Gastric-derived lactobacillus fermentum mucus LAF-09 and application thereof

Abstract

The application discloses a stomach-derived limosilactobacillus fermentum LAF-09 and application thereof, and the preservation number of the stomach-derived limosilactobacillus fermentum LAF-09 is GDMCC No. 65656. The inventor of the application screens a stomach-derived limosilactobacillus fermentum LAF-09 from stomach mucosa tissue of healthy people, the strain has strong acid resistance and high proliferation capacity in the stomach, has strong adhesion properties to stomach epithelial GSE-1 cells, can be effectively applied to prevent and treat gastric ulcers caused by indomethacin, and has no obvious side effects.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and more specifically, this invention relates to a gastric-derived fermenting Lactobacillus mucinus LAF-09 and its applications. Background Technology

[0002] Peptic ulcer disease (PUD) is a common gastrointestinal disease, generally defined as a mucosal tear in the stomach or duodenum with a diameter greater than 3-5 mm and a depth reaching the submucosa. Helicobacter pylori infection and the use of nonsteroidal anti-inflammatory drugs (NSAIDs) are currently the main contributing factors to PUD. With the widespread adoption of Helicobacter pylori eradication therapy and improved sanitation worldwide, the prevalence of PUD is declining, but the prevalence of NSAID-related gastric ulcers is increasing. Although NSAIDs have a clear risk of causing ulcers, due to their good anti-inflammatory, analgesic, and antipyretic effects, they are the second largest class of drugs after anti-infectives, currently accounting for approximately 8% of global prescriptions. It is estimated that more than 30 million people take NSAIDs daily. However, in addition to their anti-inflammatory and analgesic effects, NSAIDs also cause numerous side effects, with gastrointestinal reactions being the most prominent. It has been calculated that the incidence of gastric ulcers in people taking NSAIDs is 40 times higher than in the general population; and among people who take NSAIDs long-term, approximately 40% of patients are found to have peptic ulcers under endoscopy. NSAIDs have become the second leading cause of peptic ulcers, second only to Helicobacter pylori infection.

[0003] Currently, the clinical approach to preventing NSAID-related gastric ulcers is often through concurrent administration of proton pump inhibitors (PPIs). In recent years, due to the increasing number of NSAID prescriptions, the use of PPIs, such as omeprazole, has also increased. PPIs are characterized by their potent inhibition of gastric acid secretion; long-term use can significantly alter the gastric pH, leading to dysbiosis and potentially exacerbating gastric damage caused by NSAIDs.

[0004] Recent research has aimed to prevent and treat NSAID-related gastric ulcers using non-antibiotic substances such as probiotics, prebiotics, plant extracts, bioactive proteins, and polysaccharides. However, current probiotic sources are mainly from the gut or dairy products, and the highly acidic environment and high peristalsis of the stomach result in extremely low survival and colonization rates of probiotics, weakening their protective effect in the stomach and limiting their efficacy in preventing and treating NSAID-related gastric ulcers. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a strain that can be used to prevent and treat gastrointestinal diseases, especially gastric ulcers, caused by NSAIDs.

[0006] The specific technical solutions for achieving the above-mentioned objectives are as follows.

[0007] In a first aspect, the present invention provides a strain of gastric-derived fermenting lactobacillus (Limosilactobacillus fermentum) LAF-09, which has the accession number GDMCC No. 65656.

[0008] In a second aspect, the present invention provides the use of the above-mentioned *Limosilactobacillus fermentum* LAF-09, its culture, or its metabolites in the preparation of products for the prevention and treatment of gastrointestinal diseases.

[0009] A third aspect of the present invention provides the use of the above-mentioned *Limosilactobacillus fermentum* LAF-09, its culture, or its metabolites in the preparation of products for the prevention and treatment of *Helicobacter pylori*.

[0010] In a fourth aspect, the present invention provides a medicament for preventing and treating gastrointestinal diseases, the active ingredient of which is the above-mentioned *Limosilactobacillus fermentum* LAF-09, its culture or its metabolites.

[0011] The gastric-derived fermenting Lactobacillus fermentum LAF-09 described in this invention was deposited on December 20, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province), a depositary unit designated by the State Intellectual Property Office. The deposit date is December 20, 2024, and the accession number is GDMCC No. 65656.

[0012] The inventors of this invention screened a strain of *Limosilactobacillus fermentum* LAF-09 from the gastric mucosa tissue of healthy individuals. This strain has strong acid resistance and high proliferative capacity in the stomach, exhibits strong adhesion to GSE-1 gastric epithelial cells, and has an extremely high inhibitory rate on gastric mucosal ulcers induced by indomethacin. It has significant application value in the prevention and treatment of gastrointestinal diseases caused by nonsteroidal anti-inflammatory drugs.

[0013] The strain LAF-09 of this invention belongs to Limosilactobacillus fermentum and has been included in the "List of Microbial Strains that Can Be Used in Food" issued by the Ministry of Health. It has the characteristics of being safe and having no side effects, and will not produce toxic side effects during treatment, nor will it cause adverse reactions in patients. Attached Figure Description

[0014] Figure 1 This is the phylogenetic tree of Lactobacillus gastrogenous fermenting mucin LAF-09 in Example 1 of the present invention.

[0015] Figure 2 The image shows the Gram staining results of LAF-09, a gastric-derived fermenting lactobacillus, in Example 2 of this invention.

[0016] Figure 3 This is the growth curve of Lactobacillus gastrogenus LAF-09 in Example 2 of the present invention.

[0017] Figure 4 This is the hemolytic result of Lactobacillus gastrogenus fermenting mucus LAF-09 in Example 2 of the present invention.

[0018] Figure 5 This invention describes the survival of gastric-derived fermenting lactobacillus LAF-09, food-derived fermenting lactobacillus JS-5, and the standard strain ATCC14931 of fermenting lactobacillus in artificial gastric fluid at different pH values ​​in Example 3 of this invention.

