Lactobacillus plantarum capable of relieving diarrhea and regulating flora and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a plant lactobacillus capable of relieving diarrhea and regulating flora and application thereof. The application provides a microorganism, a nucleotide sequence of 16S rDNA of the microorganism is shown as SEQ ID NO:1. The application further provides a plant lactobacillus capable of relieving diarrhea and regulating flora, the plant lactobacillus is preserved in CGMCC, and a preservation number thereof is CGMCC No.26430. The microorganism or the plant lactobacillus provided by the application has strong acid resistance and bile salt resistance, can significantly relieve diarrhea symptoms, and has better intestinal flora regulation capacity.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a plant lactobacillus that can alleviate diarrhea and regulate gut flora, and its applications. Background Technology

[0002] The human gut is home to a vast number of diverse and complex microorganisms. Primarily composed of species such as Bacteroides, Prevotella, Fasciola, Rochetomyces, Trichophyton, and Sartella, these microorganisms participate in various metabolic, immune, and endocrine regulatory activities, influencing host physiological functions in multiple ways, including nutrient absorption, energy supply, and lipid metabolism. Under normal conditions, the gut microbiota interacts and coordinates to maintain a complex gut microbial ecological balance. Diarrhea, a common intestinal disease, is mainly characterized by increased stool volume, and its causes include alterations in the gut microbiota.

[0003] Probiotics, as live bacterial microecological preparations, play an indispensable role in maintaining the balance of the intestinal microecology. Probiotics engage in metabolic activities in the intestinal microenvironment, synthesizing beneficial metabolites and promoting the production of certain cytokines, thereby inhibiting the growth and reproduction of harmful pathogens. The efficacy mechanism of probiotics depends on the interaction between the host's microbiome or the immune-active cells of the intestinal mucosa. Currently known mechanisms of action on the intestinal microbiome include: lowering intestinal pH, producing bactericidal substances such as organic acids (lactic acid, acetic acid, butyric acid), H2O2, and bacteriocins; aggregation of pathogenic microorganisms; adhesion to the cell surface of the mucosa; competition for fermentation substrates or receptors; enhancing the barrier effect of the intestinal mucosa; releasing protective metabolites (arginine, glutamine, short-chain fatty acids, conjugated linoleic acid); binding to metabolically toxic metabolites; immunological mechanisms; or regulating intestinal motility and mucus production. Currently, those skilled in the art have attempted to use probiotic preparations to regulate the intestinal flora to alleviate diarrhea.

[0004] Chinese patent application 202210665133.5 discloses a lactic acid bacteria oral liquid with the function of relieving and treating diarrhea and its preparation method. The lactic acid bacteria oral liquid is prepared by fermenting Lactobacillus rhamnosus with a live bacteria count of over 3 billion CFU / mL. The Lactobacillus rhamnosus has the preservation number CCTCC No. M2022337. By consuming the lactic acid bacteria oral liquid, the number of lactic acid bacteria and bifidobacteria in the human intestine can be significantly increased, the flora structure can be optimized, and the body's immunity can be improved. Furthermore, the lactic acid bacteria oral liquid can effectively relieve and treat diarrhea in adults with significant effects.

[0005] Chinese patent application 202210089886.6 discloses the application of Bacteroides fragilis in improving and / or treating diarrhea. Through different mouse diarrhea model experiments, it is demonstrated that Bacteroides fragilis ZY-312 or its inactivated strain with accession number CGMCC No.10685 has the effect of improving and treating diarrhea. The inactivated Bacteroides fragilis powder, in different concentration formulations, has good effects on improving infectious or non-infectious diarrhea, and has no side effects on the body. It has good prospects for consumption and application, and provides a good health care and diarrhea improvement product suitable for human consumption.

[0006] There is still a need in this field for more microorganisms or probiotics that can alleviate diarrhea and regulate gut microbiota. Summary of the Invention

[0007] Biological Preservation Information:

[0008] Biomaterials: RH03163

[0009] Classification and nomenclature: Lactiplantibacillus plantarum

[0010] Preservation period: January 9, 2023

[0011] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0012] Accession number: CGMCC No.26430.

