Application of Limosilactobacillus fermentum WMSN-2 in the prevention of necrotizing enterocolitis in newborns

Abstract

This invention discloses the application of *Limosilactobacillus fermentum* WMSN-2 in the prevention of necrotizing enterocolitis in newborns, belonging to the field of probiotics. The *Limosilactobacillus fermentum* WMSN-2 strain provided by this invention possesses antibacterial activity, antibiotic sensitivity, and good tolerance to gastrointestinal fluids. It also has preventive and therapeutic effects on necrotizing enterocolitis in newborns, reducing the disease activity index, improving survival rate, and alleviating small intestinal shortening and villus damage. The *Limosilactobacillus fermentum* WMSN-2 strain provided by this invention can be used to prepare drugs for the prevention and treatment of necrotizing enterocolitis in newborns, showing broad application prospects.

Description

Technical Field

[0001] This invention relates to the application of Limosilactobacillus fermentum WMSN-2 in the prevention of necrotizing enterocolitis in newborns, which falls under the field of probiotics. Background Technology

[0002] Necrotizing enterocolitis (NEC) is a common and critical gastrointestinal disease in newborns, characterized by intestinal mucosal edema, hemorrhage, and intestinal necrosis. NEC is most common in premature infants, accounting for more than 90% of all cases. It has a high mortality rate, and surviving infants often suffer from serious complications such as short bowel syndrome, intestinal stenosis, and neurological sequelae, which reduce the quality of life of these children and increase the social burden.

[0003] Although NEC has been recognized clinically for many years, its core pathogenesis remains unclear. Current research on the mechanisms of NEC mainly focuses on intestinal barrier function and gut microbiota dysbiosis. The human and animal gut contains a vast community of microorganisms that adhere to the intestinal mucosa, forming a biological barrier. Studies have shown that the gut microbiota in the feces of children with NEC is disordered, with a reduced abundance of lactobacilli. Lactic acid bacteria are the most commonly used probiotics, playing an important role in protecting the host from harmful organisms, enhancing the host's immune system, and reducing metabolic disorders. *Limosilactobacillus fermentum* is a Gram-positive bacterium of the genus *Lactobacillus*. Many *Limosilactobacillus fermentum* species can enhance the body's immune response and prevent epidemic gastrointestinal and upper respiratory tract infections. However, whether *Limosilactobacillus fermentum* has a potential regulatory role in neonatal necrotizing enterocolitis remains unknown. Summary of the Invention

[0004] This invention provides a strain of fermenting *Lactobacillus mucilaginosus* (… Limosilactobacillus fermentum WMSN-2 was deposited on October 23, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 63904), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0005] The present invention provides a microbial preparation containing the aforementioned fermenting Lactobacillus mucinus WMSN-2.

[0006] In one embodiment, the content of *Lactobacillus mucinus* WMSN-2 in the microbial preparation is ≥1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0007] The present invention also provides a composition containing the aforementioned fermenting Lactobacillus mucinus WMSN-2.

[0008] In one embodiment, the composition is a drug.

[0009] In one embodiment, the drug further comprises a pharmaceutically acceptable carrier.

[0010] In one embodiment, the pharmaceutically acceptable carrier includes a drug carrier or a drug excipient.

[0011] In one embodiment, the pharmaceutical excipient comprises excipients and / or additives.

[0012] In one embodiment, the pharmaceutical excipients include anti-adhesives, penetration enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, encapsulating agents, absorbents, humectants, solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, pH adjusters, binders, disintegrants, fillers, lubricants, wetting agents, integrators, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and anti-flocculation agents, filter aids, and release inhibitors.

[0013] In one embodiment, the dosage form of the drug includes granules, capsules, tablets, pills, or oral liquid.

[0014] The present invention also provides the use of fermented Lactobacillus mucinus WMSN-2 in the preparation of medicaments for the prevention and / or treatment of neonatal necrotizing enterocolitis.

[0015] In one implementation, the application includes at least one of (1) to (4):

[0016] (1) Alleviate weight loss and reduce disease activity index in patients with neonatal necrotizing enterocolitis;

[0017] (2) Improve the survival rate of neonatal necrotizing enterocolitis patients;

[0018] (3) Relieves symptoms of small bowel shortening;

[0019] (4) Relieve damage to ileal villi.

