CN113480672B

CN113480672B - Exopolysaccharide of bacillus and application thereof

Info

Publication number: CN113480672B
Application number: CN202110836209.1A
Authority: CN
Inventors: 吴正钧; 韩瑨; 夏玮; 王晓花; 游春苹
Original assignee: Bright Dairy and Food Co Ltd
Current assignee: Bright Dairy and Food Co Ltd
Priority date: 2021-07-23
Filing date: 2021-07-23
Publication date: 2023-03-17
Anticipated expiration: 2041-07-23
Also published as: CN113480672A

Abstract

The invention discloses an extracellular polysaccharide of Paenibacillus and application thereof, wherein the extracellular polysaccharide is produced by inoculating a Paenibacillus sp strain with the preservation number of CGMCC NO.8333 to wheat bran for fermentation; the average weight molecular weight of the exopolysaccharide is 300,800-451,200 daltons, and the exopolysaccharide is an acidic heteropolysaccharide consisting of glucuronic acid, glucose and fucose in a molar ratio of 1.55-1.60; the main chain of the exopolysaccharide is composed of 1, 3-linked glucose residues, 1, 3-linked fucose residues, 1,3, 4-linked fucose residues and 1, 4-linked glucuronic acid residues, a branch point is positioned at the O-4 position of the 1,3, 4-linked fucose residues, a branch chain is composed of terminal linked glucuronic acid residues and is composed of a repeating structural unit shown in a formula I; in addition, the extracellular polysaccharide has a certain immunoregulation effect, and has good application prospects in food, medicine and related fields.

Description

Exopolysaccharide of bacillus and application thereof

Technical Field

The invention belongs to the field of microbial fermentation, and particularly relates to exopolysaccharide of bacillus sp and application thereof.

Background

Since the 60 s of the 20 th century, polysaccharide is considered as a broad-spectrum nonspecific immunostimulant, which can enhance the cellular immunity and humoral immunity of host cells, such as activating macrophages, T cells, B cells, NK cells and the like, activating complement, inducing interferon generation and the like, has the function of activating the nonspecific defense function of human bodies, and has good curative effects on antivirus, antitumor, antiradiation and the like.

According to the source, polysaccharides can be divided into animal polysaccharides, plant polysaccharides and microbial polysaccharides, wherein the microbial polysaccharides are produced by microorganisms metabolizing carbohydrates, usually, the metabolizing base materials are artificially synthesized, such as MRS, TYC and the like, and relatively few researches on the synthesis of microbial polysaccharides using natural base materials, especially agricultural processing byproducts (such as wheat bran and the like) are conducted.

On the other hand, in the field, the types of the microbial exopolysaccharides which are mature and can be popularized and applied in practice are not many, and the needs of researching new exopolysaccharides exist in production and scientific research so as to enrich the types of the microbial exopolysaccharides with excellent performance and wide application.

Disclosure of Invention

The invention aims to provide an extracellular polysaccharide of Paenibacillus and application thereof, wherein the extracellular polysaccharide has various biological effects and has good application prospects in the fields of food, medicine and the related fields. The invention mainly solves the technical problems through the following technical scheme.

The invention provides an extracellular polysaccharide of Paenibacillus, which is produced by inoculating a Paenibacillus sp strain with the preservation number of CGMCC NO.8333 to wheat bran for fermentation. The strain is preserved in China general microbiological culture Collection center (CGMCC) in 2013, 10 months and 14 days, and the preservation address is as follows: west road No. 1, north chen, chaoyang district, beijing, zip code: 100101.

Preferably, the average weight molecular weight of the exopolysaccharide of the paenibacillus is 300,800-451,200 daltons.

Preferably, the exopolysaccharide is an acidic heteropolysaccharide consisting of glucuronic acid, glucose and fucose in a molar ratio of 1.55-1.60.

Preferably, the exopolysaccharide has a backbone composed of 1, 3-linked glucose residues, 1, 3-linked fucose residues, 1,3, 4-linked fucose residues and 1, 4-linked glucuronic acid residues, a branch point located at the O-4 position of the 1,3, 4-linked fucose residue, and a branch chain composed of terminally linked glucuronic acid residues.

