CN110004140A - A method of improving curdlan Screening of strain with high productivity efficiency - Google Patents
A method of improving curdlan Screening of strain with high productivity efficiency Download PDFInfo
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Abstract
The present invention relates to a kind of methods for improving curdlan Screening of strain with high productivity efficiency, belong to Microbial Breeding technical field, mainly comprise the steps that (1) produces the culture of curdlan starting strain；(2) the NTG mutagenesis of starting strain；(3) aniline blue plate screening culture medium is prepared；(4) NaI and NaIO is added in aniline blue screening and culturing medium3；(5) mutagenesis bacterium solution is in aniline blue and NaI and NaIO3Primary dcreening operation on double screening flat board culture mediums；(6) 50mL/250mL shake flask fermentation secondary screening, according to the thick yields screening superior strain of measurement；(7) the secondary secondary screening of 75mL/500mL shake flask fermentation, according to measurement curdlan yields screening superior strain.The method of the present invention adds chemicals NaI and NaIO in plate screening culture medium3, the principle that elemental iodine kills acidogenic bactria is generated under acidic environment using it, screening is increased and obtains the probability of high yield curdlan bacterial strain, improve flat screen bacterium efficiency, shorten the sieve bacterium period.
Description
Technical field
The present invention relates to Microbial Breeding technical fields, and in particular to one kind can be improved curdlan Screening of strain with high productivity
The method of efficiency.
Background technique
Curdlan (Curdlan) is a kind of exocellular polysaccharide of microbial fermentation production, is provided simultaneously with plant polyose and closes
At the peculiar property of polymer substance and safe and non-toxic, by the research and development of decades, it is applied to multiple industry necks
Domain.Curdlan at home and abroad has the huge market demand, the application demand in the fields such as food, chemical industry, health care, medical treatment and day
All to increase, the main market share is captured by the U.S. and Japan, and the curdlan research level in China is mainly in laboratory research
In the stage, although existing factory starts to carry out large-scale industrial production, its yield and quality still has one compared with external product
Gap is determined, so take effective research method is to promote the yield and quality of curdlan and reach the standard of commercially producing
It is necessary to.
Induced mutations breeding is a kind of breeding mode that can effectively obtain superior strain, it is with nondirectional gene mutation
Wild type strain is improved on basis, obtains the excellent species that can stablize heredity and generate more useable products.Mutation breeding
The plate screening of mutant strain is very important link in the process, and traditional plate screening has blindness, randomness, to screening
There are biggish obstruction and limitation to superior strain, therefore, establishes effective plate screening method to improve sieve bacterium efficiency, shorten sieve
The bacterium period is most important.
Aniline blue is a kind of biological stain, it can combine with curdlan specificity and form blue complex, meanwhile,
Aniline blue can be also displayed in blue in acid condition.When using plating medium screening curdlan superior strain, benzene is utilized
Amine indigo plant shows the principle of blue in conjunction with curdlan, uses the coloring agent as indicator, screening efficiency can be improved.
Acid can be produced while producing glue by producing curdlan bacterial strain, and the carbon source of culture medium can be consumed by producing acid, influence curdlan
Yield, the yield of curdlan can be improved by producing acid and reducing or do not produce glue, thus the present invention propose to pass through it is flat in aniline blue screening
NaI and NaIO are further added on plate3Concentration, iodide ion and iodate ion have proton (H in acid+) under conditions of participation,
Reaction generates elemental iodine (I2), elemental iodine has fatal toxic action to somatic cells, can kill production acid and produce proton (H+) bacterium
Body, therefore generate H+More, that is, producing the more thallus of acid can be in the elemental iodine (I contained2) culture medium in be killed, and produce acid
The thallus for producing acid less or not can then survive in this context, and the thallus for producing curdlan, produce sour fewer, yield
Higher probability is bigger, therefore, by this method, can greatly improve the efficiency for screening high yield curdlan bacterial strain.
Summary of the invention
The purpose of the present invention is establishing a kind of effective plate screening method, curdlan Screening of strain with high productivity can be improved
Efficiency improves the mutagenesis screening efficiency of curdlan superior strain, shortens the sieve bacterium period.
