Preparation method of probiotic composition as well as preparation and application of probiotic composition

Abstract

The invention provides a preparation method of a probiotic composition as well as a preparation and an application of the probiotic composition. The preparation method comprises the following steps: respectively putting a bacterial strain of plant lactobacillus GOLDGUT-LP1024, a bacterial strain of casei paracasei GOLDGUT-LC12345, a bacterial strain of lactobacillus reuteri GOLDGUT-LR99, a bacterial strain of pediococcus acidilactici GOLDGUT-PA0755 and a bacterial strain of casei rhamnosus GOLDGUT-L818 into a sterile MRS culture solution for activation; carrying out continuous passage for multiple times to enable the strain to enter a logarithmic growth phase, and carrying out enlarged culture; centrifugally collecting the bacterial liquid to obtain bacterial sludge of each strain; adding a protective agent into the bacterial sludge of each strain, freeze-drying, crushing and sieving to obtain freeze-dried powder of each strain; and mixing the freeze-dried powder of each strain with the first lactic acid bacteria compound powder and the second lactic acid bacteria compound powder in parts by weight to obtain the probiotic composition with the effect of relieving the antibiotic-related diarrhea.

Description

Technical Field

[0001] This application belongs to the field of microbial technology, and in particular relates to a method for preparing a probiotic composition, its formulation and application. Background Technology

[0002] Antibiotics are a core tool in modern medicine for treating infectious diseases, greatly improving human health and average life expectancy. Consequently, they are widely used in clinical diagnosis and treatment, animal husbandry, and other fields, with global antibiotic use showing a continuous upward trend. The human gut microbiota is a complex micro-ecosystem that deeply participates in multiple physiological processes, including host immune development, metabolic regulation, intestinal barrier maintenance, and pathogen resistance. However, broad-spectrum antibiotics lack precise targeting; while killing pathogens, they also non-selectively inhibit or kill beneficial symbiotic microorganisms in the gut. This leads to gut microbiota dysbiosis, a common problem after antibiotic use, with antibiotic-associated diarrhea being one of the most typical adverse reactions. Therefore, probiotic-based gut microbiota intervention strategies have become a research hotspot in the prevention and treatment of gastrointestinal diseases.

[0003] Antibiotic use leads to a decline in gut microbiota diversity and an imbalance in gut microbiota structure. This disruption to the gut microbiota is persistent, and the dominant gut microbiota is difficult to quickly recover to its original state after drug withdrawal. This can cause damage to the intestinal barrier function and inflammatory responses, becoming a core cause of antibiotic-associated diarrhea and posing numerous challenges to clinical intervention. Current interventions for antibiotic-associated diarrhea still have significant limitations. Traditional antidiarrheal drugs can only relieve diarrhea symptoms and cannot fundamentally repair the gut microbiota imbalance and mucosal barrier damage caused by antibiotics. While probiotics have been shown to have the potential to regulate the gut microbiota, current probiotic preparations are mostly single strains or simple compound strains with limited adaptability and colonization capabilities. Furthermore, existing culture and preservation processes fail to fully account for the physiological differences of different probiotics, making it difficult to effectively maintain strain activity and resulting in insufficient stability of their efficacy in relieving antibiotic-associated diarrhea. Summary of the Invention

[0004] This application provides a method for preparing a probiotic composition, its formulation, and its application. By implementing differentiated supplementation and personalized culture condition control based on the characteristics of different probiotic strains, matching strain-specific protective agent treatment and low-temperature freeze-drying process, grading and pulverizing according to bacterial morphology and characteristics, and combining stepwise precise compounding based on strain ratio, particle size, and flowability, supplemented by low-temperature centrifugation purification of bacterial sludge preparation, the viable activity and biological function of each strain are maximized, ensuring the uniformity, stability, and colonization ability of the probiotic composition. Ultimately, it achieves the purpose of effectively regulating the host's intestinal flora structure, improving intestinal flora diversity, repairing the intestinal mucosal barrier, and regulating the body's inflammatory response, thereby effectively alleviating antibiotic-associated diarrhea. At the same time, it provides standardized and reproducible preparation process support for the application of this probiotic composition in the fields of pharmaceuticals, health products, and food.

[0005] In a first aspect, embodiments of this application provide a method for preparing a probiotic composition, wherein the probiotic composition is prepared by the following method: S1. The following strains were activated in sterile MRS medium at 37°C for 18-24 hours: Lactobacillus plantarum GOLDT-LP1024, Lactobacillus paracasei GOLDT-LC12345, Lactobacillus reuteri GOLDT-LR99, Pediococcus lactis GOLDT-PA0755, and Lactobacillus rhamnosus GOLDT-L818. They were then passaged 2-3 times to induce the strains to enter the logarithmic growth phase and then expanded into larger quantities. S2, at 2℃-8℃, 6000 Centrifuge the bacterial culture at 12000 rpm for 15 minutes. After 30 minutes, the mycelial sludge of each strain was collected; after adding a protective agent to the mycelial sludge of each strain, it was freeze-dried, pulverized and sieved to obtain freeze-dried powder of each strain; S3. Select 0.8-1 parts of *Lactobacillus plantarum* GOLDT-LP1024 bacterial powder, 0.2-0.3 parts of *Lactobacillus paracasei* GOLDT-LC12345 bacterial powder, 1.5-2 parts of *Lactobacillus reuteri* GOLDT-LR99 bacterial powder, 0.1-0.2 parts of *Pediococcus lactis* GOLDT-PA0755 bacterial powder, and 0.3-0.4 parts of *Lactobacillus rhamnosus* GOLDT-L818 bacterial powder, and thoroughly mix them with 2-3 parts of the first lactic acid bacteria compound powder and 3-4 parts of the second lactic acid bacteria compound powder to obtain the probiotic composition, wherein... The first type of lactic acid bacteria compound powder is obtained by mixing Lactobacillus acidophilus NCFM powder, Bifidobacterium animalis subsp. lactis B1-04 powder, Lactobacillus plantarum Lp116 powder, Bifidobacterium bifidum Bb-06 powder and Bifidobacterium longum subsp. infantis Bi-26 powder. The second type of lactic acid bacteria compound powder is obtained by mixing Bifidobacterium longum subsp. longum BORI powder, Lactobacillus acidophilus AD031 powder, Bifidobacterium bifidum BGN4 powder, Bifidobacterium animalis subsp. lactis AD011 powder, and Bifidobacterium longum subsp. infantis IBS007 powder. Preferably, the MRS culture medium comprises the following components: 9.0-11.0 g peptone, 9.0-11.0 g beef extract, 4.0-6.0 g yeast extract, 18.0-22.0 g glucose, 0.8-1.2 mL Tween 80, 1.8-2.2 g K₂HPO₄·7H₂O, 4.0-6.0 g CH₃COONa·3H₂O, 1.8-2.2 g triammonium citrate, 0.08-0.12 g MgSO₄·7H₂O, and 0.04-0.06 g MnSO₄·4H₂O. After weighing the above components according to the required amounts, dissolve them in 1000 mL of distilled water by heating, adjust the pH to 6.0-6.4, dispense into containers, autoclave at 121°C for 15-20 min, and cool before use.

[0006] Preferably, the freeze-drying in step S2 includes: dispensing the emulsified bacterial suspension of each single strain into freeze-drying trays, quickly placing them in an ultra-low temperature environment for pre-freezing until the bacterial suspension is completely frozen; then transferring them into a freeze dryer for drying to obtain single-strain bacterial powder.

[0007] In some embodiments, the probiotic composition has a live bacteria count ≥ 2.5 × 10¹¹ CFU / g. In some embodiments, the expanded culture in step S1 includes: inoculating the bacterial solution into the fermenter at an inoculation rate of 3%-8%, continuing to culture and ferment for 20-40 hours, and then rapidly cooling the bacterial solution to 15℃-25℃ after fermentation. During the fermentation process, the pH value of the bacterial solution in the fermenter is 6.0-7.5, and the culture temperature is 35℃-39℃. In some embodiments, the activation in step S1 includes: adding supplements to the sterile MRS culture medium; the supplements include one or more combinations of nitrogen source supplements, carbon source supplements, and buffers, and the mass-volume percentage of the supplements in the sterile MRS culture medium does not exceed 1%; The nitrogen source supplement includes one or more combinations of yeast extract, beef extract, fish peptone, soybean peptone, and corn steep liquor powder; the carbon source supplement includes one or more combinations of trehalose and glucose; and the buffer includes one or more combinations of triammonium citrate and sodium acetate. In some embodiments, the collection of bacterial sludge from each strain in step S2 includes: after centrifuging the bacterial solution, discarding the supernatant, adding sterile PBS buffer to the precipitate, gently stirring and washing, and then centrifuging again under the same centrifugation conditions. This washing process is repeated 1-3 times to collect bacterial sludge from each strain. The pH value of the sterile PBS buffer is 7.0-7.4.

