Trends in Food Science & Technology 10 (1999) 356±365
Review
The prevalence and
control of spoilage
yeasts in foods and
beverages
V. Loureiro and A. Queroly

Departamento de BotaÃnica e Engenharia BioloÂgica,
Instituto Superior de Agronomia, Universidade
TeÂcnica de Lisboa, 1349-017 Lisboa Codex, Portugal
(1fax: 351 21 363 50 31; e-mail: vloureiro@isa.utl.pt)
y
Departamento de Biotecnologia, Instituto de
AgroquõÂmica y Tecnologia de Alimentos (CSIC), P.O.
Box 73, 46100 Burjassot, Valencia, Spain (2fax: 34 96
363 63 01; e-mail: aquerol@iata.csic.es)
Consumers in developed countries have a more critical
attitude about what they eat and drink as a consequence of
modern life. Food microbiologists are facing a huge chal-
lenge regarding food `freshness' implicit in the consumer's
demand for more natural products. Food with less severe
processing, that is additive-free, safer, with satisfactory shelf
life and easy to prepare is sought because of higher con-
sciousness about nutrition and health. Besides the develop-
ment of new methods and preservation concepts, great
advances in the ®elds of chemistry, biochemistry, micro-
biology, hygiene and food safety have occurred in recent
decades. But are such developments properly used to
respond to the challenge to food mycology? We will sum-
marize the e€ects on food of yeast spoilage activity and
we will re¯ect on how this activity can be monitored by
food microbiologists. # 2000 Published by Elsevier Science
Ltd.
The overviews on food spoilage yeasts published
about 30±40 years ago [1,2] reported that food-borne
yeasts had no signi®cance in public health, being regarded
as harmless for the consumers, even when ingested in high
amounts through fermented foods (e.g. lambic beer,
0924-2244/99/$ - see front matter Copyright # 2000 Published by Elsevier Science Ltd.
PII: S 0 92 4 - 2 24 4 ( 0 0 ) 0 0 02 1 - 2
ke®r, kuomiss, some raw milk smear ripened cheeses,
etc.). However, Fleet [3], mentioning works of the
1980s, refers for the ®rst time, to the need to re-evaluate
the impact of food-borne yeasts on public health/safety.
This author cites occasional reports of gastro-enteritis
caused by the ingestion of foods in which the suspected
etiological agents were yeasts. In addition, a report is
cited referring to increasing evidence for the develop-
ment of allergies and negative e€ects in humans due to
yeasts, which has led to the coming up of lay literature
promoting the virtues of `yeast-free' diets. Thomas [4]
also cites a reference stating that the exposure to anti-
gens from Saccharomyces cerevisiae may contribute to
in¯ammatory bowel disease. In a recent review on new
and emergent yeast pathogens, Hazen [5] states, as well,
that in the medical ®eld, the infections caused by yeasts
are increasingly signi®cant, and the author attributes such
situations to, among other factors, immunosuppressive
therapeutic regimens, long-term catheterization, broad-
spectrum antibiotic use, and longer survival of immuno-
logically compromised individuals. He states that some
yeasts previously thought as innocuous (e.g. Candida
utilis, Saccharomyces cerevisiae and Candida lipolytica)
are capable of damaging the human body. Although the
cases reported are rare and occur in extremely peculiar
situations, we have to admit the possibility that the
presence of yeasts in foods, either spoilers or not, may
assume an increasing protagonism in public health and
should be a growing concern for microbiologists and
food technologists.
The spoilage yeasts of foods and drinks have also
gained, in recent years, an increasing importance in
food technology, being responsible for signi®cant eco-
nomic losses [4]. However, reports on these losses are
still rare. For reasons of commercial con®dentiality, the
incidence and economic cost of industrial outbreaks of
yeast spoilage remain unreported [3]. According to this
author and also based on our experience of outbreak
costs, these are considerably high and involve large legal
disputes involving manufacturers, suppliers of raw
materials and packaging, and, often, retailers.
In broad terms, the comprehensive overviews on
spoilage yeasts published in the second half of the 20th
century were focused, essentially, in the qualitative
description of the main yeast species present in foods
and beverages. Barnett et al. [6], listed nearly 120 yeast
species, belonging to 30 genera, as being associated with
 V. Loureiro, A. Querol / Trends in Food Science & Technology 10 (1999) 356±365
foods, which represented around 25% of those accepted
by the authors and 50% of the recognized genera.
According to Pitt and Hocking [7], most of these 120
species grow poorly, if at all, in properly formulated
foods, as they are intolerant to reduced water activity,
heat processing or preservatives. These authors consider
that only about 10 species of yeastsÐDekkera brux-
ellensis, Issatchenkia orientalis, Debaryomyces hansenii,
Kloeckera apiculata, Pichia membranifaciens, Zygo-
saccharomyces bailii, Zygosaccharomyces bisporus, Sac-
charomyces cerevisiae, Zygosaccharomyces rouxii, Schizo-
saccharomyces pombe and Candida holmii (Saccharomyces
exiguus)Ðare responsible for the spoilage of foods that
have been processed and packaged according to the
normal standards of good manufacturing practice
(GMP).
Tudor and Board [8] in their excellent comprehensive
overview on food spoilage yeasts considered, besides the
`top-ten' spoilage yeasts listed by Pitt and Hocking [7], a
group of `second-division yeasts' constituted by 18 spe-
cies and one genus of common food contaminant yeasts,
which can be opportunist spoilers. These authors con-
sidered it important to investigate the interactions
between these species and acid-tolerant spoilage bac-
teria, considering the production of a wide range of raw
products such as ready-to-use salads, dairy desserts, etc.
