Gastric mucosa repair promoting polysaccharide prepared from lactobacillus reuteri CCFM1453

Abstract

The invention discloses a polysaccharide prepared from lactobacillus reuteri CCFM1453 and used for promoting gastric mucosa repair, and belongs to the technical field of microorganisms and medicines. The lactobacillus reuteri CCFM1453 is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number of the lactobacillus reuteri CCFM1453 is GDMCC No: 65608. The lactobacillus reuteri CCFM1453 has the advantages that the gastric mucosa tissue form is improved, and the gastric mucosa injury is relieved; the content of gastric mucosal injury markers PGE2 and TFF3 is obviously reduced; the content of inflammatory factors such as IL-6 and IL-8 is reduced; the content of IL-10 and other anti-inflammatory factors is increased; the gastric mucosa is protected and inflammation is relieved by regulating and controlling key signal channels such as STAT3 and SOCS3.

Description

Technical Field

[0001] The invention relates to a polysaccharide for promoting gastric mucosal repair prepared by a strain of Lactobacillus reuteri CCFM1453, belonging to the fields of microbial technology and medical technology. Background Art

[0002] Nonsteroidal anti-inflammatory drugs exert their antipyretic, analgesic and anti-inflammatory effects by inhibiting cyclooxygenase activity and reducing prostaglandin synthesis, but they frequently cause gastric mucosal damage, which is usually related to the effect of drugs on the gastric mucosal barrier, including interference with the gastric mucosal protective mechanism, such as reducing mucus secretion and increasing gastric acid secretion, making the gastric mucosa more vulnerable to damage. As a typical manifestation of gastric mucosal inflammatory response, the occurrence and development of gastritis is closely related to the imbalance of mucosal protective mechanism, activation of proinflammatory factors and aggravation of oxidative stress. At the pathological level, persistent inflammation is prone to progress to gastric ulcer, bleeding or perforation, which seriously damages gastric homeostasis; at the functional level, gastric acid secretion disorders and mucosal repair disorders can lead to poor nutrient absorption, and then induce systemic complications such as anemia and metabolic disorders; in addition, chronic gastritis is closely related to precancerous lesions such as intestinal metaplasia and dysplasia, which significantly increase the risk of gastric cancer transformation.

[0003] The difficulty of treating gastric mucosal injury caused by NSAIDs is that it is necessary to reduce the side effects on the gastric mucosa while maintaining the efficacy of the drug. This may require the development of new drug delivery systems or the combined use of gastric mucosal protectants. Probiotic exopolysaccharides have good biocompatibility and usually do not react with drugs. They are a good choice for adjuvant treatment of gastritis caused by NSAIDs. Screening probiotic exopolysaccharides that have the effect of relieving gastritis and developing corresponding probiotic exopolysaccharide products not only has far-reaching scientific significance, but also has great practical value. This research field can provide new strategies for the treatment of gastric mucosal injury, especially gastric mucosal injury caused by NSAIDs, while promoting the innovation and development of postbiotic products, which has an impact that cannot be ignored on promoting human health and improving the quality of life. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an application of extracellular polysaccharide of Limosilactobacillus reuteri CCFM1453 in a product for alleviating gastric mucosal damage.

[0005] The present invention provides a strain of Limosilactobacillus reuteri CCFM1453, wherein the Limosilactobacillus reuteri CCFM1453 was deposited in Guangdong Provincial Microbiological Culture Collection Center on December 11, 2024, with a deposit number of GDMCC NO: 65608.

[0006] The Limosilactobacillus reuteri CCFM1453 has cells that appear as slightly irregular, curved bacilli with rounded ends under a microscope. The colonies after inoculation on MRS medium are white, with a moist and smooth surface and neat edges.

[0007] The Limosilactobacillus reuteri CCFM1453 is a Gram-positive bacterium, facultatively anaerobic, thermophilic, with an optimum growth temperature of 35 - 40 °C and an optimum growth pH of 6.0 - 7.0.

[0008] The present invention also provides a method for preparing the exopolysaccharide of Limosilactobacillus reuteri CCFM1453, which includes the following steps: inoculate the Limosilactobacillus reuteri CCFM1453 into a fermentation medium for cultivation to obtain a culture solution, collect the obtained culture supernatant, add 4 volumes of absolute ethanol to precipitate the exopolysaccharide, place it at room temperature for 24 h, and then centrifuge at 8000 rpm for 10 min to separate the crude polysaccharide. Dissolve the crude polysaccharide completely in deionized water at a concentration of 10 mg / ml, add trichloroacetic acid to make its final concentration 4% (m / v) and place it at 4 °C for 12 h, centrifuge at 6000 rpm at 4 °C for 15 min, and take the supernatant. Then transfer it into a dialysis bag (1000 Da) and dialyze at 4 °C for 48 h, changing the water every 12 h. Determine the polysaccharide content in the solution by the phenol-sulfuric acid method. Freeze-dry the dialyzed polysaccharide.

[0009] In one embodiment, the fermentation conditions are: fermentation temperature 25 - 40 °C, fermentation time 10 - 30 h; preferably, the fermentation temperature is 37 °C and the fermentation time is 18 - 24 h.