Preferably, the exopolysaccharide consists of a repeating structural unit represented by formula I.

The invention also provides application of the extracellular polysaccharide of the bacillus in the fields of food and medicine.

The invention also provides application of the exopolysaccharide of the Paenibacillus in preparing an immunomodulatory drug.

Preferably, the paenibacillus exopolysaccharide concentration is less than 100. Mu.g/mL.

Compared with the prior art, the invention has the positive improvement effects that:

the technical scheme of the invention discloses the exopolysaccharide of the paenibacillus with a definite and unique structure for the first time, and the exopolysaccharide is safe and has various biological effects, such as enhancing the phagocytic function of macrophages and promoting the macrophages to release cytokines, so that the exopolysaccharide has obvious technical advantages as a high molecular substance generated by microorganisms, and has good application prospects in the fields of food, medicine and related fields.

Drawings

FIG. 1 is gel chromatography elution curve diagram of crude product of CGMCC No.8333 exopolysaccharide

FIG. 2 is a high performance gel filtration chromatogram of CGMCC No.8333 exopolysaccharide

FIG. 3 is 1H-NMR chromatogram of CGMCC No.8333 exopolysaccharide

FIG. 4 is a 13C-NMR chromatogram of CGMCC No.8333 exopolysaccharide

FIG. 5 is H-H COSY chromatogram of CGMCC No.8333 exopolysaccharide

FIG. 6 is HSQC chromatogram of CGMCC No.8333 exopolysaccharide

FIG. 7 is TOCSY chromatogram of CGMCC No.8333 exopolysaccharide

FIG. 8 is HMBC chromatogram of CGMCC No.8333 exopolysaccharide 1

FIG. 9 shows HMBC chromatogram of CGMCC No.8333 exopolysaccharide

FIG. 10 shows NOESY chromatogram of CGMCC No.8333 exopolysaccharide

FIG. 11 shows the effect of CGMCC No.8333 exopolysaccharide on RAW264.7 cells

FIG. 12 shows the effect of CGMCC No.8333 exopolysaccharide on phagocytic ability of RAW264.7 cells

FIG. 13 shows the effect of CGMCC No.8333 exopolysaccharide on NO release of RAW264.7 cells

FIG. 14 shows the effect of CGMCC No.8333 exopolysaccharide on TNF-alpha secretion of RAW264.7 cells

FIG. 15 shows the effect of CGMCC No.8333 exopolysaccharide on IL-1 beta secretion of RAW264.7 cells

FIG. 16 shows the effect of CGMCC No.8333 exopolysaccharide on IL-6 secretion of RAW264.7 cells

Detailed Description

The exopolysaccharide of the Paenibacillus is produced by inoculating a Paenibacillus (Paenibacillus sp.) strain with the collection number of CGMCC NO.8333 to wheat bran for fermentation.

In the invention, the wheat bran is prepared manually, the prepared wheat bran for fermentation comprises the wheat bran and water, and the mass percentage of the wheat bran is 1-5%, preferably 2-4%, and more preferably 3%. The preparation method of the wheat bran for fermentation can comprise the following steps: adding wheat bran and distilled water into a triangular flask, uniformly mixing, heating and boiling, sterilizing at 95-125 ℃ for 5-25 min, and cooling to obtain the wheat bran-distilled water.

The average weight molecular weight of the exopolysaccharide of the paenibacillus is 300,800-451,200 daltons, and the exopolysaccharide is an acidic heteropolysaccharide consisting of glucuronic acid, glucose and fucose in a molar ratio of 1.55-1.60. The main chain of the exopolysaccharide is composed of 1, 3-linked glucose residues, 1, 3-linked fucose residues, 1,3, 4-linked fucose residues and 1, 4-linked glucuronic acid residues, a branch point is positioned at the O-4 position of the 1,3, 4-linked fucose residues, a branch chain is composed of terminal linked glucuronic acid residues, and the exopolysaccharide is composed of a repeating structural unit shown in a formula I.

