Lactobacillus mucilaginosus extracellular polysaccharide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of lactobacillus extracellular polysaccharide, in particular to fermented lactobacillus mucus extracellular polysaccharide as well as a preparation method and application thereof. The main chain of the exopolysaccharide comprises an acetylglucosamine residue, a glucose residue and a galactose residue; the main chain of the exopolysaccharide is composed of acetylglucosamine residues, glucose residues and galactose residues according to the molar ratio of 1: (3.78-3.84): (2.06-2.12); the exopolysaccharide disclosed by the invention can stimulate the phagocytic activity of macrophages and the release of NO and ROS, and can promote the expression of proinflammatory factors (TNF-alpha, IL-6 and IL-1beta) and anti-inflammatory factors (IL-10) of the macrophages at the same time; the M1 polarization of the macrophages is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of lactobacillus exopolysaccharide, in particular to fermented mucus lactobacillus exopolysaccharide and a preparation method and application thereof. Background Art

[0002] Since the 1960s, polysaccharides have been considered to be a broad-spectrum non-specific immunostimulant that can enhance the cellular and humoral immune functions of host cells, such as activating macrophages, T cells, B cells and NK cells, activating complement and inducing the production of interferon. Its function is to activate the body's non-specific defense function and has good therapeutic effects in anti-virus, anti-tumor, and anti-radiation.

[0003] Polysaccharides can be divided into animal, plant, and microbial polysaccharides based on their sources. Microbial polysaccharides, produced by microbial metabolism of carbohydrates, have been less studied due to their relatively low production. Exopolysaccharides, a class of naturally occurring macromolecular substances synthesized and secreted into the environment by microorganisms, are composed of multiple monosaccharide molecules that are dehydrated and polymerized, linked by various forms of glycosidic bonds, forming long, linear or branched chains. Microbial exopolysaccharides themselves create a suitable environment for microbial survival. They participate in the formation of the extracellular matrix (ECM), the medium that microbial cells adhere to their environment, forming the microbial capsule and providing a relatively stable living environment. Furthermore, exopolysaccharides serve as a means for microorganisms to store nutrients. With advances in science and technology, an increasing number of exopolysaccharides have been discovered and characterized. The rich diversity of exopolysaccharides has led to their diverse applications. With the continuous isolation, characterization, and utilization of exopolysaccharides, they have been utilized in various fields, including medicine, food, materials, cosmetics, and agriculture.

[0004] Lactobacillus mucilaginosus fermentation has become a hot topic for research and development due to its diverse beneficial effects, such as enhancing intestinal immunity, promoting nutrient absorption, regulating cholesterol and lactose metabolism, lowering blood pressure, and providing antioxidant properties, as well as its broad application prospects. Taking Lactobacillus mucilaginosus fermentation as an example, there are currently very few reports on the structural characterization and functional exploration of the exopolysaccharides of this strain. Therefore, the structural characterization and functional determination of the exopolysaccharides produced by Lactobacillus mucilaginosus fermentation MRS is an important scientific issue that needs to be addressed urgently and is also an important indicator for evaluating the application potential of this strain. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide an exopolysaccharide produced by fermentation of Lactobacillus mucilaginosus and a preparation method and application thereof, so as to solve the problem that there is little research in the prior art on the exopolysaccharide produced by fermentation of Lactobacillus mucilaginosus.

[0006] To achieve the above-mentioned purpose and other related purposes, the present invention is achieved through the following technical solutions.

[0007] The first aspect of the present invention is to provide an exopolysaccharide of fermented Lactobacillus mucilaginosus, wherein the main chain of the exopolysaccharide comprises acetylglucosamine residues, glucose residues and galactose residues; the main chain of the exopolysaccharide comprises acetylglucosamine residues, glucose residues and galactose residues in a molar ratio of 1:3.78-3.84:2.06-2.12.

[0008] The second aspect of the present invention provides a method for preparing the exopolysaccharide of fermented Lactobacillus mucilaginosus as described above, comprising the following steps: fermenting, extracting, and purifying the fermented Lactobacillus mucilaginosus to obtain the exopolysaccharide.

[0009] The third aspect of the present invention provides the use of the fermented Lactobacillus mucilaginosus exopolysaccharide as described above in preparing products.

[0010] The fourth aspect of the present invention provides a product comprising the above-mentioned fermented Lactobacillus mucilaginosus exopolysaccharide; preferably, further comprising a carrier or auxiliary material.

[0011] As described above, the fermented Lactobacillus mucilaginosus exopolysaccharide of the present invention and its preparation method and application have the following beneficial effects:

[0012] The present invention discloses for the first time a novel structure of exopolysaccharide synthesized by Lactobacillus fermentum CGMCC No. 17321 in MRS. The exopolysaccharide with this structure can stimulate the phagocytic activity of macrophages and the release of NO and ROS, and simultaneously promote the expression of pro-inflammatory factors (TNF-α, IL-6, IL-1β) and anti-inflammatory factors (IL-10) in macrophages, thereby having the purpose of bidirectional immune regulation and promoting M1 polarization of macrophages. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Gel column chromatography of exopolysaccharide of Lactobacillus fermentum CGMCC No.17321;

[0014] Figure 2 PMAA profile of exopolysaccharide A of Lactobacillus fermentum CGMCC No.17321;

[0015] Figure 3 EI-MS spectrum of exopolysaccharide A of Lactobacillus fermentum CGMCC No.17321 (corresponding to chromatographic peaks 1-8 in the PMAA spectrum from top to bottom);

[0016] Figure 4 1H-NMR spectrum of exopolysaccharide of Lactobacillus fermentum CGMCC No.17321;

[0017] Figure 5 13C-NMR spectrum of exopolysaccharide of Lactobacillus fermentum CGMCC No.17321;

[0018] Figure 6 HSQC spectrum of exopolysaccharide of Lactobacillus fermentum CGMCC No.17321;

[0019] Figure 7 HMBC profile of exopolysaccharide of Lactobacillus fermentum CGMCC No.17321;

[0020] Figure 8 COSY spectrum of exopolysaccharide of Lactobacillus fermentum CGMCC No.17321;

[0021] Figure 9 TOCSY spectrum of exopolysaccharide of Lactobacillus fermentum CGMCC No.17321;

[0022] Figure 10 NOESY spectrum of exopolysaccharide of Lactobacillus fermentum CGMCC No.17321;

[0023] Figure 11 HSQC-TOCSY spectrum of exopolysaccharide of Lactobacillus fermentum CGMCC No.17321;

[0024] Figure 12 Cytotoxicity test of exopolysaccharide of Lactobacillus fermentum CGMCC No.17321;

[0025] Figure 13 Effects of exopolysaccharide of Lactobacillus fermentum CGMCC No.17321 on phagocytic activity of cells;

