CN113755407B - Paenibacillus mucilaginosus, extracellular polysaccharide prepared by same and application of extracellular polysaccharide in preparation of microbial flocculant - Google Patents

Abstract

The invention belongs to the preparation of microbial polysaccharide, and in particular relates to bacillus mucilaginosus, extracellular polysaccharide prepared by the bacillus mucilaginosus and application of the extracellular polysaccharide in the preparation of microbial flocculant. The Latin name is Paenibaci llus mucilaginosus, and the collection number is CGMCC No.23105, and the Paenibacillus mucilaginosus is inoculated into an activation culture medium for activation; inoculating the activated strain into a fermentation medium containing carbon source, nitrogen source and nutrient elements, culturing under stirring to obtain fermentation liquor, and collecting extracellular polysaccharide from the fermentation liquor. The invention solves the technical problems of large dosage, low flocculation capacity and the like of the microbial flocculant prepared by the prior art, and has the advantages of good flocculation capacity of the prepared extracellular polysaccharide, easy control of fermentation process, short period and the like.

Description

Paenibacillus mucilaginosus, extracellular polysaccharide prepared by same and application of extracellular polysaccharide in preparation of microbial flocculant

Technical Field

The invention belongs to the preparation of microbial polysaccharide, and in particular relates to bacillus mucilaginosus, extracellular polysaccharide prepared by the bacillus mucilaginosus and application of the extracellular polysaccharide in the preparation of microbial flocculant.

Background

Flocculation is a solid-liquid separation technology with low cost and simple operation, and is widely applied in the fields of water quality control, fermentation recovery, impurity removal and the like. With the development and population growth of modern society, the importance of flocculation becomes increasingly apparent: at present, various untreated or improperly treated wastewater discharge amounts far exceed the bearing capacity of natural environment, such as Yangtze river basin with rich total water resources, besides Yangtze river dry flow and large reservoirs with strong self-cleaning capacity of water bodies, most lakes, small reservoirs and plain water nets have the problem of water quality deficiency, and according to estimation, 58% of water returns to natural water areas in the form of polluted water, so that the water quality is difficult to improve, and flocculation technology can be used for in-situ protection and restoration of ecological environment and landscape, and also can be used for water quality treatment of drinking water plants and sewage plants, removal of sand, humus and part of pathogenic bacteria, or treatment of wastewater from sugarcane industry, animal husbandry and other sources; based on the aggregation effect of flocculation on organics and microbial cells, the industries such as microalgae fermentation, monoclonal antibody production and the like have the same requirement on flocculation, and are used for solving a key bottleneck of limiting the microbial culture and fermentation industry, namely the high cost of harvesting products from a liquid culture medium; the flocculation technology can also be used for the extraction process of plant small molecules, and can remove macromolecular impurities such as polysaccharide, protein and the like.

The microbial flocculant is a natural high molecular polymer with flocculation activity produced by a microbial fermentation technology, and the microbial flocculant mainly exists in the form of an extracellular polymer taking polysaccharide, protein and nucleic acid as main components and has the characteristics of safety, biodegradability and the like.

Compared with common inorganic polymer flocculant, such as polyacrylamide and its derivative, the microbial flocculant has biocompatibility and no secondary pollution, such as cancer and neurotoxicity. The microbial flocculant with polysaccharide as a main component has the structural characteristics of high molecular weight and various functional groups, can adsorb inorganic matters and organic matters, destabilize the inorganic matters and aggregate the organic matters, realize solid-liquid separation, has outstanding flocculation activity and color removal capability, is generally nontoxic and harmless, has biodegradability, is expected to be a substitute of artificial polymer flocculant, and is a hot spot direction for developing novel green flocculant. But the microbial flocculant has the characteristics of low yield, large dosage and low flocculation efficiency. Research shows that the higher the uronic acid content in the polysaccharide, the larger the molecular weight, the higher the flocculation activity.

The background art searched by the applicant includes:

the method for producing the microbial polysaccharide fermentation liquor by using the paenibacillus mucilaginosus disclosed in the patent document with the publication number of CN102952834 has a fermentation period of 60 hours, and the polysaccharide content in the fermentation liquor can reach 7.5g/L by adopting an anthrone colorimetric method. The structure of the polysaccharide was not examined.

