A strain of fermenting *Lactobacillus mucinus* and its application

Abstract

This invention relates to the field of microbial technology and discloses a strain of *Lactobacillus fermentans* and its applications. The preservation number of the *Lactobacillus fermentans* is CGMCC No. 37245. This strain exhibits excellent antibacterial ability, effectively inhibiting intestinal pathogens; it has a strong ability to degrade dietary fiber or indigestible sugars (such as fructooligosaccharides, β-glucan, galactooligosaccharides, etc.) and can effectively proliferate in the intestinal environment; the *Lactobacillus fermentans* CGMCC No. 37245 of this invention produces a high yield of hydrogen peroxide during fermentation, making this strain potentially useful in preventing antibiotic-associated diarrhea and traveler's diarrhea; furthermore, the *Lactobacillus fermentans* CGMCC No. 37245 of this invention can relieve both constipation and diarrhea, demonstrating a bidirectional regulatory function on intestinal motility.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a fermenting strain of *Lactobacillus mucinus* and its applications. Background Technology

[0002] Constipation and diarrhea are the most common functional bowel disorders in clinical practice, seriously affecting patients' quality of life. Probiotics, as an important means of regulating intestinal microecology, improving intestinal motility and immune homeostasis, have been widely used as an adjunct therapy for functional gastrointestinal disorders. Among them, Bifidobacteria (such as...) Bifidobacterium animalum subsp. milk BB-12 and Lactobacillus strains have become mainstream probiotic products in the market due to their high safety and wide application.

[0003] However, existing probiotics still face multiple technical bottlenecks in practical applications: First, most commercial probiotics have limited ability to utilize complex carbon sources, making it difficult to proliferate effectively in the intestinal environment dominated by dietary fiber or indigestible sugars (such as fructooligosaccharides, β-glucan, galactooligosaccharides, etc.), resulting in low colonization efficiency and short duration of action; Second, their antibacterial ability is insufficient, failing to effectively inhibit the excessive growth of intestinal pathogens (such as Staphylococcus aureus, Escherichia coli, Salmonella, etc.), limiting their application in infectious diarrhea; Third, they have poor gastrointestinal tolerance, with many strains showing significantly reduced survival rates in simulated gastrointestinal environments (low pH gastric acid, high concentration of bile salts), making it difficult for them to reach the intestines and exert their functions; In particular, single function is a common defect of current probiotics: some strains can improve constipation but may aggravate diarrhea, or only have anti-inflammatory effects without prokinetic effects, making it difficult to achieve "bidirectional regulation". For example, CN120290408A discloses a strain of *Lactobacillus fermentum* BM01 with laxative effects, but its function is limited to improving constipation; CN119120325A discloses a strain of *Lactobacillus fermentum* A10 that produces a high amount of extracellular polysaccharides, but its core application is in anti-oxidation and does not involve the regulation of intestinal motility. Therefore, the current ability of probiotics to regulate complex intestinal dysfunctions such as coexisting constipation and diarrhea or alternating irritable bowel syndrome (IBS) is extremely limited.

[0004] Ideal probiotics should possess the comprehensive ability to bidirectionally regulate intestinal motility, simultaneously inhibit inflammation and pathogens, and stably colonize in the complex intestinal environment. Currently, no strain can simultaneously meet all of these requirements. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of existing technologies, such as the single function of probiotics, poor inhibitory effect on intestinal pathogens, weak ability to degrade dietary fiber, and inability to achieve bidirectional regulation of constipation and diarrhea. This invention provides a fermenting strain of *Lactobacillus mucinus* and its applications. This strain not only has excellent antibacterial spectrum and dietary fiber utilization ability, but more importantly, it can achieve bidirectional regulation of constipation and diarrhea, filling a gap in existing technologies.

[0006] To achieve the above objectives, the first aspect of the present invention provides a strain of fermenting *Lactobacillus mucinus* (… Limosilactobacillus fermentum The preservation number of the fermenting Lactobacillus mucinus is CGMCC No. 37245.

[0007] A second aspect of the present invention provides a probiotic agent, wherein the probiotic agent comprises the fermenting lactobacillus described in the present invention.

[0008] A third aspect of the present invention provides the application of the fermented Lactobacillus mucinus or probiotic agent described herein in the preparation of formulations for regulating the intestinal microecological environment.

[0009] The fourth aspect of the present invention provides the use of the fermented Lactobacillus mucinus or probiotic agent described in the present invention in the preparation of formulations for inhibiting intestinal pathogens.

[0010] The fifth aspect of the present invention provides the use of the fermented Lactobacillus mucinus or probiotic agent described herein in the preparation of a formulation for bidirectional regulation of intestinal function.

[0011] Preferably, the bidirectional regulation of intestinal function includes relieving constipation and inhibiting diarrhea.

