Plantarum and application thereof in degrading indole in intestinal tract

Abstract

The present application relates to the field of functional microorganism screening and application technology, in particular to a plant lactobacillus and its application in degrading intestinal indole. The plant lactobacillus has a preservation number of CCTCC NO: M20232619. The plant lactobacillus VHProbi O03 provided in the present application has a strong degrading effect on intestinal indole and can significantly degrade indole in vitro. The strain has strong tolerance to artificial gastric juice; is sensitive to common antibiotics such as erythromycin and ampicillin, does not produce hemolysin, cannot dissolve blood cells, and has good biological safety; has strong antioxidant capacity and cholesterollowering capacity. The plant lactobacillus VHProbi O03 can be used for preparing health care products or drugs for removing uremic toxins and relieving chronic kidney disease, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of probiotic screening and application technology, specifically relating to a strain of Lactobacillus plantarum and its application in degrading intestinal indole. Background Technology

[0002] Chronic kidney disease (CHD) is a common chronic kidney disease characterized by progressive kidney damage. Its treatment requires significant healthcare resources, making it crucial to use medications to prevent or delay its progression to end-stage renal disease. Studies have shown that indoxyl sulfate (IS), a protein-bound small molecule compound, is one of the uremic toxins. IS is formed when dietary tryptophan is metabolized by tryptophan-containing bacteria in the intestines, producing indole, which is then absorbed and oxidized and sulfonated in the liver. Over 90% of IS in the blood binds to albumin, making it difficult to remove through conventional hemodialysis and peritoneal dialysis; it is primarily excreted through the kidneys. In healthy individuals, serum indoxyl sulfate concentrations are almost zero. However, in chronic kidney disease, decreased renal excretion leads to IS accumulation. Studies have confirmed that in renal insufficiency, especially when serum creatinine is ≥265 μmol / L, serum indoxyl sulfate levels can reach 0.8-1.0 mg / dL. Indophenol sulfate accumulated in the body enters various tissues and organs through blood circulation, thereby causing damage to the kidneys and other organs, such as promoting glomerular sclerosis, renal tubular interstitial fibrosis, and accelerating renal failure; causing cardiovascular diseases, heart failure, and coronary atherosclerosis; and leading to damage to the central nervous system and cognitive impairment.

[0003] Studies show that gut indole-producing bacteria break down dietary tryptophan, which is the sole source of protein-bound toxins (IS) in the body. Therefore, reducing the production of protein-bound toxins is crucial for slowing disease progression during end-stage renal failure. Restricting protein intake and modulating the gut microbiota are two possible approaches. However, due to side effects such as malnutrition and poor adherence, restricting protein intake is not a long-term option for reducing protein-bound toxin production in patients with chronic kidney disease. Modulating the gut microbiota is a broader and more effective approach, primarily using prebiotics and probiotics.

[0004] Probiotics are a class of live microorganisms that improve gut microbiota balance and benefit the body, and can colonize the human gut. Because gut microbiota is closely related to chronic renal failure, some researchers have begun to reduce isocyanate (IS) production by intervening in the growth of pathogenic bacteria and reducing the absorption of uremic toxins, focusing on using gut bacterial preparations to interfere with the metabolism and production of IS and PCS. Studies have confirmed that probiotic-fermented camel milk can effectively alleviate nephron pathological damage, slow the progression of chronic renal failure in rats, and improve their vitality. For example, taking Bifidobacterium preparations can inhibit the growth of Escherichia coli and reduce the degradation of tryptophan by E. coli, significantly reducing IS levels in patients with chronic renal failure. The Huazhong University of Science and Technology team discovered that inulin-type fructans reduce indole production in the intestines of peritoneal dialysis patients by regulating gut microbiota and reducing the abundance of indole-producing bacteria. Therefore, targeting metabolic pathways in gut bacteria, screening for usable probiotics, and degrading indole in the gut to reduce the production level of uremic toxin indole sulfate and alleviate the damage of indole sulfate to the body are important strategies for effectively alleviating symptoms and prolonging the lives of patients with chronic renal failure. Summary of the Invention