Patent document with publication number of CN102628065 discloses a production method and application of a microbial flocculant, which utilizes geotrichum candidum seeds to ferment for 3-5 days to obtain fermentation liquor, the fermentation period is long, the structure of polysaccharide is not detected, 0.1g of the microbial flocculant is needed to be added into 50ml of karst water, and the addition amount of the microbial flocculant is large.

Applicant has no literature report in the prior art that is the same or similar to the present application.

Disclosure of Invention

The invention aims to provide paenibacillus mucilaginosus.

It is a second object of the present invention to provide an extracellular polysaccharide prepared by using Paenibacillus mucilaginosus.

The third object of the present invention is to provide a method for producing an extracellular polysaccharide.

The fourth object of the invention is to provide the application of extracellular polysaccharide in the preparation of microbial flocculant.

The invention has the following overall technical concept:

paenibacillus mucilaginosus, the Latin name of the strain is Paenibacillus mucilagino sus, and the preservation number is CGMCC No.23105.

The strain in the invention is submitted to China general microbiological culture collection center (CGMCC) with the collection number of CGMCC No.23105 in the North-West-Lu No.1 and No. 3 of the North-Yangyang area of Beijing city at the 8 th month 2 day of 2021. The classification is suggested as Paenibacillus mucilaginosus Paenibacillus mucilaginosus.

The strain is obtained by sampling, separating and purifying the wetland of the Heshui lake in Hebei province by the applicant, and the spore is elliptic, and the sporangium is slightly expanded and is approximate to the center. The colony is round, transparent and convex, and is sticky.

The cell morphology and physical and chemical characteristics of the strain in the invention are as follows:

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extracellular polysaccharide prepared by using bacillus mucilaginosus comprises the following chemical components: glucose: galactose: glucuronic acid molar ratio = 2:1:2:1, the structural formula of the polysaccharide is as follows:

the structural general formula is as follows:

→{3)-α-D-Manp-(1→3)-4-Suc-β-D-Manp-(1→3)-β-D-Glcp-(1→4) -β-D-GlcpA-(1→4)[4，6-Pyr-β-D-Galp-(1→3)]-β-D-Galp-(1→} _n →

where n=10 ⁴ -10 ⁶ Suc, succinic acid group, pyr acid group.

A method for preparing an extracellular polysaccharide, the method comprising the steps of:

A. inoculating the Paenibacillus mucilaginosus with Latin name Paenibacillus mucilaginosus and preservation number of CGMCC No.23105 into an activation culture medium for activation;

B. inoculating the strain activated in the step A into a fermentation medium containing a carbon source, a nitrogen source and nutrient elements, and culturing under stirring to prepare a fermentation broth;

C. and (c) collecting extracellular polysaccharide from the fermentation broth prepared in the step (B).

The application of extracellular polysaccharide in the preparation of microbial flocculant.

The invention has the following concrete and technical conception:

the 16S rRNA gene sequence of the strain is shown as SEQ No.1.

In order to facilitate the industrialized production, the preferred technical implementation means is that the strain activated in the step A is inoculated into a seed culture medium for expanded culture to prepare seed liquid, and the seed liquid is inoculated into a fermentation culture medium for culture according to the inoculation amount of 4-10% by volume to prepare fermentation liquor.

In order to realize better activation effect, the more preferable technical realization means is that the Latin name is Paenibacillus mucilaginosus, the collection number is CGMCC No.23105, the bacillus mucilaginosus is inoculated to an activation culture medium, and the activation is carried out for 24-48 hours under the condition of 28-40 ℃.

In order to control the culture end point conveniently, the preferred technical implementation means is that the seed liquid is prepared under the condition that the OD value is more than or equal to 1.0 when the wavelength is 550 nm.

As the viscosity of the fermentation liquid is higher and higher along with the time in the fermentation process, the preferable technical implementation means is that the culture condition in the step B is that the temperature is 28-40 ℃, the stirring rotation speed is 100-300 r/min and the culture time is 16-50 hours in order to ensure the dissolved oxygen level in the fermentation process and to facilitate the growth of thalli and the synthesis of end products.

In order to conveniently control the fermentation end point in the culture process, the preferred technical implementation means is that the fermentation is stopped when the viscosity of the fermentation liquid in the step B is more than or equal to 500 cp.

