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Breeding of acetoin high-tolerance bacterial strain and acetoin fermentation production with bacterial strain

An acetoin, tolerance technology, applied in the field of bioengineering, can solve the problems of heavy pollution, difficult to achieve product quality and high cost

Inactive Publication Date: 2014-03-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The production methods of 3-hydroxybutanone include: chemical synthesis method, enzymatic conversion method and microbial fermentation method. At present, it is mainly produced by chemical synthesis, and industrialization has been realized; although the chemical synthesis method is simple and easy to operate, it has heavy pollution, high cost and raw materials. Petroleum constraints, product quality is difficult to meet the requirements of edible spices
There is no report on industrial production of 3-hydroxybutanone by microbial fermentation

Method used

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  • Breeding of acetoin high-tolerance bacterial strain and acetoin fermentation production with bacterial strain
  • Breeding of acetoin high-tolerance bacterial strain and acetoin fermentation production with bacterial strain
  • Breeding of acetoin high-tolerance bacterial strain and acetoin fermentation production with bacterial strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] (1) Nitrosoguanidine mutagenesis:

[0050] ① Preparation of bacterial suspension

[0051] Introduce 0.2 mL of the original strain Bacillus amyloliquefaciens FMME044 into 50 mL of seed medium, culture on a shaker at 37°C for 14 hours to the mid-to-late logarithmic period, and then insert 30% of the inoculum into a new medium for 6 hours to allow the cells to be cultured synchronously. Take 10 mL of the culture solution in a 50 mL centrifuge tube, collect the cells by centrifugation, wash with 0.1 mol / L pH6.0 phosphate buffer three times, and finally add 7 mL of buffer to resuspend the cells. Take 0.7mL of the bacterial suspension and add it to a 5mL centrifuge tube, a total of 8 tubes are set aside.

[0052] ② Mutagenesis reaction

[0053] Add 0.3mL NTG mother solution to 5mL centrifuge tubes containing 0.7mL bacterial suspension, mix well, and let stand for 30min and 45min.

[0054] ③ Termination reaction

[0055]Centrifuge the centrifuge tube containing the treatme...

Embodiment 2

[0066] The screened FMME044 strains were identified according to "Microbial Taxonomy" for their physiological and biochemical characteristics (see Tables 1 and 2 below):

[0067] Table 1 strain (FMME044) and Bacillus subtilis colony morphological characteristics comparison

[0068]

[0069] Table 2 Physiological and biochemical identification results of strain (FMME044)

[0070]

Embodiment 3

[0072] Step 1: Media Preparation

[0073] Seed medium (g / L): glucose 10, beef extract 5, peptone 5, sodium chloride 5, initial pH 7.0;

[0074] Fermentation medium (g / L): glucose 140, peptone 12.5, yeast powder 12.5, KH 2 PO 4 3,K 2 HPO 4 3. MgSO 4 ·7H 2 O0.4, initial pH7.0;

[0075] Step 2: Seed Preparation

[0076] The seed medium in a 500mL Erlenmeyer flask was 50mL, and sterilized at 121°C for 15min. The strains were preserved in glycerol tubes with a final concentration of 15%, and 200 μL of the preserved bacteria solution was taken into 50 mL of seed culture medium for seed culture, and cultured at 37° C. and 200 rpm for 12 hours.

[0077] Step 3: Shake Flask Fermentation Culture

[0078] The seed medium was inserted into 50mL fermentation medium with 10% inoculum size to carry out fermentation culture. Ferment at 37° C. and 200 rpm for 48 hours, take the fermented liquid and centrifuge, collect the supernatant and measure the content of acetoin in the fermente...

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Abstract

The invention discloses breeding of an acetoin high-tolerance bacterial strain and acetoin fermentation production with the bacterial strain, and belongs to the technical field of bioengineering (the field of microorganism). The breeding procedures are as follows: (1) nitrosoguanidine mutagenesis: taking Bacillus amyloliquefaciens FMME044 screened and stored by the laboratory as an original strain to prepare a bacterial suspension, taking reaction with nitrosoguanidine in different concentrations, after a while, stopping reaction and then carrying out post-cultivation; (2) domestication: adding acetoin with different concentrations in domestication culture media to prepare acetoin fermentation liquors with different concentrations, gradually increasing the acetoin concentrate to carry out domesticating cultivation, and directly coating the domesticated bacterial suspensions on a panel containing high-concentration acetoin; (3) screening: obtaining the acetoin high-tolerance bacterial strain which is the single colonies grown on the panel containing the high-concentration acetoin, and then selecting the single colonies to carry out fermentation verification, performing secondary verification on the high-producing strains to screen out a bacterial strain with higher concentration of acetoin than the original strain, and naming the bacterial strain B. amyloliquefaciens E-11. The invention further discloses a method for producing acetoin with the bacterial strain. The yield of acetoin produced by the method with 40 h fermentation is 63 g / L.

Description

technical field [0001] The breeding of acetoin high tolerance strain and the fermentation of the strain to produce acetoin belong to the field of bioengineering. Background technique [0002] 3-Hydroxybutanone (acetoin) is a widely used and lovable edible spice. It is a commonly used spice variety in the world. It is mainly used for the production of cream, yogurt and other spices. The national standard GB2760-86 stipulates that it is Permitted food spices. 80% content of 3-hydroxybutanone, commonly known as "vinegar hum", is an extremely important variety in wine flavoring. In the pharmaceutical industry, 3-hydroxybutanone, as a chiral compound, can be used to synthesize rare drugs and drug intermediates, such as 4-chloro-4,5-dimethyl-1,3-dioxolane- 2-keto (CDMDO) and lempicillin hydrochloride. CDMDO is an important pharmaceutical intermediate, which is mainly used for the modification of penicillin, ampicillin and other antibiotics to synthesize optically active chiral ...

Claims

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Application Information

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IPC IPC(8): C12N15/01C12N1/20C12P7/26C12R1/07
Inventor 吴静罗秋玲邬敏辰
Owner JIANGNAN UNIV
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