[0012] Through the above technical solution, the present invention achieves at least the following beneficial effects: (1) Unique bidirectional intestinal regulation function: The fermented Lactobacillus mucinus CGMCC No. 37245 of this invention can significantly relieve constipation (significantly improved intestinal peristalsis in a zebrafish constipation model). p <0.01), and can also effectively inhibit diarrhea (reducing the number of neutrophils, increasing intestinal fluorescence intensity, and inhibiting inflammatory factors). tnf-α and il-6 It has the function of bidirectionally regulating intestinal motility.

[0013] (2) Broad-spectrum and highly effective antibacterial ability: The fermented Lactobacillus mucinus CGMCC No.37245 of the present invention has an inhibition rate of more than 90% against a variety of pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, and Salmonella that cause intestinal infections, and also has a significant inhibitory effect on Candida albicans. It has a broad antibacterial spectrum and has great potential in the prevention and treatment of infectious diarrhea.

[0014] (3) Excellent dietary fiber utilization: The fermented Lactobacillus mucinus CGMCC No.37245 of the present invention has outstanding utilization of various difficult-to-digest sugars such as β-glucan, isomaltooligosaccharide, and chondroitin sulfate, which enables it to gain a competitive advantage in the intestinal environment dominated by dietary fiber and achieve efficient colonization and proliferation.

[0015] (4) High hydrogen peroxide synthesis capacity: The fermented Lactobacillus mucinus CGMCC No.37245 of the present invention has a hydrogen peroxide production of up to 17.40±0.52μg / mL after 24h of fermentation. This characteristic makes it uniquely promising for the prevention of antibiotic-associated diarrhea caused by dysbiosis and traveler's diarrhea caused by foreign pathogens.

[0016] Biological Preservation The strain provided by this invention is classified as *Lactobacillus fermentans*. Limosilactobacillus leaven It was isolated from gut microbiota samples of healthy young volunteers in the Beijing area and deposited on December 30, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37245. Attached Figure Description

[0017] Figure 1 This is a colony morphology diagram of strain CCNH192 on an MRS plate.

[0018] Figure 2 This is a Gram-stained image of the cell morphology of strain CCNH192 (100× objective lens).

[0019] Figure 3 This is a standard curve for the detection of hydrogen peroxide (H2O2).

[0020] Figure 4 This is a graph showing the hydrogen peroxide (H2O2) production of strain CCNH192 at different fermentation times. Detailed Implementation

[0021] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0022] In this invention, "CCNH192" is the number of the fermenting Lactobacillus mucinus provided by the inventors during the research process, while "CGMCC No.37245" is the preservation number of the strain. The two represent the same strain, and their numbers can be used interchangeably in the following text.

[0023] The first aspect of this invention provides a strain of fermenting *Lactobacillus mucinus* (… Limosilactobacillus fermentum The preservation number of the fermenting Lactobacillus mucinus is CGMCC No. 37245.

[0024]

[0025] A second aspect of the present invention provides a probiotic agent, wherein the probiotic agent comprises the fermenting lactobacillus described in the present invention.

[0026] According to some embodiments of the present invention, the viable count of the fermenting *Lactobacillus mucinus* is ≥10 based on the total mass of the probiotic agent. 11 CFU / g.

[0027] In this invention, the probiotic agent can be a liquid or solid agent, and there is no particular limitation on the preparation method. It can be prepared by conventional freeze-drying. Specifically, the preparation method of the probiotic agent includes: taking out the glycerol cryopreservation tube of *Lactobacillus fermentatus* from -80℃ for thawing and recovery; inoculating the recovered bacteria into MRS medium and incubating statically at 34℃-37℃ for 18-24h; inoculating into MRS liquid medium at an inoculation rate of 0.2-2 (v / v)% and incubating for 8-12h; and then... Transfer the inoculum to a fermenter at a rate of 2-5 (v / v)% and incubate for 12-24 hours. During fermentation, add sodium carbonate solution to maintain the pH at 6-6.5. Centrifuge to collect the bacterial sludge, and add an equal mass of a protective agent to the sludge. The protective agent consists of 1-6 wt% skim milk powder, 4-10 wt% trehalose, 1-3 wt% monosodium glutamate, 0.5-1 wt% glycerol, 4-6 wt% glucose, and 4-12 wt% maltodextrin. After mixing and stirring evenly, pre-freeze at -80℃ and then freeze-dry to obtain solid bacterial powder.

[0028] A third aspect of the present invention provides the application of the fermented Lactobacillus mucinus or probiotic agent described herein in the preparation of formulations for regulating the intestinal microecological environment.

[0029] The fourth aspect of the present invention provides the use of the fermented Lactobacillus mucinus or probiotic agent described in the present invention in the preparation of formulations for inhibiting intestinal pathogens.

[0030] In this invention, the enteric pathogens include, but are not limited to, Escherichia coli (Escherichia coli). Escherichia coli Staphylococcus aureus ( Staphylococcus aureus ),salmonella( Salmonella enterica ) and Candida albicans ( Candida albicans At least one of the following.

