CN108060187A - A kind of polysaccharide and its preparation method and application - Google Patents
A kind of polysaccharide and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of polysaccharide with the special rheological equationm of state and its preparation method and application.The polysaccharide is new construction compound of polysaccharide, and by D glucose, D mannoses, L fucoses, D galactolipins and D glucuronic acids are 3 in molar ratio:3:2:1:1 composition.The polysaccharide is generated by the soil series bacillus NUST16 of deposit number CCTCC No.M2016542.The polysaccharide can be directly dissolved in cold water, adjust the aqueous solution rheological equationm of state, play an important role of significantly to increase solution viscosity, can tolerate the Na of solution middle and high concentration+、K+、Ca2+And Mg2+, can increase solution viscosity several times by simply heating cooling treatment, and property is stablized, and can form heat-convertible gel, emulsifier, thickener, stabilizer or gelling agent can be used as to be applied to the industrial circles such as food or cosmetics.
Description
Technical field
The invention belongs to microbial technology fields, are related to a kind of polysaccharide and its preparation method and application.
Background technology
Microbial exopolysaccharides can be used as thickener, suspending agent or stabilizer, extensive use because of its special rheological equationm of state
In industries such as food, cosmetics.Microbial exopolysaccharides with typical rheology characteristic such as xanthans (Xanthan gum) can
Increase the viscosity of solution, and with certain heat resistance, appropriate viscosity kept stable after increasing temperature；Gellan gum
(Gelan gum) aqueous solution is in Ca2+Irreversible brittleness gel can be formed under existence condition；Thermal gels (Curdlan gum)
It is not soluble in water, but its suspension is heated to be formed the elastic gel of thermal reversion after 55 DEG C, 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 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
It rings, the advantages that production technology is 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 is 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.
There are certain limiting factors in the application for Microbial exopolysaccharides at present.For example, it is glued to increase xanthan gum solution
Degree can only can not be realized by improving the additive amount of xanthans by simple aftertreatment technology；There are Ca in solution2+Or Mg2 +Limit the usage amount of gellan gum；Heat treatment process with higher temperature can then be influenced be subject to thermal gels.Therefore, to passing
System Microbial exopolysaccharides carry out chemical modification or find that novel microbial exocellular polysaccharide seems particularly necessary.
The study found that series bacillus microorganism belonging to genus has the ability for generating new exocellular polysaccharide.Soil series bacillus
Bacillus is classified as when being originally found, after renamed as soil series bacillus (Paenibacillus
edaphicus).Soil series bacillus plays an important role 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 utilization (Sheng X F.Growth promotion of the plant to 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, 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 journals, 2003,43 (2):162-168.).It has confirmed
It is similary with the colloid series bacillus that soil series bacillus affiliation is very near that there is the ability for generating pod membrane, and the pod
Film is exocellular polysaccharide, and with 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 new exocellular polysaccharide.
The content of the invention
One of the objects of the present invention is to provide a kind of polysaccharide with the special rheological equationm of state, structural formula is：
N is 50-2000.
The second object of the present invention is the preparation method for providing above-mentioned polysaccharide, and the polysaccharide is by deposit number CCTCC
The soil series bacillus NUST16 productions of No.M2016542, are as follows：
Step 1, after the soil series bacillus NUST16 of deposit number CCTCC No.M2016542 being inoculated into sterilizing
In culture medium, 25-35 DEG C of shaking table shake culture forms seed culture fluid；
Step 2, seed culture fluid is inoculated into the culture medium after sterilizing by 0.5%-20% inoculum concentrations, 25-35 DEG C is shaken
Bed shake culture obtains the zymotic fluid of extracellular polysaccharide；
Step 3, precipitating reagent A is added in zymotic fluid, the precipitation in zymotic fluid is filtered or centrifugation, it is dry after up to solid
Thick many candies, the precipitating reagent A are 95%-100% ethyl alcohol, 95%-100% methanol, 95%-100% isopropanols and 95%-
One or more in 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 zymotic fluid is 2~4:1, the precipitating reagent A are 95%-
Two or more solvents in 100% ethyl alcohol, 95%-100% methanol, 95%-100% isopropanols and 95%-100% acetone by etc.