The invention also provides application of the extracellular polysaccharide of the paenibacillus in the fields of food and medicine. Researches show that the microbial exopolysaccharide has biological activity, such as immunological activity, anti-tumor, antioxidant, anti-tumor, intestinal flora regulation, etc., and can be applied in the fields of food, medicine, etc. Immunomodulation is one of the most important biological activities of polysaccharides and is a non-specific class of immunomodulators. The immune system is a protection system for the body to resist the invasion of pathogenic microorganisms and eliminate variant cells in the body and mainly comprises immune organs, immune cells and immune molecules. In a normal organism, the immune system maintains a dynamic balance, so that the organism is kept stable. However, when the immune system is disturbed, a series of diseases are induced, and the normal physiological functions of the body are impaired. Researches show that the immunoregulation effect of the exopolysaccharide on organisms mainly comprises the steps of activating macrophages, activating immune cells such as B cells and T cells, entering the cells through cell surface receptor recognition, improving the phagocytic and secretory capacity of the cells, activating complement, activating a reticuloendothelial system and promoting the development of immune organs. In the invention, the exopolysaccharide synthesized by the paenibacillus bovis CGMCC NO.8333 has NO cytotoxicity when the concentration is lower than 100 mug/mL, can activate RAW264.7 cells and enhance the phagocytosis capability of the cells, improves the expression of NO, TNF-alpha, IL-1 beta and IL-6 cytokines by activating the RAW264.7 cells, and has good immunoregulation effect.

The following examples further illustrate the above-described embodiments without, therefore, limiting the invention to the scope of the examples described. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions.

In the examples below, all the starting materials are commercially available and meet the relevant national standards.

Example 1 preparation of Paenibacillus exopolysaccharides

1. Materials and methods

(a) Preparation of seeds (fermentation strain): dissolving the freeze-dried powder of the paenibacillus CGMCC No.8333 by using a small amount of sterile distilled water, marking a ring by using an inoculating ring on a solid culture medium containing 1% (w/w) of skim milk and 1.5% (w/v) of agar (purchased from Chinese pharmacy group, china), carrying out aerobic culture at 30 ℃ for 48h, taking out, selecting a single colony by using the inoculating ring, inoculating into 10mL of liquid culture medium containing 10% (w/w) of skim milk, and carrying out shaking culture at 30 ℃ and 180rpm for 24h to obtain seeds for fermentation.

(b) Preparing a wheat bran culture medium: adding 1.8g wheat bran (commercially available) and 58.2mL distilled water into a 250mL triangular flask, mixing, heating to boil, sterilizing at 110 deg.C for 15min, and cooling to room temperature to obtain the required wheat bran culture medium.

2. Preparation of paenibacillus extracellular polysaccharide crude product

The paenibacillus CGMCC No.8333 seeds are aseptically inoculated into the wheat bran culture medium according to the inoculum size of 3 percent (v/v, the volume percent of the seed liquid accounts for the fermentation liquid, the same is applied below), and the fermentation liquid is obtained after shaking culture at 26 ℃ and 180rpm for 48 hours. Centrifuging the fermentation liquid at 15,000rpm for 10min to obtain the supernatant, heating and boiling for 10min, cooling to room temperature, slowly adding anhydrous ethanol (purchased from the national drug group, china) with three times of volume, refrigerating and standing for 24h, taking out, centrifuging at 15,000rpm for 10min to obtain the precipitate, completely re-dissolving with a small amount of distilled water, and freeze-drying to obtain the crude product A of the paenibacillus extracellular polysaccharide.

3. Preparation of Paenibacillus exopolysaccharide (single component)

250mg of the crude Paenibacillus exopolysaccharide A is dissolved in 25mL of Tris-HCl buffer solution (50 mM, pH7.6) (purchased from the national drug group, china), loaded on a DEAE-Sepharose Fast Flow (purchased from GE, USA) chromatographic column which is pre-loaded and well balanced by a constant Flow pump, and is subjected to isocratic elution by sequentially using Tris-HCl buffer solution (50 mM, pH7.6), buffer solution containing 0.2mol/L NaCl and 0.4mol/L NaCl, wherein the elution speed is 1.5mL/min, and eluent (8 mL per tube) is collected by an automatic part collector (purchased from Shanghai Qingpu Huxi apparatus factory, china).

