All About Thiolized Yeast

Background

The Thiolized^® process enhances a yeast’s ability to biotransform compounds found in malt and hops to unleash thiols — flavor- and aroma-active compounds reminiscent of grapefruit, passion fruit, and guava. Many brewers who include dry hopping in their processes are already familiar with the concept of biotransformation, or the development of new aromas and flavors due to yeast interacting with hop compounds.

We group biotransformation events into two categories:

1. the conversion of aroma compounds
2. the release of aroma compounds from precursor forms
   

The Thiolized technology is focused on the second category, working to give brewers access to the biotransformation of thiol precursors. As it turns out, typical yeast biotransformation of thiol precursors is very inefficient in wort, but by employing these Thiolized strains in fermentation, brewers can expect a significant increase in thiol output. More on that later.

What are thiols and where do they come from?

Volatile thiols are highly impactful aroma compounds that evoke grapefruit, passion fruit, and guava and are found in a variety of tropical fruits, wine grapes, and hops. These thiol compounds exist in two forms — free forms, which are highly aromatic and volatile, and precursor forms (i.e., glutathione- and cysteine-bound thiols). The precursor forms, abundant in malt, are non-aromatic and require yeast with β – lyase biotransformation activity to release them. Hop varieties vary widely, not only in the amount of thiol compounds, but also the percentage that are in the non-volatile precursor form. New World hop varietals, specifically Southern Hemisphere hops, appear to be the highest in free thiols, which contribute to their characteristic tropical fruit aromas. The precursor for 3‑sulfanyl-1-hexanol (3SH, but also referred to as 3MH — a thiol known for its intense grapefruit and passion fruit aromas) is abundant in barley, but does not reach sensory thresholds until converted to its free form. In beer and wort, the overwhelming majority of these thiol compounds are in precursor form (1000-fold!) and are a stockpile of aroma potential with a yeast capable of biotransforming them to the free volatile and aromatic thiol compounds.

How were these strains developed?

Our R&D team became curious about thiols and methods for making the process of freeing thiols more efficient. We dug into the literature and focused on IRC7, a gene in yeast that encodes an enzyme (β – lyase) that frees thiols from their precursor form. The problem, however, is that many brewing strains have mutations in the IRC7 gene that disable the enzyme, and those strains that do have a functional IRC7 gene don’t make the enzyme in wort because high nitrogen levels (like we see in wort) turn off the gene.

We explored the possibility of altering the IRC7 promoter (the sequence that controls the gene’s expression) to a version that would keep the gene active at all times, even in a high-nitrogen environment. By finding a way to keep the IRC7 gene functional, we should theoretically be able to unlock thiol precursors and derive more flavor and aroma from our malt and hops.

The first test

We began experimenting with our British V, a popular strain many brewers use for making classic hazy IPAs. We replaced its inactive IRC7 promoter with a highly active one, but found that the levels of 3SH remained unchanged in comparison to the non-modified British V. Unfortunately, British V has mutations that inactivate its IRC7 gene, meaning there was no chance for it to work, regardless of how much enzyme was made in that strain.

Back to the drawing board. We continued digging and discovered that the IRC7 gene in our Chico (West Coast Ale I) was functional, though it was not expressed in wort. We tried combining this IRC7 gene with the highly active promoter from the other trials and found that it worked like a charm. Using CRISPR/Cas9, we were able to add this activated IRC7 into British V, creating our first Thiolized strain: Cosmic Punch.

In search of even more thiols

The success of the Cosmic Punch tests led the team to look into other sources of β – lyases to see if we could boost thiol output even further. Our research pointed us to a β – lyase called PatB, specifically that of S. hominis, which commonly occurs on human skin as part of our natural microbiome that protects us from pathogens. In our brewing trials, the PatB from S. hominis generated the greatest amount of passion fruit and guava compounds. The bacterial PatB acts very similar to the yeast β – lyase Irc7 — it cleaves cysteine-thiol precursors found in malt and hops to let those thiols shine — but PatB is more active and more specific.

Using CRISPR/Cas9, we were able to insert patB into a classic Chico (West Coast Ale I) strain and found that the results were even more intense than just the activated IRC7 gene! The resulting strain is Star Party, which generates a massive thiol burst while maintaining the fermentation character of its famous parent strain.

