Lactobacillus rhamnosus and application thereof in prevention and treatment of colon polyps and colon cancer

Abstract

This invention belongs to the field of probiotic screening and application technology, specifically relating to a strain of *Lactobacillus rhamnosus* and its application in the prevention and treatment of colon polyps and colon cancer. The *Lactobacillus rhamnosus* is named *Lactobacillus rhamnosus* (…). Lacticaseibacillus rhamnosus S28, deposited on January 15, 2026, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2026129, exhibits strong inhibitory activity against *Streptococcus bovis* and demonstrates a strong agglutination effect with it. It can be used to prevent or treat colonic polyps and colon cancer caused by *Streptococcus bovis*. This invention also provides probiotic pellets containing *Lactobacillus rhamnosus* S28, which exhibit strong resistance, effectively protecting the probiotics from gastric digestion, ensuring their smooth arrival in the intestines, and achieving rapid disintegration within the intestines, showing broad market prospects.

Description

Technical Field

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

[0002] Colorectal cancer (CRC) is a malignant tumor with a high incidence and mortality rate worldwide. Colonic polyps are the most common precancerous lesions of colorectal cancer, and the potential risk of adenomatous polyps developing into colorectal cancer has been clearly established. Studies show that approximately 80%-95% of colorectal cancers originate from adenomatous polyps, and the histological type, size, and degree of dysplasia of the polyp are key factors influencing the risk of malignant transformation. Therefore, early prevention and intervention of colonic polyps is a core strategy for reducing the incidence of colorectal cancer.

[0003] The gut microbiota, as a core component of the human gut micro-ecosystem, is closely related to the occurrence and development of colonic polyps and colorectal cancer when its homeostasis is imbalanced. Under normal circumstances, the gut microbiota maintains the balance of the colonic microecology by participating in processes such as nutrient metabolism (e.g., fermentation of dietary fiber to produce short-chain fatty acids), vitamin synthesis, maintaining the integrity of the intestinal barrier, and regulating immune function. When the gut microbiota is imbalanced, the abundance of harmful bacteria such as Fusobacterium nucleatum, enterotoxigenic Escherichia coli, and Bacteroides fragilis increases, while the number of beneficial bacteria such as butyric acid-producing bacteria, Bifidobacteria, and Lactobacillus decreases, thereby promoting disease progression through multiple mechanisms. At the same time, dysbiosis also damages the intestinal barrier function, increases intestinal permeability, promotes the invasion of harmful substances into the submucosa, exacerbates inflammation and tissue damage, and forms a vicious cycle of "inflammation-damage-proliferation," driving polyp formation and malignant transformation.

[0004] Probiotics have a multi-dimensional mechanism of action in preventing colonic polyps and colorectal cancer, providing an effective approach for disease intervention: First, probiotics can reshape the balance of gut microbiota by competitively inhibiting the colonization and proliferation of harmful bacteria (including Streptococcus bovis and pathogenic Escherichia coli), reducing the production of harmful metabolites, and restoring gut microecological homeostasis. Second, the short-chain fatty acids (SCFAs) produced by probiotic fermentation of dietary fiber, especially butyrate, can serve as an energy source for colonic epithelial cells, while inhibiting histone deacetylase activity, inducing cancer cell apoptosis, and regulating intestinal pH to create an environment unfavorable to the survival of harmful bacteria. Third, probiotics can enhance intestinal barrier function and promote intestinal... The expression of tight junction proteins in intestinal epithelial cells reduces intestinal permeability, prevents harmful substances from invading the submucosa, and reduces the risk of inflammatory response; fourth, probiotics can regulate the host immune system, promote the secretion of anti-inflammatory cytokines (such as IL-10 and TGF-β), inhibit the activation of pro-inflammatory pathways, and enhance the body's immune surveillance and clearance of abnormal cells; fifth, some probiotic strains can directly inhibit the proliferation of polyp cells and block the progression of precancerous lesions by regulating signaling pathways such as Wnt / β-catenin and MAPK; sixth, probiotics can reduce the accumulation of harmful substances in the intestine, reduce the risk of damage to the colonic mucosa by degrading potential carcinogens and promoting their excretion.

