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

A kind of soil series bacillus and its application Download PDF

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Publication number
CN106399199B
CN106399199B CN201610975024.8A CN201610975024A CN106399199B CN 106399199 B CN106399199 B CN 106399199B CN 201610975024 A CN201610975024 A CN 201610975024A CN 106399199 B CN106399199 B CN 106399199B
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series bacillus
soil series
application
exopolysaccharide
production
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CN106399199A (en
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王世明
陈向楠
李晶
程瑞
张建法
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Nanjing University of Science and Technology
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Nanjing University of Science and Technology
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20Bacteria; Culture media therefor
    • C12N1/205Bacterial isolates
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/01Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/04Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Abstract

The invention discloses a kind of soil series bacillus and its applications.The soil series bacillus is soil series bacillus NUST16, and deposit number is CCTCC No.M2016542.Soil series bacillus strain characteristics of the invention are unique, it is able to produce exocellular polysaccharide, yield is up to 15g/L, produced exocellular polysaccharide is easily isolated and purifies, and it is unique, it can be directly dissolved in cold water, the rheological equationm of state of aqueous solution can be adjusted, have the function of significantly increasing solution viscosity, is resistant to the Na of solution middle and high concentration+、K+、Ca2+And Mg2+, handling by simple heating-cooling can be such that solution viscosity several times increase, and property is stablized, and form heat-convertible gel.

Description

A kind of soil series bacillus and its application
Technical field
The invention belongs to microorganisms technical field, it is related to a kind of soil series bacillus and its application.
Background technique
Microbial exopolysaccharides can be used as thickener, suspending agent or stabilizer because of its special rheological equationm of state, be widely applied In industries such as food, cosmetics.Microbial exopolysaccharides with typical rheology characteristic such as xanthan gum (Xanthan gum) can Increase the viscosity of solution, and there is certain heat resistance, appropriateness increases viscosity kept stable after temperature;Gellan gum (Gelan gum) aqueous solution is in Ca2+Irreversible brittleness gel is capable of forming under existence condition;Thermal gels (Curdlan gum) It is not soluble in water, but its suspension be heated to 55 DEG C after be capable of forming the elastic gel of thermal reversion, can be formed after being heated to 80 DEG C The brittleness gel of heat irreversible;Weilan gum (Welan gum) has good heat-resisting property, has and is used as the oil exploitation displacement of reservoir oil The potentiality of agent.On the other hand, compared to plant polyose and polysaccharides of marine algae, Microbial exopolysaccharides have not climate shadow The advantages that sound, production technology are easy, at low cost, convenient for largely preparing (Williams P A, Phillips D L.Introduction to food hydrocolloids[M].WILLIAMS P A,PHILLIPS D L.Handbook of hydrocolloids.Woodhead publishing Ltd.2009.).It can be seen that more outside the extracellular microbial of function admirable Sugar has facilitation to the development of people's livelihood relevant industries.
At present there is certain limiting factor in Microbial exopolysaccharides in the application.For example, viscous to increase xanthan gum solution Degree can only can not be realized by improving the additive amount of xanthan gum by simple aftertreatment technology;There are Ca in solution2+Or Mg2 +Limit the usage amount of gellan gum;Heat treatment process with higher temperature then will receive the influence of thermal gels.Therefore, to biography System Microbial exopolysaccharides, which carry out chemical modification or discovery novel microbial exocellular polysaccharide, seems particularly necessary.
The study found that series bacillus microorganism belonging to genus has the ability for generating novel exocellular polysaccharide.Soil series bacillus Bacillus is classified as when being originally found, after renamed as soil series bacillus (Paenibacillus edaphicus).Soil series bacillus has the function of promotion plant growth, and main cause may be because the bacterium can The difficult potassium utilized in degradation and fixing soil, and then promote plant to utilization (the Sheng X F.Growth promotion of potassium 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 to this, it has also been found that soil series bacillus can generate thick extracellular pod membrane, (mono- plant of silicon of such as what beautiful jade swallow for research The identification of hydrochlorate bacterium and its phylogenetic analysis [J] microorganism journal, 2003,43 (2): 162-168.).It has been confirmed that It is same as the very close colloid series bacillus of soil series bacillus affiliation that there is the ability for generating pod membrane, and the pod Film is exocellular polysaccharide, and has preferable 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 may be speculated that soil class Bacillus has the ability for generating novel exocellular polysaccharide.
Summary of the invention
One of the objects of the present invention is to provide a kind of soil series bacillus Paenibacillus edaphicus NUST16 is able to produce the exopolysaccharide with the special rheological equationm of state.