Fleet [3], in his innovative overview, concluded that
yeasts occur and grow in a greater range of products
than was previously thought. He emphasized that further
advances in understanding the spoilage yeast activities
would require a more quantitative approach to study
their occurrence and growth in speci®c products, and
more detailed investigations of their biochemical trans-
formations. This author compiled, for the ®rst time, the
levels of incidence of yeast contaminants from di€erent
food commodities, referring to the maximum levels
attained, as well as the species involved, when the
environmental conditions favour their growth and the
symptoms of spoilage begin to come up.
Deak and Beauchat [9] were the ®rst authors to, based
on the compilation from the vast literature data, have
estimated the frequencies of occurrence of yeast species in
the major types of foods. The estimation of frequencies
has been calculated considering three parameters: (1)
the number of types of food from which a given species
is detected; (2) the number of times a given species is
described from foods; and (3) a commonness index
value, varying from 1 to 8, based on the number of
known strains of a given species listed in standard
descriptions and/or which have been isolated from
foods. The estimated frequency of occurrence is then
expressed as a percentage of the sum of products of the
three entries for all species considered [9]. Although the
criteria used by the authors may be controversial and
the purpose of the work was not to evaluate the inci-
dence of yeasts in spoiled foods, the huge volume of
357
information available should be useful in developing a
methodology to evaluate the prevalence of yeasts in
foods.
A rapid scan of the works published on food altera-
tion by yeasts, since the classical review of Ingram [1] to
the state of the art book of Deak and Beuchat [9], leads
to the conclusion that little has changed, in terms of
concepts, methodologies and comprehensiveness of the
biological processes involved. From this, a question
arises: is the proportion of yeast-spoiled products higher
today than in the past? In our opinion the answer is not
easy nor, certainly, consensual. On one hand, because of
the diculty in obtaining information on yeast spoilage
outbreaks and, on the other hand, because the qualita-
tive analysis of the past (or of the present) do not enable
a sound evaluation of the true extension of food
spoilage or food depreciation caused by yeasts. A recent
example in the wine industry illustrates well this last
statement. The yeasts of the genus Brettanomyces/
Dekkera have been known for a long time as o€-
¯avour producers. However, due to their slow growth
they were seldom detected in wine microbiological con-
trol, being regarded as secondary-spoilers or, more
properly, rarely-spoilers. When Chatonnet et al. [10]
identi®ed these yeasts as the main producing agents of
`horse sweat' taint in red wines, because of their ability
to produce 4-ethylphenol from p-coumaric acid, the
detection of Brettanomyces/Dekkera and the analysis of
4-ethylphenol became much more important. As a
result, these yeasts became unexpected protagonists in
wine microbiology. Nowadays, these yeasts are recog-
nized as responsible for the depreciation of a signi®cant
proportion of red wines, particularly those produced
under poor hygiene conditions and/or matured in oak
barrels, which are a well-known ecological niche for
these species.
The extent and e€ects of yeast spoilage
Food spoilage caused by yeasts consists in the visible
or detectable alteration of physical and sensorial prop-
erties of food as a result of their activity. The most
known alterations occur in acid drinks, with or without
sugar, and are characterized by abundant gas production,
which may deform or blow up the packages, cloudiness,
sediment or pellicle formation, o€-¯avours dominated by a
slight fermentation smell (alcohol, carbon dioxide and
esters) and o€-tastes. Deterioration of food and drinks
by yeasts may present other e€ects, more or less evident,
according to the type of food (Table 1). However, in
certain cases, yeast spoilage is not so easily de®ned,
mainly in fermented foods/drinks (e.g. wines, artisanal
beers, black olives, soy sauce) where the produced
metabolites contribute to the ¯avour and aroma of the
products. As Fleet [3] stated there is only a slight line
between what is perceived as either spoilage or bene-
®cial activity.
358 V. Loureiro, A. Querol / Trends in Food Science & Technology 10 (1999) 356±365

Table 1. Principal spoilage e€ects caused by yeast activity in foods

Spoilage e€ect
Food Product Surface Discolouration Gas Haze/ Sediments Films O€-¯avours/ Texture
growth production cloudiness souring changes
Fresh vegetables x x x x
Brined vegetables x x x x x x
``Ready-to-eat'' vegetables x x x
Fresh fruits x x x x
Fruit juices x x x x x
``Ready-to-eat'' fruits x x x
Mayonnaise, salad dressings x x x x x
Wine, beer x x x x x
Soft drinks x x x x
Confectionery, jams, jellies x x x x x x x
Syrups, honey, fruit concentrates x x x x
Butter, cream x x
Cheeses x x x
Yoghurts x x x
Sliced bread x x x
Unbaked bread dough x x x
Sausages, meat products x x x x

A good example is the former example of 4-ethylphe-
nol production by Brettanomyces/Dekkera yeasts in red
wines. In fact, when this secondary metabolite is present
at levels less than about 400 g/l it does not depreciate
wine quality. It may, even, contribute to wine complexity
by imparting aromatic notes of spices, leather, smoke or
hunt, appreciated by many consumers. Nevertheless, at
levels higher than 700 g/l the wines are clearly depre-
ciated.
The growth of yeast populations in food commodities
to levels as high as 105 to 108 cells/g is not, in many
cases, a clear indicator of product deterioration. In
many cheeses these levels do not cause detectable
depreciation, as well as in cloudy beers, where they are
supposed to cause an appreciable aromatic complexity.
