The Locksmith: Utilizing Bioengineered Yeast and High Bound Thiol Precersour Hops and Phantasm Powder to Thiol Drive Beer
Researching, experimenting, and writing about biotransformation has been one of the most exciting and frustrating topics to cover over the past few years. I have brewed batch after batch of beer, attempting to piece together research from the wine and beer worlds to unlock the true potential of hops and fruit. Whether it’s incorporating wine strains as a blend with ale strains to unlock bound thiols hops (the same thiols in wine grapes like 3MH and 4MMP) or doing solo ferments with wine strains like Vin 7, QA23, 71B, and the Alchemy blends (scientifically formulated to be strong biotransformers).
Except for one trial I wrote about, where we experimented with wine yeast and a lager strain, none of the trials were good enough to scale up to anything I would be excited for people to purchase and drink. Although sometimes interesting, the result of these trials is often fermented with strong phenolic characteristics masking any unlocking of thiols that may have occurred (even when some of the wine strains were less than 10% of the total yeast pitch). It’s also likely that, although these strains were great at releasing bound compounds in grapes, the nitrogen that is in excess in the wort and limited in wine ferments, can inhibit the beta-lyase activity in beer ferments and ultimately reduce the number of unlocked thiols.
This cycle of excitement and letdown with my thiol chasing trials is part of the reason I was so interested in the work done by Omega Yeast and Berkeley Yeast this year in engineering yeast strains designed precisely for biotransformation. Using CRISPR technology (at least with Omega), they are now able to create hazy IPA yeast strains that, like many of the wine strains studied prior, have the necessary gene (IRC7) to produce the required enzyme (beta-lyase) during fermentation to unlock thiols.
The advancement in engineered yeast is exciting because now there is some potential to achieve higher thiol concentration in hop-forward beers without the clashing phenolic yeast fermentation characteristics that I was getting from the wine yeast trials. But, to increase the chances of unlocking bound thiols, the engineered ale strains not only have the IRC7 gene (for Omega yeast) or TnaA (for Berkeley yeast) required for beta-lyase activity but it’s overexpressed. Essentially, this means the gene activity is turned up or highly expressed, allowing for an even greater release of bound thiols. If a strain has just a functional beta-lyase gene, it may not be enough to reach sensory results without this overexpression.
Rather than getting into a lot of technical text (most of which was covered in a previous post on bioengineered yeast strains), below is a flow chart of sorts that helps explain how and why their power in unlocking bound thiol potential in hops.
This post covers how brewers might be able to use new products (like Phantasm powder), new techniques (like mash-hopping), and how to experiment with high thiol-bound hops. Also covered in the post is Sapwood’s collaboration beer with Omega Yeast and Phantasm (along with thiol test results from France), current understanding of thiols, and how we might be able to use the science to achieve higher thiol concentrations.
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Thiol Driving Flowchart:
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Thiol Driving Roadmap Flowchart
It’s not just hops that have an incredible amount of bound thiol potential, but wine grapes (and other fruits) can have lots of bound potential, which is where most of this research started in the first place! A new product that has begun to find its way around to breweries and Instagram is called Phantasm. Essentially, Phantasm is an extract of New Zealand Sauvignon Blanc grape skins processed into a powder that can be used at various points in the brewing process. Even after being used/pressed in the wine production process, the wine grape skins can still contain bound thiols concentrated on the skins (similar to spent dry-hops).
Because many of the free thiols from the Sauvignon Blanc grapes were released into the wine, the skins are still valuable because of the incredible number of thiol precursors, mainly the cys-3MH thiol precursor. Most bound thiol precursors in hops and wine grapes are glutathione-thiol (glut-thiol) and not cysteine-thiol (cys-thiol) conjugates. Bioegineered yeast strains can liberate bound cys-3MH precursors, which is why most of the research focuses on this class. However, hops and wine contain loads of glut-3MH precursors as well, and this post will get into how the potential of introducing glut-3MH precursors into the mash might help free even more 3MH.
Reaching out to and collaborating with Jos Ruffell, the creator and founder of Phantasm (and co-founder of Garage Project in NZ), I learned a lot about how the powder is created, how it’s been used with other breweries, and what the future of the product might entail.
Phantasm is a project that Jos has been working on for over three years. Living in New Zealand, which is known for its ultra tropical passion-fruit-like Sauvignon Blanc wines, Jos thought there might be potential for incorporating these thiol-rich wine grapes into beer. After all, one of the main thiols in these wines (3MH) is also key thiol brewers seek in hops.
Utilizing only grape skins from New Zealand vineyards already known for their thiol-rich grapes, Phantasm powder would likely have a higher potential for thiol concentrations. Some vineyards have such incredible thiol-heavy grapes that “Thiol Bomb Tanks” are utilized to create highly tropical and passion fruit Sauvignon Blanc wines that can then be used to blend into wines that need the extra thiol-fruit boost. This is a process similar to breweries who keep extremely acidic low pH pale beer in tanks that they can use to increase the sourness in beers that didn’t quite get into the acidity range they were seeking.
