Quote:

---

pinophile said:
Here are some studies I have not seen posted before elsewhere, and a few that  might have been posted,  full text links for all of them.  Please do not think this is just another tryptophan/tryptamine thread, it isn%u2019t par for the course; trust me. 

Please read this whole post, all the info, esp. what I quoted below; I think many people will be interested.  I know I will now be adding L-tryptophan to my subs!  And I plan to use TLC with spot density scanning to compare the relative amount of psilocybin and psilocin with isolate mycelium and mushrooms grown with and without L-tryptophan.  The great part, IMO, is that we can legally buy L-tryptophan hassle free, unlike trying to buy tryptamine hcl.

What these papers show is that L-tryptophan *does* increase level s of psilocybin and psilocin, and does convert to tryptamine in some cases; and tryptamine *does* increase psilocybin and psilocin levels.  Thus below I suggest simply adding L-tryptophan to the media (at specific ppm rage) may increase the alkaloids in cubes.  I need to invested this further, but the info so far is exciting (to me at least). 

In the thesis paper (first reference below) it is explained that radio-labeled tryptophan (C¹⁴) added to culture media of cube mycelium produced radio labeled psilocin and psilocybin in the mycelium.  And if it%u2019s increased in the mycelium I think it%u2019s not outrages to think it increases in the fruit bodies (as is evidenced by the other studies in this post and the Gartz studies too).  I believe an isolate was used for the thesis study, and it was obtained from Steven Pollock! (the same guy who supposedly bred penis envy); so we know the thesis and the author WeiWei Wang is the real deal.  WeiWei got his isolate for testing from Dr. S.H. Pollock, Dept. of Pharmacology, University of Texas, USA, see quotes below.  However, even if an isolate was not used I don%u2019t think it would matter much because it was shown via radioactively labeled carbon that L-tryptophan does increase/produce/induce (whatever the mode of cation is) both alkaloids we want (as does tryptamine).  So, it seems old Gartz might not have been too far off the mark even though use didn%u2019t use an isolate and didn%u2019t use the best procedures.

The studies I posted below seem to suggest what Gartz has reported in at least two of his studies is probably correct; I know there is some controversy on this topic, and esp. on Gratz.  In the first paper I cited below, and quoted from, it was found L-tryptophan produced %u201Csignificantly [increased] psilocybin production%u201D in cube mycelium.  However, in the  Gartz studies, he found addition of tryptamine hcl increased psilocin much more than psilocybin; and due to instabilities of psilocin, psilocybin is preferred alkaloid in the mushroom (AFAIU).  Thus, by adding L-tryptophan instead of tryptamine HCL we would be better off if it is true both substances increase alkaloid content (which seems to be the case).

I know people claim cubes don%u2019t benefit from addition of L-tryptophan but I%u2019m not so sure that%u2019s true after reading all the studies below.  Most discussion at shroomery about L-tryptophan is about possible conversion into tryptamine (which was found to happen according to the first thesis paper below), however, addition of L-tryptophan has also been found to be increase alkaloid levels independent of tryptamine; thus I think L-tryptophan warrants some testing with TLC for sure.

To mods: if you think you need to move this thread that%u2019s cool, IMO it%u2019s still advanced but YMMV.

To all: if you see errors in what I wrote please let me know, thanks.  And if people have data or academic info I would love to read it; I know Fastfred has posted some good images and info in the past on this topic.



Here are studies showing what I claimed above; at the end of this post I write about how to provide the same amount of L-trytophan as in the thesis below:


1. "Aspects of secondary metabolism in basidiomycetes: I biological and biochemical studies on Psilocybe cubensis and II: A survey of phenol-o-methyltransferase in speices of Lentinus and Lentinellus"
WeiWei Wang (B.Sc., National Taiwan University, 1974)
Thesis for degree of Mater of Science in the faculty of graduate studies (Dept. of Botany); Univerity of Brittish Columen, Novemenvber, 1977
(full PDF; too big to upload at > 9mb) http://circle.ubc.ca/bitstream/handle/2429/21293/UBC_1978_A6_7%20W35.pdf?sequence=1

