Composite probiotic composition and application thereof in products for improving intestinal health

Abstract

The invention provides a compound probiotic composition and application thereof in a product for improving intestinal health, the compound probiotic composition is prepared from lactobacillus rhamnosus GG, lactic acid bacteria compound powder, plant lactobacillus GOLDGUT-LP1024 and lactobacillus paracasei GOLDGUT-LC12345, and the lactobacillus rhamnosus GG, the lactic acid bacteria compound powder, the plant lactobacillus GOLDGUT-LC12345 and the lactobacillus paracasei GOLDGUT-LC12345 are added into the compound probiotic composition. The lactic acid bacteria compound powder is prepared by mixing bifidobacterium longum subsp. Longum BORI, lactobacillus acidophilus AD031, bifidobacterium bifidum BGN4, bifidobacterium animalis subsp. Lactis AD011 and bifidobacterium longum subsp. Infantis IBS007; the plant lactobacillus GOLDGUT-LP1024 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the plant lactobacillus GOLDGUT-LP1024 is CGMCC No.28801. The compound probiotic composition provided by the invention can improve the immune function and metabolism function of the intestinal tract so as to improve the health of the intestinal tract and relieve antibiotic-related diarrhea.

Description

Technical Field

[0001] This application belongs to the field of microbial technology, and in particular relates to a compound probiotic composition and its application in products that improve gut health. Background Technology

[0002] The gut is a vital digestive and immune barrier organ, and maintaining a balanced gut microbiota is fundamental to human health. As a core component of the gut microbiota, the stability of its structure and function directly impacts the integrity of the intestinal mucosal barrier, immune regulation, and metabolic homeostasis. Imbalance in the gut microbiota can easily lead to intestinal barrier damage, immune disorders, and metabolic abnormalities, subsequently inducing symptoms such as diarrhea, bloating, and intestinal inflammation. In severe cases, it can also affect systemic immune and metabolic functions, becoming a contributing factor to various gut-related diseases.

[0003] Probiotics, as core active substances for regulating the balance of the gut microbiota, are an important means of improving gut health. Probiotics can maintain gut microbiota homeostasis by regulating gut microbiota structure, enhancing intestinal mucosal barrier function, and modulating intestinal immunity. However, traditional single-strain probiotics have limitations in regulating the gut microbiota, including limited strain adaptability, single function, and difficulty adapting to different gut environments, making it difficult to meet diverse gut health regulation needs.

[0004] As research into gut health deepens, it has become increasingly clear that the diversity and stability of the gut microbiota are crucial for maintaining gut homeostasis. Single probiotic strains are insufficient to address the full range of needs for gut microecological regulation. Furthermore, gut health issues are not limited to the gut itself but are closely related to systemic immune and metabolic systems. Single-function probiotic preparations cannot achieve comprehensive improvement in gut homeostasis. Therefore, the field of gut health requires a complex probiotic composition that effectively and synergistically regulates gut microbiota structure, enhances intestinal barrier function, and modulates intestinal immunity and metabolism. Summary of the Invention

[0005] This application provides a compound probiotic composition and its application in products that improve gut health. Through the synergistic effect of multiple screened probiotic strains, it repairs the intestinal mucosal barrier, regulates the structure and metabolic function of the intestinal flora, and provides an efficient and safe technical solution for gut health management. It can effectively relieve intestinal discomfort, enhance intestinal barrier function, and help rebuild intestinal homeostasis and improve related health problems.

[0006] In a first aspect, embodiments of this application provide a compound probiotic composition and its application in products for improving gut health. The compound probiotic composition includes *Lactobacillus rhamnosus* GG, a compound lactic acid bacteria powder, *Lactobacillus plantarum* GOLDGUT-LP1024, and *Lactobacillus paracasei* GOLDGUT-LC12345. The compound lactic acid bacteria powder is composed of *Bifidobacterium longum* subsp. *longum* BORI, *Lactobacillus acidophilus* AD031, *Bifidobacterium bifidum* BGN4, *Bifidobacterium animalis* subsp. *lacto* AD011, and *Bifidobacterium longum* subsp. *infant* IBS007. The *Lactobacillus plantarum* GOLDGUT-LP1024 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28801, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] In some embodiments, the mass ratio of Lactobacillus rhamnosus GG, lactic acid bacteria compound powder, Lactobacillus plantarum GOLDGUT-LP1024 and Lactobacillus paracasei GOLDGUT-LC12345 is (2-3):(6-7):(10-11):(7-8).

[0008] In some embodiments, the total live bacteria count of the compound probiotic composition is ≥2.5 × 10⁻⁶. 11 CFU / g.

[0009] Secondly, embodiments of this application provide a compound probiotic preparation containing the compound probiotic composition described in the first aspect, as well as pharmaceutically or food-acceptable excipients.

[0010] In some embodiments, the dosage form of the formulation is a solution, powder, capsule, tablet or granule; the excipients include any one or a combination of at least two of the following: protectants, fillers, binders, emulsifiers, solvents, colorants, pH adjusters, antibacterial agents or buffers.

[0011] In some embodiments, the compound probiotic preparation is a powder, prepared by the following method: The compound probiotic preparation is obtained by mixing *Lactobacillus rhamnosus* GG powder, lactic acid bacteria compound powder, *Lactobacillus plantarum* GOLDGUT-LP1024 powder, and *Lactobacillus paracasei* GOLDGUT-LC12345 powder according to the specified mass ratio. The lactic acid bacteria compound powder is composed of *Bifidobacterium longum* subsp. *longum* BORI powder, *Lactobacillus acidophilus* AD031 powder, *Bifidobacterium bifidum* BGN4 powder, *Bifidobacterium animalis* subsp. *lactospirum* AD011 powder, and *Bifidobacterium longum* subsp. *infant* IBS007 powder. The *Lactobacillus plantarum* GOLDDUT-LP1024 bacterial powder is a freeze-dried powder obtained by fermenting and centrifuging *Lactobacillus plantarum* GOLDDUT-LP1024 strain on MRS medium and then freeze-drying the bacterial sludge.

[0012] Thirdly, embodiments of this application provide the application of the compound probiotic composition described in the first aspect in products that improve gut health.

[0013] In some embodiments, improving gut health includes enhancing gut immune function and enhancing gut metabolic function.

[0014] In some embodiments, the enhancement of gut immune function includes at least one of the following: Increase the expression levels of tight junction protein ZO-1 and mucin MUC2 in the colon; It regulates the gene expression levels of colon genes Tjp1, Muc2, Ocln, Cldn1, Gja1, SCN9A, and inflammatory factors Il6 and Il10; Increase the expression levels of colonic anti-inflammatory factors IL-4 and IL-10; It reduces the expression level of the colonic pro-inflammatory factor IL-1β.

[0015] In some embodiments, the enhancement of intestinal metabolic function includes at least one of the following: Lowering serum blood urea nitrogen (BUN) levels; Increase the expression levels of acetic acid and propionic acid in the intestine; Reduce the content of short-chain fatty acids in the intestine; Enhancing the diversity of gut microbiota; Promotes the growth of beneficial bacteria in the gut microbiota and inhibits the growth of pathogenic bacteria in the gut microbiota; Improve key pathways in short-chain fatty acid metabolism.

