The potential for Scotch Malt Whisky flavour diversification by yeast

Abstract Scotch Whisky, a product of high importance to Scotland, has gained global approval for its distinctive qualities derived from the traditional production process, which is defined in law. However, ongoing research continuously enhances Scotch Whisky production and is fostering a diversification of flavour profiles. To be classified as Scotch Whisky, the final spirit needs to retain the aroma and taste of ‘Scotch’. While each production step contributes significantly to whisky flavour—from malt preparation and mashing to fermentation, distillation, and maturation—the impact of yeast during fermentation is crucially important. Not only does the yeast convert the sugar to alcohol, it also produces important volatile compounds, e.g. esters and higher alcohols, that contribute to the final flavour profile of whisky. The yeast chosen for whisky fermentations can significantly influence whisky flavour, so the yeast strain employed is of high importance. This review explores the role of yeast in Scotch Whisky production and its influence on flavour diversification. Furthermore, an extensive examination of nonconventional yeasts employed in brewing and winemaking is undertaken to assess their potential suitability for adoption as Scotch Whisky yeast strains, followed by a review of methods for evaluating new yeast strains.


Introduction
In Scotland, the production of whisky is important for the r e venue of the country as well in attracting visitors .T here are 148 oper ational Scotc h Whisky distilleries with a contribution of £7.1 billion to the UK's economy in 2020.This results in Scotch Whisky being responsible for 77% of Scottish food and be v er a ge exports.Many of these distilleries have visitor centres, attracting over 2.2 million visitors per year (The Scotch Whisky Association 2023 ) supporting Scotland's economy and tourism.The size of a malt whisky distillery is variable, with Glenlivet and Glenfiddich having the largest production capacity of 21 000 000 LPA (litres of pure alcohol per annum) and Dornoch one of the smallest with 25 000 LPA (Gordon 2022 ).
It is not only the revenue , i.e .important for Scotland, but the country is also proud of this quality product and its long history as evidenced by its protection under the Scotch Whisky Regulation ( 2009 ).Ne v ertheless, ther e is a steady str eam of innov ation and r esearc h, with on av er a ge mor e than 12 000 ne w publications e v ery year.
Following the trend of investigating the influence of nonconventional or non-Saccharomyces yeast in wine (e .g. J olly et al. 2006J olly et al. , Roudil et al. 2019 ) ) and beer (e.g.Basso et al. 2016, Bellut and Ar endt 2019, Larr oque et al. 2021 ), r ecent r esearc h has also been initiated for Scotch Whisky (Daute 2021 ).T he fla vour of Scotch whisky emanates from several sources during the production fr om r aw materials (gr ains and water), mashing, fermentation, distillation (design and conditions), and maturation (time and cask).Ho w e v er, the c hoice of yeast str ain is one of the most im-portant factors affecting the organoleptic properties of new make spirit and young whiskies .T his is primarily due to the production of high le v els of volatile congeners including esters and higher alcohols.In more matured whiskies, the maturation conditions, including choice of oak cask and the duration of ageing, act to pr ovide desir able flavours and r educe undesir able off-flavours in the spirit (W anikawa 2020 ).W e propose that unconventional yeasts can be exploited as novel drivers for distilled spirit flavour differ entiation.This pa per r e vie ws the use of yeast in Scotc h Whisky fermentations, the effect of yeast on spirit flavour, and the potential of non-Sacc harom yces yeast for production in the future.While whisky is produced worldwide, this review focuses primarily on Scotch Malt Whisky.

