Lactobacillus reuteri CCFM1454, polysaccharide prepared from lactobacillus reuteri CCFM1454 for relieving gastritis and application of lactobacillus reuteri CCFM1454 in synergism with rhizoma polygonati

Abstract

The invention discloses lactobacillus reuteri CCFM1454, polysaccharide prepared from the lactobacillus reuteri CCFM1454 for relieving gastritis and application of the polysaccharide in synergism with rhizoma polygonati, and belongs to the technical field of microorganisms and medicines. According to the lactobacillus reuteri CCFM1454, the content of gastritis markers PGE2 and TFF3 can be remarkably reduced; the content of inflammatory factors is adjusted; the gastric mucosa is protected and inflammation is relieved by regulating and controlling key signal channels such as STAT3 and SOCS3. In addition, the compound polysaccharide extracted after the rhizoma polygonati polysaccharide is fermented by the lactobacillus reuteri CCFM1454 has a synergistic effect; active substances, such as saponin, flavone and the like, in rhizoma polygonati are converted through fermentation of lactobacillus reuteri CCFM1454, and the absorption rate and bioavailability of the rhizoma polygonati are improved. The strain and the fermentation product thereof have important potential in the development of products for relieving gastritis and repairing gastric mucosa, and can be widely applied to the fields of food, health care products, medicines and the like.

Description

Technical Field

[0001] The present invention relates to a strain of Lactobacillus mucosae CCFM1454 and a polysaccharide prepared therefrom for relieving gastritis and its application in synergistically enhancing the efficacy of Polygonatum sibiricum, belonging to the fields of microbial technology and pharmaceutical technology. Background Art

[0002] Non-steroidal anti-inflammatory drugs exert their antipyretic, analgesic and anti-inflammatory effects by inhibiting cyclooxygenase activity and reducing prostaglandin synthesis. However, they frequently cause gastric mucosal damage and gastritis at the same time. There are no specific effective treatment measures for gastritis. Its treatment aims to improve symptoms and reduce gastric mucosal inflammation. According to the mechanism of action, it can be divided into: 1. Drugs that regulate gastric acid secretion, which reduce gastric acid production by inhibiting the main enzyme for gastric acid secretion - the proton pump; 2. H2 receptor antagonists, which reduce gastric acid secretion by blocking histamine H2 receptors; 3. Drugs for eradicating Helicobacter pylori (H. pylori) to eliminate H. pylori infection; 4. Drugs for protecting the gastric mucosa, which protect the gastric mucosa by forming a protective barrier and reducing inflammation and damage; 5. According to the principle of traditional Chinese medicine syndrome differentiation and treatment, the body balance is regulated to improve gastric mucosal lesions. Postbiotics products mainly composed of probiotic exopolysaccharides have characteristics such as biological safety and stability. There has been no report on probiotic exopolysaccharide products that can relieve gastritis caused by non-steroidal anti-inflammatory drugs.

[0003] Polygonatum sibiricum is used in traditional Chinese medicine to treat gastritis. It mainly helps to improve gastritis symptoms and promote recovery through its effects of replenishing middle qi, nourishing the lungs and kidneys, and its functions of combating pathogenic microorganisms, antioxidation and anti-fatigue. Polygonatum sibiricum polysaccharide is the main active ingredient of Polygonatum sibiricum, and its bioavailability is closely related to the degree of structural modification. Probiotic exopolysaccharides have become a new focus in the intervention of gastrointestinal diseases due to their anti-inflammatory, immunomodulatory and other characteristics. Using Lactobacillus to ferment Polygonatum sibiricum directionally can also induce the strain to secrete exopolysaccharides with mucosal repair characteristics, forming a synergistic product of probiotic-modified Polygonatum sibiricum polysaccharide and bacterium-derived exopolysaccharide. In addition, in addition to polysaccharides, Polygonatum sibiricum is also rich in bioactive substances such as saponins and flavonoids. However, due to their large molecular weight and strong polarity, these components often face the technical bottleneck of low intestinal absorption rate. Screening Lactobacillus strains that not only have the effect of relieving gastritis but also have high biological conversion ability is beneficial to simultaneously play the efficacy of relieving gastritis and directionally convert active substances such as saponins and flavonoids in Polygonatum sibiricum into easily absorbable derivatives, synergistically improving the efficacy of the strain and Polygonatum sibiricum in relieving gastritis and repairing the gastric mucosa. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides an exopolysaccharide of Limosilactobacillus reuteri CCFM1454, and the application of a composite polysaccharide extracted after fermenting polygonatum sibiricum by Limosilactobacillus reuteri CCFM1454 in products for relieving gastritis.

[0005] The present invention provides a strain of Limosilactobacillus reuteri CCFM1454, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on December 11, 2024, with the deposit number GDMCC NO: 65609.

[0006] Under the microscope, the cells of the Limosilactobacillus reuteri CCFM1454 are curved bacilli with slightly irregular and round ends. The colonies after inoculation on MRS medium are white, with a moist and smooth surface and neat edges.

[0007] The Limosilactobacillus reuteri CCFM1454 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 CCFM1454, which includes the following steps: inoculating the Limosilactobacillus reuteri CCFM1454 into a fermentation medium for culture to obtain a culture solution, collecting the obtained culture supernatant, adding 4 times the volume of absolute ethanol to precipitate the exopolysaccharide, placing it at room temperature for 24 h, and then centrifuging at 8000 rpm for 10 min to separate the crude polysaccharide. The crude polysaccharide is completely dissolved in deionized water at a concentration of 10 mg / ml, trichloroacetic acid is added to make its final concentration 4% (m / v), and it is placed at 4 °C for 12 h, centrifuged at 6000 rpm at 4 °C for 15 min, and the supernatant is taken. Then it is 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 is determined by the phenol-sulfuric acid method. The dialyzed polysaccharide is freeze-dried.