This focus on using only high thiol blocks of grapes from winemakers is becoming increasingly crucial to the Phantasm process; the newest crop (2021) will include a known high thiol block, for example. To enhance the thiol potential even beyond terroir, Phantasm is improving its processing methods to concentrate the thiol and thiol precursors even more, potentially making a 3 g/L dose equivalent to an 8 g/L dose.
How much 3MH thiol potential are we talking about with Phantasm combined with a bioengineered yeast strain designed to bioconvert? While not every lot of Phantasm powder will contain the same amount of 3MH precursors (similar to how not every hop lot of the same variety will yield the same concentration of oils), you can still see the incredible potential in one tested trial at Omega Yeast. Although the average of ferments with Phantasm and Cosmic Punch™ was around 775 ppt of free thiols, the experiment charted below was on the higher end of the free 3MH average than a control wort with no Phantasm.
Now that we know that including the bound-thiol rich Phantasm powder in a beer (usually in the whirlpool along with thiolized yeast strain) can get you 3MH levels ~14 times above sensory thresholds let’s look at how utilizing specific hops in the mash might help to push these free 3MH levels even further!
The collaboration beer we brewed as the main topic of this post with Omega Yeast and Phantasm (named Locksmith) was inspired by the idea that the lock was the Phantasm powder, loaded up with cys-3MH precursors. The key was Omega’s new thioloized strain called Cosmic Punch™, designed to unlock the bound potential in hops and products like Phantasm. Not only did we want to include Phantasm in the whirlpool, but we piggybacked on some Omega trials deciding to go with Cascade as a significant mash-hop addition.
Why consider mash-hopping? At least for grains, it’s the mashing process that releases the grain 3MH precursors into the wort. Cosmic Punch™, for example, is freeing 3MH precursors only from grain higher than most heavily dry-hopped IPAs. So on the surface, it makes some sense that it might also help hop-derived precursors get into the wort, but why?
I reached out to Laurent Dagan, who has published outstanding research on bound thiol precursors. Dagan shared his thoughts on mash hopping potential by saying, “In malt, many enzymes are working during mashing; among these enzymes, there are many different peptidases. Thiol precursors we know are peptide derivatives (as glut-3MH). We can suppose (and it is true!) that these peptidases can change the balance between the different thiol precursors in the final wort, associated with a FAN change. All of which could change the release during fermentation.”
Dagan also shared that thiol precursors are highly polar, so the extraction of these cys and glut precursors is likely happening quickly on the hot side. Still, they haven’t done any studies yet to see what point in the process is best to add precursor-rich products (boil or whirlpool, for example). So, it seems that hop-derived precursors are making their way into wort when added to the kettle, but the mash might be where we can convert more of the glut precursors to cys, which is the class of precursors beta-lyase producing yeast can free. The more cys-3MH precursors we can push into the fermenter, the more potential for free 3MH!
In addition, Laurent Dagan mentioned on an MBAA podcast that a protein rest might help with optimizing thiol conversion in some unpublished data they have formed. At lower temperatures in the mash (protein rest 113℉ or ~45℃), the enzymes are activated that transform bigger cys-3MH precursors into smaller precursors. Depending on the yeast, these smaller cys-3MH thiol precursors might be more readily released during fermentation. Or, perhaps more of the glutathione are converted to cysteine during a protein rest, whether that results in more free 3MH is still in question. Further, boiling wort will likely extract more of the thiol precursors into the wort, allowing a bioengineered strain to free them (not necessarily freeing them). So, we might conclude that using high bound thiol hops (like Saaz) early into the boil as a bittering charge may also help feed these precursors into the beer.
Omega Yeast found increased levels of free 3MH in beers fermented with Cosmic Punch™ that had elevated levels of mash-hopping done, giving some evidence to this glut-to-cys precursor path. Omega maxed out their mash-hopping trials at rates close to 2 pounds per bbl (~7 g/L) with varieties known for their high bound 3MH potential (hops like Saaz, Calypso, and Cascade).
Surprisingly, Omega saw even higher free 3MH in beers fermented with Cosmic Punch™ with significant mash-hop charges compared to equivalent rates of whirlpool charges! The mash-hops likely benefit from the high enzymatic environment of the mash, making these precursors more readily available (glut to cys) for the engineered yeast to free during the early days of fermentation. It’s also possible that less 3MH conversion with whirlpool hops vs. mash-hopping is due to stripping from high polyphenol vegetal hop material pulling out sensitive thiols (and precursors) during the whirlpool.
If you wonder what the IBU implications might be from mash hopping, Omega found that ~30% utilization in their trials of what you’d expect to get from the same amount of hops used as a 60-minute addition. In other words, you would get ~30% of the expected IBUs from a 60-minute hop addition with a mash-hop addition. Utilizing high bound 3MH hops with low alpha-acid content (like Saaz) is a great way to boost thiol precursors into the wort without worrying too much about IBU increases.
