|
Josex
#cheat_code



Registered: 11/13/15
Posts: 8,995
Loc:
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: Dendrocopos] 1
#27765458 - 05/06/22 05:08 AM (1 year, 8 months ago) |
|
|
 

You know me, I can't resist a good dick holding.
|
QM33
(NOT A PUPPET!) ❤❤❤❤❤



Registered: 04/09/20
Posts: 4,739
Loc: Oregon
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: Josex]
#27765465 - 05/06/22 05:14 AM (1 year, 8 months ago) |
|
|

I'll hold your dick
|
Dendrocopos
Latin woodpecker



Registered: 11/02/21
Posts: 511
Loc: Chained in RR's cellar
Last seen: 1 year, 7 months
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: Josex] 2
#27766861 - 05/07/22 03:28 AM (1 year, 8 months ago) |
|
|
Not yet 100% but look at this shiet. Both grains from the farmer a was i talking about. Same prep.
Wheat
 
Barley

Impregnated with the same G2G jar
|
Josex
#cheat_code



Registered: 11/13/15
Posts: 8,995
Loc:
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: Dendrocopos]
#27767322 - 05/07/22 12:35 PM (1 year, 8 months ago) |
|
|
Thanks for the report, that was very interesting.
I guess you'll buy wheat from that farmer again.
|
Dendrocopos
Latin woodpecker



Registered: 11/02/21
Posts: 511
Loc: Chained in RR's cellar
Last seen: 1 year, 7 months
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: Josex]
#27767454 - 05/07/22 02:27 PM (1 year, 8 months ago) |
|
|
Yeah. I'll do more test jars before commiting. After that I'll probably score a hundred keys. It just seems weird since I know both the grains were untreated with chem, so doesn't make much sense for one to be so fucked and the other one perfect.
|
Atomsplit


Registered: 01/16/21
Posts: 1,505
Loc: SAB
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: Dendrocopos] 1
#27772650 - 05/11/22 01:10 PM (1 year, 8 months ago) |
|
|
I'll let these sit for a few more days. It appears that the 8hr lime soak helped. My previous attempts with oats resulted in metabolites pooling the bottom of the jars.
Tidalwave

PE

I'm running this again & will post results.
Thanks Josex and everyone else working on this. Hopefully we can make more progress!
Edited by Atomsplit (05/15/22 03:23 AM)
|
Josex
#cheat_code



Registered: 11/13/15
Posts: 8,995
Loc:
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: Atomsplit]
#27774284 - 05/12/22 04:04 PM (1 year, 8 months ago) |
|
|
Nice man. You got pics of spawn from those oats before doing the lime thing?
We've got a pinset and a crazy one. This is the PE culture from the last prep I posted. Likes to throw packed flushes of mean little midgets.
|
Atomsplit


Registered: 01/16/21
Posts: 1,505
Loc: SAB
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: Josex]
#27774365 - 05/12/22 05:06 PM (1 year, 8 months ago) |
|
|
Quote:
Josex said: Nice man. You got pics of spawn from those oats before doing the lime thing?
No but I can easily replicate it. 
I shoeboxed a few and it resulted in a few shrooms. Not worth the substrate.
--------------------
.
|
Hindsight
Mad Scientist


