Category Archives: News

The mysteries of diastatic brewing yeast

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It’s time for another long due update. I’m very excited to share our latest manuscript on diastatic brewing yeast (or “Saccharomyces diastaticus” as it was formerly known as)! Keep in mind that this is a preprint and the manuscript is currently under peer-review. Below I’ll summarize the main findings of our small project (that ended up having quite significant results).

Edit: The manuscript has now been published in Applied Microbiology and Biotechnology: https://link.springer.com/article/10.1007/s00253-019-10021-y

First of all, here is the link to the preprint: https://www.biorxiv.org/content/10.1101/654681v1

So as most of you are aware, the presence or absence of the STA1 gene is commonly used as a marker for diastatic yeast. However, as recent work by e.g. the Hutzler lab and Escarpment Laboratories have shown, not all strains carrying the STA1 gene are problematic (i.e. not all STA1+ strains can super-attenuate beer or ferment starch). This is of course a problem. While the PCR-based method for detecting diastatic yeast with the SD-5A/SD-6B primers is rapid, it can’t differentiate between the highly and poorly diastatic strains. I set out to find out the reasons behind the variation in diastatic ability that we have observed in STA1+ strains.

We started out by screening 15 strains of S. cerevisiae carrying the STA1 gene (thanks to Mathias Hutzler and Richard Preiss for sharing strains). We tested the diastatic ability of the strains using three different tests: 1) the ability to grow on starch agar, 2) the ability to grow in beer (wort force-fermented with a lager yeast), and 3) the ability to grow on dextrin as a sole carbon source. In all three tests, the 15 strains grouped into two distinct groups: the highly diastatic, and the poorly diastatic. This was already promising.

Next, we sequenced the open reading frame and the upstream region of the STA1 gene in all 15 strains. In simpler terms, we looked at the sequence coding the actual gene and the sequence in front of the gene (which usually controls how much of the gene is expressed into the protein it codes for). Here we observed something fascinating: all 10 of the poorly diastatic strains had a large 1162 bp deletion in the promoter of STA1 (i.e. in the upstream region / the sequence in front of the gene). Within this region that was deleted, is a transcription factor binding site (transcription factors are proteins that control the expression of the gene). We hypothesised that this deletion in the STA1 promoter decreases the expression of STA1 (i.e. less of the glucoamylase enzyme is produced), which in turn results in poor diastatic ability. It seemed very obvious at this point already that this deletion was the cause of the variable diastatic ability, but we still needed to confirm this.

To confirm the effect of the deletion, we performed CRISPR/Cas9-aided reverse engineering. Wyeast 3711 was the most diastatic strain out of all 15 that we tested (in all three tests). So we used CRISPR/Cas9 to replicate this same 1162 bp deletion in the promoter of STA1 in WY3711. We then repeated the three diastatic tests. We compared the original WY3711, the WY3711 variant with the deletion in the STA1 promoter, and WLP570 that naturally has the deletion in the STA1 promoter. The deletion resulted in decreased ability to grow in beer and on dextrin (WLP570 and the WY3711 variant with the deletion performed similarly poorly), confirming that it is indeed this 1162 bp deletion in the STA1 promoter which causes the poor diastatic ability in some of the STA1+ strains. To test our hypothesis that STA1 expression is affected, we measured STA1 mRNA levels with RT-qPCR in the same three strains as above, and witnessed 150-200 fold higher levels of STA1 mRNA in WY3711 compared to WLP570 and the WY3711 variant.

So what can we do in practice with this information? We designed a new set of PCR and qPCR primers that bind within the deleted region in the promoter. So these primers will only give a signal (produce an amplicon) if the strain has the full STA1 promoter (i.e. the strain is highly diastatic). These new primers can therefore distinguish between very active (full promoter) and slow (deletion in the promoter) diastatic strains. They can also be multiplexed with the commonly used SD-5A/6B primers! So this means that you can either use these new primers on their own, which will produce a signal for very active diastatic strains, and no signal for both poorly diastatic strains and strains lacking STA1 completely. Or, you can use these new primers in the same PCR reaction together with the SD-5A/6B primers, which will produce two bands for highly diastatic strains, one band for poorly diastatic strains, and no bands for strains lacking STA1 completely. If this sounds confusing, you can check out the image below (and two above) for some examples. Now we can use PCR to distinguish the highly diastatic and poorly diastatic strains! We tested the primers on some lager yeast cultures that we contaminated with various ratios of WY3711, and we could reliably detect WY3711 down to a concentration of 10^-4 with regular PCR and 10^-5 with qPCR using these new primers.

