The July/August 2019 issue of Zymurgy included a clone recipe of Tree Houses’ Julius. The recipe author included a dry yeast blend that is possibly used by the brewers themselves and is apparently responsible for providing the beer with a lot of it’s character. Seeing how I work at a yeast lab, I had access to some tools I could use to determine what yeast is in a can of Julius. It was also a great chance for me to get more experience with DNA extraction and PCR!

Isolation

During a recent trip to Rhode Island my partner and I stopped at Tree House to see what all the hype was about (psst, their Pilsner game is on point!) where we obtained some cans of Julius; among other things. The product was kept as cold as possible until I could get back to the lab. Most of the beer was aseptically shared so we could rouse whatever yeast had flocculated to the bottom of the can. This is the yeast sample we would be streaking to determine what strains are actually in the can.

To make it easier to isolate different yeast strains we use an agar media called Wallerstein Lab Nutrient (WLN). This media contains a dye which some yeast will take up. Some strains may look a deep green while others may take up very little and still look white. This differential media makes it easier to determine if a culture is pure or not. For quality control purposes this can help one determine if a contamination has been picked up. From an isolation standpoint it makes it easier to select each separate strain and re-plate them for further analysis.

The first streak plate came back with a very obvious mixed culture and over a couple subsequent isolation steps I had what appeared to be four different strains. The media we use also contains Ferulic Acid so we can perform some sensory evaluation to determine if a yeast is Phenolic Off-Flavour positive (POF+). One of the yeasts isolated had a very strong clove aroma coming off the plate.

Testing the Clone Yeast

The yeast strains listed in the Zymurgy recipe are: S-04, WB-06 and T-58. We acquired some pitches of dry yeast to also plate so we could have something to compare the can isolates with. A combination of these yeast seems to be commonly believed to be what is used by Tree House for their core product.

Considering how heavily hopped their beers are; using dry yeast is possibly the most economical approach. The beers are hopped before yeast even has a chance to settle out, resulting in a yeast culture that would be hard if not impossible to work with. There’s also the risk for drift if the product is used as a blend from the first generation on.

DNA Extraction and Validation

The extraction on it’s own is fairly straightforward. We use this product called “Instagene Matrix” that makes it very easy to break open the yeast and expose their DNA. This solution has a yeast colony added to it then undergoes a few heating steps to break open the cells. At this point we are “done” doing our extraction, however it doesn’t always work on the first try so we always want to validate that the DNA extraction worked.

We use Polymerase Chain Reaction (PCR) on a sample of the extracted DNA to ensure that our process works. I’m a computer scientist, so I don’t know all the ins and outs of PCR, but it basically works by having a primer that hooks up to a target piece of DNA. DNA follow certain rules and during one of the reactions it builds out the strand. PCR is a “chain reaction” because it is an exponential process. For example, with 1 piece of target DNA we would create 2 on the first iteration, 4 on the second iteration and if we were to repeat this process 30 times we’d have 1,073,741,824 bits of DNA kicking around!

Check out this youtube video which helps explain what is going on during the various steps of the PCR process.

Since all we are doing is checking to see that the DNA was properly extracted we use a primer that basically all yeast strains contain, the ITS primer. This primer is used along with a “Master Mix” that contains a bunch of other necessary components for PCR to actually work. Once everything has been prepared it’s loaded into a Thermocycler and does it’s magic for about 3 hours. After the process is done the product is loaded into a gel to see if everything worked. If it does you end up with something that looks like this.

PCR is a bit of a heart breaker and in some cases you’ll end up with nothing. Sometimes that’s a good thing, such as using PCR to detect diastatic yeast where you don’t want any. However, if you were trying to amplify something and it didn’t work, you go cry in a corner because you just wasted 8 hours of your life and have nothing to show for it. When doing research at the masters or doctoral level this can happen over and over again for months!

DNA Fingerprinting

The final step to determine if there’s any shared yeasts between the dry products and those yeasts found in the beer is to run the DNA through a fingerprinting step. Fingerprinting is a useful tool to determine if something shares similar DNA, which can be useful to determine if two yeasts are the same. The results of fingerprinting look like a DNA test that you might have seen on a crime show.

The fingerprinting performed is called “interdelta fingerprinting” and used the delta 12 and delta 21 primers which was then run on a digital capillary electrophoresis unit. See Legras and Karst (2003) for more details about this method.

Results

Looking at the resulting bands we can see a very strong similarity between lanes D1 and F1. There’s some slightly different intensities but the lines are pretty similar. The colony from this plate was also POF+ so it seems that it’s quite likely that WB-06 is one of the strains used by Tree House.

Comparing our known sources (lanes B1 and C1) there don’t appear to be any other matches with the strains purported to be part of the Tree House house culture. However, E1 and H1 do match which means that what I thought were different yeasts (based on morphology) are quite likely the same. One thing that is interesting though is there appear to be three separate yeast strains in a can of Julius!

From what I was able to see from my plating there are three unique strains in their yeast blend as well, but they don’t appear to be S-04 or T-58. However, based on the results there does indeed seem to be WB-06 in the mixed culture. It’s possible that wheat strains are good at biotransformation, which might be why it is being included. As for the other two strains, there’s other dry yeasts on the market that could be tested to determine what makes up Tree House’s secret sauce. For now though, I think I might just grow these yeasts up from colony and see what kinds of beer they make on their own 😉

Additional Resources

Polymerase Chain Reaction

• http://suigenerisbrewing.com/index.php/2017/11/22/contamination-detection-1/
• http://suigenerisbrewing.com/index.php/2017/11/24/contamination-detection-2/

Plating and Isolation Techniques

• http://suigenerisbrewing.com/index.php/2014/08/09/your-home-yeast-lab-made-easy-streak-plates/
• http://suigenerisbrewing.com/index.php/2015/05/08/purifying-yeast-from-infected-cultures-part-2/
• https://www.escarpmentlabs.com/single-post/2019/03/18/agar-plating-guide
• http://www.milkthefunk.com/wiki/Laboratory_Techniques#Techniques