[0008] In some embodiments, after adding the protective agent in step S2, the mixture is stirred at 20℃-30℃ for 25-40 minutes, and the mass ratio of the bacterial sludge of each strain to the protective agent is 1:2-3. The protective agent includes one or more combinations of carbon sources, nitrogen sources, pH adjusters, stabilizers, prebiotics, osmotic pressure regulators, amino acids, and sterile water.

[0009] Preferably, the Bifidobacterium strains include the strains corresponding to Bifidobacterium animalis subsp. lactis B1-04, Bifidobacterium bifidum Bb-06, Bifidobacterium longum subsp. infantis Bi-26, Bifidobacterium longum subsp. BORI, Bifidobacterium bifidum BGN4, Bifidobacterium animalis subsp. lactis AD011, and Bifidobacterium longum subsp. infantis IBS007. The bacterial slurry is slowly mixed with the protectant and stirred until completely emulsified.

[0010] Preferably, the protective agent added to the bacterial sludge of the corresponding strains of *Lactobacillus plantarum* GOLDGUT-LP1024, *Lactobacillus paracasei* GOLDGUT-LC12345, *Lactobacillus reuteri* GOLDGUT-LR99, and *Lactobacillus rhamnosus* GOLDGUT-L818 consists of 10%-20% protein, 0.5%-3% stabilizer, 3%-8% prebiotics, and 69%-86.5% sterile water; after mixing, it is stirred at 20℃-30℃ for 30-40 minutes until a uniform bacterial suspension is formed.

[0011] Preferably, the protective agent added to the bacterial sludge corresponding to the strain GOLDGUT-PA0755 of *Pediococcus lactis* consists of 8%-15% freeze-dried protective agent, 3%-8% osmotic pressure regulator, 0.5%-4% amino acid substances, and 73%-88% sterile water; after mixing, it is stirred at 20℃-30℃ for 20-30 minutes until it is emulsified uniformly.

[0012] Preferably, the carbon source is selected from one or more of maltose, trehalose, and glucose; the nitrogen source is selected from one or more of yeast extract, skim milk powder, and soy protein peptides; the buffer is selected from one or more of potassium dihydrogen phosphate, diammonium citrate, and sodium bicarbonate; the protein is selected from one or more of skim milk powder, whey protein, and casein; the stabilizer is selected from one or more of sodium citrate, sodium ascorbate, and glutamine; the prebiotic is selected from one or more of fructooligosaccharides, galactooligosaccharides, and stachyose; the freeze-drying protectant is selected from one or more of trehalose, sucrose, and lactose; the osmotic pressure regulator is selected from one or more of mannitol, sorbitol, and glycerol; and the amino acid is selected from one or more of monosodium glutamate, alanine, and glycine.

[0013] In some embodiments, the sieve mesh size in step S2 is 60-120 mesh; the probiotic composition contains Bifidobacterium animalis subsp. lactis B1-04 powder, Bifidobacterium bifidum Bb-06 powder, Bifidobacterium longum subsp. infantis Bi-26 powder, Bifidobacterium longum subsp. BORI powder, Bifidobacterium bifidum BGN4 powder, Bifidobacterium animalis subsp. lactis AD011 powder, and Bifidobacterium longum subsp. infantis IBS007 powder, which are obtained by pulverizing through a 60-80 mesh sieve; The Lactobacillus acidophilus NCFM powder, Lactobacillus plantarum Lp116 powder, Lactobacillus acidophilus AD031 powder, Lactobacillus plantarum GOLDDUT-LP1024 powder, Lactobacillus paracasei GOLDDUT-LC12345 powder, Lactobacillus reuteri GOLDDUT-LR99 powder, and Lactobacillus rhamnosus GOLDDUT-L818 powder were obtained by pulverizing through a 60-100 mesh sieve. The *Pediococcus lactis* GOLDGUT-PA0755 bacterial powder was obtained by pulverizing it through a 60-120 mesh sieve; wherein... The bacterial powders of each strain in the first type of lactic acid bacteria compound powder and the second type of lactic acid bacteria compound powder are pre-sieved according to the specified sieve mesh size before being mixed and compounded.

[0014] Preferably, after pulverizing and sieving, the bacterial powder is sealed and stored in an environment of -20°C.

[0015] In some embodiments, the thorough mixing in step S3 to obtain the probiotic composition includes: Select 2-3 parts of the first type of lactic acid bacteria compound powder and 3-4 parts of the second type of lactic acid bacteria compound powder, and mix them; and, Add 1.5-2 parts of *Lactobacillus reuteri* GOLDTUT-LR99 bacterial powder, 0.8-1 parts of *Lactobacillus plantarum* GOLDTUT-LP1024 bacterial powder, and 0.3-0.4 parts of *Lactobacillus rhamnosus* GOLDTUT-L818 bacterial powder, and continue mixing; and, Add 0.2-0.3 parts of Lactobacillus paracasei GOLDGUT-LC12345 bacterial powder and 0.1-0.2 parts of Pediococcus lactis GOLDGUT-PA0755 bacterial powder, and continue mixing for 5 minutes. After 15 minutes, the probiotic composition was obtained.

[0016] Secondly, embodiments of this application provide a probiotic preparation containing the probiotic composition described in the first aspect, as well as pharmaceutically or food-acceptable excipients.

[0017] Thirdly, embodiments of this application provide the use of the probiotic composition described in the first aspect in the preparation of products for relieving antibiotic-associated diarrhea, the products including pharmaceuticals, health products, or food.

[0018] This application has the following advantages over the prior art: (1) Through multi-dimensional regulation of intestinal microecology and tissue barrier, it can achieve the core role of effectively relieving antibiotic-associated diarrhea. Specifically, it can repair the damage to the colonic tissue barrier caused by antibiotics, regulate the balance of pro-inflammatory / anti-inflammatory factors in the intestine, enhance the diversity of intestinal flora α and β, restore the metabolic level of short-chain fatty acids in the intestine, promote the proliferation of beneficial bacteria and inhibit the growth of pathogenic bacteria, fundamentally improve the intestinal flora imbalance caused by antibiotics, effectively reduce diarrhea symptoms and intestinal inflammatory damage, and fully restore the normal physiological function of the intestine. (2) The preparation process and strain ratio of the probiotic composition are precisely designed to meet the intervention needs of antibiotic-associated diarrhea. Specifically, the strain differentiation and exclusive culture and protective agent treatment greatly improve the activity of live bacteria and the ability to colonize the intestine. Grading and screening and step-by-step compounding ensure the uniformity and stability of the composition. The live bacteria count is clearly defined and the mechanism of action is clear. The preparation process is quantitative, controllable and repeatable throughout. It can be adapted to the development of various product forms such as pharmaceuticals, health products and food. It provides an efficient, stable and easy-to-industrialize professional technical solution for the microecological prevention and treatment of antibiotic-associated diarrhea. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Statistical charts of diarrhea scores in multiple groups of mice provided in the embodiments of this application (normal control group (Control group); model control group (Model group); drug group (Drug group); probiotic treatment group (WLFX-1 group)). Figure 2 Statistical graphs showing the results of food intake and body weight of multiple groups of mice provided in the embodiments of this application; Figure 3 Statistical charts of serum biochemical indicators of multiple groups of mice provided for embodiments of this application; Figure 4 This is a schematic diagram of H&E staining of colon tissue from multiple groups of mice provided in the embodiments of this application; Figure 5 Immunofluorescence staining images of intestinal mucosal barrier-related proteins in multiple groups of mice provided in the embodiments of this application; Figure 6 Statistical graphs showing the expression levels of intestinal mucosal barrier-related proteins in multiple groups of mice, provided for embodiments of this application; Figure 7 Statistical graphs of colon gene expression levels in multiple groups of mice provided in embodiments of this application; Figure 8 Statistical charts of intestinal short-chain fatty acid content in multiple groups of mice provided in embodiments of this application; Figure 9 Statistical graphs of intestinal microbial diversity indices of multiple groups of mice provided in embodiments of this application; Figure 10 A schematic diagram of the gut microbiota structure of multiple groups of mice provided in the embodiments of this application; Figure 11 Stacked bar charts of relative abundance of gut microbiota in multiple groups of mice provided in embodiments of this application; Figure 12 Metagenomic heatmaps of multiple groups of mice provided in the embodiments of this application; Figure 13 This is a schematic diagram illustrating the results of differential metabolic pathway analysis of gut microbiota in mice under different treatment groups, as provided in the embodiments of this application.

[0021] In the figure, the letters a, b, c, and ab represent the results of statistical analysis. If two groups have the same letter, it means there is no significant difference (P>0.05), while if two groups have no letters in common, it means there is a significant difference (p<0.05). Detailed Implementation

[0022] The technical solution of this application will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of this application and should not be construed as specific limitations thereof.

