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Isolated Yeast (Tree House): How to Identify and Characterize?

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RevKev

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Just went to a seminar about a month back in which the researcher at my university used qPCR to quantify bacterium and is working on viral applications. The work was in the field of environmental chemistry classifying different strains of bacteria that are markers for whether water pollution at beaches was at unsafe levels. Interesting stuff.

And props to you for using your knowledge base for brewing science, something that doesn't get enough exposure to the beer community. Me being an analytical chemist I grasp methods but I am likely unhelpful unless you need work done on mass spectrometers.. I have access to a wide variety (ICP, GC, LC, FTICR and Orbitrap).

I'll stay tuned as the bioanalytical work is a guilty pleasure of mine.
 
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Kind of late to the game here but have you looked into doing a genome wide SNP fingerprint? Pretty cheap these days. I haven't done much work with yeast but it's a model organism so no doubt there are vast resources out there to do this type of work. That would let you do a phylogenetic analysis and would give a better representation of relatedness than a handful of loci via PCR. Just my 2c. Cool project!
The Siebel Institute offers these services (as I'm sure other companies do as well). I had emailed with them about a different (presumably less expensive) service and that was ~$200, so I don't think genome wide sequencing is quite at the level of homebrewer access yet!
 
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Just went to a seminar about a month back in which the researcher at my university used qPCR to quantify bacterium and is working on viral applications. The work was in the field of environmental chemistry classifying different strains of bacteria that are markers for whether water pollution at beaches was at unsafe levels. Interesting stuff.

And props to you for using your knowledge base for brewing science, something that doesn't get enough exposure to the beer community. Me being an analytical chemist I grasp methods but I am likely unhelpful unless you need work done on mass spectrometers.. I have access to a wide variety (ICP, GC, LC, FTICR and Orbitrap).

I'll stay tuned as the bioanalytical work is a guilty pleasure of mine.
I would think that would be a very helpful experimental technique for homebrewers! I know it has been extensively applied to the study of hop oil compounds in beer and I'm sure there are other applications (yeast identification, other metabolites in finished beer, etc.) that it would be useful for as well.

Just wanted to add that Scott Janish's blogsite (http://scottjanish.com/increasing-bitterness-dry-hopping/) has some great reading if you're interested in the analytical side of homebrewing.
 
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***DNA fingerprinting update***



New strains analyzed in the right gel (old gel on the left for comparison purposes). Strain 1 (WY1056) was used as a control of sorts.

New strains (all are dry yeasts):
12: S-04
13: S-05
14: S-23
15: Danstar Munich
16: W-34/70
17: T-58

Nice to see S-05 and WY1056 have a very similar banding pattern, although this just further reinforces the previous commentary that similar strains at the genetic level can produce differences at the phenotypic level (e.g. S-05 krausen behavior v. WY1056).

S-23 and W-34/70 have very similar banding patterns as well. I haven't used either yeast, so can't say much there...

***REQUEST***
I am interested in testing more yeast strains, and would be willing to mail autoclaved blotting paper foil packets (see here: http://suigenerisbrewing.blogspot.com/2017/03/new-mailer-system.html) to those with yeast strains I have not tested yet. Shoot me a PM if interested.
 

RevKev

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I would think that would be a very helpful experimental technique for homebrewers! I know it has been extensively applied to the study of hop oil compounds in beer and I'm sure there are other applications (yeast identification, other metabolites in finished beer, etc.) that it would be useful for as well.

Just wanted to add that Scott Janish's blogsite (http://scottjanish.com/increasing-bitterness-dry-hopping/) has some great reading if you're interested in the analytical side of homebrewing.
Yes I think I have stumbled upon that! I've been thinking about doing headspace injections with GC-MS to identify aromatic and volatile oils for SMaSH beers.. if I pay for the instrument time. Metabolomics is my field and I actually am working on 'leaf spray' ionization with HR-MS. I have some whole cone hops that could be cool to analyze and I bet that I could talk my PI into a little side study.. I mean for the love of beer!! (He's a big fan of craft also)

I'll have to post back when it comes summer time and I'll have some free time.. and thanks for the link.