[0016] In some embodiments, the product includes a medicine, a health supplement, or a food; the product has the effect of relieving intestinal discomfort symptoms by improving gut health, including antibiotic-associated diarrhea.

[0017] This application has the following advantages over the prior art: (1) Through the precise ratio of specific strains, the core functions of improving intestinal immune function and intestinal metabolic function can be achieved simultaneously. Specifically, it can repair the colonic tissue barrier, regulate the balance of intestinal inflammation, improve the level of intestinal metabolism, and optimize the structure of intestinal flora. It can effectively relieve intestinal discomfort symptoms such as antibiotic-associated diarrhea and comprehensively improve intestinal health. (2) The strain ratio and live bacteria count of the compound probiotic composition are clearly defined, the formulation and excipient selection are flexible, and it can be adapted to various product forms such as pharmaceuticals, health products, and food. The preparation process is clear and controllable, and the technical solution is stable and reliable. It can provide a new technical solution for intestinal health conditioning that is precise, efficient and easy to industrialize. Attached Figure Description

[0018] 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.

[0019] Figure 1 Statistical chart of diarrhea scores of mice in different treatment groups provided in the embodiments of this application (normal control group (Control group); model control group (Model group); drug group (Drug group); Lactobacillus plantarum GOLDGUT-LP1024 group; compound probiotic group (WLFX-2 group)). Figure 2 A statistical graph showing the results of food intake and body weight of mice in different treatment groups provided in the embodiments of this application; Figure 3 Statistical graph of serum biochemical indicators of mice in different treatment groups provided in the embodiments of this application; Figure 4 Schematic diagram of H&E staining of colon tissue from mice in different treatment groups provided in the embodiments of this application; Figure 5 Immunofluorescence staining images of intestinal mucosal barrier-related proteins in mice from different treatment groups provided in the embodiments of this application; Figure 6 A statistical graph showing the expression levels of intestinal mucosal barrier-related proteins in mice from different treatment groups as provided in the embodiments of this application; Figure 7 A statistical graph showing the colon gene expression levels of mice in different treatment groups provided in the embodiments of this application; Figure 8 A statistical chart showing the results of intestinal short-chain fatty acid content in mice under different treatment groups provided in the embodiments of this application; Figure 9 A statistical chart showing the results of intestinal microbial diversity index in mice under different treatment groups provided in the embodiments of this application; Figure 10 A schematic diagram of the gut microbiota structure of mice in different treatment groups provided in the embodiments of this application; Figure 11Stacked bar chart of relative abundance of gut microbiota in mice from different treatment groups provided in the embodiments of this application; Figure 12 A schematic diagram showing the fecal metagenomic analysis results of mice in different treatment groups 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.

[0020] 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

[0021] 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.

[0022] The culture media involved in the following examples are as follows: MRS solid medium (g / L): peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, glucose 20g / L, Tween 80 1mL / L, K2HPO4 7H2O 2g / L, Triammonium Citrate 2g / L, MgSO4 7H2O 0.1g / L, MnSO4 0.05 g / L 4H2O, 15 g / L agar powder.

[0023] MRS broth medium (g / L): peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, glucose 20g / L, Tween 80 1mL / L, K2HPO4 7H2O 2g / L, Triammonium Citrate 2g / L, MgSO4 7H2O 0.1g / L, MnSO4 0.05 g / L of 4H2O; add the above ingredients to 1000 ml of distilled water, heat to dissolve, adjust the pH to 6.2 ± 0.2, dispense into containers, and autoclave at 121°C for 15 min for later use.

[0024] This application provides a compound probiotic composition comprising Lactobacillus rhamnosus GG, a compound lactic acid bacteria powder, Lactobacillus plantarum GOLDGUT-LP1024, and Lactobacillus paracasei GOLDGUT-LC12345, wherein the compound lactic acid bacteria powder is a mixture of Bifidobacterium longum subsp. BORI, Lactobacillus acidophilus AD031, Bifidobacterium bifidum BGN4, Bifidobacterium animalis subsp. lactis AD011, and Bifidobacterium longum subsp. infantis IBS007. Among them, *Lactobacillus plantarum* GOLDGUT-LP1024 is a preserved strain, classified and named *Lactobacillus plantarum*. Lactiplantibacillus plantarum It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28801, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0025] Example 1: Screening, purification, identification, and preservation of *Lactobacillus plantarum* GOLDGUT-LP1024: 1) Strain screening: Take 25g of pickled kimchi and place it in a homogenizing bag containing 225mL of physiological saline. Use a sterile tapping homogenizer to homogenize for 2 minutes to prepare a 1:10 sample homogenate. Take 1mL of the sample homogenate and add it to 9mL of physiological saline. Vortex to prepare a 1:10 homogenate. - ¹Diluent; perform serial dilutions, taking an appropriate dilution gradient (10). -4 -10 -9 Dilute 50-200 μL of the solution and spread it evenly on MRS solid medium. Incubate at 37°C for 48-72 h.

[0026] 2) Strain purification: Use an inoculation loop to pick up a single colony and streak it on MRS solid medium. Incubate at 37°C for 48-72 hours. Repeat this step 3-5 times.

[0027] 3) Strain identification: Single colonies of the isolated and purified bacteria were inoculated into MRS broth medium and cultured at 37°C for 16-18 hours. The bacterial sludge was collected by centrifugation and subjected to 16S rDNA sequencing. 16S rDNA sequencing included: obtaining the strain's genomic DNA using a microbial genomic DNA extraction kit or the classic extraction method (CTAB method); performing standardized PCR amplification using universal primers for bacterial 16S rDNA; and sequencing using a next-generation sequencing platform. The sequencing results were compared with mainstream microbial gene databases such as NCBI GenBank, SILVA, and EzBioCloud to identify *Lactobacillus plantarum*. The strain was identified as *Lactobacillus plantarum* and named *Lactobacillus plantarum* GOLDGUT-LP1024.

[0028] 4) Strains preservation: Select pure cultures of Lactobacillus plantarum GOLDGUT-LP1024 single colonies and preserve them at -80℃ for a long time.

[0029] Example 2: Preparation of *Lactobacillus plantarum* GOLDGUT-LP1024 bacterial powder: After thawing the preserved *Lactobacillus plantarum* GOLDGUT-LP1024, it was inoculated into 20 mL of MRS broth and cultured at 37°C for 18-24 h. Culture was stopped when the OD600 (optical density at 600 nm, a measure of cell concentration in microbial (bacteria, fungi) culture) reached 8-11, yielding the primary seed culture. The primary seed culture was then inoculated into 250 mL of MRS broth and cultured at 37°C for 6-10 h. Culture was stopped when the OD600 reached 4-6, yielding the secondary seed culture. The secondary seed culture was then inoculated into fresh MRS broth and cultured at 37°C for 6-10 h to obtain the fermentation broth. The fermentation broth was centrifuged, the bacterial sludge was collected, a cryoprotectant was added, the mixture was thoroughly mixed, and freeze-dried to obtain a viable count of 3 × 10⁻⁶ cells / mL. 11 The above CFU / g of bacterial powder.

