CN108060187B

CN108060187B - Polysaccharide and preparation method and application thereof

Info

Publication number: CN108060187B
Application number: CN201610975230.9A
Authority: CN
Inventors: 王世明; 陈向楠; 李晶; 程瑞; 张建法
Original assignee: Nanjing University of Science and Technology
Current assignee: Nanjing University of Science and Technology
Priority date: 2016-11-07
Filing date: 2016-11-07
Publication date: 2021-04-27
Anticipated expiration: 2036-11-07
Also published as: CN108060187A

Abstract

The invention discloses a polysaccharide with special rheological property, a preparation method and application thereof. The polysaccharide is a polysaccharide compound with a new structure and consists of D-glucose, D-mannose, L-fucose, D-galactose and D-glucuronic acid according to the molar ratio of 3:3:2:1: 1. The polysaccharide is produced by Paenibacillus tumefaciens NUST16 with the preservation number CCTCC No. M2016542. The polysaccharide can be directly dissolved in cold water, can adjust the rheological property of the aqueous solution, has the obvious effect of increasing the viscosity of the solution, and can resist high-concentration Na in the solution⁺、K⁺、Ca²⁺And Mg²⁺The solution can be increased in viscosity by several times through simple heating-cooling treatment, has stable properties, can form a thermoreversible gel, and can be used as an emulsifier, a thickener, a stabilizer or a gelling agent in the industrial fields of food or cosmetics and the like.

Description

Polysaccharide and preparation method and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and relates to polysaccharide and a preparation method and application thereof.

Background

The microbial exopolysaccharide can be used as a thickening agent, a suspending agent or a stabilizing agent due to the special rheological property, and can be widely applied to the industries of food, cosmetics and the like. The microbial extracellular polysaccharide with typical rheological characteristics, such as Xanthan gum (Xanthan gum), can increase the viscosity of a solution and has certain temperature resistance, and the viscosity is basically stable after the temperature is moderately increased; gelan gum (Gelan gum) in Ca²⁺Is capable of forming an irreversible brittle gel in the presence of a solvent; thermal gel (Curdlan gum) is insoluble in water, but it is suspendedThe suspension can form a thermally reversible elastic gel after being heated to 55 ℃, and can form a thermally irreversible brittle gel after being heated to 80 ℃; welan gum (Welan gum) has good heat resistance and has potential as an oil displacement agent for oil exploitation. On the other hand, compared with plant polysaccharides and marine algae polysaccharides, the microbial extracellular polysaccharides have the advantages of weather resistance, simple production process, low cost, convenience in mass preparation and the like (Williams P A, Phillips D L]WILLIAMS P A, PHILLIPS D L.handbook of hydrocolloids.Woodhead publishing Ltd.2009.). Therefore, the microbial exopolysaccharide with excellent performance has a promoting effect on the development of related industries of the livelihood.

At present, the microbial exopolysaccharide has certain limiting factors in application. For example, the viscosity of the xanthan gum solution can only be increased by increasing the addition amount of the xanthan gum and cannot be increased by a simple post-treatment process; presence of Ca in solution²⁺Or Mg² ⁺The usage amount of the gellan gum is limited; the heat treatment process with the higher temperature is affected by the thermal gelation. Therefore, it is necessary to chemically modify the conventional exopolysaccharide of the microorganism or to discover a novel exopolysaccharide of the microorganism.

