CN106434495A - Rhizobium pusense and method for preparing beta-1,3 glucan fermenting liquid by rhizobium pusense - Google Patents
Rhizobium pusense and method for preparing beta-1,3 glucan fermenting liquid by rhizobium pusense Download PDFInfo
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Abstract
The invention belongs to microorganism fermentation, and in particular relates to rhizobium pusense and a method for preparing beta-1,3 glucan fermenting liquid by the rhizobium pusense. The method comprises the process steps of inoculating the rhizobium pusense CGMCC No.12954 into a culture solution which is sterilized and containing carbon source, nitrogen source and necessary nutritional substances, performing aerated stirring fermentation, finishing fermentation when the viscosity of the culture solution is not increased or the fermentation cycle is less than or equal to 48 hours, and the like. By the method, the problems of tedious process, long fermentation cycle, low product viscosity and the like in the prior art are solved. The method has the advantages of high yield, high product viscosity, high water solubility, short fermentation cycle, low cost and the like.
Description
Technical field
The invention belongs to fermentable, particularly relate to a kind of Bodhisattva root nodule bacteria and its produce β -1,3 dextran fermentation liquid
Method.
Background technology
β -1,3 glucosans are that a class has with β -1, the glucose polymerisation with multiple biological activities that 3 glucosans connect
Thing.Efficacy performance in application experiment is：Moisturizing, antiinflammatory, defying age, wrinkle removing, anti-dandruff, jaundice eliminating, quickening are repaired, are increased skin
Elasticity, auxiliary antibacterial, enhancing human body immunity power, auxiliary hyperglycemic, blood fat reducing, improve gastrointestinal function etc..Be widely used in medicine,
The multiple fields such as food, cosmetics, animal feed additive.
Glucosan with β -1,3 glucosans most physiologically active, β -1,3 glucosans are widely present in various funguses and plant
In (as Lentinus Edodess, Ganoderma and Herba bromi japonici), it is the primary efficacy material that they play health-care effect.
In nineteen forties, doctor Pillemer finds first and reports that yeast cell wall has a kind of material to have
The effect of enhance immunity.Further study show that through Tu Lun university doctor Diluzio afterwards, improve in yeast cell wall and exempt from
Epidemic disease power material is a kind of polysaccharide β -1,3 glucosans, and isolates this material from bakery yeast.
The polysaccharide that beta glucan active structure is made up of glucose unit, their great majority are by β -1,3 combinations,
This is Fructus Vitis viniferae sugar chain connected mode.It can activated macrophage and neutrophils etc., therefore can improve leukin, thin
Born of the same parents' mitogen and distinct antibodies content, stimulate body immune system comprehensively.So body just have more be ready to resist microorganism
The disease causing.β -1,3 glucosans can make the ability that injured body lymphocyte produces cytokine (IL-1) just recover rapidly
Often, effectively adjust human body immune function.Many experiments show, β -1, and 3 glucosans can promote internal IgM antibody to produce, to improve
Humoral immunization ability.This glucosan activating cell can excite the non-specific defense mechanism of host, therefore applies in tumor, infection disease
Deeply attract attention with controlling treatment trauma aspect.Through specific step extraction and without endotoxin β -1,3 glucosans are assert in U.S. FDA is
The material of safety, can make an addition to normal food, and many report display mouse are administered orally yeast β -1,3 glucosans, can increase strong peritoneum
Cell antibacterial phagocytosiss.Additionally, glucosan still has removing free radical, radioprotective, dissolving cholesterol, prevention hyperlipemia effect
And the infection that opposing filterable viruss, funguses, antibacterial etc. cause, therefore can be widely used for the industries such as medicine, food, cosmetics, mesh
Front yeast dextran realizes industrialization, has broad application prospects.
Traditional β -1,3 glucosan preparation methods are to separate from yeast cell wall to be obtained, and product not readily dissolves in water, tradition
There is complex process in technique, control loaded down with trivial details shortcoming, technology controlling and process is unstable.
The patent documentation that applicant retrieves includes:
A kind of edaphic bacilluss ZX09 is disclosed, with edaphic bacilluss ZX09 life in the document of Patent No. 201010146371.2
Water solublity beta glucan producing and preparation method thereof and the application on reducing blood glucose.Edaphic bacilluss ZX09 is in the salt of Shandong region
Separate in soil and obtain.Can be grown well with sucrose or Lactose for carbon source, it is possible to use carbon source be D-Glucose, D-Fructose,
D-MANNOSE or D- xylose are it is impossible to be maltose using carbon source.Fermentation period is 48-72 hour, the fermentation after fermenting 48 hours
Liquid highest viscosity can reach 20000mPa s, and (NDJ-1RotationalViscometer, Rotor are 3, RPM is 6, Shanghai sky
Flat instrument plant).The problem one that the prior art exists is that strain can not utilize maltose；Two is that (48-72 is little for fermentation period length
When)；Three is the viscosity relatively low (highest viscosity can reach 20000mPa s) of fermentation ends after fermentation liquid；Four is fermentation not to be received
Rate illustrates.
A kind of agrobacterium radiobacter mutant is disclosed, this strain warp in the document of Patent No. 201210281755.4
Cultivate 90-100 hour in 500L fermentation tank, obtain final product β -1, the fermentation liquid of 3 beta-dextran contents more than 4%.Above-mentioned prior art is deposited
Problem one be fermentation period length (90-100 hour)；Two is that the water solublity to fermentation liquid and viscosity do not carry out quantifying explanation；
Three is to only disclose using sucrose or glucose as carbon source.
Disclose one kind in Patent No. 201510123040.X and be directly produced β -1,3 Portugal oligosaccharide using curdlan fermentation liquid
Method, it is disclosed that following technical scheme：Obtain curdlan fermentation first with edaphic bacilluss ATCC 31749 ventilating fermentation
Liquid, is carried out to curdlan fermentation liquid after thermokalite inactivation treatment, as one of nutrient media components cultivating Trichoderma harzianum, adds nitrogen source
After trace element adjuvant, Trichoderma harzianum obtains through aerlbic culture and is rich in β -1, the Trichoderma harzianum fermentation liquid of 3 endoglucanases；
This Trichoderma harzianum fermentation liquid mixes and carries out catalytic hydrolysis reaction with the fresh curdlan fermentation liquid without thermokalite inactivation treatment again,
Separation and Extraction is not carried out to enzyme liquid and reaction substrate, directly obtain β -1,3 Portugal's oligosaccharide semifinished products.The problem that the prior art exists
One is complex production process, wayward；Two is that processing time is long；Three be not the technical parameter to β -1,3 Portugal oligosaccharide and
Fermentation yield illustrates.
