Probiotic compositions for improving diarrhea and their uses

Abstract

This invention provides a probiotic composition for improving diarrhea and its uses. The probiotic composition of this invention comprises space-occupying bacteria, organic acid-producing bacteria, and oxygen-depleting bacteria, wherein the oxygen-depleting bacteria are *Weizmannii*, the organic acid-producing bacteria are *Lactobacillus* or *Lactobacillus lactis*, and the space-occupying bacteria are *Bifidobacterium*. The probiotic composition of this invention can effectively improve diarrhea and is therefore suitable for the prevention and / or treatment of diarrhea-related functional gastrointestinal disorders.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a probiotic composition for improving diarrhea and its use in the preparation of products for the prevention and / or treatment of diarrhea. Background Technology

[0002] In the field of gastrointestinal dysfunction, diarrhea is a highly prevalent disease that seriously affects quality of life. Its pathological mechanisms are closely related to the dysfunction of the "gut-brain axis" and the imbalance of the intestinal microecology.

[0003] Currently, antidiarrheal drugs are commonly used in clinical practice for symptomatic management of diarrhea, but they also have the problem of failing to correct underlying dysbiosis and intestinal barrier damage. Overall, traditional drugs have single targets and are difficult to restore the inherent functional homeostasis of the intestine.

[0004] Given the limitations of chemical drugs, probiotics and other microecological regulators have become a research hotspot, but current technologies still face significant bottlenecks. First, the efficacy of single strains is limited. Most studies focus on evaluating the efficacy of single strains, but the target sites of single strains are limited, making it difficult to systematically and comprehensively regulate the intestinal environment. Second, the synergistic effect of simple bacterial combinations is insufficient. Current approaches mostly involve simple strain stacking or the application of single-type products, lacking systematic research and validation of synergistic effects between different bacterial genera and strains.

[0005] Therefore, providing a probiotic composition that can effectively improve functional gastrointestinal disorders, especially diarrhea, is a technical problem that urgently needs to be solved. Summary of the Invention

[0006] Based on the above-mentioned prior art, the purpose of this invention is to provide a probiotic composition for improving diarrhea and its uses.

[0007] In a first aspect, the present invention provides a probiotic composition for improving diarrhea, wherein the probiotic composition comprises occupying bacteria, organic acid-producing bacteria, and oxygen-depleting bacteria.

[0008] In this invention, the term "oxygen-depleting bacteria" refers to a class of probiotics that can effectively consume oxygen through their own metabolism in the gastrointestinal environment, thereby reducing the local redox potential (Eh) and creating and maintaining a suitable anaerobic environment for the growth of beneficial anaerobic bacteria.

[0009] In this invention, the oxygen-depleting bacteria are preferably Weizmann's bacteria.

[0010] According to some embodiments of the present invention, the Weizmania is selected from Weizmannia coagulans.

[0011] In this invention, the term "organic acid-producing bacteria" refers to a class of probiotics that can produce and secrete organic acids (e.g., lactic acid, acetic acid, propionic acid, butyric acid, etc.) by metabolizing carbohydrates in the gastrointestinal environment. Organic acid-producing bacteria lower the pH value of the intestinal environment by secreting organic acids, thereby directly or indirectly inhibiting the growth of harmful bacteria and regulating the balance of the intestinal flora.

[0012] In this invention, the organic acid-producing bacteria are preferably Lactobacillus and / or Lactobacillus lactis.

[0013] According to some embodiments of the present invention, the lactobacillus is selected from *Lactobacillus acidophilus*, *Lactobacillus casei*, *Lactobacillus paracasei*, *Lactobacillus crispatus*, *Lactobacillus delbrueckii* ssp. bulgaricus*, *Lactobacillus fermentum*, *Lactobacillus gasseri*, *Lactobacillus shelveticus*, *Lactobacillus johnsonii*, *Lactobacillus plantarum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Lactobacillus salivarius*. One or more of *Lactobacillus salivarius*, *Lactobacillus sakei*, and *Lactobacillus curvus*.

[0014] According to some embodiments of the present invention, the lactobacillus is selected from one or more of Lactiplantibacillus plantarum, Lactiplantibacillus plantarum subsp. argentoratensis, Lactiplantibacillus pentosus, Lactiplantibacillus paraplantarum, Lactiplantibacillus fabifermentans, Lactiplantibacillus tropicalus, and Lactiplantibacillus nakhonrathomensis.

[0015] In this invention, the term "occupying bacteria" refers to a class of probiotics that can competitively adhere to intestinal mucosal epithelial cells and occupy intestinal ecological sites, thereby preventing or reducing the colonization of pathogenic and opportunistic pathogens.

[0016] In this invention, the occupant bacteria is preferably Bifidobacterium.

[0017] According to some embodiments of the present invention, the Bifidobacterium is selected from one or more of Bifidobacterium animalis subsp. lactis, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium longum.

[0018] According to some embodiments of the present invention, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, and *Bifidobacterium*.

[0019] Preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, and *Bifidobacterium* in an effective live bacteria concentration ratio of (0.6-1.4):(0.6-9.4):(10-20):(0.6-9.4).

[0020] According to some embodiments of the present invention, the Bifidobacterium is selected from Bifidobacterium longum subsp. infantis or Bifidobacterium animalis subsp. lactis;

[0021] Preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Bifidobacterium longum* subsp. *infantii*, and *Bifidobacterium animalis* subsp. *lactobacterium* in an effective live bacteria concentration ratio of (0.6-1.4):(0.6-9.4):(10-20):(0.6-1.4):(4-8).

[0022] According to some embodiments of the present invention, the Lactobacillus acidophilus includes a first Lactobacillus acidophilus and a second Lactobacillus acidophilus, wherein the first Lactobacillus acidophilus and the second Lactobacillus acidophilus are different strains from each other;

[0023] Preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus* var. *m-1*, *Lactobacillus acidophilus* var. *m-2*, *Lactobacillus plantarum*, *Bifidobacterium longum* subsp. *infantii*, and *Bifidobacterium animalis* subsp. *lactobacterium* in an effective live bacteria concentration ratio of (0.6-1.4):(4-8):(0.6-1.4):(10-20):(0.6-1.4):(4-8) in that order.

[0024] According to some embodiments of the present invention, the *Weizmannia coagulans* is selected from at least one of *Weizmannia coagulans* BC99, *Weizmannia coagulans* HY08866, *Weizmannia coagulans* HY08867, and *Weizmannia coagulans* HY08874.

[0025] It should be noted that *Weizmannia coagulans* and *Heyndrickxia coagulans* are names of the same bacterium from different classification and nomenclature periods; therefore, they have the same meaning in this invention. For example, *Weizmannia coagulans* HY08866 is also *Heyndrickxia coagulans* HY08866, and both refer to the same strain.

