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Method for producing active antimicrobial substance HSAF by optimizing lysobacter enzymogenes OH11 with response surface method

A technology of producing lysobacterium lysobacterium and response surface method, which is applied in the field of optimizing the production of active antibacterial substance HSAF by lysobacterium lysobacterium OH11 by response surface method, which can solve the problem of low yield, large gap in industrial production level, and prevention of commercial promotion and application, etc. problem, to achieve the effect of low cost and wide source

Inactive Publication Date: 2017-09-01
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although the genetic regulation mechanism of HSAF biosynthesis has been fully understood, its yield is still low (only 30-60mg / L), which is far from the level of industrial production, which prevents its further commercial promotion and application

Method used

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  • Method for producing active antimicrobial substance HSAF by optimizing lysobacter enzymogenes OH11 with response surface method
  • Method for producing active antimicrobial substance HSAF by optimizing lysobacter enzymogenes OH11 with response surface method
  • Method for producing active antimicrobial substance HSAF by optimizing lysobacter enzymogenes OH11 with response surface method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: Single factor investigation of HSAF fermentation medium

[0053] Take out the glycerin tube of L.enzymogenesOH11 strain in the ultra-low temperature refrigerator at -80°C, melt it on ice, pour it into a strain bottle containing 5mL of LB culture solution, cultivate it overnight at 200rpm at 28°C, and dip the bacterial solution with an inoculation loop to draw on a solid plate Lines were cultured statically in a 30°C incubator for 2 days. Stretch the single colony strain on the plate in 50mL LB culture medium, shake and culture at 200rpm at 28°C for 12h to OD 600 =1.5, that is, the seed liquid is obtained.

[0054] According to the inoculum amount of 1.0%, it was inserted into the fermentation medium, cultured at 28° C. and 150 rpm for 48 hours, and three parallel experiments were set up for each group of experiments. The single factor test is carried out on 5g / L yeast powder, 2.5g / L glucose and 0.5g / L NaCl as the basic medium, only need to replace the co...

Embodiment 2

[0058] Embodiment 2: Optimization of HSAF fermentation medium

[0059] According to the single factor test, select the highest value as the center point of BBD test design (that is, 0 level), and establish the factor level table. And taking the concentration of L.enzymogenes OH11 metabolite HSAF as the response value, use Design Expert software to optimize according to the BBD design principle, as shown in Table 1.

[0060] Table 1 BBD test design and results of HSAF fermentation medium components

[0061]

[0062]

[0063] According to the response surface test results in Table 1, multiple regression fitting was carried out to obtain the effect of HSAF output in the fermentation broth on soybean flour (X 1 ), glucose (X 2 ), CaCl 2 (X 3 ) Concentration quadratic multiple regression fitting equation: Y=-334.00+67.50X 1 +6.25X 2 -36.25X 3 -10.00X 1 x 2 -5.00X 1 x 3 +22.50X 2 x 3 -69.75X 1 2 -97.25X 2 2 -52.25X 3 2 , R 2 = 0.9863.

[0064] Through the ...

Embodiment 3

[0065] Example 3 PB test screens significant influencing factors affecting HSAF shake flask fermentation

[0066]According to the fermentation characteristics of L.enzymogenesOH 11 and the research basis of our laboratory, 6 culture factors (inoculum volume, liquid volume, initial pH, culture temperature, shaker speed, fermentation cycle) were selected as the factors for the PB test investigation, and each factor was respectively Take two levels of high (+1) and low (-1), and design 12 groups of experiments with the concentration of HSAF in the fermentation broth as the response value, as shown in Table 2.

[0067] Using Design Expert software to carry out regression analysis on the PB test results, the order of significance of the influence of each fermentation factor on the concentration of HSAF is: fermentation period (X 6 )>Cultivation temperature (X 4 )>Liquid volume (X 2 )>Initial pH(X 3 )>Inoculation amount (X 1 )>Shaker speed (X 5 ); wherein the fermentation cycle...

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Abstract

The invention discloses a method for producing active antimicrobial substance HSAF by optimizing lysobacter enzymogenes OH11 with response surface method. The method comprises: strain activation, seed liquid culture, fermentation culture, and extraction and detection of HSAF in fermentation broth. A fermentation medium comprises following raw materials according to a ratio: 8.00g / L of soy flour, 7.89g / L of glucose and 0.72g / L of CaCl2. The liquid containing amount, the culture temperature and the fermentation period are 22%, 26 DEG C and 58h respectively. Inert substances are added into HSAF extraction process to eliminate an emulsion problem, so that the fermentation liquid HSAF can be accurately quantitative, and ultimately the yield of HSAF can be up to 440.26mg / L, which is 15 times that produced under conventional conditions (10% TSB, 29.24mg / L). The method has the advantages of being extensive in raw materials, low in cost, capable of effectively reducing the production cost of HSAF, and providing important technical parameters for the future production of HSAF.

Description

technical field [0001] The invention belongs to the technical field of microbial fermentation engineering, and in particular relates to a method for optimizing the production of active antibacterial substance HSAF by the enzyme-producing Lysobacterium OH11 by a response surface method. Background technique [0002] At present, the prevention and control methods of crop diseases and insect pests in agricultural production mainly rely on chemical control, and the use of a large number of chemical pesticides will not only bring adverse effects on soil, water and the atmosphere, but will also inevitably lead to serious "3R" (residual Risidue) , Resurgence of harmful organisms, drug resistance of pathogens) and "three causes" (teratogenic, carcinogenic, mutagenic) crises. Biological pesticides refer to pesticides made from living organisms, biological metabolites, or biologically specific genes. At present, agricultural antibiotics (Jinggangmycin, abamectin, etc.), microbial pest...

Claims

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Application Information

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IPC IPC(8): C12P17/18C12R1/01
Inventor 汤宝刘凤权赵延存徐会永赵杨扬陈贤
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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