Lactobacillus mucilaginosus YF28 and application thereof

Abstract

The invention discloses lactobacillus mucus YF28 and application thereof, and belongs to the technical field of microorganisms. The fermented lactobacillus mucus YF28 is separated from Hu sheep manure, is classified and named as the fermented lactobacillus mucus YF28, and has the preservation number of CCTCC (China Center For Type Culture Collection) NO: M 20252433. The fermented lactobacillus mucilaginosus YF28 has good tolerance in the gastrointestinal environment and can reach the intestinal tract to play a role. The bacterial strain has strong intestinal adhesion capacity, is easy to form a probiotic biological membrane, and is beneficial to intestinal health. The fermented lactobacillus mucus YF28 can promote peristalsis, enrich the abundance of Akkermansia bacteria and bacteriophage thereof, restore the water content of excrement to a normal level, and significantly promote intestinal health. The invention has an application prospect in the development of drugs or health-care foods with the function of relieving constipation and related gastrointestinal symptoms.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a fermenting Lactobacillus mucinus YF28 and its applications. Background Technology

[0002] Studies have shown that prolonged accumulation of feces in the intestines of constipated patients may lead to the spread of harmful bacteria, resulting in damage to the intestinal mucosa and exacerbating Akkermansia spp. (…). Akkermansia Imbalance of beneficial gut microbiota such as (Zhang, Y., et al . Gastrointestinal Dysmotility Predisposes to Colitis throughRegulation of Gut Microbial Composition and Linoleic Acid Metabolism. Advanced science (Weinheim, Baden-Wurttemberg, Germany) , 2024, 11(20): e2306297.). Akkermansia It is a Gram-negative bacterium that colonizes the mucosal layer of the intestines of humans and animals and plays an important role in maintaining intestinal health. Akkermansia Its unique survival strategy means it doesn't rely on dietary fiber from the host for its primary energy source, but instead specializes in degrading and utilizing mucin secreted by intestinal epithelial cells as its carbon and nitrogen sources. While degrading mucin, it stimulates host cells to produce more mucin (such as Muc2) and thickens the mucus layer (Ioannou, A., et al .Akkermansia muciniphila: biology, microbial ecology, host interactions and therapeutic potential. Nature reviews Microbiology , 2025, 23(3):162-177.).

[0003] Studies have shown that probiotics, such as lactic acid bacteria, exhibit significant efficacy in relieving gastrointestinal disorders such as constipation. Probiotics have a wide range of sources, with animal sources being one of the important methods for screening lactic acid bacteria. Animal intestinal mucosa, contents, and feces contain a relatively rich variety and quantity of lactic acid bacteria, and animal-derived strains that have adapted to the gastrointestinal environment may exhibit better probiotic characteristics (Shao, Y., et al . Gut Microbiome Analysis and Screening of LacticAcid Bacteria with Probiotic Potential in Anhui Swine. Animals, 2023, 13(24):3812.). Therefore, it is hoped that strains with good probiotic properties can be screened from animal intestinal contents or feces, laying the foundation for the development of foods, functional foods and drugs that can relieve constipation and related gastrointestinal symptoms.

[0004] Fermented Lactobacillus mucinus ( Limosilactobacillus fermentum Lactobacillus fermentum is a Gram-positive facultative anaerobic bacillus belonging to the genus Lactobacillus, widely distributed in the oral cavity and intestinal environment. As a commonly used strain in the food industry, this bacterium can improve the digestibility of dairy products and produce probiotic activity. Currently, no enrichment of Lactobacillus fermentum has been observed. Akkermansia Reports on the activity of beneficial gut microbiota, etc. Summary of the Invention

[0005] The purpose of this invention is to provide a new strain of fermenting Lactobacillus mucinus that can regulate the intestinal microecology and effectively prevent or treat constipation, and to apply it to the development of medicines or functional foods that relieve constipation.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention isolated and screened a novel strain suspected to be lactic acid bacteria from sheep feces. Based on microbiological characteristics and 16S rDNA gene comparison, the strain was identified as belonging to the order Lactobacillus (Lactobacillus). Lactobacillales Lactobacillus family ( Lactobacillaceae Lactobacillus ( ) Lactobacillus ), fermenting mucinous lactobacillus strain ( Limosilactobacillus fermentum Therefore, it was named *Lactobacillus fermentum*. Limosilactobacillus fermentum YF28.

