**1. Introduction**

Aflatoxins family includes a great number of lipophilic molecules produced by aerobic mi‐ croscopic fungi belonging to the genus *Aspergillus*. The chapter describes their chemical structure, chemical and physical properties, and aspects related to their presence in food and commodities. Aflatoxins presence in food is considered a real and severe risk to con‐ sumers for their toxicity. Aflatoxins levels and frequency of foods natural contamination as reported in the scientific literature are briefly analyzed. Focus is given to the different food‐ stuffs that may be at risk of contamination by *Aspergillus* and the subsequent accumulation of aflatoxins in the food chain. Bioavailability and bioaccessibility of aflatoxins will be dis‐ cussed considering that these unwanted molecules can be assumed by the humans with the diet. Bioaccessibility, that deals with the fraction of micro-nutrients released from the food matrix during digestion and gastro-intestinal available for absorption, will be discussed with reference to aflatoxins bioaccessibility of during the digestion process, considering the relationships between the food matrix and its influences on aflatoxins fate. Bioavailability of the aflatoxins assumed from the diet depends on their stability during digestion, since they are released from the food matrix (bioaccessibility) and on the efficiency of their passage through the gastro-intestinal mucosa. The term bioavailability includes the concepts of availability to the absorption, metabolism, distribution of nutrients to tissues and bioactivity and indicates the fraction of micro-nutrients absorbed by the body and the speed with which these molecules are absorbed and made available at their site of action. Despite of the practical difficulties in measuring the distribution and bioactivity of aflatoxins on a specific human body organ, the bioavailability is the fraction of an oral dose of a compound or pre‐ cursor of an active metabolite that reaches the bloodstream. Bioaccessibility includes the en‐ tire sequence of events that take place during the digestion of food material that can be assimilated by the body through the epithelial cells of the gastro-intestinal mucosa. Aflatox‐ ins are often present in very small amounts or in traces and, for this reason, a part of the

© 2013 Santini and Ritieni; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Santini and Ritieni; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

chapter addresses the advanced new chromatographic and spectrometric methods descri‐ bed in the literature and applied to research, that can reveal, even in trace amounts, aflatox‐ ins in biological fluids as free form or as by-products, e.g. non-covalent adducts.

**Aflatoxin MW (g/ mol) Formula Melting point**

B1 312.28 C17H12O6 268–269

B2 314.29 C17H14O6 286–289

G1 328.28 C17H12O7 244-246

G2 330.29 C17H14O7 237–240

M1 328.28 C17H12O7 299

M2 330.29 C17H14O7 293

**Figure 1.** Chemical structures of the main aflatoxins.

**Table 1.** Chemical relevant data for main aflatoxins (O'Neil, Smith, Heckelman, 2001).

**(°C)**

**IUPAC name**

2,3,6a,9a-tetrahydro-4-methoxycyclopenta(c)furo(3',2': 4,5)furo(2,3-h)(1)benzo-pyran-1,11-dione

Aflatoxins: Risk, Exposure and Remediation http://dx.doi.org/10.5772/52866 345

2,3,6aa,8,9,9aa-Hexahydro-4-methoxycyclopenta(c)furo(2',3': 4,5)furo(2,3-h)chromene-1,11-dione

7AR,cis)3,4,7a,10a-tetrahydro-5-methoxy-1H,12H-furo(3',2': 4,5)furo(2,3-h)pyrano(3,4-c)chromene-1,12-dione

> 1H,12H-furo(3',2':4,5)furo(2,3-h)pyrano(3,4-c) (1)benzopyran-1,12-dione

> (6AR-cis)-2,3,6a,9a-tetrahydro-9a-hydroxy-4 methoxycyclopenta(c)furo(3',2':4,5)furo(2,3-h) (1)benzopyran-1,11-dione

2,3,6a,8,9,9a-Hexahydro-9a-hydroxy-4 methoxycyclopenta(c)furo(3',2':4,5)furo(2,3-h)(1) benzopyran-1,11-dione

## **2. Structure and chemistry of aflatoxins**

Aflatoxins were isolated and characterized after the Turkey X desease, that caused the death of more than 100.000 turkey poultries due to the intake of a contaminated peanut meal produced in South America starting from contaminated raw material (Blout, 1961; Goldblatt, 1969).

