A strain of *Lactobacillus plantarum* with indole-degrading capabilities and its applications

Abstract

This invention belongs to the field of probiotic screening and application technology, specifically providing a *Lactobacillus plantarum* strain, with the preservation number CCTCC NO: M20221666. The *Lactobacillus plantarum* strain provided by this invention exhibits a strong degradation effect on intestinal indole, significantly degrading indole in vitro; it can effectively degrade intestinal indole. This strain has 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, demonstrating good biocompatibility. The provided *Lactobacillus plantarum* strain has strong antioxidant capacity and cholesterol-degrading ability. The *Lactobacillus plantarum* strain provided by this invention can be used alone or in combination with various probiotics to maintain human health, with broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of probiotic screening and application technology, specifically relating to a strain of *Lactobacillus plantarum* with the ability to degrade indole and its application. Background Technology

[0002] Chronic kidney disease (CKD) is a widespread chronic disease globally and has become a major public health problem affecting human health. As CKD progresses and kidney function is lost, nitrogenous waste products generated by metabolism cannot be excreted from the body in a timely manner. These waste products accumulate in the body and form toxic substances called uremic toxins.

[0003] In recent years, uremic toxins produced by gut microbiota metabolism have attracted widespread attention. Among them, indoxyl sulfate (IS) and p-cresyl sulfate (PCS) are the most studied and representative uremic toxins. Indoxyl sulfate is a protein-bound uremic toxin. Due to its high binding rate with plasma proteins, it cannot be smoothly excreted from the body during renal failure, thus posing a threat to human health. The main clinical methods for clearing uremic toxins in patients with end-stage chronic kidney disease are dialysis and kidney transplantation. However, the vast majority of indoxyl sulfate cannot be excreted through dialysis, and kidney transplantation suffers from high treatment costs, a shortage of kidney donors, and severe rejection. Adjunctive interventions for uremic toxin accumulation, such as the use of adsorbents and laxatives, also have certain drawbacks. For example, the adsorbent AST120 easily adsorbs other drugs, reducing their therapeutic effect; long-term use of laxatives can lead to intestinal flora and electrolyte imbalance. Currently, there are no safe and effective methods or drugs for clearing the uremic toxin indoxyl sulfate. Therefore, exploring new strategies for clearing uremic toxins to slow the progression of chronic kidney disease has significant scientific and clinical translational value.

[0004] In mammals, the formation of uremic indole sulfate involves two steps. First, intestinal bacteria metabolize tryptophan from the diet into indole. Subsequently, indole is converted into indole sulfate by sulfonyltransferases in the intestinal mucosa and liver. The development of chronic kidney disease (CKD) is often accompanied by metabolic disorders of the gut microbiota and its metabolites, as well as impaired intestinal barrier function. Therefore, regulating the metabolism of gut microbiota and its metabolites to intervene in the progression of CKD has significant application prospects and scientific value. Thus, a treatment strategy targeting metabolic pathways in gut bacteria, screening for beneficial bacteria in the gut, inhibiting indole production in gut bacteria, reducing the level of uremic indole sulfate in the body, and mitigating the damage caused by indole sulfate should receive widespread attention. Summary of the Invention

[0005] The purpose of this invention is to provide a novel *Lactiplantibacillus plantarum* strain and its applications. This *Lactiplantibacillus plantarum* strain was isolated from the fermentation broth of kimchi, a traditional Chinese fermentation method. It exhibits a strong ability to degrade indole, a tryptophan metabolite 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 patients with chronic renal failure. This strain can be used as an adjunct therapy for renal failure patients, reducing their suffering and prolonging their lives.

[0006] The plant lactobacillus provided in this invention is plant lactobacillus VHProbi SY, which was deposited on October 26, 2022, at the China Center for Type Culture Collection, Wuhan University, China, with accession number CCTCC NO: M20221666.

[0007] The 16S rDNA sequence of the *Lactobacillus plantarum* VHProbi SY strain provided by this invention is SEQ ID NO:3.

[0008] The Riboprinter fingerprint of the *Lactobacillus plantarum* VHProbi SY 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.