The invention proposes a kind of methods for improving curdlan Screening of strain with high productivity efficiency, comprising the following steps:
(1) culture of curdlan starting strain is produced；
(2) the NTG mutagenesis of starting strain；
(3) aniline blue plate screening culture medium is prepared；
(4) NaI and NaIO is added in aniline blue screening and culturing medium3；
(5) mutagenesis bacterium solution is in aniline blue and NaI and NaIO3Primary dcreening operation on double screening flat board culture mediums；
(6) 50mL/250mL shake flask fermentation secondary screening, according to the thick yields screening superior strain of measurement；
(7) the secondary secondary screening of 75mL/500mL shake flask fermentation, according to measurement curdlan yields screening superior strain.
In step (1), the production curdlan starting strain is to produce curdlan bacillus ATCC31749, and any can produce can
Obtaining right glue and capable of producing sour agrobacterium could be used for method of the present invention, and the present invention is not particularly limited.
In step (2), the NTG method of mutagenesis for producing curdlan starting strain are as follows: the kind for taking appropriate shake culture good
Sub- liquid centrifugation, OD6000.2~0.8, bacterial sediment is resuspended in buffer for control, is added isometric 0.1~1mg/mL's after dilution
NTG solution reacts 20~40min, and hypo solution is then added and terminates reaction, above-mentioned reactant is centrifuged, nothing is used
Simultaneously bacterial sediment is resuspended in bacterium water washing, obtains mutagenesis bacterium solution.
Wherein, the good seed liquor of suitable shake culture can be the good seed liquor of 2mL shake culture.
In the present invention, " the producing curdlan bacterial strain " refers to all bacterial strains that can generate curdlan.
In the present invention, " seed liquor " refers to the bacterium solution of the production curdlan bacterial strain of shaking flask culture.
Preferably, the condition of culture of the seed liquor are as follows: 30 DEG C, 250rpm, 18h.
In the present invention, buffer is 20mM Tris maleate buffer, pH value 8.5.
In the present invention, the reaction condition of the method for mutagenesis is 30-35 DEG C, 180-220rpm, 20-60min；Preferably,
The mutagenesis reaction condition are as follows: 30 DEG C, 200rpm, 30min.
In the step (3), the component of the aniline blue plate screening culture medium includes: the ethylene glycol of 6-9%, 0.1-
0.4% yeast powder, the agar powder of 1-2% and the aniline blue of 0.003-0.005%, pH value 7.0；Preferably, the aniline
The component of blue plate screening culture medium includes: 7% ethylene glycol, 0.2% yeast powder, 2% agar powder and 0.005%
Aniline blue, pH value 7.0.
Wherein, the aniline blue in the aniline blue plate screening nutrient media components is bacterium colony color developing agent, it can be with
Curdlan specifically binds to form blue complex.Meanwhile aniline blue can be also displayed in blue in acid condition.
In step (4), the NaI and NaIO3For two kinds of anti-drugs of chemistry, can occur that iodine list should be generated in acid condition
Matter kills the somatic cells for producing acid.
In step (4), NaI solution and NaIO are prepared according to required drug concentration ratio3Solution is individually added after sterilizing
Into sterilized aniline blue culture medium, mix, inverted plate.Wherein, (NaI is dense eventually for the NaI final concentration being added in the culture medium
Degree, that is, after culture medium is added, the concentration of NaI in culture medium) range is 0.05-0.2mol/L, NaIO3Final concentration (NaIO3It is dense eventually
Degree, that is, after culture medium is added, NaIO in culture medium3Concentration) range is 0.01-0.04mol/L.
In step (5), addition NaI and NaIO will be coated on after the mutagenesis bacterium solution doubling dilution obtained by step (2)3's
On plate screening culture medium, cultivate 4~10 days, as experimental group；Coating is not added with NaI and NaIO simultaneously3Plate screening training
Feeding base is as a control group.
Wherein, experimental group is consistent with the mutagenesis bacterium solution extension rate that control group is coated with, and bacterium solution volume is consistent.
Preferably, all addition drugs such as NaI, NaIO3Plate screening culture medium in, the numberical range of NaI content is
0.05-0.2mol/L；NaIO3The numberical range of content is 0.01-0.04mol/L.It is further preferred that contained NaI with
NaIO3Concentration (mol/L) ratio is a certain determining value.
Preferably, flat-plate bacterial colony condition of culture is 30-35 DEG C；It is further preferred that flat-plate bacterial colony condition of culture is 30 DEG C
Stationary culture.
Preferably, flat-plate bacterial colony incubation time is 4-10 days；It is further preferred that flat-plate bacterial colony incubation time is 6 days.
