Risk Analysis see Microbial Risk Analysis
S
SACCHAROMYCES
Contents
Introduction
Brewer’s Yeast
Saccharomyces cerevisiae
Saccharomyces cerevisiae (Sake Yeast)
Introduction
GG Stewart, GGStewart Associates, Cardiff, UK
Ó 2014 Elsevier Ltd. All rights reserved.
This article is a revision of the previous edition article by Yuji Oda, Kozo Ouchi, volume 3, pp. 1907–1913, Ó 1999, Elsevier Ltd.
Yeasts are classified into three groups: ascosporogenous yeasts,
basidiosporogenous yeasts, and imperfect yeasts. Saccharomyces
is the representative of ascosporogenous yeasts and historically
is the most familiar microorganism to humans. This genus was
first described by Meyen when he assigned beer yeast as
Saccharomyces cerevisiae in 1838, and it was redefined by Reess
in 1870 from the observations of ascospores and their germi-
nation. The name is derived from the Greek words sakcharon
(sugar) and mykes (fungus). The number of Saccharomyces
species has changed according to the criteria used to delimit
species, and nine species are now accepted in the genus
Saccharomyces (Table 1).
Characteristics of the Genus
The vegetative cells of Saccharomyces species are round, oval, or
cylindrical and reproduce by multilateral budding. They may
Encyclopedia of Food Microbiology, Volume 3 http://dx.doi.org/10.1016/B978-0-12-384730-0.00290-1
form pseudohyphae but not septate hyphae. The yeasts are
predominantly diploid or occasionally of higher ploidy. Asci,
which are persistent and usually transformed by direct change
from the vegetative cells, may contain one to four ascospores.
The ascospores are round or slightly oval, with smooth walls.
Conjugation occurs during or soon after germination of the
ascospores. Some strains of S. cerevisiae and its related species
used in the brewing, distilling, and baking industries hardly
form ascospores at all. Continuous selection with respect to
their practical properties seem to cause loss of sporulation
ability in these strains.
The most notable physiological characteristic of Saccharo-
myces spp. is their capacity for vigorous anaerobic or semi-
anaerobic fermentation of one or more sugars to produce
ethanol and CO2. These sugars include D-glucose, D-fructose,
D-mannose, and D-maltose except in the case of certain
mutants. Most strains of Saccharomyces can grow on D-galactose
under aerobic or anaerobic conditions; however, none of them
297
298 SACCHAROMYCES j Introduction
Table 1 The species accepted in the genus Saccharomyces
Species Authority
Saccharomyces arboricolus F.-Y. Bai and S.-A. Wang (2008)
Saccharomyces bayanus Saccardo (1895)
Saccardo var. bayanus (2000)
Saccardo var. uvarum Naumov (2000)
Saccharomyces cariocanus Naumov, James, Naumova, Louis,
and Roberts (2000)
Saccharomyces cerevisiae Meyen ex E. C. Hansen (1883)
Saccharomyces eubayanus Libkinda et al. (2011)
Saccharomyces kudriavzevii Naumov, James, Naumova, Louis,
and Roberts (2000)
Saccharomyces mikatae Naumov, James, Naumova, Louis,
and Roberts (2000)
Saccharomyces paradoxus Bachinskaya (1914)
Saccharomyces pastorianus Hansen (1904)
Kurtzman, C.P., 2005. Yeast Systematics and Phylogeny – Implications of Molecular
Identification Methods for Studies in Ecology. Springer, Berlin; Kurtzman, C.P., Fell,
J.W., 2011. The Yeast – A Taxonomic Study, fifth ed. Elsevier, Amsterdam.
utilizes lactose, pentose, alditols, and citrate as carbon sources,
assimilates nitrate as a nitrogen source, or hydrolyzes exoge-
nous urea. Among polysaccharides, starch and pectin are
exceptionally utilized by certain (not all) strains of S. cerevisiae.
