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Pretreatment process for preparing fractions of marine microorganism small molecule metabolites

A technology for marine microorganisms and metabolites, which is applied in the field of pretreatment process for the preparation of fractions of marine microbial small molecule metabolites, can solve the problems of easy-to-destroy active substances, failure to reach pretreatment, high salt content, etc., and achieve simple and efficient implementation, fast filtering effect

Inactive Publication Date: 2014-05-07
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Marine microbial fermentation broth is slightly different from terrestrial microbial fermentation broth. The salt content is higher, and small bacteria account for the vast majority. The pretreatment effect of polymer flocculation method, ethanol precipitation method, and enzymatic protein hydrolysis method is not good; to obtain activity For the extraction of small molecular metabolites, the heating and flocculation method is easy to destroy the active substances; the simple centrifugal filtration method has a low removal rate for soluble macromolecules that affect column adsorption, and cannot achieve the purpose of pretreatment

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Take 15 L of fermentation broth of a marine bacterium (Halomonas elongatus), and use a homogenizer to break up the cells at 750 bar. Add 30 ml of 10% aluminum sulfate solution to the fermented broth after wall-breaking treatment, and stir rapidly and then slowly at 40°C to flocculate the fermented broth. The fermented broth was filtered with a disc-type membrane filter with a molecular weight cut-off of 9000. The filtered fermentation broth was passed through a styrene-divinylbenzene copolymerized macroporous resin column to absorb and enrich small molecule metabolites. After the filtrate has passed through, first wash the column with 2.5 times the column bed volume of deionized water, and then use 20%, 40%, and 60% methanol aqueous solution to elute the chromatography column successively, and the elution volumes are respectively 2 times, 3 times and 2 times the column bed. The above eluate was collected and concentrated under reduced pressure using a vacuu...

Embodiment 2

[0024] Example 2: Take 50L of fermentation broth of a marine bacterium (Pseudocardia dabsonii) and use a homogenizer to break up the cells at 700 bar. Add 25ml of 20% aluminum sulfate solution to the fermented broth after the wall-breaking treatment, and stir rapidly and then slowly at 40°C to flocculate the fermented broth. The fermented broth was filtered with a disc-type membrane filter with a molecular weight cut-off of 9000. The filtered fermentation broth was passed through styrene-divinylbenzene copolymerized macroporous resin and polydivinylbenzene macroporous adsorption resin series chromatography column to adsorb and enrich small molecule metabolites. After the filtrate has passed through, first wash the column with 2 times the volume of the column bed in deionized water, and then use 20%, 40%, and 80% ethanol aqueous solution to elute the chromatography column successively, and the elution volumes are respectively 3 times, 2 times and 2 times the column bed. The a...

Embodiment 3

[0025] Example 3: Take 100 L of fermentation broth of a strain of marine bacteria (Acinetobacter ruckeri), and use a homogenizer to break up the cells at 800 bar. Add 100 ml of 15% aluminum sulfate solution to the fermented broth after wall-breaking treatment, and stir rapidly and then slowly at 35°C to flocculate the fermented broth. The fermentation broth was filtered with a disc-type membrane filter with a molecular weight cut-off of 10,000. The filtered fermentation broth is passed through a styrene-based macroporous adsorption resin and an acrylic-based macroporous adsorption resin double-layer chromatography column to adsorb and enrich small molecule metabolites in series. After the filtrate has passed through, first wash the column with deionized water of 2 times the volume of the column bed, and then use a mixture of methanol and water to perform gradient elution on the chromatography column. The elution gradient is 10-95% methanol aqueous solution to The flow rate el...

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Abstract

The invention discloses a pretreatment process for preparing fractions of marine microorganism small molecule metabolites. The pretreatment process comprises the following steps: (1) directly performing cell disruption treatment on fermentation liquor by using a physical method; (2) performing solid-liquid separation on the fermentation liquor subjected to the disruption treatment by using a tubular bowl centrifuge, or adding the fermentation liquor subjected to the disruption treatment into an aluminum sulfate solution in proportion, stirring at a certain temperature, and flocculating the fermentation liquor; (3) filtering the fermentation liquor subjected to centrifugal solid-liquid separation or fermentation liquor subjected to aluminum sulfate flocculation by adopting a disc type membrane filter; (4) adsorbing and enriching the small molecule metabolites from the filtered fermentation liquor by using a macroporous resin chromatographic column; (5) allowing the filtrate to pass through the column by using purified water after the filtrate passes through the column; (6) eluting the small molecule metabolites adsorbed to a macroporous resin carrier by using a waterborne organic solvent. Rapid filtration and concentration of the marine microorganism fermentation liquor are realized, the small molecule metabolites are enriched and are basically not lost, and the small molecule metabolite product obtained through elution is concentrated and can be directly used for chromatographic separation and preparation.

Description

technical field [0001] The invention relates to a pretreatment process for the preparation of microbial metabolites, in particular to a pretreatment process for the preparation of fractions of marine microbial small molecule metabolites. Background technique [0002] The ocean and land have completely different ecological environments, such as high salinity, high pressure, anoxic, low temperature, oligotrophic, etc. Microorganisms from special habitats may produce metabolites that are completely different from terrestrial microorganisms. It is the discovery of novel chemical structures and biological activities. A variety of new marine biological resources. Large-scale fermentation of marine microorganisms, extraction and separation of small molecule metabolites, establishment of fraction libraries of small molecule compounds, and establishment of technical and material foundations for marine microbial metabolic component research, drug activity screening, and discovery ...

Claims

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Application Information

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IPC IPC(8): C07B63/00B01J20/281
Inventor 许晨许建中夏金梅廖云莉王伟毅林翌陈建明邵宗泽单大鹏
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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