CN106399199A - Soil paenibacillus and application thereof - Google Patents
Soil paenibacillus and application thereof Download PDFInfo
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- CN106399199A CN106399199A CN201610975024.8A CN201610975024A CN106399199A CN 106399199 A CN106399199 A CN 106399199A CN 201610975024 A CN201610975024 A CN 201610975024A CN 106399199 A CN106399199 A CN 106399199A
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, 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
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
Abstract
The invention discloses soil paenibacillus and application thereof. The soil paenibacillus is soil paenibacillus NUST16, and the preservation number is CCTCC No.M2016542. According to the soil paenibacillus, the strain is unique in property, exopolysaccharide can be produced, the yield can reach 15 g/L, and the produced exopolysaccharide is easy to separate and purify and unique in property, can be directly dissolved in cold water and regulate the rheological property of an aqueous solution, has the significant effect of increasing the viscosity of the solution, can resist high-concentration Na<+>, K<+>, Ca<2+> and Mg<2+> in the solution and increase the viscosity of the solution by multiple times through simple heating-cooling treatment, is stable in property and can form thermo reversible gel.
Description
Technical field
The invention belongs to microbial technology field, it is related to a kind of soil series bacillus and its application.
Background technology
Microbial exopolysaccharides, because of its special rheological equationm of state, extensively can be applied as thickening agent, suspending agent or stabilizer
In industries such as food, cosmetics.The Microbial exopolysaccharides such as xanthan gum (Xanthan gum) with typical rheology characteristic 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 fragility gel can be formed under existence condition；Thermal gels (Curdlan gum)
Water insoluble, but its suspension can form the elastic gel of thermal reversion after being heated to 55 DEG C, can be formed after being heated to 80 DEG C
The fragility gel of heat irreversible；Weilan gum (Welan gum) has good heat-resisting property, has as the oil exploitation displacement of reservoir oil
The potentiality of agent.On the other hand, compared to vegetable polysaccharidess and polysaccharides of marine algae, Microbial exopolysaccharides have not climate shadow
Sound, production technology simplicity, low cost, it is easy to the advantages of prepare in a large number (Williams P A, Phillips D
L.Introduction to food hydrocolloids[M].WILLIAMS P A,PHILLIPS D L.Handbook of
hydrocolloids.Woodhead publishing Ltd.2009.).As can be seen here, many outside the extracellular microbial of function admirable
Sugar has facilitation to the development of people's livelihood relevant industries.
There is certain limiting factor in the application in Microbial exopolysaccharides at present.Such as, glue for increasing xanthan gum solution
Degree cannot can only be realized by simple aftertreatment technology by improving the addition of xanthan gum；There is Ca in solution2+Or Mg2 +Limit the usage amount of gellan gum；The heat treatment process with higher temperature then can be affected by thermal gels.Therefore, to biography
System Microbial exopolysaccharides carry out chemical modification or find that novel microbial extracellular polysaccharide seems particularly necessary.
Research finds, series bacillus microorganism belonging to genus has the ability producing new extracellular polysaccharide.Soil series bacillus
Be classified as bacillus when being originally found, after renamed as soil series bacillus (Paenibacillus
edaphicus).Soil series bacillus have the effect promoting plant growing, and its main cause is possibly due to this bacterium can
The difficult potassium utilizing in degraded fixing soil, and then promote utilization (the Sheng X F.Growth promotion to potassium for the plant
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, research also find soil series bacillus can produce thickness extracellular pod membrane (what beautiful jade swallow etc. one plant of silicon
The identification of hydrochlorate antibacterial and its phylogenetic analysis [J]. microorganism journal, 2003,43 (2):162-168.).Have confirmed
The colloid series bacillus very near with soil series bacillus sibship equally have the ability producing pod membrane, and this pod
Film is extracellular 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 cereuss have the ability producing new extracellular polysaccharide.
Content of the invention
An object of the present invention is to provide a kind of soil series bacillus Paenibacillus edaphicus
NUST16, can produce the exopolysaccharide with the special rheological equationm of state.
The technical scheme realizing above-mentioned purpose is：
A kind of soil series bacillus, are Paenibacillus edaphicus NUST16, and deposit number is 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 concrete steps are such as
Under：Soil series bacillus NUST16 is inoculated into after sterilizing, pH is in the culture medium of 6.0-9.0, cultivates at 25-35 DEG C.
