Method for extracting and purifying excellent probiotic strain with fermentation benefit from human body

Abstract

The invention discloses a lactobacillus plantarum BH52, which has a preservation number of CCTCC (China Center for Type Culture Collection) NO: M 20251294, and is characterized in that the preservation number of the lactobacillus plantarum BH52 is CCTCC NO: M 20251294. The strain is separated from human intestinal tracts, and is identified as plant lactobacillus through 16S rRNA (ribosomal Ribonucleic Acid) sequencing. Experiments show that the BH52 strain can efficiently degrade cholesterol and uric acid, significantly increase high density lipoprotein (HDL is increased by 29.8%), and improve kidney function indexes. The gastric acid resistance (the survival rate is 91.5% when the pH value is 3.0) and the cholate resistance (the survival rate is 94.3% when the cholate is 0.3%) are excellent, and the safety is high. The strain can be independently used or compounded for preparing functional foods or medicines for reducing blood fat and uric acid.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for extracting and purifying superior probiotic strains from the human body that have fermentation benefits. Background Technology

[0002] Metabolic syndrome (including hyperlipidemia, hyperuricemia, etc.) has become a major challenge threatening global public health. With changes in dietary structure and the increasing aging of the population, metabolic disorders are on the rise, and patients often suffer from multiple complications (such as atherosclerosis, chronic kidney disease, etc.), which seriously affect their quality of life and increase the medical burden.

[0003] The existing clinical treatment options have significant limitations: (1) prominent side effects of chemical drugs: lipid-lowering drugs are prone to causing adverse reactions such as liver toxicity and muscle damage; uric acid-lowering drugs have the risk of kidney damage and immunosuppression, which limits the safety of long-term use; (2) single function of probiotics: commercially available strains mostly focus on single metabolic pathway regulation (such as only lowering uric acid or only lowering blood lipids), which cannot meet the needs of multi-target intervention for metabolic syndrome; (3) insufficient efficiency of metabolic regulation: the existing probiotics have a weak inhibitory effect on key enzymes such as cholesterol synthesis and uric acid production, making it difficult to achieve synchronous metabolic improvement; (4) limited bioavailability: poor gastrointestinal tolerance of strains leads to low intestinal colonization rate, and insufficient secretion of functional metabolites (such as γ-aminobutyric acid and short-chain fatty acids), which weakens the actual application effect.

[0004] Therefore, there is an urgent need to develop a new type of probiotic. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention first provides a strain of Lactiplantibacillus plantarum BH52, which is deposited at the China Center for Type Culture Collection (CCTCCNO:M 20251294).

[0006] This invention also provides a probiotic preparation containing the above-mentioned live Lactobacillus plantarum BH52 bacteria, with a live bacteria count ≥1×10⁻⁶. 8 CFU / g.

[0007] In some embodiments, the dosage form is a lyophilized powder, capsule, or tablet.

[0008] The present invention also provides a compound probiotic preparation comprising the above-mentioned strains and at least one of Lactobacillus and Bifidobacterium.

[0009] The present invention also provides the application of the above-mentioned probiotic preparation in the preparation of lipid-lowering drugs.

[0010] The present invention also provides the application of the above-mentioned probiotic preparation in the preparation of uric acid-lowering drugs.

[0011] The present invention also provides the application of the above-mentioned probiotic preparation in the preparation of drugs for improving kidney function.

[0012] This invention also provides a method for culturing *Lactobacillus plantarum* BH52, comprising the following steps: (a) The strain was inoculated into MRS liquid medium containing 0.3% bile salts and incubated at 37°C for 12 h. (b) Transfer to shake-flask fermentation medium (20 g / L glucose, 5 g / L yeast extract, 2 g / L KH2PO4, pH 6.5) and culture at 37°C and 180 rpm for 24 h with shaking.

[0013] In some embodiments, the fermentation broth is centrifuged at 4000 rpm for 10 min, and then the metabolites are separated and purified by ultrafiltration membrane (molecular weight cutoff 10 kDa).

