Lactobacillus paracasei CCFM1455, polysaccharide prepared from lactobacillus paracasei CCFM1455 for promoting gastric mucosa repair and application of lactobacillus paracasei CCFM1455 in synergism with rhizoma polygonati

Abstract

The invention discloses a Lactobacillus paracasei CCFM1455 strain, a polysaccharide prepared from the Lactobacillus paracasei CCFM1455 strain for promoting gastric mucosa repair and application of the Lactobacillus paracasei CCFM1455 strain in synergism with rhizoma polygonati, and belongs to the technical field of microorganisms and medicines. According to the lactobacillus paracasei CCFM1455 exopolysaccharide disclosed by the invention, by regulating an STAT3 / SOCS3 signal channel, the levels of gastric mucosal lesion markers PGE2 and TFF3 are remarkably reduced, the expression of inflammatory factors is regulated, and gastric barrier protection and inflammation relief are realized. In addition, when the strain is used for fermenting polygonatum sibiricum, double synergistic effects can be generated: (1) macromolecular substances such as polygonatum sibiricum saponin are biologically converted, and easily absorbed micromolecular active substances such as oleanolic acid and liukucoumarin are generated; (2) polygonatum polysaccharide is converted into high-activity compound polysaccharide, so that the mucous membrane repairing synergistic effect is enhanced; therefore, the lactobacillus paracasei CCFM1455 and the fermentation derivative thereof provide an innovative solution for targeted intervention of gastric mucosal lesion and modernization of traditional Chinese medicines.

Description

Technical Field

[0001] The present invention relates to a strain of Lactobacillus paracasei CCFM1455, a polysaccharide prepared therefrom for promoting gastric mucosa repair, 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 injury at the same time. Acute gastric mucosal injury is an acute disruption of the gastric barrier caused by physical, chemical or biological stimuli, manifested as pathological changes such as congestion, edema to ulcers. If not intervened in time, it can progress to chronic inflammation or induce complications such as perforation and bleeding. Gastritis, as its typical progression form, is closely related to mucosal defense imbalance, activation of pro-inflammatory factors and oxidative stress. Long-term lesions can cause three major harms: local tissue ulceration destroys gastric homeostasis, digestive and absorption disorders lead to systemic metabolic disorders, and precancerous lesions such as intestinal metaplasia significantly increase the risk of gastric cancer. Current clinical treatments mainly focus on symptom management and mucosal repair, including acid-suppressing drugs such as proton pump inhibitors and H2 receptor antagonists, Helicobacter pylori eradication regimens, and gastric mucosal protectants. Postbiotics prepared from probiotic fermentation metabolites have the advantages of biological safety and no reaction with gastritis drugs. It is of great significance to screen postbiotics that can specifically relieve gastritis and gastric mucosal injury caused by non-steroidal anti-inflammatory drugs.

[0003] In traditional Chinese medicine, Polygonatum sibiricum is used to treat gastritis, mainly due to its effects of tonifying the middle qi, nourishing the lungs yin, and tonifying the kidneys yin. Polygonatum sibiricum polysaccharide, as the main active ingredient of Polygonatum sibiricum, its bioavailability is closely related to the degree of structural modification. Probiotic exopolysaccharides have become a new hot spot for intervening in gastrointestinal diseases due to their anti-inflammatory and immunomodulatory properties. By directed fermentation of Polygonatum sibiricum with Lactobacillus, not only can the strain be induced to secrete exopolysaccharides with mucosal repair properties, but also a synergistic product of probiotic-modified Polygonatum sibiricum polysaccharide and bacterial-derived exopolysaccharide can be formed. In addition to polysaccharides, Polygonatum sibiricum also contains rich bioactive substances such as saponins, flavonoids, and lignans. However, due to their large molecular weight and strong polarity, their absorption rate in the intestine is often low. Lactobacillus strains with high-efficiency biotransformation ability can convert these active substances into more easily absorbed derivatives, thus achieving a double breakthrough in component synergism and improved bioavailability. Oleanolic acid, imperatorin, harmaline, etc. have significant anti-inflammatory, anti-tumor and lipid accumulation inhibitory effects. It is of great significance to provide a strain that can increase the content of the above substances in Polygonatum sibiricum and synergistically enhance the efficacy of Polygonatum sibiricum in relieving gastric mucosal injury. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides an extracellular polysaccharide of Lacticaseibacillus paracasei CCFM1455, and the application of a composite polysaccharide extracted after fermenting polygonatum sibiricum by Lacticaseibacillus paracasei CCFM1455 in a product for relieving gastric mucosal injury.

[0005] The present invention provides a strain of Lacticaseibacillus paracasei CCFM1455, which was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on December 11, 2024, with the deposit number GDMCC NO: 65610.

[0006] Under the microscope, the cells of the Lacticaseibacillus paracasei CCFM1455 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 Lacticaseibacillus paracasei CCFM1455 is a Gram-positive bacterium, facultatively anaerobic, thermophilic, with an optimal growth temperature of 35-40 °C and an optimal growth pH of 6.0-7.0.

