Lactobacillus reuteri for producing tetrahydrofolic acid and application of lactobacillus reuteri in relieving ulcerative colitis

Abstract

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

Description

Technical Field

[0001] The present invention relates to a strain of Lactobacillus mucosae Roy which produces tetrahydrofolate and its application in alleviating ulcerative colitis, belonging to the field of microbial technology. Background Art

[0002] Ulcerative Colitis (UC) is a chronic and recurrent intestinal inflammatory bowel disease characterized by persistent inflammation and ulcer formation. The exact cause of UC has not been fully understood, but it is generally believed to be related to genetic, immune, and environmental factors. The symptoms of UC include diarrhea, abdominal pain, and bloody stools, etc., which seriously affect the quality of life of patients. Existing therapeutic drugs, such as Asacol and Mesalazine, etc., although can relieve symptoms, long-term use may cause side effects such as liver and kidney damage, gastrointestinal discomfort, and immunosuppression. Therefore, finding safe and effective adjuvant treatment means has always been a research hotspot.

[0003] The role of probiotics in alleviating UC has been widely studied and applied. Research shows that specific probiotics can significantly relieve UC symptoms through mechanisms such as enhancing intestinal barrier function, regulating immune response, inhibiting pro-inflammatory factors, and restoring the balance of intestinal flora. In addition, the metabolites produced by probiotics (such as short-chain fatty acids and bioactive molecules) have a significant promoting effect on intestinal health, with high safety and no obvious side effects, so it has become an important direction for adjuvant treatment of UC. Currently, the reported probiotics for UC treatment mainly play a role by producing short-chain fatty acids, regulating immune balance, or improving intestinal barrier function.

[0004] Tetrahydrofolate (THF), as the active form of folic acid, can directly promote the repair and regeneration of intestinal epithelial cells, participate in the synthesis processes of DNA and RNA, and is crucial for 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 UC treatment. However, the human body cannot synthesize folic acid de novo and must rely on exogenous supplementation or obtain it through the intestinal flora. The existing methods of supplementing synthetic folic acid have problems such as low metabolic efficiency and poor stability, and are limited in effect for people with folic acid metabolism disorders. Probiotics can directly produce THF in the intestine, 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 metabolism cycle, and improving the utilization rate of THF. Currently, there are no relevant reports on using probiotics to produce THF to alleviate UC. Therefore, screening out a strain of probiotics that can produce high yields of THF can not only directly supplement THF but also combine with the functions of probiotics themselves to further alleviate UC inflammation, providing new ideas and theoretical basis for UC treatment. Summary of the Invention

[0005] In view of the above technical problems, the first object of the present invention is to provide a strain of Limosilactobacillus reuteri CCFM1466, which is taxonomically named Limosilactobacillusreuteri, and has been deposited in Guangdong Provincial Microbiological Culture Collection Center on January 10, 2025, with a deposit number of GDMCC No: 65752, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] In one embodiment, the Limosilactobacillus reuteri CCFM1466 is derived from a healthy human fecal sample, and its 16S rDNA sequence is as shown in SEQ ID NO.1. The colony protrusions on the MRS solid culture medium are smooth, round, milky white, opaque, and have a diameter of 1 to 2 mm.

[0007] The present invention also provides a microbial preparation, which contains the Lactobacillus reuteri CCFM1466, or a fermentation liquid containing the Lactobacillus reuteri CCFM1466, or a freeze-dried powder containing the Lactobacillus reuteri CCFM1466.

[0008] In one embodiment, the preparation method of the microbial preparation is:

[0009] 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 a bacterial solution; then, the bacterial solution was centrifuged at 8000g for 20 min, the bacterial mud was collected, and resuspended with physiological saline to prepare a resuspension; then, the resuspension was fully mixed with a lyophilization protective agent to prepare a mixed solution; finally, the mixed solution was vacuum freeze-dried to prepare bacterial powder.

[0010] In one embodiment, the amount of the lyophilization protectant added is 25% to 50% of the total weight of the resuspension.

