CN101864381A - Breeding of microbial strain for producing 3-hydroxy-2-butanone by fermenting substrate glucose - Google Patents
Breeding of microbial strain for producing 3-hydroxy-2-butanone by fermenting substrate glucose Download PDFInfo
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- CN101864381A CN101864381A CN201010166987A CN201010166987A CN101864381A CN 101864381 A CN101864381 A CN 101864381A CN 201010166987 A CN201010166987 A CN 201010166987A CN 201010166987 A CN201010166987 A CN 201010166987A CN 101864381 A CN101864381 A CN 101864381A
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Abstract
The invention discloses breeding of a microbial strain for producing 3-hydroxy-2-butanone by fermenting glucose serving as a substrate, and belongs to the technical field of fermentation in bioengineering. The screened strain is classified and named Baclliussubtilis JNA, which has been preserved in China center for type culturecollection with the preservation number of CCTCC M209309. The strain capable of producing 3-hydroxy-2-butanone by fermenting the substrate glucose is screened from natural soil, is identified in the aspects of morphology and genetics, and is named Baclliussubtilis JNA. The fermentation products are subjected to qualitative identification, the content of the 3-hydroxy-2-butanone in fermentation liquor is about 50g/L through the detection of gas chromatography (GC), and helpful guidance is provided for industrial production of the 3-hydroxy-2-butanone by a microbial fermentation method.
Description
Technical field
One strain is the seed selection that fermenting substrate produces 3-hydroxyl-2-butanone microorganism strains with glucose, belongs to fermentation technical field in the biotechnology.
Background technology
3-hydroxyl-2-butanone, English 3-Hydroxy-2-Butanone by name has another name called acetoin (Acetoin), be weak yellow liquid, the long-term placement generates dimer, is slightly soluble in water and organic flux such as ethanol, propylene glycol, 15 ℃ of fusing points, 143～148 ℃ of boiling points can spontaneous combustion.
3-hydroxyl-2-butanone nature is present in the numerous food products such as corn, grape, cocoa, apple, banana, cheese, meat.Be a kind ofly be widely used, charming flavouring agent, and in other numerous industries, also be widely used.
In Application in Food: mainly as the production of spices such as cream, dairy products, sour milk and strawberry type, standard GB 2760-86 stipulates that it is to allow the food spice that uses.The 3-oxobutanol of 80% content is commonly called as " vinegar drone ", is extremely important kind in the drinks blending.This product is extremely extensive because of its range of application, and consumption is also bigger, is that milk is used, the important kind of wine in the spices.Adopting 3-hydroxyl-2-butanone photoreactive gas at pharmaceutical industry is the synthetic important medicine intermediate CDMDO of raw material, and the 3-oxobutanol can also synthetic antibacterial drug human relations Ampicillin Trihydrate hydrochloride.Can synthesize pore forming material, sterilization and sterilant at chemical industry 3-hydroxyl-2-butanone, the stablizer of chlorine-containing polymer, 3-hydroxyl-2-butanone still is the sexual hormoue of a kind of insect in addition.
3-oxobutanol production method mainly comprises chemical synthesis, enzyme transforming process and microbe fermentation method.Chemical synthesis produces that 3-hydroxyl-2-butanone exists product yield and yield is lower, and shortcoming such as environmental pollution is more serious, and quality product is difficult to reach the maximum consumer field of present 3-oxobutanol---the requirement of flavouring agent; The raw material sources of these three kinds of technologies that even more serious is are obstructed, and have caused suitable chemical method to produce 3-hydroxyl-2-butanone and have been difficult to extensive development.The enzymatic conversion method method is produced 3-hydroxyl-2-butanone efficiency of pcr product height, does not have other by product, and product has specific rotation, but will obtain relatively difficulty of a large amount of specific enzymes.Fermentative Production 3-hydroxyl-2-butanone at first cost is lower, and secondly environmental pollution is also less, and is the restriction that fermenting substrate production 3-hydroxyl-2-butanone has alleviated raw material sources greatly with glucose.At present, the method for fermentative Production 3-hydroxyl-2-butanone also mostly is in conceptual phase both at home and abroad.
It is the microorganism strains of substrate high yield 3-hydroxyl-2-butanone that the present invention screens with glucose, this bacterium is identified, determine that it is subtilis, its fermentation condition has been carried out preliminary study, the industrialization of producing 3-hydroxyl-2-butanone for microbe fermentation method provides the foundation.
