Prevention of growth and mycotoxin production in fungi from the field is usually considered the best approach to impede the harmful effects of mycotoxins on animal and human health. As mycotoxin producing molds can usually only colonize damaged parts of plants, crops must be protected against damage caused by either mechanical processes or insects. Besides, inoculum sources, such as weeds or agricultural residues, should be minimized to avoid contamination. Regarding the plants themselves, stress should be reduced, and good agricultural practices (GAP) should be followed including crop rotation, and culture and harvest in the appropriate seasons and conditions [13]. Indeed, Rousseau and Blateyron [14] also emphasized that the occurrence of OTA in wine may be decreased via appropriate vineyard management by about 80%.

For lowering pre-harvest contamination, treatment of field crops with fungicides is the traditional technique. The effect of fungicides on mold growth and mycotoxin biosynthesis is affected by several factors, including their chemical nature, rate of application, crop type, fungal species, and storage conditions [12]. The organophosphate fungicide, dichlorvos, was found to inhibit OTA production of A. sulphureus, P. verrucosum, and A. ochraceus [15,16]. Another fungicide, iprodione, has successfully been used in agricultural commodities to prevent the growth of various fungal species, including OTA producers [12], and was found to be able to decrease OTA production of A. westerdijkiae [17]. The effects of fungicide treatments on the OTA content of wines have been examined in several laboratories. In earlier studies, combinations of Euparen (a sulfamide type fungicide) and Mycodifol [18], or captan, [19] were found to be effective against black aspergilli, which colonize grape berries. Recently, Lo Curto et al. [20] observed that the application of some pesticides, such as Azoxystrobin (a strobilurin derivative) or Dinocap (a dinitrophenyl derivative), in combination with sulfur, effectively decreased the OTA content of wines. Carbendazim and Chorus were found to be ineffective in controlling sour rot caused by aspergilli [21]. However, the application of another pesticide, Switch, led to a significant decrease in incidence of black aspergilli on grapes, as confirmed in field trials carried out in France, Spain, Greece, and Italy [21,22,23]. The fungicide Switch contains cyprodinil and fludioxonil, which belong to the pyrimidine and pyrrolnitrin classes of fungicides, respectively [21]. The observation that fludioxonil can be used against aspergilli is not surprising, since pyrrolnitrin was found previously to be effective against black aspergilli [24]. In another study, the fungicides Switch, Scala (containing the pyrimidine fungicide pyrimethanil), and Mikal (containing fosetyl-Al and the dicarboximide folpel) were found to be the most effective for lowering fungal colonization and the OTA content of wines [25]. Several other fungicides have been shown to be active in reducing either fungal growth or OTA levels in grapevine, including mepanipyrim, pyrimethanil, fluazinam, and iprodione [26]. Belli et al. [23] examined the effect of 26 fungicides on A. carbonarius infection and OTA production in synthetic medium and on grapes, and found that both infection and OTA production were reduced when using cyprodinil plus fludioxonil, azoxystrobin, and penconazole. However, it should be mentioned that some fungicides were found to stimulate OTA production in grapes [27]. For example, carbendazim has been found to reduce fungal biota, but to stimulate OTA production [28], while fenhexamid, mancozeb, and copper hydroxide plus copper also enhanced infection and OTA production in grapes [23]. Recently, fusapyrone, an antifungal compound produced by Fusarium semitectum, and natamycin were found to be effective in controlling OTA-producing aspergilli and OTA levels in vineyards [29,30].

The application of insecticides that work against vectors of ochratoxin producing fungi can also be used successfully to lower OTA levels in grapes and coffee. In grapevine, a good correlation between damage caused by the grape-berry moth (Lobesia botrana) and OTA content has been found in grape berries, due to the contribution of L. botrana to berry wounds and fungal spore dissemination [31]. Field trials confirmed that a successful control of L. botrana using either biological methods or insecticides reduced fungal infection and OTA accumulation in grapes [22,26]. Moreover, insecticide treatment against L. Botrana, in combination with fungicides, contributed significantly to a reduction of OTA levels in the field [22]. Research carried out at the Interprofessionnel de la Vigne et du Vin France (ITV France) indicated that larvae of grape moth Cochylis sp. also act as vectors for the conidial dispersal of OTA-producing fungi [32]. A strict correlation was observed between the number of perforations caused by these larvae and OTA concentrations in grapes. Consequently, researchers at the Institut Coopératif du Vin (ICV) successfully used the insecticides Lufox (carbamate type insecticide containing luferunon and fenoxycarb), Decis (a pyrethroid insecticide containing delthametrin), and Bt (Bacillus thuringiensis) for lowering the OTA content of wines [33]. Bacillus thuringiensis was also found to significantly inhibit the growth of OTA-producing fungi on grapes in another study [34].