[0007] Lactobacillus fermentans YF28 was deposited on November 3, 2025 at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), with accession number CCTCC NO: M 20252433.

[0008] The culture conditions for the fermenting *Lactobacillus mucinus* YF28 are as follows: the strain is inoculated into MRS medium and cultured anaerobically at 35℃-37℃. Specifically, the strain is inoculated into MRS liquid medium, placed in an anaerobic bag, and cultured anaerobically at 37℃ for 24 h for activation; the activated strain is then streaked onto MRS solid medium, placed in an anaerobic bag, and cultured anaerobically at 37℃ for 48 h, and pure colonies are picked.

[0009] In vitro studies on tolerance to simulated gastrointestinal environments revealed that the viable count of *Lactobacillus fermentans* YF28 remained above 90% in a simulated gastric fluid environment at pH 2.5. Furthermore, after incubation for 3 hours in simulated intestinal fluid at pH 8.0, the survival rate reached 42.67%, indicating that the *Lactobacillus fermentans* YF28 provided by this invention has good tolerance to the gastrointestinal environment and can reach the intestines to exert its effects.

[0010] The results of the self-agglutination and hydrophobicity experiments showed that the aggregation rate of *Lactobacillus fermentatus* YF28 reached 81.43% at 24 h; the hydrophobicity was higher than 70%. This indicates that *Lactobacillus fermentatus* YF28 has a strong adhesion to the intestine, easily forms a biofilm, and exerts beneficial biological activities.

[0011] The Oxford cup antibacterial experiment results showed that the inhibition zone diameters of fermented Lactobacillus mucinus YF28 against Staphylococcus aureus and Escherichia coli were 17.45±1.25 mm and 11.30±0.10 mm, respectively.

[0012] Therefore, the present invention provides the application of fermented Lactobacillus mucinus YF28 in the preparation of drugs that inhibit pathogenic bacteria in the gastrointestinal tract.

[0013] Furthermore, the gastrointestinal pathogens include Staphylococcus aureus and Escherichia coli.

[0014] Validated using a constipation model, fermented Lactobacillus mucinus YF28 significantly increased the levels of Akkermansia spp. in the intestines and feces. Akkermansia The ability to measure the abundance of bacteria and their bacteriophages. Akkermansia These bacteria are beneficial bacteria in the gut and play an important role in maintaining gut health.

[0015] Therefore, this invention provides the application of fermented Lactobacillus mucinus YF28 in the preparation of drugs or health foods that regulate intestinal microecology, wherein regulating intestinal microecology includes improving intestinal... Akkermansia Abundance of bacteria and their bacteriophages. Fermenting *Lactobacillus mucinus* YF28 enriches the gut microbiota. Akkermansia Bacteria and their bacteriophages further promote gut health.

[0016] Further research showed that in a mouse model of constipation, intervention with *Lactobacillus fermentum* YF28 significantly increased intestinal motility in constipated mice, mainly manifested in: a significant shortening of the time to the first black stool, a significant acceleration of intestinal transit rate (even showing no significant difference from the normal group), and a significant increase in fecal water content and fecal quantity to normal levels. In addition to promoting intestinal motility, intervention with *Lactobacillus fermentum* YF28 also improved the intestinal motility of constipated mice. Akkermansia The abundance of bacteria and their bacteriophages was significantly increased, which was closely related to the improvement of constipation and the recovery of intestinal motility.

[0017] Therefore, this invention provides the application of fermented Lactobacillus mucinus YF28 in the preparation of drugs or health foods for relieving constipation.

[0018] Furthermore, the fermented Lactobacillus mucinus YF28 relieves constipation by promoting intestinal peristalsis and increasing fecal water content.

[0019] Furthermore, the fermented Lactobacillus mucinus YF28 relieves constipation by promoting the expression of the colonic Muc2 gene.

[0020] Furthermore, the fermented *Lactobacillus mucinus* YF28 enriches the intestinal tract. Akkermansia The bacteria and their bacteriophages can thus relieve constipation.

[0021] Furthermore, the constipation described is functional constipation. The type of constipation is determined according to the Rome III diagnostic criteria for functional constipation.