[0010] In one embodiment, the fermentation is to inoculate the seed solution of Limosilactobacillus reuteri CCFM1453 into the medium; the inoculation amount is 1 - 5% (v / v), preferably 2% (v / v).

[0011] In one embodiment, the medium contains 10 - 12 g / L of glucose, 5.0 - 7.0 g / L of yeast powder, 9.0 - 11.0 g / L of peptone, 2 - 3 g / L of anhydrous sodium acetate, 2 - 2.5 g / L of diammonium citrate, 2 - 2.6 g / L of dipotassium hydrogen phosphate, 0.05 - 0.25 g / L of manganese sulfate monohydrate, 0.1 - 0.5 g / L of magnesium sulfate heptahydrate, and 1.0 - 1.5 mL / L of Tween-80. In one embodiment, the cell concentration of Limosilactobacillus reuteri CCFM1453 in the culture solution is not less than 5.0×10 7 CFU / mL.

[0012] The present invention also provides a fermentation composition obtained by fermenting the above method.

[0013] The present invention also provides a product, which contains the above Limosilactobacillus reuteri CCFM1453 or the above extracellular polysaccharide.

[0014] In one embodiment, the product comprises a food or a health product.

[0015] In one embodiment, the product comprises a medicine or a cosmetic.

[0016] In one embodiment, the food further comprises conventional excipients.

[0017] In one embodiment, the conventional excipients include one or more of a filler, a flavoring agent, a binder, a disintegrant, a lubricant, an antacid, and a nutrient fortifier.

[0018] In one embodiment, the health product further comprises conventional excipients, and the conventional excipients include one or more of a filler, a flavoring agent, a binder, a disintegrant, a lubricant, an antacid, and a nutrient fortifier.

[0019] In one embodiment, the medicine further comprises a drug carrier and / or a pharmaceutical excipient.

[0020] In one embodiment, the pharmaceutical excipient comprises an excipient and an additive.

[0021] In one embodiment, the pharmaceutical excipient comprises at least one of a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, an antiadhesive, a chelating agent, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, a defoaming agent, a thickening agent, a clathrate, a humectant, an absorbent, a diluent, a flocculant and an anti-flocculant, a filter aid, or a release retarder.

[0022] In one embodiment of the present invention, the cosmetic comprises the above composition, a matrix raw material, and / or conventional excipients.

[0023] In one embodiment of the present invention, the matrix raw material includes oil raw materials, wax raw materials, synthetic oil raw materials, powder raw materials, gum raw materials, coagulants, and surfactants.

[0024] In one embodiment of the present invention, the conventional excipients include one or more of a humectant, a whitening agent, a flavoring agent, a binder, a lubricant, a preservative, a film agent, an antioxidant, an emulsifier, and a cosmetic nutrient additive.

[0025] The present invention also provides the use of the Lactobacillus reuteri CCFM1453 or the extracellular polysaccharide in preparing medicines for relieving gastritis and promoting gastric mucosal repair.

[0026] In one embodiment, in the medicine, the dosage of the extracellular polysaccharide of Limosilactobacillus reuteri CCFM1453 is not less than 5 mg / kg body weight.

[0027] Beneficial effects:

[0028] The extracellular polysaccharide of Limosilactobacillus reuteri CCFM1453 of the present invention has the ability to relieve host gastritis and gastric mucosal damage after oral administration, which is specifically embodied in:

[0029] (1) Relief of gastric mucosal damage: Reduce the content of PGE2 in gastric tissue and reduce gastric mucosal inflammatory response. Reduce the overexpression of TFF3 in the stomach of mice with gastric mucosal damage and promote gastric mucosal repair.

[0030] (2) Anti-inflammatory mechanism: It significantly reduces the level of inflammatory factor IL-6 in the gastric tissue of mice, alleviating the inflammatory response caused by gastric mucosal damage; significantly increases the expression of IL-10, inhibiting excessive inflammatory response; significantly reduces the level of COX-2 in the serum of mice with gastric mucosal damage, thereby inhibiting the activation of inflammatory pathways; reduces the expression of STAT3, regulates the expression of SOCS3, alleviates gastric inflammation and cell apoptosis signaling pathways, and alleviates gastric mucosal damage; inhibits the expression of MyD88, IKBα, and AKT, intervenes in the TLR4 / MyD88 / NF-κB signaling pathway, and blocks the cascade reaction of inflammatory factors; downregulates the expression of TRPV4, alleviates cell damage caused by calcium overload, and protects the integrity of the gastric mucosa; increases the expression of Bcl2, reduces cell apoptosis, and protects gastric mucosal cells.

[0031] Deposit of biological materials:

[0032] A strain of Limosilactobacillus reuteri CCFM1453, taxonomically named Limosilactobacillus reuteri, was deposited in the Guangdong Provincial Microbiological Culture Collection on December 11, 2024, with the deposit number GDMCC No: 65608, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 :Effects of different probiotic exopolysaccharides on RAW264.7 cell proliferation.

[0034] Figure 2 : Effects of exopolysaccharides from different probiotics on the expression of IL-1β mRNA, IL-6 mRNA, IL-10 mRNA, and TNF-α mRNA in LPS-induced RAW264.7 cells.