In order to facilitate the extraction of the extracellular polysaccharide in the fermentation liquid, the preferred technical implementation means is that the extracellular polysaccharide in the fermentation liquid is settled by adopting an ethanol solution with the volume percentage concentration of 80-95%, and the volume ratio of the ethanol solution to the fermentation liquid is 2-4.

In order to facilitate the growth of the strain and the synthesis of the final product, a preferred technical implementation is that the culture medium used in step A, B comprises the following components:

sucrose 20 g/L-50 g/L; 1 g/L to 2.5 g/L of potassium nitrate; 1 g/L to 2 g/L of sodium dihydrogen phosphate; 0.01 g/L to 1 g/L of calcium chloride; 0.1 g/L to 1 g/L of magnesium sulfate heptahydrate; ferrous sulfate heptahydrate 0.01 g/l-0.05 g/l; manganese sulfate monohydrate 0.001 g/L to 0.01 g/L; zinc chloride 0.001 g/L-0.01 g/L; ph=6 to 8.

The essential characteristics and remarkable technical progress of the invention are as follows:

1. the invention provides a bacillus mucilaginosus which can synthesize extracellular polysaccharide with flocculation capability.

2. The preparation method of the extracellular polysaccharide is simple and feasible, and has short fermentation period and mild conditions.

3. The extracellular polysaccharide has the advantages of high uronic acid content and large molecular weight, and the dosage required by the applicant test to reach the equivalent flocculation effect (> 98%) is basically consistent with that of polyacrylamide, and is 1ppm for kaolin suspension and 0.5ppm to 1ppm for coal dust. Has better flocculation activity, and effectively solves the technical problems of low flocculation activity, large flocculant dosage and the like of the existing product.

Drawings

FIG. 1 is a photomicrograph of a plate colony of a strain of the invention.

FIG. 2 is an infrared chromatogram of an extracellular polysaccharide component assay of the present invention.

FIG. 3 is a liquid chromatogram of an extracellular polysaccharide component assay of the present invention.

FIG. 4 is a schematic diagram of an extracellular polysaccharide according to the present invention ¹³ C nuclear magnetic resonance spectrum.

Fig. 5 is a diagram showing the test of pulverized coal suspension at different times.

FIG. 6 is a 16S rRNA sequence listing of a strain of the present invention.

The strain in the invention is submitted to China general microbiological culture collection center (CGMCC) with the collection number of CGMCC No.23105 in the North-West-Lu No.1 and No. 3 of the North-Yangyang area of Beijing city at the 8 th month 2 day of 2021. The classification is suggested as Paenibacillus mucilaginosus Paenibacillus mucilaginosus.

In order to verify the structure and flocculation activity of the extracellular polysaccharide in the present invention, the applicant conducted the following experiments:

1. polysaccharide flocculant structural analysis

(1) Infrared spectroscopic detection

The infrared scanner used was the type NICOLETIS10 (Siemens technologies). And placing the pressed potassium bromide blank on a sample rack of a sample bin of the infrared scanner, confirming collection of a reference background spectrum, and then placing a sample to be detected into the spectrometer to scan the sample. The sample dosage is 2-5 mg each time, and the result is shown in figure 2:

the infrared spectrum result shows that 3000-3700 cm ^-1 The broad absorption peaks of (2) belong to hydroxyl groups in various environments in the polysaccharide molecule; 2931cm ^-1 The peaks from C-H stretching and bending vibrations; 1100-1200 cm ^-1 The peaks of (2) belong to the C-O-C linkage, i.e., the glycosidic bond in the polysaccharide.

(2) High performance liquid chromatography detection

The extracellular polysaccharide floccuronic acid prepared by the strain of the invention is subjected to high performance liquid phase analysis after being completely subjected to acid hydrolysis by trifluoroacetic acid, PMP derivatization, chloroform extraction and water phase filtration. FIG. 3 is a liquid chromatogram of a single sugar component assay. As can be seen from FIG. 3, the extracellular polysaccharide floccuronic acid prepared by the strain of the present invention is an acidic heteropolysaccharide, and is composed of mannose, glucose, galactose and glucuronic acid in a molar ratio of 2:1:2:1.