[0026] Figure 14 Effect of exopolysaccharide of Lactobacillus fermentum CGMCC No.17321 on NO release;

[0027] Figure 15 Effect of exopolysaccharide of Lactobacillus fermentum CGMCC No.17321 on ROS release;

[0028] Figure 16 Effect of exopolysaccharide of Lactobacillus fermentum CGMCC No.17321 on TNF-α production;

[0029] Figure 17 Effect of exopolysaccharide of Lactobacillus fermentum CGMCC No.17321 on IL-10 production;

[0030] Figure 18 Effects of exopolysaccharide from Lactobacillus fermentum CGMCC No.17321 on IL-6 production;

[0031] Figure 19 Effects of exopolysaccharide from Lactobacillus fermentum CGMCC No.17321 on IL-1β production;

[0032] Figure 20 Effects of extracellular polysaccharides of Lactobacillus fermentum CGMCC No.17321 on macrophage polarization (A) Fluorescence image; (B) Fluorescence expression of marker iNOS.

[0033] in, Figure 10-18 ** indicates comparison with the control group, **P<0.01. DETAILED DESCRIPTION

[0034] In order to make the purpose of the invention, technical solutions and beneficial technical effects of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0035] The first aspect of the present invention is to provide an exopolysaccharide of fermented Lactobacillus mucilaginosus, wherein the main chain of the exopolysaccharide comprises acetylglucosamine residues, glucose residues and galactose residues; the main chain of the exopolysaccharide comprises acetylglucosamine residues, glucose residues and galactose residues in a molar ratio of 1:3.78-3.84:2.06-2.12.

[0036] In some embodiments of the present invention, the average weight molecular weight of the extracellular polysaccharide is 1.892×10 6 ~2.386×10 6 Dalton.

[0037] In some embodiments of the present invention, the exopolysaccharide is non-toxic.

[0038] In some embodiments of the present invention, the main chain of the extracellular polysaccharide is composed of 1,3-linked glucose residues, 1,4-linked galactose residues, 1,6-linked glucose residues, 1,6-linked galactose residues, 1,2,3-linked galactose residues, 1,3,6-linked glucose residues and 1,3-linked acetylglucosamine residues, and the branch chain is composed of terminal-linked glucose residues, and its branch points are respectively located at the O2 position of the 1,2,3-linked galactose residues and the O6 position of the 1,3,6-linked glucose residues.

[0039] In some embodiments of the present invention, the exopolysaccharide is composed of repeating units represented by Formula I:

[0040]

[0041] Among them, A is a 1,3,6-linked glucose residue; B is a 1,3-linked glucose residue; C is a 1,6-linked glucose residue; D is a 1,4-linked galactose residue; E is a 1,2,3-linked galactose residue; F is a 1,3-linked acetylglucosamine residue; H is a 1,6-linked galactose residue; G is a branch chain of the extracellular polysaccharide, which is a glucose residue, and its branch points are located at the O2 position of the 1,2,3-linked galactose residue and the O6 position of the 1,3,6-linked glucose residue, respectively.

[0042] In some embodiments of the present invention, the exopolysaccharide is obtained by fermentation of Lactobacillus fermentum with a preservation number of CGMCC NO.17321.

[0043] Among them, Lactobacillus fermentum was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on March 8, 2019. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0044] In some embodiments of the present invention, the fermentative Lactobacillus mucronulae exists as living cells, dried cells, or immobilized cells. Herein, "living cells" refer to Lactobacillus mucronulae having the ability to metabolize, reproduce, or replicate; "immobilized cells" refer to Lactobacillus mucronulae immobilized on a carrier and capable of carrying out vital activities (growth, development, reproduction, inheritance, metabolism, etc.) within a certain spatial range. The dried cells are obtained by freeze-drying the fermentative Lactobacillus mucronulae.

[0045] In some embodiments of the present invention, the fermentation is carried out in a culture medium, preferably, the culture medium is MRS culture medium purchased from Merck Co., Germany.

[0046] The second aspect of the present invention is to provide a method for preparing the extracellular polysaccharide of fermented Lactobacillus muciniphila as described above, comprising the following steps: fermenting, extracting, and purifying the fermented Lactobacillus muciniphila to obtain the extracellular polysaccharide.

[0047] In some embodiments of the present invention, the Lactobacillus fermentum has a preservation number of CGMCC NO.17321.

[0048] In some embodiments of the present invention, the fermentation is carried out in a culture medium, preferably, the culture medium is MRS medium.

[0049] In some embodiments of the present invention, the inoculation amount of the fermentative Lactobacillus mucilaginosus is 1×10 7-8×10 7 CFU / mL; specifically, the inoculation volume can be 1×10 7 -2×10 7 CFU / mL, 2×10 7 -6×10 7 CFU / mL, 6×10 7 -8×10 7 CFU / mL; a typical but non-limiting example is 1×10 7 CFU / mL, 2×10 7 CFU / mL, 4×10 7 CFU / mL, 6×10 7 CFU / mL, 8×10 7 CFU / mL.

[0050] In some embodiments of the present invention, the fermentation temperature is 20°C-40°C, and the fermentation time is 12-36 hours. The fermentation is anaerobic. Specifically, the fermentation temperature can be 20-30°C or 30-40°C, with typical but non-limiting examples being 20°C, 30°C, or 40°C. The fermentation time can be 12-24 hours or 24-36 hours, with typical but non-limiting examples being 12 hours, 24 hours, or 36 hours. Lactobacillus mucilaginosus is an anaerobic bacterium that can grow and reproduce rapidly under static culture conditions.

[0051] In some embodiments of the present invention, the extraction comprises the following steps: inactivating the fermentation broth obtained by fermentation, separating the supernatant, adding an alcohol compound for precipitation, separating the precipitate, mixing the precipitate with water, adding a protein precipitation reagent, allowing to stand, separating the supernatant, removing substances with a molecular weight cutoff of ≤14,000 Daltons, and drying.

[0052] In some embodiments of the present invention, the inactivation temperature is 90-100°C, and the inactivation time is 10-20 min; specifically, the inactivation temperature can be 90-95°C, 95-100°C; typically but not limitatively, for example, 90°C, 95°C, 100°C; the inactivation time can be 10-15 min, 15-20 min; typically but not limitatively, for example, 10 min, 15 min, 20 min.

[0053] In some embodiments of the present invention, the separation method is centrifugation.

[0054] In some embodiments of the present invention, the centrifugation speed is 8000-12000 g, and the centrifugation time is 8-12 min. Specifically, the centrifugation speed can be 8000-10000 g, 10000-12000 g, and typically but not limited to, for example, 8000 g, 10000 g, and 12000 g. The centrifugation time can be 8-9 min, 9-11 min, or 11-12 min, and typically but not limited to, for example, 8 min, 9 min, 11 min, and 12 min.