The beta glucan from filamentous fungis is disclosed, said method includes in the document of Patent No. 01806862.6
The saprophytic filamentous fungis of non-cause of disease of fermenting are surveyed suspension and are extracted beta glucan from suspension.What the prior art existed mainly asks
Topic one is complex production process, wayward；Two is that fermentation period is long, at least about 50 hours, preferably 96 hours；Three is not right
The viscosity index of beta glucan illustrates.
Content of the invention
An object of the present invention is to provide a kind of Bodhisattva root nodule bacteria with preferable β -1,3 glucosan production capacity
(Rhizobium pusense)CGMCC No.12954.
The second object of the present invention is to provide one kind to utilize Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC
The method that No.12954 produces β -1,3 dextran fermentation liquid.In fermentation culture using corn starch hydrolyzed solution, maltose be carbon
Source；Sweat adopts the fermentation technology that temperature section controls, and is more beneficial for the growth metabolism of strain, the method yield is substantially excellent
In prior art, and obtained β -1,3 glucosan good water solubility.
The overall technology of the present invention is conceived：
A kind of Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954.
Strain Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954 applicant in the present invention in
September submits the Chinese microorganism strain preservation conservator being located at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 on 12nd within 2016
Meeting common micro-organisms center preservation, this depositary institution abbreviation CGMCC.
Because Bodhisattva root nodule bacteria (Rhizobium pusense) the CGMCC No.12954 in the present invention is according to depositary institution
Qualification result only have Latin title at present, there is no Chinese, the Chinese of strain is usually to use this bacterium by the first
The personnel's translation planted, the Chinese nomenclature principle of this strain is as follows：
Rhizobium pusense (pu.sen9se.N.L.neut.adj.pusense belonging to Pusa,
India, the geographical origin of the type strain).
The Chinese of this strain is to name according to the geographical position of strains separation, and the meaning is the bacterium from pusa, because
Its Chinese is translated into Bodhisattva root nodule bacteria by this applicant.
The morphological characteristic of this strain and physiological and biochemical property are as follows：
Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954 system applicant in the present invention is in Hebei province
Separate in Hengshui City Hengshui Lake soil and obtain, microscope observation of cell is in shaft-like, Gram’s staining is feminine gender.Bacterial strain bacterium colony is circle
Shape, protuberance, the smooth of the edge is neat, and thalline focuses primarily upon bacterium colony central authorities, white；There is partially transparent the gluing of white in thalline surrounding
Property extracellular productses occur, with the growth of incubation time, the amount of extracellular productses is on the increase, and with bacterium colony central authorities for the center of circle to four
Week diffusion, can be utilized and include the carbon sources such as corn starch hydrolyzed solution, maltose.Fermentation broth viscosity after fermenting 48 hours can reach
30000mPa·s.
Produce β -1, the method for 3 dextran fermentation liquid using Bodhisattva root nodule bacteria, the method comprises the technical steps that：
A, Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954 is inoculated into after sterilizing and contains carbon
In the culture fluid in source, nitrogen source and necessary nutrient matter；
B, carry out air agitation fermentation；
C, when culture fluid viscosity no longer increase or fermentation period≤48 hour fermentation ends.
The particular technique design of the present invention also has：
For being more beneficial for the growth of strain, in the present invention, cultivate Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC
In the culture medium of No.12954, preferred carbon source and nitrogen source are, the carbon source in the described culture fluid of step A is selected from glucose, wheat
Bud sugar, one of corn starch hydrolyzed solution, Semen Maydiss starch or its combination；Nitrogen source be selected from Semen Glycines powder, Oleum Glycines, peptone, yeast extract,
One of ammonium sulfate or its combination.
For ease of controlling fermentation period, provide favourable growing environment for thalline, preferred technical implementation way is, described
Step A in press seed liquor：The percent by volume of culture fluid is the inoculum concentration of 12%-18% by Bodhisattva root nodule bacteria (Rhizobium
Pusense) CGMCC No.12954 accesses in the culture fluid in fermentation tank.For providing good growing environment to ensure that thalline is normal
Growth, preferred technical scheme is that inoculation in described step A is when after sterilizing, culture-liquid temp is carried out when being down to 30-32 DEG C.
Described corn starch hydrolyzed solution preferably employs following method and makes：Corn starch tap water is made into quality hundred
The mixed liquor dividing specific concentration to be 30%, is warming up to 90-95 DEG C, maintains 40 minutes.
For ease of the acquisition of the thalli growth in sweat and end-product, used by the fermentation in step A of the present invention
Culture fluid preferably employ following manner, in described step A, culture fluid is made up of the component of following weight/mass percentage composition：
Corn starch hydrolyzed solution 4.0%-4.5%；Maltose 2.0%-2.5%；Semen Glycines powder 0.3%-0.5%；Magnesium sulfate
0.1%-0.15%；Oleum Glycines 0.05%-0.15%；Balance of sterilized water；PH=8.0-8.5.
Culture fluid in step A preferably employs following manner configuration, and in described step A, culture fluid process for preparation is as follows：
Component each in culture fluid is put in fermentation tank in proportion, plus tap water, to regulation meter material volume, stirs 30-40 minute and is allowed to molten
Solution, adjusts culture fluid pH=8.0-8.5 with sodium hydroxide solution, stirs 30-40 minute mix homogeneously.
For improving yield, meeting the generation growing and being conducive to end-product of thalline, during the aerobic fementation of the present invention
Preferably fermentation condition is that step B comprises the technical steps that:
10-30 hour：30 DEG C -32 DEG C of temperature, pressure 0.03-0.05MPa, pH=7.0-7.3, ventilation 0.4-
0.5vvm；
31 hours-put tank：26 DEG C -29 DEG C of temperature, pressure 0.03-0.05MPa, pH=7.0-7.3, ventilation 0.4-
0.5vvm.
