Bacteroides simplex exopolysaccharide as well as extraction method and application thereof

Abstract

The invention discloses bacteroides simplex exopolysaccharide as well as an extraction method and application thereof, and relates to the technical field of polysaccharide. The bacteroides simplex extracellular polysaccharide is homopolysaccharide, the monosaccharide composition of the bacteroides simplex extracellular polysaccharide is mannose, and the mannose is composed of T-alpha-Manp-(1->,-> 3)-alpha-Manp-(1->,-> 2, 6)-alpha-Manp-(1->,-> 6)-alpha-Manp-(1->,-> 2)-alpha-Manp-(1->. The compound has the effect of promoting body immunity, can activate macrophages, promote proliferation of the macrophages, improve the phagocytic ability of the macrophages, promote release of nitric oxide (NO), promote generation of ROS and activate the body immunity.

Description

Technical Field

[0001] The present invention relates to the technical field of polysaccharides, and in particular to an extracellular polysaccharide of Bacteroides monomorpha and an extraction method and application thereof. Background Art

[0002] The main function of the immune system is to identify and eliminate pathogens to maintain the body's physiological balance and stability, but too strong or too low immunity can cause a variety of adverse immune reactions. Immunodeficiency is a manifestation of low immune function, which is one of the main factors leading to infection and malignant tumors. Currently, the most common method of treating immunodeficiency is the use of immunopotentiators.

[0003] Microbial exopolysaccharides (EPS) refer to long-chain macromolecular carbohydrates secreted outside the cell or closely connected to the cell during the metabolism of microorganisms. They have multiple biological activities such as regulating immunity, improving inflammation, and regulating sugar and lipid metabolism. Compared with plant polysaccharides, exopolysaccharides have the advantages of short growth cycle, easy to control growth conditions, and less affected by geography and climate. They have good development prospects and application value.

[0004] Therefore, it is important to provide an extracellular polysaccharide with good immune enhancement effect. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a Bacteroides monomorpha exopolysaccharide having a good immune enhancement effect.

[0006] The present invention also provides a method for preparing the Bacteroides monomorpha extracellular polysaccharide.

[0007] The present invention also provides a composition comprising the above-mentioned Bacteroides monomorpha exopolysaccharide.

[0008] The present invention also provides application of the Bacteroides monomorpha extracellular polysaccharide or the composition.

[0009] According to the first aspect of the present invention, a Bacteroides monomorpha exopolysaccharide is a homopolysaccharide, and its monosaccharide composition is mannose, which is composed of T-α-Manp-(1→, →3)-α-Manp-(1→, →2,6)-α-Manp-(1→, →6)-α-Manp-(1→, →2)-α-Manp-(1→.

[0010] The Bacteroides monomorpha exopolysaccharide according to the embodiments of the present invention has at least the following beneficial effects:

[0011] The Bacteroides monomorphum exopolysaccharide of the embodiment has the effect of promoting the body's immunity, can activate macrophages and promote their proliferation, improve their phagocytic ability, and at the same time promote the release of nitric oxide (NO), produce ROS, and activate the body's immunity. The Bacteroides monomorphum exopolysaccharide can not only show certain immune activity, but also make up for the shortcomings of the colonization and survival of bacteria in the body when the body directly supplements live bacteria, such as being difficult to control.

[0012] According to some embodiments of the present invention, the total sugar content of the Bacteroides monocytogenes exopolysaccharide is 97.67%±2.38%.

[0013] According to some embodiments of the present invention, the weight average molecular weight of the Bacteroides monomorpha exopolysaccharide is 6.43×10 7 Da.

[0014] According to some embodiments of the present invention, the repeatable primary structural unit of the Bacteroides monomorpha exopolysaccharide is composed of five sugar residues, T-α-Manp-(1→, →3)-α-Manp-(1→, →2,6)-α-Manp-(1→, →6)-α-Manp-(1→, →2)-α-Manp-(1→), and the inferred connection mode is shown in the following formula:

[0015]

[0016] According to some embodiments of the present invention, the molar ratio of T-α-Manp-(1→, →3)-α-Manp-(1→, →2,6)-α-Manp-(1→, →6)-α-Manp-(1→, →2)-α-Manp-(1→) is 28-32:5-8:12-16:1:12-16.

