In-depth proteomic analysis of alfalfa silage inoculated with Lactiplantibacillus plantarum reveals protein transformation mechanisms and optimizes dietary nitrogen utilization.

Alfalfa is a vital feedstock for ruminants due to its high protein content. However, concerns regarding nitrogen (N) loss in alfalfa silage from protein transformation have arisen, but the underlying molecular mechanisms remain undefined. This study investigated the patterns of protein transformation in alfalfa during fermentation through multi-omics analysis. Lactiplantibacillus plantarum treatment effectively preserved the true protein PB1 and peptide-N, by mitigating N degradation. Proteomic profiling revealed a decrease in the abundance of higher molecular weight proteins (45-70 kDa), coupled with an increase in lower molecular weight proteins (15-45 kDa), mainly located in the membrane, cytoplasm and ribosome. Meanwhile, levels of protein hydrolysates, including peptides and essential amino acids, were elevated. Among these, Ile-Pro, Pro-Val, flavorful L-Glu and the proteogenic Arg were identified as the predominant constituents. Tripeptidyl-peptidase, carboxypeptidase, and serine protease were identified as the primary plant proteases mediating protein transformation. These findings highlight the synergistic effects of plant proteases and microbial activity, mainly from L. plantarum, in transforming large proteins into peptides and amino acids during fermentation. Our findings provide insights for the targeted regulation of alfalfa protein transformation during fermentation, potentially enhancing protein utilization in ruminants.