Registered: 01/24/21
Posts: 2,706
Last seen: 9 months, 3 days
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: Atomsplit] 5
#27777884 - 05/15/22 07:58 AM (1 year, 8 months ago) |
|
|
Great test in the OP. I followed it last year but forgot to chime in so I could see all the updates. Now I have 26 pages to read back through.
But, reading the first page updates and scanning through the last pages, I have a hunch I might know what is going on here. Sorry if this has already been mentioned in an earlier post that I haven't yet read through.
There are substances produced by various bacteria that, even after the bacteria are dead, remain active. These substances are part of their evolutionary survival mechanisms, and serve to kill other organisms that compete with the bacteria. One that immediately comes to mind is spinosyn which is sold under the brand name Spinosad as a pesticide. Another which isn't a single substance but is a more general description for inhibiting substances produced by bacteria is called Bacteriocins (which are peptides), "bacteriocin-like inhibitory substances" or "BLIS" for short.
Josex'worst performing grain batch was fermented. People have fermented foods for thousands of years as a means of food preservation. This article specifically calls out lactic acid bacteria (the bacteria responsible for fermentation) as being especially skilled at producing peptides that fight food spoilage. And while this article focuses on the bacteria inhibiting properties of these peptides, other studies have been done on these same peptides that prove their inhibiting effects on spore viability and mycelium growth as well! https://pubmed.ncbi.nlm.nih.gov/30448894/
Here is another: https://www.frontiersin.org/articles/10.3389/fmicb.2016.00461/full
Here is one study proving the inhibitory affects Lactobacillus peptides have on molds and spores: https://microbiologyjournal.org/control-of-food-spoilage-molds-using-lactobacillus-bacteriocins/
I believe the reason for poor grain performance in Josex' first batch is due to the peptides produced by lactobacillus during the grain soaking process. I also think that how grain is stored prior to use (by the farmer, or anywhere in the supply chain) can have an impact on the type and amount of bacteria that grows on the grains, and as such, the peptides left behind. And while I haven't searched for any articles on the possibility of various fungus/molds leaving their own peptides behind that could negatively impact the growth of mycelium, I would guess that this too is a strong possibility.
So it stands to reason that once these substances have been produced, they are much more difficult if not impossible to remove than live bacteria, bacterial endospores, or mold spores. I did see the posts with some people saying that it is ok to use grain water agar made from the prep of grains that provided poor performance, but I think that might need more exploration/testing to be conclusive (for example, I would love to see two batches of grain water agar tested side by side - one batch made from water from fermented grain prep and the other made from water from standard grain prep). It is also possible that part of this is due to the better heat conductivity of water vs hydrated grains. But the main question is: How do you get rid of mycelium inhibiting peptides once they are produced? I googled "Bacteriocin heat stability" and took a quick look at some results. A lot more time should be spent but in one example, I found this, specifically referring to bacteriocins produced by lactic acid bacteria:
"The bacteriocin did not lose anti-listerial activity when treated at 100°C for 30 min or at 121°C for 15 min. The bacteriocin lost its antimicrobial activity after treating with trypsin, protinase-K, protease and peptidase." https://pubmed.ncbi.nlm.nih.gov/23100837/
That's the only study I have looked at so far, it didn't say if they went higher than 121C, and it didn't say if they were using pressure as well (I assume they weren't). The main point is that these peptides appear to be at least somewhat heat stable so it's possible that a PC cycle isn't enough to break them down. But note the treatments that were able to break them down. Trypsin is a digestive enzyme that you can easily buy. Proteinase-K (typo in the abstract above) was discovered as something produced by fungus and can be purchased from lab supply stores: https://en.wikipedia.org/wiki/Proteinase_K.
Since we don't know for sure if the inhibiting substances impacting our grain jars is only produced by bacteria, or can be produced by bacteria and fungus/molds, some tests would need to be done. I think Josex' fermentation grain prep is pretty conclusive that bacteria-produced peptides are inhibiting mycelium growth. It would be interesting to do another fermentation grain prep test, then treat the soaked grain with trypsin or proteinase-k, then PC the grain and see if anything changes. A lot of variables around that though.... how much to use, how long to soak, would risidual trypsin have a negative impact mycelium etc. With more research, I think a lot of those answers could be found ahead of time.
|
tedoro
ToadStool Tender



