The Effect Of Ripening Temperature On The Proteolysis Of Hard Cheese Made From Yak Milk And Investigation Into Bitter Peptide Characterization

Hard cheese was manufactured with yak milk from the Qinghai-Tibet plateau through coagulation, cutting, stirring, whey off, matting, pressing and ripening. Hard cheese made from yak milk has low moisture content, high nutrition and easily been stored. However, ripening is a slow and consequently an expensive process that is not fully predictable or controllable so that cheese quality and flavor is not assured of their internal uniformity. Therefore, the paper investigated the effect of ripening temperature on the proteolysis and starter autolysis in the hard cheese made from yak milk at 5, 10 or 15℃ for 1, 2, 3, 4, 5 and 6 months, respectively. Compositon and content of amino acid of bitter peptide in the hard cheese ripened at 5, 10 and 15℃ were detectd and analyzed at 4, 5 and 6 months. Bitter peptides in the cheese were separated by Sephadex G-25 and bitterer fraction was indentified by LC-MS/MS. The main results showed as follows:Proteolysis through the level of soluble nitrogen at p H4.6, in 12% trichloroacetic acid and 5% PTA, total free amino acids and hydrophilic peptide and hydrophobic peptide levels in the water-soluble fraction at p H 4.6 was significantly affected by ripening temperature and time. Elevated temperature could drastically increase the level of soluble nitrogen at p H4.6, in 12% trichloroacetic acid and 5% PTA and the content of total free amino acids, especially ripening temperature at 15℃ resulted in a significant increase in the level of soluble nitrogen in 12% trichloroacetic acid and in 5% PTA. RP-HPLC analysis showed that with the ripening temperature elevation, hydrophilic peptide levels in the p H4.6 soluble fractions increased and hydrophobic peptides levels decreased.Difference in bitterness between cheese after two month of aging at 5, 10 and 15℃ was distinct, but at low level. Cheese ripened at 15℃ had more bitter than Cheese ripened at 5 and 10℃. Cheese ripened at 10 and 15℃ from 4 to 6 months had moderately strong bitterness and difference was not significant(P≥0.05).Urea-PAGE electrophoretograms of the p H 4.6-insoluble cheese extracts showed relative content of residual β-casein and αs-casein gradually dropped as the ripening temperature was increased from 5 to 15℃. Falling range of residual β-casein in cheese ripened at 15℃was higher than that in cheese ripened at 5 and 10℃ and αs-CN degration degradation was stronger than β-CN’s.The activities of lactate dehydrogenase, general aminopeptidase,X-prolyl-dipeptidlyl aminopeptidase in the hard cheese from Yak milk ripened at 10 and 15℃ were higher than those in the hard cheese ripened at 5℃ and the activities of these enzymes in the cheese ripened at 15℃were higher than those in the cheese at 10℃. Thereore, starter in the cheese ripened at 15℃ showed high autolytic activity, whereas starter in the cheese ripened at 10 and 5℃ showed medium and low autolytic activity.The level of bitter peptides in the hard cheese made from yak milk ripened at 5℃and 10℃ for 4, 5 and 6 months was obviously different. The level of bitter peptides in the hard cheese ripened at 5℃ was higher than that in the hard cheese ripened at 10℃ especially at 5 months and 6 months. The level of bitter peptides in the cheese ripened at 5℃ and 15℃ for 4 and 5 months existed little difference and there was no significant difference(P≥0.05). The level of bitter peptides in the hard cheese ripened at 15℃ was higher than that in the hard cheese ripened at 5℃ at 6 months and there was significant difference(P<0.05). The results showed that with the elevation of ripening temperature, autolysis of lactic acid bacteria could reduce accumulation of bitter peptide, but it had only limited action.Percentage of content of hydrophobic animo acid of bitter peptide in the cheese ripened at 5,10 and 15℃ exceeded 67%, and content of proline, valine, leucine, isoleucine and phenylalanine were highest among the hydrophobic animo acids of bitter peptide. Content of hydrophobic animo acid of bitter peptide were highest in the cheese ripened at 15℃ and content of proline of bitter peptide was highest in the cheese at 4 and 5 months and content of leucine of bitter peptide was highest in the cheese at 6 months; Content of hydrophobic animo acid of bitter peptide were higher in the cheese ripened at 10℃ and content of leucine of bitter peptide was highest; Content of hydrophobic animo acid of bitter peptide were low in the cheese ripened at 5℃ and content of leucine of bitter peptide was highest.Bitterness of bitter peptide in the cheese ripened at 5, 10 and 15℃existed differences. When ripening temperature was elevated, bitterness of bitter peptide in the cheese got strong and bitter peptide had moderately strong bitterness. Bitterness of bitter peptide had positive correlation with hydrophobic animo acid content.Bitter peptide was separated into three fractions with different molecular weightby Sephadex G-25.Content of proline, valine, leucine and phenylalanine were highest among the hydrophobic animo acids of bitter peptide ant content of glutamic acid was highest among the hydrophilic animo acids of bitter peptide. Content of bitter and hydrophobic animo acids in the fraction Ⅱ were highest among three fractions. Bitterness of bitter peptide in the fractionⅡwas stronger than the others.14 bitter peptides in the hard cheese made from yak milk were identified by LC-MS/MS. These bitter peptides were composed of 7 to 17 amino acids, with molecular weight less than 2000 Da. Molecular weight of most of bitter peptides ranged from 800 to 1500 Da and most of bitter peptides came from degradation of β-casein.In conclusion, ripening temperature affected proteolysis during the maturation process of hard cheese made from yak milk. Especially elevated ripening temperature could accelerate maturation of cheese. Elevated ripening temperature could strengthen starter autolysis, however, autolysis of lactic acid bacteria could reduce accumulation of bitter peptide, but it had only limited action.Through investigation into bitter peptide characterization, cheese bitterness was relevant to small peptide which came from degradation of β-casein with high content of hydrophobic animo acid. The study provides theoretical reference for accelerating ripening of hard cheese made from yak milk and avoiding of formation of too much bitterness of cheese.