Food spoilage microorganisms

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Second, stipes are usually nonseptate, so that the vesicle, stipe and footcell all ... genetic pathway, occurrence in foods, and regulatory aspects of aflatoxins.
17 Aspergillus and related teleomorphs A. D. Hocking, Food Science Australia, Australia

17.1

Introduction

The genus Aspergillus was first described almost 300 years ago and is an important genus in foods, both from the point of view of spoilage, and because many species produce mycotoxins. Although a few species have been used in production of fermented food (e.g. Aspergillus oryzae in soy sauce manufacture), most Aspergillus species occur in foods as spoilage or biodeterioration fungi. They are extremely common in stored commodities such as grains, nuts and spices, and occur more frequently in tropical and subtropical than in temperate climates (Pitt and Hocking, 1997). Aspergillus species compete with Penicillium and Fusarium species for dominance in foods and food plants. Aspergilli generally grow at higher temperatures or lower water activities than Penicillia. Aspergilli also usually grow more rapidly than Penicillia, although they take longer to sporulate, and produce spores which often are more resistant to light and chemicals. Aspergillus species dominate spoilage in the tropics, whereas in temperate zones, Penicillium species are more common. Aspergillus is a genus of Hyphomycetes. The fruiting structures are distinctive: the conidiophores have large, heavy walled stipes and swollen apices, termed ‘vesicles’, which are usually roughly spherical. The cells from which spores are formed, phialides, or metulae and phialides, are produced from the vesicle (Fig. 17.1). The simultaneous production of these rows of spore-bearing cells distinguishes Aspergillus from Penicillium, as phialide production in Penicillium and related genera is always successive, not simultaneous. © 2006, Woodhead Publishing Ltd

452 Food spoilage microorganisms

Conidia

Vesicle Metulae Phialides Stipe

25 µm

Fig. 17.1 A typical head of Aspergillus ochraceus showing the structural elements: the stalk (known as the stipe) from which is formed the central vesicle, which in turn produces a layer of cells (metulae) which support the phialides from which conidia are produced.

Two other useful features are characteristic of most Aspergillus species. First, stipes are usually formed from a short cell termed a footcell within a fertile hypha. Second, stipes are usually nonseptate, so that the vesicle, stipe and footcell all form a very large single ‘cell’. Penicillium stipes are usually septate and footcells are very unusual.

17.2

Taxonomy

Aspergillus is a large genus containing more than 100 recognized species, most of which are easily grown in the laboratory. A recent taxonomy to the most common Aspergillus species, including those important in foods, is provided by Klich (2002), although the classic taxonomic reference by Raper and Fennell (1965) is still the most comprehensive, even though some of their concepts are now out of date (Samson and Pitt, 1985, 1990), and many new species have since been described (Pitt and Samson, 1993). A nomenclaturally correct classification for species within the genus Aspergillus was proposed by Gams et al. (1985), grouping species into six subgenera which are subdivided into sections. In addition to traditional morphological taxonomic techniques, secondary metabolites (Frisvad 1985, 1989), and molecular techniques (Moody and Tyler, 1990; Mullaney and Klich, 1990; Geiser et al., 1998; Tran-Dinh et al.,

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1999; Schmidt et al., 2003; Abarca et al., 2004; Varga et al., 2004; Esteban et al., 2005, and many others) have been used to clarify relationships within the genus Aspergillus.

17.2.1 Teleomorphs Teleomorphs are sexual, or perfect, states of fungi. Aspergillus anamorphs (imperfect states) are found in at least eight teleomorphic Ascomycete genera; however only three of these, Eurotium, Neosartorya and Emericella, occur in foods. All form cleistothecia. Eurotium species (previously known as the ‘Aspergillus glaucus group’) are the most common and significant of the foodborne genera with Aspergillus anamorphs. They produce bright yellow cleistothecia and pale yellow ascospores. Heads producing conidia are formed from phialides only. All species are xerophilic, and are important in the spoilage of low water activity (aw) foods and stored commodities. Neosartorya is the second most common of the Aspergillus teleomorphs found in foods. It also produces heads formed from phialides alone, but cleistothecia are white and ascospores are uncoloured. The ascospores of Neosartorya are heat resistant, so species in this genus are important in spoilage of heat-processed foods particularly fruit products (Pitt and Hocking, 1997). Emericella species are less frequently encountered in foods, and rarely cause spoilage. They produce heads with both metulae and phialides, and white cleistothecia producing red or purple ascospores. The cleistothecia are surrounded by Hülle cells, which are thick walled, highly refractile, roughly spherical cells resembling chlamydoconidia. Growth patterns in Emericella species are similar to those of Neosartorya.

17.2.2 Classification Aspergillus is classified into subgenera and sections. The separation of these relies primarily on four features: the presence of a teleomorph and its characteristics; the presence or absence of metulae; the arrangement of metulae or phialides on the vesicle; and colony colours. In species without teleomorphs, Aspergillus colony colours are dominated by conidial colour. These colours are consistently associated with particular species. A number of Aspergillus species are capable of producing mycotoxins and, of these, the aflatoxins are by far the most widely known. Many papers have been published dealing with chemistry, detection, toxicology, production, genetic pathway, occurrence in foods, and regulatory aspects of aflatoxins. The existence of other toxigenic Aspergillus species, particularly those capable of producing ochratoxin A, means that correct identification of isolates from foods and knowledge of the ecology of these moulds is particularly important.

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17.3

Significant Aspergillus mycotoxins

Over 40 species of Aspergillus have been listed as capable of producing toxic metabolites (Cole and Schweikert, 2003; Cole et al., 2003), but the Aspergillus mycotoxins of greatest significance in foods and feeds are aflatoxins (produced by Aspergillus flavus, Aspergillus parasiticus and relatively few, less common, species which are not important in foods), ochratoxin A from Aspergillus ochraceus and related species and from Aspergillus carbonarius and occasionally Aspergillus niger, sterigmatocystin, produced primarily by Aspergillus versicolor but also by Emericella species, and cyclopiazonic acid (A. flavus is the primary source, but it is also reported to be produced by Aspergillus tamarii). Citrinin, patulin and penicillic acid may also be produced by certain Aspergillus species, and tremorgenic toxins are produced by Aspergillus terreus (territrems), Aspergillus fumigatus (fumitremorgens) and Aspergillus clavatus (tryptoquivaline) (Hocking, 2001). Toxins of Aspergillus species exhibit a wide range of toxicities, with the most significant effects being long term. Aflatoxin B1 is the most potent liver carcinogen known for a wide range of animal species, including humans. Ochratoxin A and citrinin both affect kidney function. Cyclopiazonic acid has a wide range of effects (Cole, 1986), and tremorgenic toxins such as territrems affect the central nervous system. Table 17.1 lists the most significant toxins produced by Aspergillus species and their toxic effects.

