Lactobacillus reuteri as well as preparation method and application of microbial inoculum of lactobacillus reuteri

Abstract

The invention discloses a lactobacillus reuteri and a preparation method and application of a microbial inoculum of the lactobacillus reuteri, the lactobacillus reuteri subsp.reuteri OH114 is preserved in Guangdong Province Microbial Culture Collection Center (GDMCC), the preservation address is No.59 building, No.100 courtyard, Xianlie Middle Road, Guangzhou, the preservation number is GDMCC No.66528, and the preservation time is June 16, 2025. The lactobacillus reuteri newly researched and developed by the invention can effectively inhibit the growth of a plurality of enteropathogenic bacteria such as staphylococcus aureus, escherichia coli, salmonella typhimurium, clostridium difficile, clostridium perfringens, klebsiella pneumoniae, pseudomonas aeruginosa and candida albicans, regulate the balance of intestinal flora, prevent and treat adult diarrhea, and prevent and treat diarrhea. The invention provides a new scheme and way for preventing and treating infectious diarrhea, antibiotic-related diarrhea or irritable bowel syndrome diarrhea.

Description

Technical Field

[0001] This invention relates to the field of Lactobacillus reuteri technology, and in particular to a method for preparing and applying Lactobacillus reuteri and its inoculum. Background Technology

[0002] Diarrhea is an extremely common digestive system ailment, affecting a large population worldwide. Its pathogenesis is complex, involving infectious factors, antibiotic-associated gut microbiota imbalance, irritable bowel syndrome (IBS), as well as improper diet, gastrointestinal dysfunction, and drug side effects. Epidemiological statistics show that the incidence of frequent diarrhea in the general population is as high as 3-5%. Infectious diarrhea is mainly caused by intestinal pathogens such as Escherichia coli and Salmonella; antibiotic-associated diarrhea is mainly caused by gut microbiota imbalance due to the use of broad-spectrum antibiotics; while the prevalence of IBS-diarrhea (IBS-D) in the adult population is relatively low, and it is closely related to intestinal dysfunction and mucosal barrier damage.

[0003] Lactobacillus reuteri ( 罗伊氏乳杆菌 or 罗伊氏乳杆菌 As an inherent beneficial probiotic in the gut, *Lactobacillus reuteri* plays a unique role in maintaining gut health and combating diarrhea. It secretes antimicrobial substances such as reuterin, exhibiting strong inhibitory activity against various pathogenic intestinal bacteria, effectively curbing their growth and reproduction. Simultaneously, *Lactobacillus reuteri* can regulate the intestinal immune response, enhance the body's immune defense capabilities, reduce the risk of harmful microorganisms invading the gut, alleviate intestinal inflammation, and play an important role in maintaining the integrity of the mucosal barrier, helping to repair intestinal mucosa damaged by diarrhea. However, under current technological conditions, most preparations involving *Lactobacillus reuteri* exhibit significant shortcomings. Regarding strain sourcing, many preparations use strains derived from foreign populations. Due to differences in dietary structure and living environment between domestic and foreign populations, the compatibility of these strains with the gut microecological environment of Chinese people is questionable. In terms of storage stability, the protective agent systems and freeze-drying technologies used in traditional preparation processes are insufficient, making it difficult to effectively inhibit the decline in bacterial activity during storage. Conventional dosage forms are severely lacking in effective protection mechanisms against the acidic environment of the stomach, causing a large number of live bacteria to become inactive when passing through this environment, resulting in a very limited number of effective bacteria reaching the intestines. Traditional freeze-dried powders also exhibit slow release rates of live bacteria after rehydration, resulting in low bioavailability and failing to fully realize the beneficial effects of Lactobacillus reuteri.

[0004] Currently, the development of probiotic formulations for diarrhea faces numerous technical challenges. From the perspective of strain screening, there is an urgent need to construct a specific screening system tailored to the gut microbiota characteristics of the Chinese population, thereby improving the adaptability of strains to the local intestinal environment. In the field of formulation technology, in-depth research is needed on optimizing the protective agent components, precisely controlling freeze-drying parameters, and making every effort to overcome the bottleneck of live bacteria stability. Regarding dosage form design, it is essential to focus on overcoming the technical challenge of synergistic regulation of gastric acid tolerance and intestinal targeted release, thereby improving the bioavailability of probiotics. Although existing technologies have attempted to improve formulation performance through various process improvements, they have not yet fundamentally and systematically solved the problem of the technological gap between "strain adaptability - formulation stability - intestinal targeting." Therefore, developing new *Lactobacillus reuteri* strains and creating *Lactobacillus reuteri* formulations with high adaptability, high stability, and precise intervention capabilities has become a key research direction for solving the treatment challenges of adult diarrhea, and is of vital importance for improving the treatment outcomes and quality of life of adult diarrhea patients. Summary of the Invention

[0005] To address the incompatibility issues between current Lactobacillus reuteri strains and the intestinal microecological environment of Chinese people, as well as the shortcomings of protective agent systems and freeze-drying technologies prepared using traditional processes, this invention first proposes a novel Lactobacillus reuteri strain, describes the preparation method and application of this Lactobacillus reuteri, and briefly introduces its effects. The specific technical solution is as follows: This invention discloses a strain of Lactobacillus reuteri. 罗伊氏乳杆菌 罗伊氏亚种 OH114 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 66528, and deposited on June 16, 2025.

[0006] The present invention also discloses a microbial agent comprising the Lactobacillus reuteri and a pharmaceutically or food-grade acceptable carrier.

[0007] Preferably, the bacterial agent is a powder or enteric-coated capsule.

[0008] Preferably, the viable count of Lactobacillus reuteri in the bacterial agent satisfies the following condition: ≥1×10⁻⁶ in the powder. 9 CFU / serving, ≥1×10⁻⁶ CFU / serving in enteric-coated capsules 9 CFU / capsule.

[0009] This invention also discloses a method for preparing a powdered microbial agent, comprising the following steps: (0) Culture the Lactobacillus reuteri and collect the bacterial cells; (1) The bacterial cells and the bacterial cell encapsulation agent are mixed at a weight ratio of 1:1.5, wherein the bacterial cell encapsulation agent includes at least one of skim milk powder, trehalose, soybean peptone, maltose, and sodium D-isoascorbate; (2) After freeze-drying, the powder is pulverized to obtain freeze-dried powder; (3) The freeze-dried powder is mixed with pharmaceutically acceptable excipients, including fructooligosaccharides, galactooligosaccharides, additives (at least one of monosodium glutamate, lysozyme, and skim milk) and packaging materials (at least one of silica and magnesium stearate) to form a powder.