As you can see from the chart above from Omega’s research, next to the control beer with a non-thiolized British V, Cosmic Punch™ ran away with the total free 3MH thiol count. In addition, in this case, with Chinook at 2 lbs./bbl (7 g/L) in the mash vs. added in the whirlpool, increased free 3MH thiols in the beer ~20% from the non-hopped sample (which just had cys-3MH precursors from the base grain).
The surprising result above was how much lower the total free thiol content was with the whirlpool hop addition; in fact, the whirlpool beer had ~40% fewer thiols than the non-hopped control! Thiols resulting from mash-hopping would likely increase even more when using hops known to have even a higher concentration of bound cys/glut-3MH precursors. Laura Burns, Director of Research and Development at Omega Yeast, suggests the results would have likely been even higher for free 3MH when mash-hopping with higher bound thiols hop varieties than Chinook. Laura stated that in their many trials, “overall we have the biggest sensory bump with those hops known to have high precursor levels.”
How is that whirlpool hops would result in fewer thiols? Isn’t that the whole point of whirlpool hopping, to push hop compounds into the fermenter? Should we stop whirlpool hopping? It seems that for the first question, it’s likely that thiols are sensitive enough to be pulled out in the presence of a lot of hop vegetal material. Although it’s just as much hop material in the mash in the current tests, it’s like the freeing or conversion from glut-3MH to cys-3MH that is pushing the thiol precursor count in the kettle (where they likely remain because they are so polar) and there is no big whirlpool addition then working to pull them out as the hops percolate through the kettle.
Just in terms of sensory, we noticed a similar loss of perceived thiol concentrations after dry-hopping bioengineered Yeast beers during trials at Sapwood Cellars. In our experience, these strains did a seemingly great job releasing thiol precursors during fermentation, but after a heavy hit with dry-hops, the fermentation thiol character was significantly reduced. For example, in a test batch with Cosmic Punch™, comparing a sample post-fermentation and pre-dry-hop, the non-dry-hopped beer was super bright, with white grapefruit and exotic fruit aroma. However, after dry-hopping, the beer was pulled back into a more neutral and familiar IPA-like territory.
One last thing regarding mash-hopping. At Sapwood, we have been utilizing mash-hopping for a while, entirely on separate research that suggests it may benefit shelf-life. As I wrote in The New IPA, alpha and beta acids from hops are good at complexing problematic metals released into the wort during mashing that can react with oxygen later on in the brewing process. For example, the paper cited found that early-stage hopping led to reduced metal concentrations (~30% fewer iron levels, for example) and improved oxidative stabilities compared to a reference beer. The higher the pH, the better the hop acids were at complexing the metals, so it might make sense then to add your mash hops before adjusting for mash pH to give the acids a complexing head start.1
Potential Role of Hop-derived Heavy Metals
In conversations with Dr. John Paul Maye, Technical Director at Hopsteiner, upon visiting the brewery earlier this year, I asked his opinion on why this pull-back of perceived thiol flavor/aroma might be happening after dry-hopping. Although it needs to be tested, he thought it’s possible the vegetal material in dry hops themselves might be pulling out thiols as they fall through the beer (both in the kettle and fermenter). In addition, the heavy metals in hops may also be absorbing the sensitive thiols. For example, copper sulfate, sometimes used as a fungicide in Europe, can produce hops with little to no thiols.
How much potential is there for metal pickup from hops? In a study of 15 commercial beers, those that were dry-hopped had the highest levels of metals. For example, an imperial IPA had 0.21 ppm of Manganese, and two different pale ales had 0.23 ppm and 0.15 ppm, compared to an American light lager with 0.05 ppm.[note] Porter, J., & Bamforth, C. (2016). NOTE: Manganese in Brewing Raw Materials, Disposition During the Brewing Process, and Impact on the Flavor Instability of Beer. Journal of the American Society of Brewing Chemists. doi:10.1094/asbcj-2016-2638-01[/note] Manganese, it appears from the study, is a metal from hops that is particularly good at extracting into beer (potentially then absorbing free thiols).
In the paper above, the longer the hops were in contact with the beer and the warmer the dry-hop temperature, the more metal pickup. Specifically, dry-hopping at 68°F (20°C) had 0.10 mg/L more manganese than the beer dry-hopped cold at 38°F (3°C), and manganese concentrations peaked after 5-7 days. It’s not just manganese in hops that could strip thiol (although it’s the most efficient at extracting into beer), as the paper found that iron and copper were also in beers after dry-hopping at high enough levels that could lead to oxidation problems.
Along with other reasons, you may want to consider dry-hopping cool. This might help limit metal extraction from hops into your beer and potentially less thiol stripping (again, this theory would need to be tested).