The third object of the present invention is to provide above-mentioned soil series bacillus answering in producing exopolysaccharide
With.
Further, the present invention provides the method producing exopolysaccharide for the above-mentioned soil series bacillus, specifically
Step is as follows：
Step 1, soil series bacillus NUST16 is inoculated in the culture medium after sterilizing, 25-35 DEG C of shaking table concussion training
Support and form seed culture fluid；
Step 2, by 0.5%-20% inoculum concentration, seed culture fluid is inoculated in the culture medium after sterilizing, and 25-35 DEG C is shaken
Bed concussion and cultivate, obtains the fermentation liquid of extracellular polysaccharide；
Step 3, adds precipitant A in fermentation liquid, and the precipitation in fermentation liquid, through filtering or being centrifuged, obtains final product solid after being dried
Crude polysaccharides, described precipitant A is 95%-100% ethanol, 95%-100% methanol, 95%-100% isopropanol and 95%-
One of 100% acetone or multiple.
In step 1, described incubation time is 12-48h.
In step 2, described incubation time is 48-96h.
In step 3, described precipitant A is 2～4 with the volume ratio of fermentation liquid:1, described precipitant A is 95%-
Two or more solvents in 100% ethanol, 95%-100% methanol, 95%-100% isopropanol and 95%-100% acetone press etc.
The mixed solution of volume mixture.
In the present invention, described culture medium consist of sucrose 5-50g/L, peptone 0.5-5g/L, NaH2PO40.1-
5.0g/L, CaCl20.01-0.5g/L, MgCl20.01-0.5g/L, KCl 0.01-0.5g/L, FeCl20.001-0.05g/
L, CuSO40.001-0.05g/L, MnSO40.001-0.05g/L, ZnCl20.001-0.05g/L, CoCl20.001-
0.05g/L, pH are 6.0-9.0.
Preferably, the pH of described culture medium is 7.0-9.0.
Further, above-mentioned soil series bacillus, in the method producing exopolysaccharide, also include walking as follows
Suddenly：
Step 4, formation crude polysaccharides solution soluble in water for solid crude polysaccharides adds treatment fluid B acutely shaking, stand or
Centrifugation organic faciess and aqueous phase, collect aqueous phase, and rejoin treatment fluid B, repeat 3～10 times, can obtain purified polysaccharide, described
Treatment fluid B be chloroform, phenol and n-butyl alcohol mixed solution.
In step 4, the concentration of described crude polysaccharides solution is 0.25～20g/L, described crude polysaccharides solution and treatment fluid B
Volume ratio 1:(1～2).
In step 4, in described treatment fluid B, the volume ratio of chloroform, phenol and n-butyl alcohol is 1:(0.5～1):
(0.05～0.5).
The soil series bacillus strain characteristics of the present invention are unique, can produce extracellular polysaccharide, yield is up to 15g/L, institute
The extracellular polysaccharide producing is easily isolated with purification and unique, can be directly dissolved in cold water, and has the significant solution that increases and glue
The effect of degree, is resistant to the Na of solution middle and high concentration+、K+、Ca2+And Mg2+, process and can make through simple heating-cooling
Solution viscosity several times increase, and stable in properties, form heat-convertible gel.
Brief description
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 extracellular polysaccharide compound mensuration.
Fig. 3 is the impact result figure to extracellular polysaccharide viscosity for 4 kinds of inorganic salts.
Fig. 4 is the impact result figure to 1g/L extracellular polysaccharide solution viscosity for the Repeat-heating-cooling treatment.
Fig. 5 is the impact result figure to 5g/L extracellular polysaccharide solution viscosity for the Repeat-heating-cooling treatment.
Specific embodiment
With reference to embodiment and accompanying drawing, the invention will be further described.
Embodiment 1
The separation of soil series bacillus Paenibacillus edaphicus NUST16 and identification.