It has been found that microorganisms of the genus Paenibacillus have the ability to produce novel exopolysaccharides. Paenibacillus tumefaciens was first discovered and classified as Bacillus, and then renamed as Paenibacillus tumefaciens (Paenibacillus edaphilus). Paenibacillus firmus has the effect of promoting plant growth, and the main reason of the effect is probably because the Paenibacillus firmus can degrade and fix potassium which is difficult to utilize in Soil, thereby promoting the utilization of potassium by plants (Sheng X. growth promotion and secreted potassium uptake of cotton and slide a potassium liberating strain of Bacillus edition [ J ]. Soil Biology & Biochemistry,2005,37(10): 1918-1922.). In addition, it was found that Paenibacillus tumefaciens is capable of producing thick extracellular capsules (Allianthus et al. identification of a silicate bacterium and its phylogenetic analysis [ J ] Microbiol. 2003,43(2): 162-. Paenibacillus mucilaginosus, which is very closely related to Paenibacillus Agrobacterium, has been shown to have the ability to produce a capsule as well, and the capsule is an extracellular polysaccharide and has a good biofloculation (Tang J Y, et al production, purification and application of polysaccharide-based biofloculation by Paenibacillus mucilaginosus polysaccharides [ J ]. Carbohydrate Polymers,2014,113: 463-. It can therefore be presumed that Paenibacillus tumefaciens has the ability to produce novel exopolysaccharides.

Disclosure of Invention

One of the objectives of the present invention is to provide a polysaccharide with special rheological properties, which has the structural formula:

n is 50-2000.

The invention also aims to provide a preparation method of the polysaccharide, wherein the polysaccharide is produced by paenibacillus agricus NUST16 with the preservation number of CCTCC No. M2016542, and the specific steps are as follows:

step 1, inoculating paenibacillus agricus NUST16 with the preservation number of CCTCC No. M2016542 into a sterilized culture medium, and performing shake culture on a shaker at 25-35 ℃ to form a seed culture solution;

step 2, inoculating the seed culture solution into a sterilized culture medium according to the inoculation amount of 0.5-20%, and performing shaking culture at 25-35 ℃ in a shaking table to obtain extracellular polysaccharide-producing fermentation liquor;

and 3, adding a precipitator A into the fermentation liquor, filtering or centrifuging precipitates in the fermentation liquor, and drying to obtain solid crude polysaccharide, wherein the precipitator A is one or more of 95-100% of ethanol, 95-100% of methanol, 95-100% of isopropanol and 95-100% of acetone.

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

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

In the step 3, the volume ratio of the precipitant A to the fermentation liquor is 2-4: 1, and the precipitant A is a mixed solution formed by mixing more than two of 95-100% of ethanol, 95-100% of methanol, 95-100% of isopropanol and 95-100% of acetone according to equal volume.

In the invention, the culture medium comprises 5-50g/L of sucrose, 0.5-5g/L of peptone and NaH₂PO₄ 0.1-5.0g/L，CaCl₂ 0.01-0.5g/L，MgCl₂ 0.01-0.5g/L，KCl 0.01-0.5g/L，FeCl₂ 0.001-0.05g/L，CuSO₄ 0.001-0.05g/L，MnSO₄ 0.001-0.05g/L，ZnCl₂ 0.001-0.05g/L，CoCl₂0.001-0.05g/L and pH 6.0-9.0.

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

Further, the preparation method of the polysaccharide also comprises the following steps:

and 4, dissolving solid crude polysaccharide in water to form a crude polysaccharide solution, adding the treatment solution B, violently shaking, standing or centrifugally separating an organic phase and a water phase, collecting the water phase, adding the treatment solution B again, and repeating for 3-10 times to obtain the purified polysaccharide, wherein the treatment solution B is a mixed solution of chloroform, phenol and n-butyl alcohol.

In the 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 liquid B is 1 (1-2).

In the step 4, the volume ratio of the trichloromethane to the phenol to the n-butyl alcohol in the treatment liquid B is 1 (0.5-1) to 0.05-0.5. The invention also aims to provide the application of the polysaccharide in adjusting the rheological property of the aqueous solution.

Further, the above polysaccharide can be used as an emulsifier, thickener, stabilizer or gelling agent in the application of adjusting the rheological properties of an aqueous solution.

