Application of Limosilicibacillus fermentum WMSN-2 in prevention of neonatal necrotizing enterocolitis

Abstract

The invention discloses an application of Limosilicilla fermentum WMSN-2 in the aspect of preventing neonatal necrotizing enterocolitis, and belongs to the field of application of probiotics. The invention further discloses a preparation method of the Limosilicilla fermentum WMSN-2 for preventing neonatal necrotizing enterocolitis. The lactobacillus mucus WMSN-2 strain provided by the invention has bacteriostatic ability, antibiotic sensitivity and good gastrointestinal fluid tolerance, has the effect of preventing and treating neonatal necrotizing enterocolitis, can reduce the disease activity index of neonatal necrotizing enterocolitis individuals, and can be used for treating neonatal necrotizing enterocolitis. The survival rate is improved; the small intestine shortening and intestinal villus injury are relieved. The lactobacillus mucus WMSN-2 strain provided by the invention can be used for preparing medicines for preventing and treating neonatal necrotizing enterocolitis, can also be used for preparing functional foods and feeds, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to application of Limosilactobacillus fermentum WMSN-2 in preventing neonatal necrotizing enterocolitis, and belongs to the application field of probiotics. Background Art

[0002] Neonatal necrotizing enterocolitis (NEC) is a common gastrointestinal disease in newborns, characterized by intestinal mucosal edema, bleeding, and intestinal necrosis. NEC is more common in premature infants, accounting for more than 90% of the total number, with a high mortality rate. Surviving infants often have serious complications such as short bowel syndrome, intestinal stenosis, and neurological sequelae, which reduce the quality of life of infants and increase the social burden.

[0003] Although NEC has been known for many years in clinical practice, its core pathogenesis remains unclear. Currently, research on the mechanism of NEC mainly focuses on intestinal barrier function and intestinal flora imbalance. There are a large number of microorganisms in the intestines of humans and animals, which adhere to the surface of the intestinal mucosa and form a biological barrier. Studies have shown that the intestinal flora in the feces of children with NEC is disordered, among which the abundance of Lactobacillus is reduced. Lactic acid bacteria are the most commonly used probiotics and play an important role in protecting the host from harmful organisms, enhancing the host immune system, and reducing metabolic disorders. Limosilactobacillus fermentum is a Gram-positive bacterium of the genus Lactobacillus. Many fermented mucus lactobacilli can enhance the immune response of biological organisms and prevent epidemic gastrointestinal and upper respiratory tract infections. However, it is not clear whether Limosilactobacillusfermentum has a potential regulatory effect on neonatal necrotizing enterocolitis. Summary of the invention

[0004] The present invention provides a strain of Limosilactobacillus fermentum WMSN-2, which has been deposited in Guangdong Provincial Microbiological Culture Collection Center on October 23, 2023, with a deposit number of GDMCC No: 63904, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0005] The invention provides a microbial preparation containing the fermented mucus lactobacillus WMSN-2.

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

[0007] The invention also provides a composition containing the fermented mucus lactobacillus WMSN-2.

[0008] In one embodiment, the composition is a food, a medicine or a health product.

[0009] In one embodiment, the medicament further contains 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 an excipient and / or an additive.

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

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

[0014] The present invention also provides the use of fermented mucus lactobacillus WMSN-2 in preparing a medicine for preventing and / or treating neonatal necrotizing enterocolitis.

[0015] In one embodiment, 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 patients with neonatal necrotizing enterocolitis;

[0018] (3) Relieve symptoms of small intestinal shortening;

[0019] (4) Alleviate ileal villi damage.

[0020] In one embodiment, the fermented Lactobacillus mucilaginosus WMSN-2 is a living cell with biological activity or an inactivated cell obtained after treatment.

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

[0022] In one embodiment, the number of fermented Lactobacillus mucilaginosus WMSN-2 in the product is ≥1×10 9 CFU / g or 1×10 9 CFU / mL.

