CN114134188B

CN114134188B - Method for synthesizing extracellular polysaccharide by fermenting wheat bran

Info

Publication number: CN114134188B
Application number: CN202110837218.2A
Authority: CN
Inventors: 韩瑨; 刘振民; 吴正钧; 郭诚笑; 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: CN114134188A

Abstract

The invention discloses a method for synthesizing extracellular polysaccharide by fermenting wheat bran, which comprises the following steps: (1) Inoculating a Paenibacillus sp (Paenibacillus sp.) seed with the preservation number of CGMCC No.8333 to a wheat bran culture medium to ferment to obtain fermentation liquor; (2) Centrifuging the fermentation liquor, taking the supernatant, boiling, cooling, precipitating with ethanol overnight, centrifuging again, taking the precipitate, redissolving, and freeze-drying to obtain extracellular polysaccharide; the conversion rate of the extracellular polysaccharide prepared by the technical scheme is more than 18 percent; the average weight molecular weight of the macromolecular component of the extracellular polysaccharide is 300,800-451,200 daltons, the polysaccharide is an acidic heteropolysaccharide consisting of glucuronic acid, glucose and fucose in a molar ratio of 1.55-1.60.

Description

Method for synthesizing extracellular polysaccharide by fermenting wheat bran

Technical Field

The invention belongs to the field of microbial fermentation, and particularly relates to a method for synthesizing exopolysaccharide by fermenting wheat bran.

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.

Depending on the source, polysaccharides can be divided into animal polysaccharides, plant polysaccharides and microbial polysaccharides, wherein microbial polysaccharides are produced by microorganisms metabolizing carbohydrates, usually the base of the metabolism is artificially synthesized, such as MRS, TYC, etc., these bases are relatively expensive, and the metabolites usually remain chemical agents, which are not favorable for further use, in contrast to the base of the preparation from natural being relatively cheap and healthy. However, there has been relatively little research on the synthesis of microbial polysaccharides from agricultural processing by-products (e.g., wheat bran, etc.). In addition, the strain studies on the synthesis of exopolysaccharides from fermentable bran are relatively poor with respect to the fermentation species.

Therefore, there is a need to develop a method for preparing microbial exopolysaccharide by using wheat bran as a fermentation medium, which can improve the utilization rate of agricultural and sideline products and reduce the preparation cost of microbial exopolysaccharide, and can obtain new exopolysaccharide to meet the research requirement, and meanwhile, enrich the types of microbial exopolysaccharide with excellent performance and wide application.

Disclosure of Invention

The invention aims to provide a method for synthesizing exopolysaccharide by fermenting wheat bran. The method uses healthy and natural agricultural byproducts as raw materials, ensures the safety of metabolites to the maximum extent, and the prepared 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 a method for synthesizing extracellular polysaccharide by fermenting wheat bran, which comprises the following steps:

(1) Inoculating Paenibacillus (Paenibacillus sp.) CGMCC No.8333 with the preservation number of CGMCC No.8333 to a wheat bran culture medium to ferment so as to obtain fermentation liquor;

(2) Centrifuging the fermentation liquor, taking the supernatant, boiling, cooling, precipitating with ethanol overnight, centrifuging again, taking the precipitate, redissolving, and freeze-drying to obtain the exopolysaccharide.

Preferably, the inoculation amount of the Paenibacillus sp (Paenibacillus sp.) with the preservation number of CGMCC No.8333 in the step (1) is 1x10 ⁷ ～5x10 ⁷ cfu/mL。

Preferably, the wheat bran culture medium in the step (1) comprises wheat bran and water, and the mass percentage of the wheat bran is 1-5%.

Preferably, the fermentation temperature in the step (1) is 20-37 ℃, the fermentation mode is oscillation fermentation, and the oscillation speed is 100-300 rpm; the fermentation time is 12-72 h.

Preferably, the conversion rate of the exopolysaccharide prepared by the method is more than 18 percent.

Preferably, the macromolecular component of the exopolysaccharide prepared by the method has the average weight molecular weight of 300,800-451,200 daltons, and is an acidic heteropolysaccharide consisting of glucuronic acid, glucose and fucose in a molar ratio of 1.55-1.60.