17.4 Isolation, enumeration and identification Techniques for the isolation and enumeration of Aspergillus species from foods are the same as those used for other foodborne fungi and have been described in detail in Pitt and Hocking (1997) and Samson et al. (2004a). Antibacterial media containing compounds to inhibit or reduce spreading growth of moulds, such as dichloran rose bengal chloramphenicol (DRBC) agar or dichloran 18% glycerol (DG18) agar (Pitt and Hocking, 1997) are recommended for enumerating fungi in foods (Samson et al., 1992; Hocking et al., 2006). There is one medium, Aspergillus flavus and parasiticus agar (AFPA), designed specifically for detection of potentially aflatoxigenic species (Pitt et al., 1983; Pitt and Hocking, 1997). Keys and descriptions of the most common foodborne Aspergillus species can be found elsewhere (Pitt and Hocking, 1997; Klich 2002; Samson et al., 2004a). Identification of Aspergillus species requires growth on media developed for this purpose, including Czapek agar, a defined medium based on mineral salts, or a derivative such as Czapek yeast extract agar (CYA), and malt extract agar. Growth on Czapek yeast extract 20% sucrose agar (CY20S) can be a useful aid in identifying species of Aspergillus (Pitt and Hocking, 1997). © 2006, Woodhead Publishing Ltd

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Table 17.1 Significant mycotoxins produced by Aspergillus species and their toxic effects (from Hocking 2001) Mycotoxin

Toxicity

Species

Aflatoxin B1 and B2

Acute liver damage, cirrhosis, carcinogenic (liver), teratogenic, immunosuppressive Similar effects to B aflatoxins: G1 toxicity less than B1 but greater than B2 Degeneration and necrosis of various organs, tremorgenic, low oral toxicity Kidney necrosis (especially pigs), teratogenic, immunosuppressive, probably carcinogenic Acute liver and kidney damage, carcinogenic (liver) Tremorgenic (rats and mice) Tremorgenic (rats and mice) Tremorgenic Cytotoxic Feed refusal (pigs)

A. flavus, A. parasiticus, A. nomius

Aflatoxin G1 and G2

Cyclopiazonic acid

Ochratoxin A

Sterigmatocystin Fumitremorgens Territrems Tryptoquivalines Cytochalasins Echinulins

A. parasiticus, A. nomius A. flavus, A. tamarii A. ochraceus and related species, A. carbonarius, A. niger (occasional) A. versicolor, Emericella spp. A. fumigatus A. terreus A. clavatus A. clavatus Eurotium chevalieri, E. amstelodami

Unlike Penicillium species, Aspergillus species are conveniently ‘colourcoded’, and the colour of the conidia can be a very useful starting point in identification, at least to the Section level. As well as conidial colour, microscopic morphology to determine the presence of phialides only, or metulae plus phialides, and shape and size of vesicle, etc., is important in identification. Correct identification of Aspergillus species is an essential prerequisite to assessing the potential for spoilage and mycotoxin contamination in a commodity, food, or feedstuff. The Aspergillus species, including teleomorphic species, that are commonly found in foods, are described in detail below.

17.5 Teleomorphic genera with Aspergillus anamorphs 17.5.1 Genus Emericella Berk Emericella forms white cleistothecia surrounded by Hülle cells (thick walled refractile cells like chlamydoconidia) and produces purple ascospores. © 2006, Woodhead Publishing Ltd

456 Food spoilage microorganisms Conidiophores usually have short brown stipes, bear both metulae and phialides, and produce columns of dark green conidia. Only one species, E. nidulans, is common in foods. Emericella nidulans (Eidam) Vuill. Anamorph: Aspergillus nidulans (Eidam) G. Winter Emericella nidulans (Figure 17.2e) colonies may be dark grass green if conidial heads are abundantly formed, or creamish if cleistothecia and Hülle cells are present. Violet or pink soluble pigment may be produced, and the reverse of the colonies is usually brightly coloured in shades of pink, orange, brown or violet brown. This species grows rapidly at 37 °C, and has occasionally been reported to be pathogenic to humans (Horre et al., 2002) and is the cause of guttural pouch mycosis in horses (Kosuge et al., 2000; Cabanes et al., 2002). Ecology Emericella nidulans is not particularly common in foods, and has not been implicated in actual spoilage, but has been isolated from a wide variety of sources. The most common reports are from cereals and cereal products (wheat, flour and bread, barley, rice, maize and sorghum), nuts (peanuts, hazelnuts), dried beans and spices (see Pitt and Hocking, 1997). Physiology Emericella nidulans grows between 6–8 °C and 46–48 °C or even 51°C, with an optimum at 35–37 °C (Panasenko, 1967; Lacey, 1980). The minimum aw for germination is 0.80 aw at 37 °C (Ayerst, 1969), so this species is both marginally thermophilic and xerophilic, a most unusual combination. The heat resistance of E. nidulans ascospores does not appear to have been studied, but we have not encountered any instances of spoilage in heat processed foods by this species. Mycotoxins Emericella nidulans has been reported to produce sterigmatocystin, a mycotoxin more commonly associated with Aspergillus versicolor (Horie et al., 1989; Kawahara et al., 1994) and emestrin (Terao et al., 1988, 1990). Emestrin is highly toxic, with an LD50 of 13 mg/kg when given to mice intraperitoneally (Terao et al., 1988). Natural occurrence of mycotoxins in foods contaminated by this species has not been reported.

17.5.2 Genus Eurotium Link: Fr. Eurotium is widespread and a very well-known genus of Ascomycetes, forming yellow cleistothecia with smooth, cellular walls, often encrusted in red, orange or brown hyphae. The Aspergillus anamorphs have heads with phialides only, producing dull grey-green, spinose conidia. All species of Eurotium

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(a)

(b)

(c)

(d)

(e)

(f)

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Fig. 17.2 Sporing structures of some common foodborne Aspergillus species: (a) A. carbonarius; (b) A. flavus; (c) A. fumigatus; (d) A. clavatus; (e) A. nidulans; (f) A. terreus.

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458 Food spoilage microorganisms are xerophilic, so grow and sporulate best on reduced aw media such as Czapek yeast extract agar with 20% sucrose (CY20S; Pitt and Hocking, 1997). This medium (0.98 aw) encourages development of both anamorphs and teleomorphs (ascospores) plus mycelial colours which are used for species identification. Four species, Eurotium amstelodami, E. chevalieri, E. repens and E. rubrum, are exceedingly common in all kinds of reduced aw environments. Less commonly encountered are E. herbariorum, which resembles E. rubrum, and E. cristatum, which resembles E. amstelodami. All four common Eurotium species may be isolated from a similar range of commodities in which they are often the principal spoilage organisms: stored cereals and cereal products, nuts, spices, dried meat and fish products, dried fruits, bakery goods, cheese and jam (see Pitt and Hocking, 1997). The common Eurotium species generally grow optimally between 0.90 and 0.96 aw, and their minimum aw for growth is in the region 0.70–0.74. They are mesophiles, growing best between 25–30 °C, with temperature maxima just over 40 °C (see Pitt and Hocking, 1997). Eurotium repens and E. rubrum have been used as starter cultures in the manufacture of katsuobushi from bonito (Dimici and Wada, 1994), and E. repens has been used for production of fish sauce from fish meal (Hayakawa et al., 1993). Eurotium species produce chloroanisoles which may cause off-odours in food carried in contaminated shipping containers (Hill et al., 1995), isoprene, a cause of off-odour in a bakery product (Berenguer et al., 1991), ketonic rancidity in coconut (Kinderlerer, 1984; Kinderlerer and Kellard, 1984), proteolytic activity on meat (Binzel, 1980) and lipolytic activity on vegetable oils (Kuku, 1980). They may be relatively resistant to weak acid preservatives such as propionic acid and sorbate (Viñas et al., 1990; Guynot et al., 2002). A high percentage (80–100%) of Eurotium ascospores survive heating at 60 °C for 10 min, at aw 0.98 and pH 3.8, although at 70 °C for 10 min only 0.5–25% survive, and at 75 °C for 10 min, most ascospores are inactivated (Pitt and Christian, 1970). However, E. chevalieri appears to be slightly more heat resistant than the other species, with up to 0.5% surviving 10 min at 80 °C (Pitt and Christian, 1970). Conidia of these species are much less heat resistant: only 8% survived 10 min at 50 °C, 3% 10 min at 60 °C and none at 70 °C under the same conditions (Pitt and Christian, 1970). Ascospores are also more resistant than conidia to high pressure (Karatas and Ahi, 1992), microwaves (Dragoni et al., 1990) and smoke (Kersken, 1974). The four species are distinguished by their ascospore size and ornamentation, and the colours of the hyphae enveloping the cleistothecia, features which are best seen on CY20S agar after 10–14 days incubation at 25 °C. They also vary slightly in their physiology and biochemistry.

