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Method for separating and purifying microcystin

A microcystin, separation and purification technology, applied in the field of analytical chemistry, can solve the problems of high price, achieve the effect of low maintenance cost, simple process, and easy popularization

Inactive Publication Date: 2011-10-12
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In general, microcystins can only be isolated and purified from cyanobacteria cultivated in the wild or indoors, which is expensive and currently mainly depends on imports
There are not many patents on the separation and purification of microcystins from wild toxin-producing cyanobacteria or indoor cultivation of toxin-producing cyanobacteria, which needs further research and development

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Step 1: Weigh 1.0g of cyanobacteria powder (Yunnan Delinhai Biotechnology Co., Ltd.) in a 50ml centrifuge tube, add 30ml of 5% acetic acid, ultrasonicate in an ultrasonic water bath (power 100w, frequency 40KHz) at room temperature for 30min, centrifuge at 10000rpm for 15min, pour out supernatant. Add 30ml of 5% acetic acid to the residue, repeat the above operation, and combine the supernatants twice.

[0014] Step 2: The SPE column (5g / 20ml, Beijing Kanglin Science and Technology Co., Ltd.) is equilibrated with 50ml methanol and 50ml5% acetic acid successively, then the supernatant in step 1 is passed through the SPE column, washed with 50ml30% methanol, washed with 75ml It was eluted with methanol containing 0.1% TFA (trifluoroacetic acid), and the eluate was concentrated to near dryness by rotary evaporation in a water bath at 35°C, and dissolved in 2.5ml of 50% methanol.

[0015] Step 3: Mobile phase storage tank (4L, mobile phase is composed of water, methanol, t...

Embodiment 2

[0019] Step 1: Microcystis FACHB905 (purchased from China Freshwater Algae Species Bank) culture solution (in BG-11 substratum under the conditions of temperature 28 ℃, light intensity 2000lx, light-to-dark ratio 12: 12 conditions for 20 days, biological 1.56mg / ml) 500ml, centrifuged at 15000rpm for 30min. Algae cells were collected, after repeated freezing and thawing in liquid nitrogen, 25ml of 5% acetic acid was added, ultrasonicated in an ultrasonic water bath (power 100w, frequency 40KHz) at room temperature for 30min, centrifuged at 10000rpm for 15min, and the supernatant was decanted. Add 25ml of 5% acetic acid to the residue, repeat the above operation, and combine the supernatants twice.

[0020] Step 2: The SPE column (5g / 20ml, Beijing Kanglin Science and Technology Co., Ltd.) is equilibrated with 50ml methanol and 50ml5% acetic acid successively, then the supernatant in step 1 is passed through the SPE column, washed with 50ml40% methanol, washed with 100ml The elu...

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Abstract

The present invention relates to a method for separating and purifying the microcystin, belonging to the analytical chemistry technology field. The method comprises following steps: extracting the blue algae producing the microcystin with the solvent in 1:20-80, purifying the extract through a SPE column, separating and purifying the microcystin by a column chromatography system composed of a constant flow pump, a C18 chromatography column and an automatic collector. The mobile phase is composed of 35-50% of water, 50-65% of methanol and 0.05-0.1% of acid. The purified microcystin-RR(MC-RR) or microcystin-LR(MC-LR) is analyzed by a HPLC. The method has simple art, low costs for operation and maintenance and easy popularization. The purity of the purified microcystin is more than 95%, thus satisfying requirement for carrying out correlational studies of blue algae.

Description

technical field [0001] The invention relates to a method for separating and purifying microcystin, belonging to the technical field of analytical chemistry. Background technique [0002] Microcystins (MCs) are a class of cyclic peptide toxins with hepatotoxicity, and are the most common cyanotoxins in freshwater blooms. ), Oscillatoria (or Planktothrix)), Nostoc, Anabaenopsis, Hapalosiphon, etc. The presence of microcystins in water has led to the death of wild animals and domestic animals worldwide. Recent studies have shown that microcystins are a potential threat to human health. The acute and chronic toxicity of microcystins has prompted extensive research on its detection methods, toxicology, detoxification in drinking water, accumulation in aquatic organisms, migration and degradation in the environment, all of which require purified microcystins. Cystin. [0003] Microcystin consists of 7 amino acids with a molecular weight of 800-1100Da. Its structure is character...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/56C07K1/16C07K1/14
Inventor 耿志明王澎刘蔼民
Owner JIANGSU ACAD OF AGRI SCI
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