The most important aflatoxins, among the about 13 compounds so far identified, are the afla‐ toxin B1 and B2, the aflatoxin G1 and G2 and the aflatoxin metabolic byproducts M1 and M2. The four major aflatoxins are called B1, B2, G1, and G2 based on their fluorescence under UV light (blue or green) and relative chromatographic mobility during thin-layer chromatography. Fig‐ ure 1 shows the chemical structures of the main aflatoxins. Their chemical structure incorpo‐ rates dihydrofuran and tetrahydrofuran moieties coupled to a substituted coumarin. They are produced by a polyketide pathway by many strains of *Aspergillus flavus* and *Aspergillus parasit‐ icus*; in particular, *Aspergillus flavus* is a common contaminant in agriculture. *Aspergillus bomby‐ cis, Aspergillus ochraceoroseus*, *Aspergillus nomius*, and *Aspergillus pseudotamari* are also aflatoxinproducing species, but they are encountered less frequently (Goto, Wicklow, Ito, 1996; Klich, Mullaney, Daly, Cary, 2000; Peterson, Ito, Horn, Goto, 2001). Table 1 gives some relevant chem‐ ical properties of these compounds. Aflatoxin B1 is considered the most toxic and is produced, together with aflatoxin B2 by both *Aspergillus flavus* and *Aspergillus parasiticus*. Aflatoxin G1 and G2 are produced exclusively by *Aspergillus parasiticus*. While the presence of *Aspergillus* spp. in food products does not always indicate harmful levels of aflatoxins are also present, it does im‐ ply a significant risk in consumption. Aflatoxins M1 and M2 were originally discovered in the milk of cows which fed on moldy grain. Aflatoxin M1 has been observed also in the fermenta‐ tion broth of *Aspergillus parasiticus*. These compounds are products of a conversion process in the animal's liver that try to make these molecules more hydrophilic to be easily excreted from body via the kidney. Aflatoxin M1 is a metabolite of aflatoxin B1 in humans and animals where exposure at ng levels can come from mother's milk. Similarly, aflatoxin M2 is a metabolite of aflatoxin B2 in milk of cattle fed on contaminated food (Tara, 2005). Other metabolites can de‐ rive from these main ones, like Aflatoxicol, that forms by biological reduction of aflatoxin B1 (Pawlowski, Schoenhard, Lee, Libbey, Loveland, Sinnhuber, 1977). The levels considered safe for these compounds are reported in Table 2. Aflatoxin B1 is the most potent natural carcinogen known, and is probably also the most studied aflatoxin being often the major aflatoxin pro‐ duced by toxigenic strains (Squire, R. A. 1981). For this reason, it is also the best studied: in a large percentage of the papers published the term aflatoxin can be assumed to refer to aflatoxin B1. However, many other aflatoxins (e.g., P1. Q1, B2a, and G2a) have been described, especially as mammalian biotransformation products of the major metabolites (Heathcote, Hibbert, 1978).


**Table 1.** Chemical relevant data for main aflatoxins (O'Neil, Smith, Heckelman, 2001).

chapter addresses the advanced new chromatographic and spectrometric methods descri‐ bed in the literature and applied to research, that can reveal, even in trace amounts, aflatox‐

Aflatoxins were isolated and characterized after the Turkey X desease, that caused the death of more than 100.000 turkey poultries due to the intake of a contaminated peanut meal produced in South America starting from contaminated raw material (Blout, 1961; Goldblatt, 1969).