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Eurotium amstelodami L. Mangin, Anamorph: Aspergillus vitis Novobr. Physiology The minimum aw for growth has been reported as 0.70 at 25 °C (Armolik and Dickson, 1956), or 0.75 aw (Wheeler and Hocking, 1988), with an optimal aw near 0.96 (Scott, 1957), or 0.90 to 0.93 aw (Avari and Allsopp, 1983). Eurotium amstelodami grows optimally at 33–35 °C (Domsch et al., 1980), with a maximum at 43–46 °C (Blaser, 1975). Mycotoxins No known toxins have been identified from Eurotium amstelodami. Identification Ascospores of Eurotium amstelodami have wide, irregular ridges or flanges and rough walls. Colony colours consist only of yellow from the cleistothecia and dull green from the conidia. Eurotium chevalieri L. Mangin, Anamorph: Aspergillus chevalieri L. Mangin Physiology Eurotium chevalieri grows optimally at 30–35 °C (Domsch et al., 1980) and 0.94–0.95 aw (Pitt and Hocking, 1977). Its maximum temperature is 40–43 °C (Blaser, 1975), and minimum aw is near 0.71 at 33 °C (Ayerst, 1969), although a higher minimum aw (0.74 aw at 25 °C at pH 3.8) was reported by Pitt and Christian (1968). Mycotoxins Eurotium chevalieri has been reported to produce echinulin and neoechinulin, which cause feed refusal in swine (Vesonder et al., 1988). However, other tests for toxicity of Eurotium chevalieri have been negative (Frisvad and Samson, 1991; Adebajo and Oyesiku, 1994). Identification The cleistothecia of E. chevalieri are enveloped in yellow to orange vegetative hyphae, and produce distinctive ascospores shaped like pulley wheels, 4.5– 5.0 µm long, smooth walled, with two prominent, parallel, sometimes sinuous, longitudinal flanges.

Eurotium repens de Bary, Anamorph: Aspergillus reptans Samson & W. Gams Physiology Eurotium repens grows from 4–5 °C to 38–40 °C, with an optimum near 25– 27 °C (Panasenko, 1967; Gonzalez et al., 1988). The minimum aw for © 2006, Woodhead Publishing Ltd

460 Food spoilage microorganisms germination of E. repens may be as low 0.69 aw in glucose/fructose media (Andrews and Pitt, 1987), or 0.72 aw on media of neutral pH, at temperatures of 20 to 25 °C (Snow, 1949; Armolik and Dickson, 1956; Magan and Lacey, 1984a) with optimal growth between 0.90 and 0.95 aw (Andrews and Pitt, 1987). Mycotoxins Mycotoxins are not known to be produced by this species (Frisvad and Samson, 1991). Identification Eurotium repens is distinguished from other Eurotium species by its smooth walled ascospores that have no ridges or flanges, and usually have no longitudinal furrow. Hyphal and reverse colours are yellow to orange, never red. Eurotium rubrum Jos. König et al., Anamorph: Aspergillus rubrobrunneus Samson & W. Gams Physiology Growth temperatures for Eurotium rubrum are probably similar to those reported for E. repens. The minimum aw for germination has been reported as 0.70–0.73 aw (Snow, 1949; Armolik and Dickson, 1956; Wheeler et al., 1988), with an optimum aw for growth near 0.94 aw (Avari and Allsopp, 1983). Mycotoxins Several reports have been published which indicate that Eurotium rubrum produces a range of toxic compounds. However well-documented confirmation of toxicity is still lacking (Frisvad and Samson, 1991). Identification Eurotium rubrum colonies on CY20S usually show areas of brilliant red or rusty colours after 10 days or more incubation. Ascospores are 5.0–6.0 µm long, and show a definite longitudinal furrow and low, minutely roughened ridges. Eurotium herbariorum Link, Anamorph: Aspergillus glaucus Link This resembles E. rubrum in many features, and although far less common than Eurotium rubrum, is nevertheless widespread. E. herbariorum is more xerophilic, and produces larger ascospores (commonly 6–8 µm) than E. rubrum. On media of pH 3.8 containing glucose/fructose as the controlling solute, ascospores of E. herbariorum germinated at 0.74 aw after 19 days, the shortest lag time of any of the common species. Conidia of Aspergillus glaucus germinated at 0.75 aw in 14 days (Pitt and Christian, 1968). E. herbariorum ascospores are more heat resistant than the common, smaller-spored Eurotium © 2006, Woodhead Publishing Ltd

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species. When heated in 5° Brix grape juice, E. herbariorum ascospores showed a D70 of 2.5 min and a z value of 9.1 C°; in 65 °Brix concentrate, the D70 was 5.2 min and z value 7.1 C° (Splittstoesser et al., 1989). 17.5.3 Genus Neosartorya Malloch & Cain Neosartorya produces cleistothecia with cellular walls like Eurotium, however, the walls and ascospores are colourless or white, not yellow. Like those of Eurotium, Neosartorya anamorphs produce vesicles bearing phialides only, but vesicles are small and pyriform (pear-shaped), enlarging towards the apices. About 10 species and varieties are currently accepted (Malloch and Cain, 1972; Kozakiewicz, 1989), which mainly inhabit soil and decaying vegetation. All are thermoduric or thermophilic, none is a xerophile. The well-known human pathogen Aspergillus fumigatus is closely related (Girardin et al., 1995), and occasional pathogenicity to humans has been reported. Neosartorya isolates should be handled with care. The main importance of Neosartorya species in food spoilage is the very high heat resistance of their ascospores. N. fischeri is the main Neosartorya species significant in foods. Neosartorya fischeri (Wehmer) Malloch & Cain Ecology Neosartorya fischeri was reported from cans of strawberries which had been opened and incubated at 25 °C for 5 days, and this was the first indication that this species was a potential problem in acid canned foods (Kavanagh et al., 1963). No visible spoilage had occurred, although spores had withstood the canning process of 12 min at 100 °C. This species had been isolated from canned strawberries in Ireland for 9 years out of 10 between 1958 and 1968, despite increases in the length and severity of the canning process used (McEvoy and Stuart, 1970). In our laboratory, N. fischeri has been isolated on numerous occasions from heat-treated strawberry purée and also from spoiled canned strawberries, and a sports drink. It was the most common species isolated in a survey of heat-resistant fungi in fresh Australian strawberries (Hocking, unpublished). Spotti et al. (1992) also found N. fischeri in heat-treated fresh fruit. Isolation of Neosartorya fischeri from pasteurized fruit juices (Jesenska and Petrikova, 1985; Scott and Bernard, 1987) and fruit powders (Beuchat, 1992) has been reported several times, but only occasionally from spoiled product (Splittstoesser and Splittstoesser, 1977). Neosartorya fischeri is rarely reported from foods which have not been heat treated or processed. Physiology Ascospores of this species are very heat resistant. Kavanagh et al. (1963) reported that ascospores of an isolate more recently identified as Neosartorya

