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Chen et al. Microbiome Res Rep 2025;4:6 https://dx.doi.org/10.20517/mrr.2024.38 Page 3 of 14

proportion of unconjugated bile acids. Due to the stronger hydrophobicity of unconjugated bile acids, they
are less readily ingested by the gut and are discharged together with feces. Consequently, with fewer bile
acids available, the liver is stimulated to synthesize more bile acids from cholesterol to maintain the
[8]
enterohepatic bile acid balance . Additionally, the compositional changes in bile acids influenced the
farnesoid X receptor (FXR) signaling pathway . The FXR, which is activated by bile acids, acts as a
[9]
[10]
transcription factor that functions as a ligand-activated receptor . Different bile acids are capable of acting
as messenger molecules to activate or inhibit FXR, making the distribution and variation of the bile acid
composition a material basis for the impact of BSH on FXR expression. The control of bile acid transport is
affected by the expression of genes encoding ileal bile acid-binding proteins and organic solute transport
proteins α/β (OSTα/β), which are both substantially expressed in the ileum when FXR is activated .
[11]

Previous research has found that Lactobacillus johnsonii (L. johnsonii) commonly possesses a greater
number of BSH genes . However, the BSH activity and its capacity to regulate cholesterol metabolism in
[12]
L. johnsonii are not yet well-defined. L. johnsonii CCFM1376 was isolated from a fecal sample of an adult
female and identified as L. johnsonii by 16S rDNA sequencing results. This study focuses on the high BSH
activity strain, L. johnsonii CCFM1376, to evaluate its ability to alleviate hypercholesterolemia in mice.
Additionally, the study explores the pathways through which it may ameliorate hypercholesterolemia by
modulating bile acid metabolism.

METHODS
Quantitative determination of BSH activity
The activated bacterial suspension was inoculated into 20 mL of MRS medium at a 1% volume ratio and
statically cultured at 37 °C for 24 h. Post-fermentation, the broth underwent a 10-minute centrifugation
process at 10,000 × g and a temperature of 4 °C, which effectively separated the bacterial cells. The cell pellet
was washed twice and recentrifuged and resuspended the pellet in physiological saline. Adjust the bacterial
suspension concentration to an optical density (OD) of 1 at a wavelength of 600 nm to standardize the
concentration of the bacterial suspensions. In addition, 1 mL sample of the bacterial suspension with
adjusted concentration was treated with an ultrasonic disrupter (Ningbo Xinzhi Biotechnology Co., Ltd.) for
3 min (with a duty cycle ratio of 2:3). Post-treatment, the solution underwent a second centrifugation for
10 min at 10,000 × g and 4 °C to precipitate the cellular fragments, yielding a clear cell-free extract.
Subsequently, 0.1 mL of the supernatant was combined with an equal volume of a 200 mM conjugated bile
salt solution and 1.8 mL of a 0.1 M phosphate buffer at pH 6.0, followed by a 30-minute incubation period
at 37 °C with constant agitation. After incubation, a portion of 0.5 mL was extracted from the mixture and
subsequently combined with an equal volume of a 15% trichloroacetic acid solution to cease the reaction
process, followed by thorough mixing and centrifugation at the maximum speed of the centrifuge for
10 min at 4 °C to collect the supernatant. Subsequently, 0.1 mL of the supernatant was combined with
1.9 mL of ninhydrin reagent, stirred well, and heated in boiling water for 15 min. After cooling to ambient
temperature, the mixture’s absorbance was then determined at a wavelength of 570 nm. Standard curves
were prepared using glycine and taurine, respectively. The total BSH enzyme activity is defined as the
amount of substrate, in micromoles per minute per milliliter, that is hydrolyzed by the crude enzyme to
produce amino acids from conjugated bile salts per unit volume per unit time, with the unit expressed as
μmol (min·mL) . The calculation formula is: TA = 4/3 Caa, where Caa represents the concentration of
-1
amino acids .
[13]

Animal experiments
The animal study was approved by the Jiangnan University Experimental Animal Management and Animal
Welfare Ethics Committee (JN.No20230415c1000801[117]). After a week-long acclimation, thirty-two 4-
week-old male C57BL/6J mice were evenly allocated into four distinct groups: the control group, the model