A strain of Lactobacillus reuteri MeetU capable of antagonizing Helicobacter pylori and repairing mucosal barrier damage and its application

Abstract

The present invention provides a strain of Lactobacillus reuteri MeetU that can antagonize Helicobacter pylori and repair mucosal barrier damage and its application. The Lactobacillus reuteri MeetU is deposited in the China Center for Type Culture Collection, with a preservation date of December 11, 2023, and a preservation number of CCTCC NO: M 20232531. The MeetU can effectively antagonize Helicobacter pylori and repair mucosal barrier damage. Experiments show that it is acid-resistant and bile-resistant, has good safety, and can effectively inhibit Helicobacter pylori, Escherichia Ferguson, Vibrio parahaemolyticus and Cryptococcus neoformans. In addition, it can also increase the metabolic production of butyric acid and isobutyric acid, reduce the adhesion of Helicobacter pylori, and effectively repair gastric mucosal barrier damage caused by Helicobacter pylori. Finally, MeetU can also repair genital mucosal barrier damage caused by Gardnerella, especially when used in combination with Lactobacillus rhamnosus BB520, the two have a synergistic effect, which can significantly improve the repair effect.

Description

Technical Field

[0001] The invention belongs to the technical field of Lactobacillus reuteri, and in particular relates to a Lactobacillus reuteri MeetU capable of antagonizing Helicobacter pylori and repairing mucosal barrier damage and an application thereof. Background Art

[0002] Helicobacter pylori (Hp) often parasitizes in gastric mucosal tissue. After infection, it mainly causes diseases such as chronic gastritis and peptic ulcer. It is closely related to diseases such as gastric cancer and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. It is listed as a Class I biological carcinogen by the World Health Organization (WHO). Globally, about half of the people have been infected with Helicobacter pylori. The infection rate of Helicobacter pylori varies greatly in different countries and regions and among different ethnic groups. It is mainly closely related to the socioeconomic level, population density, public health conditions and water supply. The infection rate of Helicobacter pylori increases with age. Infection with Helicobacter pylori is the basic cause of the disease. Many people are infected with Helicobacter pylori in childhood, and adults can also be infected with Helicobacter pylori. Before symptoms appear, Helicobacter pylori can colonize the gastric mucosa for many years. Most people infected with Helicobacter pylori do not have peptic ulcers. It is not clear why only some people develop peptic ulcers after infection. Some people may not have obvious symptoms or may not develop the disease after being infected with Helicobacter pylori. The symptoms of Helicobacter pylori infection vary depending on the site of infection. Newly infected people may experience acute gastritis. About 30% of Helicobacter pylori infected people develop chronic gastritis, 10% to 20% develop peptic ulcers, and a few develop gastric cancer and MALT lymphoma. The treatment of Helicobacter pylori is usually based on antibiotics, which have significant side effects.

[0003] Mucosal barrier damage includes gastrointestinal mucosal barrier damage and genital mucosal barrier damage. There are many reasons for gastric mucosal damage. The most common cause is Helicobacter pylori infection. Common causes and mechanisms of intestinal mucosal barrier damage include ischemia and hypoxia, ischemia and reperfusion, inflammatory mediators, bacterial endotoxins, intestinal flora imbalance, immune abnormalities, nutritional disorders and total parenteral nutrition, severe trauma, systemic and gastrointestinal severe infections, etc. Reproductive tract mucosal barrier damage is usually caused by pathogenic bacteria. Clinically, the occurrence, development and outcome of many diseases are related to mucosal barrier damage. Therefore, how to repair mucosal barrier damage safely, effectively and without toxic side effects requires further research. Summary of the invention

[0004] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a strain of Lactobacillus reuteri MeetU that can antagonize Helicobacter pylori and repair mucosal barrier damage and its application.

[0005] Technical solution: To achieve the above-mentioned invention object, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a strain of Lactobacillus reuteri capable of repairing mucosal barrier damage, wherein the Lactobacillus reuteri is Lactobacillus reuteri MeetU (Limosilactobacillus reuteri MeetU), which is deposited in the China Center for Type Culture Collection on December 11, 2023, and the deposit number is CCTCC NO: M20232531.

[0007] In a second aspect, the present invention provides a composition comprising the Lactobacillus reuteri MeetU.

