Some like it cold: VTT’s cold-tolerant yeast strains for wine and cider production

by Frederico Magalhães

Yeast, like all living organisms, have a narrow range of temperature at which their growth is optimal.  Temperatures below this range tend to slow down or even stop metabolic processes, while higher temperatures can cause cell death.

For many fermentation processes, different temperatures significantly impact product quality.

Ale beer is fermented at a relatively warm temperature (18-24°C) while lager fermentations require lower temperatures (5-15°C). Similarly, red wine is generally fermented at a warmer temperature than the white wine. Fermentation at different temperatures is only possible due to the availability of a variety of yeast species/strains that are adapted to specific temperature conditions.

The yeast Saccharomyces cerevisiae is the most commonly used species for baking, wine, cider and ale fermentations, but is sensitive to low temperatures. When there is a requirement for very low temperature fermentation this species is commonly found in hybrid form with a cold-tolerant species. This is the case for lager yeast.

Through hybridization, lager yeast was able to combine the good fermentative behavior of S. cerevisiae and the cold tolerance of the wild yeast S. eubayanus.

In 2015, VTT generated the first new lager brewing yeast strains in 500 years, proving that it is possible to generate hybrids in a targeted way for specific industrial processes. We recently applied the knowledge obtained in that work to create new yeast strains for the production of cider and wine.

The cold-tolerant Saccharomyces eubayanus

Cold fermentations have some advantages for cider and wine making. At such temperatures, the risk of contamination is low, reducing the need to use sulphite as a preservative. In addition, the aroma profile of white wines is generally improved by using lower fermentation temperatures. White wine and cider are already fermented in relatively cold conditions; therefore, the use of a cold tolerant yeast can potentially improve the process.

The wild yeast S. eubayanus has proven to be one of the most cold-tolerant yeasts known and is partly responsible for the success of the lager beer. In addition, previous results suggest that it contributes to a more diverse aromatic profile and it has good ability for fructose transport. Fructose represents 2/3 of the fermentable sugars in apple juice and half of the sugars in grape must. Due to the total sugar concentration being considerably higher in grape must, incomplete fructose utilization often becomes a limiting step. Therefore, the cold tolerant S. eubayanus has several phenotypic traits relevant for cider and wine making. However, the use of this species is so far limited to lager beer (in hybrid form) and to the spontaneous fermentation of Mudai (a traditional Patagonian beverage prepared with the seeds of the monkey puzzle tree).

New hybrid strains tolerate a wide range of temperatures

We successfully crossed this cold tolerant yeast with a S. cerevisiae wine strain, and tested the fermentative potential in cider and wine making. One of the most important observations was that the new hybrid yeasts tolerated a wide range of temperatures (10-37°C). The lower range allows the yeast to efficiently ferment at low temperatures, while the higher temperatures allow the yeast to resist the stressful conditions associated with the large-scale production in active dry yeast form.

The wider range of temperatures also means that it can serve multiple purposes. Temperature greatly impacts the aromatic properties of cider and wine, and therefore, the aromatic properties can be modulated by simply altering fermentation temperature.

Significantly more pleasant ciders

To test the new hybrid strains we first fermented apple juice at 10 and 20°C. The hybrid yeast outperformed the parental wine yeast. Particularly at 10°C, the wine strain was seriously inhibited by the low temperature. In addition, the hybrids produced more esters, giving a fruity/floral flavour to the ciders. Importantly, unpleasant volatile compounds produced by the wild parent were also eliminated through hybridization.

The sensorial properties of the ciders were evaluated by a consumer panel at the Functional Food Forum of the University of Turku, confirming that the ciders produced by the hybrids were perceived to be significantly more pleasant than those produced by the cold tolerant parent alone.

Wines with more complex and exotic aromatic profiles

To test the potential of the hybrid yeast for wine making, we fermented Sauvignon Blanc and Maccabeu grape musts at laboratory scale. The fermentation performance was comparable to commercial wine yeast strains but with a stronger, more complex and exotic aromatic profile, as described by a panel of expert tasters.

Furthermore, the best performing hybrid yeast was tested for its capacity to maintain high viability after drying and rehydration. The results were very positive with viabilities comparable or even higher than yeast strains commercialized in dried form.

Improved baking yeasts next

We have demonstrated that hybridization is a very powerful technique to improve yeast performance and phenotypic diversity for applications where targeted genetic modification is not yet accepted.  After successfully applying this technique to beer, wine and cider production, we are now assessing hybrids for their potential use by the baking industry. This is being done with the support of a Key Projects grant from the Academy of Finland.

Cold tolerance is also a critical issue in baking. The advent of frozen dough use in recent years represents a challenge for baking yeast. Yeast lose viability when stored frozen, and the poor heat conductivity of dough means that yeast can be exposed to quite low temperatures during the proofing stage of baking. A yeast strain able to perform well at a wider range of temperatures might aid in making loafs of bread evenly fermented after freezing.