In coffee, the coffee berry borer (also called broca), Hypotenemus hampei, has been shown to be a vector of A. ochraceus [35]. Insecticides controlling these insects could be successful in lowering the OTA contamination of coffee beans. However, such trials have not been carried out, partly due to the resistance of the coffee berry borer against some pesticides (e.g., Endosulfan), and also to the high toxicity of these insecticides. Other approaches are also used to control the coffee berry borer in the field, including cultural and manual methods, traps, or biological control using toxin-producing Bacillus thuringiensis strains [36], entomopathogenic fungi (e.g., Beauveria bassiana), or the insect parasitoids Cephalonomia stephanoderis or Prorops nasuta [37]. Such approaches could also reduce the OTA content of coffee beans. However, the insect parasitoid Prorops nasuta, which has been introduced from Africa to many coffee-producing countries in an attempt to control the coffee berry borer, has also been shown to carry another OTA producing mold, Aspergillus westerdijkiae [38]. These results raise the possibility that this insect parasitoid might be disseminating an ochratoxin-producing fungus in coffee plantations. Promising results were also obtained in grape fields using yeasts as a biological control agents. In particular, good results were obtained with Cryptococcus laurentii and Aureobasidium pullulans, and with a strain of Hanseniaspora uvarum [26]. In field trials, an Aureobasidium pullulans strain was found to be an effective biocontrol agent of A. carbonarius, reducing both severity of Aspergillus rots and OTA accumulation in wine grapes [39]. This isolate was also able to degrade OTA in in vitro experiments. Other epiphytic yeasts, including Candida guilliermondii, Acremonium cephalosporium [40], Issatchenkia orientalis, Metschnikowia pulcherrima, Issatchenkia terricola, and Candida incommunis [41] have also been found to be effective in preventing rots caused by Aspergillus niger in wine grapes. Antifungal metabolites isolated from the culture fluids of Bacillus pumilus inhibited the production of OTA [42].

Biological control using non-toxigenic Aspergillus niger isolates against toxin-producing black aspergilli has also been applied successfully in grapes [43]. On the other hand, coinfection with toxigenic black aspergilli and either Penicillium janthinellum or Eurotium amstelodami increased OTA content. A biocontrol rhizobacterial strain of Bacillus subtilis AF 1 has also been found to lyse A. niger hyphae, and was suggested to be used as a biological control agent against black mold [44].

Plant breeding is traditionally used to improve the resistance of the host plants to fungal infection. Such attempts are promising, e.g., in the case of Fusarium infection of wheat and corn. Kernels of several varieties of wheat, rye, and barley were found to have different resistance levels to fungal attack and OTA accumulation. Thus, varieties with stronger resistance to fungal invasion during storage could be selected [45]. However, to our knowledge, breeding has not been used to increase the resistance of cereals to OTA accumulation. There is also limited information on the susceptibility levels of grape varieties to infection and on OTA accumulation caused by black aspergilli [46]. In in vitro experiments, 3 of the 12 tested varieties, namely ‘‘Bianco di Alessano’’, ‘‘Pampanuto’’, and ‘‘Uva di Troia’’, showed low OTA contamination after artificial infection with a mixture of five OTA-producing strains, whereas the most susceptible variety was Cabernet Sauvignon [46]. In the case of coffee, also only limited data are available regarding the resistance of different cultivars to OTA accumulation. However, intensive research has been carried out to identify and breed cultivars resistant to the attack of coffee berry borer [47], which presumably will lead to lower OTA levels in these coffee cultivars. Some Coffea species are naturally resistant to the coffee berry borer, with Coffea abeokutae, Coffea excelsa, and Coffea kapakata being the most resistant [48].

Other preharvest management approaches, including biocompetitive exclusion or genetic engineering, which has been successfully used to lower aflatoxin levels of corn, cotton, and peanuts, and fumonisin levels in corn, respectively, have not yet been applied in practice to lower ochratoxin levels in agricultural products [49].

