CN107760608B - A kind of mutagenic strain of efficient production low molecule pulullan polysaccharide and its application - Google Patents
A kind of mutagenic strain of efficient production low molecule pulullan polysaccharide and its application Download PDFInfo
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Abstract
Mutagenic strain and its application the invention discloses a kind of efficient production low molecule pulullan polysaccharide, belong to strain mutagenesis technical field.The present invention provides a kind of Aureobasidium pullulans UVMU6 1 (Aureobasidium pullulans) of efficient production low molecule pulullan polysaccharide, and culture presevation number is CGMCC No.13178.The present invention also provides a kind of application of Aureobasidium pullulans in the pulullan polysaccharide production of low molecular weight.The pulullan polysaccharide production capacity of bacterial strain of the present invention is high, the yield of pulullan polysaccharide can be effectively improved, reduce production cost, using during the strain fermentation with relatively low viscosity, production concentration is high in zymotic fluid, effectively reduce fermentation costs, the production method mild condition of the pulullan polysaccharide of bacterial strain provided by the invention production, the advantages that environmental-friendly and Product Green is natural.
Description
Technical field
Mutagenic strain and its application the present invention relates to a kind of efficient production low molecule pulullan polysaccharide, belong to strain mutagenesis
Technical field.
Background technology
Pulullan polysaccharide (pullulan) is also pullulan, pullulan, pullulan or mould sugar of growing sturdily, is to go out
Exocellular polysaccharide caused by the short mould fermentation of stalk of bud, is tasteless and odorless white powder.Pulullan polysaccharide chemical composition is mainly by α
The poly- maltotriose of~Isosorbide-5-Nitrae~glucoside key connection, molecular weight 4.8 × 104~2.2 × 106(commodity pulullan polysaccharide is averaged
Molecular weight 2 × 105, about it is made of 480 maltotrioses).The bacterial strain of bioanalysis synthesis pulullan polysaccharide is mainly Propiram
Yeast (mould, the A.pullulans of the short stalk of bud of growing sturdily), raw materials for production include glucose, sucrose, starch, corn and other related lifes
Substance.China is food additives new varieties in approval pulullan polysaccharide in 2006.In Japan, pulullan polysaccharide is together with starch
It is classified as without the additive for using limitation.Either acute, subacute, chronic test and change heterologous experiment all show general
Shandong orchid polysaccharide does not cause any biology toxicity and abnormality, can be degraded by microorganisms utilization in nature, will not cause ring
It pollutes in border.Above-mentioned good characteristic causes pulullan polysaccharide to have in fields such as cosmetics, medicine, food, chemical industry, environmental protection, pesticides
Extensive purposes.
Although the production of pulullan polysaccharide has industrialized, by manufacturer is few etc., factors are restricted, commercially available price
Higher (230,000/ton, 300,000/ton of import price), limits the application of pulullan polysaccharide.Also have in terms of production technology following
Several important rooms for promotion, can effective reduced cost.Wherein main cause is that product yield is relatively low：Due to culture solution after production sugar
The problems such as body is sticky, oxygen supply is difficult, and pulullan polysaccharide is by strains for degrading after longer fermentation times produces pulullan polysaccharide work at present
The sugared concentration of skill is about 60g/L or so.A large amount of works have been done for the raising of yield in Shandong Fu Ruida bio tech ltd
Make, and 118g/L (ZL201210443770.4) has been arrived in fermentation yield promotion, the promotion of yield being capable of effective compressed products
Fermentation costs and separation costs.The greatest problem of limitation pulullan polysaccharide product fermentation concentration is the viscosity of polysaccharide after fermentation
Height, basic reason are that the molecular weight of polysaccharide is relevant.The pulullan polysaccharide of different molecular weight ranges has different viscosityes,
Therefore the pulullan polysaccharide production bacterial strain of acquisition low molecular weight, low sticky degree, for improving the yield of pulullan polysaccharide, reduce general
The production cost of Shandong orchid polysaccharide has great importance.Through retrieving both at home and abroad without by mutagenic obtained efficient production low molecule
The breeding screening technique of pulullan polysaccharide and the technique using this method production low molecule pulullan polysaccharide.
