The complete elimination of aflatoxins in human and animal food, while desirable, is ex-tremely difficult as they have the potential to arise in a wide range of agricultural products.
Risk assessments have been carried out for aflatoxin (see, for example, IARC, 1993). Because aflatoxin B1is a genotoxic carcinogen, most agencies, including the Joint Expert Committee on Food Additives (JECFA) and the US Food and Drug Administration, have not set a tol-erable daily intake (TDI) figure. In common with other dietary carcinogens, it is generally accepted that amounts in food should be reduced to the lowest levels that are technologically possible. Regulations have been set for human food and animal feed in many countries (FAO, 2004). In the EC, aflatoxin is strictly controlled by maximum permissible limits (Table 6.5) and by recommendations for sampling. As an example of what can be achieved over time, regulation of aflatoxin B1in animal feedingstuffs in the UK since the 1980s (Anon, 1982) has been effective in steadily reducing amounts of aflatoxin M1in milk, as shown by regular surveillance (Ministry of Agriculture, Fisheries and Food, 1980, 1987, 1993).
6.3 OCHRATOXIN A
6.3.1 Chemical properties
The ochratoxins are a group of structurally related compounds of which ochratoxin A is the most important and most commonly occurring. They consist of a polyketide-derived dihydroiso-coumarin moiety linked through the 12-carboxy group to phenylalanine (Fig. 6.2).
Ochratoxin A is a colourless crystalline compound, exhibiting blue fluorescence under UV light. It crystallises from benzene to give a product melting at 90◦C containing one molecule of benzene. This can be removed under vacuum at 120◦C to give a solid melting at 168◦C. It crystallises in a pure form from xylene. The sodium salt is soluble in water. In the acid form it is moderately soluble in polar organic solvents such as chloroform, methanol and acetonitrile, and dissolves in dilute aqueous sodium bicarbonate. It yields phenylalanine and an optically active lactone acid, ochratoxin α on acid hydrolysis. Reaction in methanol and hydrochloric acid yields the methyl ester, while methylation with diazomethane gives theo-methyl methyl ester. It can be stored in ethanol for at least a year under refrigeration and protected from light.
Ochratoxin A is a moderately stable molecule and will survive most food processing to some extent. This has been reviewed by Scott (1996) and the accompanying volume contains a series of papers covering the fate of ochratoxin A during malting and brewing (Baxter, 1996), during bread making (Subirade, 1996), as a result of processing in cereals (Alldrick, 1996), during processing of coffee (Viani, 1996), during processing of meat products (Gareis, 1996) and during processing in animal feed (Scudamore, 1996). During a study of extrusion no more than 40% of ochratoxin A was destroyed under the harshest treatments employed (Scudamoreet al., 2004). In a study of the fate of ochratoxin A in the processing of whole wheat grains during milling and bread production the mycotoxin concentrated in the bran and was reduced in the flour while most ochratoxin A survived subsequent baking into bread (Scudamoreet al., 2003).
In biological systems, ochratoxin A will bind to serum albumin and for the majority of its lifetime within the human body, OTA remains bound to the plasma protein human serum albumin (Perryet al., 2003).
6.3.2 Analytical methods
Sensitive and reliable analytical methods have been developed with detection limits better than 1 μg/kg. Extraction solvents used for ochratoxin A have included mixtures of chloroform plus orthophosphoric acid, ethyl acetate plus phosphoric acid and acetonitrile plus water. Thin layer chromatography has been widely used (e.g. Krogh and Nesheim, 1982) but this has now been mainly replaced by HPLC- based methods. Clean-up of acid extracts can be achieved by back-partition into sodium bicarbonate although immunoaffinity columns are now often used as the main clean-up stage.
A number of methods have been examined by inter-laboratory testing often as part of the AOAC testing programme. Those developed and tested for cereals include determination of ochratoxin A in barley, wheat bran and rye (Larsson and M¨oller, 1996), in barley using immunoaffinity columns (Entwisleet al., 2000), in wheat (Scudamore and McDonald, 1998) and in baby foods (Burdaspalet al., 2001). Methods for roasted coffee have also used im-munoaffinity clean-up (Entwisleet al., 2001). The discovery of the presence of ochratoxin A in wine and beer has led to the development and testing of methods specific for these bev-erages (Viscontiet al., 2001a; Leitner et al., 2002). McDonald et al. (2003) tested a method for determination of ochratoxin A in currants, raisins, sultanas, mixed dried fruit and dried figs using acidified methanol for extraction. Post-column pH shift by the addition of 1.1 M ammonia solution to the column eluant enhances fluorescence.
Methods have also been developed to determine the occurrence of ochratoxin A in bio-logical fluids such as plasma and urine (Gilbertet al., 2001) and human milk (Miraglia et al., 1995) and this has enabled estimated dietary intakes for babies to be calculated.