[0035] Figure 3 : Flow chart of the mouse experiment.

[0036] Figure 4 : Effects of exopolysaccharides extracted from Lactobacillus mucosae CCFM1453 on the contents of gastric mucosal injury markers PGE2 and TFF3 in mice.

[0037] Figure 5 : Effects of exopolysaccharides extracted from Lactobacillus mucosae CCFM1453 on the contents of inflammatory markers IL-6, IL-8, IL-10, and COX-2 in gastritis mice.

[0038] Figure 6 : Effects of exopolysaccharides extracted from Lactobacillus mucosae CCFM1453 on the alleviation of gastric mucosal injury through different pathways.

[0039] Figure 7 : Pathological sections of the promotion of gastric mucosal repair by exopolysaccharides of Lactobacillus mucosae CCFM1453.

[0040] "*" indicates a statistically significant difference from the Model group (P < 0.05), "**" indicates a significant statistical difference from the Model group (P < 0.01), "***" indicates an extremely significant statistical difference from the Model group (P < 0.001); "****" indicates an extremely significant statistical difference from the Model group (P < 0.0001). Specific embodiments

[0041] The present invention will be further described below in conjunction with specific embodiments.

[0042] The mouse macrophages (RAW264.7) involved in the following examples were purchased from: Shanghai Cell Bank.

[0043] The BALB / c mice involved in the following examples were purchased from Vital River Laboratories.

[0044] The Lactobacillus mucosae CCFM1453, Lactobacillus mucosae RC14, Lactobacillus mucosae 45B1, Lactobacillus mucosae 12M3, and Lactobacillus mucosae 215 involved in the following examples were self-screened strains from the Food Biotechnology Center of Jiangnan University.

[0045] The culture media involved in the following examples are as follows:

[0046] MRS liquid medium: 5.0 g / L of yeast extract, 10.0 g / L of beef extract, 10.0 g / L of peptone, 20.0 g / L of glucose, 2.0 g / L of anhydrous sodium acetate, 2.0 g / L of diammonium hydrogen citrate, 2.6 g / L of dipotassium hydrogen phosphate, 0.25 g / L of manganese sulfate monohydrate, 0.5 g / L of magnesium sulfate heptahydrate, and 1 mL / L of Tween-80, pH 6.2 - 6.4.

[0047] MRS solid medium: 5.0 g / L of yeast extract, 10.0 g / L of beef extract, 10.0 g / L of peptone, 20.0 g / L of glucose, 2.0 g / L of anhydrous sodium acetate, 2.0 g / L of diammonium hydrogen citrate, 2.6 g / L of dipotassium hydrogen phosphate, 0.25 g / L of manganese sulfate monohydrate, 0.5 g / L of magnesium sulfate heptahydrate, 1 mL / L of Tween-80, and 20.0 g / L of agar, pH 6.2 - 6.4.

[0048] MRS simplified liquid medium: 10 g / L of glucose, 5.0 g / L of yeast extract, 10.0 g / L of peptone, 2 g / L of anhydrous sodium acetate, 2 g / L of diammonium hydrogen citrate, 2.6 g / L of dipotassium hydrogen phosphate, 0.05 g / L of manganese sulfate monohydrate, 0.1 g / L of magnesium sulfate heptahydrate, and 1 mL / L of Tween-80, pH 6.2 - 6.4.

[0049] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin and streptomycin mixed solution (penicillin content in the mixed solution is 10000 U / mL, streptomycin concentration is 10 mg / mL).

[0050] Example 1: Cell resuscitation and culture

[0051] First, take out the cryopreserved mouse macrophages (RAW264.7), quickly thaw them in a 37°C water bath, then centrifuge at 1000 r / min for 3 min, discard the supernatant, add an appropriate volume of cell culture medium to resuspend the cells, place them in a culture dish, and put them in a 37°C incubator containing 5% CO2 for culture. When the cells grow and recover their vitality and reach 70% - 80% confluence after 1 - 2 days, cell passage is carried out.

[0052] Example 2: Screening of Lactobacillus mucosae CCFM1453 and extraction of exopolysaccharide

[0053] (1) Screening of Lactobacillus mucosae CCFM1453

[0054] The sample was derived from cow's milk. After pretreatment, the sample was stored in 20% glycerol at -80°C in a refrigerator. After taking it out and thawing, it was mixed well and 0.5 mL of the sample was added to 4.5 mL of physiological saline, and gradient dilution was carried out with physiological saline. The appropriate gradient dilution solution was spread on MRS solid medium and cultured at 37°C for 48 h. The typical colonies of Limosilactobacillus reuteri were picked and streaked and purified on MRS solid medium. Single colonies were picked and transferred to MRS liquid medium for enrichment and preserved with 30% glycerol to obtain the strain. The genomic DNA of the strain was extracted and 16S rDNA was amplified and sequenced (performed by Suzhou Genewiz Biotechnology Co., Ltd.). The 16S rDNA sequencing results were determined to be Limosilactobacillus reuteri by NCBI sequence alignment, and it was named Limosilactobacillus reuteri CCFM1453, which was deposited in the Guangdong Provincial Microbial Culture Collection Center on December 11, 2024, with the deposit number GDMCC No: 65608.