[0008] In a third aspect, the present invention provides the use of the Lactobacillus reuteri MeetU and the composition in the preparation of a product for repairing mucosal barrier damage.

[0009] As a specific embodiment, the mucosal barrier damage includes gastrointestinal mucosal barrier damage and reproductive tract mucosal barrier damage.

[0010] As a specific embodiment, the mucosal barrier damage is gastric mucosal barrier damage caused by Helicobacter pylori.

[0011] In a fourth aspect, the present invention provides use of the Lactobacillus reuteri MeetU and the composition in the preparation of an antibacterial agent.

[0012] As a specific embodiment, the antibacterial agent can inhibit Helicobacter pylori, Escherichia fergusonii, Vibrio parahaemolyticus and / or Cryptococcus neoformans.

[0013] In a fifth aspect, the present invention provides a composition, comprising Lactobacillus reuteri MeetU and Lactobacillus rhamnosus BB520, wherein Lactobacillus rhamnosus BB520

[0014] (Lacticaseibacillus rhamnosus BB520), deposited in China Center for Type Culture Collection, deposited on December 28, 2023, deposited with CCTCC NO: M 20232713. Preferably, in the composition, the ratio of the amount of live bacteria of Lactobacillus reuteri MeetU to Lactobacillus rhamnosus BB520 is (1-5): (1-5), more preferably 1:1.

[0015] In a sixth aspect, the present invention provides the use of the composition comprising Lactobacillus reuteri MeetU and Lactobacillus rhamnosus BB520 in the preparation of a product for repairing genital mucosal barrier damage.

[0016] Furthermore, the genital tract mucosal barrier damage is genital tract mucosal barrier damage caused by Gardnerella.

[0017] The products described in the present invention include food, antibacterial agents or medicines.

[0018] Beneficial effects: Compared with the prior art, the present invention provides a new strain of Lactobacillus reuteri MeetU, which can effectively antagonize Helicobacter pylori and repair mucosal barrier damage. Experiments show that the Lactobacillus reuteri MeetU is acid-resistant and bile-resistant, has good safety, and can effectively inhibit Helicobacter pylori, Escherichia Ferguson, Vibrio parahaemolyticus and Cryptococcus neoformans. In addition, it can also increase the metabolic production of butyric acid and isobutyric acid, reduce the adhesion of Helicobacter pylori, and effectively repair gastric mucosal barrier damage caused by Helicobacter pylori. Finally, MeetU can also repair genital mucosal barrier damage caused by Gardnerella, especially when used in combination with Lactobacillus rhamnosus BB520, the two have a synergistic effect, which can significantly improve the repair effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the biological evolution tree of the MeetU strain.

[0020] Figure 2 This is a Gram-stained microscope image of the MeetU strain.

[0021] Figure 3 MeetU inhibits Helicobacter pylori adhesion to human gastric mucosal cells GES-1.

[0022] Figure 4 MeetU metabolizes short-chain fatty acid content.

[0023] Figure 5 The effect of MeetU on the gene expression of gastric mucosal tight junction proteins ZO-1 and Occludin.

[0024] Figure 6 The effect of MeetU on the gene expression of gastric mucins MUC-1 and MUC-2.

[0025] Figure 7 The effect of MeetU on the gene expression of tight junction proteins ZO-1 and Occludin in reproductive tract mucosa.

[0026] Figure 8 The effect of MeetU on the gene expression of reproductive tract mucins MUC-1 and MUC-2. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0028] Example 1 Screening of highly active cultivable Gram-trophic strains

[0029] In order to obtain strains that are beneficial to the human body, healthy intestinal samples of young people with healthy physical index but BMI index ≤ 15 were selected and spread on MRS agar medium after gradient dilution. The same sample was spread twice in parallel and cultured in anaerobic and 5% oxygen environments, respectively, and the culture temperature was 35-37°C. Single colony strains were selected from MRS agar medium and placed in 15ml MRS liquid medium. The selected single colony was placed in a 35-37°C shaker for 8-10h to obtain fermentation broth. By detecting the OD600 of the fermentation broth and comparing the amount of fermentation broth with a microscope, the samples with poor liquid fermentation effect or no obvious bacterial amplification were discarded, and 0.5ml of the remaining fermentation broth was taken for strain sequencing and identification. 2ml of the fermentation broth was added to 2ml of 30%-40% glycerol solution for precooling and freezing to -80°C for storage of alternative strains. Gram-positive strains with higher OD600 during the fermentation process were selected as alternative strains, with a total of 85 strains, numbered Y1-Y85.