[0030] Example 3: Preparation of compound probiotic preparation: 1) Preparation of the compound probiotic composition: Lactobacillus rhamnosus GG powder, lactic acid bacteria compound powder, Lactobacillus plantarum GOLDGUT-LP1024 powder and Lactobacillus paracasei GOLDGUT-LC12345 powder are mixed according to the mass ratio to obtain the compound probiotic composition. The lactic acid bacteria compound powder is composed of 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. The Lactobacillus plantarum GOLDGUT-LP1024 powder is a freeze-dried powder obtained by fermenting and centrifuging Lactobacillus plantarum GOLDGUT-LP1024 strain on MRS medium and then freeze-drying the bacterial sludge.

[0031] In some embodiments, the mass ratio of Lactobacillus rhamnosus GG, lactic acid bacteria compound powder, Lactobacillus plantarum GOLDGUT-LP1024 and Lactobacillus paracasei GOLDGUT-LC12345 is (2-3):(6-7):(10-11):(7-8).

[0032] In some embodiments, the total live bacteria count of the compound probiotic composition is ≥2.5 × 10⁻⁶. 11 CFU / g.

[0033] 2) Added excipients: The compound probiotic preparation contains a compound probiotic composition, as well as pharmaceutically or food-acceptable excipients. The dosage form of the preparation is a solution, powder, capsule, tablet or granule; the excipients include any one or a combination of at least two of the following: protectants, fillers, binders, emulsifiers, solvents, colorants, pH adjusters, antibacterial agents or buffers.

[0034] The present invention also provides the use of the compound probiotic composition as described above in products for improving gut health.

[0035] In some embodiments, improving gut health includes enhancing gut immune function and enhancing gut metabolic function.

[0036] In some embodiments, the enhancement of intestinal immune function includes at least one of the following: increasing the expression levels of tight junction protein ZO-1 and mucin MUC2 in the colon; regulating the gene expression levels of colon genes Tjp1, Muc2, Ocln, Cldn1, Gja1, SCN9A, and inflammatory factors Il6 and Il10; increasing the expression levels of colonic anti-inflammatory factors IL-4 and IL-10; and decreasing the expression level of colonic pro-inflammatory factor IL-1β.

[0037] In some embodiments, the enhancement of intestinal metabolic function includes at least one of the following: reducing the expression level of blood urea nitrogen (BUN) in serum; increasing the expression levels of acetic acid and propionic acid in the intestine; reducing the content of short-chain fatty acids in the intestine; increasing the diversity of bacterial strains in the intestine; promoting the growth of probiotics and inhibiting the growth of pathogenic bacteria in the intestine; and enhancing key pathways of short-chain fatty acid metabolism.

[0038] In some embodiments, the product includes a medicine, a health supplement, or a food; the product has the effect of relieving intestinal discomfort symptoms by improving gut health, including antibiotic-associated diarrhea.

[0039] Example 4: Determination of the continuous gastrointestinal fluid tolerance of Lactobacillus plantarum GOLDGUT-LP1024: Currently, most strains have only been tested for survival rates in gastric and intestinal fluids alone, without simulating continuous exposure to these fluids during actual digestion. Continuous gastrointestinal fluid tolerance experiments can assess the survival rate of probiotics after continuous exposure to simulated gastric and intestinal fluids, which is crucial for determining whether probiotics can survive in the human digestive tract and exert their beneficial effects.

[0040] The gastrointestinal fluid tolerance of the strain was evaluated using a simulated continuous gastrointestinal digestion method. (1) Test sample: Lactobacillus plantarum GOLDGUT-LP1024 lyophilized powder.

[0041] (2) Preparation of simulated gastrointestinal fluid: Simulated gastric fluid: 0.35% (w / v) pepsin was added to 0.1 mol / L sodium dihydrogen phosphate buffer as solvent, and the pH was adjusted to 3.0±0.1 with 1 mol / L hydrochloric acid. It was then filtered through a 0.22 μm sterile filter membrane for sterilization. Simulated intestinal fluid: 0.3% (w / v) ox bile salt and 0.1% (w / v) pancreatin were added to 0.1 mol / L disodium hydrogen phosphate buffer as solvent, and the pH was adjusted to 8.0±0.1 with 1 mol / L sodium hydroxide. It was then filtered through a 0.22 μm sterile filter membrane for sterilization.

[0042] (3) Other reagents: sterile physiological saline, MRS solid culture medium, sterile centrifuge tubes, pipettes, etc.

[0043] (4) Experimental procedure: ① Simulate continuous digestion by gastrointestinal juices: After digesting the lyophilized bacterial strain powder in simulated gastric juice (0.35% pepsin, pH 3.0) for 1.5 h, the digested gastric juice sample was transferred to simulated intestinal juice (0.3% bile salts, 0.1% pancreatic enzyme, pH 8.0) for further digestion for 3 h; intestinal juice samples were obtained after digestion.

[0044] ② Plate counting: The number of viable bacteria in gastric and intestinal fluid samples was determined using the plate count method, and the continuous survival rate (%) of gastric and intestinal fluids was calculated. The calculation formula is: continuous tolerance survival rate of gastric and intestinal fluids = C1' / W1 × 100%, where W1 is the number of viable bacteria in gastric fluid after 0 h of culture, and C1' is the number of viable bacteria in intestinal fluid after 3 h of culture.

[0045] ③Experimental conclusions: After continuous digestion with gastrointestinal fluid, the survival rate of *Lactobacillus plantarum* GOLDGUT-LP1024 was 78%, indicating that *Lactobacillus plantarum* GOLDGUT-LP1024 has good gastrointestinal tolerance.

[0046] Example 5: The effect of the compound probiotic composition on improving diarrhea symptoms and intestinal health in mice with antibiotic-associated diarrhea: (1) Experimental animals: Mice, model C57BL / 6J, male, 8 weeks old, weighing (20±2) g, were purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd. A normal control group (Control group); a model control group (Model group); a drug group (Drug group); a *Lactobacillus plantarum* GOLDGUT-LP1024 treatment group (LP1024 group); and a compound probiotic treatment group (WLFX-2 group) were established. All experimental protocols and procedures in this study were approved by the Ethics Committee of Hainan University (No.: HNUAUCC-2023-00179).

[0047] (2) Chemical reagents: Cefotaxime acid, purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Loperamide hydrochloride, purchased from Xi'an Janssen Pharmaceutical Co., Ltd.; 4% paraformaldehyde, purchased from Wuhan Sewell Biotechnology Co., Ltd.; Inflammatory cytokine detection kit, purchased from Shanghai Xinyu Biotechnology Co., Ltd.; Short chain fatty acid standard, purchased from Stanford Chemicals, USA.

[0048] (3) Housing environment: All mice were housed in an SPF-grade animal room with a room temperature of 23±1℃ and a relative humidity of 52%–60%. They were given an acclimatization diet for 2 weeks (12h / 12h light and dark alternation, free access to food and water).