[0019] Figure 6 The survival rates of Lactobacillus fermentans LAF-09, Lactobacillus fermentans JS-5, and the standard strain ATCC14931 of Lactobacillus fermentans in artificial gastric fluid at pH 2.5 in Example 3 of this invention.

[0020] Figure 7 Fluorescent staining to measure the viability of gastric-derived fermenting lactobacillus LAF-09, food-derived fermenting lactobacillus JS-5, and the standard strain ATCC14931 of fermenting lactobacillus in PBS and artificial gastric fluid at pH 2.0 in Example 3 of this invention.

[0021] Figure 8 This is the result of adhesion of gastric epithelial cells GES-1 by gastric-derived fermenting lactobacillus LAF-09, food-derived fermenting lactobacillus JS-5, and the standard strain of fermenting lactobacillus ATCC14931 in Example 4 of the present invention.

[0022] Figure 9The results of the dilution spotting of Lactobacillus fermentum LAF-09, Lactobacillus fermentum JS-5, and Lactobacillus fermentum standard strain ATCC14931 adhering to GES-1 cells in Example 4 of this invention are shown.

[0023] Figure 10 The adhesion rate of gastric-derived fermenting lactobacillus LAF-09, food-derived fermenting lactobacillus JS-5, and fermenting lactobacillus standard strain ATCC14931 to GES-1 cells in Example 4 of this invention.

[0024] Figure 11 The image shows a scanning electron microscope image of GES-1 cells adhered to by LAF-09 of gastric fermentation lactobacillus and the standard strain ATCC14931 of fermentation lactobacillus in Example 4 of this invention.

[0025] Figure 12 The relative adhesion rates of gastric-derived fermenting lactobacillus LAF-09, food-derived fermenting lactobacillus JS-5, and fermenting lactobacillus standard strain ATCC14931 to inhibit Helicobacter pylori adhesion to GES-1 cells in Example 5 of this invention.

[0026] Figure 13 The inhibition zones of the bacterial suspensions, supernatants, and bacterial cells of *Lactobacillus fermentatus* LAF-09 (gastrointestinal origin), *Lactobacillus fermentatus* JS-5 (food-derived), and the standard strain of *Lactobacillus fermentatus* ATCC14931 against *Helicobacter pylori* in Example 5 of this invention are shown.

[0027] Figure 14 The images show the inhibition zones of the bacterial suspension, supernatant, and bacterial cells of Lactobacillus gastroenterogenus LAF-09 in Example 5 of this invention against some pathogenic gastrointestinal bacteria.

[0028] Figure 15 This is the result of gross gastric damage in mice after intervention with Lactobacillus mucinus LAF-09 and standard strain ATCC14931 in Example 6 of the present invention.

[0029] Figure 16 The above are the HE staining results of mouse gastric mucosa tissue after intervention with LAF-09 and standard strain ATCC14931 in Example 6 of this invention.

[0030] Figure 17 The ulcer index score of the gastric mucosa tissue of mice after intervention with LAF-09 and the standard strain ATCC14931 in Example 6 of this invention.

[0031] Figure 18The ulcer inhibition rate of mouse gastric mucosa tissue after intervention with gastric-derived fermenting Lactobacillus mucinus LAF-09 and standard strain ATCC14931 in Example 6 of this invention. Detailed Implementation

[0032] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0033] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0034] In some embodiments of the present invention, a strain of gastric-derived fermenting lactobacillus LAF-09 is disclosed, with accession number GDMCC No. 65656.

[0035] The *Limosilactobacillus fermentum* strain of this invention was isolated from the gastric mucosa tissue of healthy individuals. It exhibits extremely strong colonization and reproduction capabilities within the stomach, as well as exceptional acid resistance, tolerating an environment with a pH of 2 (currently studied strains are mostly derived from feces and dairy products, which cannot tolerate the extreme environment of the stomach, resulting in poor colonization and inadequate preventative and therapeutic effects). It also demonstrates strong adhesion to GES-1 gastric epithelial cells. Both the bacterial suspension and supernatant produce significant inhibition zones against *Helicobacter pylori* and some gastrointestinal pathogens. Furthermore, it can reduce gastric bleeding and damage caused by indomethacin and significantly inhibit gastric mucosal ulcers induced by indomethacin. Therefore, *Limosilactobacillus fermentum* can be used to prevent and treat digestive tract diseases (such as peptic ulcers) caused by nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin.

[0036] In other embodiments of the present invention, the use of the above-mentioned *Limosilactobacillus fermentum* LAF-09, its culture, or its metabolites in the preparation of products for the prevention and treatment of gastrointestinal diseases is disclosed.

[0037] In one embodiment, the gastrointestinal disease is caused by a nonsteroidal anti-inflammatory drug.

[0038] In one embodiment, the nonsteroidal anti-inflammatory drug is indomethacin.

[0039] In one embodiment, the gastrointestinal disease is a peptic gastric ulcer.

[0040] In one embodiment, the product is a pharmaceutical or food product.

[0041] In other embodiments of the present invention, the use of the above-mentioned Limosilactobacillus fermentum LAF-09, its culture or its metabolites in the preparation of products for the prevention and treatment of Helicobacter pylori is disclosed.

[0042] In one embodiment, the product is a pharmaceutical or food product.

[0043] In some other embodiments of the present invention, a drug for preventing and treating gastrointestinal diseases is disclosed, the active ingredient of which is the above-mentioned *Limosilactobacillus fermentum* LAF-09, its culture or its metabolites.

[0044] In one embodiment, the gastrointestinal disease is caused by a nonsteroidal anti-inflammatory drug.

[0045] In one embodiment, the nonsteroidal anti-inflammatory drug is indomethacin.