[0008] The present invention also provides a method for preparing the extracellular polysaccharide of Lacticaseibacillus paracasei CCFM1455, which includes the following steps: inoculating the Lacticaseibacillus paracasei CCFM1455 into a fermentation medium for cultivation to obtain a culture solution, collecting the obtained culture supernatant, adding 4 volumes of absolute ethanol to precipitate the extracellular polysaccharide, 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, adding trichloroacetic acid to make its final concentration 4% (m / v) and placing it at 4 °C for 12 h, centrifuging at 6000 rpm at 4 °C for 15 min, and taking the supernatant. 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] The present invention also provides a method for extracting composite polysaccharides from fermented polygonatum sibiricum by lactobacillus paracasei CCFM1455, 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 out polygonatum polysaccharides. Freeze-dry the polygonatum polysaccharides. Inoculate the lactobacillus paracasei CCFM1455 into a polygonatum polysaccharide fermentation medium for culture to obtain a culture solution, collect the obtained culture supernatant, add 4 volumes of absolute ethanol to precipitate composite polysaccharides, leave at room temperature for 24 h, and then centrifuge at 8000 rpm for 10 min to separate out 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.

[0010] In one embodiment, the hot water extraction is as follows: place the polygonatum sibiricum powder in a certain amount of water and water bath at 80 °C for 2 h, repeat 2 times and collect the supernatant.

[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. Preferably, it contains 5 g / L of polygonatum polysaccharides, 5 g / L of glucose, 5 g / L of yeast extract powder, 0.1 g / L of magnesium sulfate heptahydrate, and 0.05 g / L of manganese sulfate monohydrate.

[0012] In one embodiment, the method for preparing polygonatum polysaccharides is as follows: 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 out polygonatum polysaccharides. Freeze-dry the polygonatum polysaccharides for standby. The hot water extraction is as follows: place the polygonatum sibiricum powder in a certain amount of water and water bath at 80 °C for 2 h, repeat 2 times and collect the supernatant.

[0013] In one embodiment, in the culture solution used for preparing the exopolysaccharides, the cell concentration of lactobacillus paracasei CCFM1455 is not less than 5.0×10 7 CFU / mL.

[0014] The present invention also provides a product, which contains the exopolysaccharides of the above-mentioned lactobacillus paracasei CCFM1455, and / or the composite polysaccharides after fermentation of polygonatum sibiricum by lactobacillus paracasei CCFM1455.

[0015] In one embodiment, the product includes food, health products, drugs or cosmetics.

[0016] In one embodiment, the food includes the above composition and conventional excipients.

[0017] In one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0018] In one embodiment, the health product includes the above composition and conventional excipients, and the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0019] In one embodiment, the health product is used to assist in protecting the gastric mucosa.

[0020] In one embodiment, the drug contains the above composition, drug carrier and / or pharmaceutical excipients.

[0021] In one embodiment, the pharmaceutical excipients include excipients and additives.

[0022] 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.

[0023] In one embodiment of the present invention, the cosmetics include the above composition, matrix raw materials and / or conventional excipients.

[0024] In one embodiment of the present invention, the matrix raw materials include oil raw materials, wax raw materials, synthetic oil raw materials, powder raw materials, gum raw materials, coagulants, and surfactants.

[0025] In one embodiment of the present invention, the conventional excipients include one or more of humectants, whitening agents, flavoring agents, binders, lubricants, preservatives, film formers, antioxidants, emulsifiers, and cosmetic nutritional additives.

[0026] In one embodiment, the product is the fermented product of Lactobacillus paracasei CCFM1455 after fermentation in a medium containing polygonatum polysaccharide.

[0027] In one embodiment, the formulation of the culture medium containing polygonatum polysaccharide includes: 1 - 10 g / L of polygonatum polysaccharide, 1 - 5 g / L of glucose, 1 - 10 g / L of yeast extract powder, 0.1 g / L of magnesium sulfate heptahydrate, and 0.05 g / L of manganese sulfate monohydrate; preferably, 5 g / L of polygonatum polysaccharide, 5 g / L of glucose, 5 g / L of yeast extract powder, 0.1 g / L of magnesium sulfate heptahydrate, and 0.05 g / L of manganese sulfate monohydrate.

[0028] In one embodiment, the inoculation amount of Lactobacillus paracasei CCFM1455 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 fermentation composition obtained by fermenting by the above method.

[0031] The present invention also provides the application of the exopolysaccharide of the above Lactobacillus paracasei CCFM1455, or the fermentation composition in the preparation of a medicine for relieving gastritis and promoting gastric mucosa repair.

[0032] In one embodiment, in the medicine, the dose of the exopolysaccharide of Lactobacillus paracasei CCFM1455 is not less than 5 mg / kg body weight.

[0033] The present invention provides a method for improving the bioavailability and utilization rate of triterpenoid saponins and flavonoid compounds in polygonatum by fermentation conversion.

[0034] In one embodiment, inoculate Lactobacillus paracasei CCFM1455 into a culture medium containing polygonatum for fermentation; the culture medium containing polygonatum polysaccharide is a polygonatum fermentation medium, and the polygonatum fermentation medium contains 10 - 20 g / L of polygonatum, 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 Lactobacillus paracasei CCFM1455 of the present invention has the ability to relieve host gastritis and gastric mucosa damage when orally administered, specifically manifested as:

[0037] (1) Relief of gastric mucosal injury: The degree of edema in gastric tissue is reduced, and the infiltration of inflammatory cells is significantly decreased; the contents of gastric mucosal injury markers PGE2 and TFF3 are decreased.

