**1. General principles**

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The term mycotoxin was used for the first time in 1961 in the aftermath of a veterinary crisis in England, during which thousands of animals died. The disease was linked to a peanut meal, incorporated in the diet, contaminated with a toxin produced by the filamentous fungus *Aspergillus flavus* (Bennet & Klich, 2003; Richard, 2007).

In general, mycotoxins are low-molecular-weight compounds that are synthetized during secondary metabolism by filamentous fungi; their chemical structure may range from simple C4 compounds to complex substances (Paterson & Lima, 2010).

Mycotoxins are natural contaminants in raw materials, food and feeds. Mould species that produce mycotoxins are extremely common, and they can grow on a wide range of substrates under a wide range of environmental conditions; they occur in agricultural products all around the world (Bennet & Klich, 2003). Many mycotoxins may be toxic to vertebrates and other animal groups and, in low concentrations, some of them can cause autoimmune illnesses, and have allergenic properties, while others are teratogenic, carcinogenic, and mutagenic (Bennet & Klich, 2003; Council for Agricultural Science and Technology [CAST], 2003).

Apparently, mycotoxins have no biochemical significance on fungal growth; they may have developed to provide a defense system against insects, microorganisms, nematodes, animals and humans (Etzel, 2002).

Exposure to mycotoxins may occur through ingestion, inhalation, and dermal contact, and it is almost always accidental. Most cases of mycotoxicoses (animals and humans) result from eating contaminated food. Human exposure can be direct *via* cereals or indirect *via* animal products (e.g. meat, milk and eggs) (CAST, 2003).

Most mycotoxins are relatively heat-stable within the conventional food processing temperature range (80–121°C), therefore so little or no destruction occurs under normal cooking conditions, such as boiling and frying, or even following pasteurization (Milicevic et al., 2010). The stability of mycotoxins during food processing has been reviewed in the work by Bullerman & Bianchini (2007). In general, the application of a food process reduces mycotoxin concentrations significantly, but does not eliminate them completely. The food processes that have been examined include physical treatments (cleaning and milling) and thermal processing (e.g. cooking, baking, frying, roasting and extrusion). The different treatments have various effects on mycotoxins, and those that utilize the highest temperatures have the greatest effects: roasting or cooking at high temperatures (above 150 °C) appear to reduce mycotoxin concentrations significantly (Bullerman & Bianchini, 2007).

toxygenic fungi. Moisture and temperature are two factors that have a crucial effect on fungal proliferation and toxin biosynthesis (Bryden, 2007; Paterson & Lima, 2010). The incidence and level of mycotoxin contamination are closely related to the geographic position and to seasonal factors as well as to the cultivation, harvesting, stocking, and

165

Mycotoxin contaminations can be divided into the one that occurs in the developing crop (preharvest) and the one that develops after maturation (post-harvest). In the pre-harvest period, preventive measures are included in good agronomic practices, such as the careful use of insecticides and fungicides, irrigation to avoid moisture stress, harvesting at maturity and improvement by genetic resistance to fungal attack. During the post-harvest period, the control of the moisture and temperature of the stored commodity will largely determine the degree of fungal activity and consequently the mycotoxin synthesis (Bryden, 2007). Treatments with chemicals, including sodium bisulfite, ozone, and ammonia, acids and bases, represent an opportunity to control fungal growth and mycotoxin biosynthesis in stored grains (Bozoglu, 2009; Magan, 2006; Magan & Aldred, 2007). In recent years, a good control of mycotoxigenic fungi has been achieved using plant products (e.g. extracts and essential oils) as environmental

friendly fungicides (Nguefacka et al., 2004; Reddy et al., 2010; Thembo et al., 2010).

inhibit fungal growth and mycotoxin production (Bianchini & Bullerman, 2010).

effects) and pharmacologically active substances (Milicevic et al., 2010; Zain, 2011).

to aflatoxin and fumonisins (Mazzoni et al., 2011; Wogan, 1992).

