Author Topic: Increasing Alkaloid Content of Liquid Psilocybe Cubensis Mycelium Culture  (Read 229 times)

Vesp

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Increasing Alkaloid Content of Liquid Psilocybe Cubensis Mycelium Culture
« on: June 12, 2010, 12:29:28 AM »
I haven't yet done the research into this nearly as much as I would like, as I haven't had much time since I've had this idea but I wanted to post the idea before I forgot it, which happens fairly often, I believe.

I noticed that the nutrients, temperature, and other variables of the liquid cultures of Ergot can greatly affect the alkaloid content it produces in a liquid culture medium. Everything from type of carbon source to pH, phosphate, arsinic, etc levels effect it. Even levels of oxygen can affect what types of alkaloids are formed.

I assume that it is pretty likley that one could go about turning the P. Cubensis mycelium, which generally produces very low, and fairly useless levels of alkaloids, into something that might be useful and rather interesting to play with, as well as alter the levels/ratio of psilocybin and psilocin + other alkaloids.

I will look into more information for this later, but I figure altering temperature, adding certain precursors and finding a high alkaloid strain should really make a big difference as well as amount of oxygen, etc.  Really just trying out a lot of the things they've done to increase ergot alkaloids ought to help increase alkaloids, or at least lead one in the right direction -- assuming there is a similiar mechanism behind it. I'll update this more later on when/if I find more information about it.

and as for the possible question of: why not just grow mushrooms to be easy? I have no idea this interests me more.
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Vesp

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Re: Increasing Alkaloid Content of Liquid Psilocybe Cubensis Mycelium Culture
« Reply #1 on: June 12, 2010, 05:42:05 AM »
So far I have found that apparently only the stages from the knotting mycelium to the fruiting of the mushroom contains alkaloids, while the myc that is not knotting contains none to little.
Knotting is where a mycelium ball starts to form on the surface, and will lead into pinning, I believe?

At least this is for when they are grown on solid substrate -- I have not heard anything about liquid cultures, other then this seemingly sick stuff from an erowid page:
Quote
To extract the mycelium, simply add ~500g of Cranberry Juice mixed with
1/2tsp of ascorbic acid crystals (vitamin C) to each jar of
well-permeated mycelium and shake well.  Add a glass marble as an
agitator if you like.  The acidified liquid will remove the active
components, leaving behind the rye grains.  Pour off the liquid and
enjoy.  The extracted liquid should be a milky consistency with a thick,
frothy layer of mycelium.  If you want to get all the material, then do
a second extraction with another cup of juice.
 
It is probably an aquired taste, but I have found the resulting brew to
be quite palatable, and have never had any nausea using this method.
The onset of the trip occurs in about 20-30 minutes.
--  http://www.erowid.org/plants/mushrooms/mushrooms_cultivation16.shtml

Given that this next quote is for tub substrates, and not in solution like I am thinking/wanting gives me some ideas:

Quote
The major pinning triggers are in order of importance, full colonization, a decrease in CO2 levels due to increased air exchange(not gas exchange which is minimal), a steady rate of evaporation from the substrate or casing layer, and lastly, light.

Hyphal knots form best in 100% humidity, but I didn't list that because it's not a pinning trigger, but rather an environmental condition that is necessary. That's why we use casing layers. The casing helps to provide the 100% humidity right at the surface of the substrate where the hyphal knots form.

I have seen no correlation with temperature drop whatsoever. In the summer, my growing chambers are 10 or more degrees warmer than the open shelves I incubate on due to the heating effects of the lights. Even with a temperature increase, I still get wall to wall pinsets, so I don't consider temp drop relevant at all to tropical species. Other growers disagree of course, but that's just my observation after many years.

Full colonization of the substrate is the number 1 pinning trigger. Full colonization can be when the mycelium reaches the physical border of the container they are in, or when they run up against a biological border, such as a contaminant species. Either way, they see they have colonized all of what is available to them, so they then enter the next phase, which is reproduction.

There must be evaporation of moisture from the substrate for pins to form. A waterlogged substrate will just sit there forever without pinning. Even in 99% humidity, as long as you provide fresh air, moisture will be evaporating away from the substrate, and this is necessary for pinning. We mist to replenish the lost moisture, then allow it to dry slightly before misting again. This keeps the moisture content high, and keeps the humidity at the casing surface near 100%, but at the same time provides the evaporation of moisture that is a very important pinning trigger.