[0038] (2) Anti-inflammatory mechanism: Significantly reduce the levels of inflammatory factors IL-6 and IL-8 in the gastric tissue of mice, relieve the inflammatory response caused by gastric mucosal injury and gastritis; significantly increase the expression of IL-10, inhibit the excessive inflammatory response; significantly reduce the COX-2 level in the serum of mice with gastric mucosal injury and gastritis, thereby inhibiting the activation of the inflammatory pathway; reduce the expression of STAT3, regulate the expression of SOCS3, relieve gastric inflammation and the cell apoptosis signaling pathway, and relieve gastritis; inhibit the expression of MyD88, intervene in the TLR4 / MyD88 / NF-κB signaling pathway, and block the cascade reaction of inflammatory factors; increase the expression of Bcl2, reduce apoptosis, and protect gastric mucosal cells.

[0039] 2. Lacticaseibacillus paracasei CCFM1455 has the ability to degrade macromolecular substances such as steroidal saponins, triterpenoid saponins, flavonoids, and lignans in Polygonatum sibiricum. Using Lacticaseibacillus paracasei CCFM1455 to ferment Polygonatum sibiricum can produce oleanolic acid, licoricidin, harmaline, etc. with higher activity and bioavailability.

[0040] 3. After fermentation of Lacticaseibacillus paracasei CCFM1455 with Polygonatum sibiricum polysaccharide, a synergistic promoting effect is produced in relieving gastritis and gastric mucosal injury:

[0041] (1) Enhanced anti-inflammatory mechanism: The complex polysaccharide of fermented Polygonatum sibiricum significantly reduces the levels of PGE2, IL-6, and IL-8 in the stomach compared with using Polygonatum sibiricum polysaccharide alone, and has a stronger inhibitory effect on the inflammatory response.

[0042] (2) The complex polysaccharide after fermenting Polygonatum sibiricum is superior to the single polysaccharide in terms of inflammation inhibition and gastric mucosal repair.

[0043] Therefore, the complex polysaccharide extracted from the exopolysaccharide of Lacticaseibacillus paracasei CCFM1455 and the fermented Polygonatum sibiricum polysaccharide has great application prospects in products for relieving gastritis and gastric mucosal damage.

[0044] Biological material preservation

[0045] A strain of Lactobacillus paracasei CCFM1455, taxonomically named Lactobacillus paracasei, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 11, 2024, with the collection number GDMCC No: 65610, and the collection address is Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 :Effects of different probiotic exopolysaccharides on RAW264.7 cell proliferation.

[0047] Figure 2 :Effects of different probiotic extracellular polysaccharides on the expression of IL-1βmRNA, IL-6mRNA and TNF-αmRNA in LPS-induced RAW264.7 cells.

[0048] Figure 3 : Flowchart of mouse experiments.

[0049] Figure 4 :Effects of extracellular polysaccharide extracted from Lactobacillus paracasei CCFM1455 on gastric mucosal injury and the contents of PGE2 and TFF3, markers of gastritis in mice.

[0050] Figure 5 : Effects of extracellular polysaccharides extracted from Lactobacillus paracasei CCFM1455 on the contents of inflammatory markers IL-6, IL-8, IL-10, and COX-2 in mice with gastric mucosal injury.

[0051] Figure 6 :Effects of exopolysaccharides extracted from Lactobacillus paracasei CCFM1455 on alleviating gastric mucosal injury through different pathways.

[0052] Figure 7 : Effects of complex polysaccharides extracted from polygonatum sibiricum fermented by Lactobacillus paracasei CCFM1455 on the contents of inflammatory markers PGE2, IL-6, and IL-10 in mice with gastric mucosal injury.

[0053] Figure 8 :Pathological sections of Lactobacillus reuteri CCFM1455 exopolysaccharide promoting gastric mucosal repair.

[0054] “*” indicates statistical difference compared with the Model group (P<0.05), “**” indicates significant statistical difference compared with the Model group (P<0.01), “***” indicates extremely significant statistical difference compared with the Model group (P<0.001); “****” indicates extremely significant statistical difference compared with the Model group (P<0.0001). Detailed implementation manners

[0055] The present invention will be further described below in conjunction with specific embodiments.

[0056] The mouse macrophages (RAW264.7) involved in the following examples were purchased from: Shanghai Cell Bank.

[0057] The BALB / c mice involved in the following examples were purchased from Vital River Laboratories.

[0058] The Lactobacillus paracasei CCFM1455, Lactobacillus paracasei P40, Lactobacillus paracasei Z4L8, Lactobacillus paracasei Z11, and Lactobacillus paracasei NT521 involved in the following examples were self-screened strains from the Food Biotechnology Center of Jiangnan University.

[0059] Polygonatum sibiricum was purchased from Beijing Tongrentang Co., Ltd., China.

[0060] The culture media involved in the following examples are as follows:

[0061] 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 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.

[0062] 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 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.

[0063] 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 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.

[0064] Polygonatum polysaccharide fermentation medium: Add 5 g / L of polygonatum polysaccharide, 5 g / L of glucose, 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 it at 115 °C for 20 min to prepare the culture solution for polygonatum polysaccharide fermentation.

