CN106244639A - The application in improving Aureobasidium pullulans polymalic acid yield of the short chain alcohol molecule - Google Patents
The application in improving Aureobasidium pullulans polymalic acid yield of the short chain alcohol molecule Download PDFInfo
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Abstract
The present invention relates to the application in improving Aureobasidium pullulans polymalic acid yield of a kind of short chain alcohol molecule, particularly relate to short chain alcohol molecule in the method improving Aureobasidium pullulans polymalic acid yield, comprise the steps: that Aureobasidium pullulans is inoculated in seed culture medium by (1), temperature is that 23 30 DEG C of concussions are cultivated, and obtains activated seed liquid；(2) gained activated seed liquid in step (1) being inoculated in fermentation medium, temperature is 23 30 DEG C of fermentation culture.The method is according to the growth of Aureobasidium pullulans cell and polymalic acid biosynthesis pathway, use short chain alcohol molecule additive, set up orientation regulating strategy simple to operate, the carbon metabolism flow realizing cell growth and polymalic acid synthesis flows to regulation, polymalic acid fermentation yield can be increased substantially and cell produces acid yield, and the method has the advantages such as fermentation costs is low, yield is high, Technical Economy is strong, can be applicable to polymalic acid fermentation industry and amplify production.
Description
Technical field
The invention belongs to fermentation arts, be specifically related to short chain alcohol molecule in improving Aureobasidium pullulans polymalic acid yield
Application.
Background technology
Aureobasidium pullulans (Aureobasidum pullulan) is a class yeast-like fungus, and main metabolic produces poly-Fructus Mali pumilae
The metabolites such as acid, pulullan polysaccharide, melanin, short stalk mycin.Wherein polymalic acid (Polymalic acid) is with L-Fructus Mali pumilae
Acid is polymerized in vivo for only monomer, is a kind of novel completely biodegradable macromolecular material, has the biology of height
The advantages such as the compatibility, biological degradability and Bioabsorbable, can be used for medicine sustained and controlled release carrier, organizational project, food processing
In field.
Polymalic acid is generally secreted into extracellular by Aureobasidium pullulans, but specifically its biosynthesis and transporting pathway are the most unclear
Chu, therefore lacks effective control measures and promotes the excess synthesis of polymalic acid.(the patents such as existing granted patent such as Qiao Changsheng
Grant number ZL201210594880.0) invention a kind of synthetic medium (sucrose, sodium nitrate, potassium dihydrogen phosphate, potassium chloride, sulphuric acid
Manganese, bitter salt, calcium carbonate, ammonium nitrate, aspartic acid, leucine, threonine, valine, histidine, cytosine,
Adenine, the compounding composition of choline, compounded by the optimization of aminoacid, choline and purine pyrimidine somatomedin) can be used for improving
The fermentation yield of polymalic acid, but this medium component is complicated, and fermentation costs is high, is worth without industrial applications.Therefore, it is badly in need of building
The fermentation control strategy that vertical industrialization is simple and feasible, it is achieved produce acid yield in sweat raising polymalic acid yield and cell
Purpose.
Summary of the invention
In view of this, it is an object of the invention to provide short chain alcohol molecule in improving Aureobasidium pullulans polymalic acid yield
Application, for improve polymalic acid fermentation yield method deficiency, by adding short chain alcohol molecule in the fermentation medium, have
Effect regulation cell growth and polymalic acid anabolism stream, it is provided that utilize short chain alcohol molecule to improve Aureobasidium pullulans polymalic acid and produce
The method of amount.
For reaching above-mentioned purpose, the present invention provides following technical scheme:
1, short chain alcohol molecule application in improving Aureobasidium pullulans polymalic acid yield.
Further, described short chain alcohol molecule is the one in methanol, ethanol, propylene glycol or glycerol.
