Food Spoilage Flora
GG Khachatourians, University of Saskatchewan, Saskatoon, SK, Canada
Ó 2014 Elsevier Ltd. All rights reserved.
This article is a revision of the previous edition article by George G. Khachatourians, Dilip K. Arora, volume 1, pp 228–237, Ó 1999, Elsevier Ltd.
Current forecasts and concerns about global food security and
safety place significant focus on fungal contamination and
spoilage of foods and their impact on food security. In addi-
tion, consequences to humans and food-producing animals’
health and welfare are associated closely with problems of
fungi that require mitigation. The control of fungal food
contamination, disposal of spoiled foods, and prevention and
mitigation of consequential health issues will require signifi-
cant amount of resources and investments. Fundamental to all
of these concerns is proper identification and rapid detection
tools. Major advancements in detection techniques and their
scaledown or automation have allowed for early detection and
identification. The transformation of these advancements must
become more economical, reliable, and quick. These tech-
niques and tools then will help identify fungi and yeasts from
foods of greater complexity and variety. The sophisticated
instrumentation unavailable for parts of the developing world
or remote places in the meantime should be supported through
conventional methods. Comparative evaluation of protocols
will remain our challenge as will ease in operation, cost,
sensitivity, specificity, speed, and reproducibility. Lastly,
molecular methods also must address the detection and iden-
tification of nonculturable and nonviable molds.
Foodborne fungi and their mycotoxins affect a quarter of
the world’s food creating a considerable loss both in their
quantity and nutritional quality. In humans and animals,
exposure to these compounds are mutagenic, teratogenic,
hepato- or nephrotoxic, and carcinogenic and affect develop-
ment. Certain species of the genera, Fusarium, Aspergillus, and
Penicillium, are most important because of their ability to
produce secondary metabolites, aflatoxin, fumonisin, ochra-
toxin, trichothecenes such as deoxynivalenol, T-2 toxin and
nivalenol, and zearalenone. These concerns necessitate regular
monitoring of fungal propagule presence from planting, har-
vesting, storage, and even processing of agricultural products
from any corner of the world.
Fungi are ubiquitous and found in many foods and ingre-
dients and are global in their presence. Filamentous fungal
genera, Alternaria, Aspergillus, Botrytis, Cladosporium, Fusarium,
Geotrichum, Monilia, Manoscus, Mortierella, Mucor, Neurospora,
Oidium, Oosproa, Penicillium, Rhizopus, and Thamnidium, often
are found on meat products as much as on grains. Although the
obvious moldy growth on foods is noticed leading to their
rejection, in the first instance, the detection of contamination
must occur much earlier.
Taxonomic characterization is aided by microscopy,
biochemistry, and genetic techniques as the identification of
filamentous fungi is an evolving endeavor. Biochemical iden-
tification of filamentous fungi and yeasts found in spoilage of
foods can be based on genomics, transcriptomics, proteomics,
metabolomics, and phenomics. The level of spoilage can vary
depending on the type of food, relative humidity, and other
physical conditions. Chemical characteristics of ascomycetes
yeasts and certain filamentous food spoilage fungi have been
244 Encyclopedia of Food Microbiology, Volume 1
based mainly on physiological and chemical tests involving cell
wall biopolymers, quantitative profiles of sterols, total fatty
acids (FAs), and pyrolysis of triphospho-pyridine. This section
outlines methods in the identification of yeast and filamentous
fungi and discusses the values of biochemical markers, the
immunoassay, isozymes, and automated systems over molec-
ular detection techniques.