[0065] The preparation method of polygonatum polysaccharide is as follows: Crush polygonatum, add 4 volumes of absolute ethanol after hot water extraction to precipitate polygonatum polysaccharide, let it stand at room temperature for 24 h, and then centrifuge at 8000 rpm for 10 min to separate polygonatum polysaccharide. Freeze-dry the polygonatum polysaccharide. The hot water extraction is to place the polygonatum powder in water at a ratio of 1:5 (m / v), water bath at 80 °C for 2 h, repeat 2 times and collect the supernatant.

[0066] 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).

[0067] Example 1: Cell resuscitation and culture

[0068] 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 d, perform cell passage.

[0069] Example 2: Screening of Lactobacillus paracasei CCFM1455 and extraction of exopolysaccharide

[0070] (1) Screening of Lactobacillus paracasei CCFM1455

[0071] Collect fecal samples from Inner Mongolia. After pre-treating the samples, store them in a -80 °C refrigerator in 20% glycerol. Take them out and thaw, mix well and pipette 0.5 mL of the sample into 4.5 mL of physiological saline, perform gradient dilution with physiological saline, select an appropriate gradient dilution solution and spread it on MRS solid medium, culture at 37 °C for 48 h, pick the typical colonies of Lactobacillus paracasei and streak-purify them on MRS solid medium, pick single colonies and transfer them to MRS liquid medium for enrichment, and preserve them with 30% glycerol to obtain the strain. Extract the genomic DNA of the strain and amplify and sequence 16S rDNA (performed by Suzhou Genewiz Biotechnology Co., Ltd.). The 16S rDNA sequencing results are determined to be Lactobacillus paracasei by NCBI sequence alignment and named Lactobacillus paracasei CCFM1455.

[0072] (2) Extraction of exopolysaccharides from Lactobacillus paracasei CCFM1455, Lactobacillus paracasei P40, Lactobacillus paracasei Z4L8, Lactobacillus paracasei Z11, and Lactobacillus paracasei NT521

[0073] 1) Streak and resuscitate Lactobacillus paracasei CCFM1455 from the preservation tube, culture it in an MRS solid medium in a 37°C water bath constant temperature incubator for 24 - 48 h to obtain single colonies; pick the single colonies and inoculate them into an MRS liquid medium, culture at 37°C for 12 - 18 h to obtain culture solution 1;

[0074] 2) Inoculate culture solution 1 into an MRS liquid medium at an inoculation amount of 2% (v / v), culture at 37°C for 12 h to obtain a seed solution;

[0075] 3) Inoculate the seed solution into an MRS simplified liquid medium at 2 - 5% (v / v) respectively for expansion culture, culture at 37°C for 18 - 24 h to obtain bacterial liquid a.

[0076] 4) The supernatant obtained by centrifuging bacterial liquid a under the conditions of 8000 r / min for 30 min is added with 4 volumes of absolute ethanol to precipitate the exopolysaccharide, left at room temperature for 24 h, and then centrifuged 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 left 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.

[0077] Example 3: Fermentation of Polygonatum sibiricum by Lactobacillus paracasei CCFM1455 to Extract Complex Polysaccharides

[0078] Dip an inoculation loop into the bacterial liquid of Lactobacillus paracasei CCFM1455 and streak it on an MRS solid medium, culture it in an inverted position at 37°C for 48 h; take single colonies into an MRS liquid medium, culture aerobically at 37°C for 18 h, mix well, and then inoculate the bacterial liquid into a new MRS liquid medium for culture at an inoculation amount of 2% (v / v). Repeat this operation continuously for 3 times to finally obtain an activated bacterial liquid.

[0079] The obtained activated bacterial solution was inoculated into the polygonatum polysaccharide fermentation medium at an inoculum size of 2% (v / v), and cultured with shaking at 200 rpm at 37 °C for 24 h. At 24 h of culture, the fermentation broth was taken, and the supernatant obtained by centrifuging the bacterial solution at 8000 r / min for 30 min was added with 4 volumes of absolute ethanol to precipitate the complex polysaccharide, which was left standing 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 standing at 4 °C for 12 h, then 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 complex polysaccharide was freeze-dried.

[0080] Example 4: Effect of extracellular polysaccharide extracted from Lactobacillus paracasei CCFM1455 on the proliferation of mouse macrophage RAW264.7

[0081] The specific steps are as follows:

[0082] (1) Take 100 μL of mouse macrophage RAW264.7 in the logarithmic growth phase and inoculate it 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 it adheres to the wall, a blank group, a control group, and a probiotic extracellular polysaccharide treatment group are set;

[0083] The blank group contains only cell culture medium and no mouse macrophage RAW264.7;

[0084] The control group contains cell culture medium and mouse macrophage RAW264.7 but no probiotic extracellular polysaccharide;

[0085] The probiotic extracellular polysaccharide treatment group contains cell culture medium with different extracellular polysaccharides obtained in Example 2 and mouse macrophage RAW264.7.

[0086] Preparation of cell culture medium containing probiotic extracellular polysaccharide: The freeze-dried extracellular polysaccharide was resuspended in cell culture medium (the amount of the resuspended extracellular polysaccharide was equivalent to the amount of extracellular polysaccharide extracted from the bacterial solution fermented to a concentration of 5.0×10 7 CFU / mL) to obtain cell culture medium containing probiotic extracellular polysaccharide.

