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

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/

More articles related to hops & biotransformation

How Do Hop Cannons Work?

Are Yeast Driven Beers The Next Hop Craze?

Are Yeast Driven Beers The Next Hop Craze?

So are Yeast Driven Beers the Next Hops Craze? Ok so Neil so what does this title even mean? The subject was suggested by Chris, to write an article about.

My interpretation; hops experimentation has reached a certain level of maturity in brewing. We have many styles of IPA (India pale ale) such as

  • Double IPA
  • Triple IPA
  • Belgium IPA
  • Black IPA
  • New England IPA
  • Milkshake IPA
  • White IPA

And so on. There exists accepted forms of dry hopping which have developed from traditional methods, to the advent of the hop rockets to some great research and writing by the likes of Scott Janish.

There’ll be more written on the subject and great studies on hops and brewing in the future. However, I feel a lot of the basics have been covered, understood and universally accepted by the brewing community.

Then we come to yeast, which I believe is where the next evolution in brewing will come from. I’ve started writing more on the subject of yeast. Plus, about my appreciation of Milk the Funk (MTF) wiki and the accompanying MTF facebook group.

This article aims to be a jumping off point for further research, introducing the basics of how yeast can be used to make new and unique beers.

The Discovery of Kveik

Yeast evolution in brewing has already started. I think one of the main drivers was the discovery of Kveik yeast by the outside brewing world, of this obscure Norwegian yeast. This article is a great look at how the brewing community came to know about kveik .

It’s possible to ferment Kveik yeasts at crazy temperatures. With some strain working at 38 -42°C (100 – 107°F). It can finish fermenting in 2 days and still be a clean beer. It’s fair to say Kveik, took the brewing world by storm.

The rapid rise in use and acceptance of Kveik yeast in commercial brewing. I believe opened many brewers’ eyes to the myriad of possibilities, when it comes to yeast and brewing.

The Love for Brett – Yeast Driven Beers Are the Next Hop Craze

Yes, I accept Belgium has been doing wild fermentations for centuries with the brewing of Lambic beers.  However, the use of coolships and the capturing of wild yeast in craft brewing is on the rise around the world.

Brewers are sharing their experiments, with techniques being refined, adopted and used to make commercial beers. Furthermore, since the early 2000’s, craft breweries have been looking to yeasts such as Brettanomyces bruxellensis and Brettanomyces anomalus for creating beers with unique profiles.

But it is safe to say, the adoption of these yeasts is on the rise as more brewers discover the versatility they offer. Also, the informed craft drinking public understand and enjoy these beers plus, there is a market for them.

Classification Taken from Milk the Funk (MTF) – Yeast Driven Beers Are the Next Hop Craze

When it comes to putting yeasts into categories for this article it was difficult so, I looked to MTF for guidance. Here is what we have:

Mixed Fermentation

This is fermentation involving a combination of Saccharomyces (brewer’s yeast), Brettanomyces (wild yeast), Lactobacillus (lactic acid bacteria abbreviated to LAB) and Pediococcus (lactic acid bacteria) or other microbes not necessarily associated with brewing.

These beers don’t necessarily have to be sour but, might be tart due to some acetic acid production. The primary fermentation is completed by a Saccharomyces and/or Brettanomyces yeast in most cases.

Spontaneous Fermentation / Coolships

We are back to lambic beers here. A generally accepted definition of spontaneous fermentation is the inoculation of wort for fermentation by local ambient microbes. Brewers use a vessel called a coolship, please see the picture below.

Yeast Driven Beers

Coolship Brewing – Yeast Driven Beers

The coolship should be shallow to allow a large surface-to-volume ratio. This allows for more affective cooling but also makes it easier for microbes to inoculate said wort.

The wort will be left to cool overnight and exposed to the air. Where native yeast and bacteria are introduced to the wort. One term commonly used to describe these beers is “wild ale”.

In Belgium (and now adopted elsewhere) these beers are produced between autumn and spring, when ambient night temperatures are around -3.9 to 8°C (25-46°F).

The reason being these temperatures are ideal for cooling the wort. Plus, some people suggest these times are best for the ambient microbial balance, as the summertime has more acetic acid bacteria.

Wort Souring

A process of mixed fermentation where lactic acid bacteria, usually Lactobacillus is pitched before the primary fermentation yeast to produce lactic acid to sour the wort. Typical non-sour beer has a final pH between 3.8 and 4.6.

Whilst these sour beers will have a pH range between 3.0 and 3.7. The most popular method to sour wort is kettle souring. Typically, the wort is left in the brew kettle overnight, up to 3 days.

