Method for preparing questin by utilizing ocean aspergillus flavipes HN4-13 bacterial strain and application of questin

A technology of Aspergillus flavusus and emodin, which is applied in the field of preparation of emodin-8-methyl ether by using Aspergillus flavusus HN4-13 strain and its application

Active Publication Date: 2015-09-30
JIANGSU MARINE RESOURCES DEV RES INST LIAN YUNGANG
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are few reports on the active substances of marine fungi against aquatic pathogens

Method used

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  • Method for preparing questin by utilizing ocean aspergillus flavipes HN4-13 bacterial strain and application of questin
  • Method for preparing questin by utilizing ocean aspergillus flavipes HN4-13 bacterial strain and application of questin
  • Method for preparing questin by utilizing ocean aspergillus flavipes HN4-13 bacterial strain and application of questin

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0024] Example 1 , a method for isolating and purifying antibacterial active substances from the fermentation product of Aspergillus flavusus HN4-13 strain

[0025] (1) Preparation of fermentation broth of HN4-13 strain: Ferment HN4-13 strain in a 500 mL triangular flask, with a volume of 200 mL per bottle, and a total of 100 bottles were fermented; the medium composition was: glucose 3%, peptone 0.3%, KCl 0.25%, NaH 2 PO 4 0.1%, NaCl 3%, distilled water (adjust the pH to 10 with NaOH); the fermentation conditions are: the bacterial age of the seed liquid is 24 h, the inoculum size is 4.5%, the temperature is 32°C, the shaker speed is 160 rpm / min, and the fermentation time is On the 8th day, the fermentation product of the HN4-13 bacterial strain was obtained; the fermentation product was filtered through 8 layers of gauze, and the fermentation broth was obtained after discarding the cells;

[0026] (2) Preparation of the antibacterial active extract: the fermentation bro...

Embodiment 2

[0032] Example 2 , the structural identification of compound HY2

[0033] Mass spectrometry (ESI-MS) and NMR methods ( 1 H-NMR, 13 C-NMR) to identify the structure of compound HY2. Compound HY2 is orange-red crystal (methanol). Cationic ESI-MS in m / z : 285.1 gives [M + H] + ( image 3 ), suggesting that the molecular weight of the compound is 284, combined with 1 H spectrum, 13 C spectrum suggests its molecular formula C 16 h 12 o 5 . Ultraviolet spectrum: 221 nm, 251 nm, 289 nm, 444 nm, showing that it is anthraquinones. 1 H-NMR (600 MHz, DMSO- d 6 ) δ 13.41 (br s, 1H, OH-1), 7.41 (br s, 1H, H-4), 7.16 (br s, 1H, H-2), 7.11 (br s, 1H, H-5), 6.78 ( br s, 1H, H-7), 3.88 (s, 3H, 8-OCH 3 ), 2.39 (s, 3H, H-11) ( Figure 4 ). 13 C-NMR (150 MHz, DMSO- d 6 ) δ 186.4 (C, C-9), 183.1 (C, C-10), 170.8 (C, C-6), 164.1 (C, C-8), 162.2 (C, C-1), 146.8 (C, C -3), 137.2 (C, C-10a), 132.6 (C, C-4a), 124.6 (CH, C-2), 119.4 (CH, C-4), 114.9 (C, C-9a), 112.0 (C, C-8...

Embodiment 3

[0034] Example 3 , Inhibitory activity of emodin-8-methyl ether against four aquatic pathogenic Vibrio species

[0035] Preparation of indicator bacteria solution: Vibrio harveyi ( Vibrio Harvey ), Vibrio eelium ( Vibrio anguillarum ), Vibrio parahaemolyticus ( Vibrio parahaemolyticus ) and Vibrio cholerae ( Vibrio cholerae ) respectively made spore suspension (10 7 individual / mL). Inoculate the spore suspension into a sterilized 500 mL triangular flask containing 200 mL of beef extract peptone medium according to the inoculum amount of 5%, and shake it in a constant temperature shaking incubator at a temperature of 37°C and a speed of 160 r / min. Cultivate for 1 day to obtain the indicator bacteria solution.

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Abstract

The invention discloses a method for preparing questin by utilizing an ocean aspergillus flavipes HN4-13 bacterial strain. The method comprises the following steps: conducting separation and purification on antibacterial active substances generated through fermentation of the aspergillus flavipes HN4-13 bacterial strain according to silica-gel column chromatography, Sephadex LH-20 column chromatography, preparation of high performance liquid chromatography (PHPLC) and the like by taking vibrio harveyi as an indicator bacterium, so as to obtain a vibrio harveyi-preventing active compound HY2; indentifying the structure of the active compound HY2 according to spectra data such as ESI-MS, 1H-NMR and 13C-NMR, so as to determine that the active compound HY2 is questin. The questin is obtained through separation of a metabolic product of aspergillus flavipes HN4-13; experiments show that the questin can perform a certain inhibiting function of the pathogenic bacteria, such as vibrio harveyi, vibrio anguillarum, vibrio parahaemolyticus and vibrio cholerae, of aquatic products, and can be used for preparation of medicine preventing pathogenic vibrio of the aquatic products.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a method for extracting active ingredients from marine fungal fermentation products, in particular to a method for extracting emodin-8-methyl ether (questin) from fermentation products of Aspergillus flavusus HN4-13 strain , the present invention also relates to the application of the questin prepared by the aforementioned method in the preparation of antibacterial drugs. Background technique [0002] In the past two decades, my country's aquatic product breeding industry has achieved rapid development, and the output of aquatic products ranks first in the world, but various animal diseases continue to appear. Vibriosis (Vibriosis) caused by Vibrio bacteria Vibrio harveyi, Vibrio eel, Vibrio parahaemolyticus, Vibrio cholerae, etc. is the main pathogenic bacteria that cause diseases of aquatic animals such as farmed fish, shrimp, crab and shellfish . At present, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/66C07C50/34C07C46/10A61K31/122A61P31/04C12R1/66
CPCA61K31/122C07C46/10C12P7/66C07C50/34Y02A50/30
Inventor 郭雷王聪许福泉
Owner JIANGSU MARINE RESOURCES DEV RES INST LIAN YUNGANG
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