[0013] To achieve the above technical objectives, the technical solution provided by this invention is as follows:

[0014] In a first aspect, the present invention provides a microorganism whose 16S rDNA nucleotide sequence is shown in SEQ ID NO:1.

[0015] In some embodiments, the microorganism is *Lactobacillus plantarum*.

[0016] Secondly, the present invention provides a plant lactobacillus that can alleviate diarrhea and regulate gut microbiota, wherein the nucleotide sequence of the 16S rDNA of the plant lactobacillus is shown in SEQ ID NO:1.

[0017] In some embodiments, the *Lactobacillus plantarum* is deposited at CGMCC with accession number CGMCC No. 26430.

[0018] Thirdly, the present invention provides a microbial inoculant, the microbial inoculant comprising the microorganism or the *Lactobacillus plantarum* in its surviving or dead form, as well as its isolates and / or secretions.

[0019] In some embodiments, the viable form includes any biologically active *Lactobacillus plantarum*, such as: culture medium, culture medium extract, whole bacteria, whole bacteria extract, fermentation broth, and live bacteria in fermentation broth extract.

[0020] Conversely, it is a form of death.

[0021] In some embodiments, the microbial agent is obtained by culturing in a culture medium suitable for the growth of Lactobacillus plantarum.

[0022] Fourthly, the present invention provides a method for culturing the microorganism or the *Lactobacillus plantarum*, the method comprising the following steps:

[0023] (1) The microorganism or the plant lactobacillus or its isolate is inoculated into a basic culture medium for primary culture to obtain a primary culture;

[0024] (2) The primary culture was transferred to a basal medium for secondary culture to obtain a secondary culture;

[0025] (3) The secondary culture is transferred to an optimized culture medium for tertiary culture to obtain the microorganism or the culture of Lactobacillus plantarum.

[0026] In some embodiments, the primary and secondary culture conditions are 36.5-37.5°C for 16-20 hours.

[0027] In some embodiments, the inoculum amounts of the primary culture in step (2) and the secondary culture in step (3) are 4-6% (v / v, volume ratio); and / or

[0028] In some embodiments, the pH of the culture in step (3) is 3.8-4.2.

[0029] Fifthly, the present invention provides the use of the microorganism, the Lactobacillus plantarum, and / or the microbial agent in the preparation of products for relieving diarrhea and / or regulating gut microbiota.

[0030] In some embodiments, the product includes food, medicine, pharmaceutical ingredients, or food ingredients.

[0031] In some embodiments, the food includes health products, condiments, dairy products, beverages, biscuits, frozen drinks, confectionery, alcoholic beverages, or food additives.

[0032] Compared with the prior art, the *Lactobacillus plantarum* RH03163 strain provided by the present invention has at least the following beneficial effects:

[0033] (1) It has strong acid resistance. The acid resistance delay time is 2.0h under pH 3.0 conditions and 2.22h under pH 2.0 conditions.

[0034] (2) It has strong tolerance to bile salts. The time to tolerance to bile salts is less than 1 hour at a bile salt concentration of 0.3%-1.5%, and the time to tolerance to bile salts is 0.34 hours at a bile salt concentration of 0.3%.

[0035] (3) Significantly relieved diarrhea symptoms. Compared with the model control group, the number of diarrhea episodes was reduced by about 7, which was significantly less than that of the model control group, and it had a good anti-diarrheal effect.

[0036] (4) It has a better ability to regulate intestinal flora. Compared with the solvent control group, after 14 days of application, the number of Enterobacteriaceae and Enterococci in the intestinal flora decreased significantly, while the number of Lactobacillus increased significantly. Attached Figure Description

[0037] Figure 1 A schematic diagram of the morphology of candidate colonies on a neutral red plate is shown.

[0038] Figure 2 The results of macroscopic morphological identification of candidate colonies are shown.

[0039] Figure 3 The results of the microscopic morphological identification of the candidate colonies are shown. Scale bar is 5 μm. Detailed Implementation

[0040] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0041] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0042] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0043] The term "survival form of Lactobacillus plantarum" as used in this article refers to Lactobacillus plantarum possessing at least one of the following properties: culturability, transcription capability, metabolic activity, and cell membrane integrity. Culturability refers to the bacteria's ability to form colonies on a culture medium; transcription capability refers to the bacteria's ability to produce mRNA; metabolic activity refers to the bacteria's ability to exhibit life phenomena related to its metabolism, such as redox capacity, respiration, the presence of active enzymes, and substrate uptake; and cell membrane integrity refers to the absence of damage to the bacterial cell membrane.