[0005] The purpose of this invention is to provide a novel strain of *Lactiplantibacillus plantarum* and its applications. The provided *Lactiplantibacillus plantarum*, isolated from the fermentation broth of naturally fermented sauerkraut, exhibits a strong ability to degrade indole, a metabolite of tryptophan in the intestines. Indole is a precursor to indole sulfate, a protein-bound uremic toxin. Reducing indole levels thereby decreases the indole sulfate level in patients, alleviating symptoms in those with renal failure. This strain can be used as an adjunct therapy for renal failure patients, preventing further complications and prolonging their lifespan.

[0006] The plant lactobacillus provided in this invention is VHProbi O03 (Lactiplantibacillus plantarum VHProbi O03), which was deposited at the China Center for Type Culture Collection on December 21, 2023, with accession number CCTCC NO: M20232619.

[0007] The Riboprinter fingerprint of the *Lactobacillus plantarum* VHProbi O03 strain provided by this invention is as follows: Figure 3 As shown; its RAPD fingerprint spectrum is as follows. Figure 4 As shown, the rep-PCR fingerprint pattern is as follows: Figure 5 As shown, the MALDI-TOF-MS protein fingerprint is as follows: Figure 6 As shown.

[0008] The application of the *Lactobacillus plantarum* strain VHProbi O03 provided in this invention in the preparation of pharmaceuticals with indole degradation function.

[0009] The present invention also provides the use of the aforementioned *Lactobacillus plantarum* VHProbi O03 strain in the preparation of a medicine for clearing uremic toxins and alleviating chronic kidney disease.

[0010] The application of the *Lactobacillus plantarum* strain VHProbi O03 provided in this invention in the preparation of pharmaceuticals with cholesterol-lowering functions.

[0011] The application of the *Lactobacillus plantarum* strain VHProbi O03 provided in this invention in the preparation of pharmaceuticals with antioxidant functions.

[0012] The present invention also provides an article for degrading intestinal indole and alleviating chronic kidney disease, comprising live Lactobacillus plantarum VHProbi O03 and / or its fermentation products.

[0013] The product in question is a health product or a medicine.

[0014] The *Lactobacillus plantarum* VHProbi O03 strain provided by this invention exhibits a strong degradation effect on intestinal indole, significantly degrading it in vitro with a degradation rate of 51.58 ± 4.15% after 48 hours. This strain demonstrates strong tolerance to artificial gastrointestinal fluid; it is sensitive to common antibiotics such as erythromycin and ampicillin, does not produce hemolysin, and cannot lyse blood cells, exhibiting good biocompatibility. Furthermore, *Lactobacillus plantarum* VHProbi O03 possesses strong antioxidant and cholesterol-degrading capabilities, achieving a DPPH clearance rate of 25.57%; its bacterial suspension and fermentation supernatant showed clearance rates of 58.92% and 91.02% for HRS, respectively; the fermentation supernatant exhibited an anti-lipid peroxidation inhibition rate of 41.48%; and a cholesterol degradation rate of 20.40%. The *Lactobacillus plantarum* VHProbi O03 strain has broad application prospects in the production of drugs for clearing uremic toxins and alleviating chronic kidney disease. Attached Figure Description

[0015] Figure 1 This is a growth curve of strain O03 in a medium containing indole.

[0016] Figure 2 A carbon source metabolism map of strain O03;

[0017] Figure 3 Fingerprint of Riboprinter strain O03;

[0018] Figure 4 RAPD fingerprint of strain O03;

[0019] Figure 5 The rep-PCR fingerprint of strain O03;

[0020] Figure 6 The MALDI-TOF-MS protein fingerprint of strain O03. Detailed Implementation

[0021] The *Lactobacillus plantarum* VHProbi O03 provided by this invention meets regulatory requirements, and long-term use poses no risk of side effects or overdose. Polyphasic taxonomic identification has confirmed that *Lactobacillus plantarum* VHProbi O03 is a newly discovered strain. The *Lactobacillus plantarum* VHProbi O03 provided by this invention exhibits a strong degradation effect on intestinal indole and can significantly degrade indole in vitro. Using this strain alone, without the need for combination with prebiotics and / or other probiotics, can alleviate chronic kidney disease, demonstrating significant application value.