[0030] Example 2 In this example, the strains screened in Example 1 were screened for acid resistance and bile salt resistance.

[0031] The 85 bacterial strains in Example 1 were screened for acid and bile salt resistance. After adding MRS culture medium containing 0.3% ox bile salts and MRS culture medium with pH=3.5 to two 96-well sterile ELISA plates, 10 μL of oleic acid was added to each well for oil sealing. The 85 bacterial strains in Example 1 were inoculated into each well according to the number and cultured. The OD values ​​of the culture medium and the blank control in each ELISA plate before inoculation, 2 h of culture, 4 h of culture, 8 h of culture, and 16 h of culture were recorded. According to the changes in the OD values ​​of the strains on the two culture media, the strains with significantly increased OD values ​​and normal growth in the MRS culture medium containing 0.3% ox bile salts and the MRS culture medium with pH=3.5 were screened as candidate strains with high acid and bile salt resistance, and there were 5 strains in total.

[0032] Example 3 Identification, preservation and collection of bacterial strains

[0033] The 5 strains in Example 2 were taxonomically identified by 16S rRNA sequencing. The PCR amplification primer sequences were: 8F: 5'-AGAGTTTGATCCTGGCTCA-3'; 1510R: 5'-GGTTACCTTGTTACGACTT-3'. The PCR amplification products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing and identification. The obtained 16SrDNA gene sequences were compared in Genebank, and one strain belonging to the currently known probiotic species was selected and named MeetU. The other strains were Staphylococcus or Bacillus. The nucleotide sequence of the 16S rDNA of the MeetU strain is Sequence 1 in the sequence table; after 16S rDNA gene comparison, the similarity rate with Limosilactobacillus reuteri in Genebank reached 99%; combined with microbial system identification, the MeetU strain was determined to be a reuteri mucus Lactobacillus, named as Reuteri mucus Lactobacillus MeetU (Limosilactobacillus reuteri MeetU), its 16S rDNA is shown in SEQ ID NO.1, and the biological evolution tree is shown in Figure 1 As shown, the Gram staining result is Gram-positive, such as Figure 2 shown.

[0034] (1) Preservation of strains

[0035] After streaking the selected MeetU strain on MRS agar medium for 48 hours, a single colony was selected and placed in 10 ml liquid culture medium. The culture medium was placed in a constant temperature culture at 37°C for 12-16 hours, and the OD of the culture medium was measured. 600 When the value is ≥0.6, add glycerol solution in a 1:1 volume ratio to the culture medium. The concentration of the glycerol solution is 30%-40%. Mix well with a pipette and dispense into 2 ml sterile cryopreservation tubes. Precool at 4°C for 2 hours, then prefreeze at -20°C for 4 hours, and finally transfer to a -80°C refrigerator or store in liquid nitrogen.

[0036] (2) Collection of strains

[0037] The biologically identified Limosilactobacillus reuteriMeetU was deposited in the China Center for Type Culture Collection (CCTCC) on December 11, 2023, with the deposit number CCTCC NO:M 20232531. The deposit address is Wuhan University, Wuhan City, Hubei Province, People's Republic of China.

[0038] Example 4 Hemolysis test of Lactobacillus reuteri MeetU

[0039] The preserved Lactobacillus reuteri MeetU was inoculated into 5mL MRS at a 2% inoculation rate, Enterococcus faecalis (β-hemolytic, CICC23658, purchased from the China Industrial Microbiological Culture Collection Management Center) was used as a positive control, and a blank culture medium was used as a negative control. All strains were cultured anaerobically at 37°C for 12 hours in MRS culture medium to obtain activated strains. 2.5μL of each activated strain was inoculated on a Columbia blood plate (Shanghai Chromagar Microbiology Technology Co., Ltd.), with 3 parallels set up for each group. After anaerobic culture at 37°C for 48 hours, observation was performed. A hemolytic ring with a clear boundary and completely transparent was formed around the colony of the positive control strain, which was β-hemolysis; the culture medium around the colony of Lactobacillus reuteri MeetU did not change, which was γ-hemolysis, that is, no hemolysis. Therefore, there is no risk of hemolysis when applied to the human body.