Realize the technical solution of above-mentioned purpose are as follows:
A kind of soil series bacillus is Paenibacillus edaphicus NUST16, deposit number CCTCC No.M2016542, on October 08th, 2016 in China typical culture collection center (CCTCC) preservation, preservation address is Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University collection.
The second object of the present invention is to provide the cultural method of above-mentioned soil series bacillus NUST16, and specific steps are such as Under: soil series bacillus NUST16 is inoculated into after sterilizing, in the culture medium that pH is 6.0-9.0, is cultivated at 25-35 DEG C.
The third object of the present invention is to provide above-mentioned soil series bacillus answering in production exopolysaccharide With.
Further, the present invention provides above-mentioned soil series bacillus in the method for production exopolysaccharide, specifically Steps are as follows:
Step 1, soil series bacillus NUST16 is inoculated into the culture medium after sterilizing, 25-35 DEG C of shaking table concussion training It supports and forms 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 is shaken Bed shake culture, obtains the fermentation liquid of extracellular polysaccharide;
Step 3, precipitating reagent A is added in fermentation liquid, the precipitating in fermentation liquid is filtered or is centrifuged, up to solid after drying Thick many candies, the precipitating reagent A are 95%-100% ethyl alcohol, 95%-100% methanol, 95%-100% isopropanol and 95%- One or more of 100% acetone.
In step 1, the incubation time is 12-48h.
In step 2, the incubation time is 48-96h.
In step 3, the volume ratio of the precipitating reagent A and fermentation liquid is 2~4:1, and the precipitating reagent A is 95%- Two or more solvents in 100% ethyl alcohol, 95%-100% methanol, 95%-100% isopropanol and 95%-100% acetone by etc. Mixed solution made of volume mixture.
In the present invention, the group of the culture medium becomes sucrose 5-50g/L, peptone 0.5-5g/L, NaH2PO4 0.1- 5.0g/L, CaCl20.01-0.5g/L, MgCl20.01-0.5g/L, KCl 0.01-0.5g/L, FeCl2 0.001-0.05g/ L, CuSO40.001-0.05g/L, MnSO40.001-0.05g/L, ZnCl20.001-0.05g/L, CoCl2 0.001- 0.05g/L, pH 6.0-9.0.
Preferably, the pH of the culture medium is 7.0-9.0.
Further, above-mentioned soil series bacillus further includes walking as follows in the method for production exopolysaccharide It is rapid:
Step 4, by solid Thick many candies formation Thick many candies solution soluble in water, treatment fluid B is added and acutely shakes, standing or It is centrifugated organic phase and water phase, collects water phase, and rejoin treatment fluid B, repeats 3~10 times, purified polysaccharide can be obtained, it is described Treatment fluid B be chloroform, phenol and n-butanol mixed solution.
In step 4, the concentration of the Thick many candies solution is 0.25~20g/L, the Thick many candies solution and treatment fluid B Volume ratio 1:(1~2).
In step 4, in the treatment fluid B, the volume ratio of chloroform, phenol and n-butanol is 1:(0.5~1): (0.05~0.5).
Soil series bacillus strain characteristics of the invention are unique, are able to produce exocellular polysaccharide, yield is up to 15g/L, institute The exocellular polysaccharide of production is easily isolated and purifies and unique, can be directly dissolved in cold water, and has that significant to increase solution viscous The effect of degree is resistant to the Na of solution middle and high concentration+、K+、Ca2+And Mg2+, handling by simple heating-cooling can make Solution viscosity several times increase, and property is stablized, and form heat-convertible gel.
Detailed description of the invention
Fig. 1 is the flat-plate bacterial colony of soil series bacillus Paenibacillus edaphicus NUST16 bacterial strain and micro- Shape appearance figure.
Fig. 2 is the high-efficient liquid phase chromatogram of exocellular polysaccharide compound mensuration.
Fig. 3 is influence result figure of 4 kinds of inorganic salts to exocellular polysaccharide viscosity.
Fig. 4 is influence result figure of the Repeat-heating-cooling treatment to 1g/L exocellular polysaccharide solution viscosity.
Fig. 5 is influence result figure of the Repeat-heating-cooling treatment to 5g/L exocellular polysaccharide solution viscosity.
Specific embodiment
Below with reference to embodiment and attached drawing, the invention will be further described.
Embodiment 1
The separation and identification of soil series bacillus Paenibacillus edaphicus NUST16.