[0031] The fifth aspect of the present invention provides the use of the fermented Lactobacillus mucinus or probiotic agent described herein in the preparation of a formulation for bidirectional regulation of intestinal function.

[0032] Preferably, the bidirectional regulation of intestinal function includes relieving constipation and inhibiting diarrhea.

[0033] More preferably, the diarrhea includes infectious diarrhea and / or inflammatory diarrhea, such as traveler's diarrhea and antibiotic-associated diarrhea.

[0034] In this invention, antibiotic-associated diarrhea refers to the situation where, after taking antibiotics, the drug kills not only harmful bacteria but also beneficial bacteria in the gut, leading to an imbalance in the gut microbiota. Pathogenic bacteria then proliferate, causing diarrhea. Traveler's diarrhea refers to the situation where travelers encounter unfamiliar pathogens (such as Escherichia coli, Salmonella, etc.). Due to a lack of immunity, these foreign pathogens colonize the intestines and cause inflammation and diarrhea.

[0035] In this invention, the fermented Lactobacillus mucinus described herein can also be used to produce fermented foods, including solid foods, liquid foods, and semi-solid foods; the fermented foods include, but are not limited to, dairy products, soy products, and fruit and vegetable products, wherein the dairy products include, but are not limited to, milk, sour cream, and cheese, and the fruit and vegetable products include, but are not limited to, cucumbers, carrots, beets, celery, and cabbage.

[0036] The present invention will be described in detail below through embodiments.

[0037] In the following examples, the composition of the MRS solid culture medium includes: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL Tween-80, 0.5 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 20 g / L agar, pH 6.2, and the balance being deionized water.

[0038] The MRS liquid culture medium consists of: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL Tween-80, 0.5 g / L magnesium sulfate, 0.25 g / L manganese sulfate, pH 6.2, and the remainder is deionized water.

[0039] The composition of LB liquid medium includes: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, and the balance being deionized water, pH 6.8.

[0040] YPD liquid culture medium consists of 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and the remainder is deionized water.

[0041] Unless otherwise specified, all reagents and materials used in the following examples and comparative examples are commercially available products purchased from reputable chemical or biological reagent and material suppliers, and all reagents are of analytical grade.

[0042] Example 1 Strains are isolated, purified, identified, tested for performance, and preserved biologically.

[0043] (a) Isolation and purification of strains Fecal samples were collected from healthy infant volunteers in the Beijing area. Using culturamics technology, bacterial strains were isolated under anaerobic conditions at 37°C on MRS medium. Specifically, 1g of fresh fecal sample was weighed and placed in 9mL of sterile physiological saline, shaken thoroughly, centrifuged at 900rpm for 1min to remove particulate matter, and the supernatant was collected. Cells were counted using a cell counter, and based on the viable cell count, the samples were spread onto different culture plates and incubated in an anaerobic workstation for 48h. After colonies had grown on the plates, the isolated single colonies were spread onto new MRS solid medium plates and streaked three times to obtain pure cultures. Single colonies with different morphologies were repeatedly streaked for purification until a pure culture was obtained, which was designated strain CCNH192.

[0044] (II) Strain identification (1) Morphological identification: Observation of the colony morphology of strain CCNH192 revealed that its colonies on MRS plates were round, with neat, smooth, and raised edges. The colonies were relatively small, about 1 mm in diameter, and exhibited a stringy morphology (the colony morphology of strain CCNH192 is shown in Figure 1). Figure 1 (As shown); then, Gram staining was performed on strain CCNH192, and the staining results and cell morphology were observed under a microscope. The cells were short rods of varying lengths, arranged singly, in pairs, or in short chains (the cell morphology of strain CCNH192 after Gram staining is shown in the figure). Figure 2 (As shown).

[0045] (2) Molecular identification Strain CCNH192 was sequenced, and the sequencing results were compared with those in the NCBI database. It was found that the 16S rDNA of strain CCNH192 (as shown in SEQ ID No: 1) is similar to that of *Lactobacillus fermentans* (…). Limosilactobacillus fermentum The homology between the strain CCNH192 and *Lactobacillus fermentatus* reached 99.79%. Based on morphological characteristics, the strain was identified as *Lactobacillus fermentatus*. Limosilactobacillus fermentum The selected strains were inoculated into MRS medium and cultured for 24 hours to preserve the bacteria.

[0046] (III) Biological Preservation The strain provided by this invention is classified as *Lactobacillus fermentans*. Limosilactobacillus leavenIt was isolated from gut microbiota samples of healthy young volunteers in the Beijing area and deposited on December 30, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37245.