Preferably, the backbone of the macromolecular component of the exopolysaccharide prepared by the method 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 located at position O-4 of 1,3,4-linked fucose residues, and the branch chain is composed of terminally linked glucuronic acid residues and is composed of repeating structural units represented by formula I.

The invention also provides application of the exopolysaccharide prepared by the method for synthesizing the exopolysaccharide by fermenting the wheat bran in the fields of food and medicine.

The invention also provides application of the extracellular polysaccharide prepared by the method for synthesizing the extracellular polysaccharide by fermenting the wheat bran in preparing the immunomodulatory drugs.

Preferably, the macromolecular components of the exopolysaccharides are not cytotoxic at concentrations below 100 μ g/mL.

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

the technical scheme of the invention discloses a method for preparing exopolysaccharide by fermenting paenibacillus bovis by taking wheat bran as a culture medium for the first time. On one hand, the technical scheme uses healthy and natural agricultural byproducts as raw materials, ensures the safety of metabolites to the maximum extent, and adopts a simple culture medium formula and a single strain fermentation process, so that the preparation method is simpler and more feasible. On the other hand, the macromolecular components of the extracellular polysaccharide prepared by the technical scheme have definite and special structures and also have various biological effects, such as enhancing the phagocytic function of macrophages and promoting the macrophages to release cytokines, so that the extracellular polysaccharide has obvious technical advantages when used as a macromolecular substance generated by microorganisms, and has good application prospects in the fields of food, medicine and the 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 the 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 from 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

A method for synthesizing exopolysaccharide by fermenting wheat bran comprises the following steps:

(1) Inoculating a Paenibacillus sp (Paenibacillus sp.) seed with the preservation number of CGMCC No.8333 to a wheat bran culture medium to ferment to obtain fermentation liquor;

In the method for synthesizing the extracellular polysaccharide, the collection number of the Paenibacillus (Paenibacillus sp.) with CGMCC No.8333 is 1x10 ⁷ ～5x10 ⁷ cfu/mL, preferably 2X10 ⁷ ～4x10 ⁷ cfu/mL, more preferably 3X10 ⁷ cfu/mL. The bacillus-like 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.

in the invention, the fermentation substrate is a wheat bran culture medium which is artificially prepared, the prepared wheat bran culture medium comprises 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 culture medium 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 source of the wheat bran is not specially limited, and the wheat bran can be obtained by adopting a commercial product.

In the invention, a wheat bran culture medium of bacillus (Paenibacillus sp.) is fermented at the fermentation temperature of 20-37 ℃, preferably 23-30 ℃, and more preferably 26 ℃. The fermentation time is 12 to 72 hours, preferably 24 to 60 hours, and more preferably 48 hours.

In the present invention, the preferred mode of fermentation is shaking fermentation at a shaking speed of 100rpm to 300rpm, preferably 150rpm to 250rpm, and more preferably 180rpm.

According to the invention, the proportion of the wheat bran converted into the extracellular polysaccharide in the fermentation liquid of the paenibacillus CGMCC No.8333 is improved through the mutual influence among the parameters of the inoculation amount, the content of the wheat bran, the oscillation speed, the culture temperature and the fermentation time. When the inoculation amount is too small, the strain propagation speed is low, so that the number of the live bacteria participating in the metabolism and synthesis of the exopolysaccharide is small, and the conversion efficiency of the exopolysaccharide is reduced. When the wheat bran content is lower than the preferred range value, substances for stimulating the generation of related metabolic synthesis enzymes by strains are correspondingly reduced, which also causes obvious reduction of the synthesis efficiency of the exopolysaccharides. In the present invention, the conversion rate of exopolysaccharide is more than 18% in the optimized range of fermentation parameters.

In the invention, the average weight molecular weight of the exopolysaccharide synthesized by fermenting wheat bran with bacillus like CGMCC NO.8333 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 extracellular polysaccharide consists of 1,3-connected glucose residues, 1,3-connected fucose residues, 1,3,4-connected fucose residues and 1,4-connected glucuronic acid residues, the branch point is positioned at O-4 position of 1,3,4-connected fucose residues, the branch chain consists of terminal connected glucuronic acid residues, and the main chain consists of a repeating structural unit shown in a formula I.