[0011] In one embodiment, the content of Lactobacillus reuteri CCFM1466 in the microbial preparation is at least 5×10 9 CFU / g or 5×10 9 CFU / mL.

[0012] The present invention also provides a method for culturing the Lactobacillus reuteri CCFM1466, which comprises culturing the Lactobacillus reuteri CCFM1466 in a culture medium at 35-40°C.

[0013] In one embodiment, the method is to culture the Lactobacillus reuteri CCFM1466 in MRS medium at 37 °C for at least 18 h.

[0014] The present invention also provides the use of the Lactobacillus reuteri CCFM1466 or the microbial preparation in the preparation of a drug for preventing and / or alleviating colitis.

[0015] In one embodiment, in the drug, the content of Lactobacillus reuteri CCFM1466 is ≥5×10 9 CFU / g or 5×10 9 CFU / mL.

[0016] In one embodiment, the drug contains the Lactobacillus reuteri CCFM1466, as well as a drug carrier and / or pharmaceutical excipients.

[0017] In one embodiment, the drug carrier includes microcapsules, microspheres, nanoparticles and / or liposomes.

[0018] In one embodiment, the pharmaceutical excipients include excipients and / or additives.

[0019] In one embodiment, the excipients include binders, fillers, disintegrants and / or lubricants.

[0020] In one embodiment, the additives include solubilizers, cosolvents, cosolvents and / or preservatives.

[0021] In one embodiment, the dosage form of the drug is powder, granule, capsule, tablet, pill or oral liquid.

[0022] The present invention also provides a food or health product containing the Lactobacillus reuteri CCFM1466.

[0023] The present invention also provides the use of the Lactobacillus reuteri CCFM1466 in the preparation of fermented foods.

[0024] The present invention also provides the use of the Lactobacillus reuteri CCFM1466 in the preparation of products containing tetrahydrofolic acid.

[0025] Beneficial effects:

[0026] 1. The Lactobacillus reuteri CCFM1466 screened by the present invention has the characteristic of producing tetrahydrofolic acid (THF), and the THF content in its fermentation supernatant reaches 94.453 ng / mL, which is significantly higher than that of other strains of the same genus and species.

[0027] 2. The Lactobacillus reuteri CCFM1466 screened by the present invention can significantly increase the content of THF in the colon and show significant potential for alleviating ulcerative colitis, which is specifically reflected in:

[0028] (1) Lactobacillus reuteri CCFM1466 significantly increased the content of THF in the colon;

[0029] (2) Lactobacillus reuteri CCFM1466 significantly alleviated DSS-induced weight loss;

[0030] (3) Lactobacillus reuteri CCFM1466 significantly alleviated the colon shortening caused by DSS;

[0031] (4) Lactobacillus reuteri CCFM1466 significantly improved DSS-induced colonic tissue pathology;

[0032] (5) Lactobacillus reuteri CCFM1466 significantly reduced the levels of IL-1β, IL-6 and TNF-α in DSS-induced colon tissue.

[0033] (6) Lactobacillus reuteri CCFM1466 significantly increased the level of IL-10 in DSS-induced colon tissue.

[0034] The strain has important application value in the fields of functional food and intestinal health, and can be used to prepare products for preventing and / or treating colitis.

[0035] Biomaterial Deposit

[0036] Limosilactobacillus reuteri CCFM1466, taxonomically named Limosilactobacillus reuteri, was deposited in the Guangdong Provincial Microbiological Culture Collection on January 10, 2025, with the deposit number GDMCC No: 65752, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the colony morphology of Lactobacillus reuteri CCFM1466.

[0038] Figure 2 This is a graph showing the THF production capacity of Lactobacillus reuteri CCFM1466.

[0039] Figure 3 This is a graph showing the effect of Lactobacillus reuteri CCFM1466 on the content of THF in the colon contents of colitis mice.

[0040] Figure 4Effect diagram of Lactobacillus mucosae CCFM1466 on body weight changes in colitis mice.

[0041] Figure 5 Effect diagram of Lactobacillus mucosae CCFM1466 on colon length in colitis mice.

