Method for realizing high yield of bile salt hydrolase from probiotics by utilizing intestinal selective pressure and application

Abstract

The invention provides a method for realizing high yield of bile salt hydrolase by probiotics by utilizing intestinal selective pressure and application, and the method comprises the following steps: changing the intestinal selective pressure by high fat diet to promote adaptive evolution of bifidobacterium animalis H22B656 to obtain a mutant strain for efficiently producing bile salt hydrolase. Directional breeding is carried out through a sterile mouse model and intestinal tract selection pressure, the mutant strain with the bile salt hydrolase production capacity and the functional effect of the probiotics remarkably enhanced is screened out, and a new strategy is provided for functional probiotic development and accurate micro-ecological intervention of metabolic diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of bile salt hydrolase production, and relates to a method and application for realizing high-yield bile salt hydrolase production by probiotics using intestinal selection pressure. Background Art

[0002] Bile salt hydrolase (BSH) is an important functional enzyme in probiotics, which can catalyze the decomposition of conjugated bile salts into free bile acids and glycine or taurine. Its physiological function has an important impact on host metabolism and health. For example, BSH activity can reduce serum cholesterol levels and regulate bile acid metabolism, thereby affecting the intestinal microecological balance and the occurrence of metabolic diseases. In addition, probiotics producing highly efficient BSH enzymes have broad application prospects in the food industry, the development of functional foods, and the intervention of intestinal diseases.

[0003] However, in the complex intestinal microecological environment, the survival and functional expression of probiotics are often affected by the competition of other intestinal flora and the inhibition of metabolites, which limits the development and application of probiotics producing highly efficient BSH enzymes. Summary of the Invention

[0004] Based on the above, the purpose of the present invention is to provide a method and application for realizing high-yield bile salt hydrolase production by probiotics using intestinal selection pressure, which can promote the preferential growth of specific functional strains and enhance their functional expression, and is particularly significant in the efficient production of BSH enzymes.

[0005] The technical solution adopted by the present invention to achieve the technical purpose is as follows:

[0006] The present invention provides a strain for efficiently producing bile salt hydrolase, named H22B656-W5S9, which was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on January 2, 2025. The deposit address is: 5th Floor, Institute of Microbiology, Guangdong Academy of Sciences, Building 59, No. 100, Xianlie Middle Road, Guangzhou. The deposit number is GDMCC No: 65712, and the taxonomic name is Bifidobacterium animalis.

[0007] Preferably, the strain H22B656-W5S9 is a mutant strain of Bifidobacterium animalis H22B656, and the bile salt hydrolase activity of the strain H22B656-W5S9 and the expression level of bile salt hydrolase in the transcriptome are higher than those of Bifidobacterium animalis H22B656. The Bifidobacterium animalis H22B656 was deposited at the Guangdong Microbial Culture Collection Center (GDMCC) on January 2, 2025. The deposit address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. The deposit number is GDMCC No: 65711, and the taxonomic name is Bifidobacterium animalis.

[0008] The present invention also provides a method for achieving high-yield bile salt hydrolase production by probiotics using intestinal selection pressure, including: changing the intestinal selection pressure through a high-fat diet to promote the adaptive evolution of Bifidobacterium animalis H22B656, and obtaining a mutant strain with high-efficiency bile salt hydrolase production. The Bifidobacterium animalis H22B656 was deposited at the Guangdong Microbial Culture Collection Center (GDMCC) on January 2, 2025. The deposit address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. The deposit number is GDMCC No: 65711, and the taxonomic name is Bifidobacterium animalis.

[0009] Preferably, in the above method, germ-free mice are used as the domestication vector.

[0010] More preferably, the domestication time is 7 weeks.

[0011] More preferably, the high-fat diet provides 60% of the energy from fat.

[0012] Preferably, the bile salt hydrolase activity of the mutant strain and the expression level of bile salt hydrolase in the transcriptome are higher than those of the original strain.

[0013] The present invention also provides the application of the above-mentioned strain with high-efficiency bile salt hydrolase production in the preparation of a drug for preventing or improving non-alcoholic fatty liver.

