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OfflineScoutScientific
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Medicinal Mushroom Testing * 3
    #28009037 - 10/21/22 09:42 AM (1 year, 3 months ago)

My name is Shawn, I'm a chemist and have worked in pharma for 20+ years.  I started my own analytical testing lab.  In working on a number of projects I started seeing requests for determining the amount of compounds (terpenes) in medicinal mushroom extracted powders.  I was intrigued and have been thinking about medicinal mushrooms ever since.  That was nearly 2 years ago.  Fast forward to this past week and I’m ready to share some of my initial findings with others.

A disclaimer.  I own a company that offers analytical testing and I have some tests that relate to mushrooms.  I’m not promoting that service here.  I am looking for insight that can help me better perform this work and get some ideas for the different directions I can take my research and company.

I started my work with Lions’ Mane, but I’m interested in working with other medicinal mushrooms.  You can read the full “Mushroom Testing Case Study” in the link below, but in general I purchased some extracted mushrooms, and it was my intention to compare them for their terpene content.  What I found was that a couple of them were fake.  I’m sure this information is not surprising to some in this community.  Note, I will not share the brands/vendors.

https://www.scoutscientific.com/services/mushroom-testing/

For the samples where I did see peaks (compounds) in the HPLC, they compare favorably to a mushroom I extracted myself.  I also collected their UV-Spectra and Mass Spec information.  As such, I believe I’ve identified most of the peaks (compounds) in these fruiting body samples.  The possible 40+ compounds in fruiting body are well known and I’m not discovering anything new here.  My questions:

1) A few of the peaks are at similar ratios to each other (in height/area) regardless of sample.  As of now, I believe this simply means that the terpene ratio amounts in the mushroom are the same (similar).  Some of the compounds (in the middle of the chromatogram) are always different.  I’d like to understand why.  Could the amount and types of terpenes vary in Lion’s Mane based on where it’s grown?  What it’s grown on?  The weather?  How it’s extracted?  The strain?

2) I understand how extracted powders are produced.  Next, I’m working towards testing commercial Lion’s Mane tinctures (again from Amazon).  I’d like to understand the choices of extraction media (water, ethanol) and why glycerin seems to be used in many of the commercial tincture products.  Thoughts?  Lastly, any idea of the number of fake products for tinctures as compared to extracted powders?  Seems like it should be higher, since water/glycerin mixtures without mushrooms are easier to produce.

3) Is heavy metals content as big of a concern as it should be?

4) Reviewing my work, do you have any questions that I should address on the website?


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OfflineLand TroutM
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Re: Medicinal Mushroom Testing [Re: ScoutScientific] * 1
    #28009100 - 10/21/22 10:48 AM (1 year, 3 months ago)

Cool, nothing more to add.  Thanks for jumping in here with us!


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Invisiblebakedbeings
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Re: Medicinal Mushroom Testing [Re: Land Trout] * 1
    #28009224 - 10/21/22 12:20 PM (1 year, 3 months ago)

:whathesaid:


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OfflineTheUsualSuspect
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Re: Medicinal Mushroom Testing [Re: bakedbeings] * 2
    #28009580 - 10/21/22 04:03 PM (1 year, 3 months ago)

In reading a lot of the mushroom powder sales literature, many discuss if they are using just the fruiting body or also the ground up mycelium to produce the powder. I would expect fungi perfecti to be using a different substrate mix than om mushrooms. If someone were just using fruiting body, or only mycelium, or a mix of both, or a mix of both and different substrates, thats where the difference is coming in. The mushroom mixes you tested missing the critical compounds and that seem fake might just be powdering up fruiting bodies. Maybe the sellers dont even know.

I'm just taking a wild guess at the cause.

In any event its pretty cool to have you on the board.

Do you have the capacity to test for psilocybin and psilocin content and ratios of a dried sample? Have you considered offering that as a service? I think there are a lot of changes in progress across the country in legalizing mushrooms and similarities in how the legal weed business evolved vs how a legalized mushroom business would evolve. I wouldnt be surprised if that type of testing gets incorporated into legal framework at a state level.

/ edit 
And as far as you being a chemist with 20 years exp wondering why people on etsy and amazon are using x or y chemical in their solutions, you have to think, these sellers might just be some dude mixing up powders they bought off ebay in their garage on the work bench they just disassembled the lawn mower on. The suppliment business im sure has to be full of 'hold my beer and watch this' type people. I wouldnt expect actual science there.