[0005] In summary, the progression of colonic polyps to colorectal cancer is closely related to gut microbiota dysbiosis and the pathogenic effects of Streptococcus bovis, and probiotics can effectively intervene in this process through multiple mechanisms. Currently, there are no reports on reducing the risk of colonic polyps and colorectal cancer by using Lactobacillus rhamnosus to inhibit hemolytic streptococci. Therefore, developing targeted prevention technologies based on Lactobacillus rhamnosus has significant clinical value and application prospects for reducing the risk of colorectal cancer and alleviating the global cancer burden. Summary of the Invention

[0006] The purpose of this invention is to provide a novel strain of Lactobacillus rhamnosus (Lactobacillus casei). Lacticaseibacillus rhamnosus 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] This invention relates, in one aspect, to a type of Lactobacillus rhamnosus, named Lactobacillus rhamnosus (… Lacticaseibacillus rhamnosus Strain S28 was deposited on January 15, 2026, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M2026129.

[0008] The 16S rDNA sequence of Lactobacillus rhamnosus strain S28 provided by this invention is SEQ ID NO: 1.

[0009] This invention also relates to the application of Lactobacillus rhamnosus strain S28 in the preparation of Streptococcus bovis inhibitors.

[0010] The present invention also relates to the use of Lactobacillus rhamnosus strain S28 in the preparation of medicaments for the prevention or treatment of colon polyps or colon cancer.

[0011] The present invention also relates to a probiotic sphere containing Lactobacillus rhamnosus strain S28, comprising a shell and an inner core.

[0012] The components of the outer shell and their mass fractions are as follows: 5 parts gelatin, 5 parts seaweed oligosaccharide, 5 parts carrageenan, 7 parts glycerin, and 80 parts water.

[0013] The components and their mass fractions of the inner core are as follows: 10 parts of Lactobacillus rhamnosus S28 bacterial powder and 90 parts of hydrogenated vegetable oil with a melting point of 45℃.

[0014] This invention also relates to the use of the probiotic pellets in the prevention or treatment of colon polyps or colon cancer.

[0015] The *Lactobacillus rhamnosus* S28 provided by this invention has a strong inhibitory effect on *Streptococcus bovis*. The Oxford cup experiment confirmed that the size of the inhibition zone was 15.42 ± 0.10 mm, and the flocculation experiment showed that *Lactobacillus rhamnosus* S28 could produce a strong aggregation effect with *Streptococcus bovis*, with an aggregation rate of 53.22% ± 1.78%, which is beneficial to inhibiting the colonization of *Streptococcus bovis* in the intestine and reducing the risk of *Streptococcus bovis*-induced colon polyps and colon cancer.

[0016] Lactobacillus rhamnosus S28 is sensitive to common antibiotics and has good biocompatibility. It possesses strong antioxidant capabilities, with a DPPH radical scavenging rate of 72.13% and a HRS radical scavenging rate of 90.30%±4.44%. The supernatant and bacterial suspension showed significant inhibition rates of 67.42% and 55.31% against lipid peroxidation, respectively. It also exhibits a cholesterol degradation rate of 72.19%±5.28%. The surface hydrophobicity of Lactobacillus rhamnosus S28 cells is 90.36%±3.90%. It has an adhesion index of 68.39±2.76 against Caco-2 cells, effectively promoting the proliferation of Akkermansia muciniphila.

[0017] The probiotic pellets provided by this invention exhibit strong resistance, effectively protecting the probiotics from gastric digestion and ensuring their successful arrival in the intestines. The *Lactobacillus rhamnosus* S28 within the probiotic pellets maintains a survival rate of over 95% during its shelf life. Furthermore, the probiotic pellets disintegrate in 6 minutes, enabling rapid disintegration in the intestines.

