The objective of this study was to evaluate the effects of Lactiplantibacillus plantarum or cellulase on the fermentation characteristics and bacterial community of mixed alfalfa (Medicago sativa L., AF) and Leymus chinensis (LC) silage. The harvested alfalfa and Leymus chinensis were cut into 1–2 cm lengths by a crop chopper and they were thoroughly mixed at a ratio of 3/2 (wet weight). The mixtures were treated with no addition (CON), Lactiplantibacillus plantarum (LP, 1 × 10⁶ cfu/g fresh material), cellulase (CE, 7.5 × 10² U/kg fresh material) and their combination (LPCE). The forages were packed into triplicate vacuum-sealed, polyethylene bags per treatment and ensiled for 1, 3, 5, 7 and 30 d at room temperature (17–25 °C). Compared to the CON groups, all the additives increased the lactic acid content and decreased the pH and ammonia nitrogen content over the ensiling period. In comparison to the other groups, higher water-soluble carbohydrate contents were discovered in the CE-inoculated silages. Compared to the CON groups, the treatment with LPCE retained the crude protein content and reduced the acid detergent fiber content. The principal coordinate analysis based on the unweighted UniFrac distance showed that individuals in the AF, LC, CON and LPCE treatment could be significantly separated from each other. At the genus level, the bacterial community in the mixed silage involves a shift from Cyanobacteria_unclassified to Lactobacillus. Lactobacillus dominated in all the treatments until the end of the silage, but when added with Lactiplantibacillus plantarum, it was more effective in inhibiting undesirable microorganisms, such as Enterobacter, while reducing microbial diversity. By changing the bacterial community structure after applying Lactiplantibacillus plantarum and cellulase, the mixed silages quality could be further improved. During ensiling, the metabolism of the nucleotide and carbohydrate were enhanced whereas the metabolism of the amino acid, energy, cofactors and vitamins were hindered. In conclusion, the relative abundance of Lactobacillus in the mixed silage increased with the addition of Lactiplantibacillus plantarum and cellulase, which also improved the fermentation quality.

Most oat forage has low water-soluble carbohydrates (WSC), which may be the main limited factor for silage fermentation safely, but oat is rich in cellulose and hemicellulose; therefore, we assume that xylanase and cellulase as additives can reduce the content of cellulose and xylan in oat silage, increase the microbial fermentable sugar content, and improve the fermentation quality of the silage. After wilting, oats were treated as follows: (i) distributed water (CK); (ii) silages inoculated with xylanase (X); (iii) silages inoculated with cellulase (C), ensiling for 3 days (early stage of silage) and 60 days (late stage of silage), respectively, after ensiling 60 days for a 5-day aerobic exposure study. The pH, neutral detergent fiber (NDF), and acid detergent fiber (ADF) were significantly reduced by xylanase and cellulase treatment during the late stage of silage, and the concentration of lactic acid, acetic acid, and ammonia nitrogen increased remarkably. The WSC content reached its peak with xylanase treatment during the late stage of silage. The content of crude protein (CP) was not affected by additives but by the silage period; CP and ether extract (EE) significantly increased during the late stage of silage compared to the early stage. After ensiling, the bacterial community showed that xylanase and cellulase treatment increased the relative abundance of lactic acid bacteria. Lactobacillus has a higher relative abundance with cellulase treatment after 60 days of ensiling; this can effectively reduce the pH of silage and ensure long-term, stable storage of silage. Cellulase and xylanase increased bacterial diversity during aerobic exposure and improved the aerobic stability of silage significantly. This study indicated that different additives and silage periods had significant effects on chemical compositions, fermentation quality, and bacterial community; meanwhile, both additives improved the aerobic stability of silage. In summary, when the WSC of oat is low, cellulase and xylanase have good effects as silage additives, and the comprehensive effect of cellulase is more prominent.