[0026] The first and second *Lactobacillus acidophilus* are each independently selected from at least one of *Lactobacillus acidophilus* LA-5, *Lactobacillus acidophilus* CS003, *Lactobacillus acidophilus* DDS-1, *Lactobacillus acidophilus* HY01039, *Lactobacillus acidophilus* HY00760, and *Lactobacillus acidophilus* HY01043; and / or,

[0027] The *Lactobacillus plantarum* is selected from at least one of *Lactobacillus plantarum* HY02946, *Lactobacillus plantarum* HY05181, and *Lactobacillus plantarum* HY00050; and / or,

[0028] The *Bifidobacterium longum* infant subsp. *longum* is selected from at least one of *Bifidobacterium longum* infant subsp. *CS004*, *Bifidobacterium longum* infant subsp. *MP09089*, and *Bifidobacterium longum* infant subsp. *MP09270*; and / or,

[0029] The *Bifidobacterium lactis* subsp. *animal* is selected from at least one of *Bifidobacterium lactis* subsp. *animal* BB-12, *Bifidobacterium lactis* UABla-12, *Bifidobacterium lactis* subsp. *animal* HY11786, *Bifidobacterium lactis* subsp. *animal* MP15099, *Bifidobacterium lactis* subsp. *animal* HY09279 and *Bifidobacterium lactis* HY10036.

[0030] According to some embodiments of the present invention, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003.

[0031] Preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium longum* subsp. *infantica* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (10-20):(0.6-1.4):(0.6-1.4):(0.6-1.4).

[0032] More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 15:1:1:1.

[0033] According to some embodiments of the present invention, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacterium* BB-12, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or HY08866, and *Lactobacillus acidophilus* CS003.

[0034] Preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (10-20): (4-8): (0.6-1.4): (0.6-1.4): (0.6-1.4).

[0035] More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 15:6:1:1:1.

[0036] According to some embodiments of the present invention, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Lactobacillus acidophilus* LA-5, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003.

[0037] Preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Lactobacillus acidophilus* LA-5, *Bifidobacterium longum* subsp. *infantica* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (10-20): (4-8): (4-8): (0.6-1.4): (0.6-1.4): (0.6-1.4).

[0038] More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Lactobacillus acidophilus* LA-5, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 15:6:6:1:1:1.

[0039] Or more preferably, the probiotic composition comprises *Lactobacillus plantarum* HY00050, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Lactobacillus acidophilus* LA-5, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 15:6:6:1:1:1.

[0040] Or more preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Lactobacillus acidophilus* LA-5, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 20:8:8:1.4:1.4:1.4.

[0041] In a second aspect, the present invention provides a product for preventing, alleviating, improving and / or treating diarrhea, comprising the probiotic composition according to the first aspect of the present invention.

[0042] According to some embodiments of the present invention, the product further comprises excipients; the excipients are selected from one or more of pharmaceutical excipients, food excipients, and health product excipients.

[0043] In this invention, the terms "pharmaceutical excipient," "pharmaceuticalally acceptable excipient," or similar terms generally refer to substances that facilitate administration of the active agent to a subject and absorption by the subject, and which may be included in the medicament of this invention without causing significant adverse toxicological effects on the patient.

[0044] In this invention, the term "food additives" or similar terms generally refer to auxiliary materials added during food processing, other than the main raw materials, to improve the quality, processing technology, preservation performance, or nutritional value of the food. They typically do not constitute the main body of the food, but play a crucial role in the final product's appearance, taste, stability, and safety.

[0045] In this invention, the term "health supplement excipient" or similar terms generally refer to auxiliary materials other than the active ingredient in health foods (dietary supplements), used to transform the active ingredient into a dosage form suitable for consumer use and to ensure the stability, safety and effectiveness of the product.

[0046] According to some embodiments of the present invention, non-limiting examples of the excipients may be selected from one or more of excipients, fillers, flow aids, diluents, instant solvents, and disintegrants. Specifically, they are selected from water, NaCl, physiological saline solutions, lactated Ringer's solution, ordinary sucrose, ordinary glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions, alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and dyes, etc. Such articles can be sterilized, and those skilled in the art will recognize that other excipients may also be used in the present invention.

[0047] According to some embodiments of the present invention, the constipation is selected from functional constipation, slow transit constipation, outlet obstruction constipation, drug-induced constipation, metabolic-endocrine constipation, low volume constipation, dysbiosis constipation, and diarrhea-predominant irritable bowel syndrome.

[0048] According to some embodiments of the present invention, the product is selected from one or more of pharmaceuticals, food, and health products.

[0049] Thirdly, the present invention provides the use of the probiotic composition according to the first aspect of the present invention in the preparation of products for the prevention and / or treatment of diarrhea.

[0050] According to some embodiments of the present invention, the diarrhea is selected from one or more of diarrhea-predominant irritable bowel syndrome, acute diarrhea, chronic diarrhea, osmotic diarrhea, secretory diarrhea, exudative diarrhea, motility diarrhea, antibiotic-associated diarrhea, and infectious diarrhea.

[0051] According to some embodiments of the present invention, the product is selected from one or more of pharmaceuticals, food, and health products.

[0052] Compared with the prior art, the present invention has at least the following beneficial effects:

[0053] This invention overcomes the limitations of existing probiotic products, such as single-function strains, limited targets, and lack of synergistic technology, and develops a probiotic composition suitable for a variety of complex gastrointestinal disorders.

[0054] This invention uses in vitro experiments to determine the inhibition rate of fermentation broth of strains with potential to alleviate diarrhea against Escherichia coli and Staphylococcus aureus. Considering both overall antibacterial effect and species richness, the antibacterial rate was measured using single strains, dual-strain combinations, and hexa-strain combinations. The results showed that as the number of combined strains increased, the overall antibacterial effect gradually increased. Furthermore, most combined strains exhibited similar inhibitory trends against Staphylococcus aureus and Escherichia coli, with the hexa-strain combination showing the best overall antibacterial effect.

[0055] The present invention also demonstrated through in vitro experiments that the tetravalent, pentavalent, and hexavalent bacterial compositions of the present invention can reduce the average fecal water content of mice and have a significant effect on improving antibiotic-associated diarrhea and infectious diarrhea. Detailed Implementation

[0056] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0057] The method for detecting viable bacteria count in this example is as follows:

[0058] ① F1 generation breeding:

[0059] Mycelium powder: Weigh 0.1g of mycelium powder into a 15mL centrifuge tube, add 4.9mL of MRS or TPY broth and mix well;

[0060] Glycerin bacteria: Take glycerin bacteria stored at -80℃, thaw them, and inoculate them into MRS or TPY broth at an inoculation rate of 4% (Lactobacillus or Lactobacillus lactis) or 10% (Bifidobacterium or Weizmannii) and mix well. Incubate anaerobically at 37℃ for 24-48 hours.