[0010] Preferably, the raw material ratio of the powder is 25-35 parts freeze-dried powder, 20-30 parts fructooligosaccharide, 20-30 parts galactooligosaccharide, 50-80 parts additives, and 60-100 parts packaging material.

[0011] Preferably, the contents of each component in the bacterial cell encapsulation agent are: 10wt% skim milk powder, 8wt% trehalose, 7wt% soybean peptone, 6wt% maltose, and 1.5wt% sodium D-isoascorbate.

[0012] This invention also discloses a method for preparing an enteric-coated capsule-type bacterial agent, comprising the following steps: (1) Collect live cells by centrifugation after fermentation of the Lactobacillus reuteri culture; (2) The bacterial cells and the bacterial cell encapsulation agent are mixed at a weight ratio of 1:1.5, wherein the bacterial cell encapsulation agent includes at least one of skim milk powder, trehalose, soybean peptone, maltose, and sodium D-isoascorbate; (3) Freeze-dry the bacterial cells containing the bacterial cell encapsulation agent; (4) The freeze-dried product is pulverized to obtain freeze-dried powder; (5) The freeze-dried powder is mixed with enteric microcapsule encapsulation material (at least one of hydroxypropyl methylcellulose phthalate, corn starch, and magnesium stearate), and sterile water is added to prepare a suspension. Enteric microcapsules are then prepared by spray drying. (6) The enteric microcapsules are mixed with enteric capsule excipients (at least one of microcrystalline cellulose, fructooligosaccharides, and silicon dioxide) and filled into enteric capsules to obtain the product.

[0013] Preferably, the enteric microcapsule encapsulation material contains 5 wt% hydroxypropyl methylcellulose phthalate, 20 wt% corn starch, and 1 wt% magnesium stearate; the enteric capsule excipient contains 30 wt% microcrystalline cellulose, 25 wt% fructooligosaccharides, and 2 wt% silicon dioxide.

[0014] The present invention also includes the use of the Lactobacillus reuteri or the bacterial agent in the preparation of products for the treatment or prevention of diarrhea.

[0015] This invention also discloses a product for treating or preventing diarrhea, regulating intestinal flora balance, or inhibiting intestinal pathogens, wherein the product contains the *Lactobacillus reuteri* or the bacterial agent, and the intestinal pathogens include *Staphylococcus aureus*, *Escherichia coli*, *Salmonella typhimurium*, *Clostridium difficile*, *Clostridium perfringens*, etc. Compared with the prior art, the present invention has at least the following beneficial effects: 4. Outstanding strain function and excellent core performance: The Roche strain OH114 disclosed in this invention has been experimentally verified to maintain a certain number of viable bacteria in the pH range of 2-6, and its survival ability is significantly enhanced in weakly acidic to neutral environments (pH 4-6). It can tolerate the human stomach acid environment and the weakly acidic environment recommended by adults. Its performance is superior to existing commercial strains, ensuring that the strain can successfully pass through the stomach to reach the intestines and colonize stably. It solves the technical pain point that traditional probiotics are easily inactivated in the stomach acid environment, effectively ensuring that it can play a role in the gastrointestinal tract during diarrhea, helping to colonize the intestines and exert an anti-diarrheal effect.

[0016] 5. Dosage Forms Adapted to Different Scenarios, Meeting Diverse Needs: This invention designs two core dosage forms—powder and enteric-coated capsules—to cater to the usage habits of different groups. The powder can be directly mixed with everyday foods such as milk, yogurt, and meal replacement powder, making it suitable for children, the elderly, and other people with swallowing difficulties. The enteric-coated capsules utilize enteric encapsulation technology, which avoids the release of bacterial strains in the stomach, allowing them to dissolve only in the intestines. This further enhances the survival rate of the bacteria upon reaching the intestines, making it suitable for adult diarrhea treatment scenarios that require precise targeting of the intestines, effectively expanding the scope of application and efficiency of the bacterial agents.

[0017] 6. High stability of live bacteria and significant therapeutic effect: Through optimized formulation of bacterial encapsulation agent and freeze-drying process, the bacterial powder can achieve a live bacteria count of ≥1×10⁻⁶ per 0.5g packet. 9 CFU enteric-coated capsules can achieve a live bacteria count of ≥1×10⁻⁶ per capsule. 9 CFU; According to mouse experiments, Lactobacillus reuteri has a better antidiarrheal effect than the commercial drug levofloxacin. It can effectively inhibit the colonization of pathogenic bacteria in the intestine, restore the balance of intestinal flora, shorten the time of diarrhea relief, and improve the characteristics of defecation, providing a high-quality probiotic preparation for the clinical treatment of diarrhea in adults. Attached Figure Description

[0018] Figure 1 This is the result of anaerobic culture of Lactobacillus reuteri OH114 strain streaked on MRS solid plates in an embodiment of the present invention; Figure 2 This is the growth curve of Lactobacillus reuteri OH114 strain in the embodiments of the present invention; Figure 3 The results of the antibacterial experiment of Lactobacillus reuteri OH114 in the embodiments of the present invention; Figure 4This refers to the results of the acid and bile salt resistance test of Lactobacillus reuteri OH114 in the embodiments of the present invention; Figure 5 This is a flowchart of the mouse diarrhea model treatment experiment in this embodiment of the invention; Figure 6 This is a comparison of the improvement of diarrhea symptoms in different groups during human trials in this embodiment of the invention. Detailed Implementation

[0019] Lactobacillus reuteri is a major probiotic native to the adult gut. This invention has discovered a new strain of Lactobacillus reuteri, namely Lactobacillus reuteri OH114. This strain can inhibit a variety of common harmful bacteria in the gut and has good intestinal colonization ability, acid resistance and bile salt resistance.

[0020] The Lactobacillus reuteri ( 罗伊氏乳杆菌罗伊氏亚种 OH114 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 66528 and deposit date of June 16, 2025.