The inverse situation also occurs, when yeast counts are
less than 105 cells/g and visible spoilage e€ects may be
detectable. This is the case when yeast strains are able to
form cell clusters in bottled white wines, suggesting
incorrect ®ltration, or in certain solid foods (e.g. skinless
sausages, sliced bread, etc.) where the presence of yeast
colonies on the surface gives colour spots, white or pink,
according to the yeast species present.
In conclusion, the establishment of microbiological,
chemical, physical or sensorial parameters to evaluate
food spoilage caused by yeasts should account for the
nature of the food and on the type of e€ect resulting
from their activity.
Limitations of food mycology
Food spoilage is a competitive process in which, as in
other forms of competition, the race tends to go to the
strongest at the start and quickest o€ the mark [1].
When a food commodity is spoiled by yeasts, it means
that they were previously present as contaminants and
environmental conditions made yeasts stronger and
quicker than bacteria and moulds. The prevention of
such spoilage processes, with fewer chemicals and less
severe preservation processes, requires a clear under-
standing of the problem and for that, the food micro-
biologist should possess tools to evaluate the whole
microbiological ecosystem. In fact, many of the prob-
lems caused by yeasts results from the much higher
attention paid to bacteria and moulds, which are more
signi®cant in terms of public health. Their inhibition
opens the way to yeast activity. But have the food
microbiologists the tools that allow them to be su-
ciently aware of the microbiological problems of a food
commodity?
According to Mossel et al. [11] in a code of good
manufacturing and distribution practices (GMDPs)
two ecological distinct groups of measures are essential:
(i) limiting contaminants by selecting good quality raw
materials, monitoring the proper functioning of pro-
cesses designed to control microbes, and preventing
cross contamination; and (ii) limiting colonization by
arresting or retarding microbial growth.
If we take a closer look at these measures, some of
them have a rather subjective character, as the `good
quality raw materials' or `monitoring the proper func-
tioning of processes' that are concepts with a degree of
demand directly related with the technological level of
the companies and with the scienti®c and cultural level
of their technical sta€. Is the microbial ecology of foods
able to render objective these concepts? The concept of
`arresting or retarding growth' depends as well on the
ability of the microbiologist to monitor the growth
kinetics of mixed populations in the processing and dis-
tribution chain. Is this possible? If yes, to what extent
can he do it?
 V. Loureiro, A. Querol / Trends in Food Science & Technology 10 (1999) 356±365
The above mentioned questions are not usually
raised by the food microbiologists or technologists
working in the industry, particularly when it concerns
yeast populations, who rely on the usual micro-
biological techniques and on the implemented HACCP
systems. Their doubts only come up when an outbreak
occurs. The limitations of the techniques are then
recognized and they become concerned, improving the
methodologies temporarily. But once the e€ect of the
outbreak is gone the usual unworried attitude comes
back.
Despite the scienti®c innovation that occurred in the
past years on methods to identify microorganisms, the
majority of the standard methods of food analyses, as
well as other microbiological methods used in the food
industry, keep using the classical steps established
several decades ago: (i) maceration/blending of the
sample; (ii) decimal dilution of the homogenate; (iii)
plating of the dilutions onto appropriate agar media or
inoculation into multiple tubes containing liquid media;
and (iv) isolation, puri®cation, and identi®cation of
colonies. Sometimes, in addition, selective or pre-
enrichment techniques are necessary prior to plating
(low levels of contamination or stressed cells) but with a
more frequent use for bacteria than for yeast or moulds.
Nevertheless, these procedures do not provide a `real
image' of the microbial ecosystem of foods. A recent
review [12] made for microorganisms in general, pre-
sents a detailed discussion of the main limitations and
errors of the classical procedures which, as a rule, are
not generally questioned. For this reason we will focus
only on the main technical limitations to isolate and
enumerate yeast that, in our opinion, do really occur in
food and beverage industries.
Isolation and enumeration media for food-borne
yeasts
As a rule, the general isolation and enumeration
media for food-borne yeasts are the same as those used
for food-borne moulds. Basically, these are complex
media, nutritionally rich, containing an energy source
(e.g. glucose, fructose, sucrose), a digested protein (e.g.
peptone, tryptone, casitone), a complex vitamin supple-
ment (e.g. yeast extract, malt extract), one or more
antibiotics against bacteria (oxytetracycline, chlortetra-
cycline, chloramphenicol), a compound to inhibit the
most rapidly spreading moulds (e.g. rose Bengal,
dichloran), sometimes a pH indicator (e.g. bromocresol
green), agar or not, depending on its use as solid or
broth media. Many studies have demonstrated that
these media are generally superior to the earlier acidi®ed
media (with organic or inorganic acids to get a pH value
around 3.5) in terms of yeast recovery [13]. Unfortu-
nately, they have a great limitation: they do not distin-
guish between the dangerous spoilage yeasts and the
others, which we call `innocent yeasts'.
359
Selective and/or di€erential media: a promising future
The most comprehensive approaches on the use of
selective and/or di€erential media for isolation or enu-
meration of particular yeast species have been conducted
in the brewing and wine industries, where the goal is to
develop media that selectively di€erentiate Saccharo-
myces cerevisiae, spoilage species within the genus Sac-
charomyces, and spoilage species in other genera [3].
The best example of this approach is the use of the
classical Lysine agar, largely used in brewing industry,
to assess the contamination level of beer with non-
Saccharomyces species, since the former cannot utilise
lysine as a nitrogen source. Other media with similar
purposes have been developed, such as Schwarz di€er-
ential medium and Lin's wild yeast di€erential agar.
The most used strategy to design speci®c media to
isolate/enumerate yeast resistant to a given compound
has been to supplement a general-purpose medium with
that compound. Thus, selective media have been devel-
oped to assess the presence in foods of yeasts resistant
to ethanol, preservatives and reduced water activity.
