In-depth Proteomic Analysis of Alfalfa Silage Inoculated with Lactiplantibacillus plantarum Reveals Protein Transformation Mechanisms and Optimizes Dietary N Utilization

Background: Alfalfa is a crucial feed source for ruminants, especially dairy cows, and is rich in protein (16-24% dry matter). Concerns regarding alfalfa silage nitrogen (N) loss have emerged from protein transformation, but the molecular mechanism underlying this transformation remains poorly understood. Hence, there is an urgent need to elucidate the protein transformation mechanism and optimize dietary N utilization to enhance ruminant protein nutrition. Results: We established a comprehensive pipeline including protein components, peptides and amino acids (AAs) to characterize the protein transformation process and available N preservation. We found that Lactiplantibacillus plantarum treatment preserved more of the true protein fraction PB1 and peptide-N and prevented further degradation, as reflected by decreased NH 3 -N contents. Proteomic profiling revealed that protein alteration is driven by the interplay of the ensiling process and L. plantarum treatment. Tripeptidyl-peptidase, carboxypeptidase and serine protease are major plant endogenous proteases that exhibit optimal activity for protein transformation during ensiling. The transformation degree was influenced by subcellular localization and functional properties. Proteins residing in the membrane and ribosomal structures and participating in cellular processes, catalytic activities, and metabolic processes display heightened sensitivity to transformation. During ensiling process, L. plantarum treatment further downregulated proteins located in the membrane and cytoplasm. The protein transformation degree was also reflected in the intermediates of the transformation. Thus, we investigated the peptide composition of alfalfa silages by determining their amino acid sequences. Among these, Ile-Pro and Pro-Val emerged as the predominant constituents. Additionally, L. plantarum treatment increased the bioactive peptide level via dipeptidyl peptidase IV inhibition, angiotensin-converting enzyme inhibition, and antioxidative activity. Next, we provided a complete AA profile, highlighting the dominance of the flavorful L-Glu and the proteogenic Arg in the silages treated with L. plantarum . Conclusions: We present pioneering data to comprehensively unravel the mechanism of protein transformation, providing valuable resources by revealing the true protein composition serving as an alfalfa silage protein library and emphasizing the functional aspects of the proteins within it. Our findings shed light on how the utilization of rumen-available N in alfalfa silage contributes to environmental sustainability and reduces feeding costs by decreasing N excretion.