[0044] Conversely, it is the "death form of Lactobacillus plantarum".

[0045] As used in this article, "separate material" refers to microbial material in which a specific microbial individual is isolated from a population or a mixed microbial population and exists in a certain medium. This medium includes fermentation broth supernatant, fermentation broth precipitate, bacterial suspension, lyophilized powder, etc.

[0046] The term "secretions" as used in this article refers to the products secreted by specific microbial individuals during their growth or metabolism, including metabolic products such as organic acids, bacteriocins, aromatic substances such as acetaldehyde, extracellular polysaccharides, and γ-aminobutyric acid.

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Such structures and techniques have also been described in many publications.

[0048] The main materials and reagents involved in this invention are:

[0049] MRS medium: yeast peptone 10 g / L, beef meal 8 g / L, yeast extract 4 g / L, potassium dihydrogen phosphate 2 g / L, maltose 5 g / L, citric acid monohydrate 2 g / L, sodium acetate 5 g / L, anhydrous glucose 20 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, Tween-80 0.6 g / L, calcium carbonate 10 g / L, neutral red 0.05 g / L, tomato juice 10 ml / L, agar powder 20 g / L (optional for liquid medium), adjust pH to 6.0; sterilize at 115℃ for 30 min.

[0050] Basic culture medium: 20.0 g / L anhydrous glucose, 10.0 g / L yeast peptone, 5.0 g / L yeast extract, 5.0 g / L citric acid monohydrate, 5.0 g / L sodium acetate, 2.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, and 0.2 g / L manganese sulfate. Weigh and dissolve according to the formula ratio. Adjust the pH of the culture medium to 6.60 ± 0.02. Sterilize at 115℃ for 30 min before use.

[0051] Optimized culture medium: Anhydrous glucose 30.0 g / L, yeast extract 25.0 g / L, yeast peptone 10.0 g / L, L-malic acid 3.0 g / L, citric acid monohydrate 2.0 g / L, potassium dihydrogen phosphate 2.0 g / L, calcium chloride 0.5 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.2 g / L. Weigh according to the formula ratio, put into a container, dissolve, sterilize at 115℃ for 30 min. After sterilization, the temperature of the culture medium is reduced to the incubation temperature, and the pH value is adjusted to 6.20±0.02.

[0052] Example 1: Screening and Identification of Strains

[0053] 1.1 Screening of strains

[0054] (1) Separation:

[0055] Take 1.0 mL of infant stool sample and add it to 4.0 mL of MRS diluent, then serially dilute 10-fold to 10. -6 Take appropriate dilution gradients and spread them onto MRS solid culture medium plates, then incubate in an anaerobic gas-generating bag at 37°C for 24 hours.

[0056] (2) Purification

[0057] Select single colonies with typical characteristics of the target strain (round, moist and glossy colony morphology with neat edges; rod-shaped cells arranged in pairs or parallel lines, no spores, and Gram-positive staining), and streak them in modified MRS medium for purification. Repeat this process three times until the colony characteristics in the streak plates are consistent.

[0058] (3) Microscopic examination

[0059] Two single colonies were picked from each purified plate, smeared, Gram stained, and their color and cell shape were observed under a microscope.

[0060] The results showed that under anaerobic conditions, the colonies were round, moist, and glossy, about 3 mm in diameter, with neat edges. They appeared pink on plates containing neutral red and had a distinct calcification zone. They were Gram-positive, rod-shaped, and arranged in pairs or parallel lines, with a length-to-width ratio of 4-6. Figure 1 ).

[0061] (4) Submission for testing and identification

[0062] The isolated strain was sent to the China Industrial Microbial Culture Collection Center for 16S rDNA sequence identification. The identification result was Lactiplantibacillus plantarum, Lactiplantibacillus plantarum RH03163, which is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 26430.

[0063] Macroscopic morphological identification results: In MRS medium, after anaerobic incubation at 37℃ for 24 h, the colonies were white, round, moist, opaque, and had neat edges. Figure 2 ).