[0061] ② F2 generation culture: After mixing the F1 generation bacterial culture, transfer it to MRS or TPY broth at the same inoculation amount and anaerobic static culture at 37℃ for 24h-48h.

[0062] ③ Preparation of 1% solid culture medium: Weigh 52.24g of MRS broth and 36.65g of TPY broth into 1L beakers, add 10g of agar powder to each, and add 1L of distilled water. Mix well, dispense into 400mL glass bottles, autoclave at 118℃ for 15min, and then place in a 50℃ water bath for later use.

[0063] ④ Dilution and Sample Addition: Add 450 μL of PBS to each well of a 96-well plate. After mixing the F2 generation bacterial culture, perform a tenfold serial dilution with PBS (i.e., add 50 μL of bacterial culture to 450 μL of PBS). Take 10 μL of the diluted sample. 5 10 6 and 10 7 Add 100 μL of each dilution solution to the center of a sterile Petri dish, pour in about 15 mL of MRS or TPY semi-solid culture medium and shake well. After drying, incubate upside down at 37°C for 48-72 h.

[0064] ⑤ After incubation, select plates with colony counts between 30-300 CFU for counting to determine the viable count:

[0065] viable count = average colony count × dilution factor

[0066] Example 1: Screening of Functional Bacteria

[0067] I. Study on the inhibitory effects of different strains on pathogenic bacteria

[0068] 1. Candidate strains

[0069] Candidate strains are shown in Table 1.

[0070] The applicant's (Hangzhou Yuanda) proprietary strains were stored at -80°C in preservation tubes containing 15% (v / v) glycerol. Commercially purchased strains were in powder form or isolated from products.

[0071]

[0072]

[0073] 2. Experimental Methods

[0074] (1) Culture of strains

[0075] ① F1 generation: Take out 25 glycerol tubes of the test strains from the -80℃ freezer. After the glycerol tubes are thawed, mix the bacterial solution and add the bacterial solution to MRS or TPY liquid medium at an inoculation rate of 4%-10% (v / v). Incubate at 37℃ in an anaerobic workstation for 24-48 hours.

[0076] ② F2 generation: Gently mix the cultured F1 generation bacterial suspension and transfer it to 6 mL of MRS or TPY liquid medium at the same inoculum size. Incubate at 37℃ in an anaerobic workstation for 24-48 hours. After two generations of continuous activation, each candidate strain is combined into single-strain, dual-strain, triple-strain, quadri-strain, pentazocine, and hexa-strain groups. The components of the multi-strain groups are from different species and combined in equal proportions. The final concentration of each group composition is 1E+08.

[0077] (2) Antibacterial test

[0078] Escherichia coli:

[0079] ① Preparation of Escherichia coli plates

[0080] Pathogenic bacteria culture: Escherichia coli was activated and transferred to BHI liquid medium at an inoculation rate of 4% until the early stable phase. The concentration was adjusted to 1.0E+06 CFU / mL. 100 μL was spread on BHI solid plates and dried for later use.

[0081] ②Drilling holes in flat plates

[0082] Use a 1mL pipette tip to make three holes evenly on each E. coli plate, and four holes on each CK plate.

[0083] ③ Interaction experiment with Escherichia coli

[0084] Add 100 μL of the experimental group to each well of the experimental plate, with 3 replicates per group. Add MRS liquid medium and TPY liquid medium to the CK plate, with 2 replicates for each. Let stand for 1 hour to allow complete infiltration into the plate, then place it upright in an incubator at 37°C and incubate aerobically for 24 hours.

[0085] ④ Inhibition zone determination

[0086] The diameter of the inhibition zone on the experimental plate and the control plate was measured using a ruler, and the inhibition rate of the target bacteria against Escherichia coli was calculated.

[0087] The relative inhibition rate (%) of the target bacteria against Escherichia coli = (diameter of the inhibition zone of the target bacteria - diameter of the inhibition zone of the control group) / diameter of the inhibition zone of the control group

[0088] Staphylococcus aureus:

[0089] ①Preparation of Staphylococcus aureus bacterial culture

[0090] At an inoculation rate of 4%, Staphylococcus aureus was activated and transferred to BHI liquid medium until the early stable phase. The concentration was adjusted to 1.0E+04 CFU / mL, and 5 mL was transferred to a shaker tube for later use.

[0091] ②Preparation of Staphylococcus aureus culture medium

[0092] Weigh 67.4g of Staphylococcus aureus chromogenic medium powder, add 1L of distilled water or deionized water, stir and heat to boiling until completely dissolved, and place in a 50℃ water bath for later use.

[0093] ③ Interaction experiment with Staphylococcus aureus

[0094] Add 100 μL of the experimental group to the shake tube containing Staphylococcus aureus and mix well. Repeat the process three times per group. Add an equal volume of MRS or TPY liquid medium to the CK tube. Incubate at 37°C for 20 min. Mix well again. Take 50 μL of the mixture from each shake tube and add it to the center of a petri dish. Pour approximately 15 mL of Staphylococcus aureus chromogenic medium into each dish, shake well, air dry, and invert the dish to incubate aerobically at 37°C for 24 h.

[0095] ④ Determination of antibacterial rate

[0096] Viable bacteria counts were performed on Staphylococcus aureus plates to calculate the inhibition rate of the target bacteria against Staphylococcus aureus.

[0097] Relative inhibition rate against Staphylococcus aureus (%) = 1 - viable count of experimental plate / viable count of CK plate

[0098] 3. Experimental Results

[0099]

[0100]

[0101] As can be seen from the inhibition rates of single strains against pathogenic bacteria in Table 2, Lactobacillus acidophilus, Weizmann's coagulans, Lactobacillus plantarum, Bifidobacterium (Bifidobacterium longum subsp. infantis and Bifidobacterium animalis subsp. lactis) and Lactobacillus rhamnosus all showed certain inhibitory abilities against Escherichia coli and Staphylococcus aureus.

[0102] To further determine the inhibitory effects of different strain combinations on *Escherichia coli* and *Staphylococcus aureus*, the inventors randomly combined the single strains listed in Table 2 to prepare compositions containing dual, triple, quadrivalent, pentavalent, and hexavalent probiotics, and tested the inhibitory effects of different compositions on pathogenic bacteria. Statistical analysis of the inhibition rates of the dual to hexavalent compositions on pathogenic bacteria revealed that the dual and triple compositions did not show a significant increase in the inhibition rate compared to single strains. However, the quadrivalent, pentavalent, and hexavalent probiotic compositions showed a positive correlation with the increase in the number of probiotic strains, exhibiting a certain dose-dependent effect. The quadrivalent probiotic composition showed inhibition rates of 50-92% against both *Staphylococcus aureus* and *Escherichia coli*, the pentavalent composition showed inhibition rates of 50-93%, and the hexavalent composition showed inhibition rates of 50-95%.