[0009] In one embodiment, the fermentation 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.

[0010] The present invention also provides a method for extracting composite polysaccharides from fermented polygonatum sibiricum by Lactobacillus reuteri CCFM1454, comprising the following steps: pulverize polygonatum sibiricum, add 4 volumes of absolute ethanol after hot water extraction to precipitate polygonatum polysaccharides, leave at room temperature for 24 h, and then centrifuge at 8000 rpm for 10 min to separate the polygonatum polysaccharides. Freeze-dry the polygonatum polysaccharides. Inoculate Lactobacillus reuteri CCFM1454 into the polygonatum polysaccharide fermentation medium for cultivation to obtain a culture solution, collect the obtained culture supernatant, add 4 volumes of absolute ethanol to precipitate the composite polysaccharides, leave at room temperature for 24 h, and then centrifuge at 8000 rpm for 10 min to separate the crude composite polysaccharides. Dissolve the crude polysaccharides completely in deionized water at a concentration of 10 mg / ml, add trichloroacetic acid to make its final concentration 4% (m / v) and place 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 composite polysaccharides.

[0011] In one embodiment, the polygonatum polysaccharide fermentation medium contains 1 - 10 g / L of polygonatum polysaccharides, 1 - 5 g / L of glucose, 1 - 10 g / L of yeast extract powder, 0.05 - 0.25 g / L of manganese sulfate monohydrate, and 0.1 - 0.5 g / L of magnesium sulfate heptahydrate.

[0012] In one embodiment, the cell concentration of Lactobacillus reuteri CCFM1454 in the culture solution is not less than 5.0×10 7 CFU / mL.

[0013] The present invention also provides a product, which contains the above-mentioned Lactobacillus reuteri CCFM1454 or the above-mentioned exopolysaccharides.

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

[0015] In one embodiment, the health product is used for assisting in protecting the gastric mucosa.

[0016] In one embodiment, a drug or a cosmetic.

[0017] In one embodiment, the food includes the above composition and conventional excipients.

[0018] 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.

[0019] In one embodiment, the health product comprises the above composition and conventional excipients.

[0020] In one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0021] In one embodiment, the drug comprises the above composition, a pharmaceutical carrier, and / or pharmaceutical excipients.

[0022] In one embodiment, the pharmaceutical excipients comprise excipients and additives.

[0023] In one embodiment, the pharmaceutical excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, or release retardants.

[0024] In one embodiment of the present invention, the cosmetic comprises the above composition, matrix raw materials, and / or conventional excipients.

[0025] In one embodiment of the present invention, the matrix raw materials include oil raw materials, wax raw materials, synthetic oil raw materials, powdery raw materials, gum raw materials, coagulants, and surfactants.

[0026] In one embodiment of the present invention, the conventional excipients include one or more of humectants, whitening agents, flavoring agents, binders, lubricants, preservatives, film agents, antioxidants, emulsifiers, and cosmetic nutritional additives.

[0027] In one embodiment, the product is the fermented product of Lactobacillus reuteri CCFM1454 after fermentation in a medium containing polygonatum polysaccharide.

[0028] In one embodiment, the inoculum amount of the seed liquid of Lactobacillus reuteri CCFM1454 when inoculating in a medium containing polygonatum polysaccharide is 1-5% (v / v), preferably 2% (v / v).

[0029] In one embodiment, the fermentation conditions are: fermentation temperature 25-40 °C, fermentation time 10-30 h; preferably, fermentation temperature 37 °C, fermentation time 18-24 h.

[0030] The present invention also provides a fermented composition obtained by fermenting by the above method.

[0031] The present invention also provides the use of the exopolysaccharide of Limosilactobacillus reuteri CCFM1454 or the fermentation composition in the preparation of a medicament or health product for relieving gastritis and promoting gastric mucosa repair.

[0032] In one embodiment, in the medicament or health product, the dosage of the exopolysaccharide of Limosilactobacillus reuteri CCFM1454 is not less than 5 mg / kg body weight.

[0033] The present invention provides a method for increasing the content of oleanolic acid and / or harmaline in Polygonatum sibiricum, which is to add Limosilactobacillus reuteri CCFM1454 to a medium containing Polygonatum sibiricum for fermentation.

[0034] In one embodiment, the medium containing Polygonatum sibiricum contains 1-10 g / L of Polygonatum sibiricum powder, 1-10 g / L of yeast extract powder, 0.05-0.25 g / L of manganese sulfate monohydrate, and 0.1-0.5 g / L of magnesium sulfate heptahydrate.

[0035] Beneficial effects:

[0036] 1. The exopolysaccharide of Limosilactobacillus reuteri CCFM1454 of the present invention has the ability to relieve host gastritis and gastric mucosa damage when orally administered, specifically manifested as: reducing the degree of edema and inflammatory infiltration in the gastric tissue; decreasing the content of PGE2 and TFF3, which are gastric mucosa damage markers, in the gastric tissue.

[0037] The anti-inflammatory mechanism of Limosilactobacillus reuteri CCFM1454 is as follows: significantly reducing the levels of inflammatory factors IL-6 and IL-8 in the gastric tissue of mice, relieving the inflammatory reaction caused by gastritis; significantly increasing the expression of IL-10, inhibiting the excessive inflammatory reaction; significantly reducing the COX-2 level in the serum of gastritis mice, thereby inhibiting the activation of the inflammatory pathway; reducing the expression of STAT3, regulating the expression of SOCS3, alleviating the gastric inflammation and the cell apoptosis signal pathway, and relieving gastritis; inhibiting the expression of MyD88, intervening in the TLR4 / MyD88 / NF-κB signal pathway, and blocking the cascade reaction of inflammatory factors; down-regulating the expression of TRPV4, reducing the cell damage caused by calcium overload, and protecting the integrity of the gastric mucosa; increasing the expression of Bcl2, reducing cell apoptosis, and protecting gastric mucosal cells.

