CN106399199B - A kind of soil series bacillus and its application - Google Patents

Abstract

The invention discloses a soil Paenibacillus and its application. The soil Paenibacillus is Paenibacillus soil NUST16, and the deposit number is CCTCC No.M2016542. The soil Paenibacillus strain of the invention has unique properties, can produce exopolysaccharide, and the yield can reach 15g/L, the produced exopolysaccharide is easy to separate and purify, has unique properties, can be directly dissolved in cold water, and can adjust the flow of the aqueous solution. It has the effect of significantly increasing the viscosity of the solution, and can withstand high concentrations of Na ⁺ , K ⁺ , Ca ²⁺ and Mg ²⁺ in the solution. After a simple heating-cooling treatment, the solution viscosity can be increased several times, and The properties are stable and form thermally reversible gels.

Description

A kind of soil Paenibacillus and its application

technical field

The invention belongs to the technical field of microorganisms, and relates to a soil Paenibacillus and its application.

Background technique

Due to its special rheological properties, microbial exopolysaccharides can be used as thickeners, suspending agents or stabilizers, and are widely used in food, cosmetics and other industries. Microbial exopolysaccharides with typical rheological characteristics, such as Xanthan gum, can increase the viscosity of the solution and have a certain temperature resistance. Irreversible brittle gel can be formed in the presence of Ca ²⁺ ; thermal gel (Curdlan gum) is insoluble in water, but its suspension can form a thermally reversible elastic gel when heated to 55 °C, and can be formed when heated to 80 °C Thermally irreversible brittle gel; Welan gum has good heat-resistant properties and has the potential to be used as an oil-displacing agent for petroleum development. On the other hand, compared with plant polysaccharides and marine algal polysaccharides, microbial exopolysaccharides have the advantages of not being affected by climate, simple production process, low cost, and easy mass preparation (Williams PA, Phillips DL. Introduction to food hydrocolloids[M] .WILLIAMS PA, PHILLIPS D L. Handbook of hydrocolloids. Woodhead publishing Ltd. 2009.). It can be seen that microbial exopolysaccharides with excellent performance can promote the development of industries related to people's livelihood.

At present, there are certain limitations in the application of microbial exopolysaccharides. For example, in order to increase the viscosity of xanthan gum solution, it can only be achieved by increasing the amount of xanthan gum added, but not through a simple post-treatment process; the presence of Ca ²⁺ or Mg ^{2 +} in the solution limits the amount of gellan gum used; The heat treatment process at temperature is affected by thermal gelation. Therefore, it is particularly necessary to chemically modify traditional microbial exopolysaccharides or discover new microbial exopolysaccharides.

The study found that Paenibacillus microorganisms have the ability to produce novel exopolysaccharides. Paenibacillus soil was originally classified as Bacillus genus and was later renamed Paenibacillus edaphicus. Soil Paenibacillus has the effect of promoting plant growth, the main reason may be that the bacteria can degrade and fix potassium that is difficult to use in the soil, thereby promoting the utilization of potassium by plants (Sheng X F. Growth promotion and increased potassium uptake of cotton and rape by a potassium releasing strain of Bacillus edaphicus [J]. Soil Biology & Biochemistry, 2005, 37(10): 1918-1922.). In addition, the study also found that soil Paenibacillus can produce thick extracellular capsules (He Linyan et al. Identification and phylogenetic analysis of a silicate bacterium [J]. Journal of Microbiology, 2003, 43 (2 ): 162-168.). It has been confirmed that Paenibacillus colloids, which are very closely related to Paenibacillus soil, also have the ability to produce capsules, and the capsules are exopolysaccharides and have better biological flocculation (Tang J Y, et al. Production). , purification and application of polysaccharide-based bioflocculant by Paenibacillus mucilaginosus [J]. Carbohydrate Polymers, 2014, 113: 463-470.). Therefore, it can be speculated that Paenibacillus soil has the ability to produce novel exopolysaccharides.

SUMMARY OF THE INVENTION

One of the objectives of the present invention is to provide Paenibacillus edaphicus NUST16, which can produce water-soluble exopolysaccharides with special rheological properties.