[0021] The Lactobacillus reuteri OH114 has the following properties: (1) The Lactobacillus reuteri OH114 of the present invention is effective against Staphylococcus aureus ( 葡萄球菌属 金黄色 ATCC 6538), Escherichia coli ( 大肠杆菌 ATCC 25922), Salmonella typhimurium ( 沙门氏菌属 鼠伤寒 ATCC 14028), Clostridium difficile ( 艰难梭菌 ATCC 9689), Clostridium perfringens ( 产气荚膜梭菌 ATCC 13124), Klebsiella pneumoniae ( 肺炎克雷伯菌 ATCC700603), Pseudomonas aeruginosa ( 铜绿假单胞菌 ATCC 27853), Candida albicans ( 念珠菌属 白色 It has a good inhibitory effect on various enteropathogenic bacteria such as ATCC 10231, and the antibacterial effect is enhanced as the proportion of culture supernatant increases; (2) The Lactobacillus reuteri OH114 of the present invention has the ability to produce hydrogen peroxide and the color change time after exposure to air is <20 min; (3) The Lactobacillus reuteri OH114 of the present invention has a strong ability to produce lactic acid; (4) The Lactobacillus reuteri OH114 of the present invention has good acid and bile salt resistance characteristics and can maintain a high number of viable bacteria in pH 2-6 environment and 0.3% bile salt concentration; (5) The adhesion rates of Lactobacillus reuteri OH114 of the present invention to human colonic epithelial cells Caco-2 and human small intestinal epithelial cells IEC-6 were 18.3% and 16.7%, respectively, which were significantly higher than those of the control strain Lactobacillus acidophilus LA-5, and it has a stronger intestinal colonization ability.

[0022] The *Lactobacillus reuteri* OH114 of this invention can be formulated into powder or enteric-coated capsules for the treatment of diarrhea in adults. The preparation method of the powder or enteric-coated capsules includes:

[0023] (1) Collection of bacterial cells: After fermentation, centrifuge at 12,000 rpm immediately and complete the process within 10 hours to collect live bacterial cells. The entire process is carried out under aseptic conditions. (2) Cell encapsulation: Add an encapsulation agent consisting of 10wt% skim milk powder, 8wt% trehalose, 7wt% soybean peptone, 6wt% maltose, and 1.5wt% sodium D-isoascorbate. The ratio of the encapsulation agent to the bacterial sludge is 1.5:1. Stir evenly and operate under aseptic conditions. (3) Freeze-drying: Pre-freeze to -45℃, then raise the temperature to -25℃ and hold for 30 minutes, continue to raise the temperature to -15℃ and hold for 30 minutes, then raise the temperature to 25℃ at a frequency of 5℃ each time, each heating process takes about 2.5 hours, and each temperature is held for 30 minutes. The freeze-drying time is 36-48 hours. (4) Pulverization: Pulverize the freeze-dried product to obtain a milky white to light brown powder or granular freeze-dried powder with a special odor of lactic acid bacteria, weigh and store it; (5) Preparation of enteric microcapsules: The freeze-dried powder was mixed with enteric encapsulation material containing 5wt% hydroxypropyl methylcellulose phthalate, 20wt% corn starch and 1wt% magnesium stearate, and sterile water was added to make a suspension. Enteric microcapsules were prepared by spray drying with an inlet air temperature of 180℃ and an outlet air temperature of 80℃. (6) Formulation: Powder: The freeze-dried powder is mixed with excipients such as fructooligosaccharides and galactooligosaccharides and packaged. Each 0.5g package contains ≥1×10⁻⁶ live bacteria. 9 CFU; Enteric-coated capsules: The enteric-coated microcapsules are mixed with excipients containing 30 wt% microcrystalline cellulose, 25 wt% fructooligosaccharides, and 2 wt% silica, and filled into size 0 enteric-coated capsules. Each capsule contains ≥1×10⁻⁶ live bacteria. 9 CFU.

[0024] The key to the bacterial agent disclosed in this invention lies in containing *Lactobacillus reuteri* OH114 disclosed in this invention. Other components can be added according to requirements or dosage form. It is understood that *Lactobacillus reuteri* OH114 disclosed in this invention, as a type of intestinal probiotic, can be used directly as the active ingredient of the bacterial agent of this invention; alternatively, the fermented product of *Lactobacillus reuteri* can be used as the active ingredient of the bacterial agent of this invention, for example, it can be the culture medium of *Lactobacillus reuteri* or its fermented milk products, such as dairy products prepared using the excellent lactic acid production ability of *Lactobacillus reuteri* OH114 of this invention.

[0025] Furthermore, the *Lactobacillus reuteri* OH114 disclosed in this invention also possesses some common characteristics of existing *Lactobacillus reuteri* strains. Therefore, referring to existing *Lactobacillus reuteri* strains, the *Lactobacillus reuteri* OH114 of this invention can also be used to prepare food, health products, or food additives. Moreover, food, health products, or food additives containing the *Lactobacillus reuteri* OH114 of this invention can inhibit the growth of intestinal pathogenic bacteria such as *Staphylococcus aureus*, *Escherichia coli*, *Salmonella typhimurium*, *Clostridium difficile*, *Clostridium perfringens*, *Klebsiella pneumoniae*, *Pseudomonas aeruginosa*, and *Candida albicans*, and have therapeutic or preventative effects on adult diarrhea and regulation of intestinal flora balance.

[0026] It should be noted that the powder product of this invention can be taken orally directly, and the enteric-coated capsules can be precisely targeted to the intestines for release, used to balance the intestinal flora and prevent and improve adult infectious diarrhea, antibiotic-associated diarrhea, or diarrhea caused by irritable bowel syndrome. The invention will be described in detail below with reference to specific embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and should not be construed as limiting the invention.

[0027] Example 1: Isolation and Identification of Lactobacillus reuteri OH114 The culture medium involved in this example is as follows: MRS solid medium (g / L): peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, sodium acetate 5.0 g / L, diamine citrate 2.0 g / L, Tween-80 1.0 g / L, dipotassium hydrogen phosphate 0.4 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.29 g / L, calcium carbonate 20.0 g / L, agar 15.0 g / L.

[0028] MRS liquid culture medium (g / L): peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, sodium acetate 5.0 g / L, diamine citrate 2.0 g / L, Tween-80 1.0 g / L, dipotassium hydrogen phosphate 0.4 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.29 g / L, calcium carbonate 20.0 g / L.

[0029] 1. Isolation of strains The *Lactobacillus reuteri* OH114 was isolated from gut microbiota samples of healthy adults. Specifically, it was obtained from fresh fecal samples of healthy university students from a comprehensive university in Dalian (none of the subjects had metabolic diseases, gastrointestinal diseases, or infectious diseases, and had not used antibiotics, probiotics, or immunosuppressants within 3 months). This sample was collected during the construction of the gut microbiota bank.

