Russell I. (ed.) Whisky. Technology, Production and Marketing

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supply of the vitamins that humans require in their diet: pantothenate, ribo-

flavin, thiamine, etc. However, under the anaerobic conditions of fermenta-

tion, brewery and distillery yeasts are unable to synthesize the unsaturated

fatty acids and sterols that are essential structural components of cell mem-

branes. Since the amounts naturally present in the wort are insufficient for

anaerobic growth, aeration of wort at the tim e of pitching the yeast allows the

yeast ino culum to synthesize a reserve of these compounds under aerobic

conditions.

Flavour congeners are produced as by-products of the fermentation of

sugars to alcohol. Although only the principal types of congener are shown

in Table 4.1, Nykanen and Suomalainen (1983) listed approximately 400 fla-

vour metabolites formed during alcoholic fermentation by yeast and contri-

buting to the flavour of beers, ciders, wines and distilled spirits. These

compounds vary in their importance to the quality of the final product. For

any alcoholic beverage the actual amount of each compound is less important

than its aroma threshold – the concentration detectable by a trained sensory

panel. However, volatility is also important in the case of distilled beverages,

and the volatility of flavour metabol ites influences the amount collected in the

spirit fraction for malt whisky or at the spirit plate of a continuous still.

Fermentability of wort

Part of malt analysis, measurement of fermentability and prediction of spirit

yield are also relevant to the fermentation stag e. A standard hot-water extra ct

(method 2.15 of Baker, 1991) is used as experimental wort, whic h is not boiled

in order to preserve the activity of malt enzymes during the test ferm entation

by the M strain of distiller’s yeast. This is derived from the original distilling M

strain of 1952 (see Fowell, 1967), but since improved strains introduced later

were still named M, its properties have not been constant over the last 50

118 Whisky: Technology, Production and Marketing

Table 4.1

Products of yeast fermentation (modified from Suomalainen,

1971)

Alcohols Acids Esters Others

Ethanol Acetic Ethyl acetate and any

other combination of

acids and alcohols on

the left

Carbon dioxide

Propanol Caproic Acetaldehyde

Butanols Caprylic Diacetyl

Amyl alcohol Lactic Hydrogen sulphide

Glycerol Pyruvic

Phenylethanol Succinic

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years. The standard method specifies the use of commercial M yeast cake, but

since that is not necessarily a pure culture there is some advantage in using a

guaranteed pure culture obtained by plating out commercial M yeast to obtain

single colonies.

From the measured fall in gravity over 44 hours’ incubation at 338C, a

percentage fermentable extract in actual distillery practice can be calculated

(Bathgate, 1989; Bringhurst et al., 1996; Dolan, 2000). From that figure a pre-

diction of the spirit yield per tonne of malt can be derived – an essential aspect

of analysis of malt and cereal, but a figure largely dependent on the efficiency

of starch hydrolysis during both mashing and fermentation.

Yeast structures

The outermost layer of the cell is a rigid wall of fibrous b(1-3)- and (1-6)-

glucans with an outer, more amorphous, layer of mannan, mainly a(1-4) but

with a(1-2) and a(1-3) side chains. Since the scars left by separation of succes-

sive buds are composed of chitin, a polymer of N-ace tyl glucosamine, an

increasing proportion of chitin develops in the wall s of older cells. The cell

wall constitutes 15–25 per cent of the dry weig ht of the cell, and has many

functions (Stratford, 1992): physical protection, osmotic stability, support for

wall-bound enzymes (e.g. inve rtase), cell–cell adhesion (e.g. flocculation), and

as a selective permeability barrier. Although it is convenient to regard the wall

as porous to nutrients and metabolites, larger polysacc haride and polypeptide

molecules are blocked by the pore size of the wall (de Nobel and Barnett,

1991).