I may have some Belle Saison and some wild fellers in the coming months
 

The_Dirty_Spring

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It's hard to tell with those images, but is that T-58 close to the Tree House isolate? I've noticed that Tree House yeast starters smell very Belgian...
 
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It's hard to tell with those images, but is that T-58 close to the Tree House isolate? I've noticed that Tree House yeast starters smell very Belgian...
Wow, nice catch! I wasn't even thinking about that as a possibility, but I re-ran the samples side-by-side (Julius on the left, T-58 on the right):



There are a couple small differences, the main one just above the smallest ladder band (100 bp) in the T-58 sample, but wow, pretty darn close. It doesn't seem likely that they are adding yeast at canning right?
 

The_Dirty_Spring

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I've suspected TH is using something (at least partially) Belgiany based on the esters I get in starters, and the well-noted "bubblegum" character of fresh TH. Perhaps a blend including T-58 fermented at lower temps? That said, I would not be at all shocked if they mix in other strains at canning to protect their IP--assuming it could be done in a way not to affect the quality of the beer inside. I've seen others claim an isoamyl-y banana flavor in their harvested yeasts.

Forgive my ignorance... Does the Julius sequence show any indication of being multiple strains, or is that not possible to tell with the PCR? What do you make of the differences between the Julius and Double Shot sequences? Thanks so much!
 

phillip092

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I've suspected TH is using something (at least partially) Belgiany based on the esters I get in starters, and the well-noted "bubblegum" character of fresh TH.
This was my speculation as well, I cultured yeast from a can of haze, brewed two batches with it at the same time/temp, the only difference was I accidentally under pitched one of the two batches. The underpitched batch was supposed to be an IPA with a TON of hops, I couldn't taste anything but earthy/clove like belgian esters. The other one had hints of the same esters but they weren't nearly as prominent as the under pitched batch. I've since used it again, fermented on the cold side and tried to over pitch, still had hints of those same esters but turned out okay.

When I initially noticed the belgian-like esters, the first thing that came to mind was the possibility that they were adding yeast at canning, but I kinda dismissed that idea because I thought that you'd have to be really cynical to intentionally sabotage your beer in a way that could harm it, just to prevent home-brewers from culturing their yeast....especially since Nate used to be a homebrewer himself. It's definitely interesting to see other people propose that idea though.

On another note, I've recently cultured more of the yeast from a can of green, and I do not notice any of the belgian-like aroma or taste in the cultured starter. I have not used it to brew a batch yet though.

Forgive my ignorance... Does the Julius sequence show any indication of being multiple strains, or is that not possible to tell with the PCR? What do you make of the differences between the Julius and Double Shot sequences? Thanks so much!
I am also curious to know this, is there any way to tell if there are two different strains of yeast? and if so, is there any way to tell them apart?

***REQUEST***
I am interested in testing more yeast strains, and would be willing to mail autoclaved blotting paper foil packets (see here: http://suigenerisbrewing.blogspot.com/2017/03/new-mailer-system.html) to those with yeast strains I have not tested yet. Shoot me a PM if interested.
I have a few yeast that I would be willing to mail, would you be willing to test cultures from other breweries as well? Or just commercial yeast?
 
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This was my speculation as well, I cultured yeast from a can of haze, brewed two batches with it at the same time/temp, the only difference was I accidentally under pitched one of the two batches. The underpitched batch was supposed to be an IPA with a TON of hops, I couldn't taste anything but earthy/clove like belgian esters. The other one had hints of the same esters but they weren't nearly as prominent as the under pitched batch. I've since used it again, fermented on the cold side and tried to over pitch, still had hints of those same esters but turned out okay.

When I initially noticed the belgian-like esters, the first thing that came to mind was the possibility that they were adding yeast at canning, but I kinda dismissed that idea because I thought that you'd have to be really cynical to intentionally sabotage your beer in a way that could harm it, just to prevent home-brewers from culturing their yeast....especially since Nate used to be a homebrewer himself. It's definitely interesting to see other people propose that idea though.

On another note, I've recently cultured more of the yeast from a can of green, and I do not notice any of the belgian-like aroma or taste in the cultured starter. I have not used it to brew a batch yet though.



I am also curious to know this, is there any way to tell if there are two different strains of yeast? and if so, is there any way to tell them apart?