In step (6), the method for the shake flask fermentation secondary screening are as follows: select the bacterium colony grown on screening flat board culture medium, connect
Kind is in 50mL/250mL (50mL/250mL refers to that the volume of fermentation medium in 250mL shaking flask is 50mL) Medium of shaking flask fermentation
Interior, 250rpm, 30 DEG C, constant-temperature shaking culture 96h obtains fermentation liquid.According to the thick preferred superior strain of yield of measurement.
Wherein, the bacterium colony total sample number selected on control group plate should be with the bacterium colony total sample number selected on experimental group plate
Unanimously.
Preferably, the range of the shake flask fermentation condition of culture is 30-35 DEG C, 200-250rpm, 48-96h；It is further excellent
Selection of land, the shake flask fermentation condition of culture are as follows: 30 DEG C, 250rpm, 96h.
In step (6), the thick determination of yield method of curdlan in the fermentation liquid are as follows: take 10mL fermentation liquid in 50mL from
In heart pipe, 9000rpm is centrifuged 5min, and precipitating is cleaned with 95% ethyl alcohol, stood after mixing well, and is centrifuged, and repeats ethyl alcohol and cleaned
Journey 2 times, centrifugation is deposited in 60 DEG C of baking ovens and dries overnight, and weigh M, and calculating slightly proposes yield: it is thick mention yield g/L=M/10 ×
1000。
In step (7), the method for the shaking flask secondary fermentation secondary screening are as follows: select the bacterium grown on screening flat board culture medium
It falls, is inoculated in 50mL/250mL (50mL/250mL refers to that the volume of fermentation medium in 250mL shaking flask is 50mL) or 75mL/
In 500mL (75mL/500mL refers to that the volume of fermentation medium in 500mL shaking flask is 75mL) Medium of shaking flask fermentation, 250rpm,
30 DEG C, constant-temperature shaking culture 96h obtains fermentation liquid, according to the measurement preferred superior strain of curdlan yield.
In the present invention, the component of the shake flask fermentation secondary screening and the secondary secondary screening culture medium of shake flask fermentation includes: 9% sugarcane
Sugar, 0.5% (NH4)2HPO4, 0.15% KH2PO4, 0.1% MgSO4·7H2O, 0.3% yeast powder, pH are adjusted to 7.0.
In step (7), the measurement curdlan throughput method are as follows: take 5mL fermentation liquid, 3mol/LNaOH solution is added
75mL is stirred well to glue and is completely dissolved.30min, 9000rpm are stood, 10min is centrifuged, takes supernatant.With 3mol/L HCl solution
75mL is mixed with alkaline-sol, is uniformly mixed.After standing 10min, 9000rpm is centrifuged 5min.Precipitating 150mL distilled water
Three times, each 9000rpm is centrifuged 5min for washing.It is deposited in 60 DEG C of drying boxes to dry overnight, weigh M, can be calculated right glue and produces
Amount: yield g/L=M/5 × 1000.
In the present invention, the standard of the preferred superior strain is that the thick yield of curdlan or yield are higher than starting strain.
Curdlan is slightly mentioned to mutant strain of the yield higher than starting strain as direct mutation bacterial strain, the calculating of positive mutation rate
Mode are as follows: positive mutation rate (%)=(direct mutation bacterial strain number ÷ bacterium total sample number in bacterium sample) × 100%.
Its difference is compared in the positive mutation rate of experiment with computing group and control group, analysis, and experimental group positive mutation rate should be greater than control group.
In the present invention, " the direct mutation bacterial strain " refers to that curdlan slightly mentions all mutant bacterias that yield is higher than starting strain
Strain.
The present invention innovatively proposes that acid can be produced while producing glue by producing curdlan bacterial strain, and production acid is more can be certain
The secretory volume that curdlan is influenced in degree, is added chemicals NaI and NaIO in plate screening culture medium3, iodide ion and
For iodate ion under conditions of having proton (H+) participation, reaction generates elemental iodine (I2), iodine has somatic cells fatal
Toxic action, cell can be killed, H+ is more, that is, I-IO can contained by producing the more bacterium of acid from the foregoing, it will be observed that generating3 -Training
It supports and is killed in base, and produce the fewer bacterium of acid and be more possible to survive in this context, for producing curdlan bacillus, produce
It is bigger that the few bacterial strain of acid produces a possibility that glue is more.