[0023] The present invention also provides application of the fermented mucus lactobacillus WMSN-2 in preparing food, functional food, health care product or feed.

[0024] Beneficial effects:

[0025] The present invention provides a new use of a fermented Lactobacillus mucoides WMSN-2 strain in a drug for preventing and protecting neonatal necrotizing enterocolitis. The present invention verifies through experiments that the fermented Lactobacillus mucoides WMSN-2 strain has an effect on reducing the disease activity index of patients with neonatal necrotizing enterocolitis, improving the survival rate, and alleviating the shortening of the small intestine and the damage to the intestinal villi, and has a better effect than the positive control strain Lactobacillus rhamnosus GG (LGG). The fermented Lactobacillus mucoides WMSN-2 strain provided by the present invention can be used to prepare drugs, pharmaceutical compositions, functional foods and feeds for preventing and protecting neonatal necrotizing enterocolitis, and broadens the application field of the fermented Lactobacillus mucoides WMSN-2 strain.

[0026] Biomaterial Deposit

[0027] Limosilactobacillus fermentum WMSN-2, taxonomically named Limosilactobacillus fermentum, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2023, with the deposit number GDMCC No: 63904, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the colony morphology of fermented Lactobacillus mucilaginosus WMSN-2.

[0029] Figure 2 This is the electrophoresis identification diagram of the fermented mucus Lactobacillus WMSN-2 amplified using Lactobacillus-specific primers.

[0030] Figure 3 is the survival rate of fermentative Lactobacillus mucilaginosus WMSN-2 in simulated digestive fluid.

[0031] Figure 4The changes in the disease activity index of neonatal necrotizing enterocolitis mice treated with fermented Lactobacillus mucin WMSN-2 were shown in Figure 2. Con represents the sterile saline control group, WMSN-2 represents the fermented Lactobacillus mucin WMSN-2 control group, Model represents the neonatal necrotizing enterocolitis model group, WMSN-2+M represents the fermented Lactobacillus mucin WMSN-2 treatment group for neonatal necrotizing enterocolitis, and LGG+M represents the positive control group for neonatal necrotizing enterocolitis treated with Lactobacillus rhamnosus GG. *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0032] Figure 5 The survival rate of neonatal necrotizing enterocolitis mice treated with fermented Lactobacillus mucilaginosus WMSN-2 was changed. *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0033] Figure 6 The fermentation of Lactobacillus mucilaginosus WMSN-2 was used to prevent the change of small intestine length in necrotizing enterocolitis. *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0034] Figure 7 The fermentation of Lactobacillus mucilaginosus WMSN-2 was used to prevent the pathological changes of ileum tissue in necrotizing enterocolitis. *: P < 0.05, **: P < 0.01, ***: P < 0.001. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with the accompanying drawings and specific examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions in the art or according to the conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used in the text have the same meanings as those familiar to those skilled in the art.

[0036] Lactobacillus rhamnosus GG (ATCC 7469) was obtained from the American Type Culture Collection (ATCC); Lactobacillus mucosus WMSN-2 was isolated from the feces of healthy infants who were exclusively breastfed in Wuxi, Jiangsu Province.

[0037] MRS medium: 10 g soy peptone, 5 g beef extract, 5 g yeast powder, 20 g glucose, 1 mL Tween-80, 2 g sodium dihydrogen phosphate, 5 g anhydrous sodium acetate, 2 g triammine citrate, 0.02 g manganese sulfate, 0.1 g magnesium sulfate, 1 L distilled water, adjust the pH to about 6.2, 15 g agar, sterilize at 121°C for 15 min.