The Paenibacillus edaphicus NUST16 is Paenibacillus edaphilus NUST16, the preservation number is CCTCC No. M2016542, the Paenibacillus edaphus NUST is preserved in China Center for Type Culture Collection (CCTCC) 10 months 08 days 2016, and the preservation address is the preservation center of Wuchang Lojiashan university in Wuhan, Hubei province.

The polysaccharide of the invention is a polysaccharide compound with a new structure, can be directly dissolved in cold water, has the obvious effect of increasing the viscosity of the solution, and can tolerate high-concentration Na in the solution⁺、K⁺、Ca²⁺And Mg²⁺The solution viscosity can be increased by multiple times through simple heating-cooling treatment, and the gel has stable property and can form the thermoreversible gel. The polysaccharide can be produced by fermenting Paenibacillus edae NUST16, the preparation method is simple and convenient, the produced extracellular polysaccharide is easy to separate and purify and has unique properties, and the yield can reach 15 g/L. The polysaccharide of the present invention can be used as an emulsifier, thickener, stabilizer or gelling agent in the industrial fields of foods, cosmetics, and the like.

Drawings

FIG. 1 is a plate colony and micrograph of a strain.

FIG. 2 is a high performance liquid chromatogram of the determination of exopolysaccharide components.

FIG. 3 is an infrared spectrum of exopolysaccharide.

FIG. 4 is the 1H NMR spectrum of exopolysaccharide.

FIG. 5 is the 13C NMR spectrum of exopolysaccharide.

FIG. 6 shows the total ion flow pattern of gas phase-mass spectrum after methylation treatment of extracellular polysaccharide and the structural formula corresponding to the retention peak.

FIG. 7 is a graph showing the effect of 4 inorganic salts on the viscosity of exopolysaccharides.

FIG. 8 is a graph showing the effect of repeated heating-cooling treatments on the viscosity of a 1g/L exopolysaccharide solution.

FIG. 9 is a graph showing the effect of repeated heating-cooling treatments on the viscosity of a 5g/L exopolysaccharide solution.

Detailed Description

The present invention will be described in more detail with reference to the following examples and the accompanying drawings.

Example 1

Isolation and characterization of Paenibacillus edaphilus NUST 16.

(1) Isolation of Paenibacillus edaphilus exaphilus NUST16

The Paenibacillus edaphilus NUST16 is separated from coastal brown soil in Jiangsu saline city. The separation method comprises the following steps:

screening strains: dividing the soil sample intoWeighing 1g of soil sample in each group for 10 groups, adding the soil samples into the prepared liquid culture medium, putting the mixture into a constant-temperature shaking incubator for culture for 2d at the temperature of 30 ℃ at 200rpm for enrichment, taking 1mL of bacterial liquid from each group of conical flasks, inoculating the bacterial liquid into a new liquid culture medium again, and culturing for 2d for enrichment again under the same conditions. Adding 1mL of enriched bacteria liquid from each group into 9mL of sterile water, mixing to obtain bacteria suspension, diluting by 10 times gradient, and taking 10^-6-10^-4The diluted bacterial suspension is spread on a solid medium and cultured at 30 ℃.

Purifying the strain: after the bacterial colony grows, the coated plate culture 2d is used for selecting a single bacterial colony to mark on a new solid culture medium according to the morphological characteristics of the bacterial colony, the bacterial colony is cultured at the temperature of 30 ℃, the single bacterial colony is selected again to mark after the bacterial colony grows, the process is repeated for 3-4 times, and the bacterial colony growing on a culture dish is the bacterial colony in a single form so that the bacterial strain is completely purified. And then selecting a colony number with extracellular polysaccharide-producing characteristics such as colorless transparency, translucency, viscosity and the like, respectively inoculating the colony number into a liquid culture medium, culturing for 2-4d under the conditions of 200rpm and 30 ℃, and if the fermentation liquor is sticky, identifying whether the extracellular fermentation product belongs to polysaccharide. The strain of which the identified fermentation product is polysaccharide is the required exopolysaccharide-producing strain and is named NUST 16.

