CN110283735A - The breeding and production of one plant height production 3-hydroxy-2-butanone bacterium - Google Patents
The breeding and production of one plant height production 3-hydroxy-2-butanone bacterium Download PDFInfo
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- CN110283735A CN110283735A CN201810224667.8A CN201810224667A CN110283735A CN 110283735 A CN110283735 A CN 110283735A CN 201810224667 A CN201810224667 A CN 201810224667A CN 110283735 A CN110283735 A CN 110283735A
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Abstract
The invention discloses the breedings and production of a kind of high yield 3-hydroxy-2-butanone bacterium.Belong to technical field of bioengineering.Bacterial strain of the present invention is that soil sample dilution spread under the fruit tree of campus is screened and carried out form by VP experiment and Physiology and biochemistry is identified, screens the bacillus amyloliquefaciens that a plant height produces 3-hydroxy-2-butanone using spectrophotometry.The present invention discloses a kind of methods using bacterial strain production 3-hydroxy-2-butanone, produce 3-hydroxy-2-butanone using this method, yield reaches 42g/L.
Description
Technical field
One plant height produces the breeding and production of 3-hydroxy-2-butanone bacterium, belongs to bioengineering field.
Background technique
3-hydroxy-2-butanone is also known as 3- hydroxy-2-butanone (acetoin), has apparent rouge perfume (or spice) feature, is a kind of by public wide
The general flavorant used, is often added in the production of cream, dairy products, Yoghourt, and as cheese, coffee, nut fragrance
Reinforcing agent；In liquor industry, the 3-hydroxy-2-butanone of 80% content is commonly called as " vinegar drone ", can make the important species in drinks blending；In pharmacy
Industry, 3-hydroxy-2-butanone can be used for the synthesis of drug, as 3-hydroxy-2-butanone can be used to synthesize chloro- 4, the 5- dimethyl -1,3- of medicine intermediate 4-
Dioxolane -2- ketone (CDMDO)[3], to improve drug effect, mitigate side effects of pharmaceutical drugs；In tobacco business, 3-hydroxy-2-butanone, 1-
The substances such as amylene -3- alcohol have typical odor characteristic, are the main matters of special aroma in Flue-cured tobacco with special flavoring aroma[4]；In addition,
In IT industry, 3-hydroxy-2-butanone derivative with optical activation can be used as the important component of liquid crystal material[5].Meanwhile 3-hydroxy-2-butanone conduct
A kind of platform chemicals are widely used in other industry, 2004, U.S. Department of Energy be defined as 30 kinds preferentially utilize it is flat
One of platform compound[6]。
Summary of the invention
The object of the present invention is to provide a kind of screening technique of high yield 3-hydroxy-2-butanone bacterial strain and interests bacterial strain production second are even
Relation by marriage.
Technical solution of the present invention.
1, in the screening technique of high yield 3-hydroxy-2-butanone bacterium.
Take the soil of surface about 3cm thickness with scuppit under fruit tree, soil sampling about 50g is packed into small beaker and saves.
Enrichment culture: taking 10g soil to be dissolved in 90mL sterile water and suspension be made, dilute using this bacteria suspension as mother solution gradient
It releases to 10-8, take 10-6、10-7、10-8Dilution bacterial suspension inoculation in LB liquid medium, on 37 DEG C, the shaking table of 150r/min
Cultivate 12-24h.
Bacterial strain primary dcreening operation: the bacterium solution gradient dilution that enrichment is obtained to 10-8, take 10-6、10-7、10-8Dilution bacteria suspension coating
Onto screening and culturing medium, cultivated for 24 hours in 37 DEG C of incubators.The bacterium colony grown on observation culture medium flat plate, and according to bacterium colony appearance
Rough classification is done, the bacterium colony of picking different shape carries out plate streaking separation on screening and culturing medium, is up to obtaining single colonie
Only.
Bacterial strain secondary screening: picking single bacterium falls within the row V-P experiment after culture 12h on 37 DEG C of shaking tables in seed culture medium, will be positive
Property strain inoculated into preliminary fermentation culture medium, culture measures 3-hydroxy-2-butanone yield with spectrophotometry afterwards for 24 hours at 520nm,
Middle number LXF04 producing strain highest.
2, the selection of a kind of high yield 3-hydroxy-2-butanone bacterial strain according to claim 1, the identification method of selected bacterial strain
It is as follows.
(1) Morphological Identification
Shape, dry and wet, color, edge, surface, the transparency of bacterium colony.With optical microscopy and electron microscope observation cell shape
Whether shape, size have gemma, flagellum etc.；
(2) Physiology and biochemistry is identified
Gram staining experiment, Starch Hydrolysis test, aerobism test, gelatin liquefaction test, catalase test.