Detecting the polysaccharide content in each tube of eluent by a sulfuric acid-phenol method, merging and collecting a third component peak (185-205 tubes in figure 1, the abscissa is the number of the tube in figure 1, and the ordinate is the light absorption value), putting into a dialysis bag with the molecular weight cutoff of 14,000 daltons, dialyzing with deionized water for 72 hours to remove buffer salt, changing water once every 12 hours, and freeze-drying to obtain the polysaccharide with a single component, namely the paenibacillus extracellular polysaccharide, namely EPS-1.

EXAMPLE 2 preparation of Paenibacillus exopolysaccharides

1. Materials and methods

(a) Preparation of seeds (fermentation strain): the same as in example 1.

(b) Preparing a wheat bran culture medium: and (3) adding 0.6g of wheat bran and 59.4mL of distilled water into a 250mL triangular flask, uniformly mixing, heating and boiling, placing at 125 ℃ for sterilization for 5min, and cooling to room temperature to obtain the required wheat bran culture medium.

2. Preparation of paenibacillus extracellular polysaccharide crude product

Aseptically inoculating paenibacillus CGMCC No.8333 seeds with the inoculation amount of 5 percent into the wheat bran culture medium, and performing shaking culture at 37 ℃ and 300rpm for 12h to obtain fermentation liquor. Centrifuging the fermentation liquor at 15,000rpm for 10min, taking the supernatant, heating and boiling for 10min, cooling to room temperature, slowly adding anhydrous ethanol with three times of volume, refrigerating and standing for 24h, taking out the product, centrifuging at 15,000rpm for 10min, taking the precipitate, completely re-dissolving the precipitate with a small amount of distilled water, and freeze-drying to obtain a paenibacillus extracellular polysaccharide crude product B.

3. Preparation of Paenibacillus exopolysaccharide (single component)

The crude product B of Paenibacillus exopolysaccharide is separated by the method described in reference example 1, and the exopolysaccharide EPS-2 with a single component of Paenibacillus is obtained.

Example 3 preparation of Paenibacillus exopolysaccharides

1. Materials and methods

(a) Preparation of seeds (fermentation strain): the same as in example 1.

(b) Preparing a wheat bran culture medium: adding 3g of wheat bran and 57mL of distilled water into a 250mL triangular flask, uniformly mixing, heating and boiling, placing at 95 ℃ for sterilization for 25min, and cooling to room temperature to obtain the required wheat bran culture medium.

2. Preparation of paenibacillus extracellular polysaccharide crude product

Aseptically inoculating paenibacillus CGMCC No.8333 seeds with the inoculum size of 1% in the wheat bran culture medium, and performing shaking culture at 20 ℃ and 100rpm for 72h to obtain a fermentation liquid. Centrifuging the fermentation liquor at 15,000rpm for 10min, taking the supernatant, heating and boiling for 10min, cooling to room temperature, slowly adding anhydrous ethanol with three times of volume, refrigerating and standing for 24h, taking out the product, centrifuging at 15,000rpm for 10min, taking the precipitate, completely re-dissolving the precipitate with a small amount of distilled water, and freeze-drying to obtain a paenibacillus extracellular polysaccharide crude product C.

3. Preparation of Paenibacillus exopolysaccharide (single component)

The crude product C of Paenibacillus exopolysaccharide is separated by the method described in reference example 1, and the exopolysaccharide EPS-3 with a single component of Paenibacillus is obtained.

Example 4 Paenibacillus exopolysaccharide unicity validation

Weighing 5mg of EPS-1, EPS-2 and EPS-3 samples, respectively dissolving in 5mL of ultrapure water to prepare 1mg/mL polysaccharide solution, filtering with a 0.45 μm filter membrane, and introducing into Agilent 1100 high performance liquid chromatograph (from Agilent, USA) for analysis under the following chromatographic conditions: RID detector, column TSK-Gel G6000PWXL (available from Tosoh Biotech Co., ltd., japan) as mobile phase of 0.1mol/L NaNO ₃ And (3) solution. The flow rate was set at 0.6mL/min, the column temperature was set at 35 ℃ and the amount of sample was set at 20. Mu.L, and the purity (unity) was judged from the peak pattern.