However, these media have, generally, the disadvantage
to require longer incubation periods (up to one week)
than the normal media, given the stress induced on the
yeast cells. Among these media, standard malt agar
supplemented with 0.5% of acetic acid is currently used
to detect preservative-resistant yeasts. Numerous solid
media have been formulated for isolation and enu-
meration of fungi capable of growing at low aw values.
However, none is completely selective for xerotolerant
yeasts and only one, dichloran 18% glycerol (DG18)
agar, is commercially available [13]. Di€erent approa-
ches based on the evaluation of product susceptibility to
spoilage yeast have been proposed to design shelf life
tests [14]. These are based on pre-enrichment proce-
dures, which promote the emergence of dangerous
yeasts to the product stability.
Di€erential media seem to be much more promising
than selective media, because they do not involve, gen-
erally, extended incubation times. However, there are
very few di€erential media for food-borne yeasts and
much less than those for food-borne bacteria. One
strategy used to design yeast di€erential media is based
on the capacity of the yeasts to hydrolyse certain poly-
mers such as polysaccharides, proteins, pectins and
lipids. Some of these media, although with small speci-
®city, have been utilized to detect the presence of
hydrolytic yeasts in food where these compounds are
present in high-levels [3].
Perhaps the best succeeded di€erential medium for
yeast ever designed has been the medium of Chaskes
and Tyndall [15] to distinguish the non-food pathogen
Cryptococcus neoformans. The authors' strategy was
based on the species unique capacity to produce, under
appropriate environmental conditions, dark pigments
from l-2,3-dihydroxyphenylalanine (l-Dopa). This
360 V. Loureiro, A. Querol / Trends in Food Science & Technology 10 (1999) 356±365
feature is so speci®c and consistent among the strains of
this species that it has been adopted by yeast taxono-
mists as a relevant characteristic of the species. The
same authors, recently succeeded in distinguishing several
species of Cryptococcus based on their capacity to pro-
duce brown pigments from several aminophenols and
diaminobenzenes. Surprisingly this promising strategy
to design di€erential culture media for yeast, to our
knowledge, was never exploited again, until it was retaken
by the work of Carreira and Loureiro [16]. They di€er-
entiated Yarrowia lipolytica based on the production of
brown pigments from tyrosine, the detection of this
species being possible within 24 hours.
The selective properties of triarylmethane dyes and
di€erential responses of yeasts isolated from meat prod-
ucts have been also explored [17]. The results presented
by these authors suggest that this approach is promising
for developing new di€erential media for yeast isolation
and presumptive identi®cation.
The use of conjugated substrates for speci®c detection
of b-glucosidase enzyme on solid media was recently
exploited to design a chromogenic medium for the
detection of Debaryomyces hansenii from intermediate
moisture foods [18]. Using a similar strategy the same
research group had already developed a di€erential
medium (under patent protection) to detect Kluyver-
omyces marxianus and K. lactis, based on the expression
of the enzyme b-galactosidase [19].
The di€erent ability of various spoilage yeasts to grow
in a mineral medium containing glucose and formic
acid, as the only carbon and energy sources, was
recently exploited to design a selective/di€erential medium
(under patent registration) to Zygossacharomyces bailii
and Z. bisporus [20]. The nature of the acid and its
concentration as well as the pH of the medium, asso-
ciated with the incorporation of an acid-base indicator
ensured that only dangerous species gave positive
responses within 48 hours. Another selective and di€er-
ential medium (also under patent registration), designed
for species of Dekkera/Brettanomyces, is based on the
capacity of these species to produce 4-ethylphenol from
p-coumaric acid, which is very easily detectable by its
peculiar odour. In this medium, ethanol is the carbon
and energy source and the typical production of acetic
acid by these species is detected by the presence of an
acid-base indicator [21].
The new strategies developed recently create the pos-
sibility to build, in the near future, a set of di€erential
media for the main spoilage yeasts found in foods and
provide excellent opportunities to improve the control
of these contaminant microorganisms by the industry.
Yeast typing/identi®cation
Yeasts have been classi®ed on the basis of morphologi-
cal, sexual, biochemical, and physiological properties for
more than 50 years. Physiological properties, primarily,
serve to describe and to identify species and, to a very
minor extent, genera. The tests most used for routine
identi®cation purposes are fermentation of, and growth
on, carbon sources, growth on nitrogen sources,
requirements of vitamins, growth at various tempera-
tures and on media with a high content of sugar or
sodium chloride, hydrolysis of urea and resistance to
antibiotics [22]. This author emphasized that there is not
a single standardized method for many of these tests
and their results are often dependent on the techniques
employed. In addition to this weakness, many results of
the tests are variable for di€erent strains of the same
species, giving rise to frequent misidenti®cation. As
referred by von Arx [23], this type of approach resulted
in heterogeneous taxa, as well as the description of
identical species under di€erent names. This author also
referred to the description of numerous super¯uous
`species' distinguished mainly by the assimilation pat-
terns resulting in a classi®cation, which could only be
applied by a few specialists. Besides all the mentioned
limitations, it is generally necessary to conduct about
50±100 tests in order to reliably identify yeasts at the
species level, and one to two weeks, at least, are required
to obtain a ®nal result. Obviously, this identi®cation
scheme cannot be routinely utilized by the food industry
and various miniaturized and simpli®ed identi®cation
methods, based on yeast response to a few carefully
selected tests, have been developed [9]. However, they
are based on the same approach of the classical method
of yeast identi®cation, being time consuming, even when
procedures are automated and computerized, and con-
ducting, often, to false or equivocal identi®cations. To
overcome these diculties, alternative faster typing
methods have been developed, based, among other
things, on analysis of total proteins or long-chain fatty
acids of the yeast cell or based on its isoenzyme pro®le.
