Preparation And Hypoglycemic Mechanism Of Soybean Peptides By Ultrasonic Pretreatment With Enzymatic Hydrolysis

Type 2 diabetes has gradually become the most prevalent non-infectious chronic disease affecting human health.Most therapeutic drugs for type 2 diabetes have certain side effects,so it is of great significance to study their interaction with foods.Soybean protein is an important plant protein source with reasonable composition of amino acid,rich source and low price.Recent studies have shown that proteolytic peptides have hypoglycemic effects.In this study,ultrasound-assisted enzymatic hydrolysis of soybean protein isolate was optimized to produce soybean hypoglycemic peptides.The inhibitory rate of enzymatic hydrolysates on?-glucosidase was used as the evaluation index to screen the experimental enzymes.Soybean protein was pretreated by ultrasonic to increase the yield of hypoglycemic peptides,and the ultrasonic pretreatment process and enzymatic hydrolysis process were optimized.The effect of ultrasound pretreatment on the secondary structure of soybean protein was studied by spectrophotometry.The hypoglycemic peptide of soybean was isolated,purified and identified by liquid chromatography-mass spectrometry.To test the safety and stability of soybean hypoglycemic peptides,acute oral toxicity and chemical stability tests were conducted.The effects of soybean hypoglycemic peptides on an organism were explored in alloxan type diabetic mice.The molecular interaction between two soybean hypoglycemic peptides and?-glucosidase was studied using molecular docking simulations,and the molecular mechanism of?-glucosidase activity inhibition by soybean hypoglycemic peptides was elucidated.The research results are as follows:?1?Five enzyme were screened using inhibitory rate of enzymatic hydrolysates on?-glucosidase as the evaluation index,and trypsin was determined to be the best experimental enzyme to prepare the soybean hypoglycemic peptide.Ultrasound pretreatment of the soybean protein isolate increased inhibition of?-glucosidase by the soybean hydrolysis product from 45.2%to 51.6%,indicating pretreatment of soybean protein by ultrasonography could improve the inhibitory activity of?-glucosidase in soybean proteolytic products.Ultrasonic pretreatment was optimized using the inhibitory rate of?-glucosidase as the evaluation index;through single-factor tests and response surface tests,the optimal process parameters of ultrasonic pretreatment were an ultrasonic time of 20.92 min,an ultrasonic power of 245.94 W,and an ultrasonic substrate concentration of 6.27%.Likewise,enzymatic hydrolysis after ultrasonic pretreatment was optimized using the inhibitory rate of?-glucosidase,through single-factor tests and response surface tests,the optimal technological parameters of enzymatic hydrolysis were an enzymatic hydrolysis time of 22.02 min,an enzyme amount of 6416.61 U/g,and a substrate concentration of 2.10%.?2?Structural changes in soybean proteins following ultrasonic pretreatment were studied by fluorescence spectroscopy,Raman spectroscopy,Fourier transform infrared spectroscopy,and attenuated total reflectance mid-infrared spectroscopy.The results of fluorescence spectrum showed that structure of soybean protein isolate did not break,but the secondary structure changed.The results of Raman spectroscopy and attenuated total reflection mid-infrared spectroscopy showed that after ultrasonic pretreatment,the content of?-helix increased gradually,the content of?-folding decreased gradually,the angle of?-turn increased,and reached the peak in 20 minutes.Fourier transform infrared spectroscopy also showed that the secondary structure of soybean protein isolate changed greatly after ultrasonic pretreatment in 20 minutes.The results showed that the secondary structure of soybean protein isolate was more suitable for trypsin hydrolysis and hypoglycemic peptide release after ultrasonic pretreatment in 20 min.?3?The acute oral toxicity test of the soybean hypoglycemic peptide showed that the polypeptide had no toxic side effects.Stability tests have shown that soybean hypoglycemic peptides are more stable under extreme conditions such as high temperature,low pH,and high pH or after simulated intestinal digestion.The results of experiments on mice with the soybean hypoglycemic peptide with a molecular weight of less than 5000 Da showed that a dosage of 228mg/kg reduced postprandial blood glucose.Weight of treated diabetic mice recovered slightly,water and food intake decreased significantly.?4?Ultrafiltration,732 cation exchange chromatography and glucan gel chromatography were used to purification and separation of ultrasound-assisted enzymatic hydrolysis of soybean protein.Liquid phase mass spectrometry was used to structurally characterize the soybean peptides with the highest inhibition activities of?-glucosidase.The peptide sequence with an m/z of 319.55 and?-glucosidase IC₅₀0 of 20.45?mol/L was Gly-Ser-Arg;the peptide sequence with an m/z of 346.55 and?-glucosidase IC₅₀0 of 42.22?mol/L was Glu-Ala-Lys.?5?The binding mode and mechanism of the tripeptides Gly-Ser-Arg and Glu-Ala-Lys and human?-glucosidase were studied by molecular docking.Based on the crystal structure of humane?-glucosidase,the active site residues of the tripeptides Gly-Ser-Arg and Glu-Ala-Lys form 4 and 5 hydrogen bonds,respectively,and the ligand and receptor molecules have strong interactions.Both tripeptides fit well in the active pocket of a human?-glycosidase,suggesting strong van der Waals forces and anion-?interactions between the tripeptide molecules and?-glycosidase enzymes.These stabilizing effects,in combination with the above experiments,show three peptides-Gly-Ser-Arg,and Glu-Ala-Lys are potential inhibitor of?-glycosidase and should be extensive researched.