Edited by TheUsualSuspect (10/21/22 04:07 PM)


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OfflineScoutScientific
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Re: Medicinal Mushroom Testing [Re: TheUsualSuspect] * 2
    #28010455 - 10/22/22 09:41 AM (1 year, 3 months ago)

I agree if a company is using fruiting body vs mycelium or a mix of both I should see differences and it's something I'll investigate further.  Thank you for the idea! Because I can identify the compounds and it's known via literature that certain compounds only appear in one form vs another, it can be simple to figure out too.  In this case however, two of them are just grain or starch and do not contain ANY Lion's Mane compound, regardless of source.  The Beta-Glucan testing I did backs this up too.

I've been asked a couple times about psilocybin and psilocin testing.  I'm actually interested in it and I could do this, however I have a couple hurdles.  First, the reference standards materials for this are crazy expensive.  I couldn't absorb this cost and it would have to be included in the analysis which makes it much more costly for potential clients.  If I knew I could get lots of clients then maybe I could spread it out.  I could also work on isolating and purifying the compounds myself, but then there's the fine line of my second point - legality.  I'm simple not sure where the analysis of these compounds is right now with my home state of North Carolina.  I'm sure there are plenty of experts here that can help.  :cool:

There is certainly a variety of dietary supplement businesses out there.  Without the regulations or oversight (sadly), it's a wild west of mix up stuff and sell it as the cure to everything.  Right now I could throw a stick and hit 10 people needing a chemist to formulate their "all natural, no organics, herbal cosmetics" startup.  Not saying it's not possible, but you're competing with multi-billion dollar corporations with multi-million dollar research budgets.

I've helped more than one company to not buy massive wholesale stock products by testing the sample materials the company sent them.  It's a win for me and makes me feel good to help them avoid making a big mistake.  It's a bummer for them as they thought they had the deal of the century.


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Invisiblejoze
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Re: Medicinal Mushroom Testing [Re: ScoutScientific] * 1
    #28015814 - 10/25/22 12:02 PM (1 year, 3 months ago)

Quote:

ScoutScientific said:
1) A few of the peaks are at similar ratios to each other (in height/area) regardless of sample.  As of now, I believe this simply means that the terpene ratio amounts in the mushroom are the same (similar).  Some of the compounds (in the middle of the chromatogram) are always different.  I’d like to understand why.  Could the amount and types of terpenes vary in Lion’s Mane based on where it’s grown?  What it’s grown on?  The weather?  How it’s extracted?  The strain?

2) I understand how extracted powders are produced.  Next, I’m working towards testing commercial Lion’s Mane tinctures (again from Amazon).  I’d like to understand the choices of extraction media (water, ethanol) and why glycerin seems to be used in many of the commercial tincture products.  Thoughts?  Lastly, any idea of the number of fake products for tinctures as compared to extracted powders?  Seems like it should be higher, since water/glycerin mixtures without mushrooms are easier to produce.

3) Is heavy metals content as big of a concern as it should be?

4) Reviewing my work, do you have any questions that I should address on the website?



Thank you for sharing your work with us. I look forward to hearing more from you on these forums.

1. I think it's likely to assume that some of the terpenes are produced as a result of the substrate. I know nothing about this topic, so this is a hypothetical example, but I would not be surprised if mushrooms grown on conifers had a higher pinene content than those grown on hardwoods. Other environmental factors, both abiotic and biotic, would affect metabolite content as well. Given that many of these terpenes could have anti-viral or anti-bacterial properties in humans, I think it's safe to assume some mushrooms use these terpenes for similar purposes. So mushrooms growing in conditions with lots of bacterial pathogens could feasibly express a higher amount of these defensive terpenes. How well the mushrooms are extracted could also be affecting the end result. Are some of these terpenes you're looking at volatile?

2. I can't really comment on this much, but I think that glycerin is usually used to thicken it and make it easier to use in a dropper compared to an aqueous solution.

3. Who knows. I think the research on this is pretty sparse. The heavy metal content of the mushrooms is really going to be determined by the heavy metal content of their substrate, but maybe some species of fungi are hyper-accumulators.

4. I think your website is very thorough. :thumbup:


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OfflineScoutScientific
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Re: Medicinal Mushroom Testing [Re: joze]
    #28017474 - 10/26/22 11:53 AM (1 year, 3 months ago)

Quote:

joze said:
Thank you for sharing your work with us. I look forward to hearing more from you on these forums.

1. I think it's likely to assume that some of the terpenes are produced as a result of the substrate. I know nothing about this topic, so this is a hypothetical example, but I would not be surprised if mushrooms grown on conifers had a higher pinene content than those grown on hardwoods. Other environmental factors, both abiotic and biotic, would affect metabolite content as well. Given that many of these terpenes could have anti-viral or anti-bacterial properties in humans, I think it's safe to assume some mushrooms use these terpenes for similar purposes. So mushrooms growing in conditions with lots of bacterial pathogens could feasibly express a higher amount of these defensive terpenes. How well the mushrooms are extracted could also be affecting the end result. Are some of these terpenes you're looking at volatile?

