A strain of Pediococcus pentosaceus and its application in the prevention or treatment of colonic polyps and colon cancer

Abstract

This invention belongs to the field of functional probiotic screening and application technology, specifically involving a novel Pediococcus pentosaceus strain ( Pediococcus pentosaceus The *Pediococcus pentosaceus* strain was deposited on January 15, 2026, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M202026128. This strain can significantly inhibit the growth of *Streptococcus bovis*, which induces colonic polyps and colon cancer, and exhibits a flocculation effect with *Streptococcus bovis*, thus having important application value for the prevention and treatment of colonic polyps and colon cancer.

Description

Technical Field

[0001] This invention belongs to the field of functional probiotic screening and application technology, specifically involving a strain of Pediococcus pentosaceus and its application in the prevention and treatment of colon polyps and colon cancer. Background Technology

[0002] Colorectal cancer (CRC) often originates from the malignant transformation of colonic polyps. Normal colonic mucosa gradually develops into adenomatous polyps under the combined influence of genetic and environmental factors. With the accumulation of gene mutations, these polyps eventually develop into invasive cancer, a process that typically takes 5-10 years. Timely intervention with polyps can reduce the risk of colorectal cancer by 60%-70%, therefore, prevention and early intervention with polyps are key to controlling colorectal cancer.

[0003] The gut microbiota plays a central role in maintaining colonic health. When the microbiota is imbalanced, harmful bacteria (such as Fusobacterium nucleatum and enterotoxin-producing Escherichia coli) increase while beneficial bacteria decrease. This, in turn, promotes polyp formation and malignant transformation through multiple mechanisms, including inducing chronic inflammation, producing toxic metabolites, and disrupting the intestinal barrier. Among these, Streptococcus bovis is one of the "driver bacteria" most closely associated with colorectal cancer. Its colonization rate is significantly increased in the tissues of colorectal cancer patients, and it promotes polyp malignancy through epithelial adhesion, activation of β-catenin signaling, DNA damage, and exacerbation of microbiota dysbiosis. Early detection of Streptococcus bovis can serve as a potential biomarker for colorectal cancer screening.

[0004] Probiotics have shown clear potential in preventing colonic polyps and colorectal cancer. Their mechanisms of action include: regulating gut microbiota balance and inhibiting the colonization of harmful bacteria; producing short-chain fatty acids (such as butyrate), enhancing barrier function and inducing apoptosis in cancer cells; strengthening intestinal tight junctions and reducing permeability; regulating immune responses and inhibiting pro-inflammatory pathways; directly inhibiting polyp cell proliferation; degrading potential carcinogens; and neutralizing bacterial toxins.

[0005] The transformation of colonic polyps into cancer is the result of the combined effects of genetics, environment, and gut microbiota imbalance. Streptococcus bovis and gut microbiota dysbiosis are key driving factors, and probiotics can intervene in this process through multiple pathways. Therefore, developing probiotic-based targeted prevention technologies has significant clinical value and application prospects for blocking polyp malignant transformation and reducing the incidence of colorectal cancer. Summary of the Invention

[0006] The purpose of this invention is to provide a novel strain of Pediococcus pentosaceus ( Pediococcus pentosaceus This strain can significantly inhibit the growth of *Streptococcus bovis*, which induces colonic polyps and colon cancer, and produces a flocculation effect with *Streptococcus bovis*, thus having important application value for the prevention and treatment of colonic polyps and colon cancer.

[0007] The *Pediococcus pentosaceus* provided in this invention is named *Pediococcus pentosaceus* P20 (… Pediococcus pentosaceusP20 was deposited on January 15, 2026, at the China Center for Type Culture Collection, Wuhan University, China, with accession number CCTCC NO: M2026128.

[0008] The 16S rDNA sequence of Pediococcus pentosaceus P20 provided by this invention is SEQ ID NO: 1.

[0009] This invention also relates to the application of Pediococcus pentosaceus strain P20 in the preparation of Streptococcus bovis inhibitors.

[0010] The present invention also relates to the use of Pediococcus pentosaceus strain P20 in the preparation of medicines for the prevention or treatment of colon polyps and colon cancer.

[0011] The present invention also relates to a probiotic pellet, wherein the probiotic pellet contains the Pediococcus pentosaceus strain P20.

[0012] The probiotic pellets consist of an outer shell structure and an inner core structure, wherein the inner core structure contains the Pediococcus pentosaceus strain P20.

[0013] The components and their mass fractions of the probiotic crystal shell structure are as follows: 15 parts gelatin, 2 parts seaweed oligosaccharide, 15 parts carrageenan, 7 parts glycerol, and 60 parts water.

[0014] The components and their mass fractions of the probiotic crystal core structure are as follows: 5 parts of Pediococcus pentosaceus P20 bacterial powder and 95 parts of hydrogenated vegetable oil with a melting point of 45℃.

