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, form pseudohyphae but not septate hyphae. The yeasts are
basidiosporogenous yeasts, and imperfect yeasts. Saccharomyces predominantly diploid or occasionally of higher ploidy. Asci,
is the representative of ascosporogenous yeasts and historically which are persistent and usually transformed by direct change
is the most familiar microorganism to humans. This genus was from the vegetative cells, may contain one to four ascospores.
first described by Meyen when he assigned beer yeast as The ascospores are round or slightly oval, with smooth walls.
Saccharomyces cerevisiae in 1838, and it was redefined by Reess Conjugation occurs during or soon after germination of the
in 1870 from the observations of ascospores and their germi- ascospores. Some strains of S. cerevisiae and its related species
nation. The name is derived from the Greek words sakcharon used in the brewing, distilling, and baking industries hardly
(sugar) and mykes (fungus). The number of Saccharomyces form ascospores at all. Continuous selection with respect to
species has changed according to the criteria used to delimit their practical properties seem to cause loss of sporulation
species, and nine species are now accepted in the genus ability in these strains.
Saccharomyces (Table 1). 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
Characteristics of the Genus ethanol and CO2. These sugars include D-glucose, D-fructose,
D-mannose, and D-maltose except in the case of certain
The vegetative cells of Saccharomyces species are round, oval, or mutants. Most strains of Saccharomyces can grow on D-galactose
cylindrical and reproduce by multilateral budding. They may under aerobic or anaerobic conditions; however, none of them

Encyclopedia of Food Microbiology, Volume 3 http://dx.doi.org/10.1016/B978-0-12-384730-0.00290-1 297