[0040] Example 5 The ability of Lactobacillus reuteri MeetU to inhibit pathogenic bacteria

[0041] Lactobacillus reuteri RC-14 and Lactobacillus reuteri DSM17938 were selected as control test strains of Lactobacillus reuteri MeetU, and were inoculated in 50 ml of MRS liquid medium, respectively. After culturing at 37°C for 16 h, the fermentation broth was centrifuged at 10000 r / min for 10 min, and the supernatant and bacteria were collected respectively. The bacteria were washed twice with 0.9% sterile saline, and then prepared into OD 600 It is a bacterial suspension of 0.5-0.8, stored at 4°C for later use.

[0042] Helicobacter pylori SS1, Escherichia fergusonii ATCC 35469, and Vibrio parahaemolyticus ATCC 17802 were selected as test strains for in vitro antibacterial experiments, and the fermentation supernatant and bacterial suspension of each strain were subjected to an inhibition zone test using the agar punch method. Metronidazole solution with a concentration of 0.05 mg / mL was used as a positive control for inhibiting Helicobacter pylori SS1, erythromycin solution with a concentration of 0.05 mg / mL was used as a positive control for inhibiting Escherichia fergusonii ATCC 35469 and Vibrio parahaemolyticus ATCC 17802, and amphotericin B with a concentration of 0.05 mg / mL was used as a positive control for Cryptococcus neoformans ATCC 32609. The MRS culture medium was adjusted to have the same pH value as the supernatant as a blank control. The antibacterial results of each strain are shown in Table 1.

[0043] Table 1 Evaluation of antibacterial ability

[0044]

[0045] Note: a is significantly different from RC-14, b is significantly different from DSM17938

[0046] Compared with the positive control group and the blank control group, the bacterial suspension and supernatant of Lactobacillus reuteri MeetU had significant antibacterial activity against Helicobacter pylori, Escherichia ferguson, Vibrio parahaemolyticus and Cryptococcus neoformans, and the antibacterial ability of Lactobacillus reuteri MeetU suspension against the three pathogenic bacteria of Helicobacter pylori, Vibrio parahaemolyticus and Cryptococcus neoformans was significantly better than that of Lactobacillus reuteri RC-14. The antibacterial ability of Lactobacillus reuteri MeetU suspension and fermentation supernatant against Escherichia ferguson, Campylobacter jejuni and Cryptococcus neoformans was significantly better than that of Lactobacillus reuteri DSM17938. It can be seen that the fermentation metabolites of Lactobacillus reuteri MeetU contain antibacterial components, and the bacteria can still inhibit pathogenic bacteria, so it can be used in intervention preparations to inhibit pathogenic bacteria and regulate bacterial flora.

[0047] Example 6 Effect of Lactobacillus reuteri MeetU in removing Helicobacter pylori from human gastric mucosal cell GES-1

[0048] In a 96-well plate, cells (10 4 1×10 cells / well) were washed with PBS for 3 times and 0.05 mL of Helicobacter pylori SS1 (1×10 8 CFU / mL was resuspended in RPMI-1640) and cultured at 37°C and 5% CO2 for 2 h. The cells were then washed three times with PBS to remove the unadsorbed Helicobacter pylori. Then 0.05 mL of live or dead lactic acid bacteria (1×10 8 CFU / mL was resuspended in RPMI-1640, and the dead bacteria were heat-inactivated at 85°C for 3h), and then cultured in an incubator at 37°C and 5% CO2 for 1h; after washing with PBS 5 times, 200μL urease reagent was added and cultured for 3h, and then the absorbance was measured at a wavelength of 550nm using an ELISA reader. The negative control was the OD value measured when only cells were present, and the positive control was the OD value measured when Helicobacter pylori and cells coexisted, and the adhesion of Helicobacter pylori was defined as 100%.

[0049] Adhesion force of Helicobacter pylori = (OD of experimental group - OD of negative control) / (OD of positive control - OD of negative control) x 100%.