Although the prevention of mycotoxin contamination in the field is the main goal of the agricultural and food industries, the contamination of various commodities with Aspergillus or Penicillium isolates and their mycotoxins is unavoidable under certain environmental conditions. Postharvest strategies aim at lowering fungal contamination and consequently, the mycotoxin content of agricultural products during storage, handling, processing, and transport. Such strategies include the improvement of drying and storage conditions, the use of chemical and natural agents, and irradiation.

During harvest, the following factors influence OTA contamination of cereals [50]: weather before and during harvest, time before drying, efficiency of drying machinery, physical state of grains, temperature at harvest, fungal competition, cleanliness of harvesters, and transport. Once in store, cereals must be regularly inspected and kept under safe storage conditions. Some important factors for keeping grain safe are: cleanliness of storage containers, absence of structural leaks, condensation, and temperature [50].

Regarding grape products, the code put forward by the Office International de la Vigne et du Vin (OIV) of sound viticultural practices for the minimization of OTA levels in vine-based products includes hygiene of the containers, measures to avoid fruit fly infestation, avoidance of overstacking, sorting, and drying conditions [51]. To decrease OTA content in wines, the removal of rotten grapes prior to crushing and pressing should be carried out. Since the OTA content of damaged berries was higher than that of undamaged ones, selecting grapes seems to be the best and natural way to limit OTA occurrence in wine [52].

In coffee, managing the risk of OTA contamination involves key factors, including good hygiene practices along the production chain, rapid drying, and avoiding the re-wetting of coffee by ensuring clean and dry storage and transportation (http://www.coffee-ota.org/4_1_prevention.asp). Coffee postharvest manufacturing is carried out using two processes. The dry method consists of a natural drying stage (in the sun) or an artificial drying stage, followed by mechanical dehulling. In the wet method, cherries are pulped, and the resulting beans are dried and dehulled. Pulping does not result in any significant change in the OTA content. However, there is a difference between fermentative and physical mucilage removal, the latter resulting in a substantial drop in OTA levels. After hulling, only traces are found in both cases. Afterwards, recontamination during the storage stage could lead to OTA production and accumulation [53].

Mycotoxin production is dependent on a number of factors, e.g., water activity of the stored product, temperature, gas composition, the presence of chemical preservatives, and microbial interactions. An integrated approach for controlling several of these factors could provide a much more effective control of deterioration without requiring extreme control of any one factor. Water availability or moisture content is one of the most important factors in the prevention of fungal growth and mycotoxin production [54]. It has been observed that grain stored at a moisture content equivalent to a water activity of 0.70 (<14.5% moisture by weight) or less will not be subject to spoilage and mycotoxin formation [13]. Temperature also influences fungal contamination during storage.

Some chemical preservatives, such as potassium sorbate or calcium propionate, are capable of preventing OTA contamination in cereal products, including bread. [55,56]. Several other antimicrobial food additives have been found to inhibit the growth or OTA production, or both, of fungi, including methyl para-hydroxybenzoic acid, propyl-paraben, and sodium propionate [57,58]. A new strategy under study is the use of antioxidants, such as vanillic acid or 4-hydroxybenzoic acid [59], and essential oils extracted from plants, such as Thymus vulgaris, Aframomum danielli [60,61], cinnamon, and clove leaf [62], which affect mold growth and OTA synthesis. Other essential oils extracted from plants (thyme, cinnamon, marigold, spearmint, basil, quyssum, caraway, and anise) [63], and those of oregano, mint, basil, sage, and coriander, have also been found to inhibit the growth of ochratoxigenic fungi, while oregano and mint oils also inhibited OTA production in an A. westerdijkiae isolate [64]. It was suggested that the inhibitory effects exerted by spices and herbs may rely, at least in part, on phenolic compounds, such as coumarins and flavonoids [65]. Indeed, flavonoids, including rutin, quercetin, and caffeic acid were found to inhibit both growth and OTA synthesis in A. carbonarius [66]. Additionally, alkaloids produced by Piper longum, and components of sesame oil and turmeric have also been found to suppress both fungal growth and ochratoxin production in a number of OTA-producing aspergilli [67].