[0015] This invention also relates to a method for preparing the probiotic pellets, comprising the following steps: (1) Preparation of the core solution: Dissolve hydrogenated vegetable oil at 45°C, add Pediococcus pentosaceus P20 bacterial powder, and stir with a stirrer at 300 rpm for 10 minutes to mix and disperse the bacterial powder in the hydrogenated vegetable oil. Then pour it into the inner core solution storage tank of the capsule making machine. Set the stirring speed of the inner core storage tank to 8 rpm and the temperature to 45°C. (2) Preparation of the shell solution: Gelatin and carrageenan are dissolved at 75°C, then glycerin and seaweed oligosaccharides are added, and the mixture is dissolved again at 75°C. The solution is then stirred until homogeneous and poured into the outer shell solution tank of the capsule making machine, with the temperature of the outer shell tank set to 75°C. The outer shell solution tank and the inner core solution tank of the capsule making machine have concentric nozzles. (3) Preparation of probiotic pellets: The pump speed was controlled at 600 mL / h and the pressure in the inner core tank was 11 kPa. During the titration process, the solution in the outer shell completely enveloped the solution in the inner core tank. The solution was then dropped into a tank containing a liquid paraffin solution at a temperature controlled at 10°C, which caused the solution to gel and form continuous core-shell spheres. The resulting spheres were then dried at room temperature (25°C) with ventilation until the moisture content was below 1.5%, yielding probiotic spheres with a diameter of 1.2 mm.

[0016] The *Pediococcus pentosaceus* P20 strain provided by this invention is sensitive to common antibiotics, exhibits good biosafety, and possesses strong antioxidant function. Its scavenging rates against DPPH and HRS free radicals are 90.46%±3.36% and 73.46%±1.28%, respectively. The anti-lipid peroxidation inhibition rates of its supernatant and bacterial suspension are 85.65%±3.15% and 77.69%±1.06%, respectively. The *Pediococcus pentosaceus* P20 strain also achieves a cholesterol degradation rate of 65.12%±2.03% and a cell surface hydrophobicity of 42.35%±2.28%.

[0017] The Pentosacchariphyte P20 strain exhibits a strong inhibitory effect on Streptococcus bovis, with an inhibition zone size of 18.44 ± 0.28 mm and an aggregation rate of 53.32% ± 0.79% with Streptococcus bovis. This is beneficial for inhibiting the colonization of Streptococcus bovis in the intestine and reducing the risk of Streptococcus bovis-induced colonic polyps and colon cancer.

[0018] The Pediococcus pentosaceus P20 can effectively promote the proliferation of Akkermansia myxophilus. After adding 50% Pediococcus pentosaceus P20 lysate to MRS medium, the viable count of Akkermansia myxophilus increased by 17.63%.

[0019] The Pediococcus pentosaceus P20 has an adhesion index of 36.35±1.06 to Caco-2 cells, which can inhibit the inflammatory response of intestinal epithelial cells Caco-2 caused by Streptococcus bovis, downregulate the level of pro-inflammatory cytokines and upregulate the level of anti-inflammatory cytokines, and can be used to prevent or treat the development of colonic polyps and colon cancer caused by Streptococcus bovis.

[0020] The probiotic pellets provided by this invention effectively protect *Pediococcus pentosaceus* P20 within them, ensuring its successful arrival in the intestines. Accelerated stability testing confirmed that the survival rate of *Pediococcus pentosaceus* P20 is greater than 95% during its shelf life, and the probiotic pellets can withstand digestion by artificial gastric juice at pH 3.0, with a *Pediococcus pentosaceus* P20 survival rate as high as 95.23% ± 2.46%. The probiotic pellets disintegrate rapidly in the intestines within 7 minutes. These probiotic pellets have broad market prospects. Attached Figure Description

[0021] Figure 1 This is a colony diagram of Pediococcus pentosaceus P20; Figure 2 This is a microscopic image of Pediococcus pentosaceus P20 stained with crystal violet. Figure 3 RAPD fingerprint of Pediococcus pentosacchari P20; Figure 4 The rep-PCR fingerprint of Pediococcus pentosacchari P20; Figure 5 The size of the inhibition zone of Pediococcus pentosate P20 against Streptococcus bovis; Figure 6 The graph shows the flocculation effect of Pediococcus pentosaccharide P20 on Streptococcus bovis. Column A represents the flocculation effect of co-culturing Pediococcus pentosaccharide P20 and Streptococcus bovis, column B represents the flocculation effect of culturing Streptococcus bovis alone, and column C represents the flocculation effect of culturing Pediococcus pentosaccharide P20 alone. Figure 7 This diagram illustrates the cytokine-induced inflammatory response regulated by Pediococcus pentosaceus P20, where (A) represents the pro-inflammatory cytokine IL-1β, (B) represents the anti-inflammatory cytokine IL-10, and (C) represents the anti-inflammatory cytokine IL-6. Detailed Implementation

[0022] The *Pediococcus pentosaceus* P20 strain provided by this invention is a newly discovered strain that can significantly inhibit the growth of *Streptococcus bovis*, which induces colon polyps and colon cancer risk, and produces a flocculation effect with *Streptococcus bovis*. It has important application value for the prevention and treatment of colon polyps and colon cancer.

[0023] The applicant deposited the Pediococcus pentosaceus P20 at the China Center for Type Culture Collection of Wuhan University on January 15, 2026, with accession number CCTCC NO: M2026128.

[0024] The screening method described in this invention is not limited to the embodiments described. 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. The invention will now be described in detail with reference to specific embodiments.

[0025] Example 1: Isolation, screening and identification of Pediococcus pentosaceus P20 This invention isolates and screens a lactic acid bacterium with strong acid resistance from naturally fermented cheese samples collected from Tongliao City, Inner Mongolia Autonomous Region, and names it strain P20.

[0026] 1.1 Identification of colony and cell morphology The P20 strain was inoculated onto MRS agar medium and anaerobically cultured at 37°C for 24 days. The colonies of the P20 strain are shown below. Figure 1 As shown, a single colony is white, with a smooth and moist surface, neat edges, and a raised center. The colony diameter is approximately 1.5 to 2.0 mm.