Biochemical Diagnostic Markers
FAs, Proteins, and Isozymes
FAs composition can differentiate between fungi. Among the
foodborne fungi, the presence of neutral lipids, glycolipid, and
phospholipid fractions and that of omega 3 and omega 6 of
FAs and their relative amounts (C16 and C18) help with
species identification. FA profiles have helped yeast and fila-
mentous fungal taxonomists to differentiate members of
Schizosaccharomyces, Nadasonia, Aspergillus, Mucor, and Penicil-
lium. The cellular FA composition of wine spoilage strains of
Torulaspora delbreuckii and Zygosacharomyces bailli have been
a useful differentiating tool. Saccharomyces cerevisiae and other
wine-associated yeasts species have been differentiated by
capillary gas chromatography (GC), which is an easy, quick,
and inexpensive method. This method has been applied to
determine the causes of ‘stuck’ fermentation in a South African
food and beverages industry. Similarly, these methods have
been applied successfully to monitor the fungal contaminants
in the bioprotein pilot plants in South Africa. In the case of
Rhodosporidium, FA and sterol (FAST, for 20 FAs and seven
sterols) profiles have been used for the rapid differentiation of
species and intraspecific variation to determine the identity of
1740 fungal isolates collected from Finland.
Proteins can be used for the identification and separation of
fungal isolates, mating types, and formae speciales and for the
determination of spoilage species. Protein profiles may vary
depending on the growth and metabolic conditions. Detection
of common molds from contaminated foods using protein
profiling has potential difficulties and profiling needs simpli-
fication, standardization, and automation.
Isozymes are protein enzymes, which have similar and often
identical enzymatic properties with different amino acid
sequences. Because various amino acids create net charge
differences, isozymes can be detected by electrophoresis.
Isozymes can be used to identify fungal isolates based on
different alleles of a single gene locus (allozymes), multiple loci
coding for a single enzyme, and those with posttranslational
modifications. The use of isozymes as a tool allows for the
analysis of several fungal samples that are relatively simple.
Although detection of isozymes allows a genetic interpretation
of variations in alleles and loci, they are not practical for the
detection of food contaminating fungi.
Electrophoresis is a commonly used technique for the iden-
tification of isozymes. Whether using polyacrylamide or starch
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 BIOCHEMICAL AND MODERN IDENTIFICATION TECHNIQUES j Food Spoilage Flora
and isoelectric focusing gels, isozymes can be separated and used
for ‘fingerprinting’ of fungal and yeast proteins. Contemporary
electrophoresis systems permit for (1) a large number of enzymes
to be detected from a single or several fungi and (2) the detection
of allozymes and isozymes. Functional assays can be combined
with nondenaturing electrophoretic techniques to generate
effective zymography. Here, enzyme activity (e.g., amylases,
proteases, and polygalacturonidases) can be visualized directly on
a polyacrylamide gel containing appropriate substrates resulting
in discrete banding patterns. The use of isozyme analysis for
identification and examination of food-contaminating fungi is
simple. In brief, starch- or polyacrylamide-based gel is boiled and
poured into a mold to form the gel. After the gel cools, a sample
containing the enzymes is run according to defined current for
voltage, amperage, and time in a buffer. After electrophoresis, the
gel can be processed and tested for the particular enzyme activity.
Examples of assays are (1) slicing of the gel and assay for particular
activity, (2) staining of the gel for transformation of a chromo-
genic substrate (e.g., o-nitrophenylated sugars), and (3) over-
laying of the gel with a gel substrate (e.g., casein, albumin, starch,
etc.). With automated systems, many more gels under identical
conditions can be run, processed, and read in a single day.
The main drawback for isozyme analysis is that a large
number of staining systems is required for comparative studies,
especially if multiple genetic loci coding for enzymes are
involved. Additionally, with some fungi, difficulties arise if they
are difficult to grow, or the amount of material and time
requirements discourage isozyme analysis.
Fungal Metabolite Profiling
Fungal metabolites are synthesized in response to internal
needs or as a consequence of externally directed signals. Some
of the latter function as pigments, toxins, antibiotics, and
signaling molecules. The general term ‘extrolites’ is used to
describe these and can be volatile or nonvolatile secondary
metabolites, organic acids, extracellular enzymes, mycotoxins,
and other bioactive compounds. For example, only three of the
approximately 90 food-spoiling Penicillium species are able to
produce the secondary metabolite penicillin.