Several reports have dealt with the antifungal properties of various lactic acid bacteria [68]. These bacteria are of special interest as biopreservation organisms because they have a long history of use in food and have been designated or generally regarded as safe. Lactic acid bacteria produce antimicrobial compounds, including organic acids, like lactic acid and acetic acid, hydrogen peroxide, cyclic dipeptides, phenyllactic acid, 3-hydroxylated fatty acids, bacteriocins, and low-molecular-weight proteinaceous compounds. Additionally, these bacteria compete with other species by acidifying the environment and rapidly depleting nutrients. They are also promising tools for controlling ochratoxigenic fungi in various food products [68].

Another possibility of inhibiting the growth of mycotoxigenic fungi is the application of modified atmospheres or gases, such as CO₂ [69], N₂, CO, and SO₂ for the protection of cereal grain from fungal spoilage and mycotoxin contamination during the postharvest period. Modified atmosphere storage has been examined for the storage of moist grain, especially for animal feed. Studies with P. verrucosum and A. ochraceus with up to 50% CO₂ suggest that spore germination is not markedly affected, although germ tube extension, and hence colonization, is significantly inhibited by 50–75% CO₂, especially at water activities above 0.90 [69]. Paster et al. [70] reported that OTA production by A. ochraceus was completely inhibited by >30% CO₂ on agar-based media after 14 days, suggesting that there are differences between mycotoxigenic species. This suggests that for efficient storage of moist cereals CO₂ concentrations of >50% need to be achieved rapidly to prevent OTA contamination in storage or during transport [62]. Postharvest control of grape rot caused by black aspergilli (among other fungi) has also been successfully carried out using acetaldehyde vapors [71].

Irradiation of fresh fruits, including grapes and figs, can significantly decrease fungal counts [72]. γ-irradiation has also been used successfully to decompose OTA in liquid media [73].

Recently, fungicide treatment of stored products have also been claimed as a promising tool for the prevention of OTA accumulation in agricultural products [74]. A particularly effective treatment for cereals, nuts, fruits, and spices was suggested to be a combination of fungicides, e.g., prothioconazole with trifioxystrobin, tebuconazole with trifloxystrobin, or tebuconazole with prothioconazole.

Knowledge of the key critical control points during the harvesting, drying, and storage stages of the cereal production chain is essential for the development of effective pre- and postharvest prevention strategies. Ecological studies on the effect of environmental factors, which are marginal for growth and mycotoxin production, have been identified for P. verrucosum and A. ochraceus in relation to cereal production and for A. carbonarius in relation to grapes and wine production [75]. Magan [75] and Magan and Aldred [62] gave detailed accounts of pre- and postharvest control strategies for mycotoxin contamination of food in the context of an HACCP (Hazard Analysis and Critical Control Points) framework. Critical control points were identified at different stages of the coffee, cereal, and wine production chains, and these HACCP approaches have been used successfully to control OTA levels in these agricultural products [25,62,75,76,77].

In the European Union, dilution with non-contaminated foodstuffs is forbidden. The physical methods used for mycotoxin detoxification include cleaning, mechanical sorting, and separation (e.g., filtering), heat treatment, ultrasonic treatment, and irradiation. During the cleaning process of contaminated grain, dust, husks, hair, and shallow particles are separated from the grain. During mechanical sorting and separation, the clean product is separated from mycotoxin-contaminated grains, while washing procedures using water or sodium carbonate solution can also result in some reduction of mycotoxins in grains. OTA is a moderately stable molecule. It can survive most food processing, such as roasting, brewing, and baking to some extent [78,79]. Ensiling was found to reduce the OTA content of barley [80]. The scouring of wheat can lead to a reduction of more than 50% in OTA concentration, while milling hard wheat to produce white flour resulted in an approximately 65% reduction, and a further 10% decrease occurred during baking [81].

