Ginkgo biloba endophyte bacillus velezensis polysaccharide with remarkable anti-allergic activity and application of ginkgo biloba endophyte bacillus velezensis polysaccharide

Abstract

The invention discloses polysaccharide of gingko endophyte bacillus velezensis with remarkable anti-allergic activity and application of the polysaccharide. The polysaccharide is prepared by the following steps: fermenting ginkgo endophyte bacillus velezensis, centrifuging, concentrating, extracting by adopting an alcohol precipitation method to obtain crude polysaccharide, redissolving with distilled water, and freeze-drying in vacuum. The polysaccharide of the ginkgo biloba endophyte bacillus velezensis can well inhibit release of beta-HEX in RBL-2H3 cells, reduce the release amount of histamine, effectively down-regulate the release level of IL-4 cell factors and reduce the expression level of tumor necrosis factors TNF-alpha during allergy, has remarkable anti-allergic activity, can effectively relieve allergy or inflammatory response, and can be used for preparing a medicine for treating allergy. The ginkgo endophyte can be used for research and development of anti-allergic active drugs and development of functional food and animal food, solves the problems that plant-derived anti-allergic substances are low in content, high in price, tedious in separation and the like, and has important significance on exploration and utilization of the ginkgo endophyte.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a Ginkgo biloba endophyte B. velezensis polysaccharide with significant anti-allergic activity and an application thereof. Background Art

[0002] Endophytes are widely present in the tissues and organs of the host and are non-toxic to the host. Endophytes are rich in species and are mainly divided into endophytic bacteria, endophytic fungi and endophytic actinomycetes. Due to their good physical and chemical properties and biological functions, such as anti-tumor, antibacterial, antioxidant and biocontrol properties, endophytes are widely used in pharmaceutical research and development, food processing, agricultural production and daily chemical industries. However, there are few reports and studies on the anti-allergic effects of ginkgo, and there are no related studies on the prevention and treatment of allergies in animals. In addition, endophytes can develop physiological functions that are the same or similar to those of the host during long-term evolution and can produce metabolites with the same or similar biological activity as the host. Therefore, the use of endophytes isolated from ginkgo can solve the problems of low content, high price and cumbersome isolation of plant-derived anti-allergic substances.

[0003] In recent years, most researchers have focused on antimicrobial activities in ginkgo endophytes. For example, endophytes isolated from ginkgo have demonstrated significant antibacterial activity against plant pathogens, while others have isolated endophytic fungi with antibacterial properties against apple rot. In general, ginkgo endophytes can secrete one or more substances with antimicrobial activity. Using TLC and platelet aggregation inhibition assays, seven endophytic fungi were identified as producing ginkgolide C or its analogs. However, there are currently no reports on the antiallergic activity of ginkgo endophytes and their polysaccharides, or on the development of products for animal allergy control. Summary of the Invention

[0004] The purpose of the present invention is to provide a Ginkgo biloba endophyte B. velezensis polysaccharide with significant anti-allergic activity and application thereof.

[0005] Through the present invention, it is found that the polysaccharide produced by fermentation of ginkgo endophytes has good anti-allergic activity, which fills the gap in the anti-allergic biological activity of ginkgo endophytes, can alleviate the problems of low content, high price and cumbersome separation of plant-derived anti-allergic substances, broaden the application scope of ginkgo endophytes, and has good application prospects.

[0006] The present invention is achieved through the following technical solutions.

[0007] The invention discloses a ginkgo endophyte polysaccharide with significant anti-allergic activity. The ginkgo endophyte B. velezensis polysaccharide is tested for β-HEX release by cell experiments and verified by anti-allergic experiments.

[0008] The method for preparing the Ginkgo biloba endophyte B. velezensis polysaccharide having significant anti-allergic activity comprises the following steps:

[0009] (1) Pretreatment of fermentation broth of Bacillus velezensis endophytes from Ginkgo biloba: The endophytes B. velezensis isolated from Ginkgo biloba were activated for two generations and inoculated into NB medium for fermentation;

[0010] (2) Extraction of crude polysaccharides from the endophytic bacteria B. velezensis of Ginkgo biloba: centrifuging the fermentation liquid of the endophytic bacteria B. velezensis obtained in step (1), removing the bacterial cells and retaining the supernatant; rotary evaporating and concentrating the supernatant, and extracting crude polysaccharides from the endophytic bacteria B. velezensis of Ginkgo biloba by alcohol precipitation;

[0011] (3) Purification of crude polysaccharide from ginkgo endophyte Bacillus Velez subtilis: The crude polysaccharide from ginkgo endophyte Bacillus Velez subtilis was washed, and after ethanol volatilization, the polysaccharide precipitate was redissolved in distilled water, the pigment was removed, the polysaccharide was dialyzed, and vacuum freeze-dried to obtain the polysaccharide from ginkgo endophyte Bacillus Velez subtilis.

