CN106086092B - A kind of fermentation process of α-ketoglutaric acid and pyruvic acid coproduction - Google Patents
A kind of fermentation process of α-ketoglutaric acid and pyruvic acid coproduction Download PDFInfo
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- CN106086092B CN106086092B CN201610726130.2A CN201610726130A CN106086092B CN 106086092 B CN106086092 B CN 106086092B CN 201610726130 A CN201610726130 A CN 201610726130A CN 106086092 B CN106086092 B CN 106086092B
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/50—Polycarboxylic acids having keto groups, e.g. 2-ketoglutaric acid
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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Abstract
The invention discloses the fermentation process of a kind of α-ketoglutaric acid and pyruvic acid coproduction, belong to fermentation engineering field.The present invention is little in view of the commercial value difference of α-ketoglutaric acid and pyruvic acid, therefore two kinds of carboxylic acids are carried out coproduction.In carboxylic acid coproduction fermentation process, controls suitable glycerol initial concentration and flow acceleration promotes the accumulation of thalli growth and carboxylic acid.The present invention can greatly improve the substrate transformation rate, total acid yield, production intensity, shorten fermentation period, achieve the purpose that energy-saving consumption-reducing, increase economic efficiency.
Description
Technical field
The present invention relates to the fermentation process of a kind of α-ketoglutaric acid and pyruvic acid coproduction, belong to fermentation engineering field.
Background technique
α-ketoglutaric acid (α-ketoglutaric acid, abbreviation α-KG) and pyruvic acid (Pyruvic acid) are cells
Two kinds of important short chain carboxy acid's molecules, can be widely used in the fields such as food, medicine, chemical industry in metabolic process.Currently, this
Two kinds of organic acids make in chemical industry synthesis there are noxious material in the industrial production mainly by the method for chemical industry synthesis
With, yield is low etc., disadvantages, these drawbacks limit product in the use in the fields such as medicine, food.Microbe fermentation method can benefit
Multiple product is produced with a variety of renewable carbon sources, Product Safety not only can be improved, but also can reduce in production process
Pollution.
Solution rouge Asia Lip river yeast (Y.lipolytica) WSH-Z06, which is one plant, to accumulate α -one penta by sole carbon source of glycerol
The thiamine auxotroph superior strain of diacid.But it can be along with a certain amount of third during it accumulates α-ketoglutaric acid
The accumulation of ketone acid, this makes in fermentation for the purpose of to accumulate α-ketoglutaric acid, and the phase needs by adding sulfuric acid after fermentation
The conversion of pyruvate α-ketoglutaric acid that the method for control pH accumulates early period.But do so and have the disadvantage that: fermentation period prolongs
It is lower that long, sulfuric acid addition will increase new impurity, the substrate transformation rate；In the industrial production, these drawbacks can all amplify, to life
Production is made troubles.
Summary of the invention
Present invention aims to overcome that the deficiency of above-mentioned fermentation process, a kind of raising the substrate transformation rate is provided and shortens production
The α-ketoglutaric acid in period and the fermentation process of pyruvic acid coproduction.
The present invention realizes that technical solution used by above-mentioned purpose includes the following steps:
(1) actication of culture: the secondary seed solution of preparation solution rouge Asia Lip river yeast WSH-Z06；
Fed-batch cultivation: seed liquor being connected in fermentation medium by 10% inoculum concentration and carries out fermented and cultured, in earlier fermentation,
Using the calcium carbonate in fermentation medium as pH buffer, when pH drops to 3.0 naturally, with 20%Ca (OH)2Control pH
Maintain 3.0；During the fermentation, when glycerol concentration drops to 20~30g/L, add by the flow velocity constant speed stream of 43.75g/h pure sweet
Oil to total glycerol concentration is 150g/L, and 144h terminates to ferment.
Can be in embodiment in one kind of the invention, strain is inoculated into eggplant type bottle inclined-plane kind from glycerol tube by step (1)
It is activated in sub- culture medium, after 28 DEG C of cultures for 24 hours, the seed of activation is connected to seed culture medium, after 28 DEG C of culture 18h, by level-one
Seed liquor is connected in seed culture medium by 0.5% inoculum concentration, after 28 DEG C of culture 18h.