[0038] 2. The Limosilactobacillus reuteri CCFM1454 of the present invention has the characteristics of degrading steroidal saponins, triterpenoid saponins, flavonoids, lignans and other substances in polygonatum. After fermentation by Lactobacillus reuteri CCFM1454, substances such as oleanolic acid and salsapine with higher activity and bioavailability are produced in polygonatum.

[0039] 3. After co-fermentation of Limosilactobacillus reuteri CCFM1454 and Polygonatum sibiricum polysaccharide, the present invention can synergistically promote the relief of gastritis and gastric mucosal damage symptoms:

[0040] (1) Enhanced anti-inflammatory mechanism: The complex polysaccharides of fermented Polygonatum sibiricum significantly reduced the levels of IL-6 and IL-8 in the stomach compared with the use of Polygonatum sibiricum polysaccharides alone, and had a stronger inhibitory effect on inflammatory responses.

[0041] (2) The complex polysaccharides after fermentation of Polygonatum sibiricum performed better than single polysaccharides in inhibiting inflammation and repairing gastric mucosa.

[0042] Therefore, the complex polysaccharide extracted from the extracellular polysaccharide of Lactobacillus reuteri CCFM1454 and fermented Polygonatum sibiricum polysaccharide has great application prospects in products for relieving gastritis and gastric mucosal damage.

[0043] Biomaterial Deposit

[0044] A strain of Limosilactobacillus reuteri CCFM1454, taxonomically named Limosilactobacillus reuteri, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 11, 2024, with the deposit number GDMCC No: 65609, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 :Effects of different probiotic exopolysaccharides on RAW264.7 cell proliferation.

[0046] Figure 2 :Effects of different probiotic extracellular polysaccharides on the expression of IL-1βmRNA, IL-6mRNA, and IL-10mRNA in LPS-induced RAW264.7 cells.

[0047] Figure 3 : Flowchart of mouse experiments.

[0048] Figure 4: Effects of exopolysaccharides extracted from Lactobacillus mucosae CCFM1454 on the contents of gastritis markers PGE2 and TFF3 in gastritis mice.

[0049] Figure 5 : Effects of exopolysaccharides extracted from Lactobacillus mucosae CCFM1454 on the contents of inflammatory markers IL-6, IL-8, IL-10, and COX-2 in gastritis mice.

[0050] Figure 6 : Effects of exopolysaccharides extracted from Lactobacillus mucosae CCFM1454 on alleviating gastritis through different pathways.

[0051] Figure 7 : Effects of compound polysaccharides extracted from fermented Polygonatum sibiricum by Lactobacillus mucosae CCFM1454 on the contents of gastritis markers PGE2, TFF3 and inflammatory markers IL-8, IL-6, and COX-2 in gastritis mice.

[0052] Figure 8 : Pathological sections of gastritis alleviated by exopolysaccharides of Lactobacillus mucosae CCFM1454.

[0053] "*" indicates a statistically significant difference from the Model group (P < 0.05), "**" indicates a highly 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). Detailed implementation methods

[0054] The present invention will be further elaborated below in combination with specific embodiments.

[0055] The mouse macrophages (RAW264.7) involved in the following examples were purchased from: Shanghai Cell Bank.

[0056] The BALB / c mice involved in the following examples were purchased from Vital River Laboratories.

[0057] The Lactobacillus mucosae CCFM1454, 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.

[0058] Polygonatum sibiricum was purchased from Beijing Tongrentang Co., Ltd., China.

[0059] The media involved in the following examples are as follows:

[0060] MRS liquid medium: yeast powder 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, and Tween-80 1 mL / L, pH 6.2 - 6.4.

[0061] MRS solid medium: yeast powder 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL / L, and agar 20.0 g / L, pH 6.2 - 6.4.

[0062] MRS simplified liquid medium: glucose 10 g / L, yeast powder 5.0 g / L, peptone 10.0 g / L, anhydrous sodium acetate 2 g / L, diammonium hydrogen citrate 2 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.05 g / L, magnesium sulfate heptahydrate 0.1 g / L, and Tween-80 1 mL / L, pH 6.2 - 6.4.

[0063] Polygonatum polysaccharide fermentation medium: add polygonatum polysaccharide 5 g / L, glucose 5 g / L, yeast extract powder 5 g / L, manganese sulfate monohydrate 0.05 g / L, magnesium sulfate heptahydrate 0.1 g / L. After adjusting the pH to 6.8 - 7.2, sterilize at 115 °C for 20 min to prepare the culture solution for polygonatum polysaccharide fermentation.

[0064] The preparation method of polygonatum polysaccharide is as follows: crush polygonatum, extract with hot water at 80 °C at a solid-liquid ratio of 1:5 (m / v) for 2 h, then add 4 volumes of anhydrous ethanol to precipitate polygonatum polysaccharide, place at room temperature for 24 h, and then centrifuge at 8000 rpm for 10 min to separate out polygonatum polysaccharide.

[0065] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin and streptomycin mixed solution (the penicillin content in the mixed solution is 10000 U / mL, and the streptomycin concentration is 10 mg / mL).

[0066] Example 1: Cell resuscitation and culture

[0067] 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 recover their vitality and grow for 1 - 2 days to reach 70% - 80% confluence, cell passage is carried out.

