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Zhao et al. Microbiome Res Rep. 2025;4:28 https://dx.doi.org/10.20517/mrr.2025.12 Page 3 of 13

Metabolic function of BSH
BSH plays a pivotal role in BA metabolism by deconjugating bile salts, thereby altering their
physicochemical properties and influencing their absorption and reabsorption in the intestine.
Deconjugation is a key step in the enterohepatic circulation of BAs, as it affects the composition of the BA
pool and impacts processes such as lipid emulsification, cholesterol metabolism, and gut microbial ecology.
BSH is traditionally understood to function by hydrolyzing amide bonds in conBAs, releasing free BAs and
their respective amino acid residues . However, recent studies have uncovered that BSH can also catalyze
[5]
the conjugation of unconBAs with amines to form bacterial bile acid amidates (BBAAs). This discovery
challenges the conventional understanding of BSH functions and provides new insights into its broader role
[14]
in BA metabolism .
Distribution of BSH in the gut microbiota
BSH is widely distributed among gut bacteria species, with activity levels varying depending on bacterial
strain and environmental conditions. BSH genes have been identified in Lactobacillus, Bifidobacterium,
Clostridium, Enterococcus, Bacteroides, Listeria, Brucella, and Xanthomonas, which are typically associated
with high BSH activity . Advances in next-generation sequencing and bioinformatics have significantly
[15]
enhanced our understanding of the BSH gene in the human gut microbiome. These studies indicate that
BSH genes are predominantly expressed in members of the phyla Firmicutes, Bacteroidetes, Actinobacteria,
Proteobacteria, and Euryarchaeota, with Lactobacillus species showing the highest BSH activity in the
human gut [9,16] . Despite these findings, the structural and functional diversity of BSH enzymes across
different microbial taxa remains insufficiently understood.

Evaluation methods for changes in gut microbial BSH
The expression and activity of microbial BSH are typically evaluated by measuring changes in the
composition and concentration of the BA pool, BSH gene expression levels, or in vitro enzymatic activity.
Four common tools are used for these measurements: mass spectrometry, ninhydrin colorimetry, microbial
sequencing, and activity probes [Table 1]. Mass spectrometry offers high sensitivity and specificity, enabling
the precise quantification of BSH activity by measuring the concentrations of conBAs and unconBAs to
determine the hydrolysis rate [17,18] . Ninhydrin colorimetry is a traditional method that detects amino acids
[19]
released by BSH activity through a colorimetric reaction . Although less specific and accurate than modern
techniques, it remains a simple and cost-effective approach for evaluating enzyme activity in vitro.
Microbial sequencing can infer changes in BSH expression by analyzing homologous gene sequences, copy
numbers, or the relative abundance of BSH-expressing bacterial taxa [9-11,16,20-22] . Moreover, recent
developments in activity-based probes have significantly improved the precision of BSH activity
measurement [23,24] . Probes such as Ch-AOMK bind specifically to BSH active sites and enhance quantitative
analysis via mass spectrometry. These probes hold great potential for non-invasive diagnostics and
therapeutic monitoring in conditions such as inflammatory bowel disease (IBD) .
[24]

CHANGES IN MICROBIAL BSH IN MASLD
Microbial BSH plays a pivotal role in the pathophysiology of MASLD. MASLD is characterized by excessive
fat accumulation in the liver, and recent evidence suggests that alterations in gut microbial BSH may
influence disease progression through modulation of BA metabolism. This section summarizes the changes
in microbial BSH across the disease spectrum, from MASLD to its more severe forms including MASH and
liver fibrosis [Table 2].

MASLD
Both clinical and animal studies consistently report a significant reduction in microbial BSH in MASLD.
Two clinical studies from the USA and China showed that a significantly reduced abundance of BSH genes