2, the method that short chain alcohol molecule improves Aureobasidium pullulans polymalic acid yield is utilized, it is characterised in that include following
Step:
(1) seed culture
Aureobasidium pullulans (Aureobasidium pullans) CCTCC M2012223 is inoculated in seed culture medium, temperature
Degree is cultivated for 23-30 DEG C of concussion, obtains activated seed liquid；Described seed culture medium is glucose 60-80g/L, ammonium sulfate 2-4g/L,
KH2PO4 0.05-0.2g/L、ZnSO4 0.05-0.2g/L、MgSO40.05-0.2g/L, Semen Maydis pulp 0.5-2g/L and CaCO320-
50g/L；
(2) fermentation culture
Step (1) gained activated seed liquid is inoculated in fermentation medium, and temperature is 23-30 DEG C of fermentation culture；Described
Ferment culture medium is 2-4g/L, KH for sugar 90-100g/L, nitrogen source2PO4 0.05-0.2g/L、ZnSO4 0.05-0.2g/L、MgSO4
0.05-0.2g/L, Semen Maydis pulp 0.5-2g/L, CaCO3The short chain alcohol molecule of 20-50g/L, by volume part meter 0.1-3%.
Further, in step (1), the inoculation of described Aureobasidium pullulans is according to the volume of Aureobasidium pullulans Yu seed culture medium
Carry out for 1:100-1000；Described concussion is cultivated as to transfer described Aureobasidium pullulans in the shaking flask equipped with described seed culture medium
In, under revolution 180-300rpm, cultivate 36-96h.
Further, in step (2), the inoculation of described activated seed liquid is according to the volume of activated seed liquid Yu fermentation medium
Ratio is carried out for 1:10-20.
Further, in step (2), described sugar is glucose, xylose, corn stalk hydrolysis or ligno-cellulose hydrolysate
In at least one；Described nitrogen source is the one in ammonium sulfate, ammonium chloride, potassium nitrate or sodium nitrate.
Further, in step (2), described short chain alcohol molecule is the one in methanol, ethanol, propylene glycol or glycerol.
Further, in step (2), described fermentation culture is the one in shake flask fermentation technique or ferment tank technique.
Further, described shake flask fermentation technique is particularly as follows: be inoculated in described activated seed liquid equipped with described fermentation culture
In the shaking flask of base, under revolution 180-300rpm, cultivate 96-150h.
Further, described ferment tank technique is particularly as follows: be inoculated in described activated seed liquid equipped with described fermentation training
Support in the fermentation tank of base, under the conditions of revolution 300-1000rpm, ventilation ratio 1:0.8-1:1.6, cultivate 60-200h.
The beneficial effects of the present invention is: the invention provides short chain alcohol molecule and improving Aureobasidium pullulans polymalic acid product
Application in amount, specifically provides the method utilizing short chain alcohol molecule to improve Aureobasidium pullulans polymalic acid yield, the method root
According to the growth of Aureobasidium pullulans cell and polymalic acid biosynthesis pathway, use short chain alcohol molecule additive, set up simple to operate
Orientation regulating strategy, it is achieved cell growth and polymalic acid synthesis carbon metabolism flow flow to regulation, it is possible to increase substantially poly-
Malic acid fermentation yield and cell produce acid yield, and the method to have that fermentation costs is low, yield is high, Technical Economy is strong etc. excellent
Point, can be applicable to polymalic acid fermentation industry and amplifies production.
Detailed description of the invention
Below the preferred embodiments of the present invention are described in detail.
