Silibinin reduces in vitro methane production by regulating the rumen microbiome and metabolites

Rui Liu; Yueyu Shen; Haokai Ma; Yang Li; Modinat Tolani Lambo; Baisheng Dai; Weizheng Shen; Yongli Qu; Yonggen Zhang

doi:10.3389/fmicb.2023.1225643

Silibinin reduces in vitro methane production by regulating the rumen microbiome and metabolites

Front Microbiol. 2023 Aug 23:14:1225643. doi: 10.3389/fmicb.2023.1225643. eCollection 2023.

Authors

Rui Liu¹, Yueyu Shen², Haokai Ma¹, Yang Li¹, Modinat Tolani Lambo¹, Baisheng Dai³, Weizheng Shen³, Yongli Qu^{4

5}, Yonggen Zhang¹

Affiliations

¹ College of Animal Science and Technology, Northeast Agricultural University, Harbin, China.
² Beijing Sunlon Livestock Development Company Limited, Beijing, China.
³ College of Electrical Engineering and Information, Northeast Agricultural University, Harbin, China.
⁴ College of Animal Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, China.
⁵ Key Laboratory of Low-carbon Green Agriculture in Northeastern China of Ministry of Agriculture and Rural Affairs, Daqing, China.

Abstract

This study used Silibinin as an additive to conduct fermentation experiments, wherein its effects on rumen gas production, fermentation, metabolites, and microbiome were analyzed in vitro. The silibinin inclusion level were 0 g/L (control group), 0.075 g/L, 0.15 g/L, 0.30 g/L, and 0.60 g/L (experimental group). Fermentation parameters, total gas production, carbon dioxide (CO₂), methane (CH₄), hydrogen (H₂), and their percentages were determined. Further analysis of the rumen microbiome's relative abundance and α/β diversity was performed on the Illumina NovaSeq sequencing platform. Qualitative and quantitative metabolomics analyses were performed to analyze the differential metabolites and metabolic pathways based on non-targeted metabolomics. The result indicated that with an increasing dose of silibinin, there was a linear reduction in total gas production, CO₂, CH₄, H₂ and their respective percentages, and the acetic acid to propionic acid ratio. Concurrent with a linear increase in pH, when silibinin was added at 0.15 g/L and above, the total volatile fatty acid concentration decreased, the acetic acid molar ratio decreased, the propionic acid molar ratio increased, and dry matter digestibility decreased. At the same time, the relative abundance of Prevotella, Isotricha, Ophryoscolex, unclassified_Rotifera, Methanosphaera, Orpinomyces, and Neocallimastix in the rumen decreased after adding 0.60 g/L of silibinin. Simultaneously, the relative abundance of Succiniclasticum, NK4A214_group, Candidatus_Saccharimonas, and unclassified_Lachnospiraceae increased, altering the rumen species composition, community, and structure. Furthermore, it upregulated the ruminal metabolites, such as 2-Phenylacetamide, Phlorizin, Dalspinin, N6-(1,2-Dicarboxyethyl)-AMP, 5,6,7,8-Tetrahydromethanopterin, Flavin mononucleotide adenine dinucleotide reduced form (FMNH), Pyridoxine 5'-phosphate, Silibinin, and Beta-D-Fructose 6-phosphate, affecting phenylalanine metabolism, flavonoid biosynthesis, and folate biosynthesis pathways. In summary, adding silibinin can alter the rumen fermentation parameters and mitigate enteric methane production by regulating rumen microbiota and metabolites, which is important for developing novel rumen methane inhibitors.

Keywords: metabolite; methane; microbiome; rumen fermentation; silibinin.