Bifidobacterium longum subsp. longum strain and probiotic prills comprising the strain and applications thereof

Abstract

This invention belongs to the field of probiotic screening and application technology, specifically relating to a strain of *Bifidobacterium longum* subsp. *longum* and probiotic pellets containing this strain, as well as their applications. The *Bifidobacterium longum* subsp. *longum* has the accession number CCTCC NO: M2026130 and can significantly inhibit the growth of *Streptococcus pyogenes*, and can efficiently flocculate with *Streptococcus pyogenes*. The probiotic pellets provided by this invention have strong acid resistance, enteric solubility, and storage stability, and can be widely used for the prevention or treatment of *Streptococcus pyogenes*-induced colonic polyps and colon cancer, showing broad prospects.

Description

Technical Field

[0001] This invention relates to the field of functional probiotic screening and application technology, specifically providing a strain of Bifidobacterium longum subsp. longum and probiotic pellets containing this strain, as well as their applications. Background Technology

[0002] Colorectal cancer is a common malignant tumor of the digestive tract, and its incidence and mortality rates in my country are rising year by year, making it the fifth leading cause of cancer death in the country. In 2020, newly diagnosed colorectal cancer cases in China accounted for 23.6% of new cases globally, and colorectal cancer deaths accounted for 30.3% of all related deaths worldwide, far exceeding the world average. Furthermore, due to the lack of obvious early symptoms, most cases are diagnosed at an advanced stage. The 5-year relative survival rate for early-stage colorectal cancer patients is over 90%, while the relative survival rate for late-stage patients is less than 10%. Therefore, early detection and treatment are of great significance for patient recovery.

[0003] Colorectal cancer is a multifactorial disease with a multi-stage development process, and the factors influencing its occurrence and development vary across different regions and populations. Polyps, especially adenomatous polyps, are potential culprits of colorectal cancer, often having an insidious onset and silently lurking in the intestines. In my country, due to the lack of obvious endoscopic features of polyps, improper endoscopic procedures, or poor bowel preparation in patients, the detection rate of adenomas is low, at only 18%. Even after polyp removal, recurrence cannot be prevented, requiring patients to undergo repeated endoscopic examinations and treatments, causing suffering for patients and increasing the burden on social medical insurance.

[0004] The process of colon polyps transforming into colon cancer can typically take 5-10 years, providing ample time for intervention. Reports indicate that the probability of colon polyps developing into colon cancer in my country ranges from 1.4% to 20.4%. While colon cancer is not highly prevalent in my country, rapid economic development, changes in dietary structure, unhealthy lifestyles, high-fat, low-fiber diets, and environmental pollution are leading to an increasing trend of colon polyps being discovered at younger ages. Furthermore, a significant number of colon cancer patients also have colon polyps. If colon polyps are not addressed, the probability of them developing into colon cancer increases dramatically over time.

[0005] Increasing evidence suggests a link between colon polyps, colon cancer, and gut microbiota dysbiosis. Reshaping gut microbiota through probiotics is a novel approach for treating and preventing colon cancer. The human gut contains approximately 40 trillion microorganisms, forming the gut microbiota. These microorganisms create a symbiotic system with intestinal cells, maintaining a dynamic balance and ensuring a stable intestinal environment. Studies have shown that an imbalance in the gut microbiota disrupts the balance between pro-tumor pathogens and anti-tumor beneficial bacteria, leading to a decrease in beneficial bacteria and an increase in pathogens. Pathogenic bacteria secrete various toxic factors that damage intestinal epithelial cells, causing chronic inflammation. During inflammation, they release various cytokines and chemokines, activating inflammation-related signaling pathways and thus promoting colon cancer development. Currently, with ongoing research both domestically and internationally on the relationship between gut microbiota, probiotics, and colon polyps and cancer, regulating gut microbiota through probiotics to prevent and treat intestinal cancers has become a novel strategy.

[0006] Mounting evidence suggests a link between colonic polyps and colon cancer and gut microbiota dysbiosis, with restoring normal gut microbiota through probiotics being a novel approach for treating and preventing colon cancer. These methods have proven highly promising in experimental models of colon cancer. A significant research report published in the journal *Cancers* in 2023, titled "Gut Microbiotain Colorectal Cancer: Biological Role and Therapeutic Opportunities" (Cancers 2023, 15, 866), details pathogenic bacteria that can cause intestinal polyps and colon cancer, including biliary streptococci, Streptococcus pyogenes, Bacteroides fragilis, Clostridium perfringens, and Streptococcus pyogenes. Therefore, screening probiotics that can inhibit pathogenic bacteria causing intestinal polyps and colon cancer is of significant research importance and represents a new research hotspot in this field. Summary of the Invention

[0007] The purpose of this invention is to provide a new strain of Bifidobacterium longum subspecies (Bifidobacterium longum subspecies) Bifidobacterium longum subsp. Longum (and probiotic pellets containing this strain and their applications.)

[0008] This invention relates, in one aspect, to a long subspecies of Bifidobacterium longum, named Bifidobacterium longum subspecies Y03 ( Bifidobacterium longum subsp. Longum It was deposited on February 2, 2026, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M2026310.

[0009] The RAPD fingerprint of *Bifidobacterium longum* subspecies Y03 is as follows: Figure 1 As shown, the rep-PCR fingerprint pattern is as follows: Figure 2 As shown.

[0010] The 16S rDNA sequence of the *Bifidobacterium phytoendogenum* subspecies Y03 is SEQ ID NO: 1.