[0014] Finally, this invention provides an application of the fermentation product of Lactobacillus plantarum BH52, wherein the product contains γ-aminobutyric acid ≥250mg / L, total short-chain fatty acids ≥40mmol / L, and has a synergistic function of lowering uric acid.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: This invention marks the first isolation of *Lactiplantibacillus plantarum* BH52. It exhibits multi-target synergistic effects: simultaneously lowering lipids and uric acid, with superior efficacy compared to single-function strains; enhanced metabolite effects: γ-aminobutyric acid (GABA) possesses neuroprotective functions, and short-chain fatty acids can regulate the intestinal microecology; industrialization potential: the fermentation process is stable, and GABA production reaches commercial standards (>200 mg / L), indicating significant market value.

[0016] Preservation Instructions The plant lactobacillus ( Lactiplantibacillus plantarum BH52, isolated from the human body, was deposited at the China Center for Type Culture Collection on June 6, 2025, and classified as *Lactobacillus plantarum*. Lactiplantibacillus plantarum BH52, accession number: CCTCC NO:M 20251294, address: Wuhan University, Wuhan, Hubei, China. Detailed Implementation

[0017] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0018] Example 1: Isolation, Screening and Preservation of Strains Materials and Methods 1. Sample Source Fresh stool samples were collected from 10 Healthy China volunteers (aged 25-45, with no history of gastrointestinal diseases or long-term antibiotic use, and who signed informed consent forms). The samples were placed in sterile sampling tubes, immediately placed in an ice box (4°C), and transported to the laboratory for storage at -80°C for later use.

[0019] 2. Sample processing and isolation culture Serial dilution: Weigh 1 g of fecal sample, add 9 mL of sterile saline, and vortex thoroughly to prepare 10-1 dilutions. -1 Homogenize and serially dilute to 10. -8 (The diluent is 0.85% sterile saline).

[0020] Selective culture medium: Modified MRS solid medium containing 0.3% bile salts and 1.0 g / L cholesterol (formula: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L glucose, 1 mL / L Tween 80, 5 g / L sodium acetate, 2 g / L diammonium citrate, 2 g / L K2HPO4, 0.58 g / L MgSO4·7H2O, 0.25 g / L MnSO4·4H2O, 15 g / L agar, pH 6.2~6.4) was autoclaved (121℃, 15 min), cooled to 50℃, mixed well, and poured into plates.

[0021] Isolation and culture: Take 10 -4 -10 -6 100 μL of each diluted homogenate was evenly spread on a selective medium and incubated in an anaerobic incubator (37℃, 85% N2, 10% H2, 5% CO2) for 48 h. Round, smooth colonies with uniform morphology and a diameter of 1-2 mm were picked and purified by streaking three times to obtain single colonies.

[0022] 3. Initial screening: Cholesterol degradation capacity test Strain activation: Purified single colonies were inoculated into MRS liquid medium (cholesterol- and bile-free) and incubated at 37°C for 12 h (activation for 2 generations).

[0023] Fermentation culture: Take the activated bacterial solution and transfer it to MRS liquid medium containing 1.0 g / L cholesterol (250 mL Erlenmeyer flask, 50 mL liquid volume) at an inoculation rate of 1%, and culture at 37℃ and 120 rpm for 24 h with shaking.

[0024] Cholesterol content determination: An enzymatic reagent kit (Nanjing Jiancheng Bioengineering Co., Ltd., catalog number A111-1) was used, and the steps were as follows: (1) After the culture was completed, the mixture was centrifuged at 4℃ and 4000 rpm for 10 min, and the supernatant was collected; (2) The remaining cholesterol content was determined by cholesterol oxidase-peroxidase coupling method according to the kit instructions; (3) Cholesterol degradation rate calculation formula: Degradation rate (%) = (Initial cholesterol content − Residual cholesterol content) / Initial cholesterol content × 100% The initial cholesterol content is the theoretical value of cholesterol added during the preparation of the culture medium.

[0025] 4. Secondary screening and molecular identification Secondary screening: For strains with a degradation rate >40% in the initial screening, the above fermentation culture and cholesterol determination were repeated 3 times, and the average value was taken as the final degradation rate.

