Lactobacillus reuteri producing tetrahydrofolate and application thereof in relieving ulcerative colitis

Abstract

The application discloses a lactobacillus reuteri strain producing tetrahydrofolic acid and application thereof in relieving ulcerative colitis, and belongs to the technical field of microorganisms. The lactobacillus reuteri CCFM1466 screened by the application not only has the ability of producing tetrahydrofolic acid, but also shows excellent effect in relieving colitis, and has important application value in the fields of functional food and intestinal health, and can be used for preparing products for preventing and / or treating colitis.

Description

Technical Field

[0001] This invention relates to a tetrahydrofolate-producing Lactobacillus reuteri strain and its application in alleviating ulcerative colitis, belonging to the field of microbial technology. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, relapsing inflammatory bowel disease characterized by persistent inflammation and ulceration. The exact cause of UC is not fully understood, but it is generally believed to be related to genetic, immune, and environmental factors. Symptoms of UC include diarrhea, abdominal pain, and bloody stools, severely impacting patients' quality of life. Existing treatments, such as acetaminophen and mesalazine, can relieve symptoms, but long-term use may lead to side effects such as liver and kidney damage, gastrointestinal discomfort, and immunosuppression. Therefore, finding safe and effective adjunctive therapies remains a hot research topic.

[0003] The role of probiotics in alleviating ulcerative colitis (UC) has been extensively studied and applied. Research shows that specific probiotics can significantly alleviate UC symptoms through mechanisms such as enhancing intestinal barrier function, regulating immune responses, inhibiting pro-inflammatory factors, and restoring gut microbiota balance. Furthermore, the metabolites produced by probiotics (such as short-chain fatty acids and bioactive molecules) have significant promoting effects on gut health, exhibiting high safety and no obvious side effects, thus becoming an important direction for adjunctive treatment of UC. Currently, probiotics reported for UC treatment mainly exert their effects through the production of short-chain fatty acids, regulation of immune balance, or improvement of intestinal barrier function.

[0004] Tetrahydrofolate (THF), as the active form of folic acid, can directly promote the repair and regeneration of intestinal epithelial cells and participate in the synthesis of DNA and RNA, playing a crucial role in cell regeneration and repair. By regulating one-carbon metabolism, THF can not only balance the body's immune function but also effectively reduce intestinal inflammation, providing a new target for the treatment of ulcerative colitis (UC). However, the human body cannot synthesize folic acid de novo and must rely on exogenous supplementation or acquisition through the gut microbiota. Existing methods of supplementing folic acid synthesis suffer from low metabolic efficiency and poor stability, and have limited effectiveness in individuals with folic acid metabolism disorders. Probiotics can directly produce THF in the gut, bypassing the multi-step conversion process of traditional folic acid metabolism, directly providing the active form of folic acid and participating in the one-carbon metabolic cycle, thus improving the utilization rate of THF. Currently, there are no reports on using probiotics to produce THF to alleviate UC. Therefore, screening for a probiotic strain that can produce high levels of THF could not only directly supplement THF but also, combined with the functions of the probiotic itself, further alleviate UC inflammation, providing new ideas and theoretical basis for UC treatment. Summary of the Invention

[0005] To address the above-mentioned technical problems, the first objective of this invention is to provide a strain of *Lactobacillus reuteri* (…). Limosilactobacillus reuteri CCFM1466, taxonomically named Limosilactobacillus reuteri It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 10, 2025, with accession number GDMCC No: 65752, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] In one embodiment, the *Lactobacillus reuteri* ( Limosilactobacillus reuteri CCFM1466 was derived from fecal samples from healthy individuals. Its 16S rDNA sequence is shown in SEQ ID NO.1. On MRS solid culture medium, the colonies are smooth, round, milky white, opaque, and 1-2 mm in diameter.

[0007] The present invention also provides a microbial preparation, wherein the microbial preparation contains *Lactobacillus reuteri* CCFM1466, or a fermentation broth containing *Lactobacillus reuteri* CCFM1466, or a lyophilized powder containing *Lactobacillus reuteri* CCFM1466.