[0046] The Helicobacter pylori involved in the following embodiments of the present invention is Helicobacter pylori SS1 from the National Type Culture Collection Center (NTCC); the standard strain of Lactobacillus fermentum ATCC 14931 is from the American Type Culture Collection (ATCC), with accession number ATCC 14931.

[0047] The culture media involved in the following embodiments of the present invention are as follows:

[0048] MRS solid medium (g / L): peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast extract 5g / L, diammonium hydrogen citrate 2g / L, K2PO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4 0.05 g / L, Tween 80 1mL / L, agar 20g / L.

[0049] MRS liquid culture medium (g / L): peptone 10g / L, beef extract 10g / L, glucose 20g / L, sodium acetate 2g / L, yeast extract 5g / L, diammonium hydrogen citrate 2g / L, K2PO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4 0.05 g / L, Tween 80 1mL / L.

[0050] Helicobacter pylori culture medium (g / L): 70 mL / L sterile defibrinated sheep blood, 3 g / L yeast extract, 12 g / L casein, 5 g / L animal tissue digest, 3 g / L beef extract, 1 g / L corn starch, 5 g / L sodium chloride, 13.5 g / L agar, pH 7.3 ± 0.2 (25℃).

[0051] BHI solid medium (g / L): peptone 10g / L, dehydrated calf brain extract 12.5g / L, dehydrated calf heart extract 5g / L, sodium chloride 5g / L, glucose 2g / L, disodium hydrogen phosphate 2.5g / L, agar 20g / L, pH 7.4±0.2 (25℃).

[0052] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0053] Example 1: Isolation and Identification of Lactobacillus gastroenterogenus Fermentans LAF-09

[0054] In this embodiment, a strain of *Limosilactobacillus fermentum* LAF-09 was isolated and identified from gastric mucosal tissue of healthy individuals. The specific steps included:

[0055] 1. Separation

[0056] (1) Gastric mucosal tissue obtained by biopsy forceps under gastroscopy from healthy individuals was used as a sample. After being crushed by a grinding rod, it was spread on MRS solid culture medium and placed in a constant temperature anaerobic or aerobic incubator at 37℃ for 24h.

[0057] (2) After cultivation, bacilli were selected based on the color, size, and edge shape of the colonies. Colonies were picked up with an inoculation loop and streaked for purification. Finally, a bacillus strain was obtained and named LAF-09. The strain was stored in a -80°C freezer with 30% glycerol.

[0058] 2. Molecular biological identification

[0059] Using the genomic DNA of strain LAF-09 as a template, 16S rDNA fragment amplification and sequencing were performed using primers 27F (5'-AGAGTTTGATCCTG GCTCAG-3', SEQ ID NO:3) and 1492R (5'-CTACGGCTACCTTGTTACGA-3', SEQ ID NO:4). The reaction system (20 μL: universal primer 27F 1 μL, universal primer 1492R 1 μL, Taq enzyme 10 μL, template 2 μL, ddH2O 6 μL; reaction program: 95℃ 5 min; 95℃ 10 s, 55℃ 30 s, 72℃ 30 s, 30× cycles; 72℃ 5 min; 12℃ 2 min) was analyzed by sequencing. The sequencing results were searched and compared for similarity in GenBank using BLAST from the National Center for Biotechnology Information (NCBI). The results showed that the 16S rDNA sequence of this strain (including SEQ ID NO:4) was consistent with the desired sequence. Sequence ID NO:1 and Sequence ID NO:2 are the 16S rDNA sequences of *Limosilactobacillus fermentum*, a novel strain of *Limosilactobacillus fermentum*, named *Limosilactobacillus fermentum* LAF-09. A 16S rDNA phylogenetic tree was constructed using MAGE 7.0 software, and the results are as follows: Figure 1 As shown.

[0060] The methods for extracting genomic DNA are as follows:

[0061] a. Inoculate strain LAF-09 into MRS liquid medium and incubate overnight.

[0062] b. Take 1 mL of the bacterial suspension that has been cultured overnight into a 1.5 mL centrifuge tube, centrifuge at 10000 r / min for 2 min, discard the supernatant, and obtain the bacterial precipitate.

[0063] c. Add 110 μL of buffer (20 mM Tris, pH 8.0; 2 mM Na2-EDTA; 1.2% Triton) and 70 μL of lysozyme solution, and treat at 37°C for at least 30 min.

[0064] d. Add 20 μL of Proteinase K solution to the tube and mix well.

[0065] e. Add 220 μL of buffer GB, vortex for 15 seconds, incubate at 70°C for 10 minutes until the solution becomes clear, then briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0066] f. Add 220 μL of anhydrous ethanol and shake thoroughly for 15 seconds. Flocculent precipitate may appear at this time. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0067] g. Add the solution and flocculent precipitate obtained in step f to an adsorption column CB3 (place the adsorption column in the collection tube), centrifuge at 12,000 rpm (~13,400×g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into the collection tube.

[0068] h. Add 500 μL of buffer GD to the adsorption column CB3 (please check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (~13,400×g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into the collection tube.

[0069] i. Add 600 μL of washing buffer PW to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (~13,400×g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into the collection tube.

[0070] j. Repeat step i.

[0071] k. Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (~13,400 × g) for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any residual washing liquid in the adsorption material.

[0072] 1. Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 μL of elution buffer TE dropwise to the middle of the adsorption membrane, incubate at room temperature for 2-5 min, centrifuge at 12,000 rpm (~13,400×g) for 2 min, and collect the solution into the centrifuge tube.