[0050] The results of inhibiting the adhesion of Helicobacter pylori to human gastric mucosal cells GES-1 by Lactobacillus reuteri MeetU and Lactobacillus reuteri DSM17938 are shown in 3. The results show that Lactobacillus reuteri MeetU has a strong effect of reducing the adhesion of Helicobacter pylori that has adhered to GES-1. The live bacteria can reduce the adhesion of Helicobacter pylori to 6.5%, and the heat-inactivated Lactobacillus reuteri MeetU can reduce the adhesion of Helicobacter pylori to 12.7%, and the effect of reducing the adhesion of Helicobacter pylori is significantly better than that of DSM17938. Therefore, live or heat-inactivated Lactobacillus reuteri MeetU can be used in the removal preparation after Helicobacter pylori infection.

[0051] Example 7 Detection of metabolic short-chain fatty acids

[0052] Based on gas chromatography-mass spectrometry (GC-MS), the short-chain fatty acids in the fermentation products of Lactobacillus reuteri MeetU were detected. The acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid standards were prepared into ten gradient standard concentrations of 0.02-500 μg / mL using ether. Weigh 1 mL of fermentation broth into an EP tube, add 15% phosphoric acid solution (50 μL), internal standard solution (100 μL) and ether solution (400 μL) in turn, mix well and centrifuge to obtain the supernatant. The supernatant was mixed with 85% orthophosphoric acid (20 μL / mL) and placed at 4°C for 1 hour. Finally, the supernatant was centrifuged and transferred to a gas chromatography bottle, and the concentration of short-chain fatty acids was detected using GC-8A. The results are as follows. Figure 4 As shown in the figure, compared with Lactobacillus reuteri RC-14 and DSM17938, except for butyric acid and isobutyric acid, there was no significant difference in the metabolic production of other short-chain fatty acids. However, the amount of butyric acid and isobutyric acid produced by Lactobacillus reuteri MeetU was significantly higher than that of RC-14 and DSM17938. Butyric acid is a fatty acid necessary for the repair of damaged mucosa, so MeetU helps to supplement the butyric acid needed by the human body.

[0053] Example 8 Repairing effect of Lactobacillus reuteri MeetU on gastric mucosal barrier damage caused by Helicobacter pylori

[0054] Twelve male and female mice of 3 weeks of age were selected, of which 6 were blank control group (BL1 group) and the remaining 18 were used to establish the Helicobacter pylori infection mouse model. They were randomly divided into three groups, 6 in each group, with equal numbers of males and females in each group, namely positive control group (BL2 group), intervention group (TE group) and intervention control group (TE2 group). In addition to normal diet, the blank control group and the positive control group were given 50 μL of normal saline by intragastric administration every day, and the intervention group was given 50 μL of bacterial suspension (containing about 3.0×10 live Lactobacillus MeetU) by intragastric administration every day. 7CFU), and the control group was given 50 μL triple antibiotic suspension (containing omeprazole 0.6 mg, amoxicillin 0.7 mg, and metronidazole 0.5 mg) by intragastric administration every day. After 14 days of intervention, the gene expressions of gastric mucosal tight junction proteins ZO-1 and Occludin were measured by real-time fluorescence quantitative PCR as indicators of gastric mucosal physical barrier function, and the gene expressions of gastric mucins MUC-1 and MUC-2 were measured as indicators of gastric mucosal chemical barrier function. The results were as follows: Figure 5 , Figure 6 shown.

[0055] The results showed that Helicobacter pylori infection reduced the gene expression of ZO-1, Occludin, MUC-1, and MUC-2 in the gastric mucosa, significantly reduced the physical barrier function and chemical barrier function of the gastric mucosa, and could lead to rapid deterioration of the gastric mucosal barrier function, ultimately hindering normal absorption and secretion activities. After intervention with Lactobacillus reuteri MeetU, the gene expression of ZO-1, Occludin, MUC-1, and MUC-2 in the gastric mucosa was significantly repaired and restored, while antibiotic treatment did not achieve this effect, indicating that Lactobacillus reuteri MeetU can significantly increase the thickness of the colonic mucus layer by increasing the expression of mucin, thereby reducing the contact area between pathogenic bacteria and gastric mucosal epithelial cells on the gastric mucosa and strengthening the chemical intestinal barrier function of the colon.