[0008] In one embodiment, the preparation method of the microbial preparation is as follows: Lactobacillus reuteri CCFM1466 was inoculated into MRS medium and cultured at 37°C for 12 h until the end of the logarithmic growth phase to obtain bacterial suspension. Subsequently, the bacterial suspension was centrifuged at 8000 g for 20 min, and the bacterial sludge was collected and resuspended in physiological saline to prepare a resuspension. Then, the resuspension was thoroughly mixed with a freeze-drying protectant to prepare a mixture. Finally, the mixture was freeze-dried under vacuum to prepare bacterial powder.

[0009] In one embodiment, the amount of the freeze-drying protectant added accounts for 25% to 50% of the total weight of the resuspended liquid.

[0010] In one embodiment, the content of *Lactobacillus reuteri* CCFM1466 in the microbial preparation is at least 5 × 10⁻⁶. 9 CFU / g or 5×10 9 CFU / mL.

[0011] The present invention also provides a method for culturing the *Lactobacillus reuteri* CCFM1466, which involves culturing the *Lactobacillus reuteri* CCFM1466 in a culture medium at 35-40°C.

[0012] In one embodiment, the method involves culturing the *Lactobacillus reuteri* CCFM1466 in MRS medium at 37°C for at least 18 hours.

[0013] The present invention also provides the use of the Lactobacillus reuteri CCFM1466 or the microbial preparation in the preparation of medicaments for the prevention and / or relief of colitis.

[0014] In one embodiment, the drug contains ≥5 × 10⁻⁶ Lactobacillus reuteri CCFM1466. 9 CFU / g or 5×10 9 CFU / mL.

[0015] In one embodiment, the drug contains the *Lactobacillus reuteri* CCFM1466, as well as a drug carrier and / or pharmaceutical excipients.

[0016] In one embodiment, the drug carrier comprises microcapsules, microspheres, nanoparticles, and / or liposomes.

[0017] In one embodiment, the pharmaceutical excipient comprises excipients and / or additives.

[0018] In one embodiment, the excipient comprises a binder, a filler, a disintegrant, and / or a lubricant.

[0019] In one embodiment, the additive comprises a solubilizer, cosolvent, latent solvent, and / or preservative. In one embodiment, the dosage form of the drug is powder, granules, capsules, tablets, pills, or oral liquid.

[0020] The present invention also provides food or health products containing the aforementioned *Lactobacillus reuteri* CCFM1466.

[0021] The present invention also provides the application of the aforementioned *Lactobacillus reuteri* CCFM1466 in the preparation of fermented foods.

[0022] The present invention also provides the application of the aforementioned *Lactobacillus reuteri* CCFM1466 in the preparation of products containing tetrahydrofolate.

[0023] Beneficial effects: 1. The *Lactobacillus reuteri* CCFM1466 strain screened in this invention has the characteristic of producing tetrahydrofolate (THF). The THF content in its fermentation supernatant reaches 94.453 ng / mL, which is significantly higher than other strains of the same genus and species.

[0024] 2. The *Lactobacillus reuteri* CCFM1466 screened in this invention can significantly increase the THF content in the colon and shows significant potential for alleviating ulcerative colitis, specifically in the following ways: (1) Lactobacillus reuteri CCFM1466 significantly increased the content of THF in the colon; (2) Lactobacillus reuteri CCFM1466 significantly alleviated weight loss caused by DSS; (3) Lactobacillus reuteri CCFM1466 significantly reduced colonic shortening caused by DSS; (4) Lactobacillus reuteri CCFM1466 significantly improved DSS-induced colonic histopathology; (5) Lactobacillus reuteri CCFM1466 significantly reduced the levels of IL-1β, IL-6 and TNF-α in DSS-induced colon tissue.

[0025] (6) Lactobacillus reuteri CCFM1466 significantly increased the level of IL-10 in DSS-induced colon tissue.

[0026] This strain has important applications in the field of gut health and can be used to prepare products for the prevention and / or treatment of colitis.

[0027] Preservation of biological materials Lactobacillus reuteri ( Limosilactobacillus reuteri CCFM1466, taxonomically named Limosilactobacillus reuteri It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 10, 2025, with accession number GDMCC No: 65752, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0028] Figure 1 This is a colony morphology diagram of Lactobacillus reuteri CCFM1466.