[0068] Example 2: Screening of Limosilactobacillus reuteri CCFM1454 and Extraction of Exopolysaccharide

[0069] 1. Screening of Limosilactobacillus reuteri CCFM1454

[0070] The samples were from infant feces. After pretreatment, the samples were stored in -80°C refrigerator in 20% glycerol. After taking out and thawing, they were mixed well and 0.5 mL of the sample was pipetted into 4.5 mL of physiological saline, and gradient diluted with physiological saline. The appropriate gradient dilution was selected and 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. The single colonies were picked and transferred to MRS liquid medium for enrichment, and stored in 30% glycerol to obtain the strains. The genomic DNA of the strains 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, named Limosilactobacillus reuteri CCFM1454, and deposited in Guangdong Microbial Culture Collection Center on December 11, 2024, with the deposit number GDMCC No: 65609.

[0071] 2. Extraction of exopolysaccharides from Limosilactobacillus reuteri CCFM1454, Limosilactobacillus reuteri RC14, Limosilactobacillus reuteri 45B1, Limosilactobacillus reuteri 12M3, and Limosilactobacillus reuteri 215

[0072] 1) Streak and resuscitate Limosilactobacillus reuteri CCFM1454 from the preservation tube, and culture it in a 37°C water bath constant temperature incubator using MRS solid medium for 24 - 48 h to obtain single colonies; pick the single colonies and inoculate them into MRS liquid medium, and culture at 37°C for 12 - 18 h to obtain culture solution 1;

[0073] 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 the seed solution;

[0074] 3) Inoculate the seed liquid into the simplified MRS liquid medium at 2-5% (v / v) respectively for amplification culture, and culture at 37 °C for 18-24 h to obtain bacterial liquid a.

[0075] 4) The supernatant obtained by centrifuging the bacterial liquid a at 8000 r / min for 30 min was added with 4 times the volume of absolute ethanol to precipitate the extracellular polysaccharide. 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. 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. The polysaccharide content in the solution was determined by the phenol-sulfuric acid method. The dialyzed polysaccharide was freeze-dried.

[0076] Example 3: Extraction of compound polysaccharide from Polygonatum sibiricum by fermentation of Lactobacillus mucosae reuteri CCFM1454

[0077] Dip an inoculation loop into the bacterial liquid of Lactobacillus mucosae reuteri CCFM1454 and streak it on the MRS solid medium, and culture it inverted at 37 °C for 48 h; pick a single colony into the MRS liquid medium and culture it aerobically at 37 °C for 18 h. After mixing, take the bacterial liquid and inoculate it into a new MRS liquid medium for culture according to the inoculation amount of 2% (v / v). Repeat this operation continuously for 3 times to finally obtain the activated bacterial liquid.

[0078] Inoculate the obtained activated bacterial liquid into the Polygonatum sibiricum polysaccharide fermentation medium at an inoculation amount of 2% (v / v). The extraction method of Polygonatum sibiricum polysaccharide is as follows: Crush Polygonatum sibiricum, extract it with hot water at 80 °C for 2 h, then add 4 times the volume of absolute ethanol to precipitate Polygonatum sibiricum polysaccharide, leave it at room temperature for 24 h, and then centrifuge at 8000 rpm for 10 min to separate Polygonatum sibiricum polysaccharide. Freeze-dry the Polygonatum sibiricum polysaccharide. Shake-culture at 200 rpm at 37 °C for 24 h. After the fermentation is completed, take the fermentation broth. The supernatant obtained by centrifuging the bacterial liquid at 8000 r / min for 30 min was added with 4 times the volume of absolute ethanol to precipitate the compound polysaccharide, leave it at room temperature for 24 h, and then centrifuge 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. 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. The polysaccharide content in the solution was determined by the phenol-sulfuric acid method. The dialyzed compound polysaccharide was freeze-dried.

[0079] Example 4: Effect of exopolysaccharides extracted from Lactobacillus reuteri CCFM1454 on the proliferation of mouse macrophages RAW264.7

[0080] The specific steps are as follows:

[0081] (1) Take 100 μL of mouse macrophages RAW264.7 in the logarithmic growth phase and inoculate them into 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 probiotic exopolysaccharide treatment group;

[0082] The blank group contains only cell culture medium without mouse macrophages RAW264.7;

[0083] The control group contains cell culture medium and mouse macrophages RAW264.7 but no probiotic exopolysaccharide;

[0084] The probiotic exopolysaccharide treatment group contains cell culture medium with different exopolysaccharides obtained in Example 2 and mouse macrophages RAW264.7.

[0085] 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.

[0086] (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.

[0087] 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%.

[0088] 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 CCFM1454, 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.

[0089] 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 extracellular polysaccharide concentration for subsequent cell experiments.

[0090] Example 5: Effect of extracellular polysaccharide extracted from Lactobacillus mucosae CCFM1454 on the expression level of inflammatory factor mRNA in RAW264.7 cells intervened by LPS

[0091] The specific steps are as follows:

[0092] (1) Seed RAW264.7 cells at a density of 1.5×10 5 cells / mL on a 6-well plate and culture the cells overnight until they adhere. Discard the old medium, rinse 3 times with PBS, and set up control group 1 and model group 1;

[0093] Add 2 mL of cell medium to control group 1;

[0094] Add 2 mL of cell medium containing 1 μg / mL LPS to model group 1.

[0095] (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 a control group, a model group, and a treatment group:

[0096] The control group is, after changing the medium in control group 1, add 2 mL of ordinary cell medium;

[0097] The model group is, after changing the medium in model group 1, add 2 mL of ordinary cell medium;

[0098] The treatment group is grouped as follows: Refer to the method of Example 2 to prepare extracellular polysaccharides of Lactobacillus mucosae CCFM1454, Lactobacillus mucosae RC14, Lactobacillus mucosae 45B1, Lactobacillus mucosae 12M3, and Lactobacillus mucosae 215 respectively, and resuspend them with cell medium for standby (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).