They do not produce starch-like compounds. Their ubiquinone
is exclusively Q-6, but this feature is common in the genera
Kluyveromyces, Torulaspora, and Zygosaccharomyces.
Identification of Saccharomyces Species
Yeasts are usually classified by the characteristics of microscopic
appearance, sexual reproduction, and physiological features,
including (1) fermentation of certain sugars semianaerobi-
cally; (2) assimilation of various compounds each as sole
carbon or nitrogen source; (3) growth without an exogenous
supply of certain vitamins; (4) growth in the presence of 50%
or 60% (w/w) glucose; (5) growth at 37
C; (6) growth in the
presence of cycloheximide; (7) splitting of fat, production of
starch-like polysaccharides, hydrolysis of urea; and (8) forma-
tion of acid.
Saccharomyces sensu stricto
Saccharomyces sensu stricto species, including S. cerevisiae,
Saccharomyces bayanus, Saccharomyces paradoxus, and Saccharo-
myces pastorianus, are phylogenetically closely related in the
genus Saccharomyces. The species S. cerevisiae, S. bayanus,
Saccharomyces eubayanus, and S. pastorianus are specifically found
in the environments of wineries and breweries. The relative
genome sizes of these three species are estimated to be 1.00,
1.15, and 1.46, respectively. Saccharomyces paradoxus is exclu-
sively isolated from natural sources such as tree exudates, soil,
and Drosophila. Cells of S. paradoxus are small in size and readily
form asci compared with the other three species. Effective
separation of the Saccharomyces sensu stricto species is compli-
cated because these species often have apparently identical
morphological, physiological, and serological properties. The
four species have been differentiated from each other by DNA
reassociation studies. Strains with 80–100% overall homology
of base sequences are considered as belonging to the same
species, while the strains of distantly related taxa show
homology of less than 30%. Among the four species,
S. pastorianus reveals 53% homology to S. cerevisiae and 72%
homology to S. bayanus, suggesting an intermediate position
between two unrelated species, S. cerevisiae and S. bayanus
(see Saccharomyces: Brewer’s Yeast).
Since species division within the Saccharomyces sensu stricto
group were clarified at the molecular level, it became possible to
determine those physiological responses necessary for separa-
tion of the four taxa. Saccharomyces bayanus and S. eubayanus are
the only species of the genus that can grow in the absence of
vitamins. Maximum growth temperature immediately distin-
guishes S. bayanus, S. eubayanus, and S. pastorianus, which never
grow at above 35
C, from S. cerevisiae and S. paradoxus, which
grow at 37
C, and often at up to 40–42
C. An active fructose
transport system is present in the group of S. bayanus,
S. eubayanus, and S. pastorianus, while fructose uptake is reduced
in S. cerevisiae and S. paradoxus. Saccharomyces cerevisiae is
distinguished from S. paradoxus with respect to the assimilation
by S. cerevisiae of D-mannitol and fermentation of maltose.
Further details of Saccharomyces species differences, particularly
as they apply to brewer’s yeast species, can be found in Chapter
Saccharomyces: Brewer’s Yeast.
Saccharomyces sensu lato
Saccharomyces kudriavzevii and Saccharomyces mikatae are unusual
members of the genus as judged from narrow fermentative
profiles and the ability to grow in the presence of 0.1% cyclo-
heximide. Assimilation of ethylamine, cadaverine, and lysine
can differentiate these two species.
Saccharomyces cerevisiae is characterized by much lower
G þ C values (34.7–36.6%) than other Saccharomyces species
(39.3–41.9%), but do not grow in the presence of 0.1%
cycloheximide. Saccharomyces arboricolus differs from S. bayanus
in the assimilation of glycerol as a sole carbon source and
ethylamine, cadaverine and lysine as sole nitrogen sources.