[0027] P20 cells appear spherical under a microscope, are Gram-positive, and are arranged in short chains or clusters. An optical microscope photograph of these cells is shown below. Figure 2 As shown.

[0028] 1.2 Molecular biological identification 1.2.1 16S rDNA gene sequence analysis 1) Genomic DNA extraction Follow the instructions in the Tiangen Bacterial Genomic DNA Extraction Kit (catalog number: DP302).

[0029] 2) 16S rDNA gene amplification Primer sequences: 27F: AGAGTTTGATCCTGGCTCA; 1492R: GGTTACCTTGTTACGACTT.

[0030] The 16S rDNA sequence of strain P20 was obtained by sequencing, which is SEQ ID NO:1. The specific sequence is as follows:

[0031] The sequence was compared with the NCBI database, and the P20 strain was preliminarily identified as Pediococcus pentosaceus.

[0032] 2.3 MALDI-TOF-MS detection of ribosomal protein expression in strains Fresh bacterial culture was inoculated into MRS liquid medium at an inoculum rate of 0.1%, and cultured at 37°C and 150 rpm for 48 hours. The bacterial cells were then collected, washed four times with sterile water, and air-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 air-drying, 1 μL of matrix solution was added to cover the sample again. After air-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, causing the proteins in the sample to ionize. The ions were accelerated through the flight tube under an electric field of 10–20 kV, and the molecular weight of the proteins was determined based on their flight time to the detector. Protein fingerprints were obtained using Autofms 1000 software (Autof Analyzer v1.0). The strain P20 was identified as *Pediococcus pentosaceus*.

[0033] 2.4 Identification using RAPD and rep-PCR fingerprinting 2.4.1 RAPD fingerprint identification 1) Primer sequence: 5'- - GAGGGTGGCGGTTCT-3'; 2) RAPD reaction system Taq DNA polymerase (5 U / μL) 0.2 μL, 10× Buffer (containing Mg) 2+ 2 μL of primer (10 uM), 1 μL of dNTPs (2.5 mM), 0.8 μL of DNA template, 2 μL of sterile double-distilled water, and 14 μL of dNTPs (2.5 mM).

[0034] 3) Electrophoresis A 1.5% agarose gel plate was prepared, with a DL2000 DNA Marker used as a result control. Electrophoresis was performed at a constant voltage of 100V for 80 min, and the electrophoresis pattern was finally detected using a gel imaging system. The RAPD fingerprint of strain P20 is shown below. Figure 3 As shown.

[0035] 2.4.2 rep-PCR fingerprinting 1) Primer sequence: CTACGGCAAGGCGACGCTGACG.

[0036] 2) Rep-PCR reaction system 0.2 μL of r Taq DNA polymerase; 2 μL of 10×Ex Taq DNA Buffer; 1 μL of primers (10 uM); 2 μL of dNTPs (2.5 mM); 2 μL of DNA template; 12.8 μL of sterile double-distilled water.

[0037] 3) Electrophoresis The DL2000 DNA Marker was used as a result control. Amplification results were detected at 100 V for 80 min. The rep-PCR fingerprint of strain P20 is shown below. Figure 4 As shown.

[0038] 2.5 Salinity tolerance test Under aseptic conditions, 190 μL of MRS 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 P20 was 3%.

[0039] In summary, based on the colony morphology, MALDI-TOF, and molecular biological identification results, strain P20 is a newly discovered Pediococcus pentosaccharis strain, and is named Pediococcus pentosaccharis P20. Pediococcus pentosaceus P20).

[0040] On January 15, 2026, the applicant deposited the aforementioned Pediococcus pentosaceus P20 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCCNO: M2026128.

[0041] Example 3: Antibiotic resistance test of Pediococcus pentosaceus P20 1) Antibiotic preparation Ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, and tetracycline were all prepared as stock solutions of 2048 μg / mL and stored at -20℃ for later use. Before use, the stock solutions were serially diluted 2-fold with BSM liquid medium to prepare the working solutions, with concentrations ranging from 1 to 1024 μg / mL in 11 different gradients.

[0042] 2) Preparation of inoculum Take an appropriate amount of fresh bacterial culture (24h, 37℃ incubation), centrifuge at 5000rpm for 5min, wash once with sterile physiological saline, resuspend the bacterial cells with the same volume of physiological saline, and then dilute 50 times to obtain the inoculum.

[0043] 3) Determination of the minimum inhibitory concentration (MIC) of antibiotics against Pediococcus pentosaceus P20 using the microbroth dilution method. a. Add antibiotic-free MRS liquid medium to the first column of a 96-well plate as a negative control. Add 190 μL of MRS liquid medium containing different concentrations of antibiotics to columns 2 through 12 in sequence. Then, inoculate each column with 10 μL of the above inoculum solution to make 3 parallel wells, and use 1 well without bacterial culture as a blank.

[0044] b. Add 50μL of paraffin oil to cover and prevent moisture evaporation.

[0045] c. After incubating the 96-well plate at 37℃ for 24 hours, remove it and measure the OD. 600 The MIC values ​​of antibiotics against the strains were calculated using the results over 24 hours. The specific results are shown in Table 1.

[0046] Table 1. Antibiotic MIC values ​​of Pediococcus pentosaceus P20

[0047] MIC is measured in μg / mL.

