猪宰后肌肉体系中μ-calpain及肌原纤维蛋白理化特性的变化规律

doi:10.3864/j.issn.0578-1752.2015.12.016

摘要/Abstract

摘要： 【目的】研究猪宰后1—168 h，肌肉体系中μ-calpain及肌原纤维蛋白理化特性变化规律，并探究肌肉持水性变化机理，为冷鲜肉汁液流失控制提供理论依据。【方法】取宰后1 h内的猪背最长肌，在4℃条件下分别成熟6、12、24、72、120和168 h，通过活性电泳（casein zymography）对μ-calpain活性定量分析，对肌原纤维小片化指数（myofibril fragmentation index，MFI）进行过程测定，利用SDS-PAGE变性凝胶电泳分析肌原纤维蛋白降解聚合情况，研究宰后肌肉的成熟与肌原纤维蛋白的结构变化规律；通过测定肌原纤维蛋白表面疏水性和溶解性，考察肌原纤维蛋白水合力的变化，利用加压滤纸法测定肌肉的持水性（water-holding capacity，WHC），并借助低场核磁共振技术（low field nuclear magnetic resonance，LF-NMR）定性定量考察肌肉3种不同状态水（结合水、不易流动水及自由水）的分布和迁移。【结果】宰后1—24 h，μ-calpain活性升高，肌原纤维蛋白未发生明显降解，MFI无显著变化（P＞0.05）。宰后24 h肌肉进入成熟期，μ-calpain活性逐渐下降，MFI值显著升高（P＜0.05），肌原纤维蛋白显著降解，肌肉持水性随之改变。肌肉成熟过程中，μ-calpain作用于肌肉蛋白导致蛋白质结构伸展并降解，一方面会有低分子量的蛋白生成，增加蛋白质的比表面积，使其溶解度增加；另一方面也有疏水残基的暴露及蛋白的交联聚合，导致蛋白溶解度降低，表面疏水性增加，这两者之间的竞争结果决定了降解后蛋白质的水合特性。其中，宰后24—120 h，μ-calpain适度降解肌原纤维蛋白，溶解度显著升高（P＜0.05），使肌肉中不易流动水P₂₂升高（r=0.286，P＜0.01），自由水P₂₃下降（r=-0.246，P＜0.05）；肌原纤维蛋白内部疏水性残基的暴露引起表面疏水性显著升高（P＜0.05），致使肌肉自由水P₂₃升高(r=0.319，P＜0.01)；LF-NMR-T₂结果表明结合水P₂₁与不易流动水P₂₂的相关系数为r=-0.890（P＜0.01），不易流动水P₂₂与自由水P₂₃的相关系数为r=-0.360（P＜0.01），表明结合水和自由水会“态变”为不易流动水，蛋白结合水随着蛋白质的高级结构被破坏，结合水被释放，转变为不易流动水，同时肌肉蛋白的降解导致位于肌纤维细胞外的自由水流回肌纤维细胞内部，肌肉持水性升高。宰后120—168 h，肌原纤维蛋白的高度降解，引发低分子量蛋白相互聚集，表面疏水性和溶解度下降，肌肉中不易流动水P₂₂降低（P＜0.05），自由水P₂₃升高（P＜0.05），不易流动水“态变”成自由水，肌原纤维结构内的不易流动水逐渐流向肌原纤维外的空隙，变为自由水，从而造成新的汁液流失，肌肉持水性下降。【结论】宰后24—120 h，μ-calpain介导的肌原纤维蛋白降解，使蛋白溶解度和表面疏水性增加，水合力上升，肌肉中结合水和自由水“态变”为不易流动水，肌肉持水性升高。宰后120—168 h，肌原纤维蛋白进一步降解，低分子量蛋白发生交联聚合，蛋白水合力降低，肌肉中不易流动水“态变”成自由水成为汁液流失。

关键词: 猪；宰后成熟；&mu, -钙激活酶；肌原纤维蛋白；水合特性；持水性；水分迁移

Abstract: 【Objective】Variations in μ-calpain and physico-chemical characteristics of myofibrillar proteins in relation to the water-holding capacity in postmortem porcine muscle (1-168 h) were investigated. The study was expected to provide theoretical supports to regulate the unacceptable high drip loss of muscle and meat postmortem.【Method】Samples of porcine M. longissimus dorsi was collected for μ-calpain activity by casein zymography, myofibril fragmentation index, SDS-PAGE, myofibrillar protein surface hydrophobicity and solubility, water-holding capacity analysis. And low-field nuclear magnetic resonance relaxation T₂ (LF-NMR T₂) analysis of water mobility and distribution was also carried out.【Result】During the 1 h to 24 h postmortem, μ-calpain activity increased, while no significant changes of myofibrillar proteins degradation and MFI values were observed. Significant decreases of μ-calpain activity, with the increases in MFI occurred at 24 h postmortem, which revealed the conversion of muscle into meat. Proteolysis of muscle proteins by μ-calpain, on the one hand gave a rise in protein solubility because of more low molecular proteins emerged, on the other hand led to higher surface hydrophobicity and lower solubility due to hydrophobic groups in protein exposure to its surface and protein aggregation. Both contributed to the protein hydration characteristics. Changes of protein hydration capacity affected the attributes of muscle water populations. During the 24 h to 120 h postmortem, proteolysis degradation of myofibrillar proteins by μ-calpain occurred. The improvements of protein solubility led to the decreased population of free water P₂₃ (r=-0.246, P＜0.05）and increased population of immobilized water P₂₂(r=0.286, P＜0.01). Meanwhile, the exposure of hydrophobic residues resulted in increases of surface hydrophobicity, which was accompanied with decreases of the population of free water P₂₃ (r=0.319, P＜0.01). T₂ relaxation patterns data indicated that protein-associated water was released into the intra-myofibrillar spaces, which was supported by a high correlation between bound water P₂₁ and immobilized water P₂₂ (r=-0.890, P＜0.01). Free water and bound water converted to immobilized water. Therefore, porcine muscle water-holding capacity improved during this stage. During the 120 h to 168 h postmortem, ongoing degradation of myofibrillar proteins gave a rise in MFI values. Decreases of surface hydrophobicity and solubility caused the increased population of free water, and the decreased population of immobilized water. Immobilized water converted to free water. Thereby a flow of water from intra- to extra-myofibrillar water spaces occurred, and it can be lost as drip and water-holding capacity decreased.【Conclusion】The obtained data strongly supported that the postmortem μ-calpain was essential to changes of myofibrillar proteins hydration capacity, which moreover was found to affect the muscle 3 water populations distribution and mobility. During the 24-120 h, μ-calpain contributed to proteolysis of muscle proteins, increased proteins hydration capacity. Bound water and free water converted to immobilized water and porcine water-holding capacity improved. During the 120-168 h, low molecular proteins aggregated because of ongoing degradation, which led to the decreases of proteins hydration capacity. Immobilized water converted to free water, leading to the increases of drip loss.

Key words: porcine, postmortem aging, μ-calpain, myofibrillar proteins, hydration characteristics, water-holding capacity, water mobility

魏秀丽，谢小雷，张春晖，李侠，王春青. 猪宰后肌肉体系中μ-calpain及肌原纤维蛋白理化特性的变化规律[J]. 中国农业科学, 2015, 48(12): 2428-2438.

WEI Xiu-li, XIE Xiao-lei, ZHANG Chun-hui, LI Xia, WANG Chun-qing . The Variations in μ-Calpain and Physico-Chemical Characteristics of Myofibrillar Proteins in Postmortem Porcine Muscle[J]. Scientia Agricultura Sinica, 2015, 48(12): 2428-2438.

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