A pure strain of lactobacillus is introduced, where it consumes sugars in the wort transforming them into lactic acid. This is how sour beers get its tart flavor many of us love.

Typically, he wort is boiled after the target pH has been reached to pasteurize the wort, stop the souring process, it’s then cooled and sent to FV where the primary yeast is added for fermentation.

There are other methods to sour wort, such as commercial yeasts developed to ferment the glucose in the wort into lactic acid in FV (fermentation vessel). One such yeast is Fermo Acid Brew which I’ve written about here.

Once the souring yeast has finished, the primary yeast for fermentation is added to finish off the fermentation to the desired final gravity.

Brettanomyces – Yeast Driven Beers Are the Next Hop Craze

I learnt something new today, Brettanomyces is Greek for “British Fungus”! Brewers use the term Brett or Bretta. It was an important yeast for producing the desirable characteristics in English ales in the 17th century and earlier.

However, from the 1800’s onwards, Brett was seen as a spoilage yeast. Except when used in Belgian Lambic and Flanders beers. But in the last 20 years Brett has been making a comeback in craft brewing circles.

Brettanomyces, produces high levels of fruity esters which brewers seek in some styles of beer like saison, lambic and sour beers. Please see the aroma wheel below.

Yeast Driven Beers - Brett Aroma Wheel by Dr. Linda Bisson and Lucy Joseph at UC Davis.

Brett Aroma Wheel by Dr. Linda Bisson and Lucy Joseph at UC Davis.

Historically Brett was considered a “wild yeast”, due to its ability to spoil beer and for the funky aromas and flavors it produces. Some of the main descriptors include “floral”, “earthy” and “horse blanket”.

Thankfully brewers have been able to wrangle this fickle organism through culturing to produce known and desired results in finished beer. Thus allowing Brett to be used and understood by brewers with a little research using commercial strains readily available.

Lactobacillus

We brewers call this “Lacto” or abbreviate it to LAB. Lacto produces acidity and sour flavors though the formation of lactic acid.

Common beer styles produced this way are lambics, Berlinerweisse, sour brown ales gueze and gose. We covered how LAB is used in brewing in wort souring and mixed fermentation above.

The Love for Sours and Wild Yeast

Sour beers are no longer a “niche beer”, they’ve hit the mainstream. There are brewers like Wild Wave in South Korea who exclusively make sour and wild ales.

In Australia, they are several breweries known for their wild and sour ales. These include:

Wildflower – Specializes in wild yeast ales barrel age blending. Chris recently interviewed one of the owners Topher Boehm for series 2 of the podcast.

Future Mountain Brewing – Based out of Reservoir, Victoria who are well-known for their farmhouse inspired, mixed fermentation and barrel aged beers

Dollar Bill brewing – Based out Ballarat, Central, Victoria and well-known in the wild ale space.

You have all forms of sour beer, from sour IPA’s, dark sour beers, to many styles of gose to sour fruited beers. Brazil even has its own style called Catarina Sour.

Brewers are getting really creative with these brews and there’s a wonderful sharing of knowledge. People are sharing their wild yeast captures techniques and cultures. Please see this great post by Bootleg Biology for the basics of yeast capturing.

The unique flavors these yeasts are producing is akin to the discovery and research done into many strains of Kveik. There’s still so much to discover. But the possibilities are endless and why I believe yeast driven beer are the next hop craze.

Wine Yeasts

This is a subject I’ve covered in an article at Asian Beer Network which you can read here. So, I’ll just go over the basics here.

Wine Yeast and Beer Wort – Yeast Driven Beers Are the Next Hop Craze

Many wine yeasts don’t ferment the main sugars in beer wort, maltose and maltotriose. They ferment other simpler sugars in the wort so, you’ll need to use a beer yeast to ferment the beer to your desired final gravity.

The term for using two yeasts in this manner is called “co-pitching” or “mixed fermentation”, which we looked at earlier in this article. One other way to ferment the wort with wine yeast is to use an enzyme to breakdown the complex sugars into simple ones, which the wine yeast can ferment.

Kill Factor

A lot of wine yeasts have what we call a “kill factor”. Meaning, it’ll kill other yeasts if they’re added at the same time or later. In brewing, if a wine yeast has the kill factor you need to pitch the beer yeast first and then wine yeast later.

Or…

Split the batch, with say 70% of the wort fermented by the beer yeast, and 30% fermented by the wine yeast. Again, use an enzyme to break down the complex sugars so the wine yeast can work. Once both worts have been fermented and ready, you can blend the batches together.