Nutritious in culture fluid for making, it is more beneficial for the growth metabolism of strain.Preferably technical scheme is, described
Carry out filling into hydrogen peroxide and trace element in step B in air agitation sweat；Actual conditions is as follows：
12-15 hour：Fill into trace element；
23-26 hour：Fill into hydrogen peroxide；
Trace element preferably employs the mode of being implemented as follows, and described trace element is configured to trace element in the following way
Solution：Citric acid 100g, calcium hydroxide 5g, magnesium hydroxide 7g, manganese sulfate 0.083g, zinc sulfate 0.066g, copper sulphate pentahydrate
0.025g, cobalt sulfate 0.028g, magnesium sulfate 8.6g, ferrous sulfate 1.8g, boric acid 0.006g, plus distilled water is settled to
Sterilize after 1000ml；The addition of trace element solution is that every 1000L culture fluid adds trace element solution 1L.
Hydrogen peroxide preferably employs the mode of being implemented as follows, and the mass percent concentration of described hydrogen peroxide is 2-3%, addition
For nutrient solution volume 0.05%.
For ease of industrialized production, meet the economy of industrialized production in producing simultaneously, be preferred embodiment, step
Rapid A is by Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954 after amplification culture, according to seed liquor：
The percent by volume of fermentation regulation meter material volume culture fluid is that the inoculum concentration of 12%-18% is inoculated in culture fluid.
Described amplification culture process includes：
(1) Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954 is inoculated in shake-flask seed culture medium
Shake-flask seed is made on surface；
(2) shake-flask seed is made after amplification culture seed liquor；
(3) by the seed liquor made by seed liquor：The percent by volume of fermentation regulation meter material volume culture fluid is 12%-
18% inoculum concentration accesses the culture fluid in fermentation tank, is fermented.
The process conditions preparing shake-flask seed in described amplification culture process steps (1) are：Picking 2 ring slant strains connect
Enter in the shake-flask seed culture medium after sterilizing, make shake-flask seed through shaken cultivation, cultivation temperature is 30-32 DEG C, shaking speed
For 200-220 rev/min, cultivation cycle is 10-14 hour；
Shake-flask seed culture medium is grouped into by the group of following weight/mass percentage composition：
Glucose 2.5%-2.8%；Maltose 1.5%-2.0%；Peptone 1.0%-1.5%；Semen Maydiss starch 1.0%-
1.5%；Balance of sterilized water；PH=7.0-7.3.
Amplification culture in described amplification culture process steps (2) includes the preparation of first order seed, secondary seed successively
Preparation, wherein first order seed culture fluid is grouped into by the group of following masses percentage composition：
Glucose 2.5%-2.8%；Maltose 1.5%-2.0%；Yeast extract 0.4%-0.6%；Ammonium sulfate 0.2%-
0.3%；Balance of sterilized water；PH=7.0-7.3；
Inoculum concentration is shake-flask seed：Volume ratio=the 0.8%-1.0% of first order seed culture fluid, shaking table seed is accessed and goes out
First order seed culture fluid after bacterium makes first order seed through culture, and the process conditions in the preparation process of first order seed are cultures
30 DEG C -32 DEG C of temperature, ventilation 0.9vvm-1.0vvm, cultivation cycle 10-14 hour.
Described secondary seed culture fluid is made up of the component of following weight/mass percentage composition：
Glucose 2.5%-2.8%；Maltose 1.5%-2.0%；Semen Glycines powder 0.8%-1.0%；Ammonium sulfate 0.2%-
0.3%；Balance of sterilized water；PH=7.0-7.3；
Inoculum concentration is first order seed：Volume ratio=the 12%-18% of secondary seed culture fluid, first order seed is accessed sterilizing
Secondary seed culture fluid afterwards makes secondary seed through culture, and the process conditions in the preparation process of secondary seed are culture temperature
30 DEG C -32 DEG C of degree, ventilation 0.9vvm-1.0vvm, cultivation cycle 8h-10h.
In described step C, the viscosity measurement of culture fluid is as follows：NDJ- from Shanghai balance equipment factory
1RotationalViscometer, step is to take culture fluid 500ml to be placed in beaker, is that 3, RPM measures for 6 with Rotor, reads
Go out dial plate registration and be multiplied by coefficient to obtain final product viscosity number.
For verifying the technique effect of the present invention, applicant carried out following test：
First, according to β -1,3 glucosans have water solublity, as long as the very low concentration of solution can produce very high-viscosity property
Can, in the case of having no GB at present, reference《National food safety standard food additive xanthan gum》(GB1886.41-
2015) detection method of A.3 clause viscosity in appendix A in, evaluates β -1,3 glucan product viscosity numbers, concrete grammar is as follows：
1st, the preparation of powder-product：Final fermentation liquid is added after 95% ethanol of 2 times of volumes by fermentation ends, 6000g
Gravity centrifugation 15min obtains solid precipitation, and to constant weight, grind into powder is standby for 60 DEG C of dryings.
2nd, evaluation result
Viscosity number is more than 1500cp.
2nd, the detection of yield
The detection of yield there is no GB at present, measures and typically adopt with the following method in industry：
1st, instrument
Calorstat, analytical balance (0.001g).
2nd, detection method
Fermentation liquid is poured in the small beaker of certain volume, make its bubble-free with Glass rod stirring, with Glass rod surface
Floating, the fermentation liquid outside cup is cleaned up.The fermentation liquid ethanol extraction of 90%-95%, sedimentation, filter cloth are extracted, will
It is all thoroughly moved in culture dish after tearing up, and is then placed in dry to constant weight in 105 DEG C of baking ovens (about 4 hours), takes out and weigh.
Solid content=title sample grams/volume × 100%.
3rd, testing result
Fermentation yield using the method for the present invention is 5.0g-5.5g/L.
The substantive distinguishing features that the present invention possesses and significant technological progress are：
1 the invention discloses a kind of Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954, this strain
There is preferable β -1,3 glucosan production capacity, and can be using including glucose, maltose, corn starch hydrolyzed solution, Semen Maydiss
Starch, in interior carbon source, is easy to industrialized production and with low cost.