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462 Food spoilage microorganisms fischeri withstood boiling in distilled water for 60 min. More detailed studies of the heat resistance of Neosartorya fischeri ascospores indicate D88°C values of 1.2–7.5 min (Quintavalla and Spotti, 1993), 4.2–16.2 min (Beuchat, 1986) or 1.4 min with a z value of 5.6 C° (Scott and Bernard, 1987), indicating a higher heat resistance than Byssochlamys fulva or B. nivea (Beuchat, 1986). A D86°C value of 8 min, with a z value of 6.9 C° in phosphate buffer at pH 7 has also been reported (Suresh et al., 1996). Milder heat treatments, e.g. 15 min at 75–80 °C, cause activation of ascospores (Suresh et al., 1996). A mathematical model for the combined effect of aw, pH and redox potential on the heat resistance of N. fischeri has been published (Reichart and Mohacsi-Farkas, 1994). High-pressure treatments (600–900 MPa) for 20 min at 20 °C in apricot nectar reduced counts of Neosartorya fischeri 100-fold. At 50 °C, inactivation required less than 4 min at 800 MPa, and at 60 °C, 1–2 min at 700 MPa. Pressure resistance in distilled water was lower (Maggi et al., 1994). In our laboratory, we have observed activation of Neosartorya ascospores by pressure treatment of 600 MPa for 10 min at ambient temperature. Pressure resistance of N. fischeri ascospores varied with age, with older ascospores (7–15 weeks) showing greater resistance than younger ascospores (3–7 weeks) (Chapman et al., in press). Growth has been reported in O2 levels as low as 0.1% at 25 °C (Nielsen et al., 1989). Neosartorya fischeri is used to produce acid proteases which are widely used in cheese-making, baking and meat tenderization (Wu and Hang, 2000). Mycotoxins and pathogenicity This species produces fumitremorgens A and C and verruculogen (Beuchat et al., 1988; Nielsen et al., 1988; Frisvad and Samson, 1991). However, these compounds have not been reported in food. N. fischeri has been reported as an agent of pulmonary aspergillosis in a liver transplant recipient (Gori et al, 1998). In view of its close affinity with Aspergillus fumigatus, and its strong growth at 37 °C, perhaps this is not surprising. Identification Colonies of Neosartorya fischeri spread rapidly at both 25 and 37 °C, and are white; white cleistothecia and inconspicuous grey green Aspergillus heads are produced. The Aspergillus heads have pyriform vesicles bearing phialides only, with small, smooth-walled conidia. The ascospores are distinctive: ellipsoidal, 7–8 µm long, with two prominent longitudinal flanges, which may appear as crests under the microscope. The ascospore walls may be smooth, irregularly roughened or sometimes spinose.

17.6 Genus Aspergillus Fr.: Fr. The species of Aspergillus that cause spoilage in, or are regularly isolated from, food are described below. For simplicity, the species are dealt with in alphabetical rather than taxonomic order. © 2006, Woodhead Publishing Ltd

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17.6.1 Aspergillus aculeatus Iizuka Aspergillus aculeatus produces dark brown to black conidia. It belongs in Section Nigri, and is closely related to A. niger. It is distinguished from A. niger by Aspergillus heads bearing only phialides. Ecology Aspergillus aculeatus causes post-harvest dry rot of tomatoes (Fajola, 1979) and, with other members of Section Nigri, is involved in Aspergillus bunch rot of grapes (Jarvis and Traquair, 1984; Leong et al., 2004). It has been isolated in our laboratory from both fresh and dried grapes. Taxonomy Aspergillus aculeatus has sometimes been considered to be a variety of A. japonicus (Al-Musallam, 1980), but is considered distinct due to consistent differences in morphology and secondary metabolite production. Physiology Aspergillus aculeatus grows between about 10 and 42 °C, with an optimum near 30 °C (Leong et al., 2004). It has a high pectinolytic enzyme activity (Adisa, 1989). Mycotoxins This species produces secalonic acid D (Anderson et al., 1977) and some other minor compounds (Frisvad and Samson, 1991). Secalonic acid D has significant animal toxicity (Ciegler et al., 1980), but a role in human or animal disease, especially from this species, has not been shown. Identification Colonies are dark brown to black. Aspergillus heads of A. aculeatus bear phialides only. It is distinguished from A. japonicus, which also produces phialides only, by its larger vesicles and ellipsoidal conidia. The conidia are ellipsoidal, sometimes subspheroidal, 4–5 µm long, spinose, borne in radiate heads.

17.6.2 Aspergillus candidus Link Aspergillus candidus is the only species of Aspergillus with persistently white conidia. It is readily distinguished from all species other than A. niveus. It differs from A. niveus by producing vesicles fertile over the entire area and metulae usually more than 10 µm long. The conidia are mostly spherical, 2.5–3.5 µm diam, with smooth walls, borne in radiate heads. Ecology Aspergillus candidus occurs commonly in stored cereals, particularly wheat, and cereal products such as flour and bread. It has also been isolated from © 2006, Woodhead Publishing Ltd

464 Food spoilage microorganisms milled rice (it was particularly common in samples from Indonesia and the Philippines; Pitt et al., 1998), and various nuts including peanuts, hazelnuts, walnuts and pecans. A. candidus frequently occurs on salamis and other processed meats and dried fish (see Pitt and Hocking, 1997). Aspergillus candidus has been recommended as a starter culture of low toxicity for processed meat manufacture (Grazia et al., 1986; Spotti et al., 1994). Physiology A range of growth temperatures has been reported for A. candidus, perhaps reflecting that more than one species is encompassed by this name. Tansey and Brock (1978) reported an optimum at 45–50 °C, with a maximum of 50– 55 °C, whereas Panasenko (1967) reported a minimum, optimum and maximum near 3–4, 20–24 and 40–42 °C respectively, similar to those reported by Domsch et al. (1980) (11–13, 25–28 and 41–42 °C). We have recently studied a group of isolates from tropical dried fish, clearly identifiable as A. candidus, unable to grow at 37 °C. Aspergillus candidus is a xerophile, with a minimum aw for growth of 0.75 and an optimum greater than 0.98 (Ayerst, 1969). A. candidus is more tolerant of low O2 than most Aspergillus species, being capable of growth in 0.45% O2 (Magan and Lacey, 1984b). It is also tolerant to propionic acid used as a grain preservative (Müller et al., 1981). Mycotoxins Aspergillus candidus produces a range of secondary metabolites (Frisvad and Samson, 1991), but of these only kojic acid has significant toxicity. Its common occurrence in grain dust may pose a respiratory hazard (KrysinskaTraczyk and Dutkiewicz, 2000). 17.6.3 Aspergillus carbonarius (Bain.) Thom Aspergillus carbonarius (Fig. 17.2a), a member of Section Nigri, has only come to prominence in the past 10 years or so because of its ability to produce ochratoxin A in grapes (Abarca et al., 1994). A. carbonarius is distinguished from the more common A. niger by its larger, blacker, conidia and generally larger heads. It can also be distinguished by molecular techniques (Esteban et al., submitted). Ecology Most recent reports of A. carbonarius relate to its occurrence in grapes and its role in production of ochratoxin A (see Leong et al., 2004), although it was first reported as a cause of Aspergillus bunch rot in grapes by Gupta (1956). It has also been reported from coffee beans where it may also be a source of ochratoxin A (Taniwaki et al., 2003).

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Physiology Aspergillus carbonarius grows between 10 and 42 °C, with a maximum near 30–35 °C (Leong et al., 2004; Mitchell et al., 2004; Belli et al., 2005). The optimum aw range for growth is 0.93–0.98, depending on strain (Mitchell et al., 2004; Belli et al., 2005). However, the optimum temperature for ochratoxin A production is lower than for growth, around 15–20 °C (Esteban et al., 2004; Leong, 2005). Mycotoxins Aspergillus carbonarius produces ochratoxin A, and is the primary source of this toxin in grapes and grape products (wine, grape juice, wine vinegar, dried grapes) (see Leong et al., 2004; Leong, 2005).