The technical solution to achieve the above purpose is:

A soil Paenibacillus, Paenibacillus edaphicus NUST16, the preservation number is CCTCCNo.M2016542, has been deposited in the China Center for Type Culture Collection (CCTCC) on October 08, 2016, and the preservation address is Luojia Mountain, Wuchang City, Hubei Province Wuhan University Collection Center.

The second object of the present invention is to provide a method for culturing the above-mentioned Paenibacillus soil NUST16. The specific steps are as follows: inoculating Paenibacillus soil NUST16 into a sterilized medium with a pH of 6.0-9.0, and culturing at 25-35° C. .

The third object of the present invention is to provide the application of the above-mentioned Paenibacillus soil in the production of water-soluble exopolysaccharides.

Further, the present invention provides a method for producing water-soluble exopolysaccharide by the above-mentioned Paenibacillus soil, the specific steps are as follows:

Step 1, inoculate Paenibacillus soil NUST16 into the sterilized culture medium, shake and cultivate at 25-35° C. to form a seed culture solution;

In step 2, the seed culture liquid is inoculated into the sterilized medium according to the inoculum amount of 0.5%-20%, and shaken at 25-35° C. for cultivation to obtain a fermentation liquid producing exopolysaccharide;

Step 3, adding precipitant A to the fermentation broth, the precipitate in the fermentation broth is filtered or centrifuged, and then dried to obtain solid crude polysaccharide, and the precipitant A is 95%-100% ethanol, 95%-100% methanol , one or more of 95%-100% isopropanol and 95%-100% acetone.

In step 1, the incubation time is 12-48h.

In step 2, the culturing time is 48-96h.

In step 3, the volume ratio of the precipitant A to the fermentation broth is 2 to 4:1, and the precipitant A is 95%-100% ethanol, 95%-100% methanol, 95%-100% isopropyl alcohol. A mixed solution obtained by mixing two or more solvents in propanol and 95%-100% acetone in equal volumes.

In the present invention, the composition of the culture medium is sucrose 5-50g/L, peptone 0.5-5g/L, NaH ₂ PO ₄ 0.1-5.0g/L, CaCl ₂ 0.01-0.5g/L, MgCl ₂ 0.01- 0.5g/L, KCl 0.01-0.5g/L, FeCl ₂ 0.001-0.05g/L, CuSO ₄ 0.001-0.05g/L, MnSO ₄ 0.001-0.05g/L, ZnCl ₂ 0.001-0.05g/L, CoCl ₂ 0.001-0.05g/L, pH 6.0-9.0.

Preferably, the pH of the medium is 7.0-9.0.

Further, the method for producing water-soluble exopolysaccharide by above-mentioned Paenibacillus soil also comprises the following steps:

Step 4: Dissolve the solid crude polysaccharide in water to form a crude polysaccharide solution, add the treatment solution B and shake vigorously, stand or centrifuge to separate the organic phase and the aqueous phase, collect the aqueous phase, and re-add the treatment solution B, repeat 3 to 10 times , the purified polysaccharide can be obtained, and the treatment solution B is a mixed solution of chloroform, phenol and n-butanol.

In step 4, the concentration of the crude polysaccharide solution is 0.25-20 g/L, and the volume ratio of the crude polysaccharide solution to the treatment solution B is 1:(1-2).

In step 4, in the treatment solution B, the volume ratio of chloroform, phenol and n-butanol is 1:(0.5-1):(0.05-0.5).

The soil Paenibacillus strain of the invention has unique properties, can produce exopolysaccharide, and the yield can reach 15g/L, the produced exopolysaccharide is easy to separate and purify, has unique properties, can be directly dissolved in cold water, and has a significant increase in solution The effect of viscosity, can withstand high concentrations of Na ⁺ , K ⁺ , Ca ²⁺ and Mg ²⁺ in the solution, after a simple heating-cooling treatment, the solution viscosity can be increased several times, and the properties are stable, forming a thermally reversible gel .

Description of drawings

Figure 1 is the plate colony and microscopic topography of Paenibacillus edaphicus NUST16 strain.

Figure 2 is a high-performance liquid chromatogram for the determination of exopolysaccharide components.

Figure 3 is a graph showing the effect of four inorganic salts on the viscosity of exopolysaccharide.

Figure 4 is a graph showing the effect of repeated heating-cooling treatments on the viscosity of 1 g/L exopolysaccharide solution.