POF+ and POF-

The majority of wine yeasts are POF+ (phenol off flavors), meaning when they ferment beer, they create undesirable off-flavors. However, there are some POF- wine yeasts which are more suitable for brewing beer. Please check the main wine yeast article for more on this subject.

Wine Yeast Conclusions

The use of wine yeasts in beer is on the rise, with some commercial breweries producing beers made with wine yeast for market. It’s still a newish development, with little research available on the subject.

The main driver is wine yeasts offers the potential for new natural flavors and aromas in beer production. Some yeasts offer wonderful fruity aroma such as stone fruit which you get from K1B-116 white wine yeast or you can use L22-26 a red wine yeast, which gives off a general berry flavor.

Being Unique – Yeast Driven Beers Are the Next Hop Craze

As more breweries pop up around the globe and the competition gets fiercer, breweries look to offer beers which are unique and standout. There are a lot of heavy hopped beers on the market, it’s harder to stand out or be creative with hop forward beers.

The use of different yeast strains is not as developed in the brewing world as with hops. It’s easier to be unique or create something which can make you stand out. In the drive to get people to drink your beer, yeast exploration could be the way forward as it:

  • Offers the chance to create unique flavours
  • Works across multiple beer styles so creating something new is easy
  • Make you stand out from the hop hype / adjunct breweries

Financial Sense

There’s another advantage to exploring yeast, cost. Hops are expensive with certain varieties in demand being extremely costly. Plus, the more hops you use the less sellable beer you have.

Now, if you go down the yeast route it can be much cheaper. You can develop your own inhouse strains and re-use them time and time again. You can also end up with more “sellable” beer from every batch if you’re not heavily hopping the beer.

If a brewer can produce beer cheaper, plus, still be unique as well as sold for a premium it’s a win-win. These yeast driven beers are still seen as special by the consumers, as they are unique. Yes, they take a few extra steps to produce but, it makes selling them for a premium price point is acceptable.

If you can reuse the yeasts, then the price point of the beers will be attractive, especially when compared to heavy hopped beers.

Australian Breweries Playing in the Yeast Driven Beers Space

There has been a number of breweries in Australia that have also been pushing the envelope within the yeast driven beers space. Chris was able to catch up with Topher Boehm from Wildflower just recently for the upcoming series 2 of the podcast, where he discussed their unique business model and beer offerings specialising in brewing with wild yeast and barrel age blending.

Wildflower Brewing & Blending

Other breweries include Slow Lane who specialise in yeast driven European ales, and Harvest Berry Mountain, who under Master Brewer Neal Cameron, has adopted old traditional brewing methods by doing open fermentations of their beers. These two breweries are also expected to appear on the podcast, as well as some behind the scenes video footage of their operations (stay tuned for this). And let’s not forget some of the bigger guys such as Van Dieman Brewing, La Sirene, Future Mountain & Dollar Bill Brewing as well.

Slow Brewing & Harvest Berry Mountain

Yeast Driven Beers Are the Next Hop Craze – Conclusions

So, there you have it. My thoughts on yeast driven beers. As I say this article is to be a jumping off point for your own further research. I’ve my own experiments ongoing right now.

For example, two weeks ago, I took some wort from regular stout brew and put 100 litres in a pilot FV and pitched Fermo Acid Brew into it.

I let the beer sour, it went to pH 3.9, which not particularly low but I didn’t pre-acidify the wort and also the mash temperature was quite high.

Then pitched some BE-134 Fermentis yeast in the wort and let it ferment out, it’s a Saison yeast. I added some coconut and dried hibiscus flower just before terminal gravity. It’s crashing now, but so far really happy with the results.

I’ve ordered some wine yeasts and will be playing with wort streams in the future. To see what flavor profiles, I get. I’m brewing in Shanghai and I don’t know of another local brewery experimenting with wine yeast. I could offer something unique to the city.

I’m excited to trial these beers and see what feedback I get. I’ll share my experiences and results in future articles. Will yeast driven beer be the next hop craze? I don’t know but there’s one way to find out and that’s…just brew it.

One final Note

When working with new yeasts and microbes be careful of cross contamination. When you bring something new into a brewery, you need have proper measures in place.

For example, in some breweries their sour program is completely separate from the normal production facility, with their own beer hose and packaging line.

Further Content on the Topic

Check out Series 1 Episode with Avi Shayevitz from Lallemand brewing who gives in-depth insights about the importance of yeast in the brewing process and exciting prospects yeast will play in the future of craft beer.