beverages

Review Saccharomyces species in the Production of Beer

Graham G. Stewart

The International Centre for Brewing and Distilling, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, Scotland, UK; [email protected]

Academic Editor: Edgar Chambers IV Received: 20 October 2016; Accepted: 25 November 2016; Published: 2 December 2016

Abstract: The characteristic flavour and aroma of any beer is, in large part, determined by the yeast strain employed and the wort composition. In addition, properties such as flocculation, wort fermentation ability (including the uptake of wort sugars, amino acids, and peptides), ethanol and osmotic pressure tolerance together with oxygen requirements have a critical impact on fermentation performance. Yeast management between fermentations is also a critical brewing parameter. Brewer’s yeasts are mostly part of the genus Saccharomyces. Ale yeasts belong to the species Saccharomyces cerevisiae and lager yeasts to the species Saccharomyces pastorianus. The latter is an interspecies hybrid between S. cerevisiae and Saccharomyces eubayanus. Brewer’s yeast strains are facultative anaerobes—they are able to grow in the presence or absence of oxygen and this ability supports their property as an important industrial microorganism. This article covers important aspects of Saccharomyces molecular biology, physiology, and metabolism that is involved in wort fermentation and beer production.

Keywords: amino acids; ethanol; fermentation; malt; peptides; Saccharomyces species; sugars; wort; yeast

1. Introduction The flavour and aroma of any beer is, in large part, determined by the yeast strain employed together with the wort composition. In addition, yeast properties such as flocculation, fermentation ability (including the uptake of wort sugars, amino acids, small peptides, and ammonium ions), osmotic pressure, ethanol tolerance, and oxygen requirements have a critical impact on fermentation performance. Proprietary strains, belonging to individual breweries, are usually jealously guarded and conserved. However, this is not always the situation. In Germany, for example, most of the beer is produced with only four individual yeast strains and approximately 65% of it is produced with a single strain sourced from the Weihenstephan Brewing School, which is located in the Munich Technical University. No satisfactory definition of yeast exists that encompasses commonly encountered properties such as alcoholic fermentation and growth by budding. The latter is not unusual in yeast and nearly all brewer’s yeast strains multiply by budding (Figure1). Brewer’s yeast cultures predominantly come from the genus Saccharomyces—a minority of non-Saccharomyces yeast cultures employed in brewing will be discussed in Section8. Yeast is cultured in an acidic aqueous sugary solution called wort prepared from barley malt and other cereals such as corn (maize), wheat, rice, sorghum, and also cane and beet sugar. The cells absorb dissolved sugars, simple nitrogenous matter (amino acids, ammonium ions, and small peptides), vitamins, and ions through their plasma membrane. Subsequently, they employ a series of reactions known as metabolic pathways (glycolysis, biosynthesis of cellular constituents, etc.) and use these nutrient materials for growth and fermentation. It is important to emphasise that the primary products of glycolysis are: ethanol, glycerol, and carbon dioxide (Figure2).

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Figure 1. An electron micrograph of a budding yeast cell containing bud and birth scars. Figure 1. An electron micrograph of a budding yeast cell containing bud and birth scars. Figure 1. An electron micrograph of a budding yeast cell containing bud and birth scars. D-Glucose Figure 1. An electron micrograph ofD-Glucose a budding yeast cellATP containing bud and birth scars. Glucose 6-phosphate ADPATP Glucose 6-phosphate ADP FructoseD-Glucose 6-phosphate ATP ATP Fructose 6-phosphate ADPATP FructoseGlucose 1, 6-diphosphate 6-phosphate ADP Fructose 1, 6-diphosphate ADP Glyceraldehyde 3-phosphate Fructose 6-phosphate ATP Glyceraldehyde 3-phosphate Dihydroxy-acetone phosphate ADP Fructose 1, 6-diphosphate + Dihydroxy-acetone phosphate NAD Glycerol + Glyceraldehyde 3-phosphate NADHNAD 2 Glycerol 1, 3-Diphosphoglycerate ADPNADH2 Dihydroxy-acetone1, 3-Diphosphoglycerate phosphate 3-Phosphoglycerate ADPATPNAD+ Glycerol 3-Phosphoglycerate ATPNADH2 1,2-Phosphoglycerate 3-Diphosphoglycerate H2O ADP Phosphoenolpyruvate2-Phosphoglycerate H2O 3-Phosphoglycerate ADPATP Phosphoenolpyruvate Pyruvate ADPATP 2-Phosphoglycerate H O CO Pyruvate ATP2 2 Acetaldehyde CO Phosphoenolpyruvate NADH2 2 Acetaldehyde NADADP+ Ethanol NADH2 Pyruvate NADATP+ Ethanol CO2 Acetaldehyde Figure 2. Formation of ethanol, glycerol, and CO2 from glucoseNADH by2 the Embden‐Meyerhof‐Parnas NAD+ Figure 2. Formation of ethanol, glycerol, andEthanol CO2 from glucose by the Embden‐Meyerhof‐Parnas Figure(EMP) 2. Pathway.Formation of ethanol, glycerol, and CO from glucose by the Embden-Meyerhof-Parnas (EMP) Pathway. 2 (EMP)Figure Pathway. 2. Formation of ethanol, glycerol, and CO2 from glucose by the Embden‐Meyerhof‐Parnas Saccharomyces cerevisiae (ale yeast) has the ability to take up a wide range of sugars, for example, (EMP) Pathway. glucose,Saccharomyces fructose, mannose,cerevisiae (ale galactose, yeast) has sucrose, the ability maltose, to take maltotriose, up a wide range and raffinoseof sugars, (infor example,part). In glucose,Saccharomyces fructose, cerevisiae mannose,(ale galactose, yeast) has sucrose, the ability maltose, to take maltotriose, up a wide and range raffinose of sugars, (in forpart). example, In addition,Saccharomyces as will be describedcerevisiae (ale later, yeast) a sub has‐species the ability of S. to cerevisiae take up, aSaccharomyces wide range of diastaticus sugars, for, is example, able to glucose,addition, fructose, as will mannose, be described galactose, later, a sucrose, sub‐species maltose, of S. maltotriose, cerevisiae, Saccharomyces and raffinose diastaticus (in part)., is In able addition, to utiliseglucose, dextrins fructose, (partially mannose, hydrolysed galactose, starch). sucrose, Also, maltose,Saccharomyces maltotriose, pastorianus and (lager raffinose yeast) (in strains part). are In as willutilise be describeddextrins (partially later, a sub-species hydrolysed ofstarch).S. cerevisiae Also, ,SaccharomycesSaccharomyces pastorianus diastaticus (lager, is able yeast) to utilise strains dextrins are ableaddition, to utilise as willthe disaccharidebe described melibioselater, a sub (glucose—galactose)‐species of S. cerevisiae in addition, Saccharomyces to the sugardiastaticus spectra, is abletaken to (partiallyable to hydrolysed utilise the disaccharide starch). Also, melibioseSaccharomyces (glucose—galactose) pastorianus (lager in addition yeast) to strains the sugar are able spectra to utilise taken the uputilise by S. dextrins cerevisiae. (partially This melibiose hydrolysed utilisation starch). property Also, Saccharomyces can be used pastorianusin diagnostic (lager tests yeast) to distinguish strains are up by S. cerevisiae. This melibiose utilisation property can be used in diagnostic tests to distinguish disaccharidebetweenable to utiliseale melibiose and the lager disaccharide (glucose—galactose) yeast strains. melibiose The (glucose—galactose)enzymatic in addition hydrolysis to the sugarin ofaddition starch, spectra toas the takenwould sugar up occur spectra by S. during cerevisiae. taken between ale and lager yeast strains. The enzymatic hydrolysis of starch, as would occur during Thismashingup melibiose by S. (Figurecerevisiae. utilisation 3), This leads property melibiose to a fermentable can utilisation be used medium inproperty diagnostic (wort)can be tests thatused to consists in distinguish diagnostic of a between numbertests to distinguishof ale simple and lager mashing (Figure 3), leads to a fermentable medium (wort) that consists of a number of simple yeastsugars—glucose,between strains. ale The and enzymatic fructose, lager yeast hydrolysissucrose, strains. maltose, of The starch, enzymatic and as maltotriose would hydrolysis occur together during of starch, with mashing unfermentable as would (Figure occur3 dextrins.), leadsduring to a sugars—glucose, fructose, sucrose, maltose, and maltotriose together with unfermentable dextrins. fermentableThemashing predominant medium(Figure sugars 3), (wort) leads in thatmost to a consists brewer’sfermentable of worts a numbermedium are: glucose, of(wort) simple maltose, that sugars—glucose, consists and maltotriose of a number fructose, (Table of 1)simple sucrose, [1]. The predominant sugars in most brewer’s worts are: glucose, maltose, and maltotriose (Table 1) [1]. maltose,sugars—glucose, and maltotriose fructose, together sucrose, with maltose, unfermentable and maltotriose dextrins. together The with predominant unfermentable sugars dextrins. in most The predominant sugars in mostMalt brewer’sA djunctworts are: glucose,Hot maltose, Wort Tank and maltotriose (Table 1) [1]. brewer’s worts are: glucose, maltose,Malt and(rice maltotriose,A corndjunct, wheat) (Table1Hot)[1 Wort]. Tank Mill (rice, corn, wheat) Plate Cooler Mill Cereal Cooker Malt Adjunct PlateHot Wort Cooler Tank Cereal(rice, corn Cooker, wheat) Mash Mixer Yeast + 02 Mill Mash Mixer YeastPlate +Cooler 0 Cereal Cooker 2 Syrup Hops Fermenter Lauter Tun or Syrup Hops Fermenter LauterMashMash FilterTun Mixer or Yeast + 02 Mash Filter Kettle Centrifuge Spent Grain Syrup Hops CentrifugeFermenter Lauter Tun or Kettle Spent Grain Aging Mash Filter Aging Packaging Bright BeerKettle Centrifuge (bottle, can, keg) Carbonation Filtration PackagingSpent Grain BrightTank Beer (bottle, can, keg) Carbonation FiltrationAging Tank PackagingFigure 3.Bright The typicalBeer brewing process. (bottle, can, keg) Carbonation Filtration Figure 3. TheTank typical brewing process.