[0017] According to some embodiments of the present invention, the molar ratio of T-α-Manp-(1→, →3)-α-Manp-(1→, →2,6)-α-Manp-(1→, →6)-α-Manp-(1→, →2)-α-Manp-(1→) is 30-31:6-7:14-15:1:14-15.

[0018] According to some embodiments of the present invention, the molar ratio of T-α-Manp-(1→, →3)-α-Manp-(1→, →2,6)-α-Manp-(1→, →6)-α-Manp-(1→, →2)-α-Manp-(1→) is 30.16:6.72:14.23:1:14.56.

[0019] According to the second aspect of the present invention, the method for preparing the above-mentioned Bacteroides monomorpha exopolysaccharide comprises the following steps:

[0020] The Bacteroides monotheca culture supernatant is sequentially subjected to alcohol precipitation, protease hydrolysis, protein removal, first dialysis, anion exchange column chromatography, second dialysis, gel column chromatography, and third dialysis to obtain the Bacteroides monotheca extracellular polysaccharide.

[0021] The preparation method according to the embodiment of the present invention has at least the following beneficial effects:

[0022] The preparation method of the embodiment has simple process, low requirements on technical personnel, and low cost.

[0023] According to some embodiments of the present invention, the preparation method includes pretreatment.

[0024] According to some embodiments of the present invention, the pretreatment includes: isolating the bacteria after expanding the culture of the Bacteroides monocytogenes.

[0025] According to some embodiments of the present invention, the expanded culture comprises: inoculating the Bacteroides monomorpha into a Bacteroides monomorpha culture medium and anaerobic culturing at 37° C. The anaerobic culture time is 48 hours.

[0026] According to some embodiments of the present invention, the culture medium is an MRS broth medium containing L-cysteine ​​(the concentration of L-cysteine ​​is 0.5 g / L). Of course, those skilled in the art can also select other culture media for culturing Bacteroides monomorpha in the art, including but not limited to the above-mentioned MRS broth medium containing L-cysteine.

[0027] According to some embodiments of the present invention, the method for separating bacteria comprises centrifugation.

[0028] According to some embodiments of the present invention, the alcohol precipitation comprises: adding ethanol to the culture supernatant of the Bacteroides monothecae to a final concentration of 70% to 90%, standing at 4° C. overnight, and separating the precipitate. The method of separating the precipitate comprises centrifugation.

[0029] According to some embodiments of the present invention, the protease comprises papain, and the added amount of the papain is 1.8 mg / mL to 2.2 mg / mL.

[0030] According to some embodiments of the present invention, the protein removal method includes removing the protein using a Sevag reagent, wherein the Sevag reagent is prepared by mixing chloroform and n-butanol in a volume ratio of 4:1.

[0031] According to some embodiments of the present invention, the molecular weight cutoff of the dialysis bag used in the first dialysis, the second dialysis and / or the third dialysis is 3000-4000 Da.

[0032] According to some embodiments of the present invention, the anion exchange column is a DEAE cellulose DE-52 anion exchange column.

[0033] According to some embodiments of the present invention, during the anion exchange column chromatography, water (per 20g of anion exchange column filler, the amount used is 376mL volume), 0.5M NaCl aqueous solution (per 20g of anion exchange column filler, the amount used is 376mL volume), 1M NaCl aqueous solution (per 20g of anion exchange column filler, the amount used is 376mL volume) and 2M NaCl aqueous solution (per 20g of anion exchange column filler, the amount used is 376mL volume) are used for gradient elution. The flow rate is 1mL / min.

[0034] According to some embodiments of the present invention, the gel column is a Sephacryl S-500HR gel column. The separation range is 4×10 4 ~2×10 7 .

[0035] According to some embodiments of the present invention, the eluting agent used in the gel column chromatography is water, and the flow rate is 0.5 mL / min.

[0036] A composition according to the third aspect of the present invention comprises the Bacteroides monomorpha exopolysaccharide as described in the first aspect of the present invention. Since the composition adopts all the technical solutions of the Bacteroides monomorpha exopolysaccharide of the above embodiment, it has at least all the beneficial effects brought by the technical solutions of the above embodiment.

[0037] According to some embodiments of the present invention, the composition preferably includes other ingredients having an immunoenhancing effect so as not to affect the effect of the Bacteroides monocytogenes exopolysaccharide.

[0038] According to some embodiments of the present invention, the composition includes pharmaceutically acceptable excipients.