[0064] Microscopic morphological identification results: In MRS medium, after anaerobic culture at 37℃ for 24 h, the bacteria were rod-shaped, 0.6-0.8 μm × 1.1-1.9 μm, arranged singly or in pairs, and Gram-positive. Figure 3 ).

[0065] 1.2 Identification of strains

[0066] (1) Identification of physiological and biochemical characteristics

[0067] The physiological and biochemical identification in this embodiment was completed by the China Industrial Microbial Culture Collection Center, and the results are shown in Table 1.

[0068] Table 1. Identification of Physiological and Biochemical Characteristics

[0069]

[0070] "+" represents a positive result, "w" represents a weak positive result, and "-" represents a negative result.

[0071] (2) Identification of 16S rDNA sequence

[0072] The 16S rDNA sequence identification result is shown as SEQ ID NO: 1: 1 AACGAACTCT GGTATTGATTGGTGCTTGCA TCATGATTTA CATTTGAGTG51 AGTGGCGAAC TGGTGAGTAA CACGTGGGAAACCTGCCCAG AAGCGGGGGA101 TAACACCTGG AAACAGATGC TAATACCGCA TAACAACTTGGACCGCATGG151 TCCGAGCTTG AAAGATGGCT TCGGCTATCA CTTTTGGATG GTCCCGCGGC201GTATTAGCTA GATGGTGGGG TAACGGCTCA CCATGGCAAT GATACGTAGC251 CGACCTGAGAGGGTAATCGG CCACATTGGG ACTGAGACAC GGCCCAAACT301 CCTACGGGAG GCAGCAGTAGGGAATCTTCC ACAATGGACG AAAGTCTGAT351 GGAGCAACGC CGCGTGAGTG AAGAAGGGTTTCGGCTCGTA AAACTCTGTT401 GTTAAAGAAG AACATATCTG AGAGTAACTG TTCAGGTATTGACGGTATTT451 AACCAGAAAG CCACGGCTAA CTACGTGCCA GCAGCCGCGG TAATACGTAG501GTGGCAAGCG TTGTCCGGAT TTATTGGGCG TAAAGCGAGC GCAGGCGGTT551 TTTTAAGTCTGATGTGAAAG CCTTCGGCTC AACCGAAGAA GTGCATCGGA

[0073] 601 AACTGGGAAA CTTGAGTGCA GAAGAGGACA GTGGAACTCC ATGTGTAGCG

[0074] 651 GTGAAATGCG TAGATATATG GAAGAACACC AGTGGCGAAG GCGGCTGTCT

[0075] 701 GGTCTGTAAC TGACGCTGAG GCTCGAAAGT ATGGGTAGCA AACAGGATTA

[0076] 751 GATACCCTGG TAGTCCATAC CGTAAACGAT GAATGCTAAG TGTTGGAGGG

[0077] 801 TTTCCGCCCT TCAGTGCTGC AGCTAACGCA TTAAGCATTC CGCCTGGGGA

[0078] 851 GTACGGCCGC AAGGCTGAAA CTCAAAGGAA TTGACGGGGG CCCGCACAAG

[0079] 901 CGGTGGAGCA TGTGGTTTAA TTCGAAGCTA CGCGAAAGAAC CTTACCAGGT

[0080] 951 CTTGACATAC TATGCAAATC TAAGAGATTA GACGTTCCCT TCGGGGACAT

[0081] 1001 GGATACAGGT GGTGCATGGT TGTCGTCAGC TCGTGTCGTG AGATGTTGGG

[0082] 1051 TTAAGTCCCG CAACGAGCGC AACCCTTATT ATCAGTTGCC AGCATTAAGT

[0083] 1101 TGGGCACTCT GGTGAGACTG CCGGTGACAA ACCGGAGGAA GGTGGGGATG

[0084] 1151 ACGTCAAATC ATCATGCCCC TTATGACCTG GGCTACACAC GTGCTACAAT

[0085] 1201 GGATGGTACA ACGAGTTGCG AACTCGCGAG AGTAAGCTAA TCTCTTAAAG

[0086] 1251 CCATTCTCAG TTCGGATTGT AGGCTGCAAC TCGCCTACAT GAAGTCGGAA

[0087] 1301 TCGCTAGTAA TCGCGGATCA GCATGCCGCG GTGAATACGT TCCCGGGCCT

[0088] 1351 TGTACACACC GCCCGTCACA CCATGAGAGT TTGTAACACC CAAAGTCGGT

[0089] 1401 GGGGTAACCT.