Registered: 02/06/15
Posts: 2,204
Last seen: 1 day, 2 hours
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: Hindsight] 1
#27778002 - 05/15/22 10:09 AM (1 year, 8 months ago) |
|
|
Quote:
Hindsight said: Great test in the OP. I followed it last year but forgot to chime in so I could see all the updates. Now I have 26 pages to read back through.
But, reading the first page updates and scanning through the last pages, I have a hunch I might know what is going on here. Sorry if this has already been mentioned in an earlier post that I haven't yet read through.
There are substances produced by various bacteria that, even after the bacteria are dead, remain active. These substances are part of their evolutionary survival mechanisms, and serve to kill other organisms that compete with the bacteria. One that immediately comes to mind is spinosyn which is sold under the brand name Spinosad as a pesticide. Another which isn't a single substance but is a more general description for inhibiting substances produced by bacteria is called Bacteriocins (which are peptides), "bacteriocin-like inhibitory substances" or "BLIS" for short.
Josex'worst performing grain batch was fermented. People have fermented foods for thousands of years as a means of food preservation. This article specifically calls out lactic acid bacteria (the bacteria responsible for fermentation) as being especially skilled at producing peptides that fight food spoilage. And while this article focuses on the bacteria inhibiting properties of these peptides, other studies have been done on these same peptides that prove their inhibiting effects on spore viability and mycelium growth as well! https://pubmed.ncbi.nlm.nih.gov/30448894/
Here is another: https://www.frontiersin.org/articles/10.3389/fmicb.2016.00461/full
Here is one study proving the inhibitory affects Lactobacillus peptides have on molds and spores: https://microbiologyjournal.org/control-of-food-spoilage-molds-using-lactobacillus-bacteriocins/
I believe the reason for poor grain performance in Josex' first batch is due to the peptides produced by lactobacillus during the grain soaking process. I also think that how grain is stored prior to use (by the farmer, or anywhere in the supply chain) can have an impact on the type and amount of bacteria that grows on the grains, and as such, the peptides left behind. And while I haven't searched for any articles on the possibility of various fungus/molds leaving their own peptides behind that could negatively impact the growth of mycelium, I would guess that this too is a strong possibility.
So it stands to reason that once these substances have been produced, they are much more difficult if not impossible to remove than live bacteria, bacterial endospores, or mold spores. I did see the posts with some people saying that it is ok to use grain water agar made from the prep of grains that provided poor performance, but I think that might need more exploration/testing to be conclusive (for example, I would love to see two batches of grain water agar tested side by side - one batch made from water from fermented grain prep and the other made from water from standard grain prep). It is also possible that part of this is due to the better heat conductivity of water vs hydrated grains. But the main question is: How do you get rid of mycelium inhibiting peptides once they are produced? I googled "Bacteriocin heat stability" and took a quick look at some results. A lot more time should be spent but in one example, I found this, specifically referring to bacteriocins produced by lactic acid bacteria:
"The bacteriocin did not lose anti-listerial activity when treated at 100°C for 30 min or at 121°C for 15 min. The bacteriocin lost its antimicrobial activity after treating with trypsin, protinase-K, protease and peptidase." https://pubmed.ncbi.nlm.nih.gov/23100837/
That's the only study I have looked at so far, it didn't say if they went higher than 121C, and it didn't say if they were using pressure as well (I assume they weren't). The main point is that these peptides appear to be at least somewhat heat stable so it's possible that a PC cycle isn't enough to break them down. But note the treatments that were able to break them down. Trypsin is a digestive enzyme that you can easily buy. Proteinase-K (typo in the abstract above) was discovered as something produced by fungus and can be purchased from lab supply stores: https://en.wikipedia.org/wiki/Proteinase_K.
Since we don't know for sure if the inhibiting substances impacting our grain jars is only produced by bacteria, or can be produced by bacteria and fungus/molds, some tests would need to be done. I think Josex' fermentation grain prep is pretty conclusive that bacteria-produced peptides are inhibiting mycelium growth. It would be interesting to do another fermentation grain prep test, then treat the soaked grain with trypsin or proteinase-k, then PC the grain and see if anything changes. A lot of variables around that though.... how much to use, how long to soak, would risidual trypsin have a negative impact mycelium etc. With more research, I think a lot of those answers could be found ahead of time.
Thanks so much for this writeup. Man, you really nailed it with the "a lot of variables around that though" part at the end. You present a very convincing argument.
I've been thinking about one variable that we don't talk about much... in regards to fermentation and enzyme action... and thats our grain temps and holding times before PC'ing. Lets say I treat some grains with an enzyme (I'm testing with amylase currently) and my last rinse before I PC was in hot tap water... and I load my bags, put it in the PC, but don't get it running for a few hours.... those grains sat with enzymes at 100f.... very different than grains that didn't sit before getting PC'd... and maybe room temp water was used. Time and temperature in the process could have a huge effect on the outcome.
-------------------- -------------------- Deep pour soft agar plates-->bags of WBS-->Low Profile Monos Clean spawn thread | Put a thermometer on your PC
|
Josex
#cheat_code