[0020] In one embodiment, the fermenting *Lactobacillus mucinus* WMSN-2 is a biologically active live cell or an inactivated cell obtained after treatment.

[0021] In one embodiment, the fermenting Lactobacillus mucinus WMSN-2 strain is used in the form of a live bacterial preparation or a sterilized bacterial agent.

[0022] In one embodiment, the product contains ≥1×10⁻⁶ fermented Lactobacillus mucinus WMSN-2. 9 CFU / g or 1×10 9 CFU / mL.

[0023] Beneficial effects:

[0024] This invention provides a novel use of *Lactobacillus fermentum* strain WMSN-2 in drugs for the prevention and protection of neonatal necrotizing enterocolitis. Through experiments, this invention verifies that *Lactobacillus fermentum* strain WMSN-2 effectively reduces the disease activity index, improves survival rate, and alleviates small intestinal shortening and villus damage in patients with neonatal necrotizing enterocolitis, showing better efficacy than the positive control strain *Lactobacillus rhamnosus* GG (LGG). The *Lactobacillus fermentum* strain WMSN-2 provided by this invention can be used to prepare drugs and pharmaceutical compositions for the prevention and protection of neonatal necrotizing enterocolitis, thus broadening the application field of *Lactobacillus fermentum* strain WMSN-2.

[0025] Preservation of biological materials

[0026] Fermented Lactobacillus mucinus ( Limosilactobacillus fermentum WMSN-2, taxonomically named Limosilactobacillus fermentum It was deposited on October 23, 2023 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63904, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0027] Figure 1 The colony morphology of fermenting Lactobacillus mucinus WMSN-2.

[0028] Figure 2 Electrophoretic identification image of Lactobacillus fermentans WMSN-2 amplified using Lactobacillus-specific primers.

[0029] Figure 3 The survival rate of fermenting Lactobacillus mucinus WMSN-2 in simulated digestive fluid.

[0030] Figure 4This study investigated the changes in the disease activity index of neonatal necrotizing enterocolitis (NZOC) pups treated with *Lactobacillus mucinus* WMSN-2. In the data, Con represents the sterile saline control group, WMSN-2 represents the *Lactobacillus mucinus* WMSN-2 control group, Model represents the NZOC model group, WMSN-2+M represents the *Lactobacillus mucinus* WMSN-2 treatment group for NZOC prevention, and LGG+M represents the *Lactobacillus rhamnosus* GG positive control group for NZOC prevention. *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0031] Figure 5 The effect of fermenting Lactobacillus mucinus WMSN-2 on the survival rate of neonatal necrotizing enterocolitis pups. *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0032] Figure 6 The fermentation of Lactobacillus mucinus WMSN-2 was used to prevent changes in small intestinal length in necrotizing enterocolitis. *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0033] Figure 7 The application of fermented Lactobacillus mucinus WMSN-2 in the prevention of pathological changes in ileal tissue during necrotizing enterocolitis. *: P < 0.05, **: P < 0.01, ***: P < 0.001. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions in the art or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art.

[0035] Lactobacillus rhamnosus ( Lactobacillus rhamnosus GG (ATCC 7469) was obtained from the American Type Culture Collection (ATCC); Lactobacillus fermentum WMSN-2 was isolated from the feces of exclusively breastfed healthy infants in Wuxi City, Jiangsu Province.

[0036] MRS medium: 10g soybean peptone, 5g beef extract, 5g yeast powder, 20g glucose, 1mL Tween-80, 2g sodium dihydrogen phosphate, 5g anhydrous sodium acetate, 2g triamine citrate, 0.02g manganese sulfate, 0.1g magnesium sulfate, 1L distilled water, adjust pH to approximately 6.2, 15g agar, sterilize at 121℃ for 15min.