After we had managed to elucidate the cause behind the variable diastatic ability in STA1+ strains, I still wanted to explore the prevalence and evolutionary role of STA1. Brewers tend to link diastatic yeast with “wild yeast”. This turns out to be incorrect. After screening 1100+ publically available genome assemblies, we show that STA1 is prevalent in the ‘Beer 2’/’Mosaic Beer’ population, and surprisingly a population of yeast isolated from a village in French Guiana (mostly isolates from human feces). So STA1 is clearly linked to domesticated yeast (i.e. no wild strains possessed STA1). The link to ‘Beer 2’ was expected (the saison strains can be found from here), but the French Guiana link was definitely unexpected. The villagers produce and consume a traditional starch-rich beverage called cachiri, so maybe STA1 (allowing starch fermentation) has given the strains a fitness advantage there? Anyways, exploring that was outside the scope of this particular study, but it should definitely be clarified in the future.

If we go back to the ‘Beer 2’ strains, the Gallone et al. 2016 paper has some nice phenotypic data that can be used. I decided to look at the maltotriose use ability of all the STA1+ ‘Beer 2’ strains, and if we divide the strains into two groups based on whether they have the full or partial STA1 promoter, one could see that the strains with the full promoter have significantly higher maltotriose use ability. This hinted that STA1 possibly has some role in enabling maltotriose use in the ‘Beer 2’ strains. The main mechanism for maltotriose use in ‘Beer 1’ brewing strains is through the AGT1/MAL11 transporter. ‘Beer 2’ strains have a non-functional AGT1/MAL11, which means some other unknown mechanism(s) must be responsible. Could STA1 be the unknown mechanism?

I then decided to compare the wort fermentation ability of WY3711 with the WY3711 variant with the deletion in the STA1 promoter. In the first 24 hours of fermentation the strains performed identically. At this time it is mainly glucose that is consumed from the wort. After this time point, the variant with the deletion in the STA1 promoter slowed down significantly. Maltotriose use in particular slowed down. WY3711 had consumed around 80% of the maltotriose after 96 hours, while the variant with the deletion in the STA1 promoter had only consumed 12%. So these initial results already suggested that STA1 has a central role in enabling maltotriose use in STA1+ strains.

To further test this, we next used CRISPR/Cas9 to completely delete STA1 from three of the highly diastatic strains (WY3711 and two TUM strains). We repeated the wort fermentations, and again saw significantly reduced maltotriose use for all three deletion strains. The two TUM strains (TMU PI BA 109 and TUM 71) in particular, barely consumed any maltotriose at all after STA1 had been deleted. This confirmed that STA1 seems to enable efficient maltotriose use through extracellular hydrolysis! We think that the formation and retention of STA1, through the fusion of FLO11 and SGA1, is an alternative evolutionary strategy for efficient utilization of sugars present in brewer’s wort.

The WY3711 deletion strain still showed minimal maltotriose use, so that suggested that while STA1 is the main mechanism for maltotriose use in these strains, it is not the only one. To confirm this, we measured the uptake rate of maltotriose in these strains. This is done with radio-labelled maltotriose (14C). All three strains were able to transport maltotriose into the cell (though at levels lower than what is normally measured for brewing yeast), confirming that while STA1 appears to be the main mechanism for maltotriose use, the strains carry some low affinity maltotriose transporter(s) as well. We performed long-read sequencing of WY3711 using an Oxford Nanopore MinION, and the resulting genome assembly appeared to contain multiple copies of an MTT1-like transporter (which probably explain the maltotriose uptake ability and the low maltotriose use of the STA1 deletion strain). MTT1 is a transporter that has been shown to transport maltotriose.

So to conclude, we saw three major results here in this study. The first is probably of most use to the brewing community: The variable diastatic ability in STA1+ strains seems to be determined by a 1162 bp deletion in the STA1 promoter. Strains with the deletion are not very diastatic. You can use our newly designed PCR primers so differentiate between STA1+ strains with and without the deletion. The second result was that STA1 is not linked to wild yeast, rather it appears to be prevalent only in ‘Beer 2’/’Mosaic Beer’ strains, and surprisingly the ‘French Guiana, human’ strains. STA1 presumably gives a fitness advantage in starch-rich environments (e.g. beer). The third major result was that STA1 enables efficient maltotriose consumption in STA1+ strains. This appears to be the unknown mechanism that has enabled efficient maltotriose use in the ‘Beer 2’ strains (which otherwise have a non-functional AGT1/MAL11 transporter). STA1 therefore seems to be an alternative evolutionary route (‘domestication signature’) to enable efficient utilization of the sugars present in wort. Let me know if you have any questions or feedback, I’d be happy to help! Thanks again to Mathias Hutzler and Richard Preiss for sharing strains!