[0103] The inventors further compared and analyzed the inhibitory effects of different strains combined in quadrivalent to hexavalent probiotic combinations on pathogenic bacteria. They found that the quadrivalent, pentavalent, and hexavalent probiotic combinations containing *Lactobacillus rhamnosus* all showed inhibition rates of 50%-65% against both pathogenic bacteria, without demonstrating a significant improvement in pathogenic bacteria inhibition. The combination of *Lactobacillus rhamnosus* with other bacteria did not show a better synergistic effect. The quadrivalent probiotic combinations without *Lactobacillus rhamnosus* showed inhibition rates of approximately 85-92% against *Staphylococcus aureus* and *Escherichia coli*, the pentavalent probiotic combinations showed inhibition rates of approximately 86-93%, and the hexavalent probiotic combinations showed inhibition rates of approximately 87-95%. The synergistic effect between the strains was good, resulting in better inhibition of *Escherichia coli* and *Staphylococcus aureus*.

[0104] II. Study on Organic Acid Production and Adhesion Rate of Different Strains

[0105] Experimental methods:

[0106] (1) Acid production capacity test

[0107] ① Experimental instruments

[0108] The main instrument information for the experiment is shown in Table 3.

[0109]

[0110] ② Main reagents for the experiment

[0111] The main reagents used in the experiment are shown in Table 4.

[0112]

[0113] ③ Acid production test experiment

[0114] Adjust the viable bacteria count in each group to 10 10 Take 0.75 g of the CFU / g culture medium and place it in 35 g of MRS medium. Gently shake until the bacterial powder is completely dissolved. Insert a glass electrode, add 5 mm of liquid paraffin, and place the container in a 37°C water bath. Turn on the pH meter to monitor pH and ORP changes in real time. At the experimental endpoint, determine the total acid value of the MRS and the total amount of organic acids produced.

[0115] (2) Adhesion rate detection test method

[0116] ① Experimental instruments

[0117] The main instruments used in the experiment are shown in Table 5.

[0118]

[0119] ② Main reagents for the experiment

[0120] The main reagents used in the experiment are shown in Table 6.

[0121]

[0122] ③ Preparation of Caco-2 cell monolayers

[0123] Cell resuscitation and passage: Resuscitate Caco-2 cells and seed them in DMEM medium (containing 1% penicillin-streptomycin) with 20% FBS, and culture at 37°C and 5% CO2 until 80% confluence. After trypsin digestion, passage at a 1:3 ratio, with medium changed every 2 days.

[0124] Monolayer cell seeding: Digest cells and adjust density to 1.5 × 10⁶ cells / year. 5 10 cells / mL, seeded into 12-well plates (1 mL per well), and cultured for 2 days until a tight monolayer is formed.

[0125] ④ Preparation of bacterial suspension

[0126] Strain activation: Take a glycerol tube and inoculate it at 4% into MRS liquid medium, and anaerobically culture at 37°C for 24 h. Mix well, transfer it to MRS liquid medium at 4% and anaerobically culture at 37°C for 20 h.

[0127] Cell washing: Centrifuge at 6000×g for 5 min, collect the cells, wash twice with sterile PBS, and resuspend in DMEM complete medium without antibiotics to a final concentration of 2×10⁻⁶. 8 CFU / mL.

[0128] ⑤ Adhesion test

[0129] Co-incubation of bacterial strain and cells: Aspirate the culture medium from the Caco-2 cell wells and gently wash twice with preheated PBS. Add 1 mL of bacterial suspension to each well, and add an equal volume of cell culture medium to the control group. Perform two replicates per group. Incubate at 37°C and 5% CO2 for 2 hours (simulating intestinal adhesion time).

[0130] Remove any unattached bacteria, aspirate the bacterial suspension, and gently wash three times with preheated PBS, adding it slowly along the well wall each time to avoid disrupting the cell monolayer.

[0131] Cell lysis and collection: Add 1 mL of 1% Triton X-100 (dissolved in PBS) to each well, lyse at room temperature for 10 minutes, and gently shake to promote lysis.

[0132] Gradual dilution and plating: Take the lysis buffer and perform a 10-fold serial dilution (10... ⁻2 Up to 10 ⁻4 Take 100 μL of each dilution and spread it onto MRS agar plates, with two plates for each dilution. Incubate anaerobically at 37°C for 48 hours, and then count the colonies.

[0133] ⑥ Adhesion rate calculation

[0134] The formula for calculating adhesion rate is: Adhesion rate = Number of bacteria / Number of cells

[0135] Experimental results:

[0136] The experimental results are shown in Table 7. All strains in the table below have the ability to produce organic acids within 2 hours, but Lactobacillus and Lactobacillus showed better acid production ability. All strains in the table below have a certain degree of adhesiveness, but Bifidobacterium showed better cell adhesion.

[0137]

[0138]

[0139] Through research on the production of organic acids and cell adhesion rates of different bacterial strains, the inventors found that *Lactobacillus plantarum* and *Lactobacillus acidophilus* have a greater advantage in the production of organic acids compared to other bacterial species. Among them, *Lactobacillus plantarum* HY02946 produced 25.07 mg of organic acids at 2 hours. In terms of the overall bacterial species adhesion rate, *Bifidobacterium* has a greater advantage in the cell adhesion rate compared to other bacterial species. Among them, *Bifidobacterium longum* subsp. infantis CS004 achieved a cell adhesion rate of 9.2%.

[0140] Example 2: Screening of the proportion of probiotic compositions

[0141] 1. Experimental Design

[0142] The inventors further screened more preferred probiotic combinations from the quadruple, pentad, and hexapod compositions screened in Example 1. These included a quadruple probiotic composition containing Lactobacillus acidophilus, Bifidobacterium, Lactobacillus plantarum, and Weizmann's coagulant; a pentad probiotic composition containing Lactobacillus acidophilus, Bifidobacterium longum subsp. infantis, Bifidobacterium animalis subsp. lactis, Lactobacillus plantarum, and Weizmann's coagulant; and a hexapod probiotic composition containing two different strains of Lactobacillus acidophilus, Bifidobacterium longum subsp. infantis, Bifidobacterium animalis subsp. lactis, Lactobacillus plantarum, and Weizmann's coagulant. These combinations showed stronger inhibitory effects on pathogenic bacteria compared to other co-combined compositions. Therefore, based on the above quadruple, pentad, and hexapod compositions, further research was conducted on the ratio of different strains.