Fig. 5 is a graph showing the effect of repeated heating-cooling treatment on the viscosity of 5 g/L exopolysaccharide solution.

Detailed ways

The present invention will be described in further detail below in conjunction with the embodiments and the accompanying drawings.

Example 1

Isolation and identification of the soil Paenibacillus edaphicus NUST16.

(1) Isolation of Paenibacillus edaphicus NUST16

The soil Paenibacillus edaphicus NUST16 of the present invention is isolated from the coastal brown soil of Yancheng, Jiangsu. The separation method is:

①Strain screening: Divide soil samples into 10 groups, weigh 1g of soil samples from each group, add them to the prepared liquid medium, and put them into a constant temperature shaking incubator at 200rpm and 30℃ for 2 days for enrichment. Then, 1 mL of bacterial liquid was taken from each group of conical flasks and re-introduced into a new liquid medium, and cultured under the same conditions for 2 d and enriched again. Then take 1mL of the enriched bacterial solution from each group and add it to 9mL of sterile water, mix well to make a bacterial suspension, use a 10-fold gradient dilution, and take 10 ^-6 -10 ^-4 dilution of the bacterial suspension and spread it on. In solid medium, cultured at 30°C.

② Purification of strains: culture the coated plates for 2 days. After the colonies grow, a single colony is picked and streaked on a new solid medium according to the morphological characteristics of the colonies, and cultured at 30°C. After the colonies grow, a single colony is selected. Streaked, and this process was repeated 3-4 times until the colonies growing on the petri dish were all single-morphological colonies, so that the strain was purified completely. Then select the colonies with the characteristics of colorless, transparent or translucent, sticky and other exopolysaccharide production, inoculate them into the liquid medium respectively, and cultivate at 200rpm and 30℃ for 2-4d. If the fermentation broth becomes sticky, identify the Whether extracellular fermentation products are polysaccharides. After identification, the strain whose fermentation product is polysaccharide is the desired extracellular polysaccharide-producing strain, named NUST16.

(2) Identification of strains

a. Morphological characteristics: After culturing this strain on inorganic salt agar solid medium at 30℃ for 2-3 days, the colony is round, colorless, translucent, glossy and sticky, and not easy to provoke. The strain is rod-shaped and secretes a large amount of extracellular polysaccharides.

b. Physiological and biochemical characteristics: the strain is Gram-positive, aerobic, positive for contact enzyme reaction, fermented with glucose as carbon source to produce acid, and grows well when the medium contains 1% NaCl. The carbon sources available to the strain are sucrose, starch, glucose, and lactose. Fructose is weak in utilization. When lactose is used as the carbon source, the bacterial growth is the best, followed by sucrose, and it grows well in the organic nitrogen source peptone. The yield of exopolysaccharide was higher under alkaline conditions, and the yield of exopolysaccharide could reach 15g/L after 48h fermentation. High concentration of NaCl will inhibit the growth of the strain and reduce the production of exopolysaccharide, and the strain does not secrete pigment. In Fig. 1, A is a colony on a solid plate medium, and B is a photo of the bacteria under an optical microscope.

c. Using the genomic DNA of the strain NUST16 as the template and the universal primers for PCR amplification of the 16S rRNA gene as the primers, carry out PCR amplification to determine the complete sequence. The 16S rDNA gene sequence of the strain is shown in the sequence table. The 16S rDNA gene sequence of the strain was submitted to the GenBank database (GenBank accession number KX962167), and the online homology comparison with the sequence in the GenBank database showed that the sequence similarity between the strain NUST16 and Paenibacillus soil AB045093 was as high as 99% .

According to the morphological, physiological and biochemical tests and molecular biological analysis of the strain NUST16, the strain NUST16 was identified as Paenibacillus soil, named as Paenibacillus soil NUST16.

Example 2

Cultivation of soil Paenibacillus edaphicus NUST16 and its application in the production of water-soluble exopolysaccharides.