[0030] (1) Sample processing: Take 0.2 g of fresh fecal sample and immediately place it in a centrifuge tube containing 5 mL of sterile PBS. Vortex for 10 minutes until uniformly suspended to obtain the mother culture suspension. (2) Serial dilution: The stock bacterial suspension was serially diluted with sterile PBS to 10⁻¹, 10⁻²...10⁻ 7 Take 10⁻ 7 100 μL of diluted bacterial suspension; (3) Spread culture: Spread the above 100 μL diluted solution evenly on MRS solid medium containing calcium carbonate (formula: peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 20 g / L, sodium acetate 5 g / L, diamine citrate 2 g / L, Tween-80 1 g / L, dipotassium hydrogen phosphate 0.4 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.29 g / L, calcium carbonate 20 g / L, agar 15 g / L), and anaerobic culture at 37℃ for 18 hours; (4) Single colony selection: After culturing, observe the plate and select white colonies with a diameter of 2-3 mm, neat edges, and a transparent calcium-dissolving zone around them (indicating acid-producing characteristics). (5) Purification and scale-up culture: The selected single colonies were inoculated into MRS liquid medium and anaerobic cultured at 37°C for 24 hours. Then, the strain was purified three times by the streak plate method (anaerobic cultured at 37°C for 48 hours after each streak) to ensure the purity of the strain. (6) Preservation: The purified strain was inoculated into MRS liquid medium containing 20% ​​glycerol and frozen at -80°C for later use.

[0031] 2. Identification of strains (1) Morphological identification: The isolated strain was streaked onto an MRS solid plate and anaerobically cultured for 48 hours. Colony morphology was then observed, and the results are as follows: Figure 1 As shown, the colonies are round, grayish-white, opaque, with a raised center, smooth surface, and neat edges; after Gram staining, microscopic observation shows that they are positive short rods, without spores, and arranged singly or in pairs.

[0032] Isolation strains based on OD 600 The three-stage growth model of the value, i.e., the growth kinetic curve, is as follows: Figure 2 As shown, the culture conditions were: MRS liquid medium, anaerobic culture at 37°C.

[0033] (2) Molecular biological identification (16S rDNA gene sequencing): 1) DNA extraction: Take DNA from the logarithmic growth phase (OD) 600 Total DNA was extracted from bacterial suspensions containing 0.6–0.8 g of bacterial DNA using a bacterial genomic DNA extraction kit. 2) PCR amplification: PCR amplification was performed using universal primers for the 16S rRNA gene (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'). The reaction volume was 50 μL (containing 2 μL DNA template, 1 μL each of forward and reverse primers, 25 μL Taq enzyme, and 21 μL ddH2O). The reaction conditions were: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, and 72℃ extension for 1 minute, for a total of 30 cycles; and a final extension at 72℃ for 10 minutes. 3) Sequencing and Alignment: After verification by agarose gel electrophoresis, the PCR products were sent to a sequencing company for sequencing. The obtained 16S rRNA gene sequence was uploaded to the NCBI database and compared with known sequences using the BLAST tool. The results showed that the strain was similar to *Lactobacillus reuteri* (…). 罗伊氏乳杆菌 Because its homology with Lactobacillus reuteri was ≥99%, it was named Lactobacillus reuteri OH114. (3) Determination of growth kinetic curve: The Lactobacillus reuteri OH114 was inoculated into MRS liquid medium and cultured anaerobically at 37°C. The OD of the bacterial solution was measured every 2 hours. 600 Values, plot growth curves (see) Figure 2 The results showed that the growth process of this strain was divided into a lag phase (0-4 hours), a logarithmic phase (4-16 hours), and a stationary phase (16-24 hours), with the OD during the stationary phase being... 600 The value can reach 2.83, indicating that the Lactobacillus reuteri OH114 grows vigorously in MRS medium, has good growth performance, and can adapt to large-scale production to improve production efficiency.

[0034] Example 2: Determination of biochemical characteristics of Lactobacillus reuteri OH114 1. Catalase experiment Pick a single colony from an MRS plate, place it on a clean glass slide, and add a few drops of 0.3% hydrogen peroxide solution. The appearance of a large number of bubbles immediately indicates a positive (+) result, while the absence of bubbles indicates a negative (-) result.

[0035] 2. Gelatin liquefaction experiment Pick a single colony from an MRS plate using an inoculation needle, puncture it into gelatin solid medium, and puncture to a depth of about 2 / 3 of the medium depth. Incubate anaerobically at 20°C for 7 days. Before observation, place it at 4°C for 30 minutes, then remove it to observe the results. If the gelatin does not coagulate partially or completely, it is a positive (+) result; if the gelatin still coagulates completely, it is a negative (-) result.

[0036] 3. Hydrogen sulfide experiment Pick a single colony from an MRS plate using an inoculation needle, puncture and inoculate it into lead acetate medium, and incubate anaerobically at 37°C for 24-48 hours. Observe whether a black precipitate appears. If the medium turns black, it is a positive (+) result; if the medium does not change color, it is a negative (-) result.

[0037] 4. Nitrate Reduction Experiment Pick a single colony from an MRS plate using an inoculation needle and inoculate it into nitrate medium. Incubate anaerobically at 37°C for 1–4 days. Add 0.1 mL of a mixture of equal volumes of reagents A and B to a test tube and immediately observe the color change of the liquid. A red color indicates a positive (+) result, while no color reaction after adding the reagents indicates a negative (-) result.

[0038] The biochemical reaction tests of Lactobacillus reuteri OH114 are shown in Table 1.

[0039] Table 1 Biochemical reaction test of Lactobacillus reuteri OH114

[0040] The experimental results are shown in Table 1. The Lactobacillus reuteri OH114 does not contain catalase, cannot liquefy gelatin, does not produce hydrogen sulfide, and does not reduce nitrate.

[0041] Example 3: Determination of the antibacterial activity of Lactobacillus reuteri OH114 1. Detection of lactic acid production capacity of Lactobacillus reuteri OH114 The Lactobacillus reuteri OH114 was inoculated into MRS liquid medium at a 2% inoculum and cultured anaerobically at 37°C for 36 hours. After that, the fermentation broth was centrifuged at 6000 rpm for 15 minutes at 4°C. The supernatant was filtered through a 0.22 μm filter membrane and the lactic acid concentration was determined using the Nanjing Jiancheng Lactic Acid (LD) Detection Kit (catalog number A019-2-1).