(^^^ from this thesis paper I hand typed much quotes, see the quotes below, it%u2019s worth reading, trust me)



2. "The Production of Psilocybin in Submerged Cutlure by Psilocybe cubensis"
Philip Catalfomo and V.E. Tyler, Jr.
Lloydia, vol. 27 no. 1, pp. 53-63 (1964)
(full PDF) http://web.archive.org/web/20080822075128/http://www.erowid.org/archive/rhodium/pdf/psilocybin.submerged.culture.pdf

(^^ this study found addition of tryptophan didn%u2019t increase psilocybin in mycelium, however, they tested the addition of tryptophan to a nutrient deficient media, so it%u2019s not comparable to how we grow.  This study used far less tryptophan than the first thesis above.  If my math is correct this study used 0.013 ppm of tryptophan (I assume L-tryptophan) and the thesis paper uses 0.28 ppm of L-tryptophan.  The  much lager amount of tryptophan used in the thesis, along with using nutrient sufficient media, could be why the thesis showed benefit from L-tryptophan; that and the thesis paper used an isolate that produced both psilocybin and psilocin, but this study used isolate that only produced psilocybin, no psilocin.)



3. "Biosynthesis of Psilocybin: Part II. Incorporation of Labeled Tryptamine Derivatives"
Stig Agurell and J. Lars and G. Nilsson
Acta Chemica Scandinavica 22 (1968) 1210-1218
(full PDF) http://actachemscand.dk/pdf/acta_vol_22_p1210-1218.pdf

(^^^ A good paper, but old.  It too states how application of L-tryptophan increases psilocybin.  The next two references are taken from this study, they were cited in this study as other sources of the claim that L-tryptophan increases or produces or induces psilocybin; note that the fist one below Dr. Albert Hoffmann is  a co-author)



4. Brack, A., Hofmann, A., Kalberer, F., Kobel, H. and Rutschmann, J. J. Arch. Pharm. Vol.  294 (1961) 230



5. Agurell, S., Blomkvist, S., and Catalfom, P., Acta Pharm. Suecica Vol. 3 (1966) 37



6. REVIEW: "BIOACTIVE ALKALOIDS PRODUCED BY FUNGI  I. UPDATES ON ALKALOIDS FROM THE SPECIES OF THE GENERA BOLETUS, FUSARIUM AND PSILOCYBE"
ZAFAR ALAM MAHMOOD, SYED WASEEMUDDIN AHMED, IQBAL AZHAR,  MOHAMMAD SUALEH, MIRZA TASAWER BAIG AND SMS ZOHA
Pak. J. Pharm. Sci., Vol.23, No.3, July 2010, pp.349-357
(full PFD) http://www.pjps.pk/CD-PJPS-23-3-10/Paper-21.pdf

----------------------------------------------------------

Please read what I took a long time to type, especially the larger text in the quotes:


"Aspects of secondary metabolism in basidiomycetes: I biological and biochemical studies on Psilocybe cubensis and II: A survey of phenol-o-methyltransferase in speices of Lentinus and Lentinellus"
WeiWei Wang (B.Sc., National Taiwan University, 1974)
Thesis for degree of Mater of Science in the faculty of graduate studies (Dept. of Botany); Univerity of Brittish Columen, Novemenvber, 1977

Quote:

---

Abstract

Psilocybe cubensis was cultured successfully in two media.  Medium A was devised by Catalfomo and Tyler and Medium B was a modification of a medium which has been used for ergot alkaloid production by Claviceps purpurea.  Only when the fungus was kept on Sabouraud agar plates did it subsequently produce psilocybin when transferred to liquid media.  A quantitative time-course study of psilocybin production in the two media was carried out.  Maximal production appeared on the fifth day.  The activities of an acid phosphatase, acting on psilocybin, were measured form mycelia grown in the two media.  Enzyme activity from the A culture was very high and a blue color caused by oxidation of psilocin formed in five minutes.