[0143] The strains HY02946, BB-12, LA-5, CS004, BC99, CS003, and HY08866, which showed good single-strain effects, were selected and combined in proportions of 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.8, 2, 4, 6, 8, 10, 15, 20, 25, and 30 to form the aforementioned tetrad, pentad, and hexadox strains. Experimental results showed that when the proportion of HY02946 was set to 1-25, the proportions of BB-12 and LA-5 were both set to 2-10, and the proportions of CS004, BC99 (or HY08866), and CS003 were set to 0.2... At -1.8, the inhibition rates against Staphylococcus aureus and Escherichia coli both reached 90% or higher. Among them, the six-strain combinations HY02946, BB-12, LA-5, BC99, CS003, CS004 and HY02946, BB-12, LA-5, HY08866, CS003, CS004 showed superior effects at various ratios. The inhibition rates against Staphylococcus aureus ranged from 90% to 98%, and against Escherichia coli from 90% to 99%, which were generally better than other combinations. Therefore, further optimization of the combination ratios of these two six-strain combinations was conducted. The ratios of each strain are shown in Table 8 below.

[0144]

[0145] The inhibitory effects of each formulation sample on Escherichia coli and Staphylococcus aureus were tested using the same methods as in Example 1. The experimental results were analyzed using TTEST.

[0146] 2. Experimental Results

[0147] Experimental results showed that when the ratio of the six-component combination HY02946 was set at 10-20, the ratio of BB-12 at 4-8, the ratio of LA-5 at 4-8, and the ratio of CS004, BC99, and CS003 at 0.6-1.4, the inhibition rate against Staphylococcus aureus was above 95%, and the inhibition rate against Escherichia coli was above 96%. When the ratio of HY02946:BB-12:LA-5:CS004:BC99:CS003 was 15:6:6:1:1:1, the inhibition rate against Staphylococcus aureus reached 97.13%, and the inhibition rate against Escherichia coli reached 98.33%. When the ratio of HY02946:BB-12:LA-5:CS004:HY08866:CS003 is 15:6:6:1:1:1, the inhibition rate of Staphylococcus aureus reaches 96.84%, and the inhibition rate of Escherichia coli reaches 97.13%. When the ratio of HY02946:BB-12:LA-5:BC99:CS003:CS004 is 20:8:8:1.4:1.4:1.4, the inhibition rate of Staphylococcus aureus reaches 96.19%, and the inhibition rate of Escherichia coli reaches 97.67%. When the ratio of HY02946:BB-12:LA-5:HY08866:CS003:CS004 is 20:8:8:1.4:1.4:1.4, the inhibition rate of Staphylococcus aureus reaches 96.42%, and the inhibition rate of Escherichia coli reaches 97.33%.

[0148]

[0149]

[0150]

[0151] The inhibitory effect of different hexavalent compositions on pathogenic bacteria was verified by replacing them with strains other than the hexavalent composition. In this example, Lactobacillus rhamnosus was used as an example. After replacing each strain, the pathogenic bacteria inhibition rate of the hexavalent composition was tested. The experimental results are shown in Table 10.

[0152]

[0153] The results of organic acid production from the probiotic composition (Table 11) show that, through optimization of the proportions of each probiotic in the composition, when the proportion of *Lactobacillus plantarum* HY02946 was set at 5-20, the proportion of *Bifidobacterium animalis* subsp. *lactobacter* BB-12 at 4-10, the proportion of *Lactobacillus acidophilus* LA-5 at 2-8, and the proportions of *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 / HY08866, and *Lactobacillus acidophilus* CS003 at 0.6-1.4, the amount of organic acid produced in 1-3 hours showed a significant advantage compared to compositions with other proportions. When co-cultured for 4 hours, the organic acid content reached approximately 100-200 mg. Compared to commercially available products, the probiotic composition of this invention, at the 4th hour of culture, achieved a maximum organic acid production (186.30 mg), which was 2.3 times higher than the organic acid production (80.08 mg) of commercially available products.

[0154] Regarding the cell adhesion rate, within the preferred range of each strain, the cell adhesion rate of each probiotic composition is higher than that of compositions in other ratio ranges, reaching approximately 20-23, which is higher than the 15% cell adhesion rate of commercially available products.

[0155]

[0156]

[0157]

[0158] Example 3: Experimental Study on Oxygen Deprivation and Production Promotion of Probiotic Composition

[0159] I. Oxygen Deprivation and Growth Promotion Experiment

[0160] 1. Experimental Methods

[0161] 1.1 Main reagents for the experiment

[0162] The main reagents used in the experiment are shown in Table 12.

[0163]

[0164] 1.2 Experimental Group Information

[0165] The experimental groups and information on strains / compositions in each group are shown in Table 13 below.

[0166]

[0167] 1.3 Preparation of experimental reagents

[0168] MRS liquid culture medium: Weigh 52.24 g of MRS broth and dissolve it in 1000 mL of water, then autoclave at 115°C for 20 min and store at 4°C.

[0169] 0.1% Resazurin stock solution: Weigh 0.1g of Resazurin and dissolve it in 100mL of water. Filter the solution through a 0.22μm filter membrane for sterilization and store at 4℃.

[0170] MRS+Resazurin medium: Add 90 μL of 0.1% Resazurin stock solution to 30 mL of MRS liquid medium, mix well and store at 4℃.

[0171] 1.4 Preparation of bacterial suspension

[0172] ① F1 generation culture: According to Table 13, weigh 0.1g of bacterial powder for each formula combination into a 15mL centrifuge tube, add 9.9mL MRS and mix well, and anaerobic culture at 37℃ for 24h.

[0173] ② F2 generation culture: After mixing the F1 generation bacterial culture, inoculate 4% into 3mL MRS medium and incubate anaerobically at 37℃ for 24h.

[0174] ③ Bacterial culture treatment: Mix the bacterial culture after the culture is completed, and dilute it with PBS solution to a concentration of 1×10⁻⁶. 7 CFU / mL bacterial suspension is available for use.

[0175] 1.5 Determination of growth curve

[0176] ① In the anaerobic workstation, the diluted bacterial suspensions of each group were inoculated into 3 mL of MRS + Resazurin medium at a 4% inoculation rate. A blank medium was set up as the control group, and this was recorded as T0. The culture was then incubated anaerobically at 37℃. 100 μL of the bacterial suspension from each group's T0 was added to a 96-well plate, with two replicates for each group. The color change was photographed and the OD600 value was measured using a microplate reader.

[0177] ② In a biosafety cabinet, inoculate the diluted bacterial suspensions of each group into 3 mL of MRS + Resazurin medium at a 4% inoculation rate. A blank medium is set up as the control group; this is recorded as T0. Incubate at 37°C in a constant temperature incubator. Add 100 μL of the bacterial suspension from each group's T0 well to a 96-well plate, with two replicates per group. Photograph the color changes and measure the OD600 value using a microplate reader.