2nd, the method for the present invention is adopted to prepare β -1,3 glucosans, fermentation yield and viscosity height, fermentation yield reaches 5.0g-
5.5g/L, viscosity is up to 1550-1630cp.
3rd, in sweat using stage temperature control with during fill into the technological condition for fermentation of trace element, hydrogen peroxide, make
Ferment control is stable, promotes growth and the metabolism of strain.
4th, the present invention can be obtained β -1,3 glucosans, low production cost, cycle is short by fermentation, is not subject to season and region
Restriction it is easy to manipulate, supply is stable etc., has the stronger market competitiveness and wide application prospect.
5th, β -1 produced using the method for the present invention, 3 glucosans, there is water solublity, a small amount of β -1,3 glucosans can be joined
Become high viscosity solution, can be applied in the industries such as industry, agricultural, food as thickening agent, suspending agent etc..
Strain Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954 applicant in the present invention in
September submits the Chinese microorganism strain preservation conservator being located at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 on 12nd within 2016
Meeting common micro-organisms center preservation, this depositary institution abbreviation CGMCC.
Specific embodiment
With reference to embodiments the present invention is described further, but not as a limitation of the invention, the guarantor of the present invention
The content that shield scope is recorded by claim is defined, any equivalent technical elements replacement made according to description, all without departing from
Protection scope of the present invention.
Embodiment 1
A kind of Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954.
Produce β -1, the method for 3 dextran fermentation liquid using edaphic bacilluss, the method comprises the technical steps that：
A, Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954 is inoculated into after sterilizing and contains carbon
In the culture fluid in source, nitrogen source and necessary nutrient matter；
B, carry out air agitation fermentation；
C, when culture fluid viscosity no longer increase or fermentation period≤48 hour fermentation ends.
Seed liquor is pressed in described step A：The percent by volume of culture fluid is the inoculum concentration of 12%-18% by Bodhisattva root nodule
Bacterium (Rhizobium pusense) CGMCC No.12954 accesses in the culture fluid in fermentation tank.Inoculating in described step A is
When after sterilizing, culture-liquid temp is carried out when being down to 30-32 DEG C.
Described corn starch hydrolyzed solution is adopted and is made with the following method：Corn starch tap water is made into mass percent
Concentration is 30% mixed liquor, is warming up to 90-95 DEG C, maintains 40 minutes.
In described step A, culture fluid is made up of the component of following weight/mass percentage composition：
Corn starch hydrolyzed solution 4.0%；Maltose 2.0%；Semen Glycines powder 0.3%；Magnesium sulfate 0.1%；Oleum Glycines 0.05%；Surplus
For sterilized water；PH=8.0-8.5.
In described step A, culture fluid process for preparation is as follows：Component each in culture fluid is put in fermentation tank in proportion, plus
To regulation meter material volume, stirring 30-40 minute is allowed to dissolve tap water, adjusts culture fluid pH=8.0-8.5 with sodium hydroxide solution,
Stirring 30-40 minute mix homogeneously.
Step B comprises the technical steps that:
10-30 hour：30 DEG C of temperature, pressure 0.03MPa, pH=7.0-7.3, ventilation 0.4vvm；
31 hours-put tank：26 DEG C of temperature, pressure 0.03MPa, pH=7.0-7.3, ventilation 0.4vvm.
Carry out filling into hydrogen peroxide and trace element in described step B in air agitation sweat；Actual conditions is as follows：
15 hours：Fill into trace element；
26 hours：Fill into hydrogen peroxide；
Described trace element is configured to trace element solution in the following way：Citric acid 100g, calcium hydroxide 5g, hydrogen
Magnesium oxide 7g, manganese sulfate 0.083g, zinc sulfate 0.066g, copper sulphate pentahydrate 0.025g, cobalt sulfate 0.028g, magnesium sulfate
8.6g, ferrous sulfate 1.8g, boric acid 0.006g, plus distilled water is settled to and sterilizes after 1000ml；The addition of trace element solution
Add trace element solution 1L for every 1000L culture fluid.
The mass percent concentration of described hydrogen peroxide is 2%, and addition is the 0.05% of nutrient solution volume.
Step A is after amplification culture, to press Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954
According to seed liquor：The percent by volume of fermentation regulation meter material volume culture fluid is that the inoculum concentration of 12%-18% is inoculated in culture fluid.
Described amplification culture process includes：
(1) Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954 is inoculated in shake-flask seed culture medium
Shake-flask seed is made on surface；
(2) shake-flask seed is made after amplification culture seed liquor；
(3) by the seed liquor made by seed liquor：The percent by volume of fermentation regulation meter material volume culture fluid is 12%
Inoculum concentration accesses the culture fluid in fermentation tank, is fermented.
The process conditions preparing shake-flask seed in described amplification culture process steps (1) are：Picking 2 ring slant strains connect
Enter in the shake-flask seed culture medium after sterilizing, make shake-flask seed through shaken cultivation, cultivation temperature is 30 DEG C, shaking speed is
200 revs/min, cultivation cycle is 14 hours；
Shake-flask seed culture medium is grouped into by the group of following weight/mass percentage composition：
Glucose 2.5%；Maltose 1.5%；Peptone 1.0%；Semen Maydiss starch 1.0%；Balance of sterilized water；PH=
7.0-7.3.
Amplification culture in described amplification culture process steps (2) includes the preparation of first order seed, secondary seed successively
Preparation, wherein first order seed culture fluid is grouped into by the group of following masses percentage composition：
Glucose 2.5%；Maltose 1.5%；Yeast extract 0.4%；Ammonium sulfate 0.2%；Balance of sterilized water；PH=7.0-
7.3；
Inoculum concentration is shake-flask seed：Volume ratio=0.8% of first order seed culture fluid, shaking table seed is accessed after sterilizing
First order seed culture fluid makes first order seed through culture, and the process conditions in the preparation process of first order seed are cultivation temperature 30
DEG C, ventilation 0.9vvm, cultivation cycle 14 hours.