An o vervie w of Scotc h Malt Whisky production
Scotch Malt Whisky production is strictly regulated by The Scotch Whisky Regulations ( 2009 ).It must be produced and matured in Scotland from only three ingredients: water, malted barley, and yeast, with plain caramel colouring allo w ed in some cases.When making any modifications to the production methods, it is vital to ensure that the resulting spirit has the typical aroma and taste of Scotch (The Scotch Whisky Regulations 2009 ).The production process is summarized in Fig. 1 .
Malt whisky production starts with the malting of barley to break do wn star ch and proteins into fermentable sugars and amino acids .T his occurs by letting the barley germinate and then drying (kilning) it to guarantee a stable product (Bathgate 2016 , Mosher and Trantham 2017 ).The final malt specifications are important for production efficiency , processability , spirit quality, flavour, and yeast performance (Bringhurst and Brosnan 2014, Bringhurst 2015, Mar čiulionyt ė et al. 2022 ).The malt is mashed with hot water to further break down starch via malt-derived enzymes.Use of extraneous amylolytic enzymes is not permitted (The Scotch Whisky Regulations 2009 ).
The resulting liquid (wort) is cooled (20-25 • C) and transferred into either wooden or stainless steel washbacks (fermenters), where yeast is added to start the fermentation with a common pitc hing r ate of 2-4 × 10 7 cells/ml (Watson 1981, Bringhurst and Brosnan 2014, Russell and Stewart 2014, Walker and Hill 2016 ).Commonly, the wort for Scotch Whisky has an original gravity of 1060-1080 • (Russell and Stewart 2014 ).In contrast to brewing, the wort is not boiled, allowing the further hydr ol ysis of starch and in a later stage the growth of other micr oor ganisms.During the fermentation, yeast converts malt-derived sugars (primarily maltose) into carbon dioxide, ethanol, and flavour compounds (congeners) that will tr anspir e into the final distilled product.The fermentation temper atur e rises natur all y to 33 • C thr ough the metabolic activity of the yeast (W atson 1981 , W alker and Hill 2016 ).After 30 h, the fermentation is lar gel y complete and this can be detected by monitoring a decrease in the specific gravity of the wash (fermented wort) to 975 • , resulting in a liquid with an alcohol b y v olume (ABV) of 8%-10% v/v and a drop in pH to 4.2.Most malt whisky distilleries extend the fermentation time to allow micr oor ganisms (mainl y lactic acid bacteria) to pr oduce mor e congeners (Russell andStewart 2014 , Walker andHill 2016 ).
Next, the ethanol and congeners are concentrated by a double distillation in traditional copper pot stills .T he first distillation stops when the resulting distillate's alcohol content is below 1% v/v ABV, leading to an ABV of 20%-25% v/v.This distillate fraction is r eferr ed to as 'low wines' (Nicol 2014, Piggott 2017 ).The second distillation is split into thr ee sections: for eshots/head, spirit cut/heart, and feints/tails based on the ABV and congener concentration.The feints and head cut will be recirculated and included in the next distillation.Only the spirit cut with an ABV of around 70% v/v is used for the maturation which must last for at least 3 years in oak casks (The Scotch Whisky Regulations 2009 ).Some distilleries use a triple still set-up to produce their whiskies or for special r eleases, whic h was more common in the past due to lo w er alcohol yields during fermentation (Glen 1969, Wanikawa 2020 ).Triple-distillation is commonly conducted for production of Irish whisk e ys, but an example of a distillery in Scotland where it is practised is Auchentoshan (Auchentoshan 2019 ).The previous cask use (Piggott et al. 1993, Mosedale 1995 ), as well as cask and stor a ge conditions (Cl yne et al. 1993, Spillman et al. 2004, Roullier-Gall et al. 2020 ) influence the final fla vour.T he fla vour pr ofile e volv es fr om pungent, oil y , sulphury , and sour to more mello w, vanilla, and sw eet notes which constitute the main flavour c har acteristics of Scotch Malt Whisky.

History of yeast use in Scotch Whisky
Reusing yeast in Scotch Whisky fermentation is not practised because the wort is not boiled or sterilized in any other way, which increases the risk of microbial contamination (Dolan 1976, Walker et al. 2011a, Russell and Stewart 2014, Walker and Hill 2016 ).Additionally, leaving the yeast in the wash during distillation contributes to the distinct flavour c har acteristics of the resultant spirit (Suomalainen and Lehtonen 1979 ).Today, Scotch Whisky distillers usually do not propagate their yeast, buying them instead from yeast supply companies (Walker et al. 2011b , Walker andHill 2016 ).With very few exceptions, most strains used in the distilling industry in Scotland are Saccharomyces cerevisiae .
Historicall y, spent br e wing yeast was used due to its affordability and convenience (Russell and Stewart 2014 ).Records suggest that as early as 1833, Scotch Whisky distillers produced separate yeast to increase the yield.In 1920, the Distillers Company Limited introduced the first commercially available pure standard yeast for Scotch Whisky (Frey 1930 ).This did not stop distilleries from sourcing their yeast from local breweries or producing it themselves until the 1950's.With the introduction of M strain or M-type (interspecies hybrid between S. cerevisiae and S. cerevisiae var .diastaticus ) by DCL Y east Ltd (now Kerry Biosciences) in 1952, this changed, and it became the standard distilling yeast (Watson 1981 ).At this time, yeast was used in combination with 30%-50% w/w recycled brewer's yeast.This resulted in increased alcohol yield, ov er all fermentation performance, and greater flavour complexity (Dolan 1976, Noguchi et al. 2008, Yomo et al. 2008, Walker et al. 2011a,b , Walker and Hill 2016 ).This situation changed again in the late 1990's/mid 2000 due to the closure of many of the lar ger br e weries in Scotland and subsequent r educed av ailability of br e w er's y east.As a r esult, most distilleries switc hed to r el ying mainly on using commercially available Scotch Whisky yeast (Walker et al. 2011a, Stewart et al. 2013, Walker and Hill 2016, Bathgate 2019 ).
While the M-type yeast has changed over the years, it is still declared as one of the standards in the Scotch Whisky industry together with MX (Kerry Bio-Science), Pinnacle (Mauri/AB Biotek), and DistillaMax (Lallemand Inc.).All of these strains belonging to the species of S. cerevisiae (Watson 1981, Walker et al. 2011a,b , Walker and Hill 2016 ).These contemporary distilling yeasts are well-adapted to fermenting cereal-based wort, being able to conv ert lar ger starc h-deriv ed sugars and dextrin mor e efficientl y into ethanol and additionally being better able to withstand different physical and chemical environmental stresses (Russell and Stewart 2014 ).Yeast from supply companies is provided in different formats for distilling such as dried, creamed, caked, or stabilized liquid.Each distillery selects the format based on their capability for tr ansport, stor a ge, and fermentation ca pacity (Watson 1981, Russell and Stewart 2014, Walker and Hill 2016 ).