Better quality and odor of silage and normal microbial fermentation metabolism are mostly dependent on an appropriate moisture content. The purpose of this study was to determine the effects of different moisture content gradients (50, 60, 70, and 80%) on the bacterial community, odor, and quality of alfalfa silage at 60 days by using gas chromatography–mass spectrometry (GC–MS) and electronic nose, with six replicates per group. The results showed that there were significant differences in odor response intensity among all groups, among which the 80% group had the strongest reaction to terpenoids, sulfides, and nitrogen oxides. Similarly, the different volatile organic compounds (VOCs) were mainly terpenoids, alcohols, and ketones, such as pine, camphor, and menthol (e.g., carlin and levomenthol). The dominant bacterium was Enterococcus with higher fiber, pH, and ammonia nitrogen (NH₃-N) content but poorer quality and odor (p < 0.05). The differential VOCs in the 60% group were mainly heterocyclics, esters, and phenols with fruity, floral, and sweet odors such as 2-butylthiophene and acorone. Pediococcus and Lactiplantibacillus were the dominant bacteria, with higher crude protein (CP), water-soluble carbohydrates (WSC), and lactic acid (LA) contents, as well as better quality and odor (p < 0.05). The biosynthesis of terpenoids and steroids, biosynthesis of secondary metabolites, and biosynthesis of phenylpropanoids were the main metabolic pathways of differential VOCs. In conclusion, regulating moisture content can alter bacterial community and metabolites, which will encourage fermentation and enhance alfalfa silage quality and odor.

During aerobic exposure of silage, the fatty acid and amino acid composition may alter the quality and palatability, resulting in economic losses in livestock production. The objective of this study was to evaluate the effects of Lactiplantibacillus plantarum (LP), Lenti Lentilactobacillus buchneri (LB), and a mixture of LP and LB (PB) on the fatty acids, amino acids, and antioxidant capacity of Leymus chinensis silage during aerobic exposure. The lactic acid bacteria were added at 1 × 10⁶ CFU/g. The silage treatments were opened after 60 days of fermentation, and sampled on days 0, 4, and 8 of aerobic exposure. The LB group had higher total fatty acid and polyunsaturated fatty acid content, and less decrease in amino acid content and antioxidant capacity, while the LP group had a higher monounsaturated fatty acid content but a larger decrease in all indicators after exposure. Correlation analysis showed that Lactobacillus, Cryptococcus, Penicillium, and Thermoascus were more correlated with fatty acid changes, and that Lactobacillus, Actinomyces, Clostridium, and Penicillium were more correlated with amino acid changes. In conclusion, Lentilactobacillus buchneri could effectively improve the antioxidant capacity and fatty acid and amino acid contents of Leymus chinensis silage during aerobic exposure, while Lactiplantibacillus plantarum could effectively improve the content of each index of Leymus chinensis silage at opening, but deterioration was faster during aerobic exposure.

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.

The application of fermented total mixed ration (FTMR) is an effective method to prolong the use time of feed, but the understanding of the interaction mechanism between fungal microorganisms and silage quality and aerobic stability in FTMR is still limited. This study aimed to evaluate the effects of alfalfa (Medicago sativa L.) hay to oat (Avena sativa L.) hay ratios on chemical composition, fermentation characteristics, and fungal communities during aerobic exposure of fermented total mixed ration (FTMR). The supplement levels of oat were as follows: 200 g/kg oat hay (LO), 300 g/kg oat hay (MO), and 400 g/kg oat hay (HO). The water content of the three treatments was adjusted to 50% using a sprayer. After 60 days of ensiling, the bags were opened, and the chemical composition, fermentation characteristics, and fungal communities were measured after 3, 6, 9, and 12 days of aerobic exposure. The results suggested that the LO treatment significantly (p < 0.05) increased the aerobic stability than that in other treatments. The crude protein and lactic acid content in the three treatments were significantly decreased with the extension of the aerobic exposure period. Additionally, there was a remarkable (p < 0.05) higher lactic acid content observed in the LO treatment than that in the HO treatment during the aerobic stage. The PCoA showed that the compositions of the fungal community in the HO treatment were distinctly separated from the other two treatments. Compared with HO and LO treatments, the MO treatment observed relatively higher OTU, Shannon, and Chao1 indexes. Compared with LO and MO treatments, the abundances of the genes Saccharomyces and Wallemia were greater increased and decreased in the HO treatment, respectively. Integrated correlation analysis also underscores a possible link between the fermentation characteristics, aerobic stability, and significantly altered fungal community. This study suggested that the use of FTMR in production might prolong aerobic storage time when alfalfa was fermented in a mixture with ≤30% oat.