[0055] (2) Extraction of exopolysaccharides from Limosilactobacillus reuteri CCFM1453, Limosilactobacillus reuteri RC14, Limosilactobacillus reuteri 45B1, Limosilactobacillus reuteri 12M3, and Limosilactobacillus reuteri 215

[0056] 1) Streak and resuscitate Limosilactobacillus reuteri CCFM1453 from the preservation tube, and culture it in a MRS solid medium in a water-bath thermostatic incubator at 37°C for 24 - 48 h to obtain single colonies; pick single colonies and inoculate them into MRS liquid medium, and culture at 37°C for 12 - 18 h to obtain culture solution 1;

[0057] 2) Inoculate culture solution 1 into MRS liquid medium at an inoculation amount of 2% (v / v), and culture at 37°C for 12 h to obtain a seed solution;

[0058] 3) Inoculate the seed solution into MRS simplified liquid medium for expansion culture at 2 - 5% (v / v) respectively, and culture at 37°C for 18 - 24 h to obtain bacterial solution a.

[0059] 4) The supernatant obtained by centrifuging bacterial solution a at 8000 r / min for 30 min was added with 4 volumes of absolute ethanol to precipitate the exopolysaccharide, and it was left at room temperature for 24 h, and then centrifuged at 8000 rpm for 10 min to separate the crude polysaccharide. The crude polysaccharide was completely dissolved in deionized water at a concentration of 10 mg / ml, trichloroacetic acid was added to make its final concentration 4% (m / v), and it was left at 4°C for 12 h, centrifuged at 6000 rpm at 4°C for 15 min, and the supernatant was taken. Then it was transferred into a dialysis bag (1000 Da) and dialyzed at 4°C for 48 h, changing the water every 12 h. The polysaccharide content in the solution was determined by the phenol-sulfuric acid method. The dialyzed polysaccharide was freeze-dried.

[0060] Example 3: Effect of exopolysaccharide extracted from Lactobacillus reuteri CCFM1453 on the proliferation of mouse macrophages RAW264.7

[0061] The specific steps are as follows:

[0062] (1) Take 100 μL of mouse macrophages RAW264.7 in the logarithmic growth phase and inoculate them in a 96-well plate at a concentration of 7×10 3 cells / well. The outermost circle is filled with PBS solution to prevent edge effects. After culturing for 24 h until they adhere to the wall, set up a blank group, a control group, and a postbiotic treatment group;

[0063] The blank group contains only cell culture medium without mouse macrophages RAW264.7;

[0064] The control group contains cell culture medium and mouse macrophages RAW264.7 but no probiotic exopolysaccharide;

[0065] The probiotic exopolysaccharide treatment group contains cell culture medium with different exopolysaccharides obtained in Example 2 and mouse macrophages RAW264.7.

[0066] Preparation of cell culture medium containing probiotic exopolysaccharide: Resuspend the freeze-dried exopolysaccharide in cell culture medium (the amount of resuspended exopolysaccharide is equivalent to the amount of exopolysaccharide extracted from the bacterial liquid fermented to a concentration of 5.0×10 7 CFU / mL) to obtain cell culture medium containing probiotic exopolysaccharide.

[0067] (2) Incubate the above-mentioned well plates in an incubator at 37 °C for 24 h. After the incubation, add 10 μL of CCK8 solution to each well and incubate for 2 h to measure the absorbance value (OD) at 450 nm.

[0068] Calculate the cell viability according to the following formula: Cell viability (%) = (OD value of the treatment group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%.

[0069] The effect of probiotic exopolysaccharide on cell proliferation is as Figure 1 shown. Compared with the control group, the cell proliferation rates of adding exopolysaccharides of Lactobacillus reuteri CCFM1453, Lactobacillus reuteri RC14, Lactobacillus reuteri 45B1, Lactobacillus reuteri 12M3, and Lactobacillus reuteri 215 are 90.75%, 92.24%, 87.92%, 91.70%, and 80.46% respectively.

[0070] According to the toxicity grading evaluation method of ISO 10993-5:2009, if the cell viability is greater than 70%, it can be regarded as non-toxic. The above results show that the viability of mouse macrophages RAW264.7 at the above extracellular polysaccharide concentrations is higher, all above 90%. Considering its non-cytotoxicity, the inactivated cell concentration of 5.0×10 7 CFU / mL is a suitable postbiotic concentration for subsequent cell experiments.

[0071] Example 4: Effect of extracellular polysaccharide extracted from Lactobacillus mucosae CCFM1453 on the expression level of inflammatory factor mRNA in RAW264.7 cells intervened by LPS

[0072] The specific steps are as follows:

[0073] (1) Seed RAW264.7 cells at a density of 1.5×10 5 cells / mL in a 6-well plate and culture the cells overnight until they adhere to the wall. Discard the old medium, rinse 3 times with PBS, and set up control group 1 and model group 1;

[0074] Add 2 mL of normal cell medium to control group 1;

[0075] Add 2 mL of normal medium containing 1 μg / mL LPS to model group 1.