[0011] This invention relates, in one aspect, to the application of Bifidobacterium longum subsp. Y03 in the preparation of food or health products.

[0012] This invention also relates to the application of Bifidobacterium longum subspecies Y03 in the preparation of Streptococcus pyogenes inhibitors.

[0013] This invention also relates to the use of Bifidobacterium longum subsp. Y03 in the preparation of medicines for the prevention or treatment of colon polyps and colon cancer.

[0014] The present invention also provides a probiotic pellet containing Bifidobacterium longum subsp. Y03.

[0015] The probiotic pellets consist of an outer shell and an inner core. The components and their weight percentages of the inner core are as follows: 10 parts of Bifidobacterium longum subsp. Y03 bacterial powder and 90 parts of hydrogenated oil with a melting point of 45℃. The components and their weight percentages of the outer shell are as follows: 25 parts of gelatin, 8 parts of glycerin, and 67 parts of water.

[0016] The components and their mass fractions of the inner core are as follows: 10 parts of Bifidobacterium longum subsp. Y03 bacterial powder and 90 parts of hydrogenated vegetable oil with a melting point of 45℃.

[0017] This invention also relates to a method for preparing the probiotic pellets, comprising the following steps: (1) Preparation of core solution: Dissolve hydrogenated vegetable oil at 45℃, add Bifidobacterium longum subsp. longum Y03 bacterial powder, stir with a stirrer at 300 rpm for 20 minutes to mix and disperse the bacterial powder in the hydrogenated vegetable oil, and 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 5 rpm and the temperature to 45℃. (2) Preparation of the shell solution: Gelatin is dissolved at 70°C, then glycerin and seaweed oligosaccharide are added and dissolved at 70°C. The mixture is then stirred until homogeneous and poured into the outer shell solution storage tank of the capsule making machine. The temperature of the outer shell storage tank is set to 70°C. (3) Preparation of probiotic pellets: The outer shell solution tank and the inner core solution tank of the capsule machine have concentric nozzles. The pump speed is controlled at 600 mL / h and the pressure in the inner core tank is 10 kPa, so that during the titration process, the outer shell solution completely surrounds the inner core solution and drips it 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 obtained spheres are then dried in a ventilated environment at room temperature (25°C) until the moisture content is below 3.0%, resulting in probiotic spheres with a diameter of 3.0 mm.

[0018] The *Bifidobacterium longum* subspecies Y03 provided by this invention exhibits strong inhibitory activity against the pathogenic *Streptococcus pyogenes*, with an inhibition zone diameter reaching 1.4 ± 0.1 cm. Furthermore, this strain can efficiently flocculate *Streptococcus pyogenes*, and the flocculation amount is positively correlated with time. The flocculated *Streptococcus pyogenes* is then excreted from the body with feces, regulating the number of harmful bacteria in the human body and playing an inhibitory and therapeutic role in the occurrence and treatment of colon cancer and colon polyps.

[0019] Bifidobacterium longum subspecies Y03 is sensitive to common antibiotics such as erythromycin, ampicillin, tetracycline, and clindamycin, and slightly sensitive to streptomycin and gentamicin, exhibiting good overall biocompatibility. This strain has a maximum tolerated salt concentration of 6%, a cell surface hydrophobicity of 9.11%, and an adhesion index of 0.13 to Caco-2 cells.

[0020] Bifidobacterium longum subspecies Y03 exhibits strong tolerance to the digestive tract environment. After digestion with artificial gastric and intestinal fluids, the bacterial count did not decrease significantly. It also has strong antioxidant capacity, with a scavenging rate of up to 30.10% against HRS free radicals. The supernatant showed an anti-lipid peroxidation inhibition rate of 42.41%, while the bacterial cells showed an anti-lipid peroxidation inhibition rate of 14.01%.

[0021] Lysate of *Bifidobacterium longum* subsp. Y03 significantly promoted the growth of both *Lactobacillus reuteri* and *Bifidobacterium lactis*, facilitating their proliferation in the intestine. This strain significantly reduced the protein expression levels of pro-inflammatory cytokines. Compared to the model group, mice in the Y03 group, which were orally administered *Bifidobacterium longum* subsp. Y03 suspension, showed a 61% decrease in IL-1β, a 16% decrease in IL-6, and a 45% decrease in TNF-α levels in the colon tissue, respectively.

[0022] The probiotic crystals provided by this invention exhibit strong acid resistance, effectively protecting the survival of probiotics in simulated gastric fluid. The survival rate of *Bifidobacterium longum* subsp. Y03 in the crystals is as high as 93.56±2.11%, while the survival rate of *Bifidobacterium longum* subsp. Y03 powder is only 0.77±0.03%. They also demonstrate good enteric solubility, with an average disintegration time of 6 minutes, and all crystals completely dissolve in intestinal fluid within 1 hour. Furthermore, they exhibit strong stability; after 30 days of storage, the viable bacterial count of the crystals decreased by only 0.58 orders of magnitude, while the viable bacterial count of *Bifidobacterium longum* subsp. Y03 powder decreased by 2.2 orders of magnitude. These probiotic crystals have broad application potential for the prevention or treatment of colonic polyps and colon cancer induced by *Streptococcus pyogenes*, showing great promise. Attached Figure Description

[0023] Figure 1 This is a colony morphology diagram of Bifidobacterium longum subspecies Y03; Figure 2 RAPD fingerprint of Bifidobacterium longum subspecies Y03; Figure 3 The rep-PCR fingerprint of Bifidobacterium longum subsp. Y03; Figure 4 The image shows the flocculation effect of Bifidobacterium longum subsp. Y03 on Streptococcus pyogenes. Figure 5 This diagram illustrates the regulation of cytokine-induced inflammatory responses by *Bifidobacterium longum* subspecies Y03. Detailed Implementation

[0024] The *Bifidobacterium longum* subspecies Y03 provided by this invention is a newly discovered strain that can significantly inhibit the growth of *Streptococcus pyogenes* and produce a flocculation effect with *Streptococcus pyogenes*, which has important application value for the prevention and treatment of colon polyps and colon cancer.