[0029] Figure 2 A graph showing the THF production capacity of Lactobacillus reuteri CCFM1466.

[0030] Figure 3 The graph shows the THF content in the colonic contents of mice with colitis caused by *Lactobacillus reuteri* CCFM1466.

[0031] Figure 4 The figure shows the effect of Lactobacillus reuteri CCFM1466 on the body weight changes in mice with colitis.

[0032] Figure 5 The figure shows the effect of Lactobacillus reuteri CCFM1466 on colon length in mice with colitis.

[0033] Figure 6 The image shows the effect of Lactobacillus reuteri CCFM1466 on improving colonic tissue damage in mice with colitis.

[0034] Figure 7Lactobacillus reuteri CCFM1466 as an indicator of IL-1 levels in the colon of colitis-affected mice The effect of 1β levels.

[0035] Figure 8 The effect of Lactobacillus reuteri CCFM1466 on TNF in the colon of colitis mice The effect of α level.

[0036] Figure 9 Lactobacillus reuteri CCFM1466 as an indicator of IL-1 levels in the colon of colitis-affected mice The influence of level 6.

[0037] Figure 10 Lactobacillus reuteri CCFM1466 as an indicator of IL-1 levels in the colon of colitis-affected mice Influence diagram of level 10. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments.

[0039] The strains involved in the following examples: All strains involved in this patent embodiment, including the main research object Lactobacillus reuteri CCFM1466 and other Lactobacillus reuteri used in the comparative experiment (such as L7, L52, L53, L54, L60, etc.), were isolated and screened from fecal samples of healthy adults.

[0040] The culture media involved in the following examples are as follows: MRS liquid culture medium (1L): 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, 2.0 g anhydrous sodium acetate, 0.5 g magnesium sulfate heptahydrate, 0.25 g manganese sulfate monohydrate, and 2.0 g dipotassium hydrogen phosphate. Dissolve all ingredients completely in distilled water, adjust the pH to 6.8–7.2, and sterilize at 115°C for 20 min.

[0041] MRS solid medium (1L): Prepare MRS liquid medium by adding 20 g of agar powder. Sterilize at 115℃ for 20 min.

[0042] The detection methods involved in the following embodiments are as follows: The THF content in the fermentation supernatant and colon contents of *Lactobacillus reuteri* CCFM1466 was detected by enzyme-linked immunosorbent assay (ELISA): The strain was inoculated into MRS liquid medium and incubated statically at 37°C for 24 h. After centrifugation at 12000 rpm for 5 min, the supernatant was discarded, and the bacterial sludge was washed 2-3 times with sterile physiological saline before resuspending. The bacterial suspension was diluted to 1.0 × 10⁻⁶. 8 The bacterial culture was inoculated at a concentration of CFU / mL into 5 mL of MRS liquid medium at a 2% inoculum and incubated at 37°C in the dark for 12 h. 1 mL of the bacterial culture was transferred to a 2 mL brown centrifuge tube and centrifuged at 12000 rpm for 5 min. The fermentation supernatant was collected for determining the extracellular THF content.

[0043] Take approximately 0.1 g of mouse feces and add it to pre-cooled PBS at 4°C at a weight (g):volume (mL) ratio of 1:9. Homogenize the sample using a high-throughput tissue homogenizer. Centrifuge the sample at 4°C and 12,000 rpm for 15 min, collect the supernatant, and perform THF detection according to the instructions of the THF assay kit.

[0044] Colonic histopathology: During mouse dissection, tissue samples approximately 0.5 cm from the distal end of the colon were fixed in 4% paraformaldehyde solution for 24–48 hours. H&E staining was performed by Wuhan Sewell Biotechnology Co., Ltd. Prepared sections were scanned and photographed using a Pannoramic MI1I digital slide scanner.

[0045] Cytokine assay: Approximately 0.1 g of colon tissue was added to pre-chilled PBS (4°C) at a weight (g):volume (mL) ratio of 1:9 and homogenized using a high-throughput tissue homogenizer. The homogenate was then centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was collected. The levels of cytokines TNF-α, IL-β, IL-6, and IL-10 were determined according to the kit instructions.

