Bacillus producing moisturizing and anti-inflammatory polysaccharide and application thereof

Abstract

The present invention discloses a bacillus producing moisturizing and anti-inflammatory polysaccharides and its application, and relates to the technical field of microorganisms. Bacillus sp. The extracellular polysaccharide produced by ZR001 has the effects of skin moisturizing and anti-inflammatory, and can be widely used in the fields of medicine, cosmetics and skin care products. The present invention also discloses a method for applying Bacillus Bacillus sp. ZR001 Method for producing extracellular polysaccharides by optimizing Bacillus Bacillus sp. ZR001 culture medium and culture conditions greatly increased the production of extracellular polysaccharides, up to 25.8g / L.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a bacillus producing moisturizing and anti-inflammatory polysaccharides and application thereof. Background Art

[0002] Polysaccharides are natural polymers composed of multiple monosaccharides connected by glycosidic bonds. They are widely found in plants, animals and microorganisms (Zong A, Cao H, Wang F. Anticancer polysaccharides from natural resources: A review of recent research [J]. Carbohydrate Polymers, 2012, 90 (4): 1395-1410). Among microbial polysaccharides, most polysaccharides derived from bacteria are released into the extracellular medium and are therefore classified as EPS types (Sutherland I W. Novel and established applications of microbial polysaccharides [J]. Trends in Biotechnology, 1998, 16 (1): 41-46). In both in vivo and in vitro studies, bacterial extracellular polysaccharides have shown a wide range of biological activities, such as antibacterial, antiviral, antioxidant, anti-inflammatory, anti-tumor, immunomodulatory, and hypoglycemic activities. Compared with plant polysaccharides and animal polysaccharides, bacterial EPS has the advantages of low production cost, no influence from geographical environment, climate and natural disasters, stable quality and yield, easy extraction, and large-scale industrial production through deep fermentation (Guo Min. Research Progress of Polysaccharide Production by Microbial Fermentation [J]. Bulletin of Microbiology, 2008, 35(7): 1084-1090). It has excellent application prospects in the fields of biomaterials, pharmaceuticals, food, etc.

[0003] At present, the application of Bacillus exopolysaccharides is mainly concentrated in the fields of biomedicine and ecological agriculture, and is less used in the field of daily chemicals. The research of Wang et al. has proved that Bacillus polymyxa exopolysaccharides can repair cells damaged by ultraviolet rays, providing new ideas for the development of new ultraviolet damage repair agents. However, there are no reports on the application of Bacillus exopolysaccharides in skin moisturizing and anti-inflammatory research. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a Bacillus sp. Bacillus sp. and its applications.

[0005] The present invention is specifically as follows:

[0006] The first aspect of the present invention provides a Bacillus sp. producing a moisturizing and anti-inflammatory polysaccharide. The Bacillus sp. is Bacillus sp. ZR001 was isolated from the soil of the Grasse Rose Base in Banmu Huatian, Jinan City, Shandong Province (36°12'00.00"N, 117°42'00.00"E). It was identified as Bacillus by 16S rDNA strain identification. Bacillus sp. The nucleotide sequence of its 16S rDNA is shown in SEQ ID NO.1. The bacterium is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No: 32048 and the deposit date of September 23, 2024.

[0007] The second aspect of the present invention provides an extracellular polysaccharide, wherein the extracellular polysaccharide is produced by the Bacillus Bacillus sp. produce.

[0008] The third aspect of the present invention provides a composition, the composition comprising the Bacillus Bacillus sp. Or the extracellular polysaccharide.

[0009] The fourth aspect of the present invention provides the Bacillus Bacillus sp. Or use of the extracellular polysaccharide or the composition in moisturizing and / or anti-inflammatory aspects.

[0010] The fifth aspect of the present invention provides the Bacillus Bacillus sp. Or the use of the extracellular polysaccharide or the composition in the preparation of medicines, cosmetics, and skin care products. Preferably, the medicine is an anti-inflammatory medicine.