[0025] The applicant deposited Bifidobacterium longum subspecies Y03 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China on February 2, 2026, with accession number CCTCC NO: M2026310.

[0026] The screening method described in this invention is not limited to the embodiments. Any known method capable of achieving the screening purpose can be used. The screening descriptions in the embodiments are merely illustrative of this invention and are not intended to limit the scope of protection of this invention. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and substance of this invention are within the scope of this invention.

[0027] The preparation methods of the artificial gastric fluid and artificial intestinal fluid in this embodiment of the invention are as follows: Preparation of artificial gastric juice: Weigh 5g of peptone, 2.5g of yeast extract, 1g of grape extract, and 2g of NaCl, add them to 1000mL of distilled water, adjust the pH to 3.0 with dilute hydrochloric acid, and then sterilize at 115℃ for 20min. Before use, add 3.2g of porcine mucosal pepsin, shake well to dissolve, and incubate in a 37℃ water bath for 1h to simulate human body temperature.

[0028] Preparation of artificial intestinal fluid: Weigh out 5g of peptone, 2.5g of yeast extract, 1g of glucose, 6.8g of KH2PO4, and 3.0g of ox bile salts. Add 77mL of 0.2mol / L NaOH solution and bring the volume to 1000mL. Adjust the pH to 6.8±0.1 with dilute hydrochloric acid or sodium hydroxide solution. Sterilize at 115℃ for 20min. Before use, add 1g of trypsin, shake well to dissolve, and incubate at 37℃ in a shaker for 1h to simulate human body temperature.

[0029] The present invention will now be described in detail with reference to specific embodiments.

[0030] Example 1: Isolation, screening and identification of Bifidobacterium longum subspecies Y03 1. Initial screening of lactobacilli Collect fecal samples from healthy individuals. Add 10g of the sample to 90ml of sterile saline solution and stir for 10 minutes. Then, take the supernatant and perform a series of dilutions. Take 10g of the sample... -1 10 -2 10 -3 100 μL of each of the three dilution gradients was spread onto MRS plates. The plates were anaerobic incubated at 37°C for 48 hours. After single bacteria grew on the plates, 18 strains of lactic acid bacteria with the shape of bacilli were selected by microscopic examination and named Y01, Y02, Y03, ..., Y18.

[0031] 2. Rescreening of strains with anti-Streptococcus pyogenes activity Take 5 μL of the fresh bacterial suspension of the above-mentioned primary screening strains and drop it onto an MRS plate. Make three replicates for each strain. After the droplets are dried, place them in an incubator and invert them for 48 hours to prepare lactic acid bacteria culture medium plates.

[0032] Streptococcus pyogenes BNCC185918 was activated by streaking on Sabouraud dextrose agar plates. Single colonies were then picked and transferred to Sabouraud dextrose liquid medium and incubated aerobically at 37°C for 24 hours. The colonies were then transferred at a 1% ratio to fresh BHI broth liquid medium and incubated aerobically at 37°C for another 24 hours to obtain a fresh bacterial suspension. A 0.7% agar-containing BHI broth liquid medium was then prepared and autoclaved at 121°C. When the temperature dropped to 47°C, Streptococcus pyogenes bacterial suspension was added at a 0.5% volume ratio, and the mixture was shaken well. 7 mL of this solution was poured onto the prepared lactic acid bacteria medium plate. After solidification, the plate was incubated at 37°C for 24-48 hours, and the presence of inhibition zones around the lactic acid bacteria was observed.

[0033] The results showed that among the 18 Lactobacillus strains obtained in the initial screening of this invention, strain Y03 exhibited the most significant inhibitory effect against Streptococcus pyogenes, with an inhibition zone diameter of 1.4 ± 0.1 cm. Therefore, strain Y03 was selected as the target strain.

[0034] 3. Identification of strain Y03 (1) Identification of colony morphology The colony morphology of strain Y03 is shown in the figure below. Figure 1 As shown, single colonies are opaque, glossy, and have a smooth surface; under a microscope, they appear rod-shaped.

[0035] (2) Molecular biological identification Single colonies of strain Y03 were picked from the plate and incubated in MRS broth medium at 37°C for 24 hours. Then, 500 μL of the fermentation broth was taken, and the genome of the strain was obtained according to the Tiangen Bacterial Genomic DNA Extraction Kit (catalog number: DP302). 16S rDNA gene identification, MALDI-TOF protein proteometry, RAPD fingerprinting, and rep-PCR fingerprinting were performed.

[0036] The results showed that the 16S rDNA sequence of strain Y03 was SEQ ID NO:1. BLAST alignment of this sequence with the NCBI database showed that it was similar to that of *Bifidobacterium longum* subsp. *longum* (…). Bifidobacterium longum subsp. Longum The similarity is the highest among them.