[0039] According to the fourth aspect of the present invention, the use of the Bacteroides monocytogenes exopolysaccharide as described in the first aspect or the composition as described in the third aspect in the preparation of an immunity enhancing product.

[0040] According to some embodiments of the present invention, the product is selected from a reagent, a medicine or a health food.

[0041] According to some embodiments of the present invention, the medicine is a decoction, a pill, a powder, an ointment, an granule, an oral liquid, a capsule or a tablet.

[0042] According to some embodiments of the present invention, the product has at least one of the functions A1) to A4);

[0043] A1) Promote cell proliferation;

[0044] A2) Enhance the phagocytic ability of macrophages;

[0045] A3) Promotes the secretion of NO by macrophages;

[0046] A4) Promotes the production of ROS by macrophages.

[0047] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is the elution curve of the crude extracellular polysaccharide of Example 1 after chromatography on a DEAE cellulose DE-52 anion exchange column;

[0049] Figure 2 is the elution curve of the polysaccharide component of Example 1 after Sephacryl S-500HR gel column chromatography;

[0050] Figure 3 It is the HPGPC peak diagram of EPS-1A;

[0051] Figure 4 This is the monosaccharide composition chromatogram of EPS-1A;

[0052] Figure 5 is the infrared spectrum of EPS-1A;

[0053] Figure 6 is the gas phase total ion chromatogram of methylated EPS-1A;

[0054] Figure 7 For EPS-1A 1 H spectrum;

[0055] Figure 8 For EPS-1A 13 C spectrum;

[0056] Fig. 9 is the HSQC spectrum of EPS-1A;

[0057] Fig.10 COSY spectrum of EPS-1A;

[0058] Fig.11 is the HMBC spectrum of EPS-1A;

[0059] Fig.12 The effect of EPS-1A on the proliferation of RAW 264.7 cells; * indicates P < 0.05 compared with the blank control group, ** indicates P < 0.01 compared with the blank control group;

[0060] Fig.13The effect of EPS-1A on the phagocytic ability of RAW 264.7 cells; * indicates P < 0.05 compared with the blank control group, ** indicates P < 0.01 compared with the blank control group;

[0061] Fig.14 The effect of EPS-1A on NO secretion by RAW 264.7 cells; ** indicates P < 0.01 compared with the blank control group;

[0062] Fig.15 The effect of EPS-1A on ROS production in RAW 264.7 cells; * indicates P < 0.05 compared with the blank control group, ** indicates P < 0.01 compared with the blank control group. DETAILED DESCRIPTION

[0063] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0064] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0065] In the description of the present invention, if there is a description of first, second, third, etc., it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the sequence of the indicated technical features.

[0066] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0067] "And / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0068] Unless otherwise specified, "room temperature" in the present invention means (25±5)°C.

[0069] If not otherwise specified, in the present invention, Man refers to mannose; Rha refers to rhamnose; GlcA refers to glucuronic acid; GalA refers to galacturonic acid; Glc refers to glucose; Gal refers to galactose; Ara refers to arabinose; and Fuc refers to fucose.

[0070] If not otherwise specified, the "complete culture medium" in the present invention is DMEM culture medium containing 10% serum.

[0071] The steps of the phenol-sulfuric acid method are as follows:

[0072] Under light-proof conditions, add 100 μL of 5% redistilled phenol and 500 μL of concentrated sulfuric acid to 200 μL of mannose aqueous solution or sample solution of a series of concentrations, mix well, let stand for 10 min, place in a 40°C water bath for 15 min, then place in an ice water bath for 10 min, take 200 μL of the reaction solution in a 96-well plate, set 2 replica wells for each concentration, and measure the absorbance at 490 nm.

[0073] Example 1

[0074] This example provides a method for extracting exopolysaccharides from Bacteroides monocytogenes, and the steps are as follows:

[0075] (1) Take the Bacteroides monocytogenes JCM5828 frozen at -80℃ and inoculate it into MRS broth medium containing 0.5g / L L-cysteine. After activation for 24 hours, expand the culture with a certain inoculum for two generations, and continue to culture at 37℃ and anaerobic conditions for 60 hours. After the culture is completed, take the fermentation broth and centrifuge it to remove the bacteria and collect the supernatant; boil the supernatant at 100℃ for 30min, centrifuge it, and concentrate it under reduced pressure. Add 95% ethanol to the concentrate while stirring until the final ethanol concentration is 80%, let it stand at 4℃ overnight to precipitate polysaccharides, centrifuge it, and collect the precipitate.