[0038] Example 1 Screening of fermented mucus lactobacillus WMSN-2

[0039] 1. Screening of WMSN-2 strain

[0040] Take about 5g of fresh sample (feces of healthy infants who are exclusively breastfed in Wuxi, Jiangsu Province) and collect it with a sterile tube, and immediately send it to the laboratory for strain isolation. Take 1g of sample and put it into 9mL of MRS broth medium, vortex mix it and enrich it in an anaerobic incubator at 37℃ for 48h; then take 1mL of enrichment solution in the clean bench, dilute it tenfold with sterile saline, and select 10 -6 , 10 -7 , 10 -8 Three dilution gradients, 100 μL of bacterial solution from each gradient was applied to MRS agar medium and cultured anaerobically at 37°C for 48 hours. After the culture, a plate with 50-150 single colonies was selected from the agar medium, colonies with different shapes, sizes and colors were picked, and they were streaked and purified on the MRS agar plate several times until the colonies on the entire plate had the same shape, and single colonies were picked to MRS broth medium for bacterial enrichment culture. The obtained strains were all frozen and stored at -80°C in MRS broth medium containing 40% glycerol.

[0041] 2. Identification of WMSN-2 strain

[0042] After culturing strain WMSN-2 on MRS agar medium for 48 hours, the diameter of the colonies was between 0.3-1.5 mm, the edges were neat, white, and the surface was moist and smooth. Figure 1 .

[0043] The target strain genomic DNA was extracted using the Ezup column bacterial genomic DNA extraction kit, and the extracted lactic acid bacteria genomic DNA was used as a template for PCR amplification. The PCR experiment was performed using lactobacillus-specific primers. After the PCR reaction was completed, the PCR product was taken for agarose gel detection and photography. The length of the amplified fragment was about 550 bp, as shown in Figure 2. Figure 2 Subsequently, the PCR experiment of 16S rDNA was carried out using bacterial universal primers 27F and 1492R. After the PCR reaction amplification was completed, the PCR product was taken for agarose gel detection and photography. The length of the amplified fragment was about 1.2Kbp. The primers sent the PCR product to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. The BLAST sequence comparison was performed on the NCBI website. The results showed that the sequence had a homology of more than 99% with the 16S rDNA sequence of Lactobacillus fermentum.

[0044] The sequence alignment results of strain WMSN-2 were combined with the physiological and biochemical results, and the screened lactic acid bacteria WMNS-2 was determined to be Limosilactobacillus fermentum WMSN-2.

[0045] Example 2 Confirmation of digestive juice tolerance of fermented Lactobacillus mucilaginosus WMSN-2 strain

[0046] 1. Preparation of simulated digestive fluid:

[0047] (1) Preparation of simulated saliva: After sterilizing PBS, add 3 g / L α-amylase (purchased from Sigma, USA, product 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, and filter with a 0.22 μm microporous filter membrane for sterilization to prepare simulated saliva.

[0048] (2) Preparation of simulated gastric fluid: After sterilizing PBS, add 3.0 g / L pepsin (purchased from Sigma, USA, product number: 9001-75-6, enzyme activity ≥ 2000 U / 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 filter membrane to prepare simulated gastric fluid.

[0049] (3) Preparation method of simulated intestinal fluid: After sterilizing PBS, add 3 g / L ox bile salt (purchased from Sigma, USA, product number: 48305), 0.1 g / L lipase (purchased from Sigma, USA, product number: 9001-62-1, enzyme activity ≥20,000 U / mg protein), 1 g / L trypsin (purchased from Sigma, USA, product 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 and sterilize with a 0.22 μm microporous filter membrane to prepare simulated intestinal fluid.

[0050] 2. Digestive juice tolerance test method:

[0051] The fermented mucus Lactobacillus WMSN-2 strain obtained by screening was activated and cultured in MRS medium for two generations, centrifuged and washed twice, and the cells were collected and resuspended in 1 mL of simulated saliva for 5 minutes, then the cells were centrifuged (4°C, 12000rpm, 2min) and resuspended in 2 mL of simulated gastric fluid, and incubated at 37°C for 2h. At the same time, the viable bacteria rate was counted and determined by the MRS agar medium pouring method. Each sample was repeated 3 times and the average value was calculated.