[0042] Figure 6 Effect diagram of the improvement of colon tissue damage in colitis mice by Lactobacillus mucosae CCFM1466.

[0043] Figure 7 Effect diagram of Lactobacillus mucosae CCFM1466 on IL-1β level in the colon of colitis mice.

[0044] Figure 8 Effect diagram of Lactobacillus mucosae CCFM1466 on TNF-α level in the colon of colitis mice.

[0045] Figure 9 Effect diagram of Lactobacillus mucosae CCFM1466 on IL-6 level in the colon of colitis mice.

[0046] Figure 10 Effect diagram of Lactobacillus mucosae CCFM1466 on IL-10 level in the colon of colitis mice. Detailed implementation manners

[0047] The present invention will be further described below in conjunction with specific embodiments.

[0048] The strains involved in the following embodiments:

[0049] All the strains involved in the embodiments of this patent, including the main research object Lactobacillus mucosae CCFM1466 and other Lactobacillus mucosae used in the comparative experiments (such as L7, L52, L53, L54, L60, etc.), were self-isolated and screened from fecal samples of healthy adults.

[0050] The culture media involved in the following embodiments are as follows:

[0051] MRS liquid medium (1L): peptone 10.0 g, beef extract 10.0 g, yeast extract powder 5.0 g, diammonium hydrogen citrate 2.0 g, glucose 20.0 g, Tween 80 1.0 mL, anhydrous sodium acetate 2.0 g, magnesium sulfate heptahydrate 0.5 g, manganese sulfate monohydrate 0.25 g, and dipotassium hydrogen phosphate 2.0 g. The above components were added to distilled water and completely dissolved, and the pH was adjusted to between 6.8 and 7.2, and sterilized at 115 °C for 20 min.

[0052] MRS solid medium (1 L): Add 20 g of agar powder to the prepared MRS liquid medium. Sterilize at 115 °C for 20 min.

[0053] The detection methods involved in the following examples are as follows:

[0054] The content of THF in the fermentation supernatant and colonic contents of Lactobacillus mucosae CCFM1466 was detected by enzyme-linked immunosorbent assay (ELISA): The strain was inoculated into MRS liquid medium and cultured statically at 37 °C for 24 h. Centrifuge at 12,000 rpm for 5 min, discard the supernatant, and repeat washing the bacterial pellet 2 - 3 times with sterile normal saline and then resuspend. Dilute the bacterial suspension to 1.0×10 8 CFU / mL, and inoculate it into 5 mL of MRS liquid medium at an inoculation amount of 2%, and culture it statically and protected from light at 37 °C for 12 h. Take 1 mL of the bacterial liquid into a 2 mL brown centrifuge tube, centrifuge at 12,000 rpm for 5 min, and collect the fermentation supernatant for the determination of extracellular THF content.

[0055] Take about 0.1 g of mouse feces, add pre-cooled PBS at 4 °C in a ratio of weight (g): volume (mL) of 1:9, and homogenize it using a high-throughput tissue grinder. Centrifuge the sample at 4 °C and 12,000 rpm for 15 min, collect the supernatant, and perform THF detection according to the operation instructions of the THF assay kit.

[0056] Colonic tissue pathology: When dissecting the mouse, take about 0.5 cm of tissue from the end of the colonic tissue and place it in 4% paraformaldehyde solution for fixation for 24 h - 48 h. The H&E staining was completed by Wuhan Sevier Biotechnology Co., Ltd. The prepared sections were scanned and photographed using a Pannoramic MIlI digital slide scanner.

[0057] Cytokine determination: Take about 0.1 g of colonic tissue, add pre-cooled PBS at 4 °C in a ratio of weight (g): volume (mL) equal to 1:9, and homogenize it using a high-throughput tissue grinder. Then, centrifuge at 12,000 rpm for 15 min at 4 °C to separate the supernatant. Determine the contents of cytokines TNF-α, IL-β, IL-6, and IL-10 according to the method described in the kit instructions.