(1) separation of soil series bacillus Paenibacillus edaphicus NUST16
The soil series bacillus Paenibacillus edaphicus NUST16 of the present invention is the shore in Yancheng, Jiangsu Province
Separate in extra large brown soil and obtain.Wherein detached method is：
1. bacterial strain screening：Pedotheque is divided into 10 groups, each group weighs 1g soil sample respectively and is added to the liquid training preparing
In foster base, put in 200rpm in constant-temperature shaking incubator, under the conditions of 30 DEG C, culture 2d is enriched with, then from each group conical flask
In take 1mL bacterium solution to access in new fluid medium again, under the same terms culture 2d be enriched with again once.Respectively take from each group again
After 1mL enrichment, bacterium solution is added in 9mL sterilized water, mixes and makes bacteria suspension, using 10 times of gradient dilutions, takes 10-6-10-4Dilution
The bacteria suspension of degree is coated in solid medium, cultivates at 30 DEG C.
2. bacterial strain purification：Coated flat board culture 2d, after growing bacterium colony, according to the single bacterium of colony morphology characteristic picking
Fall line on new solid medium, cultivates at 30 DEG C, selects single bacterium colony line after growing bacterium colony again, and this process repeats
3-4 time, the bacterium colony that the bacterium colony growing on culture dish is single form makes bacterial strain purification complete.Then select and have no
After the bacterium colony numbering of the extracellular polysaccharide features such as color is transparent or semitransparent, toughness, it is seeded to respectively in fluid medium,
200rpm, cultivates 2-4d under the conditions of 30 DEG C, if fermentation liquid becomes sticky, whether identification cell free fermentation product belongs to polysaccharide.Identified
After fermentation product is the bacterial strain of polysaccharide is then the extracellular polysaccharide strains needing, and is named as NUST16.
(2) identification of bacterial strain
A. morphological characteristic：Inorganic salt Solid agar culture is cultivated this bacterial strain and observed after 2-3 days for 30 DEG C, bacterium colony is in circle
Shape, colourless, translucent, surface is glossy, sticky, is difficult to provoke.Bacterial strain long bar shape, the substantial amounts of extracellular polysaccharide of secretion.
B. physiological and biochemical property：Bacterial strain Gram-positive, aerobic, catalase reacting positive, with 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, really
Sugared Utilization ability is weak, and with Lactose for thalli growth during carbon source preferably, sucrose takes second place, and grows in organic nitrogen source peptone,
Under the alkalescence condition of pH7-9, yield of extracellular polysaccharide is higher, and after fermentation 48h, yield of extracellular polysaccharide can reach 15g/L.High concentration NaCl
Strain growth can be suppressed and reduce yield of extracellular polysaccharide, bacterial strain does not secrete pigment.In Fig. 1, A is the bacterium in solid plate culture medium
Fall, B is thalline photo under an optical microscope.
C. with the genomic DNA of bacterial strain NUST16 as template, with the universal primer of the PCR of 16S rRNA gene amplification for drawing
Thing, enters performing PCR amplification, measures its complete sequence, the 16S rDNA gene order of this bacterial strain is shown in sequence table.16S rDNA by bacterial strain
Gene order is committed to GenBank data base (the GenBank number of logging in is KX962167), with the sequence in GenBank data base
Carry out online tetraploid rice, result shows, the sequence similarity of bacterial strain NUST16 and soil series bacillus AB045093 is high
Reach 99%.
Tested and molecular biological analysis according to the morphology of bacterial strain NUST16, Physiology and biochemistry, this bacterial strain NUST16 reflects
It is set to soil series bacillus, be named as soil series bacillus NUST16.
Embodiment 2
The culture of soil series bacillus Paenibacillus edaphicus NUST16 and produce water solublity extracellular
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 is 6.0, and cooling after adding 121 DEG C of sterilizing 15min of 1% Nutrient agar obtains solid medium.In agar solid
In culture medium 30 DEG C cultivate this bacterial strain 48h after it can be seen that there is bacterium colony to grow, bacterium colony is rounded, translucent, and surface is glossy, glues
Thick, it is difficult to provoke, the substantial amounts of extracellular polysaccharide of strain secretes.Bacterium colony 30mL deionized water dissolving on every 9cm flat board of scraping,
Then it is centrifuged 20min through 5000 × g, in supernatant, add 60mL ethanol-isopropanol mixed liquor (volume ratio 1:1) precipitation at room temperature, warp
Precipitation is collected by filtration and obtains final product crude polysaccharides after 40 DEG C of dryings.