Wherein, the high-efficiency gel filtration chromatogram of the EPS-1 sample is shown in figure 2 (the chromatograms of EPS-2 and EPS-3 are similar), a symmetrical chromatographic peak is presented in the retention time of 14-15min, the chromatographic peak in 21min is a mobile phase peak, and the results show that the EPS-1, EPS-2 and EPS-3 samples are all homogeneous polysaccharide components.

Example 5 determination of extracellular polysaccharide molecular weight of Paenibacillus

Dextran of different molecular weights was used as standard: STD-1 (Mw =5,000), STD-2 (Mw =12,000), STD-3 (Mw =50,000), STD-4 (Mw =270,000), STD-5 (Mw =670,000). Respectively dissolving the series of standard polysaccharides and EPS-1, EPS-2 and EPS-3 samples in a mobile phase (0.1 mol/L NaNO) ₃ Solution) to give a 1mg/mL solution, respectively 0.45. The filtration through a filter membrane of μm was carried out and analyzed by Agilent 1100 HPLC under the same chromatographic conditions as in example 4. And drawing a standard curve by taking the logarithm LgMw of the molecular weight of the standard polysaccharide as an abscissa and the retention time tR as an ordinate to obtain a linear regression equation of the logarithm of the molecular weight and the retention time. The molecular weight of the sample can be calculated from the regression equation and the results are shown in the following table.

TABLE 1 Paenibacillus extracellular polysaccharide molecular weight determination

And (4) conclusion: the average weight molecular weight of the paenibacillus exopolysaccharide is 300,800-451,200 daltons.

Example 6 determination of monosaccharide composition of Paenibacillus exopolysaccharides

(1) Hydrolysis of polysaccharide samples

2.0mg of EPS-1, EPS-2 and EPS-3 samples are respectively placed in an ampoule bottle, 3mL of 2mol/L trifluoroacetic acid (TFA) is added, and hydrolysis is carried out for 5h at 110 ℃ after sealing. And cooling the hydrolysate, carrying out reduced pressure rotary evaporation at 45 ℃ until the hydrolysate is dried, adding methanol to continue rotary evaporation, and repeating the rotary evaporation for several times to remove excessive TFA to obtain the EPS hydrolysate.

(2) Derivatization of hydrolyzed samples and mixed monosaccharide standards

The EPS hydrolysate was dissolved in 1mL of water to obtain a sample solution to be derivatized. Taking 1mL of the sample solution or a mixed standard solution of 9 monosaccharides (0.5 mg/mL, rhamnose, fucose, glucuronic acid, galactose, glucose, mannose, galacturonic acid, arabinose and xylose), adding 1mL of 0.6 mol/L NaOH solution and 1mL of 0.5mol/L PMP methanol solution, uniformly mixing to completely dissolve a solid product, and placing in a 70 ℃ oven for reaction for 100min. Cooling to room temperature, adding 0.3 mol/L HCl dropwise to adjust to neutrality, extracting with chloroform for 3 times, collecting water phase, filtering the water phase with 0.45 μm filter membrane, and analyzing by HPLC sample injection.

(3) Chromatographic conditions

Agilent 1260 high performance liquid chromatograph (available from Agilent, USA) equipped with DAD detector and Agilent Eclipse XDB-C18 column (available from Agilent, USA). The column temperature was set at 30 ℃ and the sample volume was 20. Mu.L, mobile phase acetonitrile: 0.1mol/L phosphate buffer (pH 6.8) =16 (V/V), and the detection wavelength was 250nm.

(4) Data analysis

The monosaccharide composition of the polysaccharide samples was determined with reference to retention times according to different monosaccharide standards (purchased from sigma, usa). And determining the molar ratio of the monosaccharides in the polysaccharide sample according to the peak area ratio of the monosaccharide composition. The results are shown in Table 2.

TABLE 2 molar ratio of monosaccharides in Paenibacillus exopolysaccharides

	Glucuronic acid	Glucose	Fucose sugar
				EPS-1 (in terms of molar ratio)	1.58	1	1.66
EPS-2 (in terms of molar ratio)	1.55	1	1.63
				EPS-3 (in terms of molar ratio)	1.60	1	1.72

And (4) conclusion: the exopolysaccharide of the paenibacillus is an acidic heteropolysaccharide consisting of glucuronic acid, glucose and fucose in a molar ratio of 1.55-1.60.

