Study On The Preparation And Hypoglycemic Mechanism Of Collagen Active Peptides From Cowhide

The incidence of Type 2 diabetes mellitus（T2DM）,a chronic metabolic disease,has increased significantly in recent years,and long-term chemotherapy interventions are prone to adverse side effects.Based on this,food-derived active peptides have gradually become a new idea for the development of healthy hypoglycemic functional factors due to their low side effects,wide sources,high activity and easy absorption.In general,collagen is a potential precursor of dipeptidyl peptidase Ⅳ（DPP-Ⅳ）inhibitor peptidess due to the higher content of proline（Pro）.At present,the slaughter and processing industry of cattle in China is developing rapidly,and the production of cowhide by-products is increasing steadily.Cowhide collagen is rich in hydroxyproline（Hyp）,proline（Pro）and glycine（Gly）,which may be a potential DPP-Ⅳ inhibitory peptide.In this study,collagen-based DPP-Ⅳ inhibitory peptide was prepared by double enzyme hydrolysis using cowhide collagen as a substrate.Cowhide collagen was modified by ultrasound-coupled ionic liquids to promote the release of collagen-based DPP-Ⅳ inhibitory peptides.The T2DM mice model was constructed to investigate the alleviation of cowhide collagen peptide intervention on the disorders in blood gluose and blood lipid,and insulin resistance in mice induced by hyperlipidemia/streptozotocin（high fat/STZ）and to reveal the mechanism of cowhide collagen peptide intervention in improving the insulin resistance and glucose metabolism disorders from the perspective of AMPK-mediated insulin signaling pathway and PI3K/Akt signaling pathway.The cecal contents of mice were collected to analyze the regulatory mechanism of cowhide collagen peptide intervention on intestinal microecological imbalance and metabolic disorders induced by high fat/STZ.The mixture of collagen peptide and Momordica charantia extract was prepared using collagen peptide as a substrate and supplementing with Momordica charantia extract as the functional factor to increase the hypoglycemic target of cowhide collagen peptide.Therefore,this study systematically investigated the hypoglycemic mechanism of collagen peptides based on methods such as the optimization of collagen peptide extraction,separation,purification and identification,structural characterization,in vitro simulated digestion,animal experiments and other means,and combined with techniques such as molecular docking,solid phase synthesis,fluorescence quenching,key protein expression,intestinal flora analysis and metabolomics.The main results are as follows:1.The optimal processing parameters for preparing collagen-based DPP-Ⅳ inhibitory peptides assisted by ultrasound coupled with ionic liquids and the specific amino acid sequence of the functional peptide fragments were investigated.The IC₅₀value（3.04±0.18 mg/m L）of cowhide collagen peptide was significantly lower than that of the collagen peptide prepared by the conventional method at an ultrasound power of 389 W,ultrasound time of 25 min,ionic liquid type of[EMIM][Ac],ionic liquid/collagen ratio of 0.7,collagen concentration of 0.3%and a double enzyme hydrolysis（papain and complex protease）,indicating that ultrasound coupled with ionic liquid improved the DPP-Ⅳ inhibition ability of collagen peptides.In addition,the combination of ultrasound and ionic liquids promoted the partial conversion ofβ-sheet toβ-turn in the secondary structure and enhanced the gastrointestinal digestive stability of the collagen peptide.GPVG,FGPGP,APGGAP,GPPGPT and GPVGPPG peptides obtained after separation and purification process showed vigorous DPP-Ⅳ inhibitory activity,and the inhibition mode was non-competitive.Importantly,GPVGPPG have the binding site that close to the C-terminus of DPP-Ⅳ,the highest molecular docking score and conventional hydrogen bonding content.This result was supported by the lowest IC₅₀ value for the synthetic peptide,indicating that GPVGPPG is an effective DPP-Ⅳ inhibitor derived from cowhide collagen.2.The modification of collagen structure by ultrasound-coupled ionic liquid pretreatment and its effect on the production of DPP-Ⅳ inhibitory peptides were systematically analyzed.The dual modification（IL+US）significantly increased the content of hydrophobic amino acid residue and DPP-Ⅳ inhibitory activity of the low molecular weight（<1 KDa）collagen peptide as compared to the unpretreated collagen peptide.Meanwhile,the dual modification decreased the thermal stability of collagen molecules,accelerated the exposure of tyrosine and phenylalanine residues of collagen,increased the distance of adjacent amino acid residues along the central axis of the helix structure and improved the solubility of collagen,induced the roughness of collagen surface,which increased the reaction sites between protein and hydrolases and ultimately promoted the release of DPP-Ⅳ inhibitory peptides.Ionic liquids and ultrasound tended to promote the breakage of hydrogen bonds and inhibit collagen cross-linking,respectively.These results suggested that ultrasound-coupled ionic liquids promoted the release of cowhide-derived DPP-Ⅳ inhibitory peptides by modifying the collagen