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Use of Bacillus subtilis (Ehrenberg)Cohn in preparing 3-hydroxy butanone

A technology of Bacillus subtilis and hydroxybutanone, applied in the biological field, can solve the problems of raw material source, product price and scope of use, no large-scale popularization and application, no 3-hydroxybutanone, etc., and achieve attractive application Prospect, the effect of stable fermentation performance and high yield

Active Publication Date: 2007-08-01
SHANDONG FOOD & FERMENT IND RES & DESIGN INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The difference is that the yield of enzymatic products is high, there are no other by-products, and the products have optical rotation, but it is difficult to obtain a large number of specific enzymes
[0005] Both the chemical synthesis method and the biological enzyme conversion method use diacetyl or 2,3-butanediol as raw materials, but diacetyl and 2,3-butanediol are also synthetic fragrances, not bulk chemical products, so the source of raw materials , product price and scope of use are limited, which is one of the main reasons why the two processes have not been widely applied
[0006] 3-Hydroxybutanone is an intermediate metabolite of a variety of microbial sugar metabolism, and 3-hydroxybutanone can be produced by microbial fermentation using sugar as raw material. However, according to the current literature on microbial production of 3-hydroxybutanone, most reports It is a study on the theory of microbial metabolic pathways, and a few reports involving the production of 3-hydroxybutanone are mainly mentioned as a by-product of the fermentation of diacetyl and 2,3-butanediol
At present, there is no research report on 3-hydroxybutanone as the main target product of microorganisms, and there is no report on the use of Bacillus subtilis strains to ferment and transform glucose to directly generate and obtain the target product 3-hydroxybutanone

Method used

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  • Use of Bacillus subtilis (Ehrenberg)Cohn in preparing 3-hydroxy butanone
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  • Use of Bacillus subtilis (Ehrenberg)Cohn in preparing 3-hydroxy butanone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Breeding of 3-hydroxybutanone-producing Bacillus subtilis

[0054] The starting strain SFA-W15 (a strain of Bacillus subtilis that accumulates a small amount of 3-hydroxybutanone) was inoculated in the liquid seed medium, cultured to the middle logarithmic growth phase at 37°C, the bacteria were collected by centrifugation, and washed with sterile saline for two times. Then add sterile saline to make a bacterial suspension, so that the cell concentration is 1×10 8 -10 9 pieces / ml.

[0055] Take 10ml of the above-mentioned bacterial suspension and put it into a plate, put it under a 30W ultraviolet lamp to irradiate, the irradiation distance is 15cm, and the irradiation time is 3 minutes, 5 minutes, 7 minutes, 10 minutes. Plate, cultured at 37°C for 1-2 days in the dark, picked a single colony and transplanted to the slope, cultured at 37°C for 1-2 days, inoculated the shake flask with fermentation medium after the slope matured, fermented for 60 hours at 37°...

Embodiment 2

[0058] Example 2 Breeding of high-yield 3-hydroxybutanone Bacillus subtilis CGMCC NO.1869 strain

[0059] The mutant strain SFA-U97 strain screened in Example 1 was inoculated into the liquid seed medium, cultivated to the middle logarithmic growth phase at 37°C, collected the bacteria by centrifugation, washed twice with sterile normal saline, and then added sterile normal saline to make Bacteria suspension, so that the cell concentration at 1 × 10 8 -10 9 pcs / ml, spare.

[0060] Preparation of nitrosoguanidine treatment solution: Weigh 20mg of nitrosoguanidine, place it in a 100ml sterile Erlenmeyer flask, add 2ml of acetone to dissolve it, then add 18ml of Tris buffer solution (pH6.0, 0.5mol / L) and mix well ,spare.

[0061] Take 10ml of the above-mentioned nitrosoguanidine treatment solution, add 10ml of bacterial suspension, incubate and shake at 30°C for 50-60 minutes, and take samples every 10 minutes. After sampling, first dilute 1000 times to stop the reaction, then...

Embodiment 3

[0068] Example 3 Application of Bacillus subtilis in the preparation of 3-hydroxybutanone

[0069] The sequence of steps involved in applying the method is as follows:

[0070] (1) Strain selection: select Bacillus subtilis (Bacillus subtilis) CGMCC NO.1869;

[0071] (2) slant culture activation: inoculate the strains on the slant medium, and culture it statically for 30 hours at 37°C, and set aside;

[0072] (3) Seed cultivation: the bacterial strain cultivated in step (2) was placed in 30mL liquid seed culture medium (250mL Erlenmeyer flask) with an inoculation loop under aseptic conditions, and the rotation speed was set to 160 rpm and the radius of rotation was 160 rpm. On a 40mm shaker, culture at 37°C for 12 hours to obtain seed solution;

[0073] (4) Fermentation culture:

[0074] Shake flask fermentation: inoculate the seed liquid in a shake flask (500mL Erlenmeyer flask) equipped with 80mL fermentation medium with an inoculum amount of 3% by volume, at 37°C, with a...

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Abstract

The invention discloses an application of bacillus subtilis in preparing 3-hydroxy butanone. The related bacteria has been stored now in ''microbiological bacterial preservation and management conmmite normal bacteria center of China'' since November, 23, 2006, the perservation number is CGMCC NO.1869. The application method comprises: (1) bacterial selection, (2) inclined-plane culture activation, (3) seed culturing, (4) fermentating, (5) checkinga nd collecting fermentation product. The invention is characterized by simple operation, low cost, high productivity and high- purity of 3-hydroxy butanone, no generation of united compound by butanedione and 2, 3- butanedione.

Description

technical field [0001] The invention relates to the application of a strain of Bacillus subtilis, in particular to the application of a strain of Bacillus subtilis in preparing 3-hydroxybutanone, and belongs to the field of biotechnology. Background technique [0002] 3-Hydroxybutanone (acetoin), also known as acetoin and acetylmethylmethanol, naturally exists in many foods such as corn, grapes, cocoa, apples, bananas, cheese, meat, etc., and has a unique creamy aroma. In addition, beer Flavor and cheese aroma are related to 3-hydroxybutanone. [0003] 3-Hydroxybutanone is a widely used and lovable food spice, and it is a commonly used spice variety in the world. It is mainly used as spices in cream, dairy products, yogurt and strawberry type, etc. China's national standard GB2760-86 stipulates that it is a food spice that is allowed to be used. 3-Hydroxybutanone with 80% content is commonly known as "Vinegar Om", and it is an important variety in wine flavoring. 3-Hydrox...

Claims

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Application Information

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IPC IPC(8): C12P7/26C12N1/20C12R1/125
Inventor 刘建军赵祥颖田延军张家祥韩延雷韩丽
Owner SHANDONG FOOD & FERMENT IND RES & DESIGN INST
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