Example 7 determination of mode of Paenibacillus extracellular polysaccharide ligation

(1) Methylation analysis

Uronic acid reduction: adopts carbodiimide-sodium borohydride method (EDC-NaBH) ₄ ) Reducing uronic acid, the reaction is divided into two stages, the first stage: a50 mg sample of EPS-1 was dissolved in 6mL of ultrapure water and stirred until completely dissolved. 500mg of EDC is added into the solution twice at intervals of 30min, and the pH is controlled to be between 4.5 and 4.8 by 0.1mol/L HCl, and the whole reaction process needs 3 hours. And a second stage: 8mL of 2mol/L NaBH is dripped into the system within 40min ₄ And controlling the pH value of the system to be about 7.0, continuing the reaction for 1h after the dropwise addition is finished, and putting the product into a dialysis bag (with the molecular weight cutoff of 3500 Da) for running water dialysis for 24h. The above steps are repeated 4 times, and PMP-HPLC is used to detect complete reduction of uronic acid.

Methylation: taking 20mg of the dried polysaccharide sample with completely reduced uronic acid, placing the dried polysaccharide sample in a 10mL reaction bottle, quickly adding 3mL of anhydrous dimethyl sulfoxide at room temperature, sealing, magnetically stirring for 30min, dissolving the sample with ultrasonic assistance, then quickly adding 50mg of dried NaOH powder, sealing and stirring until most of NaOH is dissolved, carrying out ice bath for 5min, slowly dropwise adding 1mL of methyl iodide within 30min, stirring at room temperature in a dark place, continuing to react for 30min, and finally adding 1mL of ultrapure water to terminate the reaction. Putting the product in a dialysis bag for running water dialysis for 24h, and repeating the steps after rotary evaporation to dryness. After methylation for many times, taking a small amount of sample for infrared spectrum detection, and if the O-H stretching vibration absorption peak of the polysaccharide sample at 3400-3000cm < -1 > disappears, indicating that the polysaccharide sample has been completely methylated; if the sample is not completely methylated, the reaction is continued until the sample is completely methylated.

Hydrolysis and acetylation: will be provided with2mg of a completely methylated EPS-1 sample is placed in an ampoule, added with 3mL of 2mol/L TFA and sealed, and after 4h of closed hydrolysis at 110 ℃, methanol is added and rotary evaporated under reduced pressure several times to completely remove TFA. After spin-drying, 3mL of ultrapure water was added for dissolution, and 50mg of NaBH was added ₄ The reaction was magnetically stirred at room temperature for 3h. After the reaction, acetic acid was added until the solution was weakly acidic (pH = 5), methanol was added and the solution was rotary evaporated to dryness, and this was repeated several times to sufficiently remove boric acid. The obtained solid was dried in an oven at 100 ℃ for 10min, 3mL of acetic anhydride was added, the reaction was carried out at 100 ℃ for 100min, and after the reaction was completed, toluene (3 mL) was added for several times to co-evaporate and remove excess acetic anhydride. The product was dissolved in chloroform (5 mL), extracted 3 times with ultrapure water (5 mL. Times.3), the chloroform layer was recovered, anhydrous sodium sulfate powder was added to remove water, the mixture was allowed to stand for 30min, evaporated to dryness under reduced pressure, dissolved in 0.5mL of chloroform, filtered through a 0.22 μm organic filter and analyzed by GC-MS.

GC-MS conditions: the instrument model is as follows: agilent 7820A/5977GC-MS (available from Agilent, USA); the type of the chromatographic column: HP-5 capillary column; temperature programming: the initial temperature is 120 ℃, the temperature is raised to 250 ℃ after the temperature is maintained for 2min, the temperature raising rate is 5 ℃/min, and the temperature is maintained for 10min; the sample inlet adopts a flow splitting mode, and the flow splitting ratio is 3; the sample size was 1. Mu.L. The ion source of the mass spectrometer is an EI source, the voltage of the ion source is 70eV, and the temperature is 180 ℃.

