Wednesday, November 23, 2011

Fermentation flavours

Fermenting in Loch Lomond distillery
Whisky wort fermentation produces ethanol, but also a variety of important flavours. Yeasts produce higher (fusel) alcohols and organic acids, which together form esters. Additionally ketones, sulphur compounds and phenols are formed. Whisky fermentation is quite similar to beer fermentation, but there are couple of important differences; the wort is not boiled, the distiller's yeast is usually propagated aerobically and the fermentations are usually not aeriated or temperature controlled (except the starting temperature). Unboiled wort allows the enzymes to continue their work and break down the oligosaccharides to increase the alcohol yield, but it also enables contamination with bacteriae and wild yeasts. If the yeast used is propagated aerobically, it is faster to start the fermentation and contains more sterols and fatty acids and thus the wort needs less oxidation or rousing.

Yeasts use simple sugars for their growth and energy metabolism. Simplified; when the yeast has oxygen, it produces water and CO from glucose, but in anaerobic conditions it turns glucose into ethanol and CO or alternatively glycerol. To reproduce, the yeast needs fatty acids, sterols and amino acids for its membranes and the organelles inside the cell. Oxygen is often needed in the production of these building materials.

When yeast is pitched into the wort, it secures its energy reserves and if there are enough nutritients, it starts to reproduce by budding. The beefing up-phase is called the lag phase, and it is shorter if the yeast has been aerobically grown as the cells are usually full of nutritients already. The budding phase is called the log phase or the exponential phase, during which yeasts reproduce usually 3-4 times increasing the cell population about ten-fold. As the cells form new organelles and cell membranes, they produce a variety of different organic acids, fats and sterols including various intermediate products, some of which leak out of the cell into the wort. After that the nutritients and oxygen fall short and the cells do not reproduce, but try to produce sufficient energy to survive from the sugars, this is called the stationary phase. As the cells start to die or drop out from the fermentation, lactic acid bacteriae start to grow on the wort producing flavours typical of their metabolism, such as lactic acid and several lactones.
Yeast growth in whisky fermentation (Ramsay & Berry 1983)

The amount of higher alcohols depends on the yeast growth; basically the more the yeast grows, the more higher alcohols are formed. Therefore aeriation of the wort, high nitrogen, and high temperature promote fusel alcohol production. Ale strains usually produce more fusel alcohols than lager strains, partly because of the higher fermentation temperatures. Fusel alcohols themselves are not a desired flavour in the wort - producing usually a sharp, solventy notes - but together with acids they form esters, which are important and desired flavour compounds in whisky as they produce various fruity and flowery notes.
Amino acidFusel alcohol
LeucineIsoamyl alcohol
IsoleucineActive amyl alcohol
Tyrosinep-hydroxyphenylethanol / tyrosol
 Table1. Aminoacids metabolise into different fusel alcohols

Ester formation depends on the amount of fusel alcohols and organic acids in the wort, but also on the activity of alcohol acetyltranferase enzymes (ATAase I and II), which in turn depends greatly on the yeast strain. Esters in the fermentation can be classified into two groups: The acetate esters (acetate+alcohol) and the ethyl esters (ethanol+fatty acid). The acetate esters are usually formed in greater amounts, but the ethyl esters can be very aromatic even in low concentrations. Common descriptors for the aromas of esters are listed in the table below. The short chain fatty acid esters (C6, C8) are formed early in the fermentation, the medium chain esters (C10,C12) quite evenly throughout the fermentation and the longer chain esters (C16) mostly at the cell-death phase. Increased cell growth usually results in lower levels of esters, due to lower levels of free fatty acids in the wort, as fats are used to build cell membranes. Organic acids are formed throughout the fermentation and at high levels they produce notes of vinegar, vomit and barnyard. The right proportion of fusel alcohols and free fatty acids or acetate is crucial when producing estery wort and avoiding the solventy off-notes from the excess alcohols and on the other hand the rancid aromas from the excess free fatty acids. An estery, fruity wort can be produced with warm long fermentations, high original gravities, high pitching rates with aerobically grown yeast and low nitrogen barley. Increased glucose levels tend to produce more short chain esters, for example isoamyl acetate with a typical banana aroma. High fermentation temperatures usually produce more acetate esters with mainly fruity aromas, but also medium-long chain ethyl esters, which can give an oily and waxy texture to the flavour.