[0048] As can be seen from the results in Table 1, the Pediococcus pentosaceus P20 provided by this invention is sensitive to common antibiotics such as erythromycin, gentamicin, streptomycin, ampicillin, tetracycline and clindamycin, and has good biosafety.

[0049] Example 4: In vitro cholesterol degradation experiment of Pediococcus pentosaceus P20 1. 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.

[0050] 2. 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%.

[0051] Inoculate fresh bacterial culture at a rate of 0.1%, and incubate statically at 37°C for 48 hours. Then, take 0.2 mL of the bacterial culture, 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>.

[0052] The results showed that the cholesterol degradation rate of Pediococcus pentosaceus P20 provided by the present invention reached 65.12% ± 2.03%.

[0053] Example 5: Determination of the antioxidant function of Pediococcus pentosaceus P20 1. Determination of the ability of Pediococcus pentosaceus P20 to scavenge DPPH and HRS free radicals 1) Preparation of PBS bacterial suspension A single colony of *Pediococcus pentosaceus* P20 in excellent growth condition was inoculated into 3 mL of MRS liquid medium and cultured at 37°C for 24 h. Using this culture as the inoculum, 2% of the colony was inoculated into 50 mL of MRS liquid medium and incubated statically for 24 h to obtain the bacterial culture. 1 mL of the bacterial culture was collected, and the cells were washed twice with 1 mL of PBS buffer, then resuspended in 2 mL of PBS solution for later use.

[0054] 2) Determination of the ability of Pediococcus pentosaceus P20 to scavenge DPPH free radicals Take 1 mL of Pediococcus pentosaceus P20 strain PBS 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. Then 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 DPPH·ethanol mixture, and the blank is zeroed using an equal volume of PBS bacterial suspension and ethanol mixture. The clearance rate is calculated using the following formula: Clearance rate % = [1 - (A...] 样品 -A 空白 ) / A 对照 ]×100%.

[0055] Using commercially available Pediococcus pentosaceus strains as controls, the results are shown in Table 2.

[0056] Table 2 DPPH free radical scavenging rate Clearance rate % 21.35%±4.68% 90.46%±3.36% As can be seen from the data in Table 2, the Pediococcus pentosaceus P20 provided by the present invention can effectively scavenge DPPH free radicals, with a scavenging rate of 90.46%±3.36%, which is significantly higher than that of the control strain.

[0057] 3) Determination of the ability of Pediococcus pentosaceus P20 to scavenge hydroxyl radicals (HRS). 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 Pediococcus pentosaceus P20 suspension. Add 100 μL of 3 mM hydrogen peroxide solution. Incubate at 37°C for 15 min, then measure the absorbance of the sample at 510 nm. The hydroxyl radical scavenging rate is calculated using the following formula: Clearance rate = (A 样品 -A 控制 ) / (A 空白 -A 控制 ) × 100%.

[0058] 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.

[0059] The results showed that the scavenging rate of HRS free radicals by the control strain *Pediococcus pentosaceus* was 18.59 ± 2.11%, while the scavenging rate of HRS free radicals by *Pediococcus pentosaceus* P20 provided by this invention was as high as 73.46% ± 1.28%, which was significantly higher than that of the control strain.

[0060] 2. Identification of lipid peroxidation resistance in Pediococcus pentosaceus P20 1) Cultivation and fermentation supernatant and bacterial suspension of *Pediococcus pentosaceus* P20: Lactic acid bacteria were cultured in MRS liquid medium at 37°C for 24 h, passaged 3 times, and centrifuged at 6000 rpm / min, 4°C for 10 min. The supernatant was collected as the fermentation supernatant. The collected bacterial cells were washed 3 times by centrifugation at 6000 rpm / min for 10 min with PBS buffer (pH 7.4). The bacterial cells were resuspended in PBS buffer to adjust the bacterial concentration to 1.0 × 10⁻⁶. 9 The bacterial suspension was obtained by measuring cells / mL.

[0061] 2) Preparation of linoleic acid emulsion: 0.1 mL linoleic acid, 0.2 mL Tween 20, 19.7 mL deionized water.

[0062] 3) 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℃ for 1.5 h, add 0.2 mL of TCA (4%) and 2 mL of TBA (0.8%) to the mixture, incubate at 100℃ for 30 min, cool rapidly, centrifuge at 4000 rpm / min for 15 min, collect the supernatant and measure the absorbance at 532 nm, which is A; the control group is A0, which is 0.5 mL of distilled water instead of the sample.

[0063] Inhibition rate / % = (A0 - A) / A0 × 100%.

[0064] Note: A represents the absorbance of the sample group; A0 represents the absorbance of the control group. Using commercially available Pediococcus pentosaceus as a control, the results are shown in Table 3.

[0065] Table 3. Inhibition rate of anti-lipid peroxidation Control Pediococcus pentosaceus 35.36%±2.01% 25.48%±3.37% Pediococcus pentosaceus P20 85.65%±3.15% 77.69%±1.06% As can be seen from the data in Table 3, the lipid peroxidation inhibition rates of the supernatant and bacterial suspension of Pediococcus pentosus P20 provided by the present invention were 85.65%±3.15% and 77.69%±1.06%, respectively, both significantly higher than those of the control Pediococcus pentosus strain.

