Ergotamina in coltura liquida

[Ergot]

Ciao a tutti,

Recentemente ho studiato e sono venuto a conoscenza di Claviceps Paspali, un ceppo di Claviceps che produce rese relativamente elevate di ergotamina.
Ora sto sognando la possibilità di trovare un ceppo del genere in natura, e vorrei sapere le vostre opinioni su quanto sarebbe fattibile allevare un ceppo del genere nel mio umile biolab.
Ci sono alcune informazioni sulla coltivazione di claviceps su erowid, e sembrano piuttosto complete. Tuttavia, il web mi lascia piuttosto all'oscuro delle esperienze che altri colleghi sognatori possono aver avuto.
Trovo spesso menzionato il Paspali (Stevens e Hall): si tratta di una specifica varietà coltivata o coltivabile ad alta produzione di ergotamina?

Nel mio sogno andrei a cercare alcuni campioni sul campo, li trasferirei in condizioni sterili su piastre di agar e, a seconda di come si evolvono, li metterei in un barattolo a bocca larga o in un bioreattore improvvisato.
un bioreattore di fortuna per la propagazione in coltura liquida.

Qualcuno è disposto a condividere informazioni o riflessioni?

Grazie!

 

[HerrHaber]

La C. purpurea può essere acquistata su etsy a un prezzo decente, ma solo un piccolo numero di semi infetti, comunque sufficiente per avviare diverse colture.

 

[madmoney69]

Nel forum di Thevespiary ci sono alcune discussioni importanti sulla coltivazione della segale cornuta.

 

[Field7]

1) Easy if you know what you're doing.

2) Many Universities have strain banks. Some sell samples.

3) "Stevens and Hall"? I'd guess a substrain.

4) Erowid- no. Vespiary- no. Shroomery- yes.

If you buy a 50lb bag of rye, you're likely to find ergot on some of the rye berries. Could be cleaned up on agar plates, grown in a liquid culture, and then transferred to spawn jars (made from that 50lbs of rye you just bought).

*Easy if you know what you're doing. Big IF for a nube.

If you're thinking "LSD"... there's a bit more to master.

You're talking Mycology (Microbio) lab skills and Organic Chemistry lab skills.

Fill out your FAFSA, cause you're going to college. (Not necessary, but it wouldn't hurt.)

 

[archae]

If you don't have academical access to such strains, you are in for a world of pain. First of all, wild strains are mostly atrocious at producing ergotamine under saprophytic conditions, and you would need an extraordinary amount of luck, mutagenesis, or even both, to even manage to get anywhere. Actually, no need for "secondly". If hyou can even pull this off, hats off to you. Mutagenesis and screening are a pain in the ass and take a shit load of time (years, even with an actual lab). It isn't impossible, both for any practical purposes it doesn't make any kind of sense

 

[mycelium]

Most of the alkaloid content in C. paspali is lysergic acid, not ergotamine , and the culture can be purchased from mycology research centers

 

[archae]

True, but you will need proper accreditations before being allowed to buy from them. And they do check thoroughly

 

[Osmosis Vanderwaal]

As was said a few infected grains can be pulled out of anyb ag of cereal grains. The lowest level of accepted nomenclature is subspecies, ssp. Strains are not recognized by any serious mycologist. Paspali is a species. I don't deal in fungi imperfecti, but its very likely that a liquid culture wouldnt poduce any ergot alkaloids at all. Ever heard of st. Anthony's fire? Me either. i could be wrong, but i think the best way would be to grow some wheat and inuculate it, instead of lcs indoors. Maybe you could use an lc as an inoculant

 

[archae]

You're correct in saying that it would be more convenient to produce sclerotiae by infecting rye berries, as it's how a good chunk of the ergotamine is still produced today. However, infecting rye ears requires a spore suspension, not a liquid culture. It's really easy to produce spores however, as claviceps purpurea produces them even on grain cultures. Most procedures call for colonizing sterile hydrated grains with the mycelium (as would be done with most fungi) and keeping at room temp for around 1 month. After that, the mycelium should have produced conidiae and spores can be harvested by suspending the grains in sterile water and filtering them out

 

[Osmosis Vanderwaal]

Now were talking! I sold Morchella cultures ( morel mushrooms) which are both sclerotia formers and mycohizzal mushrooms. Also Psilocybe mexicana which are hallucinogenic scleratia fungus, both of these mushroom producensporocarps directly from the scleratia. Ive also worked with Ustilago maydis ( HUATLICOCHE, corn smut) which has an Amazing life cycle, which requires 2 different sporulations to reproduce, but the native American method of growing it is to cut thencorn stalk near the base and kickndirt on the wound. If corn smut has grown there, there are spores in the dirt.
I was considering a paint brush and a little water with the Claviceps mixed in it, much like selectively breeding cannabis, my thought being that it effects the cereal grain, it may need to infect the pistol of the grain ( corn smut does, but thats after it has grown on the lower stem, produced an entirely different sexual spore, and that is what infects the pistols)

 

[archae]

I wasn't aware the corn smut lifecycle was fun like that! If you want to infect the ears, it has to be done before they close/are fertilized. What is done on an industrial grade is to use a spiky handle which has been dipped into the spore solution and to lightly pierce the ears before fertilization even begins, that way you have a higher success rate as you introduce the spores directly where they need to go.
There are articles in the book I mentioned (I think, or it was somewhere else) which describe the exact window in the wheat reproductive cycle where this is most efficient.
As for why it has to happen before fertilization, normally the spore lands onto the stigma, germinates, and produces mycelium to reach the ovary and form the sclerotia. By piercing the ears with small needles/spikes, you bring the spores directly into the ovary, where it needs to be.