It is the cell membrane (cytoplasmic membrane) that prevents the leakage of

soluble components of the cytoplasm into the surrounding medium , but it is

equally effective in the opposite direction and prevents the free diffusion of

nutrients into the cell. True diffusion across the membrane, at a rate deter-

mined by molecular size, solubility in membrane lipids and concentration

difference between the culture medium and cytoplasm, is limited to a few

simple compounds such as ethanol and carbon dioxide (Slaughter, 2002).

For all other compounds, specific permease enzymes are required for the

import of nutrients and export of metabolites. Transport may be by facilitated

diffusion, where the energy is provided by the concentration difference across

the membrane, or by active transport, requiring en ergy input by the cell in

addition to the specific transport system (Young, 1996; Slaughter, 2002).

Saccharomyces species grow by budding. Only one bud scar is shown in

Figure 4.1: if that actually is the only scar on the cell, it represents the ‘birth

scar’ left after detaching from its mother cell. If so, the first bud produced by

the new yeast cell will be at the other end – not necessarily exactly opposite the

birth scar, but more probably near the point where the nucleus is closest to the

cell wall, to control bud initiation. The first stage is local softening, by precisely

directed b(1-6)-glucanase, of the glucan that gives the cell wall its rigidity.

Osmotic pressure within the cell causes swelling, the first microscopically

visible evidence of bud development. Subsequently, synthesis of new cell

Chapter 4 Yeast and fermentation 119

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wall, membrane, and nuclear and cytoplasmic material is precisely pro-

grammed over the generation time of the cell and the fully formed daughter

cell detaches from its mother for both to start their next cell cycle indepen-

dently. The process was illus trated by Matile et al. (1969) in an excellent suc-

cession of drawings of electron micrographs. Although parts of their text have

been overtaken by more recent research, the drawings are still perfectly valid

to complement Wheals’ (1987) review of the cell generation cycle.

Various organelles exist with in the cytoplasm (Rose and Harrison, 1991).

The most important are shown in Figure 4.1: the nucleus, mitochondria and

vacuole. These also are enclosed within membranes of the same structure and

function as the cytoplasmic membrane. In the resting stage depicted in Figure

4.1 the vacuole exists as a single organelle, the largest within the cell, but

during the generation cycle the vacuole fragments and some units migrate

to the developing bud before the entry of the divided nucleus. Obviously it

is essential that the nuclear genetic material is replicated accurately during cell

division, but it is equally important that units of divided mitochondria and

vacuole enter the developing bud at the appropriate stage of cell division

(Matile et al., 1969). Among the biochemical functions of the vacuole are the

recycling of proteins and storage of glycogen as an energy reserve, but it also

appears to have the mechanical function of forcing into the developing bud its

share of the replicated nucleus.

Mitochondria are essential organelles for the oxidative growth of yeast

(Nagley et al., 1977) and are well developed in aerobically grown baking or

120 Whisky: Technology, Production and Marketing

Figure 4.1

Principal structures of an aerobically grown cell of distilling yeast Saccharomyces

cerevisiae. BS, bud scar; CW, cell wall; CM, cytoplasmic membrane (cell membrane);

Mt, mitochondrion; N, nucleus; V, vacuole.

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distilling yeast (Figure 4.1). Among other activities, these organelles are asso-

ciated with the enzyme systems of oxidative metabolism (Nagley et al., 1977;

O’Connor-Cox et al., 1996). The importance of mitochondria to fermentation is

discussed further below, under oxygen metabolism, but Rose and Harrison

(1991) provide more general biological information on these and other orga-

nelles of the yeast cell.

Carbohydrate metabolism

There are three important aspects of carbohydrate metabolism during fermen-

tation:

1. Uptake of the fermentable sugars from the wort

2. Production of ethanol and flavour congener s within the yeast cells

3. Excretion of the metabolites as flavour of the wash and ultimately of the

whisky.

Table 4.2 shows the comp osition of all-malt wort, but it has been realized

since the 1960s that these sugars are not utilized simulta neously. Although

present in the greatest amou nt, maltose is not utilized initially by the yeast.

The specific permease for glucose is a constitutive enzyme that is immediately

available at pitching, whereas the maltose transport system is inducible.

