Isolation,Purification,Synthesis And Functionl Activity Of Bitter Taste Peptides From Yak Milk Hard Cheese

The raw material of the study was hard cheese made from yak milk in Tianzhu Tibetan Area of Gansu Province.The bitter peptide was extracted by chloroform-methanol method.The chromatographic technique was used to determine the optimal separation and purification conditions and the structure was studied in detail.The functional activity of the water soluble peptide?alcohol soluble peptide and post-synthesis bitter peptides has been studied to understand the relationship to cheese ripening time and temperature.The aim is to provide a basis for the development of bitter peptide functional products in yak milk hard cheese.The main results of this study are as follows:1.The optimal separation conditions of Sephadex G-25 gel chromatography were as follows: the loading concentration was 53 mg/m L,the loading amount was 3m L,and the flow rate was 0.7 m L/min.Under this condition,the comprehensive evaluation value was 96.007.And finally,four groups of peaks were obtained by RP-HPLC(1D,2D),and the bitterness values were: 1.19±0.04,1.45±0.07,3.93±0.15,1.4±0.02;2.The bitter peptide isolate was subjected to solid phase synthesis chemical synthesis after the sequence was determined: TPVVVPPF(P-1)L,LPPTVMFPPQ(P-2),EPVLGPVRGPFPII(P-3),and the molecular weights were968.20 Da,1126.38 Da,respectively.The purity of 1490.81 Da was 97.631%,95.573%,99.091%.3.The primary structure of the three bitter peptides was obtained and synthesized in vitro.The sequences of p-1,P-2 and P-3 were compared with those of NCBI and BIOPEP databases.The amino acid sequences of the three peptides were found to be less homologous to known bitter peptides,with the highest values reaching 55.6% and42.9%.92.8%.Therefore,P-1 and P-2 were judged to be novel bitter peptides.And the minimum threshold of bitterness of the three bitter peptides was 1.8889,1.1000,and 1.8571,respectively.P-2 and P-3 have antioxidant activities in ?-CN f(210-223)and f(166-174),respectively,and P-3 is a peptide in yak milk(M91-02162).4.The IC50 values of ACE in vitro in bitter peptides P-1,P-2 and P-3 were:0.3416 mg/m L,0.3866 mg/m L,0.2469 mg/m L,and the ACE inhibition rates were87.5% and 84.72%,92.361% respectively.The sites of action of ACE inhibitory activity in bitter peptides P-2 and P-3 are ?-CN f(179-188)and f(195-208).5.In yak milk hard cheese,the DPPH free radical scavenging rate increased from3.97%±1.25% to 28.93%±0.33% during the process of ripening for 120-180 d at5-15°C.The diameter of the inhibition zone of Staphylococcus aureus increased from13.22 mm ± 0.12 mm to 16.02 mm ± 0.09 mm,and the diameter of the inhibition zone of E.coli increased from 13.35 mm ± 0.07 mm to 16.41 mm ± 0.05 mm.The effect of maturity time and temperature was significant(p<0.05).The biological activity of the water-soluble peptide in the hard cheese of yak milk increased significantly with the increase of maturity time and temperature(p<0.05).6.In yak milk hard cheese,the ACE inhibitory activity is in the range of59.20%±1.40% to 72.33%±2.32% during the period of 5-15°C and mature 120-180 d,and it increases with the ripening time.,showing a trend of decreasing first and then rising,and the difference was significant(p<0.05).As the maturity temperature increased,the ACE inhibitory activity showed a significant downward trend(p<0.05).