Moreover, biological control represents a new opportunity in control strategies: there is evidence that *Bacillus* sp., propionic acid bacteria and lactic acid bacteria (LAB) are able to

Mycotoxins are toxic to vertebrates and humans at low concentrations. Mycotoxicoses in humans or animals have been characterized as food or feed related, non-contagious, non-

Mycotoxins have various acute and chronic effects on humans and animals, depending on the species. Within a given species, the impact of mycotoxins on health is influenced by age, sex, weight, diet, exposure to infectious agents, and the presence of other mycotoxins (synergistic

The majority of mycotoxins currently known are grouped, according to their toxic activity, under chronic conditions as mutagenic, carcinogenic or teratogenic. Grouping according to their site of action results in hemo-, hepato-, nephron-, dermato-, neuro- or immunotoxins

The most important mycotoxins worldwide are aflatoxins, fumonisins, ochratoxins, deoxinyvalenol and zearalenone. Carcinogenic properties have been recognized with regard

Aflatoxin B1 (AFB1) has been linked to human primary liver cancer, in which it acts synergistically with HBV infection and it has been classified as a carcinogen in humans (Group 1 carcinogen). Fumonisin B1, the most abundant of the numerous fumonisin analogues, was classified as a Group 2B carcinogen (possibly carcinogenic to humans) (Zain,

The potential role of dietary factors to counteract the toxic effects of mycotoxins has been reviewed by Galvano et al. (2001): the effect of antioxidants, food components and additives on reducing toxicity, by decreasing toxin formation and enhancing the metabolism, has been

A mixture of mycotoxins may occur simultaneously, depending on the environmental conditions and substrate availability (Milicevic et al., 2010). Therefore it can be expected that

transport conditions (Milicevic et al., 2010).

**1.3 Toxicology and health** 

(Niessen, 2007).

reported.

2011; Wild & Gong, 2010).

transferable, and non-infectious (Zain, 2011).

It has been estimated that 25% of the world's crops are affected by fungal growth, and commodities may be, both pre- and post-harvest, contaminated with mycotoxins. The mycotoxins that can be expected in food differ from country to country in relation to the different crops, agronomic practices and climatic conditions (Bryden, 2007). Since climate changes affect the growth of mycotoxigenic fungi, mycotoxin production is also influenced (Magan et al., 2003).

Currently, more than 400 mycotoxins are known. Scientific attention has mainly focused on those that have proven to be carcinogenic and/or toxic in humans and animals. Five classes of mycotoxins are considered the most significant in agriculture and in the food industry: aflatoxins (aflatoxin B1), ochratoxins (ochratoxin A), fumonisins (fumonisin B1), zearalenone, and patulin which are derived from polyketide (PK) metabolism, and trichothecenes (deoxynivalenol), whose biosynthetic pathway is of terpenoid origin. PKs are metabolites that are derived from the repetitive condensation of acetate units or other short carboxylic acids, via an enzymatic mechanism that is similar to that responsible for fatty acid synthesis (Huffman et al., 2010).

Aflatoxin, ochratoxin, fumonisin, trichothecene, zearalenone and patulin are the most widespread mycotoxins in animal feed and human food. The chemical structure, biosynthetic pathway, mycotoxigenic fungi, the influence of environmental factors and toxicology will be briefly described for each class. Zearalenone will not be dealth with in the present work as, because of its hormonal activity, there is considerable knowledge about ZEA and its derivatives which can be found in the literature on growth hormones.

#### **1.1 Toxigenic fungi**

*Aspergillus*, *Alternaria*, *Claviceps, Fusarium, Penicillium* and *Stachybotrys* are the recognized genera of mycotoxigenic fungi (Milicevic et al., 2010; Reddy et al., 2010). Many of these genera are ubiquitous and, in some cases, apparently have a strong ecological link with human food supplies. The natural fungal flora associated with food production is dominated by the *Aspergillus, Fusarium* and *Penicillium* genera (Sweeney & Dobson, 1998). *Fusarium* species are pathogens that are found on cereal crops and other commodities, and they produce mycotoxins before, or immediately after, the harvest. Some species of *Aspergillus* and *Penicillium* are also plant pathogens or commensals, but these genera are more commonly associated with commodities and food during drying and storage (Pitt, 2000).