Beneficial effect of the present invention further include: shorten the sieve bacterium period, improve flat screen bacterium efficiency, establish effectively industrial breeding
New method improves the situation that aniline blue plate background color becomes blue.
Detailed description of the invention
It is 7.0,6.5,6.0,5.5, NaI and NaIO Fig. 1 shows medium pH gradient in the present invention3Concentration (mol/L) ratio
Example is 0.05/0.01, the bacterium colony growing state of 0.075/0.015,0.1/0.02.
Fig. 2 indicates medium pH gradient in the present invention: 7.0,6.0, NaI and NaIO3Concentration (mol/L) ratio is 0.1/
The bacterium colony growing state of 0.02,0.15/0.03,0.2/0.04.
Fig. 3 indicate the present invention in NTG concentration be 0.3mg/mL, plate screening medium pH be 6.0, the NaI of addition with
NaIO3The curdlan of mutagenic strain slightly proposes yield when concentration is than for 0.1/0.02mol/L, and abscissa represents control group and experiment
The strain number of group bacterial strain (Fig. 3 a is control group, and Fig. 3 b is experimental group).
Fig. 4 indicate the present invention in NTG concentration be 0.5mg/mL, plate screening medium pH be 6.0, the NaI of addition with
NaIO3The curdlan of mutagenic strain slightly proposes yield when concentration is than for 0.1/0.02mol/L, and abscissa represents control group and experiment
The strain number of group bacterial strain (Fig. 4 a is control group, and Fig. 4 b is experimental group).
Fig. 5 indicate the present invention in NTG concentration be 0.7mg/mL, plate screening medium pH be 6.0, the NaI of addition with
NaIO3The curdlan of mutagenic strain slightly proposes yield when concentration is than for 0.1/0.02mol/L, and abscissa represents control group and experiment
The strain number of group bacterial strain (Fig. 5 a is control group, and Fig. 5 b is experimental group).
Specific embodiment
In conjunction with following specific embodiments and attached drawing, invention is further described in detail.Implement process of the invention, item
Part, experimental method etc. are among the general principles and common general knowledge in the art in addition to what is specifically mentioned below, the present invention
There are no special restrictions to content.
Embodiment 1 plate screening culture medium NaI and NaIO3The determination of concentration
According to different drug ratios by NaI and NaIO3The aniline blue screening and culturing medium that pH is 6.0, screening and culturing is added
Based component are as follows: ethylene glycol 7%, yeast powder 0.2%, aniline blue 0.005%；NaI and NaIO3Concentration (mol/L) ratio: 0.05/
0.01,0.075/0.015,0.1/0.02,0.15/0.03,0.2/0.04, agar 2%pH are adjusted to 7.0；By setting out for non-mutagenesis
It is coated on the plate of above-mentioned condition after bacterial strain bacterium solution doubling dilution and is not added with the plate of drug, bacterium colony growing state such as Fig. 1 institute
Show, the bacterium colony number statistical conditions on different condition plate are as shown in table 1.Control group flat-plate bacterial colony number is than experimental group flat-plate bacterial colony
Number is more, illustrates that experimental group plate has certain lethal effect；Control group flat-plate bacterial colony not only becomes blue, and also large area becomes blue to bottom plate,
And although experimental group flat-plate bacterial colony has all become blue, bottom plate, which has had no, becomes Lan Xianxiang, so NaI/NaIO is added3It can solve bacterium colony
It grows aniline blue plate bottom plate in process color and becomes blue problem, so that exclude strain causes bottom plate to become blue phenomenon because producing acid.
With the raising of drug concentration, the bacterium colony number on plate is fewer, and drug fatality acts on stronger, NaI and NaIO3It is dense
Lethal effect is suitable for when degree ratio is 0.1/0.02mol/L, and colony growth rate and growth conditions are relatively suitable, so optional
Select the plate screening for carrying out mutagenesis bacterium colony under this condition.