Timing of Dry-Hop Additions and Thiol Losses
The timing of the hop addition may also play a role when determining the potential for thiol-loss to vegetal hop material on the hot side of the process. The chart supplied below by Omega Yeast shows a higher total thiol concentration in beer when using fewer hops (.5 lbs/bbl vs. 2 lbs/bbl) when adding dry hops to beer on day one of fermentation. At least in this test, the additional amount of hop vegetal material may have helped to pull out or strip thiols. I wonder if the active fermentation (which would move around the hop material) might help pull out additional thiols, which is especially true when there is a greater amount of hops (2 lbs/bbl) in the beer. At least for this test, it appears that a little goes a long way for an increase in thiols with active fermentation dry-hopping when using a bioengineered yeast strain like Cosmic Punch™.
On the flip side, 3MH levels increased without active fermentation on day 7 of fermentation when little, if any, CO2 production is likely taking place. However, as you’ll see later in the post with our samples for The Locksmith, we saw a reduction in 3MH with even a small dry-hop charge with the less vegetal material Cryo Hops® from Yakima Chief.
So, the Omega Yeast test implies that there might be a benefit related to higher thiol concentrations with a small ½ lbs/bbl (~2 g/L) dry-hop during active fermentation (likely best during the first few days of fermentation). It’s important to take into consideration the impact of hop-creep with active fermentation dry-hopping. You don’t need to worry about off-flavors like diacetyl at this stage because the yeast cell rates are high and healthy. However, you might need to worry about subtle final gravity swings with active fermentation dry-hopping. The enzymes released into the wort from hops at this stage can work to free up dextrins and leave you with a dryer beer. It’s not a massive drop in gravity, but you could see a decline of ~.2 Plato. Another impact of active fermentation dry-hopping that Laura Burns mentioned is that it might also reduce the dissolved CO2 in the beer (by creating nucleation points), which can benefit yeast health as yeast don’t like CO2.
Looking at our Locksmith recipe and going with the theory that hops may be pulling out thiols, we chose to go with a low dry-hop charge in the collaboration of only 22 pounds (in a 10 bbl batch) of Mosaic Cryo Hops® (usually we would dry-hop with 2x this amount in a DIPA). The logic is that a smaller charge could help reduce thiol losses, and Cryo, with less vegetal material (and likely fewer metal concentrations), might strip fewer released 3MH thiols. Lastly, choosing to dry-hop post-fermentation might also help prevent a loss of thiols as the Omega test saw much higher concentrations with the day 7 dry-hop vs. the early active fermentation charge.
Oxygen and Thiols
As mentioned at the start of this post, we wanted to take the collaboration of The Locksmith a step further in terms of analyzing the beer by sending samples to a lab called Nyseos in France to get tested for thiols (3MH, 3S4MP, and 4MMP). We were instructed to dose our sample cups with metabisulfite to help protect the thiols during transit. After adding the metabisulfite, we then froze the samples and shipped them to be analyzed via LC (not GC). Thiols, because of their low concentrations, are hard to test for and only a handful of labs even offer the service. The Nyseos lab uses very robust and controlled processes that allow them to quantify thiols accurately.
I don’t want to go too far off-topic, but two things come to mind when learning that thiols are so sensitive to oxygen: 1) how important avoiding oxygen ingress is in beers chasing high thiol counts and 2) can beers with high total thiol concentrations potentially be more shelf-stable than low thiol beers due to their sensitivity to oxygen?
Again, leaning on the knowledge and experience of Laura Burns at Omega, it was interesting to hear that in multiple samples they sent to Nyseos to get tested for thiols, those that were not treated with metabisulfite came back with samples all over the place. The frustrating results were likely the result of any oxygen ingress absorbing the thiols.
Although additional research should be done in this area, it seems plausible that because thiols are so susceptible to oxygen, they may also be acting as one of the first waves of defense against oxygen (similar to sulfites). This can potentially mean that when using products like Phantasm and bioengineered yeast strains designed to free thiol precursors, you could be extending the hop-forward shelf-life in your beer. Because the thiol concentrations can be as high as +1,000 ppt (for free 3MH), even with some oxygen ingress and eventual thiol absorption, you could still be above the 3MH threshold and the total thiols taking the oxygen hit, not other important hop-forward compounds like free monoterpene alcohols or the eventual malt staling aldehydes (post-fermentation hop oils might also help reduce staling aldehydes).
At least one academic paper we can look at for insight into thiols, oxygen, and shelf-life. The chart below shows the concentration of free 3MH in the beers studied during the natural aging of six top-fermented Belgian beers. As you can see, the thiols are almost entirely absent after one year and as quickly as six months in some samples. The reduction in thiols is likely due to their “high propensity to undergo oxidation, nucleophilic additions or substitutions.” The paper’s authors add that “antioxidant components (ascorbic acid, and sulfites) added either in the brewhouse or at bottling can help to preserve thiols.2
Interestingly, you can see from the chart pasted below, when lager beer was spiked with cys-3MH precursors, there was a conversion of the bound 3MH (cys-3MH) to free 3MH with time in the bottle. Although the conversion was relatively small (ranging from 0-19% conversion across the tests), you can see how increasing the thiol precursors in the beer could benefit its flavor longevity, especially when using a yeast strain designed to free those thiols. So, free thiols are likely to oxidize post-packaging (to a lesser extent if using metabisulfites), but thiol precursors can also continue to be released into a free state during aging.
