Białka mięśniowe jako wskaźniki jakości mięsa wieprzowego wychładzanego ze zróżnicowaną szybkością oraz mięsa bydła z wadą DFD
Authors: Mikołajczak Beata 
Published: March 2024

Introduction Meat is a major source of protein in the human diet. It has a high nutritional value due to the presence of all exogenous amino acids and bioactive peptides. In order to meet consumers’ increasing demands the meat industry attempts to provide excellent quality products characterised by optimal water-holding capacity and tenderness. Muscle proteins undergo significant changes during post-slaughter glycolysis. The rate of these changes is determined by various factors, especially by the cooling process and the occurrence of the DFD defect. The rate of the cooling process is a significant factor necessary to obtain meat of high, repeatable quality and to ensure its durability. Pre-slaughter stress factors such as variable ambient temperature and glucose deficiency are indicated as the main causes of the DFD quality defect. The author of the study assumed that proteins reflect changes occurring during the conversion of muscles into meat, that they may be potential indicators of its quality, and that they determine water-holding capacity and tenderness. There are few scientific publications providing data on changes in pork proteins depending on the rate of the cooling process and the occurrence of the DFD quality defect in beef. The aim of the study was to analyse muscle proteins as potential quality indicators of pork cooled at various rates and of beef with DFD defects, with a special focus on their influence on the water-holding capacity and tenderness of the meat. Material and methods The study was conducted on the longest thoracic and lumbar muscle (musculus longissimus thoracis et lumborum – LTL) of pigs and cattle. The pork cooling process was conducted at different rates: A – 0.12◦C/min, B – 0.15◦C/min, C – 0.27◦C/min. The following measurements were made: the pH value, electrical conductivity, glycogen and lactic acid content, the weight loss during storage and after thermal treatment. The size of the centrifugal drip as well as the shear force and work were assessed. One-dimensional electrophoresis was applied to separate proteins in SDS-polyacrylamide gels (SDS-PAGE). Selected pork proteins were identified with the western blot method. The beef of normal RFN quality and with a DFD defect was assessed on the basis of its pH value, colour, water-holding capacity, and tenderness. Extract proteins were analysed with SDS-PAGE electrophoresis and high-resolution tandem mass spectrometry coupled with high performance liquid chromatography UHPLC-Q-TOF-MS/MS. Results The pork cooled at different rates was characterised by a normal course of post mortem glycolysis. Meat samples B (0.15◦C/min) had the lowest pH at all terms of analysis and the lowest content of glycogen after 2 h but the highest content of lactic acid (2 h). The cooling at a rate of 0.27◦C/min induced shrinkage, which resulted in a lower quality of pork, manifested by reduced water-holding capacity and tenderness. The electrophoretic analysis of the meat protein profile revealed that samples B had a significantly higher share of the myosin band after 24 h and the actin band after 6 days than in samples C, which were cooled at the fastest rate. After 6 days of storage there was a higher share of two bands with molecular weights of 148-153 kDa (amylo-alpha-1,6-glucosidase) and ∼47 kDa (creatine kinase/phosphoglycerate kinase) in the centrifugal drip fraction of sample C. The Western blot analysis of meat proteins showed that the cooling process caused significant changes in the content of titin, myosin, and troponin T at all terms of analyses. The share of titin, troponin T, and GAPDH in the centrifugal drip fraction was significantly influenced by the pork cooling rate. The beef with a DFD deviation was characterised by a significantly higher pH, darker colour, better tenderness, and water-holding capacity than the RFN samples. The results of the electrophoretic analysis enabled distinction between the RFN and DFD meat on the basis of the protein profile. In comparison with the RFN meat samples, the meat with the DFD defect was distinguished by the presence of a band with a mass of 2,400 kDa and a significantly higher share of protein bands within the ranges of 1,200-400 kDa and ∼68 kDa. The analysis and then the identification of proteins and peptides in the meat extracts of different quality with mass spectrometry coupled with ultra-high-performance liquid chromatography (UHPLC-Q-TOF-MS/MS) enabled the identification of proteins as potential quality indicators of the beef with DFD defects. Conclusions The research showed that the quality and profile of pork proteins were influenced by the cooling process. The cooling of samples C at a rate of 0.27◦C/min resulted in meat shrink, which significantly deteriorated the water-holding capacity and tenderness of the meat. It is recommended to use the maximum cooling rate of 0.15◦C/min to ensure the desirable quality traits of pork. Myosin heavy chains (MHC) are particularly important in explaining the causes of cooler shrink. Bands with a mass greater than 250 kDa, which were observed as early as after 45 minutes post mortem, pointed to an increase in the cross-linking of MHC. This effect is most likely to have been caused by the interaction with degradation products or other proteins. It favoured the stiffening of the muscle fibre structures. The centrifugal drip proteins, such as titin T2 and titin T1 degradation products, amylo-alpha-1,6-glucosidase, an unidentified band directly above phosphorylase b, 6-phosphofructokinase, pyruvate kinase, and lactate dehydrogenase, may be potential indicators assessing the quality of pork cooled down at different rates. The cooling process caused significant changes in muscle proteins assessed with the western blot analysis, titin, myosin heavy chains, troponin T, and GAPDH. The changes resulted from the proteolysis, degradation, and aggregation processes, which influenced the structure of the cytoskeleton of muscle fibres through various protein-protein interactions. The observed changes in the proteins influenced the quality of pork, its water-holding capacity, and tenderness. The UHPLC-Q-TOF-MS/MS technique and the analysis based on the multivariate OPLS-DA model enabled the identification of 12 proteins of the greatest significance for the differentiation between normal quality beef (RFN) and beef with the DFD defect. The group of potential protein and peptide indicators of the RFN and DFD meat quality included alpha- and beta-enolase, cysteine- and glycine-rich protein, protein of the third PDZ and LIM domains, 6-phosphofructokinase, heat shock proteins beta-1 and beta-6, L-lactate dehydrogenase A chain, creatine kinase M-type, myoglobin, protein-1-binding phosphatidylethanolamine, and troponin-T.