[0056] Example 9: Repair effect of Lactobacillus reuteri MeetU on genital mucosal barrier damage caused by Gardnerella

[0057] Two-month-old female rats were selected, and the infection model mice were genital tract infection Gardnerella infection model mice according to the grouping and intervention scheme listed in Table 2. They were randomly divided into groups, with 5 mice in each group. The Lactobacillus rhamnosus BB520 strain used was deposited in the China Center for Type Culture Collection (CCTCC) on December 28, 2023, with the deposit number CCTCC NO:M 20232713, and the deposit address is Wuhan University, Wuhan, Hubei Province, People's Republic of China. In addition to normal diet, the blank control group and the positive control group were given 20 μL of normal saline daily to irrigate the reproductive tract, the metronidazole group was given 20 μL of vitamin suspension daily to irrigate the reproductive tract (containing metronidazole 0.3 mg), and the other groups were given 20 μL of the corresponding bacterial suspension daily to irrigate the reproductive tract, and the amount of live bacteria in 20 μL was about 1.0×10 7CFU. In the TE3 and TE4 groups, the two bacteria were mixed and resuspended in a 1:1 ratio, and the concentration of live bacteria was the same as that in the TE1 and TE2 groups. After 14 days of intervention, the gene expression of the genital mucosal tight junction proteins ZO-1 and Occludin was used as an indicator of the physical barrier function of the genital mucosa by real-time fluorescence quantitative PCR technology, and the gene expression of the genital mucins MUC-1 and MUC-2 was detected as an indicator of the chemical barrier of the genital mucosa. The results were as follows: Figure 7 , Figure 8 shown.

[0058] Table 2 Experimental groups

[0059] serial number Group Experimental mice BL1 Blank control group Healthy mice BL2 Positive control group Infection model mice TE0 Metronidazole Infection model mice TE1 Lactobacillus reuteriMeetU Infection model mice TE2 Lactobacillus rhamnosus BB520 Infection model mice TE3 Lactobacillus mucilaginosus MeetU + Lactobacillus rhamnosus BB520 Infection model mice TE4 Lactobacillus reuteri RC-14 + Lactobacillus rhamnosus BB520 Infection model mice

[0060] The results showed that compared with the blank control group, Gardnerella infection of the genital mucosa led to a significant decrease in the gene expression of ZO-1, Occludin, MUC-1, and MUC-2, that is, the expression of genes related to tight junction proteins and mucins was reduced, and the mechanical and chemical barriers of the mucosa were destroyed. The intervention treatment with the antibiotic metronidazole increased and restored the gene expression of ZO-1, Occludin, MUC-1, and MUC-2 in the genital mucosa, but the effect was not significant. Lactobacillus mucosus MeetU and Lactobacillus rhamnosus BB520 significantly increased the gene expression of ZO-1, Occludin, MUC-1, and MUC-2 after intervention in model mice, indicating that the two strains can repair the damaged mechanical and chemical barriers of the genital mucosa, alleviate the normal absorption and secretion activities of the genital mucosa, increase the expression of mucins, thereby significantly increasing the thickness of the mucosal mucus layer, and strengthening the chemical intestinal barrier function of the colon. After Lactobacillus reuteri MeetU was combined with Lactobacillus rhamnosus BB520, the expression of genes related to tight junction proteins and mucins in the reproductive mucosa was significantly improved compared with other groups, indicating that the composite bacteria of Lactobacillus reuteri MeetU and Lactobacillus rhamnosus BB520 can significantly repair the genital mucosal barrier damage caused by Gardnerella, the strains can cooperate with each other, and this combination method is superior to other combination methods.

[0061] Example 10 Industrial fermentation of Lactobacillus reuteri MeetU and preparation of freeze-dried powder

[0062] In order to facilitate the wider application of Lactobacillus reuteri MeetU and facilitate industrial expansion of culture, the freeze-dried powder preparation process is as follows:

[0063] (1) Culture medium configuration.