[0022] Pharmaceutical compositions can be prepared using *Lactobacillus fermentum* YF28 of the present invention. These pharmaceutical compositions contain a pharmaceutically effective dose of live *Lactobacillus fermentum* YF28, with each single dose typically containing approximately 10 [units of something - likely a specific strain]. 8 ~10 10 CFU. In addition, the pharmaceutical composition may contain a suitable drug carrier and can be formulated into capsules, powders, etc. The pharmaceutical composition can be used for enrichment. Akkermansia The abundance of bacteria and their bacteriophages, prevention and treatment of constipation, or improvement of constipation-related gastrointestinal symptoms.

[0023] Specifically, the present invention provides a pharmaceutical composition for the prevention or treatment of constipation, the pharmaceutical composition comprising an effective dose of fermenting Lactobacillus mucinus YF28 and a pharmaceutically acceptable carrier.

[0024] Furthermore, the effective dose of *Lactobacillus fermentum* YF28 in the pharmaceutical composition is 10... 8 ~10 10 CFU.

[0025] Furthermore, the pharmaceutical composition is formulated as an oral formulation.

[0026] The fermentation of Lactobacillus mucilaginosus YF28 of the present invention can be used to prepare food or food additives, etc., and the food or food additives can be used for enrichment. Akkermansia The abundance of bacteria and their bacteriophages can relieve constipation and improve the user's health.

[0027] Specifically, the present invention provides a health food for regulating intestinal flora or relieving constipation, comprising fermented Lactobacillus mucinus YF28 as an active ingredient and food science acceptable excipients.

[0028] Furthermore, the health food can be in the form of a beverage containing fermented Lactobacillus mucinus YF28.

[0029] As a specific embodiment of the present invention, the present invention provides a microbial preparation, wherein the microbial preparation is a powder obtained by suspending Lactobacillus fermentum YF28 in a sucrose solution with a mass-volume percentage of 30% and then freeze-drying.

[0030] The beneficial effects of this invention are as follows: This invention provides a novel strain of *Lactobacillus mucinus* YF28, which, through in vitro probiotic performance testing and animal model validation, demonstrates its ability to effectively enrich *Lactobacillus mucinus*. Akkermansia The abundance of bacteria and their bacteriophages can alleviate constipation. *Lactobacillus fermentum* YF28 exhibits good tolerance in the gastrointestinal environment and can reach the intestines to exert its effects. This strain has strong intestinal adhesion ability, easily forming probiotic biofilms, which are beneficial to intestinal health. *Lactobacillus fermentum* YF28 can promote intestinal peristalsis and enrich... Akkermansia The abundance of bacteria and their bacteriophages can restore fecal water content to normal levels, significantly promoting intestinal health. The fermented *Lactobacillus mucinus* YF28 provided by this invention enriches... Akkermansia The abundance of bacteria and their phages, and their application in relieving constipation, represent a novel approach to improving constipation symptoms and are of great significance for constipation treatment. They also show promise in the development of drugs or health foods that alleviate constipation and related gastrointestinal symptoms. Attached Figure Description

[0031] Figure 1 The image shows the isolation and identification of Lactobacillus fermentum from sheep feces. The top image shows the colony morphology, and the bottom image shows the morphology of the strain after Gram staining. The scale bar is 10 μm.

[0032] Figure 2 This study investigates the in vitro simulated gastrointestinal environment tolerance of fermenting Lactobacillus mucinus YF28.

[0033] Figure 3 To determine the aggregation ability of fermenting Lactobacillus mucinus YF28.

[0034] Figure 4 Hydrophobicity determination of fermenting Lactobacillus mucilaginosus YF28.

[0035] Figure 5 The effect of fermented Lactobacillus mucinus YF28 on body weight gain in constipated mice is shown in the figure. Different letters a and b in the figure represent significant differences between groups. P <0.05).

[0036] Figure 6 The effect of fermented Lactobacillus mucinus YF28 on food intake in constipated mice.

[0037] Figure 7 The effect of fermented Lactobacillus mucinus YF28 on the number of fecal particles in constipated mice within 5 hours is shown in the figure. Different letters a and b in the figure represent significant differences between groups. P <0.05).

[0038] Figure 8 The effect of fermented Lactobacillus mucinus YF28 on the time to first black stool in constipated mice is shown in the figure. Different letters a, b, and c represent significant differences between groups. P <0.05).