[0014] Preferably, the strain can increase the level of HDL-C, reduce the content of MDA and the level of LDL-C.

[0015] More preferably, the strain can alleviate the excessive accumulation of liver fat in non-alcoholic fatty liver and repair liver tissue damage.

[0016] The beneficial effects of the present invention are as follows:

[0017] This application uses germ-free mice as a domestication vector, simulates the extreme environment under a high-fat human diet through a high-fat model diet, and changes the intestinal selection pressure to promote the adaptive evolution of the probiotic animal Bifidobacterium H22B656. Among them, the germ-free mouse model provides an ideal platform. The germ-free environment can eliminate the interference of endogenous flora and provide clear causal verification for the colonization and functional realization of specific strains. Using intestinal selection pressure, such as specific dietary interventions or chemicals, can promote the preferential growth of specific functional strains and enhance their functional expression, especially in the efficient production of BSH enzyme. To evaluate the domestication effect, this application further constructs a non-alcoholic fatty liver model to verify the differential effects of mutant strains and original strains in preventing non-alcoholic fatty liver.

[0018] This application improves the BSH enzyme production of H22B656 through directed mutagenesis, and obtains the mutant strain H22B656-W5S9 with high-efficiency BSH enzyme production, thereby laying a scientific foundation for the development and practical application of functional probiotics. This strategy not only provides theoretical support for the development of functional probiotics, but also promotes their application in the treatment of intestinal diseases and metabolic regulation, opening up a new way for precise microecological intervention. Brief Description of the Drawings

[0019] Figure 1 Showing the degradation rate of the mutant strain / original strain in the examples of the present invention (in the sample names on the abscissa, the Arabic numerals after W represent the domestication time, and the numbers after S represent the sample numbers).

[0020] Figure 2 Showing the expression levels of bile salt hydrolase of the original strain and the domesticated strain in the examples of the present invention.

[0021] Figure 3 Showing the levels of TG, TC, LDL-C, HDL-C and MDA after 8 weeks (Wilcoxon rank sum test, *P<0.05, **P<0.01, ***P<0.001).

[0022] Figure 4 Showing the morphological analysis of the liver tissues of four groups of mice. Detailed Embodiments

[0023] To illustrate the present invention more clearly, the present invention will be further described in detail below in conjunction with examples and with reference to the accompanying drawings. Those skilled in the art should understand that the specific content described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0024] Examples

[0025] I. Materials and Methods

[0026] 1.1 Materials and Reagents

[0027] 1.1.1 Isolation, identification and screening of Bifidobacterium animalis H22B656

[0028] Bifidobacterium animalis H22B656 was provided by the Tropical Probiotics Innovation and Utilization Team of the School of Food Science and Engineering, Hainan University. It was deposited in the Guangdong Microbial Culture Collection Center GDMCC on January 2, 2025. The deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou. The deposit number is GDMCC No: 65711, and the classification name is: Bifidobacterium animalis.

[0029] Bifidobacterium animalis H22B656 was cultured on MRS agar medium containing 1% cysteine ​​hydrochloride at 37°C for 48 hours. The colonies were milky white, 0.1-0.2 cm in diameter, with a smooth, moist, raised surface and neat edges. They were rod-shaped when observed under an optical microscope.

[0030] Identification of isolates: DNA of isolates was extracted using the kit. PCR system and procedure were then performed to amplify the 16S rRNA gene of each sample. The amplified products were electrophoresed using 0.8% agarose gel electrophoresis. Sequencing was completed by Qingdao Pengxiang Biotechnology.

[0031] The sequences of the isolates were compared with those of NCBI, and the information of the highest query cover and identifier in the output results was selected as the identification result of the isolate at the species level.

[0032] 16S rRNA amplification primers

[0033]

[0034] The 16S rRNA sequencing results of the strain are shown in SEQ ID NO.3.