[0049] (4) Animal grouping: After 2 weeks of acclimatization, 40 mice were randomly divided into 5 groups of 8 mice each. The details of each group are as follows: Control group: 0.85% saline was administered by gavage twice a day for 7 consecutive days to establish the model; 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 to establish the model; after successful model establishment, 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 to establish the model; after successful model establishment, loperamide hydrochloride 1 mg / kg was administered by gavage once a day in the morning for 7 consecutive days. LP1024 group: Cefotaxime 500 mg / kg was administered by gavage twice daily for 7 consecutive days to establish the model; after successful model establishment, 200 μL of *Lactobacillus plantarum* GOLDGUT-LP1024 bacterial suspension (1×10⁻⁶) was administered by gavage every morning. 9 CFU / each); WLFX-2 group: Cefotaxime 500 mg / kg was administered by gavage twice daily for 7 consecutive days to establish the probiotic model; after successful model establishment, 200 μL of the probiotic solution (1×10⁻⁶) of the compound probiotic composition described in Example 3 was administered by gavage every morning. 9 CFU / each).

[0050] (5) Indicator detection and analysis: 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 3500 rpm for 45 minutes at 4℃. 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.

[0051] ① Measurement of body weight, food intake, water intake, and diarrhea score: Starting from the first day of modeling, the body weight of the mice was recorded daily. During the modeling period, the food intake and water intake of the mice were recorded every other day. During the treatment period, the food intake and water intake of the mice were recorded daily. Diarrhea scores were recorded and photographs were taken daily. The mouse diarrhea score table is shown below: Mouse Diarrhea Scoring Scale

[0052] like Figure 1 As shown, feces are the most obvious characteristic for judging the severity of diarrhea. Three days after cefotaxime administration by gavage, all groups except the control group began to show varying degrees of diarrhea, accompanied by symptoms such as lethargy, reduced activity, inactivity, increased water intake, and increased defecation frequency, which worsened with prolonged administration. In severe cases, anal redness and swelling and fecal adhesion were observed. After modeling, all model animals developed severe diarrhea, with loose and soft feces, and the diarrhea status score reached its peak, confirming the successful establishment of the diarrhea mouse model. Figure 1 As shown, starting from day 8, the Model group received intragastric administration of normal saline to simulate a natural recovery state. On day 13 of administration, the diarrhea scores of the Lactobacillus plantarum GOLDGUT-LP1024 group, the WLFX-2 group (compound probiotic group), and the Drug group were all significantly lower than those of the Model group (p<0.05). Among them, the diarrhea score of the WLFX-2 group (compound probiotic group) decreased more significantly, and was closer to the level of the Control group, suggesting that both the compound probiotic composition and Lactobacillus plantarum GOLDGUT-LP1024 alone can relieve diarrhea symptoms, but the intervention effect of the compound probiotic composition is better.

[0053] like Figure 2As shown in Figure A, regarding food intake, on day 3, the Control group mice showed significantly higher intake than the cefotaxime-induced Model group and all intervention groups. By day 13, food intake in all groups showed an increasing trend, with the Drug group and WLFX-2 group (compound probiotic group) showing significantly higher food intake than the Control group (p<0.05). Furthermore, the WLFX-2 group showed a better recovery in food intake than the Bacillus plantarum GOLDGUT-LP1024 group, further demonstrating the advantages of the compound probiotic composition in improving the feeding status of mice. Figure 2 As shown in B, there were no significant differences in body weight among the groups of mice throughout the study.

[0054] ② Serum immune cytokines and blood urea nitrogen (BUN) assay: 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. Serum was then collected. The levels of pro-inflammatory cytokines IL-1β, anti-inflammatory cytokines IL-10 and IL-4, and blood urea nitrogen (BUN) in the serum were detected using an ELISA kit.

[0055] The expression of immune cytokines can regulate inflammatory responses. They are divided into pro-inflammatory cytokines and anti-inflammatory cytokines, which mutually restrict each other and are in a dynamic equilibrium. To investigate the effect of probiotics on improving the immune balance of 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 and analyzed.

[0056] The results are as follows Figure 3 A and Figure 3 As shown in Figure B, the serum levels of anti-inflammatory cytokines IL-4 and IL-10 in the Model group mice were significantly lower than those in the Control and Drug groups (p<0.05). After intervention, the IL-4 (pg / mL) level in the WLFX-2 group (compound probiotic group) was significantly higher than that in the Model group (p<0.05), while the Lactobacillus plantarum GOLDGUT-LP1024 group only showed an increasing trend (p>0.05). At the IL-10 (pg / mL) level, both the Lactobacillus plantarum GOLDGUT-LP1024 group and the WLFX-2 group were able to increase the expression level of IL-10 (p>0.05), indicating that Lactobacillus plantarum GOLDGUT-LP1024 and the compound probiotic composition prepared as described above have certain anti-inflammatory capabilities, with the compound probiotic composition showing a more significant effect in enhancing the expression of anti-inflammatory factors. Figure 3As shown in Figure C, compared to the Control group, the serum IL-1β (ng / L) level in the Model group mice was significantly increased, while the level in the Lactobacillus plantarum GOLDGUT-LP1024 group was decreased compared to the Model group (p > 0.05). This indicates that Lactobacillus plantarum GOLDGUT-LP1024 and the compound probiotic composition prepared as described above have certain anti-inflammatory capabilities, thereby improving the immune system disorder induced by cefotaxime.

[0057] Blood urea nitrogen (BUN) is a metabolic waste product of the body. Normally, it is filtered and excreted by the kidneys. High serum BUN levels indicate excessive kidney burden and impaired kidney function. Quantitative analysis of serum BUN levels in mice revealed that... Figure 3 As shown in D, the serum BUN (mg / mL) level in the Model group mice was significantly higher than that in the Control group. The Drug group and the Lactobacillus plantarum GOLDGUT-LP1024 group showed a significant decrease compared to the Model group (p<0.05), while the WLFX-2 group showed a decreasing trend compared to the Model group (p>0.05). This indicates that loperamide, Lactobacillus plantarum GOLDGUT-LP1024, and the compound probiotic composition prepared as described above reduced the renal burden after cefotaxime administration.

[0058] ③ Histopathological observation of colon tissue: Distal colon tissue from mice was taken, washed with PBS, fixed in 4% (w / v) paraformaldehyde, and after gradient dehydration, the sample was 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 tissue and pathological colon tissue.

[0059] H&E staining results of mouse colon as follows Figure 4 As shown, the colon of mice in the Control group exhibited normal histological characteristics. The mucosal epithelium remained intact, the intestinal glands were abundant and tightly arranged, the crypt structures were clearly visible, and the structure of each layer of the mucosa was intact. Compared with the Control group, the colonic crypts of mice in the Model group were disordered, some mucosal epithelial cells were sloughed off, goblet cells between epithelial cells were significantly reduced (green arrows in the figure), and a large number of inflammatory cells infiltrated (black arrows in the figure). After treatment with loperamide, the Lactobacillus plantarum GOLDT-LP1024 group, and the above-prepared compound probiotic composition via gavage, the colonic pathological features of fewer goblet cells and disordered crypt structure were significantly improved. Furthermore, no large number of inflammatory cells infiltrated were observed in the Drug group, the Lactobacillus plantarum GOLDT-LP1024 group, and the WLFX-2 group. Among them, the WLFX-2 group had intact epithelial structure and clearly visible crypts, showing the best treatment effect. According to histological observation, the above-mentioned compound probiotic treatment containing Lactobacillus plantarum GOLDT-LP1024 can reduce intestinal inflammation.