The most important aflatoxins, among the about 13 compounds so far identified, are the afla‐ toxin B1 and B2, the aflatoxin G1 and G2 and the aflatoxin metabolic byproducts M1 and M2. The four major aflatoxins are called B1, B2, G1, and G2 based on their fluorescence under UV light (blue or green) and relative chromatographic mobility during thin-layer chromatography. Fig‐ ure 1 shows the chemical structures of the main aflatoxins. Their chemical structure incorpo‐ rates dihydrofuran and tetrahydrofuran moieties coupled to a substituted coumarin. They are produced by a polyketide pathway by many strains of *Aspergillus flavus* and *Aspergillus parasit‐ icus*; in particular, *Aspergillus flavus* is a common contaminant in agriculture. *Aspergillus bomby‐ cis, Aspergillus ochraceoroseus*, *Aspergillus nomius*, and *Aspergillus pseudotamari* are also aflatoxinproducing species, but they are encountered less frequently (Goto, Wicklow, Ito, 1996; Klich, Mullaney, Daly, Cary, 2000; Peterson, Ito, Horn, Goto, 2001). Table 1 gives some relevant chem‐ ical properties of these compounds. Aflatoxin B1 is considered the most toxic and is produced, together with aflatoxin B2 by both *Aspergillus flavus* and *Aspergillus parasiticus*. Aflatoxin G1 and G2 are produced exclusively by *Aspergillus parasiticus*. While the presence of *Aspergillus* spp. in food products does not always indicate harmful levels of aflatoxins are also present, it does im‐ ply a significant risk in consumption. Aflatoxins M1 and M2 were originally discovered in the milk of cows which fed on moldy grain. Aflatoxin M1 has been observed also in the fermenta‐ tion broth of *Aspergillus parasiticus*. These compounds are products of a conversion process in the animal's liver that try to make these molecules more hydrophilic to be easily excreted from body via the kidney. Aflatoxin M1 is a metabolite of aflatoxin B1 in humans and animals where exposure at ng levels can come from mother's milk. Similarly, aflatoxin M2 is a metabolite of aflatoxin B2 in milk of cattle fed on contaminated food (Tara, 2005). Other metabolites can de‐ rive from these main ones, like Aflatoxicol, that forms by biological reduction of aflatoxin B1 (Pawlowski, Schoenhard, Lee, Libbey, Loveland, Sinnhuber, 1977). The levels considered safe for these compounds are reported in Table 2. Aflatoxin B1 is the most potent natural carcinogen known, and is probably also the most studied aflatoxin being often the major aflatoxin pro‐ duced by toxigenic strains (Squire, R. A. 1981). For this reason, it is also the best studied: in a large percentage of the papers published the term aflatoxin can be assumed to refer to aflatoxin B1. However, many other aflatoxins (e.g., P1. Q1, B2a, and G2a) have been described, especially as mammalian biotransformation products of the major metabolites (Heathcote, Hibbert, 1978).

ins in biological fluids as free form or as by-products, e.g. non-covalent adducts.

**2. Structure and chemistry of aflatoxins**

344 Aflatoxins - Recent Advances and Future Prospects

**Figure 1.** Chemical structures of the main aflatoxins.


**4. Frequency and levels of contamination in food**

is also important to assess the possible contamination.

**5. Aflatoxins in food and commodities**

while those that do may produce more than 106

Klich, 1987).

Aflatoxins have received greater attention than any other mycotoxins because of their dem‐ onstrated potent carcinogenic effect in susceptible laboratory animals and their acute toxico‐ logical effects in humans. Many countries have attempted to limit exposure to aflatoxins by imposing regulatory limits on commodities to be used as food and feed. The two species of *Aspergillus* fungi, aflatoxin producing, are especially found in areas with hot and humid cli‐ mate. Since aflatoxins are known to be genotoxic and carcinogenic, exposure through food should be kept as low as possible. Aflatoxins have been also associated with various diseas‐ es, such as aflatoxicosis. Aflatoxin B1 is the most common in food, and has the most potent genotoxic and carcinogenic effects. Aflatoxin M1 is a major metabolite of aflatoxin B1 in hu‐ mans and animals, which may be present in milk from animals fed with aflatoxin B1 conta‐ minated feed. Aflatoxins can occur in foods, such as groundnuts, treenuts, maize, rice, figs, grapes, raisins, and other dried foods, spices and crude vegetable oils, and cocoa beans, as a result of fungal contamination before and after harvest. The biosynthesis and the occurrence of aflatoxins is influenced by environmental factors; consequently the extent of contamina‐ tion varies with geographic location, agricultural and agronomic practices. The susceptibili‐ ty of commodities to fungal invasion during preharvest, storage, and/or processing periods

From the mycological perspective, there are great qualitative and quantitative differences in the toxigenic abilities displayed by different strains within each aflatoxigenic species. For ex‐ ample, only about half of *Aspergillus flavus* strains produce aflatoxins (Klich, Pitt, 1988),