Ester Name
Odor or occurrence
Allyl hexanoate
Benzyl acetate
pear, strawberry, jasmine
Bornyl acetate
Butyl butyrate
Ethyl acetate
nail polish remover, model paint, model airplane glue
Ethyl butyrate
banana, pineapple, strawberry
Ethyl hexanoate
pineapple, waxy-green banana
Ethyl cinnamate
Ethyl formate
lemon, rum, strawberry
Ethyl heptanoate
apricot, cherry, grape, raspberry
Ethyl isovalerate
Ethyl lactate
butter, cream
Ethyl nonanoate
Ethyl pentanoate
Geranyl acetate
Geranyl butyrate
Geranyl pentanoate
Isobutyl acetate
cherry, raspberry, strawberry
Isobutyl formate
Isoamyl acetate
pear, banana (flavoring in Pear drops)
Isopropyl acetate
Linalyl acetate
lavender, sage
Linalyl butyrate
Linalyl formate
apple, peach
Methyl acetate
Methyl anthranilate
grape, jasmine
Methyl benzoate
fruity, ylang ylang, feijoa
Methyl butyrate (methyl butanoate)
pineapple, apple, strawberry
Methyl cinnamate
Methyl pentanoate (methyl valerate)
Methyl phenylacetate
Methyl salicylate (oil of wintergreen)
Modern root beer, wintergreen
Nonyl caprylate
Octyl acetate
Octyl butyrate
Amyl acetate (pentyl acetate)
apple, banana
Pentyl butyrate (amyl butyrate)
apricot, pear, pineapple
Pentyl hexanoate (amyl caproate)
apple, pineapple
Pentyl pentanoate (amyl valerate)
Propyl acetate
Propyl hexanoate
blackberry, pineapple, cheese, wine
Propyl isobutyrate
Terpenyl butyrate
 Table2. Common esters and their aromas. 
Diacetyl is an important flavour compound producing slick, buttery mouthfeel from concentrations of about 1ppm and at higher concentrations butterscotch or even cheesy flavours, and is usually considered as an off-note. It arises from the nitrogen metabolism during the exponential phase as the cells convert aminonoacids into ketones (such as diacetyl) and back to different aminoacids, but in the late stationary and the cell-death phases the cells use ketones in their metabolism as the sugars are running low. Brewers and distillers usually allow a diacetyl-rest period after the active fermentation to clear the wort of excess ketones. Too short fermentation time usually results in excess diacetyl. Heating, for example during distilling, increases the formation of diacetyl from other ketones. Diacetyl is quite volatile with a boiling point of 88C and very hard to remove from the spirit even with column distillation.

Sulphur mining in an active volcano, Java (from
Yeast metabolism produces many sulphur compounds, mostly sulphur dioxide (SO, burnt matches). SO is easily reduced to hydrogen sulphide (HS, rotten eggs), which is very volatile and easily carried out of the wort if sufficient CO is formed. Slow fermentations due to for example low temperature, low pitching rate, contamination or unhealthy yeast could fail to produce enough CO, which leads to high levels of HS in the wort. Some highly aromatic sulphur compounds such as dimethylsulphide (DMS) and  -trisulphide (DMTS), dimethylsulphoxide (DMSO), S-methyl methionine (SMM), dithiapenthyls (DTPOH, DTPA) and various mercaptans originate mostly from the malt, but are metabolised by yeast and their concentrations can be either elevated or decreased during fermentation. Methione and cysteine are amino acids with a sulphur chain, which can be broken down during cell growth and energy metabolism. Starved cells can also turn into catabolic state (autophagosytosis), in which they break down their cell organnelles (and amino acids in the process) to produce energy, and this produces excess sulphur. This is probably why anaerobically grown brewer's yeast together with distiller's yeast produces more sulphur compounds than either one used alone. Starved brewer's yeast (cropped from the brewery, not from an aerobic propagation or a lab) produces over twice as much aromatic sulphur compounds than fresh yeast of the same strain. Distiller's yeast used alone produces slightly less aromatic sulphur than a common ale yeast, probably because of its better nutritional state. The aromatic sulphur compounds are not necessarily off-notes, but are in fact needed for full-bodied and complex aromas (in the right proportions, of course).

