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

[isomerization]

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!"

Very cool! I look forward to picking up a vial.

 

[Warthaug]

As always, I'm late to the party. I'm Bryan of Sui Generis Brewing, which has been cited here a little. Anyways, some replies to some previous posts...

Using a PCR method (https://suigenerisbrewing.blogspot....howComment=1490284392639#c8540474511060838192), I was able to amplify the ITS region (http://sites.biology.duke.edu/fungi/mycolab/primers.htm) of this yeast (my band is ~800 bp) ... I then sequenced the amplicon, and used BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to search for similar deposited sequences. I think this is where I've run into a wall, as the best hits I get back are only ~89% identical (they are S. cerevisiae).

With that said, I went back and looked at the DNA chromatogram a bit closer, and there is a suspicious amount of overlapping peaks.

89% homology is awfully low for an ITS sequence; I typically see 99-100% homology for yeast. Did you trim your sequence to remove poorly resolved bases (usually ID'd as an 'N' in the chromatagram/sequence file)? The mixed peaks may be a sign of contamination, but assuming there are two strains and both strains are S. cerevesea, it shouldn't matter as ITS sequences are pretty well conserved between species. Unfortunatly ITS size isn't a useful measure either, as pretty much any Eukaryote (including yourself) will produce an ~800bp ITS band. As Surgork already mentioned, you cannot do strain ID with ITS and need to reply on other methods. Inter-delta is the norm, but as you've already experienced, it needs to be compared to a database of other fingerprints to ID the strain.

Unsurprised that Vermont Ale resembles WY1968 on the level of DNA sequence as the source material we used to isolate Vermont Ale from uses a strain that was rumored to originally have British origins. I'd say that's where the similarities start and end! WY1968 is a lower attenuator, high flocculator, produces decent amounts of diacetyl and has a fruity but restrained ester profile. Vermont Ale is a higher attenuator (typically around 80%), low flocculator, does not produce diacetyl in any appreciable quantities and has a massive ester profile of peaches/apricots.

This is a good example of why even interDelta can be of only modest value. It doesn't take much to alter ester profiles or flocculation (and therefore attenuation) characteristics of yeast - there are many examples out there of single point mutations doing just that. interDelta can let you follow the parentage of yeasts, but some strain-specific changes can be "below detection" for even this method. Full genome sequencing is about the only way to get to that level of resolution, but the cost of that is still a bit beyond what is practical for the home brewer.

Just my $0.02

Bryan

 

[Biobrewer]

As always, I'm late to the party. I'm Bryan of Sui Generis Brewing, which has been cited here a little. Anyways, some replies to some previous posts...




89% homology is awfully low for an ITS sequence; I typically see 99-100% homology for yeast. Did you trim your sequence to remove poorly resolved bases (usually ID'd as an 'N' in the chromatagram/sequence file)? The mixed peaks may be a sign of contamination, but assuming there are two strains and both strains are S. cerevesea, it shouldn't matter as ITS sequences are pretty well conserved between species. Unfortunatly ITS size isn't a useful measure either, as pretty much any Eukaryote (including yourself) will produce an ~800bp ITS band. As Surgork already mentioned, you cannot do strain ID with ITS and need to reply on other methods. Inter-delta is the norm, but as you've already experienced, it needs to be compared to a database of other fingerprints to ID the strain.



This is a good example of why even interDelta can be of only modest value. It doesn't take much to alter ester profiles or flocculation (and therefore attenuation) characteristics of yeast - there are many examples out there of single point mutations doing just that. interDelta can let you follow the parentage of yeasts, but some strain-specific changes can be "below detection" for even this method. Full genome sequencing is about the only way to get to that level of resolution, but the cost of that is still a bit beyond what is practical for the home brewer.

Just my $0.02

Bryan

Well said Bryan, this is useful information that's worth far more than $0.02!

 

[isomerization]

Regarding the ITS sequencing, I think it was a problem on the sequencing end, rather than a true heterogenous population. But, as many have pointed out on this thread now, ITS sequencing wasn't going to be helpful in this situation anyways. So I dropped it and switched to interdelta PCR, which I was not aware of until I started playing around with this strain.

Personally, I think interdelta PCR was very helpful here not because it revealed shared parental lineage, but because it didn't! It appears to suggest that the yeast isolated from a Tree House can is different than any of the other traditionally used (by homebrewers at least) NE IPA yeasts. I think that's pretty cool!