Besides, recent progress in molecular biology has led to
the development of new techniques for yeast identi®ca-
tion and characterization. These include, for example,
RFLP of mitochondrial DNA, chromosomal DNA
electrophoresis, ribosomal DNA restriction analysis,
and RAPDs. However, most of these techniques are
useless for the routine identi®cation of yeasts and,
usually, there is no available database to identify a high
number of species. The majority of these techniques
were applied to characterize only some genera, so we
cannot use them as a general method to yeast identi®-
cation. Because of the relevance that such methods can
have to the food industry in the future, some of them
will be described in general terms.
Long-chain fatty acids
The use of fatty acid pro®ling as a biomarker has not
been successfully used to type spoilage yeasts in the
food industry. Certain problems have been suggested:
dependence of total fatty acid composition on yeast
 V. Loureiro, A. Querol / Trends in Food Science & Technology 10 (1999) 356±365 361

growth conditions, low variability of yeast fatty-acid
composition, time-consuming and technique costs, need
of highly skilled sta€ to perform it, old fashioned tech-
nique, etc. However, such prejudices are not real,
because it is possible to standardize growth conditions
(even in plate media) in order to minimize the variation
of total lipid composition, the results can be obtained
within two days after yeast isolation and puri®cation (it
may take longer if the strains are slow-growing species),
the technique is not more expensive than most of other
rapid typing techniques available [24]. We consider that
this technique has the potential to be used in the food
industry, because fatty acid pro®ling permits the typing
of most of the relevant species in the food industry, to
separate species according to their spoilage potential
and to trace yeast contamination in bottling plants, as
shown in the wine industry Malfeito-Ferreira et al. [23].
The principal advantage of this approach is to separate
the food-borne yeasts into three major groups based on
the presence of linoleic (C18:2) and linolenic (C18:3)
fatty acids (see Table 2), which have, for most of the
food industry, a technological meaning. Thus, the pres-
ence of most potential spoilage yeasts can be indicated
by signi®cant amounts of C18:2 and absence of C18:3
fatty acids in yeast cells; the presence of C18:2 and
C18:3 fatty acids in yeast cells probably re¯ects the
presence of yeast species normally associated with poor
hygiene or insucient use of preservatives; the absence
of polyunsaturated C18 acids suggests the presence of
fermentative strains which can be spoilers, such as those
belonging to the Saccharomyces genus. To those who
are not familiar with this technique, it may seem dicult to
implement it at industrial level. However, most diculties
are more apparent than real. In fact, the number of
dominating species in various food commodities seldom
exceeds three and the number of minor species is below
10±12 species [9]. Under these circumstances it is not
dicult to select yeast colonies on agar plates, based on
morphological characteristics, and to estimate, satisfac-
torily, the relative proportion of the di€erent species of
a given food. The single signi®cant diculty for the
food industry is the absence of a database of long-chain
fatty acid pro®les for the main food contaminant yeasts.
However, several research laboratories already have
such data; the diculty is easy to overcome.
Electrophoretic isoenzyme pro®les
The di€erences found in amino acid sequences of
enzymes from di€erent organisms re¯ect organismal
genetic divergence. Amino acid substitutions can often
be detected from the extent of migration shown by
enzymes on electrophoretic gels and the corresponding
patterns are termed zymograms. These zymograms, also
termed electrophoretic isoenzyme pro®les, have been
shown to be a useful yeast taxonomic tool and have
been used to type clinical isolates of yeast pathogens.
With this approach, Duarte et al. [25] grouped 35 yeast
strains into the four currently recognized species of
Saccharomyces sensu stricto (S. cerevisiae, S. bayanus, S.
pastorianus/S. carlsbergensis and S. paradoxus), demon-
strating its value for identi®cation purposes and showing
the usefulness of the technique for a rapid and sensitive
identi®cation of strains from this industrially important
group of yeasts.
Bearing in mind that, after strain puri®cation, this
technique takes about 48 hours to be performed, does

Table 2. Separation of the yeast species in three groups according to their polyunsaturated C18 fatty acids composition
Group I [C18:2 (ÿ); C18:3 (ÿ)]
Hanseniaspora uvarum
Hanseniaspora guilliermondi
Hanseniaspora vineae
Saccharomyces bayanus
Saccharomyces cerevisiae
Saccharomyces chevalieri
Saccharomyces exiguus
Saccharomyces pastorianus
Saccharomyces unisporus
Saccharomycodes ludwigii
Schizosaccharomyces octosporus
Schizosaccharomyces pombe
a
Teleomorphs of the species described in the 4th edition of ``The Yeasts. A Taxonomic Study'' [22].
