Effects of lactic acid bacteria inoculants on the nutrient composition, fermentation quality, and microbial diversity of whole-plant soybean-corn mixed silage

Abstract

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.

Keywords: Lactobacillus buchneri; Lactobacillus plantarum; aerobic stability; microbial community; silage.

Figure 1

Dynamics of mixed silage temperature (°C) versus ambient temperature during aerobic exposure (A) and the time when the silage temperature was 2°C below ambient temperature (B). Dynamics of [(C) LAB; (D) Yeast; (E) Mold] during aerobic exposure. Different letters indicate significant differences (p < 0.05), and the same or no letters indicate non-significant differences (p > 0.05).

Figure 2

Venn diagram (A) and nonmetric multidimensional scaling (B) of the bacterial community at the operational taxonomic unit level. FM, fresh material; CK, no additive; LP, L. plantarum; LB, L. buchneri; LPB, L. plantarum + L. buchneri. 14, 28, and 56 indicate 14, 28, and 56 days of ensiling, respectively.

Figure 3

(A) The relative abundance (%) of bacterial phyla (at least 1% in one group) in FM and forage oat silage at the phylum level. (B) The relative abundance (%) of bacterial phyla (at least 1% in one group) in FM and forage oat silage at the genus level. (C) Linear discriminant analysis effect size (LEfSe) was used to assess differences in microbial communities between FM and silage (LDA score > 4.0). The length of the histogram represents the LDA score of different species. FM, fresh material; CK, no additive; LP, L. plantarum; LB, L. buchneri; LPB, L. plantarum + L. buchneri. 14, 28, and 56 indicate 14, 28, and 56 days of ensiling, respectively.

Figure 4

This heatmap displays Pearson’s correlations between genus-level microbial communities and fermentation parameters. Positive and negative correlations are marked in red and blue, respectively. *p < 0.05; **p < 0.01; ***p < 0.001. NH₃-N, ammonia nitrogen; LA, lactic acid; AA, acetic acid; PA, propionic acid.

Figure 5

Tax4Fun analysis was used to elucidate the functional metabolic profile of the bacterial community in silage fermented for 56 days. (A) Level 1 metabolic pathways; (B) Level 2 Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologue functional predictions of the relative abundances of the top 20 metabolic function. *p < 0.05; **p < 0.01; ***p < 0.001. CK, no additive; LP, L. plantarum; LB, L. buchneri; LPB, L. plantarum + L. buchneri. 56 indicates 56 days of ensiling.