Isolation,purification And Activity Of Shrimp Powder Polypeptides From Antarctic Krill

Antarctic krill are small zooplankton living in the waters of Antarctica.Their huge population is of great significance for maintaining the ecological balance of the southern ocean.Because of the particularity of its living environment,Antarctic krill is rich in active substances,such as active polypeptides.Polypeptides are a class of compounds whose molecular weight is between protein and amino acid.Studies have shown that most polypeptides have antioxidant,antibacterial,blood pressure and lipid lowering effects to some extent,so the research on polypeptides has always been a hot topic in scientific research.In this study,krill powder was used as raw material to obtain polypeptide components by enzymatic hydrolysis of protease,and polypeptides with different molecular weight ranges were obtained by ultrafiltration technology.By comparing four antioxidant index comprehensive evaluation of the influence of molecular weight range of peptide antioxidant activity,by Oxford cup method to evaluate the influence of molecular weight range of peptide antimicrobial activity,and USES the high-speed counter-current chromatography for separation and purification,finally through the amino acid analysis to explore antioxidant activity and antibacterial activity and the relationship between the composition.The purpose of this study was to obtain polypeptides with optimal antioxidant activity and antimicrobial activity.Firstly,the degree of protein hydrolysis was taken as the evaluation basis,and the hydrolysis effect of neutral protease,papain and complex enzyme on the Antarctic krill powder was compared.Finally,complex enzyme was selected as the protease for the enzymatic hydrolysis of Antarctic krill powder.The digestion process with enzyme quantity is 2000 u/g,feed liquid than 1:10,enzymolysis temperature 50℃,pH 7.5,enzymolysis time 4 h.It will be destroyed after enzyme digestion liquid ultrafiltration separation intercept out five peptides,and the Mdetermination of antioxidant index of these peptides,the results show that the separation of peptides has a better removal of superoxide anion and DPPH free radical,hydroxyl free radical,and the ability of chelating Fe²⁺,of which the molecular weight range in 5¹0 KDa peptide antioxidant activity is best.When the concentration of polypeptide was 10mg/mL,the radical scavenging rate of superoxide anion reached 21.28%,DPPH radical scavenging rate was 86.52%,hydroxyl radical scavenging rate was 53.49%,and Fe2+chelation rate was 85.35%.However,the antibacterial study on the ultrafiltration separated Antarctic krill polypeptide showed that the polypeptide with the molecular weight range of 1³KDa had certain antibacterial effect,and when thepolyconcentration was 50mg/mL,it had obvious inhibition on the growth ofstaphylococcus aureus.For production,its bacteriostatic sphere size was 30.18mm,which had a weak inhibitory effect on the growth of vibrio parahaemolyticus,and its bacteriostatic sphere size was 8.41mm,but had no significant inhibitory effect onescherichia coli and bacillus subtilis.The polypeptides with molecular weight ranging from 5¹0KDa were named e-op,and were separated and purified by high speedcountercurrent chromatography.Chloroform-butanol-methanol-water=4:0.75:3:2as the two-phase solvent system,the lower phase is fixed phase and the upper phase is mobile phase.Reversal,speed 900r/min;Constant temperature water bath temperature is 25℃;The mobile phase flow rate was 1.5mL/min,and the sample concentration was20mg/mL.The DPPH radical scavenging rate of component P2 was the highest（90.33%）,and the Fe²⁺chelation rate and superoxide anion radical scavenging rate of component P3 were the highest（92.33%and 37.18%）.The hydroxyl radical scavenging rate of P5 component was the highest,70.11%.Amino acids of P2,P3 and P5 were analyzed,and the relationship between the types of amino acids with higher content and the clearance rate was compared,so as to infer that hydrophobic amino acids played a major role in scavenging DPPH free radicals and hydroxyl free radicals.Basic amino acids play a leading role in scavenging superoxide anion radicals and Fe²⁺chelation. Purity identification by chromatography showed that P2,P3 and P5 were all single peaks with good purification effect.The peptides with molecular weight ranging from 1³KDa were named E-BP and purified by high speed countercurrent chromatography.The final E-BP condition was as follows:methyl tert-butyl ether:acetonitrile:water=2:3:3 as a two-phase solvent system,with the lower phase as the stationary phase and the upper phase as the mobile phase.Reversal,speed 900r/min;Constant temperature water bath temperature is 25℃;The mobile phase flow rate was 1.0mL/min,and the injection concentration was 15mg/mL.Under these conditions,two components P1 and P2 were obtained,among which P1 had the best antibacterial activity.When the polypeptide concentration was50mg/mL,the antibacterial zone for staphylococcus aureus was 32.16mm,and the antibacterial zone for vibrio parahaemolyticus was 10.34mm.The amino acid analysis of P1 component indicated that Pro、Cys and Met were related to their antimicrobial activities.The purity of component P1 was determined to be a single peak with good purification effect.