[0023] Unless otherwise specified, all experimental materials used in the embodiments of this invention are conventional experimental materials in the art and can be purchased through commercial channels. The experimental mice used in this invention were male, C57BL / 6J, 8 weeks old, weighing (20±2) g, and were purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd. All experimental protocols and operations provided in the embodiments of this invention were approved by the Ethics Committee of Hainan University (No.: HNUAUCC-2023-00179). The drugs used in this invention, cefotaxime acid, were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; loperamide hydrochloride was purchased from Xian Janssen Pharmaceutical Co., Ltd.; 4% paraformaldehyde was purchased from Wuhan Sewell Biotechnology Co., Ltd.; the inflammatory cytokine detection kit was purchased from Shanghai Xinyu Biotechnology Co., Ltd.; and short-chain fatty acid standards were purchased from Stanford Chemicals, USA. The probiotics used in this invention were all provided by Shenzhen Baoshijian Biotechnology Co., Ltd., and the strains included: Lactobacillus acidophilus NCFM, Bifidobacterium animalis subsp. lactis B1-04, Lactobacillus plantarum Lp116, Bifidobacterium bifidum Bb-06, Bifidobacterium longum subsp. infantis Bi-26, Bifidobacterium longum subsp. BORI, Lactobacillus acidophilus AD031, Bifidobacterium bifidum BGN4, Bifidobacterium animalis subsp. lactis AD011, Bifidobacterium longum subsp. infantis IBS007, Lactobacillus plantarum GOLDGUT-LP1024, Lactobacillus paracasei GOLDGUT-LC12345, Lactobacillus reuteri GOLDGUT-LR99, Pediococcus lactis GOLDGUT-PA0755, and Lactobacillus rhamnosus GOLDGUT-L818.

[0024] Among them, Lactiplantibacillus plantarum GOLDGUT-LP1024 is a deposited strain, classified and named Lactiplantibacillus plantarum, deposited at the China General Microbiological Culture Collection Center (CGMCC) at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo. 28801 and deposit date of October 27, 2023.

[0025] Among them, Lactobacillus paracasei GOLDGUT-LC12345 is a deposited strain, classified and named Lactobacillus paracasei, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCCNo.36872 and deposit date of December 1, 2025.

[0026] Example 1: Preparation of the first type of lactic acid bacteria compound powder: Take Lactobacillus acidophilus NCFM powder, Bifidobacterium animalis subsp. lactis B1-04 powder, Lactobacillus plantarum Lp116 powder, Bifidobacterium bifidum Bb-06 powder, and Bifidobacterium longum subsp. infantis Bi-26 powder, mix and compound them according to the preset weight parts to obtain the first lactic acid bacteria compound powder.

[0027] Example 2: Preparation of the second type of lactic acid bacteria compound powder: Take Bifidobacterium longum subsp. BORI powder, Lactobacillus acidophilus AD031 powder, Bifidobacterium bifidum BGN4 powder, Bifidobacterium animalis subsp. lactis AD011 powder, and Bifidobacterium longum subsp. infantis IBS007 powder, mix and compound them according to the preset weight parts to obtain the second lactic acid bacteria compound powder.

[0028] Example 3: Preparation of *Lactobacillus plantarum* GOLDTUT-LP1024 powder, *Lactobacillus paracasei* GOLDTUT-LC12345 powder, *Lactobacillus reuteri* GOLDTUT-LR99 powder, *Pediococcus lactis* GOLDTUT-PA0755 powder, and *Lactobacillus rhamnosus* GOLDTUT-L818 powder: (1) *Lactobacillus plantarum* strain GOLDGUT-LP1024, *Lactobacillus paracasei* strain GOLDGUT-LC12345, *Lactobacillus reuteri* strain GOLDGUT-LR99, *Pediococcus lactis* strain GOLDGUT-PA0755, and *Lactobacillus rhamnosus* strain GOLDGUT-L818 were activated in sterile MRS culture medium at 37℃ for 18-24 h, and then passaged 2-3 times to induce the strains to enter the logarithmic growth phase, and then expanded the culture; among them, The expanded culture includes: inoculating the bacterial solution into the fermenter at an inoculation rate of 3%-8%, continuing to culture and ferment for 20-40 hours, and then rapidly cooling the bacterial solution to 15℃-25℃ after fermentation; during the culture and fermentation process, the pH value of the bacterial solution in the fermenter is 6.0-7.5, and the culture temperature is 35℃-39℃. Activation includes: adding supplements to sterile MRS culture medium; the supplements include one or more combinations of nitrogen source supplements, carbon source supplements, and buffers, wherein the mass-volume percentage of the supplements in the sterile MRS culture medium does not exceed 1%; the nitrogen source supplements include one or more combinations of yeast extract, beef extract, fish peptone, soybean peptone, and corn steep liquor powder; the carbon source supplements include one or more combinations of trehalose and glucose; and the buffers include one or more combinations of triammonium citrate and sodium acetate.

[0029] (2) At 2℃-8℃, 6000 Centrifuge the bacterial culture at 12000 rpm for 15 minutes. After 30 minutes, mycelial sludge from each strain was collected; a preservative was added to the mycelial sludge of each strain, followed by freeze-drying, pulverizing, and sieving to obtain freeze-dried powder of each strain; among which, Collect the bacterial sludge of each strain, including: after centrifuging the bacterial solution and discarding the supernatant, adding sterile PBS buffer to the precipitate, gently stirring and washing, and then centrifuging again under the same centrifugation conditions. Repeat the washing 1-3 times to collect the bacterial sludge of each strain. The pH value of the sterile PBS buffer is 7.0-7.4. After adding the protective agent, stir at 20℃-30℃ for 25-40 minutes. The mass ratio of the bacterial sludge of each strain to the protective agent is 1:2-3. The protective agent includes one or more combinations of carbon source, nitrogen source, pH adjuster, stabilizer, prebiotic, osmotic pressure regulator, amino acid substances and sterile water. The sieve mesh size is 60-120 mesh. The probiotic composition contains *Bifidobacterium animalis* subsp. lactis B1-04 powder, *Bifidobacterium bifidum* Bb-06 powder, *Bifidobacterium longum* subsp. infantis Bi-26 powder, *Bifidobacterium longum* subsp. BORI powder, *Bifidobacterium bifidum* BGN4 powder, *Bifidobacterium animalis* subsp. lactis AD011 powder, and *Bifidobacterium longum* subsp. infantis IBS007 powder, all obtained by pulverizing through a 60-80 mesh sieve. The probiotic composition also contains *Lactobacillus acidophilus* NCFM powder, *Lactobacillus plantarum* Lp116 powder, *Lactobacillus acidophilus* AD031 powder, and *Lactobacillus plantarum* GOLDGUT-... The LP1024 bacterial powder, Lactobacillus paracasei GOLDGUT-LC12345 bacterial powder, Lactobacillus reuteri GOLDGUT-LR99 bacterial powder, and Lactobacillus rhamnosus GOLDGUT-L818 bacterial powder are obtained by pulverizing through a 60-100 mesh sieve; the Pediococcus lactis GOLDGUT-PA0755 bacterial powder is obtained by pulverizing through a 60-120 mesh sieve; wherein, the bacterial powders of each strain in the first type of lactic acid bacteria compound powder and the second type of lactic acid bacteria compound powder are pre-sieved according to the aforementioned sieve mesh size before being mixed and compounded.

[0030] Example 4: Preparation of the probiotic composition: Weigh out 2-3 parts by weight of the first type of lactic acid bacteria compound powder prepared in Example 1, 3-4 parts by weight of the second type of lactic acid bacteria compound powder prepared in Example 2, and 0.8-1 parts by weight of *Lactobacillus plantarum* GOLDT-LP1024 powder, 0.2-0.3 parts by weight of *Lactobacillus paracasei* GOLDT-LC12345 powder, 1.5-2 parts by weight of *Lactobacillus reuteri* GOLDT-LR99 powder, 0.1-0.2 parts by weight of *Pediococcus lactis* GOLDT-PA0755 powder, and 0.3-0.4 parts by weight of *Lactobacillus rhamnosus* GOLDT-L818 powder prepared in Example 3. First, mix the first type of lactic acid bacteria compound powder and the second type of lactic acid bacteria compound powder; then add Lactobacillus reuteri GOLDGUT-LR99 bacterial powder, Lactobacillus plantarum GOLDGUT-LP1024 bacterial powder, and Lactobacillus rhamnosus GOLDGUT-L818 bacterial powder and continue mixing; finally, add Lactobacillus paracasei GOLDGUT-LC12345 bacterial powder and Pediococcus lactis GOLDGUT-PA0755 bacterial powder and mix for 5-15 minutes to obtain a probiotic composition with a live bacteria count ≥2.5×10¹¹CFU / g.

[0031] Example 5: Preparation of probiotic preparations: Using the probiotic composition prepared in Example 4 as the active ingredient, pharmaceutically or food-acceptable excipients are added in conventional proportions. The excipients may be one or more combinations of prebiotics, maltodextrin, lactose, mannitol, citric acid, steviol glycosides, etc. By thoroughly mixing the probiotic composition with excipients and using conventional preparation processes in the food or pharmaceutical fields according to the dosage form requirements, it can be made into dosage forms such as powder, granules, hard capsules, chewable tablets, and solid beverages, thus obtaining probiotic preparations containing effective active probiotics. The content of the probiotic composition in the preparation meets the relevant quality standards for pharmaceuticals, health products, or food.