FigureFigure 3. 3.The The typical typical brewing brewing process.

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Table 1. Typical sugar spectrum of wort. Table 1. Typical sugar spectrum of wort.

PercentPercent Composition Composition GlucoseGlucose 10–15 10–15 FructoseFructose 1–2 1–2 SucroseSucrose 1–2 1–2 MaltoseMaltose 50–60 50–60 MaltotrioseMaltotriose 15–20 15–20 DextrinsDextrins 20–30 20–30

2. The The Malting Malting and and Brewing Processes and Wort Composition The beer production process is outlined inin FigureFigure3 3.. Malting, Malting, mashing, mashing, and and fermentation/aging fermentation/aging are essentially enzymaticenzymatic processes. processes. The The major major brewing brewing raw raw material material is malt is malt usually usually (not always)(not always) from frombarley barley and it and contains it contains extract componentsextract components (starch, proteins,(starch, proteins, etc.) and etc.) enzymes and (amylases,enzymes (amylases, proteases, proteases,glucanases, glucanases, etc.). However, etc.). malt However, is an expensive malt is rawan expensive material and raw unmalted material cereals and unmalted (adjuncts) cereals [2] are (adjuncts)extensively [2] used are inextensively most brewing used countries.in most brewing countries. The exception to to this use of adjuncts is Germany, where the Purity Law (also called the Reinheitsgebot), was was adopted in Bavaria in 1516. As As Germany Germany unified, unified, Bavaria campaigned for adoption ofof this this law law nationally. nationally. The The only only ingredients ingredients that could that becould used be in Germanyused in Germany for the production for the productionof beer were of water, beer barley,were water, and hops. barley, The and principal hops. purpose The principal of the lawpurpose was to of prevent the law price was competition to prevent pricewith bakerscompetition for wheat with and bakers rye and for towheat ensure and the rye availability and to ensure of bread the [ 3availability]. When yeast of wasbread described [3]. When by yeastAntonie was van described Leewenhoek by Antonie in 1680 van and Leewenhoek its role in fermentation in 1680 and was its role described, in fermentation it was added was described, to modern itversions was added of the to Reinheitsgebot. modern versions It is of worth the Reinheitsgebot. noting that Norway It is worth and Greece noting also that commonly Norway subscribeand Greece to alsothis lawcommonly [2]. subscribe to this law [2]. The objectives of of malting are are to develop a spectrum of enzymes in cereals such as barley and wheat. These enzymes enzymes are able to to hydrolyse hydrolyse the cereal cereal constituents constituents in order order to produce produce a a fermentable fermentable extract called wort (Figure 44).). AsAs alreadyalready discussed,discussed, wortwort isis aa mediummedium thatthat willwill supportsupport yeastyeast growthgrowth and fermentation with with beer as the end product. It It is is important important that that beer beer is is drinkable (beer is not usually supped, it it is drunk!) and and it it exhibits exhibits a a number number of of stability stability properties, properties, i.e., i.e., flavour, flavour, physical, physical, foam,foam, and and biological biological characteristics characteristics [4]. [4 ].Malt Malt contributes contributes a large a large number number of materials of materials to wort to wort [5]. The [5]. principalThe principal wort wort components components (not (not the theonly only ones) ones) are are free free amino amino nitrogen nitrogen (FAN), (FAN), fermentable fermentable sugars, sugars, and unfermentable dextrins.

Barley

Steeping Grain hydration

Germination Enzyme generation

Kilning Drying and curing

Malt

FigureFigure 4. 4. TypicalTypical barley malting process.

Compared to other media used in the production of fermentation alcohol (both industrial and Compared to other media used in the production of fermentation alcohol (both industrial and potable), wort is by far the most intricate. Therefore, when yeast is pitched (inoculated) into wort it potable), wort is by far the most intricate. Therefore, when yeast is pitched (inoculated) into wort it is is introduced into a complex environment because it consists of simple sugars, dextrins, amino acids, introduced into a complex environment because it consists of simple sugars, dextrins, amino acids, peptides, proteins, vitamins, ions, nucleic acids, and other constituents too numerous to mention. peptides, proteins, vitamins, ions, nucleic acids, and other constituents too numerous to mention. One of the major advances in brewing science during the past 40 years has been the elucidation of One of the major advances in brewing science during the past 40 years has been the elucidation of the the mechanisms by which yeast cells utilise in an orderly manner, the plethora of wort nutrients [1,6]. mechanisms by which yeast cells utilise in an orderly manner, the plethora of wort nutrients [1,6]. FAN is the sum of the individual wort amino acids, ammonium ions, and small peptides (di‐ and tripeptides). FAN is an important general measure of yeast nutrients which constitute the yeast assimilable nitrogen during brewery fermentations [6]. Wort also contains the sugars sucrose,

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FAN is the sum of the individual wort amino acids, ammonium ions, and small peptides (di- and tripeptides). FAN is an important general measure of yeast nutrients which constitute the yeast assimilable nitrogen during brewery fermentations [6]. Wort also contains the sugars sucrose, fructose, glucose, maltose, and maltotriose, together with dextrin material. A typical percentage sugar spectrum of brewer’s wort is shown in Table1.

3. Wort Fermentation The objectives of wort fermentation are to consistently metabolise wort constituents into ethanol, carbon dioxide, and other fermentation products in order to produce beer with satisfactory quality and stability. Another objective is to produce yeast crops that can be confidently re-pitched into subsequent brews [7]. It is noteworthy that brewing is the only major fermentation process that recycles its yeast culture from one fermentation to another. Fermentation processes such as most potable and industrial distilled alcohol production, viticulture, saké brewing, and cider production only use their yeast culture once. As will be discussed later, the management of brewer’s yeast culture between fermentations is a critical part of the brewing process. It is important to jealously protect the quality of cropped yeast because it will be used to pitch a subsequent wort fermentation and will, therefore, have a profound effect on the quality of the beer resulting from it (see Section7). Over the years, considerable effort has been devoted to studies on the biochemistry and genetic/molecular biology of brewer’s yeast (together with industrial strains in general). The objectives of these studies have been two-fold:

• To learn more about the biochemical and genetic makeup of brewing yeast strains and • To improve the overall performance of such strains, with particular emphasis on broader substrate utilisation capabilities, increased ethanol production, and improved tolerance to environmental stress conditions such as temperature, elevated osmotic pressure, and ethanol, and finally to understand the mechanism(s) of flocculation.

There are several differences in the production of ale and lager beers, one of the main ones being the characteristics of the ale and lager yeast strains employed and the fermentation temperatures. Considerable research by many breweries and institutions on this topic has been conducted [8–10] and the typical differences between the ale and lager yeast strains have been established (Table2). With the advent of molecular biology-based methodologies, gene sequencing of ale and lager brewing strains has shown that S. pastorianus is an interspecies hybrid between S. cerevisiae and S. eubayanus, with homologous relationships to one another and also to Saccharomyces bayanus, a yeast species used in wine fermentation and identified as a wild yeast in brewing fermentations.

Table 2. Differences between ale and lager yeast strains.