Summary of the invention
The object of the present invention is to provide: screening from natural soil can be the bacterial strain that fermenting substrate produces 3-hydroxyl-2-butanone with glucose, and it has been carried out the evaluation of morphology and genetics aspect, with this bacterium called after Bacllius subtilis JNA.Tunning has been carried out the qualitative and quantitative detection analysis, for the industrialization of Production by Microorganism Fermentation 3-hydroxyl-2-butanone provides useful guide.
Technical scheme of the present invention: a kind of method of producing 3-hydroxyl-2-butanone by microbial fermentation, be in initial fermention medium, with the bacterial strain that screens from nature soil serves as to produce bacterial strain, at liquid amount is that 37 ℃, 350r/min, air flow are to cultivate 130h under the condition of 1L/min in the 7L fermentor tank of 4L, by material composition in gas chromatography mass spectrometry (GC-MS) the qualitative detection fermented liquid and then the content by material in the gas-chromatography detection by quantitative fermented liquid, filtering out can be the bacterial strain that fermenting substrate produces 3-hydroxyl-2-butanone with glucose.It can be the bacterial strain that fermenting substrate produces 3-hydroxyl-2-butanone with glucose that the present invention successfully screens a strain, identify by 16S rDNA, with its called after Bacllius subtilis JNA, be preserved in Chinese typical culture collection center on December 21st, 2009, deposit number is: CCTCC M 209309.
Main agents:
Acetoin is available from U.S. TCI company
Determining of 3-hydroxyl-2-butanone analytical procedure:
3-hydroxyl-2-butanone standard specimen is analyzed by gas-chromatography on the PEG-20M post, and retention time is 12.7min.Used gas chromatograph is day island proper Tianjin (Shimaduz), chromatographic column: PEG-20M 30M I.D.0.32mm
Determine that concrete chromatographic condition is: chromatographic column: PEG-20M 30M I.D.0.32mm; Headspace sampling, sample equilibrium temperature: 70 ℃; Starting time: 30min; Sample introduction temperature: 200 ℃; Detector temperature: 250 ℃; Temperature programming: 40 ℃ of (5min)-180 ℃, 10 ℃/min heats up.
The screening of purpose bacterial strain: with the sample enrichment in seed culture medium (glucose 20g/L, yeast extract paste 5g/L, peptone 10g/L, sodium-chlor 10g/L PH 7.0) that collects, 37 ℃ of 150r/min shaking table 10h get different dilution coatings and cultivate.Choose the separation of on plate isolation substratum (yeast extract paste 5g/L, peptone 10g/L, sodium-chlor 10g/L, agar powder 18g/LPH 7.0), ruling of single bacterium colony, cultivate 1d-2d for 37 ℃, microscopy, bacterial strain to be tried is preserved in the slant preservation substratum, and it is standby to make primary dcreening operation; To cultivate the seed of logarithmic phase with seed culture medium, join in the fermention medium that contains 15% glucose, place shaking culture 130h under 37 ℃, 150r/min condition then by 5% inoculum size.Collect fermented liquid, centrifugal, get supernatant liquor in order to detecting.
The evaluation of purpose bacterial strain: adopt 16S rDNA order-checking, the dna sequence dna that include at this sequence and American National bioinformation center (NCBI) is compared, the result shows that the 16S rDNA sequence similarity of itself and Bacillus subtilis strain BIHB332 is the highest, be 99%, be the subspecies of Bacillus subtilis, with its called after: Bacillus subtilis JNA.
The qualitative detection of product: adopt gas chromatography mass spectrometry (GC-MS) to identify whether contain 3-hydroxyl-2-butanone in the product.Measure 3-hydroxyl-2-butanone standard specimen GC-MS collection of illustrative plates respectively, the GC-MS collection of illustrative plates of fermented liquid after with ethyl acetate extraction, and carry out atlas analysis, determine whether contain 3-hydroxyl-2-butanone in the conversion fluid.
The detection by quantitative of product: the growing amount that detects product 3-hydroxyl-2-butanone in the fermented liquid with vapor-phase chromatography (GC).
Beneficial effect of the present invention: with the bacterial strain that from nature soil, screens as strains tested, with starting strain on the slant medium activation after, be inoculated in the triangular flask of the 250mL that the 50mL seed culture medium is housed, 37 ℃, cultivate 6h on the shaking table of 150r/min, add in the initial fermention medium by 5% inoculum size then, at liquid amount is that 37 ℃, 350r/min, air flow are to cultivate 130h under the condition of 1L/min in the 7L fermentor tank of 4L, collect fermented liquid, centrifugal, get supernatant liquor in order to detecting; Get the 5ml supernatant liquor, detect 3-hydroxyl-2-butanone content in the fermented liquid with vapor-phase chromatography (GC).Domesticly produce 3-hydroxyl-2-butanone with microbe fermentation method and be scarcely out of swaddling-clothes, the present invention lays the foundation for microbial method fermentation product 3-hydroxyl-2-butanone.