Toxigenic moulds are known to produce one or more of these toxic secondary metabolites. However, not all moulds are toxigenic and not all secondary metabolites from moulds are toxic. Many fungi produce several mycotoxins simultaneously, especially *Fusarium* species.

Moreover, recent studies have demonstrated that the necrotrophic pathogens of wheat, *Stagonospora nodorum*, *Pyrenophora tritiirepentis* and *Alternaria alternata,* are also capable of synthesizing an array of mycotoxic compounds during disease development (Solomon, 2011).

Nowadays, the identification and quantification of mycotoginenic fungi are carried out by PCR. Diagnostic PCR-based systems are now available for all of the most relevant toxigenic fungi: producers of aflatoxins, trichotecenes, fumonisins and patulin (Niessen, 2007; Paterson, 2006). The primers for mycotoxin pathway sequences have been reviewed in the work by Paterson (2006).

#### **1.2 Influence of environmental factors on mycototoxin production**

The production of mycotoxins is highly susceptible to temperature, moisture, water activity (aw), pH and oxygen concentration, the same environmental factors that affect the growth of

It has been estimated that 25% of the world's crops are affected by fungal growth, and commodities may be, both pre- and post-harvest, contaminated with mycotoxins. The mycotoxins that can be expected in food differ from country to country in relation to the different crops, agronomic practices and climatic conditions (Bryden, 2007). Since climate changes affect the growth of mycotoxigenic fungi, mycotoxin production is also influenced

Currently, more than 400 mycotoxins are known. Scientific attention has mainly focused on those that have proven to be carcinogenic and/or toxic in humans and animals. Five classes of mycotoxins are considered the most significant in agriculture and in the food industry: aflatoxins (aflatoxin B1), ochratoxins (ochratoxin A), fumonisins (fumonisin B1), zearalenone, and patulin which are derived from polyketide (PK) metabolism, and trichothecenes (deoxynivalenol), whose biosynthetic pathway is of terpenoid origin. PKs are metabolites that are derived from the repetitive condensation of acetate units or other short carboxylic acids, via an enzymatic mechanism that is similar to that responsible for fatty

Aflatoxin, ochratoxin, fumonisin, trichothecene, zearalenone and patulin are the most widespread mycotoxins in animal feed and human food. The chemical structure, biosynthetic pathway, mycotoxigenic fungi, the influence of environmental factors and toxicology will be briefly described for each class. Zearalenone will not be dealth with in the present work as, because of its hormonal activity, there is considerable knowledge about

*Aspergillus*, *Alternaria*, *Claviceps, Fusarium, Penicillium* and *Stachybotrys* are the recognized genera of mycotoxigenic fungi (Milicevic et al., 2010; Reddy et al., 2010). Many of these genera are ubiquitous and, in some cases, apparently have a strong ecological link with human food supplies. The natural fungal flora associated with food production is dominated by the *Aspergillus, Fusarium* and *Penicillium* genera (Sweeney & Dobson, 1998). *Fusarium* species are pathogens that are found on cereal crops and other commodities, and they produce mycotoxins before, or immediately after, the harvest. Some species of *Aspergillus* and *Penicillium* are also plant pathogens or commensals, but these genera are more commonly

Toxigenic moulds are known to produce one or more of these toxic secondary metabolites. However, not all moulds are toxigenic and not all secondary metabolites from moulds are toxic. Many fungi produce several mycotoxins simultaneously, especially *Fusarium* species. Moreover, recent studies have demonstrated that the necrotrophic pathogens of wheat, *Stagonospora nodorum*, *Pyrenophora tritiirepentis* and *Alternaria alternata,* are also capable of synthesizing an array of mycotoxic compounds during disease development (Solomon, 2011). Nowadays, the identification and quantification of mycotoginenic fungi are carried out by PCR. Diagnostic PCR-based systems are now available for all of the most relevant toxigenic fungi: producers of aflatoxins, trichotecenes, fumonisins and patulin (Niessen, 2007; Paterson, 2006). The primers for mycotoxin pathway sequences have been reviewed in the

The production of mycotoxins is highly susceptible to temperature, moisture, water activity (aw), pH and oxygen concentration, the same environmental factors that affect the growth of

ZEA and its derivatives which can be found in the literature on growth hormones.

associated with commodities and food during drying and storage (Pitt, 2000).