[0073] SEQ ID NO:1

[0074] GGGGGGGCTATACATGCAAGTCGAACGCGTTGGCCCAATTGATTGATGGTGCTTGCACC

[0075] TGATTGATTTTGGTCGCCAACGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCC

[0076] CAGAAGCGGGGGACAACATTTGGAAACAGATGCTAATACCGCATAACAGCGTTGTTCGC

[0077] ATGAACAACGCTTAAAAGATGGCTTCTCGCTATCACTTCTGGATGGACCTGCGGTGCATT

[0078] AGCTTGTTGGTGGGGTAACGGCCTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACT

[0079] GATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGG

[0080] AATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTT

[0081] TCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACACGTATGAGAGTAACTGTTCATACGTT

[0082] GACGGTATTTAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTA

[0083] GGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGAGAGTGCAGGCGGTTTTCTAAGT

[0084] CTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGATAACTTGAGT

[0085] GCAGAAGAGGGTAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAA

[0086] CACCAGTGGCGAAGGCGGCTACCTGGTCTGCAACTGACGCTGAGACTCGAAAGCATGG

[0087] GTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTT

[0088] GGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGGAGT

[0089] ACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGC

[0090] ATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCCAAC

[0091] CCTAGAGATAGGCGTTTCCTTCGGGAACGCAATGACAGTGTGCATGGTCGTCGTCAGCT

[0092] CGTGTCGTGAGAATGTTGGGTAAGTCCCGCAACGAGCCGCAACCCTTGTTACTAGTTGC

[0093] AAGCATTAAGTTT

[0094] SEQ ID NO:2

[0095] AAATCCCTTATGCGGCTGGCTCCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAACT

[0096] CTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTG

[0097] ATCCGCGATTACTAGCGATTCCGACTTCGTGCAGGCGAGTTGCAGCCTGCAGTCCGAAC

[0098] TGAGAACGGTTTTAAGAGATTTGCTTGCCCTCGCGAGTTCGCGACTCGTTGTACCGTCCA

[0099] TTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCT

[0100] TCCTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGTA

[0101] ACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGAC

[0102] GACCATGCACCACCTGTCATTGCGTTCCCGAAGGAAACGCCCTATCTCTAGGGTTGGCG

[0103] CAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCA

[0104] CCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAG

[0105] GCGGAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGC

[0106] ACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCG

[0107] AGTCTCAGCGTCAGTTGCAGACCAGGTAGCCGCCTTCGCCACTGGTGTTCTTCCATATAT

[0108] CTACGCATTCCACCGCTACACATGGAGTTCCACTACCCTCTTCTGCACTCAAGTTATCCA

[0109] GTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACTTAGAAAACCGC

[0110] CTGCACTCTCTTTACGCCCAATAAATCCCGGATAACGCTTGCCACCTACGTATTACCGCG

[0111] GCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTAAATACCGTCAACGTATGAACAGTT

[0112] ACTCTCATACGTGTTCTTCTTTACAACAGAGCTTTACGAGCGAAACCCTTCTTCACTCAC

[0113] Example 2: Characteristics of Lactobacillus mucinus LAF-09 of gastric origin

[0114] In this embodiment, the following performance studies were conducted on Limosilactobacillus fermentum LAF-09, which was isolated and identified in Example 1:

[0115] 1. Gram staining

[0116] The steps are as follows: After smearing the bacterial culture, dry and fix it; stain with ammonium oxalate crystal violet for 1 minute and then wash with water; remove residual water with iodine solution, and wash with water after about 1 minute; shake off the water on the slide, wash the slide with alcohol until no purple color appears, and immediately wash with water; stain with safranin solution for 1-2 minutes and then wash with water.

[0117] After drying, observe under a microscope, such as Figure 2 As shown, the results indicate that *Limosilactobacillus fermentum* LAF-09 is Gram-positive.

[0118] 2. Growth curve

[0119] (1) Inoculate LAF-09 of gastric-derived fermenting lactobacillus fermentum, which was frozen at -80℃, into MRS liquid medium and culture it at 37℃ for 24h with aerobic conditions. The recovered bacterial suspension was passaged 2-3 times in the same manner.

[0120] (2) Take 1 mL of culture medium into a 1.5 mL centrifuge tube, centrifuge at 8000 rpm for 3 min, discard the supernatant, collect the bacterial pellet, and resuspend it with MRS until the OD is adjusted. 600 The value is 1.

[0121] (3) Take 10uL of the resuspended solution obtained in step (2) and add it to 990uL of MRS. Seed it in a 96-well plate at 200uL / well.

[0122] (4) The 96-well plate was aerobically incubated at 37°C, and the OD value was measured at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h and 24h.

[0123] The results are as follows Figure 3 As shown, under constant temperature and aerobic conditions at 37℃, the culture in MRS medium reached the end of the logarithmic growth phase after about 16 hours.

[0124] 3. Hemolytic

[0125] (1) Inoculate LAF-09 of gastric-derived fermenting lactobacillus fermentum, which was frozen at -80℃, into MRS liquid medium and culture it at 37℃ for 24h with aerobic conditions. The recovered bacterial suspension was passaged 2-3 times in the same manner.

[0126] (2) Weigh out BHI solid culture medium, heat to dissolve, and autoclave at 121.5℃ for 15 min. When the temperature of the culture medium drops to 45℃, add whole blood at a ratio of 7 mL of sterile defibrinated sheep blood to 93 mL of culture medium and mix well. Coagulate to obtain BHI blood agar plates.

[0127] (3) Use an inoculation loop to streak the revived and passaged Lactobacillus mucinus culture from (1) and incubate at 37°C under aerobic conditions for 24 hours.

[0128] The results are as follows Figure 4 As shown, no hemolysis ring appeared around LAF-09 grown on BHI blood agar plates, indicating γ-hemolysis, i.e., no hemolysis.

