冰温贮藏对羊肉中蛋白质磷酸化水平的影响

Effects of Controlled Freezing Point Storage on the Protein Phosphorylation Level

【目的】通过测定冰温贮藏及冷藏过程中肌肉肌浆蛋白与肌原纤维蛋白的磷酸化水平,分析冰温贮藏对蛋白质磷酸化水平的影响,为肉类冰温贮藏品质调控提供理论依据。【方法】选取大尾寒羊与小尾寒羊杂交公羊的背最长肌于冷藏和冰温条件下成熟,分别在0.5 h、12 h、24 h、3 d、5 d、9 d取样测定蛋白激酶活性、肌浆蛋白与肌原纤维蛋白的磷酸化水平。采用蛋白激酶活性测定试剂盒、运用酶联免疫的方法检测各处理组蛋白激酶的活性随贮藏时间的变化;通过SDS-PAGE电泳、荧光染色的方法得到全蛋白染色与磷酸化染色的肌浆蛋白与肌原纤维蛋白条带,运用Quantity One软件分析各处理组各处理时间的肌浆蛋白及肌原纤维蛋白的磷酸化水平。【结果】贮藏初期(0.5—12 h),冰温组蛋白激酶活性显著升高(P<0.05),冷藏组蛋白激酶活性则显著降低(P<0.05),贮藏12 h—9 d过程中,冰温组蛋白激酶活性均显著高于冷藏组(P<0.05)。两处理组蛋白激酶活性均随贮藏时间的延长而逐渐降低,且冰温组蛋白激酶活性均显著高于冷藏组(P<0.05)。对肌浆蛋白染色效果图中分布于15—250 k Da的17个蛋白条带逐一进行分析,结果表明贮藏温度极显著影响肌浆蛋白各蛋白条带的磷酸化水平(P<0.001)。冷藏组肌浆蛋白整体磷酸化水平先升高后降低,贮藏第3天达到最大值,冰温组肌浆蛋白整体磷酸化水平先降低后升高,最小值出现在贮藏第24天,贮藏第12天至3天时,冷藏组肌浆蛋白整体磷酸化水平高于冰温组(P<0.05),贮藏第9天时,冰温组肌浆蛋白整体磷酸化水平高于冷藏组(P<0.05)。对肌原纤维蛋白染色效果图中分布于15—250 k Da的20个蛋白条带逐一进行分析,结果表明不同贮藏温度对所有肌原纤维蛋白条带的磷酸化水平均产生极显著影响作用(P<0.001)。两处理组肌原纤维蛋白整体磷酸化水平均随贮藏时间的延长呈现先升高后降低的趋势,冷藏组与冰温组最大值分别出现在24 h和12 h,贮藏初期(0.5—12 h),两处理组间肌原纤维蛋白整体磷酸化水平无显著差异(P<0.05),贮藏24 h至贮藏期结束,冷藏组肌原纤维蛋白整体磷酸化水平始终高于冰温组(P<0.05)。【结论】冰温贮藏通过影响蛋白激酶的活性来调控蛋白质的磷酸化水平,进而通过影响糖酵解途径、肌肉收缩及骨架蛋白降解来间接调控肉品质。

【Objective】 The aim of this study was to investigate the effect of controlled freezing point on protein phosphorylation level, so as to provide a new theoretical basis for controlling meat quality by controlled freezing point. 【Method】 The longissimus doris（LD） of large tail×small tail crossed sheep were obtained and divided into two groups： refrigeration group and controlled freezing point group. Each LD was assigned to 6 storage times（0.5 h, 12 h, 24 h, 3 d, 5 d and 9 d）. Kit for protein kinase activity measurement and enzyme linked immunosorbent assay were applied to detect protein kinase activity. A gel-based phosphoproteomic study was performed. And SDS-PAGE electrophoresis and fluorescence staining were applied to gain the protein bands of sarcoplasmic proteins and myofibrillar proteins stained by SYPRO Ruby and Pro-Q Diamond. A software named Quantity One was used to analyze the protein phosphorylation level by measuring the fluorescence intensity.【Result】 At 12 h, the protein kinase activity of controlled freezing point group significantly increased（P〈0.05） and that of refrigeration group significantly decreased. The protein kinase activity of the two groups were reduced from 12 h to 9 d, which was significantly higher in controlled freezing group compared with refrigeration group（P〈0.05）. Seventeen protein bands distributed from 15-250 k Da in each sarcoplasmic protein sample were analyzed and the results suggested that storage temperature influenced（P〈0.001） the protein phosphorylation level of all sarcoplasmic proteins significantly. The total phosphorylation level of sarcoplasmic proteins in refrigeration group increased at first and then decreased while the sarcoplasmic proteins in controlled freezing point group showed an opposite variation trend. The phosphorylation level of 3 d was the highest, while the lowest was 24 h. Globally, the sarcoplasmic proteins of refrigeration group had the higher total phosphorylation level than controlled freezing point group during 12 h to 3 d. However, the total phosphorylation level of the sarcoplasmic proteins of controlled freezing point was higher than refrigeration group on 9 d. Twenty protein bands distributed from 15-250 k Da in each myofibrillar protein sample were analyzed and the results suggested storage temperature influenced the protein phosphorylation level of all myofibrillar proteins very significantly（P〈0.001）. The total phosphorylation level of myofibrillar proteins increased firstly and then decreased in two groups. And the highest phosphorylation level of 24 h in refrigeration group and 12 h in controlled freezing point group was the highest. Total phosphorylation level of myofibrillar protein had no significant difference between two groups from 0.5 h to 12 h. However, total phosphorylation level of myofibrillar protein was higher in refrigeration group compared with controlled freezing group from 24 h to the end of storage.【Conclusion】 The controlled freezing point storage regulated the phosphorylation level of protein by affecting the activity of protein kinase, which can indirectly regulate the meat quality by influencing the glycometabolism, muscle contraction and degradation of skeleton protein.