[0066] Example 6 Hydrophobicity test of Pediococcus pentosaceus P20 cell surface 1. Preparation of the test bacterial suspension: Purified Pediococcus pentosaceus P20 colonies were picked and inoculated into freshly prepared MRS liquid medium and incubated at 37℃ for 24–48 h. Then, 1% (v / v) of the inoculum was added to MRS liquid medium and incubated at 37℃ for another 24–48 h. The cells were then centrifuged at 6000×g for 10 min, collected, and washed twice with sterile physiological saline. The cells were then resuspended in 1 mL of sterile 0.1 M KNO3 solution to obtain the test bacterial suspension.

[0067] 2. Surface hydrophobicity determination: Add 50 μL of the above bacterial suspension to 2450 μL of 0.1 M KNO3 and record the OD value. 600 For A0, mix 1.5 ml of bacterial suspension with 500 μL of xylene and let stand at room temperature for 10 min (at which point a two-phase system is formed). Vortex the two-phase system for 2 min and then let it stand for 20 min to reform the aqueous and organic phases. Carefully pipette the aqueous phase (avoiding the organic phase) and measure the absorbance A1 at 600 nm. Calculate the cell hydrophobicity using the following formula, and take the average of three measurements.

[0068] Hydrophobicity % = (A0 - A1) / A1 × 100%.

[0069] The results showed that the surface hydrophobicity of Pediococcus pentosaceus P20 cells provided by the present invention was 42.35% ± 2.28%.

[0070] Example 7: Inhibitory effect of Pediococcus pentosaceus P20 on pathogenic Streptococcus bovis The Streptococcus bovis used in this example are Streptococcus bovis ATCC33317 and Streptococcus bovis CICC 21604. They were cultured in brain heart infusion broth medium supplemented with 5% (v / v) fetal bovine serum at 37°C for 16-24 hours. Pediococcus pentosaceus P20 was inoculated into MRS medium at a 1% inoculation rate and incubated at 37°C for 24 hours.

[0071] Using the pouring method and Oxford cups, 3 mL of pathogenic bacterial suspension (1.5 mL of Streptococcus bovis ATCC33317 suspension + 1.5 mL of Streptococcus bovis CICC 21604 suspension) was added to 300 mL of melted and slightly cooled BHI solid medium at a ratio of 1:100.

[0072] Four Oxford cups were evenly placed in a round petri dish. The prepared BHI solid medium was poured in and allowed to solidify. The Oxford cups were then removed, leaving four wells in each dish. One well served as a blank, and 150 μL of Pediococcus pentosaceus P20 bacterial suspension was added to the other three wells. After incubating at 37°C for approximately 24 hours, the antibacterial effect was observed and photographed.

[0073] The results showed that the inhibition zone produced by *Pediococcus pentosaceus* P20 was 18.44 ± 0.28 mm in size. (Image of the inhibition zone is available in the image below.) Figure 6 The results show that Pediococcus pentosaceus P20 has a strong inhibitory effect on Streptococcus bovis.

[0074] Example 8: Experiment on the flocculation effect of Pediococcus pentosaceus P20 on Streptococcus bovis 1. Preparation of bacterial suspension Inoculate *Pediococcus pentosaceus* P20 at a 1% inoculum (volume ratio) onto MRS broth medium and incubate at 37°C for 24 hours. Stop incubation to obtain fresh bacterial suspension. Centrifuge the fresh bacterial suspension at 8000 rpm for 10 minutes and collect the bacterial cells. Wash the bacterial cells twice with pH 7.0 phosphate buffer, then resuspend the bacterial cells in pH 7.0 phosphate buffer until the initial absorbance (OD600) of the bacterial suspension is between 0.5 and 0.6. Set aside for later use.

[0075] 2. Preparation of pathogenic bacteria suspension Inoculate *Streptococcus bovis* ATCC33317 and *Streptococcus bovis* CICC 21604 bacterial suspensions into BHI broth medium at a 1% inoculation rate (v / v). Incubate aerobically at 37°C for 24 h, then stop incubation to obtain fresh bacterial suspension. Centrifuge the fresh bacterial suspension at 8000 rpm for 10 min and collect the bacterial cells. Wash the bacterial cells twice with pH 7.0 phosphate buffer, then resuspend the bacterial cells in pH 7.0 phosphate buffer, adjusting the initial absorbance (OD600) of the suspension to between 0.5 and 0.6, and set aside for later use.

[0076] 3. Coagulation rate determination Take 100 μL of *Pediococcus pentosaceus* P20 bacterial suspension and pathogenic bacteria suspension and measure the initial absorbance (OD600). Then, mix equal volumes of *Pediococcus pentosaceus* P20 bacterial suspension and pathogenic bacteria suspension, shake well, and let stand at room temperature. Perform three replicates for each sample. After 6 hours, take 100 μL of the supernatant suspension and measure the absorbance (OD600). 600 Calculate the agglomeration rate (R) using the following formula. 共 ): R 共 =1-2A / (A0+B0)

[0077] In the formula: R 共The agglutination rate is %, A is the absorbance of the mixed bacterial suspension, A0 is the initial absorbance of the Pediococcus pentosaceus P20 suspension, and B0 is the initial absorbance of the pathogenic bacterial suspension.

[0078] Flocculation effect Figure 6 As shown, Pediococcus pentosaceus P20 can effectively flocculate Streptococcus bovis, with a flocculation rate of 53.32% ± 0.79%. Therefore, Pediococcus pentosaceus P20 can reduce the load of Streptococcus bovis in the colon, and the flocculated bacteria are excreted through feces, thereby reducing the risk of Streptococcus bovis inducing colonic polyps and colon cancer.