Invention content
The present invention, which is directed to, lacks the mutagenic obtained general Shandong of efficient production low molecule in the production industry of existing pulullan polysaccharide
The breeding screening technique of blue polysaccharide and the technique using this method production low molecule pulullan polysaccharide, the present invention provides a kind of high
The mutagenic strain of effect production low molecule pulullan polysaccharide and its application, and in particular to a kind of to utilize commercialization bacterial strain Aureobasidium pullulans
The mutagenic bacteria of low molecular weight pulullan polysaccharide that (A.pullulans CGMCC 3.933) oriented evolution and mutagenesis screening obtain
Strain, Aureobasidium pullulans UVMU6-1 (Aureobasidium pullulans), culture presevation number be CGMCC No.13178 with
And the method using mutagenic strain high-efficiency fermenting production pulullan polysaccharide.
A kind of Aureobasidium pullulans UVMU6-1 (Aureobasidium of efficient production low molecule pulullan polysaccharide
Pullulans), which it is commonly micro- to be preserved in China Committee for Culture Collection of Microorganisms on October 24th, 2016
Bio-Centers, deposit number are CGMCC No.13178.
Aureobasidium pullulans UVMU6-1 (Aureobasidium pullulans) of the present invention (number by culture presevation
CGMCC No.13178) it is to be obtained by Aureobasidium pullulans (A.pullulans) CGMCC 3.933 by mutagenesis screening.
The mutagenesis and screening method of Aureobasidium pullulans UVMU6-1 of the present invention is：
The bacteria suspension of Aureobasidium pullulans (A.pullulans) CGMCC 3.933 is subjected to ultraviolet light photograph under magnetic stirring
Penetrate, then bacterium solution be added in the YPD solid mediums containing 0.03~0.07% bent sharp benzene indigo plant and applies tablet, be placed in 30 DEG C,
After constant temperature is inverted culture 2 days, bacterial strain is selected, and carry out secondary screening.
The cell concentration of the bacteria suspension of Aureobasidium pullulans (A.pullulans) CGMCC 3.933 of the present invention is 105CFU/mL
~107 CFU/mL。
It is of the present invention under magnetic stirring carry out ultraviolet light irradiation, be pre-irradiation first open ultraviolet lamp 5min~
Then culture dish containing bacteria suspension is placed on magnetic stirring apparatus by 20min, in ultraviolet lamp power 10W~15W, irradiation distance
Carry out magnetic agitation and irradiation under conditions of 30cm, when 5min closes ultraviolet lamp.
Preferably, YPD solid mediums of the present invention, the ingredient containing following mass percent：0.8%~1.2%
Yeast extract, 1.5%~2.5% peptone, 1.5%~2.5% glucose if solid medium processed, add in 1.5%~2.0%
Agar powder.The YPD solid mediums sterilising conditions sterilize 25~35 minutes for 115 DEG C.
Secondary screening of the present invention is to take seed, is inoculated into YPD culture mediums, cultivates 24~36 hours, above-mentioned seed is taken to connect
In kind to the YPD sugar-free culture-mediums of 50mL, the glucose after the sterilizing of 24 hours addition 5mL 70% is cultivated, is cultivated 48 hours,
5mL zymotic fluids are taken again, boil 5~10 minutes, 12000 revs/min centrifuge 10~20 minutes, using the ethanol precipitation of 2 times of volumes,
4~10 degrees Celsius stand overnight, and 12000 revs/min of centrifugations 10~take precipitation drying after twenty minutes measure the yield of polysaccharide.Its
It is small molecule pulullan polysaccharide that bottom is sunk to during middle ethanol precipitation.