[0099] After changing the liquid in Modeling Agent Group 1, 2 mL of exopolysaccharides of Lactobacillus mucosae CCFM1454, Lactobacillus mucosae RC14, Lactobacillus mucosae 45B1, Lactobacillus mucosae 12M3, and Lactobacillus mucosae 215, which were resuspended in cell culture medium, were respectively pipetted into a 6-well plate and cultured for 24 h, with three parallels for each sample.

[0100] (3) The above-mentioned well 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 used to extract RNA and reverse transcribed into cDNA using an RT-PCR reverse transcription kit. The expression of genes in RAW264.7 cells was detected by real-time fluorescence quantitative method, and the expression levels of IL-1β mRNA, IL-6 mRNA, and IL-10 mRNA were calculated using the 2-△△Ct formula, with β-actin as the internal reference. The primers are described in Table 1 below, and the results are as Figure 2 shown.

[0101] Table 1: Primer sequences

[0102]

[0103] The results showed that, as Figure 2 known, the expression level of IL-1β mRNA in the control group was approximately 1, and after LPS intervention, the expression level in the model group increased to 128.78; the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 significantly reduced the expression level of IL-1β mRNA in RAW264.7 cells to 26.24, 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 expression of IL-1β mRNA caused by modeling than the exopolysaccharide of CCFM1454.

[0104] The expression level of IL-6 mRNA in the control group was approximately 1, and after LPS intervention, the expression level in the model group increased to 48.64; the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 significantly reduced the expression level of IL-6 mRNA in RAW264.7 cells to 34.32, 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 expression of IL-6 mRNA caused by modeling than the exopolysaccharide of CCFM1454, and some even caused an increase in the expression of IL-6 mRNA after intervention.

[0105] 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 reuteri CCFM1454 significantly increased the expression level of IL-10mRNA in RAW264.7 cells to 5.90, while the exopolysaccharides of other Lactobacillus reuteri RC14, Lactobacillus reuteri 45B1, Lactobacillus reuteri 12M3, and Lactobacillus reuteri 215 did not have a more prominent effect on the increased expression of IL-10mRNA caused by modeling than the exopolysaccharide of CCFM1454.

[0106] It can be seen from this that the exopolysaccharide extracted from Lactobacillus reuteri CCFM1454 reduces the expression of IL-1βmRNA and IL-6mRNA in RAW264.7 cells under LPS intervention and increases the expression of IL-10mRNA. It is proved that the exopolysaccharide of Lactobacillus reuteri CCFM1454 has the effect of anti-inflammatory in macrophages RAW264.7.

[0107] Example 6: Effect of exopolysaccharide extracted from Lactobacillus reuteri CCFM1454 on the levels of PGE2 and TFF3 in the gastric tissue of mice with gastritis

[0108] The specific steps are as follows:

[0109] Preparation of exopolysaccharide 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 and purify it to obtain the exopolysaccharide powder of Lactobacillus reuteri.

[0110] Experimental grouping: Take 35 healthy male BALB / c mice at 8 weeks of age, randomly divide them into 7 cages, with 5 mice in each cage. The 7 cages are: 1 cage of pseudo-sterile control group (p-Control), 1 cage of pseudo-sterile model group (p-Model), 1 cage of pseudo-sterile exopolysaccharide of Lactobacillus reuteri CCFM1454 group (p-CCFM1454eps), 1 cage of control group (Control), 1 cage of model group (Model), 1 cage of exopolysaccharide of Lactobacillus reuteri CCFM1454 group (CCFM1454eps), and 1 cage of Yangshen group (p-pc).

[0111] Procedure:

[0112] (1) After the mice were acclimated for one week, except for the control group, the remaining groups were gavaged with acetylsalicylic acid (0.2 mL of 50 mg / mL acetylsalicylic acid) at a daily dose of 400 mg / kg for 7-day induction of chronic gastritis in BALB / c mice (7 - 14 days);

[0113] (2) From day 13 to day 25, all pseudo-sterile groups used the method of intragastric administration of quadruple antibiotics to establish a pseudo-sterile mouse model. The quadruple antibiotics were: vancomycin 100 mg / kg; neomycin 200 mg / kg; ampicillin 200 mg / kg; metronidazole 200 mg / kg, and the intragastric administration dose was 0.2 mL;

[0114] (3) From day 15 to day 25, the p-CCFM1454eps group and the CCFM1454eps group were intragastrically administered with the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 at a daily dose of 50 mg / kg (0.2 mL, 6.25 mg / mL). The control group and the model group were intragastrically administered with an equal volume of normal saline as a control; the positive control group was intragastrically administered with 30 mg / kg of hydrotalcite until the end of the experiment on day 25. All groups had free access to water and food, and the experimental procedure was as Figure 3 shown.

[0115] After the experiment, the mice were sacrificed, and the gastric tissue was removed and stored in paraformaldehyde for H&E staining. The results were as Figure 8 shown. Compared with the blank group, superficial erosion was visible in the model group, the mucosal layer and the lamina propria mucosa were loose and edematous, with a little inflammatory infiltration, and the lamina propria was loose and edematous. After repair with CCFM1454eps, 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 significantly decreased.

[0116] After the experiment, the mice were sacrificed and blood was taken from the eyeballs. After standing for 40 min, the blood was centrifuged at a speed of 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 content of PGE2 in the gastric tissue of mice through an ELISA kit. The results were as Figure 4 shown:

[0117] Compared with the p-Control group (3.28 pg / mg), the content of PGE2 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 CCFM1454 significantly reduced the content of the gastric inflammation marker PGE2 in the mice compared with the p-Model group, decreasing to 5.49 pg / mg; compared with the Control group (5.17 pg / mg), the content of PGE2 in the gastric tissue of the Model group increased significantly to 9.37 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 significantly reduced the content of the gastric inflammation marker PGE2 in the mice compared with the Model group, decreasing to 3.79 pg / mg.