[0178] ③ Take out the bacterial suspensions at 3h, 6h, 9h and 12h of anaerobic and aerobic culture. Set up a blank culture medium as the control group. The anaerobic group was operated in the anaerobic workstation and the aerobic group was operated in the biosafety cabinet. Take 100μL of bacterial suspension from each group and add it to a 96-well plate. Make two replicates for each group. Take pictures to record the color change and detect the OD600 value with an ELISA reader.

[0179] 2. Experimental Results

[0180] The experimental results are shown in Table 14 below.

[0181]

[0182] In their research experiments on oxygen deprivation and growth promotion of probiotic compositions, the inventors discovered that *Weizmannii* in the probiotic compositions of this invention exhibits a better oxygen deprivation and growth-promoting effect on the probiotic compositions under aerobic conditions than other bacteria. As shown in Table 14, the experimental results indicate that under anaerobic conditions, the presence or absence of *Weizmannii* in the composition has virtually no effect on the growth of the strains. However, under aerobic conditions, the addition of *Weizmannii* significantly promotes the growth of the compositions. For example, in groups G25 (containing BC99) and G25-2 (containing HY08866), after 12 hours of incubation, the OD values ​​of both groups were significantly higher. 600 The values ​​were 0.03051 and 0.02963, respectively. Compared to G25-1 (which does not contain Weizmannia), the OD values ​​of the composition were... 600 The value was only 0.01782. Furthermore, when other strains, such as *Lactobacillus rhamnosus*, were used to replace *Weizmannii* in the probiotic composition, the composition's growth was not promoted under aerobic conditions; for example, the OD600 value of G25-3 after 12 hours of incubation was not significantly different from that of G25-1. This indicates that *Weizmannii* in the composition can effectively exert its oxygen-scavenging effect, thus promoting the synergistic growth of other probiotics in the composition.

[0183] II. Dissolved Oxygen Saturation Detection Experiment

[0184] 1. Experimental Methods

[0185] 1.1 Experimental Conditions

[0186] The dissolved oxygen and dissolved oxygen saturation of the probiotic composition were measured using a Hach 1130DO. The specific experimental conditions are shown in Table 15 below.

[0187]

[0188] 1.2 Experimental Grouping

[0189] The experimental groups are shown in Table 16 below.

[0190]

[0191] 2. Experimental Results

[0192]

[0193] The experimental results showed that when the compositions of each experimental group were cultured for 60 minutes, the composition containing Weissella reduced the dissolved oxygen by about 75%-80%, while the composition without Weissella reduced the dissolved oxygen by only about 32%-34%; when the compositions of each experimental group were cultured for 95 minutes, the composition containing Weissella reduced the dissolved oxygen by about 97%-99%, while the composition without Weissella reduced the dissolved oxygen by only about 63%-64%. This indicates that the biological oxygen consumption of Weissella is significant, and it can effectively play the role of oxygen capture, promoting the synergistic growth of other probiotics in the probiotic composition.

[0194] Example 4: Verification of the efficacy of probiotic composition against diarrhea (AAD)

[0195] 1. Experimental method

[0196] 1.1 Experimental materials

[0197] (1) The key experimental reagents are shown in Table 18, the configuration information of the triple antibiotic is shown in Table 19, and the strain information is shown in Table 20.

[0198]

[0199]

[0200]

[0201] (2) Experimental grouping

[0202] The animal grouping information is shown in Table 21.

[0203]

[0204] 1.2 Animal system

[0205] (1) Experimental animals

[0206] C57BL / 6J mice, male, 6 weeks old. Purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK(Zhe) 2024-0001, animal certificate number: 20251016Abzz01699990630.

[0207] (2) Administration plan

[0208] An AAD mouse model was constructed by combining three antibiotics (clindamycin + ampicillin + streptomycin). After the model was successfully constructed, different test products were used for therapeutic administration, and the antibiotics were not discontinued during the treatment period to observe the effects of the test substances on AAD mice. The specific grouping design is as follows:

[0209] ① NC group: Normal control group, intragastrically administered with 0.9% normal saline;

[0210] ②MC group: model control group, administered triple antibiotic mixture solution by gavage throughout the course (10mL / kg·BW / day);

[0211] ③AW group: Antibiotic discontinuation group, after 3 days of gavage administration of triple antibiotic mixed solution (10mL / kg·BW / day), treatment was discontinued;

[0212] ④ All other experimental groups were administered a triple antibiotic mixture solution by gavage (10 mL / kg·BW / day) throughout the entire course of treatment.

[0213] Treatment began 5 hours after modeling each day. The treatment group was given the corresponding test sample by gavage, while the NC and MC groups were given an equal volume of physiological saline by gavage. The drugs were administered once a day until the end of the experiment.

[0214] 1.3 Observation and Indicator Testing

[0215] (1) Weight monitoring

[0216] After the animals have completed their acclimatization period, their body weight is measured daily to guide the dosage of medication.

[0217] (2) Fecal moisture content test

[0218] Weigh the empty glass bottle and record its weight as W. 空 Mice were placed in metabolic cages, and the number of fecal pellets was recorded. At the same time, mouse feces were collected in glass bottles. After 1 hour of collection, the weight of the feces, including the bottle, was measured as W1. The mouse feces were then placed in a forced-air drying oven at 90°C for 5 hours and the weight of the feces, including the bottle, was measured as W2.

[0219] Fecal wet weight = wet weight - empty bottle weight = W1 - W 空

[0220] Fecal dry weight = Dry weight - Empty bottle weight = W2 - W 空

[0221] Fecal moisture content = (W1-W2) / W1×100%.

[0222] (3) Stool characteristics score

[0223] The stool examination scoring criteria are as follows:

[0224] ① The stool is oval-shaped, with distinct particles, hard in texture, and brownish in color. (Score = 1)

[0225] ② The stool is sausage-shaped, smooth, soft, and yellowish in color. (Score = 2)

[0226] ③ The feces have blurred edges or no fixed shape, are soft and moist, and are yellowish in color. Score = 3

[0227] 1.4 Data Statistics

[0228] All data from this experiment were entered into Excel for corresponding calculations. GraphPad Prism software was used for statistical analysis of the experimental data. P < 0.05 was considered statistically significant. The experimental results are expressed as Mean ± SEM (standard error).

[0229] 2. Experimental Results

[0230] 2.1 Improvement of clinical symptoms in mice by the test sample

[0231] Prior to administration, the model group mice exhibited typical diarrhea symptoms. Throughout the administration period, the fecal symptom score of the model control group was consistently higher than that of the normal control group, indicating that the model was stable and suitable for efficacy evaluation.