[0012] The specific operation of the alcohol precipitation method for extracting crude polysaccharides from the ginkgo endophyte Bacillus Velez subtilis is as follows: the concentrated supernatant is fully mixed with ethanol, allowed to stand overnight, and the precipitate is collected after centrifugation.

[0013] The NB culture medium was prepared according to the following components: 3 g / L beef extract, 5 g / L peptone, and 2.5 g / L glucose.

[0014] Preferably, the fermentation conditions of the Ginkgo endophyte B. velezensis are 110-220 rpm, 25-37° C., and 24-72 h.

[0015] Preferably, the inoculation amount of the Ginkgo endophyte B. velezensis inoculated into the NB culture medium is 1%.

[0016] The pigment is removed by adsorption with macroporous resin.

[0017] The invention relates to an application of the ginkgo endophyte Bacillus velezensis polysaccharide in the preparation of anti-allergic drugs, functional foods and animal foods.

[0018] The ginkgo endophyte B. velezensis polysaccharide was verified to have good anti-allergic properties by measuring β-HEX, histamine, IL-4 and TNF-α allergy indicators.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0020] (1) The present invention can solve the problems of low content, high price and complicated separation of plant-derived anti-allergic substances.

[0021] (2) The present invention effectively fills the gap in the research on the anti-allergic activity of Ginkgo biloba endophyte polysaccharides and has good application prospects.

[0022] (3) The ginkgo endophyte B. velezensis polysaccharide prepared by the present invention can effectively inhibit the release of β-HEX in RBL-2H3 cells during allergy, reduce the release of histamine, effectively downregulate the release level of IL-4 cytokine, reduce the expression level of tumor necrosis factor TNF-α, effectively relieve allergic or inflammatory reactions, and has significant anti-allergic activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention is a flow chart for preparing the fermentation liquid of the ginkgo endophyte B. velezensis and its polysaccharide with significant anti-allergic activity.

[0024] Figure 2 The present invention is a flow chart for verifying the activity of the fermentation liquid of the ginkgo endophyte B. velezensis and its polysaccharide with significant anti-allergic activity. DETAILED DESCRIPTION

[0025] The following examples further illustrate the specific implementation of the present invention, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are any processes not specifically described below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0026] Example 1-10 uses the principle that hyaluronidase degrades hyaluronic acid, promotes the diffusion of allergic mediators and activates immune signals, leading to the aggravation of allergic reactions, to verify the anti-allergic activity of bacteria and their polysaccharides; Example 11-20 uses the principle that the body releases β-HEX (β-hexosaminidase) only when allergic, and detects the release of β-HEX through cell degranulation experiments to verify the anti-allergic activity of bacteria and their polysaccharides; Example 21-27 uses the fact that histamine causes vasodilation, increases vascular permeability and stimulates nerve endings during allergies, IL-4 promotes IgE class conversion, induces eosinophil infiltration and stimulates mucus secretion, and TNF-α promotes inflammatory cell recruitment. The release of three substances is detected by the RBL-2H3 cell model to verify the anti-allergic activity of bacteria and their polysaccharides. Example 1-10, Example 11-20 and Example 21-27 are compared to observe the anti-allergic activity results of the three methods.

[0027] NB medium was prepared with the following components: 3 g / L beef extract, 5 g / L peptone, and 2.5 g / L glucose.

[0028] Example 1

[0029] This example utilizes the principle that hyaluronidase (HAase) degrades hyaluronic acid, promotes the diffusion of allergic mediators, and activates immune signals, leading to an aggravation of allergic reactions. This is an example of verifying the anti-allergic activity of bacteria and their polysaccharides. The specific steps are as follows:

[0030] (1) Preparation of bacterial suspension: Activate the preserved Ginkgo endophytic bacteria B. velezensis for two generations, ferment at 30℃ and 200rpm for 48h, centrifuge at 8000r / min, discard the supernatant, and retain the bacteria. Wash with prepared physiological saline and resuspend the bacteria to ensure that the final bacterial suspension OD 600nm The value is 0.7;

