Both inoculants treatment and enzyme treatment promote the reproduction of lactic acid bacteria (LAB) to produce enough lactic acid to lower pH in silage. The present study investigated the microbial community and metabolome in cellulase, Lactobacillus casei, and air treated alfalfa silage. Chopped and wilted alfalfa (first cutting, 29% dry matter) was ensiled without (CON) or with L. casei (1 × 10⁶ cfu g^–1 fresh matter) (LC) or cellulase (20,000IU, 0.5% of fresh matter) (CE) for 56 days, then exposed to air for 3 days (PO). Greater ensiling quality was observed in LC and CE, which had lower pH and higher lactic acid content than CON at 56 days of ensiling and 3 days post-oxygen exposure. Air exposure was associated with decreased lactic acid concentrations and increased yeast and mold counts in all silages. SEM showed that the structure of leaf epicuticular wax crystals were intact in fresh alfalfa, totally decomposed in CON silage, and partly preserved in CE and LC silage. Gas chromatography mass spectrometry revealed that 196 metabolites and 95 differential concentration were present in the 3 days air exposure samples. Most of these metabolites, mainly organic acids, polyols, ketones, aldehydes, are capable of antimicrobial activity. The bacterial communities were obviously different among groups and Lactobacillus developed to a dominant status in all silages. Lactobacillus became dominant in bacterial communities of LC and CE silages from days 7 to 56, and their relative abundances reached 94.17–83.93% at day 56, respectively. For CON silage, until day 56, Lactobacillus dominated the bacterial community with abundance of 75.10%. After 3 days of oxygen exposure, Lactobacillus and Enterococcus were predominant in CON, and Lactobacillus remained dominant in LC and CE silages. The results indicated that, compared to untreated silages, L. casei could be a priority inoculant for alfalfa silage to boost Lactobacillus abundance and improve fermentation quality. Our high-throughput sequencing and gas chromatography mass spectrometry results provide a deep insight into the bacterial community and metabolites in alfalfa silage.

The mixture of whole-plant soybean and whole-plant corn silage (WPSCS) is nutrient balanced and is also a promising roughage for ruminants. However, few studies have investigated the changes in bacterial community succession in WPSCS inoculated with homofermentative and heterofermentative lactic acid bacteria (LAB) and whether WPSCS inoculated with LAB can improve fermentation quality by reducing nutrient losses. This study investigated the effect of Lactobacillus plantarum (L. plantarum) or Lactobacillus buchneri (L. buchneri) on the fermentation quality, aerobic stability, and bacterial community of WPSCS. A 40:60 ratio of whole-plant soybean corn was inoculated without (CK) or with L. plantarum (LP), L. buchneri (LB), and a mixture of LP and LB (LPB), and fermented for 14, 28, and 56 days, followed by 7 days of aerobic exposure. The 56-day silage results indicated that the dry matter content of the LP and LB groups reached 37.36 and 36.67%, respectively, which was much greater than that of the CK group (36.05%). The pH values of the LP, LB, and LPB groups were significantly lower than those of the CK group (p < 0.05). The ammoniacal nitrogen content of LB was significantly lower than that of the other three groups (p < 0.05), and the ammoniacal nitrogen content of LP and LPB was significantly lower than that of CK (p < 0.05). The acetic acid content and aerobic stability of the LB group were significantly greater than those of the CK, LP, and LPB groups (p < 0.05). High-throughput sequencing revealed a dominant bacteria shift from Proteobacteria in fresh forage to Firmicutes in silage at the phylum level. Lactobacillus remained the dominant genus in all silage. Linear discriminant analysis effect size (LEFSe) analysis identified Lactobacillus as relatively abundant in LP-treated silage and Weissella in LB-treated groups. The results of KEGG pathway analysis of the 16S rRNA gene of the silage microbial flora showed that the abundance of genes related to amino acid metabolism in the LP, LB, and LPB groups was lower than that in the CK group (p < 0.05). In conclusion, LAB application can improve the fermentation quality and nutritional value of WPSCS by regulating the succession of microbial communities and metabolic pathways during ensiling. Concurrently, the LB inoculant showed the potential to improve the aerobic stability of WPSCS.

Alfalfa is conserved in silo-type bunkers in the cold and humid regions of Inner Mongolia, China. Its quality is essential to ensure a healthy and sustainable dairy production. However, the impact of environmental factors on the microbiota and fermentation products of alfalfa silage remains unclear. The present research examined changes in the microbiota and fermentation products and their association with environmental parameters in 72 samples collected from 12 farms located at 4 different latitudes and longitudes across four regions. The samples were labeled with distinct codes, A, B, and C, from the cold–rainy region, D, E, and F, from the warm–rainy region, G, H, and I from the cold–dry region, and J, K, and L from the warm–dry region. The lactic acid levels ranged from 14.25 to 24.27 g/kg of DM across all samples. The pH and concentrations of NH₃-N and butyric acid in samples A, B, and H were higher (p < 0.01) than in the other samples. Samples D and E had higher acetic acid concentrations and 1, 2-propanediol content (p < 0.01). The fresh material was dominated by Pantoea and Pseudomonas, whereas Lactobacillus was the most dominant genus in all silages, except for the B silage. The A, B, and H silages contained more Clostridium but less Lactobacillus than the other silages. The lactic acid levels were strongly associated with Lactobacillus plantarum, Weissella paramesenteroides, Lactobacillus acetotolerans, Pedobacter borvungensis, and Lactobacillus brevis (p < 0.01). In contrast, the pH and the NH₃-N and butyric acid concentrations were strongly associated (p < 0.01) with the presence of Clostridium estertheticum. A correlation analysis revealed that precipitation, temperature, longitude, and latitude were the most critical factors influencing epiphytic microbes in the fresh material. After silage fermentation, low-temperature conditions significantly affected the fermentation products and microbial community composition. In conclusion, the microbial community of silages is distinctive in cold and humid regions, and climatic parameters ultimately affect the microbiota and fermentation products. Furthermore, the findings of this study demonstrate that Illumina MiSeq sequencing combined with environmental factor assessment might provide new information about the microbiota composition and fermentation quality of silages, facilitating the achievement of high-quality silage.

Aflatoxin B1 (AFB1) is the most toxic mycotoxin and is widespread in moldy feed. The use of biological removal methods to reduce AFB1 has become a research hotspot. This study aimed to isolate lactic acid bacteria (LAB) capable of removing AFB1 from moldy feeds and assessed the removal capacity under various environmental conditions. A strain named Lactobacillus brevis DN-1 was isolated from feed samples and showed 71.38% AFB1 percent removal. Furthermore, DN-1 showed good antifungal activity against Aspergillus flavus BNCC336156 and Aspergillus parasiticus BNCC335939. The optimum growth temperature and pH of DN-1 were 37 °C and 6.0, respectively, and DN-1 grew well in the concentration range of 0–20 µg/L AFB1. Under a temperature of 20–40 °C, pH of 3.0–9.0, and anaerobic conditions, the percent removal of AFB1 was more than 60%. An analysis of the different components of DN-1 showed that cell wall adsorption was the main removal method and suggested the pathway for AFB1 removal by LAB. In addition, strain DN-1 was used as a biological preservative in artificially contaminated peanut and sunflower cakes, which significantly inhibited the growth of mold and production of AFB1. In brief, this study highlights the potential use of DN-1 as a preventive agent against aflatoxicosis via strong removal capability in the application of fermented feed or food.