The high-efficient liquid phase chromatogram that Fig. 2 forms for extracellular polysaccharide, same component retention time is identical.Wherein Fig. 2A is 10 kinds
Standard list carbohydrates and their derivative elution curve in efficient liquid phase after PMP derivatization；Fig. 2 B is soil series bacillus
The eluting in efficient liquid phase is bent through PMP derivatization for hydrolyzate after trifluoroacetic acid complete hydrolysis for the extracellular polysaccharide of NUST16
, between each eluting peak, there is not overlap in line.Through comparing it is found that D-MANNOSE (D-Man), D-Glucose aldehydic acid (D-
GlcUA), D-Glucose (D-Glc), D- galactose (D-Gal) and L-fucose (L-Fuc) are complete in A figure and B in figure retention time
Entirely consistent.D-Glucose, D-MANNOSE, L- rock algae in the extracellular polysaccharide of soil series bacillus NUST16 can be obtained through quantitative Analysis
Sugar, D- galactose and D-Glucose aldehydic acid mol ratio are 3:3:2:1:1, another kind is expressed as：Glc:Man:Fuc:Gal:GlcUA
=3:3:2:1:1.This polysaccharide is heteropolysaccharide.
Embodiment 3
The culture of soil series bacillus Paenibacillus edaphicus NUST16 and produce water solublity extracellular
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
For 7.0, cool down standby after 121 DEG C of sterilizing 20min, after adding 121 DEG C of sterilizing 15min of 1% Nutrient agar cooling obtain accordingly solid
Body culture medium.The culture medium to after sterilize for the soil series bacillus NUST16 single bacterium colony growing on picking Solid agar culture
In, 25 DEG C of concussion and cultivates 24h form seed culture fluid, then 7% seed culture fluid is added to 35 DEG C in this standby culture medium
Concussion and cultivate 48h, obtains fermentation liquid.Add 95% ethanol-acetone mixed liquor (volume ratio 1 of 3.5 times of volumes to fermentation liquid:1),
Shaken at room temperature, then collects precipitation through 5000 × g centrifugation 20min, obtains final product crude polysaccharides 10.08g after 50 DEG C of dryings.
Embodiment 4
The culture of soil series bacillus Paenibacillus edaphicus NUST16 and exopolysaccharide
Produce and purification.
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, pH is standby for cooling down after 9.0,121 DEG C of sterilizing 20min；The soil series bacillus growing on picking solid medium
In the culture medium to after sterilize for the NUST16 single bacterium colony, 35 DEG C of concussion and cultivates 36h form seed culture fluid, then 5% seed is trained
Nutrient solution is added to 32 DEG C of concussion and cultivates 72h in the culture medium after sterilizing, obtains fermentation liquid.Add 3 times of volumes to fermentation liquid
95% ethanol -100% methanol-isopropanol mixed liquor (volume ratio 1:1:1), precipitate and filter, after 45 DEG C of dryings, obtain final product crude polysaccharides
14.83g.Crude polysaccharides 10g is redissolved in deionized water to final concentration 15g/L, is subsequently adding the chloroform-benzene of 1.1 times of volumes
Phenol-n-butyl alcohol mixed liquor (volume ratio 1:0.85:0.15) vibration and then stratification, collects aqueous phase and rejoins above-mentioned mixing
Liquid, repetitive operation 5 times, the extracellular polysaccharide of Deproteinization can be obtained.The Deproteinization polysaccharide of the gained deionized water of 30 times of volumes
Through molecular cut off 3000Da semipermeable membrane dialysis 48h, then in the polysaccharide after dialysis, add 4 times of volume 95% ethanol-acetones
Mixed liquor (volume ratio 1:1), after precipitating and filtering, 50 DEG C of dryings can obtain the extracellular polysaccharide 6.47g of purification.
Embodiment 5
Application in adjusting the aqueous solution rheological equationm of state for the extracellular polysaccharide that soil series bacillus NUST16 produces.