Preparation medium, sucrose 5g, peptone 0.5g, NaH ₂ PO ₄ 0.1g, CaCl ₂ 0.01g, MgCl ₂ 0.01g, KCl 0.01g, FeCl ₂ 0.001g, CuSO ₄ 0.001g, MnSO ₄ 0.001g, ZnCl ₂ 0.001 g. Dissolve 0.001 g of CoCl ₂ in 1 L of deionized water, the pH is 6.0, add 1% nutrient agar, sterilize at 121° C. for 15 min, and cool to obtain a solid medium. After culturing the strain on agar solid medium at 30℃ for 48 hours, colonies can be seen to grow. The colonies are round, translucent, glossy, sticky and not easy to provoke. The strain secretes a large amount of exopolysaccharide. Scrape the colony on each 9cm plate and dissolve it with 30mL deionized water, then centrifuge at 5000×g for 20min, add 60mL ethanol-isopropanol mixture (volume ratio 1:1) to the supernatant to precipitate at room temperature, and collect the precipitate by filtration The crude polysaccharide was obtained after drying at 40°C.

Figure 2 is a high-performance liquid chromatogram of exopolysaccharide composition, and the same component has the same retention time. Figure 2A is the elution curve of 10 standard monosaccharides and their derivatives in high performance liquid phase after derivatization with PMP; Figure 2B is the hydrolyzate of the exopolysaccharide produced by Paenibacillus soil NUST16 after complete hydrolysis by trifluoroacetic acid The elution profile of PMP derivatized in HPLC showed no overlap between the elution peaks. After comparison, it can be found that D-mannose (D-Man), D-glucuronic acid (D-GlcUA), D-glucose (D-Glc), D-galactose (D-Gal) and L-fucose (L-Fuc) has exactly the same retention time in panels A and B. The quantitative calculation shows that the molar ratio of D-glucose, D-mannose, L-fucose, D-galactose and D-glucuronic acid in the exopolysaccharide produced by Paenibacillus soil NUST16 is 3:3:2:1 :1, another expression is: Glc:Man:Fuc:Gal:GlcUA=3:3:2:1:1. The polysaccharide is a heteropolysaccharide.

Example 3

Cultivation of soil Paenibacillus edaphicus NUST16 and its application in the production of water-soluble exopolysaccharides.

Preparation medium, sucrose 50g, peptone 5g, NaH ₂ PO ₄ 5g, CaCl ₂ 0.5g, MgCl ₂ 0.5g, KCl 0.5g, FeCl ₂ 0.05g, CuSO ₄ 0.05g, MnSO ₄ 0.05g, ZnCl ₂ 0.05g, Dissolve 0.05 g of CoCl ₂ in 1 L of deionized water, pH 7.0, sterilize at 121° C. for 20 min and cool for use, add 1% nutrient agar, sterilize at 121° C. for 15 min, and cool to obtain the corresponding solid medium. Pick a single colony of Paenibacillus soil NUST16 grown on the agar solid medium to the sterilized medium, shake and culture at 25°C for 24 hours to form a seed culture solution, and then add 7% of the seed culture solution to this standby medium. Incubate with shaking at 35°C for 48h to obtain a fermentation broth. 3.5 times the volume of 95% ethanol-acetone mixture (volume ratio 1:1) was added to the fermentation broth, shaken at room temperature, then centrifuged at 5000 × g for 20 min to collect the precipitate, and dried at 50 °C to obtain 10.08 g of crude polysaccharide.

Example 4

Cultivation of soil Paenibacillus edaphicus NUST16 and production and purification of water-soluble exopolysaccharides.

Preparation medium, sucrose 20g, peptone 3g, NaH ₂ PO ₄ 3g, CaCl ₂ 0.05g, MgCl ₂ 0.15g, KCl 0.15g, FeCl ₂ 0.03g, CuSO ₄ 0.003g, MnSO ₄ 0.05g, ZnCl ₂ 0.005g, Dissolve 0.01 g of CoCl ₂ in 1 L of deionized water, pH 9.0, sterilize at 121 °C for 20 min, and cool for later use; pick a single colony of Paenibacillus soil NUST16 grown on the solid medium into the sterilized medium, 35 The seed culture solution was formed by shaking culture at ℃ for 36 hours, and then 5% of the seed culture fluid was added to the sterilized medium for 32 hours shaking culture at 32 ℃ to obtain a fermentation broth. 3 times the volume of 95% ethanol-100% methanol-isopropanol mixture (volume ratio 1:1:1) was added to the fermentation broth, precipitated and filtered, and dried at 45°C to obtain 14.83 g of crude polysaccharide. Redissolve 10 g of crude polysaccharide in deionized water to a final concentration of 15 g/L, then add 1.1 times the volume of chloroform-phenol-n-butanol mixture (volume ratio 1:0.85:0.15), shake and then stand for stratification to collect water The above mixed solution is added to the phase again, and the operation is repeated 5 times to obtain the deproteinized exopolysaccharide. The obtained deproteinized polysaccharide was dialyzed with 30 times the volume of deionized water through a semipermeable membrane with a molecular weight cut-off of 3000 Da for 48 hours, and then 4 times the volume of 95% ethanol-acetone mixture (volume ratio 1:1) was added to the dialyzed polysaccharide. After precipitation and filtration, 6.47 g of purified exopolysaccharide was obtained by drying at 50°C.