Embodiment 1
(1) seed culture
Aureobasidium pullulans CCTCC M2012223 is inoculated in the shaking flask of the 500ml equipped with seed culture medium described in 30ml
In, inoculum concentration is 1:1000 according to the volume of Aureobasidium pullulans Yu seed culture medium, and seed culture based formulas is glucose 60g/
L, ammonium sulfate 2g/L, KH2PO4 0.05g/L、ZnSO4 0.05g/L、MgSO40.05g/L, Semen Maydis pulp 0.5g/L and CaCO320g/
L, is 23 DEG C in temperature, cultivates 96h under the conditions of revolution 180rpm, obtain activated seed liquid；
(2) fermentation culture
In in step (1), activated seed liquid is inoculated in the shaking flask of the 500ml equipped with fermentation medium described in 30ml, inoculation
Amount is 1:20 according to the volume ratio of activated seed liquid Yu fermentation medium, and fermentation medium is glucose 120g/L, ammonium sulfate 2g/
L、KH2PO4 0.05g/L、ZnSO4 0.05g/L、MgSO40.05g/L, Semen Maydis pulp 0.5g/L and CaCO320g/L, by volume part
Count 0.5% ethanol, 23 DEG C, cultivate 120h under revolution 180rpm.
Comparative example 1
Being not added with the ethanol of 0.5% in fermentation medium, remaining Step By Condition is same as in Example 1.
Polymalic acid yield and cell produce acid yield
In embodiment 1, polymalic acid yield is 30.4g/L, and relatively comparative example 1 improves 27.2%；In embodiment 1 carefully
It is 1.88g/g that born of the same parents produce acid yield (Yp/x), and relatively comparative example 1 improves 32.4%.
Embodiment 2
(1) seed culture
Aureobasidium pullulans CCTCC M2012223 is inoculated in the shaking flask of the 500ml equipped with seed culture medium described in 30ml
In, inoculum concentration is 1:1000 according to the volume of Aureobasidium pullulans Yu seed culture medium, and seed culture based formulas is glucose 60g/
L, ammonium sulfate 2g/L, KH2PO4 0.05g/L、ZnSO4 0.05g/L、MgSO40.05g/L, Semen Maydis pulp 0.5g/L and CaCO320g/
L, is 23 DEG C in temperature, cultivates 96h under the conditions of revolution 180rpm, obtain activated seed liquid；
(2) fermentation culture
In in step (1), activated seed liquid is inoculated in the shaking flask of the 500ml equipped with fermentation medium described in 30ml, inoculation
Amount is 1:20 according to the volume ratio of activated seed liquid Yu fermentation medium, and fermentation medium is glucose 90g/L, ammonium sulfate 2g/
L、KH2PO4 0.05g/L、ZnSO4 0.05g/L、MgSO40.05g/L, Semen Maydis pulp 0.5g/L and CaCO320g/L, by volume part
Count 1% ethanol, 23 DEG C, cultivate 96h under revolution 180rpm.
Comparative example 2
Being not added with the ethanol of 1% in fermentation medium, remaining Step By Condition is same as in Example 2.
Polymalic acid yield and cell produce acid yield
In embodiment 2, polymalic acid yield is 30.3g/L, and relatively comparative example 2 improves 20.8%；In embodiment 2 carefully
It is 2.43g/g that born of the same parents produce acid yield (Yp/x), and relatively comparative example 2 improves 33.5%.
Embodiment 3
(1) seed culture
Aureobasidium pullulans CCTCC M2012223 is inoculated in the shaking flask of the 500ml equipped with seed culture medium described in 30ml
In, inoculum concentration is 1:1000 according to the volume of Aureobasidium pullulans Yu seed culture medium, and seed culture based formulas is glucose 60g/
L, ammonium sulfate 2g/L, KH2PO4 0.05g/L、ZnSO4 0.05g/L、MgSO40.05g/L, Semen Maydis pulp 0.5g/L and CaCO320g/
L, is 23 DEG C in temperature, cultivates 96h under the conditions of revolution 180rpm, obtain activated seed liquid；
(2) fermentation culture
In in step (1), activated seed liquid is inoculated in the shaking flask of the 500ml equipped with fermentation medium described in 30ml, inoculation
Amount is 1:20 according to the volume ratio of activated seed liquid Yu fermentation medium, and fermentation medium is glucose 120g/L, ammonium sulfate 2g/
L、KH2PO4 0.05g/L、ZnSO4 0.05g/L、MgSO40.05g/L, Semen Maydis pulp 0.5g/L and CaCO320g/L, by volume part
Count 2.5% ethanol, 23 DEG C, cultivate 120h under revolution 180rpm.