[0022] On December 21, 2023, the applicant deposited the Lactiplantibacillus plantarum VHProbi O03 at the China Center for Type Culture Collection of Wuhan University, with accession number CCTCC NO: M20232619.

[0023] The screening method described in this invention is not limited to the embodiments. Any known method capable of achieving the screening purpose can be used. The screening descriptions in the embodiments are merely illustrative of this invention and are not intended to limit the scope of protection of this invention. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and substance of this invention are within the scope of this invention.

[0024] The present invention will now be described in detail with reference to specific embodiments.

[0025] Example 1 Isolation and screening of strains

[0026] 1. Strains Isolation

[0027] Preparation of MRS (Man Rogosa Sharpe) broth: 1L pure water, 10g peptone, 10g beef extract, 5.0g yeast extract, 5g sodium acetate, 5g glucose, 2g potassium dihydrogen phosphate, 1.0mL Tween 80, 2.0g diamine citrate, 20g calcium carbonate, 0.58g magnesium sulfate heptahydrate, 0.25g manganese sulfate heptahydrate, adjust pH to 6.2-6.5.

[0028] Prepare MRS agar medium: Add 15g agar to 1LMRS broth.

[0029] Take 1g of sauerkraut juice, dilute it with sterile physiological saline, and place it in a sterile sample bag. Mix thoroughly using a homogenizer. Take 100μL of the mixture, serially dilute it, spread it on MRS agar medium, and incubate anaerobically at 37℃ for 48h. Examine the plates for single colonies under a microscope. Based on the microscopic examination results, the applicant screened out 28 potential Lactobacillus strains, named O01, O02, ..., O28.

[0030] 2. Screening of strains capable of degrading indole in vitro

[0031] Preparation of indole solution: Prepare a 30 mg / ml indole stock solution using 70% ethanol, and sterilize it using a 0.22 μm filter membrane for later use. Dilute to the required concentration with the culture medium for the strain before use.

[0032] Strain preparation: Lactic acid bacteria streak activation. Take frozen lactic acid bacteria glycerol tubes, aseptically streak them onto MRS plates, and incubate at 37°C for 48-72 hours.

[0033] Preparation of bacterial solution for screening: After single bacteria have grown on the plate, aseptically transfer them to MRS broth and incubate at 37°C for 24 hours. Inoculate fresh MRS broth at a 1% inoculum rate, approximately 6 ml per bacterial strain.

[0034] (1) Tolerance test of strains to different concentrations of indole.

[0035] Indole culture medium was diluted to 0, 50, 100, 150, 200, 300, 400, and 600 ug / ml, and the strain was inoculated at a 1% inoculum. The growth curve of the strain was monitored using an enzyme-linked immunosorbent assay (ELISA) reader.

[0036] The results showed that the activity of the strain was not affected when the culture medium contained low concentrations of indole, and even slightly promoted the growth of the strain. When the indole content in the culture medium was close to 300 ug / ml, the activity of the strain was inhibited. At the same time, according to the data, the indole in human intestine or feces is 0.25-2.6mM, which is about 45-468 ug / ml. Therefore, 300 ug / ml can be used as the indole addition concentration to verify the indole degradation ability of the strain.

[0037] (2) Detection of the strain's ability to metabolize indole:

[0038] The Lactobacillus strains obtained from the initial screening were inoculated at a 1% inoculum into fresh culture medium with an indole concentration of 300 μg / ml. The cultures were incubated at 37°C for 48 hours. At 0 h, 24 h, and 48 h, 1.5 ml of the fermentation broth was transferred to new EP tubes and centrifuged at 10000 g / min for 3 min. The supernatant was collected, and the indole content in the supernatant was detected using an indole detection kit. For detailed procedures and result interpretation, please refer to the Indole Assay Kit instruction manual.