[0060] ④ Immunofluorescence detection of intestinal mucosal barrier-related proteins: Colonic tissue was labeled with MUC-2 and ZO-1 antibodies, and the antibodies in the two tissue samples were characterized using an EclipModCl-L ​​fluorescence microscope. Areal density is equal to the ratio of cumulative optical density value to the area of ​​tissue pixels.

[0061] Tight junction proteins are located between intestinal epithelial cells, sealing the intercellular spaces and preventing the leakage of small molecules. They play a crucial role in the formation and regulation of the epithelial barrier, modulating the selective permeation of pathogens and toxins through the mucosa. Figure 5 As shown, to further evaluate the colonic mucosal barrier function in mice, the expression of colonic tight junction protein ZO-1 and mucin MUC2 was analyzed by immunofluorescence. Compared with the control group, the blue fluorescence signal of ZO-1 in the colonic tissue of the model group mice was significantly weakened, and the red fluorescence of MUC2 was also significantly reduced, indicating that the mucosal barrier was damaged. After intervention, the fluorescence signals of the drug group, the Lactobacillus plantarum GOLDGUT-LP1024 group, and the WLFX-2 group (compound probiotic group) were all enhanced compared with the model group. Among them, the fluorescence intensity of the WLFX-2 group was closest to that of the control group, which directly reflects its better mucosal barrier repair effect.

[0062] Further quantitative analysis of the areal density results of ZO-1 and MUC2, such as Figure 6 A and Figure 6 As shown in B, the areal density of ZO-1 and MUC2 in the Model group was significantly lower than that in the Control group (p<0.05). After intervention, the areal density of all three groups was significantly higher than that of the Model group (p<0.05). Among them, the areal density of ZO-1 and MUC2 in the WLFX-2 group was not significantly different from that in the Control group, while the areal density of Lactobacillus plantarum GOLDGUT-LP1024 group was slightly lower than that in the WLFX-2 group, although it was improved. This further confirms that the compound probiotic composition is more effective than the single strain in restoring the expression of mucosal barrier proteins.

[0063] The above results indicate that loperamide, Lactobacillus plantarum GOLDGUT-LP1024, and the WLFX-2 compound probiotic composition can all upregulate the expression of ZO-1 and MUC2 to enhance the intestinal mucosal barrier function. Among them, the WLFX-2 compound probiotic composition showed a more significant repair effect through synergistic action, and its protective effect on the mucosal barrier was better than that of Lactobacillus plantarum GOLDGUT-LP1024 alone.

[0064] ⑤ RNA extraction and RT-qPCR determination of gene expression in mouse colon: 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 followed by incubation at 60°C for 30 seconds. GAPDH was used as a reference gene. Relative gene expression levels were analyzed using the 2-ΔΔCT method after qRT-PCR. Each sample was measured four times, and the average value was taken.

[0065] RT-qPCR was performed on colon tissue to assess the effects of antibiotics on tight junctions. The efficacy of different probiotic groups in treating antibiotic-induced tight junction disruption was evaluated by measuring the expression levels of genes such as Tjp1, Muc2, and Ocln in different groups. The gene expression levels of inflammatory factors such as Il6 and Il10 were measured to assess whether probiotics had a relieving effect on antibiotic-induced inflammation.

[0066] like Figure 7 As shown, compared with the Control group, the relative expression level of Tjp1 mRNA in the colon of Model group mice (e.g., Figure 7 The relative expression levels of Muc2 mRNA (e.g., A) Figure 7 The relative expression levels of B and Ocln mRNA (e.g., B) Figure 7 The relative expression levels of C and Cldn1 mRNA (e.g., C) Figure 7 The relative expression levels of D and Gja1 mRNA (e.g., D) and Gja1 mRNA (e.g., D) Figure 7The expression of E in both groups was significantly downregulated (p<0.05), suggesting that antibiotic-induced diarrhea leads to impaired expression of intestinal barrier-related genes. After intervention: In terms of relative expression levels of Tjp1 mRNA and Ocln mRNA, the WLFX-2 group (compound probiotic group) was significantly higher than the Model group (p<0.05), while the Lactobacillus plantarum GOLDGUT-LP1024 group only showed an increasing trend; in terms of relative expression level of Gja1 mRNA, the WLFX-2 group was significantly higher than the Model group (p<0.01), and the increase was significantly better than that of the Lactobacillus plantarum GOLDGUT-LP1024 group; in terms of relative expression level of Cldn1 mRNA, both groups were significantly higher than the Model group, but the expression level of the WLFX-2 group was closer to that of the Control group. The above results indicate that the compound probiotic composition has a more prominent protective effect in maintaining intestinal barrier integrity, and its synergistic regulation of multiple target barrier genes is better than that of Lactobacillus plantarum GOLDGUT-LP1024 alone.

[0067] The Nav1.7 sodium channel, encoded by the SCN9A gene, plays a crucial role in pain perception, such as... Figure 7 As shown in F, the relative expression level of SCN9A mRNA in the Model group was significantly higher than that in the Control group (p<0.05). After intervention, the SCN9A expression in the Drug group, Lactobacillus plantarum GOLDGUT-LP1024 group and WLFX-2 group was significantly lower than that in the Model group. Among them, the downregulation in the WLFX-2 group was the largest, which was closer to the level of the Control group, suggesting that the compound probiotic composition was more effective in inhibiting pain signal transmission and relieving abdominal pain symptoms.

[0068] Regarding the expression of inflammatory factor genes, such as Figure 7 G in Figure 7 H in Figure 7 I and Figure 7As shown in Figure J, the relative expression levels of pro-inflammatory factors Il6 mRNA, Il1b mRNA, and Tnf mRNA in the Model group were significantly higher than those in the Control group (p<0.05), while the relative expression level of anti-inflammatory factor Il10 mRNA was significantly lower than that in the Control group (p<0.05). After intervention: Regarding pro-inflammatory factors, Il6 and Il1b were significantly downregulated in all three groups, with the WLFX-2 group showing a highly significant downregulation of Il1b mRNA (p<0.01), which was significantly better than the Lactobacillus plantarum GOLDGUT-LP1024 group; Regarding anti-inflammatory factors, the relative expression level of Il10 mRNA was significantly higher in the WLFX-2 group than in the Model group (p<0.05), while it only showed an increasing trend in the Lactobacillus plantarum GOLDGUT-LP1024 group; The relative expression level of Tnf mRNA was significantly lower in the Lactobacillus plantarum GOLDGUT-LP1024 group than in the Model group.

[0069] The above results indicate that both Lactobacillus plantarum GOLDGUT-LP1024 and the compound probiotic composition can improve antibiotic-induced intestinal inflammation. However, the WLFX-2 compound probiotic composition exhibits a more significant intestinal protective effect by synergistically regulating the expression of inflammation and barrier genes, and its effect is superior to that of Lactobacillus plantarum GOLDGUT-LP1024 alone.

[0070] ⑥ Determination of SCFAs content in mouse intestines: Weigh 40 mg of lyophilized cecal contents, add 500 μL of saturated NaCl solution, let stand for half an hour, add 20 μL of sulfuric acid and shake. Then 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, take out 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.