[0018] In summary, the Lactobacillus rhamnosus S28 provided by this invention has no toxic effects on the body, good safety, and can be used to prevent colonic polyps and colon cancer caused by Streptococcus bovis, showing broad application prospects. Attached Figure Description

[0019] Figure 1 This is a colony diagram of Lactobacillus rhamnosus S28; Figure 2 This is a microscopic image of Lactobacillus rhamnosus S28 stained with crystal violet. Figure 3 The RAPD fingerprint of Lactobacillus rhamnosus S28; Figure 4 The rep-PCR fingerprint of Lactobacillus rhamnosus S28; Figure 5 The size of the inhibition zone of Lactobacillus rhamnosus S28 against Streptococcus bovis; Figure 6 The graph shows the flocculation effect of Lactobacillus rhamnosus S28 on Streptococcus bovis. Column A represents the flocculation effect of co-culturing strain S28 with Streptococcus bovis, column B represents the flocculation effect of culturing Streptococcus bovis alone, and column C represents the flocculation effect of strain S28 alone. Figure 7 The diagram shows the regulation of cytokine-induced inflammatory response by strain S28 of Streptococcus bovis, where (A) represents the pro-inflammatory cytokine TNF-α, (B) represents the anti-inflammatory cytokine IL-10, and (C) represents the pro-inflammatory cytokine p65. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments. For the specific methods or materials used in the embodiments, those skilled in the art can make conventional substitutions based on the technical concept of the present invention and existing technologies, and are not limited to the specific descriptions in the embodiments of the present invention.

[0021] Example 1: Isolation and Screening of Strains Sample source Harvested from traditional fermented sauerkraut in Baiyin City, Gansu Province.

[0022] Separation and Screening Initial screening Take 1g of traditional fermented sauerkraut sample, dilute it with sterile physiological saline, place it in a sterile sample bag, and 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. After single colonies grow on the plate, examine them under a microscope.

[0023] Microscopic examination revealed that the applicant identified 30 potential lactobacilli strains from the fermented sauerkraut samples, which were named S01, S02, ..., S28, S29, and S30, respectively.

[0024] Secondary screening Sterilize and cool 1L of MRS liquid culture medium, add 3.2g of porcine mucosal pepsin, shake well to dissolve, and place in a 37℃ water bath shaker for 1 hour to prepare an acid-resistant culture medium. Inoculate the 30 strains of lactobacillus obtained from the initial screening into the above acid-resistant culture medium at an inoculation rate of 6%, and anaerobically incubate at 37℃ for 48 hours. Collect the fermentation broth for bacterial count.

[0025] The results showed that strain S28 had the highest acid resistance among the 30 lactobacillus strains.

[0026] Example 2 Identification of the strain 2.1 Colony and cell morphology and microscopic features Strain S28 was inoculated onto MRS agar medium and anaerobically cultured at 37°C for 24 hours. Single colonies of strain S28 were observed as follows: Figure 1 As shown, the colonies are white, smooth, raised, opaque, and round, with a diameter of approximately 1.0-2.5 mm. Further observation of the S28 cells under a microscope yielded the following results: Figure 2 As shown, the bacteria are short rod-shaped, Gram-positive, with blunt ends, and arranged in short chains.

[0027] 2.2 Molecular identification of 16S rDNA Genomic DNA was extracted according to the procedure outlined in the Tiangen Bacterial Genomic DNA Extraction Kit (catalog number: DP302). The primer sequences used for 16S rDNA gene amplification are as follows: 27F: AGAGTTTGATCCTGGCTCA; 1492R: GGTTACCTTGTTACGACTT.

[0028] The 16S rDNA sequence of strain S28 was obtained by sequencing and is SEQ ID NO:1:

[0029] The sequence was compared with the NCBI database, and the S28 strain was preliminarily identified as Lactobacillus rhamnosus.

[0030] 2.3 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 S28 was 2%.

[0031] 2.4 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 S28 was identified as *Lactobacillus rhamnosus*.

[0032] 2.5 Identification using RAPD and rep-PCR fingerprinting 2.5.1 RAPD fingerprint identification 1) Primer sequence GAGGGTGGCGGTTCT; 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).

[0033] 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 S28 is shown below. Figure 3 As shown.

[0034] 2.5.2 rep-PCR fingerprinting 1) Primer sequence CTACGGCAAGGCGACGCTGACG.