[0039] Figure 9 The effect of fermented Lactobacillus mucinus YF28 on fecal wet weight in constipated mice within 5 hours is shown in the figure. Different letters a and b in the figure represent significant differences between groups. P <0.05).

[0040] Figure 10 The effect of fermented Lactobacillus mucinus YF28 on fecal moisture content in constipated mice is shown in the figure. Different letters a and b in the figure represent significant differences between groups. P <0.05).

[0041] Figure 11 The effect of fermented Lactobacillus mucinus YF28 on the expression level of the colonic mucin Muc2 gene in constipated mice.

[0042] Figure 12 To investigate the regulatory effect of fermented Lactobacillus mucinus YF28 on the top 20 genera in terms of relative abundance in the feces of constipated mice.

[0043] Figure 13 The effect of fermented Lactobacillus mucinus YF28 on feces of constipated mice Akkermansia The enrichment effect of bacteria.

[0044] Figure 14 In mouse feces Akkermansia Correlation analysis between bacteria and constipation-related indicators.

[0045] Figure 15 The effect of fermented Lactobacillus mucinus YF28 on the contents of the cecal colon of constipated mice Akkermansia Regulatory role of bacteriophages. Detailed Implementation

[0046] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0048] Example 1: Isolation and identification of Lactobacillus fermentation from sheep feces (1) Isolation and screening of strains Collected sheep feces samples were placed in sterile bags, sterile physiological saline was added, and the mixture was thoroughly mixed. A 10-fold serial dilution was then performed, and 100 μL of each sample was spread onto MRS solid culture media containing 0.04% bromocresol purple. The media were anaerobically incubated at 37°C for 48 h. Yellow colonies of different sizes and shapes exhibiting typical lactic acid bacteria characteristics were picked and streaked until the purified colonies showed a uniform morphology. The purified bacteria were then cultured in MRS broth, and the culture was stored in glycerol at a final concentration of 25% at -80°C.

[0049] Results: Four strains suspected to be lactic acid bacteria were isolated from the feces of Hu sheep and named L34, YF28, YF38 and YF42, respectively.

[0050] like Figure 1 As shown, when lactic acid bacteria in feces were screened on MRS medium supplemented with bromocresol purple, the colonies turned distinctly yellow. The more pronounced the yellow color, the stronger the acid-producing capacity of the strain. After the lactic acid bacteria were selected, isolated, purified, and subjected to Gram staining, the results showed that all isolated strains were blue-purple, indicating they were Gram-positive bacteria. Some strains were rod-shaped and arranged in chains of varying lengths; some strains were spherical or oval and clustered together, while others were scattered individually, indicating that different lactic acid bacteria have different morphologies.

[0051] (2) Molecular identification of 16S rDNA of the strain DNA was extracted using lysozyme and then used as a template for PCR amplification. Primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used as upstream and downstream primers to amplify the bacterial 16S rDNA gene by PCR. The gene was then sequenced, and the bacterial type was identified based on the sequencing results.

[0052] The total volume of the PCR amplification system was 50 μL (1 μL DNA template, 29.5 μL premix, 1 μL each of forward and reverse primers, and 17.5 μL dd H2O). The amplification conditions were: 94 ℃ for 4 min 30 s, 94 ℃ for 50 s, 49 ℃ for 30 s, and 72 ℃ for 1 min 20 s, for 32 cycles.

[0053] Prepare a 1% agarose gel. After performing agarose gel electrophoresis on the PCR products, send the PCR amplification products to Qingke Biotechnology Co., Ltd. for sequence detection. Then, perform BLAST comparison analysis on NCBI for the obtained DNA sequences.

[0054] Results: Based on biological characteristics and 16S rDNA gene alignment, L34, YF28, YF38, and YF42 were confirmed as *Lactobacillus fermentatus* (a type of *Lactobacillus*). Limosilactobacillus fermentum ).

[0055] Specifically, the 16S rDNA gene sequence of YF28 is shown in SEQ ID NO.1. Based on biological characteristics and 16S rDNA gene alignment, it was identified as belonging to the order Lactobacillus (Lactobacillus). Lactobacillales Lactobacillus family ( Lactobacillaceae Lactobacillus ( ) Lactobacillus ), fermenting mucinous lactobacillus strain ( Limosilactobacillus fermentum It was named *Lactobacillus fermentatus* (Fermenting Mucus Lactobacillus). Limosilactobacillus fermentum YF28 was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on November 3, 2025, with accession number CCTCC NO: M 20252433, and was identified as viable on November 10, 2025.