Much has been learned from fungal comparative metabolite
profiling and metabolomics. Metabolomics is the scientific
investigation of the unique chemical fingerprints that specific
cells leave behind and can be used in functional genomics and
organismic classification. In food spoilage fungi, as with others,
growth and cell differentiation, response to the environment,
and the production of metabolites and enzymes lead to their
chemo-diversity. The fungal metabolic profiling from
a genomic perspective includes about 6000 genes in S. cerevisiae
to more than 10 000 genes in Aspergillus sp. From the
perspective of food spoilage, metabolite profiling has become
an efficient tool for identification and taxonomic placement.
Metabolite profiling requires several tools and concepts,
including integration of high-performance analytical method-
ology, intelligent screening, efficient data-handling techniques,
and accepted core concepts of species.
Volatile compounds, such as alcohols, carbonyls, hydro-
carbons, terpenyls, and others, are produced as fungi colonize.
Fungal volatiles differ from those produced by bacteria. These
245
volatile compounds can be useful as taxonomic markers and
early indicators of food quality loss and mycotoxin production
in commodities such as grains. Different analytical methods
are applied for the separation and detection of secondary
metabolites. Some of these methods involve the use of
thin-layer chromatography, GC, high-performance liquid
chromatography, micellar capillary electrophoresis, flow
injection electro-spray mass spectrometry (MS), ultraviolet
(UV) diode array detection, and nuclear magnetic resonance
detection. Synthesis of secondary metabolites can be sensitive
to growth and environmental factors, and their identity can be
strain specific. Therefore, their diagnostic use must be consid-
ered cautiously. Secondary metabolites have been particularly
effective in identifying food spoilage fungi Penicillium, Asper-
gillus, and Fusarium. There are some pitfalls in using secondary
metabolites for identification of foodborne fungi, including (1)
highly specialized people are needed, (2) simplified procedures
are lacking, (3) some food spoilage fungal species may not
produce these in situ, and (4) other limitations can be imposed
by inefficient extraction procedures or low analytical sensitivity
or low reproducibility under different growth and metabolic
conditions. The foodborne terverticilliate penicillia is difficult to
characterize by using traditional characters. Several closely
related species of Penicillium were separated using secondary
metabolites by using diode array detection or flow injection
analysis electro-spray MS. Odoriferous fungi produce unique
combinations of volatile metabolites like alcohols, ketones,
esters, terpenes, and other hydrocarbons. Isolates of Aspergillus
and Fusarium can be distinguished based on their production of
sesquiterpenes. Similarly, a large number of Penicillium species
could be classified based on profiles of volatile metabolites –
for example, Penicillium roqueforti and Penicillium commune, in
which the latter is the most frequent contaminant of cheeses.
Volatile metabolites, however, are not used widely for identi-
fication purposes.
Immunological Techniques
Fungal antigens are used for the identification of filamentous
fungi and yeasts in food. This is particularly possible because of
the availability of monoclonal antibody technology, which has
revolutionized the development process in detection and
diagnosis of organisms. It is possible to raise isolate-, species-
and genus-specific antibodies that are sensitive and target
specific. Raising monoclonal antibodies from the infected food
materials, however, has problems tied with the isolation,
growth, and extraction of fungal antigens. The immunological
diagnosis of foodborne fungi has resulted in several advance-
ments, such as characterization of immunodominant sites and
antigenic sugars and proteins in some of the common food-
borne fungi, such as Aspergillus, Botrytis, Cladosporium, Fusarium,
Geotrichum, Monascus, Mucor, Penicillium, and Rhizopus. The
rapid detection of common food spoilage flora in foods with
Aspergillus, Penicillium, and Fusarium using immunological
techniques still is underutilized.
In addition to proteins, the recognition of fungal cell wall-
and cell surface-associated or extracellular polysaccharides (EPSs)
can be deployed using specific immunoassays with appropriate
antibodies. Thermostable EPSs of fungi contain mannose,
246 BIOCHEMICAL AND MODERN IDENTIFICATION TECHNIQUES j Food Spoilage Flora
galactose, glucose, fucose, and occasionally glucoronic acid,
which are released into the growth medium in variable quantities.
