| Jinhua ham is an outstanding representative of Chinese traditional dry-cured meat product and the crystallization of human wisdom in manufacturing dry-cured meat product.However,it is not suitable for the current production mode and market demands due to these defects of the traditional production method of Jinhua ham,such as long processing cycle,high salt content,and high safety risks.The modern production mode of"Low-temperature salting,medium-temperature dehydrating and high-temperature ripening"shortens the processing cycle and improves the security of Jinhua ham through precise control of environment temperature and relative humidity.However,the effects of changes in these processing parameters during the modern processing of Jinhua ham on the formation and accumulation of taste components and on the activities of endogenous enzymes have not been fully discussed and elucidated.Therefore,this paper investigated the changes of endogenous protease activities,evaluated the degradation of proteins by proteomics and peptidomics,and further characterized the taste substance and their contents by metabolomics during the modern manufacturing process of Jinhua ham based on the control group of traditional-processed Jinhua ham,aiming at systematically discussing the taste formation mechanism of modern-processed Jinhua ham,and at providing scientific guidance and theoretical reference for the modern production of Jinhua ham.The detailed contents and results of this study are as following:?.Study on the relationship between protein degradation and endopeptidase activities during the processing of modern-processed Jinhua hamIn order to evaluate the relationship between protein degradation and endogenous proteases activities during the processing of modern-processed Jinhua ham,biceps femoris muscles of traditional-processed Jinhua ham and modern-processed Jinhua ham were sampled at the raw ham(prior salting),the end of post-salting,the middle of drying-ripening,the later stage of drying-ripening and the post-ripening stage.The activities of endogenous proteases(calpain and cathepsin)and protein degradation were investigated during the processing of Jinhua ham,and then the relationship between proteases activities and protein degradation was further evaluated by the modeling of partial least squares regression.The results showed that the proteolysis index of modern-processed Jinhua ham gradually increased(P<0.001)from the raw ham(prior salting)to the stage of post-ripening,and that the proteolysis index of modern-processed Jinhua ham was 20.89%at the end of post-ripening.The activities of cathepsin B and cathepsin L gradually decreased(P<0.001)during the modern processing of Jinhua ham,but always maintained high residual activities.The activities of cathepsin B and cathepsin L were approximately 25.40%and 14.76%of the initial activities(raw ham)at the end of post-ripening,respectively.The results of label free proteomics indicated that myosin heavy chain,troponin,myosin light chain,glyceraldehyde-3-phosphate dehydrogenase,pyruvate kinase,?-glucan phosphorylase and ?-actinin were intensely degraded during the modern processing of Jinhua ham.Partial least squares regression analysis demonstrated that the activities of cathepsin B and cathepsin L were closely related to the degradation of myosin heavy chain,myosin light chain,glyceraldehyde-3-phosphate dehydrogenase,pyruvate kinase,?-glucan phosphorylase and ?-actinin,and that the degradation of these proteins could be responsible for the increase of proteolysis index of Jinhua ham during the modern processing.?.Study on the formation and distribution of small peptides(less than 3 kD)during the modern processing of Jinhua hamIn order to investigate the formation and distribution of small peptides during the modern processing of Jinhua ham,Bicep femoris muscles of traditional-processed Jinhua ham and modern-processed Jinhua ham were randomly sampled at the raw ham(prior salting),the later stage of drying-ripening and the post-ripening stage.The changes in the activities of dipeptidyl peptidase I and aminopeptidase were analyzed,and the formation and distribution of small peptides were evaluated by peptidomics during the modern processing of Jinhua ham.The results showed that dipeptidyl peptidase I,alanyl aminopeptidase and leucyl aminopeptidase always maintained high potential activities during the modern processing of Jinhua ham,and their residual activities were about 29.45%,10.08%and 10.20%of the initial activities(raw ham)at the end of post-ripening,respectively.Peptidomics analysis revealed that a significant increase in the number of peptides with molecular weights of 1?2 kD and 2?3 kD was observed from raw ham to later stage of dry-ripening and to the stage of post-ripening(P<0.01).The numbers of myosin-derived peptide revealed that myosin of modern-processed Jinhua ham produced a large number of 1?2 kD and 2?3 kD peptides at the later stage of dry-ripening and the stage of post-ripening.Compared with traditional-processed Jinhua ham,modern-processed Jinhua ham produced more myosin-derived peptides of 2?3 kD at the later stage of dry-ripening and the stage of post-ripening(P<0.01),while the number of peptides of 0?1 kD was not significantly different between traditional-processed Jinhua ham and modern-processed Jinhua ham(P>0.05).At the stage of post-ripening,the number of myosin-derived peptides of 1?2 kD was significantly lower in modern-processed Jinhua than in traditional-processed Jinhua(P<0.01).The activities of exopeptidase including dipeptidase I,leucyl aminopeptidase and alanyl aminopeptidase could be the important contributors to develop the difference in the accumulation of peptides with molecular weights of less than 3 kD between traditional-processed Jinhua ham and modern-processed Jinhua ham.?.Study on taste sensation and its substance basis during the processing of modern-processed Jinhua hamIn order to characterize the composition and key components of taste substance of modern-processed Jinhua ham,Bicep femoris muscles of traditional-processed Jinhua ham and modern-processed Jinhua ham were randomly sampled at the raw ham(prior