Using gas chromatography mass spectrometry and the PacBio single molecule with real-time sequencing technology (SMRT), we analyzed the detailed metabolomic profiles and microbial community dynamics involved in ensiled Medicago sativa (alfalfa) inoculated without or with the homofermenter Lactobacillus plantarum or heterofermenter Lactobacillus buchneri. Our results revealed that 280 substances and 102 different metabolites were present in ensiled alfalfa. Inoculation of L. buchneri led to remarkable up-accumulation in concentrations of 4-aminobutyric acid, some free amino acids, and polyols in ensiled alfalfa, whereas considerable down-accumulation in cadaverine and succinic acid were observed in L. plantarum-inoculated silages. Completely different microbial flora and their successions during ensiling were observed in the control and two types of inoculant-treated silages. Inoculation of the L. plantarum or L. buchneri alters the microbial composition dynamics of the ensiled forage in very different manners. Our study demonstrates that metabolomic profiling analysis provides a deep insight in metabolites in silage. Moreover, the PacBio SMRT method revealed the microbial composition and its succession during the ensiling process at the species level. This provides information regarding the microbial processes underlying silage formation and may contribute to target-based regulation methods to achieve high-quality silage production.

We studied the effects on alfalfa preservation and chemical composition of the addition of different levels of malic acid and citric acid at ensiling as well as the utilization efficiency of these 2 organic acids after fermentation. Alfalfa was harvested at early bloom stage. After wilting to a dry matter content of approximately 40%, the alfalfa was chopped into 1- to 2-cm pieces for ensiling. Four levels (0, 0.1, 0.5, and 1% of fresh weight) of malic acid or citric acid were applied to chopped alfalfa at ensiling with 4 replicates for each treatment, and the treated alfalfa forages were ensiled for 60 d in vacuum-sealed polyethylene bags (dimensions: 200 mm × 300 mm) packed with 200 to 230 g of fresh alfalfa per mini silo and an initial density of 0.534 g/cm. The application of malic or citric acids at ensiling for 60 d led to lower silage pH than was observed in the control silage (0% of malic or citric acids). Application of the 2 organic acids led to higher lactic acid concentration in alfalfa silage than in the control silage except with the application rate of 1% of fresh weight. Silages treated with both organic acids had lower nonprotein nitrogen concentrations than the control silages, and the nonprotein nitrogen concentrations in ensiled forages decreased with the increase in malic or citric acid application rates. The application of the 2 organic acid additives led to lower saturated fatty acid proportions and higher polyunsaturated fatty acid proportions in ensiled alfalfa than in the control silage. The amount of malic and citric acids degraded during ensiling of alfalfa was 1.45 and 0.63 g, respectively. At the application rate of 0.5% of fresh weight, residues of malic acid and citric acid in alfalfa silage were 11.1 and 13.6 g/kg of dry matter. These results indicate that including malic or citric acids at the ensiling of alfalfa effectively improved silage fermentation quality, limited proteolysis, improved fatty acid composition of the ensiled forage, and could provide animals with additional feed additives proven to promote animal performance. However, when the application rate of both organic acids reached 1%, the concentration of lactic acid in silages decreased notably. Additionally, 0.5 and 1% application rates also increased the yeast count in ensiled alfalfa.

Aims Ferulic acid esterase (FAE)‐producing Lactobacillus species isolated from ensiled Elymus nutans growing on the Qinghai‐Tibetan plateau were characterized, and effects of their application to the alfalfa ensiling process and the evidence to synergic effect between cellulase and FAE were investigated. Methods and Results The results of 16S rRNA gene sequence and species‐specific polymerase chain reaction amplification showed that two screened strains with high FAE activity were Lactobacillus plantarum A1 (LP) and L. brevis A3 (LBr). The optimum temperature and pH for the LP and LBr was 37°C and 6·4 respectively. The FAE exhibited a good stability at temperatures between 25 and 50°C and at pH values of 5·0–7·0. The two strains and a commercial cellulase (CE) were applied as additives to alfalfa silage. After 60 days of ensiling, the lactic acid in the control and CE groups were significantly lower than those of the other treatment groups. The neutral detergent fibre and acid detergent fibre contents in the LP group were significantly lower than those observed in the other groups. At the same time, the combination of CE and FAE‐producing lactic acid bacteria synergistically improved the fermentation quality of the silage. Conclusions The addition of the FAE‐producing strain of L. plantarum A1 to alfalfa silage improved its fermentation quality, and reduced the fibre content of the silage. Significance and Impact of the Study The screened homo‐fermentative and FAE‐producing strain of L. plantarum A1 could be a candidate strain in improving fermentation quality and fibre digestibility of ensiled forages.

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The selection of P. pentosaceus inoculants for improving silage quality at low temperature, which provides a candidate strain to make high-quality silage in regions with frigid climate.