Ale Yeast Saccharomyces cerevisiae (ale type). Typical fermentation temperature—18–25 ◦C. Maximum growth temperature—37 ◦C or higher. Does not ferment melibiose. “Top” fermenter. Lager yeast Saccharomyces pastorianus. Typical fermentation temperature—8–15 ◦C. Maximum growth temperature—34 ◦C. Ferments melibiose. “Bottom” fermenter. Beverages 2016, 2, 34 5 of 18

Gallone et al. [8] have proposed that current industrial yeasts (including brewing and distilling) can be divided into five sublinkages that are genetically and phenotypically separated from wild strains and originate from only a few ancestors through complex patterns of domestic and local divergence. Large-scale phenotyping and genome analysis further show strong industry-specific selection for stress tolerance, sugar utilization, and flavor production—details to follow. BeveragesIn 2011,2016, 2 a, 34 paper entitled “Microbe domestication and the identification of the wild genetic5 stock of 18 of lager-brewing yeast” was published [11]. This study confirmed that S. pastorianus is a domesticated yeast species created by the fusion of S. cerevisiae with a previously unknown species that has now been designateddesignatedSaccharomyces Saccharomyces eubayanus eubayanusbecause because of its of close its relationshipclose relationship to S. bayanus to S.. Theybayanus proposed. They S.proposed eubayanus S. existseubayanus exclusively exists exclusively in the forests in of the Patagonia! forests of Since Patagonia! this publication, Since thisS. publication, eubayanus has S. alsoeubayanus been isolatedhas also and been identified isolated inand Tibet identified [12] and in in Tibet the USA[12] and [13]. in The the initial USA paper[13]. The from initial Argentina paper containsfrom Argentina a draft contains genome sequencea draft genome of S. eubayanus sequence; of it isS. 99.5%eubayanus identical; it is 99.5% to the identical non-S. cerevisiae to the nonDNA‐S. fragmentcerevisiae DNA of the fragmentS. pastorianus of the genomeS. pastorianus sequence. genome This sequence. suggests This specific suggests changes specific in wortchanges sugar in wort and sulphatesugar and metabolism, sulphate metabolism, compared to compared ale strains, to that ale arestrains, critical that for are determining critical for lager determining beer characteristics. lager beer Thecharacteristics. complete DNA The complete sequence DNA of S. eubayanus sequence hasof S. recently eubayanus been has published recently been [14]. published [14]

4. Uptake and Metabolism of Wort SugarsSugars It is very important that wort sugars and the FAN complex are efficientlyefficiently takentaken upup by a brewer’s yeast culture. In In normal normal situations, situations, brewing brewing yeast yeast strains strains (both (both ale ale and and lager lager strains) strains) are are capable capable of ofutilising utilising sucrose, sucrose, glucose, glucose, fructose, fructose, maltose, maltose, and maltotriose and maltotriose in this inapproximate this approximate sequence sequence (or priority) (or priority)(Figure 5), (Figure although5), although some degree some degreeof overlap of overlap does occur. does occur.The majority The majority of brewing of brewing yeast yeaststrains strains leave leavethe maltotetraose the maltotetraose (G4) (G4)and andother other dextrins dextrins unfermented. unfermented. However, However, S. S.diastaticus diastaticus isis able able to to utilise dextrin material, material, albeit albeit inefficiently. inefficiently. Wort Wort dextrin dextrin utilisation utilisation is possible is possible by this by this yeast yeast sub‐ sub-speciesspecies due dueto the to extracellular the extracellular secretion secretion of glucoamylase. of glucoamylase. However, However, this this enzyme enzyme produced produced by byS. diastaticus,S. diastaticus, is isincapable incapable of hydrolysing of hydrolysing the α‐ the1,6α -1,6bonds bonds of dextrins of dextrins whereas whereas it is able it is to able hydrolyse to hydrolyse the α‐1,4 the bondsα-1,4 bonds[15,16]—details [15,16]—details to follow. to follow.

Figure 5. Uptake order of wort sugars by brewer’s yeast cultures.

The initial step in the utilisation of any wort sugar is either its passage intact across the yeast The initial step in the utilisation of any wort sugar is either its passage intact across the yeast plasma membrane, or its hydrolysis outside the cell membrane, followed by entry into the cells of plasma membrane, or its hydrolysis outside the cell membrane, followed by entry into the cells of some or all of the hydrolysis products (Figure 6). Maltose and maltotriose (Figure 7) are examples of some or all of the hydrolysis products (Figure6). Maltose and maltotriose (Figure7) are examples sugars that pass intact across the cell membrane, whereas sucrose and dextrins are hydrolysed by of sugars that pass intact across the cell membrane, whereas sucrose and dextrins are hydrolysed by extracellular enzymes (invertase for sucrose and glucoamylase (amyloglucosidase) for dextrins) and extracellular enzymes (invertase for sucrose and glucoamylase (amyloglucosidase) for dextrins) and the resultant simple sugars taken into the cell. An important metabolic difference between the uptake the resultant simple sugars taken into the cell. An important metabolic difference between the uptake of monosaccharides such as glucose and fructose and disaccharides such as maltose and maltotriose of monosaccharides such as glucose and fructose and disaccharides such as maltose and maltotriose uptake is that energy (ATP conversion to ADP) is required for maltose and maltotriose uptake (active uptake is that energy (ATP conversion to ADP) is required for maltose and maltotriose uptake (active transport) whereas glucose and fructose are taken up passively with no energy requirement [17]. As transport) whereas glucose and fructose are taken up passively with no energy requirement [17]. maltose and maltotriose (Table 1) are the major sugars in brewer’s wort, the ability of a brewing yeast As maltose and maltotriose (Table1) are the major sugars in brewer’s wort, the ability of a brewing strain to use these two sugars depends on the correct genetic complement. Brewer’s yeast possesses yeast strain to use these two sugars depends on the correct genetic complement. Brewer’s yeast independent uptake mechanisms (maltose and maltotriose permeases) to transport these two sugars across the cell membrane into the cell. Once inside the cell, both sugars are hydrolysed to glucose units by the α‐glucosidase system (Figure 6). The transport, hydrolysis, and maltose fermentation systems are particularly important in brewing, distilling, and baking since maltose is the major sugar component of brewing wort, spirit mash, and wheat dough. Maltose fermentation by brewing, distilling, and baking yeasts requires at least one of five unlinked MAL loci, each consisting of three genes encoding: • the structural gene for α‐glucosidase (maltase) (MALS) • maltose permease (MALT) and

Beverages 2016, 2, 34 6 of 18 possesses independent uptake mechanisms (maltose and maltotriose permeases) to transport these two sugars across the cell membrane into the cell. Once inside the cell, both sugars are hydrolysed to glucose units by the α-glucosidase system (Figure6). The transport, hydrolysis, and maltose fermentation systems are particularly important in brewing, distilling, and baking since maltose is the major sugar component of brewing wort, spirit mash, and wheat dough. Maltose fermentation by brewing, distilling, and baking yeasts requires at least one of five unlinked MAL loci, each consisting of three genes encoding:

• the structural gene for α-glucosidase (maltase) (MALS)

• Beveragesmaltose 2016 permease, 2, 34 (MALT) and 6 of 18 • an activator whose product co-ordinately regulates the expression of the α-glucosidase and •permease an activator genes. whose product co‐ordinately regulates the expression of the α‐glucosidase and permease genes.

Maltose Maltotriose Active transport (G-G) (G-G-G)

Maltose α‐glucosidase Maltotriose enzyme

GLUCOSE (G)

Yeast cell wall Metabolic Pathways

Glucose (G) Fructose (F) Invertase in the periplasmic space Yeast cell membrane between the cell wall and membrane hydrolyses sucrose to glucose and fructose which then enter the cell by facilitated diffusion

Glucose (G) Facilitated Fructose (F) Sucrose (G-F) Diffusion Figure 6. Uptake of wort sugars by a yeast cell. Beverages 2016, 2, 34 Figure 6. Uptake of wort sugars by a yeast cell. 7 of 18

Figure 8. MaltotrioseFigure (A 7.) andStructure maltose of (maltoseB) uptake and profiles maltotriose. from a 16°Plato wort. Figure 7. Structure of maltose and maltotriose.