[0039] Indole degradation rate calculation: Indole degradation rate = (initial concentration - final concentration) / initial concentration * 100%.

[0040] The results showed that at 24 hours, 11 of the 28 potential lactobacilli obtained from the initial screening had a degradation rate of more than 30% for indole, with strain O03 having the highest degradation rate of 31.13±1.70%; at 48 hours, 5 strains had a degradation rate of more than 50% for indole, with strain O03 having the highest degradation rate of 51.58±4.15%.

[0041] The growth curves of strain O03 in different concentrations of indole are as follows: Figure 1 As shown.

[0042] Example 2: Strain Identification

[0043] 1. Colony morphology identification

[0044] When strain O03 was inoculated onto MRS agar medium and anaerobically cultured at 37°C for 24 hours, single colonies of O03 were observed to be milky white with a smooth surface. Under a microscope, the bacteria appeared as short rods or curved rods, sometimes in chains, without spores or flagella.

[0045] 2. Identification of physiological and biochemical characteristics

[0046] In this embodiment, the inoculum solution is prepared as follows: Under aseptic conditions, take an appropriate amount of fresh O03 bacterial culture, centrifuge at 5000 rpm / min for 5 min, wash twice with PBS buffer, then reconstitute the bacterial culture with the same volume of PBS buffer and dilute 50 times to obtain the inoculum solution.

[0047] 2.1 Salinity Tolerance Test

[0048] Under aseptic conditions, 190 μL of BSM liquid medium with salt concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8% were added to each well of a 96-well plate, with three replicates for each salt concentration. Then, 10 μL of inoculum was added to each well. Wells without inoculation served as controls. 50 μL of autoclaved paraffin oil was added to each well to prevent moisture evaporation during culture. The plates were incubated at 37°C, and the medium was observed to ensure it did not become cloudy.

[0049] The results showed that the maximum salt concentration that strain O03 could tolerate was 7%.

[0050] 2.2 Carbon Source Metabolism Test

[0051] Carbon source metabolism experiments were performed on strain O03 using API 50CHL reagent strips. For experimental methods and result interpretation, please refer to the API 50CHL kit instructions. The identification result for strain O03 was: %ID=99.1 and T value=1. The API result identified it as *Lactobacillus plantarum*. See the attached image for details. Figure 2 .

[0052] 2.3 Glucose Acid and Gas Production Test

[0053] The culture medium formulation used in this embodiment is as follows:

[0054] Peptone 0.5g; yeast extract 0.3g; Tween 80 0.1mL; salt solution A 0.5ml; salt solution B 0.5ml; sodium acetate 0.5g; glucose 2.5g; 2% bromocresol green (w / v) 0.05mL; distilled water 100ml; pH 6.8~7.0.

[0055] Dispense the prepared culture medium into large test tubes containing inverted small test tubes, 3 mL / tube, and autoclave at 121℃ for 15 min.

[0056] Salt solution A consists of 10g KH2PO4 and 1.0g K2HPO4, dissolved in distilled water and brought to a final volume of 100mL.

[0057] Salt solution B consists of: 11.5g MgSO4·7H2O, 2.4g MnSO4·2H2O, and 0.68g FeSO4·7H2O, dissolved in distilled water and brought to a final volume of 100mL.

[0058] Under aseptic conditions, the inoculum was inoculated into the culture medium at a rate of 10%, with an uninoculated culture medium serving as a control. The top was then sealed with 2 mL of sterile liquid paraffin and incubated at 37°C for 24 hours. The color of the culture medium was then observed for any changes.

[0059] The results showed that after culturing at 37℃ for 24 hours, the culture medium changed from green to yellow, and there was no gas in the small inverted tube, indicating that strain O03 fermented glucose to produce acid but not gas.

[0060] 3. Molecular biological identification

[0061] 3.1 16S rDNA gene sequence analysis

[0062] 3.1.1 Genomic DNA Extraction

[0063] Follow the instructions in the Tiangen Bacterial Genomic DNA Extraction Kit (catalog number: DP302).