17.6.4 Aspergillus clavatus Desm. Aspergillus clavatus (Fig. 17.2d) produces blue grey colonies. Its large, long, clavate (club-shaped) vesicles bearing only phialides are distinctive. Ecology Aspergillus clavatus is particularly common in barley during malting, and can build to unacceptably high levels if malting temperatures are elevated or spontaneous heating occurs (Flannigan et al., 1984; Flannigan, 1986). In extreme cases blue green mats of A. clavatus may form on grain during malting (Shlosberg et al., 1991). Under these conditions it may produce mycotoxins and can be allergenic. It is reported to be the cause of ‘malt workers’ lung’ (Riddle et al., 1968; Flannigan, 1986). It is mostly associated with cereals, and has been reported from wheat, flour, bread and maize, but occasionally from other sources (see Pitt and Hocking, 1997). Physiology Aspergillus clavatus grows optimally near 25 °C, with a minimum of 5–6 °C, and a maximum near 42 °C (Panasenko, 1967; Northolt et al., 1978). The minimum reported aw for growth is 0.88–0.87 (Northolt et al., 1978). Mycotoxins This species produces patulin (Frisvad and Samson, 1991), which has the potential to cause ill health in animals (Gilmour et al., 1989; Shlosberg et al., 1991) and may have harmful effects in humans. Aspergillus clavatus also produces tryptoquivalones, toxins isolated from a mouldy rice sample implicated in death of a child (Büchi et al., 1977) and cytochalasin E (Glinsukon et al., 1974). Some of the effects of these toxins seen in laboratory animals are similar to those observed in farm animals. Outbreaks of mycotoxicoses associated with A. clavatus have been reported in stock fed on culms from distillery maltings in Europe, the United Kingdom, and South Africa (Flannigan and Pearce, 1994).

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466 Food spoilage microorganisms 17.6.5 Aspergillus flavus Link, A. parasiticus Speare and A. oryzae (Ahlburg) Cohn Undoubtedly, the most important group of toxigenic Aspergilli are the aflatoxigenic fungi A. flavus (Fig. 17.2b), A. parasiticus and the recently described but much less common species A. nomius, all of which are classified in Aspergillus Section Flavi (Gams et al., 1985). Aspergillus flavus and A. parasiticus are distinguished by their bright yellow green (or less commonly yellow) conidial colour and rapid growth at both 25 and 37 °C. A. flavus produces conidia which are rather variable in shape and size, with relatively thin, smooth to moderately rough, walls, with most being finely roughened. Conidia of A. parasiticus are spherical and have relatively thick, rough walls. In addition, vesicles of A. flavus are larger, up to 50 µm in diameter, and usually bear metulae, while vesicles of A. parasiticus rarely exceed 30 µm in diameter and metulae are uncommon. The suite of toxins produced by these three species is species-specific (Klich and Pitt, 1988; Kozakiewicz, 1994). A. flavus can produce aflatoxins B1, B2 and cyclopiazonic acid (CPA), but only a proportion of isolates are toxigenic. A. parasiticus produces aflatoxins B1, B2, G1 and G2, but not cyclopiazonic acid, and almost all isolates are toxigenic. A. nomius is morphologically similar to A. flavus, but like A. parasiticus, produces B and G aflatoxins without cyclopiazonic acid. Aspergillus flavus and A. parasiticus are closely related to A. oryzae and Aspergillus sojae, species that are used in the manufacture of fermented foods and do not produce toxins (Egel et al., 1994). Accurate differentiation of related species within Section Flavi is important in order to determine the potential for toxin production, and the types of toxins likely to be present. Detection and identification Rapid detection of aflatoxigenic fungi is possible using Aspergillus flavus and parasiticus agar (AFPA) (Pitt and Hocking, 1997), a medium formulated specifically for this purpose. When incubated on AFPA at 30 °C for 48–72 h, A. flavus, A. parasiticus and A. nomius produce a bright orange-yellow colony reverse which is diagnostic and readily recognized. A. flavus and related species also grow well on general purpose yeast and mould enumeration media such as DRBC agar or DG18 agar, and are relatively easily recognized. Screening isolates for aflatoxin production can also be used to differentiate the species. Cultures can be grown on coconut-cream agar and observed under ultraviolet light (Dyer and McCammon, 1994) or a simple agar plug technique coupled with thin layer chromatography (Filtenborg et al., 1983) can be used to screen cultures for aflatoxin production as an aid to identification. The combination of characteristics most useful in differentiation between the three aflatoxigenic species are summarized in Table 17.2. Ecology Aspergillus flavus is cosmopolitan, but A. parasiticus is less widespread.

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Table 17.2 Distinguishing features of Aspergillus flavus, A. parasiticus and A. nomius (from Hocking, 2001) Species

Conidia

Sclerotia

Toxins

A. flavus

Smooth to moderately roughened, variable in size Conspicuously roughened, little variation in size Similar to A. flavus

Large, globose

Aflatoxins B1 and B2, cyclopiazonic acid Aflatoxins B and G

A. parasiticus A. nomius

Large, globose Small, elongated (bullet-shaped)

Aflatoxins B and G

A. flavus and A. parasiticus have a strong affinity with nuts and oilseeds. In a survey of the mycoflora of commodities in Thailand (Pitt et al., 1993, 1994), A. flavus was one of the most commonly occurring fungal species in nuts and oilseeds, but A. parasiticus was rarely encountered. Corn, peanuts and cottonseed are the most important crops invaded by these fungi, and invasion often takes place before harvest, rather than during storage. Peanuts may be invaded while still in the ground if the crop suffers drought stress or related factors (Sanders et al., 1981; Cole et al., 1982; Pitt et al., 1991). In corn, insect damage to developing kernels allows entry of aflatoxigenic moulds, but invasion can also occur through the silks of developing ears (Lillehoj et al., 1980). Cotton seeds are invaded through the nectaries (Klich et al., 1984). Cereals and spices are common substrates for A. flavus (Pitt and Hocking, 1997), but aflatoxin production in these commodities is almost always a result of poor drying, handling, or storage, and aflatoxin levels are rarely significant. Significant amounts of aflatoxins can occur in peanuts, corn, and other nuts and oilseeds, particularly in some tropical countries where crops may be grown under marginal conditions, and where drying and storage facilities are limited (Arim, 1995; Dhavan and Choudary, 1995; Lubulwa and Davis, 1994). Physiology Aspergillus flavus has been reported to have a minimum temperature for growth near 10–12 °C, a maximum near 43–48 °C, and an optimum near 33 °C, with aflatoxins being produced from 12–40 °C (Diener and Davis, 1967; Northolt et al., 1977; Domsch et al., 1980; ICMSF, 1996). Its optimal aw for growth is 0.996 (Gqaleni et al., 1997), with a minimum aw for growth variously reported as 0.78 aw at 33 °C (Ayerst, 1969) and 0.82 at 25 °C, 0.81 at 30 °C and 0.80 at 37 °C (Pitt and Miscamble, 1995). A predictive model for A. flavus growth in relation to aw and temperature has been published (Gibson et al., 1994). Growth of A. flavus occurred over the pH range 2.1 to 11.2 (the entire range examined) at 25, 30 and 37 °C, with optimal growth © 2006, Woodhead Publishing Ltd

