The present study compared muscle protein characteristics between Sepiella maindroni (S. maindroni) and Sepia esculenta (S. esculenta). We analyzed the nutrient composition, myofibrillar protein properties, and proteomic changes in muscle tissue under freezing conditions. Amino acid analysis revealed 17 hydrolyzed amino acids in both species’ muscle tissues, with tryptophan identified as the first limiting amino acid. Essential amino acids (EAAs) constituted more than 31% of total amino acids (TAAs), aligning closely with the FAO/WHO recommended ideal protein pattern. The Essential Amino Acid Index (EAAI) for S. maindroni was high at 82.99. Twenty fatty acids were identified in both species, with total contents of C20:5n-3 (EPA) and C22:6n-3 (DHA) exceeding 40%. S. maindroni had significantly more such fatty acids than S. esculenta. During the freezing process, the total sulfhydryl content and Ca2+-ATPase activity of both species showed a decreasing trend, while carbonyl content exhibited an overall increase. After 60 days of freezing, the total sulfhydryl content and Ca2+-ATPase activity in the muscles of both species decreased to minimum levels. Compared to fresh samples, both species’ muscle tissues exhibited varying degrees of deterioration in microscopic structure after 60 days of freezing. A total of 350 differentially expressed proteins (DEPs) comprising 208 upregulated and 142 downregulated proteins were identified in S. maindroni and S. esculenta. After 60 days of freezing, 322 DEPs, including 181 upregulated and 141 downregulated proteins, were detected in both species. COG functional classification indicated that low-temperature storage affected DEPs in the muscle tissue, influencing the flavor, texture, and protein functionality of the species.
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