The invention also provides application of the exopolysaccharide synthesized by fermenting wheat bran with the paenibacillus in the fields of food and medicine. Research shows that the microbial exopolysaccharide has bioactivity, such as immunological activity, tumor resistance, oxidation resistance, tumor resistance, intestinal flora regulation, etc. and may be used in food, medicine and other fields. Immunomodulation is one of the most important biological activities of polysaccharides and is a non-specific class of immunomodulators. The immune system is a guard system for resisting invasion of pathogenic microorganisms and clearing variant cells in the organism 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 extracellular polysaccharide synthesized by fermenting wheat bran by the bacillus subtilis 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, and improves the expression of NO, TNF-alpha, IL-1 beta and IL-6 cell factors by activating the RAW264.7 cells, thereby having good immunoregulation effect.

The invention also provides application of the exopolysaccharide synthesized by fermenting wheat bran with the paenibacillus in preparing the immunoregulation medicine.

The following examples further illustrate the above embodiments, but do not therefore limit the invention within 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 Bacillus Paenii CGMCC No.8333 lyophilized powder in small amount of sterile distilled water, and making a ring by inoculating to a solution containing skimmed milk 1% (w/w) and agar 1.5% (w/v) obtained from national drug groupChina), aerobic culturing for 48h at 30 ℃, taking out, selecting a single colony by using an inoculating loop, inoculating the single colony in 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, wherein the viable count is 1x10 ⁹ cfu/mL。

(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 desired 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 supernatant, heating and boiling for 10min, cooling to room temperature, slowly adding anhydrous ethanol (purchased from Chinese medicine group, china) with three times of volume, refrigerating and standing for 24h, taking out, centrifuging at 15,000rpm for 10min to obtain precipitate, completely re-dissolving with small amount of distilled water, and freeze-drying to obtain the crude Paenibacillus extracellular polysaccharide A.

3. Calculation of conversion

The exopolysaccharide prepared as described above was weighed (0.46 g) and the conversion was calculated according to the following formula:

conversion rate of exopolysaccharide = (exopolysaccharide mass ÷ bran mass) × 100%

The conversion rate of the exopolysaccharide prepared by the method is calculated to be 25.8%.

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: adding 0.6g of wheat bran and 59.4mL of distilled water into a 250mL triangular flask, uniformly mixing, heating to boil, sterilizing at 125 ℃ 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 inoculum size of 5 percent into the wheat bran culture medium, and carrying out 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 the volume being three times that of the supernatant, refrigerating and standing for 24h, taking out the supernatant, 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. Calculation of conversion

The exopolysaccharide prepared as described above was weighed (0.11 g) and the conversion was calculated according to the following formula:

The conversion rate of the exopolysaccharide prepared by the method is 18.7 percent by calculation.

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 crude paenibacillus exopolysaccharide

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. Calculation of conversion

The exopolysaccharide prepared as described above was weighed (0.71 g) and the conversion was calculated according to the following formula:

The conversion rate of the exopolysaccharide prepared by the method is calculated to be 23.7%.

Example 4 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 2.4g of wheat bran and 57.6mL of distilled water into a 250mL triangular flask, uniformly mixing, heating and boiling, placing at 120 ℃ for sterilization for 10min, 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 4% in the wheat bran culture medium, and carrying out shaking culture at 30 ℃ and 250rpm for 36h 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, centrifuging at 15,000rpm for 10min, taking the precipitate, completely re-dissolving with a small amount of distilled water, and freeze-drying to obtain the paenibacillus extracellular polysaccharide D.

3. Calculation of conversion

The exopolysaccharide prepared as described above was weighed (0.48 g) and the conversion was calculated according to the following formula:

The conversion rate of the extracellular polysaccharide prepared by the method is calculated to be 20%.

Example 5 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 1.2g of wheat bran and 58.8mL of distilled water into a 250mL triangular flask, uniformly mixing, heating and boiling, placing at 100 ℃ for sterilizing for 20min, and cooling to room temperature to obtain the required wheat bran culture medium.

