Study On Extraction,Separation,Purification And Anti-inflammatory Property Of Protaetia Brevitarsis Larvae Protein

Up to now,the domestic and foreign reports mainly focus on the prevention and control technology and feeding management of the white star golden tortoise,and the research on the utilization of its protein and other resources is rare.The main purpose of this study was to extract and purify the larval protein of albinia japonicus,to explore the anti-inflammatory components of the protein of albinia japonicus japonicus,and to study its anti-inflammatory effect and mechanism.This study can provide a new development direction for the full exploitation and utilization of this new insect resource,as well as a new thinking and scientific basis for the research on insect protein of this new insect resource.In this study,the whole insect of the 3rd instar larvae of the white star golden tortoise was used as the raw material.The main research results are as follows:?1?Selection of extraction method and optimization of extraction conditions for the larval protein of the white star beetle.The crude protein was extracted by salt solution,alkali solution,acid solution and alcohol solution,and the crude protein content was determined by kjeldahl method.The effects of different extraction methods,different extraction time,extraction temperature,extraction pH,solid-liquid ratio,and extraction NaOH concentration on the anti-inflammatory activity of the obtained proteins were investigated with the extraction rate,anti-inflammatory activity and cell survival rate as the indexes,and the optimal extraction method and conditions for the larva protein were determined.The results showed that at 55?,the concentration of NaOH in the extraction system was 1.5%,the solid-liquid ratio was 1:15?W:V?,and the extraction time was 1.5hours.The protein extracted by alkali solution with pH of 4 had a high extraction rate and the best anti-inflammatory activity.?2?LPS-induced mouse RAW264.7 macrophages were established as an inflammatory model to evaluate the anti-inflammatory effect of the extracted protein.The contents of NO in cell culture medium were determined by Griess method,and the contents of inflammatory cytokines IL-1?,TNF-?were determined by ELISA method.The anti-inflammatory activity of the larval protein and its fractions was investigated to determine the effective anti-inflammatory components in the larval protein.The results showed that the anti-inflammatory protein of the larva of the white-stellate golden turtle could significantly inhibit the release of inflammatory cytokines,and reduce the contents of NO and pro-inflammatory cytokines IL-1?,TNF-?lep in a concentration-dependent manner within a certain range.?3?After sds-page gel electrophoresis of anti-inflammatory protein,50KD and 10KD ultrafiltration tubes were selected according to the obtained bands,and the extracted protein was separated into three components PB₁,PB₂ and PB₃ according to the differences in size.The anti-inflammatory activity of PB₃ was detected respectively,and the optimal anti-inflammatory activity of PB₃ was found.Six fractions of PB_3-1,PB_3-2,PB_3-3,PB_3-4,PB_3-5-5 and PB_3-6-6 were obtained by Sephadex G-15 gel filtration chromatography,and their anti-inflammatory activities were detected respectively.The results showed that the fraction PB_3-4-4 had the best anti-inflammatory effect.?4?Real-time fluorescence quantitative PCR was used to detect the mRNA expression of RAW264.7 macrophages,and to explore the anti-inflammatory mechanism and signaling pathway of the protein.The results showed that the anti-inflammatory protein preconditioning of RAW264.7 macrophages significantly down-regulated the mRNA expression of the inflammatory mediators iNOS,and the pro-inflammatory cytokines TNF-?,IL-1?,and IL-6.Anti-inflammatory proteins influence the expression levels of RAW264.7 macrophage molecular signals JNK,P38,TRAF6,ERK2,I?B?,NF-?B P65,ERK1,and IKK?muck.It suggests that the anti-inflammatory effect of anti-inflammatory proteins is exerted by inhibiting the expression of relevant inflammatory regulators and pro-inflammatory factors,and can influence the expression of molecular signals at the gene level.