Integrated Microbiomics and Metabolomics Reveal That Moisture Content and Lactiplantibacillus plantarum Synergistically Regulate Fermentation Quality, Microbial Community, and Metabolite Profiles of Amaranth Silage

This study investigated the effects of moisture content (80% vs. 70%) and Lactiplantibacillus plantarum inoculation on the fermentation quality, microbial community structure, and metabolite profiles of amaranth (Amaranthus hypochondriacus) silage using integrated microbiomics (full-length 16S rRNA sequencing) and untargeted metabolomics (UPLC-MS/MS). The results showed that high-moisture silage without inoculation (AhGCK) exhibited poor fermentation quality, characterized by high pH (5.09) and low lactic acid content (1.42% FM). Inoculation with L. plantarum significantly reduced pH (to 4.16) and increased lactic acid accumulation (to 3.65% FM) under high-moisture conditions. Wilting to 70% moisture combined with L. plantarum inoculation (AhSLP) achieved the best fermentation quality, with the lowest pH (4.20) and highest lactic acid (4.46% FM). Microbial community analysis revealed that Enterobacter cloacae dominated in AhGCK, whereas L. plantarum, Lentilactobacillus buchneri, and Levilactobacillus brevis became dominant after inoculation and wilting. Metabolomics identified 497 compounds across all treatments, with differential metabolites primarily enriched in pathways related to amino acid metabolism, carbohydrate metabolism, and biosynthesis of secondary metabolites such as diterpenoids and isoquinoline alkaloids. Highly significant correlations (p < 0.01) were observed between Enterobacter and cyclohexylammonium, between dTDP-3-O-methyl-β-L-rhamnose and 2-hydroxy-2H-benzo[h]chromene-2-carboxylate, between Lentilactobacillus and 3-ketosucrose (positive), and between Limosilactobacillus and 8-methylthiooctyl glucosinolate (positive), whereas Lactiplantibacillus and Escherichia Shigella showed no correlations with differential metabolites. These findings indicate that inoculating Lactobacillus plantarum at specific moisture levels (80% and 70%) promotes directed microbial community succession (as exemplified by positive correlations of Lentilactobacillus and Limosilactobacillus with beneficial metabolites) and optimized metabolite accumulation, which significantly lowers dry matter loss during fermentation and enhances the output of usable silage. This mechanism offers a practical theoretical foundation for improving amaranth silage production and boosting feed yield.