Phenols in whisky are mostly derived from peat burnt in the maltings, but some very flavour-active phenol compounds can be produced  by yeasts. Wild yeasts produce significant amounts of 4-vinyl guaiacol, which has a very potent phenolic aroma. Phenolic note has been considered an off-note in brewing and therefore the brewers have usually chosen strains that do not have a functioning gene for 4-vinyl guaiacol-production, exceptions include most hefeweisen and rauchbier yeasts and of course the lambics brewed with wild yeasts. Apparently also the commercial distiller's yeasts are lacking the "phenolic off-flavour" genes.

The picture below sums the simple reactions involved in the flavour formation during alcoholic fermentation.
Flavour formation from alcoholic fermentations. (Ramsay 1982)
References and further reading:
Bryce JH et al (ed). Distilled spirits: Production, technology and innovation. Nottingham Univ Press 2008
Piggott JR et al (ed). The science and technology of whiskies. Longman 1989
Querol A, Fleet GH (ed). The Yeast Handbook. Springer-Verlag Berlin 2006
Russell I (ed). Whisky, technology, production and marketing. Academic Press 2003
Walker GM, Hughes PS (ed). Distilled spirits, new horizons: energy, environment and enlightenment. Nottingham Univ Press, 2010
White C, Zainasheff J. Yeast. Brewers Association 2010

Monday, October 17, 2011

Malting in 1660s

Just found a nice article about malting barley (or bere) by Sir Robert Moray, probably written between 1660-1673 and published in 1739 as a part of The Memoirs of the Royal Society (ed. Mr Baddam). I will try to find something more recent to blog on later...

Friday, September 30, 2011

Yeasts: pedigree and properties

Compressed Mauri Pinnacle yeast (
Yeasts used in beverage production mostly belong to the genus Saccharomyces. There are various species of Saccharomyces, including S.bayanus, S.cariocanus, S.cerevisiae, S.eubayanus, S.kudriavzevii, S.mikitae, S.paradoxus, S.pastorianus and in some sources S.uvarum, which is usually considered as a subspecies of S.bayanus. The nomenclature and classification of species changes almost daily and therefore is not always uniform in literature. The species can be further classified into different strains and there are currently thousands of different strains of S.cerevisiae alone. Hybridization is common between the domesticated yeasts used in alcohol production. The yeasts used in whisky industry are mostly S.cerevisiae although various secondary species have been used with it. Baker's yeast is usually S.cerevisiae, lager yeast is S.pastorianus, ale yeasts include S.cerevisiae and apparently some S.bayanus strains, rum ferments primarily on S.cerevisiae and Schizosaccharomyces (with various wild yeasts) and wine industry use mostly S.cerevisiae and/or S.bayanus together with various wild yeasts (for example Kloeckera, Saccharomycodes, Schizosaccharomyces, Hansenula, Candida, Pichia and Torulopsis).

The simple Saccharomyces yeast is a single-cell fungus, containing 16 different chromosomes and because its genome is diploid, there are 32 chromosomes containing the genome (DNA). It can reproduce by budding (producing a copy of genome and cell organs and dividing into two) or mating by spores. During the evolution of yeasts used in beverage production non- or low-spore-producing yeasts became selected, because consistency of the fermentation was preferred. Therefore the strains used in beverage industry reproduce almost exclusively by budding and therefore their genomes change mostly by spontaneos mutations and rarely by mating/breeding. In addition some yeasts produced polyploid (multiple choromosome sets) or aneuploid (multiple single choromosome or parts of it) genomes, which further improved the consistency as there are more than two copies of one chromosome in case of a harmful mutation(s) and less fertile spore production. The extra chromosomes will further split and/or integrate with the other chromosomes. Put simply: it's complicated. For example the species S.pastorianus (formerly called S.carlsbergensis, S.uvarum or S.cerevisiae var Hansen, etc) widely used in lager brewing was probably formed by hybridization of an ale yeast S.cerevisiae and a wild yeast S.eubayanus and by further mixing genetic material (parts of chromosomes) with S.bayanus, which itself is a hybrid of S.cerevisiae, S.eubayanus and S.uvarum (which is also a strain of S.bayanus species). Because of the complex choromosome structure and the restricted reproduction abilities of domesticated yeasts, systematic and predictable breeding of yeasts is very hard even with the modern genetic engineering techniques.