Review on the use of hybrid yeasts for brewing

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I’m really sorry for not posting in this blog more actively. I haven’t really been homebrewing much the past year, since we moved to a new house that needed renovating. We’ve finally renovated the garage into a brewing space and home bar, so I will hopefully be posting more actively about homebrewing now. I actually even brewed two batches of beer here in the new house last weekend, and I’ll be posting an update of them along with some pictures of the new brewing space in a future post.

Besides homebrewing, I like to write about my own and other’s beer and yeast research on the blog. We have some really interesting yeast projects going on, which I hope to be able to share with you soon. In the meanwhile, we were invited to write a review article on the use of hybrid yeasts in brewing for Applied Microbiology and Biotechnology, and I’m happy to say that the article has been published online now. In it we sum up the research that has been done on the use of artificial or de novo yeast hybrids for brewing applications, and discuss what kind of benefits they have to the process. These include creating strains with improved aroma formation, fermentation rate and stress tolerance. There is also a short section on how to create these hybrids. Feel free to have a look if you are interested, the article is open access!

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Here is a link to the article: http://link.springer.com/article/10.1007/s00253-016-8007-5

Abstract:

The natural interspecies Saccharomyces cerevisiae × Saccharomyces eubayanus hybrid yeast is responsible for global lager beer production and is one of the most important industrial microorganisms. Its success in the lager brewing environment is due to a combination of traits not commonly found in pure yeast species, principally low-temperature tolerance, and maltotriose utilization. Parental transgression is typical of hybrid organisms and has been exploited previously for, e.g., the production of wine yeast with beneficial properties. The parental strain S. eubayanus has only been discovered recently and newly created lager yeast strains have not yet been applied industrially. A number of reports attest to the feasibility of this approach and artificially created hybrids are likely to have a significant impact on the future of lager brewing. De novo S. cerevisiae × S. eubayanus hybrids outperform their parent strains in a number of respects, including, but not restricted to, fermentation rate, sugar utilization, stress tolerance, and aroma formation. Hybrid genome function and stability, as well as different techniques for generating hybrids and their relative merits are discussed. Hybridization not only offers the possibility of generating novel non-GM brewing yeast strains with unique properties, but is expected to aid in unraveling the complex evolutionary history of industrial lager yeast.

Report from the 35th European Brewing Convention Congress

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I recently attended the 35th EBC (European Brewery Convention) Congress in Porto, where I held both an oral presentation entitled ‘Newly-created hybrid lager yeast strains (S. cerevisiae x S. eubayanus) outperform both parents during brewery fermentation‘ and co-authored a poster entitled ‘Non-conventional yeast as a new tool for beer flavour modification’. I’ve already written two blog posts on our new lager yeast hybrids (see here and here), so I won’t go into details on that topic here. However, here is a link to my presentation slides in case you are interested. I more or less go over the data from our recent publication, but it should hopefully be presented in a way that is easy to follow.

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The topic of our poster, i.e. the use of non-Saccharomyces yeast in brewing for increased flavour, should be a topic that is interesting for many experimental homebrewers. You can download a copy of the poster here. We did small-scale wort fermentations using 13 different non-Saccharomyces yeast species, and 3 Saccharomyces yeast species as controls. We then analyzed the concentrations of higher alcohols, esters and 4-vinylguaiacol in the resulting beers, and identified yeast species that 1) produced high amounts of esters, but 2) were also POF- (i.e. they didn’t produce 4-vinylguaiacol above the flavour threshold). This was because we were interested in applying these yeasts to beer styles where a spicy, phenolic and ‘wild’ flavour isn’t wanted. Some interesting yeasts were Kazachstania servazzi, Naumovia dairenensis, Lachancea fermentati , and Kluyveromyces marxianus.

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Many of the non-Saccharomyces yeasts were quite poor at fermenting wort (with its complex mixture of sugars and with the resulting high concentrations of ethanol), so we had the idea of using them in co-fermentations together with an ale yeast strain. To maximize the flavour contribution from the non-Saccharomyces strains, yet still ensure proper attenuation from the ale strain, we first pitched only the non-Saccharomyces strain, and added the ale strain after 24 hours of fermentation. We brewed three 30 liter batches, one control with only the ale strain, one co-fermentation with Kazachstania servazzi, and one with Naumovia dairenensis. We bottled all three batches and had the beer analysed. The beers co-fermented with the wild yeasts had significantly higher level of esters than the control beer, and had a strong fruity and floral aroma.