[0087] (2) The above-mentioned well plates were respectively incubated in an incubator at 37 °C for 24 h. After the incubation, 10 μL of CCK8 solution was added to each well and incubated for 2 h to measure the absorbance value (OD) at 450 nm.

[0088] 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%.

[0089] The effects of probiotic exopolysaccharides on cell proliferation are as Figure 1 shown. Compared with the control group, the cell proliferation rates of adding exopolysaccharides of Lactobacillus paracasei CCFM1455, Lactobacillus paracasei P40, Lactobacillus paracasei Z4L8, Lactobacillus paracasei Z11, and Lactobacillus paracasei NT521 are 91.23%, 92.05%, 89.84%, 90.62%, and 75.94% respectively.

[0090] According to the toxicity grading evaluation method of ISO 10993-5:2009, if the cell viability is greater than 70%, it can be considered non-toxic. Considering its non-cytotoxicity according to the above results, the optional inactivated cell concentration is 5.0×10 7 CFU / mL is the appropriate exopolysaccharide concentration for subsequent cell experiments.

[0091] Example 5: Effects of exopolysaccharides extracted from Lactobacillus paracasei CCFM1455 on the expression levels of inflammatory factor mRNAs in RAW264.7 cells intervened by LPS

[0092] The specific steps are as follows:

[0093] (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. Discard the old medium, rinse 3 times with PBS, and set up control group 1 and model group 1;

[0094] Add 2 mL of cell culture medium to control group 1;

[0095] Add 2 mL of cell culture medium containing 1 μg / mL LPS to model group 1.

[0096] (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:

[0097] For the control group, after changing the medium in control group 1, add 2 mL of cell culture medium;

[0098] For the model group, after changing the medium in model group 1, add 2 mL of cell culture medium;

[0099] The treatment groups were grouped as follows: The exopolysaccharides of Lactobacillus paracasei CCFM1455, Lactobacillus paracasei P40, Lactobacillus paracasei Z4L8, Lactobacillus paracasei Z11, and Lactobacillus paracasei NT521 were prepared respectively according to the method of Example 2, and resuspended with cell culture medium for standby (the amount of the resuspended exopolysaccharide was equivalent to the amount of the exopolysaccharide prepared from the bacterial liquid fermented to a concentration of 5.0×10 7 CFU / mL).

[0100] After changing the liquid in Modeling Agent Group 1, 2 mL of the exopolysaccharides of Lactobacillus paracasei CCFM1455, Lactobacillus paracasei P40, Lactobacillus paracasei Z4L8, Lactobacillus paracasei Z11, and Lactobacillus paracasei NT521 resuspended with cell culture medium were respectively aspirated and added into a 6-well plate, and cultured for 24 h, with three parallels for each sample.

[0101] (3) The above-mentioned well plates were incubated in an incubator at 37°C for 24 h, the culture supernatant was discarded, each well was quickly washed 3 times with PBS, 1 mL of cell lysate was added to each well, and pipetted repeatedly. The cell lysate was taken 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 TNF-α mRNA were calculated using the 2-△△Ct formula, where the internal reference was β-actin. The primers are described in Table 1 below, and the results are as Figure 2 shown.

[0102] Table 1: Primer sequences

[0103]

[0104] The results showed that, as Figure 2 known, the expression level of IL-1β mRNA in the control group was about 1, and after LPS intervention, the expression level in the model group increased to 128.78; the exopolysaccharide extracted from Lactobacillus paracasei CCFM1455 significantly reduced the expression level of IL-1β mRNA in RAW264.7 cells to 16.59, while the exopolysaccharides of other Lactobacillus paracasei P40, Lactobacillus paracasei Z4L8, Lactobacillus paracasei Z11, and Lactobacillus paracasei NT521 did not have a more prominent down-regulation effect on the increased expression of IL-1β mRNA caused by modeling than the exopolysaccharide of CCFM1455.

[0105] Taking the expression level of IL-6 mRNA in the control group as about 1, the expression level in the model group increased to 48.64 after LPS intervention; the exopolysaccharide extracted from Lactobacillus paracasei CCFM1455 significantly reduced the expression level of IL-6 mRNA in RAW264.7 cells to 6.07, while the exopolysaccharides of other Lactobacillus paracasei P40, Lactobacillus paracasei Z4L8, Lactobacillus paracasei Z11, and Lactobacillus paracasei NT521 did not show a more prominent down-regulation effect on the increased expression of IL-6 mRNA caused by modeling than the exopolysaccharide of CCFM1455, and some even caused an increase in the expression of IL-6 mRNA after intervention.

[0106] Taking the expression level of TNF-α mRNA 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 paracasei CCFM1455 significantly reduced the expression level of TNF-α mRNA in RAW264.7 cells to 0.21, while the exopolysaccharides of other Lactobacillus paracasei P40, Lactobacillus paracasei Z4L8, Lactobacillus paracasei Z11, and Lactobacillus paracasei NT521 did not show a more prominent down-regulation effect on the increased expression of TNF-α mRNA caused by modeling than the exopolysaccharide of CCFM1455.