[0076] (2) Incubate the above plates in an incubator at 37 °C for 24 h. After incubation, discard the old medium of the control group and the model group, rinse 3 times with PBS, and set up the control group, the model group, and the treatment group:

[0077] For the control group, after changing the medium in control group 1, add 2 mL of normal cell medium;

[0078] For the model group, after changing the medium in model group 1, add 2 mL of normal cell medium;

[0079] The treatment group is divided as follows: After changing the medium in model group 1, add 2 mL of medium containing extracellular polysaccharide of Lactobacillus mucosae.

[0080] Among them, the preparation method of the extracellular polysaccharide of Lactobacillus mucosae refers to Example 2. Resuspend the extracellular polysaccharides of Lactobacillus mucosae CCFM1453, Lactobacillus mucosae RC14, Lactobacillus mucosae 45B1, Lactobacillus mucosae 12M3, and Lactobacillus mucosae 215 with 2 mL of cell medium respectively, so that the amount of the resuspended extracellular polysaccharide is equivalent to the amount of the extracellular polysaccharide prepared from the bacterial liquid fermented to a concentration of 5.0×10 7 CFU / mL to obtain the medium containing extracellular polysaccharide of Lactobacillus mucosae.

[0081] (3) For each sample, three parallel orifice plates were incubated in an incubator at 37°C for 24 h. The culture supernatant was discarded, and each well was quickly washed 3 times with PBS. 1 mL of cell lysate was added to each well, and the cells were repeatedly pipetted. The cell lysate was aspirated to extract RNA, and it was reverse transcribed into cDNA using an RT-PCR reverse transcription kit. The expression of genes in HSF cells was detected by real-time fluorescence quantification, and the expression levels of IL-1β mRNA, IL-6 mRNA, IL-10 mRNA, and TNF-α mRNA were calculated using the 2-ΔΔCt formula, with β-actin as the internal reference. The primers are described in Table 1 below, and the results are as Figure 2 shown.

[0082] Table 1: Primer sequences

[0083]

[0084] The results showed that, as Figure 2 can be seen, taking the expression level of IL-1β mRNA in the control group as approximately 1, the expression level in the model group increased to 128.78 after LPS intervention; the exopolysaccharide extracted from Lactobacillus mucosae CCFM1453 significantly reduced the expression level of IL-1β mRNA in RAW264.7 cells to 42.12, while the exopolysaccharides of other Lactobacillus mucosae RC14, Lactobacillus mucosae 45B1, Lactobacillus mucosae 12M3, and Lactobacillus mucosae 215 did not show a more prominent down-regulation effect on the increased IL-1β mRNA expression caused by modeling than the exopolysaccharide of CCFM1453.

[0085] Taking the expression level of IL-6 mRNA in the control group as approximately 1, the expression level in the model group increased to 48.64 after LPS intervention; the exopolysaccharide extracted from Lactobacillus mucosae CCFM1453 significantly reduced the expression level of IL-6 mRNA in RAW264.7 cells to 32.66, while the exopolysaccharides of other Lactobacillus mucosae RC14, Lactobacillus mucosae 45B1, Lactobacillus mucosae 12M3, and Lactobacillus mucosae 215 did not show a more prominent down-regulation effect on the increased IL-6 mRNA expression caused by modeling than the exopolysaccharide of CCFM1453, and some even caused an increase in the expression of IL-6 mRNA after intervention.

[0086] Taking the expression level of IL-10mRNA in the control group as about 1, the expression level in the model group increased to 3.29 after LPS intervention; the exopolysaccharide extracted from Lactobacillus mucosae CCFM1453 significantly increased the expression level of IL-10mRNA in RAW264.7 cells to 6.30, while the exopolysaccharides of other Lactobacillus mucosae RC14, Lactobacillus mucosae 45B1, Lactobacillus mucosae 12M3, and Lactobacillus mucosae 215 did not have a more prominent effect on the increase in IL-10mRNA expression caused by modeling than the exopolysaccharide of CCFM1453.

[0087] Taking the expression level of TNF-αmRNA in the control group as about 1, the expression level in the model group increased to 2.03 after LPS intervention; the exopolysaccharide extracted from Lactobacillus mucosae CCFM1453 significantly increased the expression level of TNF-αmRNA in RAW264.7 cells to 0.43, while the exopolysaccharides of other Lactobacillus mucosae RC14, Lactobacillus mucosae 45B1, Lactobacillus mucosae 12M3, and Lactobacillus mucosae 215 did not have a more prominent effect on alleviating the increase in TNF-αmRNA expression caused by modeling than the exopolysaccharide of CCFM1453.

[0088] It can be seen from this that the exopolysaccharide extracted from Lactobacillus mucosae CCFM1453 reduces the expression of IL-1βmRNA, IL-6mRNA, and TNF-αmRNA in RAW264.7 cells under LPS intervention and increases the expression of IL-10mRNA. It is proved that the exopolysaccharide of Lactobacillus mucosae CCFM1453 has the effect of anti-inflammatory in macrophage RAW264.7.

[0089] Example 5: Effects of exopolysaccharide extracted from Lactobacillus mucosae CCFM1453 on the levels of PGE2 and TFF3 in the gastric tissue of mice with gastric mucosal injury

[0090] The specific steps are as follows:

[0091] Preparation of the exopolysaccharide used for gavage: Referring to the method of Example 2, collect the supernatant in the fermentation broth with a colony count of 1×10 9 CFU, extract the crude polysaccharide, remove the protein, dialyze, and freeze-dry to obtain the exopolysaccharide powder of Lactobacillus mucosae.

