Nitrites are universally acknowledged natural toxic substances that frequently lead to poisoning in humans and animals. During fermentation, certain microorganisms utilize a portion of the nitrogen element and reduce nitrates to nitrites through specific metabolic pathways. In this study, a highly effective lactic acid bacterial strain, Lactiplantibacillus plantarum A50, was isolated and screened from alfalfa silage for its remarkable ability to degrade nitrites. L. plantarum A50 exhibits exceptional nitrite removal capacity, with a degradation rate of 99.06% within 24 h. Furthermore, L. plantarum A50 demonstrates normal growth under pH values ranging from 4 to 9 and salt concentrations of 5%, displaying excellent tolerance to acidity, alkalinity, and salinity. Additionally, it undergoes fermentation using various carbon sources. Within the first 6–12 h of culture, L. plantarum A50 primarily achieves nitrite degradation through non-acidic processes, resulting in a degradation rate of 82.67% by the 12th hour. Moreover, the metabolites produced by L. plantarum A50 exhibit a synergistic interaction with acidity, leading to a nitrite degradation rate of 98.48% within 24 h. Notably, both L. plantarum A50 and MRS broth were found to degrade nitrites. Consequently, a non-targeted metabolomic analysis using LC-MS was conducted to identify 342 significantly different metabolites between L. plantarum A50 and MRS broth. Among these, lipids and lipid-like molecules, organic acids and derivatives, organic oxygen compounds, and organoheterocyclic compounds emerged as the main constituents. Lipids and lipid-like molecules, derivatives of glucose and galactose, amino acids and their derivatives, as well as organoheterocyclic compounds, are likely to play a role in nitrite elimination. Through the enrichment analysis of differential metabolic pathways using KEGG, nine distinct pathways were identified. These pathways provide essential nutrients, maintain cellular structure and function, participate in substance transport, regulate metabolic activities, and enhance resistance against pathogenic microorganisms in L. plantarum A50.

This study examined the impacts of growth stage and ensiling duration on the fermentation characteristics, nitrite content, and bacterial communities during the ensiling of alfalfa. Harvested alfalfa was divided into two groups: vegetative growth stage (VG) and late budding stage (LB). The fresh alfalfa underwent wilting until reaching approximately 65% moisture content, followed by natural fermentation. The experiment followed a completely randomized design, with samples collected after the wilting of alfalfa raw materials (MR) and on days 1, 3, 5, 7, 15, 30, and 60 of fermentation. The growth stage significantly influenced the chemical composition of alfalfa, with crude protein content being significantly higher in the vegetative growth stage alfalfa compared to that in the late budding stage (p < 0.05). Soluble carbohydrates, neutral detergent fiber, and acid detergent fiber content were significantly lower in the vegetative growth stage compared to the late budding stage (p < 0.05). Nitrite content, nitrate content, nitrite reductase activity, and nitrate reductase activity were all significantly higher in the vegetative growth stage compared to the late budding stage (p < 0.05). In terms of fermentation parameters, silage from the late budding stage exhibited superior characteristics compared to that from the vegetative growth stage. Compared to the alfalfa silage during the vegetative growth stage, the late budding stage group exhibited a higher lactate content and lower pH level. Notably, butyric acid was only detected in the silage from the vegetative growth stage group. Throughout the ensiling process, nitrite content, nitrate levels, nitrite reductase activity, and nitrate reductase activity decreased in both treatment groups. The dominant lactic acid bacteria differed between the two groups, with Enterococcus being predominant in vegetative growth stage alfalfa silage, and Weissella being predominant in late budding stage silage, transitioning to Lactiplantibacillus in the later stages of fermentation. On the 3rd day of silage fermentation, the vegetative growth stage group exhibited the highest abundance of Enterococcus, which subsequently decreased to its lowest level on the 15th day. Correlation analysis revealed that lactic acid bacteria, including Limosilactobacillus, Levilactobacillus, Loigolactobacillus, Pediococcus, Lactiplantibacillus, and Weissella, played a key role in nitrite and nitrate degradation in alfalfa silage. The presence of nitrite may be linked to Erwinia, unclassified_o__Enterobacterales, Pantoea, Exiguobacterium, Enterobacter, and Allorhizobium–Neorhizobium–Pararhizobium–Rhizobium.