[0107] It can be seen from this that the exopolysaccharide extracted from Lactobacillus paracasei CCFM1455 reduces the expression of IL-1β mRNA, IL-6 mRNA, and TNF-α mRNA in RAW264.7 cells under LPS intervention. It is proved that the exopolysaccharide of Lactobacillus paracasei CCFM1455 has the effect of anti-inflammation in macrophages RAW264.7.

[0108] Example 6: Effect of exopolysaccharide extracted from Lactobacillus paracasei CCFM1455 on the levels of PGE2 and TFF3 in the gastric tissue of mice with gastric mucosal injury

[0109] The specific steps are as follows:

[0110] 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 and purify it to obtain the exopolysaccharide powder of Lactobacillus paracasei.

[0111] 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 for the pseudo-sterile control group (p-Control), 1 cage for the pseudo-sterile model group (p-Model), 1 cage for the extracellular polysaccharide group of Lactobacillus paracasei CCFM1455 in the pseudo-sterile group (p-CCFM1455eps), 1 cage for the control group (Control), 1 cage for the model group (Model), 1 cage for the extracellular polysaccharide group of Lactobacillus paracasei CCFM1455 (CCFM1455eps), and 1 cage for the yangshen group (p-pc).

[0112] Experimental procedures:

[0113] (1) After one week of adaptation, except for the control group, the remaining 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 (days 7 - 14).

[0114] (2) From days 13 - 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. The non-pseudo-sterile groups were gavaged with an equal volume of normal saline.

[0115] (3) From days 15 - 25, the p-CCFM1455eps group and the CCFM1455eps group were gavaged with the extracellular polysaccharide extracted from Lactobacillus paracasei CCFM1455 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 aluminum magnesium carbonate 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 procedures were as Figure 3 shown.

[0116] After the experiment, the mice were sacrificed, and the gastric tissues were 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, with loose edema in the mucosal layer and lamina propria mucosae, a little inflammatory infiltration, and loose edema in the lamina propria muscularis. After repair with CCFM1455eps, the damage to the mucosal barrier in the pseudo-sterile mouse group and the normal mouse group improved, the degree of edema decreased, and the inflammatory cell infiltration significantly decreased.

[0117] After the experiment, the mice were sacrificed, and the blood was taken from their eyeballs. After standing for 40 minutes, the blood was centrifuged at a speed of 3000 r / min for 20 minutes, 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 minutes, and the skin supernatant was taken for detection using an ELISA kit.

[0118] The content of PGE2 in the gastric tissue of mice was detected using an ELISA kit, and the results were as Figure 4 shown:

[0119] 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 paracasei CCFM1455 significantly reduced the content of PGE2, a gastric mucosal injury marker, in the gastric tissue of mice compared with the Model group, decreasing to 5.62 pg / mg.

[0120] The content of TFF3 in the gastric tissue of mice was detected using an ELISA kit, and the results were as Figure 4 shown:

[0121] Compared with p-Control (29.18 pg / mg), the content of TEE3 in the gastric tissue of the p-Model group increased significantly to 87.42 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus paracasei CCFM1455 significantly reduced the content of TFF3, a gastric mucosal injury marker, in the gastric tissue of mice compared with the p-Model group, decreasing to 44.20 pg / mg.

[0122] From the above results, it can be seen that the exopolysaccharide extracted from Lactobacillus paracasei CCFM1455 has the ability to alleviate 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.

[0123] Example 7: Effects of the exopolysaccharide extracted from Lactobacillus paracasei CCFM1455 on the contents of inflammatory markers in the gastric tissue and serum of mice with gastric mucosal injury

[0124] In the following examples, the animal experiment design and gavage groups involved were the same as those in Example 6. The contents of IL-6, IL-8, IL-10, and COX-2 in the gastric tissue and serum of mice with gastric mucosal injury were detected using an ELISA kit from Nanjing Senbeijia Company as Figure 5 shown.

[0125] (1) IL-6 content in gastric tissue: Compared with p-Control (2.37 pg / mg), the IL-6 content in the gastric mucosal injury tissue of p-Model increased significantly to 7.12 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus paracasei CCFM1455 significantly reduced the IL-6 content in the gastric tissue of mice, which decreased to 3.47 pg / mg.

[0126] (2) IL-8 content in gastric tissue: Compared with p-Control (1.82 pg / mg), the IL-8 content in the gastric mucosal injury tissue of p-Model increased significantly to 6.36 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus paracasei CCFM1455 significantly reduced the IL-8 content in the gastric tissue of mice, which decreased to 3.69 pg / mg.

[0127] (3) IL-10 content in gastric tissue: Compared with p-Control (40.10 pg / mg), the IL-10 content in the gastric mucosal injury tissue of p-Model decreased significantly to 7.08 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus paracasei CCFM1455 significantly increased the IL-10 content in the gastric tissue of mice, which increased to 28.64 pg / mg; Compared with Control (53.00 pg / mg), the IL-10 content in the gastric tissue of the model group decreased significantly to 37.55 pg / mg. Oral administration of the exopolysaccharide extracted from Lactobacillus paracasei CCFM1455 significantly increased the IL-10 content in the gastric tissue of mice compared with the model group, which increased to 67.11 pg / mg.

