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Cubensis [Data Log]


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#41 Ferather

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Posted 10 January 2019 - 04:10 PM

I conclude my Cubensis test.
 
Cubensis can and will target phenols-other, and decay them as the sole carbon source (no sugar, starch or cellulose).
However to do so, it must also have the essential macro-micro nutrients present, with the carbon source.
 
The Cubensis produced it's own carbon materials from the phenols: glucose, proteins, so on.
 
----
 
Notes:
 
Double concentrate required no additional nutrients, however it's slower than without.
Sucrose, MG and YN had beneficial impact to the available pool of nutrients.
The addition of nutrients improved growth and supported adaptation.

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#42 PistolPete13

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Posted 11 January 2019 - 04:54 PM

Hello Ferather, nice thread!

 

I don't know if you were aware but the Psilocybe Cubensis genome has been mapped and is available to search at the governments mycocosm portal.

https://genome.jgi.d...ub1_1.home.html

Anyway browsing it reveals that surprisingly Cubensis does posses the genes usually only seen in white rot fungi that produce ligninolytic enzymes capable of degrading lignin including but not limited to laccase and manganese peroxidase. Not every specimen is going to express these genes fully, there has been quite a lot of research done on various basidiomycetes (oysters, button ect) that focus on gene expression of these very genes for commercial purposes.

 

One of the most significant gene regulators it turns out is nitrogen! If a cube finds itself growing in a nutrient dense manure that is obviously high in nitrogen, the nitrogen inhibits expression of these genes(why waste all that time and energy making complex enzymes you dont need, if you have a much easier and instantly available food source that you could be getting energy from right now). And in nature it would compete for an easy meal like that with many other things, once it has competed for and eaten the easy food and got all the energy it could from it as quick as possible before something else gets it, at this point there would be no nitrogen left and these enzymes would start flowing and it gets to work on the more difficult substrates. Also Manganese Sulfate has been shown to promote these genes in some species of mushroom.

 

There is a strain called 'Lizard King' that was apparently found growing on wood, might be a nice strain to play around with? A better method would be to create a multi-spore of any strain and select for individuals that do better(or even just survive at the start) on woody substrates.


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#43 Ferather

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Posted 11 January 2019 - 05:18 PM

Noooooiiicceeeee data, here is some for you (from my journal, in my signature):

 

Bacterial Growth and Morphology found in Tea

Enzymatic Degradation of Lignin in Soil

Phenolic content in Tea

 

I'm temped to put this sample to enriched wood to see how well that goes.

I've tried it on WL-Tek + tea, but it was very slow (and acidic).

 

----

 

IMG_20190111_203315.jpg IMG_20190111_203751.jpg IMG_20190111_203756.jpg


Edited by Ferather, 11 January 2019 - 05:22 PM.


#44 Mushinist

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Posted 11 January 2019 - 05:19 PM

Hello Ferather, nice thread!
 
I don't know if you were aware but the Psilocybe Cubensis genome has been mapped and is available to search at the governments mycocosm portal.
https://genome.jgi.d...ub1_1.home.html
Anyway browsing it reveals that surprisingly Cubensis does posses the genes usually only seen in white rot fungi that produce ligninolytic enzymes capable of degrading lignin including but not limited to laccase and manganese peroxidase. Not every specimen is going to express these genes fully, there has been quite a lot of research done on various basidiomycetes (oysters, button ect) that focus on gene expression of these very genes for commercial purposes.
 
One of the most significant gene regulators it turns out is nitrogen! If a cube finds itself growing in a nutrient dense manure that is obviously high in nitrogen, the nitrogen inhibits expression of these genes(why waste all that time and energy making complex enzymes you dont need, if you have a much easier and instantly available food source that you could be getting energy from right now). And in nature it would compete for an easy meal like that with many other things, once it has competed for and eaten the easy food and got all the energy it could from it as quick as possible before something else gets it, at this point there would be no nitrogen left and these enzymes would start flowing and it gets to work on the more difficult substrates. Also Manganese Sulfate has been shown to promote these genes in some species of mushroom.
 
There is a strain called 'Lizard King' that was apparently found growing on wood, might be a nice strain to play around with? A better method would be to create a multi-spore of any strain and select for individuals that do better(or even just survive at the start) on woody substrates.