Proposed development of S.pastorianus and hybrids of S.bayanus (Libkind et el 2011)
Practical classification of yeast is done by its purpose (baking, ale/lager brewing, distilling) and it is common to name strains after the lab which produces it, followed by a number; for example WH301 or WL001. Various yeast labs sell probably the same (or very very similar) yeast by a different name. The yeast strains used in beverage industry can be classified further by their abilities to ferment. Important properties of an alcohol producing yeast are flocculation, attenuation, sugar utilization, ability to work in high sugar concentrations (high gravity brewing), tolerance of alcohol, temperature and various killer factors and whether they are top or bottom croppers.

Lager flocculation
Flocculation is the yeasts' ability to clump together; ale yeast flocculates on the top of the fermentation and lager yeast onto the bottom. High flocculators clump early (about 3-5 days) in the fermentation, which might lead to low attenuation, ie part of the sugars are not metabolized to alcohol. Whisky distillers usually prefer low flocculators, because flocculated yeast is more likely to stick to the heating coils or the still surface (especially when direct heated) producing burnt flavours. Low flocculators often provide better attenuation (sugar utilization) and therefore higher alcohol yields. Filtering the wash before the distillation could be an option when using medium-high flocculators, but it is not apparently used in Scotland. The "on the lees" (ie wash containing the yeast cells) distillation is considered to enhance spirit flavour in both grain and wine spirits, most likely by increased fatty acid ester and methylketone concentrations producing oily, rancio and fruity aromas

Wort contains various sugars, mostly maltose and its oligosaccharides (maltotriose, maltotetraose, maltopentaose etc), but also glucose, fructose and sucrose. The oligo- and disaccharides (glucose, fructose, sucrose, maltose) are preferred by the yeast (figure 1) and transported inside the cell by diffusion, but maltotriose utilization depends on the yeast's ability to transport maltotriose into the cell by a spesific enzyme. Effective maltotriose uptake of a whisky yeast is important for optimal alcohol yield. Apparently most whisky yeasts (and brewer's yeasts) used contain several genes for maltotriose tranport enzymes, probably result from several hybridizations and chromosomal changes.

Figure 1. Sugar utilization in all-malt wort (IBD Blue book on yeast)

Alcohol tolerance of yeast depends on the strain and the species. Most domesticated or cultured beverage yeasts tolerate over 10% ABV ethanol concentrations as most non-saccharomyces wild yeasts stop working effectively in 1-5% ABV and die in about 10% ABV as some yeasts used for industrial fuel alcohol production can go up to 23% ABV. In whisky fermentations the factor limiting the final alcohol yield is usually the amount of sugars in the wort as whisky yeast attenuation is usually very good and the primary yeasts tolerate well the 5-8% ABV of a whisky fermentation.

The killer factors are toxins that yeasts produce against other yeast strains. Strains also develop tolerance for these toxins and there are many toxins in wild yeast fermentations, too. Brewer's and distiller's yeasts are usually quite tolerant to the most common killer factors and produce some killer factors themselves, depending on the strain. Anyway a wild yeast producing a killer toxin, which is not tolerated by the primary distiller's yeast used, might ruin the whole batch by producing a stuck fermentation or an inappropriate flavour profile.

Scotch malt whisky fermentations are not usually temperature controlled (apart from the starting temperature, which is adjusted to the ambient temperature), despite practically all lager brewers and most wine producers use temperature controlled fermetors. Yeast metabolism produces lots of heat, especially when anaerobically producing alcohol. Therefore whisky yeast must tolerate different temperatures, usually from 18-20C to over 33C. Typical whisky distillery yeasts tolerate about 32-34C depending on the ethanol concentration and although some other distilling strains can cope with up to 46C (a Finnish vodka strain), most distillers yeasts produce the best alcohol yield at 20-30C. Flavour compound formation is affected quite heavily by the fermentation temperatures; higher temperature fermentations produce less esters and more higher alcohols.