298 SACCHAROMYCES j Introduction

Table 1 The species accepted in the genus Saccharomyces reassociation studies. Strains with 80–100% overall homology
of base sequences are considered as belonging to the same
Species Authority species, while the strains of distantly related taxa show
Saccharomyces arboricolus F.-Y. Bai and S.-A. Wang (2008) homology of less than 30%. Among the four species,
Saccharomyces bayanus Saccardo (1895) S. pastorianus reveals 53% homology to S. cerevisiae and 72%
Saccardo var. bayanus (2000) homology to S. bayanus, suggesting an intermediate position
Saccardo var. uvarum Naumov (2000) between two unrelated species, S. cerevisiae and S. bayanus
Saccharomyces cariocanus Naumov, James, Naumova, Louis, (see Saccharomyces: Brewer’s Yeast).
and Roberts (2000) Since species division within the Saccharomyces sensu stricto
Saccharomyces cerevisiae Meyen ex E. C. Hansen (1883) group were clarified at the molecular level, it became possible to
Saccharomyces eubayanus Libkinda et al. (2011)
determine those physiological responses necessary for separa-
Saccharomyces kudriavzevii Naumov, James, Naumova, Louis,
tion of the four taxa. Saccharomyces bayanus and S. eubayanus are
and Roberts (2000)
Saccharomyces mikatae Naumov, James, Naumova, Louis, the only species of the genus that can grow in the absence of
and Roberts (2000) vitamins. Maximum growth temperature immediately distin-
Saccharomyces paradoxus Bachinskaya (1914) guishes S. bayanus, S. eubayanus, and S. pastorianus, which never
Saccharomyces pastorianus Hansen (1904) 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
Kurtzman, C.P., 2005. Yeast Systematics and Phylogeny – Implications of Molecular transport system is present in the group of S. bayanus,
Identification Methods for Studies in Ecology. Springer, Berlin; Kurtzman, C.P., Fell,
J.W., 2011. The Yeast – A Taxonomic Study, fifth ed. Elsevier, Amsterdam. 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
utilizes lactose, pentose, alditols, and citrate as carbon sources, by S. cerevisiae of D-mannitol and fermentation of maltose.
assimilates nitrate as a nitrogen source, or hydrolyzes exoge- Further details of Saccharomyces species differences, particularly
nous urea. Among polysaccharides, starch and pectin are as they apply to brewer’s yeast species, can be found in Chapter
exceptionally utilized by certain (not all) strains of S. cerevisiae. Saccharomyces: Brewer’s Yeast.
They do not produce starch-like compounds. Their ubiquinone
is exclusively Q-6, but this feature is common in the genera
Saccharomyces sensu lato
Kluyveromyces, Torulaspora, and Zygosaccharomyces.
Saccharomyces kudriavzevii and Saccharomyces mikatae are unusual
members of the genus as judged from narrow fermentative
Identification of Saccharomyces Species profiles and the ability to grow in the presence of 0.1% cyclo-
heximide. Assimilation of ethylamine, cadaverine, and lysine
Yeasts are usually classified by the characteristics of microscopic can differentiate these two species.
appearance, sexual reproduction, and physiological features, Saccharomyces cerevisiae is characterized by much lower
including (1) fermentation of certain sugars semianaerobi- G þ C values (34.7–36.6%) than other Saccharomyces species
cally; (2) assimilation of various compounds each as sole (39.3–41.9%), but do not grow in the presence of 0.1%
carbon or nitrogen source; (3) growth without an exogenous cycloheximide. Saccharomyces arboricolus differs from S. bayanus
supply of certain vitamins; (4) growth in the presence of 50% in the assimilation of glycerol as a sole carbon source and
or 60% (w/w) glucose; (5) growth at 37
C; (6) growth in the ethylamine, cadaverine and lysine as sole nitrogen sources.
presence of cycloheximide; (7) splitting of fat, production of
starch-like polysaccharides, hydrolysis of urea; and (8) forma-
Saccharomyces kudriavzevii
tion of acid.
Saccharomyces kudriavzevii is easily distinguishable from other
species of this genus since it is characterized by a wide assim-
Saccharomyces sensu stricto
ilative and fermentative profile, including the ability to utilize
Saccharomyces sensu stricto species, including S. cerevisiae, ethylamine-HCl, cadaverine, and lysine as sole nitrogen sour-
Saccharomyces bayanus, Saccharomyces paradoxus, and Saccharo- ces for growth as well as the ability to both assimilate and
myces pastorianus, are phylogenetically closely related in the ferment melibiose. The distinct character was already antici-
genus Saccharomyces. The species S. cerevisiae, S. bayanus, pated by molecular taxonomic studies which showed no
Saccharomyces eubayanus, and S. pastorianus are specifically found nucleotide homology between S. kudriavzevii with either
in the environments of wineries and breweries. The relative Saccharomyces sensu stricto or sensu lato strains, where DNA
genome sizes of these three species are estimated to be 1.00, homology values were never above 22%.
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 Molecular Methods to Differentiate Species
form asci compared with the other three species. Effective
separation of the Saccharomyces sensu stricto species is compli- The conventional taxonomic tests to assess physiological
cated because these species often have apparently identical features are fundamental for identification, but the results have
morphological, physiological, and serological properties. The shown to be insufficient for species delimitation and discrimi-
four species have been differentiated from each other by DNA nation of interstrain variability. The genetic basis behind many
 SACCHAROMYCES j Introduction 299

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- Figure 1 Electrophoretic karyotypes of Saccharomyces species. Chro-
morphism patterns, and rRNA gene sequencing. These methods mosomal DNA of type strains was separated in 0.8% agarose gel in
are valid additions (not replacements) to the conventional 0.5  Tris–borate EDTA (TBE) (45 mmol l1 TBE, pH 7.3, 1 mol l1 EDTA)
taxonomic tests, and those applicable to the food and beverage at 125 V and 14
C. Pulse times were 3 min for 24 h followed by 5 min
industry are described below and in Chapters Saccharomyces: for 16 h.
Saccharomyces cerevisiae and Saccharomyces: Brewer’s Yeast.