[0047] Example 2 Basic performance evaluation of strain CCNH192 (1) Evaluation of the utilization capacity of indigestible sugars If probiotics can break down various indigestible sugars, they will have many benefits in terms of health, food processing, and ecology, such as regulating the balance of intestinal flora, improving intestinal function, and enhancing the body's immunity. They can also improve the texture and flavor of food and be used to develop functional foods.

[0048] Different indigestible sugars were selected as the sole carbon source. 15 μL of the 2wt% indigestible sugar stock solution was placed in an MT2 microplate. The secondary seed culture of strain CCNH192, which had been cultured overnight, was washed twice with sterile water and resuspended until the turbidity was 75%. 150 μL of the bacterial suspension was added to the wells containing different indigestible sugars and incubated anaerobically at 37°C for 24 h. The commercially available Lactobacillus fermentum CECT5716 (purchased from Chr. Hansen (China) Co., Ltd.) was used as the control group. The absorbance value was detected using Biolog software. The higher the value, the higher the ability of the strain to utilize the substrate. The specific data are shown in Table 1.

[0049] Table 1. Utilization capacity of fermenting Lactobacillus mucilaginosus CCNH192 for different recalcitrant sugars.

[0050] Note: In Table 1, the concentration of the indigestible sugars in the different types of indigestible sugar stock solutions added to the MT2 analysis microplates was the same, which was 1.82 g / L.

[0051] Table 1 shows that *Lactobacillus fermentum* CCNH192 exhibits strong metabolic activity against various indigestible sugars, particularly against common intestinal dietary fibers such as β-glucan, isomaltooligosaccharide, and chondroitin sulfate. This suggests that the strain can gain a growth advantage in the intestinal environment based on these components, which is beneficial for its colonization and function.

[0052] (2) Evaluation of antibacterial properties The following strains were used: indicator strain *Escherichia coli* ATCC25922 (purchased from the American Center for Type Culture Collection), *Escherichia coli* CICC10421 (purchased from the China Industrial Microbial Culture Collection Center), *Staphylococcus aureus* CMCC(B)26001 (purchased from the China Medical Bacteriological Culture Collection Center), *Staphylococcus aureus* CMCC(B)26003 (purchased from the China Medical Bacteriological Culture Collection Center), *Salmonella typhimurium* ATCC14028 (purchased from the American Center for Type Culture Collection), and *Salmonella enteritidis*. Subspecies CVCC3378 (purchased from the National Veterinary Microbial Culture Collection Center) was inoculated into LB broth and incubated overnight at 37°C and 200 rpm. Indicator strains *Candida albicans* CICC1965 (purchased from the China Industrial Microbial Culture Collection Center) and *Candida albicans* ATCC10231 (purchased from the American Center for Type Culture Collection) were inoculated into YPD broth and incubated overnight at 30°C and 200 rpm. After overnight incubation, the indicator cultures were transferred to their respective fresh culture media and cultured to OD200. 600 =or viable bacteria count 10 8 CFU / mL, dilute the corresponding culture medium to 10. 5 CFU / mL was used as the indicator bacterial solution.

[0053] The CCNH192 strain was inoculated into fresh, sterile MRS liquid medium vials, with commercially available *Lactobacillus fermentum* CECT5716 (purchased from Chr. Hansen (China) Co., Ltd.) used as a control. The cultures were incubated overnight at 37°C for 24 hours. A 2% inoculum was then added to fresh MRS liquid medium and anaerobically cultured at 37°C for 24 hours. The supernatant was then centrifuged, sterilized through a membrane, and used as the test sample. The MRS liquid medium served as a negative control.

[0054] Reaction system: 100 μL fermentation broth supernatant + 100 μL indicator bacteria dilution, denoted as S1; 50 μL fermentation broth supernatant + 150 μL indicator bacteria dilution, denoted as S2.

[0055] Inhibition rate (%) = (A0 - A) / A0 × 100%, where: A0 is the OD of the negative control sample. 600 Value; A is the sample OD. 600 Values ​​(where A0 and A are the OD values ​​after culture) 600 and OD before culture 600 The results are shown in Table 2.

[0056] Table 2 Evaluation of the antibacterial properties of *Lactobacillus mucinus* CCNH192 during fermentation

[0057] As shown in Table 2, the strain CCNH192 provided by this invention generally exhibits inhibition rates of over 90% against Escherichia coli, Staphylococcus aureus, and Salmonella, and also shows significant inhibitory effects against Candida albicans. This broad-spectrum and highly effective antibacterial ability demonstrates its great potential in the prevention and treatment of intestinal infections caused by various pathogens.

