【作者】 沈瑾；

【导师】 罗欣；

【作者基本信息】 山东农业大学 ， 食品科学与工程， 2013， 博士

【摘要】 嫩度是衡量牛肉食用品质好坏的重要指标。我国的牛肉要想进入世界牛肉主流市场，改变依赖进口优质牛肉的被动局面，应该将提高牛肉嫩度作为推动牛肉产业良性发展的核心任务。牛肉的嫩度除了受肉牛年龄和品种等生理方面的影响外，还受宰后处理方式的影响。电刺激处理对宰后成熟过程中牛肉蛋白质的影响仍是在肉品界的研究热点。电刺激确实提高了宰后肌肉蛋白质的降解速度，但导致其降解的作用机制错综复杂，还需要进一步研究。近年来，蛋白质组学已经成为研究蛋白质的热门方法，尤其是差异蛋白质组学，通过寻找、筛选和鉴定与肉品质性状有相关性的标记蛋白，全面和深入洞察肉品品质的形成机理，为进一步解释肉品间品质差异的根源提供了科学依据。因此，运用蛋白质组学的方法来评估肉品品质具有十分重要的意义。本研究首先测定了牛肉品质指标，优化差异蛋白质组学的双向电泳条件，建立了牛背最长肌双向电泳凝胶体系后，利用这个体系研究了电刺激后牛背最长肌中差异蛋白质，分析鉴定这些蛋白质在电刺激影响牛肉嫩度中的作用机制，探讨机理。主要结果如下：一、电刺激后牛背最长肌食用品质指标测定选择了20头中国黄牛（延边牛×西门塔尔牛），每组10头，分成两组。依据标准工艺屠宰后，一组样品电刺激，另一组样品作为对照。分别测定了宰后2h、6h、24h、72h牛背最长肌的温度、pH值及宰后1d、3d的剪切力值。结果表明，电刺激提高了宰后早期的胴体温度，使pH值快速降低，降低了宰后1d、3d时的剪切力值，提高了嫩度。二、建立牛背最长肌双向电泳技术体系对牛背最长肌总蛋白的提取方法，包括：裂解液，上样量，胶条pH范围，蛋白质定量，染色，样品处理，聚焦条件进行优化，建立了适合牛背最长肌总蛋白提取的方法及双向电泳技术体系。结果显示，提取总蛋白时，选取裂解液中DTT浓度为60mmol/L，直接裂解法提取的蛋白点数量最多且分离效果最好，蛋白质提取后使用双向电泳配套定量试剂盒进行精确定量。由于需要研究的蛋白质范围越广且越清晰越好，双向电泳条件选择上样量为400μg，24cm，pH3-10非线性胶条进行。等电聚焦条件为：50v水化12h，100v2h，200v lh，500v30min，1000v20min，3000v20min，5000v15000vhr，使用考马斯亮蓝染色，简便快速，得到的蛋白点易于处理并进行下一步分析。三、电刺激后牛背最长肌蛋白质组学研究利用PDQuest软件分析宰后1d和3d时电刺激处理牛背最长肌中有显著降解的差异蛋白点，包括凝胶图谱比对、双向电泳分离的重复性及其效果、差异蛋白点凝胶放大及3D图比对，然后对差异蛋白点进行质谱鉴定分析、Real-time PCR及Western blot鉴定分析。结果显示，与未电刺激样品相比，在宰后1d和3d电刺激处理牛背最长肌中有12个蛋白点表达明显下调，分为9种蛋白质：肌间线蛋白（Desmin）、肌钙蛋白-T（Troponin T alpha isoform）、肌球蛋白结合蛋白H（Myosin binding protein H）；肌酸激酶（Creatine kinase，2个）、磷酸丙糖异构酶（Triosephosphate isomerase，2个）；过氧化物氧化还原酶（Peroxiredoxin-6，2个）、磷脂酰乙醇胺结合蛋白（Phosphatidylethanolamine-binding protein）；组蛋白（Histone H3.3-like isoform2）、甲基转移酶（Methyltransferase）。四、电刺激后差异蛋白质影响嫩度的作用机制分析表明，电刺激后9种蛋白质通过四条途径影响电刺激后嫩度变化，具体机制如下：1.电刺激通过糖酵解代谢途径影响嫩度电刺激后代谢酶Creatine kinase和Triosephosphate isomerase表达下调，表明电刺激加快糖酵解途径，Creatine kinase和Triosephosphate isomerase快速消耗，pH值快速下降。2.电刺激通过钙激活中性蛋白酶途径影响嫩度电刺激后骨架蛋白Desmin、Troponin T alpha isoform、Myosin binding protein H表达下调，表明电刺激激活Calpains蛋白酶体系，加速降解骨架蛋白Desmin、TroponinT alpha isoform、Myosin binding protein H，剪切力降低，嫩度提高。3.电刺激通过溶酶体途径影响嫩度电刺激后Peroxiredoxin-6表达下调，表明电刺激后pH不断下降，直到极限pH为5.4左右，肌浆中Ca2+浓度升高，Peroxiredoxin-6主要发挥Ca2+依赖的磷脂酶A2活性，导致溶酶体膜破裂，溶酶体内的水解酶释放，使肌肉蛋白水解，嫩度提高。4.电刺激通过氧化应激途径影响嫩度电刺激影响嫩度的有关氧化应激途径主要有MAPK级联途径和PI3K/Akt信号通路。（1）Peroxiredoxin-6在p38MAPK级联途径中的作用机制Peroxiredoxin-6在pH接近中性时，发挥过氧化物氧化还原酶活性，通过p38MAPK级联途径刺激增殖和抑制细胞凋亡，pH为5.0左右时发挥溶酶体中的磷脂酶A2活性。电刺激后Peroxiredoxin-6表达下调，pH不断下降，直到极限pH为5.4左右，肌浆中Ca2+浓度升高，表明Peroxiredoxin-6主要发挥Ca2+依赖的磷脂酶A2活性，通过溶酶体途径使肌肉蛋白水解，在一定程度上抑制了p38MAPK级联途径，抑制增殖，加速氧化凋亡，嫩度提高。（2）PEBP在Ras-MAPK级联途径中的作用机制电刺激后磷脂酰乙醇胺结合蛋白（PEBP）表达下调。牛屠宰后胴体内产生活性氧，激活Ras-MAPK级联途径，PEBP与Raf-1相互起作用，抑制Ras-MAPK信号传导途径。电刺激后PEBP表达下调，表明电刺激加速PEBP抑制Ras-MAPK级联途径，抑制增殖，加速氧化凋亡，嫩度提高。（3）MT在PI3K/Akt信号通路中的作用机制PI3K/Akt信号转导通路诱导细胞的增殖，避免细胞发生凋亡，并且能够激活甲基转移酶（MT）。而MT表达越低，甲基化水平越低，凋亡越快。电刺激后组蛋白和甲基转移酶下调，表明甲基转移酶表达降低，组蛋白甲基化水平降低，加速凋亡，在一定程度上抑制了PI3K/Akt信号转导通路，抑制增殖，加速氧化凋亡，嫩度提高。电刺激通过氧化应激途径影响嫩度的信号通路表明，牛肉嫩化与氧化、凋亡有密切联系；电刺激在一定程度上抑制增殖，加速氧化和凋亡，加快嫩化。具体的机制还需要进一步的实验验证。更多还原

【Abstract】 Tenderness is an important indicator for evaluation of beef quality. Only by taking beeftenderness improvement as a core task to promote the development of beef industry can wechange the passive situation in which the high-quality beef is dependent on import. Thetenderness of beef is affected not only by physiological impact of cattle age and species, butalso the way of treatment after slaughter. The process of which electrical stimulation effectsprotein changes in postmortem aging is still a hot debate in the meat science. But it is certainthat electrical stimulation does improve the rate of protein degradation in post-mortem muscle.The mechanism of electrical stimulation improving tenderness is complicated and needfurther research. In recent years, proteomics has become a popular way to study