The Establishment Of Extraction Methods Of Oyster Polypeptides And The Study On Its Immunomodulatory Effect On RAW264.7 Macrophages

Oysters are typical warm-water invertebrate bivalves,and it is a good source of trace elements,essential fatty acids,proteins,glycogen,amino acids and other nutrients.In addition to these nutrients,oysters have a variety of biological activities and medicinal values.They are medical and edible foods with health care effects.Especially oyster protein hydrolysates or oyster polypeptides prepared after protease hydrolysis can lower blood pressure and lower blood sugar,anti-tumor,anti-oxidation,reproductive health care,immunity enhancement and many other important biological activities.However,most of the oysters are currently sold fresh or simply produced and processed.Oyster resources have not been fully and effectively developed,resulting in a waste of resources.Therefore,in recent years,research scholars in the fields of nutrition and medicine have devoted their attention to the research and utilization of the biological activity of oysters,providing technical support for the utilization of high value-added active substances in oysters and the research and development of oyster polypeptides functional foods,which is the future of oysters.The development direction of the processing industry.Peptides with immunomodulatory effects is called immunologically active peptides,which have many advantages such as low molecular weight,low toxicity,low sensitivity,no side effects,and easy absorption by the human body.Therefore,the study of immunologically active peptides has become a research hotspot in related fields.In this paper,oysters were used as raw materials.The papain,flavor protease and animal protease were selected to extract three oyster polypeptides through enzymatic hydrolysis and ultrafiltration.To screening for the best hydrolase enzyme for extracting immunologically active peptides,preliminary evaluation of its immune activity on macrophages RAW264.7.Further,through single-factor and response surface experiments,the p H,amount of enzyme added,temperature,material-to-liquid ratio and time of enzymatic hydrolysis were optimized with hydrolysis as an indicator,and the best process for extracting peptides was established.The experimental results showed that compared to papain hydrolyzed oyster polypeptide(POP)and flavor protease hydrolyzed oyster polypeptide(FOP),animal protease hydrolyzed oyster polypeptide(AOP)can significantly increase the proliferation rate of RAW264.7 cells(P<0.01).The proliferation rate of AOP at a concentration of 500?g/m L was(110±1.775)%;In addition,it has been found that the three oyster peptides can activated macrophages at a concentration of 500?g/m L,and AOP can significantly enhance the phagocytosis of neutral red by macrophages.The order of impact on phagocytic activity is AOP>FOP>POP(P<0.01);At the same time,the release of macrophage NO from macrophages at 500?g/m L and50,000 ?g/m L of AOP enzymatic peptide was also the highest among the three.It rose in a concentration-dependent manner.The above results indicated that AOP had the best immune-enhancing activity.The condition for best extraction process of AOP are 3000U/g for enzyme amount,20% for material-to-liquid ratio,51? for enzymolysis temperature,4h for enzymolysis time and p H 7.6.The crude oyster polypeptide extraction was prepared by the best extraction process,and the nanofiltration concentrate was obtained by ultrafiltration separation.Then,the AOP freeze-dried powder was obtained by vacuum freeze-drying,and its physical and chemical properties were tested and analyzed.The experimental results showed that the UV maximum absorption peak of AOP Appearing at 220 nm,fourier infrared spectroscopy showed that AOP had the characteristic absorption peak of peptides;The relative molecular weight distribution of reversed-phase high performance liquid chromatography(RP-HPLC)was used to analyze the relative molecular weight distribution,and the results showed that peptides with molecular weight less than 1000 Da account for 54.3%;Liquid chromatography tandem-mass spectrometry(LC-MS/MS)identified a variety of peptide sequences.We searched and analyzed the first 16 sequences with relatively high content in AOP and found that 7 peptide sequences were all derived from copper-zinc superoxide Dismutase.this substance had been confirmed to be an enzyme involved in the body's antioxidant immune response and another peptide sequence derived from HSC70 protein,which had the function of improving the body's immunity and participating in immune regulation.Amino acid analysis results showed that hydrophobic amino acids accounted for 28.45% of hydrolyzed amino acids and 17.3% of free amino acids in AOP,respectively.In order to study the effects of different concentrations of AOP on cellular immune regulation,the effect of AOP on cell proliferation,phagocytosis of neutral red,NO release,and related immune factor gene expression(IL-6,i NOS,IL-1?,TNF-?)were analyzed.The results showed that AOP at two concentrations of250?g/m L and 1250?g/m L promoted the proliferation of RAW264.7 cells,enhanced their phagocytosis of neutral red,promoted the release of NO,and up-regulated i NOS m RNA,IL-6 m RNA,and IL-1? m RNA and TNF-? m RNA gene expression levels.The Western blot method was further used to study its immune regulation mechanism,and the results showed that AOP promoted the secretion of related immune cytokines by activating the p38 and JNK MAPK signaling pathways,thereby regulating immune activity.In summary,oyster polypeptide extraction process was established in this study,and obtained the oyster polypeptide AOP by ultrafiltration.Preliminary research showed that AOP enhanced the immune activity of RAW264.7 cells,which provided a great opportunity for the development and utilization of oyster functional food.