Registered: 11/13/15
Posts: 8,995
Loc:
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: tedoro] 1
#27778188 - 05/15/22 12:05 PM (1 year, 8 months ago) |
|
|

That was an interesting read for sure.
|
Josex
#cheat_code



Registered: 11/13/15
Posts: 8,995
Loc:
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: Josex] 3
#27779973 - 05/16/22 08:39 AM (1 year, 8 months ago) |
|
|

The pain is going to be real...
|
Hindsight
Mad Scientist


Registered: 01/24/21
Posts: 2,706
Last seen: 9 months, 3 days
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: tedoro] 1
#27780001 - 05/16/22 09:06 AM (1 year, 8 months ago) |
|
|
Quote:
tedoro said: Lets say I treat some grains with an enzyme (I'm testing with amylase currently)
Are you using amylase in attempt to eliminate the BLIS, or for something else?
I agree with your comment on how long the hydrated grains sit before PCing - it's really the same situation as how long you let the grains soak. The longer they sit (in water, or out of water but fully hydrated), the more fermentation will occur and the more BLIS will be manufactured by the bacteria.
I've been thinking a bit about what kinds of tests could be run to prove out some of these theories. I think I would break the tests up into two different groups. The first group would be trying to prove that BLIS is what is causing the poor performance. The second group would be figuring out how to eliminate it. I feel like Josex original tests have already proven the first theory but the comments about GWA contradict the theory.... but those comments didn't necessarily come from side-by-side controlled experiments. So I would want to try fermenting some grains with a ~72 hour soak, then using the water to make a batch of GWA and comparing it directly to standard grain prep GWA plates. I would want to knock up each of those plates with a plug transfer from a single donor plate culture to ensure consistency. There are a couple concerns with this test though. The first concern is that I would worry that the 72 hour soaked grain would not have as many nutrients in the water as normal prep grain which was boiled for ~22 minutes, and that this could impact the growth rates. The second concern is that even if nutrients were equal, PCing liquid agar could potentially break-down the BLIS (in a way that PCing grains might not due to stronger thermal conductivity of water vs grain).
I also read this today, "The antimicrobial property of BLIS against three indicator microorganisms (Listeria monocytogenes, Escherichia coli, and Staphylococcus aureus) remained stable upon heating at 100°C but not detectable at 121°C." Which means that PCing grain water would eliminate BLIS, but I do wonder if every part of a hydrated grain reaches 121C in a 90 minute PC cycle.
I just need to think about it a bit more. I know there are ways to test all this.
That's a killer looking tub Josex......
Edited by Hindsight (05/16/22 09:27 AM)
|
tedoro
ToadStool Tender



Registered: 02/06/15
Posts: 2,204
Last seen: 1 day, 2 hours
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: Hindsight] 1
#27780080 - 05/16/22 10:51 AM (1 year, 8 months ago) |
|
|
Quote:
Are you using amylase in attempt to eliminate the BLIS, or for something else?
Well, before you came along, we all were just trying to make shit grow cleaner, no labels were put on what our enemy was.
I'm personally interested in what the environment is in a cows gut that made these cubes evolve to thrive there, so I wanted to test enzymes. and amylase was the most prevalent one used (because of brewing). I have a huge skepticism of dietary supplements and basically assume they are all placebo sawdust capsules sold to hippies. So I'm avoiding them.
-------------------- -------------------- Deep pour soft agar plates-->bags of WBS-->Low Profile Monos Clean spawn thread | Put a thermometer on your PC
|
Hindsight
Mad Scientist