[0129] Example 3: Tolerance of gastric-derived fermenting Lactobacillus mucinus LAF-09 to artificial gastric juice

[0130] This example investigated the tolerance of *Limosilactobacillus fermentum* LAF-09, isolated and identified in Example 1, to artificial gastric juice. The method is as follows:

[0131] 1. The gastric-derived Lactobacillus fermentum LAF-09, the Lactobacillus fermentum standard strain ATCC14931, and the food-derived Lactobacillus fermentum JS-5 (isolated from fermented food slurry by the applicant using conventional isolation methods and identified as Lactobacillus fermentum) frozen at -80℃ were inoculated into MRS liquid medium and cultured aerobically at 37℃ for 24h. The recovered bacterial suspension was passaged 2-3 times in the same manner.

[0132] 2. Take 1 mL of culture medium into a 1.5 mL centrifuge tube, centrifuge at 8000 rpm for 3 min, discard the supernatant, collect the bacterial pellet, and resuspend in PBS until OD is adjusted. 600 The value is 1.

[0133] 3. Take the 1OD bacterial solution obtained in step (2), centrifuge at 8000 rpm for 3 min, discard the supernatant, and resuspend in PBS, artificial gastric fluid with pH=2.5 and pH=2.

[0134] 4. Incubate the stock solution aerobically at 37℃. Take samples at 0h and 2h, wash once with PBS and resuspend. After serial dilution, take 5uL of each sample and drop it onto MRS solid medium. Incubate at 37℃. After 24h, perform plate colony counting, record the number of viable bacteria, and calculate the survival rate at different pH values. Survival rate = (number of viable bacteria after treatment with artificial gastric juice at a certain pH / number of viable bacteria after treatment with PBS) × 100%.

[0135] The results are as follows Figures 5-7 As shown, from Figures 5-7 It can be seen that *Limosilactobacillus fermentum* LAF-09 exhibits extremely strong acid resistance. At pH 2.5, the survival rate of *Limosilactobacillus fermentum* LAF-09 was approximately 95.6%, significantly higher than that of the standard strain ATCC14931 (36.7%) and food-derived *Limosilactobacillus fermentum* JS-5 (19.6%). At pH 2.0, the *Limosilactobacillus fermentum* isolate LAF-09 showed abundant colony growth, with fluorescent staining showing predominantly green viable bacteria, while the standard strain ATCC14931 and food-derived *Limosilactobacillus fermentum* JS-5 showed only a few colonies, with fluorescent staining showing predominantly red dead bacteria.

[0136] Example 4: Ability of Lactobacillus mucinus LAF-09 from gastric fermentation to adhere to GES-1

[0137] This example tested the ability of *Limosilactobacillus fermentum* LAF-09, isolated and identified in Example 1, to adhere to GES-1 (normal human gastric mucosal epithelial cells). The specific method is as follows:

[0138] 1. Fermentation of *Lactobacillus mucinus* adhering to GES-1 cell smears and counting.

[0139] (1) The gastric-derived fermenting lactobacillus LAF-09, fermenting lactobacillus standard strain ATCC14931, and food-derived fermenting lactobacillus JS-5, which were stored at -80℃, were inoculated into MRS liquid medium and cultured in an aerobic environment at 37℃ for 24h. The recovered bacterial culture was passaged 2 to 3 times in the same way.

[0140] (2) After digesting GES-1, count the samples, in increments of 1 to 2 x 10⁻⁶. 4The concentration was 1 cell / mL, seeded in 6-well plates containing cell spreaders, and cultured in DMEM containing fetal bovine serum until a monolayer was formed.

[0141] (3) Take 1 mL of the suspension of Lactobacillus fermentum LAF-09 (gastrointestinal origin), Lactobacillus fermentum JS-5 (food source), and Lactobacillus fermentum standard strain ATCC14931 into 1.5 mL centrifuge tubes, centrifuge at 5500 rpm for 2 min, discard the supernatant, and obtain bacterial pellet. Resuspend the bacterial pellet in 1 mL PBS, wash by pipetting, centrifuge at 5500 rpm for 2 min, discard the supernatant, repeat the washing three times, and then resuspend in DMEM high-glucose medium. Adjust the OD 600 The value is 1.

[0142] (4) Take 100 μL of the resuspension obtained in step (3) and add it to a 6-well cell dish containing 900 μL of LDMED high glucose medium and 10% FBS, and incubate at 37°C for 4 h with aerobic culture.

[0143] (5) Aspirate the supernatant from the 6-well plate, wash the cells 3 times with PBS, discard the supernatant, fix with 4% polyethanol, discard the supernatant, use ophthalmic forceps to pick up cell smears, Gram stain, randomly select 5 fields of view from each smear under oil immersion, and count the fermenting mucus lactobacilli adhering to GES-1 cells.

[0144] The results are as follows Figure 8 As shown, the number of LAF-09 cells adhering to GES-1 cells was significantly greater than that of the Lactobacillus fermentum standard strain ATCC14931 and food-derived Lactobacillus fermentum JS-5.

[0145] 2. Plate count of GES-1 cells adhered to fermented *Lactobacillus mucinus*

[0146] (1) The gastric-derived fermenting lactobacillus LAF-09, fermenting lactobacillus standard strain ATCC14931, and food-derived fermenting lactobacillus JS-5, which were stored at -80℃, were inoculated into MRS liquid medium and cultured in an aerobic environment at 37℃ for 24h. The recovered bacterial culture was passaged 2 to 3 times in the same way.

[0147] (2) After digesting GES-1, count the samples, in increments of 1 to 2 × 10⁻⁶. 4 The concentration was 1 mL / well, and the cells were seeded in 6-well plates. The plates were cultured in DMEM containing fetal bovine serum until a monolayer was formed.