The mixed solution that volume mixture forms.
In the present invention, the composition of the culture medium is 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, the preparation method of above-mentioned polysaccharide, further includes following steps：
Step 4, form Thick many candies solution by solid Thick many candies are soluble in water, add in and treatment fluid B and acutely shake, standing or
Organic phase and water phase are centrifuged, collects water phase, and rejoins 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).The three of the object of the invention are in the application in the above-mentioned polysaccharide of offer in the aqueous solution rheological equationm of state is adjusted.
Further, above-mentioned polysaccharide is in the application in adjusting the aqueous solution rheological equationm of state, can be used as emulsifier, thickener,
Stabilizer or gelling agent.
Soil series bacillus NUST16 in the present invention, for Paenibacillus edaphicus NUST16, preservation
Number is CCTCC No.M2016542, is protected on October 08th, 2016 in China typical culture collection center (CCTCC)
It hides, preservation address is Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University collection.
The polysaccharide of the present invention is new construction compound of polysaccharide, can be directly dissolved in cold water, has significant increase solution viscosity
Effect, be resistant to the Na of solution middle and high concentration+、K+、Ca2+And Mg2+, can make by the processing of simple heating-cooling molten
Fluid viscosity several times increase, and property is stablized, and can form heat-convertible gel.The polysaccharide can pass through soil series bacillus
NUST16 fermenting and producings, preparation method are easy, and produced exocellular polysaccharide is easily isolated and purifies and unique, and yield can reach
15g/L.The polysaccharide of the present invention can be used as emulsifier, thickener, stabilizer or gelling agent to be applied to the industry such as food or cosmetics
Field.
Description of the drawings
Fig. 1 is the flat-plate bacterial colony of bacterial strain and microstructure figure.
Fig. 2 is the high-efficient liquid phase chromatogram of exocellular polysaccharide compound mensuration.
Fig. 3 is the infrared spectrogram of exocellular polysaccharide.
Fig. 4 is the 1H nmr spectrums of exocellular polysaccharide.
Fig. 5 is the 13C nmr spectrums of exocellular polysaccharide.
Fig. 6 exocellular polysaccharides corresponding structural formula of gas phase-mass spectrum total ion current figure and reservation peak after the processing that methylates.
Fig. 7 is influence result figure of 4 kinds of inorganic salts to exocellular polysaccharide viscosity.
Fig. 8 is influence result figure of the Repeat-heating-cooling treatment to 1g/L exocellular polysaccharide solution viscosities.
Fig. 9 is influence result figure of the Repeat-heating-cooling treatment to 5g/L exocellular polysaccharide solution viscosities.
Specific embodiment
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
The soil series bacillus Paenibacillus edaphicus NUST16 of the present invention are in the shore of Yancheng, Jiangsu Province
It is isolated in extra large brown soil.Wherein separated method is：
1. bacterial strain screening：Pedotheque is divided into 10 groups, each component also known as takes 1g soil samples 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 1mL bacterium solutions is taken to access again in new fluid nutrient medium, 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 waters after 1mL enrichments, 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 tablet culture 2d, after bacterium colony is grown, according to the single bacterium of colony morphology characteristic picking
Fall and rule on new solid medium, cultivated at 30 DEG C, select single bacterium colony line again after bacterium colony is grown, this process repeats
3-4 times, until the bacterium colony that the bacterium colony grown on culture dish is single form so that bacterial strain purifying is complete.Then select with nothing
After the bacterium colony number of the extracellular polysaccharide features such as color is transparent or semitransparent, toughness, it is seeded to respectively in fluid nutrient medium,
200rpm cultivates 2-4d under the conditions of 30 DEG C, if zymotic fluid becomes sticky, identifies whether cell free fermentation product belongs to polysaccharide.It is identified
Tunning is then needed extracellular polysaccharide strains for the bacterial strain of polysaccharide afterwards, is named as NUST16.
