Evaluation of the fermentation performance and functional properties of bacterial communities of amaranth silage supplemented with Limosilactobacillus fermentum and Latilactobacillus graminis
Authors: Zhao Muqier , Bao Jian , Wang Zhijun , Du Shuai , Gao Cuiping , Nan Dinghan , Yan Xingquan , Ge Gentu 
Published: September 2023

Abstract
Background
The objective was to determine the effects of different lactic acid bacteria (LAB) on amaranth (Amaranthus hypochondriaus) silage fermentation characteristics, changes in bacterial communities and their functional properties, and to investigate the preferential effects and mechanisms of action of Limosilactobacillus fermentum (L. fermentum) and Latilactobacillus graminis (L. graminis) on the bacterial communities of silage. LAB were screened and identified from native grasses and its naturally fermented silage, and their effects on fermentation performance and bacterial community of amaranth after 60 days of fermentation were investigated. BL1 and BL5 strains were identified as L. fermentum and L. graminis, respectively. They could grow normally at concentrations of 3.0% and 6.5% NaCl, tolerating pH and temperature ranges of 3.5 –9.0 and 4.0 – 9.0, 30 – 45 ℃ and 10 – 45 ℃, respectively. The distilled water, Lactiplantibacillus plantarum (commercial inoculant), L. fermentum, and L. graminis were inoculated into amaranth at a total of 1 × 105 cfu/g fresh material and labeled AhSCK, AhSLP, AhSLF, and AhSLG, respectively, while the fresh amaranth was labeled YLS.

Results
As expected, the water soluble carbohydrates of AhSCK was lower than AhSLG (p < 0.05). The highest number of LAB was found in AhSLF (p < 0.05). The lactic acid (LA) content of AhSLP was significantly higher (p < 0.05) than other silage. Nevertheless, acetic acid (AA) content of AhSLF was significantly different from other silage (p < 0.05). All the amaranth silage was well preserved with low pH (4.16 – 4.51) and acceptable levels of butyric acid (0.00 – 0.86%FM). At the species level, Pantoea agglomerans, Pseudomonas oryzihabitans, and Cenchrus americanus served as the dominant strains in fresh amaranth, with relative abundances lower than 1% after ensiling.. Lentilactobacillus buchneri, Levilactobacillus brevis, and L. fermentum were the dominant strains of AhSCK, AhSLP and AhSLF. Nevertheless, the predominant strains of AhSLG was Xanthomonas oryzae. Both fresh material and silage, L. fermentum, L. plantarum, Levilactobacillus brevis, Pantoea agglomerans, Pseudomonas oryzihabitans, Cenchrus americanus, Pseudomonas coleopterorum, Priestia aryabhattai, and Exiguobacterium sibiricum were significantly different in relative abundance (p < 0.01).Functional prediction analysis showed that in pyruvate metabolism, glycolysis/gluconeogenesis and amino sugar and nucleotide sugar metabolism were enriched after ensiling. AhSLF showed the most obvious trend of increasing abundance of pentose phosphate pathway, biosynthesis of secondary metabolites and biosynthesis of antibiotics, while the abundance of metabolic pathways decreased significantly (p < 0.05).

Conclusions
L. fermentum can be recommended as an additive to improve the quality of amaranth silage, as it regulates the epiphytic microbiota in silage, improving the fermentation quality and metabolic capacity of the bacterial community.

Graphical Abstract