钙蛋白酶系统与肌肉嫩度的关系

钙蛋白酶系统主要由钙蛋白酶(μ-calpain,m-calpain)及钙蛋白酶抑制蛋白(calpastatin)组成,calpain是存在于细胞质中的依赖于Ca2+的中性蛋白酶,calpastatin是钙蛋白酶的内源抑制蛋白。本文综述了钙蛋白酶系统各种酶的结构、作用、活性调节机能及其与肉质嫩度的关系。     

钙蛋白酶系统及其对肉嫩度的影响

钙蛋白酶系统作为影响肉嫩度的重要因素引起了科研人员的广泛关注。钙蛋白酶系统是高度可调的复杂系统，包括钙激活酶、钙激活酶抑制蛋白和钙激活酶激活蛋白，三者相互作用，共同调控宰后肉的嫩化。本文综述了钙蛋白酶系统的结构及功能，并在此基础上阐述了其影响肉嫩度的作用机理。     

甘肃高山细毛羊肉宰后钙激活蛋白酶活性变化的研究

为研究甘肃高山细毛羊宰后钙激活蛋白酶活性的变化,以肃南高山细毛羊为研究对象、当地藏羊为对照,对其钙激活蛋白酶活性进行提取、测定。通过分析和研究,结果表明:在宰后最初钙激活酶活性值均很低,随着宰后时间的延长,细毛羊和藏羊的钙激活蛋白酶活性值都随之增大。细毛羊的最大活性值最终达到0.601,藏羊最终达到0.708,说明最终藏羊肉的嫩度要好于甘肃高山细毛羊肉。     

钙蛋白酶系统对肉的嫩化作用研究进展

肉的嫩度是肉品质的一个重要指标,严重影响肉的食用价值和经济价值.钙蛋白酶系统通过水解肌原纤维蛋白,在宰后肌肉嫩化中起着决定性的作用.综述钙蛋白酶系统(包含钙蛋白酶、钙蛋白酶抑制蛋白和钙蛋白酶激活蛋白3部分)对肉嫩化的作用机制和研究现状,并探讨研究前景.     

滩羊肉嫩度形成中Ca2+的信号转导作用

以6月龄滩羊背最长肌为研究对象，测定4 ℃贮藏0，96，192 h时剪切力、线粒体Ca2+水平、钙蛋白酶-1及细胞凋亡效应酶-3活性。基于同位素标记相对和绝对定量技术(iTRAQ)结合多维液相色谱-串联质谱(LC-MS/MS)技术，研究贮藏期间Ca2+信号通路及凋亡调节信号转导通路蛋白质表达量的变化情况。结果表明:4 ℃贮藏期间，滩羊肉剪切力先降低后升高，线粒体Ca2+含量先升高后降低，钙蛋白酶-1活性持续降低，细胞凋亡效应酶-3活性先上升后下降。滩羊肉剪切力与Ca2+水平呈显著负相关，与钙蛋白酶-1活性呈极显著正相关，与细胞凋亡效应酶-3活性呈极显著负相关。蛋白质组学分析表明:Ca2+跨膜转运ATP酶利用ATP水解的能量驱动Ca2+跨膜运输，电压依赖性阴离子通道蛋白贮藏初期上调表达，为Ca2+流入线粒体提供渠道，导致线粒体Ca2+超载、线粒体功能紊乱，使线粒体复合体表达量上升。同时，VDAC异常表达促使CYTC释放至胞浆，最终激活细胞凋亡效应酶-3，水解结构蛋白质。另外，Ca2+可间接导致肌动蛋白表达量下调，引起细胞收缩，从而影响肉的嫩度。本研究结果证实Ca2+在肉品嫩度形成过程中起重要的信号转导作用。     

