Process consideration for biotransformation

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.

Kettle/Whirlpool Additions

Biotransformation tends to be at its peak at, or around, peak fermentation cell count. Therefore, in order to maximise the freeing of these bound forms consider utilising high precursor hops in high quantities as late as possible in the kettle, whirlpool, or early dry hop. The fundamental goal and challenge is to have as many ‘surviving’ precursors as possible for the yeast.

Kettle Hopping Technique

Hop Burst is a technique that describes the addition of a late hop addition at the end of the boil in combination with a shortened boil time (15-30 min). Brewers who use this technique often combine it with a large whirlpool addition. One premise behind this technique is the limitation of Maillard reactions in the boil (browning or heat caramelisation of the wort) while retaining as many hop precursors as possible.

Fermenter Additions

Brewers should consider adding hops with high freely available terpenes or thiols as late dry hop additions. One primary reason is that these aromatic compounds can be wasted in early fermentation due to aroma scrubbing by active fermentation. Late fermentation additions when the yeast is less active will help retain aromatics due to a decrease in volatilisation of these free terpene or thiols.

Dry Hopping Technique

Dip Hopping is a term that describes the process of steeping hop pellets in hot water for a period and adding this mixture to the fermenter at the same time as the yeast (i.e. during/post knock out). One potential benefit of this technique is an apparent increase in concentration of some terpene alcohols, such as Linalool and Geraniol, which meaningfully contribute to overall hop aroma. Overall sensory effects from dip hopping were described as increasing the olfactory and gustatory pleasantness of the beers as compared to the late hopping controls.

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.

Adjunct selections can impact free thiols and to a certain extent, lactone concentration

Hop selection and addition points will determine just how much aroma is released and retained in the beer

Yeast selection will play an important role in terpene and thiol release

Fermentation temperature as well as the interplay of esters and lactones paint a more complex biotransformation picture

Hop compound sensory is highly complex and made more interesting by the interaction between terpenes, thiols, lactones, and other compounds in beer

New research in beer/wort matrix is ongoing

Learn more

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

References

1 Molitor, R. W., Fischborn, T., Dagan, L., & Shellhammer, T. H. (2023). Examining How the Fermentation Medium Influences Thiol Expression and Its Perceived Aroma in Commercial Brewing Yeast Strains. Journal of Agricultural and Food Chemistry, 71(5), 2493-2502.