The most used whisky distiller's yeast in the latter part of the 20th century was a S.cerevisiae strain called DCL M, M-strain, Quest M, Rasse M, M-1, D1 or WH301 manufactured formerly by DCL Yeast ltd and now mostly by Kerry Biosciences (Kerry Group bought Quest Ingredients in 1998). The M-strain was introduced to Scotch whisky distilleries by DCL in 1952, but a similar Rasse M was used widely in German distilleries at least from the 1930s. The name has remained the same although the properties of the strain have changed considerably from the 1930s and there most likely is some variation between different yeast manufactures despite the same name. The M-strain is a intraspecies hybrid of S.cerevisiae (as S.cerevisiae covers the former S.diastaticus species). The first Scottish pure strain whisky yeast was developed in the mid-1920s and before the WW II DCL had pure cultures of "standard" DCL-whisky yeast, DCL S.C. (probably for sugar cane fermentations) and DCL L-3 (probably a variety of the standard DCL). Whether they were used widely in distilleries is not documented, but probably they were used in DCL grain distilleries and in some malt distilleries within a reliable transport route in adjunction with a local brewer's or baker's yeast. There is some evidence that the first pure-culture distilling yeasts were being tried in Keith already in the 1870s, but apparently they were never used in larger scale.

In continental Europe pure yeast cultures were more widely used and there were spesific strains for grain/malt worts (Rasse M, Rasse XII) and rye wort (R-strain) and even a raspberry-flavour producing strain "A". Also Fleischmann and Brown-Forman in the US had developed their own distiller's strains by the 1940s. The yeast strains of European, Asian and American distillers seem to be quite different at least by their genetics (see pic below), but there is no scientific data available for differences in spirit quality or flavour. The most similar beer yeasts compared to current Scottish whisky yeasts are probably some Belgian trappist and German hefeweisen yeasts, which are low flocculators, high attenuators, very alcohol tolerant and often produce smoky-spicy aromas associated with 4-vinyl-guaiacol production typical for S.cerevisiae var diastaticus, which is considered to have contributed strongly to the development of the M-strain from the ale-type S.cerevisiae.
Neighbour-joining tree of 63
S. cerevisiae strains (Schacherer 2009)

The M-strain ruled the Scottish whisky industry from 1960s to 1980s, although many distilleries used ale brewer's and/or baker's yeasts in adjunction with it. Before WW II most distilleries propagated their yeast on site, but during 1950s the production was largely outsourced to yeast factories and breweries. The availability of cheap (used/surplus) ale yeast diminished as lager became more popular in UK and as there were claims that using brewer's yeast dimished the alcohol yield, many distilleries started using pure cultures in the 1980s. As said before, the properties of the M-strain probably changed considerably during the latter part of 20th century, primary goals being higher alcohol yields and the preservation of traditional (or neutral) flavour profile.

Cream, compressed and dried yeast
Another significant development was the development of active dry yeast (ADY or instant dry yeast IDY) during the WW II to provide longer shelf life by basically drying the yeast into small pellets rather than just a big clump. This enabled the transportation of yeast into remote locations of Scotland, too. Some Scottish malt distilleries still use dried (95% solids) or more often compressed (25-28%) bag yeast. Cream yeast (17-23% solids) was introduced in 1983 to provide easy delivery by tank trucks and automated pitching, which was important and practical for bigger plants.

The MX-strain developed in the 1990s is a bit faster fermenter and produces a very similar flavour profile compared to the M, according to the manufacturer Kerry Group. The MX is faster and more efficient especially in high gravity worts which are preferred because of the savings in heating and water costs. Another common malt whisky yeast is Pinnacle by Mauri, which is an ethanol tolerant baker's yeast (S.cerevisiae) and actually slightly faster than MX, reaching peak fermentation speed about 1 hour earlier (at 15hours of fermentation) than MX. The grain distilleries use mostly cream yeast of undisclosed strain, produced by British Fermentation Products (BFP) or Anchor Yeast. In the table below you can find information about the yeast strain used by some Scottish distillers.