[0058] (3) Evaluation of in vitro adhesion ability Self-aggregation performance: Glycerol tubes of strain CCNH192 were inoculated into fresh MRS liquid medium at a rate of 2 (v / v)‰, with commercially available *Lactobacillus fermentum* CECT5716 (purchased from Chr. Hansen (China) Co., Ltd.) as the control group. Under anaerobic conditions, the culture was carried out overnight at 37°C for 24 h. After inoculation into fresh MRS liquid medium at a rate of 2 (v / v)% and cultured at 37°C for 24 h, the OD was measured. 600 After washing twice with physiological saline, the OD was resuspended. 600 =1, pipette 100 μL of bacterial culture into the ELISA plate and measure the initial OD. 600 The value was recorded as B0; then the bacterial culture was allowed to stand for 4 hours, and 100 μL of the upper bacterial culture was transferred to an ELISA plate to detect its OD value after standing. 600 The value is denoted as B1. The aggregation rate of the strain is calculated using the following formula: Strain self-aggregation rate (%) = 100% × (1 - B1 / B0).

[0059] Hydrophobicity: Absorbs the above-mentioned OD 600 Add 100 μL of bacterial culture with a concentration of 1 to an ELISA plate and measure the initial OD. 600 The value was recorded as B0; then 900 μL of bacterial culture was taken, 180 μL of dodecane was added, and the mixture was vortexed and allowed to stand for 20 min to separate into layers. The lower aqueous phase was then taken to measure its OD. 600 The value, denoted as B, is used to calculate hydrophobicity using the following formula: Hydrophobicity (%) = 100% × [(B0-B) / B0]. The self-aggregation of strain CCNH192 is 38.86±1.27%, and its hydrophobicity is 72.22±3.56%, while the self-aggregation of the control strain CECT5716 is 27.45±1.44%, and its hydrophobicity is 55.19±2.43%. This demonstrates that strain CCNH192 has strong in vitro adhesion ability.

[0060] Evaluation of gastric acid and bile salt tolerance: The gastric acid and bile salt tolerance of strain CCNH192 and commercially available *Lactobacillus fermentum* CECT5716 (purchased from Chr. Hansen (China) Co., Ltd.) were evaluated according to T / CNHFA435-2024, "Test Method for Gastric Juice Tolerance of Probiotics". In the gastric acid tolerance test, the simulated gastric juice pH was 3, and the incubation time was 3 hours; in the bile salt tolerance test, the bile salt content was 0.2 wt%, and the incubation time was 3 hours. The gastric acid tolerance survival rate of strain CCNH192 was 85.85 ± 3.73%, and the bile salt tolerance survival rate was 75.14 ± 2.49%; the gastric acid tolerance survival rate of strain CECT5716 was 69.21 ± 4.57%, and the bile salt tolerance survival rate was 63.83 ± 3.79%.

[0061] As can be seen from the above, the strain CCNH192 provided by this invention exhibits good self-aggregation ability and cell surface hydrophobicity, suggesting that it has good potential for intestinal epithelial adhesion. Simultaneously, its high survival rate in simulated gastric acid and bile salt environments ensures that sufficient live bacteria can reach the intestines to perform their functions.

[0062] Example 3 Evaluation of hydrogen peroxide synthesis capacity of strain CCNH192 Glycerol tubes of strain CCNH192 were inoculated into fresh, sterile MRS liquid medium, with commercially available *Lactobacillus fermentum* CECT5716 (purchased from Chr. Hansen (China) Co., Ltd.) as the control group. The cultures were incubated overnight at 37°C for 18 h. Then, 2% (v / v)% of the culture was inoculated into fresh MRS liquid medium and cultured at 37°C and 200 rpm for 15-48 h. The supernatant was then collected by centrifugation. The H2O2 production capacity of strains CCNH192 and CECT5716 was detected using the 4-aminopropylantipyrine method. Specific results are shown in [link to results]. Figure 4 .

[0063] H2O2 standard solutions: Weigh an appropriate amount of H2O2 standard, add the corresponding volume of water, and prepare H2O2 standard solutions with concentrations of 0, 0.5, 1, 1.5, 2, 5, 8, 10, 25, and 50 μg / mL. The standard curve is shown below. Figure 3 .

[0064] Depend on Figure 4 It can be seen that the strain CCNH192 provided by this invention has the highest hydrogen peroxide production, reaching 17.40±0.52 μg / mL, after 24 h of fermentation culture. This is higher than that in the literature "Screening and biological characteristics of lactic acid bacteria that inhibit Gardnerella vaginalis" and "Biosynthesis and Degradation of H2O2 by Vaginal". LactobacilliThe hydrogen peroxide production of this strain (0.7-1.5 μg / mL) reached the levels of some *Lactobacillus curlis* and *Lactobacillus japonicus*, indicating its high production potential for prevention of antibiotic-associated diarrhea and traveler's diarrhea. However, no hydrogen peroxide synthesis was detected in *Lactobacillus mucinus* CECT5716, consistent with previous reports (Martín R, Olivares M, Marín ML, et al. Probiotic potential of 3...). lactobacilli strains isolated from breast milk[J]. Journal of Human Lactation, 2005, 21(1): 8-17.).