[0032] Example 6: The effect of probiotic composition on alleviating diarrhea symptoms in mice with antibiotic-associated diarrhea: 1. Experimental animals: A total of 32 eight-week-old C57BL / 6J mice were used as the research subjects. After two weeks of acclimatization, they were divided into a normal control group (Control group) of 8 mice, a model control group (Model group) of 8 mice, a drug group of 8 mice, and a probiotic treatment group (WLFX-1 group) of 8 mice.

[0033] 2. Animal grouping: Control group: 0.85% saline was administered by gavage twice a day for 7 consecutive days; after successful modeling, 200 μL of saline was administered by gavage once a day in the morning for 7 consecutive days. Model group: cefotaxime 500 mg / kg was administered by gavage twice a day for 7 consecutive days; after successful modeling, 200 μL of normal saline was administered by gavage once a day in the morning for 7 consecutive days. Drug group: Cefotaxime 500 mg / kg was administered by gavage twice a day for 7 consecutive days; after successful modeling, loperamide hydrochloride 1 mg / kg was administered by gavage once a day in the morning for 7 consecutive days. WLFX-1 group: 500 mg / kg of cefotaxime was administered by gavage twice daily for 7 consecutive days; after successful model establishment, 200 μL of the probiotic composition prepared in Example 4 (1×10⁻⁶) was administered by gavage every morning. 9 CFU / each), once a day for 7 days.

[0034] 3. Experimental methods: The animal laboratory environment was maintained at a temperature of 23±1℃ and a relative humidity of 52%–60%, with alternating light and dark conditions for 12 hours at a time. Food intake, water consumption, body weight, and fecal condition were monitored daily during the experiment.

[0035] Probiotic intervention continued until the day before the end of the experiment. Feces from each mouse were collected and placed in 2ml sterile centrifuge tubes, immediately frozen at -80℃ for subsequent fecal metagenomic sequencing and metabolic pathway analysis. All mice were prohibited from eating feed but were allowed free access to sterile distilled water. After euthanasia, blood was collected from the orbital rim of all mice. The blood was allowed to clot at room temperature for 1 hour, then centrifuged at 4℃, 3500 rpm for 45 minutes. Serum was carefully collected using a pipette and aliquoted and stored at -80℃ for subsequent analysis. After dissection, the liver, kidneys, and spleen were collected and weighed; organ indices were expressed as organ weight / body weight (mg / g). A portion of the colon was cut and fixed in 4% paraformaldehyde solution; the remaining portion was flash-frozen in liquid nitrogen and stored at -80℃. Cecal contents were also flash-frozen in liquid nitrogen and stored at -80℃ for subsequent short-chain fatty acid determination.

[0036] 4. Index Measurement: 4.1 Measurement of body weight, food intake, water intake, and diarrhea score: From the first day of modeling, the mice's weight was recorded daily. During the modeling period, the mice's food and water intake were recorded every other day. During the treatment period, the mice's food and water intake were recorded daily. Diarrhea scores were assessed and photographs were taken daily. The mouse diarrhea status scoring table is shown below: Mouse Diarrhea Status Scoring Scale

[0037] 4.2. Histopathological observation of colon tissue: Distal colon samples were collected from mice, washed with PBS, fixed in 4% (w / v) paraformaldehyde, and after gradient dehydration, cut into 5 μm thick continuous sections for hematoxylin and eosin (HE) staining. The sections were observed under a microscope to distinguish the morphology and structure of normal and pathological colon tissue.

[0038] 4.3 Immunofluorescent protein detection: Colon tissue was labeled with MUC-2 and ZO-1 antibodies, and the antibodies in both tissue samples were characterized using an EclipModCl-L ​​fluorescence microscope. Areal density is equal to the ratio of the cumulative optical density value to the area of ​​the tissue pixels.

[0039] 4.4. Serum immune cytokines and blood urea nitrogen measurement: Blood was collected from mice via orbital sampling. After collection, the blood was allowed to stand for half an hour, then centrifuged at 3000 rpm for 20 minutes at 4°C to collect serum. The levels of IL-1β, IL-6, IL-10, TNF-α, IL-4, and BUN in the serum were detected using an ELISA kit.

[0040] 4.5 RNA extraction and RT-qPCR: RNA was extracted from mouse colon using the EastepSuper Total RNA Extraction Kit. RNA quality was assessed using a micro-UV-Vis spectrophotometer. mRNA was reverse transcribed into cDNA using a cDNA synthesis kit (ThermoFisher). The mRNA expression of Tjp1, Muc2, Ocln, Cldn1, Gja1, Il6, Il10, Il1b, Tnf, and SCN9A was measured using real-time quantitative reverse transcriptase PCR (RT-qPCR). Primers were synthesized by Qingke Biotechnology. The qRT-PCR program was set to incubate at 95°C for 30 seconds, followed by 40 cycles of incubation at 95°C for 10 seconds and then at 60°C for 30 seconds. GAPDH was used as the reference gene. After qRT-PCR, 2... -ΔΔCT The method analyzed relative gene expression levels. Each sample was measured four times and the average value was taken.

[0041] 4.6 Metagenomic sequencing: Mouse feces were collected the day before the end of the experiment, flash-frozen in liquid nitrogen, and stored at -80°C. Sequencing was performed by Beijing Novogene Technology Co., Ltd. Total DNA was extracted from mouse feces using a DNA extraction kit. The integrity of the DNA was checked by 0.8% agarose gel electrophoresis, followed by metagenomic sequencing to construct a sequencing library.

[0042] 4.7 SCFAs determination: Weigh 40 mg of lyophilized cecal contents, add 500 μL of saturated NaCl solution, let stand for half an hour, then add 20 μL of sulfuric acid and shake. Add 800 μL of n-hexane, shake thoroughly, and let stand. Then centrifuge at 12000 r / min for 15 min at 4 °C. After centrifugation, collect the supernatant, mix with 0.25 g of anhydrous sodium sulfate, and centrifuge under the same conditions. Analyze the concentrations of acetic acid and propionic acid using gas chromatography. Chromatographic conditions: pipette 1 μL of sample, split at a 10:1 ratio, with high-purity nitrogen as the carrier gas. Set the detector temperature to 220 °C, maintain the column temperature at 60 °C for 3 min, then increase the temperature to 180 °C at a rate of 3.5 °C / min and hold for 30 min.

[0043] 5. Analysis of measurement results: 5.1 Effects of different probiotic groups on diarrhea scores, food intake, and body weight in mice with antibiotic-associated diarrhea: Stool is the most direct indicator of the severity of diarrhea, such as Figure 1 As shown, with an observation period of 3-13 days, mice in the Control group (administered saline by gavage) had normal mental status and stable fecal morphology. Three days after administration of cefotaxime by gavage, mice in the Model group (no intervention only), Drug group (intervention with loperamide), and WLFX-1 group (intervention with probiotic combination) began to show varying degrees of diarrhea symptoms, specifically lethargy, reduced activity or even inactivity, significantly increased water intake, and increased defecation frequency. The severity of diarrhea continued to worsen with prolonged administration (3-7 days of modeling period), and some mice exhibited severe symptoms such as anal redness and swelling, and fecal adhesion to the anus. At the end of modeling (day 7), the diarrhea scores of the Model group, Drug group, and WLFX-1 group all reached their peak, confirming the successful establishment of the antibiotic-associated diarrhea mouse model. From day 8 onwards, the intervention period began. The Model group was administered saline by gavage to simulate natural recovery, the Drug group was administered 1 mg / kg loperamide hydrochloride by gavage, and the WLFX-1 group was administered 200 μL of bacterial solution (1×10⁻⁶) by gavage. 9 The diarrhea scores of all three groups showed a decreasing trend during the intervention period (CFU / mouse), but the Model group showed a slow decrease and remained at a high level until day 13; while the Drug group and WLFX-1 group showed a significant decrease, with diarrhea scores in both groups significantly lower than those in the Model group on day 13 (p<0.05) and close to those in the Control group. The experimental results indicate that the intervention of the probiotic composition in the WLFX-1 group can significantly alleviate the diarrhea symptoms of antibiotic-associated diarrhea mice and improve fecal characteristics.

[0044] like Figure 2As shown in Figure A, regarding food intake, on day 3, the food intake of the Model group, Drug group, and WLFX-1 group all decreased significantly due to cefotaxime intervention, significantly lower than that of the Control group (p<0.05). Subsequently, the food intake of each group gradually recovered, and by the end of modeling (day 7), it had basically returned to the same level as the Control group. After entering the intervention period, the food intake of the Model group continued to rise, and by day 11, it significantly exceeded that of the Control group (p<0.05), while the food intake of the Drug group and WLFX-1 group remained at a level similar to that of the Control group, with no significant difference (p>0.05). This suggests that cefotaxime-induced diarrhea stimulates an increase in the food demand of mice, and the probiotic composition WLFX-1 can restore food intake to normal physiological levels by alleviating diarrhea symptoms.

