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Bacillus rhizosphaerae and method using Bacillus rhizosphaerae and tobacco industry waste to produce hexyl acetate

A technology of bacillus and hexyl acetate, applied in the biological field, can solve problems such as low efficiency of hexyl acetate, complicated production process, etc., and achieve the effects of promoting industrialized production and application, low cost, easy separation and cultivation

Active Publication Date: 2017-12-01
CHINA TOBACCO YUNNAN IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the use of these catalysts can reduce equipment corrosion and environmental pollution, their production process is relatively complicated
The biosynthesis of hexyl acetate is rarely reported, and its possible reason is that the efficiency of microbial synthesis of hexyl acetate is not high, which restricts the industrial application of the biosynthesis of hexyl acetate

Method used

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  • Bacillus rhizosphaerae and method using Bacillus rhizosphaerae and tobacco industry waste to produce hexyl acetate
  • Bacillus rhizosphaerae and method using Bacillus rhizosphaerae and tobacco industry waste to produce hexyl acetate
  • Bacillus rhizosphaerae and method using Bacillus rhizosphaerae and tobacco industry waste to produce hexyl acetate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Separation of microorganisms producing hexyl acetate from tobacco industrial waste

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

[0034] LB liquid medium: 1000mL distilled water; tryptone 10g; yeast extract 5g; NaCl 10g; pH natural, sterilized;

[0035] LB liquid medium diluted 2 times: 1000mL distilled water; tryptone 5g; yeast extract 2.5g; NaCl 5g; pH natural, sterilized;

[0036] LB liquid medium diluted 4 times: 1000mL distilled water; tryptone 2.5g; yeast extract 1.25g; NaCl 2.5g; pH natural, sterilized;

[0037] LB liquid medium diluted 5 times: 1000mL distilled water; tryptone 2g; yeast extract 1g; NaCl 2g; pH natural, sterilized;

[0038] The sterilization condition of the above-mentioned culture medium is sterilized at 121° C. for 20 minutes.

[0039] Add 20g / L of agar to the above medium to prepare a solid medium for strain screening.

[0040] 2. Separation method:

[0041] Randomly extract 2 g of tobacco leaf samples in th...

Embodiment 2

[0042] Example 2 The strain identification of isolated microorganisms

[0043] (1) Culture the bacteria isolated from the selected medium in LB liquid medium, and obtain the bacterial suspension of the corresponding strain after 12 hours.

[0044] (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).

[0045]The above PCR upstream primer is 27F:5'-agagtttgatcctggctcag-3' (SEQ ID NO. 2);

[0046] The downstream primer is 1492R: 5'-cggctaccttgttacgactt-3' (SEQ ID NO. 3).

[0047] The PCR reaction system and procedures are as follows:

[0048] PCR amplification system (25μL):

[0049]

[0050] PCR amplification program:

[0051]

[0052] 30 cycles.

[0053] (3) Take 5 μL of PCR reaction solution and DNA maker 2000 respectively, and verify PCR products by gel electrophoresis.

[0054] (4) There will be PCR products with fluorescent bands ...

Embodiment 3

[0055] Example 3 Production of hexyl acetate from tobacco industry waste

[0056] The components of the fermentation medium are: glucose 20g / L, tobacco industrial waste 6g / L, pH 7.2;

[0057] Under aseptic conditions, directly inoculate an inoculation loop with the pure culture of rhizobacteria into a 250mL shake flask containing 50mL of sterilized fermentation medium, and set up a control group that does not inoculate the strain, at 140 rpm in a shaker at 37°C. After culturing for 5 days, it was detected by GC-MS.

[0058] Qualitative detection of hexyl acetate: take the above fermentation broth and centrifuge at 12000 rpm for 10 minutes, discard the precipitate, extract the supernatant of the fermentation broth with ethyl acetate to obtain ethyl acetate extract, remove part of the solvent by rotary evaporation, and use After water is removed by sodium sulfate, it is filtered with a 0.22 μm membrane, and qualitatively detected by GC-MS. Adopt DB-5 capillary column (30m×0.2...

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Abstract

The invention relates to Bacillus rhizosphaerae and a method using Bacillus rhizosphaerae and tobacco industry waste to produce hexyl acetate and belongs to the technical field of biology. Specifically, the Bacillus rhizosphaerae is obtained from tobacco in the medium term of mellowing through oligotrophic culture medium separation and is preserved in China General Microbiological Culture Collection Center on July 18th, 2017, and the preservation number of the Bacillus rhizosphaerae is CGMCC No. 14440. The Bacillus rhizosphaerae has the advantages that the hexyl acetate can be synthesized by using the Bacillus rhizosphaerae and the tobacco industrial waste as the raw materials, and the synthesized hexyl acetate is rich in fruit fragrance and widely applicable to fields such as perfume synthesizing, cosmetics and feed additives and is the important raw material and intermediate of pharmaceutical industry; the fermentation culture medium used in the synthesizing process is low cost, the large amount of waste generated in the tobacco industry can be treated, a positive effect on environment protection and reasonable resource utilization is achieved, and the Bacillus rhizosphaerae is promising in application prospect.

Description

technical field [0001] The invention belongs to the field of biological technology, and specifically relates to a rhizobacterium and its application, in particular to a strain of rhizobacteria obtained through screening and a method for fermenting and producing hexyl acetate by using the strain. Background technique [0002] Hexyl acetate, also known as n-hexyl acetate, is a colorless oily liquid with a melting point of -80°C and a boiling point of 168-171.5°C. It is miscible with solvents such as ethanol, ether, and benzene, but insoluble in water. [0003] Hexyl acetate has a pleasant fruity aroma and pear-like sweet and sour taste. Hexyl acetate has been stipulated as an allowed food flavoring substance in the national standard. It is mainly used in edible fruit flavors for the preparation of apples, pears and other fruits Type fragrance. In addition, it can also be used in organic synthesis, and can also be used as a solvent for cellulose esters and resins. [0004] At...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P7/62C12R1/07
CPCC12P7/62C12N1/205C12R2001/07
Inventor 吴丽君白晓莉王毅高锐段如敏朱杰王颖琦杨德中刘晶
Owner CHINA TOBACCO YUNNAN IND
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