[0064] 3.1.2 16S rDNA gene amplification

[0065] Primer sequences:

[0066] 27F: AGAGTTTGATCCTGGCTCA;

[0067] 1492R: GGTTACCTTGTTACGACTT.

[0068] The 16S rDNA sequence (SEQ ID NO:1) of strain O03 was obtained by sequencing and compared with the NCBI database, and strain O03 was preliminarily identified as Lactobacillus plantarum.

[0069] The sequence of SEQ ID NO:1 is as follows:

[0070]

[0071] 3.2 Riboprinter fingerprint spectrum

[0072] A single purified colony was picked up from an agar plate using a sampling stick and placed into a sample tube containing buffer. The colony was then stirred with a hand stirrer to suspend it in the buffer. After inactivation in a heater, the sample was placed into the Riboprinter system. The sample underwent DNA preparation, transfer, imaging, and data processing to obtain bacterial identification results. The identification results showed that strain O03 was *Lactobacillus plantarum*, and its Riboprinter fingerprint results are shown below. Figure 3 .

[0073] 3.3 Identification using RAPD and rep-PCR fingerprinting

[0074] 3.3.1 RAPD fingerprint identification

[0075] Primer sequence: GAGGGTGGCGGTTCT.

[0076] Table 1: RAPD Reaction System

[0077] A 1.5% agarose gel plate was prepared, with a DL2000 DNA Marker used as a control. The gel was electrophoretically stabilized at 100V for 80 min, and the electrophoresis results were detected using a gel imaging system. The RAPD fingerprint of strain O03 is shown below. Figure 4 As shown.

[0078] 3.3.2 rep-PCR fingerprinting

[0079] Primer sequence: CTACGGCAAGGCGACGCTGACG.

[0080] Table 2: Reaction system table for rep-PCR

[0081] DL2000 DNA Marker was used as a result control. Electrophoresis was performed at 100 V for 80 min to detect amplification results. The rep-PCR fingerprint of strain O03 is shown below. Figure 5 As shown.

[0082] 3.4 MALDI-TOF-MS detection of ribosomal protein expression in strains

[0083] Fresh bacterial culture was inoculated into MRS liquid medium at an inoculum rate of 0.1%. After incubation at 37°C and 150 rpm for 48 hours, the bacterial cells were collected, washed four times with sterile water, and dried. A small amount of fresh bacterial cells was then evenly coated onto a target plate in the form of a thin film. 1 μL of lysis buffer was added to cover the sample, and after drying, 1 μL of matrix solution was added to cover the sample again. After drying, the sample target was placed in a mass spectrometer for identification. The co-crystallized film formed by the sample and matrix was irradiated with a laser to ionize the proteins in the sample. The ions were accelerated through the flight tube under the action of an electric field of 10–20 kV. The molecular weight of the proteins was determined based on the different flight times to the detector. Protein fingerprints were obtained using Autofms 1000 software Autof Analyzer v1.0. The ion peaks of the main ribosomal proteins of strain O03 were: m / z 5178.112, 5193.559, and 7843.814. The identification results are as follows: Figure 6 As shown.

[0084] 3.5 Whole genome sequencing

[0085] Fresh bacterial culture was inoculated into MRS liquid medium at an inoculum volume of 0.1%, cultured at 37°C for 20 h, centrifuged at 8000 rpm for 10 min, and the bacterial cells were collected. The bacterial cells were sent to a sequencing center to obtain the complete genome sequence of the bacterium. The genome sequence has been uploaded to the NCBI gene database. The genome contains one chromosome and two plasmids, with GenBank accession numbers CP134217, CP134218, and CP134219, respectively.

[0086] Based on the colony morphology, physiological and biochemical characteristics, and molecular biological identification results of strain O03, strain O03 was identified as a new Lactiplantibacillus plantarum strain and named Lactiplantibacillus plantarum VHProbi O03.