[0064] The configuration scheme of the industrial high-density fermentation of Lactobacillus reuteri MeetU culture medium is as follows: 10g / L soy peptone, 10g / L beef extract powder, 10g / L yeast extract powder, 40g / L anhydrous glucose, 3g / L dipotassium hydrogen phosphate, 2g / L diammonium hydrogen citrate, 5g / L sodium acetate, 0.5g / L magnesium sulfate, 1g / L manganese sulfate, 1g / L L-cysteine ​​hydrochloride, and 1g / L soybean lecithin. All raw materials are food-grade raw materials.

[0065] (2) Liquid culture

[0066] After the culture medium is prepared, the pH value is adjusted to 6.5, sterilized at 121° C. for 15 minutes, and when cooled to 38° C.-39° C., the fermenter is inoculated and cultured at an inoculation rate of 3%-15%, preferably 12%. The culture temperature is 36° C.-39° C., the pH value is 5.5-6.5, and the fermentation liquid is purged with nitrogen and stirred slowly during the fermentation process.

[0067] (3) Centrifugation

[0068] After 8-12 hours of cultivation, the viable bacteria reached 10 8 CFU / mL-10 10 CFU / mL, based on OD 600 After the growth curve entered the plateau phase, the fermentation broth was quickly cooled to below 15°C and centrifuged at 5000 rpm using a tubular centrifuge to harvest the bacterial sludge.

[0069] (4) Protective agent emulsification and freeze-drying

[0070] The harvested sludge is diluted to a water content of 60%-80%, and an equal volume of sludge protective agent is added for emulsification; the composition of the sludge protective agent is: 70% water, 10% skim milk powder, 5% sucrose, 5% mannitol, 2% Tween-80, 2% betaine, 1% sodium glutamate, and 5% soluble starch. After stirring evenly, it is loaded into a vacuum freeze dryer for freeze vacuum drying, and the thickness of the freeze-dried material is ≤1cm. The temperature curve of the freeze dryer is: -50℃ for 4h, the temperature is increased by 5℃ every 2h, the temperature is 0℃ at the 24th hour, and then the temperature is increased by 3℃ every 2h, and the final maximum temperature is 30℃ and remains unchanged until the material is dry. The characteristics of the material drying are that the moisture content is less than 5% and the water activity is 0.11-0.22aw.

[0071] The activity of freeze-dried bacterial powder can reach 3.6×10 11 CFU / g, which is convenient for storage. It is also convenient for preparing preparations such as inhibiting infection by pathogenic bacteria such as Helicobacter pylori and repairing damaged gastric mucosa or reproductive tract mucosa barriers.

[0072] The implementation modes of the present invention have been described in detail above in conjunction with specific embodiments, but the present invention is not limited to the above-mentioned implementation modes, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A strain of Lactobacillus reuteri capable of repairing mucosal barrier damage, characterized in that: The Lactobacillus reuteri is Limosilactobacillus reuteri MeetU, which is deposited in the China Center for Type Culture Collection on December 11, 2023, and the deposit number is CCTCC NO: M 20232531.

2. A composition, characterized in that The composition comprises the Lactobacillus reuteri MeetU according to claim 1.

3. Use of the Lactobacillus reuteri MeetU according to claim 1 and the composition according to claim 2 in the preparation of a medicament for repairing mucosal barrier damage, characterized in that: The mucosal barrier damage includes gastrointestinal mucosal barrier damage and reproductive tract mucosal barrier damage.

4. Use of the Lactobacillus reuteri Meetu according to claim 1 and the composition according to claim 2 in the preparation of an antibacterial agent, characterized in that: The antibacterial agent can inhibit Helicobacter pylori, Escherichia Ferguson, Vibrio parahaemolyticus and / or Cryptococcus neoformans.

5. A composition, characterized in that The composition comprises the Lactobacillus reuteri MeetU and Lactobacillus rhamnosus BB520 described in claim 1, wherein the Lactobacillus rhamnosus BB520 (Lacticaseibacillusrhamnosus BB520) is deposited in the China Center for Type Culture Collection on December 28, 2023, and the deposit number is CCTCC NO: M 20232713.

6. Use of the composition according to claim 5 in the preparation of a medicament for repairing reproductive tract mucosal barrier damage.

7. The use according to claim 6, characterized in that: The genital tract mucosal barrier injury is genital tract mucosal barrier injury caused by Gardner's disease.

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