Method for preparing pectenotoxins-2 standard sample from large-scale dinophysis culturing algae fluid

A large-scale culture and scallop toxin technology, applied in the biological field, can solve the problems of low content and high price, and achieve the effects of easy operation, simple collection method, and simple and fast pretreatment

Active Publication Date: 2018-04-20
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purchase of foreign standard toxins is expensive and the content is very small, but the development of domestic rapid detection reagents requires a large number of standards
In general, there are few reports on the development of standard marine biotoxins in my country, and only one patent for the preparation of standard samples of paralytic marine biotoxins has been seen.

Method used

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  • Method for preparing pectenotoxins-2 standard sample from large-scale dinophysis culturing algae fluid
  • Method for preparing pectenotoxins-2 standard sample from large-scale dinophysis culturing algae fluid
  • Method for preparing pectenotoxins-2 standard sample from large-scale dinophysis culturing algae fluid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The method for preparing the scallop toxin-2 standard substance from the large-scale fin algae culture liquid provided by the present embodiment, the specific steps are as follows:

[0035] 1) Broken algae liquid for large-scale cultivation:

[0036] Take the large-scale culture medium of Dinophysis acuminata (DAYS01-E6, F2) cultured in the previous stage, pass through a 10-15 μm filter membrane, and freeze it under liquid nitrogen at about -196°C. After freezing completely, thaw at room temperature. After the thawing is complete, repeat the freezing and thawing process 2 more times. Take the algae liquid that has been completely thawed at room temperature, put it in ice water, and use an ultrasonic cell disruptor to disrupt the cells in an ice bath. The experimental conditions of the ultrasonic cell pulverizer are: ultrasonic power 200-325W, ultrasonic frequency 10-25kHz, total working time 15-20min, sample temperature during ultrasonic treatment 15-30°C, instrument ...

Embodiment 2

[0074] Embodiment 2 The purification result of the method provided by the invention

[0075] figure 2 is the LC-MS spectrum of the toxin analysis in the crude extract.

[0076] The chromatogram (HPLC-UV) of the toxin detected in the algae liquid is as follows image 3 As shown, collect according to the retention time of the components; remove the reagents by the conventional rotary evaporation process of the collected components, dissolve them with 2mL of methanol, analyze by high performance liquid chromatography-tandem mass spectrometer, and qualitatively detect that the peak time is 16.02 The component of min is scallop toxin-2, which was quantified by the National Research Institute of Canada standard solution (NRC CRM-PTX2-b Lot#20120516), and the purified concentration was 243.16ng / mL, with a purity of over 90%.

[0077] The LC-MS spectrum of scallop toxin-2 after purification is as follows Figure 4 shown. Among them, according to the peak time, the ion peak with a...

Embodiment 3

[0078] Embodiment 3 The present invention is applied to actual sample detection

[0079] Take the standard sample prepared by the present invention, and dilute it with methanol successively to be 2ng / mL, 5ng / mL, 10ng / mL, 20ng / mL, and 40ng / mL series concentration of standard solutions, and detect the concentration in this laboratory from November 2015 to 2016 In October, the shellfish samples collected from the shellfish breeding area in Beibu Gulf, China, and the toxin samples in seawater adsorbed by SPATT (Solid Phase Adsorption Toxin Tracking Technology) were used to make an adsorption sampling device, using HP20 large Fat-soluble marine biotoxins in seawater suspended particles adsorbed by porous resin, hereinafter referred to as SPATT samples. After 36 shellfish samples and 51 SPATT samples were pretreated, a series of standard solutions ( 2ng / mL, 5ng / mL, 10ng / mL, 20ng / mL, 40ng / mL), HPLC-MS / MS detection was carried out at the same time, and the test results showed that sc...

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Abstract

The invention discloses a method for preparing a pectenotoxins-2 standard sample from large-scale dinophysis culturing algae fluid. The method comprises the following steps: 1) fragmenting the large-scale culturing algae liquid; 2) extracting toxin; 3) performing glucan gel chromatographic column separating; 4) performing pectenotoxins-2 high performance liquid chromatography separating; 5) performing pectenotoxins-2 high performance liquid chromatography purifying; and 6) detecting uniformity and stability of a standard sample. According to the pectenotoxins-2 prepared by the high performanceliquid chromatography, pre-treatment is simple and fast, and is easily operated; a collection method is simple, the prepared standard sample is uniform and stable, and purity can achieve more than 90%.

Description

technical field [0001] The invention relates to a standard sample of shellfish toxin and a preparation method thereof, in particular to a method for preparing a standard sample of scallop toxin-2 from algae culture liquid of fin algae, belonging to the field of biotechnology. Background technique [0002] Scallop toxins (Pectenotoxins, PTXs) are a class of fat-soluble compounds with a polyether macrolide structure, showing a ring structure composed of seven small ring ethers. At the Marine Biotoxins Conference held in Berlin in 2004, FAO / IOC / WHO lists it as one of the eight types of marine biotoxins. Scallop toxin-2 (PTX2) is the most common type of scallop toxin, and its chemical formula is C 47 h 70 o 14 , the molecular mass is 859.07, [M+NH 4 ] + : m / z 876.5104. Scallop toxin-2 is mainly produced by Dinophysis spp. in marine dinoflagellates, such as Dinophysis acuta, D.fortii, D.acuminata, D.caudata, D.norvegica, D.rotundata and D.infundibulus, etc. , was first id...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/02
CPCG01N30/02G01N30/06G01N2030/062
Inventor 佟蒙蒙谭志军王玉彭吉星吴海燕郭萌萌付树林翟毓秀高寒
Owner ZHEJIANG UNIV
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