Registered: 01/24/21
Posts: 2,706
Last seen: 9 months, 3 days
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: tedoro]
#27780100 - 05/16/22 11:05 AM (1 year, 8 months ago) |
|
|
Ah yeah that makes total sense! Pretty funny coincidence that the same thing you were testing is one of only two things I've read about being able to eliminate BLIS.
|
Dendrocopos
Latin woodpecker



Registered: 11/02/21
Posts: 511
Loc: Chained in RR's cellar
Last seen: 1 year, 7 months
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: Josex]
#27780112 - 05/16/22 11:14 AM (1 year, 8 months ago) |
|
|
I just got a perfect batch of spawn and they have one thing in common. I was gone for a week and just let the to their thang. Didn't pick em up. The room they were in was constantly closed etc. Feel pretty stupid. My main problem was watery spawn.
|
tedoro
ToadStool Tender



Registered: 02/06/15
Posts: 2,204
Last seen: 1 day, 2 hours
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: Dendrocopos]
#27780152 - 05/16/22 11:50 AM (1 year, 8 months ago) |
|
|
Quote:
Dendrocopos said: I just got a perfect batch of spawn and they have one thing in common. I was gone for a week and just let the to their thang. Didn't pick em up. The room they were in was constantly closed etc. Feel pretty stupid. My main problem was watery spawn.
I'm a bit confused. What are you thinking you've learned?
-------------------- -------------------- Deep pour soft agar plates-->bags of WBS-->Low Profile Monos Clean spawn thread | Put a thermometer on your PC
|
hazyhorse
scoobin



Registered: 03/19/19
Posts: 3,820
Last seen: 9 hours, 43 minutes
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: Dendrocopos]
#27780283 - 05/16/22 01:33 PM (1 year, 8 months ago) |
|
|
yeah can you elaborate? are you saying no shake? or drier spawn? i feel i prep my shit on the drier side & still run into issues
-------------------- you're not the first to set foot here, just another =================================== i love glass petris & you can too!! posts i constantly refer back to new to mushroom cultivation?? read this!! ===================================
  🅃 🄴 🄰 🄼 🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿
|
Dendrocopos
Latin woodpecker



Registered: 11/02/21
Posts: 511
Loc: Chained in RR's cellar
Last seen: 1 year, 7 months
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: Josex] 1
#27780316 - 05/16/22 02:04 PM (1 year, 8 months ago) |
|
|
Yeah sorry. I mean that jars stayed in a pretty consent temp. Didnt swing them around, which is really a big nono with straw and i am sure it does some harm to spawn as well. I always get much more watery spawn when i leave jars outside on the table (normally theyre in a box). Etc. Just try to inoculate something. Leaving it in a closet. Shake after a few days. And leave it untill it's colonised. Don't open the closet otherwise. Maybe this was a coincidence or maybe you'll get the same results.
I am only writing this down cause this happened with a lot of different types. There were three different strains. Some rhizo some fluff. Different types of grain from Milo to wbs to wheat.
Look at it. Its fucking beautiful.
|
tedoro
ToadStool Tender



Registered: 02/06/15
Posts: 2,204
Last seen: 1 day, 2 hours
|
Re: Let's Talk Grain Prep: The Good, The Shitty and The Hardy! [Re: Dendrocopos] 1
#27780469 - 05/16/22 03:32 PM (1 year, 8 months ago) |
|
|
So I've been thinking along these lines too... steady temps for the win.
Lets say the air in a spawn jar is at nearly 100% humidity... and it begins to colonize most of the grain. As the grain becomes engulfed with mycelia, perhaps the engulfed grains internal water content begins to get regulated by the mycelia network. Then the room temperature drops by accident and the air can't hold the water - the few remaining grains are drenched with water, along with the walls of the jar.
-------------------- -------------------- Deep pour soft agar plates-->bags of WBS-->Low Profile Monos Clean spawn thread | Put a thermometer on your PC
|
|