[0065] Example 4 Short-chain fatty acid synthesis capacity of strain CCNH192 Short-chain fatty acids (SCFAs) are important metabolites produced by probiotic fermentation of dietary fiber and are crucial for maintaining gut health. For constipation, SCFAs act as softeners, stimulating intestinal peristalsis, softening stool, providing energy, and restoring intestinal motility. For diarrhea, SCFAs promote water and sodium absorption, reduce stool water content, repair the intestinal barrier, and alleviate inflammatory responses. Strain CCNH192 was inoculated into fresh MRS liquid medium with glycerol tubes. Commercially available *Lactobacillus fermentum* CECT5716 (purchased from Chr. Hansen (China) Co., Ltd.) served as a control. The cultures were incubated overnight at 37°C for 18 hours. Normalized bacterial cultures were then inoculated at a 2 (v / v)% inoculum into fresh MRS liquid medium and MRS liquid medium with inulin (without sodium acetate) as the carbon source (glucose). After static incubation at 37°C for 18 hours, the cultures were centrifuged, and the supernatant was filtered. The yields of lactic acid, acetic acid, and formic acid were detected by HPLC. Specific data are shown in Table 3.

[0066] Table 3. Evaluation of short-chain fatty acid production by fermented Lactobacillus mucilaginosus CCNH192

[0067] Note: In Table 3, "-" indicates that the substance was not detected.

[0068] As shown in Table 3, the strain CCNH192 provided by this invention can not only efficiently utilize glucose to produce acid, but also produce lactic acid, acetic acid and formic acid when using indigestible sugar inulin as the sole carbon source, proving its ability to produce beneficial short-chain fatty acids through the metabolism of dietary fiber.

[0069] Example 5 Evaluation of the bidirectional regulation of intestinal function by strain CCNH192 Zebrafish models of constipation and diarrhea were constructed to comprehensively evaluate the bidirectional regulatory function of CCNH192, with commercially available Lactobacillus fermentum CECT5716 (purchased from Chr. Hansen (China) Co., Ltd.) serving as the control group.

[0070] (1) Evaluation of constipation relief efficacy of strain CCNH192 in zebrafish model A constipation model induced by aluminum sulfate was established in wild-type AB strain zebrafish. Six groups were set up, with 30 fish in each group: normal control group, model control group, positive control group (domperidone as positive drug), and CCNH192 experimental group (2×10). 8 The study evaluated the effects of CCNH192 on intestinal peristalsis, intestinal NO fluorescence intensity, and relative expression levels of intestinal-related genes in constipated zebrafish. The control group included *Bifidobacterium animalis* subsp. *lactamase* BB12 (purchased from Chr. Hansen (China) Co., Ltd.) and commercially available *Lactobacillus fermentum* CECT5716 (purchased from Chr. Hansen (China) Co., Ltd.).

[0071] Zebrafish intestinal peristalsis measurement: After the samples were processed for a period of time, they were stained with Nile red. Ten zebrafish were randomly selected from each group and photographed under a microscope to measure the intestinal peristalsis of the zebrafish. The results are shown in Table 4.

[0072] Determination of NO fluorescence intensity in the intestine of constipated zebrafish: After the samples were processed for a period of time, NO probe staining was performed. Ten zebrafish were randomly selected from each group and photographed under a microscope. The NO fluorescence intensity in the zebrafish intestine was measured. The results are shown in Table 5.

[0073] Relative expression levels of gut-related genes: After a period of sample treatment, zebrafish from each group were collected, and the relative expression levels of gut-related genes (…) were determined using qPCR. we1 , us2a and us2b The relative expression levels of ) were determined, and the results are shown in Table 6.

[0074] Table 4. Effects of CCNH192 on a zebrafish constipation model (intestinal motility)

[0075] Table 5. Application effect of CCNH192 in zebrafish constipation model (NO fluorescence intensity)

[0076] Table 6. Effects of CCNH192 on a zebrafish constipation model (relative expression levels of gut-related genes)

[0077] Tables 4-6 show that strain CCNH192 effectively reduced NO concentration in intestinal tissue, showing a significant difference compared to the model group. Regarding the relative expression of intestinal-related genes, with the expression level in the model control group as unit 1, strain CCNH192 effectively inhibited... we1 The expression of [something] accelerates intestinal peristalsis; effectively inhibits intestinal inflammation and enhances intestinal defense. nos2a、nos2b (and its effect is better than BB12.) Strain CCNH192 can simultaneously inhibit... we1 mediated enteric nerve inhibitory pathway and nos2a / 2b Mediated inflammatory pathways significantly reduce intestinal NO levels ( p <0.001), accelerates intestinal peristalsis ( p <0.01), and its overall effect is better than that of control strains BB12 and CECT5716, indicating that it has unique advantages in the intervention of functional constipation.