proteins,especially differential proteomics. By seeking, screening and identifying marker proteinsrelated to meat quality traits, differential proteomics comprehensive and in-depth insights intothe formation mechanism of meat quality beef. This study provides a scientific basis forfurther explanation of meat quality differences. Therefore, there is a very importantsignificance with proteomics method to assess meat quality.So, in this study, in addition to the determination of beef quality indicators, research ondifferential proteomics was mainly carried out. First, the system of two-dimensionalelectrophoresis gels on M. Longissimus was established, then, the differential proteins wereidentified to analysis the relations to tenderness in this system.The main results are as follows:1. Electrical stimulation influences meat quality indexs of bullsTwenty Chinese yellow crossbred bulls were selected and dividied into two groups inwhich one group was treated with electrical stimulation and slaughtered according to standardprocess. After electrical stimulation early in postmortem in the conditioning room, thecarcasses were transferred to the cutting room, and M. Longissimus were excised from thecarcasses. The indexs of temperature, pH (2h、6h、24h、72h) and shear force (1d,3d) were determined.The results showed that early in postmortem, electrical stimulation raised thetemperature, accelerated the decreasing rate of pH, reduced shear force and improvedtenderness.2. The establishment of proteomic method in M. longissimusThe methods of total protein extraction of Chinese Yellow crossbred bulls wereoptimized including lysis, loading quantity, pH range of IPG strips, protein quantification kit,dyeing method, extracting method of total protein, focusing conditions to establish theoptimum muscle protein extraction method and proteomic method in M. longissimus.The results showed that using60mmol/L as concentration of DTT in lysates and directlysis method, protein extraction is highly efficient. Due to get the wider range of proteins, theloading sample was400μg. The procedures of isoelectric focusing were24cm, pH3-10non-linear strip, CBB staining method and50v12h,100v2h,200v lh,500v30min,1000v20min,3000v20min,5000v15000vhr.3. Proteomic analysis in M. Longissimus as a result of electrical stimulationDifferential proteins were identified with PDQuest software by analyzing two-dimensional electrophoresis map to find degradated proteins markedly, including gel mapcomparison, repeatability and resolution of two-dimensional electrophoresis, differential spotsgel comparison and3D map comparison. Then the differential protein spots were identifiedand analyzed by mass spectrometry, Real-time PCR and Western blot.The results showed that twelve protein spots were down-regulated significantly at1d and3d postmortem as a result of electrical stimulation. These proteins were identified as ninetypes: Desmin, Troponin T alpha isoform, Myosin binding protein H, Creatine kinase (two),Triosephosphate isomerase (two), Peroxiredoxin-6(two), Phosphatidylethanolamine-bindingprotein, Histone H3.3-like isoform2, Methyltransferase.4. The mechanism of differential proteins as a result of electrical stimulation on tendernessThe analysis showed that proteins of nine types affect tenderness as a result of electricalstimulation through four pathways.(1) The glycolytic metabolic pathway affecting tenderness as a result of electrical stimulationMetabolic enzymes (Creatine kinase and triosephosphate isomerase) were down- regulated as a result of electrical stimulation. This indicated that electrical stimulationaccelerated the glycolytic metabolic pathway, Creatine kinase and triosephosphate isomerasewere consumed rapidly, pH value declined rapidly and finally improved tenderness.