[0056] Example 2: In vitro screening of four strains of Lactobacillus fermentum (1) Antibacterial ability test Using the Oxford cup method, Staphylococcus aureus CMCC(B)26003 and Escherichia coli CMCC(B)44102 were cultured overnight and then diluted to 10⁻⁶. 6 CFU / mL, take 100 μL and spread it evenly on LB solid medium. Carefully place the Oxford cup and gently press it to ensure close contact between the Oxford cup and the LB medium. Let it stand for 30 min. Lactobacillus fermentum L34, YF28, YF38, and YF42, and Lactobacillus rhamnosus LGG were cultured in MRS broth for 24 h. After centrifugation at 8000 r / min for 5 min, 150 μL of the supernatant was added to each Oxford cup. The plates were then incubated at room temperature for 4–6 h until the supernatant had completely diffused into the agar. They were then transferred to an incubator and incubated at 37℃ for 24 h. The diameter of the inhibition zone (mm) was observed and measured with calipers.

[0057] Results: Antibacterial activity is an important indicator for evaluating the probiotic properties of probiotics. All four strains of Lactobacillus fermentum showed inhibitory effects against Staphylococcus aureus and Escherichia coli, and their inhibitory effects were superior to those of strain LGG. The inhibitory effects are shown in Table 1.

[0058] Table 1: Antibacterial ability of bacterial strains

[0059] Note: L34, YF28, YF38, and YF42 are all Lactobacillus fermentans ( Limosilactobacillus fermentum ).

[0060] (2) Simulating gastrointestinal fluid tolerance After culturing the strain overnight in MRS broth, centrifuge at 8000 r / min for 5 min, wash twice with PBS, resuspend in PBS, and adjust OD. 600 1. Add 1 mL of bacterial suspension to 9 mL of simulated gastric fluid (pH 2.5, containing 0.3% pepsin) and incubate at 37°C on a shaker at 150 rpm for 3 h. Separately, add 1 mL of bacterial suspension to 9 mL of simulated intestinal fluid (pH 8.0, containing 0.1% trypsin and 0.3% ox bile salts) and incubate at 37°C on a shaker at 150 rpm for 3 h. Count the colonies of the strains before and after adding the simulated gastric and intestinal fluids, and calculate the survival rate.

[0061] The results are as follows Figure 2 As shown, L34, YF28, and YF42 all had a survival rate of over 90% in simulated gastric fluid, while the survival rate dropped rapidly in simulated intestinal fluid, with YF28 having the highest survival rate of 42.67%.

[0062] (3) Determination of self-aggregation ability and hydrophobicity After culturing the strain for 24 h, the bacterial suspension was centrifuged (8000 r / min, 5 min, 4℃), washed with PBS (pH=7.4) buffer, centrifuged again, and the washing was repeated twice to remove residual culture medium. The suspension was then resuspended in PBS buffer, and the OD of the bacterial suspension was adjusted. 600 Adjust the concentration to 0.6-1.0 and let it stand at 37℃ for 24 hours. At 3, 6, and 24 hours, take 1 mL of the supernatant and measure the absorbance (OD). 600 The automatic aggregation rate is calculated using the following formula: Self-aggregation rate % = (1-A) t / A0) × 100% Note: A t OD measured at different times (3, 6, 24 h) 600 A0 is the OD measured at 0 h. 600 .

[0063] After culturing the strain for 24 h, centrifuge (8000 r / min, 5 min, 4℃), wash twice with PBS, resuspend in PBS, and adjust OD. 600To a range of 1.0 ± 0.1. Take 3 mL of bacterial suspension into a centrifuge tube, add 1 mL of xylene, shake well and mix for 2 min, then incubate at 37℃ for 1 h. Use a pipette to aspirate the upper aqueous layer and measure the absorbance OD. 600 Hydrophobicity is calculated using the following formula: Hydrophobicity% = (1-A) t / A0) × 100% Note: A t A1 represents the absorbance of different Lactobacillus fermentation strains at 1 h; A0 represents the absorbance at 0 h.