[0087] Example 3: Antibiotic resistance test of Lactobacillus plantarum VHProbi O03

[0088] The minimum inhibitory concentration (MIC) of antibiotics against *Lactobacillus plantarum* VHProbi O03 was determined using the micro-broth dilution method. The specific results are shown in Table 3.

[0089] Table 3. Antibiotic MIC values ​​of Lactobacillus plantarum VHProbi O03

[0090] MIC is measured in μg / mL.

[0091] As can be seen from the results in Table 3, the *Lactobacillus plantarum* VHProbi O03 provided by this invention is sensitive to common antibiotics such as erythromycin, ampicillin and clindamycin, and has good biosafety.

[0092] Example 4: Tolerance test of *Lactobacillus plantarum* VHProbi O03 to artificial gastric and intestinal fluids.

[0093] 1. Preparation of artificial gastric juice

[0094] Weigh out 5g of peptone, 2.5g of yeast extract, 1g of glucose, and 2g of NaCl, add them to 1000mL of distilled water, adjust the pH to 3.0 with dilute hydrochloric acid, and then sterilize at 115℃ for 20min. Before use, add 3.2g of porcine mucosal pepsin, shake well to dissolve, and place in a 37℃ water bath shaker for 1h to simulate human body temperature.

[0095] 2. Preparation of artificial intestinal fluid

[0096] Weigh out 5g of peptone, 2.5g of yeast extract, 1g of glucose, 6.8g of KH₂PO₄, and 3.0g of ox bile salts, respectively. Add them to 77mL of 0.2mol / L NaOH solution, and bring the volume to 1000mL. Adjust the pH to 6.8±0.1 with dilute hydrochloric acid or sodium hydroxide solution, and sterilize at 115℃ for 20min. Before use, add 1g of trypsin, shake well to dissolve, and place in a 37℃ water bath for 1h to simulate human body temperature.

[0097] 3. Experimental Methods

[0098] Take 2 mL of fresh bacterial culture, centrifuge at 5000 rpm / min for 5 min to collect the bacterial cells, wash the bacterial cells 3 times with physiological saline, and then resuspend them in 2 mL of physiological saline as the inoculum. Take 1 mL of the inoculum and add it to 24 mL of artificial gastric or intestinal fluid, place it in a 37℃ water bath shaker (200 rpm / min) for 3 h, take 1 mL of the sample, and detect the viable bacterial count.

[0099] The viable count method was determined according to the national standard GB4789.35-2016-Food Microbiology Examination - Lactic Acid Bacteria Examination. The viable count (Log CFU / mL) of this strain after digestion with artificial gastric or intestinal fluid is shown in Table 4.

[0100] Table 4. Viable bacterial count after digestion with artificial gastrointestinal fluid

[0101] As shown in Table 4, the viable count of *Lactobacillus plantarum* VHProbi O03 screened in this invention remained basically unchanged after digestion with artificial gastric juice. After digestion with artificial intestinal juice for 3 hours, a high viable count could still be detected, indicating that this strain has strong tolerance to gastrointestinal juice.

[0102] Example 5: Determination of the antioxidant function of Lactobacillus plantarum VHProbi O03

[0103] 1. Determination of DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) scavenging ability

[0104] Take 1 mL of *Lactobacillus plantarum* VHProbi O03 bacterial suspension, add 1 mL of 0.4 mM freshly prepared DPPH radical solution, mix well, and then incubate at room temperature in the dark for 30 min. Measure the absorbance of sample A at 517 nm, repeating the measurement three times. The control sample is prepared with an equal volume of PBS solution and a DPPH-ethanol mixture, and the blank is zeroed using an equal volume of *Lactobacillus plantarum* VHProbi O03 bacterial suspension and ethanol mixture.

[0105] The clearance rate was calculated using the following formula, and the results are shown in Table 5.

[0106] Clearance rate % = [1 - (A)] 样品 -A 空白 ) / A 对照 ]×100%.

[0107] Table 5. Scavenging effect of *Lactobacillus plantarum* VHProbi O03 suspension on DPPH free radicals.