Thus the EPS or cell surface proteins could be used to produce
polyclonal IgG antibodies in rabbits to be specifically and sensi-
tively used in a number of immunoassays.
The methodology for the use of immunological techniques
depends on methodologies, instruments, and trained human
resources. The methods included in this group of tests are mold
latex agglutination test, enzyme-linked immunosorbent assay
(ELISA), radioimmunoassay, and enzyme immunoassay for
which some commercial kits are available. Commercially
available kits include the mold latex agglutination test
(Holland Biotechnology, B. V. Leiden, the Netherlands), Pas-
torex Aspergillus Test (Eco-BioDiagnostic Pasteur, Genk, Bel-
gium), and others. The use of ELISA to detect food spoilage
fungal flora basically depends upon the particular objective of
the investigator; that is, the level of sensitivity desired, appli-
cation of the technique in pure culture or food materials, the
need for quantification, and use of poly- or monoclonal anti-
bodies as reagents. The indirect and double-antibody sandwich
ELISA has been used widely for identification and detection.
Sandwich ELISA has some advantages over other techniques as
chlorophyll and other interfering components from the plant
food materials can be removed easily. Interpretation of ELISA is
usually straightforward, although appropriate control must be
included to avoid the false-positive or false-negative results.
Multiwell ELISA formats for the detection of mold from food
are available commercially that are rapid and less time
consuming. Although immunofluorescence is considered to be
more sensitive than ELISA, it is difficult to standardize and
interpret or it is difficult because of contamination of food
samples with large numbers of nontarget microorganisms may
interfere with assay unless highly specific antibodies are used.
Evaluation of Commercial Techniques and Tests
Lack of or an incomplete reference database hinders fuller
deployment of commercially available kits for fungal and
yeasts identifications for foodborne spoilage detection. In
recent years, however, several biotechnological companies have
enhanced their database use for foodborne yeasts and have
made the diagnostic techniques automated using computers,
read-out devices, and databanks. Most of these systems are
expensive but are becoming affordable for the larger food
companies and institutions. These techniques are convenient
and useful for the identification of isolates, but they may need
1–3 days for the results. Newer methodologies or kits should be
possible as is the case for human infectious fungi and yeasts.
Among all commercially available metabolic activities
determination kits perhaps the Analytical Profile Index (API)
from bioMerieux – such as API 20C, API 50CHB (which is
designed for Bacillus spp. but can be successfully used for
yeasts and fungi), and API YEAST-IDENT (Analytab Products,
Plainview, NY, USA) – have been used for the identification of
a wide range of yeasts and fungi. To perform the test, a heavy
suspension of yeast or fungal spores are prepared and inoc-
ulated into the API test system as per instructions of the
manufacturer. The resulting biochemical reactions are read
after 2–3 days and are used for identification.
The BIOLOG identification system (Biolog Inc., Hayward
CA, USA; http://www.biolog.com/products/?product¼Microbial
ID %2F Characterization&system¼Fully Automated) is a stan-
dardized, computer-linked semi- or fully automated technology
for the identification of yeasts and fungi. The Biolog derives its
uniqueness from conventional methods for identification by
introducing a number of cometabolism tests and many assimi-
lation and oxidation assay techniques not usually common in
conventional methods. This test incorporates a wide range of
substrates and a redox dye, tetrazolium violet (TZV), as an
indicator of substrate utilization. During cellular metabolic
activity of the test substrate, nicotinamide adenine dinucleotide
(NADH) is formed and for it to be reoxidized, electrons pass
through electron transport chain (ETC) and cause an irreversible
reduction of TZV to formazan, which is purple. Because the TZV
functions independent of any ETC, it will accept electrons irre-
spective of metabolism of many of some 95 substrates (e.g.,
amino acids and other carbon or nitrogen sources). Thus, an
extensive number of substrates can be used. The formation of