Described secondary seed culture fluid is made up of the component of following weight/mass percentage composition：
Glucose 2.5%；Maltose 1.5%；Semen Glycines powder 0.8%；Ammonium sulfate 0.2%；Balance of sterilized water；PH=7.0-
7.3；
Inoculum concentration is first order seed：Volume ratio=12% of secondary seed culture fluid, first order seed is accessed after sterilizing
Secondary seed culture fluid makes secondary seed through culture, and the process conditions in the preparation process of secondary seed are cultivation temperature 30
DEG C, ventilation 0.9vvm, cultivation cycle 10 hours.
In described step C, the viscosity measurement of culture fluid is as follows：NDJ- from Shanghai balance equipment factory
1RotationalViscometer, step is to take culture fluid 500ml to be placed in beaker, is that 3, RPM measures for 6 with Rotor, reads
Go out dial plate registration and be multiplied by coefficient to obtain final product viscosity number.
Embodiment 2
The present embodiment is with the difference of embodiment 1：
Seed liquor is pressed in described step A：The percent by volume of culture fluid be 18% inoculum concentration by Bodhisattva root nodule bacteria
(Rhizobium pusense) CGMCC No.12954 accesses in the culture fluid in fermentation tank.
In described step A, culture fluid is made up of the component of following weight/mass percentage composition：
Corn starch hydrolyzed solution 4.5%；Maltose 2.5%；Semen Glycines powder 0.5%；Magnesium sulfate 0.15%；Oleum Glycines 0.15%；Remaining
Measure as sterilized water；PH=8.0-8.5.
In described step A, culture fluid process for preparation is as follows：Component each in culture fluid is put in fermentation tank in proportion, plus
To regulation meter material volume, stirring is allowed to dissolve tap water for 40 minutes, adjusts culture fluid pH=8.0-8.5 with sodium hydroxide solution, stirs
Mix 40 minutes mix homogeneously.
Step B comprises the technical steps that:
10-30 hour：32 DEG C of temperature, pressure 0.05MPa, pH=7.0-7.3, ventilation 0.5vvm；
31 hours-put tank：29 DEG C of temperature, pressure 0.05MPa, pH=7.0-7.3, ventilation 0.5vvm.
Carry out filling into hydrogen peroxide and trace element in described step B in air agitation sweat；Actual conditions is as follows：
12 hours：Fill into trace element；
23 hours：Fill into hydrogen peroxide；
Step A is by Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954 after amplification culture,
According to seed liquor：The percent by volume of fermentation regulation meter material volume culture fluid is that 18% inoculum concentration is inoculated in culture fluid.
The process conditions preparing shake-flask seed in described amplification culture process steps (1) are：Picking 2 ring slant strains connect
Enter in the shake-flask seed culture medium after sterilizing, make shake-flask seed through shaken cultivation, cultivation temperature is 32 DEG C, shaking speed is
220 revs/min, cultivation cycle is 10 hours；
Shake-flask seed culture medium is grouped into by the group of following weight/mass percentage composition：
Glucose 2.8%；Maltose 2.0%；Peptone 1.5%；Semen Maydiss starch 1.5%；Balance of sterilized water；PH=
7.0-7.3.
Amplification culture in described amplification culture process steps (2) includes the preparation of first order seed, secondary seed successively
Preparation, wherein first order seed culture fluid is grouped into by the group of following masses percentage composition：
Glucose 2.8%；Maltose 2.0%；Yeast extract 0.6%；Ammonium sulfate 0.3%；Balance of sterilized water；PH=7.0-
7.3；
Inoculum concentration is shake-flask seed：Volume ratio=1.0% of first order seed culture fluid, shaking table seed is accessed after sterilizing
First order seed culture fluid makes first order seed through culture, and the process conditions in the preparation process of first order seed are cultivation temperature 32
DEG C, ventilation 1.0vvm, cultivation cycle 10 hours.
Described secondary seed culture fluid is made up of the component of following weight/mass percentage composition：
Glucose 2.8%；Maltose 2.0%；Semen Glycines powder 1.0%；Ammonium sulfate 0.3%；Balance of sterilized water；PH=7.0-
7.3；
Inoculum concentration is first order seed：Volume ratio=18% of secondary seed culture fluid, first order seed is accessed after sterilizing
Secondary seed culture fluid makes secondary seed through culture, and the process conditions in the preparation process of secondary seed are cultivation temperature 32
DEG C, ventilation 1.0vvm, cultivation cycle 8 hours.
Remaining content is same as Example 1.
Embodiment 3
The present embodiment is with the difference of embodiment 1：
Seed liquor is pressed in described step A：The percent by volume of culture fluid be 15% inoculum concentration by Bodhisattva root nodule bacteria
(Rhizobium pusense) CGMCC No.12954 accesses in the culture fluid in fermentation tank.
In described step A, culture fluid is made up of the component of following weight/mass percentage composition：
Corn starch hydrolyzed solution 4.2%；Maltose 2.3%；Semen Glycines powder 0.4%；Magnesium sulfate 0.13%；Oleum Glycines 0.1%；Surplus
For sterilized water；PH=8.0-8.5.
In described step A, culture fluid process for preparation is as follows：Component each in culture fluid is put in fermentation tank in proportion, plus
To regulation meter material volume, stirring is allowed to dissolve tap water for 35 minutes, adjusts culture fluid pH=8.0-8.5 with sodium hydroxide solution, stirs
Mix 35 minutes mix homogeneously.
Step B comprises the technical steps that:
10-30 hour：31 DEG C of temperature, pressure 0.04MPa, pH=7.0-7.3, ventilation 0.45vvm；
31 hours-put tank：28 DEG C of temperature, pressure 0.04MPa, pH=7.0-7.3, ventilation 0.45vvm.
Carry out filling into hydrogen peroxide and trace element in described step B in air agitation sweat；Actual conditions is as follows：
14 hours：Fill into trace element；
24 hours：Fill into hydrogen peroxide；
Step A is after amplification culture, to press Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954
According to seed liquor：The percent by volume of fermentation regulation meter material volume culture fluid is that 15% inoculum concentration is inoculated in culture fluid.
