| Unacceptable water holding capacity(WHC) costs meat industry enormous economic loss annually in China. The mechanism of endopeptidases mediated proteolysis of muscle proteins, and their effects on the distribution and mobility of myowater during postmortem storage is still not clear. Therefore, to investigate the relationship between endopeptidases and WHC in muscle to improve the understanding the mechanism of myowater distribution and mobility is necessary. In the present paper, the mechanism of calpin-mediated water distribution and mobility in porcine muscle was investigated, to provide theoretical basis for the postmortem meat quality. In vitro incubation of myofibrillar protein with μ-calpain was carried out to study the relationship between the μ-calpain and gel-water. Besides, hydration properties of myofibrillar protein, and interactions between muscle protein and myowater properties during postmortem storage were investigated. Moreover, calpain specific inhibitor PD150,606 and multiple protease inhibitor MG-262 were added to muscle samples to investigate how the proteolytic systems, especially calpain, could be involved in water distribution and mobility of pork during postmortem ageing. The results were as follows:In vitro incubation of purified myofibrils with μ-calpain under postmortem-simulated conditions(pH 5.8 and 4°C) was performed. The surface hydrophobicity of myofibrillar protein treated with μ-calpain was significantly lower than that in control group(20.22 μg vs. 25.18 μg, P<0.01). The solubility of the proteins treated with μ-calpain was higher than that in control group(41.83% vs. 30.01%, P<0.01). The groups with μ-calpain incubation had a higher WHC compared to control group(93.23% vs. 89.09%, P=0.0004). The incubation treatments of μ-calpain with myofibrillar protein revealed a higher P22 value with an average of 85.84% compared to the value in control group with an average of 83.51%(P=0.04). However, the results for P23 are opposite to those for P22. Compared to control, μ-calpain-treated protein samples revealed a higher content of α-helices and a lower content of β-turns and random coil fractions. μ-Calpain resulted in a larger percentage of immobilized water and lower percentage of free water. These indicated that μ-calpain is critical to improve the α-helical structures and embeds the hydrophobic parts, which broadens the surface of protein to bind water and allows more water to be trapped in the myofibrillar protein network.The effects of μ-calpain-mediated myofibrillar proteins degradation on myowater mobility postmortem(1-168 h) in pork was investigated. During 1-24 h, rigor induced immobilized water transforming into free water, thereby WHC decreased. During 24-120 h, μ-calpain contributed to proteolysis of muscle proteins, increasing protein hydration capacity. Bound water and free water converted to immobilized water and porcine WHC improved. During 120-168 h, low molecular proteins aggregated because of ongoing degradation by μ-calpain, which led to the decreases of proteins hydration capacity. Immobilized water converted to free water, leading to the decreases in WHC.The interactions of protein-water as affected by postmortem ageing in pork were investigated. Results suggested that within 5 d ageing, an increase in α-helical structures and a decrease in β-sheets were occurred. Ageing-induced protein structural and conformational changes, embedding the hydrophobic residues and increasing the protein solubility, contributed to the free water shifting to the immobilized water, thereby resulted in the decreased drip loss. However, prolonged ageing process to 9 d resulted in the increased drip loss. The transformation of α-helices into β-structures and random coils occurred. Meanwhile, these changes also were in company with myosin tails and sarcoplasmic proteins denaturation dramatically, which caused the low ability to entrap water in meat structures. The immobilized water shifted to the free water and lost as drip.Calpain specific inhibitor PD150,606 and multiple protease inhibitor MG-262 were added to muscle samples to investigate how the proteolytic systems, especially calpain, could be involved in water distribution and mobility of pork during postmortem ageing. Increased desmin degradation by calpain after 4 d storage, allowed swelling of myofibre, which enabled meat structure to hold more water. However, calpain contributed to proteolysis of integrin causing wide gaps between myofibrils, which in-turn affected the WHC. NMR studies confirmed the transformation of immobilized water to free water, causing decline in WHC after 7 d storage. Our study indicates that proteases involve in sequential manner in the postmortem water distribution and movement, but effects of calpain on WHC is time postmortem dependent. |
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