(2) Identification of strains

a. Morphological characteristics: the strain was cultured on an inorganic salt agar solid medium at 30 ℃ for 2 to 3 days and then observed, as shown in FIG. 1. In FIG. 1, A is the bacterial colony on the solid plate culture medium, B is the photo of the thallus under the optical microscope, the bacterial colony is round, colorless, semitransparent, glossy and sticky on the surface, and is not easy to pick up. The strain has a long rod shape and secretes a large amount of extracellular polysaccharide.

b. Physiological and biochemical characteristics: the strain is gram-positive, aerobic and catalase reaction-positive, the acid is produced by fermenting with glucose as a carbon source, and the strain grows well when the culture medium contains 1% of NaCl. The carbon source which can be utilized by the strain comprises sucrose, starch, glucose and lactose, the utilization capacity of fructose is weak, the thallus grows best when the lactose is taken as the carbon source, the sucrose grows second best, the thallus grows well in organic nitrogen source peptone, the extracellular polysaccharide yield is higher under the alkaline condition of pH7-9, and the extracellular polysaccharide yield can reach 15g/L after fermentation for 48 hours. High concentration NaCl will inhibit the growth of the strain and reduce the yield of extracellular polysaccharide, and the strain will not secrete pigment.

c. The genome DNA of the strain NUST16 is used as a template, a universal primer for PCR amplification of 16S rRNA gene is used as a primer, PCR amplification is carried out, the complete sequence of the strain is determined, and the 16S rDNA gene sequence of the strain is shown in a sequence table. The 16S rDNA gene sequence of the strain is submitted to a GenBank database (the GenBank accession number is KX962167), and online homology comparison is carried out on the sequence in the GenBank database, and the result shows that the sequence similarity of the strain NUST16 and the paenibacillus agri AB045093 is as high as 99%.

According to morphological, physiological and biochemical tests and molecular biological analysis of the strain NUST16, the strain NUST16 is identified as Paenibacillus edae, and is named as Paenibacillus edae NUST 16.

Example 2

The general structural formula of the polysaccharide is shown as follows:

n is 50-2000.

1. High performance liquid chromatography

The polysaccharide component comprises D-glucose, D-mannose, L-fucose, D-galactose and D-glucuronic acid in a molar ratio of 3:3:2:1: 1. FIG. 2 is a high performance liquid chromatogram of extracellular polysaccharide composition, with the same retention time of the same component. Wherein FIG. 2A is an elution profile in the high performance liquid phase of 10 standard monosaccharides and their derivatives derivatized with PMP; FIG. 2B is the elution curve of the hydrolysate of extracellular polysaccharide produced by Paenibacillus tumefaciens NUST16 after complete hydrolysis with trifluoroacetic acid in high performance liquid phase by PMP derivatization, wherein no overlap exists between the elution peaks. By comparison, it was found that D-mannose (D-Man), D-glucuronic acid (D-GlcUA), D-glucose (D-Glc), D-galactose (D-Gal) and L-fucose (L-Fuc) were retained in the A and B panels at the same time. The molar ratio of D-glucose, D-mannose, L-fucose, D-galactose and D-glucuronic acid in the exopolysaccharide produced by the paenibacillus agricus NUST16 is 3:3:2:1:1 through quantitative calculation, and the other expression is as follows: man: Fuc: Gal: GlcUA ═ 3:3:2:1: 1. The polysaccharide is heteropolysaccharide.

2. Infrared spectroscopic analysis

The polysaccharide of the present invention has a characteristic absorption curve of a typical polysaccharide substance. FIG. 3 is the infrared spectrum of exopolysaccharide, from which the wavenumbers 3313.11, 1071.75 and 1019.68cm are known^-1Typical vibration absorption peaks for polysaccharides. Wavenumbers 2912.95,1402.00 and 1362.94cm^-1Is methyl (-CH)₃) Characteristic absorption peaks, indicating that the sample contains a small amount of methyl groups and is shifted towards lower wavenumbers by the influence of ortho C-O, which is consistent with the L-fucose (Fuc) structure. In addition, the wave number was 1595.81cm^-1Typical carboxyl C ═ O absorption peaks, which matched the structure of D-glucuronic acid (D-GlcUA).