[0011] The sixth aspect of the present invention provides a method for culturing the Bacillus Bacillus sp. The method, wherein the culture conditions are:

[0012] Shake flask seed culture: Inoculate a single colony (or glycerol tube) into a 250 ml triangular shake flask containing 50 ml of seed culture medium, and culture at 150-250 rpm and 25-35°C for 18-24 hours;

[0013] Seed tank culture conditions: temperature 25-35℃, ventilation 800-1200 L / h, rotation speed 300 -350rpm, culture period 20-28 hours;

[0014] Fermentation tank culture conditions: temperature 25-35℃, ventilation 800-1200 L / h, speed, starting at 100 rpm. When the dissolved oxygen rises, start to add sucrose and trypsin, and control the dissolved oxygen at 30%-60% by adjusting the feed rate and speed. The culture period is 40-56 hours.

[0015] Furthermore, the seed culture medium includes a solid culture medium and a liquid culture medium, wherein the components of the solid culture medium include: 8-13 g / L tryptone, 4-7 g / L yeast extract, 8-13 g / L sodium chloride, and 15-25 g / L agar; the components of the liquid culture medium include 8-13 g / L tryptone, 4-7 g / L yeast extract, and 8-13 g / L sodium chloride.

[0016] Furthermore, the components of the fermentation medium include: sucrose 40-60 g / L, tryptone 4-6 g / L, yeast extract powder 0.5-1.5 g / L, and disodium hydrogen phosphate 2-5 g / L.

[0017] A seventh aspect of the present invention provides a method for producing extracellular polysaccharides, the method comprising the following steps:

[0018] 1) Bacillus fermentation stage: the cultured Bacillus Bacillus sp. The seed solution of ZR001 is added to the fermentation tank at a rate of 1-5% and the temperature is 25-35°C. When the dissolved oxygen rises, the dissolved oxygen is controlled at 30%-60% by adjusting the feed rate and rotation speed, and the culture period is 36-60 hours.

[0019] 2) Purification stage: the culture liquid obtained in step 1) is placed in a tank, and the fermentation liquid is centrifuged at 13000-15000 rpm / min using a tubular centrifuge to remove the bacteria; diatomaceous earth is added to the liquid after centrifugation, and then plate and frame filtration is performed, and then a 0.45 um filter membrane is passed to obtain a polysaccharide solution.

[0020] In a specific embodiment of the present invention, the method for producing exopolysaccharides comprises the following steps:

[0021] 1) Bacillus fermentation stage: the cultured Bacillus Bacillus sp. ZR001 seed solution was added to the fermentation tank at a 2% inoculation rate and the temperature was 30°C. When the dissolved oxygen rose, the feed rate and rotation speed were adjusted to control the dissolved oxygen at 30%-60%, and the culture cycle was 48 hours;

[0022] 2) Purification stage: the culture liquid obtained in step 1) is tanked and fermented for 48 hours. The fermented liquid is centrifuged at 14000 rpm / min using a tubular centrifuge to remove the bacteria. After centrifugation, diatomaceous earth is added to the liquid, and then plate and frame filtration is performed, and then filtered through a 0.45 um filter membrane to obtain a polysaccharide solution.

[0023] The beneficial effects of the present invention include but are not limited to:

[0024] 1. Bacillus of the present invention Bacillus sp.ZR001 can significantly increase the yield of extracellular polysaccharides, up to 15.6 g / L, providing a new option for large-scale industrial production of extracellular polysaccharides and has great application prospects.

[0025] 2. Bacillus of the present invention Bacillus sp. The extracellular polysaccharide produced by ZR001 has skin moisturizing and / or anti-inflammatory effects and can be widely used in the preparation of medicines, cosmetics, and skin care products;

[0026] 3. The present invention also optimizes the present invention by optimizing Bacillus Bacillus sp. ZR001 fermentation medium and culture conditions improve the content of extracellular polysaccharides in the fermentation broth. By optimizing the fermentation medium and culture conditions, Bacillus Bacillus sp. The yield of extracellular polysaccharides in the ZR001 fermentation broth was further increased, with the highest yield reaching 25.8 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0028] Figure 1 Bacillus sp. in the embodiment of the present invention Bacillus sp. Flow chart of extraction of exopolysaccharide solution from ZR001 fermentation;

[0029] Figure 2 In the embodiment of the present invention, hyaluronic acid, trehalose and Bacillus Bacillus sp. Schematic diagram of the hygroscopicity results of exopolysaccharide produced by ZR001;

[0030] Figure 3 In the embodiment of the present invention, hyaluronic acid, trehalose and Bacillus Bacillus sp. Schematic diagram of the moisture retention results of exopolysaccharide produced by ZR001;