[0076] (2) Dissolve the precipitate collected in step (1) with an appropriate amount of water, add papain (final concentration is 2 mg / mL), react at 60°C with stirring for 1 hour, inactivate the enzyme at 100°C for 15 minutes, centrifuge, collect the supernatant, add 1 / 4 volume of Sevag reagent (in volume ratio, chloroform: n-butanol = 4:1), vortex mix for 10 minutes, centrifuge and take the supernatant, repeat the operation until the protein emulsion layer in the middle disappears. Evaporate the obtained supernatant to dryness, remove the organic solvent, add an appropriate amount of water to dissolve, place in a dialysis bag with a molecular weight cutoff of 3500Da, and dialyze for 48 hours. The dialyzed solution is freeze-dried to obtain crude extracellular polysaccharide.

[0077] (3) Use DEAE cellulose DE-52 anion exchange column chromatography to grade and purify the crude extracellular polysaccharides obtained in step (2). Use an appropriate amount of ultrapure water to dissolve the crude extracellular polysaccharides, centrifuge, and pass the supernatant through a 0.45μm microporous filter membrane to obtain a crude polysaccharide solution. Load the crude polysaccharide solution onto a 20g DEAE cellulose column and gradient elute with ultrapure water, 0.05M NaCl aqueous solution, 0.1M NaCl aqueous solution, and 0.2M NaCl aqueous solution in sequence (flow rate is 1.0mL / min, 8mL is collected in each tube, and 47 tubes are collected for each concentration). The components obtained after gradient elution of each eluent are named: EPS-1, EPS-2, EPS-3, and EPS-4. Use the phenol-sulfuric acid method to track the elution curve (the results are shown in Figure 1 The polysaccharide components were collected according to the elution curve, placed in a dialysis bag with a molecular weight cutoff of 3500 Da for 48 h, and the dialyzed solution was freeze-dried.

[0078] (4) Purification was performed by Sephacryl S-500HR gel column chromatography. The EPS-1 polysaccharide component collected in step (3) was dissolved with an appropriate amount of ultrapure water, loaded onto a Sephacryl S-500HR gel column, and eluted with ultrapure water (flow rate was 0.5 mL / min, 4 mL was collected in each tube, and a total of 43 tubes were collected). The elution curve was tracked by the phenol-sulfuric acid method (the results were shown in FIG. 2 ). Figure 2 According to the elution curve, the eluates collected in tubes 21, 22, 23, 24, and 25 were combined and placed in a dialysis bag with a molecular weight cutoff of 3500Da for 48 hours, and the dialyzed solution was freeze-dried to obtain a Bacteroides monomorpha exopolysaccharide with a single molecular weight (abbreviated as EPS-1A).

[0079] The component EPS-1B obtained by combining the eluates collected in tubes 31, 32, and 33 had a lower absorbance, a correspondingly lower sugar content, and a low purity, so it was not further studied.

[0080] Test Example 1

[0081] In this example, the composition and structure of the Bacteroides monocytogenes exopolysaccharide EPS-1A prepared in Example 1 were analyzed.

[0082] 1. Determination of total sugar content:

[0083] Using mannose dried to constant weight as the reference substance, the phenol-sulfuric acid method was used to determine the absorbance of EPS-1A at 490 nm. With the concentration of mannose reference substance as the horizontal axis and the absorbance value as the vertical axis, a standard curve was drawn and a regression equation was fitted. The absorbance of EPS-1A was substituted into the regression equation to calculate the total sugar content of EPS-1A.

[0084] The regression equation obtained by fitting is y=6.319x+0.104(R2 =0.998). The total sugar content of EPS-1A was calculated to be 97.67%±2.38%.

[0085] 2. Purity and molecular weight determination:

[0086] High performance gel permeation chromatography (HPGPC) was used to determine the molecular weight of EPS-1A. Dextran standards of different molecular weights and EPS-1A were taken separately, and water was added to prepare a 1 mg / mL solution. After passing through a microporous filter membrane, the solution was analyzed by high performance liquid chromatography. A linear equation was established with the retention time of dextran standards of different molecular weights as the horizontal axis and the lg logarithm of the molecular weight (lgMw) as the vertical axis. Substitute the retention time of EPS-1A into the linear equation to calculate the molecular weight of EPS-1A. The chromatographic conditions are as follows:

[0087] Instrument: Shimadzu LC-2050C high performance liquid chromatograph;

[0088] Chromatographic column: PolySep-GFC-P4000 (300×7.8 mm, Phenomenex);

[0089] Mobile phase: ultrapure water;

[0090] Flow rate: 1.0 mL / min;

[0091] Injection volume: 20 μL;

[0092] Detector: Shimadzu ELSD-16 evaporative light scattering detector.

