Preparation And Structural Identification Of Rana Bone Flesh Small Peptide And Its Immunomodulatory Effect On RAW264.7 Macrophages

Rana,also known as Snow clams,is abundant in the Changbai Mountain area in northeast China.The Rana,has very high medicinal value and nutritional value,which makes it the most economical kind of economic frog.Bone and flesh of rana are rich in protein and are a good source of protein.In this study,the first step was to use bone and flesh of rana as raw material to prepare Rana bones flesh polypeptide by enzymatic method,and to determine the technological conditions for the preparation of Rana bones flesh polypeptide by the degree of hydrolysis and the yield of the peptide.Then the Rana bones flesh polypeptide prepared under optimal conditions is separated and purified by ultrafiltration,gel chromatography and other techniques.Afterwards,the five components obtained by gel chromatography are collected and screened separately,and then the amino acid sequence is identified by a high-resolution liquid chromatography-mass spectrometry instrument,and a dominant sequence is selected.Finally,the isolated and purified Rana bones flesh small peptides were studied for immunomodulation.The main findings are as follows:1.Using pre-treated Rana bones flesh as raw materials,using the degree of hydrolysis and the yield of peptides as indicators,papain,alkaline protease,neutral protease,trypsin,pepsin were used to treat Rana bones flesh under their optimal conditions.The proteolysis test of Rana bones flesh revealed that papain was better than other proteases.Among them,the degree of hydrolysis of papain was 18.31 ±0.25%.2.The single factor experiment was used to examine the effects of pH,temperature,enzymatic hydrolysis time and enzyme addition(E / S)on the degree of hydrolysis of Rana bone flesh peptide yield,and orthogonal experiments were carried out with the degree of hydrolysis as an indicator to determine.The optimal process conditions for papain to hydrolyze the Rana bones and flesh are: pH 7,temperature55 ?,enzymolysis time 4h,enzyme addition amount 6%,and the degree of hydrolysis under this condition is 25.41 ± 0.22%.3.Centrifugal filtering the prepared Rana bones flesh(0.45?m filter membrane),and then separate them by ultrafiltration,and then pass through the ultrafiltrationmembranes of 5000 Da,3000Da,and 1000 Da in turn,and finally obtain a small peptide mixture of Rana bones flesh under 1000 Da.Afterwards,it was further separated and purified by gel chromatography technology,and the influence of sample volume and sample concentration on gel chromatography separation and purification was investigated.The optimal separation conditions were determined as follows:sample volume 4 mL,sample concentration 20 mg/mL.Separation and purification by gel chromatography can obtain five components,which are then enriched and freeze-dried.Through the RAW264.7 Macrophages proliferation experiment and macrophage phagocytosis of neutral red experiment,the third component has the best immune activity effect;the third component is subjected to liquid phase tandem mass spectrometry(LC-MS/MS)to determine its fragment structure And determine the sequence and collect secondary spectra.The dominant amino acid sequences obtained are: LVDFLR(Leu-Val-Asp-Phe-Leu-Arg),DLTDYLMK(Asp-Leu-Thr-Asp-Tyr-Leu-Met-Lys),SALEQTER(Ser-Ala-Leu-Glu-GlnThr-Glu-Arg),LYDQHLGK(Leu-Tyr-Asp-Gln-His-Leu-Gly-Lys),STHPHFVR(SerThr-His-Pro-His-Phe-Val-Arg).Their molecular weights are: 381.7Da,499.7Da,467.2Da,327.5Da,487.3Da.5.Immunomodulating the third component sample.Through experiments,it was found that the small peptide of Rana bones and flesh can promote cell proliferation in the range of 75-600?g / mL,and the cell activity decreased at a concentration of1200?g / mL.According to the RAW264.7 Macrophages proliferation experiment inhibited by cyclophosphamide(CTX),the small peptide of Rana bones and flesh has no obvious effect on relieving cyclophosphamide inhibition of cell proliferation.In the experiment of macrophage phagocytosis of neutral red,it was found that the cell phagocytosis ability increased with the increase of dose,and the effect was significant when the concentration of the small peptide of Rana bones and flesh was 150-600 ?g /ml(P <0.05).The effects of Rana bones and flesh small peptide on the secretion of TNF-? and IL-2 by LPS-activated RAW264.7 Macrophages were measured.The results showed that the small peptide of Rana bone and flesh can promote the secretion of TNF-? and IL-2 by RAW264.7 Macrophages,which in turn can promote immunity.