BrewEd Ressource center: All you need to know about biotransformation

Yeast Interaction with Hops

Usually when brewers use the term biotransformation, they are referring specifically to hop biotransformation – the ability of yeast to interact with and modify flavor compounds derived from hops. When active yeast interacts with hops in the fermenter, it can increase aroma by releasing highly sensory active compounds from sensory inactive precursors, or change the flavor characteristics by modifying specific hop-derived flavor compounds.

Most of the discussion around biotransformation has focused specifically on terpenes, and polyfunctional thiols, both of which can contribute floral, fruity, and tropical flavours and aromas that form an important part of the flavor profile of many modern IPA styles. Other flavor compounds such as lactones and hop-derived esters may also play a supporting role in biotransformation sensory attributes.

Optimizing Biotransformation

Biotransformation can have a real impact on the flavor of the finished beer. The goal of this Biotransformation Resource Center is to help brewers understand the mechanisms involved in biotransformation to achieve specific flavor outcomes. Optimal biotransformation is achieved through careful consideration of yeast strain, hop variety, and brewing process.

Discover how different yeast strains interact with terpenes, thiols, and other flavor compounds, and how you can optimize your brewing process to increase biotransformation. You will also find information about our current biotransformation research projects and other technical resources.

What are thiols? 

Polyfunctional thiols refer to thiols that also have other functional groups such as alcohol, ketone or acids. Polyfunctional thiols play an important role in the organoleptic properties of wine and beer by imparting tropical fruit aromas. In the beer industry, “polyfunctional thiols” are often referred to simply as “thiols” for simplicity, and we use these terms interchangeably on this website.

Sources of Thiols

Thiols are an important part of the aroma profile of hops. In hops, thiols can be present as a free (aromatic) thiol or as an odorless precursor that is bound to an amino acid such as cysteine, glutathione, or glycine. Thiol precursors are also derived from malt.

The majority of thiol precursors from hops and malt are bound to glutathione (GLU), with a smaller amount bound to cysteine (Cys).

Want to know which hop varieties are high in either free or bound thiols? Check out this resource.

Thiol Biotransformation

Brewing yeast strains possess the IRC7 gene, which expresses a cysteine desulfhydrase β-lyase enzyme that is implicated in the release of free aromatic thiols from their amino acid bound precursors. Cysteine-bound thiols are imported into the cell where cysteine is enzymatically cleaved by this β-lyase. Cysteine is then used for yeast metabolism and the free volatile thiol is secreted from the cell where it contributes to beer aroma. Glutathione-bound thiols must also be imported into the yeast cell but require an additional enzymatic step (via a transpeptidase) before being able to be cleaved by a lyase, thereby lowering their release efficiency. As Glutathione-bound thiols are the largest pool of bound thiols contributed by hops, this means that the majority of thiol precursors are not released by normal yeast metabolism. This presents an opportunity for brewing scientists to explore better ways to exploit the Glu-bound thiol pool.

Additionally, specific thiols can be modified by yeast metabolism to produce different compounds with unique aromas. For example, 3-sulfanylhexanol (3SH) can be esterified by the yeast to form 3-sulfanylhexanyl acetate (3SHA). The sensory threshold of the latter is nearly 10-fold lower than the former, and can only come about through yeast esterification.

Further complicating the matter, in brewing yeast there exists more complete and less complete versions of the IRC7 gene (‘long’ and ‘short’ versions), differing levels of intact promoter regions (which effectively ‘turn on’ the gene), and differing allelic profiles (how the gene is carried on different chromosomes) – all of these things affect the level of activity of this β-lyase enzyme.

We have characterized Lallemand Brewing yeast strains for their thiol biotransformation potential by measurement of free thiols and thiol precursors in an identical wort fermented with different LalBrew Premium strains. A summary of the most up to date strain characterization for biotransformation potential is available here.

Want to know more about the latest in thiol biotransformation research? Check out our R&D resources page.

Thiol flavor impact and sensory threshold

Thiols in beer are usually described as having a tropical fruit flavor, including passionfruit, grapefruit and guava. Thiols have an incredibly low sensory threshold of nanograms per litre. The reason ethanethiol is added to gas is because we can smell it at levels one hundred million times lower than levels of ethanol. Not surprising then, if too many free thiols are created you can get some more undesirable characteristics, such as vegetal, sweaty, rubbery, and over-ripe fruit in your beer.  

How to optimize thiol biotransformation

Thiol biotransformation can be optimized through careful consideration of the hop variety, yeast strain, and brewing process.

The following chart is a guide to help brewers optimize thiol biotransformation during the brewing process in order to achieve specific hop aroma profile. Hop recommendations are not exhaustive, contact your hop supplier for recommendations for other varieties with similar free/bound thiol profiles. Yeast recommendations may be updated based on current R&D and commercial feedback.

