Preparation,Identification Of Goat Milk Protein Peptide And Its Mechanism Of Hypoglycemic

The incidence of type 2 diabetes mellitus（T2DM）is rapidly increasing on a global scale,exerting a significant impact on public health.Current pharmaceutical treatments exhibit shortcomings,such as high costs,numerous side effects,and drug resistance.In this context,the enormous potential of dietary protein peptides for the prevention and treatment of T2DM has garnered considerable attention.Compared to cow’s milk,goat’s milk offers higher fat digestibility,more easily absorbed proteins,lower lactose content,and a richer profile of vitamins and minerals.However,research and development related to goat’s milk and its derivatives have lagged behind cow’s milk,resulting in lower product value.Additionally,current knowledge about the role of goat’s milk protein peptides in blood sugar reduction is relatively limited.Therefore,a comprehensive investigation into the function of goat’s milk protein peptides in blood sugar control is of paramount importance.Bioinformatics analysis predicts that goat’s milk may contain potential hypoglycemic peptide segments capable of inhibiting the DPP-IV enzyme and promoting the release of endogenous GLP-1 in the intestine.However,the instability of dietary protein peptides severely limits their application,resulting in lower bioavailability.Furthermore,the mechanisms underlying goat’s milk protein peptide intervention/treatment of T2DM remain unclear.Therefore,it is essential to delve deeper into these issues to provide substantial guidance for the development of safe and innovative T2DM treatment strategies.In this study,bioinformatics was employed to predict the presence of goat milk proteins containing protein peptides with enhanced blood glucose-lowering activity.To optimize the release of these hypoglycemic peptides,in silico tools were utilized to forecast suitable proteinases for the hydrolysis of goat cheese proteins（GCA）and whey proteins（GWP）,which were subsequently validated through in vitro experiments.Following this,goat cheese protein peptides（GCAPS）and whey protein peptides（GWPPG）were further isolated and purified to assess their activity stability.Nanoencapsulation technology was implemented to enhance the stability and bioavailability of peptides.Finally,a comprehensive investigation of the blood glucose-lowering effects and mechanisms of action of goat milk protein peptides was conducted by combining animal experiments,molecular dynamics simulations,and in vitro activity verification.The primary research findings are as follows:（1）In this study,bioinformatics tools were used to predict the potential biological activity and types of proteolytic enzymes for goat milk proteins.The theoretical results indicated a strong potential for blood glucose-lowering activity within the primary sequence of goat milk proteins.Actual experiments were conducted to assess the potential of alkaline protease,neutral protease,trypsin,bromelain,pepsin,papain,and proteinase K to release blood glucose-lowering peptides.Additionally,the impact of different proteases on the hydrolysis degree（DH）,physicochemical properties,protein structure,emulsification properties,and blood glucose-lowering activity of GCA and GWP were investigated.The results showed that the hydrolysates of GCA with papain and bromelain exhibited higher DH values and lower molecular weights.Conversely,the hydrolysates of GWP with papain and alkaline protease showed higher DH and lower molecular weights.Compared to GCA and GWP,the average particle size,Zeta potential,and emulsification properties of different hydrolysates were significantly reduced（P<0.05）,and endogenous fluorescence spectra exhibited a redshift.Blood glucose-lowering experiments revealed that the hydrolysates of GCA,including papain（GCAP）,bromelain,and proteinase K,exhibited higherα-glucosidase,α-amylase,and DPP-IV enzyme inhibition activities.The hydrolysates with papain（GWPP）,alcalase,and proteinase K of GWP exhibited good DPP-IV enzyme andα-amylase inhibition activities,consistent with the computer predictions.