| Diabetes mellitus has become one of the most serious chronic diseases.Food protein-derived glucose-regulating peptides have high tissue affinity and specificity,low side effects,and can interact with glucose-regulating receptors,enzymes and biomolecules."Paguong" is a fermented meat product made from whole Tibetan pork,which is the fruit of Tibetan people’s practice of preserving and transporting meat.Some studies have been conducted to analyze the nutritional composition and flavor substances of "Palgong" in the Linzhi region of Tibet,but no research on the active peptides in "Palgong" has been reported at home and abroad.During the fermentation process of "Palgong",the proteins are gradually enzymatically dissolved to form a variety of active peptides and amino acids,which is an excellent source of bioactive peptides.Based on these,the crude peptides were prepared by gastro-pancreatic digestion and water-soluble extraction using "Pagong" muscle as the experimental material,and the physicochemical properties of "Pagong"-derived crude peptides were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and laser diffraction techniques.The glucose-modulating peptides were separated and purified by Sephadex G-25 dextran gel and reversed-phase chromatography,followed by the determination of α-amylase and α-glucosidase inhibitory activities of each fraction,and the analysis of amino acid composition of each fraction by HPLC-phenyl isothiocyanate(PITC)pre-column derivatization.The sequence identification was performed by liquid chromatography/tandem mass spectrometry(LC/MS).The peptide sequences were screened by Peptide Ranker,Expasy-Peptide Cutter,BIOPEP-UWM database,Pep Draw and other bioinformatics tools,and the structure,energy parameters and charge distribution of the screened peptide sequences were optimized by quantum chemical computation techniques.The active site of the glucose regulating peptide was deduced through the calculation of the structure optimization,energy parameters,charge distribution and bond length information of the screened peptide sequences by quantum chemistry.This study fills the gap in the functional study of "pagong" food,and investigates the mechanism of blood glucose regulation by the active peptide in meat products,which provides theoretical basis for the development,modification and synthesis of functional food or drug with blood glucose regulating peptide.The results showed that the digestibility of "pagong" was 46.41±1.23%after gastric enzyme treatment;the digestibility increased significantly to79.11±1.85% after gastric enzyme/pancrease treatment.The extraction rate of digestion method was more than 10 times than that of water solubility method,and the soluble peptide content of digestion product was significantly higher than that of water solubility method(P < 0.05).After SDS-PAGE electrophoresis,it was found that the main components of crude protein C of "Pagong" muscle were serum albumin,β-subunit of ATP synthase and β-enol protein.The protein bands in the lane of both the crude peptide WY digested by gastric trypsin and the crude peptide S extracted by water solubilization were very shallow,with a high degree of proteolysis,and all the macromolecular proteins were broken down into peptides of 11 k Da or less.The results of particle size and distribution showed that there was a significant difference in particle size before and after digestion of "Pagong" muscle,with the largest volume mean particle size D [4,3]and surface area mean particle size D [3,2] for C and the smallest volume mean particle size D [4,3] for WY.Sephadex G-25 gel column was used to separate WY and S.The molecular weight order of G-25 fraction of WY was WY-Ⅰ > WY-Ⅱ > WY-Ⅲ.The molecular weight order of G-25 fraction of S was S-1 > S-Ⅱ.The results of particle size and distribution changes showed that the particle size results of S-Ⅰand S-Ⅱ were similar,and the two fractions WY-Ⅱ and WY-Ⅲ had more regular particle shape and concentrated particle size distribution.For α-amylase inhibition activity size order: WY-Ⅰ>WY-Ⅱ>WY>WY-Ⅲ>S-Ⅱ>S-Ⅰ>S,α-glucosidase inhibition activity size order: WY-Ⅱ>WY>WY-Ⅰ>S-Ⅱ>WY-Ⅲ>S>S-Ⅰ,only the α-glucosidase inhibition activity of WY-Ⅱ was higher than60%.Three fractions of WY-Ⅱ were collected by using YMC*GEL ODS C18 column,and their hydrophobicity was WY-Ⅱ-3>WY-Ⅱ-2>WY-Ⅱ-1.The order of inhibition activities of α-amylase and α-glucosidase were all: Acarbose >WY-Ⅱ-3 > WY-Ⅱ-2 > WY-Ⅱ-1.The inhibition rates of α-amylase andα-glucosidase of the three fractions were greater than 60%,and the inhibition ofα-amylase and α-glucosidase increased with the enhancement of hydrophobicity.Based on the results of hypoglycemic activity,WY-II-3 was selected for sequence identification.A total of 86 peptide sequences were identified,and the main protein sources were smooth muscle actin α2 and myosin-4.A total of 12 peptide sequences with a score >0.60 were screened in the Peptide Ranker program,and the sequences were structurally optimized by Gaussian 09.The total energy size in the peptide molecules was in the following order: T1>T12>T2>T9>T8>T10>T7>T6>T5>T11>T3>T4.HOMO orbitals were mostly distributed on the upper arginine Arg,proline Pro,lysine Lys,leucine Leu,asparagine Asn,tryptophan Trp and methionine Met.LUMO orbitals are mainly distributed on the phenyl ring of the C-terminal amino acids-COOH,proline Pro and phenylalanine Phe.Taking into account the HOMO,charge distribution and bond length of the peptide molecule,the active sites of the molecule were inferred,T1 on Arg at-C104H105,T2 on Arg at-C106H108,T3 on Lys at-C200H201,T4 on Pro at-C4H5,T5 on Lys at-C176H177,T6 on Leu on-C10H12,T7 on-C35H37 on Pro,T8 on-C104H105 on Arg,T9 on-C14H15 on Arg,T10 on-C130H131 on Trp,T11 on-C9H12 on Met,and T12 on-C120H122 on Arg. |