[0045] like Figure 2 As stated in section B, regarding body weight, throughout the entire study period (3-13 days), the body weight of mice in the Control group, Model group, Drug group, and WLFX-1 group all showed a slow and stable growth trend. Although the Model group experienced a temporary slowdown in body weight gain due to diarrhea during the modeling period, statistical analysis showed that there was no significant difference in body weight among the groups (p>0.05). This indicates that although gavage administration of cefotaxime can significantly induce diarrhea in mice and affect food intake, it does not have a substantial adverse effect on the overall growth status of mice, and the intervention of the probiotic composition does not interfere with the normal body weight gain of mice.

[0046] 5.2 Effects of different groups of probiotics on serum biochemical indicators in mice with antibiotic-associated diarrhea: The expression of immune cytokines regulates inflammatory responses and is divided into pro-inflammatory cytokines and anti-inflammatory cytokines, which mutually restrain each other and exist in a dynamic equilibrium. To investigate the effect of probiotics on improving immune balance in mice, the levels of the pro-inflammatory cytokine IL-1β and the anti-inflammatory cytokines IL-4 and IL-10 in mouse serum were quantitatively measured using an ELISA kit. The results are as follows: Figure 3 A in Figure 3 B in Figure 3 The C triad is shown in the figure; serum blood urea nitrogen (BUN) levels were simultaneously measured to assess renal burden, and the results are as follows. Figure 3 The D subgraph is shown in the diagram.

[0047] like Figure 3As shown in Figure A, the serum IL-4 level (pg / mL) was highest in the Control group and significantly lower in the Model group, showing a statistically significant difference (p<0.05). The serum IL-4 level in the WLFX-1 group rebounded and was significantly higher than that in the Model group (p<0.05), while the levels in the Drug group were similar to those in the WLFX-1 group. This indicates that the probiotic composition can improve cefotaxime-induced immunosuppression by upregulating the expression of the anti-inflammatory factor IL-4.

[0048] like Figure 3 As shown in Figure B, the serum IL-10 (pg / mL) level in the Control group remained at a high level, while it was significantly lower in the Model group, showing a significant difference from the Control group (p<0.05). The serum IL-10 levels in the Drug group and WLFX-1 group rebounded, showing an upward trend compared to the Model group. However, statistical analysis showed no significant difference between the two groups and the Model group (p>0.05), indicating that the probiotic composition has a weaker regulatory effect on IL-10 than on IL-4, and further optimization of the intervention effect is still needed.

[0049] like Figure 3 As shown in Figure C, the serum IL-1β (ng / L) level was lowest in the Control group, while it was significantly elevated in the Model group due to cefotaxime induction, showing a significant difference from the Control group (p<0.05). The Drug group showed a significant decrease after intervention, with a statistically significant difference from the Model group (p<0.05). The serum IL-1β level in the WLFX-1 group was decreased, although it did not show a significant difference from the Model group (p>0.05), but it still showed a clear downward trend. This indicates that although the probiotic composition has a weaker inhibitory effect on the pro-inflammatory factor IL-1β than loperamide hydrochloride, it can still alleviate the inflammatory response to some extent.

[0050] Blood urea nitrogen (BUN) is a metabolic waste product of the body, normally excreted through filtration by the kidneys. High serum BUN levels indicate increased kidney burden and decreased kidney function. Figure 3 As shown in D, the serum BUN (mg / mL) level in the Control group remained at a low level, while it was significantly increased in the Model group, showing a significant difference from the Control group (p<0.05). After intervention, the serum BUN levels in the Drug group and WLFX-1 group were significantly reduced, showing a statistical difference from the Model group (p<0.05) and completely recovering to the same level as the Control group. This indicates that both the probiotic composition and loperamide hydrochloride can effectively reduce the metabolic burden on the kidneys of mice caused by cefotaxime and protect kidney function.

[0051] In summary, the probiotic composition WLFX-1 can improve the immune imbalance induced by cefotaxime antibiotics by upregulating the expression of the anti-inflammatory cytokine IL-4 and downregulating the level of the pro-inflammatory cytokine IL-1β, while reducing the metabolic burden on the kidneys, providing experimental evidence on the immunomodulatory and organ-protective aspects for alleviating antibiotic-associated diarrhea.

[0052] 5.3. Observe the effect of probiotics on the colonic tissue of mice with diarrhea by H&E staining: H&E staining results of mouse colon as follows Figure 4 As shown, the colon of mice in the Control group exhibited typical normal histological characteristics: the mucosal epithelial cells were neatly arranged, continuous, and without sloughing; the intestinal glands were abundant and densely and regularly arranged; the crypts were of uniform depth and clearly distinguishable structure; the boundaries between the mucosa, submucosa, and muscularis propria were clear; and the tissue structure of each layer was intact, with no signs of inflammatory cell infiltration or pathological damage. Compared with the Control group, the colon tissue of mice in the Model group (cefotaxime model without intervention) showed significant pathological changes: the crypt structure was severely disordered, with some crypts deformed, broken, or even missing; the continuity of the mucosal epithelial cells was interrupted, and local areas of epithelial sloughing exposed the lamina propria; the number of goblet cells between epithelial cells was significantly reduced (the green arrows in the figure clearly indicate the areas of reduced goblet cells); at the same time, a large number of inflammatory cells (such as neutrophils and lymphocytes) were observed to aggregate and infiltrate the lamina propria and submucosa (the black arrows in the figure clearly indicate the areas of inflammatory cell infiltration), consistent with the typical pathological manifestations of colonic inflammation in mice with antibiotic-associated diarrhea.

[0053] After treatment with the Drug group (loperamide intervention) and the WLFX-1 group (probiotic composition intervention), the pathological damage of the colon was significantly improved: the disordered colonic crypt structure of mice in both groups was significantly relieved, and most crypts returned to regular shape and normal depth; the shedding of mucosal epithelial cells was reduced, and the continuity of the epithelium was basically restored; the number of goblet cells between epithelial cells was significantly increased compared with the Model group, and the mucosal secretory function gradually recovered; and the amount of inflammatory cell infiltration in the lamina propria and submucosa of both groups was greatly reduced, and no large amount of inflammatory cell aggregation was observed.

[0054] Histological observations showed that both the probiotic composition and loperamide in the WLFX-1 group effectively repaired cefotaxime-induced colonic tissue damage. The WLFX-1 group showed a more normal level of recovery in goblet cell count and crypt structure integrity, further confirming that it can alleviate intestinal inflammation by reducing pathological damage to the intestinal mucosa and inhibiting inflammatory cell infiltration.

[0055] 5.4. Observe the effects of probiotics on intestinal mucosal barrier-related proteins in mice using immunofluorescence staining: Tight junction proteins (such as ZO-1) and mucins (such as MUC2) are core components of the intestinal mucosal barrier: ZO-1 maintains barrier integrity by sealing the intercellular spaces of epithelial cells, while MUC2, as the main mucin secreted by goblet cells, constitutes the intestinal mucus layer to defend against pathogen invasion. To assess the function of the colonic mucosal barrier in mice, the expression localization of ZO-1 and MUC2 was observed by immunofluorescence staining, and their areal density was quantitatively analyzed.

[0056] Immunofluorescence results as follows Figure 5 As shown, the red fluorescence signals of ZO-1 and MUC2 in the colon of mice in the Control group were strong and continuously distributed, indicating that their expression levels were high and uniformly distributed. The fluorescence signals of ZO-1 and MUC2 in the Model group (cefotaxime model group) were significantly weakened, and their expression levels were significantly reduced. The fluorescence signals of the Drug group (loperamide intervention) and the WLFX-1 group (probiotic intervention) were significantly restored, with the expression intensity of MUC2 being closer to that of the Control group.

[0057] Quantitative analysis results as follows Figure 6 As shown, this further confirms the above trend: Figure 6 As shown in Figure A, for MUC2 areal density, the Control group had the highest level, while the Model group significantly decreased to the lowest level (p<0.05); the MUC2 areal density of both the Drug group and the WLFX-1 group was significantly higher than that of the Model group (p<0.05), and the WLFX-1 group recovered to a level that was not significantly different from that of the Control group. Figure 6 As shown in B, for ZO-1 areal density, the Control group maintained a high level, while the Model group significantly decreased (p<0.05); the ZO-1 areal density of the Drug group and the WLFX-1 group were significantly higher than that of the Model group (p<0.05), with the ZO-1 areal density of the WLFX-1 group being slightly higher than that of the Control group.

[0058] In summary, both loperamide and the probiotic combination WLFX-1 can significantly improve cefotaxime-induced intestinal mucosal barrier damage by upregulating the expression of ZO-1 and MUC2 in the colon. Among them, WLFX-1 has a better repair effect on MUC2, which can completely restore it to normal physiological level, thereby effectively enhancing intestinal barrier function.

[0059] 5.5. Determining the effect of probiotics on gene expression in mouse colon using RT-qPCR: To assess the effects of antibiotics on intestinal tight junctions and inflammatory responses, the mRNA expression levels of tight junction-related genes (Tjp1, Muc2, Ocln, Cldn1, Gja1), pain-related genes (SCN9A), and inflammatory cytokine genes (Il6, Il1b, Il10) in colonic tissue were detected by RT-qPCR. Results are as follows: Figure 7 As shown, compared with the control group, the relative expression levels of Tjp1 mRNA, Muc2 mRNA, Ocln mRNA, Cldn1 mRNA, and Gja1 mRNA in the colon of Model group mice were significantly reduced (p<0.05), suggesting that cefotaxime severely disrupts the tight junction structure of the intestine.