[0035] 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.

[0036] 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 S28 is shown below. Figure 4 As shown.

[0037] In summary, based on the colony morphology, MALDI-TOF, and molecular biological identification results, strain S28 is a newly discovered *Lactobacillus rhamnosus* strain, and is named *Lactobacillus rhamnosus* S28. Lacticaseibacillus rhamnosus S28).

[0038] On January 15, 2026, the applicant deposited the aforementioned *Lactobacillus rhamnosus* S28 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCCNO: M2026129.

[0039] Example 3: Inhibitory effect of Lactobacillus rhamnosus S28 on pathogenic Streptococcus bovis. The Streptococcus bovis used in this example are Streptococcus bovis ATCC 33317 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. Lactobacillus rhamnosus S28 was inoculated into MRS medium at a rate of 1% and incubated at 37°C for 24 hours.

[0040] Using the pouring method and Oxford cups, 3 mL of pathogenic bacterial suspension (1.5 mL of Streptococcus bovis ATCC 33317 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.

[0041] 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 Lactobacillus rhamnosus S28 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.

[0042] The results showed that the inhibition zone produced by *Lactobacillus rhamnosus* S28 was 15.42 ± 0.10 mm in size. (Image of the inhibition zone is available in the image below.) Figure 5 The results show that *Lactobacillus rhamnosus* S28 has a strong inhibitory effect on *Streptococcus bovis*.

[0043] Example 4: Experiment on the flocculation effect of Lactobacillus rhamnosus S28 on Streptococcus bovis 1. Preparation of bacterial suspension Lactobacillus rhamnosus S28 was inoculated into MRS broth medium at an inoculum volume of 1% (v / v). The medium was incubated at 37°C for 24 hours, after which the culture was stopped to obtain a fresh bacterial suspension. The fresh bacterial suspension was centrifuged at 8000 rpm for 10 minutes to collect the bacterial cells. The bacterial cells were washed twice with pH 7.0 phosphate buffer, and then resuspended in pH 7.0 phosphate buffer until the initial absorbance OD of the bacterial suspension was reached. 600 It should be between 0.5 and 0.6, and kept in reserve.

[0044] 2. Preparation of pathogenic bacteria suspension At a 1% inoculum (volume ratio), *Streptococcus bovis* ATCC 33317 and *Streptococcus bovis* CICC 21604 bacterial suspensions were inoculated into BHI broth medium and cultured aerobicly at 37°C for 24 h. The culture was then stopped to obtain fresh bacterial suspensions. The fresh bacterial suspensions were centrifuged at 8000 rpm for 10 min to collect the bacterial cells. The bacterial cells were washed twice with pH 7.0 phosphate buffer, and then resuspended in pH 7.0 phosphate buffer to adjust the initial absorbance (OD) of the suspension. 600 It should be between 0.5 and 0.6, and kept in reserve.

[0045] 3. Coagulation rate determination Take 100 μL of Lactobacillus rhamnosus S28 bacterial suspension and pathogenic bacterial suspension to determine the initial absorbance (OD). 600Then, equal volumes of *Lactobacillus rhamnosus* S28 suspension and pathogenic bacteria suspension were mixed, shaken well, and allowed to stand at room temperature. Each sample was tested in triplicate. After 6 hours, 100 μL of the supernatant suspension was taken to measure the absorbance (OD). 600 Calculate the agglomeration rate (R) using the following formula. 共 ): R 共 =1-2A / (A0+B0)

[0046] 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 Lactobacillus rhamnosus S28 bacterial suspension, and B0 is the initial absorbance of the pathogenic bacterial suspension.

[0047] Flocculation effect Figure 6 As shown, *Lactobacillus rhamnosus* S28 can effectively flocculate *Streptococcus bovis*, with a flocculation rate of 53.22% ± 1.78%. Therefore, *Lactobacillus rhamnosus* S28 can reduce the *Streptococcus bovis* load in the colon, and the flocculated bacteria are excreted through feces, thereby reducing the risk of *Streptococcus bovis* inducing colonic polyps and colon cancer.

[0048] Example 5: Adhesion of Lactobacillus rhamnosus S28 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.