[0052] Subsequently, the artificial gastric juice containing bacteria that had been digested for 2 hours was centrifuged again (4°C, 12000rpm, 2min) and resuspended in 2mL simulated intestinal fluid and incubated at 37°C for 2h. Finally, the bacterial suspension was diluted and inoculated on MRS agar and cultured under anaerobic conditions at 37°C for 36-48h. The viable bacterial rate was determined by counting the MRS agar medium pouring method. The survival rates of the strains in simulated gastric juice and simulated intestinal juice were calculated according to the following formula.

[0053] Survival rate (%) = LogN1 / LogN0×100;

[0054] Among them, N1 represents the number of live bacteria in the strain system after treatment with simulated gastric fluid or simulated intestinal fluid; N0 represents the initial number of live bacteria in the strain system.

[0055] The results are as follows Figure 3 As shown, the survival rate of Lactobacillus mucilaginosus WMSN-2 under simulated human digestive fluid conditions was as high as 54.28%, which was better than that of Lactobacillus rhamnosus GG with a survival rate of 52.24%.

[0056] From the above results, it can be seen that the fermented mucus Lactobacillus WMSN-2 provided by the present invention has excellent gastrointestinal fluid tolerance, can enter the human intestine in a living state, and survive in the gastrointestinal organs of animals including humans, exerting health effects, and the above characteristics are the basis of the strain as a probiotic.

[0057] Example 3 Evaluation of the antibacterial effect of fermented mucus lactobacillus WMSN-2

[0058] 1. Preparation of strain fermentation broth

[0059] The strain stored in the glycerol tube was first activated by streaking on MRS agar plates 2-3 times, and then a single colony was picked and expanded in MRS broth medium for 18 h (cultured under anaerobic conditions at 37 °C). The culture medium was adjusted with distilled water to a bacterial concentration of 1×10 9 CFU / mL, centrifuge at 4°C, 8000r / min for 20min, and collect the supernatant as the fermentation supernatant of the bacteria.

[0060] 2. Effect of strains in inhibiting pathogenic bacteria

[0061] The Oxford cup method was used to detect the inhibitory effect of fermented Lactobacillus mucilaginosus WMSN-2 on several major intestinal pathogens (including Staphylococcus aureus ATCC 25923, Salmonella enterica ATCC 14028, Escherichia coli ATCC 25922, and Listeria monocytogenes ATCC 13932). Specifically, the culture medium was incubated at 37°C for 16-24 hours (about 1×10 9 CFU / mL) were spread on their respective solid culture media, and the Oxford cup was carefully placed on the culture medium, and 200 μL of the above bacterial supernatant was added to each cup. After culturing at 37°C for 24 hours, the diameter of the inhibition zone was measured. The inhibition zone diameter was scored as follows: ≤7mm (none, -); 7-15mm (weak, +), >15mm (strong, ++). Penicillin (30mg / mL) was used as a positive control. The corresponding liquid culture medium was used as a negative control. Each sample was repeated 3 times and the average value was calculated. The experimental results are shown in Table 1. It can be seen from Table 1 that the fermentation supernatant of Lactobacillus mucosa WMSN-2 screened by the present invention has a strong antibacterial effect.

[0062] Table 1 Inhibitory effects of strains on different pathogenic bacteria

[0063]

[0064] From the above results, it can be seen that the fermented mucus Lactobacillus WMSN-2 provided by the present invention has a better effect of inhibiting pathogenic bacteria than Lactobacillus rhamnosus GG, and this characteristic is the basis for the strain to be used as a probiotic.