Prepare the extracellular polysaccharide of purification by embodiment 4, then under room temperature, it is extremely final concentration of that deionized water dissolves this polysaccharide
6.5g/L, solution viscosity is 3755mPa.s after measured, is designated as initial viscosity.Successively compound concentration be respectively 20g/L KCl,
40g/L NaCl、80g/L MgCl2With 100g/L CaCl2Solution, dissolves extracellular polysaccharide extremely respectively with 4 kinds of solution under room temperature condition
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 all exceedes the 80% of initial viscosity.Additionally, preparation mixed inorganic salt solution, wherein KCl, NaCl, MgCl2And CaCl2Concentration is equal
For 25g/L, under room temperature, to final concentration 6.5g/L, mensure viscosity number is 3055mPa.s to dissolving extracellular polysaccharide, is initial concentration
81.4%.
Fig. 3 is NaCl, KCl, the CaCl adding variable concentrations under room temperature condition in solution respectively2And MgCl2When extracellular many
Sugar juice viscosity B coefficent situation.As can be seen that polysaccharide solution viscosity is maintained to initially when salinity is up to 100g/L
More than the 80% of viscosity, and Ca2+And Mg2+Solution will not be made to form gel, this has greatly to the unit operation under high salt conditions
Help.Understand, the extracellular polysaccharide that soil series bacillus NUST16 produces has preferable work on adjusting the aqueous solution rheological equationm of state
With, particularly when containing high salt concentration ion in solution, still being able to dramatically increase solution viscosity and keep stable.
Embodiment 6
Application in adjusting the aqueous solution rheological equationm of state for the extracellular polysaccharide that soil series bacillus NUST16 produces.
Prepare the solid extracellular polysaccharide of purification by embodiment 4, under room temperature, deionized water dissolving extracellular polysaccharide is to final concentration
5g/L, and measure viscosity for 2760mPa.s, and it is designated as initial viscosity.500mL polysaccharide solution is taken to be placed in 10min in boiling water bath, so
Take out rapidly and be cooled to room temperature (25 DEG C) afterwards, after cooling, polysaccharide solution has mobility, does not form gel, through measurement viscosity be
19640mPa.s, is 7.08 times of initial viscosity.This solution is replaced in 10min solution viscosity in boiling water bath decline, then
Being cooled to room temperature measuring viscosity is 20100mPa.s, continues to be repeated 3 times, and 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
Fixed, solution clear, no color change, and do not form heat irreversible gel all the time.
The extracellular polysaccharide solution for 1g/L for the Fig. 4 is cooled to the viscosity B coefficent situation of room temperature after 95 DEG C of heat treatment 10min.Can
To find out, after simple heat treatment, solution viscosity is improved to 850mPa.s by 235mPa.s, and viscosity is the 3.62 of before processing
Times.After 5 repetitive operations, viscosity keeps stable.Fig. 5 is the extracellular polysaccharide solution of 5g/L after 100 DEG C of heat treatment 10min
It is cooled to the viscosity B coefficent situation of room temperature.As can be seen that after simple heat treatment, solution viscosity by 2760mPa.s improve to
19640mPa.s, viscosity is 7.08 times of before processing.After 5 repetitive operations, viscosity remains in that stable.This characteristic can
Greatly reduce polysaccharide usage amount, or reduce unit operation easier during pre-treatment.Understand, soil series bacillus NUST16
The extracellular polysaccharide producing is added in solution, can increase exponentially solution viscosity further by the operation of simple heating-cooling,
And keep stable through viscosity is repeated several times, heat irreversible gel will not be formed, this extracellular polysaccharide stable in properties is described, has
Relatively broad using value.
SEQUENCE LISTING
<110>Institutes Of Technology Of Nanjing
<120>A kind of soil series bacillus and its application
<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 (10)
1. a kind of soil series bacillus, are Paenibacillus edaphicus NUST16, and deposit number is
CCTCCNo.M2016542.
2. application in producing exopolysaccharide for the soil series bacillus as claimed in claim 1.