[0118] The content of TFF3 in the gastric tissues of mice was detected by ELISA kit, and the results were as Figure 4 shown:

[0119] Compared with the p-Control group (29.18 pg / mg), the content of TEE3 in the gastric tissues of the p-Model group increased significantly to 87.42 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 significantly reduced the content of the gastritis marker TFF3 in the gastric tissues of mice, decreasing to 11.22 pg / mg; compared with the Control group (58.53 pg / mg), the content of TFF3 in the gastric tissues of the Model group increased significantly to 92.58 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 significantly reduced the content of the gastritis marker TFF3 in the gastric tissues of mice compared with the Model group, decreasing to 80.74 pg / mg.

[0120] From the above results, it can be seen that the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 has the ability to relieve the accumulation of PGE2 and TFF3 contents in the gastric tissues of gastritis mice, with obvious improvement compared with the model group, and the improvement effect is better than that of the drug.

[0121] Example 7: Effect of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 on the contents of inflammatory markers in the gastric tissues and serum of gastritis mice

[0122] In the following examples, the animal experiment design and gavage groups involved are the same as those in Example 6. The contents of IL-6, IL-8, IL-10, and COX-2 in the gastric tissues and serum of gastritis mice were detected using the Elisa kit from Nanjing Senbeijia Company as Figure 5 shown.

[0123] (1) Content of IL-6 in gastric tissues: Compared with the p-Control group (2.37 pg / mg), the content of IL-6 in the gastritis tissues of the p-Model group increased significantly to 7.12 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 significantly reduced the content of IL-6 in the gastric tissues of mice, decreasing to 0.88 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 the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 significantly reduced the content of IL-6 in the gastric tissues of mice compared with the model group, decreasing to 5.30 pg / mg.

[0124] (2) IL-8 content in gastric tissue: Compared with the p-Control group (1.82 pg / mg), the IL-8 content in the gastric tissue of the p-Model group with gastritis significantly increased to 6.36 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 significantly reduced the IL-8 content in the gastric tissue of mice, decreasing to 1.12 pg / mg. Compared with the Control group (3.06 pg / mg), the IL-8 content in the gastric tissue of the Model group significantly increased to 7.16 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 significantly reduced the IL-8 content in the gastric tissue of mice compared with the model group, decreasing to 6.60 pg / mg.

[0125] (3) IL-10 content in gastric tissue: Compared with the p-Control group (40.10 pg / mg), the IL-10 content in the gastric tissue of the p-Model group with gastritis significantly decreased to 7.08 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 significantly increased the IL-10 content in the gastric tissue of mice, increasing to 32.20 pg / mg. Compared with the Control group (53.00 pg / mg), the IL-10 content in the gastric tissue of the Model group significantly decreased to 37.55 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 significantly increased the IL-10 content in the gastric tissue of mice compared with the model group, increasing to 70.89 pg / mg.

[0126] (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 significantly increased to 4.85 ng / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 significantly reduced the COX-2 content in the serum of mice, decreasing to 3.64 ng / mg. Compared with the Control group (3.58 ng / mg), the COX-2 content in the serum of the Model group significantly increased to 7.23 ng / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 significantly reduced the COX-2 content in the serum of mice compared with the model group, decreasing to 4.65 ng / mg.

[0127] Based on the comprehensive results of relevant biochemical indicators in animal tissues and sera, it can be known that the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 can reduce the contents of inflammatory factors IL-6, IL-8, IL-10, and COX-2 in the gastric tissues and sera of gastritis mice, alleviating inflammation to combat gastritis. When 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, which may change the absorption pattern of polysaccharides and make polysaccharides more easily absorbed.

[0128] Example 8: Effects of the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454 on the expression of key target genes in the gastric tissues of gastritis mice

[0129] The animal experiment design, gavage groups, and RNA extraction and detection methods involved in the following examples are the same as those in Example 6. The primers for the key genes STAT3, SOCS3, TRPV4, MyD88, and Bcl2 in the gastric tissues of gastritis mice are described in Table 2 below, and the gene expression results are shown in Figure 6 .

[0130] Table 2: Primer sequences

[0131]

[0132] (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, tumors, and immune responses. Due to the influence of modeling, the expression level of STAT3 mRNA in the gastric tissues of mice 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 gavage with the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454, the relative expression levels of STAT3 mRNA in the p-CCFM1454eps group and the CCFM1454eps group were 2.21 and 0.94.

[0133] (2) SOCS3: SOCS3 belongs to the SOCS protein family and is involved in the regulation of inflammatory cytokines, insulin resistance, and glucose metabolism. The downregulation of SOCS3 alleviates the severity of intestinal inflammation. Due to the influence of modeling, the expression level of SOCS3 mRNA in the gastric tissues of mice 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 gavage with the exopolysaccharide extracted from Lactobacillus mucosae CCFM1454, the relative expression levels of SOCS3 mRNA in the p-CCFM1454eps group and the CCFM1454eps group were 1.49 and 0.19.

[0134] (3) TRPV4: TRPV4 is a widely expressed non-selective cation channel that plays an important role in various physiological and pathological processes. The expression level of TRPV4 mRNA in the gastric tissue of mice was upregulated from 1.00 in the control group to 4.73 in the p-Model group and 15.31 in the Model group due to modeling. However, after intragastric administration of the exopolysaccharide extracted from Lactobacillus reuteri CCFM1454, the relative expression levels of TRPV4 mRNA in the p-CCFM1454eps group and the CCFM1454eps group were 1.29 and 1.73.