[0042] 2. Detection of hydrogen peroxide production capacity of Lactobacillus reuteri OH114 MRS solid plates containing 0.25 mg / mL LTMB and 0.01 mg / mL horseradish peroxidase were prepared, and the plates were placed in the logarithmic growth phase (OD) phase. 600 The *Lactobacillus reuteri* OH114 bacterial suspension (value = 0.6~0.8) was evenly spread on the surface of an agar plate and anaerobically incubated at 37°C for 48 hours. After exposure to air, the colony color change was observed. *Lactobacillus acidophilus* ATCC 33820 was used as a positive control. The time it took for colonies to change from colorless to blue was recorded. Hydrogen peroxide production capacity was assessed based on the color change time (<10 min = high production, 10~20 min = medium production, >20 min = low production).

[0043] This example demonstrates the determination of the hydrogen peroxide production capacity of the Lactobacillus reuteri OH114 and the Lactobacillus acidophilus ATCC 33820.

[0044] Table 2 Results of hydrogen peroxide capacity test

[0045] The experimental results are shown in Table 2. The discoloration time of both *Lactobacillus reuteri* OH114 and *Lactobacillus acidophilus* ATCC 33820 after exposure to air was less than 20 min, indicating that they belong to medium hydrogen peroxide production strains. This proves that *Lactobacillus reuteri* OH114 has hydrogen peroxide-mediated antibacterial ability comparable to that of currently commercial probiotic strains, and can replace existing strains for the prevention and treatment of intestinal infections.

[0046] 3. Antibacterial activity test of Lactobacillus reuteri OH114 The inhibitory effect of *Lactobacillus reuteri* OH114 on common pathogens associated with adult diarrhea was detected using a 96-well plate serial dilution method combined with CFU counting. Target strains included *Staphylococcus aureus*. 金黄色葡萄球菌 ATCC6538), Escherichia coli ( 大肠杆菌 ATCC 25922), Salmonella typhimurium ( 鼠伤寒沙门氏菌 ATCC14028), Clostridium difficile ( 艰难梭菌 ATCC 9689), Clostridium perfringens ( 梭菌属 产气荚膜 ATCC 13124), Klebsiella pneumoniae ( 肺炎克雷伯菌 ATCC 700603), Pseudomonas aeruginosa ( 铜绿假单胞菌 ATCC 27853), Candida albicans ( 白色念珠菌 ATCC10231).

[0047] The specific steps are as follows: In a 96-well plate, an equal volume of the above eight bacterial solutions (each with a bacterial count of 10) was added to each well. 5 CFU was then mixed with different proportions (10%, 20%, 30%) of the *Lactobacillus reuteri* OH114 culture supernatant, with a total volume of 200 μL. Culture medium was used as a positive control instead of the *Lactobacillus* supernatant. The 96-well plate was incubated at 37°C for 12 hours. After incubation, the bacterial cultures from each group were serially diluted 10-fold using the CFU counting method and spotted onto the corresponding solid culture medium (TSA medium for aerobic bacteria such as *Staphylococcus aureus* and *Salmonella typhimurium*, CCFA medium for anaerobic bacteria such as *Clostridium difficile*, and Sabouraud dextrose agar for *Candida albicans*). Once the colonies reached a suitable size for counting, they were counted and analyzed.

[0048] Experimental results are as follows Figure 3 As shown, Figure 3 Staphylococcus aureus (a), Escherichia coli (b), Salmonella typhimurium (c), Clostridium difficile (d), Clostridium perfringens (e), Klebsiella pneumoniae (f), Pseudomonas aeruginosa (g), and Candida albicans (h).

[0049] from Figure 3 The results showed that *Lactobacillus reuteri* OH114 could inhibit the growth of several common harmful bacteria associated with adult diarrhea, including *Staphylococcus aureus* (a), *Escherichia coli* (b), *Salmonella typhimurium* (c), *Clostridium difficile* (d), *Clostridium perfringens* (e), *Klebsiella pneumoniae* (f), *Pseudomonas aeruginosa* (g), and *Candida albicans* (h). As the proportion of *Lactobacillus reuteri* OH114 culture supernatant increased from 10% to 30%, the inhibitory effect on each bacterium showed a gradually increasing trend, providing evidence of its antibacterial activity for application in the treatment of adult diarrhea.

[0050] Example 4: Determination of the acid and bile salt tolerance of Lactobacillus reuteri OH114 1. Acid resistance test of Lactobacillus reuteri OH114 Prepare MRS acid gradient solutions containing 0.2% NaCl: set pH values ​​of 2, 3, 4, 5, and 6 respectively to simulate the acidic environment of the adult intestine, followed by filtration for sterilization. Culture the *Lactobacillus reuteri* OH114 to the logarithmic growth phase (OD2). 600 =0.6~0.8), take 6 parallel bacterial cultures, and measure the OD of each culture. 600 Simultaneously, CFU counts were performed and recorded as the initial bacterial count. For each sample, 1000 μL of bacterial culture was added to 9000 μL of the corresponding pH acid gradient solution, incubated at 37°C for 2 hours (corresponding to the intestinal residence time), and then CFU counts were performed.

[0051] 2. Bile salt tolerance test of Lactobacillus reuteri OH114 MRS culture media containing 0.2% NaCl and bile salt concentrations of 0.1%, 0.3%, 0.5%, 0.7%, and 1.0% were prepared to simulate the adult intestinal bile salt environment, followed by filtration sterilization. The *Lactobacillus reuteri* OH114 culture was cultured to the logarithmic growth phase, and six parallel bacterial suspensions were taken for OD measurement. 600 Simultaneously, CFU counts were performed and recorded as the initial bacterial count. 1000 μL of bacterial culture was added to 9000 μL of culture medium corresponding to the bile salt concentration for each sample. After incubation at 37°C for 4 hours (simulating intestinal transit time), CFU counts were performed.