a near-complementary sequence exists, and if two priming

Electrophoretic Karyotypes sites are sufficiently close, then PCR amplifies the fragment
Chromosomal patterns resolved by pulse field gel electropho- between them. A number of fragments of various sizes may
resis are called electrophoretic karyotypes. By comparing the be produced; formed patterns are specific for the particular
results from this method and DNA reassociation, it has been DNA template used. This technique is suitable for typing
demonstrated that electrophoretic karyotypes of the two strains and identification of microorganisms, but several problems
are identical when DNA sequence homology is over 85%, while are present. First, the whole patterns of electrophoresis
low DNA relatedness corresponds to completely different are not always the same in independent experiments, and
chromosomal patterns. Since similar, but not identical, only the reproducible bands should be scored. Second, the
karyotypes are not interpreted as either different species or results are affected by the nucleotide sequence of the primer
polymorphisms in the same species, karyotyping is not as used.
reliable as DNA base sequence comparisons, but is undoubt- After the PCR products have been resolved, genetic distance
edly an important adjunct. Electrophoretic karyotypes can serve is calculated manually as the number of different bands
as a rapid, inexpensive, and relatively easy first approach for between two patterns divided by the sum of all bands in the
evaluation of a group of physiologically similar strains. same patterns. A value of 0 indicates that the two strains had
The general feature for ascosporogenous yeasts is the identical patterns, and a value of 1 indicates that the two
presence of one to five bands of chromosomal DNA larger strains had completely different patterns. The dice matrix
than 1000 kb as in S. kudriavzevii (Figure 1), whereas in obtained from these data is used to construct an unrooted
most Saccharomyces species, chromosomes smaller than dendrogram.
1000 kb are observed. Chromosomes of Saccharomyces sensu
stricto species were resolved into 12–16 bands in the range
Ribosomal RNA Gene Analysis
200–2200 kb. None of the other species contains chromo-
somes smaller than 300 kb. The patterns of Saccharomyces The analysis of rRNA genes, which can elicit exact data without
sensu stricto species are similar and are distinguishable from pairing two samples, is one of the promising methods among
the other species at a glance. A multivariate analysis of the these tools applied to the phylogenetic study of yeast. In
polymorphisms in the numbers and molecular weights of S. cerevisiae, the co-transcribed genes for small (17S–18S), 5.8S,
chromosomes has revealed that the Saccharomyces sensu stricto and large (25S–28S) rRNA and 5S rRNA genes occur as tan-
strains could be separated into four clusters that correspond demly repeated units on chromosome XII. Sequence compar-
to the four species. 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
Random Amplified Polymorphic DNA Polymerase sequence of Saccharomyces species has been almost completely
Chain Reaction sequenced and their relationships investigated in detail, but the
entire region of 18S rRNA is not simply and rapidly determined
Analysis by RAPD-PCR involves the use of small random by anyone.
primers and low stringency primer annealing conditions to The region spanning the internal transcribed spacers
amplify arbitrary fragments of template DNA. The single (ITSs) and the entire 5.8S rRNA gene is amplified by PCR
primer will anneal at any point on the genome where 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
 SACCHAROMYCES j Introduction 301