Ha, I knew that was you researcher!
And sorry OP for bustin in on your thread, nice experiment btw!
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#45 Ferather

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Posted 11 January 2019 - 05:22 PM

My threads are "open" to all, type whatever you like, as long as it's related to the thread, and not random.

 

:tongue:


Edited by Ferather, 11 January 2019 - 05:23 PM.

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#46 PistolPete13

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Posted 11 January 2019 - 07:28 PM

 

Ha, I knew that was you researcher!
 

 

:wink: aka Peter65199

 

That second paper you linked I have read before, they found that cubensis secretes manganese peroxidase(MnP). I have a few other papers documenting cubensis secreting laccase, but with the genome you do not need to rely on research now to find out what they can and cant produce. A quote from the paper;

 

Compared to laccase, MnP causes greater degradation of phenolic lignin due to its higher redox potential.

 

This is the first time I have come across this thread so still thinking about it, but are the tannins contained in tea not a potential problem?  May be why you got slow growth?

 

If you are going to enrich the wood it might be an idea to starve it of nitrogen, also manganese sulfate is super cheap in the garden center.

 

I can tell you something interesting I dont think anyone else will know.....I found that laccase is used to degrade indigo into anthranillic acid, in fact there has been an effort to screen many types of ligninolytic enzyme producing microbes for their ability to break down indigo dye(a pollutant). And to detect the presence of laccase they dye a submerged culture purple with indigo, and see how quickly the microbe can decolorize it.

 

The degradation of the textile dye indigo with purified laccases from the fungi Trametes hirsuta (THL1 and THL2) and Sclerotium rolfsii (SRL1) was studied. All laccases were able to oxidize indigo yielding isatin (indole-2,3-dione), which was further decomposed to anthranilic acid (2-aminobenzoic acid).

 

https://www.ncbi.nlm...pubmed/11500206

 

So what..... Have a look at this paper!

 

 

This is the first report that shows that medium modification through serine or anthranilic acid supplementation affects the content of indole compounds in the resulting biomass. The concentration of the additive added to the medium is also important in the context of quantitative and qualitative content of these compounds in the resulting biomass. It indicates that modification of medium for mycelial cultures may be crucial for the potential production of lyophilized biomass containing specific indole compounds at suitable amounts that can be used in the supplementation of indole derivatives.

https://akademiai.co...1326.2017.00325

 

The witnessed increased indole levels across the board, for example when the medium was supplemented they started seeing bisporus producing  decent levels of L-tryptophan and tryptamine which is normally only ever seen in tiny nearly undetectable trace amounts, sometimes.

 

The catch is anthranillic acid is a scheduled chemical, nearly impossible for mere mortals to obtain! But indigo is cheap.

 

Sorry for off-topic ferather but I thought you may be interested!


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#47 Ferather

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Posted 12 January 2019 - 10:06 AM

No that's all totally in-on topic, continue to post away. By random I mean more like posting images of mold on cheese or something (lol). What makes you say tannin is an issue?

 

Edit, found some data which back's up your data, in terms of nitrogen and manganese peroxidase, here, very interesting.

 

====

 

[Recipe A]: 100g Water, 2.4g Agar, 1.6g Black Tea (0.6g Soluble), 0.8g Sucrose, 0.04g MG.
[Recipe B]: 100g Water, 4g Agar, 3.2g Black Tea (1.2g Soluble). I used hard water *.
 
IMG_20181118_232405.jpg IMG_20181122_133240.jpg IMG_20181125_115425.jpg
IMG_20190111_203315.jpg IMG_20190112_155452.jpg IMG_20190112_155626.jpg
 
Some agar is lost to the removed tea and bag.
* 150g microwaved down to 100g.

Edited by Ferather, 12 January 2019 - 11:30 AM.


#48 PistolPete13

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Posted 12 January 2019 - 10:59 PM

Regarding tea I was under the impression it has very high levels of tannins which are antifungal, I had a very brief look at Mycocosm and I could not locate any tannase encoding genes in the cubensis genome. I might have a better look tonight, but unlike the wood lovers which break down tannins and grow quite well on tea. It seems the price you are going to pay for the benefits of tea with cubensis, is going to be slow growth.