[0058] Example 1: Isolation, screening, strain identification and culture of Lactobacillus mucosae CCFM1466

[0059] 1. Isolation and screening

[0060] Take about 0.5 g of fecal samples from healthy adults, perform 10-fold serial dilutions with sterile normal saline, and sequentially prepare 10 -2 、10 -3, 10 -4 , 10 -5 , 10 -6 Diluent. Then, 100 μL of 10 -4 , 10 -5 , 10 -6 diluent was spread on MRS solid medium and cultured at 37 °C for 48 h. The colony morphology was observed and recorded; Dilution-coated plates with colony counts in the range of 30 - 300 were selected, and colonies with different morphologies were picked for streak isolation. After culturing at 37 °C for 48 h, the above operation was repeated until pure single colonies with consistent morphology were obtained. The pure colonies on MRS solid medium were picked and inoculated into 5 mL of MRS liquid medium and cultured under anaerobic conditions at 37 °C for 18 h; 1 mL of the bacterial solution was taken into a sterile centrifuge tube, centrifuged at 8000 rpm for 3 min, the supernatant was discarded to obtain the bacterial cells, and the bacterial cells were resuspended in 30% glycerol solution and stored in a -80 °C refrigerator.

[0061] 2. Strain identification

[0062] 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 alignment and identified as Limosilactobacillus reuteri, and named Limosilactobacillus reuteri CCFM1466.

[0063] 3. Cultivation

[0064] Limosilactobacillus reuteri CCFM1466 was inoculated on MRS solid medium and cultured at 37 °C for 48 h, and its colony morphology was observed. Figure 1 Shows the colony morphology of Limosilactobacillus reuteri CCFM1466 after culturing on MRS solid plate medium for 48 h. The colonies are round, white convex, and the surface edges are smooth.

[0065] Example 2: Ability of different Limosilactobacillus reuteri to produce THF

[0066] Limosilactobacillus reuteri CCFM1466 obtained in Example 1 and other Limosilactobacillus reuteri were inoculated into 5 mL of MRS liquid medium at an inoculation amount of 2%, and cultured statically at 37 °C for 24 h. 1 mL of the bacterial solution was taken and placed in a 2 mL brown centrifuge tube, centrifuged at 12,000 rpm for 5 min at 4 °C, and the fermentation supernatant was collected, with the blank medium as the reference. According to the instructions of the tetrahydrofolate assay kit, the THF content produced by Limosilactobacillus reuteri was measured (the analysis results are shown in Table 1 and Figure 2 ).

[0067] Table 1 THF production of different Limosilactobacillus reuteri

[0068]

[0069] Example 3: Effect of Limosilactobacillus reuteri on body weight changes in DSS-induced colitis mice

[0070] (1) Limosilactobacillus reuteri CCFM1466 and Limosilactobacillus reuteri L7 were respectively inoculated into MRS liquid medium, and after culturing at 37 °C for 18 h, a bacterial suspension was prepared, and the bacterial solution concentration was adjusted to 5×10 9 CFU / mL.

[0071] (2) Forty 6-week-old SPF-grade male C57BL / 6J mice were randomly divided into 4 groups (10 mice in each group), namely the control group, the model group, the Limosilactobacillus reuteri CCFM1466 intervention group (CCFM1466), and the Limosilactobacillus reuteri L7 intervention group (L7). The experimental period was 14 days. The first 7 days were the adaptation period, and all mice were given a standard diet and free drinking water; the 8th - 14th days were the intervention period. The control group was gavaged with 200 μL of normal saline every day, the model group was gavaged with 200 μL of normal saline every day and drank an aqueous solution containing 3% DSS, the CCFM1466 group was gavaged with 200 μl of 5×10 9 CFU / mL Limosilactobacillus reuteri CCFM1466 and drank an aqueous solution containing 3% DSS, and the L7 group was gavaged with 200 μL of 5×10 9 CFU / mL Limosilactobacillus reuteri L7 and drank an aqueous solution containing 3% DSS.