(1) separation of soil series bacillus Paenibacillus edaphicus NUST16
Soil series bacillus Paenibacillus edaphicus NUST16 of the invention is in the shore of Yancheng, Jiangsu Province It is isolated in extra large brown soil.The method wherein separated are as follows:
1. bacterial strain screening: pedotheque being divided into 10 groups, each component also known as takes 1g soil sample to be added to prepared liquid training It supports in base, is put into constant-temperature shaking incubator the culture 2d under the conditions of 200rpm, 30 DEG C and is enriched with, then from each group conical flask In take 1mL bacterium solution to be accessed in new fluid nutrient medium again, under the same terms cultivate 2d be enriched with again once.It is respectively taken from each group again Bacterium solution is added in 9mL sterile water after 1mL enrichment, and bacteria suspension is made in mixing, using 10 times of gradient dilutions, takes 10-6-10-4Dilution The bacteria suspension of degree is coated in solid medium, is cultivated at 30 DEG C.
2. bacterial strain purifies: coated plate culture 2d, after growing bacterium colony, according to the single bacterium of colony morphology characteristic picking It falls on new solid medium and crosses, cultivated at 30 DEG C, select single bacterium colony scribing line again after growing bacterium colony, this process repeats 3-4 times, until the bacterium colony that the bacterium colony grown on culture dish is single form makes bacterial strain purifying complete.Then it selects with nothing After the bacterium colony number of the extracellular polysaccharide feature such as the transparent or semitransparent, toughness of color, it is seeded in fluid nutrient medium respectively, 200rpm cultivates 2-4d under the conditions of 30 DEG C, if fermentation liquid becomes sticky, identifies whether cell free fermentation product belongs to polysaccharide.It is identified Post-fermentation product is that the bacterial strain of polysaccharide is then needed extracellular polysaccharide strains, is named as NUST16.
(2) identification of bacterial strain
A. morphological feature: on inorganic salts Solid agar culture 30 DEG C cultivate this bacterial strain 2-3 days after observe, bacterium colony is in circle Shape, colourless, translucent, surface is glossy, sticky, is not easy to provoke.The long rod shape of bacterial strain, a large amount of exocellular polysaccharide of secretion.
B. physiological and biochemical property: bacterial strain Gram-positive, aerobic, catalase reacting positive, using glucose as carbon source through fermentation Produce acid, well-grown when containing 1%NaCl in culture medium.The available carbon source of bacterial strain has sucrose, starch, glucose, lactose, fruit Sugared Utilization ability is weak, and thalli growth is best when using lactose as carbon source, and sucrose takes second place, and grows in organic nitrogen source peptone, Yield of extracellular polysaccharide is higher under the alkaline condition of pH7-9, and yield of extracellular polysaccharide can reach 15g/L after the 48h that ferments.High concentration NaCl It can inhibit strain growth and reduce yield of extracellular polysaccharide, bacterial strain does not secrete pigment.In Fig. 1, A is the bacterium on solid plate culture medium It falls, B is the photo of thallus under an optical microscope.
It c. is to draw with the universal primer of the PCR amplification of 16S rRNA gene using the genomic DNA of bacterial strain NUST16 as template Object carries out PCR amplification, measures its complete sequence, the 16S rDNA gene order of the bacterial strain is shown in sequence table.By the 16S rDNA of bacterial strain Gene order is committed to GenBank database (the GenBank number of logging in is KX962167), with the sequence in GenBank database Carry out online tetraploid rice, the results showed that, the sequence similarity of bacterial strain NUST16 and soil series bacillus AB045093 are high Up to 99%.
According to the morphology of bacterial strain NUST16, Physiology and biochemistry test and molecular biological analysis, bacterial strain NUST16 mirror It is set to soil series bacillus, is named as soil series bacillus NUST16.
Embodiment 2
The culture of soil series bacillus Paenibacillus edaphicus NUST16 and water-soluble extracellular in production Application in polysaccharide.
Prepare culture medium, sucrose 5g, peptone 0.5g, NaH2PO40.1g, CaCl20.01g, MgCl20.01g, KCl 0.01g, FeCl20.001g, CuSO40.001g, MnSO40.001g, ZnCl20.001g, CoCl20.001g is dissolved in 1L and goes In ionized water, pH 6.0, cooling obtains solid medium after 1% nutrient agar, 121 DEG C of sterilizing 15min are added.In agar solid It is cultivated after this bacterial strain 48h it can be seen that there is bacterium colony to grow for 30 DEG C on culture medium, bacterium colony is rounded, and translucent, surface is glossy, glues It is thick, it is not easy to provoke, a large amount of exocellular polysaccharide of strain secretes.The bacterium colony 30mL deionized water dissolving on every 9cm plate is scraped, Then it is centrifuged 20min through 5000 × g, 60mL ethyl alcohol-isopropanol mixed liquor (volume ratio 1:1) precipitation at room temperature, warp is added in supernatant Precipitating is collected by filtration and after 40 DEG C of dryings up to Thick many candies.