Although the study here used a Belgian strain, I would guess that using a strain like Omega’s Cosmic Punch™ (or Berkeley’s Tropics strain) designed to free bound thiols with active beta-lyase activity would release substantially more thiols during aging. On top of that, the starting concentration of free thiols (especially if using high 3MH precursor hops or using Phantasm) would be incredibly higher. For example, Omega found free 3MH nearly twice as high as the beers tested in this study, and that was just from malt precursors (no hops). Although I would like to see a bioengineered strain tested for thiols throughout aging like in this study, if the majority of the Belgian beers had free 3MH above the threshold after two months in the bottle, the bioengineered strain would likely remain above the threshold for much longer at the same time potentially freeing addition bound 3MH during aging. All to say, I’m theorizing that using high thiol precursor products in combination with a strain like Cosmic Punch™ would result in a punchy aromatic beer with a more lasting flavor/aroma than a typical fermented IPA.
So, to sum up, the oxygen aspect of heavy thiol beers, it seems plausible that ignoring the sensory part altogether, high free thiol producing beers may act as the second wave of oxidation protection after the sulfites have been used up. Essentially, brewing high thiol beers, avoiding oxygen during dry-hopping and packaging, and even considering adding small amounts of metabisulfite at dry-hopping could all be ways of extending the fresh hop-forward shelf-life in IPAs.
Role of Grains in Thiol Precersour Potential and Shelf-life Extension
When researching the book, The New IPA, it was interesting to learn that even protein-derived thiols from malts may serve a protective role in hazy beers, similar to how free thiols like 3MH might. Essentially, sulfites are the primary antioxidant in beer (produced naturally during fermentation or added post-fermentation via metabisulfite), and these protein-derived thiols and free hop thiols can act as a secondary antioxidant to encourage redox stability (oxidation).
As it relates to malts, it’s challenging to know the total thiol content, but it has been found that the reduced and total thiol content in beer was highly correlated with total protein contents. However, it may not be as simple as just increasing total protein content, as not all proteins have the same antioxidant capabilities.
To test out the antioxidative properties of the stable LTP1 protein (stable here means it is a protein that can withstand the brewing process better than other proteins), a paper experimented with three Australian lagers, one fresh off the production line (“fresh”), one aged for 12-weeks at 86ºF (30°C) (“aged”) and another for five years at 68ºF (20°C) (“vintage”). The authors found that the Australian lager characteristics remained in the fresh and vintage beers but were lost in the aged beer.3 The flavor stability of the fresh and vintage beers was then correlated with the presence of the LTP1 protein. Essentially, the authors concluded that the presence of thiol-rich LTP1 indicates that the protein can play a prominent role in maintaining the redox balance of beer from its free radical scavenging and antioxidant capacity in both fermentation and packaged beer. Using the study results, you can make a case for enhancing LTP1 proteins in hazy, hoppy beers to try for flavor stability after packaging.
Interestingly, the role of the LTP1 protein in promoting head retention may be partly because it’s rich in the same free thiols that might also play a role in beer stability. Structurally, the LTP1 protein consists of 8 cysteine residues, and this high content of thiol cysteine in the protein is likely the basis for its antioxidant effects. Similarly, with cysteine-bound thiols in hops (the thiol precursors), they too could act as an antioxidant when freed.
So, to increase the protein-derived thiols to help with stability, we should be using grains high in the LTP1 protein. One possible way to do this is by brewing with a high percentage of under-modified grains (big fan of chit malt). 4
To speculate even further, I wonder too if malted grains that are under-modified (like chit malt) might be higher in the LTP1 protein (leading to potential higher shelf-life) and one of the higher cys-3MH precursor grains which a bioengineered strain can then act on and free. At least for The Locksmith beer, we brewed for this post (which included chit malt), it was one of the highest thiol beers tasted to date compared to various other beers Omega has tested.
Laurent Dagan continues to research malt and thiol precursors, and I look forward to reading his results. So far, they have done experiments testing 15 different malts, and the barley malts were all higher than non-barley (rice, sorghum, and wheat). As you can see from the chart presented in an online presentation from Dagan below, not all barley malts are equal precursor potential. It seems that less a grain lightly kilned malts help to retain these precursors.
In an October 2021 MBAA podcast, Laura Burns described that in various ferments they have done at Omega yeast, the grain with the most sensory impact of freed thiols with their Cosmic Punch™ strain has been a local wind-malt variety. Specifically, the wind-malt was with a local roaster in Indiana that produces the malt by avoiding kilning altogether as it’s dried in the sun instead. So wind-malts might be a good base malt to experiment with when using these beta-lyase-producing yeast strains. Mecca Grade Estate Malt in Oregon is one of the few wind-malts I’ve seen for sale. In general, though, Laura explained that barley malts were still “bringing a lot of precursor in,” and they have also found (not sure why), but when “wheat is in the beer beyond 20%, you start to lose the thiol impact.” This could potentially be that malted wheat just doesn’t have as much precursor thiol potential, which is what was found in the one variety in Dagan’s tests above, along with rice and sorghum malts.