[0064] The results are as follows Figure 3 and Figure 4 As shown, the four *Lactobacillus* strains exhibited similar aggregation abilities, with YF28 showing the highest aggregation rate at the initial 3 and 6 hours, reaching 81.43% at 24 hours. Meanwhile, L34, YF28, and YF38 exhibited high hydrophobicity, all exceeding 70%. Self-aggregation is the phenomenon of bacteria spontaneously forming clusters; strains with strong self-aggregation abilities typically also have strong adhesion abilities, which is beneficial for colonization in the intestine. Surface hydrophobicity affects the physical and chemical properties of bacterial cells; higher hydrophobicity indicates the presence or embedding of hydrophobic components on the bacterial surface, facilitating adhesion between bacterial cells and host intestinal epithelial cells. The results suggest that YF28 possesses excellent potential for intestinal colonization and adhesion.

[0065] Example 3: Evaluation of the constipation-relieving effect of Lactobacillus fermentum in a mouse constipation model (1) Preparation of experimental strains for gavage Lactobacillus fermentum YF28 was activated by incubation in MRS broth at 37°C for 24 h, followed by centrifugation at 8000 r / min at 4°C for 5 min. The resulting bacterial cells were washed twice with PBS and then dissolved in a 30% sucrose solution (w / v, 30 g sucrose dissolved in 100 mL water) to prepare a strain with a concentration of 2.5 × 10⁻⁶. 9 A bacterial suspension of CFUs / mL was prepared and stored at -80°C for later use. A 30% sucrose solution was used as a cryoprotectant for the bacterial cells.

[0066] (2) Experimental design of constipated mice Sixty healthy male BALB / c mice (Shanghai Silex Biotechnology Co., Ltd.), aged 4-5 weeks, were housed in an animal facility. After a week of pre-feeding, the mice were randomly divided into three groups: a blank control group (NCD), a constipation model group (Lop), and a Lactobacillus fermentum treatment group (YF28). Each group consisted of three cages with four mice per cage, for a total of 12 mice per group. The intervention period lasted 18 days. All mice were fed a basal diet with free access to water and food. The experimental environment was maintained at 22±2℃, with a 12-hour day / 12-hour night cycle. The experimental animal research protocol was approved by the Animal Ethics and Welfare Committee of Zhejiang University of Traditional Chinese Medicine (ethics number: IACUC-20240415-24). The mouse experiments strictly adhered to the institutional guidelines established by the Animal Ethics and Welfare Committee of Zhejiang University of Traditional Chinese Medicine.

[0067] Blank control group (NCD): Sterile saline was administered by gavage, followed by normal saline 0.5 h later.

[0068] Constipation model group (Lop): Loperamide hydrochloride (Lop) 0.1 mL / 10g bw was administered by gavage, followed by sterile saline by gavage 0.5 h later.

[0069] Lactobacillus fermentum treatment group (YF28): Lop 0.1 mL / 10g.bw was administered by gavage, followed by Lactobacillus fermentum YF28 0.1 mL / 10g.bw by gavage 0.5 h later.

[0070] (3) Changes in mouse body weight and food intake The initial weight of the mice was measured the day before the formal intervention, and the weight and food intake of the mice were recorded every 4 days.

[0071] The results are as follows Figure 5 and Figure 6 As shown: During the intervention period, there was no significant difference in feed intake among the various mouse groups. Compared with the CON group, the Lop group had a significantly lower body weight, and YF28 significantly slowed down the body weight loss in Lop-induced constipated mice.

[0072] (4) Time of excretion of the first black feces in mice On day 15 after gavage, mice were tested for black feces. The experimental method was as follows: On the evening of day 14, mice were fasted but allowed free water for 16 hours. On day 15, the Lop and YF28 groups were gavaged with 0.1 mL / 10g bw (10 mg / kg bw), while the NCD group was gavaged with the same volume of physiological saline. 30 minutes later, all mice in the experimental groups were gavaged with black ink, and timing was started immediately. At the same time, the mice were placed in metabolic cages and immediately resumed normal diet. The time of the first black feces excreted by each mouse was observed and recorded. The time of the first black feces excreted by the last mouse in the model group was used as the end time. Treatment groups that exceeded the time of the first black feces excreted by the model group were considered ineffective. The number of feces, total fecal weight (wet weight), and fecal water content were also recorded within 5 hours. Method for testing fecal moisture content: After drying the feces at 110℃ for 5 hours, place them in a desiccator to cool and weigh them. Fecal moisture content % = (wet weight - dry weight) / wet weight × 100%.