468 Food spoilage microorganisms over a broad range from pH 3.4–10 (Wheeler et al., 1991). Conidia of Aspergillus flavus are not heat resistant, with a D60°C of 1 min at neutral pH and high aw, with z values from 3.3 to 4.1C° (ICMSF, 1996). Mycotoxins Aspergillus flavus is the main source of aflatoxins, the most important mycotoxins in the world’s food supplies. Aflatoxins are produced in foods only by A. flavus and A. parasiticus. Although A. nomius and a few other Aspergillus species can produce aflatoxins, they do not occur in food. The four major naturally produced aflatoxins are known as aflatoxins B1, B2, G1 and G2, with ‘B’ and ‘G’ referring to the blue and green fluorescent colours produced under UV light. Aflatoxins are both acutely and chronically toxic to animals, including man. They produce four distinct effects: acute liver damage; liver cirrhosis; induction of tumours; and teratogenic effects (Stoloff, 1977). More detailed information about aflatoxins and their effects can be found elsewhere (see Pitt and Hocking, 1997; Hocking, 2001). Because of their high toxicity, low limits for aflatoxins in foods and feeds have been set by many countries (van Egmond and Jonker, 2004). Cyclopiazonic acid is produced by some strains of Aspergillus flavus. CPA is an indole tetramic acid which can occur in naturally contaminated agricultural commodities and compounded animal feeds. It is acutely toxic to rats and other test animals, causing severe gastrointestinal and neurological disorders (Nishie et al., 1985). Its role in human health remains unclear. Aspergillus oryzae (Ahlburg) Cohn is closely related to A. flavus, and produces colonies of similar or slightly smaller size on the standard media. However, colonies of A. flavus remain green as they age, whereas those of A. oryzae are more floccose and turn olive brown as they age. Aspergillus oryzae is of great economic importance, as it forms the basis of much of the fermented food industry in Japan and other parts of Asia. Tane koji, prepared by growing A. oryzae on cooked rice, provides a source of enzymes used in the production of shoyu (soy sauce), miso, hamanatto and other important Oriental products, which are mostly used as food flavourings (Hesseltine, 1965; Hesseltine and Wang, 1967; Beuchat, 1987; Cook and Campbell-Platt, 1994). A. oryzae does not produce aflatoxins, but some strains may produce cyclopiazonic acid (Orth, 1977; EPA, 1997).

17.6.6 Aspergillus fumigatus Fresen. Aspergillus fumigatus (Fig. 17.2c) forms velvety, bluish colonies bearing characteristic, well-defined columns of conidia. Growth at 37 °C is exceptionally rapid. The conidial heads are also diagnostic: elongated vesicles bear crowded phialides which bend to be roughly parallel to the stipe axis. Conidia are spherical to subspheroidal, 2.5–3.0 µm in diameter, with finely roughened or spinose walls. Care should be exercised in handling cultures of this species because it is a recognized human pathogen. © 2006, Woodhead Publishing Ltd

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Ecology The prime habitat for A. fumigatus is decaying vegetation, in which it causes spontaneous heating (Cooney and Emerson, 1964). Reported food-related sources include stored commodities in the tropics, cocoa beans during and after fermentation, oilseeds, cereal grains, spices, stored eggs and spices. It is quite common on cured and processed meats, especially in the tropics (see Pitt and Hocking, 1997). It has been reported as one cause of ‘Rio’ offflavours in coffee (Liardon et al., 1992). Physiology The most important physiological character of A. fumigatus is its thermophilic nature: its minimum growth temperature is near 12 °C, the optimum 40– 42 °C and maximum near 55 °C (Panasenko, 1967; Ayerst, 1969; Evans, 1971; Domsch et al., 1980). A. fumigatus is a marginal xerophile, with a minimum aw for growth of 0.82 aw near 40 °C (Ayerst, 1969). Mycotoxins Aspergillus fumigatus produces fumitremorgens, verruculogen and gliotoxin and although a role in animal disease seems likely (Frisvad and Samson, 1991; Land et al., 1993; Boudra and Morgavi, 2005; Frisvad et al., 2006), little has been published about the possible production or significance of these toxins in foods. Gliotoxin may be important in the invasion of human, animal and bird lungs by A. fumigatus (Richard et al., 1994; Lewis et al., 2005).

17.6.7 Aspergillus niger Tiegh. Aspergillus niger forms easily recognized black or dark brown colonies, as do the closely related species A. carbonarius, which produces conidia 7– 10 µm in diameter and A. awamori, which produces finely roughened conidia. A. awamori is used in food fermentations and is perhaps a domesticated form of A. niger. A. niger and closely related species (A. carbonarius, A. awamori, A. aculeatus and A. japonicus) all belong in Aspergillus Section Nigri. Ecology Aspergillus niger is more prevalent in warmer climates, both in field situations and stored foods. The black spores apparently provide protection from sunlight and UV irradiation, providing a competitive advantage in such habitats. A. niger is very frequently isolated from sun-dried products such as vine fruits (King et al., 1981; Leong et al., 2004) where it may produce ochratoxin A. Aspergillus niger is by far the most common Aspergillus species responsible for post-harvest decay of fresh fruit, including grapes, in which it is a principal agent of Aspergillus bunch rot (Nair, 1985; Snowdon, 1990), apples, pears, peaches, citrus, figs, strawberries, mangoes and melons (Barkai-Golan, 1980; © 2006, Woodhead Publishing Ltd

470 Food spoilage microorganisms Snowdon, 1990) and black mould rot of onions (Snowdon, 1991). Aspergillus niger is among the most common fungi isolated from nuts (peanuts, pecans, pistachios, hazelnuts, walnuts, kola nuts, coconut and copra). Cereals and oilseeds are also frequent sources, especially maize, but A. niger can be isolated from almost any type of stored commodity. Dried, smoked and cured fish and meat products are other common sources (see Pitt and Hocking, 1997). Physiology Growth temperatures for Aspergillus niger are minimum, 6–8 °C, maximum, 45–47 °C and optimum 35–37 °C (Panasenko, 1967). A. niger is a xerophile: Ayerst (1969) reported germination at 0.77 aw at 35 °C. A. niger is able to grow down to pH 2.0 at high aw (Pitt, 1981). Mycotoxins Until relatively recently, Aspergillus niger was regarded as a benign fungus, and has been widely used in food processing. It is categorized as ‘generally regarded as safe’ by the US Government. However, in 1994 Abarca et al. (1994) reported that some A. niger isolates can produce ochratoxin A. Although ochratoxin A production is not common, it has been confirmed by other researchers (Accensi et al., 2004; Belli et al., 2004; Leong, 2005). 17.6.8 Aspergillus ochraceus K. Wilh. Aspergillus ochraceus (Fig. 17.1) is the most commonly occurring species in Aspergillus Section Circumdati. A. ochraceus produces yellow brown (ochre) colonies. The vesicles are spherical, bearing densely packed metulae and phialides with small, smooth, pale brown conidia. This species rarely grows at 37 °C (Pitt and Hocking, 1997). Ecology Aspergillus ochraceus is widely distributed, particularly in dried foods, including dried fish, various dried beans and pulses, nuts and oilseeds (see Pitt and Hocking, 1997). It is less frequently reported from cereals (rice, barley, wheat, maize). The presence of this species in green coffee beans may lead to ochratoxin A production (Mantle and Chow, 2000; Taniwaki et al., 2003). Rots in garlic have been reported to be caused by Aspergillus ochraceus, but the species responsible is A. alliaceus (Snowdon, 1991). Physiology Aspergillus ochraceus grows between 8 and 37 °C, with the optimum 24– 31 °C (ICMSF, 1996). The minimum aw for growth is 0.77 at 25 °C (Pitt and Christian, 1968), with the optimum 0.95–0.99 (ICMSF, 1996). NaCl concentrations up to 30% (w/v), equivalent to 0.77 aw, are reportedly tolerated (Domsch et al., 1980). On coffee-based media, optimal conditions for germination and growth were observed at 0.95–0.99 aw and 20–30 °C, with © 2006, Woodhead Publishing Ltd