The process conditions preparing shake-flask seed in described amplification culture process steps (1) are：Picking 2 ring slant strains connect
Enter in the shake-flask seed culture medium after sterilizing, make shake-flask seed through shaken cultivation, cultivation temperature is 31 DEG C, shaking speed is
210 revs/min, cultivation cycle is 12 hours；
Shake-flask seed culture medium is grouped into by the group of following weight/mass percentage composition：
Glucose 2.6%；Maltose 1.7%；Peptone 1.3%；Semen Maydiss starch 1.3%；Balance of sterilized water；PH=
7.0-7.3.
Amplification culture in described amplification culture process steps (2) includes the preparation of first order seed, secondary seed successively
Preparation, wherein first order seed culture fluid is grouped into by the group of following masses percentage composition：
Glucose 2.6%；Maltose 1.7%；Yeast extract 0.5%；Ammonium sulfate 0.25%；Balance of sterilized water；PH=
7.0-7.3；
Inoculum concentration is shake-flask seed：Volume ratio=the 0.8%-1.0% of first order seed culture fluid, shaking table seed is accessed and goes out
First order seed culture fluid after bacterium makes first order seed through culture, and the process conditions in the preparation process of first order seed are cultures
31 DEG C of temperature, ventilation 0.95vvm, cultivation cycle 12h.
Described secondary seed culture fluid is made up of the component of following weight/mass percentage composition：
Glucose 2.7%；Maltose 1.7%；Semen Glycines powder 0.9%；Ammonium sulfate 0.25%；Balance of sterilized water；PH=7.0-
7.3；
Inoculum concentration is first order seed：Volume ratio=15% of secondary seed culture fluid, first order seed is accessed after sterilizing
Secondary seed culture fluid makes secondary seed through culture, and the process conditions in the preparation process of secondary seed are cultivation temperature 31
DEG C, ventilation 0.95vvm, cultivation cycle 9h.
Remaining content is same as Example 1.
Embodiment 4
The present embodiment is with the difference of embodiment 1：
Seed liquor is pressed in described step A：The percent by volume of culture fluid be 13% inoculum concentration by Bodhisattva root nodule bacteria
(Rhizobium pusense) CGMCC No.12954 accesses in the culture fluid in fermentation tank.
In described step A, culture fluid is made up of the component of following weight/mass percentage composition：
Corn starch hydrolyzed solution 4.1%；Maltose 2.1%；Semen Glycines powder 0.35%；Magnesium sulfate 0.11%；Oleum Glycines 0.08%；Remaining
Measure as sterilized water；PH=8.0-8.5.
In described step A, culture fluid process for preparation is as follows：Component each in culture fluid is put in fermentation tank in proportion, plus
To regulation meter material volume, stirring is allowed to dissolve tap water for 32 minutes, adjusts culture fluid pH=8.0-8.5 with sodium hydroxide solution, stirs
Mix 32 minutes mix homogeneously.
Step B comprises the technical steps that:
10-30 hour：31 DEG C of temperature, pressure 0.035MPa, pH=7.0-7.3, ventilation 0.42vvm；
31 hours-put tank：27 DEG C of temperature, pressure 0.035MPa, pH=7.0-7.3, ventilation 0.42vvm.
Carry out filling into hydrogen peroxide and trace element in described step B in air agitation sweat；Actual conditions is as follows：
13 hours：Fill into trace element；
24 hours：Fill into hydrogen peroxide；
Step A is after amplification culture, to press Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954
According to seed liquor：The percent by volume of fermentation regulation meter material volume culture fluid is that 13% inoculum concentration is inoculated in culture fluid.
The process conditions preparing shake-flask seed in described amplification culture process steps (1) are：Picking 2 ring slant strains connect
Enter in the shake-flask seed culture medium after sterilizing, make shake-flask seed through shaken cultivation, cultivation temperature is 31 DEG C, shaking speed is
210 revs/min, cultivation cycle is 11 hours；
Shake-flask seed culture medium is grouped into by the group of following weight/mass percentage composition：
Glucose 2.6%；Maltose 1.6%；Peptone 1.1%；Semen Maydiss starch 1.2%；Balance of sterilized water；PH=
7.0-7.3.
Amplification culture in described amplification culture process steps (2) includes the preparation of first order seed, secondary seed successively
Preparation, wherein first order seed culture fluid is grouped into by the group of following masses percentage composition：
Glucose 2.6%；Maltose 1.6%；Yeast extract 0.45%；Ammonium sulfate 0.22%；Balance of sterilized water；PH=
7.0-7.3；
Inoculum concentration is shake-flask seed：Volume ratio=the 0.8%-1.0% of first order seed culture fluid, shaking table seed is accessed and goes out
First order seed culture fluid after bacterium makes first order seed through culture, and the process conditions in the preparation process of first order seed are cultures
31 DEG C of temperature, ventilation 0.95vvm, cultivation cycle 12 hours.
Described secondary seed culture fluid is made up of the component of following weight/mass percentage composition：
Glucose 2.6%；Maltose 1.6%；Semen Glycines powder 0.85%；Ammonium sulfate 0.22%；Balance of sterilized water；PH=7.0-
7.3；
Inoculum concentration is first order seed：Volume ratio=13% of secondary seed culture fluid, first order seed is accessed after sterilizing
Secondary seed culture fluid makes secondary seed through culture, and the process conditions in the preparation process of secondary seed are cultivation temperature 31
DEG C, ventilation 0.95vvm, cultivation cycle 8.5h.
Remaining content is same as Example 1.
Embodiment 5
The present embodiment is with the difference of embodiment 1：
Seed liquor is pressed in described step A：The percent by volume of culture fluid be 17% inoculum concentration by Bodhisattva root nodule bacteria
(Rhizobium pusense) CGMCC No.12954 accesses in the culture fluid in fermentation tank.
In described step A, culture fluid is made up of the component of following weight/mass percentage composition：
Corn starch hydrolyzed solution 4.4%；Maltose 2.4%；Semen Glycines powder 0.45%；Magnesium sulfate 0.14%；Oleum Glycines 0.13%；Remaining
Measure as sterilized water；PH=8.0-8.5.
In described step A, culture fluid process for preparation is as follows：Component each in culture fluid is put in fermentation tank in proportion, plus
To regulation meter material volume, stirring is allowed to dissolve tap water for 38 minutes, adjusts culture fluid pH=8.0-8.5 with sodium hydroxide solution, stirs
Mix 38 minutes mix homogeneously.