Mutagenesis and screening method of the present invention is specific as follows：
Take bacteria suspension (the unit cell concentration 10 of 10mL Aureobasidium pullulans (A.pullulans) CGMCC 3.9335~107
CFU/mL) in the culture dish of diameter 9cm, ultraviolet light irradiation is carried out under magnetic agitation.Ultraviolet lamp power 10W~15W, irradiate away from
From 30cm, pre-irradiation opens ultraviolet lamp 5min~20min in advance makes its stabilization, and the culture dish containing bacteria suspension then is placed in magnetic
On power blender, simultaneously timing is stirred, covers culture dish lid when 5min, close ultraviolet lamp, 100 μ L bacterium solutions is taken to add in
Tablet is applied in containing YPD (blue containing the sharp benzene of 0.03~0.07% song) solid medium, is placed in 30 DEG C, constant temperature is inverted culture
After 2 days, using the colony colour depth, surface texturisation as screening index, filter out color depth, bacterium colony surface wettability, colony diameter with
The higher bacterial strain of thickness ratio, and it is numbered.It is accessed again containing being cultivated on Nysfungin culture medium, carries out answering for next step
Sieve and genetic stability verification, and determine the yield of pulullan polysaccharide.
The method of above-mentioned genetic stability detection is：Take the bacterium that can efficiently produce low molecule pulullan polysaccharide screened
Strain, is inoculated into the YPD culture mediums of 5ml, after cultivating 48 hours, 100 μ L is taken to be inoculated into the fresh YPD culture mediums of 5ml, so
It is iteratively repeated 10 times or more, carries out above-mentioned secondary screening verification afterwards, stable yield is tested for later stage fermentation.
The present invention also provides a kind of Aureobasidium pullulans UVMU6-1 of efficient production low molecule pulullan polysaccharide in Propiram
Application in polysaccharide production.
The application, includes the following steps：
Step 1：The seed of Aureobasidium pullulans UVMU6-1 is taken to be inoculated into YPD culture mediums to cultivate, is then seeded into YPD
In sugar-free culture-medium, the biomass sugar after addition sterilizing obtains culture seed after culture；
Step 2：The culture seed that step 1 obtains is taken to be inoculated into fermentation medium, adjusts pH value, and into culture medium
Air and biomass sugar are persistently added, measures the concentration of polysaccharide after fermentation.
Preferably, the YPD culture mediums described in step 1 of the present invention, the ingredient containing following mass percent：0.8~
1.2% yeast extract, 1.5%~2.5% peptone, 1.5%~2.5% glucose.
Preferably, the fermentation medium described in step 2 of the present invention includes the ingredient of following mass percent：2%~4%
Biomass sugar, 0.5%~1% yeast extract and 1%~2% peptone.
Preferably, the biomass sugar described in step 2 of the present invention, from starch, stalk, straw, straw, corn or rice
Chaff.
Preferably, the biomass sugar described in step 2 of the present invention is glucose, sucrose or fructose.
Preferably, the condition of the culture described in step 1 and step 2 is：30 DEG C of fermentation temperature, pH value 4 of fermenting, fermentation
400 revs/min of tank rotating speed, air mass flow 2L/ minutes.
The application (fermenting and producing pulullan polysaccharide) is as follows：
Seed is taken, is inoculated into the YPD culture mediums of 2~3mL, is cultivated 24~36 hours；Above-mentioned seed is taken to be inoculated into 50mL
YPD sugar-free culture-mediums in, add the glucose after the sterilizing of 5mL70%, cultivate 36~48 hours.Take above-mentioned culture seed
It is inoculated into and contains 2~4% glucose, in the fermentation medium of 0.5~1% yeast extract and 1~2% peptone, utilize hydrochloric acid and ammonia
Water adjusts pH value, 400 turns, pH 4.0, air mass flow 4L/min cultures, and glucose is persistently added into culture medium.Culture
After 100 hours, the concentration of polysaccharide is measured.
Advantageous effect of the present invention：
1st, the present invention carries out mutation breeding using Aureobasidium pullulans (A.pullulans) CGMCC 3.933 of commercialization, and
Establish the efficiently screening technique of production low molecule pulullan polysaccharide bacterial strain and the Aureobasidium pullulans screened using this method
UVMU6-1 (culture presevation number CGMCC No.13178), and the efficiently production low molecule pulullan polysaccharide that ferments.
