Mechanism Of The Alterations Of Water-holding Capacity And Hydration In Meat And Meat Products Induced By Protein Oxidation

The global meat industry as a whole, and the Chinese meat industry in particular, has evolved to become a major agricultural enterprise. Both the safety and quality of meat are important aspects of the meat industry. For example, moisture loss during meat processing, handling and storage impacts the economy and consumer acceptance of finished products. Water accounts for approximately 75% of the weight in lean muscle tissue and is directly responsible for juiciness in cooked meat and meat products. Of all environmental factors, protein oxidation has been recognized as a cause for reduced water binding in processed meat. Studies in the past decade have clearly demonstrated that much of the quality deterioration in muscle foods results from oxidative modification of protein by reactive oxygen species. However, the role of oxidation in the overall water-binding at the molecular level by muscle proteins or by the myofibril architecture has not been elucidated. In this dissertation study, we propose a new paradigm suggesting that oxidation could be a key causative factor affecting the overall ability of muscle proteins/myofibrils to bind water or myofrbrils to withhold water. Five experiments were conducted to reach this purpose with Fenton system (FeCl₃/ascorbate/H₂O₂, pH 6.2) simulating oxidative condition in situ.To determine the impact of protein oxidation on the pattern of water diffusion and retention in pork muscle (experiment 1), pork longissimus muscle was oxidized at 4°C at pH 6.2. Oxidation with higher concentration of H₂O₂ significantly enhanced (P < 0.05) hydration of muscle samples, but caused pronounced declines in water-holding capacity and product yield. The changes coincided with marked increases in the protein carbonyl content, TBARS formation, and cross-linking of both myofibrillar and sarcoplasmic proteins. Dye-tracing tests showed that the enhanced hydration was due to facilitated water diffusion into muscle tissue. This result was strongly corroborated by microscopic images that illustrated enlargements of intercellular spacing, i.e., gaps, in oxidized muscle tissue which served as canals for water diffusion.To investigate the mechanism of hydration and water binding in NaCl/pyrophosphate marinated, oxidative stressed pork muscle (experiment 2), pork longissimus muscle samples were subjected to three oxidation and marination conditions with 0.1 or 0.6 mol/L NaCl in the presence or absence of pyrophosphate. Protein oxidation, measured by the carbonyl and tryptophan fluorescence changes, enhanced hydration but increased cooking loss of meat. Light microscopy revealed a dense muscle structure characterized by swollen fibers and reduced intercellular spacing in certain oxidized muscle samples marinated with 0.6 mol/L NaCl and 15 mmol/L PP. However, oxidized fibers were more susceptible to transverse shrinkage than non-oxidized fibers upon cooking, in agreement with the dynamic ultrastructural changes in myofibrils observed using phase contrast microscopy. These findings provide a further understanding of the complex impact of oxidation on meat hydration and water-binding.To determine brine–induced ultrastructural changes and protein extraction from oxidatively stressed myofibrils (experiment 3), porcine longissimus myofibrils were exposed to a hydroxyl radical–oxidizing system for 1–12 h. Chemical analyses (sulfhydryls, disulfide bonds, carbonyls) indicated mild protein oxidation along with almost 40% loss of protein extractability in low-ionic-strength brines (≤0.4 mol/L NaCl, 10 mmol/L pyrophosphate, pH 6.2). Upon graded brine irrigation (0.2→0.6 mol/L NaCl) with pyrophosphate, the swelling of myofibrils and the dissolution of the A-band of oxidized myofibrils were less pronounced than those of non–oxidized. This restriction of myofibril swelling, caused largely by disulfide cross-linkages formed between oxidized myosin tails, was positioned on the transversely expansible thick filaments, reflecting a significant role and susceptibility of intra- as well as inter-myofilamental structures.To determine oxidation-induced changes in actomyosin dissociation (experiment 4), isolated natural actomyosin was oxidized in hydroxyl radical–oxidizing system for 6 or 12 h. Protein oxidation was evidenced by the formation of disulfide-cross-linked polymers, corresponding to the loss of myosin and actin bands in SDS–PAGE and elevated myosin ATPase activity. The intrinsic viscosity of oxidized actomyosin had a weaker response to pyrophosphate (PP)-Mg²⁺ than that of nonoxidized actomyosin, indicating the suppression of actomyosin dissociation. Moreover, oxidized actomyosin showed a lack of small particles (<10 nm) in solution and weaker binding to PP-Mg²⁺ when compared with non-oxidized samples, which further suggested a reduced myosin-PP interaction and subsequent dissociation of the actomyosin complex.To examine the effect of pyrophosphate on myosin oxidation and myosin gelling properties (experiment 5), purified porcine myosin was oxidized in hydroxyl radical–oxidizing solutions containing 0.5 mol/L NaCl with or without pyrophosphate (PP) and magensium for 12 or 24 h. Chymotryptic digestion and SDS–PAGE showed that PP could prevent HMM from hydroxyl radical attack, leading to the reduction of disulfide cross-linked polymers, but produce little effect on the LMM portion. Heating promoted cross-linkage in both myosin head and tail portions after oxidation, while PP-Mg²⁺ treated myosin sample still largely kept the HMM portion being protected. Also, PP-Mg²⁺ reduced the decline in proportion of some non-sulfur-containing amino acid such as tyrosine and lysine and protein surface charge after oxidation, suggesting it may influence the water-binding capacity of protein. DSC thermogram and the change in storage modulus during myosin gelation proved the promoting effect of PP-Mg²⁺ on the thermal stability of oxidized myosin S1 and S2 and the gel stength, which was suppressed by oxidation. In addition to these consequence of oxidation, it was also found heating changed the mode of myosin cleavage by chymotrypsin.Overall, this study elucidated the mechanism of the alterations in water-holding capacity of meat or meat products by protein oxidation, providing further understanding of how oxidation casues moisture loss in muscle and the deterioration of meat products'succulence. The findings should help the development of effective antioxidant formulations and processing strategies to maximize pork meat product quality, especially juiciness, tenderness, oxidative stability, and the overall shelf-life.