[0052] Experimental results are as follows Figure 4 As shown, the *Lactobacillus reuteri* OH114 maintains a certain number of viable bacteria in the pH range of 2-6, and the number of viable bacteria gradually increases with increasing pH. The survival ability of *Lactobacillus reuteri* OH114 is significantly enhanced in weakly acidic to neutral environments (pH 4-6), indicating that it has strong acid resistance and can tolerate the acidic environment in the stomach and survive stably in the weakly acidic environment of the adult intestine, which can provide a guarantee for exerting its effect through the gastrointestinal barrier when treating diarrhea. At a bile salt concentration of 0.3% (a common level in the adult intestine), its viable bacteria count is maintained at a high level (225,000 CFU / mL), proving that it can tolerate the intestinal bile salt environment, which helps it colonize the intestine and exert its anti-diarrheal effect.

[0053] Example 5: Determination of intestinal adhesion ability of Lactobacillus reuteri OH114 Adhesion ability is a key component of the probiotic properties of probiotics. For Lactobacillus reuteri, which is used to treat adult diarrhea, strong adhesion ability helps it colonize the intestine, compete for space with diarrhea-associated pathogens, and prevent pathogens from invading intestinal epithelial cells.

[0054] 1. Adhesion of Lactobacillus reuteri OH114 strain to human colonic epithelial cells Caco-2 Human colonic epithelial cells (Caco-2) were purchased from the Cell Bank of the Chinese Academy of Sciences. Caco-2 cells were cultured in cell culture flasks until approximately 80% confluence. Then, 1 mL of 0.25% trypsin-EDTA solution was added for 2 minutes to digest the cells, followed by gentle pipetting to prepare a cell suspension. 3 mL of DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-dextrose antibiotics was added and mixed thoroughly. 2 mL of the cell suspension (approximately 4 × 10⁻⁶ cells / mL) was then taken and mixed. 4 (cells / mL) were seeded into 6-well plates and cultured at 37°C and 5% CO2 until a monolayer was formed.

[0055] Take the logarithmic growth phase (OD) 600The *Lactobacillus reuteri* OH114 culture medium (0.6~0.8 g / mL) was centrifuged at 3000 rpm for 10 minutes at 4°C. After discarding the supernatant, the cells were resuspended in sterile PBS and washed three times. An equal volume of DMEM high-glucose medium was added to prepare a bacterial suspension, which was then spread on MRS plates for viable cell counting. *Lactobacillus acidophilus* LA-5 was used as a control strain. For the adhesion experiment, the culture medium in the 6-well plates was first removed, and the cells were washed three times with 2 mL of sterile PBS in each well. 2 mL of the bacterial suspension was added, and the plates were gently shaken to distribute the cells evenly. The plates were then incubated at 37°C in a 5% CO2 incubator for 2.5 hours. After incubation, the bacterial culture was aspirated, and the cells were washed three times with sterile PBS. 0.5 mL of trypsin-EDTA solution was added to digest the cells. The digestion solution was collected and 1 mL of sterile PBS was added to resuspend the cells. 1 mL of 0.05% Triton X-100 was added to lyse the cells. After dilution, the cells were spread on MRS plates for counting, and the adhesion rate was calculated according to the formula: Adhesion rate = (Number of viable cells after adhesion / Number of viable cells before adhesion) × 100%.

[0056] 2. Adhesion assay of Lactobacillus reuteri OH114 to human small intestinal epithelial cells IEC-6 Human small intestinal epithelial cells IEC-6 were purchased from the Cell Bank of the Chinese Academy of Sciences. IEC-6 cells were cultured in cell culture flasks until approximately 80% confluence. The cells were then digested with 1 mL of 0.25% trypsin-EDTA, and a cell suspension was prepared by adding 3 mL of DMEM medium containing 10% FBS and 1% penicillin-dextrose antibiotics. 2 mL of this suspension was seeded into 6-well plates (4 × 10⁻⁶). 4 (cells / mL), and cultured in a 37℃, 5% CO2 incubator until the cells form a monolayer.

[0057] After centrifugation and washing, the Lactobacillus reuteri OH114 bacterial suspension was resuspended in DMEM medium. After viable cell count, adhesion experiments were performed simultaneously with the Lactobacillus acidophilus LA-5 control strain: the cell plate was washed three times with sterile PBS, the bacterial suspension was added, and the plate was incubated at 37°C for 2.5 hours. Subsequently, the cells were digested with trypsin, lysed with Triton X-100, and counted to calculate the adhesion rate.

[0058] Table 3 Cell adhesion results

[0059] The cell adhesion test results are shown in Table 3. The adhesion rate of Lactobacillus reuteri OH114 to human colonic and small intestinal epithelial cells was significantly higher than that of the control strain, indicating that it has a stronger intestinal colonization ability. It can inhibit the adhesion and invasion of diarrhea pathogens (such as Escherichia coli and Salmonella) by occupying epithelial cell adhesion sites, and can provide a functional basis for the treatment of adult diarrhea.

[0060] Example 6: Preparation of Lactobacillus reuteri OH114 bacterial powder and enteric-coated capsules 1. Preparation of Lactobacillus reuteri OH114 bacterial powder (1) Collection of bacterial cells: After fermentation, Lactobacillus reuteri OH114 was immediately separated by high-speed centrifugation. The centrifugation speed was set to 12,000 rpm and the operation time was completed within 10 hours. Live bacterial cells were collected by centrifugation. The whole process was carried out under strict aseptic conditions.

[0061] (2) Embedding of bacterial cells: Embedding agent is added to embed the bacterial cells. The formula of the embedding agent is: 10wt% skim milk powder, 8wt% trehalose, 7wt% soybean peptone, 6wt% maltose, 1.5wt% sodium D-isoascorbate. The mass ratio of the embedding agent to the bacterial sludge is 1.5:1. Stir evenly and operate under strict aseptic conditions throughout the process.

[0062] (3) Freeze-drying: Pre-freeze by lowering the temperature to -45°C, then raise the temperature to -25°C and hold for 30 minutes, then raise the temperature to -15°C and hold for 30 minutes, then raise the temperature to 25°C at a rate of 5°C each time. Each heating process takes about 2.5 hours and is held at each temperature for 30 minutes. The freeze-drying time is 36-48 hours.

[0063] (4) Pulverization: After pulverizing the freeze-dried product, the freeze-dried powder is a milky white to light brown powder or granules with a special odor of lactic acid bacteria. Weigh the freeze-dried powder and store it.

[0064] 2. Preparation method of Lactobacillus reuteri OH114 enteric-coated capsules (1) Preparation of enteric microcapsules: The bacterial powder was mixed with the enteric encapsulation material, which included 5 wt% hydroxypropyl methylcellulose phthalate, 20 wt% corn starch and 1 wt% magnesium stearate. After mixing, sterile water was added to make a suspension. Enteric microcapsules were prepared by spray drying, with the inlet air temperature set at 180℃ and the outlet air temperature at 80℃.

