Bacillus and method for applying bacillus to producing isovaleric acid

A technology of bacillus and isovaleric acid, applied in the biological field, can solve the problems of needing a catalyst, restricting the application of isovaleric acid, cumbersome reaction steps, etc., and achieves the effects of easy separation and cultivation, promotion of industrial production and application thereof, and low cost

Active Publication Date: 2018-05-08
CHINA TOBACCO YUNNAN IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is mainly used in the application of a strong catalyst, using isoamyl alcohol or isovaleraldehyde as a raw material, and is prepared through an oxidation reaction. This method not only requires a catalyst, but also has cumbersome reaction steps and takes a long time.
The biosynthesis of isovaleric acid is rarely reported, which may be due to the unexplored microorganisms for synthesizing isovaleric acid, or because the efficiency of microbial synthesis of isovaleric acid is not high, which restricts the biosynthesis of isovaleric acid in industry. Applications

Method used

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  • Bacillus and method for applying bacillus to producing isovaleric acid
  • Bacillus and method for applying bacillus to producing isovaleric acid
  • Bacillus and method for applying bacillus to producing isovaleric acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Separation of microorganisms producing isovaleric acid from tobacco industrial waste

[0033] 1. Culture medium and culture conditions for separation:

[0034] 10 times (or 8 times, or 12 times) diluted LB liquid medium: 1000mL distilled water; tryptone 1g; yeast extract 0.5g; NaCl 1g; pH natural, sterilized;

[0035] The sterilization condition of the above-mentioned culture medium is sterilized at 121° C. for 20 minutes.

[0036] Add 20g / L of agar to the above medium to prepare a solid medium for strain screening.

[0037] 2. Separation method:

[0038] Randomly extract 2 g of tobacco leaf samples in the middle stage of alcoholization, cut them into pieces under aseptic conditions, soak them in 150 mL of sterile normal saline, and culture them with shaking for 60 minutes at a temperature of 37 ° C and a speed of 180 rpm. Bacteria single-layer gauze filter, take the filtrate, centrifuge to discard the supernatant, and resuspend with 5mL sterile water to ob...

Embodiment 2

[0039] Example 2 The strain identification of isolated microorganisms

[0040] (1) Culture the bacteria isolated from the selected medium in LB liquid medium diluted 10 times, and obtain the bacterial suspension of the corresponding strain after 12 hours.

[0041] (2) Take 500 μL of bacterial suspension, centrifuge and discard the supernatant, extract genomic DNA, and use the extracted genomic DNA as a template for polymerase chain reaction (PCR).

[0042] The above PCR upstream primer is 27F:5'-agagtttgatcctggctcag-3' (SEQ ID NO.2);

[0043] The downstream primer is 1492R: 5'-cggctaccttgttacgactt-3' (SEQ ID NO.3).

[0044]The PCR reaction system and procedures are as follows:

[0045] PCR amplification system (25μL):

[0046]

[0047] PCR amplification program:

[0048]

[0049] 30 cycles.

[0050] (3) Take 5 μL of PCR reaction solution and DNA maker 2000 respectively, and verify PCR products by gel electrophoresis.

[0051] (4) Send the PCR product with a fluores...

Embodiment 3

[0052] Example 3 Production of isovaleric acid from tobacco industrial waste

[0053] The medium components with tobacco industrial waste as nutrients are: glucose 20g / L, tobacco industrial waste (tobacco stem: debris: tobacco powder 50:30:20) 5g / L, pH value 7.0~7.4;

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Abstract

The invention relates to bacillus and a method for applying the bacillus to producing isovaleric acid. The bacillus is separated from a tobacco leaf on an alcoholization stage through a selective culture medium, and is already preserved in the general microbiological center of the China microbe preservation management committee on September 13, 2017, and the preservation number is CGMCC No.14609.The strain takes the waste of the tobacco industry as the raw material to synthesize the isovaleric acid, and the isovaleric acid is a colorless sticky liquid, and has a sweet fruit aroma and the fragrance of the vaccinium uliginosuma after being highly diluted. A fermentation culture medium used in the process of synthesizing the isovaleric acid through the bacillus is low in cost, a large quantity of wastes produced in the tobacco industry are also treated, the method plays positive roles in protecting the environment and reasonably utilizing the resources, and has an excellent application prospect.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a bacillus and its application, in particular to a bacillus obtained by screening and a method for fermenting and producing isovaleric acid using the strain. Background technique [0002] Isovaleric acid, also known as 3-methylbutyric acid, is a colorless viscous liquid with a melting point of -29.3°C and a boiling point of 176.5°C. It is miscible with alcohol, ether, and chloroform. [0003] Isovaleric acid has a pungent rancid taste, and highly diluted flavors are sweet, fruity, and huckleberry-like. Isovaleric acid is naturally present in valerian oil, vanilla oil, hop oil, bay leaf oil, spearmint oil, etc., and is allowed to be used in edible spices in my country's national standard. Isovaleric acid is not only used in the preparation of cheese and cream flavors, but also in small amounts in the preparation of fruit flavors. It can also be used in the manufa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/02C12P7/40C12R1/07
CPCC12N1/02C12N1/20C12P7/40
Inventor 向海英李雪梅刘晶曾婉俐许力蒋佳芮邓乐乐张建铎张涛宋春满张海波
Owner CHINA TOBACCO YUNNAN IND
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