InThe order expression to investigate of MALS the and MAL MALT‐gene is regulatedcassettes further,by maltose a strain induction containing and repression two MAL2 by and glucose. two MAL4WhenThe expression glucosegene copies concentrations of MALSwas constructedand areMALT high (>10usingis regulated mg/L), hybridisation the by MAL maltose genes techniques inductionare repressed, [21]. and The and repression onlywort when(16°Plato) by 40%– glucose. Whenfermentation50% glucose of the glucose concentrations rate was has compared been taken are to high up a strain from (>10 thethat mg/L), wort only will thecontained maltoseMAL genesone and copy maltotriose are of repressed, MAL2 uptake. As expected, and commence only the when 40%–50%overall(Figure fermentation of5). the glucose rate has with been the strain taken containing up from multiple the wort MAL will genes maltose was considerably and maltotriose faster than uptake commencethe strainSeveral (Figure containing ale 5and). lager the yeastsingle strains MAL2 have copy been (Figure used to9). explore The principal maltose andreason maltotriose for this uptakefaster fermentationmechanisms inrate wort. was For due example, to an aincreased 16°Plato allrate‐malt of wortmaltose was uptake fermented and in subsequent a 30 liter static metabolism vessel at compared15 °C (Figure to the 8). yeastUnder strain these containing conditions, a singlelager strainsMAL2 copyutilised (Figure maltotriose 9). more efficiently than ale strains, whereas maltose utilisation efficiency was not dependent on the type of brewing strain [18,19]. This supports the proposal that20 maltotriose and maltose possess independent, but closely linked, MAL2/MAL2; uptake (permease) systems [18,19].16 In addition, this consistentMAL4/MAL4 difference between ale and lager strains supports the observation that ale strains appear to haveMAL2/mal2; greater difficulty than lager strains to completely ferment wort, particularly12 high gravity wort (>16°Plato)mal4/mal4 [20]. Plato

Plato

8 Degree 4

Degree 0 0 24487296 Fermentation time time (hours) (hours) *Shaking fermentationsNote: Shaking at fermentations 21°C at 21 °C Figure 9. Fermentation profile of a 16°Plato wort and a diploid yeast strain containing multiple maltose (MAL) genes.

5. Carbohydrate Metabolism It has already been described that S. diastaticus is a subspecies of S. cerevisiae. It differs from the parental species by producing the extracellular enzyme glucoamylase (α‐1,4‐glucan glucohydrolase) [22]. This enzyme removes successive glucose units from the non‐reducing ends of dextrins (partially hydrolysed starch). Using hybridisation techniques, three non‐allelic genes have been identified and characterised from strains of S. diastaticus [23]. Two DEX genes (DEX1 and DEX2) have been identified that codes for the production of S. diastaticus glucoamylase, with glucose being the sole hydrolysis product. A third dextrinase gene (DEX3) was found to be allelic to STA3, a gene reported to control starch fermentation in Saccharomyces sp. [24,25]. Using hybridisation techniques [21], a number of diploid strains have been developed, each being either heterozygous or homozygous for the individual DEX genes. Wort fermentations with the DEX containing cultures showed that in addition to the wort sugars (glucose, fructose, sucrose, maltose, and maltotriose), the dextrins (G4–G15) were partially fermented. There was residual

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Several ale and lager yeast strains have been used to explore maltose and maltotriose uptake mechanisms in wort. For example, a 16◦Plato all-malt wort was fermented in a 30 liter static vessel at 15 ◦C (Figure8). Under these conditions, lager strains utilised maltotriose more efficiently than ale strains, whereas maltose utilisation efficiency was not dependent on the type of brewing strain [18,19]. This supports the proposal that maltotriose and maltose possess independent, but closely linked, uptake (permease) systems [18,19]. In addition, this consistent difference between ale and lager strains supports the observation that ale strains appear to have greater difficulty than lager strains to ◦ completelyBeverages ferment 2016, 2, 34 wort, particularly high gravity wort (>16 Plato) [20]. 7 of 18

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Figure 8. Maltotriose (A) and maltose (B) uptake profiles from a 16°Plato wort. Figure 8. Maltotriose (A) and maltose (B) uptake profiles from a 16◦Plato wort.

In order to investigate the MAL‐gene cassettes further, a strain containing two MAL2 and two Figure 8. Maltotriose (A) and maltose (B) uptake profiles from a 16°Plato wort. InMAL4 order gene to copies investigate was constructed the MAL-gene using cassettes hybridisation further, techniques a strain [21]. containing The wort two (16°Plato)MAL2 and ◦ two MAL4fermentationIngene order copiesrate to investigatewas was compared constructed the MAL to a ‐straingene using cassettes that hybridisationonly further, contained a strain one techniques containing copy of MAL2 [ 21two]. .MAL2 TheAs expected, wortand two (16 thePlato) fermentationoverallMAL4 fermentation gene rate wascopies compared rate was with constructed the to strain a strain usingcontaining that hybridisation only multiple contained MALtechniques onegenes copy [21].was ofconsiderablyTheMAL2 wort. As(16°Plato) faster expected, than the overallthefermentation strain containing rate rate was with thecompared thesingle strain to MAL2 a strain containing copy that only(Figure multiple contained 9). MALThe one copyprincipalgenes of wasMAL2 reason considerably. As expected,for this fasterthefaster than fermentationoverall fermentation rate was rate due with to the an strain increased containing rate multipleof maltose MAL uptake genes wasand considerably subsequent faster metabolism than the strain containing the single MAL2 copy (Figure9). The principal reason for this faster fermentation comparedthe strain to thecontaining yeast strain the singlecontaining MAL2 a singlecopy MAL2(Figure copy 9). (FigureThe principal 9). reason for this faster rate wasfermentation due to an increasedrate was due rate to of an maltose increased uptake rate of and maltose subsequent uptake metabolism and subsequent compared metabolism to the yeast strain containingcompared to a the single yeastMAL2 strain containing20copy (Figure a single9). MAL2 copy (Figure 9). MAL2/MAL2; 1620 MAL4/MAL4 MAL2/MAL2;MAL2/mal2; 1216 MAL4/MAL4mal4/mal4 Plato MAL2/mal2; Plato

mal4/mal4 128 Plato

Plato Degree

48 Degree Degree 4 0 Degree 0 24487296 0 Fermentation time time (hours) (hours) 0 24487296 Note: Shaking fermentations at 21 °C *Shaking fermentationsFermentation at 21 time°C time (hours) (hours) Figure 9. Fermentation* Shakingprofile of fermentations a Note:16°Plato Shaking atwort fermentations 21°C and a at diploid 21 °C yeast strain containing multiple maltoseFigure ( MAL9. Fermentation) genes. profile of a 16°Plato wort and a diploid yeast strain containing multiple Figure 9. Fermentation profile of a 16◦Plato wort and a diploid yeast strain containing multiple maltose maltose (MAL) genes. (5.MAL Carbohydrate) genes. Metabolism 5. Carbohydrate Metabolism It has already been described that S. diastaticus is a subspecies of S. cerevisiae. It differs from the 5. Carbohydrate Metabolism parentalIt hasspecies already by producing been described the extracellular that S. diastaticus enzyme is a subspeciesglucoamylase of S. ( cerevisiaeα‐1,4‐glucan. It differs glucohydrolase) from the It[22].parental has This already enzyme species been byremoves producing described successive the extracellular that glucoseS. diastaticus units enzyme from glucoamylase theis anon subspecies‐reducing (α‐1,4‐ endsglucan of S.of glucohydrolase) dextrins cerevisiae (partially. It differs [22]. This enzyme removes successive glucose units from the non‐reducing ends of dextrins (partially fromhydrolysed the parental starch). species Using byhybridisation producing techniques, the extracellular three non‐allelic enzyme genes glucoamylase have been identified (α-1,4-glucan and characterisedhydrolysed starch).from strains Using hybridisationof S. diastaticus techniques, [23]. Two three DEX non‐ allelicgenes genes (DEX1 have and been DEX2 identified) have and been glucohydrolase)characterised [22 from]. This strains enzyme of S. removesdiastaticus successive [23]. Two DEX glucose genes units (DEX1 from and the DEX2 non-reducing) have been ends of identified that codes for the production of S. diastaticus glucoamylase, with glucose being the sole dextrinsidentified (partially that hydrolysed codes for the starch). production Using of S. hybridisation diastaticus glucoamylase, techniques, with three glucose non-allelic being the genessole have hydrolysis product. A third dextrinase gene (DEX3) was found to be allelic to STA3, a gene reported hydrolysis product. A third dextrinase gene (DEX3) was found to be allelic to STA3, a gene reported to control starch fermentation in Saccharomyces sp. [24,25]. to control starch fermentation in Saccharomyces sp. [24,25]. Using hybridisation techniques [21], a number of diploid strains have been developed, each Using hybridisation techniques [21], a number of diploid strains have been developed, each beingbeing either either heterozygous heterozygous or or homozygoushomozygous for thethe individualindividual DEX DEX genes. genes. Wort Wort fermentations fermentations with with thethe DEX DEX containing containing cultures cultures showed showed thatthat in addition to to the the wort wort sugars sugars (glucose, (glucose, fructose, fructose, sucrose, sucrose, maltose,maltose, and and maltotriose), maltotriose), thethe dextrinsdextrins (G4–G15) werewere partiallypartially fermented. fermented. There There was was residual residual