[0148] (3) Take 1 mL of the suspension of Lactobacillus fermentum LAF-09 (gastrointestinal origin), Lactobacillus fermentum JS-5 (food source), and Lactobacillus fermentum standard strain ATCC14931 into 1.5 mL centrifuge tubes, centrifuge at 5500 rpm for 2 min, discard the supernatant, and obtain bacterial pellet. Resuspend the bacterial pellet in 1 mL PBS, wash by pipetting, centrifuge at 5500 rpm for 2 min, discard the supernatant, repeat the washing three times, resuspend in DMEM medium, and adjust the OD. 600 The value is 1.

[0149] (4) Take 100 μL of the resuspension obtained in step (3) and add it to a 6-well cell dish containing 900 μL of LMEM medium and 10% FBS, and incubate at 37°C for 4 h with aerobic culture.

[0150] (5) Aspirate the supernatant from the 6-well plate, wash the cells 3 times with PBS, discard the supernatant, add 200uL of sterile ultrapure water to each well, lyse the cells for 20 minutes, gently scrape the cell culture surface with a cell scraper, and collect cell debris and bacterial suspension by pipetting.

[0151] (6) After serially diluting the above-obtained suspension, spread it onto MRS plates and incubate under aerobic conditions for 24 hours. Count the colonies on the plates and calculate the adhesion rate. Adhesion rate (%) = (number of colonies on the plate / number of bacteria added to the well) × 100%.

[0152] The results are as follows Figures 9-10 As shown, the results indicate that compared with the standard strain ATCC14931 of *Lactobacillus fermentatus* and the *Lactobacillus fermentatus* isolate JS-5 from food sources, *Lactobacillus fermentatus* LAF-09 from the gastric origin exhibited stronger adhesion to GES-1 cells, indicating that *Lactobacillus fermentatus* LAF-09 from the gastric origin adheres more readily to gastric epithelial cells than the standard strain ATCC14931 and the *Lactobacillus fermentatus* isolate obtained from fermented foods.

[0153] 3. Scanning electron microscopy of GES-1 cells adhering to fermenting *Lactobacillus mucilaginosus*

[0154] (1) Human gastric-derived fermenting lactobacillus LAF-09 and fermenting lactobacillus standard strain ATCC14931, stored at -80℃, were inoculated into MRS liquid medium and cultured in an aerobic environment at 37℃ for 24h. The recovered bacterial culture was passaged 2 to 3 times in the same way.

[0155] (2) After digesting GES-1, count the samples, in increments of 1 to 2 x 10⁻⁶. 4The concentration was 1 cell / mL, seeded in 6-well plates containing cell spreaders, and cultured in DMEM containing fetal bovine serum until a monolayer was formed.

[0156] (3) Take 1 mL of the suspension of Lactobacillus fermentum LAF-09 and the standard strain ATCC14931 of Lactobacillus fermentum into 1.5 mL centrifuge tubes, centrifuge at 5500 rpm for 2 min, discard the supernatant, and obtain bacterial pellet. Resuspend the bacterial pellet in 1 mL PBS, wash by pipetting, centrifuge at 5500 rpm for 2 min, discard the supernatant, repeat the washing three times, and then resuspend in DMEM high glucose medium and adjust the OD. 600 The value is 1.

[0157] (4) Take 100 μL of the resuspension obtained in step (3) and add it to a 6-well cell dish containing 900 μL of DMED high-glucose medium and 10% FBS, and incubate at 37°C for 4 h.

[0158] (5) Aspirate the supernatant from the 6-well plate, wash the cells three times with low-temperature PBS, discard the supernatant, immerse the smear in 2.5% glutaraldehyde, and fix at 4°C in the dark for 3-5 days.

[0159] (6) After the fixed time is up, wash the slides three times with PBS in a 6-well plate. After washing, dehydrate with a gradient of 30% ethanol, 50% ethanol, 70% ethanol, 80% ethanol and 90% ethanol for 5-10 min, and dehydrate with 100% ethanol three times for 10-15 min each time.

[0160] (7) Dry the sample using a critical point dryer.

[0161] (8) The sample was made conductive by vacuum sputtering.

[0162] (9) Scanning electron microscopy was used to observe the adhesion of LAF-09 and ATCC14931 to GES-1 cells.

[0163] The results are as follows Figure 11 As shown, there is a clear adhesion structure between the gastric-derived fermenting LAF-09 and GES-1 cells, indicating that LAF-09 can adhere to GES-1 cells. However, there is no connection structure between the standard strain ATCC14931 and GES-1 cells.

[0164] Example 5: Inhibitory effect of Lactobacillus gastroenterogenosa fermentans LAF-09 on gastrointestinal pathogens

[0165] 1. Fermented *Lactobacillus mucinus* inhibits *Helicobacter pylori* adhesion GES-1

[0166] Experimental Groups:

[0167] ① Blank control group

[0168] ②HP group

[0169] ③ Experimental group: including prevention group and competition group

[0170] The specific method is as follows:

[0171] (1) The fermented Lactobacillus fermentum LAF-09, the fermented Lactobacillus fermentum standard strain ATCC14931, and the food-derived fermented Lactobacillus fermentum JS-5 stored at -80℃ were inoculated into MRS liquid medium and cultured in an aerobic environment at 37℃ for 24h. The recovered bacterial culture was passaged 2 to 3 times in the same way.

[0172] (2) Take a 96-well plate and add 100 μL of DMEM medium containing 10% FBS to each well. Shake the 96-well plate horizontally to mix. Digest with GES-1 and count the cells, using 1–2 x 10⁻⁶ cells / well. 4 Cells were seeded at a concentration of [number] cells / mL in 96-well plates, ensuring even distribution on the culture surface. The plates were incubated at 37°C for 24 hours.

[0173] (3) When the cells grow to 80% of the area of ​​the monolayer, discard the cell supernatant, wash with PBS and aspirate.

[0174] (4) Resuspend fermenting Lactobacillus mucinus and Helicobacter pylori in DMEM medium containing 10% FBS to make the cell multiplicity of infection (MOI) 1:1000.