Step B comprises the technical steps that:
10-30 hour：31 DEG C of temperature, pressure 0.045MPa, pH=7.0-7.3, ventilation 0.45vvm；
31 hours-put tank：28 DEG C of temperature, pressure 0.045MPa, pH=7.0-7.3, ventilation 0.45vvm.
Carry out filling into hydrogen peroxide and trace element in described step B in air agitation sweat；Actual conditions is as follows：
12-15 hour：Fill into trace element；
23-26 hour：Fill into hydrogen peroxide；
Step A is after amplification culture, to press Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954
According to seed liquor：The percent by volume of fermentation regulation meter material volume culture fluid is that 17% inoculum concentration is inoculated in culture fluid.
The process conditions preparing shake-flask seed in described amplification culture process steps (1) are：Picking 2 ring slant strains connect
Enter in the shake-flask seed culture medium after sterilizing, make shake-flask seed through shaken cultivation, cultivation temperature is 32 DEG C, shaking speed is
215 revs/min, cultivation cycle is 13 hours；
Shake-flask seed culture medium is grouped into by the group of following weight/mass percentage composition：
Glucose 2.7%；Maltose 1.8%；Peptone 1.4%；Semen Maydiss starch 1.4%；Balance of sterilized water；PH=
7.0-7.3.
Amplification culture in described amplification culture process steps (2) includes the preparation of first order seed, secondary seed successively
Preparation, wherein first order seed culture fluid is grouped into by the group of following masses percentage composition：
Glucose 2.7%；Maltose 1.8%；Yeast extract 0.55%；Ammonium sulfate 0.28%；Balance of sterilized water；PH=
7.0-7.3；
Inoculum concentration is shake-flask seed：Volume ratio=1.0% of first order seed culture fluid, shaking table seed is accessed after sterilizing
First order seed culture fluid makes first order seed through culture, and the process conditions in the preparation process of first order seed are cultivation temperature 30
DEG C, ventilation 1.0vvm, cultivation cycle 13h.
Described secondary seed culture fluid is made up of the component of following weight/mass percentage composition：
Glucose 2.7%；Maltose 1.8%；Semen Glycines powder 0.95%；Ammonium sulfate 0.28%；Balance of sterilized water；PH=7.0-
7.3；
Inoculum concentration is first order seed：Volume ratio=17% of secondary seed culture fluid, first order seed is accessed after sterilizing
Secondary seed culture fluid makes secondary seed through culture, and the process conditions in the preparation process of secondary seed are cultivation temperature 30
DEG C, ventilation 0.95vvm, cultivation cycle 9.5h.
Remaining content is same as Example 1.
Applicant is detected, correlated results is such as to the fermentation yield in embodiment 1-5 and prepared product viscosity
Under：
| Fermentation yield (g/L) | The product viscosity (cp) of fermentation preparation | |
| Embodiment 1 | 5.0 | 1550 |
| Embodiment 2 | 5.2 | 1600 |
| Embodiment 3 | 5.4 | 1580 |
| Embodiment 4 | 5.5 | 1650 |
| Embodiment 5 | 5.28 | 1630 |
Claims (17)
1. a kind of Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954.
2. produce β -1 using Bodhisattva root nodule bacteria, the method for 3 dextran fermentation liquid is it is characterised in that the method includes following technique
Step：
A, Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954 is inoculated into after sterilizing and contains carbon source, nitrogen
In the culture fluid of source and necessary nutrient matter；
B, carry out air agitation fermentation；
C, when culture fluid viscosity no longer increase or fermentation period≤48 hour fermentation ends.
3. according to claim 2 produce β -1 using Bodhisattva root nodule bacteria, the method for 3 dextran fermentation liquid it is characterised in that
Carbon source in the culture fluid of described step A is selected from one of glucose, maltose, corn starch hydrolyzed solution, Semen Maydiss starch
Or its combination；Nitrogen source is selected from one of Semen Glycines powder, Oleum Glycines, peptone, yeast extract, ammonium sulfate or its combination.
4. according to claim 2 produce β -1 using Bodhisattva root nodule bacteria, the method for 3 dextran fermentation liquid it is characterised in that
Seed liquor is pressed in described step A：The percent by volume of culture fluid is the inoculum concentration of 12%-18% by Bodhisattva root nodule bacteria
(Rhizobium pusense) CGMCC No.12954 accesses in the culture fluid in fermentation tank.
5. according to claim 3 produce β -1, the method for 3- dextran fermentation liquid using Bodhisattva root nodule bacteria, its feature exists
Adopt in described corn starch hydrolyzed solution and make with the following method：Corn starch tap water is made into mass percent concentration is
30% mixed liquor, is warming up to 90-95 DEG C, maintains 40 minutes.
6. produce β -1, the side of 3 dextran fermentation liquid using Bodhisattva root nodule bacteria according to any one of claim 3 or 5
Method, in described step A, culture fluid is made up of the component of following weight/mass percentage composition：
Corn starch hydrolyzed solution 4.0%-4.5%；Maltose 2.0%-2.5%；Semen Glycines powder 0.3%-0.5%；Magnesium sulfate 0.1%-
0.15%；Oleum Glycines 0.05%-0.15%；Balance of sterilized water；PH=8.0-8.5.
7. according to claim 6 produce β -1 using Bodhisattva root nodule bacteria, the method for 3 dextran fermentation liquid it is characterised in that
In described step A, culture fluid process for preparation is as follows：Component each in culture fluid is put in fermentation tank in proportion, plus tap water is extremely
Regulation meter material volume, stirring 30-40 minute is allowed to dissolve, and adjusts culture fluid pH=8.0-8.5 with sodium hydroxide solution, stirs 30-
40 minutes mix homogeneously.
8. according to claim 2 produce β -1 using Bodhisattva root nodule bacteria, the method for 3 dextran fermentation liquid it is characterised in that
Described step B comprises the technical steps that:
10-30 hour：30 DEG C -32 DEG C of temperature, pressure 0.03-0.05MPa, pH=7.0-7.3, ventilation 0.4-0.5vvm；
31 hours-put tank：26 DEG C -29 DEG C of temperature, pressure 0.03-0.05MPa, pH=7.0-7.3, ventilation 0.4-0.5vvm.