[0009] The *Lactobacillus plantarum* VHProbi SY strain provided by this invention can be used to prepare products with cholesterol-lowering functions.

[0010] The *Lactobacillus plantarum* VHProbi SY strain provided by this invention can also be used to prepare products with antioxidant functions.

[0011] The *Lactobacillus plantarum* strain VHProbi SY of the present invention can be used to prepare products that degrade indole.

[0012] The present invention also provides a probiotic preparation comprising Lactobacillus plantarum VHProbi SY strain and / or its fermentation products.

[0013] The probiotic preparation further comprises any one or more combinations of Lactobacillus acidophilus, Bacillus coagulans, Pediococcus pentosus, Lactobacillus rhamnosus, Lactobacillus paracasei, Bifidobacterium lactis, Lactobacillus salivarius, Lactococcus lactis, Lactococcus lactis, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus helveticus, Enterococcus lactis, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus fermentum, Streptococcus thermophilus, and Lactobacillus curvatureus.

[0014] The *Lactobacillus plantarum* VHProbi SY strain provided by this invention exhibits a strong degradation effect on intestinal indole, significantly degrading it in vitro; it effectively degrades intestinal indole, with a degradation rate of 58.14% after 48 hours. This strain shows 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, demonstrating good biocompatibility. The provided *Lactobacillus plantarum* VHProbi SY strain also possesses strong antioxidant and cholesterol-degrading capabilities, achieving scavenging rates of 89.02% and 90.12% for DPPH and HRS free radicals, respectively, and a cholesterol degradation rate of 78.79%. The anti-lipid peroxidation inhibition rates of the fermentation supernatant, bacterial cells, and intracellular extracts of *Lactobacillus plantarum* VHProbi SY are 72.99%, 82.82%, and 89.09%, respectively.

[0015] The *Lactobacillus plantarum* VHProbi SY strain provided by this invention has an adhesion rate as high as 55.79, which is beneficial for its colonization in the intestine and its probiotic effects. This strain can be used alone or in combination with various probiotics to maintain human health, and has broad application prospects. Attached Figure Description

[0016] Figure 1 The growth curve of *Lactobacillus plantarum* VHProbi SY in indole-containing medium;

[0017] Figure 2 This is a colony morphology diagram of *Lactobacillus plantarum* VHProbi SY.

[0018] Figure 3 Riboprinter fingerprint of Lactobacillus plantarum VHProbi SY;

[0019] Figure 4 RAPD fingerprint of Lactobacillus plantarum VHProbi SY;

[0020] Figure 5 The rep-PCR fingerprint of Lactobacillus plantarum VHProbi SY;

[0021] Figure 6MALDI-TOF-MS protein fingerprint of Lactobacillus plantarum VHProbi SY. Detailed Implementation

[0022] Based on polymorphic taxonomic identification, the *Lactiplantibacillus plantarum* strain obtained in this invention is a novel strain, named *Lactiplantibacillus plantarum* VHProbi SY, and was deposited on October 26, 2022, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M20221666.

[0023] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings.

[0024] Example 1: Screening of bacterial strains

[0025] 1. Strains Isolation

[0026] To prepare 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.

[0027] Prepare MRS agar medium: Add 15g agar to 1LMRS broth.

[0028] Take 1g of fresh, naturally fermented kimchi broth, dilute it with sterile physiological saline, place it in a sterile sample bag, homogenize it using a homogenizer, take 100μL of the mixture, serially dilute it, spread it on MRS agar medium, and incubate it anaerobically at 37℃ for 48h. Examine the plates for single colonies under a microscope. Based on the microscopic examination results, the applicant screened out 12 potential lactic acid bacteria single colonies, named A1, A2, ..., A11, A12.

[0029] 2. In vitro screening of strains capable of degrading indole

[0030] 2.1 Preparatory work

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

[0032] Strain preparation: Single colonies of selected or selected lactic acid bacteria are streaked to activate them. Take frozen lactic acid bacteria glycerol tubes, aseptically streak them onto MRS plates, and incubate at 37°C for 48–72 h.