[0026] Molecular identification: Extraction of bacterial genomic DNA (Bacterial Genomic DNA Extraction Kit, Tiangen Biotech, catalog number YDP302).

[0027] PCR amplification of the 16S rRNA gene (primers: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3'). Reaction system (50 μL): 25 μL 2×Taq Master Mix, 2 μL each of forward and reverse primers (10 μmol / L), 2 μL template DNA, 19 μL ddH2O; amplification conditions: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1.5 min (35 cycles), and 72℃ final extension for 10 min. After verification by 1% agarose gel electrophoresis, the PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequences were submitted to the NCBI database for BLAST comparison to determine the species.

[0028] 5. Preservation The selected dominant strain (number BH52) was deposited at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, Hubei, China) on June 6, 2025, and classified as *Lactobacillus plantarum*. Lactiplantibacillus plantarum BH52, accession number: CCTCC NO: M 20251294.

[0029] result: A total of 32 strains of lactic acid bacteria were isolated. After initial screening, 12 strains had a degradation rate of >30%, and after rescreening, 3 strains had a degradation rate of >45%. The cholesterol degradation rate and molecular identification results are shown in Table 1.

[0030] Table 1. Cholesterol degradation rate and identification results of strains in the primary and secondary screening.

[0031] Note: Different letters in the superscript of the data in the same column indicate significant differences (Duncan's new multiple range method, p<0.05; uppercase letters indicate p<0.01, lowercase letters indicate p<0.05).

[0032] Results showed that 32 strains of lactic acid bacteria were successfully isolated from 10 human fecal samples. Three strains of Lactobacillus plantarum with high degradation rate were obtained by targeted screening in MRS medium containing bile salts and cholesterol. Among them, strain BH52 had the highest degradation rate (51.6%±2.3%), which was significantly higher than LP-03 (43.8%±1.9%, p<0.05) and LP-07 (46.2%±2.1%, p<0.05).

[0033] Example 2: Optimization of culture process and separation of metabolites Materials and Methods 1. Culture medium formulation Seed culture medium: MRS liquid medium (cholesterol-free and cholesterol-free), pH 6.8.

[0034] Fermentation medium: glucose 20 g / L, yeast extract 5 g / L, KH2PO4 2 g / L, pH 6.5.

[0035] 2. Cultivation conditions Seed culture: static culture at 37℃ for 12 h (activation 2 generations).

[0036] Fermentation culture: The seed culture was transferred to the fermentation medium (250 mL Erlenmeyer flask, 50 mL liquid volume) at a 5% inoculation rate and cultured at 37℃ and 180 rpm for 24 h with shaking.

[0037] 3. Separation and purification The fermentation broth was centrifuged at 4000 rpm for 10 min to collect the bacterial cells, washed three times with physiological saline, and then freeze-dried under vacuum to obtain live bacterial powder.

[0038] The supernatant was purified by ultrafiltration (molecular weight cutoff 10 kDa), and then lyophilized and stored at -20°C.

[0039] 4. Detection Indicators Viable bacteria count: Plate count method (GB 4789.35-2016).

[0040] γ-Aminobutyric acid (GABA): High performance liquid chromatography (HPLC, Agilent 1260, ZORBAX SB-C18 column, mobile phase acetonitrile: 0.1% TFA aqueous solution = 3:97, detection wavelength 338 nm).

[0041] Short-chain fatty acids (SCFAs): Gas chromatography-mass spectrometry (GC-MS, Agilent 7890B-5977A, HP-INNOWax column, methyl esterification derivatization).

[0042] The results are shown in Table 2.

[0043] Table 2. Fermentation Indicator Results

[0044] Data Analysis: The GABA content was significantly higher than that of similar strains. Lactiplantibacillus plantarum WCFS1 (124 mg / L, p<0.01); The total SCFAs content was 84% ​​higher than that of WCFS1 (28.4 mmol / L) (p<0.01).