[0035] The comparison results are as follows:

[0036]

[0037] 1.1.2 Culture medium and reagents

[0038] TPY liquid medium; phosphate buffer (PB); phosphate buffered saline (PBS); 200 mM sodium glycodeoxycholate solution, 200 mM sodium taurodeoxycholate solution; 0.15 g / mL trichloroacetic acid solution;

[0039] Amino acid standard solution:

[0040] Concentration (mM) 0 0.2 0.4 0.6 0.8 1.0 Amino acid solution (1 mM, μl) 0 300 600 900 1200 1500 Distilled water μl 1500 1200 900 600 300 0

[0041] 1% Ninhydrin Citric Acid Solution: 0.25 g of ninhydrin is dissolved in 25 ml of 0.5 M citrate buffer;

[0042] Ninhydrin Color Reagent: 25 ml of 1% ninhydrin citric acid buffer + 65 ml of 30% glycerol + 10 ml of 0.5 M citrate buffer.

[0043] 1.2 Domestication of Animals and Isolation of Strains

[0044] The germ-free mice (GF mice, 7 weeks old) used in the experiment were purchased from Shenzhen Jingtuo Biotechnology Co., Ltd., Guangdong Province. The GF mice were divided into three groups: control group (Con, n = 4), model group (Mod, n = 4), and domestication group (Dom, n = 4). After all the mice were transferred into the germ-free breeding chamber, they were first fed adaptively for 1 week, and irradiated and sterilized maintenance feed for experimental mice was provided. After the adaptation period ended, the mice in the domestication group were intragastrically administered probiotic H22B656 (200 μL, 10 9 CFU) for 3 consecutive days, while the mice in the control group and the model group were intragastrically administered an equal amount of sterile normal saline. In terms of feed, the control group continued to be provided with irradiated and sterilized maintenance feed, while the model group and the domestication group were gradually provided with irradiated and sterilized high-fat feeds with different fat contents according to the progress of the experiment (purchased from Shuyu Biotechnology (Shanghai) Co., Ltd., high-fat feed: 60% fat: SYHF60-1; 45% fat: SYHF45-1; 30% fat: the fat content was adjusted to 30% in the above feed formula; maintenance feed: SY10001F-1). The formal experiment lasted for 7 weeks. At the beginning of the 1st, 3rd, and 5th weeks, the feed fat contents of the model group and the domestication group were gradually increased: 30% fat + 0.2% additional cholesterol, 45% fat + 0.2% additional cholesterol, 60% fat + 0.2% additional cholesterol, until the end of the experiment.

[0045] To obtain the mutant strains in the GF mice of the domestication group, fresh feces of the domestication group mice were collected at the end of the 1st week, 1.5 mL of normal saline was added, and the samples were ground and broken using a grinder. The processed samples were serially diluted with normal saline to 10 -4 、10 -5 、10 -6The concentration was determined and coated onto the TPY solid medium. After culturing the samples at 37°C for 48 hours, colonies with appropriate dilution gradients were selected, and several strains were randomly picked and inoculated into the TPY liquid medium, numbered as W1S1, W1S2,.... After overnight culturing the strains at 37°C, they were passaged three times for later use. Meanwhile, 200 μL of the culture solution was taken, an equal volume of 50% glycerol aqueous solution was added, and after mixing, it was stored frozen at -80°C in the refrigerator for use in subsequent verification tests. This subculture operation was repeated at the 3rd, 5th, and 7th weeks.