purple formazan can be read visually or in a microplate reader
with a filter cutoff of 600 nm. The results are compared with
Biolog 8, a database for yeasts. The specificity and sensitivity of the
test system depends on growth and metabolism of yeast or fungal
species. The Biolog FF MicroPlateÔ is the first broad-based rapid
identification and characterization product designed for fila-
mentous fungi and yeast. Not all fungal genera identified are
found to be food spoilage associated; however, they include
species of Aspergillus, Penicillium, Fusarium, Alternaria, Mucor,
Gliocladium, Cladosporium, Paecilomyces, Stachybotrys, Trichoderma,
Zygosaccharomyces, Acremonium, Beauveria, Botryosphaeria, Botrytis,
Candida, and Geotrichum. The Biolog system has been evaluated
for correct identification of 21 species (72 strains) of yeasts of
foods and wine origin; S. cerevisiae, Debaryomyces hansenii, Yar-
rowia lipolytica, Kluyveromyces marxianus, Koeckera apiculata, Dek-
kera bruxellensis, Schizosaccharomyces pombe, Zygosaccharomyces
bailii, and Zygosaccharomyces rouxii were identified correctly 50%
of the time and Pichia membranaefaciens 20% of the time. A test
of 46 strains of yeasts representing 14 species by automated
Biolog and ATB32C systems correctly identified most, with
Biolog 38 strains and with the ATB 30 strains. BioMeriex 32 C
strips for the identification of many foodborne yeasts identified
most yeast isolates with 95% or greater accuracy.
The Biolog System protocol is simple and includes (1) the
strain of the interest is cultivated on a simple agar medium
(available from Biolog), (2) cells are removed from the surface
of the agar and suspended in sterile water at specified density,
(3) cell suspension is inoculated into each of the 96 wells of the
Biolog MicroPlate, and (4) the MicroPlate is then incubated at
26  C for 24–72 h until a sufficient metabolic pattern is found.
For species identification, the MicroPlate must be read with the
Biolog MicroStation Reader. Currently, 267 species of yeast
have been identified by the Biolog System (for details, see the
published literature and protocols from Biolog).
Molecular Techniques
A most useful book entitled Biodiversity of Fungi: Inventory
and Monitoring Methods edited by Mueller, Bills, and Foster
(2004) is a remarkable compilation of standard protocols and
 BIOCHEMICAL AND MODERN IDENTIFICATION TECHNIQUES j Food Spoilage Flora
commercial product vendors, morphological data, growth
media formulas, and molecular methods for discriminating
fungal taxa and monitoring species and diversity. Advances in
molecular techniques employed in the detection of fungi in the
environment as a result of presentations at a special interest
group meeting convened during the International Mycological
Congress (IMC9) in Edinburgh, United Kingdom, August 2010.
Some of the latest diagnostic techniques employed in the
detection of fungi include fluorescence in situ hybridization,
DNA array technology, multiplex tandem polymerase chain
reaction (PCR), and Padlock probe technology with rolling circle
amplification and loop-mediated isothermal amplification are
presented. There is always a need for timely and accurate diag-
nostics in the context of food spoilage fungal tests because of
issues of sensitivity, accuracy, robustness, acceptability, and cost.
Despite many novel technologies being available, challenges
remain to identify as yet the nonculturable fungi, to detect
cryptic species, and to characterize the assemblage and diversity
of fungal communities. Next-generation sequencing (NGS) and
pyrosequencing approaches should prove to be useful in
enlarging the scope of molecular detection studies.
Molecular techniques for the identification of food spoilage
fungi and yeasts are versatile because of (1) the ability of these
tests to recognize genomic differences, (2) the speedier appli-
cation of methods through innovative new tools, and (3) the
inclusively for considering ecological or processing source in
tracking these agents. Several yeast and fungal genomes,
mitochondrial DNA, and other plasmids, killer factors, and Ty-
elements have been sequenced completely. These DNA
sequences in combination should further assist in the spoilage
fungal detection and identification as presented in the next
section.
Electrophoretic Karyotyping
Fungal genomes vary in size and are between 6 and 40 Mb.