Finally, I thought I’d do a quick summary of some topics that could be relevant for homebrewers:

  • The ‘kettle hop aroma’ mystery, Praet T et al.

I missed this presentation myself, as it was parallel to the session I was having a presentation in, but I’ve seen a variation of this presentation at an earlier brewing congress. They have revealed that oxygenated sesquiterpenoids are formed during wort boiling from hop oils, and that these give the beer ‘spicy’, ‘woody’ and ‘hoppy’ notes. So these are hop aromas that require boiling, and cannot be achieved from dry hopping.

  • Protein thiols and sulfite, Lund M et al.

Protein thiols and sulfite can act as antioxidants, and their presence in beer can improve beer flavour stability. They noticed that the concentrations of these in wort can be increased by supplementing proteases to the mash. Protease supplementation also increased the flavour stability of the resulting beers. Perhaps flavour stability can be improved by optimising the mashing conditions (e.g. utilizing the proteases already found in the malt)?

  • Genetic metabolism of hop terpenoids by yeast in beer, Tristam P et al.

I missed this presentation myself, so am going only by comments from my colleague and the abstract. Apparently they have looked at how various hop essential oil compounds are metabolised by the yeast during fermentation. They found that the ATF1 gene is required for the biotransformation of linalool and geraniol to their acetate esters, and the OYE2 gene is required for the biotransformation of geraniol to citronellol. This means that different yeast strains (depending on their genetic background and the activity of the corresponding enzymes) may produce beers with different hop aroma profiles!

  • Influence of dry hopping on changes in the key aroma compounds of pale lager beer, Stingl S et al.

They had studied how various hop compounds are transferred to the beer during dry hopping, and looked at how the ratio of linalool to myrcene in the beer affects the aroma. Apparently the transfer of linalool to beer is very rapid during dry hopping, with maximum concentrations reached within an hour. Myrcene transfer is much slower, and it takes several days to reach the maximum concentration. At high concentrations, myrcene is thought to have an unpleasant aroma, so a short dry hop time (e.g. 1-4 days) might actually be preferable.

  • A high throughput monitoring of phenotypic changes in Brewer’s yeast during serial repitching, Kocar N et al.

They had done some studies on what physiological, genetic and proteomic changes occur during serial repitching. They did 16 repitching cycles at industry-scale and 31 repitching cycles at laboratory-scale, and it seems like you can repitch around 15 times without any big changes in physiology, karyotype or proteome. So don’t be scared to reuse your yeast a couple of times (this requires good sanitation practices of course)!

  • Beyond iso-alpha acids, Shellhammer T et al.

They studied how oxidized hop acids and hop polyphenols affect the IBU value and beer bitterness. They notices that oxidized alpha acids, i.e. humulinones, that transfer from the hops to the beer while dry hopping, not only affects the IBU value of the beer but also the perceived bitterness. They are perceived as less bitter that iso-alpha acids though. Some commercial heavily dry-hopped beers even had higher bitterness contribution from the humulinones than the iso-alpha acids. So dry hopping does increase bitterness!

  • Bitterness impact of common brewing spices, O’Neill C et al.

They studied how various common brewing spices affected the measured and perceived bitterness. Especially cinnamon seems to increase IBU and perceived bitterness. Coffee beans and coriander also seem to increase the perceived bitterness. So keep that in mind when adding spices to your beer!

  • Aroma contributions from Simcoe and Hallertau Mittelfrüh hops to beer using different hopping regimes, Sharp D et al.

They had looked at and compared how different hopping regimes (kettle hopping, whirlpool hopping and dry hopping) and two hop cultivars (Simcoe and Hallertau Mittelfrüh) affect the perceived aroma and concentration of various hop oil compounds in the beer. For a homebrewer, it was no surprise that Simcoe gave more tones of tropical fruit, citrus, stone fruit and pine compared to HM. Dry hopping and whirlpool hopping seem to give similar effects, which is something to keep in mind when planning your hop schedule.

 

There were probably many more interesting presentations (and I decided to leave out the posters from here as well), but unfortunately I wasn’t able to see them all due to the triple parallel sessions during the conference. All in all it was a very interesting conference with lots of interesting researchers and research topics! Already looking forward to the next one! Please leave a comment if you have any questions, and I can try to answer them as best as I can.