[0037] Example 1: Screening of fermenting Lactobacillus mucinus WMSN-2

[0038] 1. Screening of WMSN-2 strains

[0039] Approximately 5g of fresh sample (feces from a healthy, exclusively breastfed infant in Wuxi City, Jiangsu Province) was collected in a sterile tube and immediately sent to the laboratory for bacterial isolation. 1g of sample was added to 9mL of MRS broth medium, vortexed, and incubated at 37℃ in an anaerobic incubator for 48h to enrich the sample. Then, 1mL of the enriched solution was extracted in a laminar flow hood and serially diluted tenfold with sterile physiological saline. 10... -6 10 -7 10 -8 Three dilution gradients were used, with 100 μL of bacterial culture from each gradient plated onto MRS agar medium and anaerobically incubated at 37°C for 48 h. After incubation, plates with 50-150 single colonies were selected from the agar medium. Colonies with different morphologies, sizes, and colors were picked and streaked multiple times on MRS agar plates for purification until the colonies on the entire plate had a uniform morphology. Single colonies were then picked and enriched in MRS broth medium. All obtained strains were stored frozen at -80°C in MRS broth medium containing 40% glycerol.

[0040] 2. Identification of WMSN-2 strain

[0041] After culturing strain WMSN-2 on MRS agar medium for 48 hours, the colonies were 0.3-1.5 mm in diameter, round with neat edges, white in color, and moist and smooth in surface. Figure 1 .

[0042] Genomic DNA of the target strain was extracted using the Ezup column-based bacterial genomic DNA extraction kit. The extracted lactic acid bacteria genomic DNA was used as a template for PCR amplification. PCR experiments were performed using lactobacillus-specific primers. After the PCR reaction, the PCR product was examined and photographed on an agarose gel. The amplified fragment length was approximately 550 bp. (See attached image.) Figure 2 Subsequently, PCR experiments on 16S rDNA were performed using universal bacterial primers 27F and 1492R. After the PCR reaction amplification was completed, the PCR product was examined and photographed using agarose gel electrophoresis. The amplified fragment length was approximately 1.2 kbp. The primers were used to send the PCR product to Shanghai Sangon Biotech Co., Ltd. for sequencing. BLAST sequence alignment was performed on the NCBI website, and the results showed that the sequence had more than 99% homology with the 16S rDNA sequence of Lactobacillus fermentum.

[0043] By combining the sequence alignment results and physiological and biochemical results of strain WMSN-2, the screened lactic acid bacteria WMNS-2 was identified as *Lactobacillus fermentum*. Limosilactobacillus fermentumWMSN-2.

[0044] Example 2: Confirmation of the digestive fluid tolerance of Lactobacillus mucinus strain WMSN-2

[0045] 1. Preparation of simulated digestive fluid:

[0046] (1) Preparation of simulated saliva: After sterilizing PBS, add 3 g / L α-amylase (purchased from Sigma-Aldrich, USA, catalog number: 9000-90-2, enzyme activity 300-1500 U / mg protein), 6.2 g / L NaCl, 2.2 g / L KCl, 0.22 g / L CaCl2 and 1.2 g / L NaHCO3, filter with a 0.22 μm microporous membrane to sterilize, and prepare simulated saliva.

[0047] (2) Preparation method of simulated gastric juice: After sterilizing PBS, add 3.0 g / L pepsin (purchased from Sigma-Aldrich, USA, catalog number: 9001-75-6, enzyme activity ≥2000U / mg protein), 3 g / L NaCl, 1.1 g / L KCl, 0.15 g / L CaCl2 and 0.6 g / L NaHCO3, adjust the pH value to 2.5 with 1 mol / L HCl, filter and sterilize with a 0.22 μm microporous membrane to prepare simulated gastric juice.

[0048] (3) Preparation method of simulated intestinal fluid: After sterilizing PBS, add 3 g / L ox bile salt (purchased from Sigma-Aldrich, USA, catalog number: 48305), 0.1 g / L lipase (purchased from Sigma-Aldrich, USA, catalog number: 9001-62-1, enzyme activity ≥20,000 U / mg protein), 1 g / L trypsin (purchased from Sigma-Aldrich, USA, catalog number: 9002-07-7, enzyme activity 1,000-2,000 U / mg protein), 5 g / L NaCl, 0.6 g / L KCl, 0.3 g / L CaCl2 and 0.6 g / L NaHCO3, adjust the pH value to 8.0 with 0.1 mol / L NaOH, and then filter it with a 0.22 μm microporous membrane to sterilize it, thus preparing simulated intestinal fluid.

