Biotransformation Basics: Yeast & Hop interactions, and the use of exogenous enzymes

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Introduction

Before we dive into the topic of biotransformation a brief history lesson first. By the end of the 18th century, the first India Pale Ale (IPA) was made in the United Kingdom and it was characterized for being a beer style produced with higher amounts of hops and higher alcohol content compared to the typical values ​​of its predecessor style, the Pale Ale, in order to maintain its shelf-life during the long exportation to India. Nowadays, IPAs have re-born being reinterpreted and modernized by craft brewers, generating a wide variety of derived styles, such as American Pale Ale (APA), New England IPA (NEIPA) or Sour IPA, but always having a particular thing in common, the dominant presence of hop flavors.

Beyond all this, the selection of the yeast strain is an important factor when designing an IPA recipe, or any of its derivate styles. Each strain is able to produce unique profiles through the aroma compounds produced during fermentation, proving a neutral or even a more fruity character, contributing directly to the organoleptic profile of the resulting beer. Attenuation is also an important aspect for the body and bitterness perception, as well as the flocculation rate, which has an impact on the clarity and flavor of the finished product.

The Role of Brewing Yeast in Biotransformation

Recent brewing research showed that different yeast strains can influence beer flavor and aroma by interacting with specific compounds derived from hops, a phenomenon known as biotransformation. The term Biotransformation is a buzzword nowadays in brewing, which can be sometimes confusing due to the complex biochemical processes involved. It is defined as ‘the chemical modification made by an organism on a compound’. Although this term is commonly used in other fields than brewing, from the brewer’s perspective, it refers to the interaction of two ingredients: yeast and hops.

Brewing yeast produces two different enzymes during fermentation: β-glucosidase and β-lyase, which will be discussed in the following lines, both playing a role in biotransformation with the release of aromatic compounds or volatiles.

The role of β-glucosidase

β-glucosidase is an enzyme able to cleave glycosides, a compound found in hops that do not contribute to beer aroma per se. As a result, the glycoside molecule is broken via hydrolysis into two parts: monoterpene alcohols and glucoses. An example is shown in Figure 1, where a non-volatile terpenyl glycoside is hydrolyzed through the β-glucosidase activity resulting in the release of a monoterpene alcohol (linalool) and a glucose molecule. There are many monoterpene alcohols that impart diverse flavors, such as citrus, fruity or floral, and higher levels of terpenes are associated with greater overall hop aroma intensity (OHAI).

However, β-glucosidase activity depends on each yeast strain’s genetic background. The addition of exogenous and concentrated β-glucosidase enzymes was studied (Sharp et al., 2017), and demonstrated the interesting potential to enhance biotransformation reactions.

The role of β-lyase

As shown in Figure 3, Lallemand Brewing has characterized numerous strains of the LalBrew® Premium line, where β-glucosidase and β-lyase enzymatic activities have been identified. In this study, β-glucosidase was measured as secreted enzyme activity using a standard chemical glycoside substrate, whereas β-lyase activity was measured by growth on selective media containing a specific sulfur-based precursor.β-lyase is an enzyme responsible for the release of volatile sulfur compounds called polyfunctional thiols, or mercaptans, which are usually associated with tropical aroma. Thiols are aromatic compounds found in hops and represent about 1% of the total hop oil content. Despite their low concentration, their contribution to the aroma in beer is significant due to their low detection threshold. In addition, hop also contains thiol precursors which do not impart any flavor but, through β-lyase activity, these highly aromatic compounds can be released and perceived by the consumer (see Figure 2).

Exogeneous enzymes to promote Biotransformation: An innovative tool for craft brewers

Recent studies showed that exogenous enzymes have a considerably higher glycosidic activity compared to yeast-derived enzymes (Sharp et al., 2017). As shown in Figure 4, an enzymatic preparation high in β-glucosidase activity was able to hydrolyze almost 100% of the substrate whereas yeast samples hydrolyzed about 10% in the same period of time. This would suggest that the release of hop-derived monoterpene alcohols is greater when using exogenous preparations.

However, the enzymatic performance will depend on the nature of the enzyme, which will be affected by fermentation conditions such as glucose concentration, pH and temperature. Enzymatic preparations obtained from filamentous fungi, such as Aspergillus niger, have shown more resistance to low pH and high temperatures, which could be an interesting asset in the production of beer styles such as Sour IPAs or high-temperature fermentations with kveik yeast, which is nowadays used in the production of NEIPAs as well as fast-fermented ale styles.