Stream plus sweet can be started in embodiment, when glycerol concentration drops to 26g/L in step (2) in one kind of the invention
Oil.
The group of the seed culture medium becomes (g/L): glucose 20, soybean protein 10, potassium dihydrogen phosphate 1.0, seven water
Magnesium sulfate 0.5.20g/L agar is added in solid medium.
The fermentation medium group becomes (g/L): glycerol 50, ammonium sulfate 3, potassium dihydrogen phosphate 3, epsom salt 1.2,
Sodium chloride 0.5, dipotassium hydrogen phosphate 0.1, thiamine hydrochloride 6 × 10-7, sodium acetate 6, calcium carbonate 10.
The present invention is little in view of the commercial value difference of α-ketoglutaric acid and pyruvic acid, therefore two kinds of carboxylic acids are joined
It produces, the substrate transformation rate, total acid yield, production intensity can be greatly improved, shorten fermentation period, reach energy-saving consumption-reducing, improve warp
The purpose for benefit of helping.In carboxylic acid coproduction fermentation process, controls suitable glycerol initial concentration and flow acceleration promotes thallus
The accumulation of growth and carboxylic acid.
Detailed description of the invention
Each Parameters variation situation in 1 fermentation process of Fig. 1 embodiment, dry cell weight (■)；α-ketoglutaric acid (●)；Pyruvic acid
(○)；Glycerol (▲)；pH(△).
Each Parameters variation situation in 2 fermentation process of Fig. 2 embodiment, dry cell weight (■)；α-ketoglutaric acid (●)；Pyruvic acid
(○)；Glycerol (▲)；pH(△).
Each Parameters variation situation in 3 fermentation process of Fig. 3 embodiment, dry cell weight (■)；α-ketoglutaric acid (●)；Pyruvic acid
(○)；Glycerol (▲)；pH(△).
Specific embodiment
It elaborates below with reference to embodiment to a specific embodiment of the invention.
The group of seed culture fluid becomes (g/L): glucose 20, soybean protein 10, potassium dihydrogen phosphate 1.0, epsom salt
0.5；20g/L agar is added in solid medium.
Fermentation medium group becomes (g/L): glycerol 50, ammonium sulfate 3, potassium dihydrogen phosphate 3, epsom salt 1.2, sodium chloride
0.5, dipotassium hydrogen phosphate 0.1, thiamine hydrochloride 6 × 10-7, sodium acetate 6, calcium carbonate 10.
The measurement of dry cell weight: accurate 1mL fermentation liquid of drawing in 50mL volumetric flask, be added 15mL 2mol/L hydrochloric acid with
CaCO in fermentation liquid3Sufficiently reaction is uniformly mixed with after deionized water constant volume, and OD value is measured at 570nm.According to OD570:
Dry cell weight (g/L)=1:0.223 calculates dry cell weight.
α-ketoglutaric acid, pyruvic acid and the measurement with glycerol: high performance liquid chromatography (HPLC).Instrument: Agilent 1260
High performance liquid chromatograph (matches UV-vis detector, differential refraction detector), chromatographic condition: chromatographic column: Aminex HPX-
87H ion exchange column, mobile phase: 5mM H2SO4, flow velocity: 0.6mL/min, column temperature: 40 DEG C, sample volume: 10 μ L.
UV detector wavelength: 210nm (detection α-ketoglutaric acid and pyruvic acid), differential refraction detector: detection glycerol.Sample system
Standby: 1mL fermentation liquid is centrifuged 5min under 12,000rpm, and supernatant handles by dilution appropriate and after 0.22 μ L membrane filtration,
Carry out efficient liquid phase chromatographic analysis.
The substrate transformation rate (%)=(α-ketoglutaric acid yield+output of pyruvic acid) * 100/ total glycerol concentration, individually produces α-
When ketoglutaric acid, output of pyruvic acid takes 0.
Intensity (g/ (L*h))=(α-ketoglutaric acid yield+output of pyruvic acid)/fermentation period is produced, α -one is individually produced
When glutaric acid, output of pyruvic acid takes 0.