μ-Calpain和钙离子对牛肉肌原纤维部分骨架蛋白的影响

基于有关牛肉嫩化机制的钙激活酶、Ca++、溶酶体组织蛋白酶理论,本试验模拟尸僵后牛肉的内在环境和成熟温度,将纯化后的钙激活酶Ⅰ(μ-Calpain)、内源性钙激活酶Ⅰ专一抑制剂(Calpastatin)、肌原纤维和钙激活酶Ⅰ外源性抑制剂抑亮酶肽(Leupetin)用于6个不同的处理组合,反应不同时间后,分别做变性聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹分析(Western-blotting).结果表明,Ca++(100μM)对肌原纤维没有降解作用,而在含Ca++(100μM)的反应体系中,μ-Calpain对肌间线蛋白(Desmin)和肌钙蛋白T(Troponin-T)有明显的降解作用,且降解产物和宰后牛肉自然成熟条件下的降解产物类似;Calpastatin不能完全抑制μ-Calpain的活性;离体反应和自然成熟的牛肉中的肌动蛋白都没有发生变化.以上结论表明Ca++很可能是通过激活μ-Calpain而间接发挥对肌原纤维的降解作用,而溶酶体组织蛋白酶在牛肉成熟的早期对嫩度的提高贡献不大.     

内源蛋白酶在肉嫩化中的作用(综述)

本文阐述了与肉成熟有关的两类蛋白酶,即钙激活蛋白酶和溶酶体组织蛋白酶,并分别就两类酶的分类、影响酶活性的因素、在肉嫩化中的作用、分离提纯及活性测定方法等作了介绍.     

钙离子对钙激活蛋白酶mRNA表达、活性及其对肌肉蛋白降解的影响

本实验分别以牛肉肌束和分化为肌细胞的L6成肌细胞为研究对象,研究钙离子对离体肌束中钙激活蛋白酶活性、蛋白分解以及活体肌肉(肌细胞)中钙激活蛋白酶mRNA水平表达的影响。结果表明500μmol/L以内的钙离子处理肌束,可以提高肌束中μ-钙激活蛋白酶的活性,当高于此浓度时,μ-钙激活蛋白酶因发生自动降解而失活。但是钙离子对m-钙激活蛋白酶活性影响不大,只有钙离子浓度达到10mmol/L时才会导致自动降解而失活。钙离子可以加速肌肉中蛋白质的降解,且浓度越高,降解越明显;钙离子处理活体肌细胞,在没有产生明显细胞死亡的情况下,可导致μ-钙激活蛋白酶和m-钙激活蛋白酶mRNA水平表达的增加。由本研究的结果初步推断钙离子不仅可以激活钙激活蛋白酶的酶活性,提高分解肌肉蛋白的能力,在刚刚屠宰的肉(肌肉未死亡)中进行钙离子嫩化也可以导致钙激活蛋白酶表达量的增加。     

宰后成熟对牛肉品质的影响

本文通过对牛肉中粗钙激活因子（依钙蛋白酶CAF）的活性及肌原纤维小片化指数、胶原含量的测定,说明牛肉中依钙蛋白酶的活性随宰后时间的延长面下降,直影响到肉嫩度的变化、成熟时间越长,肌原纤维小片化程度越高,肌原纤维结构破坏越来在重,越有利于提高肉的嫩度。     

钙蛋白酶系统磷酸化对肉嫩度的影响研究进展

嫩度是肉品食用品质中最重要的品质特性之一,钙蛋白酶系统在宰后成熟过程中对改善肌肉嫩度起到主要作用。研究表明,磷酸化修饰参与钙蛋白酶系统复杂的活性调控过程。本文概述了μ-/m-钙蛋白酶和钙蛋白酶抑制蛋白磷酸化研究的最新进展,根据它们的磷酸化对其结构和功能的影响,探讨钙蛋白酶系统磷酸化修饰对宰后肉嫩化的作用机制,并对其在肉品质中的应用研究进行展望。     