[0046] Example 1: Isolation, screening, identification, and culture of *Lactobacillus reuteri* CCFM1466 1. Separation and screening Take approximately 0.5 g of fecal sample from a healthy adult and perform 10-fold serial dilutions with sterile physiological saline to prepare 10... -2 10 -3 10 -4 10 -5 10 -6 Diluent. Then take 100 μL of each of the following solutions: 10 -4 10 -5 10 -6Diluted solutions were plated on MRS solid medium and incubated at 37°C for 48 h. Colony morphology was observed and recorded. Plates with colony counts between 30 and 300 were selected, and colonies of different morphologies were streaked for isolation. After incubation at 37°C for 48 h, the above steps were repeated until pure single colonies with consistent morphology were obtained. Pure colonies from the MRS solid medium were inoculated into 5 mL of MRS liquid medium and incubated anaerobically at 37°C for 18 h. 1 mL of the bacterial culture was transferred to a sterile centrifuge tube and centrifuged at 8000 rpm for 3 min. The supernatant was discarded to obtain bacterial cells, which were then resuspended in 30% glycerol solution. Store in an 80℃ refrigerator.

[0047] 2. Strain identification The isolated strain was subjected to PCR amplification, and the PCR product was sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. The sequencing results were submitted to the NCBI database for nucleic acid sequence comparison, and it was identified as *Lactobacillus reuteri*, and named *Lactobacillus reuteri* (…). Limosilactobacillus reuteri (CCFM1466)

[0048] 3. Cultivate Lactobacillus reuteri ( Limosilactobacillus reuteri CCFM1466 was inoculated into MRS solid medium and cultured at 37°C for 48 h. Its colony morphology was then observed. Figure 1 Showing Lactobacillus reuteri ( Limosilactobacillus reuteri The colony morphology of CCFM1466 after 48 h of culture on MRS solid plate medium is as follows: the colonies are round, white, raised, and have smooth edges.

[0049] Example 2: The ability of different *Lactobacillus reuteri* strains to produce THF The *Lactobacillus reuteri* obtained in Example 1 were respectively... Limosilactobacillus reuteri CCFM1466 and other *Lactobacillus reuteri* strains were inoculated at a 2% inoculum into 5 mL of MRS liquid medium and incubated at 37°C for 24 h. 1 mL of the bacterial culture was then transferred to a 2 mL brown centrifuge tube and centrifuged at 12000 rpm for 5 min at 4°C. The fermentation supernatant was collected, and the THF production of *Lactobacillus reuteri* was determined according to the tetrahydrofolate assay kit instructions (analytical results are shown in Table 1 and...). Figure 2 ).

[0050] Table 1. THF production of different *Lactobacillus reuteri* strains

[0051] Example 3: Effect of *Lactobacillus reuteri* on body weight changes in DSS-induced colitis mice (1) *Lactobacillus reuteri* CCFM1466 and *Lactobacillus reuteri* L7 were inoculated into MRS liquid medium and cultured at 37°C for 18 h to prepare bacterial suspensions. The bacterial suspension concentration was then adjusted to 5 × 10⁻⁶. 9 CFU / mL.

[0052] (2) Forty 6-week-old SPF-grade male C57BL / 6J mice were randomly divided into four groups (n=10 per group): control group, model group, *Lactobacillus reuteri* CCFM1466 intervention group (CCFM1466), and *Lactobacillus reuteri* L7 intervention group (L7). The experimental period was 14 days. The first 7 days were the adaptation period, during which all mice were given a standard diet and free access to water. The intervention period was from the 8th to the 14th day. The control group was given 200 μL of physiological saline by gavage daily, the model group was given 200 μL of physiological saline by gavage daily and drank an aqueous solution containing 3% DSS, and the CCFM1466 group was given 200 μL of 5×10⁵ DSS by gavage daily. 9 L7 group received CFU / mL Lactobacillus reuteri CCFM1466 via oral administration of a 3% DSS solution, and 200 μL of the solution was administered daily via gavage at a dose of 5 × 10 mL. 9 CFU / mL Lactobacillus reuteri L7 and drinking an aqueous solution containing 3% DSS.