[0090] The selected strain was identified as *Lactiplantibacillus plantarum*, and named *Lactiplantibacillus plantarum* strain RH03163.

[0091] Example 2: Culture of Lactobacillus plantarum strain RH03163

[0092] • Resuscitation of frozen bacterial strains

[0093] Take the cryopreservation tubes of the bacterial culture stored in the low-temperature freezer and immediately place them in a 37°C water bath to thaw the bacterial culture for 15-30 seconds, until all the solids in the cryopreservation tubes have melted.

[0094] • Level 1 training

[0095] Transfer 1 mL of the revived bacterial culture to 10 mL of basal culture medium and incubate at 37±0.5℃ for 16-20 h.

[0096] Secondary training

[0097] The bacterial suspension obtained from the primary culture was transferred to 100 mL of basal medium at an inoculation rate of 4%, with a liquid volume of 40-60%. The medium was incubated at 37±0.5℃ with shaking at 100 rpm for 16-20 h. The pH of the bacterial suspension was measured to be 3.8-4.2, and the yield was 1.00-1.30%.

[0098] Three-tier training

[0099] The bacterial suspension obtained from secondary fermentation was transferred to 300 mL of optimized culture medium at an inoculum rate of 4%, with a liquid volume of 40-60%. The medium was incubated at 37±0.2℃ with shaking at 100 rpm for 16-20 h. The pH of the bacterial suspension was determined to be 3.8-4.2, and the yield was 1.20-1.50%.

[0100] After three stages of culture, the viable count of *Lactobacillus plantarum* RH03163 in bacterial suspension was (8-9) × 10⁻⁶. 9 CFU / mL.

[0101] Example 3: Resistance of Lactobacillus plantarum RH03163 strain to adverse environments in the digestive tract

[0102] Activation conditions for the strain in this embodiment:

[0103] Single colonies obtained by streaking RH03163 strain were aseptically inoculated into 4 mL of MRS liquid medium and cultured at 37°C for 16 h; all 4 mL of bacterial culture were inoculated into 100 mL of MRS liquid medium and cultured at 37°C for 20 h to obtain the activated seed culture.

[0104] 2.1 Acid Resistance Test

[0105] The third-generation activated strains were inoculated into MRS liquid medium at a certain ratio (bacterial culture: medium = 1 mL: 3 mL) into MRS medium at pH 3.0 and pH 2.0. After incubation at 37°C for 2 hours, the inoculation was carried out at a rate of 3% into fresh MRS liquid medium at pH 6.5. OD was measured every 10 minutes using a multi-functional microplate reader. 600 Value, until OD 600 The assay was stopped when the value increased by 0.3 units. The OD values ​​of the strain were calculated in pH 3.0, pH 2.0, and normal culture media. 600 The time required for the value to increase by 0.3 units is called the acid tolerance lag time. Acid tolerance lag time (h) for the tested strain = LT2 - LT1. OD of the acid-treated medium. 600 The time required for the value to increase by 0.3 units (expressed as LT2) in OD2 of normal pH culture medium. 600 The time required for the value to increase by 0.3 units (denoted by LT1).

[0106] The results of the acid tolerance delay time experiment showed that the growth of strain RH03163 was delayed under both pH 3.0 and pH 2.0 conditions compared with that in normal MRS medium. As shown in Table 2, the delay time was 2.22 h under pH 2.0 conditions and 2 h under pH 3.0 conditions, indicating that strain RH03163 has a strong tolerance to acid.

