The Influence Of Key Endogenous Proteases On The Peptides Formation During Dry-cured Meat Processing And Function Evaluation Of Peptides

Dry-cured meat products,such as dry-cured pork coppa and dry-cured ham,with a long history and culture,are famous and classic meat products.The unique processing process contributes unique flavor and rich nutrients.Endogenous proteases could hydrolyze muscle proteins to produce small fragments,such as peptides,which is the basis for the formation of flavor substances and functional active substances in dry-cured meat products.At present,many studies focus on characterization of flavor substances,umami peptides,antioxidant peptides,and angiotensin converting enzyme(ACE)inhibitory peptides of dry-cured meat products.The potential relationship between endogenous proteases and key flavor substances in dry-cured meat products,and platelet-activating factor acetyl hydrolase(PAF-AH)inhibitory peptide that was formed under the influence of endogenous proteases have not been fully elucidated.In this study,the proteolysis,key flavor substances and potential PAF-AH inhibitory peptides under the influence of endogenous proteases during dry-cured coppa processing were investigated as following:(1)Calpain remained active in the raw meat,salting and drying stages,whereas was denatured in the subsequent stages.At post-ripening,cathepsin B,L,H and D residual activity were 61.38%,74.14%,32.15% and 48.49% of the initial activity,respectively.Dipeptidyl peptidase Ⅰ(DPP Ⅰ)and alanine aminopeptidase(AAP)residual activity were 14.24% and 40.00% of the initial activity at post-ripening.Compared with raw meat,the proteolysis index(PI)and peptides content of drycured coppa significantly increased at post-ripening,which were 9.63% and 5.96%,respectively.As sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis(Tricine-SDS-PAGE)showed endopeptidases strongly hydrolyze muscle proteins,such as myosin heavy chain,actin,and 3-phosphoglyceraldehyde dehydrogenase,resulting in many low-molecular protein fragments and peptides rapidly accumulated.DPP Ⅰ,and AAP play an important role in the subsequent hydrolysis of low-fraction protein fragments and peptides into small peptides.(2)Sweet,bitter,sour and umami taste-activity amino acids increased 2.81-fold,4.48-fold,3.45-fold and 3.19-fold in final product compared to those in raw meat.Among them,Glu,Lys,Val,Ala and Leu,with taste activity value(TAV)more than 1,which were key taste-active amino acids in dry-cured coppa.A total of 47 volatile compounds were identified during dry-cured coppa processing.According to PLS analysis results,a total of 18 volatile compounds were identified as key volatile compounds with variable importance projection(VIP)more than 1 in the final product,mainly including hexanal,nonanal,octanal,benzaldehyde,3-methylbutyric acid,2-methylpropionic acid and ethyl caprylate.Controlling p H,water activity(Aw)and moisture content,partial correlation analysis was preformed between endogenous proteases and key flavor substances.The result showed that endogenous proteases were closely related to the formation of key flavor substances,such as such as cathepsin B and D,DPP Ⅳ,and RAP exerted a strong influence on Lys.At the same time,they also strongly influence hexanal.RAP not only significantly affected the accumulation of key taste-activity amino acids,but also affected the accumulation of ethyl octanoate,2,3-pentanedione,and 2,3-octanedione perhaps by regulating the accumulation of octanoic acid and Leu.(3)Based on PAF-AH inhibitory activity and free radical scavenging ability.A total of 30 potential anti-PAF-AH peptides were separated and identified from dry-cured-pork-coppa-derived peptides by ultrafiltration and G-15 Sephadex combined with nano liquid chromatography tandem mass spectrometry(nano-LC-MS/MS).Predicted by simulation in silico,LTDKPFL,VEAPPAKVP,KVPVPAPK,IPVPKK and PIKRSP were identified the most potential PAF-AH inhibitory peptides.The terminal specificity analysis of inhibitory peptides indicated that arginine aminopeptidase(RAP),AAP,DPP Ⅰ and tripeptidyl peptidase Ⅰ(TPP Ⅰ)might be the key endogenous proteases on affecting the formation of PAF-AH inhibitory peptides.Molecular docking showed that hydrogen bonds,salt bridges and attractive charges were the main drivers of potential inhibition of peptide interactions with amino acid residues in the active center of PAF-AH.