2nd, for mutagenic strain CGMCC No.13178 of the invention compared with conventional pulullan polysaccharide produces bacterium, Propiram is more
Sugared production capacity is more high, and thus effectively improves the yield of pulullan polysaccharide, reduces production cost, and have when fermenting compared with
Low viscosity, production concentration is high in zymotic fluid.It is high using the method for the present invention production pulullan yield, utilize biology base grape
Sugar ferments, and the yield of the pulullan polysaccharide of acquisition is up to 135g/L, and the Pu Lu of fermentation acquisition is carried out using biology base sucrose
The yield of blue polysaccharide reaches 127g/L.
3rd, the production method mild condition of the pulullan polysaccharide of bacterial strain production provided by the invention, environmental-friendly and product
The advantages that green natural.
Description of the drawings
Fig. 1 is the thin plate chromatography figure that Propiram digests pulullan polysaccharide；
(1, digest standard sample；2, standard sample；3, maltotriose；4, enzymolysis product；5, product).
Specific embodiment
With reference to specific embodiment, the present invention will be further described, but the present invention should not be limited by the examples.
Involved culture medium prescription is as follows in following embodiment：
YPD culture mediums：Ingredient containing following mass percent：0.8%~1.2% yeast extract, 1.5%~2.5% egg
White peptone, 1.5%~2.5% glucose.
Fermentation medium:Ingredient containing following mass percent：2~4% glucose, 0.5~1% yeast extract and 1~
In the fermentation medium of 2% peptone, pH value is adjusted using hydrochloric acid and ammonium hydroxide.
The present invention is illustrated according to following optimization formula carries out following experiment, other ranges can also realize the present invention
Technique effect, specific formula is as follows：
YPD culture mediums：Ingredient containing following mass percent：1% yeast extract, 2% peptone, 2% glucose.
Fermentation medium:Ingredient containing following mass percent：3% glucose, 0.75% yeast extract and 1.5% albumen
In the fermentation medium of peptone, pH value is adjusted using hydrochloric acid and ammonium hydroxide.
Embodiment one：The efficiently mutagenesis and screening of production low molecule pulullan polysaccharide bacterial strain
Take 3.933 bacteria suspension (unit cell concentrations 10 of 10ml Aureobasidium pullulans (A.pullulans) CGMCC5~
107CFU/mL) in the culture dish of diameter 9cm, ultraviolet light irradiation is carried out under magnetic agitation.Ultraviolet lamp power 10W~15W shines
Distance 30cm is penetrated, pre-irradiation opens ultraviolet lamp 5min~20min in advance makes its stabilization, then puts the culture dish containing bacteria suspension
In on magnetic stirring apparatus, simultaneously timing is stirred, covers culture dish lid when 5min, closed ultraviolet lamp, take 100 μ L bacterium solutions
It is added to containing tablet is applied in YPD (blue containing the sharp benzene of 0.03~0.07% song) solid medium, is placed in 30 DEG C, constant temperature falls
After putting culture 2 days, using the colony colour depth, surface texturisation as screening index, color depth, bacterium colony surface wettability, bacterium colony are filtered out
Diameter and the higher bacterial strain of thickness ratio, and it is numbered.It accesses on YPD culture mediums and cultivates again, carry out answering for next step
Sieve and genetic stability verification, and determine the yield of pulullan polysaccharide.
Above-mentioned secondary screening method：Seed is taken, is inoculated into the YPD culture mediums of 3ml, is cultivated 24~36 hours；Take above-mentioned seed
It is inoculated into the YPD sugar-free culture-mediums of 50ml, cultivates the glucose after the sterilizing of 24 hours addition 5mL70%, culture 48 is small
When, then 5mL zymotic fluids are taken, it boils 5~10 minutes, 12000 revs/min centrifuge 10~20 minutes, are sunk using the ethyl alcohol of 2 times of volumes
It forms sediment, 4~10 degrees Celsius stand overnight, and 12000 revs/min of centrifugations 10~take precipitation drying after twenty minutes measure the yield of polysaccharide.