[0055] In some embodiments of the present invention, the volume ratio of the supernatant to the alcohol compound is 1:3-5; the present invention has no special limitation on the selection of the alcohol compound, and any alcohol compound that can precipitate the supernatant can be used. Specifically, the alcohol compound is ethanol.

[0056] In some embodiments of the present invention, the amount of the protein precipitation reagent added is 5-9% of the final concentration.

[0057] In some embodiments of the present invention, the protein precipitation reagent is trichloroacetic acid.

[0058] In some embodiments of the present invention, the standing temperature is 4-8°C, and the standing time is 12-24h; specifically, the standing temperature can be 4-6°C, 6-8°C; typically but not limitatively, for example, 4°C, 6°C, 8°C; the standing time can be 12-18h, 18-24h; typically but not limitatively, for example, 12h, 18h, 24h.

[0059] In some embodiments of the present invention, the removal of substances with a molecular weight cut-off of ≤14,000 Daltons is performed by dialysis, with a dialysis time of 72-120 hours and a water change frequency of preferably every 8 hours. Preferably, the dialysis time is 72 hours.

[0060] In some embodiments of the present invention, the present invention has no particular limitation on the drying method, and any drying method commonly used in the art can be used. Specifically, the present invention uses freeze-drying.

[0061] In some embodiments of the present invention, the purification comprises the following steps: separating and purifying the extracted substances, combining and collecting the elution products of the component peaks, removing substances with a molecular weight cutoff of ≤14,000 Daltons and drying to obtain fermented mucus Lactobacillus exopolysaccharide.

[0062] In some embodiments of the present invention, the separation and purification method is selected from gel column chromatography; preferably, the filler used in the gel column chromatography is Sepharose 6 Fast Flow gel filler, and the size of the gel column is D2.6 cm × 30 cm; the present invention has no special limitation on the source of Sepharose 6 Fast Flow gel filler, and a commercially available Sepharose 6 Fast Flow gel filler commonly used by those skilled in the art can be used. Specifically, the manufacturer of the Sepharose 6 Fast Flow gel filler is Cytiva.

[0063] In some embodiments of the present invention, the removal of substances with a molecular weight cut-off of ≤14,000 Daltons is performed by dialysis, with a dialysis time of 72-120 hours and a water change frequency of preferably every 8 hours. Preferably, the dialysis time is 72 hours.

[0064] In some embodiments of the present invention, the present invention has no special limitation on the eluent and elution rate of elution, and the eluent and elution rate commonly used in the art can be used. Specifically, the eluent for elution is selected from NaCl solution, and the elution rate is 0.1-0.3 ml / min; preferably, the concentration of the eluent is 0.1-0.3 M.

[0065] The third aspect of the present invention is to provide the use of the above-mentioned fermented Lactobacillus mucilaginosus exopolysaccharide in preparing products.

[0066] In some embodiments of the present invention, the product has one or more of the following features:

[0067] B1) immune regulation;

[0068] B2) stimulates the phagocytic activity of macrophages and the release of NO and ROS, and simultaneously promotes the expression of pro-inflammatory factors (TNF-α, IL-6, IL-1β) and anti-inflammatory factors (IL-10) in macrophages;

[0069] B3) Promotes M1 polarization of macrophages.

[0070] The fourth aspect of the present invention is to provide a product comprising the above-mentioned fermented Lactobacillus mucilaginosus exopolysaccharide; preferably, further comprising a carrier or auxiliary material.

[0071] In some embodiments of the present invention, the product is selected from any one of food, medicine, health care product, and cosmetics.

[0072] As used herein, the term "carrier" includes any solvent, stabilizer, or combination thereof, known to those skilled in the art. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in immunomodulatory products is contemplated. As used herein, the term "excipient" includes any solvent, etc., as appropriate for the specific intended dosage form. Except insofar as any conventional excipient is incompatible with the Lactobacillus muciniphilus fermentation of the present disclosure, such as by producing any adverse biological effect or by interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, its use is also contemplated by the present disclosure.

[0073] In this article, food generally refers to substances that can be eaten or drunk by humans, including processed foods, semi-finished products, food ingredients, and items that are traditionally both food and Chinese medicinal materials. Food does not include items used for therapeutic purposes.

[0074] In this article, cosmetics refer to daily chemical industrial products that are applied to the human body surface such as skin, hair, nails, lips, etc. by smearing, spraying or other similar methods for the purpose of cleaning, protecting, beautifying and modifying.

[0075] Herein, when the product is a drug, the carrier or excipient is a pharmaceutically acceptable carrier or excipient.

[0076] "Pharmaceutically acceptable" means that the drugs do not produce adverse, allergic or other untoward reactions when properly administered to animals or humans.

[0077] "Pharmaceutically acceptable carriers or excipients" should be compatible with the active ingredient, that is, they can be mixed with the active ingredient without significantly reducing the effect of the drug under normal circumstances.

[0078] The present invention is further described below by way of examples, but the scope of the invention is not limited thereto.

[0079] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In the embodiments, if specific conditions are not specified, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for all reagents or instruments, they are conventional products that can be obtained by commercial purchase. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any method, equipment, and material of the prior art similar or equivalent to the methods, equipment, and materials in the embodiments of the present invention can also be used to realize the present invention.

[0080] Example

[0081] Example 1

[0082] 1. Materials and Methods

[0083] Preparation of seeds (fermentation strain): Lyophilized powder of Lactobacillus fermentum CGMCC No. 17321 was dissolved in a small amount of sterile distilled water, and a loop of the culture was streaked onto MRS solid medium (purchased from Merck Co., Germany) using an inoculating loop. The culture was anaerobically cultured at 37°C for 48 h, and then a single colony was picked with an inoculating loop and placed into 10 mL of MRS liquid medium (purchased from Merck Co., Germany). The colony was evenly dispersed in MBS liquid medium using a vortex shaker, anaerobically cultured at 37°C for 48 h, and then inoculated into MRS liquid medium at a 2% (v / v) inoculum. After anaerobically cultured at 37°C for 24 h, the culture was centrifuged at 15,000 rpm for 10 minutes, the supernatant was discarded, the bacteria were washed twice with sterile distilled water, and then suspended in sterile distilled water equal to the original culture volume to obtain seeds for fermentation. The bacterial concentration of the seed solution was 2×10 9 CFU / mL.