[0037] MALDI-TOF protein spectroscopy identification, RAPD fingerprinting ( Figure 2 ) and rep-PCR fingerprint ( Figure 3 The identification results also showed that strain Y03 was a subspecies of Bifidobacterium longum (Bifidobacterium longum). Bifidobacterium longum subsp. Longum ).

[0038] In summary, based on the colony morphology and molecular biological identification results, the Y03 strain obtained by this invention is identified as *Bifidobacterium longum* subsp. *longum*, and is therefore named *Bifidobacterium longum* subsp. *longum* Y03. Bifidobacterium longum subsp. Longum Y03).

[0039] On February 2, 2026, the applicant deposited the above-mentioned Bifidobacterium longum subspecies Y03 at the China Center for Type Culture Collection of Wuhan University, Wuhan, China, with accession number CCTCC NO: M2026310.

[0040] Example 2: Physiological characteristics analysis of Bifidobacterium longum subspecies Y03 1. Biosafety (1) Antibiotic preparation: Ampicillin, clindamycin, erythromycin, streptomycin, tetracycline and gentamicin were all prepared into stock solutions of 2048 μg / mL and stored at -20℃ for later use. When using, the stock solutions were serially diluted 2 times with MRS liquid medium to prepare the working solutions. The serial dilution concentrations were 1 to 1024 μg / mL, a total of 11 gradients.

[0041] (2) Preparation of inoculum: Take an appropriate amount of fresh bacterial culture (24-48h, cultured at 40℃), centrifuge at 5000rpm for 5min, wash once with sterile physiological saline, then resuspend in the same volume of physiological saline and dilute 50 times to obtain the inoculum.

[0042] (3) The minimum inhibitory concentration (MIC) of antibiotics against *Bifidobacterium longum* subsp. *Y03* was determined by the micro-broth dilution method. In the first column of a 96-well plate, MRS liquid medium without antibiotics was added as a negative control. In columns 2 through 12, 190 μL of MRS liquid medium containing different concentrations of antibiotics were added sequentially. Then, 10 μL of the above inoculum was inoculated into each well, creating three parallel wells, with one well without bacterial culture as a blank. 50 μL of paraffin oil was then added to cover the wells and prevent moisture evaporation. The 96-well plate was incubated at 40°C with shaking for 48 h. The turbidity was observed visually to determine bacterial growth. The MIC values ​​of antibiotics against *Bifidobacterium longum* subsp. *Y03* were calculated. The results are shown in Table 1.

[0043] Table 1. Antibiotic MIC values ​​(μg / mL) of Bifidobacterium longum Y03

[0044] As can be seen from the results in Table 1, the Bifidobacterium longum subspecies Y03 provided by this invention is sensitive to common antibiotics such as erythromycin, ampicillin, tetracycline and clindamycin, and slightly sensitive to streptomycin and gentamicin, with good overall biosafety.

[0045] 2. Salt tolerance Under aseptic conditions, the inoculum was inoculated at a rate of 10% into 5 mL MRS liquid culture medium with salt concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8%, respectively. The uninoculated 5 mL MRS liquid culture medium served as a control. The medium was incubated at 37°C with shaking, and the condition of the medium was observed to determine whether it became turbid.

[0046] The results showed that the maximum salt tolerance concentration of Bifidobacterium longum subspecies Y03 was 6%.

[0047] 3. Cell surface hydrophobicity (1) Preparation of the test bacterial solution: The purified Bifidobacterium longum subsp. Y03 colonies were picked and inoculated into freshly prepared MRS liquid medium and cultured at 40℃ with shaking for 24-48 h. Then, the inoculum was added to MRS liquid medium at a rate of 1% (V / V) and cultured at 40℃ with shaking for another 24-48 h. After centrifugation at 6000 rpm for 10 min, the bacterial cells were collected and washed twice with sterile physiological saline. The bacterial cells were then resuspended in 1 mL of sterile 0.1 MkNO3 solution to obtain the test bacterial solution.

[0048] (2) Surface hydrophobicity determination: Add 2450 μL of 0.1 MkNO3 to 50 μL of the above bacterial suspension and record OD600 as A0. Mix 1.5 mL of the bacterial suspension with 500 μL of xylene and let it 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 aspirate the aqueous phase (avoiding the organic phase) and measure the absorbance A1 at 600 nm. Formula for calculating cell hydrophobicity: Hydrophobicity (%) = (A0-A1) / A1×100%.

[0049] The average value was taken from three experiments.

[0050] The results showed that the surface hydrophobicity of Bifidobacterium longum subspecies Y03 provided by the present invention was 9.11%.

[0051] 4. Adhesion to intestinal Caco-2 cells (1) Cell preculture Caco-2 cells were resuscitated in liquid nitrogen and cultured to the required density. 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 × 10⁻⁶ cells. 5 Cells / mL. Then, 500 μL of cell suspension was seeded into 24-well plates 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.

[0052] (2) Preparation of bacterial suspension Fresh bacterial culture of Bifidobacterium longum subsp. Y03 was washed twice with pH 7.0 phosphate buffer, then resuspended in the same volume of RPMI-1640 medium, and the absorbance was adjusted to OD600 between 0.8 and 1.0.

[0053] (3) Cell culture Add 500 μL of bacterial suspension to a prepared 24-well plate containing Caco-2 cells and co-culture in a carbon dioxide incubator for 2 h; wash three times with pH 7.0 phosphate buffer to remove unadhered bacteria.

