Preparation Of Hypoglycemic Peptide By Immobilized Enzyme And Evaluation Of Peptide Biological Activity

As living standards rise,the incidence of diabetes is getting higher and higher,posing a serious threat to human life and health.bioactive peptides as a new type of hypoglycemic drug,have less side effects and higher application value in the prevention and treatment of diabetes.The traditional enzymatic hydrolysis method for the preparation of bioactive peptides requires a large amount of free protease that activity is unstable.At present,brown rice has become the best food for diabetics.It is rich in nutrients and helps the pancreas to secrete insulin and reduce blood sugar.Therefore,in this study,brown rice was used as raw material to prepare hypoglycemic peptide by immobilized enzyme,and its hypoglycemic activity was detected.Polyvinylidene fluoride microporous membrane?F membrane?was used as the carrier to fix trypsin?TR?by covalent binding method.Response surface test was used to optimize the immobilization process of trypsin.The surface characteristics of immobilized enzyme were analyzed by SEM and FTIR.A kinetic model of hydrolysis degree for preparation of brown rice hypoglycemic peptide by immobilized enzyme was established.To establish a method for isolation and purification of hypoglycemic peptide from brown rice.Ultrafiltration,ion exchange,gel filtration chromatography and RP-HPLC were used successively,followed by the analysis of the amino acid sequence of the hypoglycemic peptides by LC-MS,then molecular docking was carried out and the inhibition kinetic model was established,to study the action mechanism and type of hypoglycemic peptides from the molecular level.Finally,To study the relationship between intestinal flora and diabetes mellitus,the hypoglycemic effect of hypoglycemic peptide on alloxan hyperglycemic mouse,growth status,relative abundance of intestinal flora and microecological balance were measured in vivo.The test results are as follows:The optimal process of trypsin immobilized as follows:glutaraldehyde concentration of 2.11%,trypsin concentration of 6.43 mg/mL,temperature of23.57?.At this conditions,predicted trypsin activity recovery was 63.1947%,and the verified trypsin activity recovery was 62.25%.Immobilized trypsin optimum temperature of 55?,the optimum pH value of 8.5,thermal and pH stability are higher than the free trypsin.At the same time,after repeated use for 6 cycles,the immobilized trypsin still retained 67.82%of the original trypsin activity.Compared with free trypsin,the storage stability of immobilized trypsin increased by 18.00%.According to the SEM images,a large number of spherical protrusions were observed on the surface of the immobilized trypsin carrier,and the characteristic peaks of the amide band in the stretch vibration of the peptide bond in trypsin appeared at 1100-1000cm^-1 on the FTIR spectroscopy.The hydrolysis capacity of free trypsin and immobilized trypsin was analyzed by single factor test.At the optimal hydrolysis conditions for immobilized trypsin,trypsin amount of 520 cm²/g,substrate concentration of 6 mg/mL,temperature of55?,pH 8.5,reaction time of 50 min,degree of hydrolysis could reach 24.72%,up63.38%compared with the free trypsin hydrolysis.The degree of hydrolysis retained72.34%of the initial degree of hydrolysis after repeated use for 5 cycles.The kinetic model of immobilized enzyme controlled enzymatic hydrolysis:DH=4.5767 ln[1+?5.2121 E₀/S₀-0.5032?t].The hypoglycemic activity of different polypeptide components was measured with?-glucosidase inhibition rate as the evaluation index,in which P-II-B-b-2component had the highest activity and IC50 was 0.097 mg/mL.The amino acid sequence of polypeptides was inferred from the secondary mass spectra.Polypeptides with molecular weight of 362.99 could be Gly-Met-Arg,476.11 included Asn-Trp-Arg,Gly-Asp-Lys-Arg and Asp-Gly-Lys-Arg,and 589.12 included Lys-Arg-Glu-Arg,Pro-Trp-Met-Arg,Val-Trp-Glu-Arg and Cys-Arg-Gly-Pro-Arg.By molecular docking,the peptides could bind tightly to the?-glucosidase core and inhibit the activity of?-glucosidase.The IC50 of Gly-Asp-Lys-Arg with the best inhibitory effect was 0.039 mg/mL.Finally,by studying the inhibition kinetics of Gly-Asp-Lys-Arg on?-glucosidase,it was found that K_m remained unchanged,while V_m decreased gradually,belonging to the non-competitive inhibition type,and the inhibition constant K_m=5.405×10^-3mol/L.After 35 days of polypeptide treatment,the blood glucose level of mouse with alloxan hyperglycemia was significantly decreased,and the diabetic symptoms,such as hair color,activity,reaction ability,survival rate and appetite were significantly improved.Through the isolation and identification of the dominant intestinal bacteria in mouse,the intestinal flora includes streptococcus,enterobacter,micrococcus,pseudomonas,vibrio,etc.The number of harmful bacteria increased and the number of probiotics decreased in the intestinal tract of hyperglycemic mouse.With the treatment of brown rice polypeptide,the blood glucose of hyperglycemic mouse decreased,and the intestinal microecological balance was improved significantly.