Small update

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Sorry again for the inactivity. Not much interesting has happened on the homebrew front. We’ve made slight progress with our homebrewery renovation, as we’ve put up some 160mm ventilation pipes and installed an inline exhaust fan in order to transfer the steam generated from the boil outside. The fan seems really powerful, which is a good sign, but we still need to test it by boiling some water in our boil kettle. We will most likely need to adjust the vent hood, so that it is closer to the top of the kettle. We’ve also started drawing cables for the electrics, and pipes for the water will be next. After that we have some thorough cleaning to do and painting the walls. We hope to be able to brew our first batch in the new homebrewery in May. Can’t wait!

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At work I have lots of interesting new results as well, which I hopefully will be able to post about later during the year. I’m also looking into generating some new S. cerevisiae × S. cerevisiae intraspecific hybrids for homebrew use. A combination of WLP001 and WLP002 sounds good doesn’t it? Or a hybrid of Conan and WLP644 for a really fruity IPA? Anyways, the possibilities are endless (in theory at least).

How new yeast species are inspiring a revolution in brewing

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Note, this is a repost of an article I wrote for VTT’s Industrial Biotechnology Blog.

Lager beers – sometimes crisp & light pilsners, sometimes dark & malty doppelbocks, have a common denominator: They are all produced using the lager yeast Saccharomyces pastorianus, the workhorse of the lager brewing industry. This yeast is known for its tolerance to lower temperatures, and brewers take advantage of this when producing lager beers.

These beers typically have a ‘clean’ flavour profile (i.e. lack of yeast character) you see, and by fermenting the beer at colder temperatures, the yeast produces less flavour-active by-products.

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Recent analysis of lager brewing yeast genomes has revealed that the many hundreds of strains used in the brewing industry are, in fact, all closely related – more like multiple variants of the same strain than individual strains. Brewers have essentially been using the same strain to brew lager beers for probably 500 years. This is in stark contrast to the other fermented beverage industries, ale, whiskey, wine, cider and so on, where a rich and diverse collection of individual yeast strains is taken for granted.

Therefore, there is huge potential for introducing diversity into the lager brewing industry by generating new strains of lager yeast.

But before one can create new lager yeast it is important to understand what exactly the lager yeast is…

It has been known for some time that lager yeast is actually a hybrid species – more like a mule than the proverbial workhorse. It was clear that one parent was the well-known ale yeast Saccharomyces cerevisiae. It wasn’t until recently that the other side of the family, Saccharomyces eubayanus, was discovered. This discovery has allowed for the improved characterization of lager yeasts, and also opened up the possibility to create new tailor-made lager yeast strains. This is possible through mating of selected strains from the two parent species. These new strains could, e.g. produce unique flavours or ferment the beer more efficiently.

This is exactly what has been the focus of our ongoing research projects at VTT.

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The research team. From left to right: Brian Gibson, Kristoffer Krogerus, Virve Vidgren and Frederico Magalhães in VTT’s pilot brewery.

Screening perfect parents to mate

There are four main challenges in generating new lager yeasts: To select suitable parent strains. To get the parents to mate. To separate the hybrid cells from the parents. And finally, to confirm that they actually are hybrids.

We began by screening a range of ale yeast strains, from both VTT’s Culture Collection and commercial yeast suppliers, for beneficial fermentation properties. Once suitable parent ale yeast strains had been identified, the next step was to try to mate them with a strain of S. eubayanus, the other parent of lager yeast.

Before mating, the parent strains still had to be modified with selection markers, so that any hybrid cells could be isolated from the population. We did this by selecting spontaneous auxotrophic mutants of the parent strains, i.e. cells that weren’t able to grow on media lacking certain amino acids. This meant the hybrid cells could be selected by their ability to grow on media lacking these certain amino acids. Mating was then attempted by simply mixing populations of both parent strains, and letting them grow for a couple of days.

Seub_cells© VTT/Ulla Holopainen

After isolating some potential hybrid cells, their hybrid status was confirmed through various PCR tests, which showed whether DNA from both parent strains was present in them. After confirmation that we had produced our own lager yeast hybrids, we wanted to compare them to the parent strains in an actual wort fermentation.

To our pleasant surprise, all hybrid strains performed better than both parent strains, fermenting faster and reaching higher ethanol contents!

The hybrid strains also inherited beneficial properties from both parent strains, such as strong flocculation, cold tolerance and maltotriose utilization.

These first results suggest that this technique is suitable for producing new lager yeast strains with unique properties. These new strains also have the benefit of being non-GMO, which currently at least remains a necessity for brewers.

We are continuing our attempts to find and create perfect lager yeast hybrids at VTT. Our research will especially pay attention to flavour formation and determining how their genetic composition is reflected in their physiology.