Compared with classical karyotyping of fungal chromosomes
by cytochemical methods, electrophoretic karyotyping (EK)
uses pulsed-field gel electrophoresis (PFGE), in which chro-
mosomes can migrate through a series of reorienting arrange-
ments in an electric field to eventually align according to their
sizes and numbers. Initially, PFGE method was used to inves-
tigate the chromosomal content of yeasts as an alternative to
karyotyping and further developed to be a powerful technique
for studying the electrophoretic karyotype of filamentous fungi.
Chromosomes of many industrially important yeasts and fungi
have been characterized in this manner. Isolates or strains of
certain species show distinct EK ‘fingerprint’ differences.
Contour-clamped homogenous electric field gel electropho-
resis, for example, is used to separate intact, chromosome-size
DNA of different species of Saccharomyces and Zygosacchar-
omyces. Strains of the same Saccharomyces species, as expected,
have similar electrophoretic karyotypes. Furthermore, differ-
ences between individual chromosomal bands can be
performed to show strain-specific chromosome-length poly-
morphism. Strains’ karyotype differences by DNA–DNA
hybridization techniques have conspecificity, permitting the
study of genetic diversity of a given yeast species and species
identification and taxonomy. This technique, however, rarely is
247
used for the contemporary identification of food spoilage
yeasts or fungi.
RFLP and DNA Amplification Techniques
Restriction fragment-length polymorphic (RFLP) analyses, even
though not as widely used today for spoilage analysis, involves
isolation of fungal DNA, its cleavage by DNA restriction
enzymes, and agarose gel electrophoresis to test fragments’
polymorphisms by their size and banding patterns. DNA from
such gels can be transferred onto suitable membranes by
electro-blotting process and identified for the presence of
hybridizable gene-specific probes. The emerging result can be
viewed and used for identification and taxonomy. RFLP anal-
yses require a substantial amount of time (several days). An
alternative to this is to use a PCR technique and employ
random-amplified polymorphic DNA (RAPD) analysis. This
method requires a nanogram quantity of fungal DNA that
would results in the formation of a number of DNA sequence
amplifications from one set of primers of arbitrary nucleotide
sequence. The product of amplification that would generate
DNA bands in an electrophoretic gel, which should vary in size
and sequence. This type of analyses has been useful in the
RAPD analysis of wine yeast. PCR-based techniques, such as
RAPD, amplified fragment-length polymorphism, DNA
amplification fingerprinting, and random amplified micro-
satellite sequence (RAMS) can also be used for identifying DNA
markers.
Genomic variability among S. cerevisiae strains using RAPD
analysis, PCR fingerprinting, and restriction enzyme analysis of
the internal or nontranscribed spacer regions (ITS and NTS,
respectively) has been performed. This approach has shown the
identity of spoilage-causing yeast in a survey of yeasts present in
certain production chains of mayonnaises to be Z. bailii strains.
The combined typing techniques are useful in discriminating
yeast species involved in food spoilage in that they help trace
back to the origin of a spoilage outbreak.
Critical Evaluation of Techniques
Assessment of fungal contamination or spoilage flora of
ingredients and processed foods is an essential part of any food
safety and food quality assurance or control programs.
Enumeration of viable yeasts and filamentous fungi associated
with fermented foods and beverages is also important. In some
instances, the counts of total fungal load in a commodity are
needed. For example, exact counts of viable molds (e.g., in
spices, dried vegetables, human and animal food, frozen and
fresh vegetables and meat) and toxigenic ones, especially if
combinations of toxins are suspected is a regulatory necessity.
It is in the latter context that critical evaluation of rapid and
reliable techniques is most valued. Moreover, in commodities
that have been damaged or deteriorated to the point of
inability to recover any viable fungal cells, the molecular
methodology becomes the sole source of identity and level of
hazard evaluation.
Molecular methods based on DNA are commonplace and
have changed our ability to detect and identify a wide variety of
248 BIOCHEMICAL AND MODERN IDENTIFICATION TECHNIQUES j Food Spoilage Flora
fungi and yeasts in foods. The PCR-based methods can amplify
a limited amount of a nucleic acids with a high degree of
sensitivity. Finally, methods for the removal of the interfering
material within foods can affect these amplification test
systems to add power to the detection of fungi.
