Isolation,Purification And Structural Research Of Fluorinated Peptides From Antarctic Krill(Euphausia Superba)

Antarctic krill(Euphausia superba)is the carrier of the development of emerging industries in the 21st century.High fluoride abnormalities in muscles are the key to limiting the development and utilization of their food.Because organisms have different absorption and bioavailability of different forms of fluorine,the existing researches mainly focus on the quantitative analysis of fluorine,and the morphological analysis of fluorine is still in the preliminary exploration stage.The binding state of Antarctic krill muscle to fluorine is also in the hypothesis stage.The mechanism of fluorine formation in Antarctic krill is still unclear.This study is based on the combination of various forms of fluoride in the muscle of Antarctic krill.The Antarctic krill muscle is used as the experimental raw material.The response surface method was used to optimize the trypsin enzymatic hydrolysis process to extract protein from the muscle.Then,using the fluorine content of the peptide as an indicator,a series of techniques such as ultrafiltration,Sephadex G-25 Sephadex chromatography and high performance liquid chromatography were used to separate and purify the Antarctic krill protein peptide.The purified fluoropeptide is subjected to molecular weight determination,amino acid analysis,and protein identification.The aim was to develop a new method for isolating the fluoride protein of Antarctic krill,and to further analyze its binding form and structure through protein identification and to explain the combination of Antarctic krill protein and fluorine,and lay a foundation for the future realization of high value utilization of Antarctic krill protein.,the specific research content:1.The study on enzymatic hydrolysis of Antarctic krill proteinTaking the amino nitrogen content in the antarctic krill hydrolysate as an indicator,the optimum enzymatic hydrolysis of trypsin,subtilisin,papain,pepsin,alkaline protease and flavor protease was determined by formaldehyde potentiometric titration.Under the condition of the proteolytic effect of Antarctic krill,the best biological enzyme for enzymatic hydrolysis of Antarctic krill protein was screened out.Based on the single factor test,Amino nitrogen content in enzymatic hydrolysate was used as the response value,and the temperature,time and pH of the enzymatic hydrolysis were selected as independent variables.The three-factor and three-level Box-Bohnken response surface analysis method was used to optimize the enzymatic hydrolysis process.The results showed that trypsin was the ideal biological enzyme for enzymatic hydrolysis of Antarctic krill protein;the response surface analysis method was used to establish a quadratic multiple regression equation:Y=51.07-0.83A+1.72B-2.06C+0.25AC+1.15BC-3.06A~2-2.77B~2-3.69C~2,the model p<0.0001,and the missing term p>0.05,indicating that the model is extremely significant and stable;the best conditions obtained by model analysis are:enzymatic hydrolysis temperature 44.48?,enzymatic hydrolysis time 8.52 h,pH 7.88,considering the practical application feasibility,we obtained the amino nitrogen content of 50.96±1.07 mg/g at the enzymatic hydrolysis temperature of 45.0?,hydrolysis time of 8.5 h and pH 7.9,which is basically consistent with the theoretical prediction value of 51.57 mg/g.2.Isolation and purification of fluoropeptides after enzymatic hydrolysis of Antarctic krillThe enzymatically hydrolyzed peptide components were analyzed by SDS-PAGE gel electrophoresis.According to the analysis results,the ultrafiltration membrane separation device with suitable molecular weight was screened for primary purification of molecular weight desalination and impurity removal,and the filtrate was freeze-dried.Crude extract of peptide.The crude peptide extract was further separated and purified by Sephadex G-25 Sephadex chromatography and high performance liquid chromatography(HPLC).The purified polypeptide solution was freeze-dried in a freeze dryer for 24 h to obtain polypeptides of different molecular weights.The content of fluorine in each part was determined by ion chromatography(IC),and the molecular weight of each part was determined by ultra performance liquid chromatography-quadrupole electrostatic field orbitrap tandem mass spectrometry(UHPLC-Q/Orbitrap MS).The results showed that the molecular weight range of Antarctic krill protein was above 15 kD,and the main molecular weight range was 15 kD~45 kD.The molecular weight of trypsin peptide was below 25 kD,and the main molecular weight distribution was below 5 kD.The enzymatic hydrolysate was purified by a 30 kD ultrafiltration membrane separation device.The three fractions were further separated and purified by Sephadex G-25 Sephadex chromatography and high performance liquid chromatography(named AKP-II,AKP-I,AKP-III,respectively),and the polypeptide AKP-III is of high purity with only one main peak.The three-part peptide was determined by ion chromatography.The fluorine content of the three-part peptide was AKP-I 495.04±2.12?g/g,AKP-II1219.52±1.11?g/g,AKP-III 209.44±2.17?g/g,which accounted for 26%,63%,and 11%of the total fluorine,respectively.The molecular weight of the three-part peptide was determined by ultra performance liquid chromatography-quadrupole electrostatic field orbitrap tandem mass spectrometry,and the molecular weight of AKP-I was 1000.Below Da,the molecular weight of AKP-II is around 2000 Da,and the molecular weight of AKP-III is up to 6000 Da.3.Study on the structure and physicochemical properties of Antarctic krill fluoropeptideThe purified three-part polypeptide(AKP-I,AKP-II,AKP-III)was steamed under vacuum at 50?,and the highest-fluorinated polypeptide AKP-II gradually changed from a colorless transparent liquid to a milky white suspension.The liquid indicates that there are differences in the physical and chemical properties of the three parts of the polypeptide.The AKP-II is greatly affected by temperature and pressure,and the fluorine in the Antarctic krill protein may be combined in the form of mineral salts.LC-MS/MS was used to identify the amino acid sequence fragment of the highly purified fluoropeptide AKP-III-F6.The NCBI(Euphausia superba)protein database was used to search for four proteins according to the matching value score of more than 100 points.Clock,tropomyosin,ATP synthase subunit 6(mitochondrion),Hsp70A),by analyzing the four proteins,combined with the physical and chemical properties of the fluoride protein,under the existing data,the Antarctic krill protein binding fluorine may be related to the Clock protein.Phosphorylation is related to the formation of stable biological minerals(FHAP)by phosphorylation to maintain normal metabolism and life activities.