[0084] 2. Preparation of exopolysaccharide from fermented Lactobacillus mucilaginosus

[0085] 2.1 Preparation of crude polysaccharide: Lactobacillus fermentum CGMCC No. 17321 seeds were aseptically inoculated into MRS at an inoculum rate of 2% (v / v, seed solution volume percentage of fermentation solution, the same below) and anaerobically cultured at 30°C for 24 h to obtain a fermentation solution. The fermentation solution was heat-inactivated and centrifuged at 10,000 g for 10 min. The supernatant was removed and precipitated by adding three volumes of anhydrous ethanol. The solution was centrifuged again at 10,000 g for 10 min. The precipitate was dissolved in water and trichloroacetic acid was added to a final concentration of 7%. The solution was refrigerated and allowed to stand overnight. The solution was centrifuged again at 10,000 g for 10 min. The supernatant was placed in a dialysis bag with a molecular weight cutoff of 14,000 Daltons and dialyzed for 72 h. The water was changed every 8 h. The solution in the bag was freeze-dried to obtain crude polysaccharide A.

[0086] 2.2 Preparation of extracellular polysaccharide: 100 mg of crude polysaccharide A was dissolved in 5 mL of NaCl (0.2 M) solution and loaded onto a Sepharose 6 Fast Flow (purchased from GE, USA, also known as General Electric Company) chromatography column (D2.6 cm × 30 cm). The column was eluted isocratically with 0.2 M NaCl solution at a flow rate of 0.25 mL / min, and 1 mL was collected in each tube. The polysaccharide content in each collection tube was determined by the sulfuric acid-phenol method, and the elution product of the peak F1 was combined and collected ( Figure 1 ), placed in a dialysis bag with a molecular weight cutoff of 14,000 Daltons and dialyzed for 72 hours, with water changed every 8 hours. The solution in the bag was freeze-dried to obtain extracellular polysaccharide A.

[0087] Example 2

[0088] 1. Materials and Methods

[0089] Preparation of seeds (fermentation strains): same as in Example 1.

[0090] 2. Preparation of fermented mucus lactobacillus agent

[0091] 2.1 Preparation of Crude Polysaccharide: Lactobacillus fermentum CGMCC No. 17321 seeds were aseptically inoculated at a 0.5% (v / v) inoculum in MRS and anaerobically cultured at 20°C for 36 h to obtain a fermentation broth. The fermentation broth was heat-inactivated and centrifuged at 12,000 g for 8 min. The supernatant was removed and precipitated by adding three volumes of anhydrous ethanol. The precipitate was centrifuged again at 12,000 g for 8 min. The precipitate was dissolved in water and trichloroacetic acid was added to a final concentration of 9%. The precipitate was refrigerated and allowed to stand overnight. The supernatant was again centrifuged at 12,000 g for 8 min. The supernatant was transferred to a dialysis bag with a molecular weight cutoff of 14,000 Daltons and dialyzed for 72 h. The water was changed every 8 h. The solution in the bag was freeze-dried to obtain crude polysaccharide B.

[0092] 2.2 Preparation of extracellular polysaccharide: same as Example 1.

[0093] Example 3

[0094] 1. Materials and Methods

[0095] Preparation of seeds (fermentation strains): same as in Example 1.

[0096] 2. Preparation of fermented mucus lactobacillus agent

[0097] 2.1 Preparation of Crude Polysaccharide: Lactobacillus fermentum CGMCC No. 17321 seeds were aseptically inoculated into MRS at a 4% (v / v) inoculum and anaerobically cultured at 40°C for 12 h to obtain a fermentation broth. The fermentation broth was heat-inactivated and centrifuged at 8,000 g for 12 min. The supernatant was removed and precipitated by adding three volumes of anhydrous ethanol. The precipitate was centrifuged again at 8,000 g for 12 min. The precipitate was dissolved in water and trichloroacetic acid was added to a final concentration of 5%. The precipitate was refrigerated and allowed to stand overnight. The supernatant was centrifuged again at 8,000 g for 12 min. The supernatant was transferred to a dialysis bag with a molecular weight cutoff of 14,000 Daltons and dialyzed for 72 h. The water was changed every 8 h. The solution in the bag was freeze-dried to obtain crude polysaccharide C.

[0098] 2.2 Preparation of extracellular polysaccharide: same as Example 1.

[0099] Example 4

[0100] 1. Materials and Methods

[0101] Preparation of seeds (fermentation strains): same as in Example 1.

[0102] 2. Preparation of fermented mucus lactobacillus agent

[0103] 2.1 Preparation of Crude Polysaccharide: Lactobacillus fermentum CGMCC No. 17321 seeds were aseptically inoculated into MRS at a 3% (v / v) inoculum and anaerobically cultured at 25°C for 18 h to obtain a fermentation broth. The fermentation broth was heat-inactivated and centrifuged at 11,000 g for 9 min. The supernatant was removed and precipitated by adding three volumes of anhydrous ethanol. The precipitate was centrifuged again at 11,000 g for 9 min. The precipitate was dissolved in water and trichloroacetic acid was added to a final concentration of 5%. The precipitate was refrigerated and allowed to stand overnight. The supernatant was again centrifuged at 11,000 g for 9 min. The supernatant was transferred to a dialysis bag with a molecular weight cutoff of 14,000 Daltons and dialyzed for 72 h. The water was changed every 8 h. The solution in the bag was freeze-dried to obtain crude polysaccharide D.

[0104] 2.2 Preparation of extracellular polysaccharide: same as Example 1.

[0105] Example 5

[0106] 1. Materials and Methods

[0107] Preparation of seeds (fermentation strains): same as in Example 1.

[0108] 2. Preparation of fermented mucus lactobacillus agent

[0109] 2.1 Preparation of Crude Polysaccharide: Lactobacillus fermentum CGMCC No. 17321 seeds were aseptically inoculated at a 1% (v / v) inoculum in MRS and anaerobically cultured at 35°C for 30 h to obtain a fermentation broth. The fermentation broth was heat-inactivated and centrifuged at 9,000 g for 11 min. The supernatant was removed and precipitated by adding three volumes of anhydrous ethanol. The precipitate was centrifuged again at 9,000 g for 11 min. The precipitate was dissolved in water and trichloroacetic acid was added to a final concentration of 5%. The precipitate was refrigerated and allowed to stand overnight. The supernatant was centrifuged again at 9,000 g for 11 min. The supernatant was transferred to a dialysis bag with a molecular weight cutoff of 14,000 Daltons and dialyzed for 72 h. The water was changed every 8 h. The solution in the bag was freeze-dried to obtain crude polysaccharide E.

[0110] 2.2 Preparation of extracellular polysaccharide: same as Example 1.

