Part One

Yeast: properties and

management
Yeast: an overview
A. Speers1, J. Forbes2
1The International Centre for Brewing & Distilling, Heriot-Watt University,
1
Edinburgh, Scotland, UK; 2Dalhousie University, Halifax, NS, Canada

1.1Yeast species/strains used in brewing and distilling

The formal classification of brewing yeasts over the past 50years has changed enough
that many brewing scientists (and most brewers!) avoid using the current genus and
species to identify their yeast and simply label them as either ale or lager strains. These
yeasts are used to produce most beers that is, either ales or lagers. Ale is nor-
mally made with Saccharomyces cerevisiae that rises to the top of the fermenter at the
cessation of fermentation while lager is made with S. carlsbergensis, which settles to
the bottom of the tank towards the end of the fermentation. In the past, Barnett, Payne
and Yarrow (1983) stated that both types of yeast should be characterized as variants
of S. cerevisiae. However, the strains differ in their DNA profiles, ability to ferment
melibiose, (ale strains lack melibiase activity) and their maximum growth temperature
(lager strains do not grow above 34C (Webb, 1977)) and for these reasons, Stewart
(1990) has argued that the two types of yeast should be classified as separate species.
Additionally, the increasing importance of a third species, Brettanomyces, has been
recognized following the massive growth of the craft brewing movement in the United
States. Brets, as they are termed in the industry, are used in various stages in the
production of lambic-type beers. They are considered a spoilage yeast in lager and
ale fermentations as they produce volatile phenolic flavours and acetic acid due to
their ability to produce off flavours by the production of volatile phenols (Libkind
etal., 2011), their ability to produce acetic acid (Wijsman, van Dijken, van Kleeff, &
Scheffers, 1984) and their ability to over attenuate products below 1Plato (Kumara &
Verachtert, 1991). Those involved with the wine industry have spent significant
amounts of time and money learning to isolate and characterize Brettanomyces spp. to
develop better methods of early detection and eradication (Conterno, Joseph, Arvik,
Henick-Kling, & Bisson, 2006; Dias etal., 2003; Oevelen, Spaepen, Timmermans,
& Verachtert, 1977). Despite the large amount of negative attention Brettanomyces
receives, this interesting microbe has been shown to contribute favourable organolep-
tic qualities to a number of products and to be of use in several industrial applications.
Belgian lambic beer producers have promoted the unique organoleptic characteris-
tics of Brettanomyces species in concert with other microbes for hundreds of years to
produce a beer that is crisp, acidic and refreshing (De Keersmaecker, 1996; Oevelen
etal., 1977). However, in comparison to ale and lager yeast less is known about Bret-
tanomyces species employed in brewing.
Since the early 2000s the advances in molecular biology have added to our under-
standing of the lager yeasts (Libkind etal., 2011; Walther, Hesselbart, & Wendland,

Brewing Microbiology. http://dx.doi.org/10.1016/B978-1-78242-331-7.00001-0

Copyright 2015 Elsevier Ltd. All rights reserved.
4 Brewing Microbiology

2014). It appears that a newly discovered and sequenced species, S. eubayanus, and
S. cerevisiae have combined to form the hybrid lager yeast genome. It is hypothe-
sized that materials containing S. eubayanus strains were imported from Patagonia to
Europe where hybridization events have occurred to form the S. carlsbergensis prog-
eny, but more recent studies suggest that the origin of the S. eubayanus strain may be
Asia (Bing, Han, Liu, Wang, & Bai, 2014).
Two types of lager yeast are in common use in the brewing industry. The first, Group I,
the so-called Saaz type (i.e. Unterhefe No. 1 isolated by the Carlsberg brewery in 1883)
is principally a triplod strain with an almost complete copy of the Saccharomyces cere-
visiae genome and slightly more than a diploid copy of S. eubayanus genome (Walther
etal., 2014). These same researchers noted that the Group II lager (i.e. the Froberg type,
Weihenstephan WS34/70) has a tetraplod with roughly two copies of chromosomes from
S. cerevisiae and two from S. eubayanus. It has been suggested that the low fermentation
temperatures (e.g. as low as 5C) that Group I lagers were exposed to may have driven
the difference between Group I and II lager yeasts (Walther etal., 2014).

