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Sustainable method for obtaining tetrodotoxin

A technology of tetrodotoxin and toxin, which is applied in the direction of using bacteria, methods based on microorganisms, biochemical equipment and methods, etc., can solve problems such as long-term survival of animals without mentioning, and achieve the effect of maintaining mucus production

Inactive Publication Date: 2013-01-16
NEMERPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these applications do not mention the addition of the nematode worm slime to the fermentation process, nor do they mention the long-term survival of the animals due to the continuous influx of deep sea water

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Preparation of Optimal Environments for Long-Term Survival of Nemeroder Worms in Aquariums

[0043] Nestemorpha were kept in glass aquariums with continuous flow seawater from a depth of 45 m, salinity range 3.1-3.4%, temperature range 7-16°C (range within a year). The water flow must not be lower than 2dl / min. Water is filtered in a way that does not affect water quality. No added substances. Aquariums were kept in the dark for 15 hours / day. No direct light was applied other than daylight for the rest of the day. The aquarium contains shells and stones used as hiding places for the worms.

Embodiment 2

[0045] Preparation of mucus from nematode worms

[0046] Worms were collected from the aquarium into clean and dry glass bowls (25ml). The worms were washed repeatedly with the same medium (sea water) by washing the worms with 10 lm of fresh seawater, performing tactile stimulation with a soft plastic pipette (10 ml), aspirating the same water. This stimulating manipulation is followed by mucus production. Approximately 20 ml of liquid seawater containing the mucus was collected with a soft plastic pipette and transferred to a sterile vial.

Embodiment 3

[0048] Biosynthesis of TTX from the mucus of Nemeroder worms

[0049] Liquids containing seawater and mucus from nematode worms are filtered in a step-by-step operation. First, filter roughly through standard filter paper to remove organic material such as algae or preserved material. Second, filter by manual pressure through a 0.4 micron syringe filter (filter of any diameter). Third, manually pressurize through a 0.2 micron syringe filter (filter of any diameter). All steps were performed according to the filter manufacturer's standard procedures.

[0050] The liquid samples were then transferred to a solid phase extraction (SPE) column Evolute ABN (manufacturer Sorbent AB, Sweden). Preparation / activation of the column (example: volume 200mg / 6ml - 50 microns) was performed using the following:

[0051] 1.6ml MeOH

[0052] 2.6ml sterile water

[0053] 3. Sample application (up to a volume of 15 ml) and collection again. This process washes proteins and organic molecule...

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PUM

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Abstract

A sustainable biosynthesizing of tetrodotoxin (TTX) based on seed culture of Vibrio spp is obtained from the mucus of various species of nemerteans (ribbon worms) (phylum Nemertea). The indispensable organisms are kept alive which extends access to crude material and makes the procedure economically and ecologically sustainable.

Description

technical field [0001] The present invention generally relates to a sustainable method for the extraction of tetrodotoxin (TTX) from the mucus of a specific species of Nexecta worm. More specifically, the present invention relates to an ecological method of repeatedly utilizing worms to obtain mucus, allowing the worms to remain alive and maintain a continuous source of mucus production, resulting in high yields of tetrodotoxin. Background technique [0002] Tetrodotoxin (TTX) is a highly potent neurotoxin also known as the causative agent of pufferfish toxicity. Furthermore, not all puffer fish species are venomous, and several are only slightly or moderately venomous. The toxicity of pufferfish species varies depending on tissue or organ, geography, season and sex. TTX is not limited to puffer fish, but is widely distributed in various types of animals, for example, California salamander Tarichi torosa, goby Gobius criniger, Atelopus frog, gastropod molluscs Charonia sau...

Claims

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Application Information

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IPC IPC(8): C12P17/18
CPCC12N1/20C12P17/188C12R1/63C12N1/205C12R2001/63
Inventor 玛琳·斯特兰德皮尔·桑德博格
Owner NEMERPHARMA
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