Effects of ensiling temperature on fermentation quality, enzyme activities, and metabolomic profiles in Pediococcus acidilactici L10-inoculated native grass silage

In cold regions, the efficient preservation of native grass is often constrained by low temperatures during the ensiling process. This study investigated the fermentation quality, enzyme activity patterns, and metabolomic profiles of native grass silage inoculated with Pediococcus acidilactici L10 under varying temperature conditions. All silage samples were inoculated with Pediococcus acidilactici L10 and stored at three temperatures (25℃, 15℃, or 5℃), with three biological replicates per treatment. Integrated analyses were conducted over a 60-day period, including measurements of fermentation parameters and enzyme activities on days 3, 7, 15, 30, and 60, as well as untargeted metabolomic analysis on day 60. Ensiling temperature significantly influenced the fermentation characteristics of the L10-inoculated silage system. Compared with the 25℃ treatment, the 15℃ and 5℃ treatments exhibited lower final pH values, greater lactic acid accumulation during the mid-to-late fermentation stages, lower ammonia-nitrogen content, and higher residual water-soluble carbohydrate content. Enzyme activity analysis indicated that the activities of hexokinase, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase activities varied with ensiling temperature and were associated with patterns of lactic acid production and carbohydrate utilization. Metabolomic analysis conducted on day 60 revealed temperature-associated differences in metabolites related to soluble carbohydrates, amino acid metabolism, and lipid metabolism. In particular, the 15℃ treatment exhibited a more favorable overall profile among the tested temperatures within the L10-inoculated silage system, accompanied by higher relative abundances of D-galactose, stachyose, L-proline, and caprylic acid. Overall, these results indicate that ensiling temperature plays a critical role in shaping fermentation dynamics and metabolic profiles in L10-inoculated native grass silage, and provide a foundation for future studies evaluating inoculant performance under low-temperature conditions.