1.2Yeast cell structure

Yeast is the most important part of the brewing fermentation process. Yeast converts
sugar to alcohol, carbon dioxide and other compounds that influence the flavour and
aroma of beer. Brewers yeast is a eukaryote and belongs to the kingdom Fungi. By
some scientific classifications, all beer-brewing strains of yeast are placed in the genus
Saccharomyces (sugar fungus) and species cerevisiae (Walker, 1998). However, the
brewing industry uses a classification which divides yeast into two types: ale yeast
(S. cerevisiae) and lager yeast (S. carlsbergensis). The distinction is kept so as to
separate yeasts used to make ales from those used to make lagers (Briggs, Boulton,
Brooks, & Stevens, 2004).
Most of the organisms in the kingdom Fungi are multicellular; however, yeast is a
single-cell organism. A single yeast cell measures about 510m in diameter and is
usually spherical, cylindrical or oval in shape (Boulton & Quain, 2001, pp. 5360).
Yeast occurs in single, pairs, chains and clusters (Stewart & Russell, 1998). Figure 1.1
is a simplified diagram of yeast cell structure. The cell wall is a barrier that is mostly
composed of carbohydrates surrounding the cell (Boulton & Quain, 2001). It is a rigid
structure which is 250nm thick and constitutes approximately 25% of the dry weight
of the cell (Stewart & Russell, 1998). There are three cross-linked layers comprising
the cell wall (Figure 1.2). The inner layer is a chitin (a long-chain polymer of an
N-acetylglucosamine) layer, composed mostly of glucans; the outer layer is mostly
mannoproteins while the intermediate layer is a mixture of both the inner and outer
layer (White & Zainasheff, 2010).
To reproduce asexually, a yeast cell clones itself, thereby creating a new daughter
cell. Cell separation is achieved when the layers of the cell wall separate, leaving the
bud scar on the mother cell and the birth scar on the daughter cell (Stewart & Russell,
1998). The bud scar is composed mainly of chitin. The average ale yeast cell will
not bud more than 30 times over its lifetime while lager yeast will bud only 20 times
before they are unable to bud further (Wyeast Laboratories, 2009).
Yeast: an overview 5

Bud

Actin Nucleus
cable

Secretory Endoplasmic
vesicles reticulum

Fat
Mitochondroin
globule

Golgi

Cell
wall
Vacuole

Cell
membrane
Figure 1.1 Main features of a typical yeast cell (Stewart & Russell, 1998).

*3,&:3 *3,&:3

*F *F
2XWVLGHZDOO 3LU&:3
&HOOZDOO
,QVLGHZDOO
&KLWLQ &KLWLQ
Figure 1.2 Molecular organization of the cell wall of S. cerevisiae. GPI-CWP are GPI-
dependent cell wall proteins, Pir-CWP are pir proteins on the cell wall and 1-6-Glc are
glucan molecules, which are highly branched. Therefore, they are water soluble, which tethers
GPI-CWPS to the cell wall (Kils, Mol, Hellingwerf, & Brul, 2002).

The plasma membrane is a semipermeable lipid bilayer between the cell wall and
the inside of the cell. There are several distinct roles that the plasma membrane car-
ries out such as to provide a barrier to free diffusion of solutes, to catalyse specific
exchange reactions, to store energy dissipation, to provide sites for binding specific
molecules involved in metabolic signalling pathways and to provide an organized sup-
port matrix for the site of enzyme pathways involved in the biosynthesis of other
cell components (Hazel & Williams, 1990). The plasma membrane is quite fluid and
6 Brewing Microbiology

flexible due to its constituents of lipids, sterols and proteins. Additionally, these con-
stituents allow for the creation of a daughter cell.
The formation of double bonds in fatty acids controls their level of saturation. The
saturation level determines the ease and extent of hydrogen bonding that can occur
between fatty acids (Briggs etal., 2004). Membrane fluidity is necessary for proper
membrane function. Lipid bilayers are by their nature fluid and that fluidity is deter-
mined by the extent to which the lipids bind to one another (White & Zainasheff,
2010). By controlling the level of saturation in their lipid membranes, yeast cells are
able to maintain proper membrane fluidity at different temperatures, which is important
during fermentation. Without proper aeration yeast cells are unable to control mem-
brane fluidity through to the end of fermentation which leads to halted fermentations
and off-flavours of the final product (White & Zainasheff, 2010).
The cytoplasm is that portion of the cell enclosed by the plasma membrane and
excluding other membrane-bound organelles. It is an aqueous colloidal liquid contain-
ing a multitude of metabolites (Briggs etal., 2004). The cytoplasm contains intercel-
lular fluid known as the cytosol. The cytosol contains enzymes involved in anaerobic
fermentation that enable the cell to convert glucose into energy immediately after it
enters the cell (White & Zainasheff, 2010).
The mitochondrion is an organelle where aerobic respiration occurs. Mitochondria
consist of a double membrane that is the location of the conversion of pyruvate (a
metabolic compound) and the tricarboxylic acid cycle. The nucleus stores the cell
DNA and is delineated by a lipid membrane that envelopes the nucleus and is similar
to the plasma membrane. The cell uses mRNA to transfer the information out into the
cytoplasm for use in protein synthesis (White & Zainasheff, 2010).
The vacuole is a membrane-bound structure that stores nutrients and is also where the
cell breaks down proteins. Brewers yeast vacuoles are large enough to be seen through
light microscopy (White & Zainasheff, 2010). The major site for proteolysis is the cell
vacuole. Much of the regulation of both specific and nonspecific proteolysis involves
the sequestration of target proteins into vacuoles where they are exposed to proteinases
(Briggs etal., 2004). The endoplasmic reticulum is a network of membranes and is
usually where the cell manufactures proteins, lipids and carbohydrates for membranes
and secretion (White & Zainasheff, 2010). Other microbodies are mainly made up by
glycogen bodies and lipid granules (Boulton & Quain, 2001).

