The aim of this study was to gain deeper insights into the dynamics of fermentation parameters and the bacterial community during the ensiling of high-moisture alfalfa. A commercial lactic acid bacteria (YX) inoculant was used as an additive. After 15 and 30 days of ensiling, the control silage (CK) exhibited a high pH and a high concentration of ammoniacal nitrogen (NH₃-N); Enterobacter and Hafnia-Obesumbacterium were the dominant genera. At 60 d, the pH value and the concentration of NH₃-N in CK silage increased compared with 15 and 30 d, propionic acid and butyric acid (BA) were detected, and Garciella had the highest abundance in the bacterial community. Compared with CK silage, inoculation of YX significantly promoted lactic acid and acetic acid accumulation and reduced pH and BA formation, did not significantly reduce the concentration of NH₃-N except at 60 d, and significantly promoted the abundance of Lactobacillus and decreased the abundance of Garciella and Anaerosporobacter, but did not significantly inhibit the growth of Enterobacter and Hafnia-Obesumbacterium. In conclusion, high-moisture alfalfa naturally ensiled is prone to rot. Adding YX can delay the process of silage spoilage by inhibiting the growth of undesirable microorganisms to a certain extent.

Lactic acid bacteria (LAB) can produce many kinds of antifungal substances, which have been widely proven to have antifungal activity. In this study, 359 strains of LAB were screened for antifungal activity against Aspergillus niger (A. niger) using the 96-well microtiter plate method, and three showed strong activity. Of these, ZZUA493 showed a broad-spectrum antifungal ability against A. niger, Aspergillus oryzae, Trichoderma longibrachiatum, Aspergillus flavus and Fusarium graminearum. ZZUA493 was identified as Lactobacillus plantarum. Protease treatment, the removal of hydrogen peroxide with catalase and heat treatment had no effect on the antifungal activity of the cell-free supernatant (CFS) of ZZUA493; organic acids produced by ZZUA493 appeared to have an important role in fungal growth inhibition. The contents of lactic acid, acetic acid and phenyllactic acid in the CFS tended to be stable at 48 h, and amounted to 28.5, 15.5 and 0.075 mg/mL, respectively. In addition, adding ZZUA493, as an ingredient during their preparation, prolonged the shelf life of Chinese steamed buns. Overall, ZZUA493 appears to have good potential as a fungal inhibitor for food preservation.

This study assessed the effects of Lactiplantibacillus plantarum (ZZU203), cellulase-producing Bacillus methylotrophicus (CB), or their combination (ZZU203_CB) on the fermentation parameters of alfalfa after 10 and 60 days of ensiling. Additionally, the bacterial community compositions were analyzed using absolute quantification 16S-seq (AQS). The results showed that CB silage displayed a higher lactic acid (LA) concentration at 10 d, a higher abundance of Lactobacillus, and lower abundance of Pediococcus, Enterococcus, and Weissella than those in the control (CK) silage. Compared with CK silage, the ZZU203 silage increased LA concentration, fructose and rhamnose concentrations, and the abundance of Lactobacillus, and decreased pH value, ammoniacal nitrogen, acetic acid, neutral detergent fiber and acid detergent fiber concentrations, and the abundance of Pediococcus, Enterococcus, Weissella, Hafnia, and Garciella after 60 days of ensiling. In addition, ZZU203 and ZZU203_CB silage had a similar silage quality and bacterial community, while the inoculation of ZZU203_CB significantly promoted LA accumulation and the numbers of Lactobacillus at 10 d compared with ZZU203 silage. Therefore, ZZU203 or a combination of ZZU203 and CB can be used as potential silage additives to improve the silage quality of alfalfa.

To investigate the effects of lactic acid bacterial (LAB) inoculants and vacuuming on the fermentation quality and bacterial community, alfalfas were ensiled with or without a commercial LAB YX or Lactobacillus plantarum strain ZZUA493 for 10, 30, 60, and 90 days while undergoing either vacuum (V) or nonvacuum (NV) treatment. At 90 days, analysis of the microbial community by high-throughput sequencing was performed, and contents of aflatoxin B1 and deoxynivalenol (DON) mycotoxins in alfalfa silage were determined. In all inoculated alfalfa silage, irrespective of V or NV treatment, lactic acid (LA) content increased, pH (p < 0.05), and ammonia nitrogen (p < 0.05) content decreased, and no butyric acid was detected. Lactobacillus or Pediococcus became the dominant genus, and the abundance of Garciella decreased in alfalfa silage with the addition of either inoculant. The LAB inoculants YX and ZZUA493 helped reduce the mycotoxin content in alfalfa silage. The abundance of Garciella in the control and DON content in all alfalfa silage groups were higher (p < 0.05) in NV than V. In summary, LAB inoculants and vacuuming had a positive influence on alfalfa silage quality, and LAB inoculants were effective in reducing mycotoxins in silage alfalfa.