I have a few yeast that I would be willing to mail, would you be willing to test cultures from other breweries as well? Or just commercial yeast?
The short answer is, I was not expecting multiple strains in the can of Julius I harvested from, so I propagated in DME before streaking for isolation and genetic analyses. Next time (might be a bit since I am in Kansas!) I will streak out the dregs of a can and analyze 10-15 colonies from there. With that said, I dont think they are adding anything nefarious at canning. I am tasting a friends all-Citra APA made with this yeast tomorrow.

The DNA fingerprinting is super cheap and easy for me to do, so I am more sure than happy to test any samples sent my way!
 

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I've also seen that WLP 644 Sacch Trois may perform well in the New England style. Do you have access to test that strain in the next iteration?
 
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Apimyces

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Wait, are you saying the yeast companies rip off their customers and commercialize their private strains?
 
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THE PLOT THICKENS



So, a buddy of mine brought a can of Doppelganger over last week (very pineapple forward), so we harvested the dregs and before propagating them, I struck out the yeast present (left image in pic above). I didn't notice this until after running the PCR, but you can clearly make out different colored yeast colonies, reflecting the diversity of strains present. I am streaking out my Julius isolate stock to see if something similar occurs.

Analyzing the DNA fingerprinting, there clearly appears to be 4 species present in the 10 colonies I picked! The majority of which contains a novel fingerprint signature (for my strains). The other 3 include the T58-like sample previously ascribed to the Julius strain, something very similar to S-04 and something somewhat similar to WY1272 (and WY1332 which looked the same) minus the high MW bands.

I will know more once I look at the streaked out Julius stock, but my friend used it to make an all Citra APA that turned out very nice. So whatever's in there makes beer!
 

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I've been quietly following along as well.
 

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Fascinating, great job on your work.

I can't wait to see what becomes of this thread
 
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At this point I have more questions to answer than when I started, sounds like science! What are the chances that this was unintentional on the part of Tree House? Will need to analyze more/different cans to see if this is a trend or consistent practice. I also plan to make small starters with each of the different isolated colonies to see if any sensory clues jump out.
 

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Nothing is unintentional with Tree House. The question is, are all strains pitched together, some later, and/or some at packaging? Incredibly fascinating. Thanks for taking us on this journey!
 

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I wonder what happens if you isolate yeast from the outside of a hop. Come to think of it, hop with its strong abiotic properties would be a good vector for inoculating yeasts.

Wild Hopping, as it were.
 
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Okay, so was able to get my hands on another TH beer (Green). This one was a bit on the old side (by taste, the date was smudged) and didn't have nearly as much viable yeast as the other 2 cans I've streaked (only 3 colonies on first plate, normally have close to 100). I also went back and analyzed the Julius glycerol stock I have in the -80C freezer. Unfortunately, it looks like I took a single colony to make the stock, as all 5 colonies have the same fingerprint.



First lane is a new yeast (Wyeast Forbidden Fruit), then next 5 lanes are different Julius colonies from main stock and then 8 colonies from Green colonies. As you can see all DNA fingerprints appear to be consistent with the 4 possible strains we saw in the Doppelganger can (gel on the right re-posted for convenience). This also means that the Julius yeast I (and my buddy) have used to make beer with is probably just S-04, haha!

My next plan is to streak out a new TH can (after centrifuging dregs to concentrate yeast) and analyze upwards of 50-100 colonies. This would enable a prediction as to what ratios are being used. Its possible TH uses different ratios for different beers. Also makes me want to re-check the Double Shot yeast stock (I saved a heterogeneous stock for everything after the Julius stock).

I'm also very interested to figure out what the red square strain is, as this consistently (n=2) has been the most abundant strain in the cans I've analyzed. I have saved this strain by itself, will need to brew a NE IPA with it soon. I have more yeast coming from 2 different people soon, so stay tuned!
 

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This also means that the Julius yeast I (and my buddy) have used to make beer with is probably just S-04, haha!
That had been my suspicion for some time now. The stouts and Bright are probably S-05 since they were using that early on.
 

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Re: Green, that beer has much less house yeast character than J, Haze, etc. It seems to be strong component of clean neutral yeast like Bright (probably S-05) with smaller portion of the unidentified "house" strain. Excited to see how the red square performs on its own.
 