[0031] (2) Preparation of crude polysaccharides: The activation, fermentation and centrifugation steps were carried out according to the method (1), the bacterial cells were removed and the supernatant was retained; the supernatant was concentrated by rotary evaporation using a rotary evaporator, and the crude polysaccharide of the endophytic fungus B. velezensis was extracted by alcohol precipitation; the obtained supernatant concentrate was thoroughly mixed with 80% ethanol and allowed to stand overnight, and the precipitate was collected by centrifugation the next day;

[0032] (3) Purification of crude polysaccharide from Ginkgo endophyte Bacillus velezensis: The crude polysaccharide from Ginkgo endophyte Bacillus velezensis was washed, and after ethanol volatilization, the polysaccharide precipitate was redissolved with distilled water. The pigment in the crude polysaccharide solution was adsorbed using a macroporous resin, dialyzed for 72 hours, and vacuum freeze-dried (-50°C, 48 hours) to obtain the Ginkgo endophyte B. velezensis polysaccharide with significant anti-allergic activity.

[0033] (4) Hyaluronidase inhibition test:

[0034] Experimental and control groups were set up respectively. Sample bacterial suspension (50 μg / mL polysaccharide solution) was added to tube A (absorbance value of experimental group) and tube B (absorbance value of blank in experimental group) of the experimental group; the same volume of distilled water was added to tube C (absorbance value of control group) and tube D (absorbance value of blank in control group) of the control group. Hyaluronidase (acetic acid / sodium acetate buffer was used as solvent to prepare the concentration of 500 U / mL) was added to tubes A and C respectively, and 500 μL acetate buffer (pH=5.6) was added to tubes B and D respectively, and kept warm at 32°C for 15 minutes. 100 μL was added to each of the four tubes. CaCl2 solution (2.5 mol / L) was added and kept at 32°C for 15 min. Sodium hyaluronate solution (AC buffer diluted to 0.5 mg / mL) was added to tubes A and C respectively, and 500 μL acetate buffer (pH = 5.6) was added to tubes B and D respectively. The mixture was kept at 32°C for 20 min and then kept at room temperature for 10 min. Distilled water was added to the four tubes respectively, and then 100 μL of NaOH solution (5 mol / L) was added to each tube, followed by 500 μL of Prepare acetylacetone solution (mix 50 mL of 1.0 mol / L sodium carbonate solution with 3.5 mL of acetylacetone solution) in a boiling water bath for 5 min, immediately place in an ice bath for 10 min, and allow to stand at room temperature for 10 min. Finally, add 1 mL of P-DAB color developer (0.8 g of p-dimethylaminobenzaldehyde dissolved in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol and mix well). Thoroughly mix the solutions in each tube, add 4 mL of anhydrous ethanol, and allow to stand at room temperature. Measure the absorbance at 530 nm using a spectrophotometer.

[0035] Example 2

[0036] During the preparation of bacterial suspension, the temperature of the entire bacterial suspension was 25°C, the fermentation time was 36 hours, the rotation speed was 200 rpm, and the OD of the bacterial suspension was 0. 600nm The value was controlled at 0.8, and the other operations were the same as those in Example 1.

[0037] Example 3

[0038] During the preparation of bacterial suspension, the temperature of the entire bacterial suspension was 28°C, the fermentation was 24 hours, the rotation speed was 150 rpm, and the OD of the bacterial suspension was 0. 600nm The value was controlled at 0.7, and the rest of the operations were the same as those in Example 1.

[0039] Example 4

[0040] During the preparation of bacterial suspension, the temperature of the entire bacterial suspension was 37°C, the fermentation time was 72 hours, the rotation speed was 220 rpm, and the OD 600nm The value was controlled at 0.7, and the rest of the operations were the same as those in Example 1.

[0041] Example 5

[0042] During the preparation of the bacterial suspension, the entire bacterial suspension was fermented at a temperature of 35° C. for 24 h at a rotation speed of 180 rpm. The bacterial suspension was resuspended to control the OD value at 1.0. The remaining operations were the same as those in Example 1.

[0043] Example 6

[0044] During the preparation of the bacterial suspension, the entire bacterial suspension was fermented at a temperature of 37° C. for 48 h at a rotation speed of 150 rpm. The bacterial suspension was resuspended to control the OD value at 0.6. The remaining operations were the same as those in Example 1.