[0049] 2. Digestive fluid tolerance test method:

[0050] The selected *Lactobacillus fermentata* strain WMSN-2 was activated and cultured for two generations in MRS medium. After centrifugation and washing twice, the cells were collected, resuspended in 1 mL of simulated saliva for 5 min, then centrifuged (4℃, 12000 rpm, 2 min) and resuspended in 2 mL of simulated gastric fluid, and incubated at 37℃ for 2 h. Viability was determined using the MRS agar pour method. Each sample was repeated three times, and the average value was calculated.

[0051] Subsequently, the artificially inoculated gastric fluid, digested for 2 hours, was centrifuged again (4°C, 12000 rpm, 2 min) and resuspended in 2 mL of simulated intestinal fluid, and incubated at 37°C for 2 hours. Finally, the bacterial suspension was diluted and inoculated onto MRS agar, and cultured anaerobically at 37°C for 36–48 hours. Viability was determined using the MRS agar pour method. The survival rates of the strains in simulated gastric and intestinal fluids were calculated using the following formula.

[0052] Survival rate (%) = LogN1 / LogN0 × 100;

[0053] Where N1 represents the number of viable bacteria in the bacterial strain system after treatment with simulated gastric or intestinal fluid; N0 represents the initial number of viable bacteria in the bacterial strain system.

[0054] The results are as follows Figure 3 As shown, the survival rate of Lactobacillus fermentum WMSN-2 under simulated human digestive fluid conditions was as high as 54.28%, which was better than that of Lactobacillus rhamnosus GG, which had a survival rate of 52.24%.

[0055] As can be seen from the above results, the fermenting Lactobacillus mucinus WMSN-2 provided by the present invention has excellent gastrointestinal fluid tolerance, can enter the human intestine in a live state, and survive in the gastrointestinal organs of animals, including humans, and exert health benefits. The above characteristics are the basis for the strain to be a probiotic.

[0056] Example 3 Evaluation of the antibacterial effect of fermented Lactobacillus mucinus WMSN-2

[0057] 1. Preparation of fermentation broth for bacterial strains

[0058] The strain preserved in glycerol tubes was first streaked onto MRS agar plates 2-3 times for activation. Then, single colonies were picked and cultured in MRS broth for 18 hours (anaerobic culture at 37°C). The culture medium was then adjusted to a cell concentration of 1×10⁻⁶ cells / mL with distilled water. 9 Centrifuge at 8000 r / min for 20 min at 4℃ for CFU / mL, and collect the supernatant, which is the fermentation supernatant of the bacteria.

[0059] 2. The effect of the strain on inhibiting pathogenic bacteria

[0060] The inhibitory effect of fermented *Lactobacillus mucinus* WMSN-2 on several major enteropathogenic bacteria (including *Staphylococcus aureus* ATCC 25923, *Salmonella enterica* ATCC 14028, *Escherichia coli* ATCC 25922, and *Listeria monocytogenes* ATCC 13932) was detected using the Oxford cup method. Specifically, the bacteria were incubated at 37°C for 16–24 h (approximately 1 × 10⁻⁶ mmol / L). 9Different pathogenic bacteria (CFU / mL) were spread onto their respective solid culture media. Oxford cups were carefully placed on the media, and 200 μL of the bacterial supernatant was added to each cup. After incubation at 37°C for 24 h, the diameter of the inhibition zone was measured. The inhibition zone diameter was scored as follows: ≤7 mm (none, -); 7-15 mm (weak, +); >15 mm (strong, ++). Penicillin (30 mg / mL) was used as a positive control. The corresponding liquid culture medium was used as a negative control. Each sample was repeated three times, and the average value was calculated. The experimental results are shown in Table 1. As can be seen from Table 1, the fermentation supernatant of *Lactobacillus mucinus* WMSN-2 screened by this invention has a strong antibacterial effect.