Chapter 4 Yeast and fermentation 121

Table 4.2

Approximate sugar composition of all-malt wort, expressed as

percentage of total carbohydrate (Palmer, 1989). Carbohydrate

constitutes approximately 90% of total solids of wort

Monosaccharide (hexose)

Fructose 1%

Glucose 10%

Disaccharide

Maltose 46%

Sucrose 5%

Tri- and tetra-saccharide

Maltotriose 15%

Maltotetraose 10%

Wort carbohydrates not fermentable by distilling yeast

Maltopentaose and higher

dextrins

13% (at collection in washback; reduced by

continuing enzymic action during fermentation)

Similar figures apply to grain distillery worts (Pyke, 1965).

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Inducible enzymes are synthesized only in the presence of their substrate, but

in addition, synthesis of maltose permease is not activated until the glucose

concentration falls below a threshold concentration. Similarly, maltotriose

transport is not induced until maltose concentration in the wort has fallen

sufficiently. Therefore the uptake of fermentable sugars is normally as

shown in Figure 4.2.

The mode of operation of the transport system also varies with different

sugars. The uptake of the main carbohydrate nutrients of a typical malt wort is

shown in Figure 4.3. Glucose and fructose are transported intact through the

cell membrane (shown in this diagram correctly as a double layer of lipid) by

facilitated diffusion with their appropriate permease, which is located within

the membrane (Meaden, 1993). Before reaching the cell membrane, sucrose is

hydrolysed within the cell wall by a wall-bound invertase system, and it is the

resulting glucose and fructose that are transported into the cell. Maltose and

maltotriose enter the cell by specific ene rgy-requiring active transport systems,

which phosphorylate these sugars. A detailed explanation of modern concepts

of the structure and mode of operation of permease enzymes in yeast is pro-

vided by Slaughter (2002).

Aerobically the sugar is fully oxidized to carbon dioxide and water, releas-

ing the same amount of energy as obtained by chemical combustion of glu-

cose, but in a biological system the energy is released and stored in non-lethal

122 Whisky: Technology, Production and Marketing

Figure 4.2

Utilization of principal wort sugars by distillery yeast. G, glucose; FS, fructose þ

sucrose; M2, maltose; M3, maltotriose; M4, maltotetraose. The horizontal bars show

the duration of the relevant permease activity.

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amounts as energy-rich compounds. Although others are possible, the normal

energy store is the energy-rich bond coupling a third phosphate group to

adenosine diphosphate (ADP) to form the triphosphate, ATP. Anaerobically

only partial oxidation is possible, to pyruvic acid. Details of the fermentative

metabolism of hexose sugars are available in all biochemistry textbooks, and

only a basic outline of the Embde n–Meyerhof–Parnas metabolic pathway is

shown in Figure 4.4.

For simplicity, few of the enzymes of the co-ordinated sequence of the path-

way are named in Figure 4.4. The first steps of the pathway involve phosphor-

ylation of sugar, an energy-requiring process requiring two molec ules of ATP

per molecule of hexose. However, maltose is already phosphorylated during

transport and before hydrolysis to glucose, so the first pho sphorylation step of

the metabolism of hexose is unnecessary. Also, whatever the original structure

of the hexose, it must be isomerized to fructose-6-phosphate. Only with the

specific structure of fructose can the 1,6 diphosphate of the following step be

split to two identical molecules of triose phosphate (glyceraldehyde 3-phos-

phate).

Phosphorylation does not necessarily require the energy of ATP; later in the

pathway phosphorylation by inorganic phosphate occurs with the reduction of

nicotinamine adenine dinucleotide (NAD) to NADH

. (An alternative name

for NAD, as used in some textbooks and research papers, is diphosphopyr-

idine nucleotide, DPN.) In Figure 4.4 the reaction is shown more correctly as

NAD

reduced to NADH as glyceraldehyde 3-phosphate is phosphorylated to

1,3-diphosphoglycerate. Subsequent reactio ns involv e transfer of phosphate to

Chapter 4 Yeast and fermentation 123

Figure 4.3

Transport of fermentable sugars into Saccharomyces cerevisiae . F, fructose; G, glucose;

GP, glucose phosphate; M2, maltose; M3, maltotriose; S, sucrose; CW, cell wall; CM,

cytoplasmic membrane.