[0073] Constipation is characterized by impaired bowel function, with main symptoms including difficulty in defecation, infrequent bowel movements, and hard, dry stools. Therefore, the state of the stool is an important criterion for diagnosing constipation. Results as follows: Figures 7 - 10 As shown: Compared to the NCD group, the Lop group showed a significant reduction in fecal particle count and fecal moisture content, indicating the success of the constipation model established by Lop. In contrast, YF28 intervention significantly increased fecal volume and significantly restored the decrease in moisture content induced by Lop. The fecal wet weight of the YF28 group was significantly higher than that of the NCD and Lop groups. These results demonstrate that YF28 intervention significantly improved fecal condition in mice compared to the Lop group.

[0074] Furthermore, after treatment with loperamide hydrochloride, the time to excretion of the first black stool in the Lop model group was significantly prolonged, consistent with constipation characteristics. After YF28 intervention, the time to excretion of the first black stool in mice was significantly shortened. A shorter time to excretion of the first black stool indicates more normal intestinal motility, suggesting that YF28 has a constipation-relieving effect.

[0075] (5) Mouse intestinal mucin Muc2 Relative expression level measurement RNA was extracted from approximately 1 cm of mouse colon tissue using the Trizol method, then purified and resuspended in RNase-free deionized water. cDNA was synthesized according to the manufacturer's instructions, and the reaction mixture was incubated at 50 °C and 85 °C. RT-qPCR reactions were established in 384-well plates using primers... ​ -F (5′-ATGCCCACCTCCTCAAAGAC-3′) and ​ -R (5′-GTAGTTTCCGTTGGAACAGTGAA-3′) served as the upstream and downstream primer pair. ​Genes are amplified; ​ As an internal reference gene, primers were used. ​ -F (5′-AGGTCGGTGTGAACGGATTTG-3′) and ​ -R (5′-TGTAGACCATGTAGTTGAGGTCA-3′) served as the upstream and downstream primer pair. ​ The gene is amplified; and this is done in the dark on ice.

[0076] The total volume of the PCR amplification system was 10 μL (1 μL DNA template, 5 μL premix, 0.2 μL each of forward and reverse primers, and 3.6 μL dd H2O). The amplification conditions were: 95 ℃ for 4 min 30 s, 95 ℃ for 5 s, 55 ℃ for 30 s, 72 ℃ for 1 min 20 s, for 39 cycles.

[0077] The mucin Muc2 secreted by goblet cells is crucial for colonic motility. Results are as follows... ​ As shown, Lop-induced constipation in mice led to a decrease in colonic Muc2 gene expression, but YF28 showed a trend of promoting an increase in colonic Muc2 gene expression (P = 0.075). This suggests that *Lactobacillus mucinus* YF28 exerts its constipation-relieving effect by promoting colonic Muc2 gene expression in constipated mice.

[0078] Example 4: Evaluation of the effects of fermented Lactobacillus mucin on a mouse constipation model ​ The promoting effect of genus 16S rDNA sequencing: Total DNA was extracted from fecal samples of mice in each experimental group of Example 3 using the QIAamp DNA Stool Mini Kit. The quality of the extracted DNA was assessed using a Thermo nanodroplet spectrophotometer and agarose gel electrophoresis. The 16S rDNA gene V3-V4 region was amplified and sequenced using the Illumina Hi Seq PE250 platform, and the data was analyzed using the Meiji Cloud platform.

[0079] The results are as follows ​ As shown, at the genus level, compared with the NCD group, the abundance of the genera *norank_o_Clostridia_UGG_014*, *Rikenellaceae_RC9_gut_group*, and *Faecalibaculum* increased in the Lop group, while... ​ , ​ , ​ Reduced abundance. YF28 intervention reversed constipation-induced bowel movement disorders. ​ , Rikenellaceae_RC9_gut_group, ​ and​ Variations in genera abundance. For example... ​ As shown, the YF28 intervention group was significantly enriched. ​ bacteria, and such ​ As shown, and ​ Significantly positively correlated with fecal particle number (r = 0.593, P = 0.007), significantly negatively correlated with time to excretion of the first black feces (r = -0.621, P = 0.01), and not significantly correlated with fecal wet weight and fecal water content; suggesting significant enrichment in the YF28 intervention group. ​ The bacteria were positively correlated with the relief of constipation in mice.