Phenotypic characterization is more important for us homebrewers anyways, but I think its still cool to ask and try to answer these types of questions.

 

[xerotime]

I forgot to add that the Julius isolate is certainly unique via this DNA fingerprinting method!

So, what this really means is that I need more yeast samples to analyze, I'm looking into figuring out an easy way to mail samples through the postal service (only costs a stamp!), if anyone has suggestions and is interested in sending yeast that is not on my current image, please let me know!!

I'd really like to see how Fermentis S-04 and S-05 compare. If you want to PM me your address, I'll just order a pack of each and have them shipped to you.

 

[isomerization]

I'd really like to see how Fermentis S-04 and S-05 compare. If you want to PM me your address, I'll just order a pack of each and have them shipped to you.

PM sent. I also picked up T-58, S-23, W-34/70 and Danstar Munich dry yeasts to look at as well ($6 including shipping for all 4 since they're old).

 

[5mooth0perator]

As always, I'm late to the party. I'm Bryan of Sui Generis Brewing, which has been cited here a little. Anyways, some replies to some previous posts...




89% homology is awfully low for an ITS sequence; I typically see 99-100% homology for yeast. Did you trim your sequence to remove poorly resolved bases (usually ID'd as an 'N' in the chromatagram/sequence file)? The mixed peaks may be a sign of contamination, but assuming there are two strains and both strains are S. cerevesea, it shouldn't matter as ITS sequences are pretty well conserved between species. Unfortunatly ITS size isn't a useful measure either, as pretty much any Eukaryote (including yourself) will produce an ~800bp ITS band. As Surgork already mentioned, you cannot do strain ID with ITS and need to reply on other methods. Inter-delta is the norm, but as you've already experienced, it needs to be compared to a database of other fingerprints to ID the strain.



This is a good example of why even interDelta can be of only modest value. It doesn't take much to alter ester profiles or flocculation (and therefore attenuation) characteristics of yeast - there are many examples out there of single point mutations doing just that. interDelta can let you follow the parentage of yeasts, but some strain-specific changes can be "below detection" for even this method. Full genome sequencing is about the only way to get to that level of resolution, but the cost of that is still a bit beyond what is practical for the home brewer.

Just my $0.02

Bryan

Single point mutations can be huge, just look at teosinte vs corn, it only took like four mutations, but you wouldn't recognize thm as the same species. Same for grapes, people did some sequencing and it turns out many of the French grapes are literally siblings.

I think it helps though to know when you have essentially the same strain producing different flavors vs a strain that is close to a different species.

 

[Redlantern]

This has got to be the most interesting conversation despite 80% of it going straight over my head.

I am glad there are guys like you out there so I can brew.

 

[trav77]

Single point mutations can be huge, just look at teosinte vs corn, it only took like four mutations, but you wouldn't recognize thm as the same species. Same for grapes, people did some sequencing and it turns out many of the French grapes are literally siblings.

I think it helps though to know when you have essentially the same strain producing different flavors vs a strain that is close to a different species.

It took only a small number of mutations to get rid of the rock-hard glume in teosinte and make it edible. After that there was an incredible amount of de novo variation and selection by humans to get to what is now corn.

But yes, a single point mutation can be huge in certain cases.

 

[trav77]

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!

 

[RevKev]

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.

 

[isomerization]

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!

 

[isomerization]

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.

 

[isomerization]

***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]

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]

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...

 

[TheHairyHop]

I wonder how WLP007 compares

 

[isomerization]

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]

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!

 

[TheHairyHop]

T-58 is known for staying powdery in suspension, right? They could be using it to up the haze around canning time... maybe?

 

[phillip092]

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?

 

[isomerization]

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!

 

[The_Dirty_Spring]

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?

 

[RevKev]

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?

This guy has WLP644 that I will be putting in a German juice bomb of an IPA

 

[isomerization]

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?

I am hopefully acquiring 12 strains from Bryan/warthaug (http://suigenerisbrewing.blogspot.com/) through his yeast exchange program in the next month or so. WLP644 is on there!

 

[Apimyces]

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

 

[isomerization]

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!

 

[Horseflesh]

I continue to love this thread.

 

[stpug]

I've been quietly following along as well.

 

[TheHairyHop]

The plot has indeed become quite thick