 

 

Testing the effects of tannin rich plant extracts on mycelium and contaminant growth

Tea and oak extract based MEA does appear to inhibit the growth of some airborne bacteria and fungi. Standard MEA is surprisingly resistant to contamination as evidenced by the relatively small number of CFUs on the control plate exposed to open air for 15 minutes. Oak and tea extracts have an inhibitory effect on the growth of Psilocybe allenii, however, this effect may be useful to aid in the selection of tissues well suited to digesting tannin rich substrates. The low growth in the tea extract plate may be the result of too much tea, perhaps the recipe could be tuned further.

 

https://www.shroomer...Number/25219070

 

Thanks for the link btw, there are few more leads in there for potential gene regulators!

 

@Ferather do you believe that growing cubensis on wood will produce more potent mushrooms? In nature the wood lovers are much more potent. I am not saying it will, but would love to know your thoughts on the matter.



#49 Ferather

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Posted 13 January 2019 - 07:08 AM

Phenols are thankfully more generic than specific enzymes (as such). As far as I know, all phenols decay to oxygen, and do not strictly need enzymes to break down.

Technically speaking, any enzyme or reaction that produces radicals or has oxidative effects will breakdown materials that breakdown to oxygen.

 

This is the same for active materials, which are also phenolic compounds, they breakdown to oxygen regardless of enzymes.

Another thing to consider is the pH, and the effective pH of enzymes and relevant reactions.

 

Mycelium produce antioxidants, not only to grow in oxygen, but also to utilize it (like wearing gloves for the oven).

 

http://www.chem.ucal...4/ch24-3-5.html

https://en.wikipedia...xidative_stress

 

----

 

Active mycelium:
 
Baeocystin:  [C11]--[H15]--[N2]--[O4]--[P]
Norbaeocystin: [C10]--[H13]--[N2]--[O4]--[P]
 
Psilocybin:  [C12]--[H17]--[N2]--[O4]--[P]
Psilocin:  [C12]--[H16]--[N2]--[O]
 
 
From above:
 
"Psilocybin is a tryptamine compound with a chemical structure containing an indole ring linked to an ethylamine substituent. It is chemically related to the amino acid tryptophan, and is structurally similar to serotonin."
"Psilocybin is a member of the tryptophan-based compounds that originally functioned as antioxidants in earlier life forms before assuming more complex functions in multicellular organisms."
 
"Psilocin is relatively unstable in solution due to its phenolic hydroxy (-OH) group. In the presence of oxygen it readily forms bluish and dark black degradation products"
"Most species of psilocybin-containing mushrooms bruise blue when handled or damaged due to the oxidization of phenolic compounds"
 
"Psilocin is broken down by the enzyme monoamine oxidase. Some psilocin is not broken down, and forms a glucuronide"
 
 
Notes:
 
Nor-baeocystin are analogs of psilocybin, meaning single or various elements are added or removed.
It appears they can be phosphorylated or dephosphorylated (cleaved, added, a cycle?).
All four compounds contain carbon, hydrogen, nitrogen and oxygen.
 
Serotonin:  [C10]--[H12]--[N2]--[O]

Edited by Ferather, 13 January 2019 - 07:31 AM.


#50 PistolPete13

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Posted 13 January 2019 - 09:33 PM

First off, I was not 100% sure what the T-gel you were referring to was. And I have to say after finding out, WELL DONE!!! I dont know if you get a lot of kudos for that(or if many have even tried it) but you deserve it (maybe one day people will catch on and thank you).

 

Regarding the non-enzymatic oxidation of tannin, it does happen but a lot slower than the life of an average plate. But surely that does not even matter! Growth is slowed by the anti-nutritional properties of tannin, it is not stopped. From what I see you have stopped the growth(to a large extent) of unwanted microbes, and your desired culture still grows. A lot of antibiotics slow growth also, that is a win in my book. Sure most white rot fungi will grow just as quick on that as normal agar, because they posses the necessary enzymes to break down tannins and use them for carbon. I searched through the cubensis genome and there are no tannase or polyphenol oxidase in sight, and without them breaking down that polymer is not as easy as a simple phenol.

 

Also technically the actives are not phenols, they posses a phenolic hydroxy group, as you pointed out above. Which basically means they have an (OH) attached to the aromatic ring. It increases acidity, and also makes it reactive towards things like acids and water that have H+ (protons) to donate or share respectively. It does not make them behave at all like simply phenols.