[0078] (2) Evaluation of the antidiarrheal efficacy of strain CCNH192 in zebrafish model Zebrafish were raised in aquarium water at 28℃ (water quality: 200mg of quick-dissolving sea salt added per 1L of reverse osmosis water, conductivity 450-550μS / cm; pH 6.5-8.5; hardness 50-100mg / L of CaCO3).

[0079] Staphylococcus aureus was cultured in nutrient broth at 37°C and 200 rpm. A Staphylococcus aureus-induced diarrhea model was established using 4-day-first-f (dpf) transgenic neutrophil-positive green fluorescent zebrafish (MPX). Six groups were set up, with 30 fish in each group: a normal control group, a model control group, a positive control group (1000 μg / mL vancomycin as a positive control), and a CCNH192 experimental group (2 × 10⁶ fish). 8 The control group consisted of CFU / mL positive strain BB12 and commercially available Lactobacillus fermentum CECT5716 (purchased from Chr. Hansen (China) Co., Ltd.). The bacteria were cultured in 6-well plates with a volume of 3 mL per well.

[0080] Evaluation of intestinal neutrophil count: After 24 hours of treatment at 28℃, samples were given Staphylococcus aureus and treated for another 6 hours. Ten zebrafish were randomly selected from each group and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software to analyze the number of neutrophils in the zebrafish intestine. The results are shown in Table 7.

[0081] Evaluation of intestinal fluorescence intensity: After 8 hours of treatment at 28℃, Nile red was administered as a fluorescent indicator of intestinal contents to each group. Treatment continued for 16 hours, after which the samples and Nile red were washed away, and samples were administered again in water solution. A zebrafish diarrhea model was established by administering Staphylococcus aureus, and treatment continued for 30 hours. Ten zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software to analyze the intestinal fluorescence intensity of the zebrafish. The results are shown in Table 8.

[0082] Fluorescence intensity of Staphylococcus aureus in the intestine: 24 h after sample administration, each experimental group was given water-soluble Staphylococcus aureus labeled with VybrantDiocell-labeling solution to establish a zebrafish diarrhea model. After further treatment at 28℃ for 6 h, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Data were analyzed using NIS-Elements D 3.20 advanced image processing software, and the results are shown in Table 9.

[0083] Relative expression levels of gut-related genes: 24 hours after sample administration, each experimental group was given Staphylococcus aureus in water to establish a zebrafish diarrhea model. After another 6 hours of treatment, total RNA was extracted from zebrafish in each group using a pre-loaded magnetic bead universal RNA extraction kit. The concentration and purity of total RNA were determined using a UV-Vis spectrophotometer. 2.00 μg of total RNA from the zebrafish sample was used to synthesize 20.0 μL of cDNA according to the instructions of the cDNA first-strand synthesis kit, and the results were detected by q-PCR. β- act , tnf-α and il-6 Gene expression. (Using...) β-actin As an internal reference for gene expression, calculation tnf-α and il-6 The relative RNA expression levels of the genes are shown in Table 10.

[0084] Table 7. Effects of CCNH192 on a zebrafish diarrhea model (number of neutrophils in diarrhea)

[0085] Table 8. Application effect of CCNH192 in zebrafish diarrhea model (intestinal fluorescence intensity)

[0086] Table 9. Application effect of CCNH192 in zebrafish diarrhea model (Staphylococcus aureus fluorescence intensity)

[0087] Table 10. Effects of CCNH192 on a zebrafish diarrhea model (relative expression levels of gut-related genes)

[0088] Note: In Table 4-10, "-" represents other experimental groups and control groups. P All values ​​are compared with the model control group.

[0089] As shown in Tables 7-10, strain CCNH192 effectively reduced the number of neutrophils in the intestine, and its performance was superior to that of control strains BB12 and CECT5716; strain CCNH192 effectively improved Staphylococcus aureus-induced diarrhea and increased intestinal fluorescence intensity, with significantly better effects than control strains BB12 and CECT5716; strain CCNH192 effectively reduced the fluorescence intensity of Staphylococcus aureus in the intestine and significantly downregulated it. tnf-α and il-6 The overall effect was significantly better than that of the control strains BB12 and CECT5716.

[0090] This indicates that strain CCNH192 can effectively reduce the inflammatory response induced by Staphylococcus aureus and improve immune homeostasis. In the model control group, rapid intestinal peristalsis led to the rapid expulsion of Nile Red-labeled intestinal contents, resulting in a significant decrease in intestinal fluorescence intensity; in the CCNH192 intervention group, the intestinal fluorescence intensity was significantly increased (…). p <0.001 indicates that it effectively inhibits pathological diarrhea and restores intestinal emptying homeostasis. Combined with its effects on Staphylococcus aureus colonization, neutrophil count, and pro-inflammatory factors (…),… tnf-α、il-6 The significant inhibitory effect of the strain CCNH192 indicates that it exerts its antidiarrheal effect through a multi-target synergistic mechanism of "antibacterial-anti-inflammatory-motility modulation".