Beverages 2016, 2, 34 8 of 18 been identified and characterised from strains of S. diastaticus [23]. Two DEX genes (DEX1 and DEX2) have been identified that codes for the production of S. diastaticus glucoamylase, with glucose being the sole hydrolysis product. A third dextrinase gene (DEX3) was found to be allelic to STA3, a gene reported to control starch fermentation in Saccharomyces sp. [24,25]. Using hybridisation techniques [21], a number of diploid strains have been developed, each being either heterozygous or homozygous for the individual DEX genes. Wort fermentations with the DEX containing cultures showed that in addition to the wort sugars (glucose, fructose, sucrose, maltose, and maltotriose), the dextrins (G4–G15) were partially fermented. There was residual dextrin in the fermentedBeverages worts 2016, 2, because 34 the glucoamylase from S. diastaticus strains was unable hydrolyse8 the of 18α (1-6) branch points, only α(1-4) linkages. dextrin in the fermented worts because the glucoamylase from S. diastaticus strains was unable Beer produced with DEX-containing yeast strains has been studied. Glucose concentration in the hydrolyse the α(1‐6) branch points, only α(1‐4) linkages. pasteurizedBeer beer produced increased with duringDEX‐containing storage yeast for three strains months has been at studied. room temperature—this Glucose concentration indicates in that thisthe pasteurized glucoamylase beer increased is not heat during sensitive storage [for24]. three Also, months overall at room beer temperature—this flavour produced indicates by these DEX-containingthat this glucoamylase yeast strains is not was heat unacceptablesensitive [24]. Also, because overall it containedbeer flavour 4-vinylguaiacol produced by these (clove-like), DEX‐ estery,containing sulphury, yeast and strains musty was aromas unacceptable [25]. Nevertheless, because it contained the brewing 4‐vinylguaiacol industry (clove has‐ continuedlike), estery, to be interestedsulphury, in the and glucoamylases musty aromas [25]. produced Nevertheless, by S. diastaticus the brewingstrains. industry Consequently, has continued to recombinant be interested DNA (rDNA),in the plasmids, glucoamylases and transformationproduced by S. diastaticus techniques strains. have Consequently, been employed recombinant to clone DNADEX (rDNA),genes into brewingplasmids, strains and [26 ].transformation In order to enhance techniques glucoamylase have been activity,employed the to glucoamylase clone DEX genes gene into from brewingAspergillus strains [26]. In order to enhance glucoamylase activity, the glucoamylase gene from Aspergillus niger niger was also cloned into a brewing yeast along with the STA2 gene. The A. niger glucoamylase has was also cloned into a brewing yeast along with the STA2 gene. The A. niger glucoamylase has the the advantage of possessing debranching activity, unlike the yeast enzyme, so that it can hydrolyse advantage of possessing debranching activity, unlike the yeast enzyme, so that it can hydrolyse more moreof of the the wort wort dextrins dextrins [26]. [26 This]. This enzyme enzyme is also is sensitive also sensitive to pasteurisation to pasteurisation conditions. conditions. TheThe cloned cloned amylolytic amylolytic strains strains have have receivedreceived approval by by the the U.K. U.K. Agriculture Agriculture and andHealth Health Ministries,Ministries, who who concluded concluded in in 1995 1995 that there there were were no no food food safety safety reasons reasons why this why yeast this should yeast not should not bebe usedused [[27].27]. The The reduced reduced dextrin dextrin beer beer produced produced with this with cloned this clonedyeast was yeast called was Nutfield called Lyte Nutfield Lyte (Figure 10) 10 and) and was was the the first first beer beer in the in world the worldto be produced to be produced from this fromgenetically this geneticallymodified yeast modified to yeastreceive to receive approval. approval. After After considerable considerable deliberation, deliberation, it was it wasdecided decided that thatspecific specific labelling labelling was was unnecessary.unnecessary. However, However, a back a back label label to Nutfield to Nutfield Lyte Lyte bottles bottles explained explained the the process process by by which which itit hadhad been been produced and the advantages of doing so. Reaction to this beer from the general public has been produced and the advantages of doing so. Reaction to this beer from the general public has been neutral neutral or positive. Media reports were largely factual and supportive. There was some criticism from or positive.the environmental Media reports lobby. were This largely criticism factual has not and diminished supportive. over Therethe years was and some the criticismproduction from of the environmentalmodified foodstuffs lobby. This (including criticism alcoholic has not diminished beverages) overin Europe the years has andbeen, the and production still is, severely of modified foodstuffsrestricted (including [28]. alcoholic beverages) in Europe has been, and still is, severely restricted [28].

Figure 10. Nutfield Lyte. Figure 10. Nutfield Lyte. 6. Uptake and Metabolism of Free Amino Nitrogen (FAN) 6. Uptake and Metabolism of Free Amino Nitrogen (FAN) FAN is a general measure of a yeast culture’s assimilable nitrogen efficiency. It is a good index FANfor yeast is a generalgrowth and measure consequently of a yeast fermentation culture’s assimilable efficiency nitrogenand sugar efficiency. uptake [29]. It is Wort a good FAN index is for yeastessential growth for and the consequently formation of fermentationnew yeast amino efficiency acids, the and synthesis sugar of uptake new structural [29]. Wort and FAN enzymatic is essential for theproteins, formation cell proliferation, of new yeast and amino cell viability acids, the and synthesis vitality. Lastly, of new FAN structural levels have and a enzymaticdirect influence proteins, on beer flavour compounds (for example, higher alcohols, carbonyls, and esters). There are differences between lager and ale yeast strains with respect to wort assimilable nitrogen uptake characteristics [30]. Nevertheless, with all brewing strains the amount of wort FAN content required by yeast under normal brewery fermentation conditions is directly proportional to yeast growth and also affects certain aspects of beer maturation. There has been considerable problems regarding the minimal wort FAN required to achieve satisfactory yeast growth and fermentation performance in conventional gravity (10–12°Plato) wort, considered to be 130 mg

Beverages 2016, 2, 34 9 of 18 cell proliferation, and cell viability and vitality. Lastly, FAN levels have a direct influence on beer flavour compounds (for example, higher alcohols, carbonyls, and esters). There are differences between lager and ale yeast strains with respect to wort assimilable nitrogen uptake characteristics [30]. Nevertheless, with all brewing strains the amount of wort FAN content required by yeast under normal brewery fermentation conditions is directly proportional to yeast growth and also affects certain aspects of beer maturation. There has been considerable problems regarding the minimal wort FAN required to achieve satisfactory yeast growth and fermentation performance in conventional gravity (10–12◦Plato) wort, considered to be 130 mg FAN/L. For rapid attenuation of high gravity wort (>16◦Plato), increased levels of FAN are required [31]. However, optimum wort FAN levels differ from fermentation to fermentation and from yeast strain to yeast Beverages 2016, 2, 34 9 of 18 strain. Furthermore, the optimum FAN values are dependent on different wort sugar levels and typeFAN/L. [6]. For rapid attenuation of high gravity wort (>16°Plato), increased levels of FAN are required Studies[31]. However, on the optimum uptake of wort wort FAN nitrogen levels bydiffer brewer’s from fermentation yeast strains to began fermentation over 50 and years from ago. yeast During the 1960s,strain to Margaret yeast strain. Jones Furthermore, and John the Pierce, optimum working FAN values in the are Research dependent Department on different of wort the sugar Guinness Brewerylevels in and Park type Royal, [6]. London, conducted notable studies on nitrogen metabolism during brewing, mashing,Studies and fermentation on the uptake [of32 wort]. They nitrogen reported by brewer’s that yeast the absorption strains began and over utilisation 50 years ago. of During exogenous nitrogenousthe 1960s, wort Margaret compounds Jones and and John their Pierce, synthesis working intracellularly in the Research are controlled Department by of three the Guinness main factors: Brewery in Park Royal, London, conducted notable studies on nitrogen metabolism during brewing, • totalmashing, wort and concentration fermentation of [32]. assimilable They reported nitrogen that the absorption and utilisation of exogenous nitrogenous wort compounds and their synthesis intracellularly are controlled by three main factors: • the concentrations of individual nitrogenous compounds and their ratio and • the• competitivetotal wort concentration inhibition ofof theassimilable uptake nitrogen of these components (mainly, but not exclusively, amino acids)• the via concentrations various permease of individual systems nitrogenous [33]. compounds and their ratio and • the competitive inhibition of the uptake of these components (mainly, but not exclusively, amino A uniqueacids) classificationvia various permease of amino systems acids [33]. according to their rates of consumption during brewing wort fermentationA unique classification has been established. of amino acids There according are four to uptaketheir rates groups of consumption of amino acids.during Three brewing groups of wortwort amino fermentation acids are has taken been upestablished. at different There stages are four of fermentationuptake groups and of amino the fourth acids. Three group groups consists of onlyof one wort amino amino acid, acids prolineare taken (the up at largest different concentration stages of fermentation amino acidand the in fourth wort), group which consists is not of taken up duringonly one brewing amino acid, fermentations proline (the largest (Figure concentration 11). This isamino because acid in of wort), the anaerobic which is not conditions taken up that prevailduring late inbrewing the fermentation fermentations (oxygen (Figure is11). necessary This is because for proline of the uptake). anaerobic When conditions this classification that prevail was late in the fermentation (oxygen is necessary for proline uptake). When this classification was developed, the methodology employed liquid chromatography for measuring individual amino acids developed, the methodology employed liquid chromatography for measuring individual amino and wasacids iconic. and was Similar iconic. measurements Similar measurements today employ today automated employ automated computerized computerized high-performance high‐ liquidperformance chromatography liquid (HPLC),chromatography and it is (HPLC), difficult and to envisageit is difficult and appreciateto envisage the and challenges appreciate that the were facedchallenges and overcome that were 50 years faced ago!and overcome Indeed, advances 50 years ago! in analytical Indeed, advances methods in haveanalytical been methods a primary have reason for ourbeen increasing a primary knowledge reason for our of increasing wort fermentation knowledge control. of wort fermentation control.