Molecular over Biochemical
With either traditional biochemical- or molecular-based tech-
niques, there are some issues in the detection of fungi in foods.
Antibody methods for the detection of certain yeast or mold
antigens rely on the principle that the presence of such antigens
always must correlate. Therefore, the accuracy of diagnosis and
identification requires that (1) the EPS always is made by the
fungal agents but not other agents or cells (animal, microbial,
or plant species); (2) the antigen or the epitope is made in
sufficient quantities to be detected by the antibodies; (3) the
antigen is accessible and stable within the food’s composition,
processing, and the environment; and (4) the antibody for test
system has high avidity and specificity. For example, with EPS
detection by immunological techniques, some cross-reacting
immunological reactions can occur. Although the EPS ELISA
of some yeasts is specific, it is not so with basidiomycetous
yeasts. The EPS of Z. bailii could be detected in a highly specific
competitive ELISA but not in a sandwich ELISA or in a latex
agglutination test. The cell surface–specific antibodies can be
used (e.g., rapid detection of Saccharomyces and Zygosacchar-
omyces species) against infectious wild yeasts rapidly and reli-
ably. With the exception of capsulated species, in which cell
wall antigens are masked, yeast cells are readily agglutinated by
specific antiserum. Some antigens are present in many asco-
mycetous yeasts and in some basidiomycetous yeasts, whereas
other antigens display more genera and species specificity.
ELISA tests for Aspergillus and Penicillium spp. are shown to
be quick, reliable, and sensitive in the testing of 161 food
samples, and the use of latex agglutination test for EPS
produced by Aspergillus and Penicillium has been performed.
This test system was tried collaboratively by nine laboratories,
and the identification results were compared with colony
counts for the detection of fungi in food samples. Eight of the
nine laboratories were able to detect 5–15 ng ml1 of purified
EPS. Fair correlation was shown between colony counts and
latex agglutination titers for cereals, spices, and animal feed,
but there was no correlation for fruit juices and walnuts, which
gave false-positive results. It is concluded that the latex agglu-
tination test is a rapid (w10–15 min to read results), simple,
and reliable quantitative method for the detection of Aspergillus
and Penicillium in cereals, spices, and animals feeds. The Mold
Reveal Kit (Eco-Bio, Genk), which is a rat monoclonal antibody
against Aspergillus galactomannan coated onto latex beads, has
been compared with Hydrogen Breath Test (HBT) Mold Latex
Agglutination Test kit (Holland Biotechnology, Laiden, the
Netherlands), an EPS-induced polyclonal antibody test. Both
kits are used for the rapid screening of food stuffs for mold
contamination. The HBT kit was negative for several fungi, and
results were not as sensitive. The sensitized latex beads detect
this EPS at a 15 ng ml1 after 5 min incubation. Of 35 common
foodborne fungi tested, 27 gave positive reactions in the latex
agglutination test, including all 16 Aspergillus and Penicillium
spp. tested. The Mold Reveal Kit was shown to be faster than
the latex agglutination test (2–5 min), and it is simple and
semiquantitative.
Advantages and Limitations of Biochemical
over Other Techniques
Although the assumption can be that binding of antibody to an
antigen is similar to that of nucleic acids, DNA-based diag-
nostics are based on principles of greater specificity and
authenticity. In contrast to immunodiagnostics, DNA has an
advantage for not only specificity of binding but also amplifi-
cation and authentication by automated sequencing. The
assumption to be satisfied, however, is that such genomic DNA
from yeasts or fungi will be present intact within or outside the
microbial agent (a cell or a spore) in partially preserved form,
interfering substances would be absent, and the sequence used
for diagnostics would be sufficiently distinct from the food
ingredients.
Both types of fungal identification tests rely on genetic and
chemical taxonomic diversity of the species. The advantage of
molecular and biochemical tests are that standard commercial
kits for performing simple tests are procedurally complex
(e.g., isolation and amplification of any DNA from most yeasts
and fungi). A number of commercial plant or fungal DNA
purification kits that utilize silica-based resins and anion
exchangers are available. Finally, the singular best advantage
here is the detection of nonviable and nonculturable fungi that
can be accomplished through the detection of a homologous
sequences search in various public repositories for culturable
agents.
