冰温贮藏对羊肉中蛋白质磷酸化水平的影响

doi:10.3864/j.issn.0578-1752.2016.22.015

Abstract

Abstract: 【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 12h 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 kDa 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 kDa 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.

Key words: controlled freezing point, protein kinase; sarcoplasmic proteins, myofibrillar proteins, protein phosphorylation

ZHANG Yan, LI Xin, LI Zheng, LI Meng, LIU Yong-feng, ZHANG De-quan. Effects of Controlled Freezing Point Storage on the Protein Phosphorylation Level[J].Scientia Agricultura Sinica, 2016, 49(22): 4429-4440.

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