[0053] During the modeling period, the weight of the mice was measured and recorded daily (results are shown in [link to results]). Figure 3 The results showed that the body weight loss in the model group mice was 19.68 ± 4.10%, compared to 16.14 ± 3.31% in the *Lactobacillus reuteri* L7 group and only 10.6 ± 4.62% in the *Lactobacillus reuteri* CCFM1466 group. The analysis indicated that the body weight loss in the CCFM1466 group was approximately 46% less than that in the model group, suggesting that *Lactobacillus reuteri* CCFM1466 has a significant alleviating effect on DSS-induced body weight loss, and its effect is significantly better than that of the L7 intervention group.

[0054] Example 4: Effect of Lactobacillus reuteri CCFM1466 on colon length in DSS-induced colitis mice The method for establishing the animal model was the same as in Example 3. After the experiment, mice were anesthetized by inhalation with 1%~1.5% isoflurane, and their colons were dissected, their length (from the end of the cecum to the anterior rectum) was measured, and photographs were taken for recording (results are shown in...). Figure 4The results showed that, compared with the control group, the colon length of the model group mice was significantly shortened, with an average length of 5.05 ± 0.34 cm. The average colon length of the mice in the *Lactobacillus reuteri* CCFM1466 group was 6.42 ± 0.14 cm, close to the 6.962 ± 0.18 cm of the control group. In contrast, the average colon length of the mice in the *Lactobacillus reuteri* L7 group was 5.20 ± 0.39 cm. This indicates that *Lactobacillus reuteri* CCFM1466 is more effective than *Lactobacillus reuteri* L7 in alleviating colon shortening caused by colitis.

[0055] Example 5: Effects of different *Lactobacillus reuteri* strains on THF content in colonic contents of DSS-induced colitis mice The animal model was established using the same method as in Example 3. Approximately 0.1 g of mouse feces was taken and added to pre-cooled PBS at 4°C at a weight (g):volume (ml) ratio of 1:9. The mixture was homogenized using a high-throughput tissue homogenizer. The sample was centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant was used for THF determination. The procedure was performed according to the instructions of the tetrahydrofolate assay kit. The results showed (…). Figure 5 The CCFM1466 strain group had the highest THF content in the colonic contents, reaching approximately 44.5 ng / ml, significantly higher than other groups. The L7 strain group had a THF content of approximately 10.2 ng / ml, also higher than the control and model groups. The control and model groups had extremely low THF content, approximately 1.2 ng / ml and 1.0 ng / ml respectively, with no significant difference between groups. This indicates that *Lactobacillus reuteri*, especially the CCFM1466 strain, has the ability to significantly increase the production of tetrahydrofolate in the colonic contents.

[0056] Example 6: Effects of *Lactobacillus reuteri* CCFM1466 on colonic tissue damage in DSS-induced colitis mice The animal model was established using the same method as in Example 3. After the experiment, mice were anesthetized with 1%–1.5% isoflurane inhalation, dissected, and approximately 0.5 cm of distal colon tissue was taken for H&E staining. The prepared sections were scanned and photographed using a Pannoramic MI11 digital slide scanner (results are shown in [link to results]). Figure 6The results showed that the DSS group mice suffered severe damage to their colonic tissue, including near-complete loss of crypt structures, large-area ulcers, extensive inflammatory cell infiltration, and significant loss of goblet cells. In contrast, intervention with *Lactobacillus reuteri* strain CCFM1466 significantly improved these pathological conditions, reduced ulceration and inflammatory cell infiltration, increased the number of intestinal glands, and maintained a relatively intact colonic mucosa. Intervention with *Lactobacillus reuteri* strain L7 also showed some improvement in colonic tissue damage, but the effect was not as good as that of *Lactobacillus reuteri* strain CCFM1466; its effect in reducing inflammatory cell infiltration and restoring crypt structures was weaker. Overall, *Lactobacillus reuteri* strain CCFM1466 had a better restorative effect on colonic tissue damage than *Lactobacillus reuteri* strain L7.