9. according to claim 8 produce β -1 using Bodhisattva root nodule bacteria, the method for 3 dextran fermentation liquid it is characterised in that
Carry out filling into hydrogen peroxide and trace element in described step B in air agitation sweat；Actual conditions is as follows：
12-15 hour：Fill into trace element；
23-26 hour：Fill into hydrogen peroxide.
10. according to claim 9 produce β -1, the method for 3 dextran fermentation liquid using Bodhisattva root nodule bacteria, its feature exists
It is configured to trace element solution in described trace element in the following way：Citric acid 100g, calcium hydroxide 5g, magnesium hydroxide
7g, manganese sulfate 0.083g, zinc sulfate 0.066g, copper sulphate pentahydrate 0.025g, cobalt sulfate 0.028g, magnesium sulfate 8.6g, sulfur
Acid ferrous iron 1.8g, boric acid 0.006g, plus distilled water is settled to and sterilizes after 1000ml；The addition of trace element solution is every
1000L culture fluid adds trace element solution 1L.
11. according to claim 9 produce β -1, the method for 3 dextran fermentation liquid using Bodhisattva root nodule bacteria, its feature exists
In described hydrogen peroxide mass percent concentration be 2-3%, addition be nutrient solution volume 0.05%.
12. sides producing β -1,3 dextran fermentation liquid using Bodhisattva root nodule bacteria according to any one of Claims 2 or 3
Method is it is characterised in that step A is through amplification culture by Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954
Afterwards, according to seed liquor：The percent by volume of fermentation regulation meter material volume culture fluid is that the inoculum concentration of 12%-18% is inoculated in culture
In liquid.
13. according to claim 12 produce β -1, the method for 3 dextran fermentation liquid using Bodhisattva root nodule bacteria, its feature exists
Include in described amplification culture process：
(1) Bodhisattva root nodule bacteria (Rhizobium pusense) CGMCC No.12954 is inoculated in shake-flask seed culture medium surface
Make shake-flask seed；
(2) shake-flask seed is made after amplification culture seed liquor；
(3) by the seed liquor made by seed liquor：The percent by volume of fermentation regulation meter material volume culture fluid is 12%-18%'s
Inoculum concentration accesses the culture fluid in fermentation tank, is fermented.
14. according to claim 13 produce β -1, the method for 3 dextran fermentation liquid using Bodhisattva root nodule bacteria, its feature exists
The process conditions preparing shake-flask seed in described amplification culture process steps (1) are：Picking 2 ring slant strains access sterilizing
In shake-flask seed culture medium afterwards, make shake-flask seed through shaken cultivation, cultivation temperature is 30-32 DEG C, shaking speed is 200-
220 revs/min, cultivation cycle is 10-14 hour；
Shake-flask seed culture medium is grouped into by the group of following weight/mass percentage composition：
Glucose 2.5%-2.8%；Maltose 1.5%-2.0%；Peptone 1.0%-1.5%；Semen Maydiss starch 1.0%-
1.5%；Balance of sterilized water；PH=7.0-7.3.
15. according to claim 11 produce β -1, the method for 3 dextran fermentation liquid using Bodhisattva root nodule bacteria, its feature exists
Amplification culture in described amplification culture process steps (2) includes the preparation of the preparation of first order seed, secondary seed successively,
Wherein first order seed culture fluid is grouped into by the group of following masses percentage composition：
Glucose 2.5%-2.8%；Maltose 1.5%-2.0%；Yeast extract 0.4%-0.6%；Ammonium sulfate 0.2%-0.3%；
Balance of sterilized water；PH=7.0-7.3；
Inoculum concentration is shake-flask seed：Volume ratio=the 0.8%-1.0% of first order seed culture fluid, shaking table seed is accessed after sterilizing
First order seed culture fluid make first order seed through culture, the process conditions in the preparation process of first order seed are cultivation temperature
30 DEG C -32 DEG C, ventilation 0.9vvm-1.0vvm, cultivation cycle 10-14 hour.
16. according to claim 13 produce β -1, the method for 3 dextran fermentation liquid using Bodhisattva root nodule bacteria, its feature exists
It is made up of the component of following weight/mass percentage composition in described secondary seed culture fluid：
Glucose 2.5%-2.8%；Maltose 1.5%-2.0%；Semen Glycines powder 0.8%-1.0%；Ammonium sulfate 0.2%-0.3%；Remaining
Measure as sterilized water；PH=7.0-7.3；
Inoculum concentration is first order seed：Volume ratio=the 12%-18% of secondary seed culture fluid, first order seed is accessed after sterilizing
Secondary seed culture fluid makes secondary seed through culture, and the process conditions in the preparation process of secondary seed are cultivation temperature 30
DEG C -32 DEG C, ventilation 0.9vvm-1.0vvm, cultivation cycle 8-10 hour.
17. according to claim 2 produce β -1, the method for 3 dextran fermentation liquid using Bodhisattva root nodule bacteria, its feature exists
In described step C, the viscosity measurement of culture fluid is as follows：NDJ- from Shanghai balance equipment factory
1RotationalViscometer, step is to take culture fluid 500ml to be placed in beaker, is that 3, RPM measures for 6 with Rotor, reads
Go out dial plate registration and be multiplied by coefficient to obtain final product viscosity number.
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| CN112358985A (en) * | 2020-11-09 | 2021-02-12 | 河北沣川生物科技有限公司 | Pradazobium and application thereof in preparation of water-soluble beta-1, 3 glucan |
| CN112358985B (en) * | 2020-11-09 | 2022-08-23 | 河北沣川生物科技有限公司 | Pradazobium and application thereof in preparation of water-soluble beta-1, 3 glucan |
| CN113943761A (en) * | 2021-12-22 | 2022-01-18 | 山东国力生物科技有限公司 | Preparation method of micromolecular beta-1, 3-glucan |
| CN113943761B (en) * | 2021-12-22 | 2022-03-22 | 山东国力生物科技有限公司 | Preparation method of micromolecular beta-1, 3-glucan |
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