Variety of yeast species and their application in alcoholic be v erages
All alcoholic be v er a ges, distilled or not, have one thing in common: yeast.The most commonly used yeast species S. cerevisiae has been used by humans for centuries (McGovern et al. 1996(McGovern et al. , 2004 ) ).The fermentation of food products was discov er ed accidentall y by gr a pes starting to spontaneousl y ferment due to natur all y occurring yeast.Micr oor ganisms , including yeasts , were discov er ed in 1680 by Antoine van Leeuwenhoek followed by further studies of fermentation in 1789 by Antoine Lavoisier (Mortimer 2000 , Chambers andPretorius 2010 ).
Yeasts belong to the kingdom of fungi and are present in the divisions of ascom ycetous, basidiom ycetous, and deuterom ycetous fungi.Often, only the subphylum of Saccharomycotina is considered as 'real' y east.Overall, y east are eukaryotic, unicellular organisms that got their name based on their ability to ferment with a meaning of 'foam' and 'to rise' (Kurtzman et al. 2011a ).For the industrial use of yeast, they are often separated into Saccharomyces spp., yeast that have been used for many years for brewing or baking and 'non-conventional' yeast or non-Saccharomyces yeast, which came into the focus of industry only relatively recently.These y easts w ere frequently branded as spoilage wild yeasts (Legan and Voysey 1991, Fleet 2011, Blomqvist and Passoth 2015, Shimotsu et al. 2015 ) and it was assumed that they were less effective in their fermentation performance than to S. cerevisiae .Table 1 summarizes the strengths and weaknesses of S. cerevisiae and non-Sacc harom yces yeasts in distilled spirits pr oduction.

Experimental data of Scotch Whisky fermentation
Exploration of novel distilling yeasts for the Scotch Whisky industry is not a new task, with early initiatives, such as by Chivas Brothers in 1981, involving the establishment of a yeast production plant to produce alternative and secondary yeast strains (Watson 1981 ).The analytical focus at that time extended to assessing the influence of different fermentation parameters, including temper atur e , suspended solids , alcohol tolerance , and bacterial contamination (Merritt 1966, 1967, Dolan 1976, Ramsay and Berry 1983, 1984, Okolo et al. 1990, Daute et al. 2021a ).The primary emphasis remained on the development of high ethanolyielding yeasts, with distillers r el ying on the distillation process to ensure the production of an acceptable spirit (Dolan 1976, Watson 1981, Berbert de Amorim Neto et al. 2009 ), or comparing different commer cial y east products , formats , and pitc hing r ates (Reid et al. 2023, Spasova et al. 2023, Waymark and Hill 2021 ).
Notably, limited attention has been given over the years to investigating the influence of yeasts on the flavour profile of Scotc h Whisky.Pr e vious r esearc h pr edominantl y explor ed distinctions among commercial S. cerevisiae yeasts (Ensor et al. 2015, Miles 2015, Ekins et al. 2018 ).Some non-distilling yeasts used in co-cultures with distilling strains demonstrated a reduction in yield but an increase in estery (fruity) flavours (Miles 2015 ).Co-fermentation with pure cultures of brewing yeast exhibited fla vour enhancement (Wanika wa et al. 2004, Noguchi et al. 2008, Yomo et al. 2008 ), while the use of bioethanol strains resulted in spirits with flavours comparable to whisky distilling yeast (Berbert de Amorim Neto et al . 2008, 2009, Daute 2021 ).
To date, very few commercial Scotch Whiskies have prominentl y featur ed the use of nonconv entional yeasts in their marketing.Sc hizosacc harom yces pombe : Glen Elgin 1998-18-year-old Special Release 2017 (Master of Malt 2021 ) and the Glenmorangie Allta, produced with a local wild yeast from Cadboll barley named Sacc haorm yces diaemath (Br oom 2019 ).Ne v ertheless, some cr aftdistillers investigate and isolate wild yeasts from the area around the distillery or their raw materials to cr eate ne w pr oducts with alternative fla vours , as observ ed at Lindor es Abbe y Distillery (Burk e et al. 2014, 2015, Walker and Hill 2016 ).
As Scotch Whisky fermentations are not sterile processes, micr oor ganisms other than the pitched distilling yeast strain influence the fermentation flavour of the new make spirit (Watson 1981 , Walker andHill 2016 ).A distilling yeast with a poor sugarto-alcohol conversion results in more residual sugars, giving other micr oor ganisms a higher chance to grow and potentially have a deleterious influence on product quality.These microorganisms enter the process through raw materials, the environment (air and dust), or production equipment: water used in different production steps can bring in low le v els of wild Bacillus spp., and Enterobacteria (Guild et al. 1985, Wilson 2014 ).Barley is a source of a wide variety of bacteria and wild yeast including Candida spp ., Cryptococcus spp ., Hansenula spp ., Rhodotorula spp ., and Sacc harom yces spp.(Flannigan 1999, Noots et al. 1999, Van Nierop et al. 2006, Justé et al. 2011 ).During malting the variety of bacteria decreases with a dominance of lactic acid bacteria.Ne v ertheless, a wide variety of wild yeast is still present, consisting among others, of Aureobasidium spp., Candida spp., Cryptococcus spp, Debaryom yces spp., Issatc henkia spp ., Kluyveromyces spp ., Pichia spp ., Table 1.Comparison of S. cerevisiae and non-Sacc harom yces yeasts for distilled spirits production.