[0093] The HPGPC peak diagram of EPS-1A is as follows Figure 3 shown.

[0094] The linear equation established is y = -0.7985x + 10.665 (R 2 =0.9964). The molecular weight of EPS-1A was calculated to be 6.43×10 7 Da.

[0095] 3. Analysis of monosaccharide composition:

[0096] The monosaccharide composition of EPS-1A was determined by high performance liquid chromatography combined with 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization. Take 50 μL of monosaccharide standard (Man, Rha, GlcA, GalA, Glc, Gal, Ara, Fuc) aqueous solution, add 450 μL of 0.5M PMP methanol solution and 0.3M NaOH aqueous solution, mix well, and place in a 70°C water bath for 30 minutes. After cooling to room temperature, add excess 0.3M HCl solution for neutralization, and then add chloroform for extraction. Repeat several times until the excess PMP is removed, and finally take the water layer solution for analysis by high performance liquid chromatography.

[0097] Take 5 mg of EPS-1A sample, add 2 mL of trifluoroacetic acid (TFA, 3M) and hydrolyze at 130 ° C for 3 hours. After the reaction tube cools for several minutes, add 5 mL of methanol and concentrate to dryness under reduced pressure. Repeat several times to remove excess TFA. Add appropriate amount of ultrapure water to dissolve the hydrolyzate, and use the monosaccharide standard PMP derivatization method to derivatize the sample and analyze it on a high performance liquid chromatograph.

[0098] The results are as follows Figure 4 shown.

[0099] EPS-1A is composed of mannose.

[0100] 4. Infrared spectrum analysis:

[0101] 4000~400cm -1 Within the wavenumber range, EPS-1A was scanned using a Fourier transform infrared spectrometer and its infrared scanning spectrum was recorded.

[0102] The results are as follows Figure 5 shown.

[0103] At 3426cm -1 The broad absorption peak at 2936 cm-1 belongs to the -OH stretching vibration. -1 The broad absorption peak at 1655 cm-1 belongs to CH stretching vibration. -1 The absorption at 1422cm belongs to the bending vibration of OH, indicating the presence of bound water in the polysaccharide. -1 and 1363cm -1 The absorption peak at -CH 2 Deformation vibration. 1139cm -1 、1047cm -1 and 979cm -1 The signal is attributed to the absorption of the pyranose ring of EPS-1A. -1 and 578cm -1 The absorption peaks at 890 cm-1 There is no obvious absorption peak nearby, indicating that EPS-1A does not contain β-configuration glycosidic bonds.

[0104] 5. Analysis of sugar chain connection mode:

[0105] The methylation experiment was used to detect and analyze the glycosidic bond connection mode of EPS-1A. EPS-1A was treated by methylation, acid hydrolysis, acetylation and other steps to obtain sugar alcohol acetate compounds, which were analyzed by GC-MS to obtain the total ion flow chart. By comparing with the corresponding databases and literature, information such as glycosidic bond type, content and ratio of various sugar residues was obtained.

[0106] The results are as follows Figure 6 And as shown in Table 1.

[0107] Table 1. EPS-1A methylation analysis results

[0108]

[0109]

[0110] BEP-1A is mainly composed of five sugar residues: T-α-Manp-(1→, →3)-α-Manp-(1→, →2)-α-Manp-(1→, →6)-α-Manp-(1→, →2,6)-α-Manp-(1→

[0111] 6. Nuclear Magnetic Resonance Analysis:

[0112] Take 30 mg of EPS-1A and repeatedly dissolve it in deuterated water and freeze-dry it, and finally dissolve it in 500 μL of deuterated water and detect it in a Bruker 600M nuclear magnetic resonance spectrometer. 1 H NMR, 13 C NMR, 1 H- 1 H correlation spectroscopy (COSY), heteronuclear single quantum correlation spectroscopy (HSQC) and heteronuclear multiple quantum correlation spectroscopy (HMBC).

[0113] The results are as follows Figures 7 to 11 And as shown in Table 2.