(2) identification of bacterial strain
A. morphological feature：It cultivates this bacterial strain on inorganic salts Solid agar culture and is observed after 2-3 days for 30 DEG C, such as Fig. 1 institutes
Show.In Fig. 1, A is the bacterium colony on solid plate culture medium, and B is the photo of thalline under an optical microscope, and bacterium colony is rounded, nothing
Color, translucent, surface is glossy, sticky, is not easy to provoke.The long rod shape of bacterial strain, the substantial amounts 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
Production is sour, 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 during using lactose as carbon source, and sucrose takes second place, and is grown 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.
C. using the genomic DNA of bacterial strain NUST16 as template, universal primer using the PCR amplification of 16S rRNA genes is draws
Object carries out PCR amplification, measures its complete sequence, the 16S rDNA gene orders of the bacterial strain are shown in sequence table.By the 16S rDNA of bacterial strain
Gene order is committed to GenBank databases (the GenBank numbers of logging in are KX962167), with the sequence in GenBank databases
Carry out online tetraploid rice, the results showed that, the sequence similarity of bacterial strain NUST16 and soil series bacillus AB045093 is 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 general structure of polysaccharide is as follows：
N is 50-2000.
1. efficient liquid phase chromatographic analysis
Polysaccharide component includes D-Glucose, D-MANNOSE, L-fucose, D- galactolipins and D-Glucose aldehydic acid, molar ratio
For 3:3:2:1:1.Fig. 2 is the high-efficient liquid phase chromatogram of exocellular polysaccharide composition, and same component retention time is identical.Wherein Fig. 2A is
Elution curve of 10 kinds of standard list carbohydrates and their derivatives after PMP derivatizations in efficient liquid phase；Fig. 2 B are soil class gemma bar
Hydrolysate elution through PMP derivatization in efficient liquid phase of the extracellular polysaccharide of bacterium NUST16 after trifluoroacetic acid complete hydrolysis
Curve, 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- galactolipins (D-Gal) and L-fucose (L-Fuc) retention time in A figures and B figures are complete
It is complete consistent.Through quantitatively can be calculated D-Glucose, D-MANNOSE, L- rock algaes in the extracellular polysaccharide of soil series bacillus NUST16
Sugar, D- galactolipins and D-Glucose aldehydic acid molar ratio are 3:3:2:1:1, another kind is expressed as：Glc:Man:Fuc:Gal:GlcUA
=3:3:2:1:1.The polysaccharide is heteroglycan.
2. infrared spectrum analysis
The polysaccharide of the present invention has the polysaccharose substance characteristic absorption curve of typicalness.Fig. 3 is the infrared light of exocellular polysaccharide
Spectrogram, it can be seen that 3313.11,1071.75 and 1019.68cm of wave number-1For polysaccharide Typical Vibration absworption peak.Wave number
2912.95,1402.00 and 1362.94cm-1For methyl (- CH3) characteristic absorption peak, show containing a small amount of methyl in sample, and
And influenced to deviate to lower wave number be subject to ortho position C-O, this coincide with L-fucose (Fuc) structure.In addition, wave number 1595.81cm-1For
Typical carboxyl C=O absworption peaks, as a result coincide with D-Glucose aldehydic acid (D-GlcUA) structure.
