Study On The Excavation And Activity Of Antimicrobial Peptides From Antarctic Krill

Antarctic krill mainly lives in the waters of Antarctica,with large biological resources,rich nutrients and high protein content.However,at present,the processed products are mainly Antarctic krill oil.The degreased Antarctic krill powder,which is rich in protein after oil extraction,has not been effectively used and is only used as animal feed and other low value-added products.Currently,the abuse of antibiotics leads to bacterial resistance,and the development speed of new drugs is far lower than the rate at which bacteria develop resistance.Research has shown that antimicrobial peptides have broad-spectrum antibacterial properties and low biological toxicity,and are not easy to induce bacterial resistance,providing new ideas and methods for solving the problem of antibiotic resistance.Therefore,this study took Antarctic krill as the experimental object,guided by the antimicrobial activity,and used two methods of enzymolysis separation and purification,peptidomics and chemical synthesis to mine antimicrobial peptides,and evaluated their safety.While realizing the high value utilization of Antarctic krill,it also explored a method of efficient mining of Antarctic krill antimicrobial peptides.The main research results obtained are as follows:1.Firstly,the basic composition of defatted Antarctic krill powder was analyzed,and its protein content was 59.66 ± 1.18%;Nine kinds of proteases were used to hydrolyze defatted Antarctic krill powder,and E.coli was used as the test strain;It was found that both animal protease hydrolysate and flavor protease hydrolysate had antibacterial activity,and the former had the best effect.After ultrafiltration,gel chromatography column separation and high performance liquid phase purification,the single peak D10 has the best antibacterial effect;Its amino acid sequence was determined to be NLYIIAVQGIKGR(NR-13)through primary structure analysis.Through chemical solid phase synthesis,the purity of antibacterial peptide can reach95.33%.Through activity verification,NR-13 can inhibit the growth of E.coli and B.megaterium,and the minimum inhibitory concentration(MIC)of both E.coli and B.megaterium is more than 512 μg/m L.The hemolytic activity and cytotoxicity experiments showed that NR-13 was present in 1×MIC,the hemolysis rate was 47.71±3.44%,indicating hemolysis;It has no cytotoxicity to human fibroblasts and human ovarian cancer cells and can promote cell proliferation.2.Based on peptidomics and bioinformatics,the antimicrobial peptides in the whole Antarctic krill and its enzymatic hydrolysate were screened and identified.The whole Antarctic krill protein and the protein in animal protease hydrolysate and flavor protease hydrolysate were extracted.The concentration and content of Antarctic krill protein were 19.28±2.92 μg/μL、38175.28±1.95 μg;The protein concentration and content in the enzymatic hydrolysate of Antarctic krill were 0.57±0.06 μg/μL、97.35±0.02 μg(animal protease);0.63 ± 0.01 μg/μL 、 107.69 ± 0.01 μg(flavor protease).Extract and purify peptides based on this,and perform LC-MS/MS detection on the purified peptides.According to the peptide sample data generated by the mass spectrometer,the whole Antarctic krill protein was identified using the Andromeda engine integrated by Max Quant,and 582 peptides were identified;The protein in the hydrolysate of Antarctic krill was identified by Mascot search,and 41 polypeptides(animal protease)and 197 polypeptides(flavor protease)were identified.Three online database,namely APD3 database,CAMPR3 database and Anti BP2 database,were used to predict and analyze 820 peptides obtained.The prediction results showed that there were 21 suspected antimicrobial peptides in the whole shrimp of Antarctic krill,and 4suspected antimicrobial peptides(one of which was a known antimicrobial peptide)in the enzymatic hydrolysate,and these four peptides were all from the flavor protease hydrolysate.20 suspected antimicrobial peptides were synthesized using chemical solid-phase synthesis with a purity of ≥95%,and all were identified as target peptides by mass spectrometry.Four highly active antimicrobial peptides ER-13,SR-18,SR-17,and SL-16 were obtained through antibacterial activity screening,with a minimum inhibitory concentration of 8 μg/m L、64 μg/m L、128 μg/m L、256 μg/m L,respectively.The four peptides are all from the whole Antarctic krill protein,and have broad-spectrum bacteriostasis.The safety evaluation of four antimicrobial peptides showed that SR-18 and ER-13 were found to be effective in 1×MIC no hemolysis.Using human fibroblasts and ovarian cancer cells as experimental cells,SR-17 and ER-13 promote the proliferation of human fibroblasts,while SL-16 and ER-13 promote the proliferation of human ovarian cancer cells during 1×MIC.