[0232]

[0233] Overall, the AB1, AB2, AB3, AB4, and AH groups showed better results than the quadrivalent and pentavalent strains, indicating that the more strains present, the better the synergistic effect. Furthermore, the TB1, TB2, FB, AB1, AB2, AB3, AB4, and AH groups all showed significant differences compared to the model group, while the antibiotic discontinuation group did not show a significant reduction in fecal characteristic scores. This suggests that the quadrivalent, pentavalent, and hexavalent strains of this invention all have excellent effects in relieving diarrhea, with the hexavalent strain showing the best effect. The study found that when any strain other than *Lactobacillus plantarum*, *Bifidobacterium*, *Lactobacillus acidophilus*, and *Weizmannii coagulans* were arbitrarily replaced in the probiotic composition, taking the replacement of *Lactobacillus rhamnosus* as an example, the AR group results showed that replacing the strains in the composition with *Lactobacillus rhamnosus* HY02078 resulted in a fecal characteristic score of only 1.68 points, which was not significantly different from the model group.

[0234] 2.2 Effect of the test sample on the water content of mouse feces

[0235] The results of fecal moisture content testing are shown in Table 23. The results showed that on day 8 after administration, the fecal moisture content of the model control group was significantly higher than that of the normal control group. On day 8 after administration, the fecal moisture content of all treatment groups except AR group was significantly lower than that of the model control group (P < 0.05).

[0236]

[0237] The data in Table 23 show that the fecal water content of the tetravalent, pentavalent, and hexavalent strains of the present invention was significantly lower than that of the model group (p < 0.05). This indicates that probiotics can not only improve the "clinical manifestations" of diarrhea (fecal characteristics score), but also fundamentally improve intestinal physiological function, promote water absorption, and reduce fecal water content. Although the AR group showed a decrease, there was no statistically significant difference compared with the model group. Overall, the data in Table 23 show that the fecal water content study obtained results consistent with the diarrhea score study.

[0238] The results in Tables 22 and 23 together demonstrate that the probiotic composition prepared from *Lactobacillus plantarum*, *Bifidobacterium*, *Lactobacillus acidophilus*, and *Weizmannii coagulans* used in this invention exhibits good synergy among the various strains, achieving a good therapeutic effect in treating diarrhea.

[0239] Example 5: Efficacy verification of probiotic composition against diarrhea (infectious diarrhea)

[0240] 1. Experimental Methods

[0241] 1.1 Experimental Materials

[0242] The experimental strain was the same as that in Example 4.

[0243] 1.2 Laboratory Animals

[0244] Select C57BL / 6N juvenile mice, SPF grade, male, 8-10g / mouse.

[0245] Animal housing conditions should be set at a room temperature of 20-22℃, humidity of 40-70%, and alternating light and dark cycles of 12 hours per day. Bedding should be changed at least twice a week, along with the feeding boxes; feeding boxes should be replaced immediately if any abnormal conditions are observed. Water bottles and stoppers should be changed and sterilized daily, and cages should be sterilized every two weeks. All cages should be autoclaved after washing.

[0246] 1.3 Experimental grouping and dosing regimen

[0247] As shown in Table 24.

[0248] The treatment procedures for each group of mice are as follows:

[0249] Pretreatment of young mice: Except for the normal control group, the other young mice were given a quadruple antibiotic drinking water (gentamicin, vancomycin, metronidazole and polymyxin) for 4 days before E. coli infection to disrupt the resident microbiota, and then replaced with normal drinking water to remove the antibiotics.

[0250] Young mice were inoculated with *E. coli*: Except for the normal control group, the remaining young mice were inoculated with *E. coli* (standard strain, CICC10413). Each young mouse was orally administered 100 μL (4 × 10⁶ μL) via gavage. 9(cfu / mL), administered once, with the vaccination time recorded as Day 0.

[0251] Administration to young mice: The probiotic group and the cefixime group were given the drug 1 day after infection, once a day for 5 consecutive days.

[0252]

[0253] 1.4 Experimental Indicators

[0254] Fecal scoring: During treatment, the fecal condition of the young mice was observed and scored daily (feces were graded into 6 levels: Grade 1 was normal stool, Grade 2 was yellow, formed, soft stool, Grade 3 was yellow, pasty stool, Grade 4 was yellow, watery, mucous stool, Grade 5 was yellow, egg-soup-like stool, and Grade 6 was completely yellow, watery stool. Grade 3 (yellow, pasty stool) and above indicated intestinal infection, Grades 4-5 indicated mild infection, and Grade 6 indicated severe infection. Symptom assessment was performed by the same person). Data are expressed as mean ± standard deviation (Mean ± SD).

[0255] Colorectal length: After the last administration, the young mice were sacrificed and the length of their colorectal length was measured.

[0256] Statistical analysis: p-values ​​were obtained according to the significance test method, where p<0.05 was considered statistically significant and p<0.01 was considered statistically significant.

[0257] 2. Experimental Results

[0258] 2.1 Stool score

[0259]

[0260] The results showed that the probiotic compositions of the present invention began to take effect on the third day of administration. The fecal scores of the AB1 and AB3 groups were significantly lower than those of the model group on the fourth day. On the fifth day, the effects of the AB1, AB2, AB3, AB4 and AH groups were all highly significant, which were better than the positive control group, demonstrating the superiority of rapid onset and strong efficacy.

[0261] However, the AR group had the slowest onset of action and the worst recovery throughout the treatment course, which again proves that replacing the probiotic composition of the present invention with other strains cannot achieve the same therapeutic effect. Furthermore, although the AR group consisted of six strains, from day 3 onwards, the effects of the present invention's quadrivalent strains TB1 and TB2, as well as the pentavalent strain FB, already reached or even exceeded those of the AR group. This further illustrates that the effectiveness of the present invention lies in the synergy between specific strains, and cannot be achieved simply by increasing the number of strains.

[0262] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or related embodiments obtained by those skilled in the art through some modifications or variations made to the above-disclosed technical content without departing from the scope of the present invention are within the scope of the present invention.

Claims

1. A probiotic composition for improving diarrhea, wherein, The probiotic composition comprises oxygen-depleting bacteria, organic acid-producing bacteria, and space-occupying bacteria; Preferably, the oxygen-depleting bacteria is Weizmann's bacterium; Preferably, the organic acid-producing bacteria are Lactobacillus and / or Lactobacillus lactis; Preferably, the occupant bacteria is Bifidobacterium.