[0108] 2. HRS clearance capacity measurement

[0109] Mix 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate, 500 μL of deionized water, and 200 μL of *Lactobacillus plantarum* VHProbi O03 suspension. Then add 100 μL of hydrogen peroxide solution (3 mM). After incubating in a water bath at 37°C for 15 min, measure the absorbance of the sample at a wavelength of 510 nm.

[0110] The hydroxyl radical scavenging rate is calculated using the following formula.

[0111] Clearance rate % = (A 样品 -A 控制 ) / (A 空白 -A 控制 ) × 100%.

[0112] Among them: A 控制 A was used as a substitute for deionized water in the sample. 空白 Deionized water was used to replace the sample and H2O2.

[0113] The specific results are shown in Table 6.

[0114] Table 6. Scavenging effect of Lactobacillus plantarum VHProbi O03 on HRS free radicals

[0115] 3. Determination of anti-lipid peroxidation capacity

[0116] Preparation of linoleic acid emulsion: 0.1 mL linoleic acid, 0.2 mL Tween 20, 19.7 mL deionized water.

[0117] Add 1 mL of linoleic acid emulsion and 1 mL of FeSO4 (1%) to 0.5 mL of PBS solution (pH 7.4), then add 0.5 mL of sample. Incubate at 37°C for 1.5 h. Add 0.2 mL of TCA (4%) and 2 mL of TBA (0.8%) to the mixture. Incubate at 100°C for 30 min, cool rapidly, centrifuge at 4000 rpm / min for 15 min, and collect the supernatant. Measure the absorbance at 532 nm to obtain A. The control group is A0, which is obtained by replacing the sample with 0.5 mL of distilled water.

[0118] Inhibition rate (%) = (A0 - A) / A0 × 100%.

[0119] Note: A represents the absorbance of the sample group; A0 represents the absorbance of the control group. The results are shown in Table 7.

[0120] Table 7. Inhibition rate of lipid peroxidation by Lactobacillus plantarum VHProbi O03

[0121] Example 6: In vitro cholesterol degradation assay using *Lactobacillus plantarum* VHProbi O03

[0122] 1. Qualitative test of bile salt enzyme activity

[0123] Preparation of MRS solid plates containing bile salts: Add 0.2% TCA, 0.2% sodium thioglycolate, 0.37 g / L CaCl2, and 1.5% agar to freshly prepared MRS liquid medium. Sterilize at 121℃ for 15 min, pour into plates, and condense for later use.

[0124] Preparation of MRS solid plates: Sterilize fresh MRS medium at 121℃ for 15 min, pour into plates and condense for later use.

[0125] The *Lactobacillus plantarum* strain VHProbi O03 was isolated and purified by streaking on MRS plates. Single colonies were identified and then streaked on MRS plates and MRS plates containing bile salts. The results were observed after incubation at 37°C for 72 hours and photographed for archiving.

[0126] The results showed that the colonies on the two streaks were indistinguishable, and no other substances were generated next to the colonies, indicating that the bile saltase activity of *Lactobacillus plantarum* VHProbi O03 was negative.

[0127] 2. Cholesterol Degradation Test

[0128] Preparation of cholesterol micelle solution: Accurately weigh 1g of cholesterol, dissolve it in anhydrous ethanol, and bring the volume to 100 mL. Filter the solution under sterile conditions using a 0.22 µm microporous membrane.

[0129] Weigh out 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 2.0 g of diammonium hydrogen citrate, 20.0 g of glucose, 1.0 mL of Tween 80, 5.0 g of sodium acetate, 0.1 g of magnesium sulfate, 0.05 g of manganese sulfate, 2.0 g of dipotassium hydrogen phosphate, 1 g of bile salts, and 1000 mL of distilled water. Adjust the pH to 7.3, sterilize at 115℃ for 30 min, and then add cholesterol solution to make the final cholesterol concentration 0.1%.