6.3.3 Occurrence in raw materials and processed foods
Ochratoxin A is produced by certain strains ofAspergillus ochraceus and related species, and byPenicillium verrucosum. There are literature reports of other Penicillia producing ochratoxin A but most of these are probably as the result of mis-identification.A. ochraceus occurs principally in tropical climates whileP. verrucosum is a common storage fungus in temperate areas such as Canada, Eastern Europe, Denmark, parts of South America and the UK. The main ochratoxin A producing fungus occurring in grapes has shown to beAspergillus carbonarius (Valero et al., 2005).
Ochratoxin A often occurs in stored cereals and has been found in other foods including coffee (Tsubouchiet al., 1988), beer (Payen et al., 1983), dried fruit (Ozay and Alperden, 1991), wine (Zimmerli and Dick, 1996), cocoa (Ministry of Agriculture, Fisheries and Food, 1980), nuts (Cooperet al., 1982), spices (Thirumala-Devi et al., 2001; Fazekas et al., 2005) and liquorice (Breschet al., 2000). A comprehensive review of the literature on the worldwide occurrence of ochratoxin has been carried out by Speijers and van Egmond (1993). In Europe an assessment of dietary intake of ochratoxin A has been undertaken under the ‘SCOOP’
programme (European Commission, 2002a). This involved collection of data on occurrence in food using methodology considered as soundly based. This together with consumer con-sumption data enabled the dietary intake of ochratoxin A to be calculated. Estimate of dietary intake on the basis of ochratoxin A level in serum/plasma has also been carried out.
6.3.4 Toxicology
Ochratoxin A is a potent carcinogenic mycotoxin that can affect kidneys, the immune system and the nervous system. The kidney is the most sensitive target organ. However, its dechloro derivative, ochratoxin B, is non-toxic. A nephrotoxic effect has been demonstrated in all mammalian species tested to date (Harwiget al., 1983). In acute toxicity studies LD50values vary greatly in different species, the dog and pig being especially susceptible.
The European Food Safety Authority (EFSA) published an opinion on ochratoxin A (EC, 2006a). They concluded that ‘although some early epidemiological data had suggested that ochratoxin A might be involved in the pathogenesis of distinct renal diseases and otherwise rare tumours of the kidneys in certain endemic regions of the Balkan Peninsula these epi-demiological data were incomplete and did not justify the classification of ochratoxin A as a human renal carcinogen’. Ochratoxin A has been found to be a potent renal toxin in all of the animal species tested, the dog being the most sensitive. The extent of renal injury is dose-dependent, but also associated with the duration of exposure, as ochratoxin A accumulates in renal tissue.
Recent scientific evidence indicates that the site-specific renal toxicity as well as the DNA damage and genotoxic effects of ochratoxin A, measured in various in vivo and in vitro studies, are most likely attributable to cellular oxidative damage. Furthermore, advanced chemical analytical procedures have failed to demonstrate the existence of specific ochratoxin A–DNA adducts.
Human exposure to ochratoxin A has been clearly demonstrated by its detection in blood (e.g. Baueret al., 1986; Breitholtz et al., 1991; Palli et al., 1999; Filali et al., 2002; Sangare-Tigoriet al., 2006) and breast milk (Miraglia et al., 1993).
6.3.5 Regulation and control
The presence of ochratoxin A in foodstuffs is clearly undesirable, although few coun-tries have introduced statutory control to date (Food and Agriculture Organisation of the United Nations, 2004). Most attention to legislation for ochratoxin has been within the EU and a summary of current limits for ochratoxin A in cereals and other products is given (Table 6.6) although limits are currently being introduced and modified as more informa-tion on occurrence and toxicology is acquired. A full descripinforma-tion of limits, recommended sampling protocols and analytical methods is provided (EC, 2005a,b).
The prevention and control of ochratoxin A in cereals depends on the crop being dried promptly at harvest and good store hygiene. In general ochratoxin A develops slowly in grain
Table 6.6 Maximum limits for ochratoxin A in raw cereal grain and finished products intended for human consumption, in the EU as of October 2006.
Product Ochratoxin A (μg/kg)
Raw cereal grains (including raw rice and buckwheat) 5.0 All products derived from cereals (including processed cereal products and
cereal grains intended for direct human consumption)
3.0
Baby foods and processed cereal-based foods for infants and young children 0.5
Dried vine fruits (currants, raisins, sultanas) 10.0
Roasted coffee beans and ground roasted coffee except soluble coffee 5.0 Wine and other wine and/or grape must based beverages 2.0 Green coffee, dried fruit other than vine fruit, beer, cocoa, cocoa products,
liqueur wines, meat products, spices, liquorice
No current limits
below 18% moisture content but this depends on temperature, grain water activity, extent of infection with P. verrucosum and the length of time the cereal remains under such conditions.
When grain is dried by ambient air the bottom layer dries first while the upper layers may remain damp for much longer. The availability of adequate drying capacity is important, hot air facilities being preferable. Even after drying, cereal bulks should be monitored regularly for temperature, moisture content and insect activity and action taken if it becomes necessary.
For grapes, the critical control points for the control of ochratoxin A in the grape-wine chain have been established (Battilaniet al., 2003).
In addition, contamination of animal feeds with ochratoxin A may result in the presence of residues in edible offal and blood serum, whereas the ochratoxin A contamination in meat, milk and eggs is negligible.