[0072] During the modeling period, the body weights of the mice were weighed and recorded daily (the results are shown in Figure 3)。The results showed that the weight loss of the mice in the model group was 19.68 ± 4.10%, while that of the mice in the Lactobacillus mucosae L7 group was 16.14 ± 3.31%, and that of the mice in the Lactobacillus mucosae CCFM1466 group was only 10.6 ± 4.62%. The analysis results showed that the weight loss in the CCFM1466 group was reduced by about 46% compared with the model group, indicating that Lactobacillus mucosae CCFM1466 had a significant alleviating effect on DSS-induced weight loss, and its effect was significantly better than that of the L7 intervention group.

[0073] Example 4: Effect of Lactobacillus mucosae CCFM1466 on the colon length of mice with DSS-induced colitis

[0074] The animal model was established in the same way as in Example 3. After the experiment, the mice were anesthetized by inhaling 1% - 1.5% isoflurane, dissected to remove the colon, and its length (from the end of the cecum to the anterior part of the rectum) was measured and photographed (the results are shown in Figure 4 )。The results showed that the colon length of the mice in the model group was significantly shorter than that of the control group mice, with an average length of 5.05 ± 0.34 cm. The average colon length of the mice in the Lactobacillus mucosae CCFM1466 group was 6.42 ± 0.14 cm, close to 6.962 ± 0.18 cm of the control group. While the average colon length of the mice in the Lactobacillus mucosae L7 group was 5.20 ± 0.39 cm. This indicated that Lactobacillus mucosae CCFM1466 was more effective than Lactobacillus mucosae L7 in alleviating colon shortening caused by colitis.

[0075] Example 5: Effect of different Lactobacillus mucosae on the THF content in the colon contents of mice with DSS-induced colitis

[0076] The animal model was established in the same way as in Example 3. About 0.1 g of mouse feces was taken and added to pre-cooled PBS at 4°C according to the ratio of weight (g): volume (ml) equal to 1:9, and homogenized using a high-throughput tissue grinder. The sample was centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was separated for the determination of THF. It was carried out according to the operation instructions of the tetrahydrofolic acid assay kit. The results showed ( Figure 5 ) that the THF content in the colon contents of the CCFM1466 strain group was the highest, reaching about 44.5 ng / ml, significantly higher than other groups; the THF content in the L7 strain group was about 10.2 ng / ml, also higher than the control group and the model group; while the THF contents in the control group and the model group were extremely low, about 1.2 ng / ml and 1.0 ng / ml respectively, and there was no significant difference between groups. This indicated that Lactobacillus mucosae, especially the CCFM1466 strain, had the ability to significantly increase the production of tetrahydrofolic acid in the colon contents.

[0077] Example 6: Effect of Lactobacillus mucosae CCFM1466 on colonic tissue injury in DSS-induced colitis mice

[0078] The method for establishing the animal model was the same as that in Example 3. After the experiment, the mice were anesthetized by inhalation of 1% - 1.5% isoflurane, dissected, and about 0.5 cm of distal colon tissue was taken for H&E staining. The prepared sections were scanned and photographed using a PannoramicMIlI digital slide scanner (the results are shown in Figure 6 ). The results showed that severe damage occurred in the colonic tissue of the DSS group mice, including the basic disappearance of the crypt structure, large-area ulcers, massive infiltration of inflammatory cells, and a large loss of goblet cells. In contrast, the intervention of Lactobacillus mucosae CCFM1466 strain significantly improved the above pathological conditions, reduced ulcers and inflammatory cell infiltration, increased the number of intestinal glands, and the colonic mucosa layer was relatively intact. The intervention of Lactobacillus mucosae L7 also had a certain improvement effect on colonic tissue injury, but the effect was not as good as that of Lactobacillus mucosae CCFM1466 strain, and its effect in reducing inflammatory cell infiltration and restoring the crypt structure was weaker. Generally speaking, Lactobacillus mucosae CCFM1466 had a better restorative effect on colonic tissue injury than Lactobacillus mucosae L7.