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the minimum aw for germination 0.80, and for mycelial growth 0.85 aw (Pardo et al., 2005). A. ochraceus grows well between pH 3 and 10, and slowly at pH 2.2 (Wheeler et al., 1991). Mycotoxins Aspergillus ochraceus was discovered to produce a mycotoxin, named ochratoxin A by van der Merwe et al. in 1965. Other minor toxins of lower toxicity were ochratoxins B and C. A. ochraceus may be a significant source of ochratoxin A in coffee (Taniwaki et al., 2003). Pardo et al. (2005) reported that maximum ochratoxin A production in coffee beans occurred at 20 °C and 0.99 aw, whereas at 10 °C, ochratoxin was not produced, regardless of aw. No ochratoxin was detected by Pardo et al. (2005) at 0.80 aw; however, Milanez and Leitao (1994) reported ochratoxin production down to 0.80 aw on beans at 25 °C. Aspergillus ochraceus also produces penicillic acid, a mycotoxin of lesser importance (Northolt et al., 1979), xanthomegnin and viomellein (Frisvad and Thrane, 2000). Recent molecular studies by Frisvad et al. (2004) indicate that a new species, Aspergillus westerdijkiae, may be a more important ochratoxin A producer than A. ochraceus. Other Aspergillus species in Section Circumdati can also produce ochratoxin A. Aspergillus sclerotiorum, Aspergillus alliaceus, Aspergillus melleus and other less common species have all been reported to produce ochratoxins (Ciegler, 1972; Varga et al., 1996; Frisvad and Samson, 2000; Frisvad et al., 2006).

17.6.9 Aspergillus penicillioides Spegazzini and A. restrictus G. Sm. Aspergillus penicillioides and A. restrictus are extremely xerophilic species. Because they grow poorly on most microbiological media, they are often overlooked. Both species grow very slowly under all standard conditions, and produce green conidia. A. penicillioides is more xerophilic than A. restrictus, and forms spathulate vesicles, fertile over more than the upper half, whereas those of A. restrictus are more rounded, and usually fertile only over the upper half, with conidia borne in distinct columns. Ecology Aspergillus penicillioides and A. restrictus are both common in a variety of low aw foods including flour, dried fruit and dried fish, spices, including pepper and dried chillies, confectionery, and low aw baked goods such as fruit cakes and puddings (see Pitt and Hocking, 1997). They are probably among the pioneer species to establish in commodities stored at low aw, and may pave the way for fungal succession by less xerophilic species such as Eurotium species (Hocking, 2003). A. restrictus is frequently isolated from house dust and may play a role in fungal allergies (Toyazaki, 2002).

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472 Food spoilage microorganisms Physiology Both species grow very poorly at high aw. The optimal aw for growth of A. penicillioides is 0.91–0.93 (Andrews and Pitt, 1987). At 25 °C, Aspergillus penicillioides is capable of germination down to 0.73 aw in laboratory media containing glucose/fructose or glycerol as principal solute. In natural substrates, A. penicillioides grows at much lower water activities: we have repeatedly isolated this species from grain and dried pet food stored at 0.68 aw for up to 6 months (our unpublished observations). Growth temperatures have not been accurately reported but growth occurs at 37 °C at lower aw (Wheeler et al., 1988), with a minimum near 15 °C at 0.95–0.90 aw (Wheeler et al., 1988). Smith and Hill (1982) reported the temperature range for growth of Aspergillus restrictus was minimum, 9 °C, optimum, 30 °C and maximum, 40 °C. Growth of this species has been observed down to 0.75 aw (Snow, 1949; Pelhate, 1968). Mycotoxins Neither species has been reported to produce mycotoxins.

17.6.10 Aspergillus sydowii (Bainier & Sartory) Thom & Church Aspergillus sydowii forms dark turquoise colonies, often with a brown reverse. It grows slowly at 25 °C and often not at all at 37 °C, produces heads with both metulae and phialides, and dark blue-green conidia. Vesicles on the larger stipes are small and club-shaped, and diminutive penicilli are also formed. Ecology Aspergillus sydowii is a widely distributed storage fungus, isolated from a variety of dried foods, particularly nuts, but also from rice, beans, spices and dried meat products (see Pitt and Hocking, 1997). Physiology Aspergillus sydowii is closely related to Aspergillus versicolor so can be expected to have similar physiological properties. The minimum for growth is near 0.78 aw (Snow, 1949; Wheeler and Hocking, 1988). 17.6.11 Aspergillus tamarii Kita Aspergillus tamarii belongs in Aspergillus Section Flavi, and resembles A. flavus and A. parasiticus, but conidia of A. tamarii are coloured olive to brown, and are larger, with thick, conspicuously roughened walls. On AFPA, A. tamarii produces a deep brown reverse coloration, in contrast to the orange yellow of A. flavus and A. parasiticus. This is a useful diagnostic aid.

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Ecology Aspergillus tamarii occurs most commonly in tropical and subtropical regions, in nuts (peanuts, pistachios, pecans, hazelnuts, walnuts, kola nuts and betel nuts), oilseeds and copra. It is occasionally isolated from other sources such as wheat and other small grains, coffee beans, soybeans, spices and dried meat and fish products (see Pitt and Hocking, 1997). Physiology The physiology of Aspergillus tamarii is similar to that of A. flavus. Ayerst (1969) reported that A. tamarii was capable of growth down to 0.78 aw at 33 °C. Mycotoxins Aspergillus tamarii does not produce aflatoxins, but it does produce some other toxic compounds, including cyclopiazonic acid (Dorner, 1983; Ito et al., 1998), and kojic acid, a less toxic compound (Ito et al., 1998). The presence of A. tamarii in high concentrations in foodstuffs is clearly undesirable. A. tamarii may have been the cause of ‘kodua poisoning’ from kodo millet seed (Paspalum scrobiculatum) in India (Rao and Husain, 1985). The alkaloid fumiclavine A was also found in kodo millet seed from that outbreak, and it may have contributed to the toxicity (Janardhanan et al., 1984).

17.6.12 Aspergillus terreus Thom Aspergillus terreus (Fig. 17.2f), Section Terrei produces rapidly growing pale brown colonies, with Aspergillus heads bearing densely packed metulae and phialides with minute conidia borne in distinctive long columns. Ecology Aspergillus terreus occurs commonly in soil and in foods, particularly stored cereals and cereal products, beans, pulses and nuts, but is not regarded as an important spoilage mould (Pitt and Hocking, 1997). Physiology Aspergillus terreus grows much more strongly at 37 °C than at 25 °C (Pitt and Hocking, 1997), but little information has been published on its physiology. The reported minimum aw for growth is 0.78 at 37 °C (Ayerst, 1969). Mycotoxins Aspergillus terreus produces a wide range of metabolites (Frisvad and Samson, 1991), the most significant of which are probably a group of tremorgenic toxins known as territrems (Ling et al., 1979). These toxins have not been implicated in human disease (Cole and Dorner, 1986; Ling, 1994).