[0060] like Figure 7 As shown in Figure A, the control group had the highest relative expression level of Tjp1 mRNA, while the model group showed a significant decrease to extremely low levels. Expression levels in both the drug and WLFX-1 groups significantly increased (p<0.05), with the WLFX-1 group showing better recovery. Figure 7 As shown in B, in terms of relative Muc2 mRNA expression, the Control group had the highest expression level, while the Model group showed almost no expression; the expression levels in both the Drug and WLFX-1 groups were significantly increased (p<0.05), and the expression level in the WLFX-1 group was significantly higher than that in the Drug group (p<0.05). Figure 7 As shown in Figure C, the control group had the highest relative expression level of Ocln mRNA, while the model group showed a significant decrease. Although the expression levels in the drug and WLFX-1 groups rebounded, there was no significant difference compared to the model group (p>0.05). Figure 7 As shown in Figure D, the relative expression level of Cldn1 mRNA was higher in the Control group and significantly lower in the Model group; the expression levels in both the Drug and WLFX-1 groups were significantly increased (p<0.05), and the expression level in the WLFX-1 group was significantly higher than that in the Drug group (p<0.05). Figure 7 As shown in Figure E, the relative expression level of Gja1 mRNA was highest in the Control group and significantly decreased to a very low level in the Model group. Both the Drug and WLFX-1 groups showed significantly increased expression (p<0.05), with the WLFX-1 group showing a significantly higher expression level than the Drug group (p<0.05). These results indicate that the probiotic composition WLFX-1 can effectively repair cefotaxime-induced intestinal barrier structure damage by upregulating the expression of multiple tight junction-related genes, with an overall effect superior to loperamide (Drug group).

[0061] The Nav1.7 sodium channel encoded by SCN9A is closely related to the perception of intestinal pain. For example... Figure 7 As shown in F, the relative expression level of SCN9AmRNA in the Model group was significantly higher than that in the Control group (p<0.05), suggesting that antibiotic-associated diarrhea is accompanied by increased sensitivity to intestinal pain. The expression in the Drug group was significantly reduced, while the expression in the WLFX-1 group was not significantly different from that in the Model group, indicating that loperamide can directly relieve intestinal pain, while the WLFX-1 group has no significant regulatory effect on pain perception.

[0062] To evaluate the anti-inflammatory effect of probiotics, the expression of pro-inflammatory factors (Il6, Il1b) and anti-inflammatory factors (Il10) was detected. Figure 7 As shown in G, the relative expression level of Il6 mRNA was significantly higher in the Model group than in the Control group; the expression was significantly decreased in both the Drug group and the WLFX-1 group. Figure 7 As shown in H, the relative expression level of Il1b mRNA was significantly higher in the Model group than in the Control group; the expression levels in both the Drug group and the WLFX-1 group were significantly reduced, with comparable effects between the two groups. Figure 7 As shown in Figure I, the relative expression level of Il10 mRNA in the Model group was significantly lower than that in the Control group; the expression level in the WLFX-1 group was significantly increased and significantly higher than that in the Drug group, suggesting that the WLFX-1 group can more effectively improve immune imbalance by upregulating the expression of anti-inflammatory factors.

[0063] In summary, the probiotic composition WLFX-1 can repair the intestinal barrier structure by upregulating the expression of tight junction-related genes (Tjp1, Muc2, Cldn1, Gja1), and alleviate intestinal inflammation by inhibiting the expression of pro-inflammatory factors (Il6, Il1b) and upregulating the expression of anti-inflammatory factors (Il10). Its overall effect is better than that of loperamide, providing molecular-level experimental evidence for its ability to alleviate antibiotic-associated diarrhea.

[0064] 5.6 Effects of probiotics on the content of short-chain fatty acids in the mouse intestine: Short-chain fatty acids (SCFAs) are core metabolites of gut microbiota, among which acetic acid and propionic acid play key roles in regulating host metabolism, intestinal function, and immune homeostasis. To assess the effects of probiotics on intestinal metabolism, the levels of acetic acid and propionic acid in the cecal contents of mice were measured. The results are as follows: Figure 8 As shown. Figure 8As shown in Figure A, the Control group had the highest acetic acid content, while the Model group (cefotaxime model group) showed a significant decrease in acetic acid content. After intervention, the Drug group (loperamide) and the WLFX-1 group (probiotic composition) both had significantly higher acetic acid (μmol / g) content than the Model group (p<0.05), effectively reversing the antibiotic-induced decrease in acetic acid levels. Figure 8 As shown in B, the propionic acid (μmol / g) content in the Control group was significantly higher than that in the other groups; the propionic acid content in the Model group was significantly reduced; although the propionic acid content in the Drug group and WLFX-1 group showed an increasing trend compared with the Model group, there was no significant statistical difference compared with the Model group (p>0.05), suggesting that the intervention had a weaker restorative effect on propionic acid than acetic acid.

[0065] The use of cefotaxime significantly reduced the levels of short-chain fatty acids (SCFAs), a metabolite of gut microbiota, and this reduction is closely related to gut microbiota dysbiosis. Intervention with the probiotic combination WLFX-1 effectively increased acetic acid levels; although the recovery of propionic acid did not show a statistically significant difference, it still showed a positive trend. Acetic acid can inhibit the proliferation of harmful bacteria and reduce pathogenic bacterial colonization by lowering intestinal pH; propionic acid, as a major energy source for colonic epithelial cells, can promote mucosal cell proliferation and differentiation, regulate mucus layer thickness, and improve intestinal barrier function.

[0066] In summary, the intake of the probiotic combination WLFX-1 helped restore the concentration of short-chain fatty acids in a mouse model of cefotaxime-induced diarrhea, and maintained intestinal homeostasis by regulating the level of intestinal metabolites, providing experimental evidence at the metabolic level for alleviating antibiotic-associated diarrhea.

[0067] 5.7 Effects of probiotics on the gut microbiota of mice with antibiotic-associated diarrhea: As shown in Figure 9, the results of gut microbiota α-diversity analysis indicate that a higher Shannon Value represents richer microbiota diversity, while a higher Simpon Value represents better microbiota evenness. The α-diversity of mouse gut microbiota was analyzed at both the species and genus levels. The results include: Species-level α-diversity: such as... Figure 9 As shown in Figure A, the Control group had the highest Shannon Value, while the Model group showed a significant decrease; the Drug group and the WLFX-1 group both had significantly higher Shannon Values ​​than the Model group, effectively improving microbial diversity. Figure 9As shown in B, the Control group had the highest Simspon Value, while the Model group showed a significant decrease. Both the Drug group and the WLFX-1 group had significantly higher Simspon Values ​​than the Model group (p<0.05). The Drug group recovered to a level where it was not significantly different from the Control group, while the WLFX-1 group remained slightly lower than the Control group. Genus-level α-diversity: (e.g., ...) Figure 9 As shown in Figure C, the Control group had the highest Shannon value, while the Model group showed a significant decrease; the Drug group and the WLFX-1 group both had significantly higher Shannon values ​​than the Model group, indicating a significant improvement in microbial diversity. Figure 9 As shown in Figure D, the Simspon Value was highest in the Control group and significantly lower in the Model group. The Simspon Values ​​of both the Drug group and the WLFX-1 group were significantly higher than those of the Model group (p<0.05). The Drug group recovered to a level with no significant difference from the Control group, while the WLFX-1 group remained slightly lower than the Control group. In summary, cefotaxime significantly reduced the α-diversity (richness and evenness) of the intestinal flora in mice, while loperamide and the probiotic combination WLFX-1 could effectively reverse this trend, significantly improving flora diversity and evenness, laying the foundation for restoring intestinal microecological homeostasis.

[0068] like Figure 10 As shown, principal coordinate analysis (PCoA) based on Bray-Curtis distance further revealed the differences in β-diversity of the gut microbiota. Figure 10 As shown in Figure A, the cumulative explained rate of PCoA1 and PCoA2 reached 64.82% (23.55% + 41.27%). At the species level, the microbial community structure of each group showed significant separation (P < 0.05). The Control group (pink) and the Model group (blue) had the greatest community distribution distance, indicating that cefotaxime gavage significantly remodeled the species-level composition of the mouse gut microbiota. After intervention with loperamide (Drug group, orange) and the probiotic combination WLFX-1 group (green), the community structure of both groups shifted towards the Control group, and the community composition was closer to the normal physiological state. Figure 10 As shown in B, the cumulative explained rate of PCoA1 and PCoA2 reached 36.97% (17.62% + 19.35%). At the genus level, the overlap of community distribution among groups was high, and the differences between groups were not statistically significant (P = 0.68). This suggests that the regulatory effect of intervention on microbial community composition is more prominent at the species level. This corresponds to the result in the α-diversity analysis that intervention can significantly improve the species-level microbial community richness and evenness. This further confirms that the probiotic composition WLFX-1 can restore the cefotaxime-induced intestinal microecological imbalance by regulating the species-level microbial community structure.