[0079] Example 9: Adhesion of Pediococcus pentosaceus P20 to Caco-2 intestinal epithelial cells 1. Cell pre-culture Human intestinal epithelial cells were resuscitated in liquid nitrogen (Caco-2) and cultured to the required quantity. When the cell density reached approximately 80%, they were digested with trypsin into a single-cell suspension and counted using a hemocytometer to obtain a cell count of 5 × 10⁶ cells / mL. 5 Cells / mL. Then, 500 μL of cell suspension was seeded into 24-well plates with cell spreaders at a seeding density of 2.5 × 10⁻⁶ cells / mL. 5 Cells / well, after overnight culture until fully adhered, discard the culture medium, rinse twice with fresh culture medium, and use for later use.

[0080] 2. Preparation of bacterial suspension Fresh Pediococcus pentosaceus P20 culture was washed twice with pH 7.0 phosphate buffer, then resuspended in an equal volume of 1640 culture medium containing 10% fetal bovine serum. The absorbance was adjusted to achieve the desired OD value. 600 It is between 0.4 and 0.5.

[0081] 3. Cell Culture Add 500 μL of bacterial suspension to a prepared 24-well Caco-2 cell plate and co-culture in a carbon dioxide incubator for 2 h; wash three times with pH 7.0 phosphate buffer to remove unadhered bacteria.

[0082] 4. Microscopic examination Cell slides were fixed with methanol for 15 min, stained with Giemsa stain for 5 min, washed thoroughly with pH 7.0 phosphate buffer, and then transferred to a glass slide. Cells were observed and counted under a microscope. Fifty cells were randomly selected, and the number of Pediococcus pentosaceus on the visible cell surface was calculated. The mean and standard deviation of the adhesion index were calculated using statistical methods.

[0083] Adhesion index = number of adhering bacteria / number of cells.

[0084] The results showed that the adhesion index of Pediococcus pentosaceus P20 to Caco-2 cells was 36.35±1.06, indicating that Pediococcus pentosaceus P20 could effectively colonize in the intestine, which was conducive to its flocculation with Streptococcus bovis, thereby exerting its probiotic effect.

[0085] Example 10: The promoting effect of Pediococcus pentosaceus P20 on beneficial intestinal bacteria 1. Preparation of Pediococcus pentosaceus P20 lysate Pediococcus pentosaceus P20 was inoculated into MRS broth medium at an inoculum volume of 1% (v / v) and cultured at 37°C for 24 h. The culture was then stopped to obtain a fresh fermentation broth. The fresh fermentation broth, along with the bacterial cells, was homogenized three times under high pressure to obtain the lysate of Pediococcus pentosaceus P20.

[0086] 2. Preparation of beneficial bacteria culture medium Prepare MRS solid medium by adding 10% (w / w), 20% (w / w), 20% (w / w), and 50% (w / w) of Pediococcus pentosaceus lysis broth to the medium, respectively. Use solid medium without lysis broth as a blank control. Autoclave the medium at 121°C for 15 min and then cool it to 50°C for later use.

[0087] 3. Detection of beneficial bacteria growth One mL of activated Akkermansia myxophilus bacterial suspension was placed in a sterile agar plate, and 15 mL of culture medium was added and mixed well. Three replicates were performed for each culture medium. After the medium cooled and solidified, the culture was anaerobically incubated at 37°C for 48 hours, and colony counting was performed to detect the growth of Akkermansia myxophilus in media containing different concentrations of lysis buffer. The results are shown in Table 4. Table 4 Viable count of Akkermansia myxophilus viable count Log CFU / mL 7.77±0.26 8.01±0.33 8.65±0.27 9.14±0.32 The results showed that *Pediococcus pentosaceus* P20 could promote the growth of *Ackermania viride*. Compared with the control, the viable count of *Ackermania viride* increased by 17.63% after adding 50% *Pediococcus pentosaceus* P20 lysate to MRS medium. This indicates that *Pediococcus pentosaceus* P20 is beneficial to the proliferation of beneficial intestinal bacteria.

[0088] Example 11: Immunomodulatory effect of Pediococcus pentosaceus P20 on intestinal cells Culture of Pediococcus pentosaceus P20 and Streptococcus bovis are shown in Example 9.

[0089] Intestinal epithelial cells Caco-2 were cultured according to Example 10. The resuscitated and passaged Caco-2 intestinal epithelial cells were cultured at a rate of 5 × 10⁶ cells / year. 6Cells were seeded in 6-well plates and, after cell adhesion, divided into three groups: one control group (no bacterial infection), and the other two groups (Streptococcus bovis + P20) with bacteria resuspended in 10% FBS DMEM medium and infected with cells at a MOI of 200. The control group included Streptococcus bovis and the experimental group. Cells were cultured at 37°C for 24 h in a 5% CO2 incubator. After culture, the supernatant was collected after centrifugation at 1000g for 20 min for the detection of cytokines IL-10, IL-6, and IL-1β. Each group was repeated in triplicate. ELISA assays were performed according to the kit instructions.

[0090] The results are as follows Figure 7 As shown, compared with the control group, the levels of pro-inflammatory cytokines IL-1β and IL-6 in the experimental group decreased by 21.29% and 41.01%, respectively, and the differences were statistically significant (*, P<0.05; *, P<0.05); compared with the control group, the level of anti-inflammatory cytokines IL-10 in the experimental group increased by 65.62%, and the difference was also statistically significant (*, P<0.05).