[0046] 1.3 In vitro verification by ninhydrin colorimetry

[0047] The ninhydrin colorimetry is a classical method for amino acid detection and has been widely used to evaluate the bile salt hydrolase (BSH) activity of bacteria. BSH is an enzyme that can hydrolyze conjugated bile salts into free bile acids and amino acids. By detecting the amount of amino acids generated in the reaction, the BSH activity of bacteria can be indirectly evaluated. Using ninhydrin colorimetry, the generated amino acids can be colored and quantified, and the absorbance value is detected by colorimetry to calculate the concentration of amino acids, thereby reflecting the BSH activity. In this application, after shaking and mixing the culture solution of the obtained mutant strains, 1 mL of the culture solution was aspirated into a centrifuge tube and centrifuged at 4°C and 3000 rpm for 10 minutes, and the supernatant was discarded. The cell pellet was washed twice with 1 mL of 0.1 M phosphate buffer (PB), and then the bacteria were resuspended with 500 μL of PBS sodium phosphate buffer solution at pH 6.0. 100 μL of the bacterial suspension was added to 100 μL of the bile salt binding solution and mixed with 800 μL of PBS buffer solution at pH 6.0 to form a reaction system; for the blank control, 100 μL of the bacterial suspension was added to 900 μL of PBS buffer solution at pH 6.0. After incubating the samples at 37°C for 30 minutes, 500 μL of trichloroacetic acid solution was immediately added, gently mixed, and allowed to stand for 3 minutes to terminate the reaction. Subsequently, it was centrifuged at 14000 rpm and 4°C for 10 minutes, the supernatant was aspirated and added to a test tube containing 1.5 mL of ninhydrin color-developing solution, and after shaking evenly, it was heated in a boiling water bath for 15 minutes. The amino acid standard solution did not need to be centrifuged, and the remaining steps were the same as above. After the reaction was completed, 200 μL of the reaction solution was aspirated into a 96-well plate, and the absorbance of the reaction solution was detected at a wavelength of 570 nm, and the amino acid concentration was calculated through the standard curve. The degradation rate calculation formula is as follows:

[0048]

[0049] Among them, C n is the detected amino acid concentration (n = 1, 2..., 60), and C 0 is the amino acid concentration bound by the conjugated bile salts.

[0050] 1.4 Transcriptome sequencing and analysis

[0051] To further evaluate the domestication effect of probiotic strains, we selected the mutant strain W5S9 (representing the strain numbered 9 in the 5th week of domestication, showing the highest bile salt hydrolase (BSH) activity in the ninhydrin colorimetric method) and the original strain H22B656, and inoculated them separately into TPY medium containing 0.3% bile salt for 12 hours of culture. After the culture, centrifuge at 4000 rpm for 30 minutes, and collect the supernatant for transcriptome detection.

[0052] The mutant strain W5S9, also known as H22B656-W5S9, was deposited at the Guangdong Microbial Culture Collection Center (GDMCC) on January 2, 2025. The deposit address is: 5th Floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. The deposit number is GDMCC No: 65712, and the taxonomic name is Bifidobacterium animalis.

[0053] Use the Qiagen RNA extraction kit to extract total RNA from the culture samples, ensuring high purity and high integrity, while avoiding genomic DNA contamination. The extracted RNA needs to be detected for its integrity by agarose gel electrophoresis, and the concentration and purity are measured using Nanodrop. For prokaryotic samples, ribosomal RNA is first removed from the total RNA and further purified by ethanol precipitation. Subsequently, the RNA is fragmented and the first-strand cDNA is synthesized using random hexamer primers. During the synthesis of the second-strand cDNA, dUTP in the reaction buffer replaces dTTP. Then, end repair, A-tailing, adapter ligation, fragment selection, USER enzyme digestion, amplification, and purification are carried out to finally obtain a strand-specific library. The library is quantified by Qubit and real-time fluorescence quantitative PCR, and the fragment size distribution is detected using a bioanalyzer. Qualified libraries are pooled according to the requirements of the effective concentration and the target data volume, and sequenced on an Illumina sequencer.

[0054] The image data generated by sequencing is converted into sequence data in fastq format through base recognition, which contains the sequence information of the sequencing fragments and the corresponding quality information. To ensure the quality and reliability of data analysis, the raw data needs to be filtered. Use Fastp (version 0.23.1) to perform quality control on the sequence data, remove adapter contamination, low-quality reads, and unidentified bases (N) to improve the accuracy of subsequent bioinformatics analysis.

[0055] To obtain the expression level of bile salt hydrolase in the transcriptome, we used salmon for annotation, and the running code is as follows: salmon quant -i mg -l A -1 test_1.fastq.gz -2 test_2.fastq.gz -o test.quant.