3.5 4.5

4.0 3.0 3.5 2.5 3.0 2.0 2.5

1.5 2.0

1.5 1.0 1.0 0.5 0.5 Proline C, Group (mmol/L) Group A, A, Group Group B (mmol/L)

0.0 0.0 0192443 48 67 72 96 Fermentation Time (hours) Group A Group B Group C Proline

FigureFigure 11. 11.Amino Amino acid acid absorption absorption pattern pattern during during wort wort fermentation. fermentation.

The Jones and Pierce amino acid classification [32,33] continues to be the basis of our current Theunderstanding Jones and of Pierce the relative amino importance acid classification of individual [32 wort,33] continuesamino acids to during be the fermentation. basis of our This current understandinginformation of has the assisted relative manipulation importance of of wort individual nitrogen wortlevels aminoby the addition acids during of supplements fermentation. such This as yeast extract or specific amino acids, particularly during high gravity brewing. However, this assimilation pattern is often specific to the conditions employed. The nutritional preferences of a particular yeast strain are paramount. The absorption and utilisation of wort amino acids has recently been re‐examined [34] and the overall Jones and Pierce classification was confirmed using contemporary analytical techniques. However, the order of methionine uptake has been transferred from Group B to Group A (Table 3).

Beverages 2016, 2, 34 10 of 18 information has assisted manipulation of wort nitrogen levels by the addition of supplements such as yeast extract or specific amino acids, particularly during high gravity brewing. However, this assimilation pattern is often specific to the conditions employed. The nutritional preferences of a particular yeast strain are paramount. The absorption and utilisation of wort amino acids has recently been re-examined [34] and the overall Jones and Pierce classification was confirmed using contemporary analytical techniques. However, the order of methionine uptake has been transferred from Group B to Group A (Table3).

Table 3. Order of wort amino acids and ammonia uptake during fermentation.

Group A Group B Group C Group D Fast Absorption Intermediate Absorption Slow Absorption Little or No Absorption Glutamic acid Valine Glycine Proline Aspartic acid Leucine Phenylalanine Asparagine Isoleucine Tyrosine Glutamine Histidine Tryptophan Serine Alanine Methionine Ammonia Threonine Lysine Arginine

It has also been reported [35] that approximately 30% of incorporated nitrogen compounds come from sources other than amino acids. Although the utilisation of small peptides by brewing yeasts was confirmed over 50 years ago, an understanding of their role in yeast nitrogen requirements is still limited. Small peptides can be used as nutritional sources of amino acids as both carbon or nitrogen sources and precursors of cell wall peptides during yeast growth, although, growth is much slower when they are the sole nitrogen source [36]. Polypeptides may also used as a substrate because yeasts can generate proteolytic enzymes extracellularly to provide additional assimilable nitrogen to the cells—see below and reference [37]. Most brewing strains transport peptides with no more than three amino acid residues but this limit is strain dependent [35]. Nevertheless, small wort peptides are an important source of assimilable nitrogen and 20%–40% of wort oligopeptides are used during fermentation. Similar to single amino acids, peptides probably contribute to beer character and flavour. Wort peptides are taken up in a specific order dependent on their amino acid composition. The determination of small (two and three amino acids) peptides has been an impediment to this area of research. However, a method to measure these small oligopeptides has now been developed [35]. In brief, the sample is deproteinised, the supernatant ultrafiltered through a membrane with a 500 Da exclusion limit, the filtrate is acid and alkaline hydrolysed, and the hydrolysate is subjected to amino acid analysis by HPLC. It has already been briefly discussed that yeast secretes proteinase into fermenting wort (mainly proteinases). Therefore, the hydrolysis of medium-sized peptides into smaller peptides occurs, which can be taken up by yeast cultures, and this activity continues throughout fermentation [35]. This fact highlights a difference between the uptake of sugars and FAN during brewing. The spectrum of wort sugars at the beginning of fermentation is fixed because after wort boiling, all malt amylases have been inactivated. Whereas, because of proteinase secretion by yeast, the spectrum of nitrogen compounds is dynamic. This is not the case during malt and grain whisky fermentations where the wort is not boiled. Consequently, the wort still contains amylase activity (along with proteinase activity) and microbial contaminants. Consequently, the spectrum of wort carbohydrate is flexible [38]. Additionally, stress effects on yeast can increase proteinase secretion and this occurs to a greater extent during the fermentation of high gravity worts [37,38]. Regarding wort fermentation by brewing yeast, the metabolism of wort sugars and FAN during fermentation has already been discussed but a plethora of other metabolic reactions also occur. As there Beverages 2016, 2, 34 11 of 18 are many reactions, they will not be discussed in detail here, but are covered in greater detail in reference [39]. As a consequence, only three reactions will be considered:

• yeast management, including the initial stages of fermentation, where the intracellular storage carbohydrate glycogen (Figure 12) is used in the presence of oxygen in order to synthesize unsaturated fatty acids (UFAs) and sterols which are incorporated into yeast membrane structures [40]. • the formation of diacetyl during the exponential stages of fermentation (Figure 13A) followed by • theBeverages later stages2016, 2, 34 of fermentation overlapping into maturation (aging/lagering). This11 involves of 18 the Beverages 2016, 2, 34 11 of 18 management (reduction) of diacetyl and other vicinal diketones (VDK—see Figures 13B and 16). • the later stages of fermentation overlapping into maturation (aging/lagering). This involves the Such• compoundsmanagementthe later stages impart(reduction) of fermentation a butterscotch of diacetyl overlapping and flavour other into vicinal in maturation beer. diketones and (aging/lagering). (VDK—see Figures This 13Binvolves and 16). the • the harvestingSuchmanagement compounds (cropping) (reduction) impart of a theof butterscotch diacetyl yeast cultureand flavour other for vicinal in reuse beer. diketones and either by (VDK—see flocculation Figures or 13B with and a 16). centrifuge, as• discussed theSuch harvesting compounds later. (cropping) impart aof butterscotch the yeast culture flavour for inreuse beer. either and by flocculation or with a centrifuge, • asthe discussed harvesting later. (cropping) of the yeast culture for reuse either by flocculation or with a centrifuge, as discussed later.