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I believe they meant that the colony that had been cultured was S-04. It seems there is more than one strain per can
You are correct. Makes sense with the side by side we did (along with WY1318), as I thought they were pretty similar.

Also note that the previous Julius colony I analyzed was likely T-58 (or very close relative), so I think all 3 cans I've analyzed so far have been mixed populations.

Re: Green, that beer has much less house yeast character than J, Haze, etc. It seems to be strong component of clean neutral yeast like Bright (probably S-05) with smaller portion of the unidentified "house" strain. Excited to see how the red square performs on its own.
I think I agree with that statement, my palate is unfortunately not the best, but Green is definitely lower on my like list when compared to Julius, Haze and Alter Ego.

I'm planning a side by side with the red square (and hoping to possibly identify it with some new yeast strains I have coming) and WY1318 in an all Citra Oat SMaSH next month.
 

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Is it possible that they are just naturally carbonating all of their beers with something similar to T-58? In the description of T-58 that is one of the recommended uses for it.

Propped up a starter from a 750 of SAP that I grabbed from the brewery. First few steps smelled totally normal but the last 2.5 step starter might have got a little warmer (had to move it to a different room) and it totally exploded everywhere (in a 5L flask) and smelled incredibly phenolic. Pitched it into a 1.068 wort (loose Swish clone). Kept
It at 65 for the first 4 days then raised it to 68 then 70. Only got to 1.022 after 8 days but it still fermenting and the sample was very peppery and phenolic (more pepper than anything).

For me one of the most distinct qualities of Treehouse is the mouthfeel. Although some people say Co2 is Co2 I sometimes wonder. As I've only ever force carbonated I haven't done a split batch side by side comparison but I might just have to. People always rave about the mouthfeel of HF and TH beers and I'm pretty sure that most if not all the HF beers are naturally carbonated....
 
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Is it possible that they are just naturally carbonating all of their beers with something similar to T-58? In the description of T-58 that is one of the recommended uses for it.

Propped up a starter from a 750 of SAP that I grabbed from the brewery. First few steps smelled totally normal but the last 2.5 step starter might have got a little warmer (had to move it to a different room) and it totally exploded everywhere (in a 5L flask) and smelled incredibly phenolic. Pitched it into a 1.068 wort (loose Swish clone). Kept
It at 65 for the first 4 days then raised it to 68 then 70. Only got to 1.022 after 8 days but it still fermenting and the sample was very peppery and phenolic (more pepper than anything).

For me one of the most distinct qualities of Treehouse is the mouthfeel. Although some people say Co2 is Co2 I sometimes wonder. As I've only ever force carbonated I haven't done a split batch side by side comparison but I might just have to. People always rave about the mouthfeel of HF and TH beers and I'm pretty sure that most if not all the HF beers are naturally carbonated....
Anything is possible at this point! I completely agree that bottle condition-derived CO2 has a "creamier" texture than kegerator-derived CO2 (with the caveat that I haven't done a real side by side, so could be biased). With that said, I'd expect there to be a lot more sediment in the cans if they were really doing that. Maybe the extra yeast are added in a secondary or brite tank set up? I don't know enough about the logistics of that to know whether that's a silly idea or not.

If they are truly fermenting with multiple strains, then they almost have to be pitching known amounts/ratios and not propagating them together. I bet that is where a lot of people have strange cultures when trying to harvest from cans. Eventually one of the strains takes over (sounds like its often the Belgian one, based on a lot of the comments I've read RE phenols).
 

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Any updates on this? I just read through the thread - fascinating stuff.

A couple things:

I know many people have commented that their Heady Topper strains have been quite Belgiany - even Jim has mentioned that you can get this character but HE knows how to ferment it so it doesn't happen with HT (I will try and search for the interview where he states this). Have you tested any strains since then that are purportedly from Alchemist or Conan?

I run a commercial brewery in KY and we have a microbiologist on site. I can definitely send you some strains to compare if you are interested. I can say from my experience with yeast providers, some *cough*wyeast*cough* are notorious for unclean pitches. Most breweries I know that have used them have since moved on. I would be curious if you have tested any recent cans of TH and have seen any change in yeasts collected.