It is identified by form and Physiology and biochemistry and determines that the bacterial strain is bacillus amyloliquefaciens, and be named asBacillus amyloliquefaciens LXF04。
3, the present invention discloses a kind of production method of 3-hydroxy-2-butanone, it is characterized in that usingBacillus amyloliquefaciens LXF04 is starting strain, produces 3-hydroxy-2-butanone using seed culture and liquid fermentation.
(1) seed culture
Condition of culture are as follows: 37 DEG C of temperature, pH7.5, shaking speed 175rpm, culture is for 24 hours；
Culture medium is in terms of g/L: glucose 60, peptone 5, yeast powder 10, KH2PO40.5, sodium acetate 2.4；
(2) fermented and cultured
Fermentation condition: inoculum concentration 5%(v/v), 37 DEG C of temperature, pH7.5, shaking speed 175rpm, cultivate 60-70h；
Fermentation medium is in terms of g/L: glucose 77.8g/L, peptone 5g/L, yeast powder 22.8g/L, NaCl 0.05g/L,
MgSO4 0.05g/L、KH2PO4 0.1g/L and sodium acetate 2.4g/L.
4, the measurement of 3-hydroxy-2-butanone yield
(1) drafting of standard curve
Oscillation after the dilution of 3-hydroxy-2-butanone sterling is shaken up to the 3-hydroxy-2-butanone solution that various concentration gradient is made.It is molten to the 3-hydroxy-2-butanone of each gradient
Liquid is separately added into creatine solution and alpha-Naphthol solution, is placed in 37 DEG C of constant incubators and cultivates 60min progress chromogenic reaction.It is aobvious
After colour response, light absorption value is measured to each test tube solution at 520nm, records data.By the extinction Value Data and 3-hydroxy-2-butanone of record
Concentration data carries out linear fit, generates standard curve (attached drawing), fit equation: y=151x+0.006, R2=0.9994；
(2) it takes bacterium solution in centrifuge tube, 10min is centrifuged under 10000r/min revolving speed, obtain supernatant and bacterial sediment, it will be upper
Bacterium solution 2mL after clear liquid dilution is added 2 drop creatines and 5 drop alpha-Naphthol solution is placed in 37 DEG C of constant incubators in test tube
Carry out chromogenic reaction.Test tube is taken out after 60min, using creatine solution, alpha-Naphthol solution and distilled water as blank, uses spectrophotometric
Meter measures light absorption value under 520nm wavelength, brings equation into, and the yield of the 3-hydroxy-2-butanone obtained accordingly is 42g/L.
Detailed description of the invention
Fig. 1 is 3-hydroxy-2-butanone standard curve.
Specific embodiment
The following are bacillus amyloliquefaciensBacillus amyloliquefaciens Breeding, identification and the fermentation of LXF04
Produce the embodiment of 3-hydroxy-2-butanone.
Embodiment 1
Take the soil of surface about 3cm thickness with scuppit under fruit tree, soil sampling about 50g is packed into valve bag and saves；Take 10g native
Earth is dissolved in 90mL sterile water and suspension is made, and is diluted to 10 using this bacteria suspension as mother solution gradient-8, take 10-6、10-7、10-8's
It dilutes bacterial suspension inoculation in LB liquid medium, cultivates 12-24h on 37 DEG C, the shaking table of 150r/min；The bacterium that enrichment is obtained
Liquid gradient dilution is to 10-8, take 10-6、10-7、10-8Dilution bacteria suspension be applied on screening and culturing medium, trained in 37 DEG C of incubators
It supports for 24 hours.The bacterium colony grown on observation culture medium flat plate, and rough classification is done according to bacterium colony appearance, the bacterium colony of picking different shape exists
Plate streaking separation is carried out on screening and culturing medium, until obtaining single colonie；Picking single bacterium is fallen in seed culture medium 37
Row V-P is tested after cultivating 12h on DEG C shaking table, and positive strain is inoculated into preliminary fermentation culture medium, and culture is for 24 hours afterwards with light splitting light
Degree method measures 3-hydroxy-2-butanone yield at 520nm, and screening produces 9 plants of 3-hydroxy-2-butanone bacterial strain altogether, wherein number LXF04 producing strain is most
It is high.