Wherein ethanol precipitation when to sink to bottom be small molecule pulullan polysaccharide.
The method of above-mentioned genetic stability detection：Take the mutagenesis that can efficiently produce low molecule pulullan polysaccharide screened
Bacterial strain is inoculated into the YPD culture mediums of 5mL, after cultivating 48 hours, 100 μ L is taken to be inoculated into the fresh YPD culture mediums of 5ml, such as
This is iteratively repeated 10 times or more, carries out above-mentioned secondary screening verification afterwards, and stable yield is tested for later stage fermentation.
The mutagenic strain obtained by above-mentioned mutagenesis and screening technique, is identified as Aureobasidium pullulans UVMU6-1
(Aureobasidium pullulans), the bacterial strain were preserved in Chinese microorganism strain preservation on October 24th, 2016
Administration committee's common micro-organisms center, deposit number are CGMCC No.13178.
Embodiment two：The measure of low molecule pulullan polysaccharide
Tunning and pulullan polysaccharide are dissolved into according to the ratio of 1g/10mL in deionized water, obtain sample solution
With pulullan polysaccharide standard solution；Maltotriose, glucose are dissolved in deionized water according to the ratio of 10mg/mL；Open up layer body
System：Acetonitrile and distilled water are according to volume ratio n-butanol：Acetic acid：Distilled water=8:3:2 ratio is uniformly mixed, and is placed in tool plug glass
It is spare in bottle.Color developing agent：The concentrated sulfuric acid and absolute ethyl alcohol are according to the V concentrated sulfuric acids：V absolute ethyl alcohol=1:6 ratio is uniformly mixed, and is attached to
It is spare in watering can.
Pulullan polysaccharide standard solution, pulullan polysaccharide hydrolyzate, sample solution, sample enzymolysis liquid are diluted 100 respectively
Times, according to pulullan polysaccharide standard solution, pulullan polysaccharide hydrolyzate, maltotriose, sample enzymolysis liquid, sample solution, grape
The sequence of sugar point sample on silica gel column chromatography plate, chromatographs in layer liquid is opened up, and drying, weight are taken out after at the top of liquid chromatography(LC) to silica gel plate
Cladding is analysed 2-3 times.Color developing agent is sprayed onto on the silica gel plate after chromatography, heats colour developing after drying on alcolhol burner, observation experiment is existing
As.Separation acquisition sample is the same with standard items, is hydrolyzed to maltotriose, the results showed that acquisition product is pulullan polysaccharide (figure
1)。
Embodiment three：Low molecule pulullan polysaccharide is efficiently produced using biology base glucose
Basal medium：1.0% (w/v) dusty yeast, 2.0% (w/v) peptone, natural pH.Propiram shake flask fermentation is trained
Support base：It is 4 that above-mentioned culture medium, which adjusts pH, and adds 70% glucose 10ml, 8 pounds of (115 DEG C) sterilizing 30min.Glucose needs
Individually sterilizing.
Above-mentioned culture medium inoculated Aureobasidium pullulans UVMU6-1, growth are used as seed after 36 hours, are inoculated into the fermentation tank of 5L
Culture.5L fermentation tanks contain following component：4.0% (w/v) glucose, 1.0% (w/v) dusty yeast, 2.0% (w/v) peptone,
Initial 4,8 pounds of (115 DEG C) sterilizing 30min of pH.30 DEG C of fermentation temperature, pH value 4 of fermenting, 400 revs/min of fermentation tank rotating speed, air
Flow 2L/ minutes.Glucose need to individually sterilize, and in fermentation process, glucose and ammonium hydroxide, fermentation ends are added into fermentation tank
Afterwards, then 5ml zymotic fluids are taken, boiled 5~10 minutes, 12000 revs/min centrifuge 10~20 minutes, are sunk using the ethyl alcohol of 2 times of volumes
It forms sediment, 4~10 degrees Celsius stand overnight, and 12000 revs/min of centrifugations 10~take precipitation drying after twenty minutes measure the yield of polysaccharide.