2. Preparation of paenibacillus extracellular polysaccharide crude product

Aseptically inoculating 2 percent of paenibacillus CGMCC No.8333 seeds into the wheat bran culture medium, and performing shaking culture at 23 ℃ and 150rpm for 60 hours 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, centrifuging at 15,000rpm for 10min, taking the precipitate, completely re-dissolving with a small amount of distilled water, and freeze-drying to obtain the paenibacillus extracellular polysaccharide E.

3. Calculation of conversion

Exopolysaccharide prepared by the above method was weighed (0.32 g) and the conversion was calculated according to the following formula:

The conversion rate of the exopolysaccharide prepared by the method is 26.7 percent by calculation.

Example 6 preparation of exopolysaccharide macromolecular Components from Paenibacillus

250mg of paenibacillus extracellular polysaccharide A is respectively 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 sequentially subjected to isocratic elution by using Tris-HCl buffer solution (50 mM, pH7.6) and buffer solution containing 0.2mol/LNaCl and 0.4mol/LNaCl, the elution speed is 1.5mL/min, and eluent (8 mL per tube) is collected by using an automatic part collector (purchased from Shanghai Qingpu Huxi apparatus, china).

Detecting polysaccharide content in eluate of each tube by sulfuric acid-phenol method, combining, collecting third component peak (185-205 tubes in figure 1, the abscissse:Sup>A is tube number and the ordinate is light absorption value in figure 1), placing into dialysis bag with cut-off molecular weight of 14,000 daltons, dialyzing with deionized water for 72h to remove buffer salt, changing water once every 12h, and freeze drying to obtain extracellular polysaccharide macromolecular component called EPS-A.

By the method, the paenibacillus extracellular polysaccharide macromolecular components EPS-B, EPS-C, EPS-D and EPS-E are respectively prepared.

Example 7 confirmation of the uniqueness of the exopolysaccharide macromolecular Components of Paenibacillus

5mg of EPS-A, EPS-B, EPS-C, EPS-D and EPS-E samples were weighed and respectively dissolved in 5mL of ultrapure water to prepare 1mg/mL of polysaccharide solution, and the polysaccharide solution was filtered through a 0.45 μm filter and then subjected to sample injection in an Agilent 1100 high performance liquid chromatograph (purchased from Agilent, USA) for analysis under the chromatographic conditions: RID detector, TSK-Gel G6000PWXL (available from Tosoh Biotech Co., ltd., japan) as chromatographic column, and 0.1mol/LNaNO as mobile phase 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 performance gel filtration chromatogram of the EPS-A sample is shown in figure 2 (the chromatograms of EPS-B, EPS-C, EPS-D and EPS-E are similar), se:Sup>A symmetrical chromatographic peak is presented in the retention time of 14-15min, the chromatographic peak of 21min is se:Sup>A mobile phase peak, and the result shows that the EPS-A, EPS-B, EPS-C, EPS-D and EPS-E samples are all homogeneous polysaccharide components.

Example 8 determination of the molecular weight of the exopolysaccharide macromolecular Components 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). The series of standard polysaccharides and EPS-A, EPS-B, EPS-C, EPS-D and EPS-E samples were dissolved in mobile phase (0.1 mol/LNaNO) ₃ Solution) 1mg/mL solution was obtained, filtered through 0.45 μm filter and analyzed by Agilent 1100 hplc under the same chromatographic conditions as in example 7. 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 according to the regression equation, and the results are shown in the following table.

TABLE 1 determination of the molecular weight of the exopolysaccharide macromolecular Components of Paenibacillus

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

Example 9 determination of monosaccharide composition of Paenibacillus exopolysaccharide macromolecular Components

(1) Hydrolysis of polysaccharide samples

2.0mg of EPS-A, EPS-B, EPS-C, EPS-D and EPS-E samples are respectively placed in ampoule bottles, 3mL of 2mol/L trifluoroacetic acid (TFA) is added, and after sealing, hydrolysis is carried out for 5h at 110 ℃. Cooling the hydrolysate, performing rotary evaporation at 45 ℃ under reduced pressure until the hydrolysate is dried, adding methanol to continue the 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 of an NaOH solution and 1mL of 0.5mol/L of a PMP methanol solution, uniformly mixing to completely dissolve a solid product, and placing in an oven at 70 ℃ 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 with 0.45 μm filter membrane, and analyzing by HPLC sample injection.