In the 2020, Lallemand Brewing launched a new enzymatic preparation, named ABV Aromazyme and developed by AB Vickers, to provide a creative tool for brewers to promote hop biotransformation during fermentation (See Figure 5). ABV Aromazyme was tested in the pre-launch period by several breweries on a global scale.

As shown in Figure 6, a candidate brewery followed this experimental design in order to check differences between a control batch versus a treated batch. A single batch was split into two different fermentation vessels, in which samples were taken for sensory purposes (All recipe details and dry-hop additions are listed in Table 1).

The beers were tasted by two different panels, one general and one expert, both in a blind tasting. As shown in Figure 7, both panels preferred the beers treated with the enzyme, being described as more hoppy, fresher, and easier to drink.

Although the use of enzymatic preparations is an innovative tool for brewers to keep experimenting in their modern recipes, the prediction of specific aroma profiles obtained by the enzymatic activity is still not easy to control due to the multiple variables playing a role. Aspects such as the hop variety used, its crop year, the selected brewing yeast strain, as well as process parameters like dry-hop additions, time of contact, pH, temperature, … all play a role in defining the organoleptic profile of the final beer. Therefore, brewers need to consider these enzymatic activities when making their choice of yeast strain and/or the possibility of the addition of exogenous enzymes depending on the beer style they are targeting.

Hops

Figure 1. Terpenyl glycoside hydrolyzed by β-glucosidase releasing a monoterpene alcohol (e.g. linalool) and a glucose molecule.

 

Figure 2. The thiol 4MSP is released from a non-aromatic cysteinylated precursor.

 

Figure 3. Relative biotransformation activities of β-glucosidase and β-lyase enzymes in LalBrew® Premium brewing yeast strains. β-glucosidase was measured as secreted enzyme activity using a standard chemical glycoside substrate. β-lyase activity was measured by growth on selective media containing a specific sulfur-based precursor. Relative activities are shown for comparison, but β-glucosidase and β-lyase activities cannot be directly compared with each other (β-lyase activity not determined for LalBrew London™).

 

Biotransformation

Figure 4. Percentage hydrolysis of octyl-β-D-glucoside in wort by purified β-glucosidase enzyme versus ale yeasts (low and high enzyme activity (Sharp et al., 2017)

 

Biotransformation

Figure 5. ABV Aromazyme,is a food-grade enzyme preparation with a strong glycosidase activity developed by AB Vickers (a subsidiary of Lallemand).

 

Biotransformation

Figure 6. Trial design with a split batch into a control versus enzyme treated.

 

Biotransformation

Figure 7. Summary of the preferred beer between the two tasting panels.

 

Biotransformation

Table 1. Dry-hops and other actions performed during the trial.

References

Günata, Z. (2002). Flavor Enhancement in Fruit Juices and Derived Beverages by Exogenous Glycosidases and Consequences of the Use of Enzyme Preparations. En: W. J. (ed), Handbook of Food Enzymology (pp. 303-330). New York: Marcel Dekker.

Lallemand (2020). Best Practices: Biotransformation. www.lallemandbrewing.com/wp-content/uploads/2020/08/LAL-bestpractices-Biotransformation2020.pdf

Lallemand (2020). Best Practices: IPA Solutions. www.lallemandbrewing.com/wp-content/uploads/2020/07/LAL-bestpractices-IPA_Solutions_digital-T.pdf

Montasell, J. (2020). Biotransformation: A story of yeast, hops and enzymes. IBD Brewer and Distiller International Magazine, August 2020, pp26-30.

Sharp, D. C., Steensels, J., & Shellhammer, T. H. (2017). The effect of hopping regime, cultivar and β‐glucosidase activity on monoterpene alcohol concentrations in wort and beer. J. Inst. Brew., 123, pp185-191.

Takoi, K. (2017). Systematic Analysis of Behaviour of Hop-Derived Monoterpene Alcohols During Fermentation and New Classification of Geraniol-Rich Flavour Hops. BrewingScience (70), 177-186.

Lallemand (2020). Biotransformation featuring new product Aromazyme with Dr. Shellhammer. https://www.lallemandbrewing.com/en/canada/live-events/biotransformation-with-new-product-aromazyme/

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