The discovery of aflatoxins in the 1960s [1], with approximately 100,000 turkey poult deaths in England, was the seminal event that made the scientific community realize that mold secondary metabolites could be responsible for food and feed safety problems. Several other mycotoxins were identified when fungi in food were more fully investigated. Ochratoxin A (OTA) was purified and characterized from Aspergillus ochraceus Wilh. strain K‑804 [2,3] isolated from sorghum grain, and proved to be acutely toxic to Pekin ducklings, mice and rats [4]. Nowadays, OTA is one of the most relevant mycotoxins, with its presence in food and feed products being regulated in many countries.

OTA is composed of a 7-carboxy-5-chloro-8-hydroxy-3,4-dihydro-3-R-methylisocoumarin (ochratoxin α) moiety and a L-β-phenylalanine molecule, which are linked through the 7‑carboxy group by an amide bond. The compound belongs to a group of fungal secondary metabolites, commonly known as ochratoxins (Figure 1), which share a similar chemical structure. Ochratoxin B (OTB), ochratoxin C (OTC), ochratoxin α (OTα), ochratoxin β (OTβ), (4‑R)- and (4‑S)-hydroxy-ochratoxin A (4R‑OH OTA and 4S‑OH OTA), 4-hydroxy-ochratoxin B (4‑OH OTB) and 10-hydroxy-ochratoxin A (10‑OH OTA) are naturally produced by certain fungi.

Of these, OTA is the predominant compound detected in agriculture commodities and the most relevant to food and feed safety. The IUPAC formula is L‑phenylalanine-N-[(5-chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyran-7-yl)carbonyl]-(R)-isocoumarin and its chemical abstract specification (CAS) is 303-47-9 [7]. OTA is a colorless crystal of empirical formula C20H18O6NCl, which has a molecular weight of 403.822 Da [8]. The melting points are 94-96 °C and 169 °C when crystallized from benzene or xylene, respectively. Maximum absorbances in (a) are 216, and 330 nm (ε 37,060 and 6050, respectively); and (b) are 214 and 332 nm (ε 37,200 and 6330, respectively) [9]. The infrared spectrum in chloroform presents major peaks at 3380, 1723, 1678 and 1655 cm⁻¹. OTA has weak acid properties with pKa₁ 4.2-4.4 and pKa₂ 7.0-7.3, from the carboxyl group of phenylalanine and from the phenolic hydroxyl group of the isocoumarin, respectively [7]. OTA produces a green-bluish fluorescence when excited by UV light (366 nm) using TLC, which changes to a dark blue fluorescence when exposed to ammonia vapors, aqueous NaHCO₃ or NaOH [10]. The fluorescence properties are commonly used for detection and identification purposes in TLC and HPLC. LC-MS can be employed to detect OTA. The identity of OTA can be confirmed by converting it to the methyl ester [11], and/or to OTα using carboxypeptidase A. OTA is highly soluble in polar organic solvents, soluble in aqueous sodium hydrogen carbonate and slightly soluble in water.

OTA is produced by several Aspergillus species and Penicillium verrucosum and P. nordicum. These penicillia are the species most authoritatively listed as OTA-producing. The P. viridicatum strains that were reported as OTA producers were reclassified as P. verrucosum by Pitt [12]. Other species such as P. chrysogenum, P. brevicompactum, P. crustosum, P. olsonii and P. oxalicum have been claimed as OTA producers [13,14]. Nevertheless, a careful confirmation of these findings is required since no other authors report the capacity of these species to produce OTA. Strains of a larger number of species are known to produce OTA in Aspergillus. Species accepted by Frisvad and co-authors [15] are listed in Table 1.

Some other species were reported incorrectly as OTA producers, due to either misidentification of the isolates in question or to the use of contaminated cultures. Others still were reclassified, having now a different scientific name. For example, the original A. ochraceus strain from which OTA was first isolated, was reclassified as A. westerdijkiae, and many other A.ochraceus strains as A. steynii [16].

The major OTA producers in food and feed products are considered to be A. alliaceus, A. carbonarius, A. ochraceus, A. steynii, A. westerdijkiae, P. nordicum and P. verrucosum [15]. These are mainly associated with agricultural crops pre-harvest, or in post harvest storage situations. P. nordicum is a high OTA producer and is isolated predominantly from certain cheeses and fermented meats. Aspergillus niger, which is a very common species, is less relevant since most of the isolates are non-ochratoxigenic [28,29] and the others usually produce small amounts of OTA [30]. However, since it is a widely used species in several biotechnological processes it can pose some safety issues in the manufacture of food grade organic acids and enzymes. The other OTA producers are rare and do not pose a great concern for food safety.