The initial glycerol concentration 50g/L of the embodiment 1 and later period adds carboxylic acid coproduction under the conditions of glycerol
Step 1: solution rouge Asia Lip river yeast WSH-Z06 is inoculated into eggplant type bottle slant activation from glycerol tube, 28 DEG C of cultures are for 24 hours
Afterwards, the seed of activation is connect into a ring to primary-seed medium with oese, after 28 DEG C of culture 18h, primary seed solution is pressed
0.5% inoculum concentration is connected in secondary seed medium, after 28 DEG C of culture 18h, then secondary seed solution is connected to by 10% inoculum concentration
In fermentation medium, in 28 DEG C of progress fermented and cultureds.First order seed cultural method are as follows: liquid amount 50mL in 500mL triangular flask, training
28 DEG C, revolving speed 200r/min of temperature is supported, 17-18h is cultivated.Secondary seed cultural method are as follows: liquid amount 10.5L in 15L seeding tank,
Inoculum concentration 0.5%, cultivates 17-18h by 28 DEG C of cultivation temperature, speed of agitator 300r/min, ventilatory capacity 1vvm.
Step 2: in earlier fermentation, by the calcium carbonate of the 10g/L in fermentation medium as pH buffer, when pH from
When so dropping to 3.0, with 20%Ca (OH)2Solution control pH maintains 3.0.
In fermentation process, controlled for 24 hours when glycerol concentration drops to 26g/L in fermentation liquid, by the flow velocity constant speed stream of 43.75g/h
Add pure glycerin that total glycerol dosage is made to reach 150g/L, during which glycerol concentration maintains 20-30g/L.144h terminates to ferment.
After fermentation, measuring total acid yield is 106.7g/L, and α-ketoglutaric acid and output of pyruvic acid are respectively 67.4g/L
And 39.3g/L.
Carboxylic acid coproduction under the conditions of the initial glycerol concentration 150g/L of embodiment 2
Step 1: solution rouge Asia Lip river yeast WSH-Z06 is inoculated into eggplant type bottle slant activation from glycerol tube, 28 DEG C of cultures are for 24 hours
Afterwards, the seed of activation is connect into a ring to primary-seed medium with oese, after 28 DEG C of culture 18h, primary seed solution is pressed
0.5% inoculum concentration is connected in secondary seed medium, after 28 DEG C of culture 18h, then secondary seed solution is connected to by 10% inoculum concentration
In fermentation medium, in 28 DEG C of progress fermented and cultureds.
Step 2: in earlier fermentation, by the calcium carbonate of the 10g/L in fermentation medium as pH buffer, when pH from
When so dropping to 3.0, with 20%Ca (OH)2Solution control pH maintains 3.0.
In fermentation process, initial glycerol concentration is 150g/L, glycerol is not added during fermentation, 144h terminates to ferment.
After fermentation, measuring total acid yield is 75.7g/L, and α-ketoglutaric acid and output of pyruvic acid are respectively 61.5g/L
And 14.2g/L.
The initial glycerol concentration 50g/L of embodiment 3 and later period add a production α-ketoglutaric acid under the conditions of glycerol
Step 1: strain is inoculated into eggplant type bottle slant activation after 28 DEG C of cultures for 24 hours from glycerol tube will be lived with oese
The seed of change connects a ring to primary-seed medium, and after 28 DEG C of culture 18h, primary seed solution is connected to two by 0.5% inoculum concentration
In grade seed culture medium, after 28 DEG C of culture 18h, then secondary seed solution is connected in fermentation medium by 10% inoculum concentration and is sent out
Ferment culture terminates fermentation after 28 DEG C of fermentation 144h.
Step 2: in earlier fermentation by the 10g/L calcium carbonate in fermentation medium as pH buffer, when under pH nature
When being down to 3.0, with 20%Ca (OH)2Control pH maintains 3.0, when pH starts rebound raising, controls pH with 4mol/L sulfuric acid
Maintain 3.0.
In fermentation process, glycerol concentration drops to 26.2g/L in fermentation liquid for 24 hours, and the flow velocity of 43.75g/h is pressed during 24-104h
Constant speed stream adds pure glycerin to total glycerol dosage to be 150g/L, and during which glycerol concentration maintains 20-30g/L, and 192h terminates to ferment.