OTA is generally stable at temperatures used during ordinary cooking. Boudra et al. [82] showed that OTA is heat stable, and it took for more than 10 hours (700 min) and 200 min to decompose 50% of OTA in dry wheat at 100 °C and 150 °C, respectively. However, due to the high temperatures used for coffee roasting, a higher percentage of destruction was observed, although contradictory results from different studies have been reported. Initial studies on the influence of the roasting process on OTA levels indicated a reduction of 77–87% [83], 80–90% [84], and 90–100% [85], although opposing values of 0–12% [86] and 2–28% [87] have also been published. These differences can be due to different spiking methods, selectivity and sensitivity values, initial contamination level, and roasting and drying conditions, or inhomogeneous toxin distribution. More recently, Blanc et al. [88] have found a loss of 84%, van der Stegen et al. [89] of more than 69%, Romani et al. [90] of more than 90%, and Pérez de Obanos et al. [91] of 13–93%. Urbano et al. [92] found that roasting at the temperature of 200 °C for 10 minutes reduced OTA content only by 22%. However, by increasing the temperature to 220 °C for 15 minutes, the OTA content was reduced by up to 94%. Ferraz et al. [93] observed a 8–98% OTA reduction in artificially contaminated coffee beans, depending on the temperature and time period of roasting. The spouted bed roasting used by these authors proved to be a very efficient procedure for OTA reduction in coffee, and its reduction depended directly on the degree of roasting. They also suggested that the low reduction in OTA content observed by some previous authors could be due to the use of a static oven, in which heat exchange is very low. In such a situation, a considerable part of the roasting time might have been used just for drying, with the beans remaining at temperatures of around 80–90 °C. Three different explanations were suggested for this reduction: physical OTA removal with the husk, isomerization at the C-3 position into another diastereomer, and thermal degradation with the possible involvement of moisture [89].

Regarding other coffee processing techniques, Wilkens and Jörissen [96] showed that cyclone cleaning of green coffee beans had little effect on the beans’ OTA concentration: although the OTA concentration in the discarded fraction was high, the dust comprised <1% of the weight of the cleaned coffee. Sorting with color sorters resulted in some reduction, and steaming caused a mean 25% reduction. During coffee brew manufacturing, the coffee grinding entails OTA losses of 20% [88]. Coffee steaming might also promote OTA removal (reduction of about 25%) [97]. Regarding brew preparation, contradictory studies exist: some authors found that all the toxin found in the coffee bean was still present in the brew [86,89], while others noted that 90–100% of the OTA was absent after this stage [85]. More recently, Pérez de Obanos et al. [91] have pointed out that, depending on the brew preparation method used, the OTA losses vary in the range 15–50%. Heilmann et al. [97] studied OTA reduction in raw coffee beans roasted industrially, and showed that levels of OTA were significantly reduced, especially in coffee decaffeinated by solvent extraction. Similar results were reported by Micco et al. [85].

A freezing (−20 °C) defrost (26 °C) process and UV and γ irradiation treatments were found to be able to destroy the conidia of mycotoxin producing fungi, but only γ-irradiation was found to destroy OTA itself [72,98,99,100].

Wine filtration through a 0.45 μm membrane showed an 80% decrease of OTA [101]. More recently, an environmentally friendly corrective measure has been developed to reduce OTA levels by repassage of contaminated musts or wines over grape pomaces having little to no OTA contamination [102]. The use of grape pomaces from red wines of the same grape variety did not affect wine quality parameters, including color intensity and health-promoting phenolic content.

Extensive detoxification of mycotoxins, including ochratoxins, could be achieved by using the intermittent ultrasonic treatment of cereal grains in an aqueous medium supplemented with alcohol and alkali in a temperature range of 12–50 °C [103,104]. The decontaminated cereal products or treated products remained largely unchanged with respect to their appearance, flavor, and nutritious value.

Another approach for removing mycotoxins from contaminated agricultural products involves the use of adsorbent materials with the capacity to tightly bind and immobilize mycotoxins. Adsorbing agents can be classified into different groups based on their origin: minerals (e.g., aluminosilicates), activated coals, biological adsorbents (e.g., yeast and bacterial cell walls or vegetal fibers), and synthetic adsorbents, including modified natural clays (e.g., grafting of quaternary ammonium groups) and synthetic resins (e.g., polyvinylpyrrolidone, cholestyramine). Such materials have been tested mainly to lower OTA contamination of wine and must. Several fining agents have been tested for their ability to remove OTA from contaminated must or wines [26,105]. Activated carbon and potassium caseinate have been reported to remove the highest amounts of OTA, although carbon also removes anthocyanins and other colored polyphenols from wine. Potassium caseinate was able to remove up to 82% of OTA when used at 150 g hl⁻¹, while activated carbon showed the highest specific adsorption capacity, owing to its high surface area per mass ratio, and the low adsorption of total polyphenols. Dumeau and Trione [106] achieved reductions in the OTA content of red wines of up to 90%, although maximum reductions led to negative effects on wine quality, such as reduction of the color intensity and of the polyphenol index of wines. Products like gelatine preparations, silica gel powder, even cellulose, gave good results. Enological decolorizing carbon was also able to remove up to 72% of OTA in red wines during a recent study carried out by Gambuti et al. [101], and the carbon did not affect either polyphenol content or the color of wine, although at this time, a decrease of several sensory odorants was observed. The effectiveness of treatment with oak wood fragments depended upon the quantity of wood chips and powder used [107]. According to the results of Olivares-Marin et al. [108], activated carbon produced from cherry stones could be used to remove up to 50% of OTA from wines, and the changes produced in the total polyphenol index and color intensity were small. Modified silica gels, dodecylammonium bentonite, KSF-montmorillonite, and chitosan beads have also been found to be useful for OTA removal from wine [109,110]. Other adsorbents, including bentonite, cellulose acetate esthers, polyvinylpyrrolidone, cholestyramine, and polygel were inefficient for OTA removal [26,101,111].