culture in a logarithmic phase of growth of 0.1 will contain inoculated strains are killer-sensitive. In sake brewing and wine
about 2  106 cells. fermentations, killer sake and wine strains were constructed by
Ascospores are induced on the sporulation media, most of the methods of backcrossing and cytoduction to overcome
which have been developed for Saccharomyces species. Young these problems.
cells grown on YM agar for 2–3 days are spread on Fowell’s or Most species of the genus Saccharomyces are ‘generally
McClary’s agar based on sodium acetate, Gorodkowa agar, or recognized as safe’ owing to the fact that many strains have been
malt extract agar, and incubated at least 4–6 weeks. Freshly applied to the food and beverage industries. There is no
isolated cells sporulate on the isolation medium and asco- confirmed report of disease in healthy humans and other warm-
spores can be observed after cultivation for about 1 month, blooded animals caused by Saccharomyces sensu stricto species.
while the cells cultured on the nutrient medium often require
certain sporulation media to convert asci. See also: Saccharomyces cerevisiae (Sake Yeast);
Saccharomyces: Saccharomyces cerevisiae; Saccharomyces:
Brewer’s Yeast.
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 Further Reading
the food industry (Table 3). The first is the production of
Barnett, J.A., 1992. The taxonomy of the genus Saccharomyces Meyen ex Reess:
industrial alcohol and alcoholic beverages, including wine,
a short review for non-taxonomists. Yeast 8, 1–23.
beer, sake, and potable spirits. Saccharomyces pastorianus Barnett, J.A., 2000. Yeasts: Characterization and Identification, third ed. Cambridge
(including Saccharomyces carlsbergensis) was initially recog- University Press.
nized as a lager brewing strain. Saccharomyces bayanus has been Benitez, T., 1996. Development of new strains for the food industry. Biotechnology
mostly associated with the wine industry. Second is the baking Progress 12, 149–163.
Evans, I.H., 1996. Yeast Protocols, Methods in Cell and Molecular Biology, vol. 53.
industry; originally, spent yeasts from the brewing and Humana Press, Ottawa.
distilling industries were used for baking, but they became James, A.H., 1997. A phylogenetic analysis of the genus Saccharomyces based on
insufficient as the baking industry expanded. Yeasts for dough 18S rRNA gene sequences: description of Saccharomyces kunashirensis sp. nov.
leavening are now propagated to meet these growing needs. and Saccharomyces martiniae sp. nov. International Journal of Systematic
Bacteriology 47, 453–480.
The third process includes the production of biomass, extracts,
Kurtzman, C.P., 1994. Molecular taxonomy of the yeasts. Yeast 10, 1727–1740.
autolyzates, and flavoring compounds. The yeast used in such Kurtzman, C.P., 2005. Yeast Systematics and Phylogeny – Implications of Molecular
processes can be either purpose-grown or a by-product of Identification Methods for Studies in Ecology. Springer, Berlin.
a related process. Kurtzman, C.P., Fell, J.W., 2011. The Yeast – A Taxonomic Study, fifth ed. Elsevier,
Saccharomyces bayanus and its anamorph, Candida holmii, are Amsterdam.
Panchal, C.J., 1990. Yeast Strain Selection. Marcel Dekker, New York.
also (along with S. cerevisiae) responsible for the leavening of Reed, G., Nagodawithana, T.W., 1991. Yeast Technology, second ed. Van Nostrand
sourdough, which is usually prepared by adding a commercially Reinhold, New York.
produced culture containing lactic acid bacteria. No other Rose, A.H., Harrison, J.S., 1987. The Yeast, Biology of Yeasts, second ed. vols. 1 and 5.
species of Saccharomyces is of commercial importance for baking, Academic Press, London.
Russell, I., Jones, R., Stewart, G.G., 1987. Yeast – the primary industrial microorganism.
although some strains of Torulaspora and Zygosaccharomyces spp.,
In: Biological Research on Industrial Yeasts, vol. 1. CRC Press, Boca Raton.
formerly accepted in the genus Saccharomyces, are used for Spencer, J.F.T., Spencer, D.M., 1997. Yeast – In Natural and Artificial Habitats.
baking and the production of miso and shoyu, respectively. Springer, Berlin.
Saccharomyces species are found in many foods and some- Vaughan-Martini, A., 1996. Synonomy of the yeast genera Saccharomyces Meyen ex
times cause spoilage. Wild strains (unwanted strains) which Hansen and Pachytichospora van der Walt. International Journal of Systematic
Bacteriology 46, 318–320.
contaminate the pure culture reduce the fermentation rate and Vaughan-Martini, A., 1996. Saccharomyces rosinii sp. nov., a new species of
diminish the quality of final beer in the brewing process. Killer Saccharomyces sensu lato (van der Walt). International Journal of Systematic
wild yeasts will dominate within a short period of time when Bacteriology 46, 615–618.