The effect of adding L-tryptophan on alkaloid production as well as the fate of tryptophan-C¹⁴ [that is a radioactive labeled form of tryptophan so it can be traced in the fungus] was also investigated.  Tryptophan stimulated significantly psilocybin production in the very beginning the B medium.  The degradation of tryptophan was different in the two media.  It was converted to kynurenine and anthranilic acid in A medium and to tryptamine in tryptophan added B medium (B%u2019 medium).  Radioactive D,L-tryptophan side chain labeled, gave labeled psilocin and psilocybin

Potassium deficiency decreased psilocybin production while a potassium supplement had no effect.  The fungus did not produce polyacetylenic compounds in the medium but ergosterol was detected as a major acetate derived metabolite when the fungus was kept on MYP agar plates and transferred subsequently to liquid media.  Psilocin has a very slight anti-biotic activity against Candida albicana whereas psilocybin has none.

Eighth species of Lentinus and Lentinellus were investigated for the occurrence of phenol-o-methyltransferase.  Only Lentinus lepideus and Lentinus ponderosus showed enzyme activity in both light and dark conditions.  The specificity of the enzyme for a number of substrates was also examined.  Of six compounds tested, methyl p-coumarate, methyl caffeate and methyl ferulate served as substrates.  The products of enzyme activity were identified by radioautography.

---

Quote:

---

Introduction

%u2026

The chemical synthesis of these two indole alkaloids has been well studied.  The biosynthesis has only been studied by feeding C¹⁴-labled precursors and from these results a pathway has been proposed.  Enzymological studies are meager.  One of the objectives of the present study was to find better growth conditions for the production of psilocybin so as to lead to further studies on enzymology of biosynthesis and psychopharmacology.
 
Psilocybin has been demonstrated to be derived form tryptophan.  And objective of this study concerned the fate of added tryptophan and of [radioactively] labeled tryptophan.  It was interesting to find out if there is any biological significance in the production of these two indole comopoundss.  A brief examination of the cultures for other obvious secondary metabolites, such as polyacetylenes, was also carried out.

---

Quote:

---

Culture (strain of cube):

Psilocybe cubensis was obtained from Dr. S.H. Pollock, Dept. of Pharmacology, University of Texas, USA.  It was reported to produce both psilocybin and psilocin.  The fungus was maintained on slants of malt extract/yeast extract/soytone (MYP, 7 gm/0.5 gm/1 gm) agar.  After growth, the cultures were covered with sterile mineral oil and stored in closed screw cap tubes at 4%u2019C.

---

Below is the culture media A and B the researches used.  When media B was mixed with L-tryptophan that was subsequently taken up the cube mycelium the L-tryptophan was and converted into tryptamine.  For both medias below (A and B), the addition of L-tryptophan increased both alkaloids, more of an increase for psilocybin.  The increase came quickly after the tryptophan was introduced.


Here is the A culture media for P.cubes; L-tryptophan was added to this media and B media:





Here is the B culture media for P.cubes, this one L-tryptophan converted to tryptamine:






If my math* is correct, in the thesis they used ~0.278 ppm of L-tryptophan, thus if we want to use L-tryptophan like they did in the thesis we would add 0.000278 grams per liter (0.278 ppm); or test using more like 0.5 to 1 ppm.  If using 0.278 ppm we will only use a small amount of L-tryptophan so weighing is hard.  It%u2019s best to measure to at last a hundredth of a gram (0.001 gram) because good jewelry and lab scales can be bought for around $75 that will weight a little as 0.001 grams. So if using 0.278 ppm as a goal we can use 7 liters (7,000 ml) of water to which we add and 0.002 grams of L-tryptophan to get about 0.028 ppm in the water; then use it to hydrate the sub (or make and use a stock solution but that%u2019s a little more math I don%u2019t feel like posting right now).

