Extraction of Toxins from Molds

[MrSamosa]

I had done a bit of research on T-2 Mycotoxicosis for a class in school not too long ago, and I found some information worthy of notice. It's nothing terribly new, but it should be sufficiently useful... It concerns T-2's solubility. :)

It is soluble in DMSO, Alcohols, Acetone, Ether, and Propylene Glycol. It is almost insoluble in water. Its vapor pressure is not very high, but when dissolved in a volatile solvent, it decreases. That's all the solvents I can remember now, but there are a few more.

So, now the question comes to mind... How would we get the crude toxin to begin with? The obvious answer is to culture the mold. Unfortunately, my experience in Biology is somewhat limited (going only to a highschool general biology class), and my Microbiology experience even more limited (informal education; just going through a sister-in-law's medical books). So, does anyone know the proper method to culture molds??

The desired Fusarium molds are ubiquitous in the soils of almost every part of the world. Corn tends to be the growing medium, hence T2 Mycotoxicosis' common name of "Moldy Corn Disease." It grows best in temperatures around 70 degrees Fahrenheit (I *think* that's somewhere in the area of 25 degrees Celsius). That's the extent of my information so far, but I would like help in developing a "theoretical" means of producing Fusarium molds and extracting the resulting toxin... This is what I am thinking:

-Get a soil sample and some corn, and mix them in with a sufficient amount of water (as Fusarium molds love water too!) to make a "nutrient slurry" (Forgive my horrible nomenclature).
-Store the slurry in the proper environment to promote growth for a month or so.
-When sufficient amounts of mold grow, pour the slurry into Acetone or some other organic solvent. This should dissolve the T-2 which is produced by the Fusarium mold, and leave everything else undissolved.
-Filter out the unwanted soil and corn, and keep the T-2 Solution.
-Distill out the T-2.

Does my logic seem right? Or would this just make an ugly mess? Also, what would be the best means of discouraging the growth of competing molds?

[Mr Cool]

Well, you wouldn't want to use acetone to dump the soup of stuff into because it'll mix too well with the water, lowering the toxin's solubility too much. Better to use ether or some other non-polar, and do a double-layer extraction a few times. This will also lower the amount of ionic salts present in your product.

The method as you described will give a mix of many fungi and bacteria, with the most common or the most vigorous colonising the nutrient first and largely preventing anything else from growing. What we need to find is a condition that the required fusarium species will tolerate, or even like, that the other common organisms will not. eg if fusarium sp can survive and grow at low temperatures, like in a fridge, then soil could be added to cold, sterilised corn/water/trace nutrient paste and then left to colonise for a few weeks/months at 5*C. Assuming, in this hypothetical example, that the desired fungus tolerates these conditions the best, it will colonise the nutrient and then it can be allowed to warm up and be put in more suitable growing conditions. Since the nutrient would already be totally colonised by fusarium sp., little else should grow. I have used this method to isolate photobacterium phosphoreum from a dead sea fish, although in this case it is easy to get a pure culture because you can see the colonies glowing, so they can be collected exclusively and moved on to clean growing medium.
Obviously the special conditions needed might not be temperature related, it could be pH, light, high salt concs., trace levels of toxins, etc.
A better way might be to provide conditions ideal for fusarium sp to grow in naturally, and wait in the hope that your corn or whatever is colonised. This way you are not deliberately adding any other organisms, and the medium should be colonised by what you want (sooner or later, you might need a few tries) if you get the conditions right for the organism. Identification of a succesfully colonised growing medium might be hard.

When you've grown a load of the mould, wizz it up (medium and all to get the mycealium, which I can never spell, and not just the fruiting bodies on the surface) in your solvent in a blender, wait for it to settle, decant, wizz with more solvent, etc. Repeat this four or five times, I would imagine. Collect the solvent washings, and evapourate.
Before you do this, save a bit of your colony, so that you don't have to go to the trouble of isolating one again.

I like microbiology (although the photobacterium experiment is probably the only serious one I've done), maybe I'll have a go at this one day...