[0069] like Figure 11 As shown, an analysis of the species composition of the gut microbiota at the genus level, focusing on the top 15 most abundant core genera, revealed that cefotaxime modeling (Model group) significantly reshaped the microbiota structure: compared to the Control group, the abundance of short-chain fatty acid-producing bacteria *Blautia* was significantly reduced in the Model group, while the abundance of conditionally pathogenic bacteria *Enterobacter* was significantly increased, which is directly related to antibiotic-induced gut microecological imbalance and barrier damage. After intervention with loperamide (Drug group) and the probiotic combination WLFX-1, the microbiota composition was significantly regulated: compared to the Model group, the abundance of beneficial bacteria *Blautia* rebounded in the Drug group, while the abundance of *Enterobacter* decreased; the regulatory effect of the WLFX-1 group was more targeted, not only significantly increasing the abundance of *Blautia* but also effectively reducing the relative abundance of conditionally pathogenic bacteria *Enterobacter*.

[0070] Blautia, a core short-chain fatty acid-producing bacterium, can maintain intestinal barrier integrity and exert anti-inflammatory effects by generating metabolites such as acetic acid and propionic acid. Enterobacter, a common opportunistic pathogen, shows that increased abundance is closely related to intestinal inflammation, decreased barrier function, and increased risk of antibiotic resistance. These results indicate that the probiotic composition WLFX-1 can restore the balance between beneficial and pathogenic bacteria by precisely regulating the abundance of core bacterial species, providing direct evidence at the microecological level for alleviating antibiotic-associated diarrhea.

[0071] Furthermore, species-level metagenomic heatmap analysis, such as Figure 12As shown, the differences in the composition of the gut microbiota at the species level among the various groups of mice are clearly demonstrated, intuitively reflecting the regulatory effects of antibiotic modeling and different intervention methods on the gut microbiota. Compared with the control group, the abundance of various conditionally pathogenic bacteria in the Model group was significantly increased: the heatmap colors of Phytobacter diazotrophicus, Citrobacter amalonaticus, and Enterobacter bugandensis were significantly darker (higher abundance), while the abundance of beneficial bacteria such as Blautia pseudococcoides was significantly decreased. This is completely consistent with the mechanism by which antibiotics disrupt the competitive inhibition of gut microbiota, leading to the excessive proliferation of pathogenic bacteria. After intervention with loperamide (Drug group), the composition of the microbiota was regulated to a certain extent: compared with the Model group, the abundance of Enterobacter bugandensis in the Drug group was significantly reduced, the abundance of Citrobacter amalonaticus returned to a level with no significant difference from the Control group, and the abundance of Phytobacter diazotrophicus also decreased, suggesting that loperamide can inhibit the proliferation of pathogenic bacteria to a certain extent. The intervention of the probiotic composition WLFX-1 showed a more precise effect on the regulation of the microecology: the probiotics administered by gavage (including Bifidobacterium bifidum, Bifidobacterium longum, Lacticaseibacillus paracasel, Lactiplantibacillus plantarum, etc.) marked in the blue box area in the heat map had a significantly higher abundance in the WLFX-1 group than in the Control and Model groups, confirming that these exogenous probiotics successfully colonized and accumulated in the mouse intestine; at the same time, the abundance of conditional pathogens such as Citrobacter amalonaticus and Phytobacter diazotrophicus was significantly reduced, returning to a level similar to that of the Control group, and the abundance of Enterobacter bugandensis also decreased significantly.

[0072] Previous studies have shown that Lactiplantibacillus plantarum and Pediococcus acidilactici can enhance barrier function by adhering to intestinal epithelial cells; Lactobacillus acidophilus can improve intestinal epithelial transport properties; Bifidobacterium bifidum can inhibit the excessive proliferation of pathogenic bacteria; Bifidobacterium longum and Bifidobacterium animalis can restore the balance of the gut microbiota through nutrient competition; Akkermansia muciniphila and Lacticaseibacillus paracasel can prevent antibiotic-associated diarrhea; and Lacticaseibacillus rhamnosus and Limosilactobacillus reuteri can alleviate gastrointestinal symptoms by enhancing the barrier and regulating immunity. Phytobacter diazotrophicus, a Gram-negative opportunistic pathogen, and Citrobacter amalonaticus and Enterobacter bugandensis, drug-resistant opportunistic pathogens, showed increased abundance, which was closely related to the occurrence and development of antibiotic-associated diarrhea. Broad-spectrum antibiotics (such as cefotaxime) disrupt the normal gut flora, reducing competitive inhibition of pathogens, allowing these drug-resistant strains to proliferate excessively under antibiotic pressure, exacerbating the microecological imbalance. In summary, the intervention of the probiotic composition WLFX-1 can effectively restore the cefotaxime-induced gut microbiota imbalance by precisely enriching beneficial bacteria and inhibiting opportunistic pathogens, providing a direct microecological basis for alleviating antibiotic-associated diarrhea.

[0073] Furthermore, differential metabolic pathway analysis of gut microbiota, such as Figure 13The results reveal the regulatory effects of cefotaxime modeling and probiotic intervention on gut microbial metabolic function. Compared with the control group, the model group showed significant remodeling of gut microbiota metabolic pathways: the log2 FoldChange (logarithmic transformation index of the difference in abundance of metabolic pathways between the two groups) of the Bifidobacterium shunting pathway and the thiamine diphosphate reuptake IV (yeast) pathway was significantly negative, indicating that these two pathways were significantly downregulated; while the log2 FoldChange of the L-carnitine degradation I pathway and the D-galacturonic acid degradation I pathway was significantly positive, indicating that these two pathways were significantly upregulated, suggesting that antibiotic modeling leads to dysfunction of gut microbial vitamin metabolism, Bifidobacterium-specific metabolism, and glucose metabolism. After intervention with loperamide (Drug group), some metabolic pathways were restored to a certain extent: compared with the Model group, the log2 FoldChange of Bifidobacterium shunting pathway and thiamine diphosphate reuptake IV (yeast) pathway in the Drug group showed an upward trend, while the log2 FoldChange of L-carnitine degradation I pathway and D-galacturonic acid degradation I pathway showed a downward trend, but the overall regulatory magnitude was limited.

[0074] The intervention of the probiotic composition WLFX-1 group showed a more significant metabolic regulatory effect: compared with the Model group, the log2 FoldChange of the Bifidobacterium shunting pathway and the thiamine diphosphate reuptake IV (yeast) pathway in the WLFX-1 group was significantly positive, indicating that these two key pathways were significantly upregulated, effectively reversing the inhibition of Bifidobacterium metabolism and vitamin B1 metabolism caused by antibiotics; at the same time, the log2 FoldChange of the L-carnitine degradation I pathway and the thiamine diphosphate reuptake IV (yeast) pathway also showed a downward trend, suggesting that the WLFX-1 group can restore the balance of sugar metabolism and carnitine metabolism in intestinal microorganisms.

[0075] The bifidobacterium diversion pathway is a core metabolic pathway specific to bifidobacteria, and its activity is directly related to the abundance and probiotic function of bifidobacteria. The thiamine diphosphate reuptake IV (Yeast) pathway is involved in the reuptake and metabolism of vitamin B1, which is crucial for maintaining intestinal energy metabolism and immune function. Abnormal upregulation of the L-carnitine degradation I pathway and the thiamine diphosphate reuptake IV (Yeast) pathway is closely related to intestinal flora imbalance and metabolic disorders. These results indicate that the probiotic composition WLFX-1 can restore cefotaxime-induced metabolic dysfunction by precisely regulating key metabolic pathways of intestinal microbiota, providing important metabolic evidence for alleviating antibiotic-associated diarrhea.

[0076] In summary, the experiment found that cefotaxime significantly reduced the α-diversity (richness and evenness) of the intestinal flora in mice and reshaped the community structure at the species level (significant differences in β-diversity). It also led to a decrease in the abundance of short-chain fatty acid-producing bacteria *Blautia*, excessive proliferation of opportunistic pathogens (such as *Enterobacter* and *Citrobacter amalonaticus*), and significant inhibition of key pathways such as Bifidobacterium metabolism and vitamin B1 regeneration. Both loperamide and the probiotic combination WLFX-1 effectively reversed these changes: improving flora diversity and evenness, shifting the community structure towards a normal state, precisely enriching beneficial bacteria such as *Blautia*, and inhibiting the proliferation of pathogenic bacteria. The regulatory effect of the WLFX-1 group was more prominent, not only promoting the colonization of exogenous probiotics but also significantly restoring the activity of Bifidobacterium distribution pathways and thiamine metabolic pathways. Ultimately, by repairing intestinal microecological imbalance and metabolic disorders, it provides core evidence at the microecological and metabolic levels for alleviating antibiotic-associated diarrhea.