*math: In the thesis they used 2.5 mg/ml to make a stock solution of L-tryptophan (into media A or B); that comes to 2,500 mg/L which is 2500 ppm.  Then they took 10 ml of the stock solution and added it to 90 ml of media A or B; thus the final ppm after dilution should be ~0.278 ppm (i.e. 0.2777777778 ppm):

(((2.5 mg/1 ml)1000)/100)/90 ml = 0.278 ppm

The more accurate equation would be to account for the purity of the L-tryptophan you can buy at the health food store; I%u2019m not sure if it%u2019s pure or not.  In the thesis L-tryptophan was sourced from Sigma, so it could be lab grade.

---

Quote:

---

pinophile said:

Quote:

---

RogerRabbit said:
Don't assume my adventure in mushroom growing started when I made an account here.

---

I wasn't assuming anything, I am sorry if it came across that way.  I wrote "AFAIK not with tryptamine, which is what Gartz used, unless you haven't written about it before?"  In other words, I was saying I have never read anything you have written before in terms of using tryptamine, even when we talked about it before.  If you don't mine could you pm me how you got a hold of tryptamine and the quantities you used and methods you used?  (not that I don't believe you; it's just that what you claim is counter to all data and studies and proof I have been able to find; and counter to what PF found when he used tryptamine). 

I know you used mod be mod over at Topia, and that you have been working with fungi for many decades and I respect you for that; but, from what you have written it seems you haven't read the studies I posted above; or you don't agree with the results, if that is the case could you explain why you don't agree with the results?

Quote:

---

I've used tryptamine, as well as tryptophan. There was no noticeable difference whatsoever.  Now that I understand more about fungi than I did back then, I simply don't see the pathways for it to be converted.  It can be metabolized, but not converted to psilocin.  Prove me wrong please, but do it, not talk about it.

---

Did you not read my posts above?  You were already proven wrong like 50 years ago.  Over 6 studies I posted show above that, in fact, tryptophan and tryptamine is converted into psilocin and psilocybin.  Two papers above detail the probable pathway (there are probably two at least); and like I showed above, it's been proven via radioactive labeled tryptophan that it does convert into tryptamine, psilocybin and psilocin (all three were labeled via conversion from the labeled l-tryptophan).  The same has been done with radioactive labeled tryptamine and even radioactive labled psilocin; both of which produced radioactive labeled psilocybin; thus it proves they all do convert into psilocybin via one pathway or another.  The psilocin used to study radioactive labeled psilocin came from Albert Hofmann and the isolate used in the main thesis I cited was from Steven Pollock

The only question IMO s if application of tryptophan means an increase in the fruit bodies.  And as Gartz showed in two different studies, tryptamine did increase both actives (granted he didn't use an isolate in one study AFAIK); thus I don't think it's a huge leap to think tryptophan can do the same if it's applied correctly (which probably means spraying pins and dunking sub in tryptophan enriched water)

See Figure 3 in this post (link) of mine for one well evidenced pathway of tryptophan into psilocybin, however, it has been sown tryptophan doesn't always convert into tyrptamine (see other references and info I posted above):

tryptophan > tryptamine > N-mytheltryptamine > N,N-dimethyltryptamine > psilocin > psilocybin.


AFAIU it has been shown (from my reading of the studies I cited) that tryptophan increased psilocybin/psilocin in mycelium independently of tryptamine conversion; and that tryptophan converted into tryptamine in some situations. 

The use of radioactively labeled tryptophan, tryptamine, psilocin (and other intermediaries) has shown application of them to media creates radio labeled psilocybin which means they all converted into psilocybin by the fungus either via the pathway above or other pathway.  See Table 1 in this post of mine for results from one study using radioactive substance and see the thesis paper for more current studies using radioactive labeled tryptophan:  http://www.shroomery.org/forums/showflat.php/Number/13203004#13203004

Quote:

---

Eating mushrooms is a great way to test this too.  I can very easily tell the difference between cubes and P cyanescens, so if a cube doubled in potency, I'd certainly be able to tell, as would anybody else.
RR

---

Maybe yes, maybe no, it depends upon lots of factors.  Testing biased upon non-quantitative methods is not the way to go IMO; if we want to prove this one way or another we need data, not first hand opinion from eating them.  That would be like testing THC by smoking cannabis, which isn't valid either.