structure.3.The regulatory mechanism of collagen peptide intervention in insulin resistance,gluconeogenesis and glycogen synthesis and its effects on T2DM were analyzed.After the intervention of collagen peptide,the symptoms of excessive drinking,overeating and weight loss were alleviated significantly.The damage of the pancreas,liver,kidney and colon was also alleviated to a greater extent.At the same time,the fasting blood glucose and oral glucose tolerance were significantly reduced,and the content of TC and LDL-C were decreased,while the HDL-C level were increased（P<0.05）,indicating that collagen peptide intervention effectively alleviated T2DM by regulating blood glucose level,improving the dyslipidemia and weakening organ damage.In addition,after collagen peptide intervention,the serum insulin resistance index（HOMA-IR1）was reduced significantly.Liver glycogen content was increased significantly in mice（P<0.01）,and p-AMPK,IRS1 and p-GSK3β protein expression was significantly up-regulated in mice liver（P<0.01）.In contrast,PEPCK and Fox O1 protein expression was significantly down-regulated（P<0.05）,indicating that collagen peptide intervention promoted glycogen synthesis through effectively activating AMPK to up-regulating insulin receptor substrate（IRS1）expression and through activating PI3K/Akt pathway by activated IRS1 to up-regulate the expression of p-GSK3βto regulate insulin sensitivity.The gluconeogenesis was inhibited by decreasing the expression of Fox O1 and PEPCK,resulting in decreased output level of hepatic glucose.4.The regulation of collagen peptide intervention on the microbiota and metabolites in the contents of the mice cecum was investigated.Collagen peptides improved the intestinal microbiota of T2DM mice.Compared to the normal mice,the gut of high-fat diet and STZ-triggered mice contained a higher abundance of Phylum Thicket,Phylum Clostridium and Lactobacillus spp.The collagen peptide intervention induced mice to form high abundances of Aspergillus phylum,Clostridium phylum,Bacteroidetes phylum and Bacteroidetes phylum while producing a microbiota structure of Lactobacillus phylum associated with the N-acetylglucosamine,N-acetyl mannose and sialic acid degradation pathways.Collagen peptides regulated intestinal microbial metabolism in T2DM mice.After diseasing（T2DM）,amino acid metabolites（methyl tyrosine,N-formyl-L-methionine and phenylacetylglycine）,fatty acids and fatty acid derivatives（guanyl succinate,salicylic acid and 9（S）-HPODE）,vitamins（tocotrienols）,ketones（quinolones）,prostaglandins F2a,6-methyladenine and AMP were increased significantly in mice intestine,while the collagen peptide（CP）intervention mitigated the increase of above metabolites.Differential metabolites were mainly enriched in steroid hormone biosynthesis,aldosterone synthesis and secretion,cancer pathways,regulation of adipocyte lipolysis,c AMP signaling pathway,oxytocin signaling pathway,lifespan regulation pathway,cortisol synthesis and secretion,aldosterone regulation of sodium reabsorption and prostate cancer.5.The effect of Momordica charantia extract（MCE）on the structural and functional properties of cowhide collagen peptides and its effect on T2DM was investigated.The thermal stability,α-amylase inhibitory activity,antioxidant activity and gastrointestinal digestive resistance of the collagen peptides were enhanced significantly after compounding with MCE（P<0.05）.The optimal ratio of CP and MCE was 1:0.35,the fluorescence quenching mode between CP and MCE was static quenching,and the type of interaction was hydrophobic interaction,which indicated that MCE enhanced the stability and antioxidant properties of CP through hydrophobic interactions and improved the ability of CP to inhibit carbohydrate absorption.In addition,compared with the single CP intervention,the CP-MCE complex significantly improved the glucose regulation process in T2DM mice,reduced fasting glucose and oral glucose tolerance in T2DM mice,enhanced the function of pancreaticβ-cells,increased the hepatic glycogen content,promoted the uniform distribution of glycogen in the liver and kidney and effectively alleviated oxidative stress.These results indicated that the complexation with MCE significantly enhanced the alleviation of cowhide collagen peptide on T2DM symptoms in mice.In summary,a new peptide（GPVGPPG）with higher DPP-Ⅳ inhibitory activity was isolated using bovine collagen as a source of highly active hypoglycemic peptides.Ultrasound-coupled ionic liquids promoted the release of DPP-Ⅳ inhibitory peptides.CP intervention could alleviate T2DM-induced dysglycemia and insulin resistance by affecting the AMPK-mediated insulin signaling pathway and PI3K/Akt signaling pathway,remodeling intestinal flora and regulating intestinal microbial metabolism.In addition,the CP-MCE complex could increase the hypoglycemic target of cowhide collagen peptide and improve the digestive resistance of the gastrointestinal tract.The hypoglycemic mechanism of cowhide collagen peptides revealed in this study may provide a theoretical basis and reference for developing efficient,sensitive and safe hypoglycemic dietary supplements and enhance the high-value utilization of cowhide collagen.