[0107] Table 2 Results of acid tolerance delay time for strain RH03163

[0108]

[0109] 2.2 Test of bile salt tolerance

[0110] The third-generation activated strains were inoculated at a 3% inoculum into MRS liquid medium containing 0.3%, 0.5%, 1.0%, and 1.5% bile salts, and into MRS medium without bile salts, respectively. OD values ​​were measured every 10 minutes using a multi-functional microplate reader. 600 Value, until OD 600 The assay was stopped when the value increased by more than 0.3 units. The OD values ​​of the strains in MRS media containing 0.3%, 0.5%, 1.0%, 1.5% bile salts, and without bile salts were calculated. 600The time required for the value to increase by 0.3 units is called the bile salt tolerance delay time. The bile salt tolerance delay time (h) for the tested strain = LT4 - LT3. OD of bile salt-containing medium. 600 Time required for a 0.3 unit increase in OD value (expressed as LT4) in bile-free medium. 600 The time required for the value to increase by 0.3 units (denoted by LT3).

[0111] The results of the bile salt tolerance delay time experiment showed that although the growth of strain RH03163 was delayed under different bile salt concentrations compared with that in normal MRS medium, the delay time was very short. As shown in Table 3, the delay time of strain RH03163 was less than 1 hour, and the delay time under 0.3% bile salt concentration was 0.34 hours, indicating strong bile salt tolerance.

[0112] Table 3 Results of bile salt tolerance delay time of strain RH03163

[0113]

[0114] Example 4: Determination of the diarrhea-relieving function of Lactobacillus plantarum RH03163 strain

[0115] Experimental animals: 4-5 week old clean-grade Kunming mice (Liaoning Changsheng Biotechnology Co., Ltd.), 18-22g, male, 30 mice in total.

[0116] Model establishment and experimental grouping: A mouse diarrhea model was established by oral gavage administration of senna leaves (0.5 mL / 20 g mice twice daily). The number of bowel movements within 6 hours was observed to reflect the model's defecation status. Two dosage groups and one control group were set up. The test substance dosage groups were 1.5 g / kg bw and 3.0 g / kg bw, administered once daily by gavage at a volume of 10 mL / kg bw for 7 consecutive days. The mouse diarrhea model was successfully established when mice exhibited significant emaciation, dirty fur, lethargy, anorexia, and watery stools.

[0117] Preparation of senna leaf decoction: Take 50g of senna leaves and 100mL of tap water, boil for about 10 minutes, filter with two layers of gauze, and evaporate and concentrate to a concentration of 1g / mL.

[0118] Table 4. Experimental results of antidiarrheal efficacy of Lactobacillus plantarum RH03163

[0119]

[0120] * indicates a significant difference compared to the model control group, p < 0.05. In g / kg, g represents the mass of the lyophilized Lactobacillus plantarum RH03163 powder, and kg represents the body weight of the mouse.

[0121] The results are shown in Table 4. At a dose of 3.0 g / kg, Lactobacillus plantarum RH03163 reduced the number of diarrhea episodes by about 7, which was significantly less than that of the model control group, demonstrating good antidiarrheal efficacy.

[0122] Example 5: Determination of the microbial community regulation function of Lactobacillus plantarum strain RH03163

[0123] Experimental animals: 40 clean-grade Kunming mice (Liaoning Changsheng Biotechnology Co., Ltd.), aged 4-5 weeks, 18-22g, male.

[0124] Experimental Procedure: After balanced feeding, mice were randomly divided into groups according to body weight and administered the sample. There were four groups, with 10 mice in each group. The experiment included three test substance dosage groups and one solvent control group. The test substance groups for *Lactobacillus plantarum* RH03163 were: a low-dose group (1 g / (kg bw·d), a medium-dose group (2 g / (kg bw·d), and a high-dose group (4 g / (kg bw·d)). Mice were administered the sample once daily by gavage at a volume of 10 mL / kg bw for 14 consecutive days. The solvent control group received an equal volume of sterilized pure water by gavage.

[0125] Before administering the test sample, 0.1 g of fecal matter from each group of mice was aseptically collected and serially diluted 10-fold to 1000 mg / L. -8 Appropriate dilutions were selected and inoculated onto different culture media. After incubation, colonies (lactobacters, enterobacteria, and enterococci) were identified and counted using colony morphology, Gram staining microscopy, and biochemical reactions to calculate the number of bacteria per gram of wet stool. Twenty-four hours after the last sample administration, stool samples were collected using the same method as before the experiment to detect the gut microbiota. Changes in the gut microbiota before and after the experiment reflected the efficacy of the test substance in regulating the intestinal flora.