[0057] Example 7: Influence of Lactobacillus reuteri CCFM1466 on IL-1 levels in colon tissue of DSS-induced colitis mice Effects of 1β levels The method for establishing the animal model was the same as in Example 3. After the experiment, the mice were fasted for 12 hours and then sacrificed. Colon tissue was removed, and a homogenate was prepared at a ratio of 1:9 (tissue weight to tissue lysis buffer). After centrifugation, the supernatant was obtained, and the IL-1 levels were measured. 1β content (see results) Figure 7 The results showed that IL-1 in the colon tissue of the control group was significantly lower. The 1β level was 4.56 ± 0.47 pg / mg; after DSS treatment, IL-1β levels in the colon tissue of the model group mice were [missing information]. 1β levels were significantly elevated to 9.23 ± 0.54 pg / mg, while intervention with *Lactobacillus reuteri* CCFM1466 significantly reduced IL-1 levels. The 1β content was reduced to 6.01 ± 0.21 pg / mg. Simultaneously, intervention with *Lactobacillus reuteri* L7 strain also reduced IL to some extent. The level of 1β was reduced, but the effect of reducing it was not significant.

[0058] Example 8: Effect of *Lactobacillus reuteri* CCFM1466 on TNF-α levels in colon tissue of DSS-induced colitis mice The method for establishing the animal model was the same as in Example 3. The method for processing colon tissue was the same as in Example 6. The results showed ( Figure 8 In the control group, the TNF-α level in the colon tissue was 4.87 ± 0.09 pg / mg. After DSS treatment, the TNF-α level in the colon tissue of the model group mice was significantly increased to 6.31 ± 0.22 pg / mg, while the intervention of Lactobacillus reuteri CCFM1466 significantly reduced the TNF-α content to 5.16 ± 0.21 pg / mg.

[0059] Example 9: Effect of *Lactobacillus reuteri* CCFM1466 on IL-6 levels in colon tissue of DSS-induced colitis mice The method for establishing the animal model was the same as in Example 3. The method for processing colon tissue was the same as in Example 6. The results showed ( Figure 9 In the control group, the IL-6 level in the colon tissue was 3.29 ± 0.19 pg / mg. After DSS treatment, the IL-6 level in the colon tissue of the model group mice was significantly increased to 5.52 ± 0.18 pg / mg, while the intervention of Lactobacillus reuteri CCFM1466 significantly reduced the IL-6 content to 3.55 ± 0.19 pg / mg.

[0060] Example 10: Effect of Lactobacillus reuteri CCFM1466 on IL-10 levels in colon tissue of DSS-induced colitis mice The method for establishing the animal model was the same as in Example 3. The method for processing colon tissue was the same as in Example 6. The results showed ( Figure 10 In the control group, the IL-10 level in the colon tissue was 5.68 ± 0.28 pg / mg. After DSS treatment, the IL-10 level in the colon tissue of the model group mice was significantly reduced to 3.00 ± 0.45 pg / mg, while the intervention of *Lactobacillus reuteri* CCFM1466 significantly increased the IL-10 content to 4.86 ± 0.35 pg / mg, which was more effective than that of *Lactobacillus reuteri* L7.

[0061] Example 11: Application of *Lactobacillus reuteri* CCFM1466 Lactobacillus reuteri CCFM1466 can be used to prepare bacterial powder. The specific preparation process is as follows: A small amount of bacterial solution from a glycerol tube is streaked onto MRS solid medium and incubated at 37°C for 48 hours to obtain single colonies. Single colonies are picked and inoculated into MRS liquid medium and incubated at 37°C for 24 hours. This process is repeated three times to obtain activated bacterial solution. The bacterial solution is then inoculated into MRS liquid medium at a 2% inoculum and incubated at 37°C for 24 hours to obtain fermentation broth. The fermentation broth is centrifuged at 12000 rpm for 10 minutes to collect bacterial sludge. After washing three times with physiological saline, the viable count is adjusted to 1×10⁻⁶. 11 CFU / mL.