FigureFigure 12. 12.Structure Structure of of glycogen. glycogen. Figure 12. Structure of glycogen. Carbohydrate Ethanol Carbohydrate Acetic Acid Ethanol Pyruvate Acetic Acid Pyruvate Ethanol Acetic Acid Enzymatic Ethanol Acetic Acid ConversionEnzymatic α-Acetohydroxy- Conversion α-Acetolactate α-Acetohydroxy-butyrate Passive α-Acetolactate butyrate DiffusionPassive Plasma Diffusion MembranePlasma Valine Iso- Non-Enzymatic leucine Membrane leucineValine Iso- DecompositionNon-Enzymatic leucine leucine Decomposition α-Acetohydroxy- α-Acetolactate α-Acetohydroxy-butyrate α-Acetolactate butyrate Diacetyl Pentanedione DiacetylA Pentanedione Diacetyl A Diacetyl Plasma Membrane Diacetyl Plasma Diacetyl Membrane Enzymatic Conversion Acetoin Enzymatic Conversion Acetoin Plasma Diffusion Plasma Butanediol Diffusion Butanediol

Acetoin Acetoin Butanediol ButanediolB B Figure 13. Diacetyl metabolism of a fermenting wort. (A) Formation of diacetyl and 2,3‐pentandione Figure 13.duringFigureDiacetyl logarithmic13. Diacetyl metabolism andmetabolism early ofstationary aof fermenting a fermenting growth wort.phases;wort. ( (A A(B) )Formation) Conversion Formation of ofdiacetyl of diacetyl diacetyl and to 2,3acetoin and‐pentandione 2,3-pentandione and 2,3‐ during logarithmic and early stationary growth phases; (B) Conversion of diacetyl to acetoin and 2,3‐ duringbutanediol logarithmic late andin the early fermentation. stationary growth phases; (B) Conversion of diacetyl to acetoin and butanediol late in the fermentation. 2,3-butanediol late in the fermentation. 7. Yeast Cropping—Flocculation and/or Centrifugation 7. Yeast Cropping—Flocculation and/or Centrifugation Flocculation is one of the major factors when considering important characteristics of yeast strainsFlocculation during brewing is one fermentations of the major [7]. factors Most brewingwhen considering yeast strains important are classified characteristics as either flocculent of yeast orstrains non‐ flocculentduring brewing (powdery) fermentations (Figure 14) [7]. [41–43]. Most brewing One definition yeast strains of flocculation are classified that as has either been flocculent used for manyor non years‐flocculent is: “the (powdery) phenomenon (Figure wherein 14) [41–43]. yeast cellsOne adheredefinition in clumps of flocculation and either that sediment has been from used thefor many years is: “the phenomenon wherein yeast cells adhere in clumps and either sediment from the

Beverages 2016, 2, 34 12 of 18

7. Yeast Cropping—Flocculation and/or Centrifugation Flocculation is one of the major factors when considering important characteristics of yeast strains during brewing fermentations [7]. Most brewing yeast strains are classified as either flocculent or non-flocculent (powdery) (Figure 14)[41–43]. One definition of flocculation that has been used for Beverages 2016, 2, 34 12 of 18 Beveragesmany years 2016, 2 is:, 34 “the phenomenon wherein yeast cells adhere in clumps and either sediment12 from of 18 mediumthe medium in which in whichthey are they suspended are suspended or rise to or the rise medium’s to the medium’s surface” [43]. surface” This [definition43]. This excludes definition medium in which they are suspended or rise to the medium’s surface” [43]. This definition excludes formsexcludes of “clumpy forms of growth” “clumpy and growth” “chain and formation” “chain formation” [44] which [44 will] which not willbe discussed not be discussed further further here forms of “clumpy growth” and “chain formation” [44] which will not be discussed further here (Figurehere (Figure 15). 15). (Figure 15).

Non‐flocculent Flocculent Non‐flocculent Flocculent Figure 14. Flocculation characteristics of Saccharomyces cerevisiae strains. Figure 14. FlocculationFlocculation characteristics of Saccharomyces cerevisiae strains.

Figure 15. Chain‐forming yeast strain. Figure 15. Chain‐forming yeast strain. Figure 15. Chain-forming yeast strain. It is important to crop a yeast culture at the end of fermentation either using a culture’s It is important to crop a yeast culture at the end of fermentation either using a culture’s flocculation characteristics (Figure 16) or with a centrifuge (Figure 17). Flocculation is a cell surface flocculationIt is important characteristics to crop a (Figure yeast culture 16) or at with the enda centrifuge of fermentation (Figure either 17). Flocculation using a culture’s is a flocculationcell surface phenomenon and the cell wall structure of a culture is critical (Figure 18). Components such as phenomenoncharacteristics and (Figure the 16cell) or wall with structure a centrifuge of a (Figure culture 17 is). critical Flocculation (Figure is a18). cell Components surface phenomenon such as mannoprotein are found distributed throughout the entire yeast wall and therefore the wall’s layered mannoproteinand the cell wall are structurefound distributed of a culture throughout is critical the (Figure entire yeast18). Componentswall and therefore such the as mannoprotein wall’s layered arrangement exhibits zones of enrichment. It is a matrix, not a sandwich! Nevertheless, the arrangementare found distributed exhibits throughoutzones of enrichment. the entire yeast It is wall a andmatrix, therefore not a the sandwich! wall’s layered Nevertheless, arrangement the mannoprotein is an integral structure that determines a yeast culture’s flocculation characteristics [7]. mannoproteinexhibits zones is of an enrichment. integral structure It is a matrix, that determines not a sandwich! a yeast culture’s Nevertheless, flocculation the mannoprotein characteristics is [7]. an integral structure that determines a yeast culture’s flocculation characteristics [7].

Figure 16. Flocculation characteristics of a brewer’s yeast strain during static fermentation and its Figure 16. Flocculation characteristics of a brewer’s yeast strain during static fermentation and its influence on wort diacetyl levels. influence on wort diacetyl levels.

Beverages 2016, 2, 34 12 of 18

medium in which they are suspended or rise to the medium’s surface” [43]. This definition excludes forms of “clumpy growth” and “chain formation” [44] which will not be discussed further here (Figure 15).

Non‐flocculent Flocculent

Figure 14. Flocculation characteristics of Saccharomyces cerevisiae strains.

Figure 15. Chain‐forming yeast strain.

It is important to crop a yeast culture at the end of fermentation either using a culture’s flocculation characteristics (Figure 16) or with a centrifuge (Figure 17). Flocculation is a cell surface phenomenon and the cell wall structure of a culture is critical (Figure 18). Components such as mannoprotein are found distributed throughout the entire yeast wall and therefore the wall’s layered

Beveragesarrangement2016, 2 , 34exhibits zones of enrichment. It is a matrix, not a sandwich! Nevertheless,13 ofthe 18 mannoprotein is an integral structure that determines a yeast culture’s flocculation characteristics [7].

Figure 16. Flocculation characteristics of a brewer’s yeast strain during static fermentation and its influence on wort diacetyl levels. Beveragesinfluence 2016, 2, on 34 wort diacetyl levels. 13 of 18 Beverages 2016, 2, 34 13 of 18

FigureFigure 17.17. A disc stackstack centrifugecentrifuge (5(5 hL/h).hL/h). Figure 17. A disc stack centrifuge (5 hL/h).

Fibrillar Layer Fibrillar Layer

Mannoprotein Mannoprotein

β Glucan β Glucan

β Glucan-Chitin β Glucan-Chitin Mannoprotein Mannoprotein Plasma Membrane Plasma Membrane

Components such as mannoprotein occur as a matrix and are Componentsdistributed throughout such as mannoprotein the entire wall occur and thereforeas a matrix the and are distributedlayered arrangement throughout shouldthe entire show wall zones and therefore of enrichment. the layered arrangement should show zones of enrichment. Figure 18. Schematic diagram of the yeast cell wall architecture. FigureFigure 18. 18. SchematicSchematic diagram diagram of of the the yeast yeast cell cell wall wall architecture. architecture. Flocculation usually occurs in the absence of cell division (not always) during late logarithmic and Flocculationearly stationary usually growth occurs phases in the and absence only under of cell rather division circumscribed (not always) environmental during late logarithmic conditions andinvolving early stationaryspecific yeastgrowth cell phases surface and onlystructures under (mainlyrather circumscribed protein and environmental carbohydrate conditions(mannan) involvingcomponents) specific and alsoyeast an cellinteraction surface of structures calcium ions (mainly [45]. Onlyprotein a microamount and carbohydrate of calcium (mannan) ions is components)necessary for andthe flocculationalso an interaction of brewer’s of calcium yeast strains. ions [45]. Cells Only can a be microamount deflocculated of as calcium a result ions of the is necessaryaddition forof thesugars flocculation such as of mannosebrewer’s yeastand strains.also treatment Cells can bewith deflocculated chelating asagents a result such of theas addition of sugars such as mannose and also treatment with chelating agents such as ethylenediamine tetraacetic acid (EDTA) that complex with Ca++ ions. Cells will usually reflocculate ethylenediamine tetraacetic acid (EDTA) that complex with Ca++ ions. Cells will usually reflocculate rapidly upon the addition of Ca++ ions [45]. ++ rapidlyAlthough upon the yeast addition separation of Ca often ions occurs[45]. by sedimentation, it may also be by flotation because of Although yeast separation often occurs by sedimentation, it may also be by flotation because of cell aggregation entrapping bubbles of CO2 as is the case of hydrophobic top‐cropping ale brewing cellyeast aggregation strains. Co entrapping‐flocculation bubbles [46] (also of CO called2 as ismutual the case agglutination of hydrophobic [47], top mutual‐cropping aggregation, ale brewing and yeastmutual strains. flocculation) Co‐flocculation is an aggregation[46] (also called process mutual between agglutination two yeast [47], strains mutual (yeast aggregation,‐bacteria andco‐ mutualflocculation flocculation) has also beenis an reported aggregation [48,49]). process When twobetween ale strains two yeast(one being strains non (yeast‐flocculent‐bacteria and cothe‐ flocculation has also been reported [48,49]). When two ale strains (one being non‐flocculent and the other weakly flocculent) are mixed together in the presence of Ca++, flocs form and the culture rapidly ++ othersettles weakly out of flocculent)suspension are or mixedrises to together the surface in the during presence static of wortCa , flocsfermentation. form and Cothe‐ flocculationculture rapidly has settlesnot been out reported of suspension in lager or yeast rises strains to the surface[50]. during static wort fermentation. Co‐flocculation has not been reported in lager yeast strains [50].