Comparative example 3
Being not added with the ethanol of 2.5% in fermentation medium, remaining Step By Condition is same as in Example 3.
Polymalic acid yield and cell produce acid yield
In embodiment 3, polymalic acid yield is 30.3g/L, and relatively comparative example 3 improves 26.7%；In embodiment 3 carefully
It is 1.88g/g that born of the same parents produce acid yield (Yp/x), and relatively comparative example 3 improves 57.7%.
Embodiment 4
(1) seed culture
Aureobasidium pullulans CCTCC M2012223 is inoculated in the shaking flask of the 500ml equipped with seed culture medium described in 30ml
In, inoculum concentration is 1:1000 according to the volume of Aureobasidium pullulans Yu seed culture medium, and seed culture based formulas is glucose 60g/
L, ammonium sulfate 2g/L, KH2PO4 0.05g/L、ZnSO4 0.05g/L、MgSO40.05g/L, Semen Maydis pulp 0.5g/L and CaCO320g/
L, is 23 DEG C in temperature, cultivates 96h under the conditions of revolution 180rpm, obtain activated seed liquid；
(2) fermentation culture
Being inoculated in activated seed liquid in step (1) equipped with in the 5L fermentation tank of 3L culture medium, fermentation medium is Fructus Vitis viniferae
Sugar 90g/L, potassium nitrate 3g/L, KH2PO4 0.2g/L、ZnSO4 0.15g/L、MgSO4 0.2g/L、CaCO330g/L, by volume
Part meter 1% ethanol, cultivation temperature 25 DEG C, rotating speed 600rpm, ventilation ratio 1:1.2, incubation time is 96h.
Comparative example 4
Being not added with the ethanol of 1% in fermentation medium, remaining Step By Condition is the same as in Example 4.
Polymalic acid yield and cell produce acid yield
In embodiment 4, polymalic acid yield is 38.8g/L, and relatively comparative example 4 improves 32%；Cell in embodiment 4
Producing acid yield (Yp/x) is 1.88g/g, and relatively comparative example 4 improves 16.9%.
Embodiment 5
(1) seed culture
Aureobasidium pullulans CCTCC M2012223 is inoculated in the shaking flask of the 500ml equipped with seed culture medium described in 60ml
In, inoculum concentration is 1:500 according to the volume of Aureobasidium pullulans Yu seed culture medium, seed culture based formulas be glucose 70g/L,
Ammonium sulfate 3g/L, KH2PO4 0.1g/L、ZnSO4 0.1g/L、MgSO40.1g/L, Semen Maydis pulp 1g/L and CaCO330g/L, in temperature
Degree is 25 DEG C, cultivate 72h under the conditions of revolution 200rpm, obtains activated seed liquid；
(2) fermentation culture
In in step (1), activated seed liquid is inoculated in the shaking flask of the 500ml equipped with fermentation medium described in 60ml, inoculation
Amount is 1:15 according to the volume ratio of activated seed liquid Yu fermentation medium, fermentation medium is xylose 95g/L, ammonium chloride 3g/L,
KH2PO4 0.1g/L、ZnSO4 0.1g/L、MgSO40.1g/L, Semen Maydis pulp 1g/L and CaCO330g/L, by volume part meter 1% the third
Glycol, 25 DEG C, cultivate 132h under revolution 200rpm.
Comparative example 5
Being not added with the propylene glycol of 1% in fermentation medium, remaining Step By Condition is same as in Example 5.
Polymalic acid yield and cell produce acid yield
In embodiment 5, polymalic acid yield is 27.2g/L, and relatively comparative example 5 improves 8.3%；Cell in embodiment 5
Producing acid yield (Yp/x) is 2.1g/g, and relatively comparative example 5 improves 13.3%.