References:

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

Magalhães F, Krogerus K, Vidgren V, Sandell M & Gibson B. (2017) Improved cider fermentation performance and quality with newly generated Saccharomyces cerevisiae × Saccharomyces eubayanus hybrids. Journal of Industrial Microbiology and Biotechnology. https://link.springer.com/article/10.1007/s10295-017-1947-7

Magalhães F, Krogerus K, Castillo S, Ortiz-Julien A, Dequin S & Gibson B. (2017) Exploring the potential of Saccharomyces eubayanus as a parent for new interspecies hybrid strains in winemaking. FEMS Yeast Research. https://doi.org/10.1093/femsyr/fox049

 

DSC_0057xThe author Frederico Magalhães is a third-year PhD student at VTT’s Bioprocess engineering team. His main research interests include studying of yeasts’ natural diversity to develop new hybrid yeasts with relevant phenotypes for the beverage and bread industry. frederico.magalhaes@vtt.fi

A Fresh Look at Old Beer

by Brian Gibson

Archaeological brewing at VTT

The work of an archaeologist is complicated by the fact that the materials under investigation are invariably imperfect. Metal artefacts rust, wood decomposes, DNA degrades, bones and clay vessels shatter. Time leaves only a suggestion of the original materials’ properties and the archaeologist is left to conclude as much as possible from the limited information available. However difficult, with the right approach one can learn much from ancient materials regardless of the size or condition. In this post I write about two of our recent archeological brewing projects: shipwreck beer and sahti.

Shipwreck beer

Shipwreck bottle C49How to interpret information from compromised samples was an issue faced by a team at VTT after receiving two bottles from the ‘Champagne wreck’ – the remains of a schooner found on the sea bed near the Åland Islands in the Baltic sea. Archaeological analysis dated the wreck to sometime in the 1840s. The bottles appeared to contain beer but sensory analysis was impossible due to extensive bacterial spoilage of the beer. Trained beer tasters described the contents in less than favourable terms: rancid, goaty, sulphuric. With such degraded samples it was difficult to imagine how the beer would have originally tasted or how it could be recreated.

The first challenge of the VTT team, led by Dr John Londesborough, was to determine if the contents were, in fact, beer. This was done through sugar analysis which revealed traces of maltose and maltotriose, which are the main sugars present in beer. Further confirmation came from collaboration with partners at the University of Munich who showed unequivocally that the samples contained hops, one of the main ingredients of beer and one which is not found in other beverages. Having established that the samples were beer, it was necessary to determine the level of sample degradation. In particular, it was crucial to determine the level of seawater contamination. The bottles recovered were sealed with corks, which offered only limited protection against the entry of seawater. By determining the concentration of sodium in the beer it was possible to calculate the level of NaCl (salt) and therefore seawater contamination, which in both bottles was approx. 30%.

Vrak Öl Sample collectionAnalysis in Munich  showed that the hop components were extensively degraded, but the concentrations and kinds of degradation compounds could be used to determine what the original compounds were. The presence of hop iso-α-acids indicated that the wort had been boiled prior to fermentation.  A relatively high level of β-acids showed that the hops were less refined than today’s varieties (β-acids have been bred out of modern hop varieties because of their harsh taste). The ethanol concentration (correcting for seawater-contamination) was a relatively mild 4.5% alcohol by volume (ABV). The fruit and floral flavours characteristic of beer were in the normal range, except that the concentrations of 2-phenylethanol were relatively high. This would have imparted a touch of rose aroma to the beers. The presence of a volatile phenol giving a slight clove aroma was also detected. This suggests that the beer may have been ale, as such flavours are not produced by lager yeast.

Detailed microbiological analysis failed to recover any viable yeast cells from the beer, though microscopy revealed abundant dead cells. Likewise, any yeast DNA that may have been present originally was absent – probably degraded enzymatically by the bacteria that appear to have been much more tolerant of the conditions in the bottles during their long storage.

Despite the many challenges faced by the VTT team that analysed the bottles, recreation of the beer, based on the physical and chemical profiling carried out at VTT (and some creative licence) was possible, and Stallhagen’s 1843 beer can now be purchased in Finnish supermarkets.  More detailed results of the study can be found in the Journal of Agriculture and Food Chemistry:

Londesborough, J., Dresel, M., Gibson, B., Holopainen, U., Mikkelson, A., Seppänen-Laakso, T., Viljanen, K., Virtanen, H., Wilpola, A., Kivilompolo, M., Hoffmann, T. & Wilhelmson, A. (2015) Analysis of beers from an 1840s’ shipwreck. Journal of Agriculture and Food Chemistry, DOI: 10.1021/jf5052943.