[0054] (4) Counting Add 300 μL of trypsin for 3 minutes, then add 700 μL of cell culture medium to stop the digestion. Repeat the pipetting process and collect the resulting solution into sterile EP tubes. Perform serial dilutions of the collected solution at 10-fold, 100-fold, 1000-fold, and 10000-fold, and plate the cells for cell counting. Simultaneously, count the cells in the control group. Calculate the adhesion ability of the tested strain using the following formula: Adhesion capacity (CFU / cells) = Total number of bacteria adhering in each culture well / Total number of cells in each culture well.

[0055] The results showed that the adhesion index of *Bifidobacterium longum* subsp. Y03 to Caco-2 cells was 0.13. This indicates that *Bifidobacterium longum* subsp. Y03 has good adhesion properties to the intestine and can colonize the gut.

[0056] Example 3: Inhibitory effect of Bifidobacterium longum subsp. Y03 on pathogenic Streptococcus pyogenes 1. Activation of the strain BHI liquid medium was used to activate Streptococcus pyogenes BNCC185918 in a shaker at 37°C for 24 hours.

[0057] Bifidobacterium longum subsp. Y03 was activated using MRS liquid culture medium and incubated at 37°C for 24 hours.

[0058] 2. The antibacterial effect of Bifidobacterium longum subsp. Y03 was verified by co-culture. Press 10 respectively 6 CFU / ml and 10 5 The inoculum size was determined by co-culturing *Bifidobacterium longum* subsp. *Y03* and *Streptococcus pyogenes* BNCC185918 in BHI medium at an inoculation volume of CFU / ml. After 48 hours, the culture was plated and the viable bacterial count was determined. *Bifidobacterium longum* subsp. *Y03* was detected using MRS medium, and *Streptococcus pyogenes* was detected using BHI liquid medium.

[0059] The results showed that after 48 hours of co-culture, the viable count of *Bifidobacterium longum* subsp. Y03 reached 3.5 × 10⁻⁶. 8 CFU / ml, while Streptococcus pyogenes had only 6.2 × 10⁻⁶ CFU / ml. 4 CFU / ml. This indicates that *Bifidobacterium longum* subsp. Y03 has a strong inhibitory effect on the growth of *Streptococcus pyogenes*.

[0060] Example 4: Flocculation effect of Bifidobacterium longum subsp. Y03 on Streptococcus pyogenes Bifidobacterium longum subsp. Y03 was inoculated into MRS liquid medium and incubated statically at 37°C for 24-48 hours. Streptococcus pyogenes BNCC185918 was inoculated into BHI broth (containing 5% newborn calf serum) and incubated at 37°C and 200 rpm for 2-3 days.

[0061] Fresh bacterial suspensions of *Bifidobacterium longum* subsp. Y03 and *Streptococcus pyogenes* were centrifuged at 8000 rpm for 10 min to collect the bacterial cells. The cells were washed twice with pH 7.0 phosphate buffer, then resuspended in pH 7.0 phosphate buffer, and the initial absorbance (OD) of the suspension was adjusted. 600 It should be between 0.5 and 0.6, and kept in reserve.

[0062] Add 300 μL of Bifidobacterium longum subsp. Y03 suspension to a 24-well ELISA plate, and then add 300 μL of Streptococcus pyogenes suspension as the experimental group. Take an equal amount of buffer solution and mix them as the blank control group. Set up 2 replicates.

[0063] Place the 24-well plate in a microplate shaker at 400 rpm and room temperature for incubation. Take photos to record the initial state of the plate and its state at each shutdown, and observe for any aggregation.

[0064] The results are as follows Figure 4 As shown, after 16 hours of incubation, the blank control group on the left showed no change, while the experimental group on the right produced obvious flocculated patches. After 24 hours of incubation, the blank control group still showed no change, while the flocculated patches in the experimental group were larger than those at 18 hours. This indicates that *Bifidobacterium longum* subsp. Y03 has a strong flocculation effect on the pathogenic *Streptococcus pyogenes*, and the amount of flocculation is positively correlated with time. The flocculated *Streptococcus pyogenes* is excreted from the body with feces, regulating the number of harmful bacteria in the human body, and playing an inhibitory and therapeutic role in the occurrence and treatment of colon cancer and colon polyps.

[0065] Example 5: Tolerance analysis of *Bifidobacterium longum* subsp. Y03 to artificial gastric and intestinal fluids. The cryopreserved *Bifidobacterium longum* subsp. Y03 strain was streaked onto MRS solid medium and cultured at 37°C for 24–48 h. After one subculture on MRS liquid medium, *Bifidobacterium longum* subsp. Y03 was inoculated into fresh MRS liquid medium at a 5% inoculum and cultured at 40°C with shaking for 24–48 h to obtain fresh bacterial culture.

[0066] Take 2 mL of fresh bacterial culture, centrifuge at 5000 rpm for 5 min to collect bacterial cells, wash the cells three times with physiological saline, and then resuspend them in 2 mL of physiological saline as the inoculum. Take 1 mL of the inoculum and add it to 9 mL of artificial gastric fluid that has been warmed for 1 h. Place the mixture in a 37℃ water bath shaker at 200 rpm for 2 h. Take 1 mL samples at 0 h and 2 h to detect the viable bacterial count. Then take 1 mL of artificial gastric fluid after 2 h of digestion and add it to 24 mL of artificial intestinal fluid. Place the mixture in a 37℃ water bath shaker (200 rpm) for 3 h. Take 1 mL samples to detect the viable bacterial count.

