| Roe occupies an important proportion in the fish by-product processing industry.It contains a variety of amino acids,fatty acids,proteins and other nutrients required by the human body.As a by-product,it has a huge output.However,at present,the development and utilization of roe is relatively scarce.Active functional peptides are a kind of health food.They have a wide range of sources,rich and diverse activities,are safe and reliable,and have great research value,It is one of the main ways to utilize by-products of aquatic products.Based on the inhibitory activity of xanthine oxidase of Bonito seed polypeptide,the optimum enzymatic hydrolysis conditions of alkaline protease were screened;The molecular weight distribution and amino acid composition of Bonito seed protease hydrolysate were studied,and its XOD inhibitory activity was evaluated by digestion in vitro;The effect of embedding on the inhibitory activity of xanthine oxidase during digestion was investigated.Combined with Autodock molecular docking,the inhibitory mechanism and structureactivity relationship between polypeptide and XOD were studied.The results are as follows:1.Preparation of XOD inhibitory peptide from bonito seed by alkaline protease hydrolysisUsing alkaline protease hydrolyzed roe,under different conditions of enzymatic hydrolysis time,temperature,p H and enzyme dosage,respectively,the degree of hydrolysis and inhibitory activity of enzymatic hydrolysate XOD were measured.Finally,the optimal reaction conditions of alkaline protease hydrolyzed roe were found out by orthogonal experiment.According to the single factor experimental results,1:10 was selected as the optimal solid-liquid ratio.When the enzymatic hydrolysis time was 2 h,the degree of proteolysis and XOD inhibitory activity were higher,so 2 h was the best enzymatic hydrolysis time for alkaline proteolysis of roe.The inhibitory activity of XOD was selected as enzymolysis temperature,enzymolysis p H and enzymolysis dosage,and orthogonal test with three factors and three levels was conducted.The results show that the ratio of solid to liquid is 1:10.The optimal enzymatic conditions were as follows: 2 h,6000 U/g,50℃,p H=10,the degree of hydrolysis of roe hydrolyzed by alkaline protease was19.28 ± 0.13.2.Effect of simulated digestion in vitro on XOD inhibitory peptide activity of Bonito seed and peptide sequence analysisIn this study,the molecular weight distribution and amino acid composition of peptide at different digestion stages were analyzed using the inhibitory activity of peptide XOD at different digestion stages as the entry point.The peptide sequence with the activity of lowering uric acid was obtained by mass spectrometry analysis of the small molecule peptide with the highest inhibitory activity.The inhibitory activity of XOD after gastric digestion was the highest(76.09±0.12%).After intestinal digestion,the inhibitory activity of XOD decreased to 53.02±0.48%.By analyzing the molecular weight distribution and amino acid composition of gastric digestive fluid,we found that the molecular weight <3 k Da polypeptide and hydrophobic amino acid played an important role in the inhibitory activity of roe polypeptide XOD.At the same time,the gastric digestive juice of roe polypeptide was isolated and purified to obtain small molecular peptides 10 k Da,10-3 k Da,3-1 k Da and 1 k Da.Their XOD inhibitory activity was measured respectively.It was concluded that small molecular peptides played an important role in XOD inhibitory activity.Finally,LC-MS /MS was used to identify the amino acid sequence of roe polypeptide with the highest XOD inhibitory activity,and four groups of small peptides with xanthine oxidase inhibitory activity were obtained,namely MMIMLEPL,MVLAAVLL,GAGGVGGLGAL and FLLPILQ.3.Effect of embedding on XOD inhibitory activity of roe polypeptide during in vitro digestionThe roe peptide was embedded in calcium alginate with good enteric solubility,and the cumulative release rate and XOD inhibitory activity of roe peptide calcium alginate microcapsules under gastric/intestinal digestion were measured;Finally,molecular docking technology was used to analyze the mechanism and structure-activity analysis of the interaction between polypeptide and XOD.It is concluded that the cumulative release rate of microspheres in intestinal fluid is as high as 88.79±0.65%,and the roe peptide microspheres can be almost completely released in intestinal fluid.At this time,the XOD inhibitory activity in intestinal fluid is 81.82±0.28%,which is significantly higher than that of non embedded roe peptide,which indicates that embedding can prevent the destruction of roe peptide from gastric fluid,and the roe peptide loaded microspheres decomposed and released in intestinal fluid have high XOD inhibitory activity. |
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