Example 5

The use of exopolysaccharides produced by Paenibacillus aeruginosa NUST16 in modulating the rheological properties of aqueous solutions.

The purified exopolysaccharide was prepared according to Example 4, and then at room temperature, the polysaccharide was dissolved in deionized water to a final concentration of 6.5 g/L. The measured solution viscosity was 3755 mPa.s, which was recorded as the initial viscosity. Concentrations of 20g/L KCl, 40g/L NaCl, 80g/L MgCl ₂ and 100g/L CaCl ₂ were prepared in turn, and the 4 solutions were used to dissolve the exopolysaccharide to a final concentration of 6.5g/L at room temperature, and the viscosity was measured. , the viscosity is 3525mPa.s, 3645mPa.s, 3042mPa.s and 3160mPa.s in turn, and the viscosity is more than 80% of the initial viscosity. In addition, a mixed inorganic salt solution was prepared, in which the concentrations of KCl, NaCl, MgCl ₂ and CaCl ₂ were all 25 g/L, the exopolysaccharide was dissolved at room temperature to a final concentration of 6.5 g/L, and the measured viscosity value was 3055 mPa.s, which was the initial concentration of 81.4%.

Figure 3 shows the changes in the viscosity of the exopolysaccharide solution when different concentrations of NaCl, KCl, CaCl ₂ and MgCl ₂ were added to the solution at room temperature. It can be seen that the viscosity of the polysaccharide solution can still maintain more than 80% of the initial viscosity when the salt concentration is as high as 100 g/L, and Ca ²⁺ and Mg ²⁺ do not make the solution gel, which is very important for the unit operation under high salt conditions. Very helpful. It can be seen that the exopolysaccharide produced by Paenibacillus soil NUST16 has a good effect on regulating the rheological properties of the aqueous solution, especially when the solution contains a high concentration of salt ions, it can still significantly increase the solution viscosity and maintain stability.

Example 6

The use of exopolysaccharides produced by Paenibacillus aeruginosa NUST16 in modulating the rheological properties of aqueous solutions.

The purified solid exopolysaccharide was prepared according to Example 4, the exopolysaccharide was dissolved in deionized water at room temperature to a final concentration of 5 g/L, and the viscosity was measured as 2760 mPa.s, which was recorded as the initial viscosity. Take 500mL of polysaccharide solution and put it in a boiling water bath for 10min, then quickly take it out and cool it to room temperature (25°C). After cooling, the polysaccharide solution is fluid and does not form a gel. The measured viscosity is 19640mPa.s, which is 7.08 times the initial viscosity. . The solution was re-placed in a boiling water bath for 10 minutes and the viscosity of the solution decreased, then cooled to room temperature and the viscosity was measured to be 20100mPa.s, and repeated 3 times. The observed phenomena were consistent, and the viscosity was 19950mPa.s, 19970mPa.s and 20550mPa.s. s. After 5 heating-cooling cycles, the viscosity of the polysaccharide solution remained stable, the solution was clear and transparent, without color change, and thermally irreversible gel was never formed.

Figure 4 shows the viscosity change of 1 g/L exopolysaccharide solution after heat treatment at 95 °C for 10 min and then cooled to room temperature. It can be seen that after a simple heat treatment, the solution viscosity increased from 235mPa.s to 850mPa.s, and the viscosity was 3.62 times that before treatment. After 5 repetitions, the viscosity remained stable. Figure 5 shows the viscosity change of 5 g/L exopolysaccharide solution after heat treatment at 100 °C for 10 min and then cooled to room temperature. It can be seen that after a simple heat treatment, the solution viscosity increased from 2760 mPa.s to 19640 mPa.s, and the viscosity was 7.08 times that before treatment. After 5 repetitions, the viscosity remained stable. This feature can greatly reduce the amount of polysaccharide used, or reduce the difficulty of unit operations during pretreatment. It can be seen that the exopolysaccharide produced by Paenibacillus aeruginosa NUST16 is added to the solution, and the viscosity of the solution can be further doubled through a simple heating-cooling operation, and the viscosity remains stable after repeated repetitions without forming a thermally irreversible gel. It shows that the extracellular polysaccharide has stable properties and has a relatively wide application value.