Yeast speciesa
GroupII [C18:2 (+); C18:3 (ÿ)]
Dekkera anomala
Dekkera bruxellensis
Dekkera naardenensis
Pichia etchellsii
Saccharomyces dairensis
Torulaspora delbrueckii
Torulaspora globosa
Yarrowia lipolytica
Zygosaccharomyces bailii
Zygosaccharomyces bisporus
Zygosaccharomyces ¯orentinus
Zygosaccharomyces mellis
Zygosaccharomyces microellipsoides
Zygosaccharomyces rouxii
Group III [C18:2 (+); C18:3 (+)]
Candida catenulata
Candida diddensiae
Candida guilliermondii
Candida norvegica
Candida parapsilosis
Candida sake
Candida rugosa
Candida tropicalis
Candida utilis
Candida zeylanoides
Cryptococcus albidus
Cryptococcus humicolus
Cryptococcus ¯avus
Cryptococcus laurentii
Cryptococcus terreus
Debaryomyces hansenii
Debaryomyces polymorphus
Issatchenkia orientalis
Issatchenkia terricola
Kluyveromyces lactis
Kluyveromyces marxianus
Kluyveromyces thermotolerans
Lodderomyces elongisporus
Metschnikowia pulcherrima
Pichia anomala
Pichia norvergensis
Pichia fermentans
Pichia guilliermondii
Pichia jadini
Pichia membranifaciens
Rhodotorula glutinis
Rhodotorula mucilaginosa
Saccharomyces kluyveri
Zygosaccharomyces fermentati
362 V. Loureiro, A. Querol / Trends in Food Science & Technology 10 (1999) 356±365
not require expensive or sophisticated equipment, is
cheaper than other rapid typing methods and is extremely
sensitive and reliable, we do not understand why it has
been forgotten by food microbiologists. In our opinion,
this technique has high potential applicability in food
industry. For this purpose it is necessary to create a
representative database of the electrophoretic isoenzyme
patterns of the most relevant yeast species in food tech-
nology.
Molecular biology techniques
In several hemiascomycetous yeasts, rRNA genes are
located in a single genomic region composed of 100±150
tandem repeats of a fragment of 9 Kb. These fragments
contain two transcriptional units, one of them (7 Kb) is
a cluster of the genes coding for the 18S, 5.8S and 25S
rRNAs and two internal transcribed spacers, ITS1 and
ITS2. The second unit, which is transcribed in the
opposite direction, corresponds to the 5S rRNA.
Recently, Esteve-Zarzoso et al. [26], proposed a new
rapid and easy method of routine yeast identi®cation
based on the restriction analysis of the 5.8S rRNA gene
and the internal transcribed spacers (ITS). They pre-
sented an initial database to identify a total of 132 yeast
species belonging to 25 di€erent genera. Using this
method it is possible to identify yeast from isolated
colonies or directly from food samples. This is the ®rst
available molecular method that presented information
to identify a high number of yeast species. Using the
same methodology, but amplifying di€erent regions
(18S rRNA and ITS1), Dlauchy et al. [27] constructed a
database of restriction fragment patterns to identify 128
species associated mainly with food, wine, beer, and soft
drinks.
Di€erent approaches are applied using PCR. The
Random Ampli®ed Polymorphic DNA (RAPD) tech-
nique is based on the ampli®cation of random segments
of DNA with a single and short (5±15 mer) primer of an
arbitrary nucleotide sequence. Due to the low annealing
temperature, the primer hybridizes at loci distributed at
random throughout the genome, allowing the ampli®-
cation of polymorphic DNA fragments (for a schematic
diagram see Refs 28 and 29). The level of di€erentiation,
either interspecies or intraspecies, depended highly on
the primers used. However, one of the most important
problems with this technique is the low stable pattern.
Otherwise, the RAPD assay is a less time-consuming
tool principally for typing organisms and has also been
shown to be suitable for the identi®cation of yeast species.
This technique has been used to discriminate between
some of the typical spoilage yeast such as Candida lipo-
lytica, C. valida, S. cerevisiae, Z. bailii and Z. rouxii [30].
Another PCR-based approach consists in the ampli®-
cation of fragments with oligonucleotides speci®c for
simple repetitive DNA sequences, named microsatellites.
Primers (GTC)5, (GAC)5, (GACA)4, the phage M13
DNA and only the M13 sequence GAGGGTGGCGG-
TTCT have been used for yeast identi®cation and charac-
terization. This technique is more reproducible than
RAPD based methods since the annealing temperature
is higher, 55 C instead of 37 C [29]. Using this technique
Baleiras Couto et al. [31] identi®ed strains of Z. bailli
and Z. bisporus present in mayonnaise and salad dressings.
The usefulness of PCR for detection of yeasts involved
in food spoilage includes assays for Dekkera/Brettano-
myces [32] and Z. bailii [33].
Gene probe in a variety of in situ hybridization or
PCR formats have been used for the detection, identi®-
cation, and characterization of speci®c DNA segments.
The versatility of gene probe analyses is demonstrated
by the many clinical and environmental applications
that have been performed. Constantly new gene probes
for food analyses are potential tools in improving the
rate of identi®cation and characterization of food-borne
or spoilage microorganisms.
Another application of gene probes is in situ hybridi-
zation. Fluorescent in situ hybridization of whole cells
with rRNA-targeted oligonucleotide probes has
increasingly become a valuable tool for the speci®c
detection of microorganisms without previous cultiva-
tion. The use of probes that speci®cally binds to a target
region in the 18S rRNA was applied in the identi®cation
of fungal pathogens such as Candida species [34] and
yoghurt spoiling yeast [35]. The dependence of this
method from sequences deposited in databases for
probe design and comparison constitutes, frequently, a
limitation of this technique. The 18S rRNA and 28S
rRNA gene sequences were applied in yeast taxonomy
studies. However, the homology of this region among
species is very high and in some cases it is dicult to
distinguish closely related species [35]. Therefore, the
application of probes for other regions could be more
promising.
Since the ®rst karyotyping performed by orthogonal-
®eld-alternation gel electrophoresis, pulsed ®eld electro-
phoresis techniques have made possible the separation
of the chromosomes of S. cerevisae and other yeasts (for
a review and food industry application, see Deak et al.