Our work will show, for the first time, that such hybrids can be created and how they can be applied in the brewing industry. The results will appear shortly in the Journal of Industrial Microbiology and Biotechology:

Krogerus, K., Magalhães, F., Vidgren, V. & Gibson, B. (2015) New lager yeast strains generated by interspecific hybridization. Journal of Industrial Microbiology and Biotechnology, in press. DOI:10.1007/s10295-015-1597-6.

Maybe someday also you have an opportunity to enjoy these new tasty lager beers in your local pub. Cheers!

Generating new lager yeast hybrids

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For my PhD thesis, I’ve been researching the flavour- and stress-related properties of brewing yeast hybrids. It has been known for some time that lager yeast (Saccharomyces pastorianus) is actually a hybrid species, and that one parent was the well-known ale yeast Saccharomyces cerevisiae. In 2011, the other side of the family, Saccharomyces eubayanus, was discovered. This discovery has allowed for the improved characterization of lager yeasts, and also opened up the possibility to create new tailor-made lager yeast strains. This is possible through mating of selected strains from the two parent species.

graphical abstractThis is exactly what I’ve been doing during the past year, and I’m happy to announce that we recently published our first results (New lager yeast strains generated by interspecific hybridization) in the Journal of Industrial Microbiology and Biotechnology. We mated a strongly flocculent production ale strain (from a brewery in the UK) with S. eubayanus, to produce lager yeast hybrids which performed better than the parent strains, and inherited beneficial properties from both. This will open up the possibility to produce a range of new lager yeast strains, with e.g. interesting flavour production and increased stress tolerance. We already have plenty of new interesting hybrid combinations that I’m looking forward to characterizing. I will post more details in a later post, but in the meanwhile feel free to read the publication if you are interested, it is Open Access!

Link to the publication: http://link.springer.com/article/10.1007/s10295-015-1597-6

Abstract:

The interspecific hybrid Saccharomyces pastorianus is the most commonly used yeast in brewery fermentations worldwide. Here, we generated de novo lager yeast hybrids by mating a domesticated and strongly flocculent Saccharomyces cerevisiae ale strain with the Saccharomyces eubayanus type strain. The hybrids were characterized with respect to the parent strains in a wort fermentation performed at temperatures typical for lager brewing (12 °C). The resulting beers were analysed for sugar and aroma compounds, while the yeasts were tested for their flocculation ability and α-glucoside transport capability. These hybrids inherited beneficial properties from both parent strains (cryotolerance, maltotriose utilization and strong flocculation) and showed apparent hybrid vigour, fermenting faster and producing beer with higher alcohol content (5.6 vs 4.5 % ABV) than the parents. Results suggest that interspecific hybridization is suitable for production of novel non-GM lager yeast strains with unique properties and will help in elucidating the evolutionary history of industrial lager yeast.

Small update

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Sorry for not having posted in a while. I’ve been quite busy with other things and have also been abroad (Berlin again). Nothing new on the homebrewing front, since we’re still in the middle of moving to a new brewing location. We have lots of nice plans though, including an update to our brewing kettle and the addition of a whirlpool kettle. More about those once we make them. In Berlin, I didn’t do much beer-related, other than a quick visit to Getränkefeinkost in Friedrichshain. I can definitely recommend a visit as they had a nice selection of German microbrews and craft beer imports (USA, Denmark, UK, Italy etc.). Oh, and they had most the craft beer in coolers! I bought a total of 12 bottles, some of which I had during the trip already. Tomorrow I’m off abroad again, this time to Gent in Belgium for a conference (Young Scientists Symposium on Malting, Brewing & Distilling). The social programme includes a visit to the Rodenbach brewery, which should be very interesting. I will try to post some kind of report from the visit, and will hopefully post more homebrewing-related posts next month as well!

Physiology of Finnish Baker’s Yeast (Suomen Hiiva)

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While ale yeast and baker’s yeast belong to the same species, Saccharomyces cerevisiae, they have over time been adapted to different functions. Brewing with baker’s yeast is not a very common practice, nor generally recommend amongst brewers, mostly because these strains might not exhibit favorable fermentation characteristics, such as production of desirable flavour compounds, adequate attenuation, and flocculation. Of the recognized beer styles, it is more or less only in Northern European Traditional ales (such as Sahti, Gotlandsdricka and Kvass) that it acceptable to ferment with baker’s yeast. As there is little information published on the physiological characteristics of Finnish baker’s yeast in brewing, I thought I’d post some recent results from my related research.