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ADP and isomerization to prepare the triose phosphate for release of its phos-

phate, generating a total of four ATP molecules for each molecule of hexose.

Since two are required to ‘pay back’ the initial use of two ATP molecules, the

overall effect of the pathway is a net gain of the energy of two ATP molecules,

which is sufficient for the biosynthetic activities of the growing cell.

124 Whisky: Technology, Production and Marketing

Figure 4.4

Summary of Embden–Meyerhof–Parnas metabolic pathway (modified from Hough et

al., 1982).

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In fermentative yeasts, subsequent re-oxidation of NADH to NAD

by acet-

aldehyde is required to allow metabolism to continue with the limited quan-

tity of NAD within the cell, and the two hydrogen a toms removed in the re-

oxidation of each molecule of NAD reduce acetaldehyde to ethanol.

Decarboxylation of pyruvate and formation of acetaldehyde and ethanol is

only one of many possible methods for recovery of NAD

: in many lactic

acid bacteria, for example, a single step of reduction of pyruvic to lactic acid

provides for re-oxidation of NADH.

The branch route to glycerol normally represents only a small part of car-

bohydrate metabolism. Pyke (1965) reported 0.25 per cent w/v in grain dis-

tillery wash, substantially above the levels of higher alcohols and esters, but its

volatility is too low for glycerol to appear in the spirit. The flavour congeners

directly produced by the main route of the EMP pathway are pyruvic acid and

acetaldehyde, but many other acids, alcohols and esters are produced as by-

products of biosynthetic activities of the yeast (Table 4.1). However, the major-

ity of these flavour congeners are by-products of biosynthesis of lipids, nucleic

acids and proteins, as shown later in this chapter.

EMP is only one of the possible pathways of energy-yielding carbohydrate

metabolism, but is the important pathway under the anaerobic conditions of

fermentation. Hough et al. (1982) provided an adequate account of the hexose

monophosphate (or pentose phosphate) pathway, krebs cycle and other meta-

bolic activities relevant to aerobic growth of brewing yeast (and therefore also

the aerobic propagation of distilling yeast), but a more detailed explanation

can be found in any biochemistry textbook.

Nitrogen metabolism

The amino acids required for yeast growth are produced by hydrolysis of malt

protein during malting, and the amount of a-amino nitrogen in the wort is

partly related to the degree of modification of the malt. The higher the a-amino

nitrogen content of the wort the greater is the amount of yeast growth (ulti-

mately above about 150–180 mg/l), reducing spirit yield by conversion of

sugar to cell mass rather than ethanol. Also, higher nitrogen levels mean

proportionally lower carbo hydrate content in the barley and malt. Given the

relatively low nitrogen content of maize and wheat, this is unlikely to be a

problem in grain distilleries, and a higher nitrogen content of barley is usually

associated with higher enzyme activity – an important property of grain dis-

tillery malt.

It is impossible for the limited space of the yeast cell membrane to contain

individual permease enzymes for each of the twenty structural amino acids of

proteins. Only a few amino acids have specific transport enzymes, and most

have to share. Although a few of the amino acids of protein structure were not

mentioned by Jones and Pierce (1964) or Pierce (1987), Table 4.3 shows their

comparison of the rate of uptake of individual amino acids – effectively a

summary of their transport systems. Only aspartic acid/asparagine, glutamic

acid/glutamine and lysine have their own specific transport enzymes. Since

Chapter 4 Yeast and fermentation 125

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arginine, serine and threonine share a single permease, the rate and amount of