And (3) data analysis: by comparing the EI-MS pattern obtained by GC-MS with the standard PMAA pattern and combining the results of monosaccharide composition, it can be determined that each sugar residue of EPS-1 after reduction is linked in the manner of 1, 3-linked fucose residue, 1,3, 4-linked fucose residue, 1, 3-linked glucose residue and 1, 4-linked glucuronic acid residue, terminal-linked glucuronic acid residue.

(2) Nuclear magnetic resonance spectroscopy

20mg of EPS-1 sample was sampled and used in a volume of 0.5mL D ₂ O dissolved, transferred to a clean nuclear magnetic tube and chromatographed on a 600MHz nuclear magnetic resonance apparatus (from Bruker, switzerland).

For is to ¹ H-NMR (FIG. 3), ¹³ After a comprehensive chromatographic analysis of C-NMR (FIG. 4), H-H COSY (FIG. 5), HSQC (FIG. 6), TOCSY (FIG. 7), HMBC (FIG. 8, FIG. 9) and NOESY (FIG. 10), it was found that the EPS-1 backbone is composed of 1, 3-linked glucansA glucose residue, a 1, 3-linked fucose residue, a 1,3, 4-linked fucose residue, and a 1, 4-linked glucuronic acid residue, the branch point being at the O-4 position of the 1,3, 4-linked fucose residue, the branch being composed of terminally linked glucuronic acid residues.

In conclusion, the paenibacillus exopolysaccharide is composed of the repeated structural unit shown in the formula I.

Effect example 1 Effect of Paenibacillus exopolysaccharides on cell growth

The concentration of RAW264.7 cells was adjusted to 1X 10 ⁴ Per mL, inoculated in 96-well cell culture plates, 200. Mu.L/well, 37 ℃,5% CO ₂ Culturing is carried out under the conditions. After the cells are attached to the wall, the culture medium is discarded, EPS-1 solutions (0, 6.25, 12.5, 25, 50, 100, 200, 400, 600. Mu.g/mL) with different concentrations are added into each well, LPS (1. Mu.g/mL) is used as a positive control, 200. Mu.L/well, and 5 parallel wells are arranged with each concentration. After 24 hours of incubation, the well plate was aspirated, 30. Mu.L of sterilized MTT solution (5 mg/mL) was added to each well, the well plate was aspirated after 4 hours of incubation, 200. Mu.L of DMSO was added to each well, the purple crystals in the well plate were sufficiently dissolved by shaking, and then the absorbance (OD) was measured at 492nm using a microplate reader (purchased from Molecular Devices, USA) and the survival rate of the cells under the administration conditions was calculated by the following equation, as shown in FIG. 11.

Survival% = OD _{Experimental group} /OD _{Control group} ×100％

When the concentration of EPS-1 is 6.25, 12.5, 25, 50 and 100 mu g/mL respectively, the survival rate of RAW264.7 cells is higher than that of a blank control group, namely 120.92 +/-1.73%, 120.637 +/-3.80%, 121.15 +/-2.37%, 116.05 +/-3.24% and 109.70 +/-3.16% respectively; when the administration concentration is increased to 200 mug/mL, the survival rate of RAW264.7 cells is reduced and is lower than that of a blank control group; when the administration concentration reached the maximum concentration, i.e., 600. Mu.g/mL, the viability of RAW264.7 cells was significantly lower than that of the blank control group, only 62.74. + -. 2.94%. The experimental result shows that the EPS-1 has no cytotoxicity to RAW264.7 cells within the range of 6.25-100 mu g/mL.

And (4) conclusion: when the concentration is lower than 100 mu g/mL, the Paenibacillus exopolysaccharide is safe to cells.