[0126] Table 5. Experimental Study on the Efficacy of Lactobacillus plantarum RH03163 in Regulating Intestinal Flora

[0127]

[0128] * indicates a significant difference compared to the model control group, p < 0.05. In lg CFU / g, CFU represents colony-forming units, and g represents the mass of wet stool from mice.

[0129] The results are shown in Table 5. On day 0, there was no significant difference in the number of viable Enterobacteriaceae in the intestines of mice in each group (P>0.05). After 14 days of gavage, the number of viable Enterobacteriaceae in the intestines of mice in the solvent control group remained basically unchanged, while the number of Enterobacteriaceae in each dose group of Lactobacillus plantarum RH03163 was significantly reduced in the low dose group compared with the solvent control group (P<0.05), and extremely significantly reduced in the medium and high dose groups (P<0.01).

[0130] On day 0, there was no significant difference in the number of live lactobacilli in the intestines of mice in each group (P>0.05); after 14 days of gavage, the number of live lactobacilli in the intestines of mice in the solvent control group remained basically unchanged, while the number of live lactobacilli in each dose group of *Lactobacillus plantarum* RH03163 was significantly increased compared with the solvent control group (P<0.01).

[0131] On day 0, there was no significant difference in the number of viable enterococci in the intestines of mice in each group (P>0.05); after 14 days of gavage, the number of viable enterococci in the intestines of mice in the solvent control group remained basically unchanged, while the number of viable enterococci in each dose group of *Lactobacillus plantarum* RH03163 was significantly lower than that in the solvent control group (P<0.01).

[0132] Comparative Example 1

[0133] This comparative study compared the diarrhea-relieving abilities of *Lactobacillus plantarum* strain RH03163 and strain HCS03001.

[0134] Experimental animals: 4-5 week old clean-grade Kunming mice (Liaoning Changsheng Biotechnology Co., Ltd.), 18-22g, male, 30 mice in total.

[0135] Mouse modeling and experimental grouping: A mouse diarrhea model was established by oral gavage administration of senna leaves. The number of diarrhea episodes within 6 hours was observed to reflect the defecation status of the model. Two dosage groups and one model control group were set up. The test substance dosage groups were 1.5 g / kg bw and 3.0 g / kg bw, administered once daily by gavage at a volume of 10 mL / kg bw for 7 consecutive days.

[0136] Table 6. Experimental results on the diarrhea-relieving ability of *Lactobacillus plantarum* strains RH03163 and HCS03-001.

[0137]

[0138] * indicates a significant difference compared to Lactobacillus plantarum HCS03-001. In g / kg, g represents the mass of Lactobacillus plantarum RH03163 lyophilized powder, and kg represents the body weight of mice.

[0139] The results are shown in Table 6, compared with Lactobacillus plantarum HCS03-001:

[0140] When administered at a dose of 1.5 g / kg, RH03163 reduced diarrhea episodes by 5.9 times, and HCS03-001 reduced diarrhea episodes by 4.8 times.

[0141] When administered at a dose of 3.0 g / kg, RH03163 reduced diarrhea episodes by 7.7 times, and HCS03-001 reduced diarrhea episodes by 5.1 times.

[0142] In summary, *Lactobacillus plantarum* RH03163 is more effective than *Lactobacillus plantarum* HCS03-001 in relieving diarrhea.

[0143] Comparative Example 2

[0144] This study compares the microbial community regulation functions of *Lactobacillus plantarum* strain RH03163 and strain HCS03001.

[0145] Experimental animals: 40 clean-grade Kunming mice (Liaoning Changsheng Biotechnology Co., Ltd.), aged 4-5 weeks, 18-22g, male.

[0146] Experimental grouping: After balanced feeding, mice were randomly divided into groups according to body weight and administered the sample. There were four groups, with 10 mice in each group. The experiment included three test substance dosage groups and one solvent control group. The *Lactobacillus plantarum* RH03163 test substance dosage groups were: low dose (1 g / (kg bw·d), medium dose (2 g / (kg bw·d), and high dose (4 g / (kg bw·d)). Mice were administered the sample once daily by gavage at a volume of 10 mL / kg bw for 14 consecutive days. The solvent control group received an equal volume of sterilized pure water by gavage.