[28]). Chromosomal polymorphisms are due to dele-
tions, insertions and translocations of long enough
DNA fragments to be detectable by electrophoresis and
have proved to be useful for the di€erentiation of species
belonging to several genera, e.g. Candida, Kluyver-
omyces, Saccharomyces, and Zygosaccharomyces (for a
review see Esteve-Zarzoso et al. [36]). However, this
technique is complex and time-consuming and does not
permit the analysis of a large number of samples; this
could be the reason why no applications to study spoilage
yeasts have been published until now.
Finally, mitochondrial DNA (mtDNA) polymor-
phisms have been extensively used to detect changes in the
organization of this genome and, as well, as an important
 V. Loureiro, A. Querol / Trends in Food Science & Technology 10 (1999) 356±365
tool in taxonomic studies. Yeasts are a group of organ-
isms with a high degree of variability in the size and
form of their mtDNA, and several methods have been
developed for the isolation of yeast mitochondrial
DNA. However, Querol et al. [37] have developed a
mtDNA restriction analysis method for Saccharomyces,
which does not require previous isolation of mitochon-
dria or puri®cation of mtDNA. This technique has also
been used successfully to characterize strains of species
other than Saccharomyces; Candida zeylanoydes and
Debaryomyces hansenii and species of the genera Brettano-
myces, Kluyveromyces and Zygosaccharomyces [36].
The rapid identi®cation of spoilage microorganisms is
of eminent importance to the food industry. Rapid
PCR-based detection techniques or in situ hybridization
can be used in the food industry as an integral part of
quality control to detect or identify yeasts. The major
problem of these techniques is the direct application to
the sample without previous yeast isolation due to the
impossibility to discriminate non-viable cells. For this
reason a negative result is conclusive but not a positive
one. Another important problem asociated with the
identi®cation of foodborne yeast consists in the speci®c
detection of strains. Discrimination at subspecies or
strain levels and the analyses of within-species genetic
polymorphisms and natural population variability have
been shown as very helpful to determine contamination
sources and processes. After the critical analysis of the
application of di€erent methods currently available for
yeast strain characterization, we conclude that the use
of chromosomal pro®le and mtDNA restriction pattern
analyses are the most useful to di€erentiate and dis-
criminate a higher number of yeast strains.
Spoilage indicators
There are di€erent reasons to perform micro-
biological analysis of a food product. The most rele-
vant, without any doubt, is to estimate the presence of
pathogenic microorganisms in foods which may put in
jeopardy the health of the consumers. However, from the
industrial point of view, other reasons must be considered
as well: the estimation of the product shelf life, the eval-
uation of the quality of raw materials, the tracing of the
route of contamination in the processing lines, etc. The
choice of the analysis to be made, that is, the micro-
biological criteria to be adopted, depends on the purpose
and on the diculty of the techniques to perform the
required analysis. This fact allowed the emergence of
the concept of `microbiological indicator', as a means of
avoiding the diculties associated with the detection of
pathogenic bacteria. In general, pathogens are present in
foods in low numbers and their identi®cation requires
the utilisation of numerous biochemical and physiologi-
cal tests, turning impracticable their detection in
industrial routine. Alternatively, other microorganisms or
groups of microorganisms, without taxonomic signi®cance
363
(e.g. faecal coliformes, enterococci, etc.) but with eco-
logical habits similar to the pathogens are detected. They
are called `index organisms' [11] and each one includes,
generally, several bacterial genera and species. Like
these markers, others are also used to evaluate the pre-
sence or activity of spoilage microorganisms in foods,
generally named as `spoilage indicators'. When measur-
ing directly the presence of spoilage microorganisms
they are called `microbiological markers' and include
groups without taxonomical signi®cance, but with one
or more relevant phenotypical characteristics (e.g. spore
formers, thermophiles, psychrotrophs, proteolytics,
lipolytics, pseudomonads, micrococci, etc.); when mea-
suring the metabolic activity, through metabolite deter-
mination (e.g. carbon dioxide, butanediol, trimethyl-
amine, etc.) or through the composition of cellular biomass
(e.g. long-chain fatty acids, electrophoretic isoenzyme
pro®les, etc.) they are named as `chemical markers';
when measuring physical parameters dependent on
microbial activity (e.g. pH, changes in the electrical
resistance, redox potential, etc.) they are called `physical
markers'. Most `spoilage indicators' used in food indus-
try were developed to characterize bacterial spoilage. To
characterize the activity of spoilage yeast there are,
however, fewer indicators, revealing the lower impor-
tance paid to yeast by food microbiologists and, also,
the lower biochemical versatility of yeast. This fact
makes the development of di€erential tests to distin-
guish the di€erent groups of yeasts relevant in food
technology more dicult. We will now discuss some of
the main `zymological indicators' (zymo=yeast) used,
or with potential to be used, in food industry.
Zymological indicators
The most common indicator is the classical `yeast and
moulds', resulting from the utilization of a general pur-
pose plating medium to enumerate microfungi (yeasts
and moulds) responsible for the contamination of food.