1.3Comparison of lager and ale yeast

The distinctions between the yeast used in ale and lager brewing are small. Tradi-
tionally, ale yeast were regarded as top fermenters that formed a frothy yeast head
on the surface of the fermenting beer, which was skimmed off to be used for sub-
sequent brews, while lager yeasts were bottom fermenters that formed little surface
head and were recovered from the bottom of the fermenter (Briggs etal., 2004).
Today, this is a less useful distinction as many types of ale yeast now have the
capacity to fall out of solution and settle at the bottom of the fermenter (Adams &
Moss, 2008).
Yeast: an overview 7

The optimal growth temperature of lager and ale yeast differs and this is reflected in
the different temperatures used for lager fermentations, 815C, and for ale fermenta-
tions, 1822C (Adams & Moss, 2008). Lager and ale yeasts can also be distinguished
by the ability of lager strains to ferment the disaccharide melibiose because they have
-d-galactosidase activity, which hydrolyses melibiose to galactose and glucose while
ale strains cannot. However, this is of no practical importance since the sugar does
not occur in wort (Briggs etal., 2004). Additionally, lager yeast strains can utilize
maltotriose more rapidly than ale strains. Lager strains utilize mixtures of galactose
and maltose simultaneously, whereas ale strains prefer to utilize maltose (Boulton &
Quain, 2001).

1.4Flocculation
One functional definition of flocculation is that it describes the ability of yeast strains
to clump together and fall out of solution. Near the end of fermentation, single cells
aggregate into clumps of thousands of cells. Different strains of yeast have different
flocculation characteristics. Some strains flocculate earlier during fermentation and
subsequently do not attenuate (i.e. finish the fermentation) normally. Flocculating too
early results in a beer that is under attenuated and sweet; however, when yeast fails to
flocculate entirely, it results in a beer that is cloudy with a yeasty flavour (Speers, 2012).
Flocculation has been studied for many years and the exact mechanism is still
debated. Cell wall composition is a key factor in the ability of adjacent cells to stick
to each other. Yeast has a thick cell wall made up of protein and polysaccharides with
a net negative surface charge due to phosphates in the cell wall (Briggs etal., 2004).
The extent of the negative charge depends on the yeast strain, phase of growth, oxygen
availability, starvation, generation number, dehydration and cell age. Yeast cells are
also hydrophobic due to exposed hydrophobic peptides and lipids (Akiyama-Jibiki,
Ishibiki, Yamashita, & Eto, 1997). The primary determinant of flocculation is the yeast
strain itself (Speers, Smart, Stewart, & Jin, 1998).
The minute differences in cell wall composition play a key role in flocculation
behaviour and determine the degree of flocculation for a strain. Factors that influ-
ence the degree of flocculation include the original gravity of the wort, temperature
of fermentation, pH of the wort, pitching rate, initial oxygen content, calcium and
inorganic ion concentration, and cell age. Additionally, anything that affects the health
and growth rate of the yeast affects flocculation (Speers etal., 1998).
The mechanism of lectin-like cellcell interactions has been established to explain
yeast flocculation (Speers etal., 1998). Lectins are a structurally diverse group of pro-
teins that are capable of binding carbohydrates while zymolectin is an anchored yeast
cell wall protein that contains one or more mannose binding sites (Boulton & Quain,
2001). This mechanism proposes that specific surface proteins known as zymolectins,
which are present on flocculent yeast cells, bind to mannose residues of mannan mol-
ecules on neighbouring cell surfaces (Speers etal., 1998). The involvement of this
proteincarbohydrate interaction was suggested by Taylor and Orton (Taylor & Orton,
1978), as flocculation can be inhibited specifically by mannose.
8 Brewing Microbiology

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