[0045] Example 7

[0046] During the preparation of crude polysaccharide, the temperature of the entire bacterial fermentation was 32°C, the fermentation was carried out for 24 hours, the rotation speed was 220 rpm, the polysaccharide was precipitated with 95% ethanol, the polysaccharide pigment was adsorbed with macroporous resin for 2 hours, and dialyzed for 24 hours. The remaining operations were the same as those in Example 1.

[0047] Example 8

[0048] During the preparation of crude polysaccharide, the temperature of the entire bacterial fermentation was 27°C, the fermentation was carried out for 72 hours, the rotation speed was 180 rpm, the polysaccharide was precipitated with 95% ethanol, the polysaccharide pigment was adsorbed using macroporous resin for 6 hours, and dialyzed for 72 hours. The remaining operations were the same as those in Example 1.

[0049] Example 9

[0050] During the preparation of crude polysaccharide, the temperature of the entire bacterial fermentation was 30°C, the fermentation was 36 hours, the rotation speed was 110 rpm, the polysaccharide was precipitated with 80% ethanol, the polysaccharide pigment was adsorbed with macroporous resin for 10 hours, and dialyzed for 36 hours. The remaining operations were the same as those in Example 1.

[0051] Example 10

[0052] During the preparation of crude polysaccharide, the temperature of the entire bacterial fermentation was 25°C, the fermentation was carried out for 72 hours, the rotation speed was 200 rpm, the polysaccharide was precipitated with 80% ethanol, the polysaccharide pigment was adsorbed with macroporous resin for 4 hours, and dialyzed for 48 hours. The remaining operations were the same as those in Example 1.

[0053] Example 11

[0054] This example utilizes the principle that β-HEX (β-hexosaminidase) is released only when the body is allergic. The release of β-HEX is detected through a cell degranulation experiment. This is another example of verifying the anti-allergic activity of bacteria and their polysaccharides. The specific steps are as follows:

[0055] (1) Preparation of fermentation supernatant: The preserved Ginkgo endophytic bacteria B. velezensis were activated for two generations in NB medium, fermented at 200 rpm and 30°C for 72 h, centrifuged at 8000 rpm, and the supernatant was retained and filtered through a 0.22 μm filter membrane, and the pH was adjusted to 7.0.

[0056] (2) Preparation of crude polysaccharides: The specific operation is as follows: activate the Ginkgo biloba endophyte B. velezensis for two generations, ferment at 28°C, 220 rpm, and ferment for 48 hours. Centrifuge the fermentation broth to remove the bacteria and retain the supernatant. Concentrate the supernatant using a rotary evaporator and extract the crude polysaccharide of Ginkgo biloba endophyte B. velezensis by alcohol precipitation. The obtained supernatant concentrate is thoroughly mixed with 80% ethanol and allowed to stand overnight. The precipitate is collected by centrifugation the next day.

[0057] (3) Purification of crude polysaccharide from Ginkgo endophyte Bacillus velezensis: The crude polysaccharide from Ginkgo endophyte Bacillus velezensis was washed, and after ethanol volatilization, the polysaccharide precipitate was redissolved with distilled water. The pigment in the crude polysaccharide solution was adsorbed with macroporous resin for 5 hours, dialyzed for 48 hours, and vacuum freeze-dried (-50°C, 48 hours) to obtain the Ginkgo endophyte B. velezensis polysaccharide with significant anti-allergic activity.

[0058] (4) β-HEX release inhibition experiment: The experiment was performed when RBL-2H3 cells grew to about 80%. The experiment was divided into three groups: negative control group (N group), experimental group (T group) and model group (M group), with five samples in each group;

[0059] First, the complete culture medium was discarded from the fully adherent cells. 250 μL of complete culture medium containing anti-dinitrobenzene monoclonal mouse antibody was added to both T and M groups to induce an allergic reaction. The same amount of DMEM complete culture medium was added to N group. The cells were placed in a cell culture incubator at 37°C and 5% CO2 for overnight culture.

[0060] The next day, the culture medium in each group of 48-well plates was discarded and the cells were washed with PBS buffer. 250 μL of fermentation supernatant and 250 μL of polysaccharide solution (50 μg / mL) were added to the T group, and equal amounts of DMEM complete medium were added to the M and N groups. The cells were placed in a cell culture incubator at 37°C and 5% CO2 for 1 hour.