Embodiment 6
(1) seed culture
Aureobasidium pullulans CCTCC M2012223 is inoculated in the shaking flask of the 500ml equipped with seed culture medium described in 90ml
In, inoculum concentration is 1:250 according to the volume of Aureobasidium pullulans Yu seed culture medium, seed culture based formulas be glucose 70g/L,
Ammonium sulfate 3g/L, KH2PO4 0.15g/L、ZnSO4 0.15g/L、MgSO40.15g/L, Semen Maydis pulp 1.5g/L and CaCO340g/L,
It is 27 DEG C in temperature, cultivates 48h under the conditions of revolution 250rpm, obtain activated seed liquid；
(2) fermentation culture
In in step (1), activated seed liquid is inoculated in the shaking flask of the 500ml equipped with fermentation medium described in 90ml, inoculation
Amount is 1:12.5 according to the volume ratio of activated seed liquid Yu fermentation medium, fermentation medium be corn stalk hydrolysis 95g/L,
Potassium nitrate 3g/L, KH2PO4 0.15g/L、ZnSO4 0.15g/L、MgSO40.15g/L, Semen Maydis pulp 1.5g/L and CaCO340g/L、
By volume part counts 1% methanol, 27 DEG C, cultivate 120h under revolution 250rpm.
Comparative example 6
Being not added with the methanol of 1% in fermentation medium, remaining Step By Condition is same as in Example 6.
Polymalic acid yield and cell produce acid yield
In embodiment 6, polymalic acid yield is 22.2g/L, and relatively comparative example 6 improves 20%；Cell in embodiment 6
Producing acid yield (Yp/x) is 1.3g/g, and relatively comparative example 6 improves 16.1%.
Embodiment 7
(1) seed culture
Aureobasidium pullulans CCTCC M2012223 is inoculated in the shaking flask of the 500ml equipped with seed culture medium described in 120ml
In, inoculum concentration is 1:100 according to the volume of Aureobasidium pullulans Yu seed culture medium, seed culture based formulas be glucose 80g/L,
Ammonium sulfate 4g/L, KH2PO4 0.2g/L、ZnSO4 0.2g/L、MgSO40.2g/L, Semen Maydis pulp 2g/L and CaCO350g/L, in temperature
Degree is 30 DEG C, cultivate 36h under the conditions of revolution 300rpm, obtains activated seed liquid；
(2) fermentation culture
In in step (1), activated seed liquid is inoculated in the shaking flask of the 500ml equipped with fermentation medium described in 90ml, inoculation
Amount is 1:10 according to the volume ratio of activated seed liquid Yu fermentation medium, and fermentation medium is ligno-cellulose hydrolysate 100g/
L, sodium nitrate 4g/L, KH2PO4 0.2g/L、ZnSO4 0.2g/L、MgSO40.2g/L, Semen Maydis pulp 2g/L and CaCO350g/L, press
Parts by volume meter 1% glycerol, 30 DEG C, cultivate 96h under revolution 180rpm.
Comparative example 7
Being not added with the glycerol of 1% in fermentation medium, remaining Step By Condition is same as in Example 7.
Polymalic acid yield and cell produce acid yield
In embodiment 7, polymalic acid yield is 20.3g/L, and relatively comparative example 7 improves 9.7%；Cell in embodiment 7
Producing acid yield (Yp/x) is 1.27g/g, and relatively comparative example 7 improves 13.4%.
The short chain alcohol molecule by volume part added in fermentation medium in the present invention is calculated as 0.1-3%；Fermentation culture is removed
Shake flask fermentation technique, it is also possible to for ferment tank technique, wherein, ferment tank process conditions are at revolution 300-
1000rpm, cultivates 60-200h under the conditions of ventilation ratio 1:0.8-1:1.6.
Finally illustrate, preferred embodiment above only in order to technical scheme to be described and unrestricted, although logical
Cross above preferred embodiment the present invention to be described in detail, it is to be understood by those skilled in the art that can be
In form and it is made various change, without departing from claims of the present invention limited range in details.
Claims (10)
1. short chain alcohol molecule application in improving Aureobasidium pullulans polymalic acid yield.