What are terpenes?

Sources of terpenes

In brewing, terpenes are typically introduced to the wort stream or beer through hops. These terpenes can be present in ‘free’ aromatic form, or ‘bound’ forms, the latter of which is glycosylated (bonded to a sugar molecule). 

Other brewing ingredients may also contain terpenes. For example, citrus peel is rich in limonene, and coriander seed contains geraniol and linalool. 

Want to know which hop varieties are high in either free or bound terpenes? Check out this resource. 

Terpene Biotransformation

Some brewing yeast strains can produce the β-glucosidase enzyme can cleave the terpene-sugar bond, freeing a sugar molecule for the yeast to consume, as well as a highly sensory active terpene molecule. The yeast secretes β-glucosidase enzyme outside the cell, so the reaction takes place in the beer itself. Additionally, specific terpenes can be modified by yeast metabolism to produce different compounds with unique aromas (i.e. geraniol conversion to beta-citronellol).

Two Types of Terpene Biotransformation

Terpene flavor impact and sensory threshold

Terpenes have a diverse range of sensory characteristics such as citrus, floral, pine, or other flavors depending on the compound. Terpenes have very low sensory thresholds, meaning that they are perceptible at very low concentrations. As a point of reference, geraniol is sensory active at <100 μg/L, whereas acetaldehyde is sensory active at 10,000-20,000 μg/L. Even at very low concentrations, terpenes contribute to overall hop intensity and enhance the perception of other positive sensory attributes such as ‘tropical’ from thiols.

How to optimize terpene biotransformation 

Terpene biotransformation can be optimized through careful consideration of the hop variety, yeast strain, and brewing process.

The following chart is a guide to help brewers optimize terpene biotransformation during the brewing process in order to achieve specific hop aroma profile. Hop recommendations are not exhaustive, contact your hop supplier for recommendations for other varieties with similar characteristics. Yeast recommendations may be updated based on current R&D and commercial feedback. For a more detailed discussion about optimizing biotransformation in the brewing process, visit our Process Considerations page.

Other Flavor Compounds

What are hop esters?

Esters are compounds that are formed by linking an organic acid to an alcohol. Esters have fruity aromas such as banana, apple, pear, or other aromas depending on the compound. Brewers are well aware of esters as a fermentation biproduct, whereby yeast enzymes catalyze the reaction of ethanol with different organic acids to form ethyl esters that contribute to the unique flavor profile of each yeast strain.

Hops also contain esters, which comprise about 15% of the total composition of hop oils. One example is 2-methyl isobutyrate (2MIB). While hop esters receive much less attention than terpenes and thiols, they play an important role in determining the unique flavor profile of different hop varieties.

Yeast interaction with hop esters

How to optimize hop ester biotransformation 

The general trends of the isobutyric esters (examples in the table above) contributed from hops are a gradual decrease during fermentation, and a corresponding increase in ethylated versions of these esters (which are virtually not present in the initial wort). Typically around 50-60% of the initial isobutyric ester in the wort at the start of fermentation will carry over to the final beer – therefore, to increase concentration of these compounds in the finished beer, target late kettle/whirlpool/dry hop additions of hop varieties high in these esters.

If the brewers target is some degree of biotransformed ester character, use late kettle/whirlpool as your addition point for high ester hops. If the converse, consider utilising the hops mentioned in the table above as dry hop only. There is not yet very much data available for how different yeast strains interact with hop esters. 

Lactones

What are lactones?

Lactones are cyclic esters formed by the condensation of an acid and an alcohol group on the same molecule. They are typically associated with aromas of stone fruit and coconut. Lactones may also contribute to a creamy mouthfeel if present in high enough concentrations.

Sources of lactones

Lactones are present in hops and are part of the distinct aroma profile of specific varieties. One notable example is Sabro hops, which have a characteristic aroma of stone fruit and coconut.

Lactones can also be formed by the yeast during fermentation using fatty acid precursors derived from malt. Oats and corn are good sources of fatty acids due to their higher lipid content.

Lactone flavor impact and sensory threshold

Lactones are usually associated with aromas of stone fruit and coconut, but the sensory characteristics vary for each specific compound. They are highly flavor active with sensory thresholds in the ppb range. Some examples of lactones found in beer are shown in the table below.

Lactones are also interesting because they have a synergistic relationship with esters and terpenes which, when combined, can increase sensory perception of stone fruit and berry characters, as well as overall intensity¹¹.​ 

Lactone Biotransformation

Fatty acids derived from malt are oxidized in the mash into hydroxycarboxylic acids. Yeast will interact with these hydroxycarboxylic acids to form lactones. Yeast may also modify lactones by interacting with their chemical structure. One potential way to increase lactones created by yeast is to use malts or grains known to be higher in certain fatty acids, such as oats and maize/corn.