[0111] Exopolysaccharide structure test of fermented Lactobacillus mucilaginosus

[0112] Example 6 Determination of Molecular Weight of Exopolysaccharide from Fermented Lactobacillus mucilaginosus

[0113] Molecular weight determination of exopolysaccharides from Lactobacillus mucilaginosus fermentation on a Viscotek TDAmax TMThe analysis was carried out on a device (Malvern Panalytical, UK). The device was equipped with two gel permeation columns (G6000 PWXL (7.8×300 mm) and G4000 PWXL (7.8×300 mm)) (TSK Tosoh Corporation, Japan) connected in series, and a Viscotek 270max detection system combined with a differential refractive index (RI) detector and a multi-angle light scattering (MALLS) detector. Standards (PEO24k, Dextran73k) (purchased from Malvern Panalytical, UK) and the samples to be tested (exopolysaccharides A, B, C, D and E) were dissolved in the mobile phase (0.1 mol / L NaNO3 solution) to obtain a 1 mg / mL solution. After filtering through a 0.22 μm Millipore filter membrane, the sample was loaded onto the above-mentioned Viscotek TDAmax. TM The analytical instrument was set to a column temperature of 35°C, an injection volume of 100 μL, a mobile phase of 0.1 mol / L NaNO₃ solution, and a flow rate of 0.8 mL / min. The system was first calibrated using data from a standard. Subsequently, the molecular weight of the sample was calculated using the instrument's built-in software (OmniSEC software). The results are shown in Table 1.

[0114] Table 1 Average weight molecular weight of exopolysaccharides of fermented Lactobacillus mucilaginosus of Examples 1-5

[0115]

[0116] Conclusion: The average molecular weight of the exopolysaccharide of Lactobacillus fermentum CGMCC No.17321 is 1.892×10 6 ~2.386×10 6 Dalton.

[0117] Example 7 Monosaccharide composition of fermented Lactobacillus mucilaginosus exopolysaccharide

[0118] The method for determining the exopolysaccharide of fermented Lactobacillus mucilaginosus comprises the following steps:

[0119] (1) Hydrolysis of polysaccharide samples

[0120] 2.0 mg of exopolysaccharide A was placed in the corresponding ampoule, 3 mL of 2 mol / L trifluoroacetic acid (TFA) was added, and the ampoule was sealed and hydrolyzed at 110°C for 5 h. After cooling the hydrolyzate, it was rotary evaporated to dryness under reduced pressure at 45°C. Methanol was added and rotary evaporation was continued. This was repeated 5 times to remove excess TFA to obtain the exopolysaccharide hydrolyzate.

[0121] (2) Derivatization of hydrolyzed samples and mixed monosaccharide standards

[0122] Dissolve the exopolysaccharide hydrolysate in 1 mL of water to obtain the sample solution to be derivatized. Take 1 mL of the sample solution or a mixed standard solution of nine monosaccharides (0.5 mg / mL, D-glucose, D-glucuronic acid, D-mannose, D-galactose, glucosamine, D-galacturonic acid, D-fructose, D-xylose, and L-arabinose, i.e., each mL of the mixed standard solution contains 0.5 mg of each monosaccharide) and add 1 mL of 0.6 mol / L NaOH solution and 1 mL of 0.5 mol / L PMP methanol solution. Mix thoroughly to completely dissolve the solid product and place in a 70°C oven for 100 min. After cooling to room temperature, add 0.3 mol / L HCl dropwise to neutralize the solution. Extract three times with chloroform, collect the aqueous phase, filter through a 0.45 μm filter, and prepare for HPLC analysis.

[0123] (3) Chromatographic conditions

[0124] An Agilent 1260 high performance liquid chromatograph (Agilent, USA) was used, equipped with a DAD detector. The chromatographic column was a Kromasil 100-5-C18 (4.6*250 mm) column (Kromasil, Sweden). The column temperature was set at 40°C, the injection volume was 10 μL, the mobile phase was acetonitrile: phosphate solution (0.1 mol / L) = 16:84 (V / V), the flow rate was 1 mL / min, and the detection wavelength was 250 nm.

[0125] (4) Data Analysis

[0126] The monosaccharide composition of the polysaccharide sample was determined by referring to the retention times of different monosaccharide standards (purchased from Sigma, USA). The molar ratio of each monosaccharide in the polysaccharide sample was then determined based on the peak area ratio of each monosaccharide composition. The results are shown in Table 2.

[0127] The determination method of monosaccharide in samples of exopolysaccharide B, C, D and E is as described above, and the determination results are shown in Table 2.

[0128] Table 2 Determination of monosaccharide composition of extracellular polysaccharides of fermented Lactobacillus mucilaginosus obtained in Examples 1-5

[0129]

[0130] Conclusion: The exopolysaccharide of Lactobacillus fermentum CGMCC No.17321 is a heteropolysaccharide composed of glucosamine, glucose and galactose in a molar ratio of 1:3.78-3.84:2.06-2.12.

[0131] Example 8 Determination of the Linking Mode of Exopolysaccharides of Fermented Lactobacillus mucilaginosus

[0132] The method for determining the connection mode of the exopolysaccharide of fermentation Lactobacillus mucilaginosus comprises the following steps:

[0133] (1) Uronic acid reduction: Uronic acid was reduced using the carbodiimide-sodium borohydride method (EDC-NaBH4). The reaction was divided into two stages. The first stage was to dissolve 50 mg of extracellular polysaccharide A in 6 mL of ultrapure water and stir until completely dissolved. 500 mg of EDC was added to the solution twice, with an interval of 30 min between the two times, and the pH was always controlled between 4.5 and 4.8 with 0.1 mol / L HCl. The entire reaction process took 3 h. The second stage was to dropwise add 8 mL of 2 mol / L NaBH4 to the above system within 40 min, and the pH of the system was controlled at around 7.0. After the addition was completed, the reaction was continued for 1 h. The product was placed in a dialysis bag (molecular weight cutoff of 3500 Da) and dialyzed with running water for 24 h. The steps of the second stage were repeated 4 times, and the complete reduction of uronic acid was detected by PMP-HPLC.