The OTA biosynthesis pathway is not completely established. However, it is known that the isocoumarin is a pentaketide which derives from the polyketide pathway and that L-β-phenylalanine derives from the shikimic acid pathway. More precisely, experimental studies with radioactive labeled precursors demonstrated that the isocoumarin is synthesized by head-to-tail condensation of five acetate units with the subsequent addition in C7 of a unit of methionine, which is subsequently oxidized to carboxyl [31,32,33]. Apparently, L-β-phenylalanine is coupled unaltered to the isocoumarin moiety since an isolated A. ochraceus enzyme fraction was able to link the two portions of the molecule [31]. Finally, the chlorine is incorporated in C5 from NaCl (although it is not known at what exact point of the biosynthesis) and probably through the action of chloroperoxidases [34]. There is little information about the genes responsible for these biosynthetic steps. Only DNA fragments that encode for some OTA polyketide synthases in P. nordicum [35], A. ochraceus [36] and A. carbonarius [37] have been identified.

OTA production depends on factors such as temperature, substrate water activity (aw), and micronutrients [9]. The optimal conditions for OTA production by A. carbonarius, A. ochraceus and P. verrucosum are 15-20 °C and 0.95-0.98 aw [38]; 25-30 °C and 0.98 aw [39]; and 24 °C and 0.95-0.99 aw [40], respectively. The culture media most used for its biosynthesis are Yeast Extract Sucrose (YES) and Czapek Yeast Autolysate (CYA) [41].

OTA has been detected in a wide variety of agricultural commodities, livestock products and processed food (Table 2). Concentrations found in the final food products are lower than those found in raw materials since some processing steps can contribute actively to its elimination. For example, (i) malting; (ii) malt fermentation; (iii) white bread and (iv) whole-bread production can contribute to reduce OTA by 56% [42]; 21% [43]; 80% [44] and 40% [44], respectively. Additionally, reductions of 35%, 71% and 83% for mild, medium and strong coffee roasting, respectively, were reported [45]. Finally, the wine-making process contributes to almost 90% reduction of OTA [46].

OTA is also detected in feed (Table 3) with the concentrations usually being higher than those in food. Processing steps such as extrusion may contribute to reduce concentrations [69]. Nevertheless, other practices can increase OTA concentrations. For example, some by-products derived from cereal processing, such as cracked grains, cereal cleanings, wheat and corn bran, are often the fractions most contaminated with OTA, and are usually directed for feed proposes [70]. Moreover, it is common practice to direct the lower contaminated commodities for human consumption while the most contaminated are used for feed.

OTA is the most toxic and relevant of the known ochratoxins. However, some consider that OTC is equally toxic to OTA [74,75] since, when ingested, it is converted rapidly into OTA, which becomes available in the bloodstream [76,77]. OTA is known primarily for its nephrotoxicity. It was nephrotoxic for all tested animals with the exception of adult ruminants [78] and it appears to be the cause of porcine nephropathy, human Balkan endemic nephropathy (BEN) and chronic interstitial nephropathy (CIN) in North Africa [79,80,81]. OTA is also classified as possibly carcinogenic to humans (group 2B) since there is evidence for experimental animals but not for humans [82]. In addition, OTA has mutagenic, teratogenic, neurotoxic, hepatotoxic and immunotoxic properties [80]. Oral LD₅₀ values are 1.0-6.0 mg/kg for pigs, 20-30 mg/kg for rats and 48-58 mg/kg for mouse [83]. In these studies, OTA also caused haemorrhages in almost all vital organs, nephrosis, and necrosis in the liver and lymphoid tissues. OTA is considered to be a cumulative toxic compound since it is easily absorb through the stomach and the small intestine but hardly eliminated through the biliary and urinary routes. Oral OTA half-lives are 35.5 days for humans, 21 days for monkeys, 72-120 hours for pigs, 55-120 hours for rats and 40 hours for mice [83]. The high elimination half-lives observed in some species are due to the strong OTA affinity to serum proteins, which limit its transfer from the blood to the hepatic and renal cells. OTA affinity to bovine serum albumin [84] and to human serum albumin [85] was observed in vitro, and its relationship with OTA excretion confirmed by Kumagai [86] who verified that albumin-deficient rats were able to eliminate this mycotoxin more quickly through urine.

OTα toxicity has not been so extensively studied. Nevertheless, some studies indicated it is essentially non-toxic. For example, OTα was ineffective as an immunosuppressor when tested in mice [87] and was considered 1,000-times less toxic than OTA in brain cells cultures [88]. Furthermore, OTα has an elimination half-live of 9.6 h in rats, which is well below that of OTA (103 h) [77]. Therefore, processes that lead to the conversion of OTA into OTα contributes substantially to reduce OTA toxic effects and, hence, are considered to be routes for OTA detoxification.