Weaknesses Strengths Weaknesses
Ferment sugars 1 Only metabolize mono-, di-, and tri-hexoses (no starch or lactose) 8   Wide variety Mostl y Cr abtr ee-negativ e 13   High stress tolerance 1 Limited genetic variability Different sugar metabolism 9 Some yeasts are opportunistically pathogenic Wide temper atur e toler ance 1 Not regarded as thermophilic 2 Selected yeasts have high alcohol production 10   Only selected yeasts recognized as generally regarded as safe (GRAS) High alcohol tolerance ( ∼14%-15% v/v) 2   Room for impr ov ement with industrial strains Different metabolic pathways 11   Limited r esearc h High sugar tolerance 3  Weak osmotolerance in some strains 2 Diversification of congener production 11   Produce low/no alcohol 9 Cr abtr ee-positiv e/fermenting in the presence of high sugar levels and oxygen 4   Cr abtr ee effect needs.To be avoided for optimal yeast pr opa gation Pr ovide ne w congeners suc h as: 4-ethylguaiacol 12   Incomplete fermentation 9   Gener all y r egarded as safe (GRAS) 5  Well-r esearc hed 6  Widely used 7  Metabolic pathways known 7  Easy to culture Rhodotorula spp.(Flannigan 1999, O'Sullivan et al. 1999, Booysen et al. 2002, Laitila et al. 2006, 2011, Justé et al. 2011 ).During mashing, the ov er all wild yeast count is dr asticall y r educed.As for bacteria, the micr oflor a consists mostl y of lactic acid bacteria, acetic acid bacteria, and Gluconobacter spp.(Guild et al. 1985, O'Sullivan et al. 1999, Wilson 2014 ).In the subsequent production step, fermentation, the added yeast will be the dominant micr oor ganism.Onl y low le v els of other wild yeast will still be pr esent, lactic acid bacteria and r ar el y acetic acid bacteria, Zymomonas spp., and Pediococcus spp.Often the concentration of these increase with extended fermentation time (Makanjuola and Springham 1984, Priest and Barker 2010, Wilson 2014 ).