Fig. 2 is the high-efficient liquid phase chromatogram of exocellular polysaccharide composition, and same component retention time is identical.Wherein Fig. 2A is 10 kinds Elution curve of the standard list carbohydrates and their derivative after PMP derivatization in efficient liquid phase;Fig. 2 B is soil series bacillus Hydrolysate of the extracellular polysaccharide of NUST16 after trifluoroacetic acid complete hydrolysis is bent through elution of the PMP derivatization in efficient liquid phase Line, there is no overlappings between each eluting peak.By comparing it can be found that D-MANNOSE (D-Man), D-Glucose aldehydic acid (D- GlcUA), D-Glucose (D-Glc), D- galactolipin (D-Gal) and L-fucose (L-Fuc) retention time in A figure and B figure are complete It is complete consistent.It quantitatively can be calculated D-Glucose, D-MANNOSE, L- rock algae in the extracellular polysaccharide of soil series bacillus NUST16 Sugar, D- galactolipin and D-Glucose aldehydic acid molar ratio are 3:3:2:1:1, another kind statement are as follows: Glc:Man:Fuc:Gal:GlcUA =3:3:2:1:1.The polysaccharide is heteroglycan.
Embodiment 3
The culture of soil series bacillus Paenibacillus edaphicus NUST16 and water-soluble extracellular in production Application in polysaccharide.
Prepare culture medium, sucrose 50g, peptone 5g, NaH2PO45g, CaCl20.5g, MgCl20.5g, KCl 0.5g, FeCl20.05g, CuSO40.05g, MnSO40.05g, ZnCl20.05g, CoCl20.05g is dissolved in 1L deionized water, pH Cooling is spare after being 7.0,121 DEG C of sterilizing 20min, and cooling obtains accordingly admittedly after 1% nutrient agar, 121 DEG C of sterilizing 15min are added Body culture medium.The soil series bacillus NUST16 single bacterium grown on picking Solid agar culture drops down onto the culture medium after sterilizing In, 25 DEG C of shake cultures form seed culture fluid for 24 hours, then 7% seed culture fluid is added in this spare culture medium 35 DEG C Shake culture 48h obtains fermentation liquid.95% ethanol-acetone mixed liquor (volume ratio 1:1) of 3.5 times of volumes is added to fermentation liquid, Then shaken at room temperature collects precipitating through 5000 × g centrifugation 20min, up to Thick many candies 10.08g after 50 DEG C of dryings.
Embodiment 4
The culture of soil series bacillus Paenibacillus edaphicus NUST16 and exopolysaccharide Production and purifying.
Prepare culture medium, sucrose 20g, peptone 3g, NaH2PO43g, CaCl20.05g, MgCl20.15g, KCl 0.15g, FeCl20.03g, CuSO40.003g, MnSO40.05g, ZnCl20.005g, CoCl20.01g is dissolved in 1L deionization In water, cooling is spare after 9.0,121 DEG C of sterilizing 20min of pH;The soil series bacillus grown on picking solid medium NUST16 single bacterium drops down onto the culture medium after sterilizing, and 35 DEG C of shake culture 36h form seed culture fluid, then 5% seed is trained Nutrient solution is added to 32 DEG C of shake culture 72h in the culture medium after sterilizing, obtains fermentation liquid.3 times of volumes are added to fermentation liquid - 100% methanol-isopropanol mixed liquor (volume ratio 1:1:1) of 95% ethyl alcohol, precipitates and filters, up to Thick many candies after 45 DEG C of dryings 14.83g.Thick many candies 10g is redissolved in deionized water to final concentration 15g/L, chloroform-benzene of 1.1 times of volumes is then added Phenol-n-butanol mixed liquor (volume ratio 1:0.85:0.15) oscillation is then allowed to stand layering, collects water phase and rejoins above-mentioned mixing Liquid, repetitive operation 5 times, the exocellular polysaccharide of available deproteinized.The deionized water of resulting 30 times of volumes of deproteinized polysaccharide Through molecular cut off 3000Da semi-permeable membrane dialysis 48h, 4 times of 95% ethanol-acetones of volume are then added into the polysaccharide after dialysis Mixed liquor (volume ratio 1:1), after precipitating and filtering, exocellular polysaccharide 6.47g that 50 DEG C of dryings can must purify.