Bacteria, for example, Lactobacillus plantarum and Oenococcus oeni, have been found to reduce thre OTA content of wine [52,112]. However, later Mateo et al. [113] observed that part of the OTA was not sorbed by O. oeni and remained in the liquid medium as on ethanol-containing media. Thus, the bacteria cannot efficiently eliminate OTA in acidic ethanol-containing beverages, such as wine. The addition of these adsorbents to red wine by up to 20 g hl⁻¹ did not modify substantially the color, but high amounts of adsorbent (≥50 g hl⁻¹) affected the color and the organoleptic properties of wines [52]. Bejaoui et al. [114] successfully used inactivated Saccharomyces strains to lower the OTA content of grape juices. Their results showed that treatments of yeasts by heating and acids significantly enhanced OTA removal from liquid media. Polysaccharides and peptidoglycans are both expected to be affected by heat and acid treatments, and the released products could offer more adsorption sites than viable cells and may increase surfaces for OTA binding. Yeast cell wall preparations and other yeasts have been found to be effective for OTA adsorption by other authors too [115,116]. There are two main classes of proteins covalently coupled to cell wall polysaccharides in S. cerevisiae, among these, GPI-dependent cell wall proteins, which are generally indirectly linked to β-1,3-glucan through a connecting β-1,6-glucan moiety, have been suggested to be responsible for OTA adsorption on the cells wall [117,118,119]. Raju et al. [120,121] have shown that OTA also binds to the glucomannan component of the cell wall. Yeast industrial byproducts have also been used successfully to bind OTA from wine [122,123].

In conclusion, many adsorbent materials have been tested for OTA detoxification; however, their activity was not as high as expected, with the exception of activated charcoal [79,124,125]. Recently, a new insoluble vegetal fiber has been developed, which is found to be able to adsorb OTA present in liquid food products [126]. Other promising adsorbent materials are modified zeolites, as they have shown good results in foodstuff decontamination [127,128,129,130]. Organophilic bentonites showed the ability to bind OTA up to 100% independently of the pH of the test medium buffer [131].

A wide variety of chemicals have been found to be effective in destroying mycotoxins. The chemicals used include various acids, bases, oxidizing agents, chlorinating or reducing agents, salts, and miscellaneous reagents, such as formaldehyde. Ammoniation is the method that has received the most attention for detoxification of aflatoxin- or ochratoxin-contaminated feeds and has been used successfully in several countries [132]. Ammoniation almost completely decomposes OTA in corn, wheat, and barley [79]. Although the ammoniation process does not lead to the formation and accumulation of toxic breakdown products of mycotoxins in agricultural products, the observed changes in sensory and nutritional qualities (e.g., the brown color of the treated cereals and a decrease in lysine and sulfur-containing amino acids), the relatively long period of aeration, and the cost restrict its use in cereals destined to be used in animal feed formulations [79,133]. Besides, in feeding experiments, some toxicity and lower nutritional values were observed when ammoniated OTA-contaminated barley was used [134]. Consequently, ammoniation was not recommended for the detoxification of OTA-contaminateed feeds [79]. However, ammoniation is an approved procedure for the detoxification of aflatoxin-contaminated agricultural commodities and feeds in some US States (e.g., in Arizona, California, Texas, Georgia, and Alabama). Additionally, in France and Senegal, ammoniation is used for mycotoxin detoxification in contaminated peanut, cotton, and maize meals [135,136,137]. The import of ammoniated peanut meal is allowed by several European Union member countries. However, the US Food and Drug Administration (FDA) does not permit interstate shipment of ammoniated cottonseed or maize.