[0091] The above results indicate that Pediococcus pentosaceus P20 can inhibit the Caco-2 inflammatory response in intestinal epithelial cells induced by Streptococcus bovis, downregulate the levels of pro-inflammatory cytokines and upregulate the levels of anti-inflammatory cytokines.

[0092] Example 12 Preparation of Pediococcus pentosaceus P20 probiotic pellets The probiotic pellets provided in this embodiment include an outer shell structure and an inner core structure. The components and their mass fractions in the outer shell structure are as follows: 15 parts gelatin, 2 parts seaweed oligosaccharide, 15 parts carrageenan, 7 parts glycerin, and 60 parts water.

[0093] The core structure contains the following components and their mass fractions: 5 parts of Pediococcus pentosaceus P20 bacterial powder and 95 parts of hydrogenated vegetable oil with a melting point of 45℃.

[0094] Activated *Pediococcus pentosaceus* P20 was inoculated into MRS broth medium and cultured at 37°C for 24 hours to obtain a seed culture. The seed culture was then inoculated into MRS broth medium at a volume ratio of 5%, and cultured at 37°C for another 24 hours. Fermentation was then stopped to obtain a fermentation broth. The fermentation broth was centrifuged at 3000 rpm for 20 minutes, and the bacterial sludge was collected. A freeze-drying protectant was added to the bacterial sludge at a mass ratio of 15%, mixed thoroughly, and then freeze-dried to obtain the bacterial powder. The viable count in the bacterial powder was determined according to the national standard GB4789.35-2016, "Microbiological Examination of Food - Lactic Acid Bacteria Examination".

[0095] The results showed that the viable count of Pediococcus pentosaceus P20 powder prepared by this invention reached 2.55E+11 CFU / g.

[0096] The outer shell solution and inner core solution are prepared separately. The inner core solution is prepared as follows: hydrogenated vegetable oil is dissolved at 45°C, bacterial powder is added, and the mixture is stirred at 300 rpm for 10 minutes to ensure the bacterial powder is evenly dispersed in the hydrogenated vegetable oil. This mixture is then poured into the inner core solution tank of the capsule making machine. The stirring speed of the inner core tank is set to 8 rpm, and the temperature is set to 45°C. The outer shell solution is prepared as follows: gelatin and carrageenan are dissolved at 75°C, then glycerol and seaweed oligosaccharides are added, and the mixture is dissolved again at 75°C. The mixture is then stirred evenly and poured into the outer shell solution tank of the capsule making machine. The temperature of the outer shell tank is set to 75°C. The outer shell solution tank and the inner core solution tank of the capsule making machine have concentric nozzles. Then, the pump speed was controlled at 600 mL / h and the pressure in the inner core tank was 11 kPa, so that the outer shell solution completely enveloped the inner core solution during the titration process. The solution was then dropped into a tank containing a liquid paraffin solution at a temperature controlled at 10°C, causing it to gel and form continuous core-shell spheres. The resulting spheres were then dried in an ventilated environment at room temperature (25°C) until the moisture content was below 1.5%, yielding probiotic spheres with a diameter of 1.2 mm.

[0097] Example 13: Resistance test of Pediococcus pentosaceus P20 probiotic pellets to artificial gastric juice 1. Preparation of artificial gastric juice 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 121℃ for 15min. Before use, add 3.2g of porcine mucosal pepsin, shake well to dissolve, and incubate in a 37℃ water bath for 1h to simulate human body temperature. 2. Probiotic pellet artificial gastric juice tolerance test 2.1 Determination of the amount of crystalloid bacteria 38 mL of physiological saline was kept in a 45°C water bath for 7 min. 2 g of probiotic crystals from Example 12 were added and incubated for 5 min. The mixture was then homogenized at 45°C for 5 min. The amount of crystals was then determined according to GB4789.35-2016 "Food Microbiology Examination - Lactic Acid Bacteria Examination".

[0098] 2.2 Artificial gastric juice digestion test Weigh 2g of the probiotic pellets from Example 12 and add them to 8mL of artificial gastric fluid that has been kept at a constant temperature (37°C for 10 min). Shake well by hand and then place in a 37°C water bath for 2 hours to digest. Filter the artificial gastric fluid away with sterile filter paper, then place the filtered pellets in a sterile container and soak in 18mL of physiological saline for 10 min. Filter again with sterile filter paper and rinse twice with sterile physiological saline. Add physiological saline to make the total weight of the pellets and saline 40g. Then determine the bacterial count according to the method for determining the bacterial count of the pellets. The survival rate of the probiotic pellets after digestion in the artificial gastric fluid is obtained by comparing the data before and after digestion.

[0099] 2.3 Tolerance test of bacterial powder to artificial gastric juice 0.04 g of Pediococcus pentosaceus P20 bacterial powder was added to 9.96 mL of artificial gastric juice that had been kept at a constant temperature (37℃ for 10 min), shaken manually to mix, and then placed in a 37℃ water bath for 2 h for digestion. The number of viable bacteria before and after digestion in the artificial gastric juice was measured. The survival rate of the bacterial powder after digestion in the artificial gastric juice was obtained by comparing the data before and after digestion.

[0100] Table 5. Survival rate of probiotic pellets after digestion with artificial gastric juice. Probiotic pellets 95.23%±2.46% Pediococcus pentosaceus P20 powder 10.92%±3.17% The data in Table 5 show that after the probiotic crystals prepared in this invention are digested with artificial gastric juice at pH 3.0 for 2 hours, the survival rate of probiotics is still as high as 95%, which can effectively protect the probiotics from digestion by artificial gastric juice and ensure that they reach the intestine smoothly.