（2）To mitigate interference from other substances in the mixture,the DPP-IV inhibitory peptides from GCA and GWP were isolated and purified using ultrafiltration,Sephadex G-15gel column,and reverse-phase high-performance liquid chromatography（RP-HPLC）.Their environmental stability and in vitro simulated gastrointestinal digestion stability were assessed,aiming to facilitate the industrial-scale production of dairy-derived peptides.Initially,ultrafiltration was employed to fractionate papain（GCAP）,bromelain,and proteinase K hydrolysates of GCA,papain（GWPP）,alcalase,and proteinase K of GWP,identifying the most suitable protease type.Results indicated that components with molecular weights<3k Da in several hydrolysates contained higher levels of peptides and exhibited favorable DPP-IV,α-glucosidase,andα-amylase inhibitory activities,particularly in the papain hydrolysates of GCA and GWP（GCAP and GWPP）.Further purification of GCAP and GWPP was conducted using Sephadex G-15 gel column and preparative RP-HPLC,revealing that GCAP-S3-P1（GCAPS）and GWPP-G2-H2（GWPPG）exhibited the lowest DPP-IV IC50values at 0.32±0.047 mg/m L and 0.34±0.02 mg/m L,respectively.Chemical stability experiments demonstrated that both GCAPS and GWPPG were sensitive to high temperatures（>80°C）and low p H conditions（2-4）,resulting in reduced stability.Under high Na Cl conditions,peptide solubility decreased,leading to aggregation and loss of activity,whereas the impact of metal ions(K⁺,Ca²⁺,Mg²⁺,Zn²⁺,and Cu²⁺)on stability was minimal.In vitro simulated gastrointestinal digestion stability experiments revealed burst release during simulated gastric digestion for both GCAPS and GWPPG.After 2 h of simulated digestion,peptide retention rates were 51.2±3.2%and 47.78±2.58%,respectively,with DPP-IV inhibition rates decreasing to 44.55±2.54%and 40.8±2.02%.During the simulated intestinal digestion phase,the degradation rate of peptides in GCAPS and GWPPG decreased,indicating sustained release in the intestines.Consequently,the use of encapsulation technology is necessary to protect the functionality of GCAPS and GWPPG,ensuring the preservation of their complete biological activity.（3）The objective of this study was to develop a nanoscale delivery system to enhance the stability and bioavailability of GCAPS and GWPPG.Liposomes（GCAPS-LS and GWPPG-LS）and proliposomes loaded with GCAPS and GWPPG were prepared using plant sterols（ergosterol,β-sitosterol,stigmasterol,and mixture of plant sterols）as substitutes for cholesterol.Results revealed that proliposomes prepared withβ-sitosterol exhibited higher encapsulation efficiency（GCAPS-NS:94.98±2.35%and GWPPG-NS:91.11±1.89%）and smaller particle sizes（GCAPS-NS:89.8±8.97 nm and GWPPG-NS:91.37±3.2 nm）compared to liposomes.Morphological examinations indicated that both liposomes and niosomes of the two peptides displayed smooth,spherical particles.FT-IR spectra confirmed the successful loading of peptides.Moreover,compared to GCAPS and GWPPG,GCAPS-LS,GWPPG-LS,GCAPS-NS,and GWPPG-NS exhibited enhanced stability under various p H,temperature,and Na Cl concentration conditions,particularly GCAPS-NS.Furthermore,GCAPS-NS significantly improved peptide retention during simulated gastrointestinal digestion,in vitro bioavailability,and DPP-IV inhibitory activity.These findings suggest thatβ-sitosterol could serve as a potential cholesterol substitute as a membrane stabilizer.Further investigations are warranted to compare and examine the actual in vivo effects of GCAPS-NS and GWPPG-NS.