[0079] Example 7: Effect of Lactobacillus mucosae CCFM1466 on the level of IL-1β in colonic tissue of DSS-induced colitis mice

[0080] The method for establishing the animal model was the same as that in Example 3. After the experiment, the mice were fasted for 12 h and then sacrificed, the colon tissue was taken out, homogenized according to the ratio of tissue weight to tissue lysate of 1:9, and the supernatant was obtained after centrifugation, and the content of IL-1β was measured (the results are shown in Figure 7 ). The results showed that the level of IL-1β in the colonic tissue of the control group was 4.56 ± 0.47 pg / mg; after DSS treatment, the level of IL-1β in the colonic tissue of the model group mice increased significantly to 9.23 ± 0.54 pg / mg, while the intervention of Lactobacillus mucosae CCFM1466 significantly reduced the content of IL-1β, reducing it to 6.01 ± 0.21 pg / mg. At the same time, the intervention of Lactobacillus mucosae L7 strain also reduced the level of IL-1β to a certain extent, but the reduction effect was not significant.

[0081] Example 8: Effect of Lactobacillus mucosae CCFM1466 on the level of TNF-α in colonic tissue of DSS-induced colitis mice

[0082] The method for establishing the animal model was the same as that in Example 3. The method for treating the colon tissue was the same as that in Example 6. The results showed ( Figure 8) The level of TNF-α in the colon tissue of the control group was 4.87 ± 0.09 pg / mg; after DSS treatment, the level of TNF-α in the colon tissue of the model group mice increased significantly to 6.31 ± 0.22 pg / mg, while the intervention of Lactobacillus mucosae CCFM1466 significantly reduced the content of TNF-α, reducing it to 5.16 ± 0.21 pg / mg.

[0083] Example 9: Effect of Lactobacillus mucosae CCFM1466 on the level of IL-6 in the colon tissue of DSS-induced colitis mice

[0084] The method for establishing the animal model was the same as that in Example 3. The method for treating the colon tissue was the same as that in Example 6. The results showed ( Figure 9 ) The level of IL-6 in the colon tissue of the control group was 3.29 ± 0.19 pg / mg; after DSS treatment, the level of IL-6 in the colon tissue of the model group mice increased significantly to 5.52 ± 0.18 pg / mg, while the intervention of Lactobacillus mucosae CCFM1466 significantly reduced the content of IL-6, reducing it to 3.55 ± 0.19 pg / mg.

[0085] Example 10: Effect of Lactobacillus mucosae CCFM1466 on the level of IL-10 in the colon tissue of DSS-induced colitis mice

[0086] The method for establishing the animal model was the same as that in Example 3. The method for treating the colon tissue was the same as that in Example 6. The results showed ( Figure 10 ) The level of IL-10 in the colon tissue of the control group was 5.68 ± 0.28 pg / mg; after DSS treatment, the level of IL-10 in the colon tissue of the model group mice decreased significantly to 3.00 ± 0.45 pg / mg, while the intervention of Lactobacillus mucosae CCFM1466 significantly increased the content of IL-10, bringing it to 4.86 ± 0.35 pg / mg, and the effect was better than that of Lactobacillus mucosae L7.

[0087] Example 11: Application of Lactobacillus mucosae CCFM1466

[0088] Lactobacillus mucosae CCFM1466 can be used to prepare bacterial powder. The specific preparation process is as follows: Take a small amount of bacterial liquid from the glycerol tube and streak it on MRS solid medium, and culture it at 37 °C for 48 h to obtain single colonies; Pick the single colonies and inoculate them into MRS liquid medium, and culture it at 37 °C for 24 h, repeat 3 times to obtain an activated bacterial liquid. Inoculate the bacterial liquid into MRS liquid medium at an inoculation amount of 2%, and culture it at 37 °C for 24 h to obtain a fermentation broth. Centrifuge the fermentation broth at 12,000 rpm for 10 min to collect the bacterial sludge, wash it 3 times with physiological saline and set aside, and adjust the viable bacteria count to 1 × 10 11 CFU / mL.