(3) Nuclear magnetic detection

The extracellular polysaccharide floccuronic acid prepared by the strain of the invention is partially hydrolyzed by trifluoroacetic acid and then is treated by D ₂ O was configured at 80mg/ml and NMR analysis was performed using TMSP as an internal standard. The one-dimensional nuclear magnetic carbon spectrum of the polysaccharide is shown in figure 4. In 13C NMR, there are 6 anomeric carbon signals and one sharp signal between delta 102-106ppm, indicating that floccuronic acid has 6 anomeric carbons and 1 pyruvate group. Chemical shifts 179.26ppm in 13C NMR were ascribed to the carboxyl carbon atom of glucuronic acid, 178.66ppm were ascribed to the carboxyl carbon atom of pyruvate, 183.91ppm and 178.52ppm were ascribed to succinyl groups, the other 2 carbon atoms of amber groups peaking at 34.76ppm and 33.26ppm, respectively.

In combination with the above analysis, it can be confirmed that the extracellular polysaccharide floccuronic acid produced by paenibacillus mucilaginosus (Paenibacillus mucilagino sus) CGMCC No.23105 has the following structural formula:

where n=10 ⁴ -10 ⁶ Suc, succinic acid group, pyr acid group.

2. Flocculation vitality test (turbidity removal rate) of polysaccharide flocculant

At present, kaolin suspension or coal dust suspension is usually used as simulated wastewater for the flocculation capability test, and the most widely applied inorganic flocculant polyacrylamide is used as a control group.

100mL of coal ash suspension (the mass concentration is 4 g/L) is prepared, a certain amount of flocculating agent is added for flocculation experiments, and the coal ash suspension without the flocculating agent is used as a blank. Magnetic stirring (rapid stirring for 3 min, slow stirring for 1 min, standing for 5 min), photographing to record the phenomenon, as shown in fig. 6, and measuring the change of absorbance. And (3) taking deionized water as a reference, sucking liquid at the position of 2cm below the liquid level, and measuring absorbance of a blank and flocculation system at 550nm to calculate flocculation activity. The flocculation activity calculation formula is:

flocculation activity= (a ₀ －A ₁ )/A ₀ ×100％

Wherein A is ₀ Absorbance of blank coal ash suspension, A ₁ The absorbance of the coal ash suspension of the experimental group. As a result, the flocculation activities of the extracellular polysaccharide floccur onic acid prepared by the strain of the present invention were 89.3%, 93.7% and 91.2%, respectively, at 0.5ppm, 1ppm and 2ppm, as shown in FIG. 5.

3. Dosage of flocculant

Through experimental comparison, the dosage required by the extracellular polysaccharide prepared by the invention to achieve the same flocculation effect (> 98%) is basically consistent with that of polyacrylamide, and the dosage is 1ppm for kaolin suspension and 0.5ppm to 1ppm for coal dust.

Detailed Description

The present invention is further described below with reference to examples, but the present invention is not limited thereto, and the claims of the present invention should be construed as being limited thereto, and any equivalent means according to the specification may be substituted without departing from the scope of the present invention.

Example 1

The method for preparing extracellular polysaccharide in this example comprises the following steps:

A. inoculating the Paenibacillus mucilaginosus with Latin name Paenibacillus mucilaginosus and preservation number of CGMCC No.23105 into an activation culture medium for activation;

B. inoculating the strain activated in the step A into a fermentation medium containing a carbon source, a nitrogen source and nutrient elements, and culturing under stirring to prepare a fermentation broth;

C. and (c) collecting extracellular polysaccharide from the fermentation broth prepared in the step (B).

The application of extracellular polysaccharide in the preparation of microbial flocculant.

The 16S rRNA gene sequence of the strain in this example is shown as SEQ No.1.

And B, inoculating the strain activated in the step A into a seed culture medium, performing expansion culture to obtain a seed solution, and inoculating the seed solution into a fermentation culture medium according to an inoculum size of 4% by volume to prepare a fermentation broth.

The activation in the step A is to inoculate the Paenibacillus mucilaginosus with the Latin name of Paenibacillus mucilaginosus and the preservation number of CGMCC No.23105 into an activation culture medium, and activate the Paenibacillus mucilaginosus for 24 hours at the temperature of 28 ℃.

The seed liquid is prepared under the condition that the wavelength is 550nm, and the culture of the seed liquid is stopped when the OD value is more than or equal to 1.0.

The culture conditions in the step B were 28℃and the stirring speed was 100 rpm, and the culture time was 16 hours.