As with bacteria, there are certain foods (e.g. meat, ®sh
and chilled products) in which, occasionally, it is useful
to detect/enumerate `psychrotroph yeasts' using the
same culture medium for `yeasts and moulds' but incu-
bating at temperatures lower than 25 C (generally 5 or
10 C).
Other common indicators are formed by groups of
yeasts tolerant to environmental stress, which are
detected/enumerated by modi®ed general purpose media,
according to the stress factor involved. Thus, `acid-
resistant yeasts' is a zymological indicator used to
characterize dangerous yeast in acid food industries,
such as acetic and lactic acid preserved pickles, olives,
mayonnaise, sauces, fruit juices, beverages stabilized
with preservatives, wines, etc. `Xerotolerant (osmophilic)
yeasts' is used for grouping yeast particularly dangerous in
industries that process low aw foods, such as sugar syrups,
fruit concentrates, jams, jellies, confectionery, bakery,
364 V. Loureiro, A. Querol / Trends in Food Science & Technology 10 (1999) 356±365
dairy desserts, dried fruits, cured meats and other meat
products, etc. The medium chosen for detection or enu-
meration of these yeasts should re¯ect the content of the
food being analysedÐglucose, fructose, sucrose, sodium
chloride [9]. Ethanol (11.4% v/v) has been successfully
used as a selective agent in a medium developed to
detect `wine-spoiling yeast' [38]. `Wild yeasts' is an
indicator commonly used in brewing, based on the fact
that brewer's yeast (and most other Saccharomyces species),
unlike other yeast genera that occur as contaminants,
cannot utilize lysine as a sole nitrogen source. Although
the absence of yeast growth on lysine agar does not
necessarily mean an absence of wild yeast, this medium
can, nevertheless, be used to monitor the presence of
contaminants in the beer processing.
Occasionally, it is of interest to detect the presence of
yeasts with speci®c hydrolytic capacities, namely `lipo-
lytic yeasts', `pectinolytic yeasts' and `proteolytic
yeasts', using appropriate culture media [9].
The species of spoilage yeasts are seldom used as
zymological indicators, given the lack of selective or
di€erential media to quantify them in a rapid way. A few
of the known examples have already been referred to in
the description of selective and di€erential culture media.
An alternative approach to the zymological indica-
tors, which has been extensively studied in bacteria
during the past 80 years, is to examine food samples for
chemical (or sensorial) evidence of past microbial activity.
The use of metabolites as indicators of spoilage is often
more convenient and faster than using microbiological
methods of examination. However, very few have
received acceptance as a means of assessing the degree of
yeast spoilage of foods. Ethanol and acetoin levels pro-
vide reliable indexes of the quality of the fruit on arrival
at the factory and of hygiene in the processing plant,
respectively [11]; analysis of carbon dioxide in the head
space of sealed culture vials was proposed for rapid
enumeration of fermentative yeasts in food, using a
selective medium and gas-chromatographic analysis
[39]; 4-ethyl-phenol can be used as a sensory or chemical
marker for Dekkera/Brettanomyces-infected wines. This
chemical marker may be regarded as a `spoilage pre-
dictor' because, as already mentioned, in low levels the
wine is not spoiled and allows the detection of Dekkera/
Brettanomyces yeasts in contaminated wines before they
deteriorate. Recently, Casas et al. [40] proposed a sim-
pli®ed test to detect, in 48 hours, `petroleum-like' o€-
odour-producing fungi for marzipan based products
preserved with sorbate. The principle of the test is based
on the ability of some yeasts (Z. rouxii and D. hansenii)
and moulds (Penicillium chrysogenum, P. simplicissimum,
P. crustosum and Aspergillus niger) to convert sorbate in
1,3-pentadiene, which is easily detectable by nose. In
addition, the test allows distinguishing Z. rouxiiÐthe
most dangerous and frequent spoilage yeast from this
type of productsÐbecause it is the only gas producer.
As yeast and moulds can produce 1,3-pentadiene much
faster in laboratory conditions than in the product, this
compound can be considered an excellent `spoilage pre-
dictor' to this type of food products.
As already mentioned, it is possible to type (identify)
the most relevant yeast to food technology based on
their total biomass fatty acid pro®les and to separate
them into three groups with technological signi®cance
according to the presence or absence of linoleic (C18:2)
or linolenic (C18:3) acids. A careful examination of
these groups (see Table 2) shows that these acids can be
used as chemical markers of the microbiological quality
of raw materials and ®nal products in many food
industries. Besides, taking into account that these acids
are detected through a chromatogram giving the total
long-chain fatty acid composition, it is possible, in
many cases, to type the species contaminating the food.
According to the experience of one of us (V.L.) this
chemical marker can be especially useful in the charac-
terization of the microbial quality of processed raw
materials, such as sugar syrups and fruit concentrates.
Here the presence of group III yeasts is not usually
dangerous, but the presence of group I and, particularly,
group II yeasts are extremely dangerous to the industries
reprocessing them (e.g. confectionery, soft drinks,
bakery, etc.).
Conclusions
In the last few decades the relevance of yeasts as food
spoilage agents has become greater. Nevertheless, the
zymological indicators used in food industry, to assess
the quality of raw materials, processing and ®nal products,
are not, at present, completely satisfactory and re¯ect
the still limited tools available in food mycology to
enumerate and to type food contaminant yeast. The
strategies to be developed to improve the present indi-
cators should consider the following aspects: (i) rather
than `spoilage indicators' it is necessary to develop
`spoilage predictors'; (ii) rather than identifying food
contaminant yeasts it is necessary to know the yeasts
properties or, as stated by Davenport [14], ``micro-
organism behaviour patterns and traits are paramount
over correct names''; (iii) the development of new
zymological indicators should be based, as a matter of
priority, on methods that lead to a `coarse distinction'
of the yeasts; the `®ne distinction', usually made by
molecular methods at an intraspeci®c level, should
complement the former ones, especially to trace routes
of contamination in processing or distribution lines; and
(iv) the de®nition of spoilage indicators/predictors
should always have in mind the concept of `ecology of
zero growth', being based on two or more determina-
tions separated in time. This last condition leads,
necessarily, to predictive mycology which, for food
spoilage yeast, is still virtually terra incognita or it is
used con®dentially by large companies.
 V. Loureiro, A. Querol / Trends in Food Science & Technology 10 (1999) 356±365
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