[0067] The viable count method was determined according to the national standard GB4789.35-2016-Food Microbiology Examination - Lactic Acid Bacteria Examination. The Log (CFU / mL) of the viable count of this strain after artificial gastric fluid and artificial intestinal fluid are shown in Table 2.

[0068] Table 2. Logarithmic values ​​of viable bacterial count after digestion in artificial gastrointestinal tract. Before digestion After digestion by artificial gastric juice After digestion by artificial intestinal fluid 8.08±0.02 8.18±0.03 8.16±0.02 Table 2 shows that the bacterial count of *Bifidobacterium longum* subsp. Y03 did not decrease significantly after digestion with artificial gastric and intestinal fluids. This indicates that this strain has strong tolerance to the digestive tract environment and possesses the potential to serve as a probiotic for the gut.

[0069] Example 6: Determination of the antioxidant function of Bifidobacterium longum subsp. Y03 1. Determination of the strain's ability to scavenge DPPH and hydroxyl radicals (HRS) A single colony of *Bifidobacterium longum* subsp. Y03 with excellent growth was inoculated into 3 mL of MRS liquid medium and cultured at 37°C for 18-20 h. Using this culture as the inoculum, 2% of the colony was inoculated into 50 mL of MRS liquid medium and incubated statically for 18 h to obtain the bacterial culture. 1 mL of the bacterial 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] (1) Determination of the ability of the strain to scavenge DPPH free radicals Take 1 mL of PBS bacterial suspension of Bifidobacterium longum subsp. Y03, 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. Then measure the absorbance of the sample A at a wavelength of 517 nm, and perform three parallel measurements. The control sample is prepared with an equal volume of PBS solution and DPPH·ethanol mixture, and the blank is zeroed with an equal volume of PBS bacterial suspension and ethanol mixture.

[0071] The clearance rate is calculated using the following formula: Clearance rate = [1 - (A sample - A blank) / A control] × 100%.

[0072] The results showed that the Bifidobacterium longum subspecies Y03 provided by this invention had a DPPH free radical scavenging rate of up to 47.73%.

[0073] (2) 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 Bifidobacterium longum subsp. longum Y03 suspension, then add 100 μL of hydrogen peroxide solution (3 mM). After incubating in a water bath at 37°C for 15 min, measure the absorbance of the sample at a wavelength of 510 nm.

[0074] The hydroxyl radical scavenging rate is calculated using the following formula: Clearance rate = (A sample - A control) / (A blank - A control) × 100%.

[0075] In this design, A control is a deionized water substitute for the sample, and A blank is a deionized water substitute for the sample and H2O2.

[0076] The results showed that the Bifidobacterium longum subspecies Y03 provided by this invention had a scavenging rate of up to 30.10% against HRS free radicals.

[0077] 2. Determination of the strain's resistance to lipid peroxidation (1) Preparation of bacterial strain culture and fermentation supernatant, bacterial cells, and intracellular extracts: Bifidobacterium longum subsp. Y03 was cultured in MRS liquid medium at 37°C for 24 h. After three passages, the culture was centrifuged at 6000 rpm for 10 min at 4°C, and the supernatant was collected as the fermentation supernatant. The collected bacterial cells were washed three times by centrifugation at 6000 rpm for 10 min with 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 LTween20, 19.7 mL deionized water.

[0079] (3) Add 1 mL of linoleic acid emulsion and 1 mL of FeSO4 (1%) to 0.1 mL of PBS solution (pH 7.4), then add 0.5 mL of Bifidobacterium longum subsp. Y03 bacterial suspension, incubate at 37℃ for 1.5 h, add 0.2 mL LTCA (4%) and 2 mL LTBA (0.8%) to the mixture, incubate at 100℃ for 30 min, cool rapidly, centrifuge at 4000 r / min for 15 min, collect the supernatant and measure the absorbance at OD 532 nm to get A; the control group is A0, which is 0.5 mL of distilled water instead of the sample.

[0080] Inhibition rate = (A0 - A) / A0 × 100%.

[0081] The results showed that the supernatant of Bifidobacterium longum subsp. Y03 provided by the present invention had an anti-lipid peroxidation inhibition rate of 42.41%; the bacterial cell had an anti-lipid peroxidation inhibition rate of 14.01%.

[0082] Example 7: The promoting effect of Bifidobacterium longum subsp. Y03 on the growth of beneficial intestinal bacteria. Probiotics, as healthy bacteria in the human body, have a significant impact on human health due to their abundance. The proportion of probiotics in healthy individuals is higher than in the general population. Analysis of fecal samples from centenarians revealed that the higher the health status of the elderly, the greater the detection probability of *Lactobacillus reuteri* and *Bifidobacterium lactis*, which are important probiotics in the human gut. This embodiment selects *Lactobacillus reuteri* and *Bifidobacterium lactis* as research subjects to investigate whether the lysate of *Bifidobacterium longum* subsp. Y03 has a promoting effect on these two bacteria.

[0083] 1. Preparation of lysate: *Bifidobacterium longum* subsp. *Y03* was inoculated into MRS broth medium at an inoculum volume of 1% (v / v). The medium was incubated at 37°C for 24 hours, and then the incubation was 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 *Bifidobacterium longum* subsp. *Y03*.