With as many as 70 fungal species genomes fully se-
quenced, in many cases the PCR detection system can identify
desired gene sequences quickly, with high specificity and in
large volumes. Reports indicate that amplification of DNA
sequences from a number of regions can be used for the
identification and differentiation of yeasts and fungi. Variable
region of the 50 end of the large nuclear ribosomal DNA (28S),
ITS sequences, intron splice sites, and RAMSs have been used
for detection and identification purposes. As for sequencing,
the best analytical tool available to do this is current NGS
technology (e.g., mass or ion torrent spectrometry that has the
capability to analyze hundreds of DNA samples in a day).
Although originally used more than a decade ago for protein
analysis, it was not available for DNA analysis until 1993
when various matrices were developed that would work with
DNA fragments as long as 100 base pairs. For practical
sequencing, however, matrix-assisted laser desorption ioni-
zation time of flight (MALDI-TOF) would have to work with
DNA fragments much longer than the current 100 base-pair
capacity. At the present time, new matrices are being studied
that could extend MALDI-TOF reach to 1000 bases, and if this
works, then this technique would be a major breakthrough for
high-throughput sequencing. Ion torrent DNA sequencing is
both cheaper and faster. Described in 2006 by Nader Pour-
mand and Ronald Davis of Stanford University, this system
determines DNA sequences through electrical detection,
measuring the release of hydrogen ions. Six years after
publishing details of the first NGS system is commercially
 BIOCHEMICAL AND MODERN IDENTIFICATION TECHNIQUES j Food Spoilage Flora
available, by 454 Life Sciences, Jonathan Rothberg and his
colleagues at the company Ion Torrent published in the
journal Nature the first results from a new desktop NGS
technology.
In spite of the power of DNA amplification methods, and
while overcoming the sensitivity limitations of direct DNA
probe assays and immunological assays, they contain inherent
limitations and problems, such as carryover contamination of
amplification products. In a typical PCR amplification reaction
with nanomolar concentrations of reagents and products, one
can estimate 1012 molecules 100 ml1 of amplification prod-
ucts to be present. A carryover of just under 10 copies of
product in 1015 l can generate false-positive results and hence
the need for extreme care. To further prevent such problems,
postamplification sterilization of amplification reaction prod-
ucts through UV irradiation, use of uracil DNA glycosylase, and
addition of psoralens and copper bis(1, 10-phenanthroline)
are encouraged. Finally, proper negative controls, such as
internal and external amplification controls and a DNA
extraction control, should be prerequisites for a reliable
spoilage fungal flora identification and detection system.
The development of real-time PCR systems in which the
reaction tubes are not opened after amplification and in-tube
monitoring of amplification is carried out has eliminated most
of the concerns of carryover. Full instrumentation is now
possible as advances in the clinical use of nucleic acid analysis
are being applied for food systems.
It is important to realize that against strengths or weak-
nesses of any one test system, the powerful approaches of
molecular or biochemical diagnostics can complement the
conventional techniques. It should be interesting to see how
future developments will shape today’s ‘modern’ techniques.
See also: Application in Meat Industry; Biochemical and
Modern Identification Techniques: Introduction; Biochemical
and Modern Identification Techniques: Food-Poisoning
Microorganisms; Enzyme Immunoassays: Overview; Food-
borne Fungi: Estimation by Cultural Techniques; Mycotoxins:
Detection and Analysis by Classical Techniques; Spoilage of
Plant Products: Cereals and Cereal Flours; Spoilage Problems:
Problems Caused by Fungi; Identification Methods:
Introduction; Enrichment; Viable but Non-culturable.
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Bridge, P.D., Arora, D.K., Reddy, C.A., Elander, R.P. (Eds.), 1998. Applications of PCR
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Deak, T., 2012. Handbook of Food Spoilage Yeasts, second ed. CRC Press, Boca
Raton, FL.
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249
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