[0071] Short-chain fatty acids, including acetic acid and propionic acid, are major metabolic products of gut microbiota and play a crucial role in regulating important biological processes, including host metabolism, intestinal function, and immunity. The short-chain fatty acid content of mouse cecal contents is shown in the figure below. Figure 8 A and Figure 8As shown in Figure B, the levels of acetic acid and propionic acid in the Model group were significantly lower than those in the Control group, decreasing to 1.04 μmol / g and 1.79 μmol / g, respectively. After treatment with loperamide hydrochloride and probiotics, the WLFX-2 group had significantly higher acetic acid levels than the Model group, reaching 1.27 μmol / g, and the WLFX-2 group had higher propionic acid levels than the Model group, reaching 2.04 μmol / g.

[0072] Antibiotics reduce short-chain fatty acids (SCFAs), which are primarily metabolic products of microorganisms. Treatment with probiotics increased the diversity of gut microbiota in diarrheal mice, promoting the production of acetic acid and propionic acid. Among SCFAs, the growth of harmful bacteria is inhibited under acidic conditions, and acetic acid, by lowering intestinal pH, reduces the number of harmful bacteria. Propionic acid is absorbed in the liver and inhibits cholesterol synthesis, strongly reducing blood cholesterol and lipid levels by regulating the expression of lipid synthase-related genes. These results indicate that the aforementioned probiotic combination helps restore the reduced SCFA concentration in a cefotaxime-induced diarrhea mouse model and may contribute to maintaining intestinal homeostasis.

[0073] ⑦ Measurement of mouse intestinal microbial diversity indices (Shannon index, Simspon index): The Shannon value is a core indicator for assessing microbial community diversity. It reflects both the richness (number of strains / species) and evenness (balance of abundance among strains) of the community. A higher value indicates a greater variety of strains and a more balanced abundance distribution, resulting in higher overall diversity. The Simspon value, on the other hand, focuses on reflecting the dominance of the community. It is commonly interpreted using a converted 1-D value. A higher value indicates a greater probability that two randomly selected individuals belong to different strains, suggesting no clearly dominant strain and better diversity and stability. Combining both values ​​provides a comprehensive assessment of the diversity characteristics of the community.

[0074] like Figure 9 A and Figure 9As shown in Figure B, at the species level, the Shannon Value of the Model group mice was significantly lower than that of the Control group, indicating a significant decrease in the richness and evenness of the gut microbiota after antibiotic treatment. After intervention with probiotics or loperamide hydrochloride, the Shannon Values ​​of the Drug group, the *Lactobacillus plantarum* GOLDGUT-LP1024 group, and the WLFX-2 group were significantly higher than those of the Model group, and there was no significant difference compared with the Control group, indicating that the intervention could effectively restore the richness and evenness of the microbiota. Meanwhile, the Simspon Value of the Model group was significantly lower than that of the Control group, reflecting that antibiotic treatment led to an increase in the dominance and a decrease in the stability of the microbiota. After intervention, the Simspon Values ​​of all groups were significantly higher than those of the Model group, suggesting a decrease in the dominance and an improvement in the diversity and stability of the microbiota.

[0075] like Figure 9 C and Figure 9 As shown in D, at the genus level, the results showed a consistent trend with those at the species level: the Shannon Value and Simspon Value of the Model group were significantly lower than those of the Control group. However, after intervention with Drug, Lactobacillus plantarum GOLDGUT-LP1024, or WLFX-2, the indices of both groups were significantly higher than those of the Model group, and there was no significant difference between the intervention group and the Control group.

[0076] The above results indicate that WLFX-2 (a complex probiotic composition) can effectively increase the Shannon Value and Simspon Value of the gut microbiota in mice with antibiotic-associated diarrhea, significantly improve the richness, evenness, and stability of the microbiota, thereby helping to restore the imbalanced gut microbiota and alleviate the symptoms of antibiotic-associated diarrhea.

[0077] ⑧ Measurement of mouse gut microbiota structure: such as Figure 10 The two PCoA diagrams shown are on the left. Figure 10 A in the middle and the right side Figure 10Figure B shows the differences in microbial community structure at the species and genus levels, respectively. The cumulative explained rates of PCoA1 and PCoA2 reached 46.43% (19.75% + 26.68%) and 48.16% (20.39% + 27.77%), respectively, which can effectively reflect the main variations in community composition. In terms of distribution characteristics, the control group is clustered in the left area in both figures, showing a clear spatial separation from the model group, indicating that the microbial community has changed significantly after the model was constructed. The intervention groups (Drug group, Lactobacillus plantarum GOLDGUT-LP1024 group, WLFX-2 group) are mainly distributed in the lower to middle areas of the figures, with some overlap between them, but they are clearly distinguished from the model group (upper right area), suggesting that different intervention methods can regulate the microbial community structure at the species and genus levels, making it deviate from the model group state and move closer to the control group. Furthermore, the significance markers P<0.05 in the figure further validated the statistical differences in microbial community composition among the six groups, and these differences remained stable at both the species and genus levels, indicating that the intervention measures had a hierarchical consistency in their regulatory effect on the microbial community.

[0078] ⑨ Mouse gut microbial abundance determination: In microbial community analysis, relative abundance refers to the proportion (usually expressed as a percentage) of a certain group (such as bacteria at the genus level) in the entire community, directly reflecting the dominance and relative importance of that group in the microbial community. Changes in the relative abundance of different groups are the core basis for determining whether intervention measures affect the microbial community structure.

[0079] like Figure 11As shown, the colored blocks on the right side of the figure represent the top 15 bacterial taxa in terms of relative abundance at the genus level, as well as "Others" (the sum of all low-abundance taxa not in the top 15), specifically including: Others: other low-abundance bacterial taxa, Lachnospiraceae_unclassified: unclassified Lachnospiraceae, Lacticaseibacillus: Lactobacillus, Escherichia: Escherichia (e.g., Escherichia coli), Akkermansia: Akkermania, Clostridium: Clostridium (e.g., Clostridium difficile), Bi fidobacterium: Bifidobacterium, Enterocloster: Clostridium, Erysipelatoclostridium: Erysipelothrix, Duncanella: Duncanella, Hungatella: Hungatella, Ligilactobacillus: Ligilactobacillus, Muribaculaceae_unclassified: Unclassified Myxobacteriaceae, Enterobacter: Enterobacter, Blautia: Blautia, Phytobacter: Phytobacter.

[0080] At the genus level, Figure 11 The distribution characteristics of the top 15 bacterial genera in relative abundance in the gut microbiota of mice in each group were presented. Compared with the Model group, the Drug group significantly increased the relative abundance of Blautia (a beneficial bacterium that produces short-chain fatty acids) while decreasing the abundance of Enterocloster. The Lactobacillus plantarum GOLDGUT-LP1024 group also significantly increased the abundance of Blautia and decreased the abundance of Enterobacter. The WLFX-2 group also showed a trend of increasing Blautia abundance while decreasing the abundance of Clostridioides (such as Clostridium difficile, which is associated with intestinal inflammation). These results indicate that although the specific bacterial targets regulated by different interventions vary slightly, they all improve the gut microbiota structure by increasing the abundance of beneficial bacteria and reducing the abundance of potentially pathogenic / harmful bacteria, thereby helping to alleviate antibiotic-associated diarrhea.