[0045] Example 3: Functional Validation and Multi-Target Mechanism Materials and Methods 1. Animal models SD rats were randomly divided into 4 groups (n=10): Normal group (negative control group): ordinary feed; Model control group: high-fat diet (45% fat) + potassium oxonate (250 mg / kg); BH52 intervention group: high-fat diet + Lactobacillus plantarum ( Lactiplantibacillus plantarum BH52 bacterial powder (1×10) 8 CFU / d); Positive control group: high-fat diet + Lactiplantibacillus plantarum WCFS1 (purchased from China Microbial Culture Collection Center) (1×10) 8 CFU / d).

[0046] 2. Detection Indicators Blood lipid indicators: TC, TG, LDL, HDL (enzymatic method, Nanjing Jiancheng).

[0047] Uric acid metabolism: serum UA (uricase method), urinary UA excretion (phosphotungstic acid method).

[0048] Kidney function: serum creatinine (Jaffe method), blood urea nitrogen (urease method).

[0049] Liver biomarkers: XOD activity (colorimetric method), MDA content (TBA method).

[0050] 3. Statistical Analysis Differences between groups were analyzed using one-way ANOVA, and multiple comparisons were performed using Duncan's method (p < 0.05 was considered significant, and p < 0.01 was considered highly significant). The results are shown in Table 3.

[0051] Table 3. Results of various experimental indicators

[0052] Note: **Compared with the model group, p < 0.01; *Compared with the model group, p < 0.05; Data showed that the activities of TC, UA, and XOD in the BH52 intervention group were significantly lower than those in the positive control group (p<0.05).

[0053] Example 4: Metabolite Enhancement and Industrial Value Materials and Methods 1. Detection Method GABA content: HPLC method (same as Example 2).

[0054] SCFAs content: GC-MS method (same as Example 2).

[0055] Antioxidant activity: DPPH free radical scavenging rate (ABTS method).

[0056] 2. Industrial parameters Fermentation cycle: 24 hours (20% shorter than traditional processes); Product yield: GABA 287 mg / L (131% higher than WCFS1); Energy consumption cost: Energy consumption per unit product was reduced by 35% (based on shake flask fermentation data). The results are shown in Table 4.

[0057] Table 4. Results of Fermentation Products

[0058] Conclusion: The fermentation products of BH52 contain significantly higher levels of GABA and SCFAs than similar strains, and also possess antioxidant activity, making them suitable for the production of functional food additives and pharmaceutical intermediates.

[0059] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of *Lactobacillus plantarum* BH52, characterized in that, The strain described is *Lactiplantibacillus plantarum* BH52, deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20251294.

2. A probiotic preparation, characterized in that, Contains live Lactobacillus plantarum BH52 bacteria as described in claim 1, with a live count ≥ 1 × 10⁻⁶. 8 CFU / g.

3. The formulation according to claim 2, characterized in that, Dosage forms include lyophilized powder, capsules, or tablets.

4. A compound probiotic preparation, characterized in that, It includes the strain of claim 1 and at least one of Lactobacillus and Bifidobacterium.

5. The use of the strain described in claim 1 or the probiotic preparation described in any one of claims 2-3 in the preparation of lipid-lowering drugs.

6. The use of the strain described in claim 1 or the probiotic preparation described in any one of claims 2-3 in the preparation of uric acid-lowering drugs.

7. The use of the strain described in claim 1 or the probiotic preparation described in any one of claims 2-3 in the preparation of a drug for improving kidney function.

8. A method for culturing *Lactobacillus plantarum* BH52, characterized in that, Includes the following steps: (a) The strain was inoculated into MRS liquid medium containing 0.3% bile salts and incubated at 37°C for 12 h. (b) Transfer to shake-flask fermentation medium (20 g / L glucose, 5 g / L yeast extract, 2 g / L KH2PO4, pH 6.5) and culture at 37°C and 180 rpm for 24 h with shaking.

9. The cultivation method as described in claim 8, characterized in that, The fermentation broth was centrifuged at 4000 rpm for 10 min, and the metabolites were separated and purified by ultrafiltration membrane (molecular weight cutoff 10 kDa).

10. An application of a fermentation product of *Lactobacillus plantarum* BH52, characterized in that, The product contains ≥250 mg / L of γ-aminobutyric acid and ≥40 mmol / L of total short-chain fatty acids.

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