Saccharomyces kudriavzevii
Saccharomyces kudriavzevii is easily distinguishable from other
species of this genus since it is characterized by a wide assim-
ilative and fermentative profile, including the ability to utilize
ethylamine-HCl, cadaverine, and lysine as sole nitrogen sour-
ces for growth as well as the ability to both assimilate and
ferment melibiose. The distinct character was already antici-
pated by molecular taxonomic studies which showed no
nucleotide homology between S. kudriavzevii with either
Saccharomyces sensu stricto or sensu lato strains, where DNA
homology values were never above 22%.
Molecular Methods to Differentiate Species
The conventional taxonomic tests to assess physiological
features are fundamental for identification, but the results have
shown to be insufficient for species delimitation and discrimi-
nation of interstrain variability. The genetic basis behind many

of these characteristics is often either poorly understood or
unknown. In the past, DNA reassociation studies have signifi-
cantly contributed to molecular taxonomy of the genus
Saccharomyces. This method was used to reestablish several
species names, reduce other names to synonyms, describe new
species, and raise the likelihood of the existence of additional
species. However, the equipment used to measure DNA asso-
ciation is highly specialized and expensive, and the amount
of data obtainable in an average week is relatively small.
Discrimination of the closely related species has been confirmed
by other molecular techniques, such as whole-cell protein
patterns, multilocus enzyme electrophoresis, fructose transport
systems, mitochondrial DNA restriction analysis (see Saccharo-
myces: Saccharomyces cerevisiae), electrophoretic karyotypes,
random amplified polymorphic DNA polymerase chain reac-
tion (RAPD-PCR) and restriction fragment length poly-
morphism patterns, and rRNA gene sequencing. These methods
are valid additions (not replacements) to the conventional
taxonomic tests, and those applicable to the food and beverage
industry are described below and in Chapters Saccharomyces:
Saccharomyces cerevisiae and Saccharomyces: Brewer’s Yeast.
Electrophoretic Karyotypes
Chromosomal patterns resolved by pulse field gel electropho-
resis are called electrophoretic karyotypes. By comparing the
results from this method and DNA reassociation, it has been
demonstrated that electrophoretic karyotypes of the two strains
are identical when DNA sequence homology is over 85%, while
low DNA relatedness corresponds to completely different
chromosomal patterns. Since similar, but not identical,
karyotypes are not interpreted as either different species or
polymorphisms in the same species, karyotyping is not as
reliable as DNA base sequence comparisons, but is undoubt-
edly an important adjunct. Electrophoretic karyotypes can serve
as a rapid, inexpensive, and relatively easy first approach for
evaluation of a group of physiologically similar strains.
The general feature for ascosporogenous yeasts is the
presence of one to five bands of chromosomal DNA larger
than 1000 kb as in S. kudriavzevii (Figure 1), whereas in
most Saccharomyces species, chromosomes smaller than
1000 kb are observed. Chromosomes of Saccharomyces sensu
stricto species were resolved into 12–16 bands in the range
200–2200 kb. None of the other species contains chromo-
somes smaller than 300 kb. The patterns of Saccharomyces
sensu stricto species are similar and are distinguishable from
the other species at a glance. A multivariate analysis of the
polymorphisms in the numbers and molecular weights of
chromosomes has revealed that the Saccharomyces sensu stricto
strains could be separated into four clusters that correspond
to the four species.
Random Amplified Polymorphic DNA Polymerase
Chain Reaction
Analysis by RAPD-PCR involves the use of small random
primers and low stringency primer annealing conditions to
amplify arbitrary fragments of template DNA. The single
primer will anneal at any point on the genome where
SACCHAROMYCES j Introduction 299
Figure 1 Electrophoretic karyotypes of Saccharomyces species. Chro-
mosomal DNA of type strains was separated in 0.8% agarose gel in
0.5  Tris–borate EDTA (TBE) (45 mmol l1 TBE, pH 7.3, 1 mol l1 EDTA)
at 125 V and 14
C. Pulse times were 3 min for 24 h followed by 5 min
for 16 h.
a near-complementary sequence exists, and if two priming
sites are sufficiently close, then PCR amplifies the fragment
between them. A number of fragments of various sizes may
be produced; formed patterns are specific for the particular
DNA template used. This technique is suitable for typing
and identification of microorganisms, but several problems
are present. First, the whole patterns of electrophoresis
are not always the same in independent experiments, and
only the reproducible bands should be scored. Second, the
results are affected by the nucleotide sequence of the primer
used.