Apply the most as claimed in claim 1, it is characterised in that described short chain alcohol molecule is methanol, ethanol, propylene glycol or the third three
One in alcohol.
3. utilize the method that short chain alcohol molecule improves Aureobasidium pullulans polymalic acid yield, it is characterised in that comprise the following steps:
(1) seed culture
Aureobasidium pullulans (Aureobasidium pullans) CCTCC M2012223 is inoculated in seed culture medium, and temperature is
23-30 DEG C of concussion is cultivated, and obtains activated seed liquid；Described seed culture medium is glucose 60-80g/L, ammonium sulfate 2-4g/L,
KH2PO4 0.05-0.2g/L、ZnSO4 0.05-0.2g/L、MgSO40.05-0.2g/L, Semen Maydis pulp 0.5-2g/L and CaCO320-
50g/L；(2) fermentation culture
Step (1) gained activated seed liquid is inoculated in fermentation medium, and temperature is 23-30 DEG C of fermentation culture；Described fermentation training
Supporting base for sugar 90-100g/L, nitrogen source is 2-4g/L, KH2PO4 0.05-0.2g/L、ZnSO4 0.05-0.2g/L、MgSO4
0.05-0.2g/L, Semen Maydis pulp 0.5-2g/L, CaCO3The short chain alcohol molecule of 20-50g/L, by volume part meter 0.1-3%.
Utilize the method that short chain alcohol molecule improves Aureobasidium pullulans polymalic acid yield, its feature the most as claimed in claim 3
Being, in step (1), the inoculation of described Aureobasidium pullulans is 1:100-according to the volume of Aureobasidium pullulans Yu seed culture medium
1000 are carried out；Described concussion is cultivated as to transfer in the shaking flask equipped with described seed culture medium by described Aureobasidium pullulans, is turning
36-96h is cultivated under number 180-300rpm.
Utilize the method that short chain alcohol molecule improves Aureobasidium pullulans polymalic acid yield, its feature the most as claimed in claim 3
Being, in step (2), the inoculation of described activated seed liquid is 1:10-according to the volume ratio of activated seed liquid Yu fermentation medium
20 are carried out.
Utilize the method that short chain alcohol molecule improves Aureobasidium pullulans polymalic acid yield, its feature the most as claimed in claim 3
Being, in step (2), described sugar is at least in glucose, xylose, corn stalk hydrolysis or ligno-cellulose hydrolysate
Kind；Described nitrogen source is the one in ammonium sulfate, ammonium chloride, potassium nitrate or sodium nitrate.
Utilize the method that short chain alcohol molecule improves Aureobasidium pullulans polymalic acid yield, its feature the most as claimed in claim 3
Being, in step (2), described short chain alcohol molecule is the one in methanol, ethanol, propylene glycol or glycerol.
Utilize the method that short chain alcohol molecule improves Aureobasidium pullulans polymalic acid yield, its feature the most as claimed in claim 3
Being, in step (2), described fermentation culture is the one in shake flask fermentation technique or ferment tank technique.
Utilize the method that short chain alcohol molecule improves Aureobasidium pullulans polymalic acid yield, its feature the most as claimed in claim 8
Be, described shake flask fermentation technique particularly as follows: described activated seed liquid is inoculated in the shaking flask equipped with described fermentation medium,
96-150h is cultivated under revolution 180-300rpm.
Utilize the method that short chain alcohol molecule improves Aureobasidium pullulans polymalic acid yield, its feature the most as claimed in claim 8
Being, described ferment tank technique is particularly as follows: be inoculated in the fermentation equipped with described fermentation medium by described activated seed liquid
In tank, under the conditions of revolution 300-1000rpm, ventilation ratio 1:0.8-1:1.6, cultivate 60-200h.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110964647A (en) * | 2019-12-24 | 2020-04-07 | 天津科技大学 | Bacterial strain for high yield of polymalic acid and method for improving yield of polymalic acid |
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