[0049] 2. Preparation of bacterial suspension Fresh Lactobacillus rhamnosus S28 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. 600 It is between 0.4 and 0.5.

[0050] 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.

[0051] 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 visible *Lactobacillus rhamnosus* on the cell surface was counted. The mean and standard deviation of the adhesion index were calculated using statistical methods.

[0052] Adhesion index = number of adhering bacteria / number of cells.

[0053] The results showed that the adhesion index of Lactobacillus rhamnosus S28 to Caco-2 cells was 68.39±2.76, indicating that Lactobacillus rhamnosus S28 can effectively colonize in the intestine, which is conducive to its flocculation with Streptococcus bovis and thus exert its probiotic effect.

[0054] Example 6: The promoting effect of Lactobacillus rhamnosus S28 on the proliferation of beneficial intestinal bacteria 1. Preparation of Lactobacillus rhamnosus S28 lysate Lactobacillus rhamnosus S28 was inoculated into MRS broth medium at an inoculum volume of 1% and cultured at 37°C for 24 hours. 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 Lactobacillus rhamnosus S28 lysate.

[0055] 2. Preparation of beneficial bacteria culture medium Prepare MRS solid culture medium by adding 10% (w / w), 20% (w / w), and 50% (w / w) Lactobacillus rhamnosus S28 lysis buffer to the medium, respectively. Use solid culture medium without lysis buffer as a blank control. Autoclave the medium at 121°C for 15 min and then cool it to 50°C for later use.

[0056] 3. Detection of beneficial bacteria growth Take 1 mL of activated Akkermansia myxophilus bacterial suspension and place it in a sterile agar plate. Pour in 15 mL of culture medium and mix well. Perform three replicates for each culture medium. After the culture medium cools and solidifies, anaerobically incubate at 37°C for 48 hours and then count the colonies to detect the growth of Akkermansia myxophilus in media containing different concentrations of lysis buffer. The results are shown in Table 1.

[0057] Table 1 Viable count of Akkermansia myxophilus culture medium blank 10% 20% 50% viable count Log CFU / mL 7.48±0.35 7.92±0.43 8.35±0.21 8.99±0.28 The results showed that *Lactobacillus rhamnosus* S28 could promote the growth of *Ackermania mutans*. Compared with the control, the viable count of *Ackermania mutans* increased by 20.19% after adding 50% *Lactobacillus rhamnosus* S28 lysate to the MRS. This indicates that *Lactobacillus rhamnosus* S28 can effectively promote the proliferation of beneficial intestinal bacteria.

[0058] Example 7: Immunomodulatory effects of Lactobacillus rhamnosus S28 on intestinal cells Reference Example 5: Culture of Lactobacillus rhamnosus S28 and Streptococcus bovis.

[0059] Intestinal epithelial cells Caco-2 were cultured according to Example 6. The resuscitated and passaged Caco-2 intestinal epithelial cells were cultured at a rate of 5 × 10⁶ cells / year. 6 Cells 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 + S28) 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 hours in a 5% CO2 incubator. After culture, the supernatant was collected after centrifugation at 1000g for 20 minutes for the detection of cytokines IL-10, p65, and TNF-α. Each group was repeated in triplicate. ELISA assays were performed according to the kit instructions.

[0060] The results are as follows Figure 7 As shown, compared with the control group, the levels of pro-inflammatory cytokines TNF-α and p65 in the experimental group decreased by 23.78% and 16.68%, respectively, and the differences were statistically significant (**, P<0.01; *, P<0.05); compared with the control group, the level of anti-inflammatory cytokine IL-10 in the experimental group increased by 74.73%, and the difference was also statistically significant (*, P<0.05). These results indicate that *Lactobacillus rhamnosus* S28 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.

[0061] Example 8 Antibiotic resistance test of Lactobacillus rhamnosus S28 1. Preparation of antibiotics 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.

[0062] 2. Preparation of inoculum Take an appropriate amount of fresh bacterial culture (cultured at 37℃ for 24 hours), centrifuge at 5000 rpm for 5 minutes, 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.

