Antibiotic active substance of Aspergillus flavus biocontrol bacterium, and its separating and purifying method

An antibacterial active substance, separation and purification technology, applied in the field of microbiology

Active Publication Date: 2014-12-03
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, since these antibacterial active substances have been used for many years, bacteria have generally developed resistance to these antibacterial active substances, so it is urgent to find new, more powerful new antibacterial active substances

Method used

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  • Antibiotic active substance of Aspergillus flavus biocontrol bacterium, and its separating and purifying method
  • Antibiotic active substance of Aspergillus flavus biocontrol bacterium, and its separating and purifying method
  • Antibiotic active substance of Aspergillus flavus biocontrol bacterium, and its separating and purifying method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: the culture condition screening of Bacillus atrophaeus antibacterial active substance

[0025] Experimental materials: Bacillus atrophaeus is used as the bacterial species; any commercially available medium can be used as the nutrient agar medium, and the composition of this medium used in the present invention is: peptone 10g, beef extract 3g, sodium chloride 5g , agar 17g, distilled water 1000 mL, pH7.2, sterilized at 121°C for 20min.

[0026] The source of Aspergillus flavus used in the test: Inoculate the Aspergillus flavus species on the potato dextrose agar slant medium (PDA), culture at a constant temperature of 30°C for 48 hours, add sterile water (containing 0.1% Tween-80 and 0.1% agar) to it, The vortex mixer oscillated to distribute the spores evenly, and adjusted the concentration of the spore suspension to 1×105 CFU / mL, which was the Aspergillus flavus spore suspension.

[0027] In order to study the influence of different culture conditions ...

Embodiment 2

[0053] Embodiment 2: the determination of crude extraction conditions

[0054] Using the optimal fermentation conditions for the antibacterial active substances screened in the above experiments, Bacillus atrophaeus was shaken and cultured in the nutrient broth medium, and the fermentation liquid was centrifuged at 4°C and 8000r / min for 20min, and the supernatant was collected and passed through 0.45μm The rough extraction experiment of antibacterial active substances was carried out after the microfilter removed the bacteria.

[0055] Ammonium sulfate fractional precipitation: remove 100 mL of bacterial fermentation supernatant, slowly add ammonium sulfate solid powder 10.6g, 22.6g, 36.1g, 51.6g, 69.7g, so that the saturation of ammonium sulfate is 20%, 40% %, 60%, 80%, 100%, and then stand overnight at 4°C, centrifuge at 8000r / min for 20min at 4°C, and collect the precipitate. Dissolve the precipitate in 2 times the volume of pH7.5 0.05mol / L phosphate buffer, transfer it in...

Embodiment 3

[0060] Embodiment 3: Separation and purification of crude extract

[0061] On the basis of obtaining the crude extract, the present invention further separates and purifies the crude extract. In order to improve the efficiency, the crude extract is first concentrated, and the dialysis bag is cut into small sections of 10-20 cm. / v) Boil in NaHCO3 and 0.01mol / L pH8.0 EDTA for 10min, rinse with distilled water, then boil in 0.01mol / L pH8.0 EDTA for 10min, rinse thoroughly with distilled water after cooling. Tighten the dialysis bag at 1 cm from one end with a thread, pour the crude protein extract to be concentrated into the dialysis bag, reserve 1 / 3 to 1 / 2 of the space, tie the other end tightly with a thread, and put it into a beaker , Sprinkle PEG20000 on the dialysis bag, let it stand at 4°C until the protein in the dialysis bag reaches a certain concentration, then carry out Sephadex G-25 desalting and DEAE Sepharose Fast Flow ion exchange chromatography purification.

[0...

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Abstract

The invention discloses an antibiotic active substance. The antibiotic active substance is from Bacillus atrophaeus, and a preparation method of the antibiotic active substance comprises the following steps: inoculating Bacillus atrophaeus into a nutritional broth medium, carrying out shaking table culturing, centrifuging the obtained cultured broth, collecting the obtained supernatant, and filtering the supernatant by a filter in order to obtain a bacterium-removed fermentation supernatant containing the antibiotic active substance. The invention further discloses a culturing, separating and purifying method of the antibiotic active substance.

Description

technical field [0001] The invention relates to an antibacterial active substance, in particular to an antibacterial active substance derived from bacillus atrophaeus, and belongs to the field of microbiology. Background technique [0002] It is well known that microorganisms can produce antibacterial active substances with antibacterial, antifungal and antiviral effects. So far, penicillin produced by Penicillium bacteria, cephalosporin produced by Cephalosporin, streptomycin produced by Streptomyces, tetracycline and erythromycin have been widely used as antibacterial active substances. [0003] However, since these antibacterial active substances have been used for many years, bacteria have generally developed resistance to these antibacterial active substances, so it is urgent to find new and more powerful new antibacterial active substances. Contents of the invention [0004] Aiming at the needs of the prior art, the present invention provides a novel antibacterial a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00C07K1/36C07K1/30C07K1/18C12R1/07
Inventor 秦文刘丁张志清叶劲松李莹露
Owner SICHUAN AGRI UNIV
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