Yeast strain improvement
The primary objectives for distilling yeast strains encompass ac hie ving a high sugar-to-alcohol conversion (exceeding 90%), minimizing the production of off-fla vours , exhibiting high-stress tolerance, ensuring high viability, and demonstrating efficient rehydration efficiency (Pretorius et al. 2003, Walker et al. 2011a ).In addition to this, further de v elopment of new Scotch Whisky distilling strains is focused on the following desired attributes: -high tolerance to ethanol, heat, low pH, osmotic pr essur e, and high sugar concentration -r a pid fermentation of the wort sugars glucose , maltose , and maltotriose -production of appropriate congeners -high flavour consistency -high viability/vitality -a short lag phase -minimal yeast biomass r equir ement -competitiveness with other microorganisms -high endurance under various transport conditions -culture stability -non-flocculent -Gener all y Recognized as Safe (GRAS) or Qualified Presumption of Safety (QPS) status Adapted from Walker et al. ( 2011a ,b ), Russell and Stewart ( 2014 ), and Walker and Hill ( 2016 ).
Four a ppr oac hes ar e commonl y emplo y ed to attain these goals in new distilling strains: natural biodiversity, selection through methods such as mutagenesis (Liu et al. 2008(Liu et al. , 2018b ) ) and hybridization/breeding (Bellon et al. 2013, Gibson et al. 2017, Gallone et al. 2019, Stewart 2019 ), adaptive evolution (Saerens et al. 2010, Gallone et al. 2016, 2018, Barbosa et al. 2018, Gibson et al. 2020 ), and genetic modification (GM)/gene editing.The current stance of the Scottish Government and public opinion opposes the use of GM crops, leading to the exclusion of these or other GMOs (genetically modified organisms) in food production (Stewart et al. 2013 , Scottish Government 2020 , Science and Advice for Scottish Agricultur e 2021 ).Consequentl y, GM and asexual hybridization methods like protoplast fusion, often considered as GM (Husby 2007 ) ar e curr entl y not emplo y ed b y the Scotch Whisk y industry for yeast strain improvement.
A common a ppr oac h in industry is to either start with an alr eady commerciall y av ailable yeast str ain, scr een a str ain collection, or collect wild samples to exploit the natural biodiversity.For example, a wide variety of Saccharomyces spp.and non-Sacc harom yces yeasts can be isolated fr om differ ent habitats (Alsammar and Delneri 2020, Hutzler et al. 2021, Pinto et al. 2022, Piraine et al. 2022, Iturritxa et al. 2023 ), with se v er al S. cerevisiae isolations often associated with human habitats (Fay and Benavides 2005 ).Different selection techniques and media have been used for the isolation of specific yeasts .T he next step involves further modification and adaptation of the selected yeast strain.For this, a combination of br eeding, m uta genesis, and ada ptiv e e volution or a combination thereof can be used.Yeast breeding can integr ate tr aits fr om differ ent str ains and, potentiall y, closel y r elated species, and this r equir es further work to stabilize the traits in the final yeast str ain (Kr ogerus et al. 2017 ).Mutagenesis involves exposing the yeast to m uta genic materials or UV-rays to elevate the m utation r ate, and r esultant yeasts ar e scr eened for specific phenotypes.Yeasts exhibiting desired traits are selected for subsequent rounds until the yeast possesses improved characteristics, which can be again bred with a different strain.A similar principle is used for directed ev olution, the y east is placed in an envir onment that a pplies an e volutionary pr essur e, suc h as steady increase of sugar concentrations to guide the direction of mutation, enhancing the yeast's survival in an artificially adjusted envir onment, and ther eby impr oving physiological tr aits like sugar metabolism or flavour de v elopment (Dequin 2001, Liu et al. 2008 ).Recentl y, the Carlsber g Researc h Labor atory has intr oduced a ne w technique called FIND-IT to accelerate the identification of yeast and other organisms with desired mutations, allowing to screen for single nucleotide pol ymor phisms (SNP) (Knudsen et al. 2022 ).
Additional pr omising av enues for further r esearc h in whisky fermentations include exploring amylolytic yeasts for more efficient starc h br eakdown (Laluce et al. 1988, Pretorius et al. 2003, Cheng et al. 2011, Walker et al. 2011a ) or further elaborating fla vour profiles , e .g. using POF + (phenolic off-fla vour positive) yeasts to impart phenolic and spicy notes (Heresztyn 1986, Coghe et al. 2004 ).Further r esearc h into non-Sacc harom yces yeasts for industrial fermentations is expected.Recent findings comparing the flavour profile of wash, low wines, and new make spirit of differ ent yeast str ains sho w ed that the k e y flavour notes are stable thr oughout these pr oduction steps .T his finding will support the de v elopment of ne w yeast str ains by r educing the time needed for sample pr epar ation by eliminating the need for a double distillation for early yeast screening rounds (Daute et al. 2023 ).Together with the finding that congener profiling of wort by gas c hr omatogr a phy-mass spectr ometry (GC-MS) giv es compar able data to the sensory e v aluation, this could further reduce the time by not requiring a sensorial e v aluation of samples in early screening steps (Daute et al. 2021b ).