[0033] Preparation of lactic acid bacteria cell solution: 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] 2.2 Tolerance test of strains to different concentrations of indole

[0035] Indole was diluted in the culture medium to concentrations of 0, 50, 100, 200, 300, 400, and 600 μg / ml, and inoculated into the strain at a 1% inoculum. The growth curves of the strains were monitored using a microplate reader. The results showed that the presence of low concentrations of indole in the culture medium did not affect the activity of the strains and even slightly promoted their growth. When the indole concentration in the culture medium approached 300 μg / ml, the activity of the strains was inhibited. Furthermore, according to available data, the concentration of indole in human intestines or feces is 0.25-2.6 mM, approximately 45-468 μg / ml. Therefore, 300 μg / ml was used as the indole concentration for verifying the indole degradation ability of the strains during screening.

[0036] 2.3 Detection of the ability of strains to metabolize indole

[0037] Fresh culture medium containing 1% of the strain was supplemented with indole to a final concentration of 300 μg / ml. At 0h, 24h, and 48h after indole addition, 1.5 ml of the medium was transferred to a new EP tube, centrifuged at 10000 g / min for 3 min, and the supernatant was collected. The indole content in the supernatant was detected using an indole detection kit. The specific procedures and result interpretation were performed according to the Indole Assay Kit instructions.

[0038] Indole degradation rate calculation: Indole degradation rate = (initial concentration - final concentration) / initial concentration × 100%.

[0039] The results showed that among the 12 potential lactic acid bacteria isolated in this invention, 8 strains exhibited an indole degradation rate exceeding 30% after 24 hours, with strain A3 showing a degradation rate of 34.42 ± 3.33% after 24 hours; 3 strains exhibited an indole degradation rate exceeding 50% after 48 hours, with strain A3 showing the highest degradation rate of 58.14 ± 1.27% after 48 hours. The growth curves of strain A3 in different concentrations of indole are shown below. Figure 1 As shown.

[0040] Example 2, Strain Identification

[0041] 1. Colony morphology identification

[0042] The A3 strain was inoculated onto MRS agar medium and anaerobically cultured at 37°C for 24 hours. The colony morphology of the A3 strain is as follows: Figure 2As shown, single colonies are milky white, with most colonies having a diameter of more than 1.0 mm and a smooth surface. Under a microscope, the bacteria appear as long rods of varying lengths, arranged in single, paired, or chain formations.

[0043] 2. Identification of physiological and biochemical characteristics

[0044] In this embodiment, the inoculum solution is prepared as follows: Under aseptic conditions, take an appropriate amount of fresh A3 strain 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.

[0045] 2.1 Salinity Tolerance Test

[0046] 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. Uninoculated wells 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 prevent turbidity. The results showed that the maximum salt concentration tolerated by strain A3 was 5%.

[0047] 2.3 Carbon source metabolism test

[0048] Carbon source metabolism experiments were performed on strain A3 using API 50CHL reagent strips. For experimental methods and result interpretation, please refer to the API 50CHL kit instructions. The A3 strain identification results were: %ID = 99.9 and T value = 0.79. The API result identified it as *Lactobacillus plantarum*. The results are shown in Table 1.

[0049] Table 1: Results of API 50 CHL assay for the strains

[0050]

[0051]

[0052] 2.4 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 the 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 A3 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 (SEQ ID NO: 1);

[0067] 1492R: GGTTACCTTGTTACGACTT (SEQ ID NO: 2).

[0068] The 16S rDNA sequence SEQ ID NO:3 of strain A3 was obtained by sequencing and compared with the NCBI database, preliminarily identifying strain A3 as *Lactobacillus plantarum*.

[0069] The specific sequence of the 16S rDNA is as follows (SEQ ID NO:3):

[0070] ACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGCATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAATAACCTGAACAGTTACTCTCAGATATGTTCTTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCAT TGCCATGGTGAGCCGTTACCCCACCATCTAGCTAATACGCCGCGGACCATCCAAAAGTGATAGCCGAAGCCATCTTTCAAGCTCGGACCATGCGGTCCAAGTTGTTATGCGGTATTAGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTGGGCAGGTTTCCCACGTGTTACTCACCAGTTCGCCACTCACTCAAATGTAAATCATGATGCAAGCACCAATCAATACCAGAGTTCGTTCGACTGC.