3. Total ion flux analysis of NMR and gas-mass spectrometry

The nuclear magnetic resonance spectrum shows that the extracellular polysaccharide contains methyl and carboxyl signals. FIGS. 4 and 5 are of exopolysaccharides, respectively¹H and¹³c nuclear magnetic resonance spectrum. As can be seen from FIG. 4(1), the high field region has δ 1.29ppm (A) and δ 1.21ppm (B) corresponding to the methyl hydrogens at the C-6 positions of the two L-fucose

And the methyl carbon at the C-6 position of L-fucose

Also as shown in FIG. 5¹³Chemical shifts δ 15.48(a) and δ 15.35ppm (b) in the C NMR spectrum correlate, further validating the conclusion of the liquid phase and infrared spectra. As can also be seen in FIG. 5, the low field region, delta 175.86ppm (C), is typical for the carbonyl carbons of carboxylic acids

The conclusion is consistent with the presence of glucuronic acid in the polysaccharide. As can be seen from FIG. 4, the low field region has a value of delta 4.50 to 5.50ppm (a to f)₃) Combining the integrated area and the coupling constant data as the hydrogen chemical shift of each anomeric carbon in the exopolysaccharide repeating unit, it can be concluded that a is D-glucuronic acid and b is_1,2Is two D-mannose, c_1,2Is two L-fucose, D is D-galactose, e is D-mannose, f_1,2,3Three D-glucose. 10 anomeric carbons¹H chemical shifts are respectively shown in FIG. 5¹³The (i-x) in the C NMR spectrum corresponds to each other one by one, and the chemical shifts are as follows: δ 98.31,99.96,101.23,100.46,100.64,102.65,103.35,101.50,101.79 and 102.65 ppm. According to chemical shift sum³J_1,2By coincidence, it can be presumed that the 10 sugar unit conformations are, in order, α -D-glucuronic acid, α -D-mannose, α -L-fucose, β -D-galactose, β -D-mannose, β -D-glucose and β -D-glucose. The connection mode between each glycoside is further deduced from methylation data (see fig. 6), and fig. 6 is a total ion flow diagram of gas-mass spectrometry analysis after methylation treatment of extracellular polysaccharide. The retained peaks A-G are compared with the standard compound substance spectrum database, and the structural formulas are respectively shown in FIG. 6, and the rest peaks are irrelevant peaks of monosaccharide and are not shown. A to G correspond to: D-GlcUA, 3-L-Fuc, D-Gal, 3-D-Glc, 2,3,6-D-Glc, 3,6-D-Man and 3-D-Man. Combining the information of fig. 2-6, the exopolysaccharide structure was finally determined.

Example 3

Paenibacillus edae is used for producing extracellular polysaccharide through fermentation of NUST 16.

Preparing a culture medium, 5g of sucrose, 0.5g of peptone and NaH₂PO₄ 0.1g，CaCl₂ 0.01g，MgCl₂ 0.01g，KCl 0.01g，FeCl₂ 0.001g，CuSO₄ 0.001g，MnSO₄ 0.001g，ZnCl₂ 0.001g，CoCl₂Dissolving 0.001g in 1L deionized water, adjusting pH to 6.0, adding 1% nutrient agar, sterilizing at 121 deg.C for 15min, and cooling to obtain solid culture medium. After the strain is cultured on an agar solid culture medium at 30 ℃ for 48 hours, a bacterial colony can be seen to grow out, the bacterial colony is round and translucent, the surface is glossy and viscous, the bacterial colony is not easy to pick up, and the strain secretes a large amount of extracellular polysaccharide. Scraping colony on each 9cm plate, dissolving with 30mL deionized water, centrifuging for 20min at 5000 Xg, adding 60mL ethanol-isopropanol mixture (volume ratio 1:1) into supernatant, precipitating at room temperature, filtering, collecting precipitate, and cooling at 40 deg.CDrying to obtain crude polysaccharide.

