Lactiplantibacillus plantarum L94 inoculation prevents clostridial spoilage and proteolysis in high-moisture oat silage via steering microbial and metabolic dynamics

High-moisture oat silage is exceptionally vulnerable to severe nutrient dissipation, extensive proteolysis, and secondary clostridial spoilage, posing a major bottleneck in forage preservation. Inoculating high-efficiency indigenous lactic acid bacteria represents a promising biotechnological strategy to accelerate acidification and stabilize silage quality. This study investigated the regulatory capacity of an epiphytic strain, Lactiplantibacillus plantarum L94, on the fermentation kinetics, bacterial community succession, untargeted metabolomic profiles, and their multi-omics interactive mechanisms in high-moisture oat silage over a 90-day ensiling period. Chopped high-moisture oats were ensiled either without inoculant (control, CK) or with L. plantarum L94 (1 × 10 5 cfu/g fresh weight). Fermentation characteristics, nitrogenous fractions, and chemical components were monitored longitudinally across 90 days. High-throughput 16 S rRNA gene sequencing and LC-MS/MS untargeted metabolomics were integrated to profile microbial succession and metabolic shifts, complemented by Spearman correlation and KEGG pathway enrichment analyses to elucidate the underlying microbial-metabolic crosstalk. Inoculation with L94 significantly accelerated initial acidification (pH declining to 3.81 by day 7) and stimulated lactic acid accumulation (peaking at 6.11% DM on day 15). L94 treatment effectively suppressed ammonia-nitrogen generation and constrained butyric acid below 0.200% DM throughout storage, entirely preventing the severe clostridial spoilage observed in the control group (where butyric acid escalated to 2.761% DM by day 90). Microbiome profiling revealed that L94 reshaped bacterial succession: Lactiplantibacillus rapidly dominated early fermentation (days 1–7), whereas the heterofermentative genus Lentilactobacillus proliferated during late fermentation (reaching 89.0% relative abundance at day 90), executing a strategic homofermentative-to-heterofermentative transition. Metabolomics identified 1,382 metabolites, demonstrating that L94 inoculation markedly downregulated key proteolytic markers (e.g., Ser-Val and N-acetyltyramine) to preserve protein integrity, while concurrently enriching functional bioactive metabolites such as Boscartin H. Correlation analysis established that Lactiplantibacillus and Lentilactobacillus abundances were positively correlated with organic acid accumulation, but negatively associated with spoilage indicators and proteolytic metabolites. KEGG enrichment confirmed that metabolic reprogramming was driven primarily by amino acid metabolism, aminoacyl-tRNA biosynthesis, and ABC transporter pathways. The indigenous inoculant L. plantarum L94 optimizes high-moisture oat silage preservation by steering beneficial bacterial succession, completely arresting clostridial spoilage, mitigating proteolysis, and promoting the accumulation of functional metabolites. These findings highlight the potential of strain L94 as a tailored bio-inoculant for sustainable high-moisture forage conservation.