Embodiment 2
Form is carried out to the LXF04 bacterial strain of breeding and Physiology and biochemistry identifies (see Table 1)
The qualification result of 1 bacterial strain of table (LXF04)
| Physiology and biochemistry identification | Experimental phenomena and conclusion |
| Colony morphological observation test | White, flat, dry, matt, surface folding;When growing in liquid medium, wrinkle mould is formed |
| Cellular morphology observation test | It is round or oval, it is single or pairs of |
| Gram's staining | It is aubergine, it is positive |
| Aerobism test | It is surface growth, it is aerobic |
| Starch Hydrolysis test | Reaction is the positive |
| Gelatin liquefaction test | Reaction is the positive |
| Catalase test | Reaction is the positive |
Embodiment 3
1, the preparation of culture medium
(1) screening and culturing medium (g/L): glucose 20, yeast powder 2, NaCl 0.5, MgSO40.5, KH2PO40.5, agar powder
20, pH value 7.0
(2) seed culture medium (g/L): glucose 10, yeast powder 1, peptone 2, (NH4)2SO46, KH2PO4 10, NaCl 0.5,
MgSO40.5, pH value 7.0
(3) fermentation medium (g/L): glucose 60, peptone 5, yeast powder 10, KH2PO40.5, pH value 7.2
2, prepared by seed
The bottled 50mL seed culture medium of 250mL triangle, 121 DEG C of sterilizing 15min take the preservation bacterium solution that 250 μ L concentration are 10% to be inoculated with
In seed culture medium, 37 DEG C, shaking speed 175rpm, culture is for 24 hours；
3, fermented and cultured
Fermentation medium is sub-packed in 250mL conical flask, and every bottle of culture medium is 30mL, adjusts pH to 7.2, and encapsulation sterilizing is cooling,
The seed culture fluid of inoculation 5%, sealing, cultivate 60-70h by 37 DEG C of temperature, shaking speed 175rpm；It takes fermentation liquid to be centrifuged, utilizes
Honourable photometry surveys 3-hydroxy-2-butanone content in supernatant, yield 42g/L.
Claims (4)
1. a kind of selection for producing 3-hydroxy-2-butanone bacterial strain, comprising the following steps:
The soil 10g of surface about 3cm thickness is taken under the fruit tree of campus, is dried for 24 hours in 80 DEG C of baking ovens, is dissolved in 90mL sterile water and is made
Suspension is diluted to 10 using this bacteria suspension as mother solution gradient-8, take 10-6、10-7、10-8Dilution bacterial suspension inoculation in LB liquid train
Base is supported, cultivates 12-24h on 37 DEG C, the shaking table of 150r/min；By the obtained bacterium solution gradient dilution of enrichment to 10-8, take 10-6、
10-7、10-8Dilution bacteria suspension be applied on screening and culturing medium, cultivated for 24 hours in 37 DEG C of incubators；It observes on culture medium flat plate
The bacterium colony grown, and rough classification is done according to bacterium colony appearance, the bacterium colony of picking different shape carries out plate on screening and culturing medium and draws
Line separation, until obtaining single colonie；Picking single bacterium is fallen in seed culture medium 12h is cultivated on 37 DEG C of shaking tables after row V-P
Experiment, positive strain is inoculated into preliminary fermentation culture medium, and culture measures second idol with spectrophotometry afterwards for 24 hours at 520nm
Relation by marriage yield, wherein number LXF04 producing strain highest.
2. a kind of selection for producing 3-hydroxy-2-butanone bacterial strain, the bacterial strain screened reflect through Physiology and biochemistry according to claim 1
It is set to bacillus amyloliquefaciens, and is named asBacillus amyloliquefaciens LXF04。
3. a kind of production method of 3-hydroxy-2-butanone, it is characterized in that usingBacillus amyloliquefaciens LXF04 is to set out
Bacterial strain produces 3-hydroxy-2-butanone with seed culture and fermented and cultured.
4.(1) seed culture
Condition of culture are as follows: 37 DEG C of temperature, pH7.5, shaking speed 175rpm, culture is for 24 hours；
Culture medium is in terms of g/L: glucose 60, peptone 5, yeast powder 10, KH2PO40.5, sodium acetate 2.4；
(2) fermented and cultured
Fermentation condition: inoculum concentration 5%(v/v), 37 DEG C of temperature, pH7.5, shaking speed 175rpm, cultivate 60-70h；
Fermentation medium is in terms of g/L: glucose 77.8g/L, peptone 5g/L, yeast powder 22.8g/L, NaCl 0.05g/L,
MgSO4 0.05g/L、KH2PO4 0.1g/L and sodium acetate 2.4g/L.
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| CN201810224667.8A CN110283735A (en) | 2018-03-19 | 2018-03-19 | The breeding and production of one plant height production 3-hydroxy-2-butanone bacterium |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113430147A (en) * | 2021-07-30 | 2021-09-24 | 千禾味业食品股份有限公司 | Bacillus villagens QH-20011 with low pH tolerance and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113430147A (en) * | 2021-07-30 | 2021-09-24 | 千禾味业食品股份有限公司 | Bacillus villagens QH-20011 with low pH tolerance and application thereof |
| CN113430147B (en) * | 2021-07-30 | 2022-11-01 | 千禾味业食品股份有限公司 | Bacillus villagens QH-20011 with low pH tolerance and application thereof |
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