3. nuclear magnetic resonance map and the analysis of the total ion current of gas phase-mass spectral analysis
Nuclear magnetic resonance map shows that the exocellular polysaccharide contains methyl and carboxyl signal.Fig. 4 and Fig. 5 is exocellular polysaccharide respectively
's1H and13C nmr spectrums.It can be seen that from Fig. 4 (1), the δ 1.29ppm (A) and δ 1.21ppm (B) of high field region are right respectively
Answer the methyl hydrogen of two L-fucoses C-6And the methyl carbon of L-fucose C-6And and Fig. 513C
Chemical shift δ 15.48 (A) in H NMR spectroscopy is related to δ 15.35ppm (B), further demonstrates the knot of liquid phase and infared spectrum
By.It is also seen that the δ 175.86ppm (C) in low field area are typical carboxylic acid carbonyl's carbon from Fig. 5Chemical shift,
This conclusion with there are glucuronic acids in polysaccharide to match.It is also known from Fig. 4, the 4.50~5.50ppm of δ in low field area (a~
f3) for the hydrogen chemical shifts of each anomeric carbon in exocellular polysaccharide repetitive unit, with reference to integral area and coupling constant data, can push away
Disconnected, a is D-Glucose aldehydic acid, b1,2For two D-MANNOSEs, c1,2For two L-fucoses, d is D- galactolipins, and e is D- sweet dews
Sugar, f1,2,3For three D-Glucoses.10 anomeric carbons1H chemical shifts respectively with Fig. 513(i-x) one in C H NMR spectroscopies is a pair of
Should, chemical shift is followed successively by：δ98.31,99.96,101.23,100.46,100.64,102.65,103.35,101.50,
101.79 and 102.65ppm.According to chemical shift and3J1,2Even summation relation can speculate that 10 sugar unit conformations are followed successively by α-D-
Glucuronic acid, α-D-MANNOSE, α-D-MANNOSE, α-L-fucose, α-L-fucose, β-D- galactolipins, β-D-MANNOSE,
β-D-Glucose, β-D-Glucose and β-D-Glucose.Connection mode between each glucosides is further by the data-speculative that methylates
Draw (see Fig. 6), Fig. 6 for exocellular polysaccharide after the processing that methylates, the total ion current figure of gas phase-mass spectral analysis.Wherein retain peak
A-G with n-compound mass spectrometric data storehouse through comparing, and structural formula is listed in Fig. 6 respectively, remaining peak is monose irrelevant peaks, not
It gives and listing.A-G is corresponded to respectively：D-GlcUA, 3-L-Fuc, D-Gal, 3-D-Glc, 2,3,6-D-Glc, 3,6-D-Man and 3-D-
Man.With reference to the information of Fig. 2-Fig. 6, exocellular polysaccharide structure is finally determined.
Embodiment 3
Soil series bacillus Paenibacillus edaphicus NUST16 fermenting and producing exocellular polysaccharides.
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 adds in 1% nutrient agar, 121 DEG C of sterilizing 15min postcoolings and obtains solid medium.In agar solid
It is cultivated on culture medium after this bacterial strain 48h it can be seen that there is bacterium colony to grow for 30 DEG C, bacterium colony is rounded, translucent, and surface is glossy, glues
It is thick, it is not easy to provoke, the substantial amounts of exocellular polysaccharide of strain secretes.The bacterium colony 30mL deionized water dissolvings on every 9cm tablet are scraped,
Then 20min is centrifuged through 5000 × g, 60mL ethyl alcohol-isopropanol mixed liquor (volume ratio 1 is added in supernatant：1) precipitation at room temperature, warp
Precipitation is collected by filtration and after 40 DEG C of dryings up to Thick many candies.
Embodiment 4
Soil series bacillus Paenibacillus edaphicus NUST16 fermenting and producing exocellular polysaccharides.
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 waters, pH
For 7.0,121 DEG C of sterilizing 20min postcoolings are spare, add in 1% nutrient agar, 121 DEG C of sterilizing 15min postcoolings obtain it is accordingly solid
Body culture medium.The soil series bacillus NUST16 single bacteriums grown on picking Solid agar culture drop 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 zymotic fluid.95% ethanol-acetone mixed liquor (volume ratio 1 of 3.5 times of volumes is added in zymotic fluid:1),
Then shaken at room temperature collects precipitation, up to Thick many candies 10.08g after 50 DEG C of dryings through 5000 × g centrifugations 20min.