Alkaline hydrogen peroxide, sodium hydroxide, and monomethylamine or ammonium with calcium hydroxide treatments have also been found to be effective methods for OTA decontamination in this matrix [79]. Nevertheless, their in vivo application is not possible due to the undesirable changes caused in the nutritional and sensory quality of the substrate [79].

Regarding other agricultural products, treatments with ethyl acetate, dichlorometane, and methylene chloride supplemented with 2% formic acid were found to be able to reduce OTA levels by up to 80% in coffee beans [97,138,139]. In cocoa, more than 98% of the OTA could be eliminated by alkaline treatment [140]. Another method is ozonization; the development of sophisticated electrochemical techniques has allowed the application of ozone (O₃) for OTA removal up to undetectable levels in foodstuffs such as grains, nuts, or vegetables [141,142]. OTA was degraded in 15 sec using 10 wt.% O₃ in an aqueous solution, with no by-products detectable by HPLC [142].

Formic, propionic, and sorbic acids and sodium hypochlorite at concentrations ranging from 0.25% to 1% have also been found to degrade OTA after an exposure of 3–24 hours [79,143]. However, alkaline hydrogen peroxide treatment with 0.05–0.1% H₂O₂ did not detoxify OTA [144]. We should mention that chemical treatment is not allowed within the European Union for commodities destined for human consumption. An alternative strategy could be the utilization of microorganisms capable of detoxifying mycotoxins in contaminated foods and feeds (see below).

Microbes or their enzymes can be applied for mycotoxin detoxification; such biological approaches are widely studied [12,129,135,137,145,146,147]. Several reports describe the OTA degrading activities of the microbial flora of the mammalian gastrointestinal tract, including the rumen microbes of cows and sheep [148,149], and the bacteria that mainly live in the caecum and large intestine of rats [150,151]. The duodenum, ileum, and pancreas have also been shown to be able to carry out this reaction in rats, whereas the activity in the liver and kidney was low [152], and was not observed in rat hepatocytes [153], nor in rabbit and rat liver [154]. Up to 12 mg kg⁻¹ of OTA in feed was estimated to be converted to ochratoxin α (OTα) in cows [149]. Thus, this species is assumed to be relatively resistant to the effects of OTA in feed. Sheep also have a good capacity to detoxify OTA before it reaches the blood [155]. Studies in mice suggested that OTA circulates from the liver into the bile and into the intestine, where it is also hydrolysed to OTα [156]. The species responsible for OTA detoxification have not yet been identified, although protozoa were suggested to take part in the biotransformation process in ruminants [155,157]. The enzymes responsible for hydrolysis to OTα in cows and rodents are carboxypeptidase A and chymotrypsin [158]. The human intestinal microflora can also partially degrade OTA [159].

Additionally, numerous other bacteria, protozoa, and fungi have been found to be able to degrade OTA since the 80s (Table 2). Some enzymes, such as carboxypeptidase A [158,160,161], lipases from Aspergillus niger [162], and some commercial proteases [163] have also been identified as being able to carry out this reaction (Figure 1).

Acinetobacter calcoaceticus has also been found to degrade OTA to OTα [164]. These authors hypothesize that an extracellular esterase enzyme is responsible for OTA degradation. The toxicity of OTA decreased since OTα is almost nontoxic [165,166], although it has been found to exhibit genotoxic effects [167]. Actually, most OTA degrading microbes have been found to be able to remove the phenylalanine moiety from OTA, which leads to the accumulation of OTα. Rhizopus isolates are also able to partially degrade OTA within ten days. However, only a R. stolonifer isolate could detoxify OTA in spiked moistened wheat [168]. More recently, Angioni et al. [169] observed that Saccharomyces cerevisiae and Kloeckera apiculata isolates were able to degrade OTA during alcoholic fermentation. The absence of OTA residues in the biomass excluded an adsorbing effect from the yeast cell walls of the strains studied, and the absence of ochratoxin α and phenylalanine suggested other degradation pathways of OTA than what was observed in most other microorganisms. Silva et al. [170] also observed the OTA degrading activities of a Lactobacillus plantarum isolate. However, degradation products of OTA were not observed, indicating that possibly OTA adsorption, instead of degradation, took place in these cases. OTA was also efficiently detoxified by some Bacillus isolates, especially by B. licheniformis CM21 [171]. Similarities between OTA degradation kinetics by Aspergillus niger and Bacillus isolates and the detection of the degradation product, OTα in the ferment broth of B. licheniformis suggest that carboxypeptidase A activity may be responsible for OTA decomposition by these isolates [171].

