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Method for separating microbe producing tetraodotoxin from puffer

A technology of tetrodotoxin and microorganisms, applied in the field of microorganisms producing tetrodotoxin, can solve the problems of low content, limited natural resources, difficulties, etc., and achieve the effects of stable genetic properties, good separation effect and simple operation

Inactive Publication Date: 2004-12-08
SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tetrodotoxin used to be a biologically active substance isolated from puffer fish ovary or liver, but extracting TTX from puffer fish ovary is not only difficult to catch puffer fish, but also has limited natural resources. For a long time, scientific and technological workers at home and abroad It is found that tetrodotoxin (TTX) exists not only in puffer fish, but also in the microorganisms that coexist with puffer fish. It is often found that marine bacteria and actinomycetes can produce tetrodotoxin, but because the obtained microorganisms produce TTX content is low Or the TTX-producing genetic traits are unstable, and their progeny tend to lose TTX-producing characteristics during artificial culture. Therefore, the research on the use of microbial sources to produce tetrodotoxin has not yet entered the application stage.

Method used

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  • Method for separating microbe producing tetraodotoxin from puffer
  • Method for separating microbe producing tetraodotoxin from puffer

Examples

Experimental program
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Effect test

Embodiment 1

[0021] The method for isolating the bacterium that produces tetrodotoxin (TTX) from pufferfish ovary can be operated according to the following steps:

[0022] (1) Select healthy red-fin oriental dolphins in the spawning period from the ocean;

[0023] (2) Scrub and disinfect the fresh red-finned oriental porpoise with alcohol, immediately dissect the fish with a sterile scalpel, thin blade and tweezers, take out the fresh ovary accurately, add cell dispersion solution of sterile saline or phosphate buffer Grinding with quartz sand for 3-5 minutes (which can make the microorganisms in the ovary fully free and dispersed, and be separated), to obtain fresh puffer fish ovary extract;

[0024] (3) For the fresh puffer fish ovary extract, use the plate dilution method to dilute with 10 -2 , 10 -3 、10 -4 Concentration, smear a flat plate on the plate with the following composition medium, and then put it in an incubator at 28 ° C for incubation; pick out a single colony and separ...

Embodiment 2

[0028] The difference from Example 1 is that, for the fresh puffer fish ovary extract, use the plate dilution method, with one drop of stock solution and 10 -1 、10 -2 Concentration dilution, smear plate on the plate of following composition culture medium, then put into 28 ℃ incubator and incubate; pick out single bacterium colony and divide pure (observed by microscope, then transplant to slant for preservation, carry out producing tetrodotoxin (TTX ) Detection and verification of biologically active substances.

[0029] The culture medium for separating and producing tetrodotoxin actinomycetes consists of:

[0030] Soluble starch 1%, sucrose 0.5%, mannitol 0.3%, KNO 3 0.1%, K 2 HPO 4 0.35%, MgSO 4 .7H 2 O 0.001% FeSO 4 .7H 2 O 0.001%, fugu ovary extract 0.01%, the balance is sea water (or artificial sea water), agar 2%, pH7.0~7.2;

[0031] When the culture medium is ready for use, add 1ml of 3% potassium dichromate solution to the 300ml culture medium that has bee...

Embodiment 3

[0033] The difference from Example 1 is that the composition of the culture medium for isolating actinomycetes producing tetrodotoxin is changed to: 1% glucose, 1% sucrose, 0.3% beef extract, 1% yeast extract, 0.3% peptone, 2.5% NaCL, pufferfish Ovary extract 0.01%, the balance is sea water (or artificial sea water), agar 2%, pH7.0-7.2;

[0034] When the culture medium is ready for use, add 1ml of 3% potassium dichromate solution to the 300ml culture medium that has been heated and melted, then add 0.5ml of 1% streptomycin solution, and 1-2 drops of ciprofloxacin, shake gently , and then pour the plate for separation and purification.

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Abstract

A method for separating tetradotoxin is carried out by microbe. It comprises 1) selecting fresh globefishes; 2) washing and disinfecting them to take out their ovaries, grinding the ovaries with cell dispersing liquid to obtain fresh ovary extract liquid; and 3) separately and thermally culturing in medium of marine bacteria and actinomice to separate and purify tetradotoxin. It operates easily with good effect, separates well to obtain strains with 90% achieving rate. The obtained microbe can produce tetradotoxin at high yield and breed ten more generations with stable heredity.

Description

technical field [0001] The invention relates to microorganisms producing tetrodotoxin (TTX), in particular to a method for isolating the microorganisms producing tetrodotoxin (TTX) from puffer fish. technical background [0002] Due to the delicious meat of puffer fish, there are many people who love to eat it, especially in Japan, it is regarded as a fashion, but poisoning incidents often occur due to wrong preparation, causing numbness, paralysis, and even death of the eaters; but on the other hand, using TTX has the effects of analgesia, sedation, convulsions, and pain relief. It has been used as a local anesthetic in clinical practice, and can be used to treat rheumatism, arthritis, and leprosy; Tetanus, cholera, typhoid, asthma, and advanced cancer all have curative effects and have great development value; Tetrodotoxin (TTX) is one of the most toxic neurotoxins found in nature, and its toxicity is equivalent to sodium cyanide 1250 times higher, can block the sodium io...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/18
Inventor 胡江春薛德林马成新刘全永王书锦
Owner SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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