[0175] (5) Treatment of each group

[0176] Blank control group: 100 μL of DMEM medium containing 10% FBS was added to the 96-well plate in step (3), and the plate was incubated at 37°C for 2 h. The supernatant was discarded, the plate was washed 3 times with PBS, and 100 μL of fresh DMEM medium containing 10% FBS was added.

[0177] HP group: 100uL of DMEM medium containing 10% FBS, incubated at 37℃ for 2h, supernatant discarded, washed 3 times with PBS, 50uL of fresh DMEM medium containing 10% FBS and 50uL of Helicobacter pylori suspension (resuspended in DMEM medium containing 10% FBS).

[0178] Prevention group: Add 50 μL of DMEM medium containing 10% FBS and 50 μL of fermented Lactobacillus suspension (resuspended in DMEM medium containing 10% FBS) to the 96-well plate in step (3), incubate at 37°C for 2 h, discard the supernatant, wash 3 times with PBS, add 50 μL of fresh DMEM medium containing 10% FBS and 50 μL of Helicobacter pylori suspension (resuspended in DMEM medium containing 10% FBS).

[0179] Competition group: 100 μL of DMEM medium containing 10% FBS was added to the 96-well plate in step (3), and the plate was incubated at 37°C for 2 h. The supernatant was discarded, and the plate was washed 3 times with PBS. 25 μL of fermented Lactobacillus suspension (resuspended in DMEM medium containing 10% FBS), 25 μL of Helicobacter pylori suspension (resuspended in DMEM medium containing 10% FBS) and 50 μL of DMEM medium containing 10% FBS were added.

[0180] (6) After incubating at 37℃ for 4 hours, the supernatant was aspirated, washed 3 times with PBS, and 100uL of urease detection reagent was added to each well. The absorbance at 540nm of each well was immediately detected by microplate reader, and the relative adhesion rate of HP (%) was calculated.

[0181] HP relative adhesion rate (%) = [(Experimental group absorbance - Control group absorbance) / (HP group absorbance - Control group absorbance)] * 100%.

[0182] The results are as follows Figure 12 As shown, in the prevention group, LAF-09 reduced the adhesion rate of Helicobacter pylori to GES-1 cells (48.26%), which was significantly higher than that in the ATCC14931 group (4.12%) and the JS-5 group (20.33%). In the competition group, the inhibition rate of LAF-09 was 41.19%, which was also significantly higher than that in the ATCC14931 group (26.61%) and the JS-5 group (35.05%).

[0183] 2. Inhibition zone experiment of LAF-09 inhibiting Helicobacter pylori

[0184] (1) The five strains of L. fermentum (Limosilactobacillus fermentum) LAF-09, L. fermentum standard strain ATCC14931, and L. fermentum JS-5 (food source) were streaked on MRS solid medium and cultured at 37℃ for 24 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37℃ for 24 h to activate them. The activation was repeated for two generations to obtain the activated solution.

[0185] (2) Adjust the activating solution to OD 600=1, then inoculate at a rate of 2% (v / v) into MRS liquid medium and incubate at 37°C for 24 h to obtain bacterial culture; centrifuge the bacterial culture at 8000g for 2 min to obtain supernatant and bacterial cells, and keep the supernatant for later use; then adjust the bacterial cells to OD using PBS and the remaining supernatant respectively. 600 =1 (for standby).

[0186] (3) The effect of the supernatant obtained in step (2), the bacterial cells resuspended in PBS, and the bacterial cells resuspended in supernatant on inhibiting the growth of Helicobacter pylori was determined by the Oxford cup method.

[0187] The results are as follows Figure 13 As shown, transparent inhibition zones appeared around the bacterial suspensions and supernatants of all three strains, indicating that LAF-09 of gastric-derived fermenting *Lactobacillus mucinus* can inhibit the growth of *Helicobacter pylori*, but there was no statistically significant difference between it and the standard strain ATCC14931 of *Lactobacillus mucinus* and the food-derived fermenting *Lactobacillus mucinus* JS-5.

[0188] The statistical results of the inhibition zone diameter of Helicobacter pylori for each strain are shown in Table 1.

[0189] Table 1. Diameter of the inhibition zone of Helicobacter pylori for each strain

[0190] Bacterial solution (mm) Supernatant (mm) Bacterial cells (mm) ATCC14931 13.00±2.00 11.00±1.00 0.00±0.00 LAF-09 13.00±1.50 11.00±1.00 0.00±0.00 JS-5 11.00±1.50 10.00±0.50 0.00±0.00

[0191] Table 1 shows that the inhibition zone size of *Limosilactobacillus fermentum* LAF-09 bacterial suspension against *Helicobacter pylori* reached 13.00±1.50 mm, while the inhibition zone size of the supernatant against *Helicobacter pylori* reached 11.00±1.00 mm. No inhibition zone was observed in the bacterial cells themselves. This indicates that *Limosilactobacillus fermentum* LAF-09 or its metabolites have an inhibitory effect on the growth of *Helicobacter pylori*. Compared to ATCC14931, this bacterium originates from the stomach and possesses the characteristic of naturally colonizing the gastric environment.

[0192] 3. Inhibition zone experiment of LAF-09 inhibiting some pathogenic gastrointestinal bacteria

[0193] (1) The five strains of L. fermentum (Limosilactobacillus fermentum) LAF-09, L. fermentum standard strain ATCC14931, and L. fermentum JS-5 (food source) were streaked on MRS solid medium and cultured at 37℃ for 24 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37℃ for 24 h to activate them. The activation was repeated for two generations to obtain the activated solution.