Bacterium colony grows number on 1 different condition plate of table
NaI and NaIO when embodiment 2NTG concentration is 0.3mg/mL3Influence to mutagenic strain the selection result
The NTG that the 20mM Tris maleate buffer for being 8.5 with pH value prepares 0.3mg/mL carries out chemical mutagenesis, plate
Screening and culturing medium are as follows: ethylene glycol 7%, yeast powder 0.2%, aniline blue 0.005%, the NaI and NaIO of addition3Concentration ratio is 0.1/
0.02mol/L, agar 2%, pH are adjusted to 7.0, and mutagenesis bacterium solution is coated with control group plate (being not added with drug) simultaneously and experimental group is flat
Plate (addition drug), control group and experimental group respectively choose 50 plants of bacterium, calculate according to selecting bacterial strain later stage fermentation and slightly mentioning output condition
16 plants of control group direct mutation bacterium, positive mutation rate 31%；24 plants of experimental group direct mutation bacterium, positive mutation rate 47%.Experimental group is just
Mutation rate is higher than control group positive mutation rate, illustrates under the experiment condition, and screening flat board adds NaI and NaIO3Can be produced from part
Bacterial strain more than acid kills, and makes to become larger a possibility that screening high yield glue ability bacterial strain.
The bacterial strain later stage fermentation liquid that control group and experimental group are selected after mutagenic strain slightly proposes yield as shown in figure 3, can by Fig. 3
Know, control group mutation rate is only 31%, and experimental group positive variation rate is 47%, significant difference (P < 0.05).
NaI and NaIO when embodiment 3NTG concentration is 0.5mg/mL3Influence to mutagenic strain the selection result
NTG concentration 0.5mg/mL, plate screening medium pH are 7.0, the NaI and NaIO of addition3Concentration ratio is 0.1/
0.02mol/L, mutagenesis bacterium solution are coated with control group plate (being not added with drug) and experimental group plate (addition drug), control group simultaneously
Respectively choose 50 plants of bacterium with experimental group, 14 plants of control group direct mutation bacterium, positive mutation rate 28%；23 plants of experimental group direct mutation bacterium, it is just prominent
Variability is 46%.
Bacterium colony number statistical conditions of the mutagenic strain on above-mentioned condition plate are as shown in table 3, and control group and experimental group are chosen
The bacterial strain later stage fermentation liquid of choosing slightly proposes yield as shown in figure 4, as shown in Figure 4, control group mutation rate is only 28%, and experimental group is just
Variability is 46%, significant difference (P < 0.05).
NaI and NaIO when embodiment 4NTG concentration is 0.7mg/mL3Influence to mutagenic strain the selection result
NTG concentration is 0.7mg/mL, and plate screening medium pH is 7.0, the NaI and NaIO of addition3Concentration ratio is 0.1/
0.02mol/L, mutagenesis bacterium solution are coated with control group plate (being not added with drug) and experimental group plate (addition drug), control group simultaneously
Respectively choose 50 plants of bacterium with experimental group, 17 plants of control group direct mutation bacterium, positive mutation rate 34%；28 plants of experimental group direct mutation bacterium, it is just prominent
Variability is 56%.Screening flat board adds NaI and NaIO3The probability for screening superior strain can be promoted.
Bacterium colony number statistical conditions of the mutagenic strain on above-mentioned condition plate are as shown in table 5, and control group and experimental group are chosen
The bacterial strain later stage fermentation liquid of choosing slightly proposes yield as shown in figure 5, as shown in Figure 5, control group mutation rate is only 34%, and experimental group is just
Variability is 56%, significant difference (P < 0.05).
The above-mentioned experimental result of comprehensive analysis, using suitable concentration is added in plate screening culture medium, (NaI concentration range is
0.05-0.2mol/L, NaIO3Concentration range is 0.01-0.04mol/L) NaI and NaIO3Method, can inhibit produce acid it is more
The growth of bacterium colony can increase when sieving bacterium from the screening flat board since bacterial strain produces acid and produces glue into certain negative correlation relationship
The probability of Producing Strain is picked out greatly, to improve positive mutation rate, achievees the purpose that improve curdlan Screening of strain with high productivity efficiency.
The present invention protects content not limit to above embodiments.Without departing from the spirit and scope of the invention, this field
Technical staff it is conceivable that variation and advantage be all included in the present invention, and with appended claims be protection model
It encloses.
Claims (10)
1. a kind of method for improving curdlan Screening of strain with high productivity efficiency, which is characterized in that the described method comprises the following steps:
(1) culture of curdlan starting strain is produced；
(2) the NTG mutagenesis of starting strain；
(3) aniline blue plate screening culture medium is prepared；
(4) NaI and NaIO is added in above-mentioned aniline blue plate screening culture medium3；
(5) mutagenesis bacterium solution is in aniline blue and NaI and NaIO3Primary dcreening operation on double screening flat board culture mediums；
(6) 50mL/250mL shake flask fermentation secondary screening, according to the thick yields screening superior strain of measurement；
(7) the secondary secondary screening of 75mL/500mL shake flask fermentation, according to measurement curdlan yields screening superior strain.
