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Method for producing lipopeptide antibiotics by optimizing bacillus subtilis with response surface method

A technology for Bacillus subtilis and lipopeptide antibiotics, which is applied in the field of optimizing the production of lipopeptide antibiotics by Bacillus subtilis by response surface methodology, can solve problems such as unclear optimal fermentation conditions, and achieve the effects of improving production efficiency and preventing and controlling diseases. Improve the ability to control plant fungal diseases, the effect of improving the ability

Pending Publication Date: 2020-06-19
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The optimal fermentation conditions for the synthesis of lipopeptide antibiotics by Bacillus subtilis are still unclear

Method used

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  • Method for producing lipopeptide antibiotics by optimizing bacillus subtilis with response surface method
  • Method for producing lipopeptide antibiotics by optimizing bacillus subtilis with response surface method
  • Method for producing lipopeptide antibiotics by optimizing bacillus subtilis with response surface method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Isolation and identification of antibacterial substances in the fermentation supernatant of Bacillus subtilis Z-5

[0045] Inoculate the Bacillus subtilis Z-5 strain stored at -80°C into the NA slant medium, and culture at a constant temperature of 37°C overnight. Inoculum size Inoculate the fermentation broth into the basal medium (glucose 20.0g, peptone 20.0g, NaH 2 PO 4 2H 2 O 2.0g, Na 2 HPO 4 2H 2 O 4.0g, MgSO 4 ·5H 2 O 0.5g, H 2 O 1L, pH 7.2-7.4), shake culture at 37°C for 48h, centrifuge at 8000r / min for 10min to recover the supernatant, filter through a 0.22μm microporous membrane, and recover the filtrate as the sterile supernatant.

[0046] In order to determine the structure of antibacterial substances synthesized by strain Z-5, it was isolated and identified. Take 50 mL of the fermentation supernatant, adjust the pH to 2.0 with 6 mol / L hydrochloric acid solution, place in a refrigerator at 4°C overnight, centrifuge at 8000 r / min for 20 min, discard t...

Embodiment 2

[0050] Determination of carbon source, nitrogen source and inorganic salt of medium

[0051] Corn flour and mannitol had the best effect on producing lipopeptide antibiotics, and the width of the antibacterial zone was 1.12cm. Considering the economic benefits of later industrial production, corn flour was selected as the best carbon source for follow-up experiments. When soybean cake powder, peptone, tryptone and yeast extract were used as nitrogen source for culture, there was no significant difference in the width of the inhibition zone of the fermentation supernatant. Considering economy and source of raw materials, soybean meal was finally selected as the best nitrogen source. The best inorganic salt is FeSO 4 ·7H 2 O. The best carbon source, nitrogen source, and inorganic salt for cultivating the Z-5 strain were corn flour, soybean cake flour, and FeSO by single factor experiments. 4 ·7H 2 O.

[0052] For corn flour, soybean meal and FeSO 4 Carry out the steepest...

Embodiment 3

[0056] Optimizing Box-Behnken Test of Medium Components

[0057] Design-Expert 8.0.6 software was used to design the Box-Behnken experiment. According to the design, there were 17 groups of experiments, of which 5 groups were center-point repeated experiments (Table 2). According to the design results, the medium was prepared and fermented, and the width of the inhibition zone of the fermentation supernatant against the pathogen of wheat take-all was detected. The results are shown in Table 2.

[0058] Table 2 Media composition Box-Behnken test design and results

[0059]

[0060]

[0061] The regression equation obtained by software analysis of the width of the antibacterial zone is:

[0062] Y=–0.29741+0.40575A+0.14825B+49.3C+0.07875AB–1.75AC–1.75BC–0.058375A 2 –0.12338B 2 –521.25C 2 (R 2 = 0.9946; R 2 -adj=0.9878)

[0063] Where Y is the predicted value of the width of the inhibition zone (cm), A, B and C are corn flour, soybean cake flour and FeSO4·7H 2 O.

...

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Abstract

The invention discloses a method for producing lipopeptide antibiotics by optimizing bacillus subtilis with a response surface method, and belongs to the field of microbial fermentation. The used bacillus subtilis is Z-5 strain; the strain is collected in China General Microbiological Culture Collection Center on 18 March, 2014, and the collection number is CGMCC NO.8925. In allusion to the strain, the response surface method is utilized to determine optimal fermentation conditions of the strain that 3.85% of corn flour, 1.57% of soybean cake powder and 0.03% of FeSO4.7H2O are added; the initial pH is 7.0; the inoculation amount is 8.5%; the rotating speed is 190 r / min; culture is performed for 33h at 37 DEG C, and meanwhile, 0.02% of NaH2PO4.2H2O and 0.04% of Na2HPO4.2H2O can also be added into a culture medium. The inhibition zone width of a supernate fermented under such conditions is larger, and thus, a foundation is laid for industrial production of the lipopeptide antibiotics.

Description

technical field [0001] The invention belongs to the field of microbial fermentation, and in particular relates to a method for optimizing the production of lipopeptide antibiotics by Bacillus subtilis by a response surface method. Background technique [0002] Plant diseases have always been one of the important restrictive factors for high-quality and high-yield crops. Among plant diseases, 70% to 80% of diseases are caused by pathogenic fungal infection. Plant fungal diseases not only directly cause the decline of crop yield and quality, but also some pathogenic fungi can secrete and produce a variety of toxins and metabolites harmful to humans and animals during the process of infecting crops, posing a great threat to the safety of agricultural products. Biological control is green and environmentally friendly, and pathogenic bacteria are not easy to develop drug resistance. The production process of biocontrol agents is relatively simple and can promote crop growth. A...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P21/00C12R1/125
CPCC07K14/32C12N1/20
Inventor 张冬冬陈晓萌张学超朱宝成
Owner HEBEI AGRICULTURAL UNIV.
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