To push the boundaries even further, we began experimenting with inserting patB into our lager strains. Through various trials we found Mexican Lager (OYL-113) to be the best candidate for Thiolizing, and thus Lunar Crush was born. Lunar Crush retains the lager fermentation behavior of its parent strain but adds a twist of tropical fruit flavor, creating lagers unlike the classics we’ve come to know over the years.

One lingering question remained: if patB was effective in Chico and Mexican Lager, how would it work in our favorite hazy strain? Using patB in British V worked well, giving rise to an even more potent thiol-producing strain: Helio Gazer. Helio Gazer produces the same sturdy haze as Cosmic Punch but with an even higher thiol output for brewers who want to really amp up the tropical qualities of their beers.

What’s the deal with mash hopping?

Using hops so early in the brewing process at well below boiling temperatures may seem counterintuitive, but it turns out that using hops in the mash provides an opportunity to convert thiol precursors into a form that makes it possible for our Thiolized strains to generate rich, tropical aromas.

In developing Cosmic Punch, we included Cascade hops in the mash for a test batch and found that 3SH levels increased nearly tenfold with Cosmic Punch in comparison to its parent strain, British V. When comparing these 3SH levels to those found during traditional whirlpool hopping, Cosmic Punch still generated about twice the free 3SH when measured in nanograms per liter (ng/L).

In other words, mash hopping gives brewers the opportunity to extract additional flavor potential from hops, especially those not typically prized for their intense aromatics. In our brewing trials, we have found Saaz, Cascade, Calypso, and Mittelfrüh to be effective as mash hops, generating a particularly pleasing thiol output. Researchers have also found high precursor levels in Apollo, Eureka, Hallertau Hallertauer, Nugget, and Hallertau Perle hops.

Unfortunately, we can’t yet say definitively which varieties of hops will be best for mash hopping. Environmental factors like temperature, elevation, and precipitation, and even lot-to-lot variance can affect the composition of a given hop variety. In general, we recommend using lower alpha acid hops to avoid over-bittering the beer and to save your expensive aroma hops for later additions where they can really shine without being lost during the boil.

Wait a second… you can get thiols from malt?

One of the most interesting discoveries in all of our research into thiols has been the emerging information about thiol precursors in malt. In one of our trials, we brewed a test batch with 100% two-row malt and added no hops, letting our Thiolized yeast run with what was available, and the results were incredible — we observed an increase of free thiols nearly ten times sensory threshold!

Much like hops, we don’t yet know which types of malt have the highest levels of thiol precursors available, but from our benchtop trials, unkilned and lightly kilned malts have shown the greatest increase in thiol output over a control (non-Thiolized) strain.

This presents an exciting opportunity for brewers to work with local maltsters and begin to discover the potential of malt terroir. Barley is abundant in thiol precursors, but as with any agricultural crop, the exact makeup of barley will vary from region to region. We recommend trying out one of our Thiolized strains in a relatively simple recipe, like a blonde or pale ale with minimal hop rates to let the yeast really shine and experience firsthand how these thiols are expressed in the finished product.

Are these strains safe?

Our engineered strains undergo thorough characterization and evaluation to confirm that the process of gene editing as well as the resulting application of the engineered strain to beer fermentation are safe for the intended use. The Irc7 β – lyase is naturally occurring in brewing yeast and the PatB β – lyase is derived from S. hominis, a bacteria that is abundant on your skin and is a natural protectant against harmful bacteria. The thiol levels in beers brewed with our Thiolized strains in beer are within the natural range found in Sauvignon blanc wine and tropical fruits like grapefruit, guava, and passion fruit. We engineered these strains using CRISPR/Cas9 and have confirmed no off target effects occurred. After the gene editing has occurred, the CRISPR/Cas9 machinery is removed. The benefit to expressing β – lyase in brewing yeast is that brewers have an option to remove the yeast, and thus remove the enzyme. Naturally the yeast will settle, autolyse, and degrade the β – lyase post fermentation. If you have any questions or concerns, feel free contact us at info@omegayeast.com.