Example 4

Preparing a culture medium, 50g of cane sugar, 5g of peptone and NaH₂PO₄ 5g，CaCl₂ 0.5g，MgCl₂ 0.5g，KCl 0.5g，FeCl₂ 0.05g，CuSO₄ 0.05g，MnSO₄ 0.05g，ZnCl₂ 0.05g，CoCl₂Dissolving 0.05g in 1L deionized water, adjusting pH to 7.0, sterilizing at 121 deg.C for 20min, cooling, adding 1% nutrient agar, sterilizing at 121 deg.C for 15min, and cooling to obtain corresponding solid culture medium. Selecting a single colony of Paenibacillus Gelatinosus NUST16 growing on an agar solid culture medium, placing the single colony into the sterilized culture medium, performing shake culture at 25 ℃ for 24h to form a seed culture solution, adding 7% of the seed culture solution into the standby culture medium, and performing shake culture at 35 ℃ for 48h to obtain a fermentation liquid. Adding 3.5 times volume of 95% ethanol-acetone mixed solution (volume ratio 1:1) into the fermentation liquid, oscillating at room temperature, centrifuging at 5000 × g for 20min, collecting precipitate, and drying at 50 deg.C to obtain 10.08g of crude polysaccharide.

Example 5

Paenibacillus edae is fermented to produce extracellular polysaccharide and purifying the polysaccharide by using NuST 16.

Preparing culture medium, 20g of cane sugar, 3g of peptone and NaH₂PO₄ 3g，CaCl₂ 0.05g，MgCl₂ 0.15g，KCl 0.15g，FeCl₂ 0.03g，CuSO₄ 0.003g，MnSO₄ 0.05g，ZnCl₂ 0.005g，CoCl₂Dissolving 0.01g in 1L deionized water, adjusting pH to 9.0, sterilizing at 121 deg.C for 20min, cooling, adding 1% nutrient agar, sterilizing at 121 deg.C for 15min, and cooling to obtain corresponding solid culture medium. Selecting a single colony of the Paenibacillus Gelatinosus NUST16 growing on the solid culture medium to the sterilized culture medium, carrying out shake culture at 35 ℃ for 36h to form a seed culture solution, adding 5% of the seed culture solution to the sterilized culture medium, and carrying out shake culture at 32 ℃ for 72h to obtain a fermentation liquid. Adding 3 times volume of 95% ethanol-100% methanol-isopropanol mixture (volume ratio 1:1:1) into the fermentation broth, precipitating, filtering, and drying at 45 deg.CThus, 14.83g of crude polysaccharide was obtained. Re-dissolving 10g of crude polysaccharide in deionized water to a final concentration of 15g/L, adding a 1.1-time volume of chloroform-phenol-n-butanol mixed solution (volume ratio is 1:0.85:0.15), oscillating, standing for layering, collecting a water phase, re-adding the mixed solution, and repeating the operation for 5 times to obtain the deproteinized extracellular polysaccharide. The deproteinized polysaccharide is dialyzed for 48h by 30 times volume of deionized water through a semipermeable membrane with the molecular weight cutoff of 3000Da, then 95% ethanol-acetone mixed solution (volume ratio is 1:1) with 4 times volume of the dialyzed polysaccharide is added, and after precipitation and filtration, purified exopolysaccharide 6.47g can be obtained by drying at 50 ℃.

Example 6

Use of exopolysaccharides for regulating the rheological properties of aqueous solutions.

