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Method for preparing biocontrol bacillus secondary metabolite surfactins

A technology of secondary metabolites and surfactant, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of long time, high cost, low yield of secondary metabolites, etc., and achieve preventive effect The effect of improving and increasing production

Pending Publication Date: 2019-12-31
天津市农业科学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the preparation method of surfactin mainly adopts the method of liquid fermentation, which has the disadvantages of long time, high cost, low yield of secondary metabolites, and insignificant effect in the practical application of live bacteria. Antibacterial colonization and biocontrol effects are of great significance

Method used

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  • Method for preparing biocontrol bacillus secondary metabolite surfactins
  • Method for preparing biocontrol bacillus secondary metabolite surfactins
  • Method for preparing biocontrol bacillus secondary metabolite surfactins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Efficient preparation of surfactins, a secondary metabolite of Bacillus veleisi TB1501

[0022] (1) Plate induction: set up two treatments, LB plate induction and LBS plate induction

[0023] Preparation of TB1501 fermentation broth: Inoculate a ring of Bacillus TB1501 into a test tube containing 5 ml of liquid LB medium (NaCl 5g / L, tryptone 10g / L, yeast extract powder 5g / L), 37°C, 150rpm, Shake culture overnight to obtain TB1501 fermentation broth.

[0024] LB plate induction: Dilute TB1501 fermentation broth 1000 times, pipette 1.5 μl, and vertically spot on the center of LB plate (NaCl 5g / L, tryptone 10g / L, yeast extract powder 5g / L, agar powder 15g / L), 37℃ , overnight cultured upside down.

[0025] LBS plate induction: Dilute TB1501 fermentation broth 1000 times, pipette 1.5 μl, and vertically spot on LBS plate (NaCl 5g / L, tryptone 10g / L, yeast extract powder 5g / L, sucrose 5g / L, agar powder 15g / L ) center, 37 ℃, overnight culture.

[0026] (2) Solvent extraction...

Embodiment 2

[0034] Efficient preparation of surfactins secondary metabolites of Bacillus subtilis NCIB 3610 and TB1340 for biocontrol

[0035] (1) Plate induction: set up two treatments, LB plate induction and LBS plate induction

[0036] Preparation of fermentation broth of strain 3610: Inoculate a ring of Bacillus 3610 and TB1340 into test tubes containing 5 ml of liquid LB medium (NaCl 5g / L, tryptone 10g / L, yeast extract powder 5g / L) respectively, 37 °C, 150rpm, overnight shake culture to obtain 3610, TB1340 fermentation broth.

[0037] LB plate induction: Dilute 3610 and TB1340 fermentation broth 1000 times, pipette 1.5 μl, and vertically spot on the center of LB plate (NaCl 5g / L, tryptone 10g / L, yeast extract powder 5g / L, agar powder 15g / L) , 37°C, overnight cultured upside down.

[0038] LBS plate induction: Dilute 3610 and TB1340 fermentation broth 1000 times, pipette 1.5 μl, and vertically spot on LBS plate (NaCl 5g / L, tryptone 10g / L, yeast extract powder 5g / L, sucrose 5g / L, aga...

Embodiment 3

[0048] Comparative Test

[0049] Preparation of conventional surfactin:

[0050] (1) Liquid fermentation: LB liquid fermentation

[0051] Preparation of TB1501 seed liquid: Inoculate a ring of Bacillus TB1501 into a test tube containing 5 ml of liquid LB medium (NaCl 5g / L, tryptone 10g / L, yeast extract powder 5g / L), 37°C, 150rpm, Shake culture overnight to obtain TB1501 seed solution.

[0052] Expansion culture: Inoculate 1 / 1000 of TB1501 seed solution into 100ml liquid LB ((NaCl 5g / L, tryptone 10g / L, yeast extract powder 5g / L) medium, 37℃, 150rpm, shaking culture for 48h The TB1501 fermentation broth containing the secondary metabolite surfactin was obtained.

[0053] (2) Solvent extraction:

[0054] Centrifuge the TB1501 fermentation broth at 12000rpm for 20min, discard the precipitate, keep the supernatant, adjust the pH of the supernatant to 2.0 with 6mol / L HCl, precipitate overnight at 4°C, centrifuge at 12000rpm for 20min, discard the supernatant and keep the precipi...

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Abstract

The invention discloses a method for preparing biocontrol bacillus secondary metabolite surfactins. The method comprises the steps of plate induction, LBS plate induction, solvent extraction, HPLC detection and the like. The biocontrol bacillus strain adopted by the method is TB1501 with a preservation number of CGMCC No.16069. According to the method, the yield of the secondary metabolite surfactins induced by sucrose is increased, and the control effect of the biocontrol bacteria is remarkably improved. Particularly, a botrytis cinerea bioassay result shows that after the sucrose is added, and the control effect of the biocontrol bacteria on the botrytis cinerea is remarkably improved. In combination with the phenomenon that the surfactins produced by the biocontrol bacteria after sucrose induction are increased, the invention speculates that the synergistic effect of the sucrose-induced biocontrol bacteria on the botrytis cinerea depends on the increase of the yield of the surfactins.

Description

technical field [0001] The invention belongs to the technical field of metabolic regulation and control of secondary metabolites of Bacillus veleisi, and relates to a method for preparing surfactins (surfactins), a secondary metabolite of Bacillus veleisi TB1501. Background technique [0002] Bacillus has strong stress resistance, fast reproduction, and strong colonization ability in the plant rhizosphere. It is an important source of microbial fungicides. At present, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens and Bacillus cereus It is the main source of microbial fungicides in my country. Its biocontrol mechanism is mainly manifested in antagonism, nutrient and ecological site competition, induction of plant disease resistance and so on. Existing studies have shown that active substances such as lipopeptide antibiotics and antibacterial proteins produced by Bacillus are the key factors for their inhibition of plant pathogenic fungi and their bioco...

Claims

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Application Information

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IPC IPC(8): C12P21/00A01P3/00C12R1/07
CPCC12P21/00
Inventor 田涛孙冰冰石皓文
Owner 天津市农业科学院
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