[0063] 3. Determination of the minimum inhibitory concentration (MIC) of antibiotics against *Lactobacillus rhamnosus* S28 using the micro-broth dilution method. 1) Add antibiotic-free MRS liquid medium to the first column of the 96-well plate as a negative control. Add 190 μL of MRS liquid medium containing different concentrations of antibiotics to columns 2 to 12 in sequence. Then, inoculate 10 μL of the above inoculum into each well, make 3 parallel wells, and use 1 well without bacterial culture as a blank.

[0064] 2) Add 50μL of paraffin oil to cover and prevent moisture evaporation.

[0065] 3) 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 2.

[0066] Table 2. Antibiotic MIC values ​​of Lactobacillus rhamnosus S28

[0067] MIC is measured in μg / mL.

[0068] As can be seen from the results in Table 2, the Lactobacillus rhamnosus S28 provided by this invention is sensitive to common antibiotics such as erythromycin, gentamicin, streptomycin, ampicillin, tetracycline and clindamycin, and has good biosafety.

[0069] Example 9: Determination of the antioxidant function of Lactobacillus rhamnosus S28 1. Determination of the strain's ability to scavenge DPPH and HRS free radicals 1) Preparation of PBS bacterial suspension A single colony of *Lactobacillus rhamnosus* S28 with excellent growth 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.

[0070] 2) Determination of the strain's ability to scavenge DPPH free radicals Take 1 mL of *Lactobacillus rhamnosus* S28 PBS 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 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%.

[0071] Using commercially available Lactobacillus rhamnosus strains as a control, the results are shown in Table 3.

[0072] Table 3 DPPH free radical scavenging rate strain Control Lactobacillus rhamnosus Lactobacillus rhamnosus S28 Clearance rate % 15.38% 72.13% Standard deviation 2.76% 4.22% As can be seen from the data in Table 3, the Lactobacillus rhamnosus S28 provided by the present invention can effectively scavenge DPPH free radicals, with a scavenging rate of 72.13%, which is significantly higher than that of the control Lactobacillus rhamnosus strain.

[0073] 3) Determination of the strain's ability 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 *Lactobacillus rhamnosus* S28 suspension. Add 100 μL of 3 mM hydrogen peroxide solution and incubate at 37°C for 15 min. Measure the absorbance of the sample at 510 nm. Use commercially available *Lactobacillus rhamnosus* as a control.

[0074] 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%.

[0075] 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.

[0076] The results showed that the *Lactobacillus rhamnosus* S28 provided by this invention had a scavenging rate of 90.30% ± 4.44% against HRS free radicals, while the scavenging rate of the control *Lactobacillus rhamnosus* was only 23.12 ± 3.43%. The scavenging effect of *Lactobacillus rhamnosus* S28 against HRS free radicals was significantly better than that of the control strain.

[0077] 2. Identification of the strain's resistance to lipid peroxidation 1) Culture and fermentation supernatant and bacterial suspension of lactic acid bacteria Lactic acid bacteria were cultured in MRS liquid medium at 37°C for 24 h. After three passages, the culture was centrifuged at 6000 rpm / min at 4°C for 10 min, and the supernatant was collected as the fermentation supernatant. The collected bacterial cells were washed three times by centrifugation at 6000 rpm / min 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 cells / min. 9 The bacterial suspension was obtained by measuring cells / mL.

[0078] 2) Preparation of linoleic acid emulsion 0.1 mL linoleic acid, 0.2 mL Tween 20, 19.7 mL deionized water.

[0079] 3) Anti-lipid peroxidation inhibition rate 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.

[0080] Inhibition rate / % = (A0 - A) / A0 × 100% Note: A represents the absorbance of the sample group; A0 represents the absorbance of the control group. Commercially available *Lactobacillus rhamnosus* was used as a control; the results are shown in Table 4.

[0081] Table 4. Inhibition rate of anti-lipid peroxidation

[0082] As can be seen from Table 4, the supernatant of Lactobacillus rhamnosus S28 provided by the present invention has an anti-lipid peroxidation inhibition rate of 67.42%, which is higher than that of the control Lactobacillus rhamnosus strain; while the bacterial suspension has an anti-lipid peroxidation inhibition rate of 55.31%, which is also higher than that of the control Lactobacillus rhamnosus strain.