AultmoreBowmore 25% (+Mauri)AberlourBen Nevis (50/50)Auchentoshan (+Mauri)
Blair AtholBruichladdichArdbegBalblairDaftmill
Bruichladdich (+Mauri)BunnahabhainAuchentoshan (+Anchor)BenromachGrain distilleries
BunnahabhainCraigellachie (+Mauri)BenrinnesCardhu

Glengoyne (+MX)Glengoyne(+M)Bowmore 75% (+MX)Glenburgie

Glen ScotiaLagavulin (+Mauri)Bruichladdich (+M)Glenmorangie (5dist, 2brew)

Highland ParkSpeyside (+M)Caol IlaImperial

Lagavulin (+Mauri)

Craigellachie (+MX)Jura

Macallan (+Mauri+brewers)


Speyside (+MX)

GlenfiddichMacallan (+M+Mauri)

Lagavulin (+M)Miltonduff


Macallan (+M+brewers)Speyburn

Strathmill (+brewers)Strathmill (+Mauri)

Yeasts used by some Scottish whisky distilleries (Udo 2006)

The use of brewer's yeast as a secondary yeast strain produces more sulphury compounds into the wash and less fatty acid esters, especially when using dry ale yeasts. As brewer's yeast attenuates or even dies earlier than distiller's strain, the use of secondary strain increases the growth of lactic acid bacteria (LAB) towards the end of fermentation, which in turn lowers the pH of the wash altering the distillation process and produces specific flavours depending on the bacteria strain. One LAB strain might produce for example vinyl-guaiacol (smoky-spicy), as another produces damascenone (floral). Practically all the LAB growth results in more esters into the new-make, especially hexanoate and octanoate and decreased ethanol yield.

Because Scottish distillers at the present time use very similar primary yeasts, the selection of the strain of distiller's yeast is a minor factor in terms of flavour profile, at least when compared with other aspects of fermentation, such as original wort gravity, fermentation time and temperature and the material and microflora of washbacks.

In the future the whisky industry is looking to develop yeast strains suitable for higher gravity worts, shorter fermentation times and better utilization of maltotetraoses and -pentoses. Hopefully the flavour issues are also considered in the process and different strains are studied for improved flavour profiles.

Bryce JH et al (ed). Distilled spirits: Production, technology and innovation. Nottingham Univ Press 2008
Dunn B, Sherlock G. Reconstruction of the genome origins and evolution of the hybrid lager yeast S.pastorianus. Genome Res 2008;18;1610-1623
Gray WD. Studies on the alcohol tolerance of yeasts. J Bacteriol 1941;42(5);561-574
Hansen R et al. Proteomic analysis of a distilling strain of Saccharomyces cerevisiae during industrial grain fermentation. Appl Microbiol Biotech 2006;72;116-125
Landry CR et al. Ecological and evolutionary genomics of S.cerevisiae. Molec Ecol 2006;15;575-591
Libkind D et al. Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast. PNAS 2011;108;35;14539-14544
Piggott JR et al (ed). The science and technology of whiskies. Longman 1989
Pretorius IS et al. Designer Yeasts for the Fermentation Industry, Food Tech Biotech 2003;41(1);3–10
Querol A, Fleet GH (ed). The Yeast Handbook. Springer-Verlag Berlin 2006
Russell I (ed). Whisky, technology, production and marketing. Academic Press 2003
Udo M. The Scottish Whisky Distilleries. Black & White 2006
Saerens SMG et al. Genetic improvement of brewer’s yeast: current state, perspectives and limits. Appl Microbiol Biotech 2010;86;1195-1212
Schacherer J et al. Comprehensive polymorphism survey elucidates population structure of Saccharomyces cerevisiae. Nature 2009;458;342-346
Sipiczki M. Interspecies hybridization and recombination in Saccharomyces wine yeasts. FEMS Yeast 2008;8;996-1007
Suomalainen, H & Lehtonen, P. The production of aroma compounds by yeast. J Inst Brew 1978;85;149-156
Walker GM, Hughes PS (ed). Distilled spirits, new horizons: energy, environment and enlightenment. Nottingham Univ Press, 2010
White C, Zainasheff J. Yeast. Brewers Association 2010