[0128] (4) COX-2 content in serum: Compared with p-Control (3.01 ng / L), the COX-2 content in the serum of p-Model increased significantly to 4.85 ng / L. Oral administration of the exopolysaccharide extracted from Lactobacillus paracasei CCFM1455 significantly reduced the COX-2 content in the serum of mice, which decreased to 3.31 ng / L; Compared with Control (3.58 ng / L), the COX-2 content in the serum of the model group increased significantly to 7.23 ng / L. Oral administration of the exopolysaccharide extracted from Lactobacillus paracasei CCFM1455 significantly reduced the COX-2 content in the serum of mice compared with the model group, which decreased to 4.85 ng / L.

[0129] Based on the comprehensive results of relevant biochemical indicators in animal sera, it can be known that the exopolysaccharide extracted from Lactobacillus paracasei CCFM1455 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, alleviating inflammation to counteract gastric mucosal injury. 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. This may be because antibiotics increase gastrointestinal permeability, which may change the absorption pattern of polysaccharides, making them more easily absorbed.

[0130] Example 8: Effect of the exopolysaccharide extracted from Lactobacillus paracasei CCFM1455 on the expression of key target genes in the gastric tissue of mice with gastric mucosal injury

[0131] 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, MyD88, and Bcl2 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 .

[0132] Table 2: Primer sequences

[0133]

[0134] (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 effect of modeling, the expression level of STAT3 mRNA in the gastric tissue 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 paracasei CCFM1455, the relative expression levels of STAT3 mRNA in the p-CCFM1455eps group and the CCFM1455eps group were 2.26 and 0.88.

[0135] (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 effect of modeling, the expression level of SOCS3 mRNA in the gastric tissue 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 paracasei CCFM1455, the relative expression levels of SOCS3 mRNA in the p-CCFM1455eps group and the CCFM1455eps group were 1.30 and 0.83.

[0136] (3) 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 modeling effect, the expression level of MyD88 mRNA in mouse gastric tissue was upregulated 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 paracasei CCFM1455, the relative expression levels of MyD88 mRNA in the p-CCFM1455eps group and the CCFM1455eps group were 1.87 and 1.08, respectively.

[0137] (4) 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. Due to the modeling effect, the expression level of Bcl2 mRNA in mouse gastric tissue was upregulated from 1.00 in the control group to 1.46 in the p-Model group. After intragastric administration of the exopolysaccharide extracted from Lactobacillus paracasei CCFM1455, the relative expression level of Bcl2 mRNA in the p-CCFM1455eps group was 2.28.

[0138] The exopolysaccharide extracted from Lactobacillus paracasei CCFM1455 can inhibit gastric mucosal injury 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.

[0139] Example 9: Effect of Lactobacillus paracasei CCFM1455-fermented polygonatum on improving the oral alleviation of gastric mucosal injury by polygonatum polysaccharide in mice

[0140] For the mouse experiment of gastric mucosal injury, the methods for constructing gastric tissue and detecting serum PGE2, IL-6, IL-8, and IL-10 were the same as those in Example 6. Groups were added: pseudo-sterile polygonatum polysaccharide + exopolysaccharide of Lactobacillus paracasei CCFM1455 (p-hjeps), pseudo-sterile exopolysaccharide of Lactobacillus paracasei CCFM1455-fermented polygonatum (p-hjfps), polygonatum polysaccharide + exopolysaccharide of Lactobacillus paracasei CCFM1455 (hjeps), and exopolysaccharide of Lactobacillus paracasei CCFM1455-fermented polygonatum (hjfps).

[0141] Among them, the preparation method of the exopolysaccharide of fermented polygonatum was referred to Example 3, and the number of colonies collected was 5×10 8The supernatant in the CFU fermentation broth was taken to extract the complex polysaccharide, followed by alcohol precipitation, protein removal, dialysis, and freeze-drying to obtain the complex polysaccharide powder. The preparation method of polygonatum polysaccharide was as follows: polygonatum was crushed, hot water extracted, followed by alcohol precipitation and centrifugation to separate the polygonatum polysaccharide. The polygonatum polysaccharide was freeze-dried. The total gavage dose of the polygonatum polysaccharide + extracellular polysaccharide of Lactobacillus paracasei CCFM1455 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 for each group was: 50 mg / kg.

[0142] It can be seen from Figure 7 that after fermenting polygonatum with Lactobacillus paracasei CCFM1455, the content of PGE2 in the p-hjfps group decreased from 14.78 pg / mg to 8.20 pg / mg compared with the p-CCFM1455eps group, and in the hjfps group decreased from 5.62 pg / mg to 4.72 pg / mg compared with the CCFM1455eps group; the content of IL-6 in the hjfps group decreased from 6.30 pg / mg to 5.26 pg / mg compared with the CCFM1455eps group; the content of IL-10 in the p-hjfps group increased from 28.64 pg / mg to 49.44 pg / mg compared with the p-CCFM1455eps group, proving that the complex polysaccharide extracted after fermentation with Lactobacillus paracasei CCFM1455 has better effects.

[0143] Combined with the in vitro experimental results, the above experimental results prove that orally administering the complex polysaccharide extracted after fermenting polygonatum with Lactobacillus paracasei CCFM1455 can exert its synergistic effect in vivo, that is, the ability of polygonatum polysaccharide to resist gastric mucosal damage is further enhanced by using the extracellular polysaccharide of Lactobacillus paracasei CCFM1455.