[0134] (2) Methylation: Take 20 mg of the dry polysaccharide sample with complete uronic acid reduction, place it in a 10 mL reaction bottle, quickly add 3 mL of anhydrous dimethyl sulfoxide at room temperature, seal it, stir it magnetically for 30 minutes, and use ultrasound to dissolve the sample, then quickly add 50 mg of dry NaOH powder, seal it and stir it until the NaOH dissolves, then ice bath it for 5 minutes, slowly add 1 mL of iodomethane dropwise over 30 minutes, stir it in the dark at room temperature and continue to react for 30 minutes, finally add 1 mL of ultrapure water to terminate the reaction. The product is placed in a dialysis bag with a molecular weight cutoff of 14,000 Daltons and dialyzed with running water for 24 hours. After rotary evaporation to dryness, repeat the methylation step. After methylation for 5 times, take a small amount of sample for infrared spectroscopy detection. If the polysaccharide sample has a wavelength of 3400-3000 cm -1 The OH stretching vibration absorption peak disappears, indicating that the polysaccharide sample has been completely methylated; if the sample has not been completely methylated, the reaction needs to be continued until the sample is completely methylated;

[0135] (3) Hydrolysis and acetylation: Place 2 mg of fully methylated exopolysaccharide A sample in an ampoule, add 3 mL of 2 mol / L TFA and seal the bottle. After hydrolysis at 110°C for 4 h, add methanol and evaporate under reduced pressure three times to completely remove TFA. After drying by rotation, dissolve in 3 mL of ultrapure water, add 50 mg of NaBH4, and react under magnetic stirring at room temperature for 3 h. After the reaction, add acetic acid until the solution becomes weakly acidic (pH = 5), add methanol and evaporate to dryness by rotation, repeat three times to fully remove boric acid. The obtained solid is dried in an oven at 100°C for 10 min, add 3 mL of acetic anhydride, and react at 100°C for 100 min. After the reaction, add toluene (3 mL) five times and co-evaporate to remove excess acetic anhydride. The product was dissolved in chloroform (5 mL) and extracted three times with ultrapure water (5 mL × 3). The chloroform layer was recovered and dehydrated by adding anhydrous sodium sulfate powder. The mixture was allowed to stand for 30 min and evaporated to dryness under reduced pressure. 0.5 mL of chloroform was added for dissolution. After filtration through a 0.22 μm organic filter membrane, GC-MS analysis was performed to obtain an EI-MS spectrum.

[0136] (4) GC-MS conditions: Instrument model: Agilent 7820A / 5977 GC-MS (purchased from Agilent, USA); Chromatographic column model: HP-5 capillary column (30 m × 0.25 mm × 0.25 μm); Temperature program: Initial temperature 120 °C, hold for 2 min, then increase to 250 °C, heating rate 5 °C / min, hold for 10 min; Injection port adopts split mode with a split ratio of 3:1; Injection volume 1 μL. Carrier gas is nitrogen; Carrier gas velocity is 40 cm / s; Mass spectrometry ion source is EI source, ion source voltage is 70 eV, temperature is 180 °C;

[0137] (5) Data analysis: The EI-MS spectra obtained by GC-MS (such as Figure 3 ) and the standard PMAA spectrum (as shown Figure 2 The results of the comparison of the reduced exopolysaccharide A and the monosaccharide composition showed that the connection mode of the sugar residues was 1,3-linked glucose residues, 1,4-linked galactose residues, 1,6-linked glucose residues, 1,6-linked galactose residues, 1,2,3-linked galactose residues, 1,3,6-linked glucose residues, terminal-linked glucose and 1,3-linked acetylglucosamine residues.

[0138] The acetylglucosamine residues in the above methylation analysis do not conflict with the glucosamine in the aforementioned monosaccharide composition. The reason is that during the sample preparation process of the monosaccharide composition, the acetyl group on the acetylglucosamine residue falls off when the polysaccharide sample undergoes acid hydrolysis, resulting in only glucosamine being detected. Therefore, the actual monosaccharide composition of the extracellular polysaccharide of fermentation Lactobacillus mucilaginosus CGMCCNO.17321 is acetylglucosamine, glucose and galactose.

[0139] Example 9 Nuclear Magnetic Resonance Spectroscopy Analysis

[0140] 20 mg of exopolysaccharide A was dissolved in 0.5 mL of D2O and transferred to a clean NMR tube. After chromatographic analysis on a 700 MHz NMR spectrometer (purchased from Bruker, Switzerland), 1H-NMR ( Figure 4 )、13C-NMR( Figure 5 )、HSQC( Figure 6 )、HMBC( Figure 7 )、COSY( Figure 8 )、TOCSY( Figure 9 )、NOESY( Figure 10 )、HSQC-TOCSY( Figure 11 ), comprehensive analysis revealed that the main chain of the extracellular polysaccharide of Lactobacillus fermentum CGMCC No.17321 was composed of 1,3-linked glucose residues, 1,4-linked galactose residues, 1,6-linked glucose residues, 1,6-linked galactose residues, 1,2,3-linked galactose residues, 1,3,6-linked glucose residues and 1,3-linked acetylglucosamine residues, and the side chains were composed of terminal-linked glucose residues, with the branching points located at the O2 position of the 1,2,3-linked galactose residues and the O6 position of the 1,3,6-linked glucose residues, respectively. The composition of the repeating unit is shown in Formula I.

[0141]

[0142] Performance Testing

[0143] Effect Example 1 Toxicity test of exopolysaccharide of fermented Lactobacillus mucilaginosus (CCK8 method)

[0144] RAW264.7 cells were cultured at 2×10 3The cells were plated at a concentration of 100 μL / well in a 96-well plate and cultured overnight at 37°C in a saturated humidity of 5% CO2. After aspirating the culture medium, 100 μL of fresh DMEM cell culture medium (purchased from GIBCO, USA) containing different concentrations of exopolysaccharide A (0, 6.25, 12.5, 25, 50, 100, 200 μg / mL) was added to the wells and incubated at 37°C in a saturated humidity of 5% CO2 for 24 h. RAW264.7 cells cultured without exopolysaccharide A and LPS served as the blank control group, and RAW264.7 cells cultured without exopolysaccharide A and only with LPS (1 μg / mL) served as the positive control group. 10 μL of CCK-8 solution (purchased from Biyuntian Biotechnology Co., Ltd., China) was added to each well and incubated at 37°C under 5% CO2 saturated humidity for 1 h. The absorbance of each well at 450 nm was measured using a microplate counter (Multiskan FC, China). This value can indirectly reflect the number of viable cells. The results are shown in Figure 2. Figure 12 shown.

[0145] Conclusion: Compared with the control group, all tested concentrations of exopolysaccharide A (6.25-200 μg / mL) significantly promoted macrophage proliferation in a dose-dependent manner. Therefore, within the range of 6.25-200 μg / mL, the exopolysaccharide of Lactobacillus fermentum CGMCC No. 17321 is safe and non-toxic.