After the PCR products have been resolved, genetic distance
is calculated manually as the number of different bands
between two patterns divided by the sum of all bands in the
same patterns. A value of 0 indicates that the two strains had
identical patterns, and a value of 1 indicates that the two
strains had completely different patterns. The dice matrix
obtained from these data is used to construct an unrooted
dendrogram.
Ribosomal RNA Gene Analysis
The analysis of rRNA genes, which can elicit exact data without
pairing two samples, is one of the promising methods among
these tools applied to the phylogenetic study of yeast. In
S. cerevisiae, the co-transcribed genes for small (17S–18S), 5.8S,
and large (25S–28S) rRNA and 5S rRNA genes occur as tan-
demly repeated units on chromosome XII. Sequence compar-
isons of the rRNA genes have shown a relatively high degree of
evolutionary conservation and have been used as bases for
inferring phylogenetic relationships. The 18S rRNA gene
sequence of Saccharomyces species has been almost completely
sequenced and their relationships investigated in detail, but the
entire region of 18S rRNA is not simply and rapidly determined
by anyone.
The region spanning the internal transcribed spacers
(ITSs) and the entire 5.8S rRNA gene is amplified by PCR
using pITS1 (50 -TCCGTAGGTGAACCTGCGG-30 ) and pITS4
300 SACCHAROMYCES j Introduction

Table 3 Application of Saccharomyces species in the food industry

1. Industrial alcohol and alcoholic beverages

2. Bakery products
3. Biomass, extracts, autolyzates, and flavoring
compounds
Figure 2 Size of ITS regions amplified from the type strains
of Saccharomyces species.

nutrient media such as yeast extract–malt extract (YM) agar,

(50 -TCCTCCGCTTATTGATATG-30 ), which are derived from which is principally composed of yeast extract and malt
conserved regions of the 18S and 28S rRNA genes, respectively. extract. Potato-dextrose agar is suitable for storage of cultures
The size is over 800 bp for Saccharomyces sensu stricto species and but is not satisfactory for detection because each species
less than 800 bp for the other Saccharomyces species (Figure 2). develops a less characteristic colony on this medium. Colonies
Furthermore, restriction analysis of ITS region allows the of Saccharomyces and some species of Hansenula and Pichia,
separation of S. cerevisiae, S. bayanus, S. eubayanus, and which ferment glucose vigorously, are simply discriminated on
S. pastorianus. These may be useful methods to identify the agar plate: When medium containing 0.5% glucose, 0.05%
Saccharomyces isolates. 2,3,5-triphenyltetrazolium chloride, and 1.5% agar is overlaid
on the agar plate and incubated for 2–3 h at 30
C, the color of
the colony changes to pink or red.
Detection, Isolation, and Cultivation Selective isolation of yeasts and estimation of viable cell
number require special techniques to repress the growth of
Composition of media for yeast detection and isolation is bacteria and fungi. The use of acidified agar (<pH 5.0) is
shown in Table 3. The presence of yeasts in food and wild adequate for isolation of yeasts from samples containing
yeast in alcohol beverages is usually investigated using bacteria. When acidified medium alone is inadequate to
eliminate bacterial growth, chloramphenicol dissolved in
ethanol (20 mg ml1) is added at a final concentration of
50 mg ml1. Unlike other antibiotics that depress bacterial
Table 2 Composition of culture media
growth, chloramphenicol can be added to the medium before
Medium Contents Percentage w/v autoclaving. Addition of either 0.2% sodium propionate or
2–3% ethanol to an acidic medium has only limited effect on
YM Yeast extract 0.3 prolonging the growth of fungi. However, it is possible to
Malt extract 0.3
isolate yeasts selectively by the difference in growth rates in the
Peptone 0.5
presence of sodium propionate.