[0071] 3.2 Riboprinter fingerprint spectrum

[0072] A single colony of purified strain A3 was collected 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. Following DNA preparation, transfer, imaging, and data processing, bacterial identification results were obtained. The identification results showed that strain A3 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 (SEQ ID NO:4).

[0076] Table 2: RAPD Reaction System

[0077]

[0078]

[0079] A 1.5% agarose gel plate was prepared, with a DL2000 DNA Marker used as a result control. The electrophoresis was performed at a constant voltage of 100V for 80 minutes, and the electrophoresis pattern was finally detected using a gel imaging system. The RAPD fingerprint of strain A3 is shown below. Figure 4 As shown.

[0080] 3.3.2, rep-PCR fingerprinting

[0081] Primer sequence: CTACGGCAAGGCGACGCTGACG (SEQ ID NO:5).

[0082] rep-PCR reaction system

[0083] Table 3: Reaction system of rep-PCR

[0084]

[0085] The DL2000 DNA Marker was used as a result control. Amplification results were detected at 100V and an electrophoresis time of 80 minutes. The rep-PCR fingerprint of strain A3 is shown below. Figure 5 As shown.

[0086] 3.4 MALDI-TOF-MS detection of ribosomal protein expression in strains

[0087] 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 A3 were: m / z 4700.168, 5893.391, and 9400.959. The identification results are as follows. Figure 6 As shown.

[0088] 3.5 Whole genome sequencing

[0089] Fresh bacterial culture was inoculated into MRS liquid medium at an inoculum rate 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 CP134220, CP134221, and CP134222, respectively.

[0090] Based on the colony morphology, physiological and biochemical characteristics, and molecular biological identification results of strain A3, strain A3 was identified as a new Lactobacillus plantarum strain, named Lactobacillus plantarum VHProbi SY (Lacticaseibacillus paracasei VHProbi SY).

[0091] Example 3: Antibiotic resistance test of Lactobacillus plantarum VHProbi SY

[0092] The specific results of determining the minimum inhibitory concentration (MIC) of antibiotics against *Lactobacillus plantarum* VHProbi SY using the micro-broth dilution method are shown in Table 4.

[0093] Table 4: Antibiotic MIC values ​​of Lactobacillus plantarum VHProbi SY

[0094]

[0095] MIC is measured in μg / mL.

[0096] As can be seen from the results in Table 4, the *Lactobacillus plantarum* VHProbi SY provided by this invention is sensitive to common antibiotics such as erythromycin, ampicillin, and clindamycin, and has good biosafety.

[0097] Example 4: Determination of the antioxidant function of *Lactobacillus plantarum* VHProbi SY

[0098] 1. Determination of the strain's ability to scavenge DPPH (1,1-diphenyl-2-trinitrophenylhydrazine)

[0099] A single colony of *Lactobacillus rhamnosus* VHProbi M12 with excellent growth was inoculated into 3 mL of MRS liquid medium and cultured at 37°C for 18-20 h. Using this culture as the inoculum, 2% of the colony was inoculated into 50 mL of MRS liquid medium and incubated statically for 18 h to obtain the bacterial culture. 1 mL of the bacterial suspension was collected, and the cells were washed twice with 1 mL of PBS buffer. The cells were then resuspended in 2 mL of PBS solution to obtain the bacterial suspension.

[0100] Take 1 mL of *Lactobacillus plantarum* VHProbi SY bacterial suspension, add 1 mL of freshly prepared 0.4 mM 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 SY bacterial suspension and ethanol mixture.

[0101] The clearance rate is calculated using the following formula:

[0102] Clearance rate % = [1-(A 样品 -A 空白 ) / A 对照 ×100%.

[0103] The results are shown in Table 5.