[0092] Experimental grouping: 35 healthy male BALB / c mice at 8 weeks of age were randomly divided into 7 cages, with 5 mice in each cage. The 7 cages were as follows: 1 cage of pseudo-sterile control group (p-Control), 1 cage of pseudo-sterile model group (p-Model), 1 cage of extracellular polysaccharide group of Lactobacillus mucosae CCFM1453 (p-CCFM1453eps), 1 cage of control group (Control), 1 cage of model group (Model), 1 cage of extracellular polysaccharide group of Lactobacillus mucosae CCFM1453 (CCFM1453eps), and 1 cage of Yangshen group (p-pc).

[0093] Experimental procedure:

[0094] (1) After one week of adaptation, except for the control group, the other groups were gavaged with acetylsalicylic acid at a daily dose of 400 mg / kg (0.2 mL of 50 mg / mL acetylsalicylic acid) for 7-day-induced chronic gastritis in BALB / c mice (7 - 14 days);

[0095] (2) From day 13 to 25, all pseudo-sterile groups were used to establish a pseudo-sterile mouse model by gavage with a combination of four antibiotics. The four antibiotics were: vancomycin 100 mg / kg; neomycin 200 mg / kg; ampicillin 200 mg / kg; metronidazole 200 mg / kg, and the gavage dose was 0.2 mL;

[0096] (3) From day 15 to 25, the p-CCFM1453eps group and the CCFM1453eps group were gavaged with extracellular polysaccharides extracted from Lactobacillus mucosae CCFM1453 at a daily dose of 50 mg / kg (0.2 mL of 6.25 mg / mL). The control group and the model group were gavaged with an equal volume of normal saline as a control. The Yangshen group was gavaged with hydrotalcite at a daily dose of 30 mg / kg until the end of the experiment (day 25). All groups had free access to water and food, and the experimental procedure was as Figure 3 shown.

[0097] After the experiment, the mice were sacrificed, and the gastric tissues were removed and preserved in paraformaldehyde for H&E staining. The results were as Figure 7 shown. Compared with the blank group, superficial erosion was visible in the model group, with loose edema in the mucosal layer and lamina propria mucosae, a little inflammatory infiltration, and loose edema in the muscularis propria. After repair with CCFM1453eps, the damage of the mucosal barrier in the pseudo-sterile mouse group and the normal mouse group improved, the degree of edema decreased, and the infiltration of inflammatory cells was significantly reduced.

[0098] After the experiment, the mice were sacrificed and eyeball blood was collected. After standing for 40 min, the blood was centrifuged at 3000 r / min for 20 min, and the blood supernatant was taken for ELISA detection. The back skin tissue was cut and ground into a homogenate according to a weight-to-volume ratio of 1:10 with PBS, centrifuged at 3000 r / min for 20 min, and the skin supernatant was taken to detect the PGE2 content in the gastric tissue of the mice by an ELISA kit. The results are as Figure 4 shown:

[0099] Compared with the p-Control group (3.28 pg / mg), the PGE2 content in the gastric tissue of the p-Model group increased significantly to 11.70 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1453 significantly reduced the content of the gastric inflammation marker PGE2 in the gastric tissue of the mice compared with the p-Model group, decreasing to 4.04 pg / mg. Compared with the Control group (5.17 pg / mg), the PGE2 content in the gastric tissue of the Model group increased significantly to 9.37 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1453 significantly reduced the content of the gastric inflammation marker PGE2 in the gastric tissue of the mice compared with the Model group, decreasing to 3.69 pg / mg.

[0100] The TFF3 content in the gastric tissue of the mice was detected by an ELISA kit. The results are as Figure 4 shown:

[0101] Compared with the p-Control group (29.18 pg / mg), the TEE3 content in the gastric tissue of the p-Model group increased significantly to 87.42 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1453 significantly reduced the content of the gastric inflammation marker TFF3 in the gastric tissue of the mice compared with the p-Model group, decreasing to 50.59 pg / mg, and the effect was better than that of hydrotalcite. Compared with the Control group (58.53 pg / mg), the TFF3 content in the gastric tissue of the Model group increased significantly to 92.58 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1453 significantly reduced the content of the gastric inflammation marker TFF3 in the gastric tissue of the mice compared with the Model group, decreasing to 87.56 pg / mg.

[0102] From the above results, it can be seen that the exopolysaccharide extracted from Lactobacillus mucosae CCFM1453 has the ability to relieve the accumulation of PGE2 and TFF3 contents in the gastric tissue of mice with gastric mucosal injury, and the improvement is obvious compared with the model group, and the improvement effect is better than that of the drug hydrotalcite.

[0103] Example 6: Effects of extracellular polysaccharides extracted from Lactobacillus mucosae CCFM1453 on the contents of inflammatory markers in gastric tissues and serum of mice with gastric mucosal injury

[0104] The animal experiment design and gavage groups involved in the following examples were the same as those in Example 5. The contents of IL-6, IL-8, IL-10, and COX-2 in the gastric tissues and serum of mice with gastric mucosal injury were detected using an Elisa kit from Nanjing Senbeijia Company as Figure 5 shown.