Pulullan yield reaches 135g/L.Original strain yield 30g/L, 350% is improved compared with original strain.
Example IV：Low molecule pulullan polysaccharide is efficiently produced using biology base sucrose
Propiram Medium of shaking flask fermentation：It is 4, and add 70% sucrose that basal medium, which adjusts pH, in embodiment three
10ml, 8 pounds of (115 DEG C) sterilizing 30min.Sucrose need to individually sterilize.Above-mentioned culture medium inoculated Aureobasidium pullulans UVMU6-1, growth
As seed after 36 hours, it is inoculated into the fermentation tank culture of 5L.5L fermentation tanks contain following component, 4.0% (w/v) sucrose,
1.0% (w/v) dusty yeast, 4,8 pounds of (115 DEG C) sterilizing 30min of the initial pH of 2.0% (w/v) peptone.30 DEG C of fermentation temperature, hair
Ferment pH value 4,400 revs/min of fermentation tank rotating speed, air mass flow 2L/ minutes.Sucrose need to individually sterilize, in fermentation process, to fermentation
Sucrose and ammonium hydroxide are added in tank, after fermentation, then takes 5ml zymotic fluids, is boiled 5~10 minutes, 12000 revs/min of centrifugations 10
~20 minutes, using the ethanol precipitation of 2 times of volumes, 4~10 degrees Celsius stood overnight, and 12000 revs/min centrifuge 10~20 points
Precipitation drying is taken after clock, measures the yield of polysaccharide.Pulullan yield reaches 127g/L.Original strain yield 30g/L, it is more former
Beginning bacterial strain improves 323%.
The measure of 5 pulullan polysaccharide molecular weight of embodiment
Pulullan polysaccharide is obtained to embodiment 3 and embodiment 4 and carries out molecular weight determination, molecule using gel permeation chromatography
Amount is less than 200,000 dalton, therefore the mutagenic strain that present invention screening obtains can efficiently produce low molecule pulullan polysaccharide.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this
The people of technology is not being departed from spirit and scope of the invention, can do various change and modification, therefore, guarantor of the invention
Shield range should be subject to what claims were defined.
Claims (8)
1. a kind of Aureobasidium pullulans (Aureobasidium pullulans) UVMU6- of efficient production low molecule pulullan polysaccharide
1, culture presevation number is CGMCC No.13178.
2. Aureobasidium pullulans (Aureobasidium pullulans) UVMU6-1 described in claim 1 is more in production Propiram
Application in sugar.
3. application according to claim 2, which is characterized in that include the following steps：
Step 1：The seed of Aureobasidium pullulans UVMU6-1 described in claim 1 is taken to be inoculated into YPD culture mediums to cultivate, Ran Houjie
In kind to YPD sugar-free culture-mediums, the biomass sugar after addition sterilizing obtains culture seed after culture；
Step 2：The culture seed that step 1 obtains is taken to be inoculated into fermentation medium, adjusts pH value, and continue into culture medium
Air and biomass sugar are added, measures the concentration of polysaccharide after fermentation.
4. application according to claim 3, which is characterized in that the YPD culture mediums described in step 1 contain following quality hundred
Divide the ingredient of ratio：0.8%~1.2% yeast extract, 1.5%~2.5% peptone, 1.5%~2.5% glucose.
5. application according to claim 3, which is characterized in that the fermentation medium described in step 2 includes following quality hundred
Divide the ingredient of ratio：2%~4% biomass sugar, 0.5%~1% yeast extract and 1%~2% peptone.
6. application according to claim 3, which is characterized in that the biomass sugar described in step 2, from starch, straw
Stalk, straw, straw, corn or rice bran.
7. application according to claim 3, which is characterized in that biomass sugar described in step 2 is glucose or sucrose.
8. application according to claim 3, which is characterized in that the condition of the culture described in step 1 and step 2 is：
30 DEG C of fermentation temperature, pH value 4 of fermenting, 400 revs/min of rotating speed, air mass flow 2L/ minutes.
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