钙激活酶激活蛋白cDNA的克隆与序列分析

钙激活蛋白酶（calpain）是动物宰后导致肉嫩化的主要蛋白酶。促进calpain活性的发挥，从而提高肉的嫩度，一直是肉类科学研究工作者的重要研究课题。最新的研究表明，钙激活酶激活蛋白（calpain activator）－钙激活蛋白酶（calpain）系统的一个新成员，是calpain活性的主要调节者。Calpain activator可以激活calpain，使其活性提高大约400倍。利用分子生物学方法克隆calpain activator,为生物肉嫩化技术奠定基础，是食品领域应用生物技术提高生产效率的又一新的课题。本文利用PCR方法克隆了calpain activator cDNA序列，并对其进行了序列分析。结果表明，该片段长1017bp,5‘－端非翻译区长39bp,3‘－端非翻译区长567bp。编码区长411bp，编码一个长137个氨基酸残基的蛋白质，其理论分子量为14298Da。与Edon Melloni et.al1998年报道的从牛脑中提取纯化的钙激活酶激活蛋白的氨基酸序列相比发现。二者仅有五个位点不同。     

Contribution of postmortem muscle biochemistry to the delivery of consistent meat quality with particular focus on the calpain system

Tenderness has been repeatedly reported as the most important quality aspect of meat. However, a number of studies have shown that a significant portion of retail meat can be considered tough. As a consequence, a significant consumer segment is willing to pay a premium for guaranteed tender meat. However, apart from measuring the shear force, there is no reliable method to predict tenderness. Most of the branded meat programs therefore attempt to ensure eating quality by controlling some of the factors that affect tenderness. Meat tenderness is determined by the amount and solubility of connective tissue, sarcomere shortening during rigor development, and postmortem proteolysis of myofibrillar and myofibrillar-associated proteins. Given the effect of postmortem proteolysis on the muscle ultrastructure, titin and desmin are likely key substrates that determine meat tenderness. A large number of studies have shown that the calpain proteolytic system plays a central role in postmortem proteolysis and tenderization. In skeletal muscle, the calpain system consists of at least three proteases, μ-calpain, m-calpain and calpain 3, and an inhibitor of μ- and m-calpain, calpastatin. When activated by calcium, the calpains not only degrade subtrates, but also autolyze, leading to loss of activity. m-Calpain does not autolyze in postmortem muscle and is therefore not involved in postmortem tenderization. Results from a number of studies, including a study on calpain 3 knockout mice, have shown that calpain 3 is also not involved in postmortem proteolysis. However, a large number of studies, including a study on μ-calpain knockout mice, have shown that μ-calpain is largely, if not solely, responsible for postmortem tenderization. Research efforts in this area should, therefore, focus on elucidation of regulation of μ-calpain activity in postmortem muscle. Discovering the mechanisms of μ-calpain activity regulation and methods to promote μ-calpain activity should have a dramatic effect on the ability of researchers to develop reliable methods to predict meat tenderness and on the meat industry to produce a consistently tender product.     

Calpain3的初步提取和对其自动降解性的研究

骨骼肌特异性的Calpain3(又称Ncl-1、p94、CAPN3)是除μ-Calpain和m-Calpain以外,另一个可能与肉类嫩化有关的Calpains成员。由于Calpain3具有很强的自动降解性,使得对其的提取纯化和依钙性的研究很难进行。本实验从牛肉背最长肌中提取到了具有一定活性的Calpain3粗酶液,通过蛋白质免疫印迹法,对Calpain3的随时间的降解变化进行了研究,并对不同Ca2+浓度下Calpain3自动降解的变化进行了比较系统的探讨,研究发现Calpian3的自动降解分为四个连续性的过程:首先降解为89、68和58kDa的中间片断,然后进一步降解为55kDa的片断并在一定时间内保持不变,其降解的速度与Ca2+浓度和反应时间成正比。     

钙激活酶与肌肉嫩化的研究

宰后肌肉嫩度的变化是在多种酶的协同作用下完成的,其中一个重要部分是钙激活酶,主要论述了钙激活酶对肌肉嫩化的作用机理、钙激活酶嫩化模型、氯化钙与肉的嫩化和影响钙激活酶活性的因素等方面的内容。     

Protease activity higher in postmortem water buffalo meat than Brahman beef

We previously demonstrated that postmortem water buffalo meat had higher tenderness than Brahman beef. In order to explain this difference in tenderness, the objective of the current study was to investigate the protease activity in these two meats. Five female crossbred water buffalo (Philippine Carabao×Bulgarian Murrah) and five female crossbred cattle (Brahman×Philippine Native) were slaughtered at 30months of age, followed by immediate sampling of Longissimus thoracis muscle for measurement of protease activity. Results showed that buffalo meat had significantly higher protease activity compared to beef (P<0.05). Furthermore, calpain inhibitor 1, a specific inhibitor of calpains 1 and 2, was the most effective inhibitor of protease activity. There was no difference in calpastatin activity, and no major differences were observed in calpains 1, 2, and calpastatin expression by Western blotting. This study suggests that higher calpain activity in early postmortem buffalo meat was responsible for the increased tenderness of water buffalo meat compared to beef.     