During colonization, we provide very small holes in the jars or tubs for gas exchange. We want a high CO2 environment during colonization, because this prevents the mycelium from consuming all of the substrate. The mycelium colonizes the substrate, but doesn't 'eat it all up' due to the high CO2 levels. During fruiting, we remove the covers to provide air exchange, which is at a much higher level then the minimal gas exchange provided during colonization. This increase in air exchange lowers the CO2 levels, and is a major pinning trigger. At this time, the mycelium begins to consume the substrate it has previously colonized, and we notice during fruiting that our substrates pull away from the sides of the container. This is not due to moisture loss, but rather due to the mycelium 'eating' the substrate and turning it into CO2, a waste product. It is easily proved that this shrinking isn't related to moisture loss, because even when we dunk a bulk substrate, it doesn't return to its pre-flush size.

Last, but not by any means least is exposure to light. Light does much more than just tell the mushrooms which way to grow. There are mechanisms in the light that stimulate the formation of hyphal knots as well, and light at the higher end of the spectrum(blue) definitely, absolutely stimulate more hyphal knots(which grow into primordia, which then morph into pins) than light at the lower end of the spectrum(red) This does not mean to get a 'mood light' with a blue lens, but rather to select lights such as metal halide, or much more economical is 'natural daylight' fluorescent that emit light at around 6,000 kelvin to 7,500 kelvin depending on the brand. Cool white fluorescent emit light at around 5,000 kelvin and the 'red' incandescents emit light at around 3,000 kelvin. The higher the light temperature in kelvin, the more stimulatory it is to hyphal knot formation. I hope this helps.
RR
- http://www.shroomery.org/forums/showflat.php/Number/6686698#6686698

At the moment, I am thinking that a fully developed liquid culture of myc with light and good ventilation will possibly lead to more alkaloids, and if they as myc act anything like ergot as myc, then sucrose would be a good source of carbon.

Quote
The best substrates for
alkaloid production are slowly metabolizable Saccharides, e.g., sucrose, maltose
and polyols (mannitol, sorbitol) (Køen et al., 1984).


for Ergot myc again:
Quote
In high alkaloid producing Claviceps strains the
optimum level of phosphate in medium ranges from 1 to 4 mM. The phosphate
is taken up by the fungus during first 2–4 days of the cultivation and it is
accumulated in the mycelium. Higher phosphate concentrations stimulate
vegetative growth of the mycelium, glucane production and inhibit alkaloid
biosynthesis.

So -- phosphate concentration seems like it might also play a roll here as well.. not sure -- and I cannot find much information on trying to induce alkaloid production in mycelial growths of Psilocybe Cubensis -- do any of you have info on it?
« Last Edit: June 12, 2010, 06:00:38 AM by Vesp »
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fnord

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Re: Increasing Alkaloid Content of Liquid Psilocybe Cubensis Mycelium Culture
« Reply #2 on: June 12, 2010, 06:04:43 AM »
Ive wondered about trying bulk morning glories,phalaris,MHRB or HBWR seeds mixed in with the substrate i think this could have some very iterestings results.

Interesting thread,cant wait to hear your friends outcome.
« Last Edit: June 12, 2010, 06:19:24 AM by fnord »

Vesp

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Re: Increasing Alkaloid Content of Liquid Psilocybe Cubensis Mycelium Culture
« Reply #3 on: June 12, 2010, 06:15:16 AM »
Oh yes -- adding tryptophan and other amino acids would also probably help as well, phosphate is probably also very important -- which would be the roll of the phalaris especially -- another idea might be to add an MAOI so that the tryptamines that do form cannot be metabolized into something else unwanted (though, I don't think that happens?)

Just a thought, if an equilibrium type thing forms - perhaps pH and phosphate play a bigger role then I was thinking  since psilocybin and psilocin are only different because of the phosphate group -- if it had higher phosphate in the nutrient solution, you may end up with a higher a psilocybin but would keep the psilocin concentration the same since it is a precursor to the psilocybin, it would just shift the equilibrium a bit, and perhaps allow for more overall alkaloids to be made?