[0081] ⑩ Mouse fecal metagenomic analysis: Mouse feces were collected the day before the end of the experiment, flash-frozen in liquid nitrogen, and stored at -80℃. Sequencing was performed by Beijing Novogene Technology Co., Ltd. Total DNA was extracted from mouse feces using a DNA extraction kit. After checking the integrity of the DNA using 0.8% agarose gel electrophoresis, metagenomic sequencing was performed to construct a sequencing library.

[0082] like Figure 12 As shown, the abundance of *Phytobacter diazotrophicus* in the gut of the Model group was higher than that in the Control and Drug groups. The abundance of *Citrobacter amalonaticus* in the gut of the Model group was higher than that in the Control group, while no significant difference was observed between the Control and Drug groups. Both groups of probiotics administered via gavage (the areas within the blue boxes in the figure) were found in the gut, and their abundance was higher than that in the Control and Model groups. They also inhibited the growth of pathogenic bacteria. Specifically, the abundance of *Enterobacter bugandensis* in the *Lactobacillus plantarum* GOLDGUT-LP1024 group and the WLFX-2 group was lower than that in the Model group, while the abundance of *Citrobacter amalonaticus* in the WLFX-2 group was lower than that in the Model group, but no difference was found compared to the Control group.

[0083] Previous studies have shown that *Lactiplantibacillus plantarum* and *Pediococcus acidilactici* have significant adhesion to intestinal epithelial cells; *Lactobacillus acidophilus* can improve intestinal epithelial barrier function and transport properties; *Bifidobacterium bifidum* can inhibit the excessive proliferation of *E. coli* and restore jejunal villus length, thereby improving dysbiosis and alleviating diarrhea symptoms; antibiotics disrupt gut microbiota, and *Bifidobacterium longum* can help restore the gut microbiota by competing with harmful bacteria and promoting the growth of beneficial microorganisms; supplementation with *Bifidobacterium animalis* can prevent antibiotic-induced changes in the gut microbiota; *Lactiplantibacillus plantarum* can alleviate antibiotic-induced intestinal inflammation by inhibiting pro-inflammatory responses and modulating the gut microbiota; *Akkermansia muciniphila* and *Lacticaseibacillus paracasei* can prevent antibiotic-associated diarrhea in mice; and *Lacticaseibacillus*... *Citrobacter rhamnosus* works by enhancing the intestinal barrier, inhibiting pathogens, and modulating immunity. Inhibiting *C. difficile* toxins can effectively prevent antibiotic-associated diarrhea. Numerous clinical studies have demonstrated that *Limosilactobacillus reuteri* can improve gastrointestinal symptoms, reduce antibiotic side effects, and restore the balance of the intestinal flora. *Phytobacter diazotrophicus* is a Gram-negative bacterium and an opportunistic pathogen. *Enterobacter bugandensis* is also an opportunistic pathogen and exhibits extensive resistance to third-generation cephalosporins, penicillins, and quinolones. Studies have reported cases of diarrhea caused by *Citrobacter amalonaticus* and found that this strain exhibits resistance to multiple antibiotics (such as β-lactams and quinolones). This finding supports the potential pathogenic role of *Citrobacter amalonaticus* in antibiotic-associated diarrhea.

[0084] The use of broad-spectrum antibiotics (such as clindamycin and cephalosporins) can disrupt the normal gut microbiota, reducing competitive inhibition of opportunistic pathogens. This may promote the overgrowth of *Enterobacter bugandensis* and *Citrobacter amaloniatus*, whose drug resistance gives them a survival advantage under antibiotic pressure, potentially further exacerbating gut microbiota imbalance and leading to diarrhea. Intervention with *Lactobacillus plantarum* GOLDGUT-LP1024 and WLFX-2 groups can reduce the abundance of harmful bacteria, increase the abundance of beneficial bacteria, restore the gut microbiota, and thus alleviate diarrhea.

[0085] Analysis of differential metabolic pathways in mouse gut microbiota: Based on the aforementioned metagenomic sequencing, further functional enrichment analysis of metagenomic sequencing was performed, such as... Figure 13 As shown, log2FoldChange (horizontal axis) reflects the fold change in metabolic pathway expression, with the color of the point corresponding to the P-value (purple indicates a lower P-value and a more significant difference), and the size of the point representing pathway abundance. The four subplots sequentially illustrate: Model group vs. Control group; Drug group vs. Model group; *Lactobacillus plantarum* GOLDGUT-LP1024 group vs. Model group; and WLFX-2 group vs. Model group, visually demonstrating the regulatory effects of antibiotic treatment and different interventions on gut microbiota functional pathways.

[0086] Compared with the Control group, the Bifidobacterium shunt pathway, unique to Bifidobacteria, was significantly downregulated in the Model group (log2FoldChange < 0, P < 0.05). This pathway efficiently produces acetic acid, which is an important support for the intestinal barrier and immune regulation. After intervention, Bifidobacterium shunt was significantly upregulated in the Lactobacillus plantarum GOLDGUT-LP1024 group compared with the Model group (log2FoldChange > 0, P < 0.05), while the upregulation in the WLFX-2 group was greater and more significant (higher log2FoldChange, lower P value), suggesting that the compound probiotic composition can more effectively activate the Bifidobacterium metabolic pathway, enhance acetic acid production, and strengthen intestinal defense function.

[0087] Thiamine diphosphate salvage IV (yeast) is a key pathway for the synthesis and recycling of the active form of vitamin B1 by microorganisms. B1 deficiency can impair energy metabolism in intestinal epithelial cells and affect barrier integrity. This pathway was significantly downregulated in the Model group (log2FoldChange < 0, P < 0.05). After intervention, this pathway was significantly upregulated in both the *Lactobacillus plantarum* GOLDGUT-LP1024 group and the WLFX-2 group compared to the Model group. However, the WLFX-2 group had a higher log2FoldChange and greater pathway abundance, indicating that the compound probiotic composition was more effective in restoring vitamin B1 metabolism and ensuring energy supply to the intestinal epithelium.

[0088] L-carnitine degradation I can produce harmful substances associated with inflammation and metabolic diseases, while elevated D-galacturonate degradation I indicates gut microbiota imbalance. Compared with the control group, both pathways were significantly upregulated in the model group (log2FoldChange>0, P<0.05). After intervention, both pathways were significantly downregulated in the WLFX-2 and Drug groups (log2FoldChange<0, P<0.05), while no significant downregulation was observed in the Lactobacillus plantarum GOLDGUT-LP1024 group. This indicates that the compound probiotic composition can more precisely inhibit harmful metabolic pathways and reduce endogenous damaging factors.

[0089] Upregulation of Bifidobacterium shunt and thiamine diphosphate salvage IV (yeast) helps enhance the gut's defense and repair capabilities (e.g., increasing acetic acid production and ensuring energy metabolism in the intestinal epithelium); while downregulation of L-carnitine degradation I and D-galacturonate degradation I can eliminate internal damaging factors (e.g., reducing inflammation-related metabolites and correcting gut microbiota imbalance). Both *Lactobacillus plantarum* GOLDGUT-LP1024 and the compound probiotic composition can remodel gut microbiota function, but the WLFX-2 compound probiotic composition, through the synergistic effect of multiple strains, has a more comprehensive and significant effect in activating beneficial pathways and inhibiting harmful pathways. Its ability to remodel gut microbiota function is superior to that of *Lactobacillus plantarum* GOLDGUT-LP1024 alone, thus more effectively alleviating antibiotic-associated diarrhea symptoms.