Effect example 2 Effect of Paenibacillus extracellular polysaccharide on phagocytic Capacity of RAW264.7 cells

The concentration of RAW264.7 cells was adjusted to 1X 10 ⁴ seed/mL, inoculation in 96 well cell culture plates, 200 μ L/well, 37 ℃,5% ₂ The culture was performed under conditions such that the culture medium was discarded after the cells were attached to the wall, and 100. Mu.L each of EPS-1 solution (0, 6.25, 12.5, 25, 50, 100. Mu.g/mL) and LPS (1. Mu.g/mL) was added to each well at different concentrations, and 5 parallel wells were provided for each concentration. After 24h of incubation, 0.08% freshly prepared neutral red solution was added, the incubator was incubated for 1h, the solution was aspirated after removal, rinsed 2 times with PBS, lysed by addition of lysis buffer (glacial acetic acid: ethanol = 1) for 1h, absorbance was measured at 492nm with a microplate reader, and the phagocytosis rate of each group was calculated by the following formula, the results of which are shown in fig. 12.

Cell phagocytosis% = OD _{Experimental group} /OD _{Control group} ×100％

When the concentration of EPS-1 is 6.25, 12.5, 25, 50 and 100 mug/mL, respectively, the phagocytosis rate of each administration treatment group is 105.63 +/-2.17%, 109.11 +/-2.43%, 109.91 +/-2.75%, 119.42 +/-1.84% and 126.25 +/-2.77%, respectively, which are higher than those of the blank control group and are significantly different. In the above concentration range, the phagocytic activity of RAW264.7 cells gradually increased with increasing administration concentration, and the phagocytic ratio of cells at an administration concentration of 100 μ g/mL approached that of the positive control group (129.03 ± 3.13%).

And (4) conclusion: the paenibacillus extracellular polysaccharide is between 6.25 and 100 mu g/mL, and can activate RAW264.7 cells and enhance the phagocytosis capacity of the cells.

Effect example 3 Effect of Paenibacillus extracellular polysaccharide on cytokine Release from RAW264.7 cells

The concentration of RAW264.7 cells was adjusted to 5X 10 ⁵ mL, inoculation in 96 well cell culture plates, 200. Mu.L/well, 37 ℃,5% ₂ Culturing is carried out under the conditions. To be thinAfter cell attachment, the culture medium was discarded, and 100. Mu.L each of EPS-1 solution (0, 6.25, 12.5, 25, 50, 100, 200, 400, 600. Mu.g/mL) and LPS (1. Mu.g/mL) at different concentrations was added to each well for 24 hours, and 2 parallel wells were set at each concentration. 50 μ L of cell supernatant was collected from each well and the corresponding cytokines were detected using NO kit, TNF-. Alpha.ELISA kit, IL-1. Beta. ELISA kit and IL-6ELISA kit (purchased from ELISA, USA), respectively.

The results are shown in FIG. 13 (NO), FIG. 14 (TNF-alpha), FIG. 15 (IL-1 beta) and FIG. 16 (IL-6), and the secretion levels of NO, TNF-alpha, IL-1 beta and IL-6 of RAW264.7 increase dose-dependently after adding different concentrations of EPS-1 for a certain period of time, which indicates that the paenibacillus exopolysaccharide can activate RAW264.7 cells, increase the expression of the above cytokines and promote the immunomodulatory effect.

The extracellular polysaccharide of Paenibacillus and its application provided by the present invention are described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims

1. Exopolysaccharide of paenibacillus bovis, which is characterized by comprising the paenibacillus bovis (with the preservation number of CGMCC NO. 8333)Paenibacillus bovis) The strain is inoculated to wheat bran for fermentation; the extracellular polysaccharide consists of a repeating structural unit shown as a formula I,

（I）

the average weight molecular weight of the extracellular polysaccharide is 300,800 to 451,200 daltons;

the exopolysaccharide is an acidic heteropolysaccharide consisting of glucuronic acid, glucose and fucose in a molar ratio of 1.55 to 1.60;

the main chain of the exopolysaccharide is composed of 1, 3-linked glucose residues, 1, 3-linked fucose residues, 1,3, 4-linked fucose residues and 1, 4-linked glucuronic acid residues, the branch point is positioned at the O-4 position of the 1,3, 4-linked fucose residues, and the branch chain is composed of terminally linked glucuronic acid residues.

2. The application of the exopolysaccharide of the Paenibacillus bovis in preparing the immune regulation medicine, wherein the concentration of the exopolysaccharide of the Paenibacillus bovis is between 6.25 and 100 mu g/mL.