[0105] (1) Content of IL-6 in gastric tissue: Compared with the p-Control group (2.37 pg / mg), the content of IL-6 in the gastric mucosal injury tissue of the p-Model group increased significantly to 7.12 pg / mg. Oral administration of extracellular polysaccharides extracted from Lactobacillus mucosae CCFM1453 significantly reduced the content of IL-6 in the gastric tissues of mice, decreasing to 3.53 pg / mg; compared with the Control group (3.54 pg / mg), the content of IL-6 in the gastric tissues of the Model group increased significantly to 7.86 pg / mg. Oral administration of extracellular polysaccharides extracted from Lactobacillus mucosae CCFM1453 significantly reduced the content of IL-6 in the gastric tissues of mice compared with the model group, decreasing to 4.35 pg / mg.

[0106] (2) Content of IL-8 in gastric tissue: Compared with the p-Control group (1.82 pg / mg), the content of IL-8 in the gastric mucosal injury tissue of the p-Model group increased significantly to 6.36 pg / mg. Oral administration of extracellular polysaccharides extracted from Lactobacillus mucosae CCFM1453 significantly reduced the content of IL-8 in the gastric tissues of mice, decreasing to 4.72 pg / mg; compared with the Control group (3.06 pg / mg), the content of IL-8 in the gastric tissues of the Model group increased significantly to 7.16 pg / mg. Oral administration of extracellular polysaccharides extracted from Lactobacillus mucosae CCFM1453 significantly reduced the content of IL-8 in the gastric tissues of mice compared with the model group, decreasing to 5.67 pg / mg.

[0107] (3) IL-10 content in gastric tissue: Compared with the p-Control group (40.10 pg / mg), the IL-10 content in the gastric mucosa damaged tissue of the p-Model group was significantly reduced to 7.08 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus reuteri CCFM1453 significantly increased the IL-10 content in the gastric tissue of mice, reaching 36.53 pg / mg. Compared with the Control group (53.00 pg / mg), the IL-10 content in the gastric tissue of the Model group was significantly reduced to 37.55 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus reuteri CCFM1453 significantly increased the IL-10 content in the gastric tissue of mice compared with the model group, reaching 43.24 pg / mg.

[0108] (4) COX-2 content in serum: Compared with the p-Control group (3.01 ng / mg), the COX-2 content in the serum of the p-Model group was significantly increased to 4.85 ng / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus reuteri CCFM1453 significantly reduced the COX-2 content in the serum of mice, to 2.70 ng / mg. Compared with the Control group (3.58 ng / mg), the COX-2 content in the serum of the Model group was significantly increased to 7.23 ng / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus reuteri CCFM1453 significantly reduced the COX-2 content in the serum of mice compared with the model group, to 4.98 ng / mg.

[0109] Based on the comprehensive results of relevant biochemical indicators in animal serum, it can be seen that the exopolysaccharide extracted from Lactobacillus reuteri CCFM1453 can reduce the contents of inflammatory factors IL-6, IL-8, IL-10 and COX-2 in the gastric tissue and serum of mice with gastric mucosal injury, and relieve inflammation to counteract gastric mucosal injury. By observing the differences between the pseudo-axenic mouse group and the normal mouse group, it was found that the intervention effect of the pseudo-axenic mouse group was better, probably because antibiotics can increase gastrointestinal permeability and may change the absorption pattern of polysaccharides, making polysaccharides more easily absorbed.

[0110] Example 7: Effect of exopolysaccharide extracted from Lactobacillus reuteri CCFM1453 on the expression of key target genes in the gastric tissue of mice with gastric mucosal injury

[0111] The animal experiment design, gavage groups, and RNA extraction and detection methods involved in the following examples are the same as those in Example 5. The primers for the key genes STAT3, SOCS3, TRPV4, MyD88, AKT, and IKBα in the gastric tissue of mice with gastric mucosal injury are described in Table 2 below, and the gene expression results are shown in Figure 6 .

[0112] Table 2: Primer Sequences

[0113]

[0114] (1) STAT3: STAT3 is a member of the Stat family of cytoplasmic transcription factors and is involved in many biological processes, including cell proliferation, survival, differentiation, and angiogenesis, as well as many pathological processes, including inflammation, tumor, and immune responses. Due to the impact of modeling, the expression level of STAT3 mRNA in mouse gastric tissue was upregulated from 1.00 in the control group to 4.09 in the p-Model group and 10.42 in the Model group. However, after intragastric administration of the exopolysaccharide extracted from Lactobacillus reuteri CCFM1453, the relative expression levels of STAT3 mRNA in the p-CCFM1453eps group and the CCFM1453eps group were 1.48 and 1.11, respectively.