[0031] Figure 4 This is a schematic diagram of the relative activity results of each cell in the cytotoxicity experiment in the embodiment of the present invention;

[0032] Figure 5 This is a schematic diagram of the relative concentration results of IL-6 in the anti-inflammatory test experiment of the embodiment of the present invention, # indicates that the difference is statistically significant (p<0.05) compared with the negative control group (NC), and * indicates that the difference is statistically significant (p<0.05) compared with the model control group (M);

[0033] Figure 6This is a schematic diagram of the relative concentration results of TNF-α in the anti-inflammatory test experiment of the embodiment of the present invention, # indicates that the difference is statistically significant (p<0.05) compared with the negative control group (NC), and * indicates that the difference is statistically significant (p<0.05) compared with the model control group (M);

[0034] Figure 7 Schematic diagram of the relative concentration results of TSLP in the anti-inflammatory test experiment of the embodiment of the present invention, # indicates that the difference is statistically significant compared with the negative control group (NC) (p<0.05), and * indicates that the difference is statistically significant compared with the model control group (M) (p<0.05). DETAILED DESCRIPTION

[0035] The present invention is described in detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present invention are purchased through commercial channels.

[0036] Unless otherwise specified, the concentration percentages in the present invention are all mass percentages.

[0037] Instruments: 15L and 50L automatic fermenters from Shanghai Guoqiang Biochemical Equipment Co., Ltd.; Shanghai Sunny Hengping process mass spectrometer; Biostar software package from National Biochemical Engineering Center; Model 722 UV-visible spectrophotometer; rotary shaker.

[0038] Example 1 Bacillus Bacillus sp. Isolation and identification of ZR001

[0039] Bacillus Bacillus sp. ZR001 was isolated from the soil of the Grasse Rose Base in Banmu Huatian, Jinan City, Shandong Province (36°12'00.00"N, 117°42'00.00"E). The isolation process was as follows: about 10g of soil at a depth of 10 to 20cm was selected and mixed with 100ml of physiological saline. It was bathed in water at 80℃ for 1h, and then 4g of NaCl was added and bathed in water at 37℃ for 2h. 1mL of soil suspension was diluted 10 times with sterile water, and 100μL of the diluted test solution was added dropwise to the LB solid culture medium, spread evenly, and inverted to culture at 37℃ overnight. The morphology of individual colonies on the culture medium was observed, including the color, size, edge of the colony, and surface roughness of the colony, and 23 strains were obtained. These strains were fermented separately, and the exopolysaccharide content was detected, and 6 initial screening strains that can produce exopolysaccharides were obtained.

[0040] The present invention screened multiple microorganisms B1, B2, B3, B4, B5, and ZR001 for fermentation respectively to obtain a strain ZR001 with high extracellular polysaccharide production. The results of the extracellular polysaccharide production of different strains after fermentation for 48 hours are shown in Table 1.

[0041] Table 1 Polysaccharide yield after 48h fermentation

[0042]

[0043] Example 2 Bacillus Bacillus sp. Fermentation of ZR001 and separation and purification of extracellular polysaccharide

[0044] Bacteria: Bacillus obtained by natural environment screening Bacillus sp. ZR001.

[0045] Seed medium: 10.0 g of tryptone, 5.0 g of yeast extract, 10.0 g of sodium chloride, and 20.0 g of agar were added to 1.0 liter of purified water and sterilized in an autoclave at 121°C for 20 min to obtain a seed solid medium. 10.0 g of tryptone, 5.0 g of yeast extract, and 10.0 g of sodium chloride were added to 1.0 liter of purified water and sterilized in an autoclave at 121°C for 20 min to obtain a seed liquid medium.

[0046] Fermentation medium: 50.0 g sucrose, 5.0 g tryptone, 1.0 g yeast extract powder, and 3.0 g disodium hydrogen phosphate were added to 1.0 liter of purified water, the pH value was adjusted to 7.2, and the fermentation medium was obtained after sterilization in an autoclave at 115°C for 25 min.

[0047] Cultivation methods

[0048] (1) Culture conditions

[0049] Shake flask seed culture: Inoculate a single colony (or glycerol tube) into a 250 ml triangular shake flask containing 50 ml of seed culture medium, and culture at 200 rpm and 30°C for 18-24 hours.

[0050] Seed tank culture conditions: temperature 30°C, ventilation volume 1080 L / h, rotation speed 300 rpm, and culture period 24 hours.