[0104] Table 5: Scavenging effect of Lactobacillus plantarum VHProbi SY on DPPH free radicals

[0105]

[0106] 2. Determination of the strain's ability to scavenge HRS

[0107] 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 SY suspension. Then add 100 μL of hydrogen peroxide solution (3 mM), incubate at 37°C for 15 min, and measure the absorbance of the sample at 510 nm.

[0108] The hydroxyl radical scavenging rate is calculated using the following formula.

[0109] Clearance rate % = (A 样品 -A 控制 ) / (A 空白 -A 控制 )×100%.

[0110] 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.

[0111] The specific results are shown in Table 6.

[0112] Table 6: Scavenging effect of Lactobacillus plantarum VHProbi SY on HRS

[0113]

[0114] 3. Identification of the strain's resistance to lipid peroxidation

[0115] 1) Preparation of bacterial strain culture and fermentation supernatant, bacterial cells, and intracellular extracts:

[0116] Lactobacillus plantarum VHProbi SY strain was cultured in MRS liquid medium at 37℃ for 24 h. After three passages, the culture was centrifuged at 6000 rpm for 10 min at 4℃, and the supernatant was collected as the fermentation supernatant. The collected bacterial cells were washed three times by centrifugation at 6000 rpm for 10 min in PBS buffer (pH 7.4). The bacterial cells were then resuspended in PBS buffer to adjust the bacterial concentration to 1.0 × 10⁻⁶. 9 The bacterial suspension was obtained by sonicating the bacterial suspension with cells / mL. The bacterial suspension was then sonicated for 20 minutes to obtain the intracellular extract.

[0117] (2) Preparation of linoleic acid emulsion:

[0118] 0.1 mL linoleic acid, 0.2 mL Tween 20, 19.7 mL deionized water.

[0119] (2) Experimental procedure

[0120] 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.

[0121] Inhibition rate / % = (A0 - A) / A0 × 100%.

[0122] Note: A is the absorbance of the sample group; A0 is the absorbance of the control group.

[0123] Table 7: Anti-lipid peroxidation inhibitory effect of Lactobacillus plantarum VHProbi SY

[0124]

[0125] Example 5: Surface hydrophobicity test

[0126] After activating the A3 strain in MRS liquid medium, 1 ml of bacterial suspension was centrifuged at 6000 rpm for 2 min, and the cells were collected and washed twice with PBS solution. The 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.

[0127] Hydrophobicity (%) = (A0 - A1) / A1 × 100%.

[0128] The results showed that the surface hydrophobicity of strain A3 was 69.18% ± 2.45%.

[0129] Example 6: In vitro cholesterol degradation assay of *Lactobacillus plantarum* VHProbi SY

[0130] 1. Qualitative test of bile salt enzyme activity

[0131] 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°C for 15 min, pour into plates, and condense for later use.

[0132] Preparation of MRS solid plates: Sterilize fresh MRS medium at 121℃ for 15 min, pour into plates and condense for later use.

[0133] Lactobacillus plantarum VHProbi SY 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.

[0134] The results showed that there was no difference between the two streaked colonies, and no other substances were generated next to the colonies, indicating that the bile saltase activity of *Lactobacillus plantarum* VHProbi SY was negative.

[0135] 2. Cholesterol Degradation Test

[0136] Preparation of cholesterol micelle solution: Accurately weigh 1g of cholesterol, dissolve it in anhydrous ethanol, and bring the volume to 100mL. Filter the solution under sterile conditions using a 0.22μm microporous membrane.

[0137] Weigh out 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 1.0mL of Tween 80, 5.0g of sodium acetate, 0.1g of magnesium sulfate, 0.05g of manganese sulfate, 2.0g of dipotassium hydrogen phosphate, 1g of bile salts, and 1000mL of distilled water. Adjust the pH to 7.3, sterilize at 115℃ for 30min, and then add cholesterol solution to make the final cholesterol concentration 0.1%.

[0138] Inoculate fresh *Lactobacillus plantarum* VHProbi SY 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 / T5009.128-2016 <Determination of Cholesterol in Food>.