[0115] (2) SOCS3: SOCS3 belongs to the SOCS protein family and is involved in the regulation of inflammatory cytokines, insulin resistance, and glucose metabolism. Downregulation of SOCS3 alleviates the severity of intestinal inflammation. Due to the impact of modeling, the expression level of SOCS3 mRNA in mouse gastric tissue was upregulated from 1.00 in the control group to 4.22 in the p-Model group and 2.22 in the Model group. However, after intragastric administration of the exopolysaccharide extracted from Lactobacillus reuteri CCFM1453, the relative expression levels of SOCS3 mRNA in the p-CCFM1453eps group and the CCFM1453eps group were 0.99 and 0.80, respectively.

[0116] (3) TRPV4: TRPV4 is a widely expressed non-selective cation channel that plays an important role in various physiological and pathological processes. Due to the impact of modeling, the expression level of TRPV4 mRNA in mouse gastric tissue was upregulated from 1.00 in the control group to 4.73 in the p-Model group and 11.50 in the Model group. However, after intragastric administration of the exopolysaccharide extracted from Lactobacillus reuteri CCFM1453, the relative expression levels of TRPV4 mRNA in the p-CCFM1453eps group and the CCFM1453eps group were 1.11 and 1.09, respectively.

[0117] (4) AKT: As one of the serine / threonine protein kinases, AKT plays a key role in the treatment of inflammatory gastric diseases. Due to the impact of modeling, the expression level of AKT mRNA in mouse gastric tissue was upregulated from 1.00 in the control group to 7.40 in the p-Model group and 8.68 in the Model group. However, after intragastric administration of the exopolysaccharide extracted from Lactobacillus reuteri CCFM1453, the relative expression levels of AKT mRNA in the p-CCFM1453eps group and the CCFM1453eps group were 1.28 and 4.13, respectively.

[0118] (5) MyD88: MyD88 is a key molecule in the TLR4 / MyD88 / NF-κB signaling pathway, which plays an important role in the occurrence and development of gastric mucosal injury. Due to the influence of modeling, the expression level of MyD88 mRNA in the gastric tissue of mice increased from 1.00 in the control group to 3.13 in the p-Model group and 2.12 in the Model group. However, after intragastric administration of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1453, the relative expression levels of MyD88 mRNA in the p-CCFM1453eps group and the CCFM1453eps group were 1.50 and 1.32, respectively.

[0119] (6) IκBα: The role of IκBα in gastric mucosal injury is mainly related to its regulation of the NF-κB signaling pathway. Due to the influence of modeling, the expression level of IκBα mRNA in the gastric tissue of mice increased from 1.00 in the control group to 2.55 in the p-Model group and 4.01 in the Model group. However, after intragastric administration of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1453, the relative expression levels of IκBα mRNA in the p-CCFM1453eps group and the CCFM1453eps group were 0.91 and 0.65, respectively.

[0120] The exopolysaccharide extracted from Lactobacillus mucosae CCFM1453 can inhibit gastric mucosal injury through the JAK / STAT3 signaling pathway, PI3K / AKT signaling pathway, NF-κB signaling pathway and MAPK signaling pathway, and these signaling pathways play important roles in cell proliferation, differentiation, immune regulation and inflammatory response.

[0121] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A Limosilactobacillus reuteri CCFM1453, which is deposited in the Guangdong Microbial Culture Collection Center with the deposit number GDMCC No: 65608.

2. A method for preparing exopolysaccharide of Lactobacillus reuteri CCFM1453, characterized in that, It is obtained by fermenting the Limosilactobacillus reuteri CCFM1453 described in claim 1 in a medium, collecting the fermentation supernatant, and performing alcohol precipitation and purification.

3. The method according to claim 2, wherein The fermentation is carried out by inoculating the seed liquid of Limosilactobacillus reuteri CCFM1453 into a medium and culturing at 25 - 40 °C for at least 10 - 30 h to obtain a fermentation broth.

4. The method according to claim 3, wherein The cell concentration of Limosilactobacillus reuteri CCFM1453 in the fermentation broth is not less than 5.0×10 7 CFU / mL.

5. The method according to claim 4, wherein The medium contains 10 - 12 g / L of glucose, 5.0 - 7.0 g / L of yeast powder, 9.0 - 11.0 g / L of peptone, 2 - 3 g / L of anhydrous sodium acetate, 2 - 2.5 g / L of diammonium hydrogen citrate, 2 - 2.6 g / L of dipotassium hydrogen phosphate, 0.05 - 0.25 g / L of manganese sulfate monohydrate, 0.1 - 0.5 g / L of magnesium sulfate heptahydrate, and 1.0 - 1.5 mL / L of Tween - 80.

6. The exopolysaccharide obtained by the method according to any one of claims 2 to 5.

7. A product containing Lactobacillus reuteri CCFM1453 as described in claim 1 or the extracellular polysaccharide as described in claim 6, characterized in that The product includes food, medicine, health products or daily chemical products.

8. The product according to claim 7, characterized in that, The medicine further contains a drug carrier and / or pharmaceutical excipients; the pharmaceutical excipients include excipients and additives; the health product is used to assist in protecting the gastric mucosa.

9. The product according to claim 8, wherein, The dosage form of the product includes tablets, granules, capsules or liquid preparations.

10. Use of the Limosilactobacillus reuteri CCFM1453 described in claim 1, or the exopolysaccharide described in claim 5, in the preparation of a medicine or health product for relieving gastric mucosal injury.

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