[0061] The old culture medium in the well plate was aspirated and washed with PBS buffer. 250 μL of complete culture medium containing DNP-BSA was added to both the T and M groups, and an equal amount of DMEM complete culture medium was added to the N group for culture. After treatment with DNP-BSA for 1 hour, the reaction was terminated in an ice bath for 10 minutes. The supernatant in the 48-well plate was aspirated and centrifuged at 4°C and 1000 rpm for 3 minutes, and the supernatant was retained for later use. The supernatant (50 μL per well) was added to the 96-well culture plate, and then 50 μL of color development solution was added to each well. After incubation at 37°C for about 1 hour, 200 μL of stop solution was added to each well to terminate the reaction. The absorbance was measured at a wavelength of 405 nm using an enzyme reader and the results were calculated by substituting the following formula:

[0062]

[0063] Example 12

[0064] During the preparation of the fermentation supernatant, the temperature of the entire bacterial fermentation was 25° C., the rotation speed was 200 rpm, the fermentation was carried out for 48 h, the pH was controlled at 6.5, and the remaining operations were the same as those in Example 11.

[0065] Example 13

[0066] During the preparation of the fermentation supernatant, the temperature of the entire bacterial fermentation was 28° C., the rotation speed was 200 rpm, the pH was controlled at 6.5, and the remaining operations were the same as those in Example 11.

[0067] Example 14

[0068] During the preparation of the fermentation supernatant, the temperature of the entire bacterial fermentation was 37° C., the fermentation was carried out for 24 h, the rotation speed was 120 rpm, the pH was controlled at 8.0, and the remaining operations were the same as those in Example 11.

[0069] Example 15

[0070] During the preparation of the fermentation supernatant, the temperature of the entire bacterial fermentation was 35° C., the fermentation was carried out for 36 h, the rotation speed was 180 rpm, the pH was 6.5, and the remaining operations were the same as those in Example 11.

[0071] Example 16

[0072] During the preparation of the fermentation supernatant, the temperature of the entire bacterial fermentation was 28° C., the fermentation was carried out for 48 h, the rotation speed was 150 rpm, the pH was 8.5, and the remaining operations were the same as those in Example 11.

[0073] Example 17

[0074] During the preparation of the fermentation supernatant, the temperature of the entire bacterial fermentation was 30° C., the fermentation was carried out for 36 h, the rotation speed was 220 rpm, the pH was 7.5, and the remaining operations were the same as those in Example 11.

[0075] Example 18

[0076] The fermentation temperature of the bacterial liquid was 35°C for 24 hours at a rotation speed of 220 rpm. During the preparation of crude polysaccharide, polysaccharide was precipitated with 80% ethanol, polysaccharide pigment was adsorbed with macroporous resin for 3 hours, and dialyzed for 36 hours. The remaining operations were the same as those in Example 11.

[0077] Example 19

[0078] The fermentation temperature of the bacterial liquid was 27°C, the fermentation time was 36 hours, and the rotation speed was 160 rpm. During the preparation of crude polysaccharide, polysaccharide was precipitated with 95% ethanol, polysaccharide pigment was adsorbed with macroporous resin for 4 hours, and dialyzed for 24 hours. The remaining operations were the same as those in Example 11.

[0079] Example 20

[0080] The fermentation temperature of the bacterial liquid was 30°C for 24 hours at a rotation speed of 150 rpm. During the preparation of crude polysaccharide, polysaccharide was precipitated with 95% ethanol, polysaccharide pigment was adsorbed with macroporous resin for 3 hours, and dialyzed for 72 hours. The remaining operations were the same as those in Example 11.

[0081] Example 21

[0082] This example utilizes the fact that during allergy, histamine causes vasodilation, increases vascular permeability, and stimulates nerve endings; IL-4 promotes IgE class switching, induces eosinophil infiltration, and stimulates mucus secretion; and TNF-α promotes inflammatory cell recruitment. The release of the three substances is detected using an RBL-2H3 cell model. This is another example of verifying the anti-allergic activity of bacteria and their polysaccharides. The specific steps are as follows:

[0083] (1) Preparation of fermentation supernatant: The preserved endophytic bacteria B. velezensis were activated for two generations in NB medium, fermented at 180 rpm and 25°C for 36 h, centrifuged at 8000 rpm, and the supernatant was retained and filtered through a 0.22 μm filter membrane, and the pH was adjusted to 7.2.