(3) Chromatographic conditions

An Agilent 1260 high performance liquid chromatograph (available from Agilent, USA) equipped with a DAD detector and an Agilent Eclipse XDB-C18 column (available from Agilent, USA) was used. 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 for the exopolysaccharide macromolecular Components of Paenibacillus

	Glucuronic acid	Glucose	Fucose sugar
				EPS-A (in terms of molar ratio)	1.58	1	1.66
EPS-B (in terms of molar ratio)	1.55	1	1.63
				EPS-C (in terms of molar ratio)	1.60	1	1.72
EPS-D (in terms of molar ratio)	1.57	1	1.65
				EPS-E (in terms of molar ratio)	1.56	1	1.70

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

Example 10 determination of mode of attachment of Paenibacillus exopolysaccharide macromoleculars

(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-A was dissolved in 6mL of ultrapure water and stirred until it was 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/LNaBH 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 complete uronic acid reduction, 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, stirring until most of NaOH is dissolved, carrying out ice bath for 5min, slowly dropwise adding 1mL of iodomethane 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 samples 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 is completely methylated; if the sample is not completely methylated, the reaction is continued until the sample is completely methylated.

Hydrolysis and acetylation: 2mg of se:Sup>A completely methylated EPS-A sample is placed in an ampoule, added with 3mL of 2mol/L TFA and sealed, and after hydrolysis at 110 ℃ in se:Sup>A closed manner for 4h, added with methanol and rotary evaporated under reduced pressure for 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, 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 increased to 250 ℃ after the temperature is maintained for 2min, the temperature increase rate is 5 ℃/min, and the temperature is maintained for 10min; the sample inlet adopts a split mode, and the split ratio is 3:1; the amount of sample was 1. Mu.L. The mass spectrum ion source 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 the monosaccharide composition, it was determined that the linkage of each sugar residue of EPS-A after reduction was 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

Taking an EPS-A sample of 20mg, adding 0.5mL D ₂ O dissolved, transferred to a clean NMR tube and chromatographed on a 600MHz NMR spectrometer (from Bruker, switzerland).

For is to ¹ H-NMR (FIG. 3), ¹³ After the chromatographic integration 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-A backbone 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 at position O-4 of 1,3,4-linked fucose residues, and the branch chain is composed of terminally linked glucuronic acid residues.

In conclusion, the paenibacillus exopolysaccharide macromolecular component consists of the repeated structural units shown in the formula I.

Comparative example 1

The inoculation amount, the wheat bran content, the oscillation speed, the culture temperature and the fermentation time in example 1 were adjusted one by one to obtain a set of wheat bran fermentation liquors prepared by different methods, and the conversion rate of exopolysaccharides in each set of the obtained fermentation liquors is shown in table 3.

TABLE 3 conversion rate of extracellular polysaccharide of fermentation broth of wheat bran prepared by different methods

As can be seen from the results shown in table 3, when the inoculation amount, the wheat bran content, the oscillation speed, the culture temperature and the fermentation time in the preparation method of the fermented milk powder are adjusted to be outside the preferred range, the paenibacillus CGMCC No.8333 can still convert the wheat bran into the exopolysaccharides, but the conversion efficiency is obviously reduced, so that the proportion of the wheat bran converted into the exopolysaccharides in the prepared fermentation broth is far lower than that of the fermentation broth prepared in the preferred range.

Effect example 1 Effect of exopolysaccharide macromolecular Components of Paenibacillus on cell growth

The RAW264.7 cell concentration was adjusted to 1X10 ⁴ The amount of the active carbon is one/mL,inoculation 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-A 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 se:Sup>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-A is 6.25, 12.5, 25, 50 and 100 mug/mL respectively, the survival rate of RAW264.7 cells is higher than that of blank control group, which is 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 reaches the maximum concentration, namely 600 mug/mL, the survival rate of RAW264.7 cells is obviously lower than that of a blank control group, and is only 62.74 +/-2.94%. The experimental result shows that the EPS-A 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 bacillus exopolysaccharide macromolecular component is safe for cells.