2. the method as described in claim 1, which is characterized in that in the step (2), the production curdlan starting strain
NTG method of mutagenesis are as follows: take the seed liquor centrifugation that shaken cultivation is good, OD6000.2~0.8, bacterial sediment is resuspended in buffer for control,
It is added after dilution in equal volume with the NTG solution of 0.1~1mg/mL of buffer solution, reacts 20~40min, be then added thio
Metabisulfite solution terminates reaction, and above-mentioned reactant is centrifuged, and is laid equal stress on outstanding bacterial sediment using sterile water washing, obtains mutagenic bacteria
Liquid.
3. method according to claim 2, which is characterized in that the buffer is 20mM Tris maleate buffer, pH value
It is 8.5.
4. the method as described in claim 1, which is characterized in that in the step (4), NaI final concentration range is 0.05-
0.2mol/L, NaIO3Final concentration range is 0.01-0.04mol/L.
5. the method as described in claim 1, which is characterized in that in the step (5), will be coated with after mutagenesis bacterium solution doubling dilution
In addition NaI and NaIO3Aniline blue Screening of Media plate on, cultivate 4~10 days, as experimental group；Coating does not add simultaneously
Add NaI and NaIO3Plate screening culture medium as a control group.
6. the method as described in claim 1, which is characterized in that the aniline blue plate screening culture medium are as follows: 7% second two
Alcohol, 0.2% yeast powder, 0.005% aniline blue, 2% agar, pH are adjusted to 7.0；Shake flask fermentation secondary screening and shake flask fermentation two
The component of secondary secondary screening culture medium includes: 9% sucrose, 0.5% (NH4)2HPO4, 0.15% KH2PO4, 0.1%
MgSO4·7H2O, 0.3% yeast powder, pH are adjusted to 7.0.
7. the method as described in claim 1, which is characterized in that in the step (6), the method for the shake flask fermentation secondary screening
Are as follows: the bacterium colony grown on screening flat board culture medium is selected, is inoculated in 50mL/250mL Medium of shaking flask fermentation, 250rpm, 30
DEG C, constant-temperature shaking culture 96h obtains fermentation liquid, according to the thick preferred superior strain of yield of measurement.
8. the method as described in claim 1, which is characterized in that in the step (7), the side of the secondary secondary screening of shake flask fermentation
Method are as follows: select the bacterium colony grown on screening flat board culture medium, be inoculated in 50mL/250mL or 75mL/500mL shake flask fermentation culture
In base, 250rpm, 30 DEG C, constant-temperature shaking culture 96h obtains fermentation liquid, according to the measurement preferred superior strain of curdlan yield.
9. the method as described in claim 1, which is characterized in that in the step (6), the thick yield of curdlan in fermentation liquid
Measuring method are as follows: take 10mL fermentation liquid in 50mL centrifuge tube, 9000rpm is centrifuged 5min, and precipitating is cleaned with 95% ethyl alcohol, filled
Divide after mixing and stand, be centrifuged, repeat ethyl alcohol cleaning process 2 times, centrifugation is deposited in 60 DEG C of baking ovens and dries overnight, weighing M, meter
Calculation slightly proposes yield: slightly mentioning yield g/L=M/10 × 1000.
10. the method as described in claim 1, which is characterized in that in the step (7), measure curdlan throughput method are as follows:
5mL fermentation liquid is taken, 3mol/L NaOH solution 75mL is added, is stirred well to glue and is completely dissolved；30min, 9000rpm are stood, from
Heart 10min, takes supernatant；It is mixed, is uniformly mixed with alkaline-sol with 3mol/L HCl solution 75mL；After standing 10min,
9000rpm is centrifuged 5min；Three times with 150mL distillation water washing, each 9000rpm is centrifuged 5min to precipitating；60 DEG C are deposited in do
Dry case is dried overnight, and weigh M, can be calculated right glue yield: yield g/L=M/5 × 1000.
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| CN102296045A (en) * | 2011-09-20 | 2011-12-28 | 云南省微生物发酵工程研究中心有限公司 | High-curdlan-yield strain and preparation method thereof |
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