Understanding Thiol Precersour Potential
Because thoil precursors can come from multiple sources (hops, fruits, and grains), it seems helpful to look at the potential from each source. Again, remember that a yeast strain that is capable of producing the enzyme beta-lyase during fermentation is needed to free the precursors to allow them to have a sensory impact on the beer.
When it comes to grain, there is still room for experimentation to understand what is increasing precursor counts, but the chart below from Omega Yeast (in Cosmic Punch™ ferments) shows the potential from just a 2-row base malt. With the thiolized Cosmic Punch™ strain (and no hops at all), the cys-3MH precursors in malt were enough to get the beer way above the free 3MH threshold of 60 ng/L getting to 546 ng/L. This is compared to a ferment with the same parent strain of OYL-011, which produced only 11 ng/L of free 3MH. It would be interesting to see the same test done with thiol-rich lightly kilned LTP1 grains included in the grist.
Although each batch of Phantasm might vary in its total cys-3MH precursor concentration, the results tested so far show that using the dried Sauvignon Blanc product with a beta-lyase producing yeast will get you at extremely high free 3MH levels. In smaller flask fermentation trials, Omega found that compared to a control wort with no Phantasm, across four ferments, the Phantasm beers averaged an increase of 441 ppt or over 130% increase in free 3MH! The highest precursor lot of Phantasm increased the free 3MH thiols 583 ppt (over 170%). It’s obvious now why we wanted to call our collaboration beer The Locksmith when you see the potential of a bioengineered yeast strained designed to unlock bound thiols and a product like Phantasm loaded with bound potential.
Again, not all hops are the same for thiol precursor potential, but for the hops that have been tested to date in the literature, below is a chart of the potential in 17 different hops. I hope the cys-3MH (and glut-3MH) figures are something that hop suppliers consider testing and advertising along with their hop oil information as our understanding of the potential in bound compounds continues to improve. Having this type of information can help us understand when and how to use certain varieties.
It’s probably important to remember that because the freeing of the bound thiols is happening inside the bioengineered yeast cell itself, it’s probably best to try and load your wort with precursors (from malt, hops, and products like Phantasm) prior or during the start of fermentation or you might miss the window for freeing 3MH. That’s not to say that releasing thiol precursors won’t happen post-primary fermentation, as the Belgian study above found, or as was the case in late dry-hopping in the Omega trials. I would assume this continual freeing would be significantly halted when the beer is stored cold, however. This also brings up the idea of using bioengineered strains to re-ferment and condition beers that might have high thiol precursors when bottling beer (mix-fermentation wine grape beers or dry-hopped sour beers, for example).
The Locksmith Recipe and Results
Collaboration with Omega Yeast and Phantasm
|OG/FG/ABV||Est. IBU||SRM||Water||Mash Temp|
|1.083/1.020/8.4%||54||4.7||~200ppm chloride/200ppm sulfate||156°F (69°C)|
|Weyermann Rye Malt||10%|
|BEST Chit Malt||5%|
|Cascade||160 grams (~2lbs/bbl or 8g/L)||Mash||~16|
|Mosaic Incognito Extract||16.5 mL (~37 IBUS)||Knockout||~37|
|Phantasm Powder||160 grams (~2lbs/bbl or 8g/L||Whirlpool (180°F or 82°C)||0|
|Mosaic® CRYO HOPS®||160 grams||Post-fermentation @ 56°F or 13°C|
|COSMIC PUNCH™ ALE||66°F or 19°C ramping up to 74°F or 23°C||7 Days|
To help us better understand how big the Phantasm’s role powder might play in this batch, I pulled ~17-gallons before the whirlpool Phantasm addition to fill a smaller half bbl Spike conical fermenter. A week into fermentation, we pulled some samples before dry-hopping to see how the Phantasm beer compared to the non-Phantasm ferment. It was highly evident that the precursors from the Phantasm impacted the beer’s sensory. The bigger batch ferment (10 bbls) was white-wine forward, with a touch of Welch’s-like white grapefruit juice, whereas the non-Phantasm beer was relatively neutral.
In general, when comparing our smaller trials fermented with Cosmic Punch to our RVA Manchester ferments, the Cosmic Punch beers (pre-dry-hop) tended to be brighter and less doughy/malty. This quality (likely from simply having more intense fruit-forward thiols released) is a nice base for dry-hopping. Although we found that dry-hopping these bioengineered strain beers can greatly impact the sensory of the thiol-ferment, whether this is by hop vegetal material stripping or hop metals absorption, or masking, the brighter, less worthy base seems to allow the dry-hops to pop more with less malt competition.
