Method for producing bacteriostatic active substances by bacillus amyloliquefaciens

A technology of amylolytic spores and antibacterial activity, applied in the field of microorganisms, can solve the problems of complex components of antibacterial substances, low content, and insufficient research on the mechanism of antibacterial substances

Active Publication Date: 2020-02-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Relevant studies have shown that Bacillus amyloliquefaciens can produce antibacterial active substances, but since this type of product depends on the control of fermentation conditions, and the composition of

Method used

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  • Method for producing bacteriostatic active substances by bacillus amyloliquefaciens
  • Method for producing bacteriostatic active substances by bacillus amyloliquefaciens
  • Method for producing bacteriostatic active substances by bacillus amyloliquefaciens

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Embodiment 1

[0042] Bacillus amyloliquefaciens was cultivated with fermentation medium, and the supernatant was acid-precipitated and organic solvent extracted for DEAE anion exchange chromatography, column: DEAE-Sepharose-Fast Flow anion chromatography column, mobile phase A: 20 mmol / L, pH 8 Tris-HCl buffer, mobile phase B: 1 mol / L NH 4 HCO 3 Tris-HCl buffer solution, injection volume: 5 mL, detection wavelength: 220 nm. After elution with 3 column volumes of buffer A, gradient elution was performed with mobile phase B at concentrations of 0%, 60%, 80%, and 100%. Using Staphylococcus aureus as the indicator bacteria, the modified Oxford cup method was used to measure the antibacterial activity of the separated peaks, and the results were as follows: figure 1 The results showed that the elution peak B detected the bacteriostatic zone, the measured bacteriostatic diameter was (19.2±0.3) mm, and the calculated bacteriostatic rate was 61.25%.

Embodiment 2

[0044] RP-HPLC separation was performed on the isolated peak B. Chromatographic conditions: Waters XBridge Prep C18 column (10×250mm, 5μm), mobile phase A: acetonitrile containing 0.1% TFA, mobile phase B: ultrapure water containing 0.1% TFA, flow rate: 0.5 mL / min, detection wavelength : 220 nm, injection volume: 0.5 mL. Equilibrate 3 times column volume with mobile phase B, select linear elution mode,. The result is as figure 2 , the elution peak D had the highest antibacterial activity, the bacteriostatic diameter was (18.0±0.2) mm, and the bacteriostatic rate was 57.62%, the elution peak F had the second most antibacterial activity, the bacteriostatic diameter was (16.2±0.3) mm, Its antibacterial rate is 48.35%. The inhibition diameter of the isolated and purified active peak is as follows: image 3 shown.

Embodiment 3

[0046] Collect the separation peaks with high antibacterial activity, choose WATERS ACQUITY UPLC chromatograph, chromatography column: BEH C18 (2.1×50 mm 1.7 μm), mobile phase A: acetonitrile containing 0.1% TFA, mobile phase B: containing 0.1% TFA Milli-Q water, flow rate 0.3 mL / min, injection volume: 5 μL, detection wavelength 220 nm, column temperature: 45 ℃, gradient elution: 0~3 min, 5% phase A 95% phase B; 3 ~15 min, 5%~95% phase A, 95%~5% phase B; 15~17 min, 95%~100% phase A, 5%~0% phase B; 17.1min, 5% phase A, 95 %B phase. The UPLC separation peaks were injected into the WATERS MALDI SYNAPT Q-TOF MS, and the positive ion mode electrospray was used to obtain the mass spectrum of the signal peaks. Such as Figure 4-7 .

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Abstract

The invention relates to a method for producing bacteriostatic active substances by bacillus amyloliquefaciens and belongs to the technical field of microbes. The method comprises the steps of culturing the bacillus amyloliquefaciens so as to obtain fermentation supernatant, and subjecting the supernatant to separating and purifying, thereby preparing a novel hexapeptide. According to the method provided by the invention, the obtained fermentation supernatant can be directly applied to bacteriostasis, three kinds of bacteriostatic substances can be obtained after further separating and purifying the fermentation supernatant and comprises the novel hexapeptide which similarly has a bacteriostatic action; and when the three kinds of bacteriostatic substances jointly act on indicator bacterium cells, the extent of damage to the cells is greater. Discovered by results of acting on indicator bacteria by pairwise combinations of the three kinds of bacteriostatic substances, lactic acid and Surfactin have a synergistic action, the lactic acid and the hexapeptide have a synergistic action, and the Surfactin and the hexapeptide have an additive action. A theoretical support is provided forcontinuous development of novel antibacterial peptides, and a new way of think is provided for efficient use of natural bacteriostatic substances.

Description

technical field [0001] The invention relates to a method for producing antibacterial active substances by Bacillus amyloliquefaciens, belonging to the technical field of microorganisms. Background technique [0002] Bacillus amyloliquefaciens belongs to Gram-positive bacteria, facultative anaerobic type, can produce lactic acid, can produce antibacterial active substances, and inhibit the growth of some spoilage and pathogenic microorganisms. Because it is non-toxic and harmless to the human body, it has better inhibitory effect on intestinal pathogenic bacteria in the intestinal tract of animals, and can be applied in the field of medical care. [0003] Relevant studies have shown that Bacillus amyloliquefaciens can produce antibacterial active substances, but because this type of product depends on the control of fermentation conditions, and the composition of antibacterial substances in fermentation products is complex, and its content is low, the mechanism of action of a...

Claims

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Application Information

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IPC IPC(8): C07K7/06C12P21/02C07K1/36C07K1/30C07K1/18C07K1/20C12P7/56C12Q1/18A61K38/08A61K31/19A61P31/04C12R1/07C12R1/445
CPCA61K31/19A61K38/08A61P31/04C07K7/06C12P7/56C12P21/02C12Q1/18A61K2300/00
Inventor 周楠迪田亚平姚佳明
Owner JIANGNAN UNIV
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