[0083] Example 10: In vitro cholesterol degradation experiment of Lactobacillus rhamnosus S28 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.

[0084] 2. Cholesterol degradation rate Weigh out 10.0 g peptone, 10.0 g beef extract, 5.0 g yeast extract, 2.0 g diammonium citrate, 20.0 g glucose, 1.0 mL Tween 80, 5.0 g sodium acetate, 0.1 g magnesium sulfate, 0.05 g manganese sulfate, 2.0 g dipotassium hydrogen phosphate, 1 g bile salts, and 1000 mL 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%.

[0085] 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 is in accordance with GB / T 5009.128-2003 "Determination of Cholesterol in Food".

[0086] The results showed that the cholesterol degradation rate of Lactobacillus rhamnosus S28 provided by the present invention reached 72.19% ± 5.28%.

[0087] Example 11 Hydrophobicity test of Lactobacillus rhamnosus S28 cell surface Preparation of test bacterial solutions Purified *Lactobacillus rhamnosus* S28 colonies were picked and inoculated into freshly prepared MRS liquid medium and cultured at 37°C for 24–48 h. Then, 1% (v / v) of the inoculum was added to MRS liquid medium and cultured at 37°C 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 solution.

[0088] Surface hydrophobicity measurement Add 50 μL of the above bacterial suspension to 2450 μL of 0.1 M KNO3 and record the OD. 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. Take the average of three measurements. Calculate the cell hydrophobicity using the following formula: Hydrophobicity % = (A0 - A1) / A1 × 100%.

[0089] The results showed that the surface hydrophobicity of Lactobacillus rhamnosus S28 cells provided by the present invention was 90.36% ± 3.90%.

[0090] Example 12 Preparation of Lactobacillus rhamnosus S28 probiotic pellets The probiotic pellets provided in this embodiment consist of an outer shell and an inner core. The components and their mass fractions of the outer shell are as follows: 5 parts gelatin, 5 parts seaweed oligosaccharide, 5 parts carrageenan, 7 parts glycerin, and 80 parts water. The components and their mass fractions of the inner core are as follows: 10 parts Lactobacillus rhamnosus S28 bacterial powder and 90 parts hydrogenated vegetable oil with a melting point of 45°C.

[0091] Activated *Lactobacillus rhamnosus* S28 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 GB 4789.35-2016, "Microbiological Examination of Food - Examination of Lactic Acid Bacteria".

[0092] The results showed that the viable count of Lactobacillus rhamnosus S28 bacterial powder prepared by this invention reached 3.0E+11 CFU / g.

[0093] The outer shell solution and the inner core solution were prepared separately. The preparation method of the inner core solution was as follows: hydrogenated vegetable oil was dissolved at 45°C, bacterial powder was added, and the mixture was stirred at 280 rpm for 10 min to make the bacterial powder evenly dispersed in the hydrogenated vegetable oil. Then, the mixture was poured into the inner core solution storage tank of the capsule making machine. The stirring speed of the inner core storage tank was set to 10 rpm and the temperature was set to 45°C.

[0094] Preparation of the outer shell solution: 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 solution is then stirred until homogeneous and poured into the outer shell solution tank of the capsule making machine, with the tank temperature set at 75°C. The outer shell solution tank and the core solution tank of the capsule making machine have concentric nozzles. The pump speed is controlled at 550 mL / h, and the core tank pressure is 9 kPa, ensuring that the outer shell solution completely surrounds the core solution during titration. The solution is then dropped into a tank containing a liquid paraffin solution at 15°C, causing gelation and forming continuous core-shell spheres. The resulting spheres are then dried at room temperature (25°C) with ventilation until the moisture content is below 2.0%, yielding probiotic spheres with a diameter of 1.5 mm.

[0095] Example 13: Resistance test of Lactobacillus rhamnosus S28 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 shaker for 1h to simulate human body temperature.