Embodiment 5
Soil series bacillus Paenibacillus edaphicus NUST16 fermenting and producings exocellular polysaccharides and polysaccharide
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 deionizations
In water, 9.0,121 DEG C of sterilizing 20min postcoolings of pH are spare, add in 1% nutrient agar, 121 DEG C of sterilizing 15min postcoolings and obtain
Corresponding solid medium.The soil series bacillus NUST16 single bacteriums grown on picking solid medium drop down onto the culture after sterilizing
In base, 35 DEG C of shake culture 36h form seed culture fluid, then 5% seed culture fluid is added in the culture medium after sterilizing
32 DEG C of shake culture 72h obtain zymotic fluid.- 100% methanol-isopropanol of 95% ethyl alcohol that 3 times of volumes are added in zymotic fluid mixes
Liquid (volume ratio 1:1:1), precipitate and filter, up to Thick many candies 14.83g after 45 DEG C of dryings.By Thick many candies 10g be redissolved in from
To chloroform-phenol-n-butanol mixed liquor (volume ratio 1 of final concentration 15g/L, then 1.1 times of volumes of addition in sub- water:0.85:
0.15) vibration and then stratification collect water and mutually rejoin above-mentioned mixed liquor, and repetitive operation 5 times can obtain deproteinized
Exocellular polysaccharide.The deproteinized polysaccharide of gained is dialysed 48h with the deionized waters of 30 times of volumes through molecular cut off 3000Da semi-permeable membranes,
Then 4 times of 95% ethanol-acetone mixed liquor (volume ratios 1 of volume are added in into the polysaccharide after dialysis:1), after precipitation and filtering,
The exocellular polysaccharide 6.47g that 50 DEG C of dryings can must purify.
Embodiment 6
Application of the exocellular polysaccharide in the aqueous solution rheological equationm of state is adjusted.
The exocellular polysaccharide of purifying is prepared by embodiment 5, 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 respectively with 4 kinds of solution under room temperature
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, prepare 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 measure viscosity number is 3055mPa.s, is initial concentration
81.4%.
Fig. 7 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 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 the unit operation under high salt conditions
It helps.It understands, the exocellular polysaccharide that soil series bacillus NUST16 is generated has on the aqueous solution rheological equationm of state is adjusted preferably to be made
With, particularly when in solution contain high salt concentration ion when, still be able to dramatically increase solution viscosity and keep stable.
Embodiment 7
Application of the exocellular polysaccharide that soil series bacillus NUST16 is generated in the aqueous solution rheological equationm of state is adjusted.
The solid exocellular polysaccharide of purifying is prepared by embodiment 5, at room temperature with deionized water dissolving exocellular polysaccharide to final concentration
5g/L, and viscosity is measured as 2760mPa.s, and it is denoted as initial viscosity.500mL polysaccharide solutions is taken to be placed in 10min in boiling water bath, so
It takes out rapidly afterwards and is cooled to room temperature (25 DEG C), polysaccharide solution has mobility after cooling, does not form gel, is through measuring viscosity
19640mPa.s, for 7.08 times of initial viscosity.The solution is replaced in 10min solution viscosities in boiling water bath to decline, then
Be cooled to room temperature measure viscosity be 20100mPa.s, continue to be repeated 3 times, it is observed the phenomenon that it is consistent, 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 do not form heat irreversible gel always.
Viscosity change situation of the exocellular polysaccharide solution that Fig. 8 is 1g/L through 95 DEG C of heat treatment 10min postcoolings to room temperature.It can
To find out, after being simply heat-treated, solution viscosity is improved by 235mPa.s to 850mPa.s, and viscosity is the 3.62 of before processing
Times.After 5 repetitive operations, viscosity keeps stablizing.The exocellular polysaccharide solution that Fig. 9 is 5g/L is after 100 DEG C are heat-treated 10min
The viscosity change situation being cooled to room temperature.As can be seen that after simple heat treatment, solution viscosity by 2760mPa.s improve to
19640mPa.s, viscosity are 7.08 times of before processing.After 5 repetitive operations, viscosity still maintains stable.This characteristic can
Unit operation difficulty when greatly reducing polysaccharide usage amount or reducing pre-treatment.It understands, soil series bacillus NUST16
The exocellular polysaccharide of generation is added in solution, and solution viscosity can be further increased exponentially by the operation of simple heating-cooling,
And it keeps stablizing by viscosity is repeated several times, heat irreversible gel will not be formed, 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 polysaccharide and its preparation method and 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 polysaccharide, structural formula are：
N is 50-2000.