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#51 Ferather

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Posted 14 January 2019 - 04:53 PM

1: Hehe yeah, I wasn't really talking about the fact T-Gel is designed to prevent infection.
2: I apologize, my phenol terming was based from here, too generic?
 
:biggrin:


#52 PistolPete13

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Posted 14 January 2019 - 06:25 PM

Don't apologize! It was a bit generic though as it is only a functional group on a much larger molecule, the actives are indoles and behave much more like indoles than phenols. In fact they are very much the same except that phenolic functional group increases the molecules acidity and makes it more reactive to certain things, so indoles bearing a phenolic hydroxyl group (OH) attached directly to the aromatic ring for example will be more acidic than other indoles. And that is because of the somewhat loosely held H in the (OH) that is attached, it can lose its H as a cation H+ and act a s proton donor (Brønsted acids are proton donors and Brønsted bases are proton acceptors). H+ is the simplest ion it is just one proton without any electrons, and acids like H2SO4 (sulfuric) HCl (hydrochloric) and so on all start with a H and the more reactive and stronger the acid is he more loosely it is holding onto its H, and the more easily it will give it up and why they are referred to as proton donors.

 

Hehe yeah, I wasn't really talking about the fact T-Gel is designed to prevent infection.

It is just I was little slow to catch on to what T-gel was when I found out, I was a little impressed! I have seen on forums recently (i am sure you have too) about people extracting antibiotics from over the counter meds, ect. And this would have to be a 100% better option IMO.


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#53 Ferather

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Posted 15 January 2019 - 04:33 PM

Ok thanks. That's great info.

 

I have seen a few posts. There is also a member on another forum who tested LME, LME + Gentamycin and T-Gel on a bacterial wild gilled polypore, Trametes betulina.

Only T-Gel worked, black tea is also much cheaper than gentamycin, in addition T-Gel needs no ME at all, other additives like ME or PD are optional.

 

https://www.shroomer...708438#25708438

https://www.shroomer...721501#25721501

https://www.shroomer...732364#25732364

 

Mycelium should, but not always, adapt to the materials, nutrients and settings of the extract, and improve with transfer. Some may stall to start with.

My first test with Cubensis stalled, however by increasing the continual oxygen level it continued and improved in growth.

 

The same can be said for any oyster, although oyster mycelium is much faster on T-Gel.

 

====

 

Now to say oxygen lots of times:
 
In anaerobic conditions, on T-Gel, oyster grew to oxygen. It also brought up materials to oxidize to oxygen.
At first growth was weak, as phenols decay to oxygen, and relevant enzymes need oxygen.
 
The oyster became thicker, more populated, and more robust to higher oxygen.
 
IMG_20180125_124225.jpg IMG_20180124_125441.jpg

 

====

 

https://mycotopia.ne...-3#entry1392779

 

The new plate (recipe B, above), has turned radial, growth is thick and populated, it's clean but not quick.
 
IMG_20190115_215545.jpg IMG_20190115_215630.jpg IMG_20190115_215828.jpg
 
The master plate (recipe A, above), has bleaching or whitening, I removed the pins.
 
IMG_20190115_215252.jpg IMG_20190115_215223.jpg

Edited by Ferather, 15 January 2019 - 05:26 PM.


#54 Ferather

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Posted 23 January 2019 - 04:45 PM

Cubensis experimental wedge to T-Gel (updated recipe, today). See my signature for the updated recipe.
I have reverted back to Intralabs food grade agar. It's 250% better, and needs less.
 
I'm using T-Gel plain, which means no optional malt extract.
 
IMG_20190123_155958.jpg

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#55 PistolPete13

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Posted 23 January 2019 - 07:47 PM

Thanks for sharing your journal! :thumbs_up:  As soon as my jars colonize I can start contributing some research....


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#56 Ferather

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Posted 24 January 2019 - 07:09 AM

I'm not so sure you can strictly 'improve' potency, if that's what you are looking for, however, it's very possible to adapt mycelium and dial in conditions that reach the genetic potency cap.

 

Some notes of mine:

 

> Mycelium need nitrogen to produce ample active materials, as well as other materials, so lets say it had a nitrogen deficiency, this would effect production and yield.

> Cubensis grown in gourmet conditions (lots of oxygen, light and humidity) bruised more than when grown in low-medium oxygen, moderate conditions.

> The physical mass of Cubensis is higher than most other actives, it's potential for nutrient capacity is actually higher than thin alternatives.