[0065] (2) Capsule filling: The enteric microcapsules are mixed with excipients, including 30 wt% microcrystalline cellulose, 25 wt% fructooligosaccharides, and 2 wt% silica. After being mixed evenly, the mixture is filled into No. 0 enteric capsules to obtain the enteric capsules of this embodiment. Each capsule contains ≥1 × 10⁻⁶ live Lactobacillus reuteri OH114 bacteria. 9 CFU.

[0066] Example 7: Treatment Experiment of Mouse Diarrhea Model Using Lactobacillus reuteri OH114 Forty 6-8 week old SPF-grade female Balb / c mice (weighing 16-20g) were randomly divided into four groups: healthy group, infected group, levofloxacin group and Lactobacillus reuteri OH114 group, with 10 mice in each group.

[0067] Reference Figure 5 The protocol established a mouse model of diarrhea caused by *E. coli* O157:H7 infection: From Day 1 to Day 3, mice were acclimatized by gavage with 0.5 mL of sterile saline daily; from Day 4 to Day 6, mice were gavage with 200 μL of *E. coli* O157:H7 bacterial suspension (1×10⁻⁶) for 3 consecutive days. 9 The CFU / mL level was measured, and the healthy group was given an equal volume of physiological saline by gavage. On Day 6, the establishment of the diarrhea model was confirmed by fecal occult blood test and pathological section observation.

[0068] Administration continued for 7 days from Day 7 to Day 13: The healthy group and the infected group were administered 200 μL of normal saline by gavage; the levofloxacin group was administered 200 μL of levofloxacin solution (10 mg / kg) by gavage; and the Lactobacillus reuteri OH114 group was administered 200 μL of bacterial suspension (1×10⁻⁶) by gavage. 10 (CFU / mL).

[0069] Mouse feces were collected on day 6 (before drug administration), day 7 (after drug administration), and day 13 (after drug administration). The feces were resuspended in 500 μL of sterile PBS and serially diluted 10-fold. 100 μL of each diluted feces was spread onto MAC agar plates (for E. coli counting) and MRS agar plates (for Lactobacillus counting). After incubation at 37°C for 24-48 hours, CFU were counted. Daily diarrhea scores (0-3 points) were recorded, with the following scoring criteria: 0 points for formed stool, 1 point for soft stool, 2 points for loose stool, and 3 points for watery stool.

[0070] Table 4 Results of Treatment Experiments in Mouse Diarrhea Model

[0071] The test results are shown in Table 4. It can be seen that on the 7th day after administration, the number of Escherichia coli in the feces of the Lactobacillus reuteri OH114 group was significantly lower than that in the infection group, while the number of Lactobacillus was significantly higher. The diarrhea score decreased from 2.8 ± 0.5 points after modeling to 0.5 ± 0.3 points. The antidiarrheal effect was better than that of the levofloxacin group, indicating that Lactobacillus reuteri OH114 can effectively inhibit the colonization of pathogenic bacteria in the intestine, restore the balance of intestinal flora, and has a therapeutic effect on adult diarrhea.

[0072] Example 8: Human safety and efficacy trial of Lactobacillus reuteri OH114 1. Test materials Active ingredients: Lactobacillus reuteri OH114 bacterial powder and enteric-coated capsules (preparation method is the same as in Experiment 6).

[0073] Bacterial powder: Bacterial cells were collected by centrifugation at 12,000 rpm, encapsulated with 10 wt% skim milk powder and 8 wt% trehalose, then freeze-dried and pulverized. Each 0.5g packet contains ≥1×10⁻⁶ live bacteria. 9CFU.

[0074] Enteric-coated capsules: Bacterial powder and enteric materials such as 5% hydroxypropyl methylcellulose phthalate are spray-dried into microcapsules, which are then filled into size 0 capsules. Each capsule contains ≥1×10⁻⁶ live bacteria. 9 CFU.

[0075] Excipients: Microcrystalline cellulose, fructooligosaccharides, etc. (all meet pharmaceutical standards), and are used after passing viable bacteria count and miscellaneous bacteria test.

[0076] 2. Test subjects Forty adult patients with acute diarrhea (aged 18-60, with ≥4 bowel movements / day and watery stools) were recruited, excluding those with infectious diarrhea and a history of probiotic allergy, and randomly divided into three groups: (1) Fungal powder group: 14 people, orally administered 0.5 g / time, 3 times a day for 5 consecutive days; (2) Enteric-coated capsule group: 14 people, orally administered enteric-coated capsules (0.5 g / time), twice a day for 5 consecutive days; (3) Control group: 12 people, who took oral excipient capsules (0.5 g / time) without probiotics three times a day for five consecutive days.

[0077] 3. Test Results (1) Safety of use Table 5. Distribution of participants in each group regarding safety status.

[0078] The test results are shown in Table 5. No adverse reactions such as nausea or vomiting occurred in any of the subjects, and their liver and kidney function indicators were normal, indicating that the preparation is safe, complies with drug quality management and safety regulations, and has few side effects.

[0079] (2) Effects on diarrhea symptoms and stool form Table 6. Distribution of participants in each group regarding changes in bowel movements

[0080] The experimental results are shown in Table 6. In the Lactobacillus reuteri powder group, most patients experienced relief from diarrhea and their stools gradually became formed. In the enteric-coated capsule group, the improvement in diarrhea was more significant, and the recovery rate of stool form was higher than that in the powder group. In the control group, the diarrhea symptoms did not improve significantly, and the loose stools persisted.

[0081] Daily diarrhea scores (0-3 points) were recorded simultaneously, with the following scoring criteria: formed stool 0 points, soft stool 1 point, loose stool 2 points, and watery stool 3 points. Diarrhea scores before and after treatment were statistically analyzed for each group, and the results are as follows: Figure 6 As stated above.

[0082] like Figure 6The results showed that both the bacterial powder group and the enteric-coated capsule group had significant therapeutic effects compared to the control group, especially the enteric-coated capsule group, which had a better therapeutic effect. This indicates that the Lactobacillus reuteri strain performed better in the enteric-coated capsule dosage form and has higher application value. It can effectively fill the gap in the field of enteric-coated capsule drugs for treating diarrhea and regulating intestinal flora balance.