[0114] Table 2. Different sugar residues in EPS-1A 1 H and 13 Assignment table of chemical shifts of C

[0115]

[0116] Test Example 2

[0117] This test example tests the effect of the Bacteroides monocytogenes exopolysaccharide EPS-1A prepared in Example 1 on the proliferation of RAW 264.7 cells. The steps are as follows:

[0118] RAW 264.7 cells in the logarithmic growth phase were seeded in 96-well plates at a cell seeding density of 2×10 4 100 μL of cell suspension was added to each well and cultured overnight. After the cells attached to the wall, the old culture medium was discarded. Complete culture medium was added to the blank control group (CON). The drug-treated groups were given EPS-1A solutions of different concentrations (25, 50, 100, 200 μg / mL) (the solvent was complete culture medium). The positive control group (LPS) was added with LPS solution (1 μg / mL; obtained by diluting PBS containing 1 mg / mL LPS with complete culture medium). 24 hours after drug administration, the culture medium was discarded, 110 μL of complete culture medium (containing 10 μL CCK-8) was added, and after incubation in a cell culture incubator for 1 hour, the absorbance was detected at 450 nm using a full-wavelength microplate reader, and the cell survival rate was calculated.

[0119] The results are as follows Fig.12 shown.

[0120] EPS-1A was able to promote the proliferation of RAW 264.7 cells in the concentration range of 25-200 μg / mL, indicating that EPS-1A had no cytotoxic effect on cells and could promote their growth.

[0121] Test Example 3

[0122] Phagocytic activity is the most critical step for macrophages to resist and eliminate pathogens. This test example uses a neutral red test to test the effect of the Bacteroides monocytogenes exopolysaccharide EPS-1A prepared in Example 1 on the phagocytic activity of RAW 264.7 cells. The steps are as follows:

[0123] RAW 264.7 cells in the logarithmic growth phase were seeded in 96-well plates at a cell seeding density of 2×10 4100 μL of cell suspension was added to each well and cultured overnight. After the cells attached to the wall, the old culture medium was discarded. Complete culture medium was added to the blank control group (CON). Different concentrations (25, 50, 100, 200 μg / mL) of EPS-1A solution (solvent was complete culture medium) were given to the drug-treated groups. LPS solution (1 μg / mL; obtained by diluting PBS containing 1 mg / mL LPS with complete culture medium) was added to the positive control group (LPS). 24 hours after drug administration, the old culture medium was discarded, the cells were washed once with PBS, and 100 μL of 0.075% neutral red solution (solvent was PBS) was added to each well in the dark. After incubation in a 37°C incubator for 45 minutes, the supernatant was discarded, the neutral red on the cell surface that was not phagocytosed was washed with PBS, and alcohol-acid lysis solution (in volume ratio, ethanol: glacial acetic acid = 1:1) was added. After being placed in the dark at room temperature for 2 hours, the absorbance was measured at 540 nm, and the relative phagocytosis rate was calculated.

[0124] The results are as follows Fig.13 shown.

[0125] EPS-1A can enhance the phagocytic ability of RAW264.7 cells, which further indicates that EPS-1A has an immunostimulatory effect on RAW264.7 cells.

[0126] Test Example 4

[0127] This test example tests the effect of the Bacteroides monocytogenes exopolysaccharide EPS-1A prepared in Example 1 on the secretion of NO by RAW 264.7 cells. The steps are as follows:

[0128] RAW 264.7 cells in the logarithmic growth phase were seeded in 96-well plates at a cell seeding density of 2×10 4 100 μL of cell suspension was added to each well and cultured overnight. After the cells adhered to the wall, the old culture medium was discarded. Complete culture medium was added to the blank control group (CON). The drug-treated groups were given EPS-1A solutions of different concentrations (25, 50, 100, 200 μg / mL) (the solvent was complete culture medium). LPS solution (1 μg / mL; obtained by diluting PBS containing 1 mg / mL LPS with complete culture medium) was added to the positive control group (LPS). 24 hours after drug administration, the culture supernatant was collected, and the NO standard curve was drawn according to the instructions of the NO kit, and the content of nitric oxide secreted by each group of cells was detected and calculated.

[0129] The results are as follows Fig.14 shown.

[0130] Compared with the blank control group, the NO secretion of RAW 264.7 cells increased significantly after administration of EPS-1A, indicating that EPS-1A has immunoenhancing activity.