**1.2 Influence of environmental factors on mycototoxin production** 

(Magan et al., 2003).

**1.1 Toxigenic fungi** 

work by Paterson (2006).

acid synthesis (Huffman et al., 2010).

toxygenic fungi. Moisture and temperature are two factors that have a crucial effect on fungal proliferation and toxin biosynthesis (Bryden, 2007; Paterson & Lima, 2010). The incidence and level of mycotoxin contamination are closely related to the geographic position and to seasonal factors as well as to the cultivation, harvesting, stocking, and transport conditions (Milicevic et al., 2010).

Mycotoxin contaminations can be divided into the one that occurs in the developing crop (preharvest) and the one that develops after maturation (post-harvest). In the pre-harvest period, preventive measures are included in good agronomic practices, such as the careful use of insecticides and fungicides, irrigation to avoid moisture stress, harvesting at maturity and improvement by genetic resistance to fungal attack. During the post-harvest period, the control of the moisture and temperature of the stored commodity will largely determine the degree of fungal activity and consequently the mycotoxin synthesis (Bryden, 2007). Treatments with chemicals, including sodium bisulfite, ozone, and ammonia, acids and bases, represent an opportunity to control fungal growth and mycotoxin biosynthesis in stored grains (Bozoglu, 2009; Magan, 2006; Magan & Aldred, 2007). In recent years, a good control of mycotoxigenic fungi has been achieved using plant products (e.g. extracts and essential oils) as environmental friendly fungicides (Nguefacka et al., 2004; Reddy et al., 2010; Thembo et al., 2010).

Moreover, biological control represents a new opportunity in control strategies: there is evidence that *Bacillus* sp., propionic acid bacteria and lactic acid bacteria (LAB) are able to inhibit fungal growth and mycotoxin production (Bianchini & Bullerman, 2010).

#### **1.3 Toxicology and health**

Mycotoxins are toxic to vertebrates and humans at low concentrations. Mycotoxicoses in humans or animals have been characterized as food or feed related, non-contagious, nontransferable, and non-infectious (Zain, 2011).

Mycotoxins have various acute and chronic effects on humans and animals, depending on the species. Within a given species, the impact of mycotoxins on health is influenced by age, sex, weight, diet, exposure to infectious agents, and the presence of other mycotoxins (synergistic effects) and pharmacologically active substances (Milicevic et al., 2010; Zain, 2011).

The majority of mycotoxins currently known are grouped, according to their toxic activity, under chronic conditions as mutagenic, carcinogenic or teratogenic. Grouping according to their site of action results in hemo-, hepato-, nephron-, dermato-, neuro- or immunotoxins (Niessen, 2007).

The most important mycotoxins worldwide are aflatoxins, fumonisins, ochratoxins, deoxinyvalenol and zearalenone. Carcinogenic properties have been recognized with regard to aflatoxin and fumonisins (Mazzoni et al., 2011; Wogan, 1992).

Aflatoxin B1 (AFB1) has been linked to human primary liver cancer, in which it acts synergistically with HBV infection and it has been classified as a carcinogen in humans (Group 1 carcinogen). Fumonisin B1, the most abundant of the numerous fumonisin analogues, was classified as a Group 2B carcinogen (possibly carcinogenic to humans) (Zain, 2011; Wild & Gong, 2010).

The potential role of dietary factors to counteract the toxic effects of mycotoxins has been reviewed by Galvano et al. (2001): the effect of antioxidants, food components and additives on reducing toxicity, by decreasing toxin formation and enhancing the metabolism, has been reported.