2. polysaccharide as described in claim 1, which is characterized in that the polysaccharide is by deposit number CCTCC No.M2016542
Soil series bacillus NUST16 production.
3. the preparation method of polysaccharide as claimed in claim 1 or 2, which is characterized in that be as follows：
Step 1, the soil series bacillus NUST16 of deposit number CCTCC No.M2016542 is inoculated into the culture after sterilizing
In base, 25-35 DEG C of shaking table shake culture forms seed culture fluid；
Step 2, seed culture fluid is inoculated into the culture medium after sterilizing by 0.5%-20% inoculum concentrations, 25-35 DEG C of shaking table shake
Culture is swung, obtains the zymotic fluid of extracellular polysaccharide；
Step 3, precipitating reagent A is added in zymotic fluid, the precipitation in zymotic fluid is filtered or centrifugation, more up to solids crude after dry
Sugar, the precipitating reagent A are 95%-100% ethyl alcohol, 95%-100% methanol, 95%-100% isopropanols and 95%-100%
One or more in acetone.
4. the preparation method of polysaccharide as claimed in claim 3, which is characterized in that 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 zymotic fluid for 2~
4:1, the precipitating reagent A are 95%-100% ethyl alcohol, 95%-100% methanol, 95%-100% isopropanols and 95%-100%
Two or more solvents in acetone are by the mixed solution mixed in equal volume.
5. the preparation method of polysaccharide as claimed in claim 3, which is characterized in that the composition of the culture medium is 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 6.0-9.0.
6. the preparation method of polysaccharide as claimed in claim 5, which is characterized in that the pH of the culture medium is 7.0-9.0.
7. the preparation method of polysaccharide as claimed in claim 3, which is characterized in that further include following steps：
Step 4, by solid Thick many candies formation Thick many candies solution soluble in water, add in treatment fluid B and simultaneously acutely shake, stand or centrifuge
Organic phase and water phase are separated, collects water phase, and rejoins treatment fluid B, repeats 3~10 times, purified polysaccharide, the place can be obtained
Manage the mixed solution that liquid B is chloroform, phenol and n-butanol.
8. the preparation method of polysaccharide as claimed in claim 7, which is characterized in that in step 4, the Thick many candies solution it is dense
It spends for 0.25~20g/L, the volume ratio 1 of the Thick many candies solution and treatment fluid B:(1~2), in the treatment fluid B, three
The volume ratio of chloromethanes, phenol and n-butanol is 1:(0.5~1):(0.05~0.5).
9. application of the polysaccharide as claimed in claim 1 or 2 in the aqueous solution rheological equationm of state is adjusted.
10. application of the polysaccharide as claimed in claim 9 in the aqueous solution rheological equationm of state is adjusted, which is characterized in that described is more
Sugar is used as emulsifier, thickener, stabilizer or gelling agent.
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| 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 |
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| WO2003102204A2 (en) * | 2002-06-03 | 2003-12-11 | Vrije Universiteit Brussel | Streptococcus thermophilus strain producing exopolysaccharide |
| CN101935623A (en) * | 2010-04-14 | 2011-01-05 | 南京理工大学 | Agrobacterium ZX09, water-soluble beta-glucan prepared from Agrobacterium ZX09 and preparation method thereof and application on reducing blood sugar |
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| WO2003102204A2 (en) * | 2002-06-03 | 2003-12-11 | Vrije Universiteit Brussel | Streptococcus thermophilus strain producing exopolysaccharide |
| CN101935623A (en) * | 2010-04-14 | 2011-01-05 | 南京理工大学 | Agrobacterium ZX09, water-soluble beta-glucan prepared from Agrobacterium ZX09 and preparation method thereof and application on reducing blood sugar |
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| 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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