[nbk2000]

Most T2 producing fungi glow under UV light, enabling detection. Aflatoxin producing fungi are endemic to peanuts, so search peanuts with a UV lamp till you find something that glows, then culture it.

Biology is very complicated, so much study at a uni library is in order, before you can successfully culture anything, let alone mass produce.

[Boob Raider]

As far as Aflatoxin is concerned ..... Aspergillus flavus is responsible for afla production when it is grown on peanuts, if it were to be grown on soy beans ... one ends up with soya sauce. Although trying to use soya sauce as a culture doesn't work as it is pasturised (sp?) but if you know someone who makes their soya sauce .... then you can make your aflatoxin :D . None of the tricothecene mycotoxins are that potent. They are mostly in the mg/Kg range (ie LD 50).
Also if you find old peanut jars ... chances are the top layer might glow bluish under UV due to aflatoxin.

<small>[ March 28, 2003, 03:04 AM: Message edited by: Boob Raider ]</small>

[Einsteinium]

Well... even if you sucessfully grows your fungi (which is a damn pain with almost any of them mostly due to contamination with aspergilius (that dreaded green mold), bacillus subtilis and the peniciliums, lemme tell ya) it will take you quite much efforts to cleanly separate the toxins from all other crap in the solvent. You definitivly want to wash the extracts several times with water, brine (NaCl/H₂O), and other mildly polar solvents like chloroform where the toxin is insoluble but the contaminants are (or where the contaminants are insoluble but the toxin is). Don't even think of distilling such a toxin as it will most likely decompose before it's boiling point, even under a good vacuum. Well, it might not but I wouldn't try it out unless having some ref about it. Anyway, if the extracted toxin is still a little contaminated with other stuff, it should still be very effective :D

[Mr Cool]

Ooh, thanks for adding another potential source of source material to my list - organically grown, raw, unsalted peanuts. Do you think the toxin/fungus might be why you sometimes get a peanut which tastes really bad?
I will also try organically grown wheat for F. Roseum.
Currently I'm growing the fungi from soil on some gowth medium that I've made from corn, a dash of oats, a vitamin tablet, and enough water to make it pasty and sticky. If anything useful happens I'll find the scrap of paper on which I wrote down exactly what I did, but I REALLY don't expect anything too useful from this attempt. Maybe just a nice mould garden :) .

</font><blockquote><font size="1" face="Verdana, Arial, Helvetica">quote:</font><hr /><font size="2" face="Verdana, Arial, Helvetica">As far as Aflatoxin is concerned ..... Aspergillus flavus is responsible for afla production when it is grown on peanuts</font><hr /></blockquote><font size="2" face="Verdana, Arial, Helvetica"> </font><blockquote><font size="1" face="Verdana, Arial, Helvetica">quote:</font><hr /><font size="2" face="Verdana, Arial, Helvetica">Well... even if you sucessfully grows your fungi (which is a damn pain with almost any of them mostly due to contamination with aspergilius (that dreaded green mold)</font><hr /></blockquote><font size="2" face="Verdana, Arial, Helvetica">Hmmm...

[Boob Raider]

Mr.Cool if you make your medium 10% saline with NaCl .... it will retard the growth of rhizzopus (sp?) type moulds. Most mycotoxin producing fungi will grow.
Like NBK and I said before UV fluoresces Aflatoxin and T-2 with a bluish color.
It is quite difficult isolating A.flavus from the other aspergillus species and the same holds true for fusarium etc. you need a microscope and a mycology handbook as there is not much difference in colony appearance to the naked eye.

[Haggis]

I'm not sure if it's uploaded and I don't have access to the FTP, but the book "Potential Military Chemical/Biological Agents and Compounds" has a nice entry on the T-2 Trichothecene Mycotoxin. It is on page 89 and 90. There is some very good information in the back of this book about general toxins, sources, symptoms, properties, protection, routes of entry, treatment and toxicity. No information on isoloation, cultivation or weaponization however.