And B, stopping fermentation when the viscosity of the fermentation liquid is more than or equal to 500 cp.

And C, adopting an ethanol solution with the volume percentage concentration of 80% to settle extracellular polysaccharide in the fermentation liquor, wherein the volume ratio of the ethanol solution to the fermentation liquor is 2.

The culture medium used in the step A, B comprises the following components:

sucrose 20 g/l; 1 g/l potassium nitrate; sodium dihydrogen phosphate 1 g/L; 0.01 g/l of calcium chloride; 0.1 g/l magnesium sulfate heptahydrate; ferrous sulfate heptahydrate 0.01 g/l; manganese sulfate monohydrate 0.001 g/l; zinc chloride 0.001 g/l; ph=6 to 8.

Example 2

This embodiment differs from embodiment 1 in that:

and B, inoculating the strain activated in the step A into a seed culture medium, performing expansion culture to obtain a seed solution, and inoculating the seed solution into a fermentation culture medium according to an inoculum size of 10% by volume to culture to obtain a fermentation broth.

The activation in the step A is to inoculate the Paenibacillus mucilaginosus with the Latin name of Paenibacillus mucilaginosus and the preservation number of CGMCC No.23105 into an activation culture medium, and activate the Paenibacillus mucilaginosus for 48 hours at the temperature of 40 ℃.

The culture conditions in the step B were a temperature of 40℃and a stirring speed of 300 rpm, and the culture time was 50 hours.

And C, adopting an ethanol solution with the volume percentage concentration of 95% to settle extracellular polysaccharide in the fermentation liquor, wherein the volume ratio of the ethanol solution to the fermentation liquor is 4.

The culture medium used in the step A, B comprises the following components:

sucrose 50 g/l; 2.5 g/l potassium nitrate; 2 g/l sodium dihydrogen phosphate; 1 g/l of calcium chloride; 1 g/L magnesium sulfate heptahydrate; ferrous sulfate heptahydrate 0.05 g/l; manganese sulfate monohydrate 0.01 g/l; zinc chloride 0.01 g/l; ph=6 to 8.

The remainder was the same as in example 1.

Example 3

This embodiment differs from embodiment 1 in that:

and B, inoculating the strain activated in the step A into a seed culture medium, performing expansion culture to obtain a seed solution, and inoculating the seed solution into a fermentation culture medium according to an inoculum size of 7% by volume to prepare a fermentation broth.

The activation in the step A is to inoculate the Paenibacillus mucilaginosus with the Latin name of Paenibacillus mucilaginosus and the preservation number of CGMCC No.23105 into an activation culture medium, and activate the Paenibacillus mucilaginosus for 36 hours at the temperature of 34 ℃.

The culture conditions in the step B were 34℃and the stirring speed was 200 rpm, and the culture time was 33 hours.

And C, adopting an ethanol solution with the volume percentage concentration of 88% to settle extracellular polysaccharide in the fermentation liquor, wherein the volume ratio of the ethanol solution to the fermentation liquor is 3.

The culture medium used in the step A, B comprises the following components:

sucrose 35 g/l; 1.7 g/l potassium nitrate; 1.5 g/l sodium dihydrogen phosphate; 0.5 g/l calcium chloride; 0.55 g/L magnesium sulfate heptahydrate; ferrous sulfate heptahydrate 0.03 g/l; manganese sulfate monohydrate 0.005 g/l; zinc chloride 0.005 g/l; ph=6 to 8.

The remainder was the same as in example 1.

Example 4

This embodiment differs from embodiment 1 in that:

and B, inoculating the strain activated in the step A into a seed culture medium, performing expansion culture to obtain a seed solution, and inoculating the seed solution into a fermentation culture medium according to an inoculum size of 5% by volume to prepare a fermentation broth.

The activation in the step A is to inoculate the Paenibacillus mucilaginosus with the Latin name of Paenibacillus mucilaginosus and the preservation number of CGMCC No.23105 into an activation culture medium, and activate for 30 hours at the temperature of 31 ℃.

The culture conditions in the step B were a temperature of 31℃and a stirring speed of 150 rpm, and the culture time was 25 hours.

And C, adopting an ethanol solution with the volume percentage concentration of 80-95% to settle extracellular polysaccharide in the fermentation liquor, wherein the volume ratio of the ethanol solution to the fermentation liquor is 2.5.