To become to have the higher 3-hydroxyl-2-butanone of value added than the cheap substrates conversion of glucose, it be important chemical material and medicine intermediate; Produce 3-hydroxyl-2-butanone with microbial method simultaneously, its than the chemical production method have reaction conditions gentleness, raw material availability height, product purity height and technology simple, be easy to advantage such as control, help environment protection simultaneously, be easy to apply.
Description of drawings
Fig. 1 bacterial strain Bacillus subtilis JNA Electronic Speculum picture
Fig. 2 3-hydroxyl-2-butanone standard substance GC-MS collection of illustrative plates
2.78min separates figure result in the place among Fig. 3 Fig. 1
Fig. 4 JNA is the GC-MS collection of illustrative plates of substrate 130h fermented liquid with glucose
3.56min separates figure result in the place among Fig. 5 Fig. 4
Fig. 6 3-hydroxyl-2-butanone standard substance GC collection of illustrative plates
Fig. 7 Bacillus subtilis JNA is substrate 130h fermented liquid GC collection of illustrative plates with glucose
Specific implementation method
Embodiment 1: the screening of bacterial strain:
The screening of purpose bacterial strain: with the sample enrichment in seed culture medium (glucose 20g/L, yeast extract paste 5g/L, peptone 10g/L, sodium-chlor 10g/L PH 7.0) that collects, 37 ℃ of 150r/min shaking table 10h get different dilution coatings and cultivate.Choose the separation of on plate isolation substratum (yeast extract paste 5g/L, peptone 10g/L, sodium-chlor 10g/L, agar powder 18g/L PH 7.0), ruling of single bacterium colony, cultivate 1d-2d for 37 ℃, microscopy, bacterial strain to be tried is preserved in the slant preservation substratum, and it is standby to make primary dcreening operation.
To cultivate the seed of logarithmic phase with seed culture medium, join in the fermention medium that contains 15% glucose, place shaking culture 130h under 37 ℃, 150r/min condition then by 5% inoculum size.Collect fermented liquid, centrifugal, get supernatant liquor in order to detecting.
Embodiment 2:3-hydroxyl-2-butanone qualitative detection:
Get the 2ml supernatant liquor, extract, adopt gas chromatography mass spectrometry (GC-MS) to identify in the extraction liquid whether contain 3-hydroxyl-2-butanone then with the 2m1 ethyl acetate.The gas chromatography mass spectrometry testing conditions: the chromatographic column temperature control program of employing is: at first at 60 ℃ of following insulation 1min, rise to 90 ℃ and keep 2min with the temperature rise rate of 6 ℃/min then, the temperature rise rate with 30 ℃/min rises to 180 ℃ at last, constant temperature 2min.Injector temperature is 250 ℃, and sample size is 1 μ L, and carrier gas is high-purity helium, and carrier gas flux 1.2mL/min, detector temperature are 240 ℃;
TraceMS mass spectrum condition: EI+ bombards the source, and full scan mode, quality of scanning scope are 30～500amu, and transmitter current is 200 μ A, and electron energy is 70eV, and mass spectrometric detection spectrum storehouse is NIRT98 spectrum storehouse;
Standard substance 3-hydroxyl-2-butanone standard substance GC-MS collection of illustrative plates and separate figure result such as Fig. 2 is shown in Figure 3, bacterial strain fermentation liquor GC-MS collection of illustrative plates as shown in Figure 4 and Figure 5.
Embodiment 3: the synthetic 3-hydroxyl of bacterial strain-2-butanone ability detects:
To activate good seed by embodiment 1 method joins in the fermention medium that contains 15% glucose by 5% inoculum size, at liquid amount is that 37 ℃, 350r/min, air flow are to cultivate 130h under the condition of 1L/min in the 7L fermentor tank of 4L, collect fermented liquid, centrifugal, get the content that supernatant liquor adopts 3-hydroxyl-2-butanone in the gas Chromatographic Determination fermented liquid.Day island proper Tianjin (Shimaduz) gas chromatograph, chromatographic column: PEG-20M 30M I.D.0.32mm; Headspace sampling, sample equilibrium temperature: 70 ℃; Starting time: 30min; Sample introduction temperature: 200 ℃; Detector temperature: 250 ℃; Temperature programming: 40 ℃ (5min)---180 ℃, the 10 ℃/min that heats up, the sample retention time is 12.7min.
