Article pubs.acs.org/JAFC
Phosphorproteome Changes of Myofibrillar Proteins at Early Postmortem Time in Relation to Pork Quality As Affected by Season Xiao Li,† Tian Fang,† Menghuan Zong,† Xiaoqin Shi,† Xinglian Xu,† Chen Dai,‡ Chunbao Li,*,† and Guanghong Zhou† †
Key Laboratory of Meat Processing and Quality Control, MOE; Key Laboratory of Animal Products Processing, MOA; and Jiangsu Synergetic Innovation Center of Meat Production, Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China ‡ Experimental Teaching Center of Life Science, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China S Supporting Information *
ABSTRACT: The effect of season on phosphorylation of myofibrillar proteins and meat quality of pork longissimus muscles was investigated. Muscle samples were obtained from 40 pork carcasses (10 for each season) at 45 min and 3 and 9 h post-mortem. Myofibrillar proteins were extracted, separated by SDS-PAGE, quantified by phosphor-specific staining, and finally identified by LC-MS/MS. Muscle pH, glycogen, and ATP were measured, and pale, soft, and exudative (PSE) meat was identified by pH value at 45 min post-mortem. A total of 23 bands were detected on SDS-PAGE gels. The phosphorylation levels of bands did not differ between PSE and normal meat. However, the phosphorylation levels of 22 bands were significantly changed by season. Nine of them showed different changes from 45 min to 9 h post-mortem, which were identified to be involved in energy metabolism and sarcomere contraction. Correlation analysis indicated the regulatory progress of these proteins during rigor mortis. These observations contribute to a better understanding of the biochemical processes for the conversion of muscle to meat varying with season. KEYWORDS: pork, myofibrillar proteins, protein phosphorylation, rigor mortis, season
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INTRODUCTION Myofibrillar proteins are the main contributors to muscle contraction during rigor mortis. Early in post-mortem, the depletion of ATP and the release of Ca2+ from the sarcoplasmic reticulum lead muscles to continuous contraction and increase the toughness of muscles.1 Anaerobic glycolysis is the only way to produce ATP in post-mortem muscles accompanied by lactate accumulation and pH decline early in post-mortem, which affect enzyme activity and protein degradation.2,3 Protein phosphorylation regulates critical biological processes.4 In recent years, with the development of phosphorproteome methodology, myofibrillar proteins have been widely studied in various kinds of muscles. In porcine muscles, the phosphorylation of myofibrillar proteins has been shown to be associated with rigor mortis and meat tenderization.1,5,6 In bovine muscle, Li et al.7 observed that electrical stimulation could accelerate the phosphorylation of myofibrillar proteins and meat tenderization. In ovine muscles, Chen et al.8 observed that the phosphorylation of some myofibrillar proteins had a significant effect on meat tenderness. Season has been shown to affect meat quality attributes, including pH decline rate and water-holding capacity.9−11 Temperature change during season transitions may induce in pigs heat or cold stresses by overexpression of some chaperones12 and finally produce PSE meat.13 In eastern China, pigs are commonly housed in pens without the maintenance of air temperature or directly outranged.14 The air temperature changes from −4 °C in winter to 40 °C in summer. As a result, the incidence of PSE meat is higher in summer than in other seasons.15,16 This could © 2015 American Chemical Society
be related to energy metabolism in live animals or post-mortem muscle. The objective of the present study was to investigate the phosphorylation of myofibrillar proteins of pork longissimus muscle in different seasons and its significance for PSE meat.
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MATERIALS AND METHODS
Sampling. A total of 40 native black pigs with the same genetic backgrounds and feeding regimens were randomly selected and slaughtered in a local slaughterhouse in January (winter), March (spring), July (summer), and November (autumn). At each time point, 10 pigs of 150−170 days old were slaughtered and longissimus lumborum (LL) muscles were removed across the last three lumbar vertebrae at 30 min post-mortem. Muscle pH values were measured in triplicate at 45 min and 3 and 9 h post-mortem by using a portable pH meter (Orion Star, Thermo). Muscle samples were divided into two groups by the initial pH values (pH45 min), that is, group A, pH45 min > 6.0, and group B, pH45 min < 6.0.9,17,18 The LL samples were packaged and kept at room temperature (18 °C) until 3 h postmortem to avoid cold shortening and then transferred to a 4 °C chiller. At 45 min and 3 and 9 h post-mortem, about 10 g of muscle samples was taken and immediately snap-frozen in liquid nitrogen and then stored at −80 °C for analyses of SDS-PAGE, glycogen, and ATP. In total, 120 samples were analyzed. Glycogen and ATP Determination. Glycogen content was determined in triplicate using a commercial kit (Jiancheng Bioengineering, Nanjing, China). Briefly, 90 mg of samples was mixed with 3-fold Received: Revised: Accepted: Published: 10287
August 18, 2015 October 12, 2015 November 8, 2015 November 8, 2015 DOI: 10.1021/acs.jafc.5b03997 J. Agric. Food Chem. 2015, 63, 10287−10294
Article
Journal of Agricultural and Food Chemistry Table 1. Summary of pH Value, Glycogen, and ATP Contents in Post-mortem Pork Grouped by Seasona p values PTb 45 min 3h 9h
spring 6.13 ± 0.34a 5.83 ± 0.19bB 5.50 ± 0.12c
summer 6.10 ± 0.29a 5.88 ± 0.25bAB 5.53 ± 0.17c
autumn 6.11 ± 0.25a 5.91 ± 0.20bAB 5.59 ± 0.08c
winter 6.28 ± 0.27a 6.07 ± 0.26bA 5.69 ± 0.14c
season 0.005
PTb
