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

Table 1. Evaluation methods for intestinal BSH activity
Target
Method Sample BSH activity indicators Pros and cons References
compounds/microbes
Mass Bile acid profiles (e.g., GCDCA, Cecal contents, Ratio of CDCA to TCDCA Pros: high accuracy, high [17,18]
spectrometry CDCA) serum, feces throughput
Cons: expensive
Ninhydrin Free amino acids released from Feces Rate of CA generation Pros: cost-effective [19]
colorimetry conjugated BAs Cons: low specificity
Microbial
sequencing
16s rRNA Bacteroides, Clostridium, Feces Relative abundance of Pros: established method [20]
Lactobacillus representative BSH- Cons: indirect reflection of
producing bacteria BSH gene expression
Bacteroides, Lactobacillus, [21]
Bifidobacterium
Bifidobacterium, Lactobacillus [22]
16s rRNA Various species Feces Relative abundance of BSH Pros: easy to perform [9,10]
genes Cons: limited by database
coverage and update
frequency
Metagenomics Various species Feces Relative abundance of BSH Pros: high accuracy, [9,16]
homologous sequences comprehensive analysis
Cons: poor data quality and
parameter consistency
Activity probe Ch-AOMK, BAL Bacteria, feces Fluorescence intensity Pros: high sensitivity [23,24]
Cons: expensive
BSH: Bile salt hydrolase; CDCA: chenodeoxycholic acid; BAs: bile acids; GCDCA: glycochenodeoxycholic acid; TCDCA: taurochenodeoxycholic
acid; CA: cholic acid; BAL: BSH-activatable luciferin.

in the gut microbiome of MASLD patients . Additionally, a metagenomic study in children with MASLD
[9]
reported that the conversion of PBAs to SecBAs was inhibited, which was supported by a marked decrease
in BSH-expressing genera (Bacteroides and Eubacterium) . Numerous animal studies have further
[25]
validated the reduction of BSH in MASLD mouse models. Modulating BA metabolism by targeting
BSH-expressing bacteria has shown beneficial effects on cholesterol, triglyceride, and glucose levels. For
instance, increasing the abundance of BSH-producing bacteria via herbal medicine elevated the levels of
unconBAs in the serum and liver. This, in turn, upregulated the expression of Ehhadh and Hadha genes,
promoting fatty acid degradation, lowering cholesterol and triglyceride levels, and improving glucose
homeostasis . He et al. demonstrated that a high-fat diet disrupted glucose homeostasis in mice and led to
[26]
a reduction in hyocholic acid (HCA), which was associated with decreased abundance of Lactobacillus and
[22]
Bifidobacterium, two key BSH-producing genera . HCA is known to improve glucose homeostasis via
distinct TGR5 and FXR signaling pathways . In addition to HCA, other unconBAs such as
[27]
ursodeoxycholic acid (UDCA) and LCA have also been found to offer metabolic benefits, including
anti-inflammatory, antioxidant, and gut barrier-protective effects. Moreover, enhancing microbial BSH
expression or activity can increase the ratio of unconBAs to conBAs. Because unconBAs are more
hydrophobic, they are more readily excreted via feces. This increases hepatic BA synthesis, particularly via
alternative pathways, thereby promoting cholesterol consumption and alleviating hepatic steatosis [28,29] .

However, not all findings are consistent. Some studies have reported increased microbial BSH expression in
MASLD. In these cases, reducing BSH expression or suppressing BSH activity was found to increase the
conBA/unconBA ratio, reduce cholesterol levels, and limit lipid accumulation. Elevated microbial BSH
activity may raise concentrations of certain cytotoxic unconBAs, such as glycodeoxycholic acid,
[11]
7-ketodeoxycholic acid, and dehydrocholic acid, which can exacerbate liver damage . Smirnova et al.
reported significantly higher BSH gene expression in MASLD patients compared to healthy individuals,