[0065] Example 4 Sensitivity of fermented mucus lactobacillus WMSN-2 to antibiotics

[0066] Seven antibiotics (kanamycin, penicillin, vancomycin, erythromycin, ampicillin, streptomycin and chloramphenicol) were dissolved in appropriate solutions and filtered. The fermented Lactobacillus mucosa WMSN-2 strain was added with different final concentrations (2, 4, 8, 16, 32, 64, 128, 256, 512 and 1024 μg / mL) of antibiotics in MRS broth and cultured anaerobically at 37°C for 24-36 hours. Each sample was repeated 3 times. After the culture was completed, the absorbance value at OD610nm in the 96-well plate was measured. MIC (minimum inhibitory concentration) is considered to be the lowest antibiotic concentration that can inhibit the growth of the strain and is used to evaluate the antibiotic resistance of the selected strain.

[0067] The results are shown in Table 2. The fermented mucus lactobacillus WMSN-2 screened out by the present invention has different degrees of sensitivity to these antibiotics.

[0068] Table 2 The sensitivity of strains to different antibiotics

[0069]

[0070] It can be seen from the above experimental results that the fermented mucus Lactobacillus WMSN-2 provided by the present invention is relatively sensitive to different antibiotics, indicating that the probiotic has the potential to be used as a live bacteria product.

[0071] Example 5 Preparation of fermented Lactobacillus mucilaginosus WMSN-2 bacterial agent

[0072] Preparation of WMSN-2 bacterial agent: The fermented mucus lactobacillus WMSN-2 was strictly cultured in MRS broth. The bacterial solution in the logarithmic growth phase was centrifuged at 4°C, 8000g for 10 minutes, the supernatant was removed, the bacterial sludge was washed twice with sterile saline, and the bacterial sludge was resuspended in 30% (v / v) glycerol, and optionally stored in a -80°C refrigerator.

[0073] Preparation of positive LGG bacterial agent: Lactobacillus rhamnosus GG is strictly cultured in MRS broth. The bacterial solution in the logarithmic growth phase is centrifuged at 4°C, 8000g for 10 minutes, the supernatant is removed, the bacterial sludge is washed twice with sterile physiological saline, and the bacterial sludge is resuspended in 30% (v / v) glycerol, and optionally stored in a -80°C refrigerator.

[0074] Optionally, WMSN-2 and positive LGG strains were diluted to 1×10 with sterile saline containing 3% (v / v) glycerol before use. 9 CFU / mL is reserved.

[0075] Example 6 Preparation of a medicament containing fermented mucus lactobacillus WMSN-2

[0076] The bacterial suspension containing viable cells of fermented mucus Lactobacillus WMSN-2 prepared in Example 3 is mixed with the auxiliary materials to obtain a liquid preparation; optionally, the liquid preparation is compressed to obtain tablets; optionally, the liquid preparation is dried to obtain a powder; optionally, the powder is used as a filler to prepare capsules.

[0077] Among them, the pharmaceutical excipients include anti-adhesive agents, penetration enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, pH regulators, adhesives, disintegrants, fillers, lubricants, wetting agents, integrators, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculants, filter aids and release retardants.

[0078] Example 7 Fermentation of Lactobacillus mucilaginosus WMSN-2 for the prevention of neonatal necrotizing enterocolitis

[0079] (1) Establishment of neonatal necrotizing enterocolitis model:

[0080] Six-day-old C57BL / 6J newborn mice were randomly divided into a saline control group (Con), a probiotic control group of Lactobacillus mucin fermentation WMSN-2 (WMSN-2), a neonatal necrotizing enterocolitis model group (Model), a Lactobacillus mucin fermentation WMSN-24 treatment group (WMSN-2+M), and a Lactobacillus rhamnosus GG treatment group (LGG+M), and controlled feeding conditions were provided. From the sixth to the ninth day, for four consecutive days, each young mouse in the WMSN-2 group and the WMSN-2+M group was given 1×10 9 CFU / mL of fermented Lactobacillus mucosa WMSN-2 bacterial solution, each young mouse in the LGG+M group was intragastrically administered with 1×10 CFU / mL of 3% glycerol. 9 50 μL of Lactobacillus rhamnosus GG bacterial solution with CFU / mL was administered to the Con group and the Model group with an equal volume of sterile saline containing 3% glycerol. On the tenth day, the neonatal necrotizing enterocolitis model was induced by gavage and rectal instillation of trinitrobenzene sulfonic acid (TNBS). TNBS was dissolved in saline, and TNBS (10 mg / g) was administered to the Model group, WMSN-2+M group and LGG+M group on the 10th day, with 25 μL of each by gavage and rectal instillation. The CON group and the WMSN-2 group pups were given an equal volume of saline. When the pups showed cachexia or wasting symptoms (if no obvious symptoms appeared, they were killed after 24 hours), they were anesthetized with 5% isoflurane, and after blood was taken from the heart, they were killed by cervical dislocation, and the ileum, colon and their contents were collected.