[0077] 6. Experimental Conclusions: This mouse experiment successfully established a cefotaxime-induced antibiotic-associated diarrhea model in mice, demonstrating that broad-spectrum antibiotics can disrupt intestinal microecological homeostasis, leading to diarrhea symptoms, colonic tissue pathological damage, intestinal barrier dysfunction, immune factor imbalance, and metabolic disorders. Specifically, this manifests as increased diarrhea scores, mucosal epithelial shedding and inflammatory infiltration, downregulation of tight junction proteins and related gene expression, increased pro-inflammatory factors and insufficient anti-inflammatory factors, and decreased short-chain fatty acid levels. Simultaneously, it is accompanied by decreased α-diversity and significant remodeling of β-diversity in the intestinal flora, decreased abundance of short-chain fatty acid-producing bacteria Blautia, excessive proliferation of opportunistic pathogens (such as Enterobacter and Citrobacter amalonaticus), and significant inhibition of key pathways such as Bifidobacterium metabolism and thiamine reuptake.

[0078] Intervention with the probiotic combination WLFX-1 can reverse the aforementioned pathological changes from multiple dimensions: significantly reducing diarrhea scores, repairing colonic tissue damage, upregulating the expression of tight junction proteins and genes such as ZO-1 and MUC2 to enhance intestinal barrier function; regulating the balance of immune factors and reducing intestinal inflammation; increasing the level of short-chain fatty acids (especially acetic acid) and improving intestinal metabolism; and effectively restoring the diversity and community structure of intestinal flora, precisely enriching beneficial bacteria, inhibiting the proliferation of pathogenic bacteria, and reversing the abnormalities of key metabolic pathways. The overall effect is better than loperamide, making up for the limitation of traditional antidiarrheal drugs that only treat the symptoms, and providing a new strategy for the clinical intervention of antibiotic-associated diarrhea that combines microecological repair, barrier protection and immune regulation.

[0079] It should be noted that this application illustrates the technical solution through the above embodiments, but this application is not limited to the above embodiments. Any improvements to this application, equivalent substitution of raw materials, and addition of excipients shall fall within the protection scope and disclosure scope of this application.

[0080] The preferred embodiments of this application have been described in detail above. This application is not limited to the specific details of the above embodiments. Various simple modifications can be made to the technical solution within the scope of the technical concept, and these modifications all fall within the protection scope of this application.

[0081] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid repetition, this application will not describe various combination methods separately.

Claims

1. A method for preparing a probiotic composition, characterized in that, The probiotic composition is prepared by the following method: S1. The following strains were activated in sterile MRS medium at 37°C for 18-24 hours: Lactobacillus plantarum GOLDT-LP1024, Lactobacillus paracasei GOLDT-LC12345, Lactobacillus reuteri GOLDT-LR99, Pediococcus lactis GOLDT-PA0755, and Lactobacillus rhamnosus GOLDT-L818. They were then passaged 2-3 times to induce the strains to enter the logarithmic growth phase and then expanded into larger quantities. S2, at 2℃-8℃, 6000 Centrifuge the bacterial culture at 12000 rpm for 15 minutes. After 30 minutes, the mycelial sludge of each strain was collected; after adding a protective agent to the mycelial sludge of each strain, it was freeze-dried, pulverized and sieved to obtain freeze-dried powder of each strain; S3. Select 0.8-1 parts of *Lactobacillus plantarum* GOLDT-LP1024 bacterial powder, 0.2-0.3 parts of *Lactobacillus paracasei* GOLDT-LC12345 bacterial powder, 1.5-2 parts of *Lactobacillus reuteri* GOLDT-LR99 bacterial powder, 0.1-0.2 parts of *Pediococcus lactis* GOLDT-PA0755 bacterial powder, and 0.3-0.4 parts of *Lactobacillus rhamnosus* GOLDT-L818 bacterial powder, and thoroughly mix them with 2-3 parts of the first lactic acid bacteria compound powder and 3-4 parts of the second lactic acid bacteria compound powder to obtain the probiotic composition, wherein... The first type of lactic acid bacteria compound powder is obtained by mixing Lactobacillus acidophilus NCFM powder, Bifidobacterium animalis subsp. lactis B1-04 powder, Lactobacillus plantarum Lp116 powder, Bifidobacterium bifidum Bb-06 powder and Bifidobacterium longum subsp. infantis Bi-26 powder. The second type of lactic acid bacteria compound powder is obtained by mixing Bifidobacterium longum subsp. longum BORI powder, Lactobacillus acidophilus AD031 powder, Bifidobacterium bifidum BGN4 powder, Bifidobacterium animalis subsp. lactis AD011 powder, and Bifidobacterium longum subsp. infantis IBS007 powder.

2. The preparation method according to claim 1, characterized in that, The probiotic composition has a live bacteria count ≥ 2.5 × 10¹¹ CFU / g.

3. The preparation method according to claim 1, characterized in that, The expanded culture described in step S1 includes: inoculating the bacterial solution into the fermenter at an inoculation rate of 3%-8%, continuing to culture and ferment for 20-40 hours, and then rapidly cooling the bacterial solution to 15℃-25℃ after fermentation. During the fermentation process, the pH value of the bacterial solution in the fermenter is 6.0-7.5, and the culture temperature is 35℃-39℃.

4. The preparation method according to claim 1, characterized in that, The activation described in step S1 includes: adding supplements to the sterile MRS culture medium; the supplements include one or more combinations of nitrogen source supplements, carbon source supplements, and buffers, and the mass-volume percentage of the supplements in the sterile MRS culture medium does not exceed 1%; The nitrogen source supplement includes one or more combinations of yeast extract, beef extract, fish peptone, soybean peptone, and corn steep liquor powder; the carbon source supplement includes one or more combinations of trehalose and glucose; and the buffer includes one or more combinations of triammonium citrate and sodium acetate.

5. The preparation method according to claim 1, characterized in that, The collection of bacterial sludge from each strain in step S2 includes: centrifuging the bacterial solution, discarding the supernatant, adding sterile PBS buffer to the precipitate, gently stirring and washing, and then centrifuging again under the same centrifugation conditions. This washing process is repeated 1-3 times to collect bacterial sludge from each strain. The pH value of the sterile PBS buffer is 7.0-7.

4.

6. The preparation method according to claim 1, characterized in that, In step S2, after adding the protective agent, the mixture is stirred at 20℃-30℃ for 25-40 minutes, and the mass ratio of the bacterial sludge to the protective agent is 1:2-3. The protective agent includes one or more combinations of carbon sources, nitrogen sources, pH adjusters, stabilizers, prebiotics, osmotic pressure regulators, amino acids, and sterile water.

7. The preparation method according to claim 1, characterized in that, The sieve used in step S2 has a mesh size of 60-120; the probiotic composition contains Bifidobacterium animalis subsp. lactis B1-04 powder, Bifidobacterium bifidum Bb-06 powder, Bifidobacterium longum subsp. infantis Bi-26 powder, Bifidobacterium longum subsp. BORI powder, Bifidobacterium bifidum BGN4 powder, Bifidobacterium animalis subsp. lactis AD011 powder, and Bifidobacterium longum subsp. infantis IBS007 powder, which are obtained by pulverizing through a 60-80 mesh sieve. The Lactobacillus acidophilus NCFM powder, Lactobacillus plantarum Lp116 powder, Lactobacillus acidophilus AD031 powder, Lactobacillus plantarum GOLDDUT-LP1024 powder, Lactobacillus paracasei GOLDDUT-LC12345 powder, Lactobacillus reuteri GOLDDUT-LR99 powder, and Lactobacillus rhamnosus GOLDDUT-L818 powder were obtained by pulverizing through a 60-100 mesh sieve. The *Pediococcus lactis* GOLDGUT-PA0755 bacterial powder was obtained by pulverizing it through a 60-120 mesh sieve; wherein... The bacterial powders of each strain in the first type of lactic acid bacteria compound powder and the second type of lactic acid bacteria compound powder are pre-sieved according to the specified sieve mesh size before being mixed and compounded.

8. The preparation method according to claim 1, characterized in that, The thorough mixing described in step S3 yields the probiotic composition, comprising: Select 2-3 parts of the first type of lactic acid bacteria compound powder and 3-4 parts of the second type of lactic acid bacteria compound powder, and mix them; and, Add 1.5-2 parts of *Lactobacillus reuteri* GOLDTUT-LR99 bacterial powder, 0.8-1 parts of *Lactobacillus plantarum* GOLDTUT-LP1024 bacterial powder, and 0.3-0.4 parts of *Lactobacillus rhamnosus* GOLDTUT-L818 bacterial powder, and continue mixing; and, Add 0.2-0.3 parts of Lactobacillus paracasei GOLDGUT-LC12345 bacterial powder and 0.1-0.2 parts of Pediococcus lactis GOLDGUT-PA0755 bacterial powder, and continue mixing for 5 minutes. After 15 minutes, the probiotic composition was obtained.

9. A probiotic preparation, characterized in that, The formulation contains a probiotic composition prepared by any one of the preparation methods of claims 1-8, and pharmaceutically or food-acceptable excipients.

10. The use of the probiotic preparation according to claim 9 in the preparation of a product for relieving antibiotic-associated diarrhea, characterized in that, The products include pharmaceuticals, health products, or food.

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