(2) The Calpains pathway affecting tenderness as a result of electrical stimulationCytoskeletal proteins (Desmin, Troponin T alpha isoform, myosin binding protein H) weredown-regulated as a result of electrical stimulation. This indicated that electrical stimulationaccelerated the Calpains pathway, Desmin, Troponin T alpha isoform, myosin binding proteinH were degradated rapidly, shear force declined rapidly and finally improved tenderness.(3) The lysosomal pathway affecting tenderness as a result of electrical stimulationPeroxiredoxin-6was down-regulated as a result of electrical stimulation. This indicatedthat electrical stimulation declined pH value rapidly until about5.4, raised sarcoplasmic Ca2+concentration and peroxiredoxin-6played a Ca2+-dependent phospholipase A2activity,resulted in the rupture of the lysosomal membrane, the release of hydrolytic enzymes, thehydrolysis of muscle proteins, finally improved tenderness.(4) The oxidative stress affecting tenderness as a result of electrical stimulationThe oxidative stress pathways of tenderization as a result of electrical stimulation mainlycomprises MAPK cascade pathway and PI3K/Akt signal pathway.1) The mechanism of peroxiredoxin-6in p38MAPK cascade pathway as a result of electricalstimulationPeroxiredoxin-6plays a peroxide oxidoreductase activity when pH is about neutral,which stimulates the proliferation and inhibition of apoptosis through p38MAPK cascadepathway. Peroxiredoxin-6was down-regulated as a result of electrical stimulation and thisindicates that electrical stimulation declined pH value rapidly until about5.4, raisedsarcoplasmic Ca2+concentration and peroxiredoxin-6played a Ca2+-dependent phospholipaseA2activity, resulted in the hydrolysis of muscle proteins and finally improved tenderness.So,electrical stimulation inhibited p38MAPK cascade pathway at some extent, inhibitedproliferation, accelerated oxidation and apoptosis and finally accelerated meat tenderization.2) The mechanism of PEBP in Ras-MAPK cascade pathway as a result of electricalstimulationPEBP was down-regulated as a result of electrical stimulation. Reactive oxygen species produced in the carcass after slaughter and activated Ras-MAPK cascade pathway. PEBPintegrated Raf-1and inhibited Ras-MAPK cascade pathway. This indicated that electricalstimulation accelerated PEBP inhibiting Ras-MAPK cascade pathway, inhibited proliferation,accelerated oxidation and apoptosis and finally improved tenderness.3) The mechanism of MT in PI3K/Akt signal pathway as a result of electrical stimulationPI3K/Akt signal transduction pathway induces proliferation, avoids apoptosis andactivates methyltransferase (MT). However, the lower expression of MT is, the lowermethylation is, the faster apoptosis is. Histone and methyltransferase were down-regulated asa result of electrical stimulation and this indicated that the expression of MT declined, thelevel of histone methylation was declined and accelerated apoptosis. So, electrical stimulationinhibited PI3K/Akt signaling pathway at some extent, inhibited proliferation, acceleratedoxidation and apoptosis and finally accelerated meat tenderization.The oxidative stress pathways of electrical stimulation indicate that meat tenderness isclosely related to oxidation and apoptosis, electrical stimulation inhibites proliferation,accelerates oxidation and apoptosis and finally accelerates meat tenderization. Specificmechanisms need to be further verified by experiments.更多还原