[0083] (3) Effects on intestinal inflammation and discomfort Table 7 Distribution of participants in each group regarding the recovery of intestinal inflammation

[0084] The results are shown in Table 7. In the bacterial powder group, patients with intestinal inflammation-related discomfort (such as abdominal pain and bloating) were generally relieved, and some completely disappeared. The enteric-coated capsule group had a better effect on relieving inflammation, and most patients' discomfort symptoms were eliminated. In the control group, intestinal inflammation and discomfort did not improve, and the symptoms persisted.

[0085] The experimental results above show that both Lactobacillus reuteri OH114 powder and enteric-coated capsules are safe and effective in treating adult diarrhea. Among them, the enteric-coated capsules, due to their intestinal-targeted release characteristics, can more effectively regulate the intestinal flora, shorten the time to stop diarrhea, and improve the characteristics of defecation, providing a high-quality probiotic preparation option for the clinical treatment of adult diarrhea.

[0086] Experimental results showed that the *Lactobacillus reuteri* OH114 disclosed in this invention is effective against *Staphylococcus aureus* (…). 金黄色葡萄球菌 ATCC 6538), Escherichia coli ( 大肠杆菌 ATCC 25922), Salmonella typhimurium ( 鼠伤寒沙门氏菌 ATCC 14028), Clostridium difficile ( 艰难梭菌 艰难 ATCC9689), Clostridium perfringens ( 产气荚膜梭菌 ATCC13124), Klebsiella pneumoniae ( 肺炎克雷伯菌 ATCC700603), Pseudomonas aeruginosa ( 假单胞菌属 铜绿 ATCC27853), Candida albicans ( 白色念珠菌 It has a good inhibitory effect on various enteropathogenic bacteria such as ATCC10231; it can adhere to intestinal epithelial cells, has a strong colonization ability, and has good acid and bile salt resistance; it can be made into powder or enteric-coated capsules to balance the intestinal flora, improve adult infectious diarrhea, antibiotic-associated diarrhea or diarrhea-type irritable bowel syndrome, relieve diarrhea symptoms and intestinal inflammation discomfort such as abdominal pain and bloating, and has no side effects.

[0087] The above description represents the preferred embodiments of the present invention. It should be noted that all reagents and materials used in the embodiments of the present invention, unless otherwise specified, are standardized products that can be obtained through conventional commercial channels. However, it should be understood that those skilled in the art can choose equivalent products from other suppliers, which does not depart from the scope of protection of the present invention. For those skilled in the art, several improvements and modifications can be made without departing from the principles described in the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus reuteri, characterized in that, Lactobacillus reuteri limosilactobacillus reuteri subsp.reuteri OH114 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 66528, and deposited on June 16, 2025.

2. A microbial agent, characterized in that, It includes Lactobacillus reuteri as described in claim 1 and a pharmaceutically or food-grade carrier.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent is in the form of powder or enteric-coated capsules, and the viable count of *Lactobacillus reuteri* in the bacterial agent satisfies the following condition: ≥1 × 10⁻⁶ in the powder. 9 CFU / serving, ≥1×10⁻⁶ CFU / serving in enteric-coated capsules 9 CFU / capsule.

4. A method for preparing the powdered microbial agent according to claim 3, characterized in that, Includes the following steps: (1) Culture the Lactobacillus reuteri and collect the bacterial cells; (2) The bacterial cells and the bacterial cell encapsulation agent are mixed at a weight ratio of 1:1.5, wherein the bacterial cell encapsulation agent contains at least one of skim milk powder, trehalose, soybean peptone, maltose, and sodium D-isoascorbate; (3) After freeze-drying, the powder is pulverized to obtain freeze-dried powder; (4) The freeze-dried powder is mixed with pharmaceutically acceptable excipients, including fructooligosaccharides, galactooligosaccharides, additives (including at least one of monosodium glutamate, lysozyme, and skim milk) and packaging materials (including at least one of silica and magnesium stearate) to form a powder.

5. A method for preparing the enteric-coated capsule-type bacterial agent according to claim 3, characterized in that, Includes the following steps: (1) Collect live cells by centrifugation after fermentation of the Lactobacillus reuteri culture; (2) The bacterial cells and the bacterial cell encapsulation agent are mixed at a weight ratio of 1:1.5, wherein the bacterial cell encapsulation agent includes at least one of skim milk powder, trehalose, soybean peptone, maltose, and sodium D-isoascorbate; (3) Freeze-dry the bacterial cells containing the bacterial cell encapsulation agent; (4) The freeze-dried product is pulverized to obtain freeze-dried powder; (5) The freeze-dried powder is mixed with enteric microcapsule encapsulation material (containing at least one of hydroxypropyl methylcellulose phthalate, corn starch, and magnesium stearate), and sterile water is added to prepare a suspension. Enteric microcapsules are then prepared by spray drying. (6) The enteric microcapsules are mixed with enteric capsule excipients (including at least one of microcrystalline cellulose, fructooligosaccharides, and silicon dioxide) and filled into enteric capsules to obtain the product.

6. The preparation method according to claim 4 or claim 5, characterized in that, The contents of each component in the bacterial cell encapsulation agent are as follows: 10wt% skim milk powder, 8wt% trehalose, 7wt% soybean peptone, 6wt% maltose, and 1.5wt% sodium D-isoascorbate.

7. The preparation method according to claim 4, characterized in that, The raw material ratio of the powder is 25-35 parts freeze-dried powder, 20-30 parts fructooligosaccharide, 20-30 parts galactooligosaccharide, 50-80 parts additives, and 60-100 parts packaging material.

8. The preparation method according to claim 5, characterized in that, The enteric-coated microcapsule encapsulation material contains 5 wt% hydroxypropyl methylcellulose phthalate, 20 wt% corn starch, and 1 wt% magnesium stearate; the enteric-coated capsule excipients contain 30 wt% microcrystalline cellulose, 25 wt% fructooligosaccharides, and 2 wt% silicon dioxide.

9. The use of Lactobacillus reuteri as described in claim 1 or the bacterial agent as described in claim 2 in the preparation of products for the treatment or prevention of diarrhea.

10. A product for treating or preventing diarrhea, regulating intestinal flora balance, or inhibiting intestinal pathogens, characterized in that, The product contains Lactobacillus reuteri as described in claim 1 or the bacterial agent as described in claim 2, wherein the enteropathogenic bacteria include Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Clostridium difficile, Clostridium perfringens, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Candida albicans.

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