[0090] (6) Experimental conclusions: Probiotics improve inflammation levels and repair the colonic barrier in mice with antibiotic-associated diarrhea (AAD) by enhancing intestinal immune function: To evaluate the ameliorative effect of probiotics on AAD, this study successfully constructed an AAD mouse model. Changes in intestinal immune function-related indicators were systematically analyzed using ELISA, H&E, immunofluorescence staining, and q-PCR. Probiotic intervention significantly increased the expression levels of tight junction proteins ZO-1 and mucin MUC2 in the colon, regulated the gene expression of Tjp1, Muc2, Ocln, Cldn1, Gja1, SCN9A, and inflammatory cytokines Il6 and Il10 in the colon, and simultaneously increased the expression levels of anti-inflammatory cytokines IL-4 and IL-10 while decreasing the expression level of pro-inflammatory cytokines IL-1β. The experimental results showed that, compared with the model group, probiotic intervention significantly reduced the severity of diarrhea in mice, effectively regulated inflammatory cytokine levels, repaired colonic tissue damage, enhanced intestinal barrier integrity, and alleviated antibiotic-induced diarrhea. Among them, the compound probiotic composition (WLFX-2) is more effective in enhancing intestinal immune function, and its regulation of tight junction proteins and inflammatory factors is significantly better than that of the single Lactobacillus plantarum GOLDGUT-LP1024.

[0091] The mechanism by which probiotics improve diarrhea in mice with antibiotic-associated diarrhea by enhancing intestinal metabolic function and regulating gut microbiota: Metagenomic sequencing was used to explore the diversity, differences, and metabolic function changes of gut microbiota in different groups of mice. It was found that compared with the Model group, probiotic intake can significantly increase the diversity of strains in the gut, promote the growth of probiotics (such as Muribaculaceae and Lachnospiraceae) in the gut microbiota, and inhibit the growth of pathogenic bacteria (such as Clostridiaceae and Peptostreptococcaceae) (p < 0.05). At the same time, it can reduce the level of blood urea nitrogen (BUN) in serum, increase the content of acetic acid and propionic acid in the gut, regulate the content of short-chain fatty acids in the gut to physiologically appropriate levels, and significantly upregulate key short-chain fatty acid metabolism pathways such as thiaminediphosphate salvage IV.

[0092] The above results indicate that probiotics can alleviate antibiotic-associated diarrhea symptoms by enhancing intestinal metabolic function and reshaping the intestinal flora structure. In particular, the compound probiotic composition prepared in Example 3 of this application can more comprehensively reshape the metabolic function of the intestinal flora, and its upregulation of short-chain fatty acid metabolic pathways and its inhibitory effect on pathogenic bacteria are superior to those of the single Lactobacillus plantarum GOLDGUT-LP1024.

[0093] 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.

[0094] 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.

[0095] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable way without contradiction. To avoid repetition, this application will not describe various combination methods separately.

Claims

1. A compound probiotic composition and its application in products for improving gut health, characterized in that, The compound probiotic composition includes Lactobacillus rhamnosus GG, lactic acid bacteria compound powder, Lactobacillus plantarum GOLDGUT-LP1024 and Lactobacillus paracasei GOLDGUT-LC12345, wherein the lactic acid bacteria compound powder is a mixture of Bifidobacterium longum subsp. BORI, Lactobacillus acidophilus AD031, Bifidobacterium bifidum BGN4, Bifidobacterium animalis subsp. lactis AD011 and Bifidobacterium longum subsp. infantis IBS007; The *Lactobacillus plantarum* GOLDGUT-LP1024 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28801, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The compound probiotic composition according to claim 1, characterized in that, The mass ratio of Lactobacillus rhamnosus GG, lactic acid bacteria compound powder, Lactobacillus plantarum GOLDGUT-LP1024 and Lactobacillus paracasei GOLDGUT-LC12345 is (2-3):(6-7):(10-11):(7-8).

3. The compound probiotic composition according to claim 1, characterized in that, The total live bacteria count of the compound probiotic composition is ≥2.5×10⁻⁶. 11 CFU / g.

4. A compound probiotic preparation, characterized in that, The formulation contains the compound probiotic composition according to any one of claims 1-3, and includes pharmaceutically or food-acceptable excipients.

5. The compound probiotic preparation according to claim 4, characterized in that, The dosage form of the preparation is a solution, powder, capsule, tablet or granule; the excipients include any one or a combination of at least two of the following: protectants, fillers, binders, emulsifiers, solvents, colorants, pH adjusters, antibacterial agents or buffers.

6. The compound probiotic preparation according to claim 5, characterized in that, The compound probiotic preparation is a powder, prepared by the following method: The compound probiotic preparation is obtained by mixing *Lactobacillus rhamnosus* GG powder, lactic acid bacteria compound powder, *Lactobacillus plantarum* GOLDGUT-LP1024 powder, and *Lactobacillus paracasei* GOLDGUT-LC12345 powder according to the specified mass ratio. The lactic acid bacteria compound powder is composed of *Bifidobacterium longum* subsp. *longum* BORI powder, *Lactobacillus acidophilus* AD031 powder, *Bifidobacterium bifidum* BGN4 powder, *Bifidobacterium animalis* subsp. *lactospirum* AD011 powder, and *Bifidobacterium longum* subsp. *infant* IBS007 powder. The *Lactobacillus plantarum* GOLDDUT-LP1024 bacterial powder is a freeze-dried powder obtained by fermenting and centrifuging *Lactobacillus plantarum* GOLDDUT-LP1024 strain on MRS medium and then freeze-drying the bacterial sludge.

7. The application according to claim 1, characterized in that, The improvement of gut health includes enhancing gut immune function and improving gut metabolic function.

8. The application according to claim 7, characterized in that, The enhancement of intestinal immune function includes at least one of the following: Increase the expression levels of tight junction protein ZO-1 and mucin MUC2 in the colon; It regulates the gene expression levels of colon genes Tjp1, Muc2, Ocln, Cldn1, Gja1, SCN9A, and inflammatory factors Il6 and Il10; Increase the expression levels of colonic anti-inflammatory factors IL-4 and IL-10; It reduces the expression level of the colonic pro-inflammatory factor IL-1β.

9. The application according to claim 7, characterized in that, The enhancement of intestinal metabolic function includes at least one of the following: Reduce serum urea nitrogen (BUN) expression levels; Increase the expression levels of acetic acid and propionic acid in the intestine; Reduce the content of short-chain fatty acids in the intestine; Enhancing the diversity of gut microbiota; Promotes the growth of beneficial bacteria in the gut microbiota and inhibits the growth of pathogenic bacteria in the gut microbiota; Improve key pathways in short-chain fatty acid metabolism.

10. The application according to any one of claims 7-9, characterized in that, The product includes pharmaceuticals, health products, or food; the product has the effect of relieving intestinal discomfort symptoms by improving intestinal health, including antibiotic-associated diarrhea.

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