Cheers!
 

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My supposition was that they may have branched off from a common lineage, more recently than not. Can't really speak to the isolation efforts of others, and I'm not saying we're isolating this strain, but we are currently in the process of isolating a new strain that is similar. Just posted about it on The Yeast Bay Facebook page:

"Everything is back in stock on the online store! Also, Brettanomyces bruxellensis - Strain TYB261 will be available to homebrewers shortly, and we'll likely be posting stock next week.

We also have yet another beta in the works that hasn't even been assigned a TYB number yet. We just streaked out the source material and are excited to get into characterizing the many isolates we'll pick. This house yeast, isolated from another brewery in the Northeastern United States, is similar to the Vermont Ale strain though the ester profile is less peach/apricot driven and more closely resembles citrus/pineapple/mango/guava. Keep your eyes peeled for updates, we're hoping to get through the isolation and characterization of this strain and make it available to commercial brewers and homebrew folks by the middle-end of May.

Cheers!"

Hi, so did you get this new strain out? What's it's number?
 
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Any updates on this? I just read through the thread - fascinating stuff.

A couple things:

I know many people have commented that their Heady Topper strains have been quite Belgiany - even Jim has mentioned that you can get this character but HE knows how to ferment it so it doesn't happen with HT (I will try and search for the interview where he states this). Have you tested any strains since then that are purportedly from Alchemist or Conan?

I run a commercial brewery in KY and we have a microbiologist on site. I can definitely send you some strains to compare if you are interested. I can say from my experience with yeast providers, some *cough*wyeast*cough* are notorious for unclean pitches. Most breweries I know that have used them have since moved on. I would be curious if you have tested any recent cans of TH and have seen any change in yeasts collected.

Cheers!
I have recently analyzed two more TH cans (Julius and Alter Ego). I only looked at 10-12 colones each (realized it would take way too long to run gels if I analyzed 50+ colonies/can). All colonies were either the blue triangle (S-04), red square (mystery) or green circle (T-58). The ratios were 14 (S-04), 5 (mystery) and 3 (T-58). Propagating the blend over several generations and then reanalyzing might provide some insight into why some people's starters from can dregs "turn" Belgiany. It could be that that strain grows faster than the others? I haven't analyzed any other breweries dregs (I live in KS, so difficult to get the NE IPAs).
 

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Any progress identifying the red square? Have you propagated and used just that yeast on a batch yet? Thanks again!
 
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Any progress identifying the red square? Have you propagated and used just that yeast on a batch yet? Thanks again!
I'm getting ready to analyze some new strains, but no evidence yet what the red square strain is. I've been talking with someone about how it could conceivably be S-33, a dry Belgian yeast that might actually be an English strain. It would fit with the other 2 primary yeasts that are dry (S-04 and T-58), just need to get down to the LHBS do buy some.

It might be a month away, but I'm going to use the red square strain in a NE IPA oat SMaSH (prob with Citra). Will update here once I do.
 

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I'm getting ready to analyze some new strains, but no evidence yet what the red square strain is. I've been talking with someone about how it could conceivably be S-33, a dry Belgian yeast that might actually be an English strain. It would fit with the other 2 primary yeasts that are dry (S-04 and T-58), just need to get down to the LHBS do buy some.

It might be a month away, but I'm going to use the red square strain in a NE IPA oat SMaSH (prob with Citra). Will update here once I do.
I can send you some money to offset your costs? I love everything about this thread and would like to support your research.
 
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I can send you some money to offset your costs? I love everything about this thread and would like to support your research.
I appreciate the offer, but time is the real limiting factor for me right now. I will say that if you have a strain you'd like analyzed, send me a PM to discuss shipping. Its also possible to order yeast and have it shipped to me if you aren't currently set up for "home wrangling".
 
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I just read in a post that TH growlers say they are naturally carbonated? I'm now wondering if the T-58 like yeast is added along with a dose of fermentable sugar under pressure. They could dry hop at this time and get bio transformation as well along with O2 scavenging. This would possibly suggest they use a less complicated yeast blend for primary.

Anyone thoughts or personal experience with something like this in a NE IPA?
 
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