---

Quote:

---

imachavel

ok now that we know that tryptophan converts into tryptamine(which is useless right?) we know that definately converts into more psilocybin? forgive me for not reading more.

---

Quote:

---

imachavel

that is awesome. I heard that tryptophan wouldn't work in sclerotia because the fungi only produced a certain number of enzymes to transform tryptophan into psilocybin. it's awesome they know this now.

---

Quote:

---

Fruitbuddy said:

Quote:

---

pinophile said:
If my math* is correct, in the thesis they used ~0.278 ppm of L-tryptophan, thus if we want to use L-tryptophan like they did in the thesis we would add 0.000278 grams per liter (0.278 ppm); or test using more like 0.5 to 1 ppm.  If using 0.278 ppm we will only use a small amount of L-tryptophan so weighing is hard.  It’s best to measure to at last a hundredth of a gram (0.001 gram) because good jewelry and lab scales can be bought for around $75 that will weight a little as 0.001 grams. So if using 0.278 ppm as a goal we can use 7 liters (7,000 ml) of water to which we add and 0.002 grams of L-tryptophan to get about 0.028 ppm in the water; then use it to hydrate the sub (or make and use a stock solution but that’s a little more math I don’t feel like posting right now).

*math: In the thesis they used 2.5 mg/ml to make a stock solution of L-tryptophan (into media A or B); that comes to 2,500 mg/L which is 2500 ppm.  Then they took 10 ml of the stock solution and added it to 90 ml of media A or B; thus the final ppm after dilution should be ~0.278 ppm (i.e. 0.2777777778 ppm):

(((2.5 mg/1 ml)1000)/100)/90 ml = 0.278 ppm

The more accurate equation would be to account for the purity of the L-tryptophan you can buy at the health food store; I’m not sure if it’s pure or not.  In the thesis L-tryptophan was sourced from Sigma, so it could be lab grade.

---

well...adding such small amounts of tryptophan will most likely have no effect if you are using grains as substrate since all grains naturally contain more tryptophan than that.

according to the USDA National Nutrient Database rye contains 0.108 g of tryptophan per 100g.

http://www.nal.usda.gov/fnic/foodcomp/search/

---

Quote:

---

pinophile said:
hey

no need to sterilize the water, just add it to the normal spray water used to spray pins/fruits or dunk water used to dunk subs.  I agree that spraying when pins are very young (new cells) is probably a good idea.

I'm not sure about how scleroita act from your question; but roots do have 'defense', they don't just suck up what is in the water.  The plant can auto-regulates uptake of nitrate N, P, Ca, Mg and other elements, etc.  In the case of plants it seems if the ionic solution is too rich the osmotic factors can bypass plant self-regulation of ion uptake.

no problem, your welcome.  I hope some folks like fastfred, workman, pinback, etc., pop in, some people who probably have a better understating than I about the topic at hand.

---

Quote:

---

imachavel said:

Quote:

---

pinophile said:
hey

no need to sterilize the water, just add it to the normal spray water used to spray pins/fruits or dunk water used to dunk subs.  I agree that spraying when pins are very young (new cells) is probably a good idea.

I'm not sure about how scleroita act from your question; but roots do have 'defense', they don't just suck up what is in the water.  The plant can auto-regulates uptake of nitrate N, P, Ca, Mg and other elements, etc.  In the case of plants it seems if the ionic solution is too rich the osmotic factors can bypass plant self-regulation of ion uptake.

no problem, your welcome.  I hope some folks like fastfred, workman, pinback, etc., pop in, some people who probably have a better understating than I about the topic at hand.

---

i know who fast fred and entropymancer are. but why doubt your understanding? you seem to have a good idea.

---

Quote:

---

VizualDistorshon said:
It was more of an epiphany than source material. I was just remembering a bio 1 college lecture I had a few semesters ago.

---

Quote:

---

13shrooms said:

---

nice to have you here