Embodiment 5
The exocellular polysaccharide that soil series bacillus NUST16 is generated is adjusting the application in the aqueous solution rheological equationm of state.
By the exocellular polysaccharide of the preparation purifying of embodiment 4, then at room temperature, with the deionized water dissolving polysaccharide to final concentration of 6.5g/L, solution viscosity is 3755mPa.s after measured, is denoted as initial viscosity.Successively compound concentration be respectively 20g/L KCl, 40g/L NaCl、80g/L MgCl2With 100g/L CaCl2Solution dissolves exocellular polysaccharide extremely with 4 kinds of solution under room temperature respectively Final concentration 6.5g/L simultaneously measures viscosity, and viscosity is followed successively by 3525mPa.s, 3645mPa.s, 3042mPa.s and 3160mPa.s, glues Degree is more than the 80% of initial viscosity.In addition, preparing mixed inorganic salt solution, wherein KCl, NaCl, MgCl2And CaCl2Concentration is equal For 25g/L, exocellular polysaccharide is dissolved at room temperature to final concentration 6.5g/L, and measurement viscosity number is 3055mPa.s, is initial concentration 81.4%.
Fig. 3 is NaCl, KCl, the CaCl for adding various concentration respectively into solution under room temperature2And MgCl2When it is extracellular more Sugar juice viscosity change situation.As can be seen that polysaccharide solution viscosity can still be maintained initially when salinity is up to 100g/L 80% or more of viscosity, and Ca2+And Mg2+Solution will not be made to form gel, this has greatly the unit operation under high salt conditions It helps.Preferably make it is found that the exocellular polysaccharide that soil series bacillus NUST16 is generated has on adjusting the aqueous solution rheological equationm of state With, especially when in solution contain high salt concentration ion when, still be able to dramatically increase solution viscosity and keep stable.
Embodiment 6
The exocellular polysaccharide that soil series bacillus NUST16 is generated is adjusting the application in the aqueous solution rheological equationm of state.
By the solid exocellular polysaccharide of the preparation purifying of embodiment 4, at room temperature with deionized water dissolving exocellular polysaccharide to final concentration 5g/L, and measuring viscosity is 2760mPa.s, and is denoted as initial viscosity.500mL polysaccharide solution is taken to be placed in 10min in boiling water bath, so It takes out rapidly and is cooled to room temperature (25 DEG C) afterwards, polysaccharide solution has mobility after cooling, and not formed gel, being measured viscosity is 19640mPa.s is 7.08 times of initial viscosity.The solution is replaced in 10min solution viscosity in boiling water bath to decline, then Being cooled to room temperature measurement viscosity is 20100mPa.s, continues to be repeated 3 times, observed phenomenon is consistent, and viscosity is followed successively by 19950mPa.s, 19970mPa.s and 20550mPa.s.Through 5 heating-cooling circulate operations, polysaccharide solution viscosity keeps steady It is fixed, solution clear, no color change, and not formed heat irreversible gel always.
The viscosity change situation that the exocellular polysaccharide solution that Fig. 4 is 1g/L is cooled to room temperature after being heat-treated 10min through 95 DEG C.It can To find out, after being simply heat-treated, solution viscosity is improved by 235mPa.s to 850mPa.s, and viscosity is 3.62 before processing Times.After 5 repetitive operations, viscosity keeps stablizing.Fig. 5 is the exocellular polysaccharide solution of 5g/L after 100 DEG C of heat treatment 10min The viscosity change situation being cooled to room temperature.As can be seen that after being simply heat-treated, solution viscosity by 2760mPa.s improve to 19640mPa.s, viscosity are 7.08 times before processing.After 5 repetitive operations, viscosity still maintains stable.This characteristic can Polysaccharide usage amount is greatly reduced, or reduces unit operation difficulty when pre-treatment.It is found that soil series bacillus NUST16 The exocellular polysaccharide of generation is added in solution, can further increase exponentially solution viscosity by the operation of simple heating-cooling, And it keeps stablizing by viscosity is repeated several times, not will form heat irreversible gel, illustrate that the exocellular polysaccharide property is stablized, have Relatively broad application value.
SEQUENCE LISTING
<110>Institutes Of Technology Of Nanjing
<120>a kind of soil series bacillus and its application
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<170> PatentIn version 3.5
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<212> DNA
<213> Paenibacillus edaphicus
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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 (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).
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