[0061] Table 1. Inhibitory effects of strains against different pathogenic bacteria

[0062]

[0063] The results above show that the fermenting Lactobacillus mucinus WMSN-2 provided by the present invention has a better inhibitory effect on pathogenic bacteria than Lactobacillus rhamnosus GG, and this characteristic is the basis for the strain to be used as a probiotic.

[0064] Example 4: Antibiotic sensitivity of fermenting Lactobacillus mucinus WMSN-2

[0065] Seven antibiotics (kanamycin, penicillin, vancomycin, erythromycin, ampicillin, streptomycin, and chloramphenicol) were dissolved in appropriate solutions and filtered. *Lactobacillus fermentum* strain WMSN-2 was anaerobically cultured in MRS broth with different final concentrations (2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 μg / mL) of antibiotics at 37°C for 24–36 h. Each sample was repeated three times. After culture, the absorbance at OD610 nm was measured in 96-well plates. The MIC (minimum inhibitory concentration) is considered the lowest antibiotic concentration that can inhibit bacterial growth and is used to assess the antibiotic resistance of selected strains.

[0066] The results are shown in Table 2. The fermenting Lactobacillus mucinus-2 strain screened by this invention showed varying degrees of sensitivity to these antibiotics.

[0067] Table 2. Susceptibility results of strains to different antibiotics

[0068]

[0069] The experimental results above show that the fermenting Lactobacillus mucinus WMSN-2 provided by the present invention is sensitive to different antibiotics, indicating that this probiotic has the potential to be used as a live bacteria product.

[0070] Example 5 Preparation of fermented Lactobacillus mucinus WMSN-2 inoculum

[0071] Preparation of WMSN-2 bacterial agent: Fermenting Lactobacillus mucinus WMSN-2 was strictly cultured in MRS broth. The logarithmic growth phase bacterial culture was centrifuged at 8000 g for 10 min at 4°C, the supernatant was discarded, and the bacterial sludge was washed twice with sterile physiological saline. The bacterial sludge was resuspended in 30% (v / v) glycerol. Optionally, it can be stored at -80°C.

[0072] Preparation of positive LGG bacterial agent: Lactobacillus rhamnosus GG was strictly cultured in MRS broth. The logarithmic growth phase bacterial suspension was centrifuged at 8000 g for 10 min at 4°C, the supernatant was discarded, and the bacterial sludge was washed twice with sterile physiological saline. The bacterial sludge was resuspended in 30% (v / v) glycerol and optionally stored at -80°C.

[0073] Optionally, before use, dilute the WMSN-2 bacterial agent and the positive LGG bacterial agent with sterile physiological saline containing 3% (v / v) glycerol to a concentration of 1×10⁻⁶. 9 CFU / mL available for use.

[0074] Example 6 Preparation of a drug containing *Lactobacillus fermentans* WMSN-2

[0075] The bacterial suspension containing live fermenting Lactobacillus mucinus WMSN-2 cells prepared in Example 3 is mixed with the required excipients to obtain a liquid formulation; optionally, it is compressed into tablets; optionally, the liquid formulation is dried to obtain a powder; optionally, the powder is used as a filler to prepare capsules.

[0076] The pharmaceutical excipients include anti-adhesives, penetration enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, encapsulating agents, absorbents, humectants, solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, pH adjusters, binders, disintegrants, fillers, lubricants, wetting agents, integrators, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and anti-flocculation agents, filter aids, and release inhibitors.

[0077] Example 7: Fermentation of Lactobacillus mucinus WMSN-2 for the prevention of necrotizing enterocolitis in newborns.

[0078] (1) Establishment of a neonatal necrotizing enterocolitis model:

