Abstract
Pseudomonas fragi and Aeromonas salmonicida are major spoilage microorganisms in refrigerated grass carp. This study systematically investigated the physicochemical and metabolomic characteristics of chilled grass carp that were artificially inoculated with P. fragi and A. salmonicida in mono- and co-culture. The results indicated that P. fragi was the dominant bacterium in the co-culture of grass carp. The P. fragi-inoculated group exhibited significantly higher levels of total volatile basic nitrogen and thiobarbituric acid reactive substances (TBARs, byproducts of lipid peroxidation) compared with the A. salmonicida group. Moreover, the TBAR levels were greater in the co-culture than in the A. salmonicida group at the end of storage. A metabolomic analysis revealed that 712, 424, and 465 differential metabolites were identified in grass carp inoculated with A. salmonicida, P. fragi, and their co-culture, respectively. The metabolic pathway enrichment showed that purine metabolism, aminoacyl-tRNA biosynthesis, glycerophospholipid metabolism, and amino acid metabolism were prevalent across all three inoculated groups. A total of 175 amino acids, peptides, and analogues were identified in the A. salmonicida group, indicating that A. salmonicida played a vital role in protein degradation. P. fragi was primarily enriched in linoleic acid metabolism and the biosynthesis of unsaturated fatty acids and fatty acids, demonstrating its advantages in lipid metabolism. Additionally, six potential spoilage biomarkers were identified, including inosine, cytidine, L-aspartic acid, L-tyrosine, Pro-Ile and PS(17:1(9Z)22:0). These results elucidated the complex and competitive interactions between A. salmonicida and P. fragi in the spoilage of grass carp, providing a scientific basis for the quality monitoring of grass carp and targeted preservation strategies.