SEQUENCE LISTING

<110> Nanjing University of Science and Technology

<120> A soil Paenibacillus and its application

<130> 666

<160> 1

<170> PatentIn version 3.5

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<211> 1241

<212> DNA

<213> Paenibacillus edaphicus

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gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgaac 1020

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Claims (10)

1. a kind of soil series bacillus (Paenibacillus edaphicus) NUST16, deposit number CCTCC No.M2016542。

2. application of the soil series bacillus as described in claim 1 in production exopolysaccharide.

3. application of the soil series bacillus as claimed in claim 2 in production exopolysaccharide, which is characterized in that Steps are as follows for concrete application:

Step 1, soil series bacillus NUST16 is inoculated into the culture medium after sterilizing, 25-35 DEG C of shaking table shake culture shape At seed culture fluid；

Step 2, seed culture fluid is inoculated into the culture medium after sterilizing by 0.5%-20% inoculum concentration, 25-35 DEG C of shaking table shake Culture is swung, the fermentation liquid of extracellular polysaccharide is obtained；

Step 3, precipitating reagent A is added in fermentation liquid, the precipitating in fermentation liquid is filtered or is centrifuged, more up to solids crude after dry Sugar, the precipitating reagent A are 95%-100% ethyl alcohol, 95%-100% methanol, 95%-100% isopropanol and 95%-100% One or more of acetone.

4. application of the soil series bacillus as claimed in claim 3 in production exopolysaccharide, which is characterized in that In step 1, the incubation time is 12-48h.

5. application of the soil series bacillus as claimed in claim 3 in production exopolysaccharide, which is characterized in that In step 2, the incubation time is 48-96h.

6. application of the soil series bacillus as claimed in claim 3 in production exopolysaccharide, which is characterized in that In step 3, the volume ratio of the precipitating reagent A and fermentation liquid is 2~4:1, the precipitating reagent A be 95%-100% ethyl alcohol, Two or more solvents in 95%-100% methanol, 95%-100% isopropanol and 95%-100% acetone by isometric mixing and At mixed solution.

7. application of the soil series bacillus in production exopolysaccharide as described in claim 2~6 is any, special Sign is that the group of the culture medium becomes sucrose 5-50g/L, peptone 0.5-5g/L, NaH₂PO₄0.1-5.0g/L, CaCl₂ 0.01-0.5g/L, MgCl₂0.01-0.5g/L, KCl 0.01-0.5g/L, FeCl₂0.001-0.05g/L, CuSO₄ 0.001- 0.05g/L, MnSO₄0.001-0.05g/L, ZnCl₂0.001-0.05g/L, CoCl₂0.001-0.05g/L, pH 6.0- 9.0。

8. application of the soil series bacillus as claimed in claim 7 in production exopolysaccharide, which is characterized in that The pH of the culture medium is 7.0-9.0.

9. application of the soil series bacillus as claimed in claim 3 in production exopolysaccharide, which is characterized in that It is further comprising the steps of:

Step 4, by solid Thick many candies formation Thick many candies solution soluble in water, treatment fluid B is added and acutely shakes, stands or is centrifuged Organic phase and water phase are separated, collects water phase, and rejoin treatment fluid B, repeats 3~10 times, purified polysaccharide, the place can be obtained Reason liquid B is the mixed solution of chloroform, phenol and n-butanol.

10. application of the soil series bacillus as claimed in claim 9 in production exopolysaccharide, feature exist In in step 4, the concentration of the Thick many candies solution is 0.25~20g/L, the body of the Thick many candies solution and treatment fluid B Product is than 1:(1~2), in the treatment fluid B, the volume ratio of chloroform, phenol and n-butanol is 1:(0.5~1): (0.05 ~0.5).