A tolerable daily intake (TDI value) of 5 ng OTA/kg bw/day is recommended by the World Health Organization since it has toxic effects and is found in human blood [80,89,90,91] and in breast milk [80,92,93,94], thus proving human exposure. Furthermore, it is recommended that OTA levels in food and feed should be reduced as much as technologically possible.

Several strategies can be employed to reduce OTA levels. The most important are preventive since they avoid the contamination of commodities in the first place. The use of (i) good agricultural practices, (ii) fungal resistant crop varieties, (iii) the correct application of fungicides and (iv) the proper storage of commodities are common measures that could, ideally, be part of a Hazard Analysis Critical Control Point (HACCP) scheme to minimize OTA at critical control points of the food supply chain.

However, fully implemented HACCP schemes are rare, and when the individual measures fail or are not in place, OTA remains in food and feed products. Decontamination or detoxification procedures can be used to remove or to reduce OTA levels. These measures, which are technologically diverse, are usually classified into physical, chemical or biological [95]. Physical methods consist of segregation, sorting, cleaning, peeling and shelling processes that aim to remove the most contaminated fractions of the commodities. They also may involve the utilization of sorbents as nutritional additives that absorb OTA hence reducing bioavailability. Chemical methods consist of the utilization of compounds to destroy OTA: some processes use ammonium (ammoniation), alkaline hydrolysis (nixtamalization), bisulphites and ozone (ozonation). These are reported generally as effective in the elimination of OTA and other mycotoxins [96]. However, the toxicological safety of the final product is not always guaranteed since some chemical residues may remain in products and the toxicity of the reaction products formed is not usually studied. Furthermore, there is a significant reduction in palatability and nutritive quality of treated products.

Biological methods use microorganisms, which can decompose, transform or adsorb OTA to detoxify contaminated products or to avoid the toxic effects when mycotoxins are ingested. These are the technologies of choice for decontamination proposes because they present several advantages from being mediated by enzymatic reactions. For example, they are very specific, efficient, environmentally friendly, and they preserve nutritive quality. However, the non-pathogenicity of the microorganism and the non-toxicity of the reaction products formed are essential [97]. More research is needed to render these methods practical, effective and economically feasible.

Several protozoal, bacterial, yeast and filamentous fungal species are able to biodegrade OTA.

Several enzymes may be involved in the microbiological degradation of OTA. However, little information is available and very few have been purified and characterized. The first reported protease able to hydrolyze OTA was carboxypeptidase A (CPA) (EC 3.4.17.1) from bovine pancreas [137]. Subsequently, a screening study which included several commercial hydrolases, verified that a crude lipase product from A. niger was able to hydrolyze OTA via the amide bond [138]. The enzyme was purified by anion exchange chromatography and was demonstrated to cleave OTA and p‑nitrophenyl palmitate, a specific lipase substrate. Several proteolytic preparations were also studied, which were involved in the hydrolysis of OTA to OTα. These included protease A from A. niger, pancreatin from porcine pancreas and to a lesser extent, Prolyve PAC from A. niger [139]. Additionally, the production and purification of an A. niger cell-free crude enzyme preparation that demonstrated a significant capacity to cleave the amide bond of OTA was reported. The OTA-degrading enzyme involved was purified by anion exchange chromatography and characterized [140]. This enzyme showed higher OTA-degrading activity then CPA at pH 7.5 and 37 °C, and was inhibited by EDTA, which is a specific inhibitor of metalloproteases. In subsequent studies [141], it was found that carboxypeptidase Y (CPY) (EC 3.4.16.1) from S. cerevisiae is also able to hydrolyze OTA with optimal activity at pH 5.6 and 37 °C (Figure 3). However, the specific activity of CPY is very low as indicated by the OTA hydrolyzation reaction being very slow. Nevertheless, after five days of incubation, CPY converted 52% of the OTA present in the reaction assay into OTα. This activity is sufficient to reduce significantly levels of OTA during wine or beer fermentation, since these processes take several days to complete. Hence, a biodegradation pathway is possible for S. cerevisiae in addition to the OTA adsorption phenomenon. It is necessary to consider that CPY is a vacuolar exopeptidase where OTA enters the yeast cells before it is catabolized. However, the S. cerevisiae wall-binding properties can make difficult OTA uptake. Further information concerning the enzymes which are able to hydrolyze OTA is presented in Table 4.