[0135] (4) 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 gastritis. The expression level of MyD88 mRNA in the gastric tissue of mice was upregulated from 1.00 in the control group to 3.13 in the p-Model group and 2.12 in the Model group due to modeling. However, after intragastric administration of the exopolysaccharide extracted from Lactobacillus reuteri CCFM1454, the relative expression levels of MyD88 mRNA in the p-CCFM1454eps group and the CCFM1454eps group were 1.37 and 1.04.

[0136] (5) Bcl2: Bcl2 is an apoptosis inhibitor that can inhibit apoptosis in various cell systems. Bcl2 reduces inflammation by weakening the activation of the NLRP1 inflammasome, attenuating caspase1 activation and the release of IL-1β. The expression level of Bcl2 mRNA in the gastric tissue of mice was upregulated from 1.00 in the control group to 1.46 in the p-Model group and 1.17 in the Model group due to modeling. However, after intragastric administration of the exopolysaccharide extracted from Lactobacillus reuteri CCFM1454, the relative expression levels of Bcl2 mRNA in the p-CCFM1454eps group and the CCFM1454eps group were 2.04 and 1.88.

[0137] The exopolysaccharide extracted from Lactobacillus reuteri CCFM1454 can inhibit gastritis through the JAK / STAT3 signaling pathway, the PI3K / AKT signaling pathway, and the NF-κB signaling pathway. These signaling pathways play important roles in cell proliferation, differentiation, immune regulation, and inflammatory responses.

[0138] Example 9: Effect of Fermented Polygonatum sibiricum by Lactobacillus reuteri CCFM1454 on Oral Relief of Gastritis in Mice by Polygonatum sibiricum Polysaccharide

[0139] For the experiment on gastritis mice, the methods for detecting PGE2, COX-2, IL-6, IL-8, and IL-10 in gastric tissue and serum were as described in Example 6. The groups added with pseudo-sterile polygonatum polysaccharide + extracellular polysaccharide of Lactobacillus mucosae CCFM1454 (p-hjeps), pseudo-sterile extracellular polysaccharide of Lactobacillus mucosae CCFM1454 fermented polygonatum extracted composite polysaccharide (p-hjfps), polygonatum polysaccharide + extracellular polysaccharide of Lactobacillus mucosae CCFM1454 (hjeps), and extracellular polysaccharide of Lactobacillus mucosae CCFM1454 fermented polygonatum extracted composite polysaccharide (hjfps) were included.

[0140] Among them, the preparation method of the fermented polygonatum extracted composite polysaccharide was referred to Example 3. The supernatant in the fermentation broth with a colony count of 5×10 8 CFU was collected, and the composite polysaccharide was extracted and deproteinized by dialysis, and then freeze-dried to obtain the composite polysaccharide powder. The preparation method of polygonatum polysaccharide was as follows: Polygonatum was crushed, extracted with hot water at 80°C for 2 h, and then 4 volumes of absolute ethanol were added to precipitate polygonatum polysaccharide. It was left at room temperature for 24 h, and then centrifuged at 8000 rpm for 10 min to separate polygonatum polysaccharide. The polygonatum polysaccharide was freeze-dried. The total gavage dose of the polygonatum polysaccharide + extracellular polysaccharide of Lactobacillus mucosae CCFM1454 group was the same as that of the single polysaccharide gavage group. The two polysaccharides were mixed at a mass ratio of 1:1 and then gavaged. The gavage dose of each group was: 50 mg / kg.

[0141] It can be seen from Figure 7 that there was no significant difference in the protein expression of gastritis markers such as PGE2 between the groups added with polygonatum polysaccharide and the extracellular polysaccharide of Lactobacillus mucosae CCFM1454. However, after adding Lactobacillus mucosae CCFM1454-fermented polygonatum, the content of IL-6 in the p-hjfps group decreased from 0.88 pg / mg to 0.55 pg / mg compared with the p-CCFM1454eps group, and in the hjfps group decreased from 5.30 pg / mg to 3.73 pg / mg compared with the CCFM1454eps group; the content of IL-8 in the p-hjfps group decreased from 1.12 pg / mg to 1.01 pg / mg compared with the p-CCFM1454eps group, and in the hjfps group decreased from 6.60 pg / mg to 3.54 pg / mg compared with the CCFM1454eps group. It was proved that the composite polysaccharide extracted after fermentation by Lactobacillus mucosae CCFM1454 had better effects. And because there was a significant difference between the p-hjeps group and the p-hjfps group, it could be proved that the fermented composite polysaccharide product had better effects than adding the extracellular polysaccharide of Lactobacillus mucosae CCFM1454 and polygonatum polysaccharide simultaneously.

[0142] The above experimental results, combined with the in vitro experimental results, prove that the complex polysaccharide extracted from fermented polygonatum sibiricum by orally administered lactobacillus reuteri CCFM1454 can exert its synergistic effect in vivo, that is, the extracellular polysaccharide of lactobacillus reuteri CCFM1454 further enhances the anti-gastritis ability of polygonatum sibiricum polysaccharide.

[0143] Example 10: Effect of Lactobacillus reuteri CCFM1454 fermented Polygonatum sibiricum on the optimization of functional components based on non-target metabolomics

[0144] Metabolite extraction:

[0145] Ferment Polygonatum sibiricum with Lactobacillus reuteri CCFM1454. The fermentation medium for Polygonatum sibiricum: add 10 g / L of Polygonatum sibiricum powder, 5 g / L of yeast extract powder, 0.05 g / L of manganese sulfate monohydrate, and 0.1 g / L of magnesium sulfate heptahydrate. After adjusting the pH to 6.8 - 7.2, sterilize at 115 °C for 20 min. Inoculate Lactobacillus reuteri CCFM1454 into the sterilized fermentation medium of Polygonatum sibiricum at 2% (v / v) and ferment at 37 °C for 24 h.