Effects of nitric oxide and oxidation in vivo and postmortem on meat tenderness

Metabolic processes in muscle tissue in vivo result in the production of reactive oxygen species and oxidative compounds including superoxide anions and nitric oxide (NO). Reactive oxygen species can react with both lipids and proteins and often have deleterious effects, contributing to the onset of ageing and senescence as well as cell death. Nitric oxide (NO) is a free radical that is constantly produced or released throughout the body by diverse tissues and is known to influence proteolytic activity in human and rodent skeletal muscle as well as being involved in regulation of calcium homeostasis in the muscle cell. The influence of nitric oxide on development of meat tenderness has been studied through postmortem manipulation and also through in vivo studies. The effect of NO on meat tenderness is postulated to be via its regulatory effects on the proteins calpain, cathepsins, ryanodine receptor channel in the sarcoplasmic reticulum (SR) and the sarcoplasmic-endoplasmic release calcium ATPase in the SR. NO is an oxidant although the effects of NO on effector proteins can be distinguished from a direct oxidation reaction. The onset of oxidation in meat postmortem is well known to produce off-odours, discolouration and unacceptable flavours associated with rancidity. Oxidation during the immediate postmortem period appears to inhibit tenderisation during ageing, probably through an inhibitory effect of oxidation on the calpain enzyme. Oxidation of muscle tissue occurring as a result of availability of oxygen during modified atmosphere packaging may also have deleterious consequences for tenderness development during storage of meat prior to retail display. In conclusion, it is proposed that postmortem meat tenderisation is influenced by skeletal muscle's release of NO pre-slaughter and the oxidation of proteases postmortem. This proposal is compatible with the existing tenderness model and will hopefully assist in increasing the accuracy of prediction of meat tenderness. Future directions for research are discussed.     

不同年龄牦牛宰后钙激活酶活性及嫩度指标变化

本研究对不同年龄牦牛宰后成熟过程中钙激活酶活性和嫩度指标（肌纤维直径、肌原纤维小片化指数（myofibrillar fragmentation index,MFI）、肌原纤维超微结构）进行测定。结果表明：成熟过程中,不同年龄牦牛肉肌纤维直径、MFI都发生显著变化（P＜0.05）,肌原纤维在成熟过程中Z线断裂,框架结构被完全破坏;钙激活酶活性在宰后的前2 d均显著下降（P＜0.05）,且成熟过程中不同年龄公牦牛的钙激活酶活性均高于母牦牛;成熟时间与钙激活酶活性、嫩度指标均成极显著相关性（P＜0.01）;牦牛宰后经过7 d的成熟,可以降低牦牛因屠宰年龄不同而产生的差异,同时也可以改善肉的嫩度;与公牦牛相比母牦牛宰后成熟时间可以适当的延长。     

电刺激对牛背最长肌中钙激活酶活性及嫩度的影响

通过对中国杂交黄牛(鲁西黄牛×西门塔尔)牛背最长肌中Calpains活性及剪切力值的分析,研究了电刺激对牛背最长肌宰后成熟过程中Calpains活性及牛肉嫩度的影响。结果表明:电刺激显著提高了μ-calpain的活性(P<0.05),降低了calpastatin对μ-calpain的抑制作用,提高了牛背最长肌的嫩度,缩短了牛肉的成熟时间;但电刺激仅在宰后成熟1h,显著提高了m-calpain的活性(P<0.05);宰后成熟24h、3d,与对照组之间差异不显著(P>0.05),试验结果证实,电刺激通过对Calpains体系的影响,改变了牛肉的成熟速度,显著的改善了牛肉的嫩度。