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.

The aim of this study was to investigate effects of wilting and Lactobacillus plantarum inoculation on the dynamics of the fermentation products, residual non-structural carbohydrates, and bacterial communities in alfalfa silage. Fresh and wilted alfalfa were ensiled with and without L. plantarum for 10, 30, 60, and 90 days. A high-throughput sequencing method for absolute quantification of 16S rRNA was adopted to determine the bacterial community composition at different ensiling periods. For the wilted silage, the bacterial community, pH value, and ammonia nitrogen concentration remained stable in the silage at 30 days. L. plantarum inoculation accelerated lactic acid fermentation and altered the predominant genus in the wilted silage as compared with the non-inoculated group. For the non-wilted group, fast consumption of water-soluble carbohydrates (WSCs) was observed at 10 days in the non-inoculated silage along with rapid growth of undesirable Hafnia. L. plantarum inoculation inhibited growth of Hafnia at 10 days in the non-wilted silage. Clostridia fermentation occurred in the non-wilted silage at 90 days, as indicated by an increased pH, formation of butyric acid (BA), and apparent abundance of genera belonging to Clostridia. L. plantarum inoculation inhibited BA accumulation and growth of Garciella in the non-wilted silage at 90 days as compared with the non-wilted silage without inoculation, but had little effect on the growth of Clostridium sensu stricto. Overall, the high moisture content of the non-wilted alfalfa silage led to rapid consumption of WSCs and growth of harmful microorganisms at the early stage of ensiling, resulting in poor fermentation quality. Wilting and L. plantarum inoculation both improved fermentation quality and inhibited the growth of spoilage microorganisms in alfalfa silage, while L. plantarum inoculation alone failed to achieve optimum fermentation quality of non-wilted alfalfa silage.

Relative quantification 16S-seq (RQS) has drawn deeper insights into bacterial community compositions in silage. However, it provides no information on dynamics of the total amount of bacterial DNA through the ensiling process and across different treatments. In this study, bacterial compositions in alfalfa silage with and without Lactobacillus plantarum inoculation after 10 and 60days of ensiling were investigated using absolute quantification 16S-seq (AQS), and bacterial composition and its interaction with fermentation properties of silage indicated by AQS and RQS were compared. Variation in total bacterial DNA amounts across different treatments and ensiling periods was illustrated by AQS. AQS indicated higher bacterial richness indices and closer correlations of these indices with fermentation properties than RQS via spearman’s correlation analyses, as well as more taxa with significance on bacterial abundance via lefse analyses. In conclusion, AQS effectively illustrated the dynamics of bacterial communities during the ensiling process.

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.