A kind of aureobasidin A high-yield bacterial strain and its application

A technology of strains and microbial strains, applied in the field of high-yielding AureobasidinA strains, can solve the problems of increasing the yield of AureobasidinA, which has not been seen, and achieve the effects of reducing large-scale production costs, strong antifungal activity, and improving production capacity

Active Publication Date: 2022-06-21
ZHEJIANG HUIDA BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 1991, Kazutoh Takesako’s team first isolated the Aureobasidium pullulans strain from the leaves of Tsushima Island. After fermentation, the AureobasidinA production was about 140 μg / ml. Since then, there has been no report on the increase in AureobasidinA production.

Method used

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  • A kind of aureobasidin A high-yield bacterial strain and its application
  • A kind of aureobasidin A high-yield bacterial strain and its application
  • A kind of aureobasidin A high-yield bacterial strain and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Acquisition of the original producing bacteria of AureobasidinA

[0051] Soil samples were taken from the lower layer of decayed leaves in Moganshan Scenic Area, Hangzhou City, Zhejiang Province, with a sampling amount of 5g, which was diluted with sterile water and then evenly spread on PDA supplemented with 50μg / ml ampicillin sodium and 50μg / ml streptomycin sulfate. On the medium, the cells were cultured upside down in a constant temperature and humidity chamber at 25°C and 60% relative humidity for 3 days. Take the plate with independent single colony distribution, and use sterile toothpicks to inoculate the creamy colonies on fresh PDA medium supplemented with 80 μg / ml ampicillin sodium and 80 μg / ml streptomycin sulfate, at 25 ° C, 60% The cells were cultured upside down in a constant temperature and humidity chamber with relative humidity for 9 days and observed every day. The colonies that were creamy white in the early stage but turned black in the la...

Embodiment 2

[0056] Example 2: Confirmation of AureobasidinA-producing ability of A. pullulans HDCC101-R13 strain

[0057] The isolated and purified strain of A. pullulans HDCC101-R13 was inoculated into PDA solid medium, and cultured upside down in a constant temperature and humidity chamber at 25°C and 60% relative humidity for 7 days. An appropriate amount of bacterial moss was taken with an inoculation loop, inoculated into 100 ml of sterile liquid seed medium, and cultured on a shaker at 25°C and 220 rpm for 48 hours. After microscopic examination, 0.4ml of seed liquid was inoculated into a 250ml conical flask containing 40ml of fermentation medium, and 5 bottles of fermentation medium were inoculated, and cultured on a shaker at 25°C and 220rpm for 8 days. After the fermentation culture, take 4 ml of the fermentation broth, mix it with 1 volume of absolute ethanol, soak it in ultrasonic for 20 minutes, and then mix it again and centrifuge it at 3000 rpm for 15 minutes. The supernatan...

Embodiment 3

[0058] Example 3: Preparation and verification of AureobasidinA high-yielding strains

[0059] 1) Take HDCC101-R13 as the original starting strain, inoculate it into PDA solid medium, and cultivate it upside down in a constant temperature and humidity chamber at 25°C and 60% relative humidity for 5 days to obtain fresh provenance.

[0060] 2) Take 1 dish of the grown solid culture bacterial moss, scrape off the bacterial moss with a sterile steel stick, transfer it to a triangular flask with glass beads and 15ml sterile saline, wrap it and place it on a shaker to shake and break up 30min to obtain a well-dispersed bacterial suspension.

[0061] 3) Take the prepared bacterial suspension and evenly coat it on the PDA solid medium with specific resistance reagent after mutagenesis treatment, and invert the culture in a constant temperature and humidity chamber at 25°C and 60% relative humidity for 4 days, then They were seeded on fresh PDA solid medium supplemented with specific...

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Abstract

The invention belongs to the field of microbial pharmacy, and discloses an Aureobasidin A high-yield bacterial strain and application thereof. The strain is Aureobasidium pullulans, which was obtained from the original strains isolated from the soil samples of Moganshan Scenic Area in Hangzhou, Zhejiang through compound mutagenesis screening, and has been preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee. The number is: CGMCC NO.20887. The fermentation level of Aureobasidin A of the strain can reach more than 990 μg / ml, and can be used as a strain for industrial production of Aureobasidin A, greatly reducing the cost of large-scale production of Aureobasidin A. In addition, the fermented liquid, bacterial suspension, clarified liquid of the fermented liquid and its concentrate of the strain all have strong antifungal activity and can be used for the preparation of antifungal agents.

Description

technical field [0001] The invention relates to the technical field of microbial pharmacy, in particular to a high-yielding strain of Aureobasidin A and an application thereof. Background technique [0002] AureobasidinA is a cyclic lipopeptide antibiotic composed of 9 amino acid molecules, which was first isolated from the culture medium of black yeast Aureobasidiumpullulans by the Japanese team of Kazutoh Takesako in 1991. Subsequent studies confirmed that AureobasidinA has very strong antifungal ability, and it can be toxic to yeast at a low concentration of 0.1-0.5 μg / ml. Fungal species susceptible to it include: Saccharomyces cerevisiae, Schizosaccharomycespombe, Candida glabrata, Aspergillus nidulans, and Aspergillus niger. The mechanism of action of AureobasidinA is to inhibit the activity of inositol phosphorylceramide (IPC) synthase, which is dependent on fungal growth, and interfere with sphingolipid synthesis, thereby further killing the strain. However, Aureoba...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/14C12P21/02C07K7/06C12R1/645
CPCC12N1/14C12P21/02C07K7/06
Inventor 彭湘屏朱进伟孙琼张敏石磊汪超高祥陈世敏
Owner ZHEJIANG HUIDA BIOTECHNOLOGY CO LTD
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