[0051] Fermentation tank culture conditions: temperature 30℃, ventilation volume 1080 L / h, speed, starting at 100 rpm. When the dissolved oxygen rises, start to feed sucrose and trypsin, and control the dissolved oxygen at 30%-60% by adjusting the feed rate and speed. The culture period is 48 hours.

[0052] (2) Cultivation process

[0053] Bacillus Bacillus sp. The extraction process of fermentation exopolysaccharide solution is as follows Figure 1 The main process includes fermentation → centrifugation → impurity removal → membrane → extracellular polysaccharide solution. The specific steps are as follows:

[0054] ① BacillusBacillus sp. Fermentation stage: the cultured Bacillus Bacillus sp. The seed solution was added to the fermentation tank at a 2% inoculation rate and the temperature was 30°C. When the dissolved oxygen rose, the dissolved oxygen was controlled at 30%-60% by adjusting the feed rate and rotation speed, and the culture period was 48 hours.

[0055] ② Purification stage: After 48 hours of fermentation, the fermentation liquid is put into the tank and centrifuged at 14000rpm / min using a tubular centrifuge to remove the bacteria; after centrifugation, diatomaceous earth is added to the liquid to remove impurities, and then plate and frame filtration is performed, and then passed through a 0.45um filter membrane to obtain a polysaccharide solution.

[0056] ③Analysis of polysaccharide content in fermentation broth: The polysaccharide content of the solution after filtration was detected by anthrone-sulfuric acid method and was 25.8 g / L.

[0057] Example 3 Efficacy Identification of Extracellular Polysaccharides

[0058] Reducing sugar determination: biochemical analyzer enzyme membrane detection method.

[0059] Polysaccharide determination: alcohol precipitation method, anthrone-sulfuric acid method.

[0060] Biomass determination: Off-line determination uses the wet volume method. After the fermentation broth is diluted a certain multiple, the absorbance is measured on a spectrophotometer to obtain the OD value.

[0061] pH and DO online determination: Mettler Toledo high temperature resistant electrode is used for online determination.

[0062] Temperature: measured online by platinum temperature electrode.

[0063] Determination of oxygen and carbon dioxide in intake and exhaust gases: The American Extrel process mass spectrometer MAX300-LG was used to perform real-time online collection and analysis of intake and exhaust gases during the fermentation process.

[0064] (1) Moisturizing test experiment

[0065] The ZR001 extracellular polysaccharide solution after fermentation and purification was vacuum freeze-dried to obtain polysaccharide powder. The experiment was conducted with reference to the moisture absorption and moisture retention test method. This experiment used two batches of polysaccharide powder, 912 and 914, for the experiment.

[0066] Hygroscopicity test: Accurately weigh 0.3 g of each of the freeze-dried sample of extracellular polysaccharide, trehalose and sodium hyaluronate (Huaxi Biotechnology, name: sodium hyaluronate (HA-TP), batch number: J201230575) dried to a constant mass, and place them in a dry plate. Place the plate in a 20°C constant temperature dryer filled with an appropriate amount of saturated ammonium sulfate solution (relative humidity 81%), and weigh the sample mass regularly. Each group and each sample were tested in parallel 3 times. The formula for calculating the sample moisture absorption rate is as follows:

[0067] Moisture absorption rate ×100%

[0068] Where: m t is the mass of the sample at time t, in g; m0 is the mass of the dry sample, in g.

[0069] Moisture retention test: Accurately weigh 0.3g of each of the extracellular polysaccharide freeze-dried sample, trehalose and hyaluronic acid dried to constant weight, add 3 times the volume of deionized water, and after the samples are evenly and completely soaked, place them in a desiccator filled with dry silica gel (relative humidity 0%), and weigh the sample mass regularly. The sample moisture retention rate is calculated as follows:

[0070] Moisture Retention Rate = ×100%

[0071] Where: m t is the mass of the sample at time t, in g; m0 is the mass of the dry sample, in g; m 水 is the mass of deionized water added, in g.