Non-con ventional y east used for distilled spirits
In the production of neutral spirits such as vodka, gin, or bioethanol, yeast selection is not a primary consideration because the final product undergoes extensive purification, and most yeast derived congeners are undesired in the final product.Consequently, efficiency becomes the primary factor, leading to the pr efer ence for highl y ada pted S. cerevisiae str ains with r obust str ess toler ance (P aule y and Mask ell 2017 , Black and Walk er 2023 , Spasova et al. 2023 ) instead of nonconventional flavourful yeast.
In contrast to Scotch Whisky production, the use of a variety of yeast strains is more commonplace in other distilled spirit industries .For example , Bourbon and Tennessee whisk e y distilleries often cultivate their own proprietary yeast strains (Smith 2017 ).Historically, after the increased availability of commercial y east, Scotch Whisk y producers hesitated to adopt this practice, deeming it economically impractical due to concerns about quality, cost, and sustainability (Walker and Hill 2016 ).
The transition to w ar ds deliberately inoculated fermentations with S. cerevisiae marked a departur e fr om the div ersity and complexity of flavours typically associated with spontaneous fermentations (Gschaedler 2017 ).While wild fermentation offers potentiall y mor e complex fla vours , it concurr entl y extends fermentation time, potentially resulting in a 40%-60% v/v decrease in alcohol yield, and higher le v els of r esidual sugars.Despite this, some distilleries prioritize flavour over yield (Fahrasmane and Ganou-P arfait 1998, Nuñez-Guerr er o et al. 2016, Portugal et al. 2017 ).Table 2 provides an ov ervie w of yeasts used in various distilled spirits production.
Pur e cultur es of non-Sacc harom yces yeasts exhibit distinct fla vour profiles , often c har acterized by higher le v els of esters or higher alcohols compared to S. cerevisiae .Ho w ever, their fermentation performance is often poorer by comparison (Dato et al. 2005, Oliv eir a et al. 2005, Arellano et al. 2008, López-Alv ar ez et al. 2012, Segura-García et al. 2015 ).T herefore , a combination of a non-Sacc harom yces str ain with a commer cial distilling y east often results in increased yield and enhanced ester notes (Duarte et al. 2012, Nuñez-Guerr er o et al. 2016 ).Optimizing non-Sacc harom yces yeast could enhance their fermentation performance, increase ABV, and introduce unique flavours (Dato et al. 2005, Oliveira et al. 2005, Arellano et al. 2008, López-Alvarez et al. 2012, Segura-García et al. 2015 ).Commer cial y east strains, belonging to S. cerevisiae , hav e under gone years of optimization, and new yeast strains with impr ov ed fermentation pr operties, suc h as MG + from AB Mauri, hav e r ecentl y been intr oduced to the market (Storr and Walker 2018 ).
Recentl y, Kv eik yeast, tr aditional Norwegian farmhouse yeast, has gained attention in br e wing due to its phenolic off-flavour negativity, high fermentation rate, tolerance to high temperatures ( > 28 • C), and classification within the S. cerevisiae clade (Preiss et al. 2018 ).This interest has extended to the distilling industry, wher e Kv eik yeast demonstr ates a fermentation pattern similar to commercial distilling yeast and a distinct flavour profile, offering the opportunity for de v elopment of ne w pr oducts (Dippel et al. 2022, Horstmann et al. 2023 ).

Non-con ventional y east used for winemaking and brewing
Non-conventional yeasts are increasingly used in the production of nonalcoholic or low-alcoholic be v er a ges, particularl y for wine and beer.Although these yeasts produce less ethanol, they contribute different and often increased levels of congeners, resulting in an altered flavour profile of these beverages (Bellut and Arendt 2019 ).
In wine and beer production, selecting starter cultures is a common practice to improve control over fermentation performance , fla vour, and the creation of specific products (Carrasco et al. 2001, Fernández-Espinar et al. 2001, Romano et al. 2003, Ribéreau-Gayon et al. 2006, Torrens et al. 2008, Chambers and Pretorius 2010, Schuller 2010, Garofalo et al. 2016, Capozzi et al. 2017, Berbegal et al. 2018, Vilela 2021 ).In the wine industry, S. cerevisiae str ains ar e the pr edominant commer cial y east starters, resulting in most r esearc h focused on S. cerevisiae (Cadière et al. 2012 , Tian T able 2. Y easts involved in the production of distilled spirits.Kluyveromyces marxianus Fruit spirit 9  A ureobasidium sp ., Kluyveromyces apiculate , Lachancea thermotoler ans , Torulaspor a delbrueckii Superscripted numbers in the table r epr esent following r efer ences: In br e wing, S. cerevisiae str ains dominate ale pr oduction, while S. pastorianus (a hybrid of S. cerevisiae and S. eubayanus ) is pr ominent in la ger pr oduction.Commerciall y offer ed str ains also include S. cerevisiae and S. uvarum (Stewart et al. 2013, Gibson et al. 2017 ).While commercial starter cultures provide consistent fermentations and flavour pr ofiles, nonconv entional yeasts offer the opportunity to diversify flavour in fermented be v er a ges (Roudil et al. 2019 , Molinet andCubillos 2020 ).The introduction of commercial non-Sacc harom yces yeasts in winemaking began in 2004 by Christian Hansen, resulting in the release of a pure Torulaspora delbrueckii strain in 2009 (Roudil et al. 2019 , P ey er 2020 ).Non-Sacc harom yces yeasts ar e often used in cocultur es or sequential fermentations together with Sacc harom yces yeasts to optimize sugar utilization, ethanol production, and wine flavour elaboration.Table 3 provides a list of nonconventional and non-Saccharomyces yeasts used in both spontaneous and controlled winemaking and br e wing.