[0084] (2) Preparation of crude polysaccharides: The Ginkgo biloba endophyte B. velezensis was activated for two generations and fermented at 26°C, 220 rpm, for 72 h. The fermentation broth was centrifuged to remove the bacteria and retain the supernatant. The supernatant was concentrated by rotary evaporation using a rotary evaporator, and crude polysaccharides from the Ginkgo biloba endophyte B. velezensis were extracted by alcohol precipitation. The resulting supernatant concentrate was thoroughly mixed with 80% ethanol and allowed to stand overnight. The precipitate was collected by centrifugation the next day.

[0085] (3) Purification of crude polysaccharide from Ginkgo endophyte Bacillus velezensis: The crude polysaccharide from Ginkgo endophyte Bacillus velezensis was washed, and after ethanol volatilization, the polysaccharide precipitate was redissolved with distilled water. The pigment in the crude polysaccharide solution was adsorbed with macroporous resin for 6 hours, dialyzed for 72 hours, and vacuum freeze-dried (-50°C, 48 hours) to obtain the Ginkgo endophyte B. velezensis polysaccharide with significant anti-allergic activity.

[0086] (4) Histamine assay: Refer to Example 11 with additional information. The experiment was divided into four groups: negative control group (N group), experimental group (T group), positive control group (Y group), and model group (M group), with five samples in each group.

[0087] First, the complete culture medium was discarded from the fully adherent cells. 250 μL of complete culture medium containing anti-dinitrobenzene monoclonal mouse antibody was added to the T, M, and Y groups to induce an allergic reaction. The same amount of DMEM complete culture medium was added to the N group. The cells were placed in a cell culture incubator at 37°C and 5% CO2 for overnight culture.

[0088] The next day, the culture medium in each group of 48-well plates was discarded and the cells were washed with PBS buffer. Group T was added with 250 μL of fermentation supernatant and 250 μL of polysaccharide solution (50 μg / mL), groups M and N were added with equal volumes of DMEM complete medium, and group Y was added with 250 μL of complete medium containing ketotifen fumarate (sterilized with a 0.22 μm syringe filter). The cells were then placed in a cell culture incubator at 37°C and 5% CO2 for 1 hour.

[0089] The old culture medium in the well plate was aspirated and washed with PBS buffer. 250 μL of complete culture medium containing DNP-BSA was added to the T, M, and Y groups, and an equal amount of DMEM complete culture medium was added to the N group for culture. After treatment with DNP-BSA for 1 hour, the reaction was terminated by ice bath for 10 minutes, the supernatant was aspirated and centrifuged at 1000 rpm for 3 minutes at 4°C, and the supernatant was retained for later use. The amount of histamine released in the supernatant containing RBL-2H3 cells was detected using a histamine enzyme-linked immunosorbent assay kit (ELISA kit);

[0090] (5) IL-4 assay: Refer to Example 11 with additional information. The experiment was divided into four groups: negative control group (N group), experimental group (T group), positive control group (Y group), and model group (M group), with five samples in each group.

[0091] First, the complete culture medium was discarded from the fully adherent cells. 250 μL of complete culture medium containing anti-dinitrobenzene monoclonal mouse antibody was added to the T, M, and Y groups to induce an allergic reaction. The same amount of DMEM complete culture medium was added to the N group. The cells were placed in a cell culture incubator at 37°C and 5% CO2 for overnight culture.

[0092] The next day, the culture medium in each group of 48-well plates was discarded and the cells were washed with PBS buffer. Group T was treated with 250 μL of fermentation supernatant and 250 μL of polysaccharide solution (50 μg / mL), while groups M and N were treated with equal amounts of DMEM complete medium. Group Y was treated with 250 μL of complete medium containing ketotifen fumarate (sterilized with a 0.22 μm syringe filter). The cells were then placed in a cell culture incubator at 37°C and 5% CO2 for 1 hour.

[0093] The old culture medium in the well plate was aspirated and washed with PBS buffer. 250 μL of complete culture medium containing DNP-BSA was added to the T, M, and Y groups, and an equal amount of DMEM complete culture medium was added to the N group for culture. After treatment with DNP-BSA for 1 hour, the reaction was terminated by ice bath for 10 minutes, the supernatant was aspirated and centrifuged at 1000 rpm for 3 minutes at 4°C, and the supernatant was retained for later use. The IL-4 enzyme-linked immunosorbent assay kit (ELISA kit) was used to detect the amount of IL-4 released in the supernatant containing RBL-2H3 cells;

[0094] (6) TNF-α assay: Refer to Example 11 with additional details. The experiment was divided into four groups: negative control group (N group), experimental group (T group), positive control group (Y group), and model group (M group), with five samples in each group.

