Advancing safe and value-added alfalfa silage: Multi-omics insights into β-carotene biosynthesis and suppression of antibiotic resistance genes

Hazardous antibiotic resistance genes (ARGs) tend to accumulate in alfalfa during the silage-making process, posing potential environmental and public health risks. Inoculation with lactic acid bacteria (LAB) has shown promise in mitigating ARGs proliferation, yet the underlying mechanisms and management implications remain poorly understood. This study aimed to elucidate the mechanisms by which LAB reduced and hindered the diffusion of hazardous ARGs by multi-omics. Alfalfa was ensiled without (CON) or with Lactiplantibacillus pentosus h (Lh) and Lactiplantibacillus plantarum PA (PA) for 7, 35, and 84 d. Results showed that β-carotene produced in the inoculated groups interfered with ARGs expression and dissemination, while simultaneously promoting the synthesis of antimicrobial compounds. This was supported by linear downregulating hazardous ARGs (r = -0.71, P < 0.01) and mobile genetic elements (r = -0.44, P < 0.05), upregulating phenyllactic acid (r = 0.40, P < 0.05) and gene of coding safe bacitracin (r = 0.69, P < 0.01). Additionally, β-carotene suppressed the expression of genes involved in β-lactam antibiotic hydrolysis (P < 0.01). These effects were attributed to the activation of a novel β-carotene biosynthetic pathway, which differed from conventional enzyme-mediated routes. The activity of this pathway in Lh and PA groups was more than 1.60-fold higher than that in the CON (P < 0.01). LentiLactobacillus buchneri enhanced key precursor pathways, while Lacticaseibacillus paracasei and phenyllactic acid were identified as the keystone species and key metabolites, respectively. Collectively, this study demonstrated that LAB inoculation suppressed ARGs enrichment and diffusion in silage through β-carotene-mediated metabolic regulation, offering mechanistic insights and practical strategies for safe, large-scale silage production and resistance gene management.