Effect example 2 Effect of extracellular polysaccharide macromolecular Components of Paenibacillus on phagocytic Activity of RAW264.7 cells

The concentration of RAW264.7 cells was adjusted to 1X10 ⁴ Per mL, inoculated in 96-well cell culture plates, 200. Mu.L/well, 37 ℃,5% CO ₂ Culturing under the condition, discarding culture solution after cell adherence, adding EPS-A solution (0, 6.25, 12.5, 25, 50, 100 μ g/mL) and LPS (1 μ g/mL) of different concentrations into each well, and setting 5 parallel wells for each concentration. After 24h of culture, 0.08% ready-prepared neutral red solution was added, the incubator was incubated for 1h, the liquid was aspirated after removal, rinsed 2 times with PBS, lysed by adding lysis buffer (glacial acetic acid: ethanol = 1:1) for 1h of lysis, the 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-A is respectively 6.25, 12.5, 25, 50 and 100 mug/mL, 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%, which are higher than 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 bacillus Paenibacillus extracellular polysaccharide macromolecular components are 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 extracellular polysaccharide macromolecular fraction of Paenibacillus on cytokine release from RAW264.7 cells

The RAW264.7 cell concentration was adjusted to 5X 10 ⁵ mL, inoculation 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 solution is discarded, and 100. Mu.L of EPS-A solution (0, 6.25, 12.5, 25, 50, 100, 200, 400, 600. Mu.g/mL) and 100. Mu.L of LPS (1. Mu.g/mL) with different concentrations are added into each well for 24h culture, wherein 2 parallel wells are arranged at each concentration. 50 μ L of cell supernatant was collected from each well and the corresponding cytokines were detected using NO kit, TNF- α ELISA kit, IL-1 β ELISA kit and IL-6ELISA kit (purchased from ELISA, USA), respectively.

The results are shown in FIG. 13 (NO), FIG. 14 (TNF-alphse:Sup>A), FIG. 15 (IL-1 betse:Sup>A) and FIG. 16 (IL-6), and show that the secretion levels of NO, TNF-alphse:Sup>A, IL-1 betse:Sup>A and IL-6 of RAW264.7 are increased dose-dependently after adding EPS-A with different concentrations for se:Sup>A period of time, which indicates that the extracellular polysaccharide macromolecular component of Paenibacillus bovis can activate RAW264.7 cells, increase the expression of the above cytokines and promote the immunomodulatory effect.

The method for synthesizing the extracellular polysaccharide by fermenting the wheat bran provided by the invention is 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. A method for synthesizing exopolysaccharide by fermenting wheat bran is characterized by comprising the following steps:

(2) Centrifuging the fermentation liquor, taking the supernatant, boiling, cooling, precipitating with ethanol overnight, centrifuging again, taking the precipitate, redissolving, and freeze-drying to obtain extracellular polysaccharide;

the exopolysaccharide is composed of a repeating structural unit shown in a formula I,

the average weight molecular weight of the macromolecular components 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 backbone of the macromolecular component 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 at position O-4 of 1,3,4-linked fucose residues, and the branch chain is composed of terminally linked glucuronic acid residues.

2. The method for synthesizing extracellular polysaccharide by fermenting wheat bran according to claim 1, wherein the inoculation amount of the Paenibacillus sp with the preservation number of CGMCC No.8333 in the step (1) is 1x10 ⁷ ～5x10 ⁷ cfu/mL。

3. The method for synthesizing exopolysaccharide by fermenting wheat bran according to claim 1, wherein the wheat bran culture medium in the step (1) comprises wheat bran and water, and the wheat bran accounts for 1-5% by mass.

4. The method for synthesizing exopolysaccharide by fermenting wheat bran according to claim 1, wherein the temperature of the fermentation in the step (1) is 20 ℃ to 37 ℃, the fermentation mode is oscillation fermentation, and the oscillation speed is 100rpm to 300rpm; the fermentation time is 12-72 h.

5. The method for synthesizing exopolysaccharide by fermenting wheat bran according to claim 1, wherein the conversion rate of exopolysaccharide prepared by the method is > 18%.

6. Use of exopolysaccharide prepared by the method for synthesizing exopolysaccharide by fermenting wheat bran according to any one of claims 1 to 5 in the field of food.