After fermentation, measuring total acid yield is 79.3g/L, and α-ketoglutaric acid and output of pyruvic acid are respectively 79.3g/L
And 0g/L.
Two kinds of fermentation methods of 1 carboxylic acid coproduction of table compare
The fermentation comparison of 2, table production α-ketoglutaric acids and carboxylic acid coproduction
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (4)
1. the fermentation process of a kind of α-ketoglutaric acid and pyruvic acid coproduction, which comprises the steps of:
(1) actication of culture: strain being inoculated into eggplant type bottle inclined-plane seed culture medium from glycerol tube and is activated, after 28 DEG C of cultures for 24 hours,
The seed of activation is connected to seed culture medium, after 28 DEG C of culture 18h, primary seed solution is connected to seed training by 0.5% inoculum concentration
It supports in base, after 28 DEG C of culture 18h, the secondary seed solution of preparation solution rouge Asia Lip river yeast WSH-Z06；
(2) fed-batch cultivation: seed liquor being connected in fermentation medium by 10% inoculum concentration and carries out fermented and cultured, in earlier fermentation,
Using the calcium carbonate in fermentation medium as pH buffer, when pH drops to 3.0 naturally, with 20%Ca (OH)2Control pH
Maintain 3.0；During the fermentation, when glycerol concentration drops to 20~30g/L, add by the flow velocity constant speed stream of 43.75g/h pure sweet
Oil to the total dosage of glycerol is 150g/L, and 144h terminates to ferment.
2. the method according to claim 1, wherein the fermentation medium forms based on g/L are as follows: glycerol
50, ammonium sulfate 3, potassium dihydrogen phosphate 3, epsom salt 1.2, sodium chloride 0.5, dipotassium hydrogen phosphate 0.1, thiamine hydrochloride 6 × 10-7,
Sodium acetate 6, calcium carbonate 10.
3. the method according to claim 1, wherein the seed culture medium forms based on g/L are as follows: glucose
20, soybean protein 10, potassium dihydrogen phosphate 1.0, epsom salt 0.5.
4. the method according to claim 1, wherein starting to flow when glycerol concentration drops to 26g/L in step (2)
Glycerol adding.
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| CN106635852B (en) * | 2016-12-07 | 2019-11-26 | 江南大学 | A kind of recombination torulopsis glabrata of cogeneration of propanone acid and α-ketoglutaric acid |
| CN108179117B (en) * | 2016-12-09 | 2020-12-29 | 天津农学院 | Method for producing brown mushroom by high-density fermentation |
| CN110499259B (en) * | 2019-07-22 | 2021-07-27 | 浙江工业大学 | Yarrowia lipolytica YW100-1 and application thereof |
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| CN101215529A (en) * | 2007-12-26 | 2008-07-09 | 江南大学 | Alpha-ketoglutaric acid high yield bacterium, screening method thereof and production of alpha-ketoglutaric acid from the same by fermentation method |
| CN102586347A (en) * | 2012-03-06 | 2012-07-18 | 江南大学 | Two-stage pH (Potential Of Hydrogen) control method for high output of alpha-ketoglutaric acid |
| CN103074232A (en) * | 2011-10-26 | 2013-05-01 | 中国农业大学 | Method and special-purposed strain used for producing alpha-ketoglutaric acid |
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Patent Citations (3)
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| CN101215529A (en) * | 2007-12-26 | 2008-07-09 | 江南大学 | Alpha-ketoglutaric acid high yield bacterium, screening method thereof and production of alpha-ketoglutaric acid from the same by fermentation method |
| CN103074232A (en) * | 2011-10-26 | 2013-05-01 | 中国农业大学 | Method and special-purposed strain used for producing alpha-ketoglutaric acid |
| CN102586347A (en) * | 2012-03-06 | 2012-07-18 | 江南大学 | Two-stage pH (Potential Of Hydrogen) control method for high output of alpha-ketoglutaric acid |
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| 《微生物发酵生产α-酮戊二酸研究进展》;郭洪伟等;《生物工程学报》;20130225;第29卷(第2期);第141-152页 |
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