[0139] The results showed that the *Lactobacillus plantarum* VHProbi SY provided by this invention achieved a cholesterol degradation rate of 78.79% (this data does not include bile salts).

[0140] Example 7: Intestinal cell adhesion test of Lactobacillus plantarum VHProbi SY

[0141] 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; stationary *Lactobacillus plantarum* VHProbi SY strains were resuspended in MRS medium to 5 × 10⁻⁶ m³ / well. 7 CFU / mL; Add 1 mL of the above strain to a six-well plate with adherent cells and incubate in a CO2 incubator for 2 h; Wash three times with PBS to remove unadhered bacteria; Add 500 μl of trypsin for 3 minutes, then add 1.5 mL of cell culture medium to stop digestion, repeatedly pipetting and collecting the resulting solution into sterile EP tubes. Perform serial dilutions of the collected solution at 10-fold, 100-fold, 1000-fold, and 10000-fold, and plate the cells for counting. Simultaneously count the cells in the blank control group. Calculate the adhesion ability of the tested strain using the following formula:

[0142] Adhesion capacity (CFU / cells) = Total number of bacteria adhering in each culture well / Total number of cells in each culture well.

[0143] The results showed that the adhesion ability of *Lactobacillus plantarum* VHProbi SY was 55.79, with a standard deviation of 1.31.

[0144] In summary, the *Lactobacillus plantarum* VHProbi SY provided by this invention is sensitive to common antibiotics, does not produce hemolysin, and has good biocompatibility. In vitro experiments have verified that *Lactobacillus plantarum* VHProbi SY has a strong ability to degrade indole, can clear uremic toxins, and alleviate chronic kidney disease.

[0145] The plant-derived Lactobacillus VHProbi SY provided by this invention can be used alone as a probiotic, or in combination with other probiotics such as Lactobacillus acidophilus, Bacillus coagulans, Pediococcus pentosus, Lactobacillus rhamnosus, Lactobacillus paracasei, Bifidobacterium lactis, Lactobacillus salivarius, Lactococcus lactis, Lactococcus lactis, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus helveticus, Enterococcus lactis, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus fermentum, Streptococcus thermophilus, and Lactobacillus curvatureus, for the purpose of maintaining human health, with broad application prospects.

Claims

1. A plant lactobacillus ( Lactiplantibacillus plantarum ), characterized in that, The preservation number of the *Lactobacillus plantarum* is CCTCC NO: M20221666.

2. The *Lactobacillus plantarum* as described in claim 1, characterized in that, The 16S rDNA sequence of *Lactobacillus plantarum* is SEQ ID NO:

3.

3. The *Lactobacillus plantarum* as described in claim 1, characterized in that, The Riboprinter fingerprint of *Lactobacillus plantarum* is shown in Figure 3, and the RAPD fingerprint is shown in Figure 4.

4. The *Lactobacillus plantarum* as described in claim 1, characterized in that, The rep-PCR fingerprint of *Lactobacillus plantarum* is shown in Figure 5, and the MALDI-TOF-MS protein fingerprint is shown in Figure 6.

5. The application of the *Lactobacillus plantarum* as described in claim 1 in the preparation of probiotic preparations with cholesterol-lowering function.

6. The use of *Lactobacillus plantarum* as described in claim 1 in the preparation of probiotic formulations with antioxidant functions.

7. The use of *Lactobacillus plantarum* as described in claim 1 in the preparation of probiotic formulations that degrade indole.

8. A probiotic preparation, characterized in that, The probiotic preparation contains live Lactobacillus plantarum as described in claim 1.

9. The probiotic preparation as described in claim 8, characterized in that, The probiotic preparation also contains any one or more of the following: Lactobacillus acidophilus, Bacillus coagulans, Pediococcus pentosus, Lactobacillus rhamnosus, Lactobacillus paracasei, Bifidobacterium lactis, Lactobacillus salivarius, Lactococcus lactis, Lactococcus lactis, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus helveticus, Enterococcus lactis, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus fermentum, Streptococcus thermophilus, and Lactobacillus curvatureus.

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