[0089] Prepare a lyophilization protectant containing 110 g / L sodium glutamate, 140 g / L skim milk, and 90 g / L trehalose (by final concentration). Add the lyophilization protectant to the bacterial sludge at a ratio of 3 times the weight of the bacterial sludge, mix well, and then perform vacuum freeze-drying to finally obtain the powder of Limosilactobacillus reuteri CCFM1466.

[0090] Example 12: Application of Limosilactobacillus reuteri CCFM1466

[0091] Limosilactobacillus reuteri CCFM1466 can be used to prepare capsules. The specific preparation process of the capsules is as follows: Dip the bacterial solution of Limosilactobacillus reuteri CCFM1466 from the glycerol tube and streak it on the MRS solid medium, and culture it at 37 °C for 48 h under anaerobic conditions to obtain single colonies; Pick the single colonies and inoculate them into the MRS liquid medium, and culture them at 37 °C for 24 h under anaerobic conditions for activation culture, and repeat this operation 3 times to obtain the activated bacterial solution. Inoculate the bacterial solution into the MRS liquid medium at an inoculation amount of 2%, and culture it anaerobically at 37 °C for 24 h to obtain a fermentation broth. Centrifuge the obtained fermentation broth at 12,000 rpm for 10 min, collect the bacterial sludge, wash the bacterial sludge 3 times with physiological saline and then set aside, and adjust the viable cell count to 1×10 11 CFU / mL.

[0092] Mix the bacterial solution and the sodium alginate solution evenly at a ratio of 1:10, pour them into a pressure-resistant bottle, and use a microcapsule granulator to drop the mixed liquid of sodium alginate and the bacterial suspension into the 3% CaCl2 solution through high-frequency oscillation to make the liquid droplet particles non-connected and uniform. After granulation, cure for 40 min. Centrifuge to obtain the sample, wash away the residual CaCl2 solution on the surface of the microcapsules with physiological saline to obtain the finished microcapsules, and store them at -20 °C.

[0093] Example 13: Application of Limosilactobacillus reuteri CCFM1466

[0094] Limosilactobacillus reuteri CCFM1466 can be used to prepare tablets. The specific preparation process of the tablets is as follows: The preparation method of the powder of Limosilactobacillus reuteri CCFM1466 is the same as that in Example 11. Mix the powder of the bacteria and skim milk by the trituration method, and press the powder on a tablet press at a pressure of 2.4 t to obtain the tablets of Limosilactobacillus reuteri CCFM1466, and its quality is controlled at 250 mg ± 10 mg.

[0095] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Limosilactobacillus reuteri CCFM1466 was deposited in Guangdong Microbiological Culture Collection Center on January 10, 2025, with the deposit number GDMCC No: 65752.

2. A microbial agent containing Lactobacillus reuteri CCFM1466 according to claim 1, characterized in that: The microbial agent contains the Lactobacillus reuteri CCFM1466 according to claim 1, or a fermentation liquid containing the Lactobacillus reuteri CCFM1466, or a freeze-dried powder containing the Lactobacillus reuteri CCFM1466.

3. The microbial preparation according to claim 2, characterized in that The method is prepared by culturing the Lactobacillus reuteri CCFM1466 described in claim 1 in a culture medium for a period of time, collecting the bacteria, mixing the bacteria with a freeze-drying protective agent, and then freeze-drying the mixture.

4. A medicine containing the Lactobacillus reuteri CCFM1466 according to claim 1.

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

6. Use of Lactobacillus reuteri CCFM1466 according to claim 1 in the preparation of a medicament for preventing and / or alleviating colitis.

7. The use according to claim 6, characterized in that: In the drug, the content of Lactobacillus reuteri CCFM1466 is ≥5×10 9 CFU / g or 5×10 9 CFU / mL.

8. Food or health product containing Lactobacillus reuteri CCFM1466 according to claim 1.

9. Use of Lactobacillus reuteri CCFM1466 according to claim 1 in the preparation of products containing tetrahydrofolate.

10. Use of the Lactobacillus reuteri CCFM1466 according to claim 1 in preparing fermented food.

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