A mixture of mycotoxins may occur simultaneously, depending on the environmental conditions and substrate availability (Milicevic et al., 2010). Therefore it can be expected that

Structurally, AFs are difurocoumarin derivatives that fluoresce under ultraviolet light. Depending upon colour of the fluorescence, AFs are divided into aflatoxin B1 and B2 (AFB1, AFB2) for blue, and G1 and G2 (AFG1, AFG2) for green (Dalvi, 1986) (Figure 1). Aflatoxin M1 and M2 (AFM1, AFM2), known as milk-AFs, are the metabolites of AFB1 and AFB2, respectively (Carnaghan et al., 1963). Other metabolites of AFB1 are aflatoxin Q1 (AFQ1) and aflatoxicol. Of the known AFs, AFB1 is the most common produced mycotoxin and the most potent; it has been reported to be the most powerful natural carcinogen in mammals

167

The biosynthesis of aflatoxins is a complex process, involving multi-enzymatic reactions. Genetic studies on the molecular mechanism of aflatoxin B1 biosynthesis have identified an aflatoxin pathway gene cluster of 70 kilobase pairs in length consisting of at least 24 identified structural genes including a positive regulatory gene as the transcription activator. The structural genes encode cytochrome P450 monooxygenases, dehydrogenases, oxidases, methyltransferases, a polyketide synthase and two unique fatty acid synthases (Yu et al., 2002).

Aflatoxins are closely related to a group of aspergilli: *A. flavus*, *A. parasiticus*, and *A. nomius*; although one report also adds a sclerotium producing strain of *A. tamarii*, which is closely related to *A. flavus*, to the list (Goto et al., 1996). Earlier reports of the production of aflatoxins by other aspergilli, penicillia or even a species of *Rhizopus*, have not been

*A. flavus* and *A. parasiticus*, which are found worldwide in the air and soil, usually infest both living and dead plants and animals, and as a consequence, aflatoxins in agricultural commodities are primarily produced by *A. flavus* and *A. parasiticus*. *A. flavus* produces only B aflatoxins, while *A. nomius* and *A. parasiticus* produce both B and G toxins (Rustom, 1997;

Aflatoxin contamination of food and feeds is a serious problem worldwide. Studies focusing on AF contamination in foodstuffs have in fact been reported in many countries, especially those in tropical and subtropical regions, such as Asia and Africa (Bankole et al., 2010;

Aflatoxin contamination can develop both in the pre- and post-harvest periods, but the highest levels are usually associated with post-harvest spoilage of food commodities, stored under inappropriate high moisture content and high temperature conditions which facilitate the rapid growth of moulds; the level of contamination depends on the plant stress, temperature, water activity, genotype, culture and storage conditions, but appropriate postharvest treatments, under dry cool settings, should control this source of contamination

As far as pre-harvest, is concerned, aflatoxigenic fungi have a complex ecology. The spores of *A. flavus* and *A. parasiticus* can germinate on the stigma surfaces of plants, and the germ tube can penetrate the developing embryo in a manner which mimics pollen germ tubes. The mycelium can establish an endotrophic relationship, which is not harmful to a healthy plant, while if the plant is stressed (e.g. drought), significant levels of aflatoxin may be produced during field growth. Under these circumstances food commodities may already be contaminated at harvesting and, even though the concentrations are never as high as

(Creppy, 2002).

**2.1 Fungi** 

Yu et al., 2002).

**2.2 Food** 

adequately confirmed (Moss, 2002a).

Shundo et al., 2009; Soubra et al., 2009).

(Moss, 2002a; Wilson & Payne, 1994).

humans and animals are exposed to a mixture rather than to individual compounds. For example, the interactive (synergistic) cytotoxic effects of Ochratoxin A (OTA), Ochratoxin B (OTB), citrinin, and patulin, which are produced by a number of *Penicillium* and *Aspergillus*  species, have recently been evaluated by Heussner et al. (2006).