[0081] (2) Treatment with fermented Lactobacillus mucilaginosus WMSN-2 alleviated weight loss and reduced the disease activity index in young mice

[0082] The disease activity index, which is the sum of the scores of weight change, diarrhea and blood in stool, is one of the most significant phenotypes of neonatal necrotizing enterocolitis and can reflect the severity of the disease. The mice were weighed at 0h, 3h, 6h, 12h and 24h after induction of NEC, and the diarrhea was observed and the blood in stool was measured using an occult blood test kit. Figure 4At 24h, compared with the Model group (the Model group had a weight loss of 13.9% and a disease activity index of 3.2), the bacterial injection groups were able to significantly alleviate the weight loss and reduce the disease activity index of NEC mice (the WMSN-2+M group had a weight loss of 11.1%, P<0.01, and a disease activity index of 2.33, P<0.01; the LGG+M group had a weight loss of 11.8%, P<0.01, and a disease activity index of 2.56, P<0.05), and the fermented mucus lactobacillus WMSN-2 had a better effect, which was 0.7% and 0.23 higher than the LGG+M group. In addition, compared with the Con group, the weight change rate and disease activity index of the mice in the WMSN-2 group were similar, indicating that the fermented mucus lactobacillus WMSN-2 would not have adverse effects on the apparent phenomena of the mice.

[0083] (3) Fermentation of Lactobacillus mucilaginosus WMSN-2 improves the survival rate of young mice

[0084] Severe NEC can lead to the death of young mice. Survival rate is another important indicator for evaluating the protection of young mice against NEC by probiotics. After NEC was induced in the Model group, WMSN-2+M group and LGG+M group, the death of young mice was observed every 1 hour, with the absence of heartbeat as the criterion. Figure 5 The Model group mice died at 6 hours, while the WMSN-2+M group and the LGG+M group mice died at 13 hours and 10 hours, respectively. The observation ended at 24 hours, and the survival rate of the Model group was only 25%, while the survival rates of the WMSN-2+M group and the LGG+M group were 40% and 37.5%, respectively, indicating that the fermented mucus lactobacillus WMSN-2 can prolong the survival time of NEC mice and improve the survival rate of NEC mice, and the improvement effect is better than that of the positive bacteria Lactobacillus rhamnosus GG.

[0085] (4) Treatment with fermented Lactobacillus mucilaginosus WMSN-2 alleviates the symptoms of small intestinal shortening in NEC mice

[0086] The most obvious pathological features of NEC are small intestinal shortening and inflammatory edema. The mice in the Con, WMSN-2, Model, WMSN-2+M, and LGG+M groups were killed, and the stomach, small intestine, and cecum were completely removed. The mesentery was carefully peeled off, and the degree of shortening of the small intestine between the stomach and cecum was evaluated with a standard ruler. Figure 6Compared with the Con group, the length of the small intestine of the young mice in the Model group was significantly shortened, from 17.2cm in the CON group to 15.1cm (P<0.001), and the inflammatory edema was more severe, while the shortening of the small intestine length of the young mice in the positive LGG+M group was significantly alleviated, from 15.1cm in the Model group to 16.1cm (P<0.05), and the length of the small intestine in the WMSN-2+M group increased from 15.1cm in the Model group to 16.5cm, and showed a better preventive and protective effect than the LGG+M group, which was 0.4cm higher than the 16.1cm of the LGG+M group (P<0.05). Compared with the Con group, the length of the small intestine of the WMSN-2 group did not change significantly, indicating that the fermented mucus lactobacillus WMSN-2 would not have adverse effects on the small intestine of young mice and showed a better preventive and protective effect than the positive control bacteria Lactobacillus rhamnosus GG.