Research by Lallemand Brewing has shown that the type and amount of lactone present in a beer is yeast strain dependent. However, the mechanisms by which brewing yeast interacts with lactones is not well understood, and it is an active area of research.

Biotransformation Process Considerations

While the biotransformation process occurs throughout fermentation, it actually begins with raw ingredient selection and choice of hop addition timing. In general, the level of free amino nitrogen (FAN) in the malt has been found to play a part in the expression of biotransformation. Read about the effects of FAN on thiol release here.

Additionally, consideration of hop addition points (hot or cold side) will have dramatic effects on aromatic and flavor, in addition to determining later yeast harvesting and repitching potential.

Crucially, knowledge of how each factor impacts the brewing process will not only help the brewer achieve their desired flavor and aromatic results but also help with overall labor and raw ingredients cost efficiency.

Hop Selection and Timing

Both terpenes and thiols come in “free” and “bound” forms and one objective of the brewer is to release these bound forms to aromatic “free” forms.  Hop addition timing depends on the type of precursors available and the mechanics of fermentation occurring at that particular addition time.

The table below gives a broad overview of what compound extraction and losses to expect at different hop addition points starting from the kettle till the end of fermentation.

Yeast Selection

Yeast strains differ in their enzymatic capabilities, thus affecting the degree to which bound hop precursors can be released to their “free” forms. Yeast selection is therefore an important part of achieving the desired aromatics.

Brewers will want to carefully consider their desired aromatics from hops (terpene or thiol leaning) and pair that will a yeast that has the appropriate level of enzymatic activity to release the aromatic compounds.It is also important to note that enzyme evaluation, especially concerning the beta-lyase enzyme is often difficult. Brewers should consider yeast selection, but also remember that hop selection, fermentation control, and nutrients all play significants roles in effective biotransformation.

Refer to the Strain Terpene and Thiol Favoring Table here to see examples of different LalBrew strains terpene or thiol favoring activity. ¹ 

Fermentation Control 

Fermentation control, be that with temperature, or CO₂ management, plays a significant role in biotransformation. There is little point in adding a large quantity of hops, only to have the beautiful aromatics dissipate due to carbon dioxide scrubbing.  

One way to minimize volatile off-gassing is by early spunding (after 24-72 hours) set to a low top pressure. Other methods of carbonation such as natural carbonation through spunding/top pressure, or in-line carbonation, will also help retain these extremely volatile compounds.  

Temperature can also impact thiol release, as recent research has also shown that generally free thiols increase at higher fermentation temperatures. Alternatively, cold contact around 4°C over 3-5 days also allows thiols to be released into the beer. Ultimately, brewers should be aware of the optimal fermentation temperature of their selected yeast strain to ensure smooth fermentation.  

Read about the recent research here. 

Nutrition and Enzyme Additions 

Nutrition additions, especially regarding malt selection, can play a role in biotransformation. Specifically, thiol precursors which are bound to the amino acids cysteine and glutathione that can be found in both malt and hops. The level of free amino nitrogen (FAN) can also impact thiol expression. All malt worts typically have enough free amino nitrogen (FAN) for healthy fermentations, but high FAN levels have been shown to correlate with a lower level of free thiols. See recent research (OSU research). 

Using adjuncts, such as wheat or oats, lessen the FAN level in the grist and thereby can assist with the increase in free thiols and tropical aromas. Read more in the thiols section.

Enzymes are another way to potentially boost biotransformation.  While no exogenous β-lyase enzyme exists, there is an exogenous β-glucosidase enzyme, AB Vickers Aromazyme™. This enzyme efficiently cleaves glycosidic bonds and can be used in conjunction with yeast strains that do not show high terpene freeing potential but have other favourable brewing characteristics. For example, a hopfenweisse fermented with LalBrew Munich Classic™, Amarillo hops, and Aromazyme™.  

One primary reason there is no commercially available beta-lyase enzyme is that beta-lyase expression occurs intracellularly and is active at yeast cytosolic pH of 6.8-7.2. As such, beta-lyase is not active at normal beer fermentation pH, which makes adding an exogenous beta-lyase enzyme directly to the beer difficult. However, there are commercial products available for increasing thiol precursors in beer, for example those derived from grape skins and other fruits. 

Biotransformation Key Takeaways 

Brewers should take a holistic approach to biotransformation, taking into consideration their desired final aroma and flavor profile, then working backwards with hop, yeast, and malt selection to maximize the most out of their ingredients and process.  

Biotransformation Resources and R&D