The purified exopolysaccharide was prepared as in example 5 and then dissolved in deionized water at room temperature to a final concentration of 6.5g/L and the solution viscosity was determined to be 3755mPa.s and noted as initial viscosity. Sequentially preparing 20g/L KCl, 40g/L NaCl and 80g/L MgCl₂And 100g/L CaCl₂The solution is prepared by respectively dissolving exopolysaccharide with 4 solutions at room temperature to a final concentration of 6.5g/L and measuring the viscosity, wherein the viscosity is 3525mPa.s, 3645mPa.s, 3042mPa.s and 3160mPa.s in sequence, and the viscosity is more than 80% of the initial viscosity. In addition, a mixed inorganic salt solution is prepared, in which KCl, NaCl, MgCl₂And CaCl₂The concentration is 25g/L, the exopolysaccharide is dissolved at room temperature to the final concentration of 6.5g/L, the viscosity value is determined to be 3055mPa.s, which is 81.4 percent of the initial concentration.

FIG. 7 shows that NaCl, KCl and CaCl with different concentrations were added to the solution at room temperature₂And MgCl₂The viscosity of the extracellular polysaccharide solution changes. 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 100g/L, and Ca²⁺And Mg²⁺The solution is not caused to form gel, which is greatly helpful for unit operation under high salt conditions. It is known that exopolysaccharides produced by paenibacillus agri NUST16 have a good effect on regulating the rheological properties of aqueous solutions, and particularly when the solutions contain high concentrations of salt ions, the solution viscosity can be significantly increased and kept stable.

Example 7

Use of exopolysaccharides produced by Paenibacillus Agrobacterium NUST16 for modulating the rheological properties of aqueous solutions.

The purified solid exopolysaccharide was prepared as in example 5, the exopolysaccharide was dissolved with deionized water to a final concentration of 5g/L at room temperature and the viscosity was determined to be 2760mpa.s and recorded as the initial viscosity. 500mL of the polysaccharide solution was placed in a boiling water bath for 10min, then quickly removed and cooled to room temperature (25 ℃ C.), after cooling the polysaccharide solution was fluid and had no gel formation, measured as a viscosity of 19640mPa.s, which was 7.08 times the initial viscosity. The solution was placed again in a boiling water bath for 10min and the viscosity of the solution was reduced, then cooled to room temperature and determined to be 20100mpa.s, and repeated 3 more times, with consistent observed phenomena, in order of 19950mpa.s, 19970mpa.s and 20550 mpa.s. After 5 times of heating-cooling circulation operation, the viscosity of the polysaccharide solution is kept stable, the solution is clear and transparent, no color change occurs, and no thermally irreversible gel is formed all the time.

FIG. 8 shows the viscosity change of 1g/L exopolysaccharide solution after heat treatment at 95 deg.C for 10min and cooling to room temperature. It can be seen that the solution viscosity increased from 235mpa.s to 850mpa.s after a simple heat treatment, which was 3.62 times the viscosity before the treatment. After 5 repetitions, the viscosity remained stable. FIG. 9 shows the viscosity change of 5g/L exopolysaccharide solution after heat treatment at 100 deg.C for 10min and cooling to room temperature. It can be seen that the solution viscosity increased from 2760mpa.s to 19640mpa.s after a simple heat treatment, which was 7.08 times the viscosity before the 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 known that the extracellular polysaccharide generated by the paenibacillus agri NUST16 is added into the solution, the viscosity of the solution can be further improved by times through simple heating-cooling operation, and the solution can be kept stable after repeated viscosity for many times without forming heat irreversible gel, which indicates that the extracellular polysaccharide has stable property and wider application value.