Many substrates support growth and aflatoxin production by aflatoxigenic molds. Natural contamination of cereals, figs, oilseeds, nuts, tobacco, and a long list of other commodities is a common occurrence (Detroy, Lillehoj, Ciegler, 1971; Diener, Cole, Sanders, Payne, Lee,

Crops can be contaminated with aflatoxins in the field before harvest (Diener, Cole, Sanders, Payne, Lee, Klich, 1987; Klich, 1987). Even more problematic is the fate of crops stored under conditions that favor mold growth. The most relevant variables to keep under control dur‐ ing the storage are considered the moisture content of the substrate and the relative humidi‐ ty of the surroundings (Detroy, Lillehoj, Ciegler, 1971; Wilson, Payne, 1994). There are many side implications of aflatoxins contamination. Aflatoxin contamination has been linked to in‐ creased mortality in farm animals and thus significantly lowers the value of grains as an ani‐ mal feed and as an export commodity (Smith, Moss, 1985). Milk products can also be an indirect source of information on aflatoxins presence in the diet, and considering the broad diffusion of these products mainly addressed to infants, children, and people affected by health conditions, the risk associated to aflatoxins M1 and M2 is relevant. When cows assume

µg/kg (Cotty, Bayman, Egel, Elias, 1994).

Aflatoxins: Risk, Exposure and Remediation http://dx.doi.org/10.5772/52866 347

**Table 2.** Aflatoxins levels limits generally considered as safe.

## **3. Biosynthesis of aflatoxins**

Many relevant aspects of aflatoxins biosynthesis and molecular biology have been studied and extensively described. The first step in the biosynthetic pathway is considered the pro‐ duction of norsolorinic acid, an anthraquinone precursor, by a type II polyketide synthase. A series of about 15 post-polyketide synthase steps follows, yielding increasingly toxigenic metabolites (Bennett, Chang, Bhatnagar, 1997; Cleveland, Bhatnagar, 1992; Hicks, Shimizu, Keller, 2002; Payne, Brown, 1998; Townsend, 1997; Trail, Mahanti, Linz, 1995). Sterigmato‐ cystin, a related dihydrofuran toxin, mutagenic and tumorigenic but less potent than afla‐ toxin (Berry, 1988), is a late metabolite in the aflatoxin pathway, and is also produced as a final biosynthetic product by a number of species like *Aspergillus versicolor* and *Aspergillus nidulans*. Analysis of the molecular genetics of sterigmatocystin biosynthesis in the genetical‐ ly tractable species *Aspergillus nidulans* has provided a useful model system. The genes for the sterigmatocystin gene cluster from *Aspergillus nidulans* have been cloned and sequenced (Brown, Yu, Kelkar, Fernandes, Nesbitt, Keller, Adams, Leonard, 1996). Cognate genes for aflatoxins pathway enzymes from *Aspergillus flavus* and *Aspergillus parasiticus* show high se‐ quence similarity to the sterigmatocystin pathway genes (Payne, Brown, 1998; Yu, Chang, Bhatnagar, Cleveland, 2000; Yu, Woloshuk, Bhatnagar, Cleveland, 2000). Genes organization for *Aspergillus flavus, Aspergillus nidulans*, and *Aspergillus parasiticus* sterigmatocystin-aflatox‐ in pathway has been studied as reported by Cary et al. (Cary, Chang, Bhatnagar, 2001) and Hicks et al. (Hicks, Shimizu, Keller, 2002).

*Aspergillus oryzae* and *Aspergillus sojae*, species that are widely used in Asian food fermenta‐ tions such as soy sauce, miso, and sake, are closely related to the aflatoxigenic species *Asper‐ gillus flavus* and *Aspergillus parasiticus*. Although these food fungi have never been shown to produce aflatoxin (Wei, Jong. 1986), they contain homologues of several aflatoxin biosynthe‐ sis pathway genes (Klich, Yu, Chang, Mullaney, Bhatnagar, Cleveland, 1995). Deletions and other genetic defects have led to silencing of the aflatoxin pathway in both *Aspergillus oryzae* and *Aspergillus sojae* (Takahashi, Chang, Matsushima, Abe, Bhatnagar, Cleveland, Koyama, 2002; Watson, Fuller, Jeens, Archer, 1999; Bennett, Klich, 2003).