[0130] Inoculate fresh *Lactobacillus plantarum* VHProbi O03 bacterial suspension at a rate of 0.1%, and incubate statically at 37°C for 48 hours. Then, take 0.2 mL of the bacterial suspension, add 1.8 mL of anhydrous ethanol, mix well, let stand for 10 minutes, centrifuge at 3000 rpm for 5 minutes, and use the supernatant to determine the cholesterol content. The cholesterol determination method follows GB / T 5009.128-2003 <Determination of Cholesterol in Food>.

[0131] The results showed that the *Lactobacillus plantarum* VHProbi O03 provided by this invention achieved a cholesterol degradation rate of 20.40% (this data does not include bile salts).

[0132] Example 7: Determination of the hydrophobicity of *Lactobacillus plantarum* VHProbi O03 on the cell surface

[0133] After activating *Lactobacillus plantarum* VHProbi O03 strain in MRS liquid medium, 1 ml of bacterial suspension was centrifuged at 6000 rpm for 2 min, and the bacterial cells were collected and washed twice with PBS solution. The bacterial cells were resuspended in 1 ml of sterile 0.1 M KNO3 solution. The bacterial suspension was diluted three times and recorded as OD600 A0. One-third volume of xylene was added to the bacterial suspension, mixed well, and allowed to stand at room temperature for 10 min (at which point a two-phase system was formed). The two-phase system was vortexed for 2 min and then allowed to stand for 20 min to reform the aqueous and organic phases. The aqueous phase was carefully aspirated, and the absorbance A1 was measured at OD600 nm. Cell hydrophobicity was calculated using the following formula, and the average value of three measurements was taken.

[0134] Hydrophobicity (%) = (A0-A1) / A1×100%.

[0135] The results showed that the surface hydrophobicity of the *Lactobacillus plantarum* VHProbi O03 strain provided by this invention was 38.95%.

[0136] Example 8: Intestinal cell adhesion test of Lactobacillus plantarum VHProbi O03

[0137] Caco-2 cells at 2×10 6 Cells / well were inoculated into six-well plates and incubated in a CO2 incubator for 24 h for cell adhesion assays; the stationary strains were resuspended in MRS medium to 5 × 10⁻⁶ m³ / well. 7 CFU / mL; 1 mL of *Lactobacillus plantarum* VHProbi O03 bacterial culture was added to a six-well plate with adherent cells and incubated in a CO2 incubator for 2 h; the cells were washed three times with PBS to remove unadhered bacteria; 500 μL of trypsin was added for digestion for 3 minutes, followed by 1.5 mL of cell culture medium to stop the digestion. The cells were repeatedly pipetted and the resulting solution was collected into sterile EP tubes. The collected solution was then serially diluted 10-fold, 100-fold, 1000-fold, and 10000-fold, and plated for cell counting. Cells in the control group were also counted simultaneously.

[0138] The adhesion ability of the tested strains was calculated using the following formula:

[0139] Adhesion capacity (CFU / cells) = Total number of bacteria adhering in each culture well / Total number of cells in each culture well.

[0140] The results showed that the adhesion ability of *Lactobacillus plantarum* VHProbi O03 was 1.72, with a standard deviation of 0.15%.

[0141] In summary, the *Lactobacillus plantarum* VHProbi O03 provided by this invention is sensitive to common antibiotics, does not produce hemolysin, and exhibits good biocompatibility. In vitro experiments have verified that *Lactobacillus plantarum* VHProbi O03 has a strong ability to degrade indole, which is beneficial for clearing uremic toxins and alleviating chronic kidney disease. The *Lactobacillus plantarum* VHProbi O03 can be widely used in the preparation of health products or pharmaceuticals that degrade intestinal indole and alleviate chronic kidney disease.

Claims

1. A plant lactobacillus, characterized in that, The aforementioned Lactobacillus plantarum ( Lactiplantibacillus plantarum The accession number for VHProbi O03 is CCTCC NO: M20232619.

2. A pharmaceutical product for degrading intestinal indole, characterized in that, The medicine contains *Lactobacillus plantarum* as described in claim 1.

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