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I performed a series of mini-fermentations (30 ml of wort) on a range of yeast strains earlier this spring for screening purposes, and I included the baker’s yeast from Suomen Hiiva, as well as two common homebrewing strains WLP002 (English Ale) and WLP380 (Hefeweizen IV). These mini-fermentations were performed at two different temperatures, 15 and 20 °C. Fermentation progress was monitored by weight loss, and after fermentation the resulting beer was analyzed for ABV% (Anton Paar Alcolyzer ME), Extract (Anton Paar DMA 5000 M), pH (Anton Paar pH ME), aroma compounds (HS-GC/FID), diacetyl (HS-GC/ECD), and phenolics (HPLC/PAD). A 15 °Plato (OG 1.060) wort was used for all fermentations, and the yeast was pitched at a rate of 2.5 g/L (~ 10 million cells/ml).

What first surprised me was how well the baker’s yeast performed during fermentation. Compared to the other two reference strains presented here, it fermented faster at both temperatures and reached a higher final attenuation. It managed to reach a final attenuation of 85% in just 5 days when fermented at 20 °C. Not bad for an all-malt wort at this strength. So the baker’s yeast can definitely give rise to adequate attenuation. Note, a pure culture of the yeast was used (starting from a similar package as in the picture above), so there was no risk of any lactic acid bacteria contamination, which otherwise is probable when using the yeast directly. The pH values of all beers were quite similar irrespective of yeast strain and temperature, and it seems like the baker’s yeast acidified the wort slightly less than the other two reference strains. In the table below you can find a summary of the Extract, ABV% and pH of the beers. In the figure below you can find a plot of the fermentation progress over time (20 °C squares; 15 °C circles).

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I have never actually used baker’s yeast for beer fermentation myself, as I’m not that big of a Sahti fan, but all beers (well mainly Sahtis) I’ve tried have had quite a ester-dominated aroma. Especially 3-methylbutylacetate (isoamyl acetate), with its prominent banana aroma, is very pronounced in, and also an integral part of, Sahti. Hence, it comes as no surprise that, compared to the other two reference strains, the baker’s yeast produced more higher alcohols and esters. It was not only 3-methylbutylacetate that was produced in large amounts (even more than the WLP380 Hefeweizen strain), but also the ethyl esters. Ethyl acetate, with its solvent-like aroma, is typically unwanted at higher concentrations in beer, but the other ethyl esters may contribute a fruity aroma to the beer. In the figure below you can see a summary of the flavour impact (i.e. the concentration of the compound in the beer divided by its flavour threshold. An impact above 1 should affect flavour, while an impact between 0.5 and 1 may affect flavour) of the various higher alcohols, esters, and acetaldehyde in the beers fermented with the different yeast strains (20 °C solid; 15 °C striped). As can be seen from the figure, only 3-methylbutanol (the precursor to the banana ester) of the higher alcohols is close to the flavour threshold. As should come to no surprise, less higher alcohols and esters were produced at a lower fermentation temperature. From these aroma compound results, it is clear that the baker’s yeast will produce a fruity, and maybe even slightly solvent-like, beer. Hence, the yeast would probably be suitable for Hefeweizens and Belgian-style beers, and a slightly lower fermentation temperature is probably recommended.sh_aroma

 

Finally we arrive at two other important aroma compounds in beer, diacetyl (butter-like aroma) and 4-vinylguaiacol (clove-like aroma; 4-VG). Diacetyl is always considered an off-flavour (well in some cases in might be acceptable, but in my opinion it is just a sign of poor fermentation practices), while 4-VG is acceptable (and even required) in some styles (such as Hefeweizens and Belgian-style beers). Diacetyl levels decrease towards the end of fermentation, and are highly dependent on fermentation dynamics, so the concentration that was measured here at the end of fermentation doesn’t say that much. The diacetyl concentration was above the flavour threshold (50 ppb) for all strains at both temperatures, which is not that surprising since the measurements were made 120 hours after pitching the yeast, and the lowest flavour impact was observed in the beer fermented with the baker’s yeast at 20 °C. This result again suggests that baker’s yeast is a good candidate for beer fermentations (and especially more rapid ones). The baker’s yeast is also POF+ (i.e. positive for producing ‘phenolic off-flavours’; the yeast produces a phenylacrylic acid decarboxylase enzyme, that decarboxylates ferulic acid (and other phenolic acids) from the wort into 4-vinylguaiacol (and other phenolic compounds)), since 4-VG was observed in the beer. The baker’s yeast produced slightly less 4-VG than WLP380 (the table below displays the percentage of ferulic acid converted into 4-vinylguaiacol; 77% is the theoretical maximum), but it still produced concentrations above the flavour threshold in an all-barley wort (worts made from wheat malts contain more ferulic acid). This slightly limits the beer styles that the baker’s yeast could potentially be used for, but again it could be used for Hefeweizens and Belgian-style beers.