uptake of each individual amino acid is approximately in proportion to their

relative amounts in the wort, but all are absorbed from wort at a sufficiently

rapid rate to satisfy the requirements of the growing cells for these amino

acids. Proline and hydroxyproline are absorbed only slowly, but meet the

requirements of the cell throughout fermentation. However, since all amino

acids listed as ‘group B’ share a single permease, they are not absorbed in

sufficient amount to meet the requirements of protein synthesis during active

growth of the yeast. All amino acids can be transported by a general amino

acid permease (GAP), but that is inactive while specific transport of the group

A and B acids is in operation. Therefore the amino acids listed as group C,

which are totally dependent on GAP for transport, could be absorbed only late

in the fermentation, too late be incorporated directly into enzyme structure

during the phase of active growth. For the development of the next generation

bud the growing yeast cell must synthesize its own weight of cell material

from the nutrients in the wort. That biosynthetic activity includes all of its

requirements of group C amino acids and the necessary supplement of the

limited uptake of group B. Since lysine cannot be used for that purpose by

Saccharomyces spp., the amino groups of aspa rtic acid/asparagine and glu-

tamic acid/glutamine provide the amino nitrogen for biosynthesis of other

amino acids.

Figure 4.5 shows a basic summary of the formation of the various keto-acid

intermediates required for synthesis of amino acids (Quain, 1988; Slaughter,

2002). Although enzyme-med iated reactions are normally reversible, some of

the steps of keto-acid biosynthesis are not. If the supply of amino nitrogen is

exhausted, obviously the keto acids cannot be converted to the intended amino

acids. However, these oxidized compounds, keto acids, represent a valuable

resource that cannot be permitted to accumulate under anaerobic conditions.

126 Whisky: Technology, Production and Marketing

Table 4.3

Classification of amino acids by their rate of absorption from wort (Jones and Pierce,

1964; Pierce, 1987)

Group A Group B Group C Group D

Rapidly absorbed from

start of fermentation

Slowly absorbed from

start of fermentation

Slowly absorbed, late in

fermentation

Absorbed slowly over

fermentation

Aspartic acid/amine Histidine Alanine Proline

Glutamic acid/amine Isoleucine Glycine Hydroxyproline

Lysine Leucine Phenylalanine

Argine, serine,

threonine

Methionine Tryptophan

Valine Tyrosine

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Hence if they cannot be converted to amino acids, the keto acids are decar-

boxylated to the equivalent aldehyde and reduced to an alcohol by analogous

reactions to the metabolism of pyruvate to ethanol. A gen eral alcohol dehy-

drogenase redu ces the aldehyde to its corresponding alcohol, although a spe-

cific decarboxylase is required for each keto acid. Therefore if nitrogen

depletion prevents conversion of a-keto isocaproic acid to leucine, isoamyl

alcohol is formed instead (Figure 4.6). This relationship between amino

acids and higher alcohols was first noted by Ehrlich in 1911, but the reactions

were not fully explained until the 1950s. It is also possible that even though

amino-N is available, the yeast may convert part of its reserve of keto acids to

higher alcohols in order to generate oxidized NAD (Quain, 1988; Slaughter,

2002). This response to a redox problem is an analogous reaction to the pro-

duction of glycerol by the Embden–Meyerhof–Parnas pathway: whereas the

pyruvate route is exactly balanced in NAD and generates a ‘prof it’ of ATP, the

glycerol branch is balanced with respect to ATP and provides oxidized NAD

for use in biosynthetic reactions under anaerobic conditions.

Diacetyl is another by-product of nitrogen metabolism. Figure 4.7 shows

that isobutanol is the higher alcohol associated with the biosynthesis of valine,

but acetolactate, an intermediate in the biosynthetic pathway, is decarboxy-

lated and reduced to acetoin and 2,3 butanediol by similar reactions to ethanol

Chapter 4 Yeast and fermentation 127

Figure 4.5

Biosynthesis of amino acids or higher alcohols by Sac charomyces cerevisiae. Pathways

generate an excess of ATP or oxidized NAD as shown.

Figure 4.6

Structural relationship between amino acids and higher alcohols.