[0144] Example 10: Analysis of the effect of fermenting polygonatum with Lactobacillus paracasei CCFM1455 on the optimization of functional components based on non-target metabolomics

[0145] Metabolite extraction:

[0146] Polygonatum was fermented with Lactobacillus paracasei CCFM1455. The polygonatum fermentation medium: add 10 g / L of polygonatum powder, 5 g / L of yeast extract powder, 0.05 g / L of manganese sulfate monohydrate, 0.1 g / L of magnesium sulfate heptahydrate. After adjusting the pH to 6.8 - 7.2, sterilization was carried out at 115 °C for 20 min to prepare the culture solution for polygonatum fermentation. Then, 2% (v / v) of Lactobacillus paracasei CCFM1455 was inoculated into it and fermented at 37 °C for 24 h.

[0147] Transfer the supernatant before and after the fermentation of Polygonatum sibiricum by Lactobacillus paracasei CCFM1455 into an EP tube, add 4 times the extraction solution (methanol:acetonitrile = 1:1 (V / V)), and the extraction solution contains an 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 put it into a sample vial for on-machine detection.

[0148] On-machine detection:

[0149] For polar metabolites, in this project, a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph is used, and the target compounds are chromatographically separated through a Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) liquid chromatography column. The liquid chromatography mobile phase A is the aqueous phase, containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia water, and the mobile phase B is acetonitrile. Sample tray temperature: 4 °C, injection volume: 2 μL. The Orbitrap Exploris 120 mass spectrometer can collect primary and secondary mass spectrometry data under the control of the control software (Xcalibur, version: 4.4, Thermo). The detailed parameters are 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).

[0150] Data processing:

[0151] After the raw data is converted into the mzXML format by the ProteoWizard software, a co-written R package is used for metabolite identification, and the database used is BiotreeDB (V3.0), and then a self-written R package is used for visualization analysis.

[0152] Consult the literature to collect the chemical formula names, molecular formulas, and molecular weights of the active substances in Polygonatum sibiricum. Combine the differentially expressed metabolites screened by non-target metabolomics, and screen the differential metabolites according to P-value < 0.05 and Log Fold Change > 2. Lactobacillus paracasei CCFM1455 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, ligustilide, harmaline, etc., as shown in Table 3.

[0153] Table 3 Changes in substances before and after fermentation

[0154]

[0155] Although these compounds had certain biological activities before fermentation, their absorption rate, utilization rate, and activity might be limited, especially the gastrointestinal absorption efficiency was low. Lactobacillus paracasei CCFM1455 transformed into new secondary metabolites, such as oleanolic acid, ligustilide, harmaline, etc. These secondary metabolites showed higher potential for biological activity, higher GI absorption ability, and better bioavailability scores. For example, oleanolic acid had significant anti-inflammatory effects and could exert anti-inflammatory activity by inhibiting the release of inflammatory mediators and the expression of cytokines; ligustilide had various pharmacological activities such as anti-tumor, anti-inflammatory, and anti-osteoporosis; harmaline could inhibit fat synthesis and thus inhibit fat accumulation.

[0156] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and decorations 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 strain of Lacticaseibacillus paracasei CCFM1455, which has been deposited in the Guangdong Microbial Culture Collection Center with the deposit number GDMCC No: 65610.

2. A method for preparing exopolysaccharide of Lactobacillus paracasei CCFM1455, characterized in that, The extracellular polysaccharide is obtained by fermenting the Lacticaseibacillus paracasei CCFM1455 described in claim 1, collecting the fermentation supernatant, and performing alcohol precipitation and purification.

3. The method according to claim 2, wherein The fermentation is as follows: inoculating the seed liquid of Lactobacillus paracasei CCFM1455 into a culture medium, and culturing at 25-40 °C for at least 10-30 h to obtain a fermentation broth; the cell concentration of Lactobacillus paracasei CCFM1455 in the fermentation broth is not lower than 5.0×10 7 CFU / mL.

4. The method according to claim 3, characterized in that The purification method includes, but is not limited to, deproteinization and / or dialysis.

5. The method according to claim 3 or 4, characterized in that, The medium includes MRS simplified liquid medium or polygonatum polysaccharide fermentation medium; the MRS simplified liquid 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.

6. The extracellular polysaccharide prepared by the method according to any one of claims 2 - 5.

7. A product containing the Lactobacillus paracasei CCFM1455 described in claim 1 or the exopolysaccharide described in claim 6, characterized in that, The product includes food, medicine, health products, or daily chemical products; the health product is used to assist in protecting the gastric mucosa.

8. The product according to claim 7, wherein, The product also includes conventional excipients; the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

9. The use of the Lacticaseibacillus paracasei CCFM1455 described in claim 1, or the extracellular polysaccharide described in claim 6, in the preparation of a medicine or health product for relieving gastric mucosal injury and / or gastritis.

10. Application of Lactobacillus paracasei CCFM1455 in improving the bioavailability of Polygonatum sibiricum, characterized in that, Inoculate the Lacticaseibacillus paracasei CCFM1455 described in claim 1 into a medium containing polygonatum for fermentation; The improvement of the bioavailability of polygonatum is to increase the content of oleanolic acid and / or fraxetin and / or harmaline; Optionally, the 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.

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