[0079] Six-day-old C57BL / 6J newborn mice were randomly divided into four groups: a saline control group (Con), a *Lactobacillus fermentum* WMSN-2 probiotic control group (WMSN-2), a neonatal necrotizing enterocolitis model group (Model), a *Lactobacillus fermentum* WMSN-24 treatment group (WMSN-2+M), and a *Lactobacillus rhamnosus* GG treatment group (LGG+M), under controlled feeding conditions. From day six to day nine, for four consecutive days, each mouse in the WMSN-2 and WMSN-2+M groups was administered 1×10⁻⁶ probiotic solution containing 3% glycerol via gavage. 9 50 μL of *Lactobacillus mucinus* WMSN-2 bacterial culture (CFU / mL) was administered orally to each LGG+M group infant mouse containing 1×10⁻⁶ CFU / mL glycerol. 9 50 μL of *Lactobacillus rhamnosus* GG culture (CFU / mL) was administered to the Con and Model groups, along with an equal volume of sterile saline containing 3% glycerol. On day 10, a neonatal necrotizing enterocolitis model was induced by gavage and rectal infusion of trinitrobenzenesulfonic acid (TNBS). TNBS was dissolved in saline, and on day 10, 25 μL of TNBS (10 mg / g) was administered to the Model, WMSN-2+M, and LGG+M groups via gavage and rectal infusion, respectively. Puppies in the CON and WMSN-2 groups received an equal volume of saline. When puppies exhibited cachexia or wasting symptoms (or were euthanized after 24 hours if no obvious symptoms were observed), they were anesthetized with 5% isoflurane, blood was drawn from the heart, and they were euthanized by cervical dislocation. The ileum, colon, and their contents were collected.

[0080] (2) Treatment with fermented Lactobacillus mucinus WMSN-2 alleviated weight loss and reduced disease activity index in young mice.

[0081] The Disease Activity Index (DIA), calculated by summing scores for weight change, diarrhea, and bloody stools, is one of the most prominent phenotypes of necrotizing enterocolitis (NEC) in newborns and reflects the severity of the disease. Young mice were weighed at 0, 3, 6, 12, and 24 hours after NEC induction to observe diarrhea and measure bloody stools using an occult blood test kit. Figure 4At 24 hours, compared with the Model group (whose body weight decreased by 13.9% and disease activity index was 3.2), the enema groups significantly alleviated the weight loss and reduced the disease activity index in NEC pups (WMSN-2+M group: body weight decreased by 11.1%, P<0.01; disease activity index: 2.33, P<0.01; LGG+M group: body weight decreased by 11.8%, P<0.01; disease activity index: 2.56, P<0.05), with the fermented Lactobacillus mucinus WMSN-2 showing a better effect, exceeding the LGG+M group by 0.7% and 0.23%, respectively. Furthermore, compared with the Con group, the WMSN-2 group showed similar rates of weight change and disease activity index in pups, indicating that the fermented Lactobacillus mucinus WMSN-2 did not adversely affect the pups' appearance.

[0082] (3) Fermentation of Lactobacillus mucinus WMSN-2 improves the survival rate of young mice

[0083] NEC can severely lead to the death of young mice, and survival rate is another important indicator for evaluating the protective effect of probiotics against NEC in young mice. After inducing NEC in the Model group, WMSN-2+M group, and LGG+M group, the death criterion for young mice was the absence of heartbeat, and the mortality of young mice was observed every 1 hour. Figure 5 In the Model group, pups died at 6 hours, while those in the WMSN-2+M and LGG+M groups died at 13 and 10 hours, respectively. With a 24-hour observation period, the final survival rate of the Model group was only 25%, while the survival rates of the WMSN-2+M and LGG+M groups were 40% and 37.5%, respectively. This indicates that fermented Lactobacillus mucinus WMSN-2 can prolong the survival time and improve the survival rate of NEC pups, and its effect is superior to that of the positive bacterium Lactobacillus rhamnosus GG.

[0084] (4) Treatment with fermented Lactobacillus mucinus WMSN-2 alleviated the symptoms of small intestinal shortening in NEC pups.

[0085] The most obvious pathological features of NEC are small intestinal shortening and inflammatory edema. Young mice in the Con, WMSN-2, Model, WMSN-2+M, and LGG+M groups were sacrificed, and their stomach, small intestine, and cecum were removed intact. The mesentery was carefully dissected, and the degree of small intestinal shortening between the stomach and cecum was assessed using a standard ruler. Figure 6Compared with the Con group, the small intestine length of the Model group was significantly shortened, from 17.2 cm in the CON group to 15.1 cm (P<0.001), and the inflammation and edema were more severe. The shortening of the small intestine length in the positive LGG+M group was significantly alleviated, from 15.1 cm in the Model group to 16.1 cm (P<0.05). The small intestine length in the WMSN-2+M group increased from 15.1 cm in the Model group to 16.5 cm, showing a better preventive and protective effect than the LGG+M group, exceeding the LGG+M group's 16.1 cm by 0.4 cm (P<0.05). Compared with the Con group, the small intestine length in the WMSN-2 group did not change significantly, indicating that the fermenting Lactobacillus mucinus WMSN-2 did not have adverse effects on the small intestine of young mice and showed a better preventive and protective effect than the positive control Lactobacillus rhamnosus GG.