The culture medium used in the step A, B comprises the following components:

sucrose 25 g/l; 1.3 g/l potassium nitrate; 1.3 g/l sodium dihydrogen phosphate; 0.3 g/l of calcium chloride; 0.3 g/l magnesium sulfate heptahydrate; ferrous sulfate heptahydrate 0.02 g/l; manganese sulfate monohydrate 0.003 g/l; zinc chloride 0.003 g/l; ph=6 to 8.

The remainder was the same as in example 1.

Example 5

This embodiment differs from embodiment 1 in that:

and B, inoculating the strain activated in the step A into a seed culture medium, performing expansion culture to obtain a seed solution, and inoculating the seed solution into a fermentation culture medium according to an inoculum size of 9% by volume to culture to obtain a fermentation broth.

The activation in the step A is to inoculate the Paenibacillus mucilaginosus with the Latin name of Paenibacillus mucilaginosus and the preservation number of CGMCC No.23105 into an activation culture medium, and activate the Paenibacillus mucilaginosus for 42 hours at the temperature of 38 ℃.

The culture conditions in the step B were a temperature of 38℃and a stirring speed of 260 rpm, and the culture time was 37 hours.

And C, adopting an ethanol solution with the volume percentage concentration of 92% to settle extracellular polysaccharide in the fermentation liquor, wherein the volume ratio of the ethanol solution to the fermentation liquor is 3.5.

The culture medium used in the step A, B comprises the following components:

sucrose 40 g/l; 2.2 g/l potassium nitrate; 1.8 g/l sodium dihydrogen phosphate; 0.08 g/L calcium chloride; 0.8 g/L magnesium sulfate heptahydrate; ferrous sulfate heptahydrate 0.04 g/l; manganese sulfate monohydrate 0.008 g/l; zinc chloride 0.008 g/l; ph=6 to 8.

The remainder was the same as in example 1.

Sequence listing

<110> Hebei Fengchuan Biotech Co., ltd

<120> Paenibacillus mucilaginosus, extracellular polysaccharide prepared therefrom and application thereof in preparation of microbial flocculant

<130> CN102952834

<160> 1

<170> SIPOSequenceListing 1.0

<210> 2

<211> 1405

<212> DNA

<213> Paenibacillus mucilaginosus (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 2

cggctggctc ccttgcgggt taccccaccg gcttcgggtg ttgtaaactc tcgtggtgtg 60

acgggcggtg tgtacaagac ccgggaacgt attcaccgcg gcatgctgat ccgcgattac 120

tagcaattcc gacttcatgc aggcgagttg cagcctgcaa tccgaactga gaccggcttc 180

taaggattcg ctccatctcg cgacttcgct tcccgttgta ccggccattg tagtacgtgt 240

gtagcccagg tcataagggg catgatgatt tgacgtcatc cccaccttcc tccggtttgt 300

caccggcagt cactctagag tgcccaactc aatgctggca actaaagtca agggttgcgc 360

tcgttgcggg acttaaccca acatctcacg acacgagctg acgacaacca tgcaccacct 420

gtcacctctg tcccgaagga ggaccctatc tctagggctt tcagagggat gtcaagacct 480

ggtaaggttc ttcgcgttgc ttcgaattaa accacatact ccactgcttg tgcgggtccc 540

cgtcaattcc tttgagtttc actcttgcga gcgtactccc caggcggagt gcttattgtg 600

tttacttcgg caccaagggt atcgaaaccc ctaacaccta gcactcatcg tttacggcgt 660

ggactaccag ggtatctaat cctgtttgct ccccacgctt tcgcgcctca gcgtcagtta 720

cagtccagaa agccgccttc gccactggtg ttcctccaca tctctacgca tttcaccgct 780

acacgtggaa ttccgctttc ctctcctgca ctcaagtctt ccagtttccg gtgcgaaccg 840

gggttgagcc ccgggcttaa acaccagact taaaaaaccg cctgcgcgcg ctttacgccc 900

aataattccg gacaacgctt gccccctacg tattaccgcg gctgctggca cgtagttagc 960

cggggctttc ttctcaggta ccgtcattcg cagagcagtt actctccacg acattcttcc 1020

ctggcaacag agctttacga tccgaaaacc ttcatcactc acgcggcgtt gctccgtcag 1080

gcttgcgccc attgcggaag attccctact gctgcctccc gtaggagtct gggccgtgtc 1140

tcagtcccag tgtggccgat caccctctca ggtcggctac gcatcgtcgc cttggtgggc 1200

cgttaccccg ccaactagct aatgcgccgc aggcccatcc gtaagccaca ggttgccccg 1260

tgtttcatga ttccggcatg caccgaaacc agctatccgg tcttagctac cgtttccggt 1320

agttatcccg atcttacagg caggttgcct acgtgttact cacccgtccg ccgctaagca 1380

ccgaagtgct ccgctcgact tgcat 1405

Claims (11)