[0131] Test Example 5

[0132] This test example tests the effect of the Bacteroides monocytogenes exopolysaccharide EPS-1A prepared in Example 1 on the production of ROS by RAW 264.7 cells. The steps are as follows:

[0133] RAW 264.7 cells in the logarithmic growth phase were seeded in 96-well plates at a cell seeding density of 2×10 4 100 μL of cell suspension was added to each well and cultured overnight. After the cells attached to the wall, the old culture medium was discarded. Complete culture medium was added to the blank control group (CON), and the drug-treated groups were given EPS-1A solutions of different concentrations (25, 50, 100, 200 μg / mL) (the solvent was complete culture medium). LPS solution (1 μg / mL; obtained by diluting PBS containing 1 mg / mL LPS with complete culture medium) was added to the positive control group (LPS). 24 hours after administration, the culture supernatant was collected and the old culture medium was discarded. The cells were washed once with PBS, and 100 μL of 10 μmol / L DCFH-DA solution was added. The cells were placed in a 37°C incubator and incubated in the dark for 30 minutes. After the incubation, the supernatant was carefully aspirated, and the cells were washed three times with PBS solution to wash away the unbound DCFH-DA on the cell surface. After 100 μL of PBS was added to resuspend the cells, the fluorescence intensity of each well was detected with a multifunctional microplate reader. The excitation wavelength was 500 nm and the emission wavelength was 525 nm when the multifunctional microplate reader was used for detection.

[0134] The results are as follows Fig.15 shown.

[0135] Compared with the blank control group, the production of ROS in RAW 264.7 cells increased significantly after EPS-1A administration, indicating that EPS-1A enhances the cells' resistance to invasion by external pathogens.

[0136] The embodiments of the present invention are described in detail above in conjunction with the embodiments, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A Bacteroides monomorpha exopolysaccharide, characterized in that The Bacteroides monomorpha extracellular polysaccharide is a homopolysaccharide, and its monosaccharide composition is mannose, which is composed of T-α-Manp-(1→, →3)-α-Manp-(1→, →2,6)-α-Manp-(1→, →6)-α-Manp-(1→, →2)-α-Manp-(1→.

2. The Bacteroides monomorpha exopolysaccharide according to claim 1, characterized in that The total sugar content of the Bacteroides monomorpha exopolysaccharide is 97.67% ± 2.38%; And / or, the weight average molecular weight of the Bacteroides monomorpha exopolysaccharide is 6.43×10 7 Da; And / or, the molar ratio of T-α-Manp-(1→, →3)-α-Manp-(1→, →2,6)-α-Manp-(1→, →6)-α-Manp-(1→, →2)-α-Manp-(1→ is 28-32:5-8:12-16:1:12-16.

3. The method for preparing the Bacteroides monomorpha exopolysaccharide according to claim 1 or 2, characterized in that: The following steps are involved: The culture supernatant of Bacteroides monotheca is sequentially subjected to alcohol precipitation, protease hydrolysis, protein removal, first dialysis, anion exchange column chromatography, second dialysis, gel column chromatography, and third dialysis to obtain the Bacteroides monotheca extracellular polysaccharide.

4. The preparation method according to claim 3, characterized in that: The proteases include papain.

5. The preparation method according to claim 3, characterized in that: The protein removal method comprises using Sevag reagent to remove protein.

6. The preparation method according to claim 3, characterized in that: The anion exchange column is a DEAE cellulose DE-52 anion exchange column; And / or, during the anion exchange column chromatography, water, 0.5M NaCl aqueous solution, 1M NaCl aqueous solution and 2M NaCl aqueous solution are sequentially used for gradient elution.

7. The preparation method according to claim 3, characterized in that: The gel column is a Sephacryl S-500HR gel column; And / or, the eluting reagent used in the gel column chromatography is water.

8. A composition, characterized in that The invention comprises the Bacteroides monomorpha exopolysaccharide according to claim 1 or 2.

9. Use of the Bacteroides monomorpha exopolysaccharide according to claim 1 or 2 or the composition according to claim 8 in the preparation of a product that enhances immunity.

10. The use according to claim 9, characterized in that: The product has at least one function from A1) to A4); A1) Promote cell proliferation; A2) Enhance the phagocytic ability of macrophages; A3) Promotes the secretion of NO by macrophages; A4) Promotes the production of ROS by macrophages.

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