3-hydroxyl-2-butanone standard substance GC schemes as shown in Figure 6, Bacillus subtilis JNA with glucose be substrate 130h fermented liquid GC collection of illustrative plates as shown in Figure 7;
Behind the bacillus subtilis 7L ferment tank 130h who screens, in the fermented liquid glucose almost detect less than, 3-hydroxyl-2-butanone content is about 50g/L.
Claims (3)
1. a strain is the seed selection that fermenting substrate produces 3-hydroxyl-2-butanone microorganism strains with glucose, it is characterized in that trying strain as confession with the bacterial strain that from nature soil, screens, produce 3-hydroxyl-2-butanone with glucose for the substrate microbial method, the 1 bacillus subtilis strain Bacllius subtilis JNA that screening obtains, this bacterium can be that substrate ferments with glucose, records 3-hydroxyl-2-butanone in fermented liquid;
(1) screening of bacterial strain:
Bacterial screening: with the sample enrichment in seed culture medium (glucose 20g/L, yeast extract paste 5g/L, peptone 10g/L, sodium-chlor 10g/L PH 7.0) that collects, 37 ℃ of 150r/min shaking table 10h get different dilution coatings and cultivate.Choose the separation of on plate isolation substratum (yeast extract paste 5g/L, peptone 10g/L, sodium-chlor 10g/L, agar powder 18g/L, PH 7.0), ruling of single bacterium colony, cultivate 1d-2d for 37 ℃, microscopy, bacterial strain to be tried is preserved in the slant preservation substratum, and it is standby to make primary dcreening operation; The picking bacterial strain is cultivated to seed culture medium from the inclined-plane, will cultivate the seed of logarithmic phase with seed culture medium, joins in the fermention medium that contains 15% glucose by 5% inoculum size, places shaking culture 130h under 37 ℃, 150r/min condition then.Collect fermented liquid, centrifugal, get supernatant liquor in order to detecting;
(2) 3-hydroxyl-2-butanone qualitative detection:
Get the 2ml supernatant liquor, extract, adopt gas chromatography mass spectrometry (GC-MS) to identify in the extraction liquid whether contain 3-hydroxyl-2-butanone then with the 2ml ethyl acetate.The gas chromatography mass spectrometry testing conditions: the chromatographic column temperature control program of employing is: at first at 60 ℃ of following insulation 1min, rise to 90 ℃ and keep 2min with the temperature rise rate of 6 ℃/min then, the temperature rise rate with 30 ℃/min rises to 180 ℃ at last, constant temperature 2min.Injector temperature is 250 ℃, and sample size is 1 μ L, and carrier gas is high-purity helium, and carrier gas flux 1.2mL/min, detector temperature are 240 ℃;
TraceMS mass spectrum condition: EI+ bombards the source, and full scan mode, quality of scanning scope are 30～500amu, and transmitter current is 200 μ A, and electron energy is 70eV, and mass spectrometric detection spectrum storehouse is NIRT98 spectrum storehouse;
(3) identification of strains
Extract the genomic dna of starting strain
Genomic dna with starting strain is a template, uses the universal primer of yeast 16S rDNA to increase
Forward primer is f:5 '-AGAGTTTGATCCTGGCTCAG-3 '
Reverse primer is r:5 '-CTACGGCTACCTTGTTACGA-3 '
PCR method is as follows: add in the 50 μ L reaction systems: each 0.5 μ L of the primer f of 10mol/L and r, and the dNTP 5 μ L of 2mmol/L, 10 * ExTaq Buffer, 5 μ L, the ExTaq archaeal dna polymerase 0.5 μ L of 5U/ μ L, template 1 μ g adds distilled water polishing 50 μ L; The PCR condition is: 95 ℃ of sex change 5min, and 56 ℃ of annealing 90S, 72 ℃ are extended 2min, and 94 ℃ of sex change 1min circulate 30 times;
Reclaim test kit purifying pcr amplification product with glue, the electrophoresis checking; Be connected to the pMD18-T carrier, Transformed E .coli JM109.Through the amicillin resistance screening, obtain positive colony.16S rDNA order-checking is given birth to worker's bio tech ltd by Shanghai and is finished, and sequencing result is compared analysis with the existing sequence of database in NCBI.