[0101] Example 14: Disintegration test of Pediococcus pentosaceus P20 probiotic pellets 1. Preparation of artificial gastric juice The preparation of artificial gastric fluid is shown in Example 13.

[0102] 2. Preparation of artificial intestinal fluid Weigh out 5 g of peptone, 2.5 g of yeast extract, 1 g of glucose, 6.8 g of KH₂PO₄, and 3.0 g of ox bile salts, respectively. Add them to 77 mL of 0.2 mol / L NaOH solution, and bring the volume to 1000 mL. Adjust the pH to 6.8 ± 0.1 with dilute hydrochloric acid or sodium hydroxide solution, and sterilize at 115℃ for 20 min. Before use, add 1 g of trypsin, shake well to dissolve, and incubate in a 37℃ water bath for 1 h to simulate human body temperature.

[0103] 3. Disintegration test Following the method and apparatus described in the "Disintegration Time Test" of the Chinese Pharmacopoeia, six probiotic crystal balls from Example 12 were taken and tested in artificial gastric fluid without baffles for 2 hours. No disintegration or cracks were found in each crystal ball. The basket was then removed, washed with a small amount of physiological water, and baffles were added to each tube. The test was then conducted in artificial intestinal fluid using the same method as described above, and the time for complete dissolution of the crystal balls was recorded.

[0104] The results showed that the probiotic pellets disintegrated in 7 minutes. Therefore, the probiotic pellets prepared in Example 12 can disintegrate rapidly in the intestine.

[0105] Example 15 Accelerated stability test of Pediococcus pentosaceus P20 probiotic pellets The probiotic crystals prepared in Example 12 were sealed and placed in an incubator at 37°C. At the same time, Pediococcus pentosus P20 bacterial powder was taken as a control. The bacterial count was measured every 5 days, and the test was conducted continuously for 30 days.

[0106] Table 6. Stability test of bacterial count at 37℃ (Log CFU / g) Probiotic pellets 11.28 11.26 11.25 11.25 11.26 11.24 11.22 Pediococcus pentosaceus P20 powder 11.48 11.13 10.54 10.00 9.62 9.12 8.26 As shown in Table 6, after 30 days of accelerated stability testing, the viable count of the probiotic pellets remained stable with a survival rate greater than 95%, while the viable count of the Pediococcus pentosaceus P20 powder decreased by 3.22 Log CFU. This demonstrates that the probiotic pellets provided by this invention can effectively improve the stability of Pediococcus pentosaceus P20 within them.

Claims

1. A strain of Pediococcus pentosaceus ( Pediococcus pentosaceus ), characterized in that, The preservation number of the *Pediococcus pentosaceus* is CCTCC NO: M2026128.

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

1.

3. The use of Pediococcus pentosaceus as described in claim 1 in the preparation of products with antioxidant functions.

4. The use of Pediococcus pentosaceus as described in claim 1 in the preparation of Streptococcus bovis inhibitor.

5. The use of Pediococcus pentosaceus as described in claim 1 in the preparation of a medicine for the prevention or treatment of colonic polyps and colon cancer.

6. A probiotic pellet, characterized in that, The probiotic pellets comprise Pediococcus pentosaceus as described in claim 1.

7. The probiotic pellets as described in claim 6, characterized in that, The probiotic pellets consist of an outer shell structure and an inner core structure, wherein the inner core structure contains Pediococcus pentosaceus.

8. The probiotic pellets as described in claim 7, characterized in that, The components and their mass fractions of the outer shell structure are as follows: 15 parts gelatin, 2 parts seaweed oligosaccharide, 15 parts carrageenan, 7 parts glycerin, and 60 parts water.

9. The probiotic pellets as described in claim 7 or 8, characterized in that, The components and their mass fractions of the inner core structure are as follows: 5 parts of Pediococcus pentosaceus powder and 95 parts of hydrogenated vegetable oil with a melting point of 45°C.

10. The method for preparing the probiotic pellets according to claim 9, characterized in that, The preparation method includes the following steps: (1) Preparation of the core solution: Dissolve hydrogenated vegetable oil at 45°C, add Pediococcus pentosaceus powder, and stir with a stirrer at 300 rpm for 10 min to mix and disperse the powder evenly in the hydrogenated vegetable oil. Then pour it into the inner core solution storage tank of the capsule making machine. Set the stirring speed of the inner core storage tank to 8 rpm and the temperature to 45°C. (2) Preparation of the outer shell solution: Gelatin and carrageenan are dissolved at 75°C, then glycerol and seaweed oligosaccharide are added and dissolved at 75°C. Then the mixture is stirred evenly and poured into the outer shell solution storage tank of the capsule making machine. The temperature of the outer shell storage tank is set at 75°C. The outer shell solution storage tank and the inner core solution storage tank of the capsule making machine are concentric nozzles. (3) Preparation of probiotic pellets: The pump speed was controlled at 600 mL / h and the pressure in the inner core tank was 11 kPa. During the titration process, the solution in the outer shell completely enveloped the solution in the inner core tank. The solution was then dropped into a tank containing a liquid paraffin solution at a temperature controlled at 10°C, which caused it to gel and form continuous core-shell spheres. The resulting spheres were then dried in an air-conditioned environment at room temperature (25°C) until the moisture content was below 1.5%, thus obtaining probiotic spheres.

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