[0084] 2. Preparation of beneficial bacteria culture medium: Prepare MRS solid culture medium and add 10% (w / w), 20% (w / w), 20% (w / w) and 50% (w / w) of Bifidobacterium longum subsp. longum Y03 lysis buffer to the culture medium respectively. Use solid culture medium without lysis buffer as blank control. Autoclave the culture medium at 121℃ for 15 min and then cool it to 50℃ for later use.

[0085] 3. Beneficial bacteria culture: Lactobacillus reuteri and Bifidobacterium lactis were inoculated into MRS medium containing lysate of Bifidobacterium longum subsp. Y03 and cultured separately. The OD value changes were detected using a microplate reader, and growth curves were generated.

[0086] The results showed that the lysate of *Bifidobacterium longum* subsp. Y03 significantly promoted the growth of the probiotics *Lactobacillus reuteri* and *Bifidobacterium lactis*, with the numbers of these two probiotics significantly increasing compared to the control group. *Lactobacillus reuteri* can promote epithelial cell repair, relieve diarrhea, and reduce constipation, while *Bifidobacterium lactis* can promote intestinal peristalsis, help excrete waste, regulate intestinal flora balance, protect the intestinal mucosal barrier, and inhibit the growth of harmful bacteria. The effective proliferation of these two probiotics in the intestine has potential benefits for patients with colon polyps and colon cancer.

[0087] Example 8: Immunomodulatory effects of Bifidobacterium longum subsp. Y03 on intestinal cells Weakened immunity is a manifestation of an imbalance in the immune system, specifically resulting in increased susceptibility to illnesses (such as colds and pneumonia). Individuals with generally weakened immune systems can improve their immunity through nutritional supplementation and exercise. In recent years, many studies have reported that proper and appropriate intake of probiotics can also enhance immune function. This example uses animal experiments to verify the effects of *Bifidobacterium longum* subsp. Y03 on the expression levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 proteins in the rectum.

[0088] Sixty mice were randomly divided into three groups of 20 each. The control group (CK) received 200 μL of PBS (pH 7.4) via gavage daily; the model group mice were induced to develop colorectal cancer by intraperitoneal injection of azomethane (AOM) / dextrose sulfate (DSS); and the Y03 group mice received 200 μL (1 × 10⁻⁶) orally once daily. 8 A suspension of *Bifidobacterium longum* subsp. Y03 (cfu / ml) was prepared. Mice were sacrificed after 20 days. The levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in mouse colon tissue were measured using a mouse TNF-α ELISA kit (mIC50536-1, Shanghai ELISA, China), an IL-6 ELISA kit (mL098430, Shanghai ELISA, China), and an IL-1β ELISA kit (mIC50300-1, Shanghai ELISA, China).

[0089] The results are as follows Figure 5 As shown, compared with the model group, the levels of IL-1β, IL-6, and TNF-α in the colon tissue of mice in the Y03 group, which were orally administered with a suspension of Bifidobacterium longum subsp. Y03, decreased by 61%, 16%, and 45%, respectively. This indicates that Bifidobacterium longum subsp. Y03 can significantly reduce the protein expression levels of pro-inflammatory cytokines and has potential application value in inhibiting the occurrence of colonic polyps and colon cancer.

[0090] Example 9 Preparation of Probiotic Spheres The probiotic pellets in this embodiment include an outer shell and an inner core. The components and their mass fractions of the outer shell are as follows: 25 parts gelatin, 8 parts glycerin, and 67 parts water. The components and their mass fractions of the inner core are as follows: 10 parts Bifidobacterium longum subsp. Y03 bacterial powder and 90 parts hydrogenated oil with a melting point of 45°C.

[0091] (1) Preparation of core solution: Dissolve hydrogenated vegetable oil at 45℃, add Bifidobacterium longum subsp. longum Y03 bacterial powder, stir with a stirrer at 300 rpm for 20 min to mix and disperse the bacterial powder in the hydrogenated vegetable oil, and 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 5 rpm and the temperature to 45℃. (2) Preparation of the shell solution: Gelatin is dissolved at 70°C, then glycerin and seaweed oligosaccharide are added and dissolved at 70°C. The mixture is then stirred until homogeneous and poured into the outer shell solution storage tank of the capsule making machine. The temperature of the outer shell storage tank is set to 70°C. (3) Preparation of probiotic pellets: The outer shell solution tank and the inner core solution tank of the capsule machine have concentric nozzles. The pump speed is controlled at 600 mL / h and the pressure in the inner core tank is 10 kPa, so that during the titration process, the outer shell solution completely surrounds the inner core solution and drips it 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 obtained spheres are then dried in a ventilated environment at room temperature (25°C) until the moisture content is below 3.0%, resulting in probiotic spheres with a diameter of 3.0 mm.

[0092] Example 10: Testing of the acid resistance, enteric solubility, and storage stability of probiotic pellets. 1. Acid resistance (1) Determination of viable count of crystal spheres 38 ml of anaerobic diluent was kept in a 45°C water bath for 7 min, 2 g of probiotic crystals from Example 9 was added, the mixture was kept in a 5 min water bath, and homogenized at 45°C for 5 min. Then the amount of crystals was determined according to GB4789.35-2016 "Food Microbiology Examination - Lactic Acid Bacteria Examination".

[0093] (2) Artificial gastric juice digestion test Weigh 2g of the probiotic pellets from Example 9 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 anaerobic diluent to make the total weight of the pellets and anaerobic diluent approximately 40g, and record the actual mass. 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 artificial gastric fluid is obtained by comparing the data before and after digestion.