[0072] The results are as follows Figures 2 - 3 As shown in the figure. The moisture retention rates of hyaluronic acid, trehalose, and extracellular polysaccharide produced by strain ZR001 decreased with time under dry conditions. Within 60 hours, the moisture retention rate of extracellular polysaccharide produced by strain ZR001 was better than that of hyaluronic acid and chitosan. At 60 hours, the moisture retention rate of extracellular polysaccharide produced by strain ZR001 was 51%, which was equivalent to hyaluronic acid and better than trehalose. After 60 hours, the moisture retention rate of hyaluronic acid>exocellular polysaccharide produced by strain ZR001>trehalose, indicating that the extracellular polysaccharide has a certain moisturizing effect.

[0073] (2) Anti-inflammatory test

[0074] Experimental principle:

[0075] Inflammation is one of the most common clinical diseases. It is a defensive response of the human body to ensure the removal of harmful stimuli and repair of damaged tissues. When human immune cells are affected by inflammatory factors, some small molecular weight soluble proteins or polypeptides that can transmit information between cells and have specific immune regulatory functions are secreted by the body itself and can participate in or cause inflammatory reactions. These substances are called inflammatory factors, including NO, TNF-α, IL-6, PGE-2, IL-1, etc. NO, IL-6, and TNF-α are all important cell active factors in inflammatory reactions. They have direct or indirect effects on cell inflammation and also affect each other.

[0076] In this experiment, LPS-induced RAW264.7 cells (cell line: RAW264.7 cells, source: Beina Bio, passage number: 8) were used as an in vitro inflammatory cell model. The inflammatory factors IL-6, TNF-α, and TSLP secreted in the cell supernatant were detected by ELISA kits to evaluate the anti-inflammatory effect of the test samples.

[0077] Experimental steps:

[0078] 1. Cytotoxicity Screening

[0079] 1.1 The cultured cells were seeded into a 96-well plate and cultured in an incubator for 18-24 h until the cells were 80% confluent.

[0080] 1.2 The original culture medium was removed, and 100 μL of sample extracellular polysaccharide dilutions of different concentrations were added to each well and exposed in the incubator for 24±0.5 h. The sample concentrations were 20%, 6%, 2%, 0.6%, 0.2%, 0.06%, 0.02% and 0.006%, respectively. The negative control group (NC) was only added with the same volume of culture medium, the model control group (M) was added with culture medium and induced with LPS, the test sample group (TA) was added with culture medium containing samples of different concentrations and induced with LPS, and the positive control group (PC) was added with culture medium containing dexamethasone and induced with LPS.

[0081] 1.3 Add 20 μL of MTT solution to each well and incubate at 37°C for 3 ± 0.5 h. Then remove the MTT solution, add 100 μL of DMSO to each well, shake in the dark for 10-15 min, and measure the absorbance at 570 nm.

[0082] The cell activity of the negative control group (Control) was taken as 100%, and the relative cell activity (Viability) of each group was calculated. The calculation formula is as follows:

[0083]

[0084] Among them, OD TA The absorbance value of the sample group to be tested was measured at a wavelength of 570 nm, OD Blank The absorbance of the negative control group (NC) was measured at 570 nm. Control The absorbance value of the model control group (M) was measured at a wavelength of 570 nm.

[0085] The results are as follows Figure 4As shown in the figure, according to the results of cell activity test, sample concentrations below 0.6% have little effect on cell activity. In order to display the experimental results more intuitively and facilitate experimental operations, 0.60% (v / v), 0.10% (v / v), and 0.01% (v / v) were selected as test concentrations for subsequent efficacy experiments.

[0086] 2. Cellular anti-inflammatory test

[0087] 2.1 For routine cell culture, inoculate the cell suspension into a 12-well cell culture plate and return to the incubator for 18-24 h.

[0088] 2.2 Remove the culture plate and discard the original culture medium in the wells. Add the test sample and LPS culture medium with concentrations of 0.6%, 0.1% and 0.01% to each well of the sample group, add LPS as a stimulator to the model group (M), add dexamethasone and LPS culture medium to the positive control (PC group), and add culture medium to the negative control group (NC group). Culture for 24±1 h.

[0089] 2.3 The supernatant was collected and stored at -80°C. Cytokines were measured using ELISA kits (Wuhan Huamei: CSB-E04639m).

[0090] The data were analyzed using SPSS and presented as mean ± SD. The differences were considered statistically significant if p < 0.05.

[0091] The relative contents of IL-6, TNF-α and TSLP in each group were calculated with the model control group (M) as 100%.

