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Breeding of microbial strain for producing 3-hydroxy-2-butanone by fermenting substrate glucose

A technology of glucose and fermentation medium, applied in microorganism-based methods, microorganisms, fermentation and other directions, can solve the problem that 3-hydroxy-2-butanone is difficult to develop on a large scale, product quality is difficult to achieve, product yield and yield Low problems, to achieve the effect of being conducive to environmental protection, easy to control, and high utilization of raw materials

Inactive Publication Date: 2010-10-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The production of 3-hydroxy-2-butanone by chemical synthesis has the disadvantages of low product yield and yield, and serious environmental pollution, and the product quality is difficult to reach the current largest consumption field of 3-hydroxybutanone - food Requirements for spices; what is more serious is that the sources of raw materials for these three processes are blocked, which makes it difficult to develop large-scale production of 3-hydroxy-2-butanone by applying chemical methods
The enzymatic conversion method produces 3-hydroxy-2-butanone with a high yield, no other by-products, and the product has optical rotation, but it is difficult to obtain a large number of specific enzymes

Method used

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  • Breeding of microbial strain for producing 3-hydroxy-2-butanone by fermenting substrate glucose
  • Breeding of microbial strain for producing 3-hydroxy-2-butanone by fermenting substrate glucose
  • Breeding of microbial strain for producing 3-hydroxy-2-butanone by fermenting substrate glucose

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: the screening of bacterial strain:

[0029] Screening of target strains: Enrich the collected samples in seed medium (glucose 20g / L, yeast extract 5g / L, peptone 10g / L, sodium chloride 10g / L pH 7.0), shake at 37°C 150r / min Bed 10h, take different dilutions to spread and culture. Pick a single colony on the plate separation medium (yeast extract 5g / L, peptone 10g / L, sodium chloride 10g / L, agar powder 18g / L pH 7.0) for streak separation, culture at 37°C for 1d-2d, and microscopic examination , the strains to be tested were preserved in the slant preservation medium for primary screening.

[0030] The seeds cultivated to the logarithmic phase with the seed medium were added to the fermentation medium containing 15% glucose according to the inoculation amount of 5%, and then placed under the condition of 37°C and 150r / min for shaking culture for 130h. The fermentation broth was collected, centrifuged, and the supernatant was taken for detection.

Embodiment 2

[0031] Embodiment 2: Qualitative detection of 3-hydroxyl-2-butanone:

[0032] Take 2ml of the supernatant, extract with 2ml of ethyl acetate, and then use GC-MS to identify whether the extract contains 3-hydroxy-2-butanone. GC-MS detection conditions: The temperature control program of the chromatographic column is as follows: first, keep warm at 60°C for 1min, then raise the temperature to 90°C at a rate of 6°C / min and keep it for 2min, and finally increase the temperature at a rate of 30°C / min. To 180 ℃, constant temperature 2min. The temperature of the injection port is 250°C, the injection volume is 1 μL, the carrier gas is high-purity helium, the flow rate of the carrier gas is 1.2mL / min, and the detector temperature is 240°C;

[0033] TraceMS mass spectrometry conditions: EI+ bombardment source, full scan mode, scan mass range of 30-500amu, emission current of 200μA, electron energy of 70eV, mass spectrometry detection spectrum library is NIRT98 spectrum library;

[00...

Embodiment 3

[0035] Example 3: Detection of the ability of the strain to synthesize 3-hydroxy-2-butanone:

[0036] According to the method of Example 1, the activated seeds are added to the fermentation medium containing 15% glucose according to the inoculum size of 5%. After culturing for 130 h under the condition of 1 min, the fermented liquid was collected, centrifuged, and the supernatant was taken to determine the content of 3-hydroxy-2-butanone in the fermented liquid by gas chromatography. Shimaduz gas chromatograph, chromatographic column: PEG-20M 30M I.D.0.32mm; headspace sampling, sample equilibrium temperature: 70°C; equilibrium time: 30min; injection temperature: 200°C; detector temperature: 250°C ℃; temperature program: 40°C (5min)---180°C, temperature rise 10°C / min, sample retention time is 12.7min.

[0037] 3-Hydroxy-2-butanone standard GC chart Image 6 As shown, Bacillus subtilis JNA takes glucose as the substrate 130h fermentation broth GC spectrum as Figure 7 shown;

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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 [0001] The invention relates to the breeding of a microbial strain which uses glucose as a substrate to ferment and produce 3-hydroxy-2-butanone, belonging to the technical field of fermentation in bioengineering. Background technique [0002] 3-Hydroxy-2-butanone, the English name is 3-Hydroxy-2-Butanone, also known as acetoin (Acetoin), is a light yellow liquid, long-term storage to form dimers, slightly soluble in water, ethanol, propylene glycol, etc. Organic solvent, with a melting point of 15°C and a boiling point of 143-148°C, capable of spontaneous combustion. [0003] 3-Hydroxy-2-butanone naturally exists in many foods such as corn, grapes, cocoa, apples, bananas, cheese, and meat. It is a widely used and popular food flavor, and it is also widely used in many other industries. [0004] Application in food: It is mainly used in the production of flavors such as butter, dairy products, yogurt and strawberry type. The national standard GB2760-86 stip...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P7/26C12R1/125
Inventor 饶志明张显林清
Owner JIANGNAN UNIV
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