2. I can't really comment on this much, but I think that glycerin is usually used to thicken it and make it easier to use in a dropper compared to an aqueous solution.

3. Who knows. I think the research on this is pretty sparse. The heavy metal content of the mushrooms is really going to be determined by the heavy metal content of their substrate, but maybe some species of fungi are hyper-accumulators.

4. I think your website is very thorough. :thumbup:




Thank you for the comments, ideas and kind words.  As of now, I'm not focused on the volatile terpenes, but if I were, I'd have to use GC-FID/MS.  I've purchased some LM tinctures.  Should be another interesting round of testing coming soon.


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OfflineDharmaForKarma
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Re: Medicinal Mushroom Testing [Re: ScoutScientific]
    #28029208 - 11/02/22 09:56 AM (1 year, 2 months ago)

In the context of ideas, this one does not appear to align with your company’s current capabilities. However, it’s our holy grail:
A home test kit to detect and measure substances of interest in magic mushrooms. This would be a game changer.


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OfflineScoutScientific
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Re: Medicinal Mushroom Testing [Re: DharmaForKarma]
    #28029390 - 11/02/22 12:56 PM (1 year, 2 months ago)

Quote:

DharmaForKarma said:
In the context of ideas, this one does not appear to align with your company’s current capabilities. However, it’s our holy grail:
A home test kit to detect and measure substances of interest in magic mushrooms. This would be a game changer.




With the right setup, either the lateral flow type or some other, could be used for detection.  Quantitation is more of a challenge.  Perhaps a colorimetric type test to give an idea of level, similar to what is used for determining the amount of chlorine in a pool or the pH.


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OfflineShu
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Re: Medicinal Mushroom Testing [Re: ScoutScientific]
    #28029924 - 11/02/22 06:42 PM (1 year, 2 months ago)

Glad to see an analytical chemist in business for themselves. I'm a retired biopharma analytical chemist of 40 years. I always wanted to work for myself but didn't have the balls for it. Best of luck to you.

Disclaimer: I have no lab experience with terpenes.

I read over the linked page and FWIW, IMHO I have a few comments.

Ideally I agree that each terpene should be identified and quantified on the product label but practically it would be a formidable task to replicate such a formulation batch to batch. Perhaps listing the major terpene or three would be sufficient / acceptable. I assume a standardized extract would be based on only one terp or on total terps. Of course raw material and intermediate certs could contain the "entire" list.

Sourcing or manufacturing and certifying reference materials (especially when considering all medicinal species) is a bitch. This is another reason to limit the number of analytes. Also, ELSD and perhaps CAD detection may alleviate the need for so many RMs, if your mobile phase permits.  Only when specific active cpds are identified and published would I consider additional quantitative reference production.

I agree that heavy metals is an important starting material test and in the cases of dried and extraction products, so is a residual solvents test.

The McCleary paper seems to be missing any discussion of b-glucan / glucose recovery of purified polysaccharides such as cellulose, curdlan, scleroglucan, yeast, wheat or barley for which they provide quantification. This silence disturbs the analytical chemist in me.

1) Not having seen your data my bet is on "how it's extracted." Consider both how the product is manufactured and how you extract the product for analysis. Ratios that don't vary imply partitioning coefficients and equilibria. Those that do vary imply variable extraction efficiencies.

2) Glycerine is likely a stabilizer and/or viscosity modifier.

3) If you're asking about the emphasis you place on HMs on your webpage then I suggest you add a stock blurb about their toxicity and ubiquity. Also, your customers are unlikely to care about the old USP test (unless you offer it).

Disclaimer: I do not know how extracted powders are produced.

4) You state that two samples "were not lion's mane" whereas what I believe you've shown is that they contain undetectable amounts of terpenes and / or glucans characteristic of your proprietary LM extract. Perhaps your subjective knowledge allows your original conclusion.


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OfflineScoutScientific
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Re: Medicinal Mushroom Testing [Re: Shu]
    #28030763 - 11/03/22 07:26 AM (1 year, 2 months ago)

Quote:

Shu said:
Glad to see an analytical chemist in business for themselves. I'm a retired biopharma analytical chemist of 40 years. I always wanted to work for myself but didn't have the balls for it. Best of luck to you.

Disclaimer: I have no lab experience with terpenes.

I read over the linked page and FWIW, IMHO I have a few comments.

Ideally I agree that each terpene should be identified and quantified on the product label but practically it would be a formidable task to replicate such a formulation batch to batch. Perhaps listing the major terpene or three would be sufficient / acceptable. I assume a standardized extract would be based on only one terp or on total terps. Of course raw material and intermediate certs could contain the "entire" list.