[0146] Transfer the supernatant before and after fermentation of Polygonatum sibiricum by Lactobacillus reuteri CCFM1454 into an EP tube, add 4 times the extraction solution (methanol: acetonitrile = 1:1 (V / V)), and the extraction solution contains isotope-labeled internal standard; vortex for 30 s and sonicate for 10 min (ice-water bath); let stand at -40 °C for 1 h; centrifuge the sample at 4 °C and 12000 rpm (centrifugal force 13800 (×g), radius 8.6 cm) for 15 min; take the supernatant and load it into a sample vial for on-machine detection.

[0147] On-machine detection:

[0148] For polar metabolites, a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph was used in this project. Chromatographic separation of the target compounds was carried out on a Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) liquid chromatographic column. The mobile phase A for liquid chromatography was an aqueous phase containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia water, and the mobile phase B was acetonitrile. The sample tray temperature was 4°C, and the injection volume was 2 μL. The Orbitrap Exploris 120 mass spectrometer was capable of collecting primary and secondary mass spectrometry data under the control of the control software (Xcalibur, version: 4.4, Thermo). The detailed parameters were as follows: Sheath gas flow rate: 50 Arb, Aux gas flow rate: 15 Arb, Capillary temperature: 320°C, Full ms resolution: 60000, MS / MS resolution: 15000, Collision energy: SNCE 20 / 30 / 40, Spray Voltage: 3.8 kV (positive) or -3.4 kV (negative).

[0149] Data processing:

[0150] After the original data was converted into the mzXML format using the ProteoWizard software, a metabolomics identification was performed using a jointly developed R package. The database used was BiotreeDB (V3.0), and then a visualization analysis was carried out using a self-developed R package.

[0151] Consult the literature to collect the chemical formula names, molecular formulas, and molecular weights of the active substances in Polygonatum sibiricum. Combining non-target metabolomics, differential metabolites were screened based on P-value < 0.05 and Log Fold Change > 1.5. Lactobacillus reuteri CCFM1454 catabolizes steroidal saponins, triterpenoid saponins, flavonoids, lignans, etc. in Polygonatum sibiricum (the smaller the change multiple before and after fermentation, the better the decomposition degree), producing secondary metabolites with higher activity and bioavailability, such as oleanolic acid, harmaline, etc., as shown in Table 3.

[0152] Table 3 Changes in substances before and after fermentation

[0153]

[0154] Before fermentation, although these compounds have certain biological activities, their absorption rate, utilization rate, and activity may be limited, especially the gastrointestinal absorption efficiency is relatively low. Lactobacillus mucosae CCFM1454 has transformed into new secondary metabolites, such as oleanolic acid, harmaline, and axillarin, etc. These secondary metabolites exhibit higher potential for biological activity, higher GI absorption capacity, and more excellent bioavailability scores. For example, oleanolic acid has significant anti-inflammatory effects and can exert anti-inflammatory activity by inhibiting the release of inflammatory mediators and the expression of cytokines; harmaline can inhibit fat synthesis and thus inhibit fat accumulation.

[0155] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone 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 CCFM1454, which is deposited in the Guangdong Provincial Culture Collection of Microorganisms with the deposit number GDMCC NO: 65609.

2. A microbial preparation containing the Limosilactobacillus reuteri CCFM1454 as claimed in claim 1.

3. A method for preparing extracellular polysaccharide of Lactobacillus reuteri CCFM1454, characterized in that, It is obtained by fermenting the Limosilactobacillus reuteri CCFM1454 as claimed in claim 1 in a culture medium, collecting the fermentation supernatant, and performing alcohol precipitation and purification.

4. The method according to claim 3, wherein The culture medium includes a fermentation medium or a polygonatum polysaccharide fermentation medium; the fermentation 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; the polygonatum polysaccharide fermentation medium contains 1 - 10 g / L of polygonatum polysaccharide, 1 - 5 g / L of glucose, 1 - 10 g / L of yeast extract powder, 0.05 - 0.25 g / L of manganese sulfate monohydrate, and 0.1 - 0.5 g / L of magnesium sulfate heptahydrate.

5. The method according to claim 4, characterized in that, The cell concentration of Limosilactobacillus reuteri CCFM1454 in the fermentation broth is not less than 5.0×10 7 CFU / mL.

6. An exopolysaccharide prepared by the method as claimed in any one of claims 3 - 5.

7. A product containing Lactobacillus reuteri CCFM1454 as described in claim 1, or the microbial preparation as described in claim 2, or the exopolysaccharide as described in claim 6, characterized in that The product includes food, medicine, health products or daily chemical products; the health products are used to assist in protecting the gastric mucosa.

8. A method for increasing the content of oleanolic acid and / or harmaline in Polygonatum sibiricum, characterized in that, The Limosilactobacillus reuteri CCFM1454 as claimed in claim 1 is added to a culture medium containing polygonatum for fermentation.

9. The method according to claim 8, wherein, The culture medium containing polygonatum contains 1 - 10 g / L of polygonatum powder, 1 - 10 g / L of yeast extract powder, 0.05 - 0.25 g / L of manganese sulfate monohydrate, and 0.1 - 0.5 g / L of magnesium sulfate heptahydrate.

10. Use of the Limosilactobacillus reuteri CCFM1454 as claimed in claim 1, or the microbial preparation as claimed in claim 2, or the exopolysaccharide as claimed in claim 6 in the preparation of a medicine or a health product for relieving gastric mucosal injury and / or gastritis.

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