SEQUENCE LISTING

<110> Nanjing university of science and technology

<120> polysaccharide, preparation method and application thereof

<130> 666

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1241

<212> DNA

<213> Paenibacillus edaphicus

<400> 1

gggtgagtaa cacgtaggca acctgcctga aggatcggga taactaccgg aaacggtagc 60

taagaccgga tagctggctc tggtgcatgc cggagtcatg aaacacggag caatctgtgg 120

cctttggatg ggcctgcggt gcattagcta gttggtgggg taacggctca ccaaggcgac 180

gatgcatagc cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact 240

cctacgggag gcagcagtag ggaatcttcc gcaatggacg caagtctgac ggagcaacgc 300

cgcgtgagtg atgaaggttc tcggatcgta aagctctgtt gccagggaag aacgtcgtgg 360

ggagtaactg ccctgcgaat gacggtacct gagaagaaag ccccggctaa ctacgtgcca 420

gcagccgcgg taatacgtag ggggcaagcg ttgtccggaa ttattgggcg taaagcgcgc 480

gcaggcggtt cattaagttt ggtgtttaag cccggggctc aaccccggtt cgcactgaaa 540

actggtgaac ttgagtgcag gagaggaaag cggaattcca cgtgtagcgg tgaaatgcgt 600

agagatgtgg aggaacacca gtggcgaagg cggctttctg gactgtaact gacgctgagg 660

cgcgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc gtaaacgatg 720

agtgctaggt gttaggggtt tcgataccct tggtgccgaa gtaaacacaa taagcactcc 780

gcctggggag tacgctcgca agagtgaaac tcaaaggaat tgacggggac ccgcacaagc 840

agtggagtat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatccc 900

cctgaaagcc ccagagatgg ggccctcctt cgggacaggg gagacaggtg gtgcatggtt 960

gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgaac 1020

ttagttgcca gcattcagtt gggcactcta agttgactgc cggtgacaaa ccggaggaag 1080

gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg tactacaatg 1140

gccggtacaa cgggaagcga agtcgcgaga tggagcgaat ccttacaagc cggtctcagt 1200

tcggattgca ggctgcaact cgcctgcatg aagtcggaat t 1241

Claims

1. A polysaccharide having the formula:

n is 50-2000.

2. The method for preparing polysaccharide according to claim 1, comprising the following steps:

3. The method for producing the polysaccharide of claim 2, wherein the culture time in step 1 is 12 to 48 hours; in the step 2, the culture time is 48-96 h; in the step 3, the volume ratio of the precipitant A to the fermentation liquor is 2-4: 1, and the precipitant A is a mixed solution formed by mixing more than two of 95-100% of ethanol, 95-100% of methanol, 95-100% of isopropanol and 95-100% of acetone according to equal volume.

4. The method for preparing the polysaccharide of claim 2, wherein the medium comprises sucrose 5-50g/L, peptone 0.5-5g/L, NaH₂PO₄ 0.1-5.0g/L，CaCl₂ 0.01-0.5g/L，MgCl₂ 0.01-0.5g/L，KCl 0.01-0.5g/L，FeCl₂ 0.001-0.05g/L，CuSO₄ 0.001-0.05g/L，MnSO₄ 0.001-0.05g/L，ZnCl₂ 0.001-0.05g/L，CoCl₂0.001-0.05g/L and pH 6.0-9.0.

5. The method for producing the polysaccharide of claim 4, wherein the pH of the medium is 7.0 to 9.0.

6. The method of producing the polysaccharide of claim 2, further comprising the steps of:

7. The method for preparing polysaccharide according to claim 6, wherein in step 4, the concentration of the crude polysaccharide solution is 0.25-20 g/L, the volume ratio of the crude polysaccharide solution to the treatment solution B is 1 (1-2), and the volume ratio of the trichloromethane, the phenol and the n-butanol in the treatment solution B is 1 (0.5-1) to 0.05-0.5.

8. Use of a polysaccharide according to claim 1 for the adjustment of the rheological properties of an aqueous solution.

9. Use of a polysaccharide according to claim 8 as an emulsifier, thickener, stabilizer or gelling agent for the adjustment of rheological properties of aqueous solutions.