（4）Using C57BL/6J mice,a high-fat diet-induced insulin resistance model was established to investigate the blood-glucose-lowering mechanism and impact on gut microbiota of orally administered free-form GCAPS and GCAPS-NS at different dosages.There is a close relationship between high fat and high sugar,and high blood sugar can affect the body’s metabolism of fat.During oral administration,various samples were monitored for their effects on mice in lipid metabolism and glucose metabolism.Results showed that compared to the blank control group,mice in the model group exhibited significant increases in body weight and liver weight（P<0.01）,along with notable elevation in serum TG,TC,LDL-C,ALT,and AST levels（P<0.01）.Blood glucose levels and hs CRP were significantly elevated while serum GLP-1 levels were significantly decreased（P<0.01）.HDL-C was substantially reduced,and hepatic lipid droplet accumulation and marked pathological changes were observed.Compared to the model control group,different doses of GCAPS demonstrated a dose-dependent attenuation of mouse obesity and blood glucose levels.However,the effects on blood lipids,liver lipids,and liver injury were not significant.Additionally,there were no significant differences in blood glucose and GLP-1 levels between low and medium doses,while high-dose GCAPS moderately improved GLP-1 levels.Compared to free-form GCAPS,GCAPS-NS significantly ameliorated mouse obesity,reduced lipid accumulation,and improved liver damage.Additionally,serum levels of TG,TC,LDL-C,ALT,and AST were significantly decreased,blood glucose levels and hs CRP were significantly reduced,and serum GLP-1 levels were significantly increased.Adipocyte vacuoles in mouse fat tissue noticeably decreased,hepatic lipid droplets were reduced,and the degree of pathological changes was significantly altered,particularly in the medium-dose GCAPS-NS group.Furthermore,compared to high-dose GCAPS（HFD+NSM）,medium-dose GCAPS-NS（HFD+NSM）reversed the composition of mouse gut microbiota,showing increased abundances of Bacteroidaceae,Akkermansia,and Lactobacillaceae,decreased abundance of Blautia,and an improved ratio of Firmicutes to Bacteroidetes.This indicated the restoration of gut microbiota imbalance caused by a high-fat diet-induced insulin-resistant diabetic state.These findings suggest that the liposomal nanoparticle encapsulation technology effectively preserved the activity stability of GCAPS,contributing to the enhancement of glucose and lipid metabolism disorder mitigation,intestinal microbiota balance,relief of hepatic injury and lipidosis,amelioration of inflammation levels,and enhancement of insulin sensitivity.The mechanism might be related to the promotion of endogenous GLP-1 secretion in the intestinal tract.These results demonstrate that GCAPS-NS effectively preserves the activity of GCAPS,improves its bioavailability,and significantly elevates glucose tolerance,insulin tolerance,and GLP-1 expression levels.GCAPS may modulate GLP-1 levels to enhance glucose metabolism and gut microbiota balance in type 2diabetic mice.（5）To identify the active DPP-IV inhibitory peptides within GCAPS,LC-MS/MS was employed for amino acid sequence identification,followed by molecular docking and molecular dynamics simulations to elucidate the mechanism of action of these DPP-IV inhibitory peptides.Peptides were synthesized via solid-phase synthesis and their DPP-IV inhibitory activities were verified.Results revealed that five novel and highly active DPP-IV inhibitory peptides,LLIPF,WPQYLK,FLPYPYY,RRHPYF,and KFPQY,exhibited IC50values of 62.23±3.44μmol/L,85.45±1.68μmol/L,50.98±1.88μmol/L,53.44±2.15μmol/L,and 108.65±2.67μmol/L,respectively.Molecular docking and dynamics simulations indicated that LLIPF,WPQYLK,FLPYPYY,and RRHPYF exhibited tighter binding to the active pocket of DPP-IV,consistent with in vitro activity.Moreover,WPQYLK,FLPYPYY,and RRHPYF inhibited the N-terminal second/site of the peptide with a proline characteristic and competed with DPP-IV competitively,while LLIPF and KFPQY exhibited a mixed-type inhibition.In vitro activity validation demonstrated that the aforementioned candidate peptides exhibited strong DPP-IV inhibitory activity,indicating their potential to lower blood glucose levels by promoting the release of endogenous GLP-1 in the intestine.