[0194] (2) Adjust the activating solution to OD600 =1, then inoculate at a rate of 2% (v / v) into MRS liquid medium and incubate at 37°C for 24 h to obtain bacterial culture; centrifuge the bacterial culture at 8000g for 2 min to obtain supernatant and bacterial cells, and keep the supernatant for later use; then adjust the bacterial cells to OD using PBS and the remaining supernatant respectively. 600 =1 (for standby).

[0195] (3) The effect of the supernatant obtained in step (2), the bacterial cells resuspended in PBS, and the bacterial cells resuspended in the supernatant on inhibiting the growth of Shigella, Candida albicans, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Salmonella was determined by the Oxford cup method.

[0196] The results are as follows Figure 14 As shown, transparent inhibition zones appeared in both the bacterial suspension and supernatant of the three strains, indicating that LAF-09 of gastric-derived fermenting Lactobacillus mucinus could inhibit the growth of Shigella, Candida albicans, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Salmonella, but there was no statistically significant difference between it and the standard strain of fermenting Lactobacillus mucinus ATCC14931 and food-derived fermenting Lactobacillus mucinus JS-5.

[0197] Example 6: Effect of Lactobacillus gastroenterologica f. m. LAF-09 on indomethacin-induced gastric ulcers.

[0198] This embodiment tested whether the gastric-derived fermenting lactobacillus LAF-09 isolated and identified in Example 1 had a relieving effect on indomethacin-induced gastric ulcers. The method is as follows:

[0199] 1. Twenty-five 6-week-old C57BL / 6 mice were randomly divided into five groups after one week of normal feeding:

[0200] Control group (n=5)

[0201] IND group (indomethacin, n=5)

[0202] Group LAF-09 (n=5)

[0203] ATCC14931 group (n=5)

[0204] PPI group (proton pump inhibitor, n=5)

[0205] 2. From day 1 to day 6, mice in the Control and IND groups were administered 0.1 mL of PBS by gavage, mice in the LAF-09 group were administered 0.1 mL of LAF-09 broth with an OD600 of 1 by gavage, mice in the ATCC14931 group were administered 0.1 mL of ATCC14931 broth with an OD600 of 1 by gavage, and mice in the PPI group were administered 0.1 mL of omeprazole at a concentration of 13 mg / kg by gavage (drug concentration approximately 2.6 mg / mL); fasting was enforced at 4 PM.

[0206] 3. On day 7, mice in the Control and IND groups were administered 0.1 mL of PBS by gavage, mice in the LAF-09 group were administered 0.1 mL of LAF-09 broth with an OD600 of 1 by gavage, mice in the ATCC14931 group were administered 0.1 mL of ATCC14931 broth with an OD600 of 1 by gavage, and mice in the PPI group were administered 0.1 mL of 13 mg / kg omeprazole by gavage. One hour later, mice in the Control group were administered 0.2 mL of PBS by gavage, and mice in the other groups were administered 0.2 mL of 40 mg / kg IND by gavage (drug concentration approximately 4 mg / mL). Six hours later, mice were dissected and their various indicators were measured.

[0207] Gross condition of the stomachs of mice in each group is as follows: Figure 15 As shown, the IND group showed obvious punctate or linear hemorrhages, and some even formed continuous hemorrhage areas; while the LAF-09 group had only a few hemorrhages, and the number of hemorrhages was significantly less than that of ATCC14931.

[0208] HE staining results of gastric mucosal tissues from each group of mice are as follows: Figure 16 As shown, the IND group exhibited significant neutrophil infiltration, epithelial cell shedding, and mucosal bleeding damage; LAF-09 can prevent a series of damages caused by indomethacin, and its effect is better than that of the standard strain ATCC14931.

[0209] The results of the gastric mucosal ulcer index scores of each group of mice are as follows: Figure 17 As shown, the ulcer index score of the LAF-09 group was 6.00±3.16, which was significantly lower than that of the IND group (22.75±4.5) and the ATCC14931 group (14.5±3.42).

[0210] The results of the gastric mucosal ulcer inhibition rate of each group of mice are as follows: Figure 18 As shown, the average ulcer inhibition rate of the LAF-09 group was 74.47%, which was significantly higher than that of the ATCC14931 group (34.81%).

[0211] In summary, the results of this embodiment indicate that *Limosilactobacillus fermentum* LAF-09 can prevent and treat gastric ulcers caused by indomethacin.

[0212] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0213] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A strain of gastric-derived fermenting lactobacillus (Limosilactobacillus fermentum) LAF-09, characterized in that, Its accession number is GDMCC No.65656.

2. The use of the gastric-derived fermenting lactobacillus LAF-09 or its culture as described in claim 1 in the preparation of a medicine for the prevention and treatment of gastrointestinal diseases.

3. The application according to claim 2, characterized in that, The gastrointestinal disease was caused by nonsteroidal anti-inflammatory drugs.

4. The application according to claim 3, characterized in that, The nonsteroidal anti-inflammatory drug is indomethacin.

5. The application according to claim 3, characterized in that, The gastrointestinal disease mentioned is peptic gastric ulcer.

6. The use of the gastric-derived fermenting lactobacillus LAF-09 or its culture as described in claim 1 in the preparation of a drug for the prevention and treatment of Helicobacter pylori.

7. A drug for preventing and treating gastrointestinal diseases, characterized in that, Its active ingredient is the gastric-derived fermenting lactobacillus LAF-09 or its culture as described in claim 1.

8. The drug for preventing and treating gastrointestinal diseases according to claim 7, characterized in that, The gastrointestinal disease was caused by nonsteroidal anti-inflammatory drugs.

9. The drug for preventing and treating gastrointestinal diseases according to claim 7, characterized in that, The nonsteroidal anti-inflammatory drug is indomethacin.

10. The drug for preventing and treating gastrointestinal diseases according to claim 7, characterized in that, The gastrointestinal disease mentioned is peptic gastric ulcer.

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