[0087] (5) Treatment with fermented Lactobacillus mucilaginosus WMSN-2 alleviates ileal villus damage

[0088] HE staining was used to observe the tissue integrity of the ileum in the CON group, WMSN-2 group, Model group, WMSN-2+M group, and LGG+M group after fixation, dehydration, staining, dewaxing, transparency, and sealing. Figure 7 The results showed that the ileum tissue structure of the Con group and the WMSN-2 group was dense, without obvious cracks, and the ileum villi were intact. In the Model group, obvious sparse and broken intestinal villi were observed, and the HE score increased significantly by 2.5 points (P<0.001). Compared with the Model group, the intestinal villi of the WMSN-2+M group and the LGG+M group were sparse and partially broken, and the HE scores decreased by about 1.2 points (P<0.05) and 1 point (P<0.05), respectively, indicating that fermented mucus lactobacillus WMSN-2 and rhamnosus lactobacillus GG have the ability to prevent and protect the ileum villi damage caused by NEC, and the villi of the WMSN-2+M group are more compact and complete than those of the LGG+M group, indicating that the preventive and protective effect of fermented mucus lactobacillus WMSN-2 is better than that of the positive control rhamnosus lactobacillus GG.

[0089] Comparative Example 1:

[0090] The antibacterial effect of the fermented mucus lactobacillus WMSN-2 screened by the present invention was compared with other fermented lactobacillus strains in the prior art. The strains J2-4, J2-5, and J2-9 were respectively 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 of Example 3, the antibacterial zone diameter score is as follows: ≤7mm (none, -); 7-15mm (weak, +), >15mm (strong, ++).

[0091] Table 3 Inhibition of Lactobacillus fermentum on pathogenic bacteria

[0092]

[0093] As shown in Table 3, the previously disclosed fermented Lactobacillus J2-4, J2-5 and J2-9 have no inhibitory effect on Escherichia coli, while the fermented Lactobacillus MWSN-2 of the present invention can better inhibit the pathogenic bacteria Escherichia coli, which has an unexpected effect.

[0094] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Limosilactobacillus fermentum WMSN-2 was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 23, 2023, with the deposit number GDMCC No: 63904.

2. A microbial preparation containing the fermented mucus lactobacillus WMSN-2 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The content of fermented mucus lactobacillus WMSN-2 in the microbial preparation is ≥1×10 9 CFU / mL or 1×10 9 CFU / g.

4. A composition comprising the fermented mucilaginous Lactobacillus WMSN-2 according to claim 1.

5. The composition according to claim 4, characterized in that The composition is food, medicine or health product.

6. A medicament containing the fermented mucus lactobacillus WMSN-2 according to claim 1, characterized in that: The drug also contains a pharmaceutically acceptable carrier.

7. Use of the fermented mucus lactobacillus WMSN-2 according to claim 1 in the preparation of a medicament for preventing and / or treating neonatal necrotizing enterocolitis.

8. The use 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 patients with neonatal necrotizing enterocolitis; (3) Relieve symptoms of shortened small intestine; (4) Alleviate ileal villi damage.

9. The use according to claim 7 or 8, characterized in that: The fermented mucus lactobacillus WMSN-2 is used in the form of a live bacterial preparation or a sterilized bacterial preparation.

10. Use of the fermented mucus lactobacillus WMSN-2 according to claim 1 in preparing food, functional food, health care product or feed.

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