[0147] Before administering the test samples, 0.1g of fecal matter from each group of mice was aseptically collected, serially diluted 10-fold, and inoculated onto different culture media at appropriate dilutions. After incubation, colonies (lactobacters, enterobacteria, and enterococci) were identified and counted using colony morphology, Gram staining microscopy, and biochemical reactions to calculate the number of bacteria per gram of wet feces. Twenty-four hours after the last sample administration, fecal matter was collected using the same method as before the experiment to detect the gut microbiota. Changes in the gut microbiota before and after the experiment reflected the efficacy of the test substance in regulating the intestinal flora.

[0148] Table 7. Results of experiments on the ability of *Lactobacillus plantarum* strains RH03163 and HCS03-001 to regulate intestinal flora.

[0149]

[0150] * indicates a significant difference compared to Lactobacillus plantarum HCS03-001, and ** indicates an extremely significant difference compared to Lactobacillus plantarum HCS03-001.

[0151] The results are shown in Table 7, compared with Lactobacillus plantarum HCS03-001:

[0152] When a dose of 1 g / kg was administered, after 14 days, RH03163 showed a decrease of 0.441 g CFU / g in Enterobacteriaceae, an increase of 0.641 g CFU / g in Lactobacillus, and a decrease of 0.821 g CFU / g in Enterococcus.

[0153] When a dose of 1 g / kg was administered, after 14 days, HCS03-001 showed a decrease of 0.031 g CFU / g in Enterobacteriaceae, an increase of 0.091 g CFU / g in Lactobacillus, and a decrease of 0.441 g CFU / g in Enterococcus.

[0154] When a dose of 2 g / kg was administered, after 14 days, RH03163 showed a decrease of 0.641 g CFU / g in Enterobacteriaceae, an increase of 1.041 g CFU / g in Lactobacillus, and a decrease of 1.391 g CFU / g in Enterococcus.

[0155] When a dose of 2 g / kg was administered, after 14 days, HCS03-001 showed a decrease of 0.04 lg CFU / g in Enterobacteriaceae, an increase of 0.31 lg CFU / g in Lactobacillus, and a decrease of 0.73 lg CFU / g in Enterococcus.

[0156] When a dose of 4 g / kg was administered, after 14 days, RH03163 showed a decrease of 1.161 g CFU / g in Enterobacteriaceae, an increase of 1.431 g CFU / g in Lactobacillus, and a decrease of 1.871 g CFU / g in Enterococcus.

[0157] When a dose of 4 g / kg was administered, after 14 days, HCS03-001 showed a decrease of 0.05 lg CFU / g in Enterobacteriaceae, an increase of 0.32 lg CFU / g in Lactobacillus, and a decrease of 0.75 lg CFU / g in Enterococcus.

[0158] In summary, *Lactobacillus plantarum* RH03163 is superior to *Lactobacillus plantarum* HCS03-001 in reducing the number of Enterobacteriaceae and Enterococci, and increasing the number of Lactobacillus, thus exhibiting a stronger function in regulating the gut microbiota.

[0159] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A plant-derived Lactobacillus that can relieve diarrhea and regulate intestinal flora ( Lactiplantibacillus plantarum ), characterized in that, The *Lactobacillus plantarum* is deposited at CGMCC with accession number CGMCC No. 26430; The method for preparing the *Lactobacillus plantarum* includes the following steps: (1) Inoculate Lactobacillus plantarum into a basal culture medium for primary culture to obtain a primary culture; (2) The primary culture was transferred to a basal medium for secondary culture to obtain a secondary culture; (3) The secondary culture was transferred to an optimized culture medium to obtain the culture of *Lactobacillus plantarum*. The conditions for the primary and secondary cultures are: incubation at 36.5-37.5°C for 16-20 h; and / or The inoculum amounts for the primary culture in step (2) and the secondary culture in step (3) are 4-6% (v / v); and / or The pH value of the culture in step (3) is 3.8-4.

2.

2. The use of *Lactobacillus plantarum* as described in claim 1 in the preparation of drugs for relieving diarrhea and / or regulating intestinal flora.

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