[0056] 1.5 In Vivo Verification Experimental Protocol for Non-Alcoholic Fatty Liver

[0057] To further verify the functional differences between the domesticated probiotic bacteria and the original strains, we established a non-alcoholic fatty liver model in SPF mice to explore the preventive effects of each strain on non-alcoholic fatty liver. A total of four groups were set up in the experiment: control group (n = 6, normal diet, gavage with 200 μl of normal saline daily), model group (n = 6, high-fat diet, gavage with 200 μl of normal saline daily), original strain group (n = 6, high-fat diet, gavage with 200 μl containing 10^9 CFU of the original strain H22B656 daily), and domesticated group (n = 6, high-fat diet, gavage with 200 μl containing 10^9 CFU of the mutant strain W5S9 daily). Both the high-fat diet and the maintenance diet were purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd. (high-fat diet: XTMRCD60, maintenance diet: A10021B). The entire experimental period was 8 weeks. After the experiment, all mice were euthanized, and serum, liver tissue, and feces were collected for subsequent analysis.

[0058] 1.6 Determination of Physiological and Biochemical Indicators

[0059] The liver tissue was thoroughly rinsed with pre-cooled sterile saline and then fixed in 4% paraformaldehyde solution or liquid nitrogen respectively. Subsequent processing of the samples, including image acquisition, section preparation, and analysis, was completed by Sevier Biotechnology Co., Ltd. in Wuhan, China. The contents of high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), total triglyceride (TC), total cholesterol (TG), and malondialdehyde (MDA) in the serum were determined according to the instructions of the ELISA kit (purchased from Shanghai Xinyu Biotechnology Co., Ltd.).

[0060] 1.7 Statistical Analysis

[0061] All statistical analyses were performed using R (version 3.6.1) software. The Wilcoxon rank sum test was used to identify physiological and biochemical indicators, and significant differences were considered based on a p-value threshold of 0.05 (*p < 0.05, **p < 0.01). The "ggplot2" package was used to generate bar charts. Differential analysis of transcriptome data was performed using the "DESeq2" package.

[0062] II. Results

[0063] 2.1 Directed Domestication Increased the BSH Yield of the Probiotic Strain H22B656

[0064] After the seven-week domestication period, 60 strains of bacteria were isolated from GF mice, and their BSH enzyme activities were uniformly determined by the ninhydrin detection method to evaluate the domestication effect. To visually quantify the BSH enzyme activity of mutant strains, we calculated the ratio of "degradation rate of mutant strains / degradation rate of original strains" to evaluate the degradation ability of mutant strains. The results showed that strains with a ratio greater than 1 had a better effect on degrading conjugated bile salts than the original strains. Results( Figure 1 ) showed that a total of 19 strains had higher BSH enzyme activity than the original strains, and the highest activity reached 1.75 times that of the original strains.

[0065] The results of transcriptome annotation showed that the expression level of bile salt hydrolase (BSH) in the domesticated strains was significantly higher than that in the original strains (Log2FC = 0.4802, p < 0.001). The above results indicate that the strategy of directional domestication of probiotics using intestinal selection pressure through a germ-free mouse model is feasible( Figure 2 ).

[0066] 2.2 The mutant strains are superior to the original strains in improving the blood lipid levels of mice with non-alcoholic fatty liver

[0067] To systematically evaluate the effects of mutant strains and original strains in preventing non-alcoholic fatty liver, we measured the contents of HDL-C, LDL-C, TC, TG, and MDA in the sera of mice using kits( Figure 3 ). The results showed that both W5S9 and H22B656 significantly increased the HDL-C level, and the HDL-C level in the W5S9 group was significantly higher than that in the H22B656 group, indicating that the mutant strains were superior to the original strains in increasing HDL-C (p < 0.05). In terms of the MDA level, both W5S9 and H22B656 significantly decreased the MDA content, and the effect of the mutant strain W5S9 was more significant. In addition, both W5S9 and H22B656 significantly decreased the LDL-C level, but there was no significant difference between them. In terms of the regulation of TC and TG levels, the mutant strains significantly decreased the TC level in the serum.

[0068] Overall, W5S9 and H22B656 showed significant effects in regulating the contents of HDL-C, LDL-C, and MDA, indicating their potential protective effects on improving blood lipid levels and antioxidant stress. Among them, W5S9 had a more significant effect in increasing HDL-C and decreasing MDA, and was superior to the original strains in preventing non-alcoholic fatty liver.