3. soil series bacillus as claimed in claim 2 produce exopolysaccharide in application it is characterised in that
Concrete application step is as follows：
Step 1, soil series bacillus NUST16 is inoculated in the culture medium after sterilizing, 25-35 DEG C of shaking table concussion and cultivate shape
Become seed culture fluid；
Step 2, by 0.5%-20% inoculum concentration, seed culture fluid is inoculated in the culture medium after sterilizing, 25-35 DEG C of shaking table shake
Swing culture, obtain the fermentation liquid of extracellular polysaccharide；
Step 3, adds precipitant A in fermentation liquid, and the precipitation in fermentation liquid, through filtering or being centrifuged, obtains final product solids crude after being dried many
Sugar, described precipitant A is 95%-100% ethanol, 95%-100% methanol, 95%-100% isopropanol and 95%-100%
One of acetone or multiple.
4. soil series bacillus as claimed in claim 3 produce exopolysaccharide in application it is characterised in that
In step 1, described incubation time is 12-48h.
5. soil series bacillus as claimed in claim 3 produce exopolysaccharide in application it is characterised in that
In step 2, described incubation time is 48-96h.
6. soil series bacillus as claimed in claim 3 produce exopolysaccharide in application it is characterised in that
In step 3, described precipitant A is 2～4 with the volume ratio of fermentation liquid:1, described precipitant A be 95%-100% ethanol,
Two or more solvents in 95%-100% methanol, 95%-100% isopropanol and 95%-100% acetone press equal-volume mixing and
The mixed solution becoming.
7. application in producing exopolysaccharide for the described soil series bacillus as arbitrary in claim 3～6, it is special
Levy and be, described culture medium consist of 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, CuSO40.001-
0.05g/L, MnSO40.001-0.05g/L, ZnCl20.001-0.05g/L, CoCl20.001-0.05g/L, pH are 6.0-
9.0.
8. soil series bacillus as claimed in claim 7 produce exopolysaccharide in application it is characterised in that
The pH of described fluid medium is 7.0-9.0.
9. soil series bacillus as claimed in claim 3 produce exopolysaccharide in application it is characterised in that
Further comprising the steps of：
Step 4, formation crude polysaccharides solution soluble in water for solid crude polysaccharides adds treatment fluid B and acutely shakes, stands or be centrifuged
Separate organic faciess and aqueous phase, collect aqueous phase, and rejoin treatment fluid B, repeat 3～10 times, can obtain purified polysaccharide, described place
Reason liquid B is the mixed solution of chloroform, phenol and n-butyl alcohol.
10. application in producing exopolysaccharide for the soil series bacillus as claimed in claim 9, its feature exists
In, in step 4, the concentration of described crude polysaccharides solution is 0.25～20g/L, the body of described crude polysaccharides solution and treatment fluid B
Amass and compare 1:(1～2), in described treatment fluid B, the volume ratio of chloroform, phenol and n-butyl alcohol is 1:(0.5～1):(0.05
～0.5).
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| CN115895930A (en) * | 2021-08-31 | 2023-04-04 | 四川大学 | Salt-tolerant bacillus and application of levan produced by same |
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| CN109136110B (en) * | 2018-09-26 | 2022-03-25 | 南京理工大学 | Phomopsis liquidambar and application thereof |
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| CN111286466B (en) * | 2018-12-07 | 2023-05-05 | 南京理工大学 | Agrobacterium and anti-inflammatory extracellular polysaccharide produced by same |
| CN111909872A (en) * | 2020-08-03 | 2020-11-10 | 南京理工大学 | Paenibacillus ZX1905, exopolysaccharide Lubcan produced by same and application of exopolysaccharide Lubcan |
| CN111909872B (en) * | 2020-08-03 | 2022-05-20 | 南京理工大学 | Paenibacillus ZX1905, extracellular polysaccharide Lubcan produced by same and application of extracellular polysaccharide Lubcan |
| CN115895930A (en) * | 2021-08-31 | 2023-04-04 | 四川大学 | Salt-tolerant bacillus and application of levan produced by same |
| CN115895930B (en) * | 2021-08-31 | 2023-10-31 | 微元合成生物技术(北京)有限公司 | Salt-tolerant bacillus and application of levan produced by same |
| CN114164241A (en) * | 2021-12-27 | 2022-03-11 | 南京理工大学 | Polysaccharide, sulfation product thereof and application thereof in preparation of anti-inflammatory drugs |
| CN114164241B (en) * | 2021-12-27 | 2024-05-03 | 南京理工大学 | Polysaccharide, sulfated product thereof and application of sulfated product in preparation of anti-inflammatory drugs |
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