[0146] Effect Example 2 Immunomodulatory Effect of Exopolysaccharide from Fermented Lactobacillus mucilaginosus

[0147] 2.1 Effect of exopolysaccharide of Lactobacillus mucilaginosus on phagocytic activity (neutral red method)

[0148] RAW264.7 cells were cultured at a rate of 1 × 10 4 The concentration of cells / well was plated in a 96-well plate and cultured overnight at 37°C and 5% CO2 saturated humidity. After aspirating the culture medium, 200 μL of fresh DMEM cell culture medium containing different concentrations (25, 50, 100 μg / mL) of extracellular polysaccharide A was added to the wells and incubated at 37°C and 5% CO2 saturated humidity for 24 hours. RAW264.7 cells cultured without extracellular polysaccharide A and LPS were used as the blank control group, and RAW264.7 cells cultured without extracellular polysaccharide A and only with LPS (1 μg / mL) were used as the positive control group. 100 μL of neutral red solution (0.1%) was added to each well and incubated at 37°C and 5% CO2 saturated humidity for 1 hour. The liquid in the well was aspirated, PBS was added for washing three times, and lysis solution (glacial acetic acid: ethanol = 1:1) was added to lyse the cells. The absorbance of each well at 540 nm was measured using a microplate counter. The results are as follows: Figure 13 shown.

[0149] Conclusion: Treatment of RAW264.7 cells with 25-100 μg / mL exopolysaccharide A significantly enhanced their phagocytic rate. The phagocytic activity of RAW264.7 cells reached its maximum at the lower treatment concentration (25 μg / mL), exceeding 150% of the blank control group. These results indicate that, at the experimental concentrations, exopolysaccharides from Lactobacillus fermentum CGMCC No. 17321 can stimulate RAW264.7 cells and enhance their phagocytic capacity.

[0150] 2.2 Effect of extracellular polysaccharide of Lactobacillus mucilaginosus fermentation on NO release

[0151] RAW264.7 cells were cultured at a rate of 1 × 10 5 The concentration of cells / well was plated in a 96-well plate and cultured overnight at 37°C under 5% CO2 saturated humidity. After aspirating the culture medium, 200 μL of fresh DMEM cell culture medium containing different concentrations (25, 50, 100 μg / mL) of exopolysaccharide A was added to the wells and incubated at 37°C under 5% CO2 saturated humidity for 24 hours. RAW264.7 cells cultured without exopolysaccharide A and LPS were used as the blank control group, and RAW264.7 cells cultured without exopolysaccharide A and only with LPS (1 μg / mL) were used as the positive control group. 50 μL of the culture supernatant was collected and the NO content was determined using a NO kit (Beijing Solebold Technology Co., Ltd., China) according to the instructions. The results are as follows: Figure 14 shown.

[0152] Conclusion: Compared with the blank control group, exopolysaccharide A significantly promoted NO release from RAW 264.7 cells in a concentration-dependent manner within the range of 25-100 μg / mL. When the concentration of exopolysaccharide A was 100 μg / mL, the maximum NO release reached 94.77 μM, which was 9.35 times that of the blank control group.

[0153] 2.3 Effect of extracellular polysaccharide of Lactobacillus mucilaginosus fermentation on ROS release

[0154] RAW264.7 cells were cultured at a rate of 1 × 10 5Cells were plated at a concentration of 100 μg / well in a black 96-well plate and cultured overnight at 37°C in a 5% CO2 saturated humidity environment. After aspirating the culture medium, 200 μL of fresh DMEM cell culture medium containing different concentrations of exopolysaccharide A (25, 50, and 100 μg / mL) was added to the wells and incubated at 37°C in a 5% CO2 saturated humidity environment for 24 hours. RAW264.7 cells cultured without exopolysaccharide A and LPS served as the blank control group, and RAW264.7 cells cultured without exopolysaccharide A but only with LPS (1 μg / mL) served as the positive control group. 100 μL of DMEM medium containing 10 μmol / L DCFH-DA (purchased from Beyotime Biotechnology Co., Ltd., China) was added to each well and incubated for another 30 minutes. The culture medium was discarded, and the well wall and cells were washed three times with DMEM (purchased from GIBCO, USA). 100 μL of DMEM was added to each well, and the fluorescence intensity of each well was measured using a multifunctional microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The results were as follows: Figure 15 shown.

[0155] Conclusion: Compared with the blank control group, exopolysaccharide A can significantly promote the release of ROS in RAW264.7 cells in the range of 25-100 μg / mL, and has a clear concentration dependence.

[0156] 2.4 Effect of exopolysaccharide from fermented Lactobacillus mucilaginosus on cytokine release from macrophages

[0157] RAW264.7 cells were cultured at 5 × 10 4 The concentration of cells / well was plated in a 96-well plate and cultured overnight at 37°C and 5% CO2 saturated humidity. After aspirating the culture medium, 100 μL of fresh DMEM cell culture medium containing different concentrations (25, 50, 100 μg / mL) of exopolysaccharide A was added to the wells and incubated at 37°C and 5% CO2 saturated humidity for 24 hours. RAW264.7 cells cultured without exopolysaccharide A and LPS were used as the blank control group, and RAW264.7 cells cultured without exopolysaccharide A and only with LPS (1 μg / mL) were used as the positive control group. The culture supernatant was collected and the levels of TNF-α, IL-10, IL-6, and IL-1β in the supernatant were determined using an ELISA kit (purchased from Anogen, Canada), and the concentrations of TNF-α, IL-10, IL-6, and IL-1β were calculated in turn using the standard curve. The results are shown in Figure 2. Figure 16-19 shown.

[0158] Conclusion: TNF-α, IL-6, and IL-1β are common pro-inflammatory factors, while IL-10 is a cytokine that plays an anti-inflammatory role. Figure 16-19As can be seen, compared with the blank control group, exopolysaccharide A at concentrations of 25-100 μg / mL significantly increased macrophage secretion of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and anti-inflammatory cytokines (IL-10) in a dose-dependent manner. These data indicate that exopolysaccharides from Lactobacillus fermentum CGMCC No. 17321 can activate RAW264.7 cells and promote the simultaneous release of anti-inflammatory and pro-inflammatory cytokines, potentially demonstrating potential dual immune regulatory activity.

[0159] 2.5 Effects of Lactobacillus mucilaginosus exopolysaccharides on macrophage polarization

[0160] RAW264.7 cells were cultured at a rate of 1 × 10 5 Cells were seeded into confocal microplates at a concentration of 100 cells / well and incubated at 37°C in a humidified atmosphere of 5% CO₂ for 24 h. After aspirating the culture medium, 1 mL of fresh DMEM containing exopolysaccharide A (100 μg / mL) was added to the wells and incubated at 37°C in a humidified atmosphere of 5% CO₂ for 24 h. RAW264.7 cells cultured without exopolysaccharide A and LPS served as a blank control, while RAW264.7 cells cultured without exopolysaccharide A but only with LPS (1 μg / mL) served as a positive control. Cells were washed twice with pre-chilled PBS, fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, and permeabilized with 0.5% Triton X-100 for 30 min at room temperature. Following this, the cells were blocked with 5% bovine serum albumin (purchased from Beyotime Biotechnology Co., Ltd., China) for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against iNOS and CD206. After extensive washing with PBS, the cells were incubated with Cy3-labeled goat anti-rabbit IgG (1:1000) in the dark for 1 h. After the PBS washing step, the cells were incubated with DAPI (1:1000) at room temperature for 10 min. Finally, the cells were washed three times with PBS and images were captured using a confocal microscope (A1R, Nikon, Japan), and the fluorescence intensity was measured. The results are shown in Figure 2. Figure 20 shown.