[0095] First, the complete culture medium was discarded from the fully adherent cells. 250 μL of complete culture medium containing anti-dinitrobenzene monoclonal mouse antibody was added to the T, M, and Y groups to induce an allergic reaction. The same amount of DMEM complete culture medium was added to the N group. The cells were placed in a cell culture incubator at 37°C and 5% CO2 for overnight culture.

[0096] The next day, the culture medium in each group of 48-well plates was discarded and the cells were washed with PBS buffer. Each of the five T groups was treated with 250 μL of the corresponding fermentation supernatant. Groups M and N were treated with an equal volume of DMEM complete medium. Group Y was treated with 250 μL of complete medium containing ketotifen fumarate (sterilized with a 0.22 μm syringe filter). The cells were then placed in a cell culture incubator at 37°C and 5% CO2 for 1 hour.

[0097] The old culture medium in the well plate was aspirated and the well plate was washed with PBS buffer. 250 μL of complete culture medium containing DNP-BSA was added to the T, M, and Y groups, and an equal amount of DMEM complete culture medium was added to the N group for culture. After treatment with DNP-BSA for 1 hour, the reaction was terminated by incubating on ice for 10 minutes. The supernatant was aspirated and centrifuged at 1000 rpm for 3 minutes at 4°C. The supernatant was retained for later use. TNF-α release in the supernatant containing RBL-2H3 cells was detected using a TNF-α enzyme-linked immunosorbent assay (ELISA) kit.

[0098] Example 22

[0099] During the preparation of the fermentation supernatant, the temperature of the entire bacterial fermentation was 28° C., 200 rpm, fermentation time was 48 h, pH was 7.5, and the remaining operations were the same as those in Example 21.

[0100] Example 23

[0101] During the preparation of the fermentation supernatant, the temperature of the entire bacterial fermentation was 32° C., 120 rpm, fermentation time was 72 h, pH was 7.0, and the remaining operations were the same as those in Example 21.

[0102] Example 24

[0103] During the preparation of the fermentation supernatant, the temperature of the entire bacterial liquid fermentation was 30°C, 120 rpm, fermentation was 24 hours, pH was 8.5, and the remaining operations were the same as those in Example 21.

[0104] Example 25

[0105] During the preparation of the fermentation supernatant, the entire fermentation temperature was 35°C, 200 rpm, and fermentation was carried out for 24 hours. During the preparation of the crude polysaccharide, the polysaccharide was precipitated with 95% ethanol, the polysaccharide pigment was adsorbed using a macroporous resin for 3 hours, and dialyzed for 12 hours. The remaining operations were the same as those in Example 21.

[0106] Example 26

[0107] During the preparation of the fermentation supernatant, the entire fermentation temperature was 27°C, 160 rpm, and fermentation was performed for 36 hours. During the preparation of the crude polysaccharide, the polysaccharide was precipitated with 95% ethanol, the polysaccharide pigment was adsorbed using a macroporous resin for 4 hours, and dialyzed for 24 hours. The remaining operations were the same as those in Example 21.

[0108] Example 27

[0109] During the preparation of the fermentation supernatant, the entire fermentation temperature was 30°C, 150 rpm, and fermentation was performed for 24 hours. During the preparation of the crude polysaccharide, the polysaccharide was precipitated with 80% ethanol, the polysaccharide pigment was adsorbed using a macroporous resin for 4 hours, and dialyzed for 24 hours. The remaining operations were the same as those in Example 21.

[0110] In Examples 1-10 of the present invention, the anti-allergic activity of the ginkgo endophyte B. velezensis and its polysaccharides was verified by using the hyaluronidase inhibition experiment, as shown in Tables 1-1 and 1-2, respectively. In Examples 11-20 of the present invention, the anti-allergic activity of the ginkgo endophyte B. velezensis and its polysaccharides was verified by using the β-HEX inhibition effect in the RBL-2H3 cell degranulation model, as shown in Tables 2-1 and 2-2, respectively. In Examples 21-27 of the present invention, the anti-allergic activity of the ginkgo endophyte B. velezensis and its polysaccharides was verified by using histamine, IL-4 and TNF-α enzyme-linked immunosorbent assay kits, as shown in Tables 3-1 and 3-2, respectively.