[0092] The results are as follows Figures 5 - 7 As shown in the figure, under the conditions of cell anti-inflammatory test, the relative contents of IL-6, TNF-α, and TSLP in the model group (M) were significantly increased compared with the negative control group (NC) (p<0.05); the relative contents of IL-6, TNF-α, and TSLP in the positive control group were significantly decreased compared with the model group (p<0.05), indicating that the model was successfully established. The relative contents of IL-6 in the sample "exopolysaccharide produced by strain ZR001" decreased by 42%, 22%, and 18% respectively at the test concentrations of 0.60% (v / v), 0.10% (v / v), and 0.01% (v / v) compared with the model group, the relative contents of TNF-α decreased by 55%, 50%, and 39% respectively, and the relative contents of TSLP decreased by 43%, 39%, and 36% respectively, and there were significant differences (p<0.05).

[0093] Under the conditions of this experiment, the sample "exopolysaccharide produced by strain ZR001" had anti-inflammatory effects at test concentrations of 0.60% (v / v), 0.10% (v / v), and 0.01% (v / v) under the test indicators of IL-6, TNF-α, and TSLP.

[0094] The above description is only an embodiment of the present invention. The protection scope of the present invention is not limited by these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent substitution, improvement, etc. made within the technical ideas and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A bacillus producing polysaccharides having moisturizing and / or anti-inflammatory effects, characterized in that: The name of the bacillus is Bacillus Bacillus sp. ZR001 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No: 32048 and the deposit date of September 23, 2024.

2. An extracellular polysaccharide, characterized in that The exopolysaccharide is produced by the Bacillus according to claim 1.

3. A composition, characterized in that The composition comprises the bacillus according to claim 1 or the exopolysaccharide according to claim 2.

4. Use of the bacillus according to claim 1, the exopolysaccharide according to claim 2, or the composition according to claim 3 in the preparation of skin moisturizing and / or anti-inflammatory medicines or cosmetics.

5. Use of the bacillus according to claim 1, the exopolysaccharide according to claim 2, or the composition according to claim 3 in preparing skin moisturizing and / or anti-inflammatory skin care products.

6. The use according to claim 4, characterized in that: The medicine is an anti-inflammatory drug.

7. A method for culturing the Bacillus according to claim 1, characterized in that: The culture conditions are: Shake flask seed culture: Inoculate a single colony or glycerol tube into a 250 ml triangular shake flask containing 50 ml of seed culture medium, culture at 150-250 rpm and 25-35°C for 18-24 hours; Seed tank culture conditions: temperature 25-35℃, ventilation 800-1200 L / h, rotation speed 300 -350rpm, culture period 20-28 hours; Fermentation tank culture conditions: temperature 25-35℃, ventilation 800-1200 L / h, speed, starting at 100 rpm. When the dissolved oxygen rises, start adding sucrose and trypsin. Control the dissolved oxygen at 30%-60% by adjusting the feed rate and speed. The culture period is 40-56 hours.

8. The method according to claim 7, characterized in that The seed culture medium includes solid culture medium and liquid culture medium, wherein the components of the solid culture medium include: 8-13 g / L tryptone, 4-7 g / L yeast extract, 8-13 g / L sodium chloride, and 15-25 g / L agar; the components of the liquid culture medium include 8-13 g / L tryptone, 4-7 g / L yeast extract, and 8-13 g / L sodium chloride.

9. The method according to claim 7, characterized in that: The components of the fermentation medium include: sucrose 40-60 g / L, tryptone 4-6 g / L, yeast extract powder 0.5-1.5 g / L, disodium hydrogen phosphate 2-5 g / L.

10. A method for producing the extracellular polysaccharide according to claim 2, characterized in that: The method comprises the following steps: 1) Bacillus fermentation stage: the seed liquid of Bacillus cultured by any method described in any one of claims 7 to 9 is inoculated into a fermentation tank at a rate of 1% to 5%, the temperature is 25 to 35° C., when the dissolved oxygen rises, the dissolved oxygen is controlled at 30% to 60% by adjusting the feed rate and the rotation speed, and the culture period is 36 to 60 hours; 2) Purification stage: the culture liquid obtained in step 1) is placed in a tank, and the fermentation liquid is centrifuged at 13000-15000 rpm using a tubular centrifuge to remove the bacteria; diatomaceous earth is added to the liquid after centrifugation, and then plate and frame filtration is performed, and then a 0.45 μm filter membrane is passed to obtain a polysaccharide solution.

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