1. The bacillus mucilaginosus is characterized in that the Latin name of the strain is Paenibacillus mucilaginosus, and the preservation number is CGMCC No.23105.

2. Extracellular polysaccharide prepared by using the paenibacillus mucilaginosus according to claim 1, characterized in that the chemical composition of the polysaccharide is mannose: glucose: galactose: glucuronic acid molar ratio = 2:1:2:1, the structural formula of the polysaccharide is as follows:

the structural general formula is as follows:

→{3)-α-D-Manp-(1→3)-4-Suc-β-D-Manp-(1→3)-β-D-Glcp-(1→4)-β-D-GlcpA-(1→4)[4，6-Pyr-β-D-Galp-(1→3)]-β-D-Galp-(1→} _n where n=10 ⁴ -10 ⁶ Suc, succinic acid group, pyr acid group.

3. The method for preparing extracellular polysaccharide according to claim 2, characterized in that it comprises the following steps:

A. inoculating the Paenibacillus mucilaginosus with Latin name Paenibacillus mucilaginosus and preservation number of CGMCC No.23105 into an activation culture medium for activation;

B. inoculating the strain activated in the step A into a fermentation medium containing a carbon source, a nitrogen source and nutrient elements, and culturing under stirring to prepare a fermentation broth;

C. and (c) collecting extracellular polysaccharide from the fermentation broth prepared in the step (B).

4. The method for preparing extracellular polysaccharide according to claim 3, wherein the step B is characterized in that the strain activated in the step A is inoculated into a seed culture medium for expansion culture to prepare a seed liquid, and the seed liquid is inoculated into a fermentation culture medium for culture according to the inoculation amount of 4-10% by volume to prepare a fermentation liquid.

5. The method of preparing extracellular polysaccharide according to claim 3, wherein the activation in the step A is to inoculate Paenibacillus mucilaginosus with Latin name Paenibacillus mucilaginosus and preservation number CGMCC No.23105 into an activation culture medium, and activate for 24-48 hours at 28-40 ℃.

6. The method for preparing extracellular polysaccharide according to claim 4, wherein the seed liquid is prepared by terminating the seed liquid culture when the OD value is not less than 1.0 under the condition of 550nm wavelength.

7. The method for producing an extracellular polysaccharide according to claim 3, wherein the culturing conditions in the step B are a temperature of 28℃to 40℃and a stirring speed of 100 to 300 rpm, and the culturing time is 16 to 50 hours.

8. The method for preparing extracellular polysaccharide according to claim 3, 4 or 7, wherein the fermentation is terminated when the viscosity of the fermentation broth is not less than 500cp in the step B.

9. The method for preparing exopolysaccharide according to claim 3, wherein the exopolysaccharide in the fermentation broth is settled by using an ethanol solution with a volume percentage concentration of 80% -95% in the step C, and the volume ratio of the ethanol solution to the fermentation broth is 2-4.

10. The method for producing an extracellular polysaccharide according to any one of claims 3 to 7, wherein the medium used in step A, B comprises the following components:

sucrose 20 g/L-50 g/L; 1 g/L to 2.5 g/L of potassium nitrate; 1 g/L to 2 g/L of sodium dihydrogen phosphate; 0.01 g/L to 1 g/L of calcium chloride; 0.1 g/L to 1 g/L of magnesium sulfate heptahydrate; ferrous sulfate heptahydrate 0.01 g/l-0.05 g/l; manganese sulfate monohydrate 0.001 g/L to 0.01 g/L; zinc chloride 0.001 g/L-0.01 g/L; ph=6 to 8.

11. Use of an extracellular polysaccharide according to claim 2 in the preparation of a microbial flocculant.

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