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474 Food spoilage microorganisms 17.6.13 Aspergillus versicolor Aspergillus versicolor grows slowly, produces both metulae and phialides from small vesicles, and green conidia. Growth at 37 °C is weak or absent. This species may produce a wide range of mycelial and reverse pigmentation, especially if cultures are incubated for 14 days or so. Ecology Aspergillus versicolor is the most important food spoilage and toxigenic species in Aspergillus Section Versicolores. It is widely distributed and has been reported from most kinds of foods. Major sources include cereals, oilseeds, nuts and pulses from both tropical and temperate regions, but it has also been found in dried meat products and hard cheeses (see Pitt and Hocking, 1997). It is one fungus responsible for decay in fresh breadfruit (Omobuwajo and Wilcox, 1989), and is also one cause of the ‘Rio’ offflavour in coffee due to the formation of trichloroanisoles (Liardon et al., 1992). Physiology Aspergillus versicolor is a mesophile, with a minimum temperature for growth of 9 °C at 0.97 aw, a maximum of 39 °C at 0.87 aw, and an optimum of 27 °C at 0.98 aw (Smith and Hill, 1982). It is also xerophilic: its minimum aw for growth is 0.78–0.80 (see Pitt and Hocking, 1997). A. versicolor has a pH minimum above pH 3.1, and a pH maximum above pH 10.2 (Wheeler et al., 1991). Mycotoxins Aspergillus versicolor is the major producer of sterigmatocystin, a carcinogen which is a precursor of the aflatoxins (Cole and Cox, 1981), but aflatoxins are not produced by this species. Natural occurrence of sterigmatocystin has been reported in rice in Japan, wheat and barley in Canada, cereal-based products in the United Kingdom (Scott, 1994) and Ras cheese (Abd Alla et al., 1996). Sterigmatocystin has also been reported from mould affected buildings and building materials (Nielsen et al., 1999; Tuomi et al., 2000; Nielsen 2003).

17.6.14 Aspergillus wentii Wehmer Aspergillus wentii (Section Wentii) produces golden brown colonies. It grows faster on reduced aw media than on CYA. It produces Aspergillus heads with both metulae and phialides on long stipes, and the conidia are relatively large (3–5 µm). Ecology Aspergillus wentii has only rarely been reported as a spoilage fungus, possibly because of its xerophilic nature. However, it appears to be relatively common

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on stored commodities, including dried fish, from tropical countries (Wheeler et al., 1986; Pitt et al., 1993, 1994, 1998). Physiology Aspergillus wentii is a xerophile that exhibits strong growth in both sugar and salt environments. The optimum aw for growth is near 0.94, with a minimum aw for germination at 25 °C of 0.73 aw in glucose/fructose, 0.75 aw in glycerol and 0.79 aw in NaCl (Andrews and Pitt, 1987). Mycotoxins Emodin is produced by Aspergillus wentii (Wells et al., 1975); however, there are no reports of toxicoses associated with A. wentii in foods or animal feeds.

17.7 Aspergillus as spoilage fungi Why are Aspergillus species so ubiquitous and successful as spoilage fungi? Their ability to grow at reduced aw and in warmer climates, on a wide range of substrates, ensures that they can be found in almost all food storage facilities. Although they are not the most xerophilic fungi (Xeromyces bisporus holds that honour), they are non-fastidious and will grow on a wide range of food and non-food substrates, including leather, paper, museum artefacts, etc. (Pitt and Hocking, 1997). In addition, the ascospores of Eurotium species are more resistant than conidia to heat, UV irradiation and high pressure, although they not as heat-resistant as ascospores of Neosartorya, Byssochlamys or Talaromyces.

17.8 Control measures Spoilage of foods by Aspergillus species falls into two general categories: specific food–fungus associations and non-specific spoilage. Where there is an association between particular species of Aspergillus and a food crop, such as A. flavus and peanuts, or black Aspergilli and grapes, measures can be taken during the growing phase of the crop to minimize fungal populations and thus contamination levels. In the case of peanuts, a biocontrol strategy involving competitive exclusion by non-toxigenic strains of A. flavus can reduce aflatoxin levels in peanuts (Cleveland et al., 2003; Pitt and Hocking, 2003). For grapes, current research is targeting reduction of inoculation levels in the vineyard by management strategies such as mulching vines, pruning to improve canopy and bunch architecture and suitable irrigation systems (Leong et al., accepted). Aspergillus penicillioides and Eurotium species are pioneer species in the

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476 Food spoilage microorganisms colonization of stored commodities. Control of spoilage of stored commodities relies largely on control of water activity. Maintenance of a dry environment (below 0.6 aw) will ensure that mould growth cannot develop. Temperature gradients and large diurnal fluctuation in temperature can cause moisture migration resulting in localized areas of high aw where fungal growth can develop. Adequate ventilation of storage structures can minimize temperature gradients and consequent moisture migration. Regular cleaning of storage facilities to remove grain residues and prevent insect infestation will minimize inoculum levels of spoilage fungi. Control of spoilage of reduced aw foods such as bakery goods and confectionary also relies on aw control, but there are other hurdles than can be used to prevent spoilage. In the food industry, weak acid preservatives such as sorbates, benzoates and propionates are commonly used to extend shelflife of cakes and bread. Preservatives are used in combination with acid pH, reduced aw and modified atmosphere packaging to extend shelf-life of baked goods (Abellana et al., 2000; Guynot et al., 2002; Gock et al., 2003). Models for growth of Aspergillus species and mycotoxin production, based on combinations of aw and temperature, such as those of Rosso and Robinson (2001), Parra and Magan (2004) and Pardo et al. (2004, 2005) can be used to predict safety and stability of foods that are susceptible to spoilage by Aspergilli.

17.9 Future trends Molecular techniques have brought great changes to our understanding of the fungal genome and to fungal taxonomy. Gene sequences are now routinely being used to establish relationships within the various sections of Aspergillus and to describe new species that are morphologically indistinguishable (Frisvad et al., 2004; Samson et al., 2004b). This will make identification of some Aspergillus species very difficult for the food microbiologist at the bench level. However, fungal genomics opens the way for a better understanding of pathways leading to the production of enzymes, useful secondary metabolites and mycotoxins. This knowledge can be exploited, either for enhanced enzyme and secondary metabolite production, or in control of fungal growth and mycotoxin production.

17.10

Conclusions

Aspergillus is one of the most important fungal genera in the spoilage of foods and animal feeds, particularly in warm-temperate climates and the tropics. Eurotium species along with A. restrictus and A. penicillioides are significant because they are the pioneering species in spoilage of stored © 2006, Woodhead Publishing Ltd

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commodities (grains and nuts), and their metabolic activities pave the way for fungal succession by less xerophilic species such as mycotoxigenic Aspergilli and Penicillium species (Hocking, 2003). Unlike Penicillium, there are few instances of associations between spoilage of specific foods and particular Aspergillus species. Although species-substrate associations such as Aspergillus flavus with peanuts and A. carbonarius with grapes are well recognized, these two species are also very common in other substrates, and conversely, other fungi are also known to cause spoilage in these commodities. The genus Aspergillus contains a number of economically significant, highly mycotoxigenic species, the aflatoxigenic species clearly being the most important from the point of view of human health, although ochratoxin production by a number of Aspergillus species is becoming increasingly important. Many of the other mycotoxins produced by Aspergillus may have roles in cancer induction and immunosuppression, but these are yet to be elucidated.

17.11

Sources of further information

There is a plethora of literature on Aspergillus species and their metabolic activities that is of relevance to food microbiologists. However, the following texts are recommended as useful compilations of information on this genus and its importance to the food industry. Hocking A D (2001), ‘Toxigenic Aspergillus species’, in Food Microbiology – Fundamentals and Frontiers, 2nd edn, Doyle M P, Beuchat L R and Montville T J, Washington DC, American Society for Microbiology, 451– 465. Klich M A (2002), Identification of Common Aspergillus Species, Utrecht, Netherlands, Centraalbureau voor Schimmelcultures. Pitt J I and Hocking A D (1997), Fungi and Food Spoilage, 2nd edn, London, Blackie Academic and Professional. Powell K A, Renwick A and Peberdy J F (1994), The Genus Aspergillus. From Taxonomy and Genetics to Industrial Application, New York, Plenum Press. Samson R A, Hoekstra E S and Frisvad J C (2004), Introduction to Foodand Airborne Fungi, 7th edn, Utrecht, Netherlands, Centraalbureau voor Schimmelcultures.

17.12

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