Again, we used Mosaic Incognito to get some bitterness without introducing lots of vegetal material that might strip thiols. Likewise, we did a small (2.2 pounds/bbl dry-hop (8 grams/liter) to get some dry-hop character again but hopefully with less pullback of thiols with the smaller charge in combination with less vegetal material in Cryo. As you can see in the chart below from our tested results, The Locksmith did see a decrease in thiols after even a small dry-hopping charge with Mosaic Cryo, especially with the passion-fruit forward 3MHA thiol. I suspect the thiol levels would have decreased even further with a bigger dry-hop charge (5+pounds/bbl), especially with T-90 pellets, as more vegetal material might pull out additional free thiols.
Sensory, you could taste this pullback, despite the thiols still well in and above their respective threshold limits in beer. I’m sure it’s partly the slight reduction from the dry-hop, but there is likely some type of free-thiol masking taking place with all the other compounds dry-hopping brings to the beer (polyphenols, hydrocarbons, terpenes, etc.). For the Locksmith, after dry-hopping, I didn’t have nearly that grapefruit juice-like taste we were getting before the dry-hop. However, the beer maintained the brightness that Cosmic Punch™ brings.
The beer has a nice fruitiness that we typically wouldn’t get with just a Mosaic dry-hop with a non-thiolized London Ale II. Overall, the beer had low sensory bitterness and finished just a touch too sweet. The combination of the two wasn’t my favorite; I think we should have tried to dry the beer out a touch more and bring a little bit more from the hot-side hopping (perhaps more Incognito). The overall hop flavor of the beer is relatively weak, but that’s to be expected with such a small dry-hopping charge. It misses that DIPA-like hop intensity that people would expect in a beer like this. However, the lower Cryo rate kept a bit more of the thiol-fruit aromatics produced during fermentation, especially when considering other trials we did that were dry-hopped much more heavily.
The Locksmith was a unique DIPA; I’m not sure I’ve ever tasted something in this type of category of IPA before. I liked it a lot! However, being sold as a DIPA, I think we’d have to up the dry-hopping, which would ultimately reduce that fermentation character we like. There may be potential in post-fermentation hop oils to go along with a smaller hop charge in beers like this to get them up to that DIPA hop-like level without introducing too much hop vegetal material.
The other interesting thing we found in this beer was the pH change brought from the Phantasm powder and the Cryo dry-hop. Post-boil (with a 5.18 mash pH), the Phantasm dropped the whirlpool pH to a low 4.78. We typically add phosphoric acid to our kettle to lower the pH to limit color pickup from Maillard reactions but didn’t in this beer because of the pH drop from Phantasm. The result was a beer with more color than we were going for. Perhaps we could have added some Phantasm as we filled the kettle to get us where we wanted to be. Because thiol precursors are so polar, they would have likely remained in the beer throughout the boil. We could have also considered adding baking soda to the whirlpool to help offset the pH drop that Phantasm brings.
As for The Locksmith’s pH, it was only 4.16 pH before dry-hopping and rose only slightly to 4.21 after the Cryo dry-hop. The slight increase is because as the vegetal material increases in the dry-hop, so too does the pH of the beer. This lower pH, much lower than most of our IPAs, also contributed to a not a DIPA vibe. Just something else to consider when experimenting with Phantasm!
Phantasm Without Beta-Lyase
I was curious how Phantasm powder might work when using it with a yeast strain not bioengineered to unlock bound 3MH thiols, so I reached out to Mike Foniok, head brewer and owner of The Establishment Brewing Company in Calgary (as they are not allowed to use these yeasts yet Canada). Mike has one of the highest-rated Phantasm beers on Untappd with a hazy DIPA called Irrational Things, so he seemed like a good person to ask!
Irrational Things (8.5% DIPA)
Malt: 2 Row, Flaked Oats, Flaked Wheat, Malted Wheat, and Chit Malt
Hops: Galaxy, Equanot, Azacca, Citra Incognito, and Mosaic Cryo
Yeast: Escarpment Labs Foggy London
The establishment utilized Phantasm in the beer by loading it in the whirlpool and then followed that up with a smaller active fermentation addition. The whirlpool addition was 1.3 lbs./bbl (5g/L) and the fermentation addition was ~0.5 lbs./bbl (1.8g/L). They didn’t want to displace the number of hops that they usually put into the whirlpool with phantasm powder; instead, they wanted it to be in addition to their regular whirlpool hopping as an additional layer of potential flavor. Since their whirlpool vessel limits how much matter they can physically put in it, they relied on Cryo Hops® and a small addition of incognito in the whirlpool (which may have preserved some thiols via less hop vegetal material).