There are different solutions to increase the likelihood of infection, such as putting a sponge at the base of the spikes/needles to increase the amount of spore solution carried at a time

 

[mycelium]

@Osmosis, according to stuff I read long ago, in Michael valentine Smith's book Psychedelic chemistry, the LC is where it's at, and the broth has lots of stuff in it, not just agar and LME. And it is extracted from the LC after however long(it has literally been 3 decades since I read it).
And Shulgin mentioned C paspali as being higher in lysergic acid
I saw, on the shroomery this year, pictures of infected paspalum grass...definitely would attempt to culture that

And as osmosis mentioned, st Anthony's fire is one reason to be extra careful with ergot. Literally the reason the Salem witch trials happened

 

[Osmosis Vanderwaal]

What is in a liquid culture is whatever you put in it. Ive boiled potatoes and added dextrose sugar for pda, bought beer brewers malt light, medium, dark, ive used grain soak water as my nutrient, added colors added sawdust for lignin, added celulose from a shredded filter disk, activated charcoal. You could put nothing in it, then it would have nothing.
having a lot of stuff in it is counter preductive usually because if the mycelia is getting plenty to eat, it won't spread as fast because its not hungry
admittedly, erdot is not my expertise, I was only interested in kingdom fungi perfecti and really basidiomycetes and ascomycetes ( mushroom sporoarps) and rut fungus have conidiospores, which is fairly rare, but I had some oddball cultures like Pestilotiopsis microspora ( the plastic eating fungus, polyeurothan specificaly) and pleurotus cystidiosus ( an oyster mushroom that makes conidiisporesnalong with basidiospores)

 

[Osmosis Vanderwaal]

Also bearing in mind psychedelic chemistry is not exactly mycology or botony. Chemists mostly have terrible aseptic culture technique. YEAH I SAID IT. And botonistsnare right there between the two. Botonists occasionally use a laminar flow hood fornbreeding orchids and stuff but a biological lab and a chemistry lab have only preacious few things in common

 

[archae]

One great resource if you want accurate, scientific information on the matter is https://www.routledge.com/Ergot-The-Genus-Claviceps/Kren-Cvak/p/book/9789057023750. I cannot attach the pdf to this message, but I will send it to anyone who wants it. I recommend anyone who wants to play around with ergot to read it in its entirety.

Beyond the need for a good strain (and yes, strain is a relevant biological term), the production of ergotamine and other related compounds requires specific conditions, such as very high sugar and citric acid concentrations, high aeration, and low iron concentration (I think, it's been a long time).

A few examples of typical medium combinations:

Example 1​

pH 4.5-6.2 (optimum 5.4)
aeration 0.5-1.51 of air/1 of medium mixing rate 75-200rpm (180)
temperature 24 °C
Carbon source: sucrose or sorbitol 70-250 g/l
citrate 5-10 g/1
Phosphate 5mM

Example 2:​

From Ergotamine Production in Submerged Culture and Physiology of Claviceps purpurea https://doi.org/10.1128/am.15.3.597-602.1967

Medium TG Inoculum:
Glucose: 100
citric acid: 10
KH2PO4: 0.5
Mg5047H20: 0.3
yeast extract: 0.1
Fe504 7H20: 0.007
Zn504: 0.006
Tap water to 1000ml, adjust to 5.2pH with ammonia
Sterilize at 110 for 20min

Medium T25 production
sucrose 200
Citric acid: 15
KH2PO4: 0.5
MgSO4-7H20: 0.25
yeast extract: 0.1
KCl: 0.12
FeSO4 7H2O: 0.007
ZnSO4: 0.006
Tap water to 1000ml, adjust to 5.2pH with ammonia
Sterilise at 110 for 20min

In this case, the inoculum/production media serve different purpose: using the inoculum, we accumulate biomass, before scaling up by a factor of 10 at most using the production medium.
For example: 1l of inoculum is prepared and incubated for 2 weeks at room temp. 100ml is used to inoculate 900ml of production medium, total volume 1l

 

[Osmosis Vanderwaal]

Stain is relevent in the sense of phenotype vs. Genotype but it's all a strain really is, a pheno. There's not one peer reviewed document that labels any fungal "strains" its not accepted by the ICBN. Talk of strains in relation to kingdom fungi is universaly rejected without prejudice

 

[archae]

Ho yeah we agree on that, it's not usually a phylogenically distinctive label, but to characterize a specific genotype/pheno it's still relevant! And by that I mean for biotechnological applications, without any will to add a lower phylogeny level. However you can make distinction between the genotypes of 2 fungal strains, in terms of comparative genomics. In my experience, the accessory genome between filamentous fungal strains can vary quite a bit. Not as much as most prokaryotes can, but hey, give the poor lads a break!