No idea.. probably not but its fun to think about.
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fnord

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Re: Increasing Alkaloid Content of Liquid Psilocybe Cubensis Mycelium Culture
« Reply #4 on: June 12, 2010, 06:22:34 AM »
Im ow banned from the site but i have a thread at drugs-forum with a decent amount of information on cubesis converting tryptamines to more interesting ones. Im sure someoen here can find it for you.

Satan

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Re: Increasing Alkaloid Content of Liquid Psilocybe Cubensis Mycelium Culture
« Reply #5 on: June 12, 2010, 09:30:24 AM »
Attached paper of interest.

Vesp

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Re: Increasing Alkaloid Content of Liquid Psilocybe Cubensis Mycelium Culture
« Reply #6 on: June 12, 2010, 10:33:09 AM »
Thank you, It says that increasing the concentration of tryptamine derivitives leads to an increase of baeocystin in the mycelia, while only small amounts of psilocybin could be found in the mycelia

From wikipedia, baeocystin it claims
Quote
...but in Magic Mushrooms Around the World, Jochen Gartz reports being aware of a study in which "10 mg of baeocystin were found to be about as psychoactive as a similar amount of psilocybin."
I don't know if that is a good source or not - but it is just something to keep in mind I guess.
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Naf1

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Re: Increasing Alkaloid Content of Liquid Psilocybe Cubensis Mycelium Culture
« Reply #7 on: June 12, 2010, 09:40:41 PM »
Production of Psilocybin in Submerged Culture by Psilocybe cubensis
Philip Catalfomo and V.E. Tyler Jr
Lloydia 27, 53-63 (1964)

http://www.erowid.org/archive/rhodium/pdf/psilocybin.submerged.culture.pdf


Vesp

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Re: Increasing Alkaloid Content of Liquid Psilocybe Cubensis Mycelium Culture
« Reply #8 on: June 12, 2010, 10:49:06 PM »
Nice find. So from their starting solution, and adjusting for what increased alkaloid content it seems to me that this solution would produce the highest alkaloid yield, on the seventh day



Glucose............... 1.0 gram
Ammonium Succinate.....0.5 gram
Phosphate..............0.1 gram
Yeast extract..........0.5 gram
Thiamine*HCl...........0.003 gram
(NH4)6Mo7O24*4H2O..... 0.05 miligram
ZnSo4*7H20.............0.3 miligram
MnCl2*4H2O.............0.35 miligram
FeSO4*7H2O.............2.5 miligram
CuSo4*5H2O.............0.5 miligram
MgSo4*7H2o.............0.5 gram
Distilled water, to make 1.o Liter
Adjust to pH 5.5 with hydrochloric acid

Seeing that some of the variables get it to range from from .5% to 1% - ish of the mycelium alone -- not counting the solution, where I don't think they found any alkaloids.

I believe psilocybin makes up roughly 1% of the weight of Psilocybe cubensis  mushroom so it seems like, at least under these conditions they are practically equivalents.

It seems to me that they left out a really important part, and maybe I skipped over it - but they did not specify what lighting their cultures had -- was it always in the dark, sitting open in the lab? constant lighting? replicated day/night?

Light I am sure would have a huge effect on alkaloid content because it is a trigger for knotting/pinning. right?


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Naf1

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Re: Increasing Alkaloid Content of Liquid Psilocybe Cubensis Mycelium Culture
« Reply #9 on: June 13, 2010, 05:57:04 AM »
Light is not really relevant as the mycellium do not need any they are usually under the ground (they do not photosynthesize they are saprophages and their food has been supplied by you), they only need around 1/100 hundredth of a second of light to trigger fruiting as they are actually photosensitive and will produce a brown pigment in the cap when exposed to light. Also to get the bastards to knot the substrate has to be fully (100%)colonized, so they are not going to knot (unless one or two fruit on the surface, but just keep shaking it). You never know though? But would imagine UV could eventually break down psilocybin lowering yields, but could also trigger alkaloid production (as a defence? or something?).

I also thought that shrooms had more than 1% ???