[0062] A freeze-drying protectant consisting of 110 g / L monosodium glutamate, 140 g / L skim milk, and 90 g / L trehalose (based on final concentration) was prepared. The freeze-drying protectant was added to the bacterial sludge at a ratio of 3 times the weight of the sludge, mixed thoroughly, and then subjected to vacuum freeze-drying to finally obtain Lactobacillus reuteri CCFM1466 bacterial powder.

[0063] Example 12: Application of Lactobacillus reuteri CCFM1466 Lactobacillus reuteri CCFM1466 can be used to prepare capsules. The specific preparation process is as follows: A bacterial suspension of Lactobacillus reuteri CCFM1466 is taken from a glycerol tube and streaked onto MRS solid medium. It is then cultured anaerobically at 37°C for 48 hours to obtain single colonies. A single colony is picked and inoculated into MRS liquid medium, and activated by anaerobic culture at 37°C for 24 hours. This process is repeated three times to obtain the activated bacterial suspension. The bacterial suspension is then inoculated into MRS liquid medium at a 2% inoculum and cultured anaerobically at 37°C for 24 hours to obtain the fermentation broth. The fermentation broth is centrifuged at 12000 rpm for 10 minutes, and the bacterial sludge is collected. The bacterial sludge is washed three times with physiological saline and then used for further processing. The viable cell count is adjusted to 1×10⁻⁶. 11 CFU / mL.

[0064] The bacterial suspension and sodium alginate solution were mixed evenly at a ratio of 1:10 and poured into a pressure-resistant bottle. Using a microencapsulation apparatus, the mixture of sodium alginate and bacterial suspension was added dropwise to a 3% CaCl2 solution via high-frequency oscillation, ensuring the droplets were uniform and free of clumping. Granulation was completed and the mixture was allowed to solidify for 40 minutes. The sample was obtained by centrifugation, and the residual CaCl2 solution on the surface of the microcapsules was washed away with physiological saline to obtain the finished microcapsules. Store at 20℃.

[0065] Example 13: Application of Lactobacillus reuteri CCFM1466 Lactobacillus reuteri CCFM1466 can be used to prepare tablets. The specific preparation process is as follows: The preparation method of Lactobacillus reuteri CCFM1466 bacterial powder is the same as in Example 11. The bacterial powder and skim milk are mixed evenly by a compounding method. The powder is then compressed into tablets using a tableting machine at a pressure of 2.4 t to obtain Lactobacillus reuteri CCFM1466 tablets, with the quality controlled at 250 mg ± 10 mg.

[0066] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. *Lactobacillus reuteri* ( Limosilactobacillus reuteri CCFM1466 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 10, 2025, with accession number GDMCC No: 65752.

2. A microbial inoculum containing *Lactobacillus reuteri* CCFM1466 as described in claim 1, characterized in that, The microbial agent contains *Lactobacillus reuteri* CCFM1466 as described in claim 1, or a fermentation broth containing *Lactobacillus reuteri* CCFM1466, or a freeze-dried powder containing *Lactobacillus reuteri* CCFM1466.

3. The microbial agent according to claim 2, characterized in that, It is prepared by culturing the *Lactobacillus reuteri* CCFM1466 as described in claim 1 in a culture medium, collecting the bacterial cells, mixing them with a freeze-drying protectant, and then freeze-drying them.

4. A drug containing the *Lactobacillus reuteri* CCFM1466 as described in claim 1.

5. The drug according to claim 4, characterized in that, The drug contains *Lactobacillus reuteri* CCFM1466, as well as a drug carrier and / or pharmaceutical excipients.

6. The use of the *Lactobacillus reuteri* CCFM1466 of claim 1 in the preparation of a medicament for the prevention and / or relief of ulcerative colitis.

7. The application according to claim 6, characterized in that, The drug contains ≥5 × 10⁻⁶ Lactobacillus reuteri CCFM1466. 9 CFU / g or 5×10 9 CFU / mL.

8. Food or health products containing *Lactobacillus reuteri* CCFM1466 as described in claim 1.

9. The use of Lactobacillus reuteri CCFM1466 as described in claim 1 in the preparation of products containing tetrahydrofolate; said products are food or health products.

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