Study on the mechanism of meat quality deterioration induced by the freezing temperature

Freezing is the most common meat preservation technique and is essential in meat production and trade circulation. Although freezing extends the shelf life of meat, the physical and chemical changes that occur during the freezing process make it less appreciated than fresh meat. The deterioration of frozen meat quality induces not only nutritional and weight loss but also a huge economic loss to the enterprise. The fundamental requirement for freezing is negative temperature. Inappropriate freezing temperatures not only affect the quality of the meat but also result in actual energy usage. Therefore, in the present study, freezing temperature, which is the primary condition for freezing, was chosen as a jumping-off point to investigate the underlying mechanism of freezing-induced quality degradation. Firstly, the longissimus thoracis (LT) muscles of the Simmental × Qinchuan crossbred cattle was used as raw material in this study. The effect of four treatments of freezing temperatures (-12 °C, -18 °C, -38 °C, and -80 °C) on the quality of meat after freezing and thawing was systematically examined. The results showed that, in comparison to fresh meat, all four frozen treatment groups demonstrated a decline in meat quality. As the freezing temperature decreased, the extent of quality deterioration decreased. The samples frozen at -80 °C had significantly better color, water holding capacity (WHC), and tenderness than the other three freezing groups (p < 0.05). Lower freezing temperatures could effectively reduce the mobility of water, slow the migration of immobile water to free water, and reduce the thawing loss and deterioration of product quality. The quality deterioration at -12 °C was the most severe, particularly the thawing loss, which reached 10.5%. There was no significant difference between the -12 °C group and the -18 °C group for the overall change in color value (ΔE), cooking loss, and tenderness. For the total change in color (ΔE) and water holding capacity, there was no significant difference between the -18 °C and -38 °C groups. The overall effect of freezing at -38 °C on meat quality is comparable to freezing at -80 °C. And after that, the nucleation temperature, phase transition time, effective freezing time, scanning electron microscopy (SEM), transmission electron microscopy (TEM), freezing shrinkage, and the number, equivalent diameter, shape, and area of ice crystals were investigated to evaluate the impact of ice crystal formation and growth on the microstructure of muscle tissue. As the temperature dropped throughout the freezing process, myowater gradually nucleated to form ice crystals, after which the ice crystals continued to grow. The nucleation rate at -12 °C was significantly lower than that at -18 °C, -38 °C, and -80 °C. At the same time, the phase transition time and effective freezing time were considerably longer than those of the three freezing temperature groups (p < 0.05). Similarly, the -80 °C freezing group formed small intracellular and extracellular ice crystals with an average equivalent ice crystal diameter of 2.44 μm. The -12 °C, -18 °C, and -38 °C treatment groups, on the other hand, produced extracellular ice crystals with an average diameter of 38.45, 33.44, and 28.54 μm, respectively. In addition, the number of ice crystals formed at a temperature of -12 °C was significantly lower than at the other three temperatures (p < 0.05). Still, their relative total area was the largest and caused the most damage to muscle fibers. Protein solubility, Ca2+-ATPase activity, DSC profile, and primary, secondary, and tertiary structure-related indicators were systematically analyzed in the current study. The findings showed that freezing disrupted the conformational structure of the myofibrillar protein, cross-linked and degraded it, and altered the protein-water interaction relationship. Protein denaturation decreased gradually at freezing temperatures of -12 °C, -18 °C, -38 °C, and -80 °C. Ca2+-ATPase activity, protein solubility, total sulfhydryl content, α-helix, hydrogen, and ionic bonding levels were significantly lower in the -12 °C freezing group than in the other three freezing temperatures groups. In contrast, dityrosine content, hydrophobic interactions, and disulfide bonds were considerably higher. The protein conformational structure was most severely damaged by -12 °C freezing; hydrophobic amino acid groups such as tryptophan were exposed, the protein-water interaction relationship was weakened, and surface hydrophobicity and hydrophobic interactions were significantly higher than those of the -18 °C, -38 °C, and -80 °C freezing treatment groups (p < 0.05). When frozen at -80 °C, the protein conformation structure was least damaged, and protein stability was highest. DIA (data-independent acquisition) proteomics was, for the first time, used to investigate the effects of different freezing temperatures on protein profiles and functions, as well as to identify biomarkers linked with quality changes. Following statistical analysis, 262 and 135 different abundance proteins (DAPs), respectively, were found in frozen meat and thawed exudates. As the freezing temperature decreased, the number of DAPs significantly decreased. 109 DAPs were generated in the frozen -12 °C sample, significantly more than the other three temperature groups. Most of the 262 unique proteins in muscle were structural proteins engaged in pathways such as tight junction, focal adhesion, etc. In addition, several enzymes participated in oxidative phosphorylation, glycolysis, and other processes. Seven proteins, including ACTN3, CTTN, and MYH1, were screened as potential biomarkers associated with changes in frozen meat quality. They were presumed to play a significant role in muscle protein structure and muscle contraction. More than 60% of the 135 DAPs identified in the thawed exudates were oxidoreductases and binding proteins. Correlation analysis between DAPs and quality traits showed that 21 proteins were significantly correlated with color (L*, a*, and b*), thawing loss, cooking loss, and shear force of frozen beef. Finally, the psoas major (PM) muscles of Belgian Blue cattle were used to confirm the effect of freezing temperature on the physicochemical properties of meat. This validation experiment adopted two freezing temperatures (-18 °C and -38 °C). The results showed that the cooking loss, color, and shear force indicators decreased after freezing compared to fresh meat. Characteristic indicators of myofibrillar protein, including protein solubility, surface hydrophobicity, and chemical bonding, also changed significantly after freezing. The water holding capacity, color, shear force, and protein properties of -38 °C frozen samples were superior to those of -18 °C frozen samples, especially the thawing loss, hydrogen bonding, and ionic bonding, which were infinitely more remarkable (p < 0.05). The present study confirmed the significant effect of different freezing temperatures on muscle's physicochemical and biological properties. The freezing temperature was unquestionably the most influential factor in the formation of ice nuclei and the growth of ice crystals, as well as the most critical factor in the deterioration of muscle quality. The growth of ice crystals during the initiation of freezing damaged the microstructure of muscle and altered the structural conformation of the myofibrillar protein, resulting in changes in the abundance of structural proteins such as ACTN3, CTTN, MYH1, and metabolic enzymes, thereby affecting muscle contraction, structural connectivity, redox, and other protein functions. The outcomes of this research will serve as a guide for controlling the quality of meat during freezing and subsequent storage and will provide data to support the development of new freezing techniques.