In terms of the aroma impact from the Phantasm in the whirlpool and during primary fermentation, Foniok noticed that the hop aroma impact in the beer pre-dry hop addition was more intense than usual in sensory when considering their whirlpool hop load was very similar to a typical Imperial NEIPA. Like with some of Sapwood’s trials with Phantasm, The Establishment saw pH drops in the whirlpool compared to their conventional whirlpool hopping methods. Below you can see the pH reduction in three consecutive batches of the beer with Phantasm added to the whirlpool. Essentially, they averaged a ~0.30 ph drop when using Phantasm at a whirlpool rate of 1.3 lbs./bbl (5g/L).
In terms of shelf-stability impact from thiols coming from the Phantasm powder, Foniok anecdotally thought this was the case. They didn’t do a warm vs. cold-stored double-blind to confirm these observations, but the beer’s hop aroma and the flavor did seem to stay at “its peak” longer than typical. In terms of dry hop matter pulling out any free 3MH thiols that may have been present on the dried wineskins, they didn’t notice as much of a pull-back as we did in some of our trials. I would assume this is partly because the number of free thiols was so much less post-fermentation using a conventional yeast strain.
To try and bring all this information together into some type of visual, below is a chart of many of the different thiol tests discussed in the post. Below the chart is the usual key points summary I like to include in posts of this length. You can see how the different potential in unlocking bound thiols compares with other products (grain precursors, mash hops, whirlpool hops, and phantasm) compared to a control. Using the science available at the time of writing for The Locksmith, we tried our best to formulate a recipe that might help push thiols as much as possible; luckily, we achieved that goal!
- Hops have both free and bound thiols. Hops that are high in free thiols (like Citra and Simcoe, both high in 4MMP) are more efficiently utilized as a dry-hop. Hops high in bound thiols (like Saaz and Calypso, both in cys-3MH) are more efficiently used early in the process, especially when paired with a bioengineered yeast strain designed to free the bound thiols (mainly cys-3MH).
- Yeast strains bioengineered to unlock bound thiols have the necessary gene (IRC7 for Omega yeast) or TnaA (for Berkeley yeast) to produce the required enzyme (beta-lyase) during fermentation to unlock thiols (going from cys-3MH to free 3MH). Not only are the genes inserted, but they are overexpressed, which means the gene activity is turned up or highly expressed, allowing for an even greater release of bound thiols.
- Phantasm is an extract of New Zealand Sauvignon Blanc grape skins (targeted for thiol-rich grapes) processed into a powder that, like hops, are high in thiols and thiol precursors (cys-3MH). Specifically, an average of Phantasm ferments with Cosmic Punch™ yeast had ~450 ppt more free 3MH than those with only grain.
- Mash-hopping as high as ~2 lbs./bbl (7 g/L) with high bound 3MH hops (like Saaz, Calypso, and Cascade) can increase free 3MH concentrations (even higher than whirlpool hopping).
- In terms of bitterness from heavy mash-hopping, Omega found ~30% utilization in their trials of what you’d expect to get from the same amount of hops used as a 60-minute addition.
- Free thiols can be pulled out and masked by heavy dry-hopping rates. Utilizing less vegetal material products like LUPOMAX™ and Cryo Hops® or post-fermentation hop oils might be ways to increase hop flavor while maintaining more free thiols.
- Active fermentation dry-hopping might also act to pull out free thiols compared to post-fermentation hopping. If an active fermentation dry-hop charge is wanted, a smaller dosage has been shown to increase free 3MH thiols than more significant charges.
- Free thiols are very sensitive to oxygen, so avoiding oxygen during dry-hopping, purging vessels/bottles/cans, and even considering the use of metabisulfite might be a way to preserve thiols freed during fermentation and dry-hopping.
- Thiols (especially in the presence of yeast strains capable of beta-lyase activity) can continue to unlock precursors during storage, suggesting they may be great bottling strains if using high bound thiol products as part of the recipe.
- Grains themselves can contain 3MH precursors, especially those lightly kilned or not kilned at all (wind malt may be exceptionally high). In one test, malted wheat was a very low source of 3MH precursors. Bioengineered yeast strains designed to unlock these thiols are essential to achieving high free 3MH, however.
- Wietstock, P., Kunz, T., & Methner, F. (2016). Influence of Hopping Technology on Oxidative Stability and Staling-Related Carbonyls in Pale Lager Beer. BrewingScience, 69, 73-84.)
- Tran, T. T., Cibaka, M.-L. K., & Collin, S. (2015). Polyfunctional thiols in fresh and aged Belgian special beers: Fate of hop S-cysteine conjugates. Journal of the American Society of Brewing Chemists, 73(1), 61–70. https://doi.org/10.1094/asbcj-2015-0130-01
- Wu, M., Clarke, F., Rogers, P., Young, P., Sales, N., O’Doherty, P., & Higgins, V. (2011). Identification of a Protein with Antioxidant Activity that is Important for the Protection against Beer Ageing. International Journal of Molecular Sciences.
- Sorensen, S.B., Bech, L./ M., Muldbjerg, M., Beenfeldt, T. and Breddam, K., Barley lipid transfer protein 1 is involved in beer foam formation. Tech. Q. Master Brew. Assoc. Am., 1993, 30, 136-145