[0091] In addition, based on the results of Example 5, CCNH192 performed well in both constipation and diarrhea models, with an overall effect superior to the control strains BB12 and CECT5716, confirming its unique advantage of "bidirectional regulation of intestinal function".

[0092] Example 6 Preparation of probiotic inoculants The activated CCNH192 seed culture was inoculated into the fermentation medium at a rate of 5% (v / v) and cultured at 37°C. During fermentation, the pH was maintained at 6.5 using a 20% sodium carbonate solution. After the culture was completed, the viable cell count in the fermentation broth reached 1.6 × 10⁻⁶. 10CFU / mL; the bacterial sludge was collected by centrifugation, and an equal mass of freeze-drying protectant (formula: 6wt% skim milk powder, 4.8wt% trehalose, 1.7wt% monosodium glutamate, 0.5wt% glycerol, 4wt% glucose, and 5.5wt% maltodextrin) was added. After mixing, the mixture was pre-frozen at -80℃ and then freeze-dried under vacuum to obtain CCNH192 probiotic powder. Testing showed that the viable count of the obtained powder reached 1.85 × 10⁻⁶. 11 CFU / g; The fermentation medium consists of: 1L deionized water, 44g glucose, 30g yeast extract, 5g sodium acetate, 0.25g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, 2g dipotassium hydrogen phosphate, 2g triammonium hydrogen citrate, 1mL Tween-80, 22g calcium carbonate, and pH 6.5.

[0093] Example 7 Preparation of fermented milk Preparation of CCNH192 bacterial agent: Activated CCNH192 was anaerobically cultured in MRS liquid medium at 37℃ for 48h; 30mL of fermentation broth was placed in a centrifuge tube and centrifuged at 4℃ and 4000rpm for 10min. The supernatant was discarded, and the precipitate was collected. After washing 2-3 times with sterile physiological saline, 12% (w / v) skim milk powder was added and mixed well. After pre-cooling at -80℃ for 4h, it was freeze-dried. The freeze-drying conditions were: cold trap temperature -70℃, drying time 24h, and vacuum degree 1Pa. After freeze-drying, the bacterial powder was immediately collected in a sterile packaging bag and sealed to obtain CCNH192 bacterial agent.

[0094] Fresh milk and white sugar were mixed and homogenized at a mass ratio of 10:1. The mixture was then sterilized at 140°C for 2 seconds and cooled to 35°C. The prepared CCNH192 inoculum was then inoculated, and the mixture was sealed and fermented at 35°C for 4 hours. Afterward, it was transferred to a 4°C refrigerator for 12 hours for post-fermentation to obtain the final fermented product (bacterial concentration 10). 8 (CFU / mL or higher).

[0095] In summary, the fermenting Lactobacillus mucinus CCNH192 provided by this invention is an excellent strain with multiple probiotic properties, including bidirectional regulation of intestinal function, broad-spectrum and potent antibacterial activity, efficient utilization of dietary fiber, and high production of hydrogen peroxide. Its comprehensive performance is significantly superior to related strains in the prior art, and it has extremely high development and application value.

[0096] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A strain of fermenting *Lactobacillus mucilaginosus* ( Limosilactobacillus fermentum ), characterized in that, The preservation number of the fermenting Lactobacillus mucinus is CGMCC No. 37245.

2. The fermented *Lactobacillus mucinus* according to claim 1, wherein, The 16S rDNA of the fermenting Lactobacillus mucinus is shown in SEQ ID No:

1.

3. A probiotic preparation, characterized in that, The probiotic agent includes the fermented Lactobacillus mucinus as described in claim 1 or 2.

4. The probiotic agent according to claim 3, wherein, The viable cell content of the fermented L. muci- dos is ≥10 11 CFU / g, based on the total mass of the probiotic bacterial agent.

5. The use of the fermented Lactobacillus mucinus according to claim 1 or 2, and the probiotic agent according to claim 3 or 4, in the preparation of formulations for regulating the intestinal microecological environment.

6. The use of the fermented Lactobacillus mucinus according to claim 1 or 2, and the probiotic agent according to claim 3 or 4, in the preparation of formulations for inhibiting intestinal pathogens.

7. The application according to claim 6, wherein, The intestinal pathogens include Escherichia coli (Escherichia coli) Escherichia coli Staphylococcus aureus ( Staphylococcus aureus ),salmonella( Salmonella enterica ) and Candida albicans ( Candida albicans At least one of the following.

8. The use of the fermented Lactobacillus mucinus according to claim 1 or 2, and the probiotic agent according to claim 3 or 4, in the preparation of formulations for bidirectional regulation of intestinal function.

9. The application according to claim 8, wherein, The bidirectional regulation of intestinal function includes relieving constipation and inhibiting diarrhea.

10. The application according to claim 9, wherein, The diarrhea includes infectious diarrhea and / or inflammatory diarrhea.

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