Sourcing or manufacturing and certifying reference materials (especially when considering all medicinal species) is a bitch. This is another reason to limit the number of analytes. Also, ELSD and perhaps CAD detection may alleviate the need for so many RMs, if your mobile phase permits.  Only when specific active cpds are identified and published would I consider additional quantitative reference production.

I agree that heavy metals is an important starting material test and in the cases of dried and extraction products, so is a residual solvents test.

The McCleary paper seems to be missing any discussion of b-glucan / glucose recovery of purified polysaccharides such as cellulose, curdlan, scleroglucan, yeast, wheat or barley for which they provide quantification. This silence disturbs the analytical chemist in me.

1) Not having seen your data my bet is on "how it's extracted." Consider both how the product is manufactured and how you extract the product for analysis. Ratios that don't vary imply partitioning coefficients and equilibria. Those that do vary imply variable extraction efficiencies.

2) Glycerine is likely a stabilizer and/or viscosity modifier.

3) If you're asking about the emphasis you place on HMs on your webpage then I suggest you add a stock blurb about their toxicity and ubiquity. Also, your customers are unlikely to care about the old USP test (unless you offer it).

Disclaimer: I do not know how extracted powders are produced.

4) You state that two samples "were not lion's mane" whereas what I believe you've shown is that they contain undetectable amounts of terpenes and / or glucans characteristic of your proprietary LM extract. Perhaps your subjective knowledge allows your original conclusion.




Thank you for your comments!

I think the goal of some of the vendors is to quantitate a few key compounds that have been found to offer the most benefit.  But I agree that batch to batch variability will be a quality nightmare if all peaks/compounds were to be monitored.

Right now the method is a simple UV method that is MS friendly (on purpose).

Extracted powders:  Grind/blend dried mushrooms into small pieces, mix with solvent (hot/cold water and/or EtOH), filter, spray dry resulting solution, collect super fine powder, package.

As for the two samples that do not contain lion's mane mushroom, they don't.  I know the convention is to say that they are undetectable by UV, but my MS didn't detect these compounds either (nothing in the MS TIC or by targeted analysis).


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Invisibleilus
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Re: Medicinal Mushroom Testing [Re: ScoutScientific] * 1
    #28032584 - 11/04/22 12:26 PM (1 year, 2 months ago)

Hey Shawn!  This is Travis from our e-mails.  I'm super happy to see you on here and hope that it will connect you with some chemists and customers.  Lots of interesting people on here.  Check out the Chemistry forum here too if it's available for you.

I'm most excited to read your research and conclusions on mycelium vs. fruiting bodies.  That has been such a hot topic in the medicinal mushroom community with not too much third party scientific involvement that doesn't have a substantial financial interest. I'm very impressed with your website and interest in all of this, you are obviously very thorough with your work and interested in actual  science and truth.

Quote:


Lastly, any idea of the number of fake products for tinctures as compared to extracted powders?  Seems like it should be higher, since water/glycerin mixtures without mushrooms are easier to produce.





In my experience it has been primarily powders that have been faked.  Even though it's easier to create a water/glycerin mixture, it's harder to replicate the taste and potency of a good tincture.  I was very surprised to have similar results to you where about half of the products I found on Amazon were fake or had a filler (even on bags that said "NO FILLER" in huge font on it!). 

I feel like one can separate it by price, nearly anything on there that is super cheap is going to be fake or heavily cut at a minimum.  When a tincture is even diluted with water or ethanol it is noticeably weaker tasting.  Also, if you're going to go through the process to make a tincture then you may as well just do it properly.  I think it's in human nature to want to cut powders.  :lol:


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OfflineScoutScientific
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Re: Medicinal Mushroom Testing [Re: ilus]
    #28037726 - 11/07/22 06:48 AM (1 year, 2 months ago)

Quote:

illustrain said:
Hey Shawn!  This is Travis from our e-mails.  I'm super happy to see you on here and hope that it will connect you with some chemists and customers.  Lots of interesting people on here.  Check out the Chemistry forum here too if it's available for you.

I'm most excited to read your research and conclusions on mycelium vs. fruiting bodies.  That has been such a hot topic in the medicinal mushroom community with not too much third party scientific involvement that doesn't have a substantial financial interest. I'm very impressed with your website and interest in all of this, you are obviously very thorough with your work and interested in actual  science and truth.




Thanks man!


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Offlinecaptaink
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Re: Medicinal Mushroom Testing [Re: ScoutScientific]
    #28038079 - 11/07/22 11:23 AM (1 year, 2 months ago)

Hopping on :smile:


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