2. The probiotic composition according to claim 1, wherein: The Weizmania bacteria mentioned are selected from Weizmannia coagulans. The lactobacillus is selected from one or more of the following: Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus crispatus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sakei, and Lactobacillus curvus. The Lactiplantibacillus is selected from one or more of the following: Lactiplantibacillus plantarum, Lactiplantibacillus plantarum subsp. argentoratensis, Lactiplantibacillus pentosus, Lactiplantibacillus paraplantarum, Lactiplantibacillus fabifermentans, Lactiplantibacillus tropicus, and Lactiplantibacillus nakhonrathomensis. The Bifidobacterium is selected from one or more of the following: Bifidobacterium animalis subsp. lactis, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium longum.

3. The probiotic composition according to claim 2, wherein, The probiotic composition contains *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, and *Bifidobacterium*. Preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, and *Bifidobacterium* in an effective live bacteria concentration ratio of (0.6-1.4):(0.6-9.4):(10-20):(0.6-9.4).

4. The probiotic composition according to claim 3, wherein, The Bifidobacteria are selected from Bifidobacterium longum infantis subsp. or Bifidobacterium animalis lactis subsp. Preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus*, *Lactobacillus plantarum*, *Bifidobacterium longum* subsp. *infantii*, and *Bifidobacterium animalis* subsp. *lactobacterium* in an effective live bacteria concentration ratio of (0.6-1.4):(0.6-9.4):(10-20):(0.6-1.4):(4-8).

5. The probiotic composition according to claim 4, wherein, The Lactobacillus acidophilus includes Lactobacillus acidophilus I and Lactobacillus acidophilus II, wherein Lactobacillus acidophilus I and Lactobacillus acidophilus II are different strains from each other; Preferably, the probiotic composition comprises *Weizmannii coagulans*, *Lactobacillus acidophilus* var. *m-1*, *Lactobacillus acidophilus* var. *m-2*, *Lactobacillus plantarum*, *Bifidobacterium longum* subsp. *infantii*, and *Bifidobacterium animalis* subsp. *lactobacterium* in an effective live bacteria concentration ratio of (0.6-1.4):(4-8):(0.6-1.4):(10-20):(0.6-1.4):(4-8) in that order.

6. The probiotic composition according to any one of claims 2 to 5, wherein, The *Wickemium coagulans* is selected from at least one of *Wickemium coagulans* BC99, *Wickemium coagulans* HY08866, *Wickemium coagulans* HY08867, and *Wickemium coagulans* HY08874; and / or, The first and second *Lactobacillus acidophilus* are each independently selected from at least one of *Lactobacillus acidophilus* LA-5, *Lactobacillus acidophilus* CS003, *Lactobacillus acidophilus* DDS-1, *Lactobacillus acidophilus* HY01039, *Lactobacillus acidophilus* HY00760, and *Lactobacillus acidophilus* HY01043; and / or, The *Lactobacillus plantarum* is selected from at least one of *Lactobacillus plantarum* HY02946, *Lactobacillus plantarum* HY05181, and *Lactobacillus plantarum* HY00050; and / or, The *Bifidobacterium longum* infant subsp. *longum* is selected from at least one of *Bifidobacterium longum* infant subsp. *CS004*, *Bifidobacterium longum* infant subsp. *MP09089*, and *Bifidobacterium longum* infant subsp. *MP09270*; and / or, The *Bifidobacterium lactis* subsp. *animal* is selected from at least one of *Bifidobacterium lactis* subsp. *animal* BB-12, *Bifidobacterium lactis* UABla-12, *Bifidobacterium lactis* subsp. *animal* HY11786, *Bifidobacterium lactis* subsp. *animal* MP15099, *Bifidobacterium lactis* subsp. *animal* HY09279 and *Bifidobacterium lactis* HY10036.

7. The probiotic composition according to claim 6, wherein, The probiotic composition comprises Lactobacillus plantarum HY02946, Bifidobacterium longum subsp. infantis CS004, Weizmannii coagulans BC99 or Weizmannii coagulans HY08866 and Lactobacillus acidophilus CS003. Preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium longum* subsp. *infantica* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (10-20):(0.6-1.4):(0.6-1.4):(0.6-1.4). More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 15:1:1:

1.

8. The probiotic composition according to claim 6, wherein, The probiotic composition contains Lactobacillus plantarum HY02946, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longum subsp. infantis CS004, Weizmannii coagulans BC99 or HY08866, and Lactobacillus acidophilus CS003. Preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (10-20): (4-8): (0.6-1.4): (0.6-1.4): (0.6-1.4). More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 15:6:1:1:

1.

9. The probiotic composition according to claim 6, wherein, The probiotic composition contains Lactobacillus plantarum HY02946, Bifidobacterium animalis subsp. lactis BB-12, Lactobacillus acidophilus LA-5, Bifidobacterium longum subsp. infantis CS004, Weizmannii coagulans BC99 or Weizmannii coagulans HY08866, and Lactobacillus acidophilus CS003. Preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Lactobacillus acidophilus* LA-5, *Bifidobacterium longum* subsp. *infantica* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of (10-20): (4-8): (4-8): (0.6-1.4): (0.6-1.4): (0.6-1.4). More preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Lactobacillus acidophilus* LA-5, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 15:6:6:1:1:

1. Or more preferably, the probiotic composition comprises *Lactobacillus plantarum* HY00050, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Lactobacillus acidophilus* LA-5, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 15:6:6:1:1:

1. Or more preferably, the probiotic composition comprises *Lactobacillus plantarum* HY02946, *Bifidobacterium animalis* subsp. *lactobacter* BB-12, *Lactobacillus acidophilus* LA-5, *Bifidobacterium longum* subsp. *infant* CS004, *Weizmannii coagulans* BC99 or *Weizmannii coagulans* HY08866, and *Lactobacillus acidophilus* CS003 in an effective live bacteria concentration ratio of 20:8:8:1.4:1.4:1.

4.

10. A product for the prevention, relief, improvement and / or treatment of diarrhea, comprising a probiotic composition according to any one of claims 1 to 9.

11. The product according to claim 10, wherein, The product also includes excipients; the excipients are selected from one or more of pharmaceutical excipients, food excipients, and health product excipients; Preferably, the excipients are selected from one or more of excipients, fillers, flow aids, diluents, quick-dissolving agents, and disintegrants.

12. Use of the probiotic composition according to any one of claims 1 to 9 in the preparation of products for the prevention, relief, improvement and / or treatment of diarrhea.

13. The product according to claim 10 or 11, or the use according to claim 12, wherein, The diarrhea is selected from one or more of the following: diarrhea-predominant irritable bowel syndrome, acute diarrhea, chronic diarrhea, osmotic diarrhea, secretory diarrhea, exudative diarrhea, motility-related diarrhea, antibiotic-associated diarrhea, and infectious diarrhea.

14. The product according to claim 10 or 11, or the use according to claim 12, wherein, The product is selected from one or more of the following: pharmaceuticals, food, and health products.

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