> Increased oxygen would increase overall oxidative stress, and the correct response would be to produce more antioxidants.

> Total physical yield should reduce if more active materials are being produced, leaving less for other materials.



#57 MycoBabe

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Posted 24 January 2019 - 11:22 AM

Hello!  Ok.. so, I honestly didnt get to read through all of this yet, bc my kid is tearing shit up downstairs ..lol

 

  But I wanted to let you know that i am very impressed! I have been growing for 10 years, and I guess I havent seen too many people break it down like that... Or maybe it's your notations...   However... Well Done.. I did download your journal too..

 

I was a microbiologist and chemist during my career...For 20 years... I'm not tooo old.. just a little old..lol...  So I am a nerd that  just looks like some stoner hippy chick... Yah, they think i stupid..lol.. However.. 20 year career as a scientist, then to disability.. I read tarot cards and meditate , and am a Reiki Master now..

 

I love to talk about the things I love... But the people all around me don't think like that.. when i google something, I have to spend days on that subject, to know everything about it..  Sorry, I am rambling right now bc I'm stoned..

 

I have been really stressed out lately... and I was having a horrible day.. But I logged in here for a couple just to browse around.. bc I need to make a room more sterile... I don't have a hood.. Long story.. But your research and attention to detail actuallly brightened my day... And I just wanted to say thank you,, 

 

I will definitely be following this.. 


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#58 Ferather

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Posted 25 January 2019 - 02:28 PM

@ MycoBabe

 

Thank you very much, I am here to help. There are plenty of things I don't know, even terms and pronunciation elude me at times.

If I do anything wrong let me know, so I can correct myself. My journal is an eBook, and subject to updates (free).

 

:hug:

 

====

 

The Cubensis to T-Gel plain (latest recipe) is working well, I have strong healthy growth.


Edited by Ferather, 25 January 2019 - 02:29 PM.


#59 PistolPete13

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Posted 25 January 2019 - 03:28 PM

I was only going to try to reach the full genetic potential.... :biggrin:

 

Thanks for the notes, I have just been reading a thesis about submerged culture that also has some interesting notes;

 

  • Catalfomo and Tyler found that higher levels of glucose resulted in higher yields of psilocybin in Psilocybe cubensis.
  • The nitrogen source is also important in revealing alkaloid-producing strains (49). The differences in nitrogen sources in MYP and Sabouraud may also account for the alkaloid production.
  • Comparing the yield of psilocybin on two different media, fungus from B-medium gave a better yield but a poorer growth rate. Abe and co-workers stress that the fact that slow growth is required for alkaloid production(54). Demain suggested that rapid growth during trophophase involves a balanced assimilation of all essential nutrients with a minimum of accumulation of metabolites.
  • Tryptophan does not appear to have a clear-cut role in regulating psilocybin biosynthesis in all species which produce it. Several workers have shown there is no consistent relationship between tryptophan addition and psilocybin production. In this work L-tryptophan stimulated psilocybin production by almost a factor of two in the very beginning in B'-medium. On the other hand, in  A'-medium, the stimulation was not so significant.
  • In this work, potassium deficient conditions decreased the psilocybin yield without changing growth or.medium pH. Potassium supplement had no effect at all.
  • In A and A' media, the fungus metabolized tryptophan mainly through kynurenine and anthranilic acid; while in B'-medium instead of kynurenine, tryptamine was formed.
    Kaplan et al.(63) concluded that the degradation of tryptophan by the kynurenine pathway did not play a significant role in the metabolism of tryptophan, under conditions of high alkaloid production in Claviceps strain SD 58.
  • Fluctuating rates of psilocybin production could be seen in both A and B media. Rahacek et al.(53) suggested in Claviceps that alkaloid production commences while tryptophan  synthetase activity is increasing. Alkaloid formation is suggested to reflect a regulatory device to keep endogenous tryptophan levels in balance.---Another reason for these fluctuations could be the heterogenous growth of the mycelium.

https://open.library...items/1.0094541


Edited by PistolPete13, 25 January 2019 - 03:32 PM.

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#60 Ferather

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Posted 27 January 2019 - 07:19 AM

T-Gel wedge to brown rice, the wedge is visibly bleaching.
 
IMG_20190127_105556.jpg IMG_20190127_105811.jpg





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