sh_diacetyl_pof

To conclude, it is evident that the Finnish baker’s yeast is perfectly usable for beer fermentations, and it comes to no surprise that it has successfully been used for traditional beer fermentation in the form of Sahti. Of the two reference strains, the baker’s yeast was closer to WLP380 (Hefeweizen IV), and it can be described as a faster-fermenting and fruitier version of it. As a side-note, it can be mentioned that the baker’s yeast flocculated very poorly, which is also similar to the behavior of WLP380. I would not recommend the use of the baker’s yeast for beer styles calling for a clean yeast aroma and no spicy phenolics, but it would be perfectly suitable for a Hefeweizen, Belgian Blond and why not even Belgian Strong Ale (Sahtis are brewed strong, so the baker’s yeast should be quite tolerant to alcohol levels reaching up to 10% ABV). It fermented quite fast at both 20 °C and 15 °C, and I would recommend a lower temperature if you are after a cleaner finish. As mentioned previously, the yeast packages found in the supermarket are all almost certainly contaminated with lactic acid bacteria, so the use of a pure culture is definitely recommended. Good luck with the brewing!

Feel free to contact me if you have any questions!

Reference for flavour thresholds:

  • Meilgaard, M., Prediction of Flavor Differences between Beers from Their Chemical Composition. Journal of Agricultural and Food Chemistry 30 (1982) 1009-1017.

Article on the differences in diacetyl formation between lager yeast strains and the role of the ILV6 gene

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I’ve been researching diacetyl formation and removal by brewing yeasts the past year(s), and we recently did some screening of various lager yeast (Saccharomyces pastorianus) strains for diacetyl production and subsequent transcriptional analysis of key genes in the isoleucine-valine catabolic pathway (diacetyl is formed as an indirect by-product of valine biosynthesis in yeast; more information available here). We did this in order to identify what genes contribute most to the differences in wort total diacetyl concentration we observed between the different strains. We observed that particularly the ILV6 gene, encoding for a regulatory subunit of the acetohydroxy acid synthase enzyme (responsible for converting pyruvate into the diacetyl precursor α-acetolactate), showed greater expression early during fermentation in a strain producing more diacetyl. To confirm the role of this ILV6 gene in controlling α-acetolactate/diacetyl formation, we overexpressed both the S. cerevisiae- and S. eubayanus-form of ILV6 in our standard production lager strain A15, and found that overexpression of either form of ILV6 resulted in an identical two-fold increase in wort total diacetyl concentration relative to a control. These results suggest that both forms of the gene influence diacetyl formation, and different levels of transcription (this could be a result of a gene-dosage effect, since different lager yeast hybrids have inherited different amounts of the parental genomes; for a recent review on the subject click here) may contribute to differences in diacetyl production in various lager yeast strains.

Link to the publication: http://onlinelibrary.wiley.com/doi/10.1002/yea.3026/abstract

E-mail me if you are interested in the article, but don’t have access to the full-text.

Abstract:

A screen of 14 S. pastorianus lager-brewing strains showed as much as a nine-fold difference in wort total diacetyl concentration at equivalent stages of fermentation of 15°Plato brewer’s wort. Two strains (A153 and W34), with relatively low and high diacetyl production, respectively, but which did not otherwise differ in fermentation performance, growth or flavour production, were selected for further investigation. Transcriptional analysis of key genes involved in valine biosynthesis showed differences between the two strains that were consistent with the differences in wort diacetyl concentration. In particular, the ILV6 gene, encoding a regulatory subunit of acetohydroxy acid synthase, showed early transcription (only 6 h after inoculation) and up to five-fold greater expression in W34 compared to A153. This earlier transcription was observed for both orthologues of ILV6 in the S. pastorianus hybrid (S. cerevisiae × S. eubayanus), although the S. cerevisiae form of ILV6 in W34 also showed a consistently higher transcript level throughout fermentation relative to the same gene in A153. Overexpression of either form of ILV6 (by placing it under the control of the PGK1 promoter) resulted in an identical two-fold increase in wort total diacetyl concentration relative to a control. The results confirm the role of the Ilv6 subunit in controlling α-acetolactate/diacetyl concentration and indicate no functional divergence between the two forms of Ilv6. The greater contribution of the S. cerevisiae ILV6 to acetolactate production in natural brewing yeast hybrids appears rather to be due to higher levels of transcription relative to the S. eubayanus form.