[0096] 2. Probiotic pellet artificial gastric juice tolerance test 2.1 Determination of the amount of crystalloid bacteria Incubate 38 mL of physiological saline in a 45°C water bath for 7 min, add 2 g of probiotic crystals from Example 12, incubate for 5 min, homogenize at 45°C for 5 min, and then determine the amount of crystals according to GB 4789.35-2016 "Food Microbiology Examination - Lactic Acid Bacteria Examination".

[0097] 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.

[0098] 2.3 Tolerance test of bacterial powder to artificial gastric juice 0.04 g of Lactobacillus rhamnosus S28 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.

[0099] Table 5. Survival rate of probiotic pellets after digestion in artificial gastric juice. sample Probiotic survival rate Probiotic pellets 90.48%±3.37% Lactobacillus rhamnosus S28 bacterial powder 15.69%±4.21% After 2 hours of digestion with artificial gastric juice, the survival rate of *Lactobacillus rhamnosus* S28 in the probiotic crystals was over 90%. This demonstrates that the probiotic crystals prepared by this invention can effectively protect probiotics from gastric juice digestion and allow them to successfully reach the intestines.

[0100] Example 14: Disintegration test of Lactobacillus rhamnosus S28 probiotic pellets 1. Preparation of artificial gastric juice The preparation of artificial gastric fluid is shown in Example 13.

[0101] 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.

[0102] 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.

[0103] The results showed that the probiotic pellets disintegrated in 6 minutes. Therefore, the probiotic pellets prepared in Example 12 can disintegrate rapidly in the intestine.

[0104] Example 15 Accelerated stability test of Lactobacillus rhamnosus S28 probiotic pellets The probiotic pellets prepared in Example 12 were sealed and placed in an incubator at 37°C. Simultaneously, bacterial powder was taken for comparison. Samples were taken every 5 days to determine the bacterial count, and the test was conducted continuously for 30 days.

[0105] Table 6. Stability test of bacterial count at 37℃ Time (d) 0 5 10 15 20 25 30 Probiotic pellets 10.39 10.37 10.35 10.30 10.33 10.31 10.30 Lactobacillus rhamnosus S28 bacterial powder 11.26 11.03 10.68 10.02 9.57 9.06 8.38 Unit: Log CFU / g.

[0106] 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 of over 95% for *Lactobacillus rhamnosus* S28, while the viable count of the freeze-dried *Lactobacillus rhamnosus* S28 powder decreased by 2.88 Log CFU. This demonstrates that the probiotic pellets provided by this invention can effectively improve the stability of *Lactobacillus rhamnosus* S28.

Claims

1. A type of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus S28, characterized in that, The Lactobacillus rhamnosus S28 was deposited on January 15, 2026, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M2026129.

2. The *Lactobacillus rhamnosus* S28 as described in claim 1, characterized in that, The 16S rDNA sequence of *Lactobacillus rhamnosus* S28 is SEQ ID NO:

1.

3. The use of Lactobacillus rhamnosus S28 as described in claim 1 in the preparation of food or health products.

4. The use of Lactobacillus rhamnosus S28 as described in claim 1 in the preparation of a Streptococcus bovis inhibitor.

5. The use of the Lactobacillus rhamnosus S28 of claim 1 in the preparation of a medicament for the prevention or treatment of colonic polyps or colon cancer.

6. A probiotic pellet, comprising a shell and an inner core, characterized in that, The inner core contains Lactobacillus rhamnosus S28 as described in claim 1.

7. The probiotic pellets as described in claim 6, characterized in that, The components of the outer shell and their mass fractions are as follows: 5 parts gelatin, 5 parts seaweed oligosaccharide, 5 parts carrageenan, 7 parts glycerin, and 80 parts water.

8. The probiotic pellets as described in claim 7, characterized in that, The components and their mass fractions of the inner core are as follows: 10 parts of Lactobacillus rhamnosus S28 bacterial powder and 90 parts of hydrogenated vegetable oil with a melting point of 45℃.

9. The use of the probiotic pellets according to any one of claims 6-8 in the preparation of a medicament for the prevention or treatment of colon polyps or colon cancer.

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