salting),the later stage of drying-ripening and the post-ripening stage.Moisture content,water distribution,sensory evaluation,and the composition and content of taste substance were investigated,and partial least squares regression analysis was employed to determine the key components which contributed to the taste attributes of modern-processed Jinhua ham.The water content of muscle tissues of modern-processed Jinhua ham significantly decreased from 74.47%in raw ham to 52.19%in final product(P<0.001).The salting and ripening did not change the T2 relaxation time and the proportion of bound water in the muscle tissues of modern-processed Jinhua ham(P>0.05),but significantly decreased the proportion of free water(P<0.001)and increased the proportion of immobilized water(P<0.001);at the end of post-ripening,the proportions of bound water,immobilized water and free water in the muscles tissues of modern-processed Jinhua ham were approximately 6.08%,84.23%and 9.69%,respectively.The attributes of color,juiciness,texture and aroma in modern-processed Jinhua ham were similar to the changes of traditional-processed Jinhua ham(P>0.05),but the intensities of taste and richness of modern-processed Jinhua ham were relatively higher than that of traditional-processed Jinhua ham(P<0.01).1H nuclear magnetic resonance(NMR)analysis showed that carnosine,anserine,isoleucine,alanine,tyrosine,phenylalanine,methionine,leucine,valine,glutamic acid,glycine,lactic acid,glycerol,creatine and creatine phosphate were the main taste components of modern-processed Jinhua ham.Analysis of partial least squares regression and taste active values revealed that anserine,creatine,creatine phosphate,glycerol and lactic acid could be the significant components to develop the attributes of taste and richness,and could be further responsible for higher scores of taste and richness of modern-processed Jinhua ham,compared with the traditional-processed Jinhua ham.?.Comparative study on protein degradation and sensory quality of premium ham and defective hamIn order to evaluate the differences of protein degradation sites and sensory quality between premium ham and defective ham,the classification of modern-processed Jinhua ham was performed according to GB/T 19088-2008,and biceps femoris muscles of premium ham and defective ham were sampled.Sensory attributes,texture parameters,protease activities and protein degradation were evaluated in both premium ham and defective ham.The results of sensory evaluation indicated that defective ham showed intense bitterness and sourness tastes,and the hardness values of defective ham were about 0.8 times than that of premium ham(P<0.01),while the adhesiveness values of defective ham were approximately 1.6 times that of premium ham(P<0.01).The proteolysis index of defective ham was approximately 24.89%,which was about 1.2 times of premium ham(P<0.01).The residual activities of cathepsin B and cathepsin B+L in defective ham were significantly higher than that of premium ham,which were approximately 1.3 and 1.2 times of premium ham,respectively(P<0.01).Dipeptidase ?(DPP ?),dipeptidase ?(DPP ?),dipeptidase ?(DPP ?)and dipeptidase ?(DPP ?)activities significantly decreased(P<0.001)from raw ham to premium ham and to defective ham;compared with other dipeptidase activities,DPP ? and DPP ? in premium ham and defective ham showed higher residual activities(P<0.01).The residual activities of DPP ? in defective ham accounted for about 42.78%of initial activities(raw ham),and DPP ? accounted for about 24.21%of initial activities;the residual activities of DPP ? in premium ham accounted for about 32.43%of initial activities,and DPP ? activities accounted for about 8.51%of initial activities.Label free quantitative proteomics revealed that more intense degradation of myosin and troponin was observed in defective ham than in premium ham(P<0.001).Partial least squares regression analysis indicated that excessive residual activities of cathepsin B and cathepsin L were strongly associated with exc essive degradation of myosin and troponin in defective ham,and that excessive degradation of myosin and troponin could accelerate the formation of intense bitterness and high adhesiveness in defective ham.The taste active values of free amino acids with bitterness were significantly lower in defective ham than those in premium ham(P<0.001),indicating that free amino acids could not be the main source of excessive bitterness in defective ham.Ile-41,Ala-87,Glu-109,Tyr-127 and Glu-140 were specific degradation sites for myosin light chain 1 of defective ham;Gln-28,Glu-29,Glu-32,Ile-37,Met-71,Phe-104,Leu-107 and Lys-116 were specific degradation sites for myosin light chain 2 of defective ham,and Pro-50,Glu-93,Tyr-132,Glu-133 and Lys-144 were specific degradation sites for myosin light chain 3 of defective ham.These excessive degradation sites of myosin light chain could accelerate the formation and accumulation of peptides in defective ham.These results further demonstrated that the excessive activities of endogenous enzymes,specifically cathepsin B,cathepsin L and dipeptidase activities could be the key factors to develop the difference in muscle protein degradation and organoleptic quality between defective ham and premium ham.In conclusion,cathepsin B and L were the key proteases during the processing of modern-processed Jinhua ham,which promoted the degradation of proteins including myosin,troponin,glyceraldehyde-3-phosphate dehydrogenase and ?-actinin,which further facilitated the formation and accumulation of free amino acids and small peptides in modern-processed Jinhua ham.Free amino acids,small peptides and organic acids were important taste substances of modern-processed Jinhua ham.However,excessive activities of cathepsin B,L and dipeptidyl peptidase could lead to excessive degradation of myosin and troponin and result in a strong bitter taste and high adhesiveness of Jinhua ham.Therefore,it is particularly important to fully understand the role of endogenous proteases and protein degradation in the formation of taste substances during the processing of Jinhua ham. |
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