[0161] Conclusion: iNOS is a common marker of macrophage M1 phenotype, and CD206 is often used to characterize the M2 phenotype of macrophages. Figure 20 As shown, iNOS expression was almost absent in the blank group, indicating that the cells were in the M0 phenotype. However, after stimulation with exopolysaccharide A, iNOS expression on the cell membrane increased significantly, reaching 60% of that in the LPS-positive control group. There was no significant difference in CD206 expression on the cell membrane compared to the blank group. These results confirm that exopolysaccharide A can promote M1 polarization of cells.

[0162] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A fermentation of Lactobacillus mucilaginosus exopolysaccharide, characterized in that The main chain of the extracellular polysaccharide comprises acetylglucosamine residues, glucose residues and galactose residues; the main chain of the extracellular polysaccharide is composed of acetylglucosamine residues, glucose residues and galactose residues in a molar ratio of 1:3.78-3.84:2.06-2.

12.

2. The fermented Lactobacillus mucilaginosus exopolysaccharide according to claim 1, characterized in that Also includes one or more of the following characteristics: a1) The average weight molecular weight of the extracellular polysaccharide is 1.892×10 6 ~2.386×10 6 Dalton; a2) the main chain of the exopolysaccharide is composed of 1,3-linked glucose residues, 1,4-linked galactose residues, 1,6-linked glucose residues, 1,6-linked galactose residues, 1,2,3-linked galactose residues, 1,3,6-linked glucose residues and 1,3-linked acetylglucosamine residues, and the side chains are composed of terminal-linked glucose residues, and their branch points are located at the O2 position of the 1,2,3-linked galactose residues and the O6 position of the 1,3,6-linked glucose residues, respectively; a3) The exopolysaccharide is non-toxic.

3. The fermented Lactobacillus mucilaginosus exopolysaccharide according to claim 2, characterized in that Also includes one or more of the following characteristics: b1) the exopolysaccharide is obtained by fermentation of Lactobacillus fermentum with a preservation number of CGMCC NO.17321; preferably, the fermentation is carried out in a culture medium, and more preferably, the culture medium is MRS culture medium; b2) The exopolysaccharide is composed of repeating units represented by formula I: Among them, A is a 1,3,6-linked glucose residue; B is a 1,3-linked glucose residue; C is a 1,6-linked glucose residue; D is a 1,4-linked galactose residue; E is a 1,2,3-linked galactose residue; F is a 1,3-linked acetylglucosamine residue; H is a 1,6-linked galactose residue; G is a branch chain of the extracellular polysaccharide, which is a glucose residue, and its branch points are located at the O2 position of the 1,2,3-linked galactose residue and the O6 position of the 1,3,6-linked glucose residue, respectively.

4. A method for preparing the exopolysaccharide of fermented Lactobacillus mucilaginosus according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: fermenting, extracting and purifying the fermented mucus lactobacillus to obtain extracellular polysaccharide.

5. The preparation method according to claim 4, characterized in that Also includes one or more of the following characteristics: 1) The accession number of the fermented Lactobacillus mucilaginosus is CGMCC NO.17321; 2) The fermentation is carried out in a culture medium, preferably, the culture medium is MRS medium; 3) The inoculation amount of the fermented Lactobacillus mucilaginosus is 1×10 7 -8×10 7 CFU / mL; 4) the fermentation temperature is 20° C.-40° C., the fermentation time is 12-36 hours; the fermentation is anaerobic fermentation; 5) The extraction comprises the following steps: inactivating the fermentation broth obtained by fermentation, separating the supernatant, adding an alcohol compound for precipitation, separating the precipitate, mixing the precipitate with water, adding a protein precipitation reagent, allowing to stand, separating the supernatant, removing substances with a molecular weight cutoff of ≤14,000 Daltons, and drying; 6) The purification comprises the following steps: separating and purifying the extracted substances, combining and collecting the eluted products of the component peaks, removing substances with a molecular weight cutoff of ≤14,000 Daltons and drying to obtain the fermented Lactobacillus mucilaginosus exopolysaccharide.

6. The method for preparing the exopolysaccharide of fermented Lactobacillus mucilaginosus according to claim 5, wherein: Also includes one or more of the following characteristics: 51) The inactivation temperature is 90-100°C and the inactivation time is 10-20 minutes; 52) The separation method is centrifugation. Preferably, the centrifugal speed is 8000-12000g and the centrifugal time is 8-12min; 53) The volume ratio of the supernatant to the alcohol compound is 1:3-5; 54) The amount of the protein precipitation reagent added is 5-9% of the final concentration; 55) The protein precipitation reagent is trichloroacetic acid; 56) The standing temperature is 4-8°C and the standing time is 12-24h; 57) The removal of substances with a molecular weight cut-off of ≤14,000 Daltons is performed by dialysis. Preferably, the dialysis time is 72-120 h, and the water change frequency is preferably once every 8 h. 61) The separation and purification method is selected from gel column chromatography; preferably, the filler used in the gel column chromatography is Sepharose 6 Fast Flow gel filler, and the size of the gel column is D2.6 cm × 30 cm; 62) The removal of substances with a molecular weight cut-off of ≤14,000 Daltons is performed by dialysis. Preferably, the dialysis time is 72-120 h, and the water change frequency is preferably once every 8 h. 63) The elution solution is selected from NaCl solution, and the elution rate is 0.1-0.3 ml / min; preferably, the concentration of the eluent is 0.1-0.3 M.

7. Use of the fermented Lactobacillus mucilaginosus exopolysaccharide according to any one of claims 1 to 3 in preparing products.

8. The use according to claim 7, characterized in that The product has one or more of the following features: B1) immune regulation; B2) stimulates the phagocytic activity of macrophages and the release of NO and ROS, and simultaneously promotes the expression of pro-inflammatory factors (TNF-α, IL-6, IL-1β) and anti-inflammatory factors (IL-10) in macrophages; B3) Promotes M1 polarization of macrophages.

9. The use according to claim 7, characterized in that The product is selected from any one of food, medicine, health care product and cosmetics.

10. A product, characterized in that The method comprises the fermented Lactobacillus mucilaginosus exopolysaccharide according to any one of claims 1 to 3; preferably, further comprises a carrier or an auxiliary material.

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