Sahti: a part of Finland’s living heritage

In 1938, fishermen off the coast of South Africa hauled up an unusual fish. This find was exceptional not because the fish was unknown to science but rather because the species was known only from the fossil record. It had been believed that this fish, the coelacanth Latimeria chalumnae had become extinct 65 million years previously. The coelacanth is one of a number of species that seem to have resisted the effects of evolution over a huge stretch of time. The gingko tree is another notable example which has survived, essentially in the same form, for 170 million years. Darwin, in the Origin of Species wrote:

“…these anomalous forms may almost be called living fossils; they have endured to the present day, from having inhabited a confined area, and from having thus been exposed to less severe competition.”

Such species are uniquely valuable as they provide a direct link to the past. One does not need to base conclusions on scraps of information from long dead and degraded material but can study the real thing in detail. Such studies provide not just a glimpse into the past but also a clearer understanding of how evolutionary forces have shaped modern species; relatives of the lobe-finned coelacanth eventually became the first land animals.  The concept of the living fossil is not necessarily restricted to biological species but can also be applied to the cultural sphere; many traditions, customs, languages, celebrations, etc. have survived unchanged over long periods and offer a unique glimpse into the past. One can even apply this concept to brewing of beer. One relevant example is Finland’s own sahti beer, which appears to have been brewed in more or less the same way for hundreds of years.

Sahti GlassSahti is one of the few ‘ancient’ beer styles still produced in Europe and differs in many respects to modern beers. The main difference is that hops are not an essential ingredient in sahti. Hops have been used to add bitterness and aroma to beer since around 800 AD. Prior to this, a diverse range of ingredients were utilized to add bitterness and balance to beer flavour. These included anise, heather, cinnamon, clove, coriander, liquorice and, as in the case of sahti, juniper. Sahti’s method of production pre-dates the use of hops and is often described as an ancient beer style for exactly this reason. In the EU, sahti is protected by ‘Traditional Speciality Guaranteed’ label, implying that commercial use of the name is restricted to beer produced according to the traditional, registered production method. But what exactly is sahti and how does it differ from modern beers? These questions motivated scientists at VTT to carry out the first comprehensive physical and chemical study of sahti beer.

By analysing samples collected throughout the traditional sahti brewing region of southern and south-western Finland, the researchers showed that, in every respect, sahti was different to commercially available lager, wheat and porter beers. All 12 sahti beers were strong with average alcohol levels of 8% ABV but also with high levels of residual sugars, in some cases more than 100g per litre. Sahti is typically brewed with baker’s yeast rather than brewer’s yeast and this imparts specific flavour characteristics to the beer. In particular, sahti beers contain a trace of clove aroma, which is normally only found in wheat beers. Like English ales, sahti beers have little or no foam – in this case probably because of the high sugar concentration and the absence of wort boiling, both of which are expected to limit foam stability. Other unique features of sahti are a very low level of bitterness due to the absence of hops and an intense fruit flavour due to esters produced by the yeast during fermentation. In some cases the concentrations of fruit compounds were more than ten times higher than in commercial beers. These compounds give sahti its intense banana aroma for example.

As sahti wort is not boiled and contains little or no hops (a natural antimicrobial agent), sahti beer tends to stay fresh for only a short period. The ephemeral nature of the product has probably contributed to its limited geographic distribution and this may, as Darwin contended for biological species, explain why this beer has not ‘evolved’ in the same way as other European beer styles.

Our study has emphasised the uniqueness of sahti beer compared to modern beers and has introduced the international brewing research community to a very individual style of beer and an important part of Finland’s living heritage. Results appear in an upcoming issue of the Journal of the Institute of Brewing:

Jukka Ekberg, Brian Gibson, Jussi Joensuu, Kristoffer Krogerus, Frederico Magalhães, Atte Mikkelson, Tuulikki Seppänen-Laakso & Arvi Wilpola (2015) Physicochemical characterization of sahti, an ‘ancient’ beer style indigenous to Finland. J Inst Brew. In press.

Brian GibsonDr. Brian Gibson has worked at VTT Technical Research Centre of Finland, Ltd. as Senior Scientist and Project Manager since 2009. Brian is responsible mainly for projects relating to brewing yeast biology, fermentation efficiency and beer quality. Current topics of interest include improvement of yeast performance through hybridization and/or adaptive evolution as well as optimization of process conditions. The underlying mechanisms (genetic, molecular, physiological) that govern yeast performance are a main topic of interest. brian.gibson@vtt.fi

Link to Brian’s publications

 

 

 

How new yeast species are inspiring a revolution in brewing

by Kristoffer Krogerus

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.

Blog Image 3The 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.

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!

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The author Kristoffer Krogerus is first-year PhD student working at VTT Industrial Biotechnology. His supervisor Dr. Brian Gibson is Senior scientist and project manager with responsibility for projects relating to brewing yeast physiology and fermentation.

 

ext-kristoffer.krogerus@vtt.fi , brian.gibson@vtt.fi

www-pages of VTT’s brewing yest development