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Method for high density production of bacillus amyloliquefaciens by combination of liquid and semisolid fermentation technologies

A technology of semi-solid fermentation and amylolysis spores, applied in the direction of microorganism-based methods, biochemical equipment and methods, chemicals for biological control, etc., can solve the problems of low bacterial volume and spore rate, and achieve the production process Economical and practical, reducing the chance of pollution and occupying a small area

Active Publication Date: 2016-04-27
ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In view of this, the present invention provides a method for combining high-density production of Bacillus amyloliquefaciens by liquid and semi-solid fermentation techniques, which solves the problems of low bacterial mass and spore rate in the industrial production of Bacillus amyloliquefaciens

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  • Method for high density production of bacillus amyloliquefaciens by combination of liquid and semisolid fermentation technologies
  • Method for high density production of bacillus amyloliquefaciens by combination of liquid and semisolid fermentation technologies
  • Method for high density production of bacillus amyloliquefaciens by combination of liquid and semisolid fermentation technologies

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Experimental program
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Embodiment 1

[0033] The production method of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HC200 comprises the following steps:

[0034] 1. Bacteria culture

[0035] Bacillus amyloliquefaciens HC200 was inoculated on the LB medium plate, and cultured at 37°C for 32 hours until the bacteria covered the plate.

[0036] LB medium: tryptone 10g, yeast extract 5g, NaCl 10g, agar 17g, dilute to 1000ml with water, pH 7.0.

[0037] 2. Liquid seed culture

[0038] Fill 400ml liquid seed culture medium in the 1000ml Erlenmeyer flask, then inoculate the bacterial classification that step 1 cultivates, the inoculation amount is the culture dish flat plate bacterial classification of 1 / 6 of each Erlenmeyer flask inoculation (petri dish specification is 90mm), 37 ℃ , 180rpm cultured for 24h to obtain seed liquid, the bacterial concentration in the seed liquid was 1.2×10 10 cfu / mL.

[0039] Liquid seed medium: take 20g of sucrose, 10g of yeast powder, K 2 HPO 4 1.2g and MgCl 2 1g, dissol...

Embodiment 2

[0055] The production method of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HC200 comprises the following steps:

[0056] 1. Bacteria culture

[0057] Bacillus amyloliquefaciens HC200 was inoculated on the LB medium plate, and cultured at 37°C for 40 hours until the bacteria covered the plate.

[0058] LB medium: tryptone 10g, yeast extract 5g, NaCl 10g, agar 17g, dilute to 1000ml with water, pH 7.0.

[0059] 2. Liquid seed culture

[0060] Fill 400ml liquid seed culture medium in the 1000ml Erlenmeyer flask, then inoculate the bacterial classification that step 1 cultivates, the inoculation amount is the culture dish flat plate bacterial classification of 1 / 6 of each Erlenmeyer flask inoculation (petri dish specification is 90mm), 37 ℃ , 180rpm cultured for 24h to obtain seed liquid, the bacterial concentration in the seed liquid was 4.0×10 10 cfu / mL.

[0061] Liquid seed medium: take 20g of sucrose, 10g of yeast powder, K 2 HPO 4 1.2g and MgCl 2 1g, dissol...

Embodiment 3

[0077] The production method of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HC200 comprises the following steps:

[0078] 1. Bacteria culture

[0079] Bacillus amyloliquefaciens HC200 was inoculated on the LB medium plate, and cultured at 37°C for 48 hours until the bacteria covered the plate.

[0080] LB medium: tryptone 10g, yeast extract 5g, NaCl 10g, agar 17g, dilute to 1000ml with water, pH 7.0.

[0081] 2. Liquid seed culture

[0082] Fill 400ml liquid seed culture medium in the 1000ml Erlenmeyer flask, then inoculate the bacterial classification that step 1 cultivates, the inoculation amount is the culture dish flat plate bacterial classification of 1 / 6 of each Erlenmeyer flask inoculation (petri dish specification is 90mm), 37 ℃ , 180rpm culture for 24h, to obtain seed liquid, the bacterial concentration in the seed liquid is 5.0×10 10 cfu / mL.

[0083] Liquid seed medium: take 20g of sucrose, 10g of yeast powder, K 2 HPO 4 1.2g and MgCl 2 1g, dissolv...

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Abstract

The present invention discloses a method for high density production of bacillus amyloliquefaciens by combination of liquid and semisolid fermentation technologies, the method is as follows: liquid fermentation, to be more specific, a bacillus amyloliquefaciens seed liquid is inoculated into a liquid fermentation medium, the inoculation amount is 1-5%, and a liquid fermentation product is obtained by culturing for 20-26h under the conditions of temperature of 35-38 DEG C, the air inlet amount of 25-35L / min and the pressure of 0.02-0.04Mpa; and semi-solid fermentation, to be more specific, the liquid fermentation product is inoculated into a semisolid fermentation medium containing coco coir powder and cassava residue powder for even mixing, and a semi-solid fermentation product is obtained by culturing for 4-6 days under the conditions of temperature of 35-38 DEG C, the humidity of 50-70% and the inoculation amount of 18-22%; and by combination of the liquid and semisolid fermentation technologies and process parameter optimization, the bacteria content of obtained bacillus amyloliquefaciens final product is 3.21*10<12>cfu / g, and the rate of sporation is up to 93%.

Description

technical field [0001] The invention relates to the field of microbial fermentation, in particular to a method for high-density production of bacillus amyloliquefaciens by combining liquid and semi-solid fermentation techniques. Background technique [0002] Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is a bacterium with broad-spectrum antibacterial activity, which has a strong ability to produce secondary metabolites, and can inhibit a variety of plant pathogenic fungi such as Fusarium oxysporum. [0003] At present, the optimization of Bacillus amyloliquefaciens fermentation at home and abroad mainly focuses on the production of antimicrobial lipopeptides, α-amylase, protease and other enzymes, and less research has been done on the optimization of the fermentation process of Bacillus amyloliquefaciens; In the research, the target of most of the research is the Bacillus amyloliquefaciens liquid submerged fermentation technology, and the research on the Bacillus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A01P3/00C12R1/07
CPCC12N1/20
Inventor 黄俊生梁昌聪刘磊汪军张建华王伟伟杨腊英郭立佳王国芬丁兆建
Owner ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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