[0080] Example 5: Evaluation of the promoting effect of Lactobacillus fermentum on Akkermansia phage in a mouse constipation model Metavirome sequencing: Myxophages collected from mice in each experimental group of Example 3 were purified and libraries constructed according to the kit instructions. Then, ~400 bp DNA target fragment libraries were selected on 2% Low Range Ultra agarose gels and sequenced using an Illumina NovaSeq 6000 (paired-end, 2 × 150 bp reads). Raw sequencing data were preprocessed and quality-controlled using Trimmomatic software to obtain high-quality reads for subsequent analysis.

[0081] In the enterovirome, bacteriophages are absolutely dominant (accounting for over 90%). Therefore, the abundance and stability of Akkermansia bacteriophages are largely regulated by complex bacteriophage-Akkermansia bacteria interactions. These interactions include lysis-latency cycles, horizontal gene transfer, and various ecological relationships ranging from parasitism to conditional symbiosis. These processes not only shape the abundance and function of Akkermansia but also play an indispensable role in maintaining healthy homeostasis of the host gastrointestinal tract (Cao, Z., ​ . The gut virome: A new microbiomecomponent in health and disease. ​ , 2022, 81 :104113).

[0082] Metavirome sequencing results as follows ​As shown, at the species level, compared with the Lop group, YF28 intervention significantly enriched Akkermansia_phage_DTMo.2021a, Privateervirus_privateer, Streptococcus_phage_Javan628, Punavirus_RCS47, Clostridium_phage_vB_CtyS-FA88, Lactobacillus_phage_JNU_P4, Phage_vB_RanS_PJN03 and other phages, and the trend also increased. However, YF28 significantly reduced the relative abundance of Enterobacter_phage_EC151, Acidithiobacillus_phage_AcaML1, Bacteroides_phage_LoVEphage, and Bacteroides_phage_p00.

[0083] Bacteriophages have a significant impact on the dynamic balance of gut microbiota. As one of the core gut microbiota, the abundance of Akkermansia is regulated by phage infection. YF28 may alleviate symptoms such as constipation in mice by enriching Akkermansia_phage_DTMo.2021a.

[0084] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A fermenting Lactobacillus mucinus YF28, characterized in that, The fermenting myxobacterium YF28 was isolated from sheep feces and classified as fermenting myxobacterium (…). Limosilactobacillus fermentum YF28, with accession number CCTCCNO: M 20252433.

2. The fermented Lactobacillus mucinus YF28 as described in claim 1, characterized in that, The culture conditions for the fermenting Lactobacillus mucinus YF28 are as follows: the strain is inoculated into MRS medium and cultured under anaerobic conditions at 35℃-37℃.

3. The application of the fermented Lactobacillus mucinus YF28 as described in claim 1 or 2 in the preparation of drugs or health foods for regulating intestinal microecology, characterized in that, The regulation of the intestinal microecology includes increasing the number of Akkermansia spp. in the intestine. Akkermansia Abundance of bacteria and their bacteriophages.

4. The use of the fermented Lactobacillus mucinus YF28 as described in claim 1 or 2 in the preparation of drugs or health foods for relieving constipation.

5. The application as described in claim 4, characterized in that, The fermented Lactobacillus mucinus YF28 promotes intestinal peristalsis, increases fecal water content, promotes colonic Muc2 gene expression, and enriches the intestinal tract. Akkermansia The bacteria and their bacteriophages can thus relieve constipation.

6. A pharmaceutical composition for preventing or treating constipation, characterized in that, The pharmaceutical composition comprises an effective dose of the fermenting Lactobacillus mucinus YF28 as described in claim 1 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, The effective dose of *Lactobacillus fermentum* YF28 in the pharmaceutical composition is 10. 8 ~10 10 CFU.

8. A microbial preparation, characterized in that, The microbial preparation is a powder obtained by suspending the bacterial weight of Lactobacillus fermentum YF28 as described in claim 1 in a sucrose solution with a mass-volume percentage of 30%, and then freeze-drying it.

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