[0069] 2.3 The mutant strains are superior to the original strains in alleviating excessive liver fat accumulation in mice with non-alcoholic fatty liver

[0070] Subsequently, we analyzed the HE-stained pathological sections of the livers of mice in the four groups. The results were asFigure 4 As shown, the pathological morphology of liver tissue is presented in the figure, including the control group, the model group, and two experimental treatment groups (H22B656 and W5S9). In the control group, hepatocytes were arranged regularly, and the structures of hepatic sinusoids and central veins were normal, without inflammatory infiltration or cell necrosis. In the model group, however, hepatocytes were arranged disorderly, accompanied by obvious apoptosis or necrosis features such as karyopyknosis and karyorrhexis, as well as dilation of hepatic sinusoids and inflammatory cell infiltration, indicating significant tissue damage. In contrast, the morphology of liver tissue in the H22B656 treatment group was improved, with relatively regular arrangement of hepatocytes and reduced inflammatory infiltration, but slight cell necrosis and dilation of hepatic sinusoids were still observed. The improvement in the W5S9 treatment group was more significant, with regular arrangement of hepatocytes, significantly reduced inflammatory cell infiltration, and the disappearance of dilation of hepatic sinusoids and cell necrosis, approaching the normal tissue morphology. The results indicate that the experimental treatment (especially W5S9) has a good repair effect on liver tissue damage, and the effect is significantly better than that of the model group.

[0071] Generally speaking, the mutant strain W5S9 after 7 weeks of directional domestication is significantly superior to the original strain H22B656 in preventing non-alcoholic fatty liver.

[0072] Obviously, the above-mentioned embodiments of the present invention are merely examples for more clearly illustrating the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, based on the above description, other different forms of changes or modifications can be made. It is impossible to list all the implementation methods here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. The strain that efficiently produces bile salt hydrolase is named H22B656-W5S9 and was deposited in the Guangdong Provincial Microbial Culture Collection Center GDMCC on January 2, 2025. The storage address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The storage number is GDMCC No: 65712, and the classification name is: Bifidobacteriumanimalis.

2. The strain for efficiently producing bile salt hydrolase according to claim 1, characterized in that Strain H22B656-W5S9 is a mutant strain of animal Bifidobacterium H22B656, and the bile salt hydrolase activity and the expression level of bile salt hydrolase in the transcriptome of strain H22B656-W5S9 are higher than those of animal Bifidobacterium H22B656. The animal Bifidobacterium H22B656 has been deposited in Guangdong Provincial Microbiological Culture Collection Center GDMCC on January 2, 2025, with the deposit address at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou, with the deposit number GDMCC No: 65711, and the classification name: Bifidobacterium animalis.

3. A method for achieving high production of bile salt hydrolase by probiotics by utilizing intestinal selection pressure, comprising: By changing the intestinal selection pressure through a high-fat diet to promote the adaptive evolution of animal Bifidobacterium H22B656, a mutant strain that efficiently produces bile salt hydrolase was obtained. The animal Bifidobacterium H22B656 was deposited in the Guangdong Provincial Microbial Culture Collection Center GDMCC on January 2, 2025. The storage address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou. The storage number is GDMCC No: 65711, and the classification name is: Bifidobacterium animalis.

4. The method according to claim 3, characterized in that Germ-free mice were used as acclimation vehicles.

5. The method according to claim 4, characterized in that The acclimatization time is 7 weeks.

6. The method according to claim 5, characterized in that The high-fat diet uses 60% fat for energy.

7. The method according to claim 3, characterized in that The bile salt hydrolase activity and the expression level of bile salt hydrolase in the transcriptome of the mutant strain were higher than those of the original strain.

8. Use of the strain capable of efficiently producing bile salt hydrolase according to claim 1 in the preparation of a drug for preventing or improving non-alcoholic fatty liver disease.

9. The use according to claim 8, characterized in that: The strain can increase the HDL-C level and reduce the MDA content and LDL-C level.

10. The use according to claim 8 or 9, characterized in that: The strain can alleviate excessive liver fat accumulation in non-alcoholic fatty liver disease and repair liver tissue damage.

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