Beverages 2016, 2, 34 14 of 18

Flocculation usually occurs in the absence of cell division (not always) during late logarithmic and early stationary growth phases and only under rather circumscribed environmental conditions involving specific yeast cell surface structures (mainly protein and carbohydrate (mannan) components) and also an interaction of calcium ions [45]. Only a microamount of calcium ions is necessary for the flocculation of brewer’s yeast strains. Cells can be deflocculated as a result of the addition of sugars such as mannose and also treatment with chelating agents such as ethylenediamine tetraacetic acid (EDTA) that complex with Ca++ ions. Cells will usually reflocculate rapidly upon the addition of Ca++ ions [45]. Although yeast separation often occurs by sedimentation, it may also be by flotation because of cell aggregation entrapping bubbles of CO2 as is the case of hydrophobic top-cropping ale brewing yeast strains. Co-flocculation [46] (also called mutual agglutination [47], mutual aggregation, and mutual flocculation) is an aggregation process between two yeast strains (yeast-bacteria co-flocculation has also been reported [48,49]). When two ale strains (one being non-flocculent and the other weakly flocculent) are mixed together in the presence of Ca++, flocs form and the culture rapidly settles out of suspension or rises to the surface during static wort fermentation. Co-flocculation has not been reported in lager yeast strains [50]. Genetic studies on yeast flocculation began over 60 years ago. Thorne [51] and Gilliland [52] independently confirmed that this phenomenon was an inherited characteristic with the flocculation phenotype being dominant over non-flocculence. The first flocculation gene (FLO gene) to be studied in detail was FLO1. Using traditional gene mapping techniques (mating, sporulation, micromanipulation, tetrad analysis, etc.) [21], it was shown that FLO1 is located on chromosome I, 33cM from the centromere on the right-hand side of the chromosome [53]. Novel genetic techniques have been developed, the principal of which is the sequencing of the Saccharomyces genome [54]. A sequenced laboratory yeast strain contains five FLO genes, four located near the chromosome telomeres FLO1, FLO5, FLO9, and FLO10 and one neither at the centromere nor at the telomeres, FLO11 [55]. At least nine genes—FLO1, FLO5, FLO9, FLO10, FLO11, FLONL, FLONS, and LgFLO in S. cerevisiae and S. pastorianus—encode flocculin proteins. The flocculin encoded by FLO11 differs from other FLO genes in that it is involved in filamentous growth, adhesion to solid surfaces, and floc formation rather flocculation per se [56]. Another FLO gene, FLO8, encodes for a transposable factor regulating the expression of other FLO genes [57]. It has already been stated that many yeast strains usually possess several FLO genes in their genome [56]. FLO genes are very long (up to 4 Kbp) because of the number of tandem repeated DNA sequences of about 100 nucleotides that are repeated 10–20 times in each gene [56]. Tandem repeated DNA sequences of the FLO genes are highly dynamic components of the genome—they change more rapidly than other parts of the genome. They enable rearrangements both between and within flocculin genes. The longer the FLO protein, the stronger the strain’s flocculation ability [56]. FLO1 has been identified as the longest FLO gene, with the most repeats and confers the strongest flocculation phenotype to a yeast strain [57]. The sedimentation performance of a brewer’s yeast strain often changes during repeated re-pitching in a brewery [39,58]. This could be due to irreversible or reversible genetic change. Alternatively, it could be due to long lasting physiological changes—perhaps modifications in yeast handling and fermentation environments (for example, the use of high gravity worts). When a genetic change, conferring a non-flocculent phenotype, occurs in a yeast culture, it will gradually become a mixture of flocculent and non-flocculent cells [39]. Often within a lager yeast population, exhibiting moderate flocculent characteristics, a more flocculent variant culture can be isolated. An example of this development was when a Canadian brewing company began brewing its beer, under contract, in breweries in the United Kingdom. Most of the contracted breweries employed vertical fermenters. However, at that time, the Canadian breweries only possessed horizontal tanks (as both fermentation and maturation vessels). This difference in tank geometry influenced the yeast culture’s sedimentation characters. In vertical fermenters, this yeast culture was too non-flocculent Beverages 2016, 2, 34 15 of 18

(powdery), with too much yeast remaining in suspension at the end of fermentation (centrifuges were not available at the time). It was thought possible that the culture contained a spectrum of isolates that exhibited various flocculation intensities. Consequently, one of the variants from this strain, with more intensive flocculation characteristics, was successfully isolated and employed in the vertical fermenters. The result was less yeast in suspension at the end of fermentation. However, care had to be taken to ensure that the flocculent variant used was not too flocculent because under-fermented wort and residual unwanted beer flavours (for example, diacetyl and other VDKs—Figure 16) could have been the result [39]. The alternative way to crop yeast for reuse and to clarify primary fermented wort is with the use of a disk stack centrifuge (Figure 17). However, when a yeast culture is passed through a centrifuge it can experience mechanical and hydrodynamic shear stresses unless the centrifuge is correctly operated under the appropriate conditions. This shear stress can cause a decrease in cell viability/vitality, cell wall damage, injury to the mitochondrial DNA leading to formation of respiratory deficient mutants, increased extracellular proteinase A levels, hazier beer, and reduced foam stability [59].

8. Non-Saccharomyces Yeasts in Brewing Many yeast species are emerging as candidates for the production of specialty beers. Innovation not only relies on obtaining new products with more complex aromatic and flavour characters, but also increasing the range of approaches to achieve different results in a growing market. Using non-Saccharomyces yeasts for pitching, in sequential or co-inoculation, in bottle conditioning, or many other strategies, has presented the possibility to unveil old and new methods to produce specialty and original beers. Brewers are now reinterpreting the role of different yeast species—previously regarded as spoilage microorganisms in uncontrolled or in the absence of good manufacturing practices—and learning to find a multitude of applications and benefits with them. Yeasts from the genera Dekkera, Hanseniaspora, Pichia, Torulaspora, Wickerhamomyces, and others, can offer a diversified enzymatic apparatus and bioconversion abilities that are allowing brewers to work with new concepts that include bioflavouring, beers with reduced calories and alcohol contents, and also various functional beers. Nonetheless, the introduction of a new yeast culture must be carefully assessed, because each microorganism is unique, and can develop a range of process adaptations and flavours in contact with different substrates and/or conditions. Finally, it is important not to ignore the importance of the ongoing craft brewery revolution that is occurring in many parts of the globe. This is a movement that is paving the way to explore such traits and possibilities in order to identify a growing market of enthusiastic and expert consumers [60]. The impact of non-Saccharomyces yeasts on the volatile components and sensory profile of beer, wine, spirits, and other fermented beverages has recently been reviewed [61].

9. Conclusions The brewing process involves the use of four major raw materials: water, barley malt, hops, and yeast. As well as focusing on brewing yeast, research has productively compared yeast’s use with the production of other products such as distilled spirits, sake, wine, baked goods, and food extract. Yeast is also employed as a model eukaryote and it is interesting to note that the 2016 Nobel Prize in Physiology or Medicine has been awarded to a Japanese cell biologist for research on autophagy (the degradation and recycling of cellular components). This research has been conducted with S. cerevisiae by Yoshinori Õhsumi [62]. Another recent example of yeast’s contribution to our knowledge of basic biology, that has been applied to medical science, concerns the structure and function of yeast mitochondria [59]. There are a group of diseases caused by dysfunctional mitochondria often as a result of mitochondrial mutation of its DNA (mtDNA). Current research removing the mtDNA from the ovaries of a diseased patient and replacing it with unmutated mtDNA from a donor to produce healthy zygotes is a novel technique with significant potential (the three parent concept) [63]. Without Beverages 2016, 2, 34 16 of 18 basic mitochondrial research on brewer’s yeast and its close relations, these medical developments would have been inhibited, impeded, and protracted [59].

Acknowledgments: The invaluable assistance and support of Anne Anstruther in developing this review paper is gratefully acknowledged. Conflicts of Interest: The authors declare no conflict of interest.

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