Glucose 1
Agar (if required) 2 Saccharomyces sensu stricto species frequently appear after
YPD Yeast extract 1 enrichment in YM broth in which glucose was added at
Peptone 2 10–20%. The addition of a suitable indicator such as brom-
Glucose 2 phenol blue permits the monitoring of changes in pH and
Agar (if required) 2 periodic adjustments if desired. Glucose and other components
Potato-dextrose agar Potato extracta 23 (volume) should be autoclaved separately to avoid browning. Less than
Glucose 2 0.1 g of sample is added to 10 ml of the modified YM broth
Agar 2 containing chloramphenicol and sodium propionate in a test
Yeast nitrogen base Yeast nitrogen base 0.67
tube. After static incubation for the desired period, about
(YNB glucose) without amino acidsb
0.1 ml obtained from near the bottom is transferred to another
Glucoseb 2
Agar (if required) 2 medium. The fermentation rate can be followed by recording
Fowell’s acetate agar Sodium acetate 0.5 the weight decrease of flasks due to the loss of the CO2. This
Agar 2 procedure is repeated several times to enrich yeasts fermenting
McClary’s acetate agar Glucose 0.1 glucose vigorously. The culture broth is successively diluted
Potassium chloride 0.18 and spread on a plate of acidified YM agar.
Yeast extract 0.25 For cultivation, Saccharomyces yeasts are generally grown on
Sodium acetate 0.82 yeast extract–peptone–dextrose (glucose) (YPD) medium (see
Agar 1.5 Table 2) rather than YM medium, at 25–30
C. The most
Gorodkowa agar (modified) Glucose 0.1
common system for aerobic growth uses liquid medium in an
Peptone 1
Erlenmeyer flask on an orbital shaker. Shaking at 150–200 rpm
Sodium chloride 0.5
Agar 2 provides efficient aeration for 100 ml volume of medium in
Malt extract agar Malt extract 5 a 300 ml flask. The growth rate varies depending on strain,
Agar 3 medium, and incubation temperature, typically within the range
of 90 min–3 h per doubling time. The cell concentration can be
a
The filtrate autoclaved for 1 h at 120
C after washed, peeled, and finely grated determined using an electronic particle counter or a hemocy-
potato (100 g) is soaked in 300 ml tap water for several hours in a refrigerator and
filtered through either cloths or membranes. tometer and microscope. Alternatively, a spectrophotometer
b
Tenfold concentrated solution is filter-sterilized and added. can be used to measure turbidity at 600 nm. Typically, a yeast

culture in a logarithmic phase of growth of 0.1 will contain
about 2  106 cells.
Ascospores are induced on the sporulation media, most of
which have been developed for Saccharomyces species. Young
cells grown on YM agar for 2–3 days are spread on Fowell’s or
McClary’s agar based on sodium acetate, Gorodkowa agar, or
malt extract agar, and incubated at least 4–6 weeks. Freshly
isolated cells sporulate on the isolation medium and asco-
spores can be observed after cultivation for about 1 month,
while the cells cultured on the nutrient medium often require
certain sporulation media to convert asci.
Importance to the Food Industry
The genus Saccharomyces is the most extensively utilized group
of yeasts for the benefit of humans. Saccharomyces cerevisiae
and related species are employed in three main processes of
the food industry (Table 3). The first is the production of
industrial alcohol and alcoholic beverages, including wine,
beer, sake, and potable spirits. Saccharomyces pastorianus
(including Saccharomyces carlsbergensis) was initially recog-
nized as a lager brewing strain. Saccharomyces bayanus has been
mostly associated with the wine industry. Second is the baking
industry; originally, spent yeasts from the brewing and
distilling industries were used for baking, but they became
insufficient as the baking industry expanded. Yeasts for dough
leavening are now propagated to meet these growing needs.
