached a very significant level(p<0.01),the effect was close to simvastatin(Simvastain),only small peptide components It has the activity of decreasing TG,while the trypsin hydrolysate has the strongest TG activity.