Rum
In contrast to whisky production, where the emphasis is on maintaining or increasing alcohol content, the wine industry seeks to lo w er alcohol le v els due to c hanges in a gricultur e leading to gr a pes with excessive sugar levels .T his results in highalcohol wines with decreased flavour complexity, higher taxation, and evolving consumer preferences (Heymann et al. 2013, King et al. 2013, Saliba et al. 2013, Varela et al. 2015 ).As gr a pe juice primarily consists of fructose rather than maltose, the find-ings of these yeast strains cannot be directly applied to whisky production.
Ne v ertheless, r esearc h has demonstr ated that non-Sacc harom yces yeasts significantl y influence flavour pr oduction and fermentation performance, offering potential for innovation in various industries (Chatonnet et al. 1992, Romano et al. 2008, Lucy Joseph et al. 2013, Sc hifferdec ker et al. 2014, Agnolucci et al. 2017, Berbegal et al. 2018 ).Given the similarity in the early production steps of Scotch Malt Whisky and beer, the knowledge gained from brewing yeast research can be more easily transferred to Scotch Malt Whisky production due to the common fermentable carbohydrate sources (Stewart et al. 2013, Bringhurst 2015, Larroque et al. 2021 ).

Examples of new yeast species for Scotch Malt Whisky production
While there were 1414 accepted yeast species in 2011 (Kurtzman et al. 2011b ), new yeasts are regularly found or reclassified.Curr entl y, ov er 2000 yeast species and over 280 yeast genera have been identified and c har acterized (Boekhout et al . 2023 ).Unfortunatel y, not all of them can be discussed in this r e vie w .T able 4 provides a summary of 10 yeast species exhibiting potential as alternative Scotch Whisky distilling yeasts, as e v aluated thr ough an analysis of current literature and research (Daute 2021 ).Selection criteria include their ability to ferment glucose and maltose, prior use in the food industry, and a well-established r esearc h bac kground.While less-known yeasts may also hold promise, starting  2020), Lallemand Br e wing ( 2021 ), Omega Yeast ( 2022 ), The Yeast Bay ( 2023 ), White Labs ( 2021 ), and Wyeast ( 2021 ). 3 Gr anc hi et al. ( 1998 ), Pretorius ( 2000 ), Torija et al. ( 2001), Rementeria et al. ( 2003), Combina et al. ( 2005 ), Di Maro et al. ( 2007), Milanovi ć et al. ( 2013 ), Wang and Liu ( 2013 ), Liu et al. ( 2016 ), andBougreau et al. ( 2019 ). 4 Van Oe v elen et al. ( 1976 , 1977 ) with easily accessible and food-approved yeasts can simplify the initial stages of exploration.
Sacc harom yces pastorianus and Schiz.pombe have been reported to produce lo w er levels of congeners compared to S. cerevisiae (Pownall et al . 2022, Benito et al. 2016, Meier-Dörnberg et al. 2017, Loira et al. 2018, Callejo et al. 2019 ).This c har acteristic could be used for lighter Scotc h Whiskies, wher e most of the flavour originates in matur ation.Alternativ el y, they could be used in other cer eal gr ain-based distilled spirits wher e lo w er le v els of congeners ar e desir ed suc h as gin or vodka.In sugarcane molasses fermentations, ho w e v er, Sc hiz.pombe is known for its congener contributions to heavy flavoured dark rums.
While a wider range of non-Saccharomyces yeast is offered for br e wing and winemaking, not all of these are able to ferment maltose .T his includes P. kluyveri , C. zemlinina , K. wickerhamii , Metsc hnikowia pulc herrima , and M. fructicola .Since these yeasts cannot effectiv el y conv ert all wort sugars, they ar e often sourced for br e wing to pr oduce low-alcohol beers (Johansson et al. 2021 ).While unsuitable for use as a pur e cultur e in Scotch Whisky fermentations, they remain viable candidates for cofermentation, particularly in combination with S. cerevisiae for spirit flavour elaboration.
Se v er al other yeast species capable of fermenting maltose hav e under gone labor atory studies in br e wing and winemaking.Due to the scarcity of publications and nonfood safety a ppr ov al, these y easts w er e not included in this r e vie w.Ne v ertheless, it is important that other glucose and maltose fermenting yeasts such Candida spp., K. dobzhanskii , L. citri , L. fermentati , M. caribbica , Sc heffersom yces stipites , Sc hiz.japonicus , Sc hw anniom yces capriottii , Starmerella meliponinorum , T. franciscae , W. subpelliculosus , or Z. rouxii (Kurtzman et al. 2011b ) are further researched to make mor e yeast biodiv ersity av ailable for the Scotc h Whisky indus-Table 4. List of 10 non-conventional yeasts with the potential to be used for Scotch Whisky fermentations.