[0086] (5) Treatment with fermented Lactobacillus mucinus WMSN-2 alleviated damage to ileal villi.

[0087] Using HE staining, the ileum from the CON group, WMSN-2 group, Model group, WMSN-2+M group, and LGG+M group underwent fixation, dehydration, staining, dewaxing, clearing, and mounting processes to observe the tissue integrity of the ileum. Figure 7 The results showed that the ileum tissue structure in the Con group and WMSN-2 group was dense, without obvious cracks, and the ileum villi were intact. The Model group showed significantly sparse and broken villi, with a significantly increased HE score of 2.5 points (P<0.001). Compared with the Model group, the WMSN-2+M group and LGG+M group had sparser and partially broken villi, with HE scores decreasing by approximately 1.2 points (P<0.05) and 1 point (P<0.05), respectively. This indicates that *Lactobacillus fermentum* WMSN-2 and *Lactobacillus rhamnosus* GG have protective effects against NEC-induced ileum villi damage. Furthermore, the villi in the WMSN-2+M group were more compact and intact than those in the LGG+M group, suggesting that the protective effect of *Lactobacillus fermentum* WMSN-2 is superior to that of the positive control *Lactobacillus rhamnosus* GG.

[0088] Comparative Example 1:

[0089] The antibacterial effect of *Lactobacillus fermentum* WMSN-2, screened in this invention, was compared with that of other *Lactobacillus fermentum* strains in the prior art. Strains J2-4, J2-5, and J2-9 are disclosed in the paper "Lactic acid bacteria with a strong antioxidant function isolated from 'Jiangshui,' pickles, and feces." Referring to the antibacterial effect evaluation method in Example 3, the inhibition zone diameter was scored as follows: ≤7mm (none, -); 7-15mm (weak, +); >15mm (strong, ++).

[0090] Table 3. Inhibition of pathogenic bacteria by Lactobacillus fermentation

[0091]

[0092] As shown in Table 3, the previously disclosed Lactobacillus fermentum J2-4, J2-5 and J2-9 have no inhibitory effect on Escherichia coli, while the Lactobacillus fermentum MWSN-2 in this invention can effectively inhibit the pathogenic Escherichia coli, which is an unexpected effect.

[0093] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of fermenting *Lactobacillus mucilaginosus* ( Limosilactobacillus fermentum WMSN-2 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 23, 2023, with accession number GDMCC No: 63904.

2. A microbial preparation containing the fermenting Lactobacillus mucinus WMSN-2 as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The content of fermenting Lactobacillus mucinus WMSN-2 in the microbial preparation is ≥1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

4. A composition containing the fermenting Lactobacillus mucinus WMSN-2 as described in claim 1.

5. The composition according to claim 4, characterized in that, The composition is a drug.

6. A drug containing *Lactobacillus fermentans* WMSN-2 as described in claim 1, characterized in that, The drug also contains a pharmaceutically acceptable carrier.

7. The use of the fermented Lactobacillus mucinus-2 according to claim 1 in the preparation of a medicament for the prevention of neonatal necrotizing enterocolitis.

8. The application according to claim 7, characterized in that, The application includes at least one of (1) to (4): (1) Alleviate weight loss and reduce disease activity index in patients with neonatal necrotizing enterocolitis; (2) Improve the survival rate of neonatal necrotizing enterocolitis patients; (3) Relieves symptoms of small bowel shortening; (4) Relieve damage to ileal villi.

9. The application according to claim 7 or 8, characterized in that, The fermented Lactobacillus mucinus WMSN-2 was used in the form of a live bacterial preparation.

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