The third process includes the production of biomass, extracts,
autolyzates, and flavoring compounds. The yeast used in such
processes can be either purpose-grown or a by-product of
a related process.
Saccharomyces bayanus and its anamorph, Candida holmii, are
also (along with S. cerevisiae) responsible for the leavening of
sourdough, which is usually prepared by adding a commercially
produced culture containing lactic acid bacteria. No other
species of Saccharomyces is of commercial importance for baking,
although some strains of Torulaspora and Zygosaccharomyces spp.,
formerly accepted in the genus Saccharomyces, are used for
baking and the production of miso and shoyu, respectively.
Saccharomyces species are found in many foods and some-
times cause spoilage. Wild strains (unwanted strains) which
contaminate the pure culture reduce the fermentation rate and
diminish the quality of final beer in the brewing process. Killer
wild yeasts will dominate within a short period of time when
SACCHAROMYCES j Introduction 301
inoculated strains are killer-sensitive. In sake brewing and wine
fermentations, killer sake and wine strains were constructed by
the methods of backcrossing and cytoduction to overcome
these problems.
Most species of the genus Saccharomyces are ‘generally
recognized as safe’ owing to the fact that many strains have been
applied to the food and beverage industries. There is no
confirmed report of disease in healthy humans and other warm-
blooded animals caused by Saccharomyces sensu stricto species.
See also: Saccharomyces cerevisiae (Sake Yeast);
Saccharomyces: Saccharomyces cerevisiae; Saccharomyces:
Brewer’s Yeast.
Further Reading
Barnett, J.A., 1992. The taxonomy of the genus Saccharomyces Meyen ex Reess:
a short review for non-taxonomists. Yeast 8, 1–23.
Barnett, J.A., 2000. Yeasts: Characterization and Identification, third ed. Cambridge
University Press.
Benitez, T., 1996. Development of new strains for the food industry. Biotechnology
Progress 12, 149–163.
Evans, I.H., 1996. Yeast Protocols, Methods in Cell and Molecular Biology, vol. 53.
Humana Press, Ottawa.
James, A.H., 1997. A phylogenetic analysis of the genus Saccharomyces based on
18S rRNA gene sequences: description of Saccharomyces kunashirensis sp. nov.
and Saccharomyces martiniae sp. nov. International Journal of Systematic
Bacteriology 47, 453–480.
Kurtzman, C.P., 1994. Molecular taxonomy of the yeasts. Yeast 10, 1727–1740.
Kurtzman, C.P., 2005. Yeast Systematics and Phylogeny – Implications of Molecular
Identification Methods for Studies in Ecology. Springer, Berlin.
Kurtzman, C.P., Fell, J.W., 2011. The Yeast – A Taxonomic Study, fifth ed. Elsevier,
Amsterdam.
Panchal, C.J., 1990. Yeast Strain Selection. Marcel Dekker, New York.
Reed, G., Nagodawithana, T.W., 1991. Yeast Technology, second ed. Van Nostrand
Reinhold, New York.
Rose, A.H., Harrison, J.S., 1987. The Yeast, Biology of Yeasts, second ed. vols. 1 and 5.
Academic Press, London.
Russell, I., Jones, R., Stewart, G.G., 1987. Yeast – the primary industrial microorganism.
In: Biological Research on Industrial Yeasts, vol. 1. CRC Press, Boca Raton.
Spencer, J.F.T., Spencer, D.M., 1997. Yeast – In Natural and Artificial Habitats.
Springer, Berlin.
Vaughan-Martini, A., 1996. Synonomy of the yeast genera Saccharomyces Meyen ex
Hansen and Pachytichospora van der Walt. International Journal of Systematic
Bacteriology 46, 318–320.
Vaughan-Martini, A., 1996. Saccharomyces rosinii sp. nov., a new species of
Saccharomyces sensu lato (van der Walt). International Journal of Systematic
Bacteriology 46, 615–618.