Maltose fermentation
Origin/use
3 Domizio et al. ( 2016), Morata et al. ( 2018), and Toh et al. ( 2020 ).In accordance with findings from prior research (Daute 2021 ), a str ategic a ppr oac h to e v aluating ne w yeast str ains for enhanced fla vour diversification in volv es se v er al steps, as depicted in Fig. 2 .First, it is crucial to assemble a diverse collection of yeast from v arying geogr a phical locations, yeast species, and yeast strains, establishing a br oad biodiv ersity.This a ppr oac h aligns with observations in S. cerevisiae , highlighting the wide diversity in the same yeast species (Sampaio et al. 2017 ).Next, the yeast strains should under go scr eening in small-scale fermentations, using platforms such as microtiter plates or anaerobic flasks, conducted under standardized conditions to allow comparison of fermentation re-sults.An essential aspect of this process is the analysis of fermentation samples using GC to measure ethanol le v els (indicator for fermentation performance) and congener production (indicator for flavour profile).Based on the analytical data, yeast strains with a desirable congener profile and ethanol production can be selected.Ensuring the safety of the chosen yeast strains is important before scaling further up, including assessing pr e vious information.From this selection, a limited number of yeast strains may be selected for further optimization if necessary.The optimization phase may involve modifications to the yeast through m uta genesis, br eeding, or ada ptiv e e v olution, follo w ed b y a rescreening of the strains.As the yeast strains progress, the evaluation should scale up, incor por ating double distillation and sensory assessments by a panel of experts in a medium-scale fermentation setting.This iter ativ e pr ocess r epeats, until the final scale-up to large-or commercial-scale fermentations.By adhering to this systematic a ppr oac h, r esearc hers can effectiv el y navigate the pr ocess of yeast strain selection and enhancement for the diversification of Scotch whisky fla vours .

Ev alua ting ne w yeast species and food safety qualification
Non-Sacc harom yces yeast seem to offer a wide variety of flavour potential for the distilled spirit industry .Unfortunately , some can also be harmful by producing biogenic amines (Visciano and Schirone 2022 ), or some can cause opportunistic infections such as Candida albicans (Caetano et al . 2023 ).To ensure that the Scotch whisky consumption is safe, all new yeast str ains, pur posel y added, m ust adher e to food safety r egulation.Ther e ar e two main different food safety approval systems: QPS from EFSA's scientific panel for the European Union and GRAS from US Food and Drug Administration (FDA).Decisions are made based on the taxonomic identification, present kno wledge, kno wn safety concerns, biogenic amines, antifungal resistance, virulence, pathogenicity, and safety concerns related to the use of the yeast such as acetaldehyde production (Miguel et al. 2022 ) .These assessments can take a long time and can be expensiv e. Ne v ertheless, this does not stop the br e wing and winemaking industry from persevering with the certification of new promising yeast species for new products (Roudil et al. 2019 ).Recent examples of ne wl y r egister ed yeast str ains ar e P. kluyveri fr om Christian Hansen (Food and Drug Administration 2020 ) or M. pulcherrima and M. fructicola from Lallemand (Food and Drug Administration 2021 ).With more and more yeast being assessed for their food safety, it can be hoped that we see further diversification in the future.
In addition to the food safety assessment, an implementation of new yeasts for Scotch Whisky also needs to adhere the Scotch Whisky Regulations ( 2009 ): Scotch whisky must 'have the aroma and taste of Scotch Whisky'.With non-Saccharomyces yeast bringing new flavours into the product, it is important to ensure that the product still tastes like Scotch whisky, which limits the possible diversification.

Conclusion
As for most distilled be v er a ges, the consider ations for Scotc h Malt Whisky pr oduction r e volv e ar ound ethanol yield and the ov er all efficiency of sugar conversion.Recent developments within the industry have witnessed distillers embracing a willingness to sacrifice ethanol yield for the creation of special-release whiskies characterized by unique and desirable fla vours .Although commercial S. cerevisiae yeast strains continue to dominate the Scotch Whisky landsca pe, ther e exists an opportunity to draw from the trends observed in winemaking and br e wing, wher e a div erse r ange of yeasts can be employed to enhance fla vour profiles .Yeasts such as other Sacc harom yces spp., D. bruxellensis , Kluyverom yces spp ., or Schiz.pombe showcasing the capability to ferment primary wort sugars, demonstrate significant potential.Ho w ever, using y easts with poorer fermentation performance compared with S. cerevisiae distiller's strains, can result both in reduced ethanol yields and an increase in unpleasant (e.g.sulphury) flavour notes.At the same time, other factors such as the stability of consistent fermentations, risks of unwanted contamination and ease of utilization, would need to be e v aluated.In addition, it is yet unknown how any c hanges in ne w make spirit flavour profiles would pair with different oak cask types and change during maturation, although this could be predicted based on the chemical composition of the new make spirit.Looking ahead, it is pr edicted ther e will be a rise in the utilization of non-conventional yeasts and cofermentation strategies aimed at further diversifying the flavour spectrum of whiskies in the coming years.Ne v ertheless, these yeasts m ust compl y with food safety regulations and the Scotch Whisky Regulations, in that the flavour profile adheres to the typical flavour of whisky.

Figure 2 .
Figure 2. Illustrating of a strategic approach to evaluating new yeast strains.