[0111] Table 1-1

[0112]

[0113] Table 1-2

[0114]

[0115] Table 2-1

[0116]

[0117] Table 2-2

[0118]

[0119] Table 3-1

[0120]

[0121] Table 3-2

[0122]

[0123] The present invention verifies that the ginkgo endophyte B. velezensis and its polysaccharides have anti-allergic activity through three different methods. According to the data in Tables 1-1, 1-2, 2-1, 2-2, 3-1 and 3-2, all three methods can verify the anti-allergic activity of endophytes and polysaccharides, and are different levels of verification of activity. The hyaluronidase inhibition experiment is a simple and rough preliminary screening, and the construction of a cell degranulation model for the β-HEX release inhibition experiment is more universal.

[0124] The above embodiments are only preferred implementation modes of the present invention and are only used to explain the present invention rather than to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection of the present invention.

Claims

1. A polysaccharide of Ginkgo biloba endophyte Bacillus Velezii, characterized in that: Prepared by the following method: (1) Preparation of crude polysaccharide: Endophytic bacteria of Ginkgo biloba B. velezensis , inoculated into NB culture medium for fermentation, centrifuged, removed the bacteria, and retained the supernatant; concentrated the supernatant, and used alcohol precipitation to extract the endophytic bacteria of Ginkgo biloba B. velezensis crude polysaccharides; (2) Purification of crude polysaccharide from ginkgo endophyte Bacillus Velez subtilis: The crude polysaccharide from ginkgo endophyte Bacillus Velez subtilis was washed, and after ethanol volatilization, the polysaccharide precipitate was redissolved in distilled water, the pigment was removed, dialyzed, and vacuum freeze-dried to obtain the polysaccharide from ginkgo endophyte Bacillus Velez subtilis.

2. The method for preparing the polysaccharide of the ginkgo endophyte Bacillus Velez subtilis according to claim 1, characterized in that: The specific steps are as follows: (1) Preparation of crude polysaccharide: Endophytic bacteria of Ginkgo biloba B. velezensis , inoculated into NB culture medium for fermentation, centrifuged, removed the bacteria, and retained the supernatant; concentrated the supernatant, and used alcohol precipitation to extract the endophytic bacteria of Ginkgo biloba B. velezensis crude polysaccharides; (2) Purification of crude polysaccharide from ginkgo endophyte Bacillus Velez subtilis: The crude polysaccharide from ginkgo endophyte Bacillus Velez subtilis was washed, and after ethanol volatilization, the polysaccharide precipitate was redissolved in distilled water, the pigment was removed, dialyzed, and vacuum freeze-dried to obtain the polysaccharide from ginkgo endophyte Bacillus Velez subtilis.

3. The method for preparing the polysaccharide of the ginkgo endophyte Bacillus Velez subtilis according to claim 2, characterized in that: The specific operation of the alcohol precipitation method for extracting crude polysaccharides from the ginkgo endophyte Bacillus Velez subtilis is as follows: the concentrated supernatant is fully mixed with ethanol, allowed to stand overnight, and the precipitate is collected after centrifugation.

4. The method for preparing the polysaccharide of the ginkgo endophyte Bacillus Velez subtilis according to claim 2, characterized in that: The NB medium was prepared according to the following components: 3 g / L beef extract, 5 g / L peptone, and 2.5 g / L glucose.

5. The method for preparing the polysaccharide of the ginkgo endophyte Bacillus Velez subtilis according to claim 2, characterized in that: The crude polysaccharide of the ginkgo endophyte Bacillus Velez subtilis is separated and purified by column chromatography to obtain the ginkgo endophyte Bacillus Velez subtilis polysaccharide.

6. The method for preparing polysaccharide from the ginkgo endophyte Bacillus Velez subtilis according to claim 2, characterized in that: The fermentation conditions of the ginkgo endophyte Bacillus Velez subtilis are 110-220 rpm, 25-37° C. and 24-72 hours.

7. The method for preparing polysaccharide from the ginkgo endophyte Bacillus Velez subtilis according to claim 2, characterized in that: Ginkgo endophytes B. velezensis The inoculum size for culture in NB medium is 1%.

8. The method for preparing polysaccharide from the ginkgo endophyte Bacillus Velez subtilis according to claim 2, characterized in that: The pigment is removed by adsorption with macroporous resin.

9. The method for preparing polysaccharide from the ginkgo endophyte Bacillus Velez subtilis according to claim 2, characterized in that: The vacuum freeze-drying conditions are: -50°C, 48h.

10. Use of the ginkgo endophyte Bacillus velezensis polysaccharide according to claim 1 in the preparation of anti-allergic drugs, functional foods and animal foods.

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