[0094] Meanwhile, as a control, 0.04 g of Bifidobacterium longum subsp. Y03 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, 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.

[0095] The results showed that, after digestion in artificial gastric juice, the survival rate of *Bifidobacterium longum* subsp. Y03 in the probiotic pellets prepared in Example 9 was as high as 93.56±2.11%, while the survival rate of *Bifidobacterium longum* subsp. Y03 powder was only 0.77±0.03%. The results indicate that the pellets prepared in this invention can effectively protect the survival of probiotics in artificial gastric juice, which lays the foundation for their subsequent entry into the intestines.

[0096] 2. Enteric-coated Following the method and apparatus described in the "Disintegration Time Test" of the Chinese Pharmacopoeia, six probiotic pellets prepared in Example 9 were taken and tested in artificial gastric fluid without baffles for 2 hours. Each pellet showed no disintegration or cracks. 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, and the time for complete dissolution of the pellets was recorded.

[0097] The results showed that the average disintegration time of the probiotic pellets was 6 minutes, and all of them could be completely dissolved in intestinal fluid within 1 hour.

[0098] 3. Storage stability Accelerated probiotic testing refers to accelerating the deactivation of probiotics under intensified temperature conditions without altering the product's failure mechanism. This allows for the achievement of necessary probiotic stability within a shorter timeframe, enabling the evaluation of probiotic reliability or lifespan indicators under normal conditions. Since probiotic pellets have a relatively long shelf life, this example uses an extreme temperature of 37°C to determine their stability. The probiotic pellets prepared in Example 9 were sealed and placed in a 37°C incubator. Samples were taken every 5 days to determine the viable cell count, and this was repeated for one month. Simultaneously, *Bifidobacterium longum* subsp. Y03 bacterial powder was used as a control. The test data are shown in Table 3.

[0099] Table 3. Stability test results of viable bacterial count at 37℃ (LOG CFU / g) Time (days) 0 5 10 15 20 25 30 Probiotic pellets 9.74 9.66 9.53 9.42 9.29 9.20 9.16 Y03 bacterial powder 10.77 10.64 10.21 9.70 9.23 8.66 8.57 As shown in Table 3, after 30 days of storage, the viable count of probiotic pellets decreased by only 0.58 orders of magnitude, while the viable count of Bifidobacterium longum subsp. Y03 bacterial powder decreased by 2.2 orders of magnitude. Therefore, compared with conventional freeze-dried bacterial powder, the stability of Bifidobacterium longum subsp. Y03 in probiotic pellets is better.

[0100] In summary, the probiotic pellets containing Bifidobacterium longum subsp. Y03 prepared by this invention have strong acid resistance, enteric solubility and storage stability, and can be widely used to prevent or treat colonic polyps and colon cancer induced by Streptococcus pyogenes, showing great promise.

Claims

1. A long subspecies of Bifidobacterium longum ( Bifidobacterium longum subsp. Longum ), characterized in that, The accession number of the long subspecies of Bifidobacterium longum is CCTCC NO: M2026310.

2. The *Bifidobacterium longum* subsp. as described in claim 1, characterized in that, The RAPD fingerprint of the *Bifidobacterium longum* subsp. *longum* is shown in Figure 2, and the rep-PCR fingerprint is shown in Figure 3.

3. The *Bifidobacterium longum* subsp. as described in claim 1, characterized in that, The 16S rDNA sequence of the *Bifidobacterium longum* subsp. *longum* is SEQ ID NO:

1.

4. The use of the long subspecies of Bifidobacterium longum as described in claim 1 in the preparation of food or health products.

5. The use of the *Bifidobacterium longum* subsp. as described in claim 1 in the preparation of *Streptococcus pyogenes* inhibitors.

6. The use of the *Bifidobacterium longum* subsp. as described in claim 1 in the preparation of a medicament for the prevention or treatment of colonic polyps and colon cancer.

7. A probiotic pellet, comprising a shell and an inner core, characterized in that, The inner core contains the long subspecies of Bifidobacterium longum as described in claim 1.

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 Bifidobacterium longum subsp. longum powder and 90 parts of hydrogenated oil with a melting point of 45℃.

9. The probiotic pellets as described in claim 8, characterized in that, The components and their mass fractions of the outer shell are as follows: 25 parts gelatin, 8 parts glycerin, and 67 parts water.

10. The method for preparing probiotic pellets according to any one of claims 7-9, characterized in that, The preparation method includes the following steps: (1) Preparation of core solution: Dissolve hydrogenated vegetable oil at 45°C, add Bifidobacterium longum subsp. longum powder, stir with a stirrer at 300 rpm for 20 minutes to mix and disperse the powder in the hydrogenated vegetable oil, and 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 5 rpm and the temperature to 45°C. (2) Preparation of the outer shell solution: Gelatin is dissolved at 70°C, then glycerin and seaweed oligosaccharide are added and dissolved at 70°C. The mixture is then stirred until homogeneous and poured into the outer shell solution storage tank of the capsule making machine. The temperature of the outer shell storage tank is set to 70°C. (3) Preparation of probiotic pellets: The outer shell solution tank and the inner core solution tank of the capsule machine have concentric nozzles. The pump speed is controlled at 600 mL / h and the pressure in the inner core tank is 10 kPa, so that during the titration process, the outer shell solution completely surrounds the inner core solution and drips it 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 obtained spheres are then dried in a ventilated environment at room temperature (25°C) until the moisture content is below 3.0%, resulting in probiotic spheres with a diameter of 3.0 mm.

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