2. the 16S rDNA according to the described bacterial strain of claim 1 analyzes, and this strain classification name is called: Bacllius subtilis, and with its called after Bacllius subtilis JNA.
3. subtilis according to claim 2, it is characterized in that this inoculation in 50mL seed culture medium (peptone 10g/L, yeast extract paste 5g/L, glucose 20g/L) in the triangular flask of 250mL, 37 ℃, 150r/min is behind the cultivation 8h, joining in the fermention medium that contains 15% glucose by 5% inoculum size, is that 37 ℃, 350r/min, air flow are to cultivate 130h under the condition of 1L/min in the 7L fermentor tank of 4L at liquid amount.Collect fermented liquid, centrifugal, get supernatant liquor in order to detecting, the content that records 3-hydroxyl-2-butanone is 50g/L.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102367448A (en) * | 2011-09-28 | 2012-03-07 | 江南大学 | Construction method of genetic engineering strain for high expression and easy purification of beta-mannanase |
| CN102876703A (en) * | 2012-09-20 | 2013-01-16 | 江南大学 | Path for moderately enhancing synthesis of acetoin produced by bacillus subtilis by utilizing regulatory protein acetolactate aminotransferase (ALsR) |
| CN103114083A (en) * | 2012-06-20 | 2013-05-22 | 江南大学 | Method for enhancing beta-mannase yield by two-stage temperature control strategy |
| CN103333842A (en) * | 2013-07-10 | 2013-10-02 | 山东省食品发酵工业研究设计院 | Bacillus subtilis producing 3-hydroxybutanone and application thereof |
| CN104404089A (en) * | 2014-12-09 | 2015-03-11 | 江南大学 | Method for improving acetoin yield by adding gluconic acid |
| CN104403984A (en) * | 2014-12-09 | 2015-03-11 | 江南大学 | Yield improvement method of acetoin by strengthening expression of bacillus subtilis glucose-6-phosphate dehydrogenase |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101008019A (en) * | 2007-01-29 | 2007-08-01 | 山东省食品发酵工业研究设计院 | Use of Bacillus subtilis (Ehrenberg)Cohn in preparing 3-hydroxy butanone |
| CN101016530A (en) * | 2007-01-29 | 2007-08-15 | 山东省食品发酵工业研究设计院 | Bacillus subtilis capable of producing high purity 3-hydroxy butanone |
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2010
- 2010-05-10 CN CN201010166987A patent/CN101864381A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101008019A (en) * | 2007-01-29 | 2007-08-01 | 山东省食品发酵工业研究设计院 | Use of Bacillus subtilis (Ehrenberg)Cohn in preparing 3-hydroxy butanone |
| CN101016530A (en) * | 2007-01-29 | 2007-08-15 | 山东省食品发酵工业研究设计院 | Bacillus subtilis capable of producing high purity 3-hydroxy butanone |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102367448A (en) * | 2011-09-28 | 2012-03-07 | 江南大学 | Construction method of genetic engineering strain for high expression and easy purification of beta-mannanase |
| CN103114083A (en) * | 2012-06-20 | 2013-05-22 | 江南大学 | Method for enhancing beta-mannase yield by two-stage temperature control strategy |
| CN102876703A (en) * | 2012-09-20 | 2013-01-16 | 江南大学 | Path for moderately enhancing synthesis of acetoin produced by bacillus subtilis by utilizing regulatory protein acetolactate aminotransferase (ALsR) |
| CN102876703B (en) * | 2012-09-20 | 2015-03-04 | 江南大学 | Path for moderately enhancing synthesis of acetoin produced by bacillus subtilis by utilizing regulatory protein acetolactate aminotransferase (ALsR) |
| CN103333842A (en) * | 2013-07-10 | 2013-10-02 | 山东省食品发酵工业研究设计院 | Bacillus subtilis producing 3-hydroxybutanone and application thereof |
| CN104404089A (en) * | 2014-12-09 | 2015-03-11 | 江南大学 | Method for improving acetoin yield by adding gluconic acid |
| CN104403984A (en) * | 2014-12-09 | 2015-03-11 | 江南大学 | Yield improvement method of acetoin by strengthening expression of bacillus subtilis glucose-6-phosphate dehydrogenase |
| CN104404089B (en) * | 2014-12-09 | 2018-02-23 | 江南大学 | A kind of method for improving 3-hydroxy-2-butanone yield by adding gluconic acid |
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Application publication date: 20101020 |