Vesp

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Re: Increasing Alkaloid Content of Liquid Psilocybe Cubensis Mycelium Culture
« Reply #10 on: June 13, 2010, 06:54:56 AM »
They may have more then 1% but the highest I have ever really read is that they have 1.5%.

"A typical dose of the rather common species, Psilocybe cubensis, is approximately 1 to 2.5 grams,[23]  while about 2.5 to 5 grams[23]  dried mushroom material is considered a strong dose. Above 5 dried grams is often considered a heavy dose."

"A typical recreational dosage is from 10–50 mg psilocybin."

having a dose of 1 gram of shrooms being about 1% alkaloids, it makes sense that ~10mg would be a dose for psilocybin. This works out well, but is of course questionable material + probably generalized.


Very interesting that they only need that little of light - i thought it not only helped to induce them to grow but also which way to grow -- so they know which way is up, or something like that -- instead of using the mechanism plants use.


Quote
Also to get the bastards to knot the substrate has to be fully (100%)colonized, so they are not going to knot

Quote
Full colonization of the substrate is the number 1 pinning trigger. Full colonization can be when the mycelium reaches the physical border of the container they are in, or when they run up against a biological border, such as a contaminant species. Either way, they see they have colonized all of what is available to them, so they then enter the next phase, which is reproduction.

Ah! So perhaps having a contaminant in the liquid culture with the myc of the shrooms would help increase alkaloid content, since it may see that it has came into contact with a biological border?

I wonder - what purpose does the alkaloids serve in the first place? I assume that it is some defense mechanism for insects or perhaps animals due to the fact that it doesn't contain much when it is underground -- protected from maggots, beatles, etc -- while above ground it does not have that protection and so it has that as a way to defend itself long enough to produce spores.

It might seem that if there were another contaminant in the jar, it would then begin to at least try and knot - then the myc solution may contain increased alkaloids because it would be trying to  prepare for its survival above ground.

Perhaps adding a small amount of yeast or something would do some good for the myc.



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Vesp

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Re: Increasing Alkaloid Content of Liquid Psilocybe Cubensis Mycelium Culture
« Reply #11 on: July 08, 2010, 09:01:50 AM »
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Vespula germanica

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Re: Increasing Alkaloid Content of Liquid Psilocybe Cubensis Mycelium Culture
« Reply #12 on: November 27, 2010, 02:17:41 PM »
The last link seems to be dead. What were you referring to?
...bzzzzzz...

Vesp

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Re: Increasing Alkaloid Content of Liquid Psilocybe Cubensis Mycelium Culture
« Reply #13 on: November 27, 2010, 07:29:28 PM »
Yeah it is, and I do not remember. Sorry.
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oldguy

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Re: Increasing Alkaloid Content of Liquid Psilocybe Cubensis Mycelium Culture
« Reply #14 on: May 30, 2011, 08:33:02 PM »
There was an article by E.B. Gold in Psychedelic Monographs, vol. 6 dealing with this subject (unfortunately I don't have easy access to the journal).  For cubensis grown on rye, he concluded that the best additive was 1 gram of dextrose per pint of water, resulting in firmer, heavier, more potent mushrooms.  More than one gram per pint made the water too syrupy and impeded shaking of the grain.

I've also heard reports that P. cubensis grown on long grain brown rice is more potent than that grown on rye.  More difficult, best to steam or cook the rice first to avoid a solid rice ball that is resistant to colonization.  Alternatively, the pressure release valve on the pressure cooker can be left open while blowing steam for 20 minutes to cook the rice before sterilizing.

wrench352

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Re: Increasing Alkaloid Content of Liquid Psilocybe Cubensis Mycelium Culture
« Reply #15 on: May 20, 2012, 10:34:53 PM »
Quote
I've also heard reports that P. cubensis grown on long grain brown rice is more potent than that grown on rye
Its the other way around rye has higher tryptamine levels,brown rice is easier to work with.

http://forums.blacklight.me/viewtopic.php?f=22&t=793
this link worked for me.You have to be signed in first to view it. its very relevent and most of it is beyond my skill set.What I took away most was addition of triptamine HCL to the medium.

also have a look here http://www.shroomery.org/9040/Tryptamine-Cubensis

I know its been a year since this thread fizziled out but I thought Id give it abump to see what shakes out


namaste'