Several aerobic or anaerobic bacteria and yeasts have been identified, which were found to cleave the phenylalanine group of the ochratoxins [147,172,173] (Table 2). Among these microbes, the detoxifying bacteria belonging to Sphingomonas sp., Stenotrophomonas nitritreducens, Stenotrophomonas, Ralstonia eutropha, and Eubacterium sp., and the detoxifying yeasts belonging to Trichosporon sp., Cryptococcus sp., Rhodotorula yarrowii, Trichosporon mucoides, and Trichosporon dulcitum have proved to be particularly efficient, since they not only ensured complete degradation of OTA, but could additionally be used safely in food products and animal feeds, which is not necessarily the case with a plurality of other mycotoxin-cleaving and/or degrading bacteria and yeasts.

Streptococcus salivarius, Bifidobacterium bifidum, Lactobacillus delbrueckii, and yogurt bacteria have completely reduced OTA levels in milk samples [174]. Varga et al. [175] examined more than 70 Aspergillus isolates for their ability to degrade OTA to OTα, which has only limited toxicity. Only A. fumigatus and some black Aspergillus isolates were found to be able to carry out this reaction. The kinetics of the degradation of OTA of an atoxigenic A. niger strain was further studied. OTA degradation was faster in solid media than in liquid cultures. A. niger could also degrade ochratoxin α to an unknown compound within some days [175]. This is a promising result because it might allow for the biological elimination of this mycotoxin and may provide a source of enzymes, which could be used for detoxification of OTA in contaminated agricultural products. Further studies have been carried out to identify the enzyme responsible for OTA degradation in A. niger CBS 120.49 [176]. It was predicted that the enzyme involved in the reaction is possibly a carboxypeptidase, as carboxypeptidase A can convert OTA to ochratoxin α. The entire cpa gene in A. niger was identified and the promoter and terminal regions of this gene were also determined. The whole cpa gene has been cloned and analyzed: a 3287 base pair (bp) long sequence was determined. This sequence contains a 1939 bp long open reading frame and a 72 bp long intron. This open reading frame encodes a 621 amino acid long protein (Figure 2). Besides the coding region, the 648 bp long promoter region and the 700 bp long terminal region were determined as well. In further experiments, the gene was transformed to a Pichia pastoris isolate; the isolated cpa gene was inserted into the pPCIZα vector, which was used in the transformation of the Pichia pastoris KM71H isolate. The results showed that this protein was not secreted into the ferment broth or was present only in low quantities. Experiments have also been initiated to transform the gene into an atoxigenic A. niger (JHC 607) and A. nidulans (SZMC 0552) isolate, without success. The results showed that these transformants were unable to degrade OTA in liquid medium. We suppose that the cpa gene was not integrated into the genome of A. niger and A. nidulans, or was not expressed in these isolates. Further studies are in progress to clarify the role of this gene in OTA degradation.

Abrunhosa et al. [177] identified 51 fungal isolates from grapes with the ability to degrade more than 80% of the OTA added to a culture medium. The most effective isolates belonged to the A. clavatus, A. ochraceus, A. versicolor, and A. wentii species. Other genera found to degrade OTA include Alternaria, Botrytis, Cladosporium, and Penicillium [177]. In the cases of A. ochraceus and A. wentii, OTα was not detected in the medium as a breakdown product of OTA [177]. Similarly, some strains of A. carbonarius, A. niger aggregate, and A. japonicus isolated from French grapes degraded more than 80% of the OTA to OTα in liquid medium [178]. Some yeasts belonging to the genera Rhodotorula, Cryptococcus, and Pichia have also been found to be able to degrade OTA [179].

We recently examined the OTA degrading and adsorbing activities of astaxanthin-producing yeast isolates (Phaffia rhodozyma and Xanthophyllomyces dendrorhous) [180]. The data indicate that besides producing astaxanthin, Ph. rhodozyma is also able to both detoxify and adsorb OTA at temperatures well above the temperature optimum for the growth of Phaffia cells. One Ph. rhodozyma and two X. dendrorhous isolates have been tested for OTA degradation. All of them were able to degrade OTA in a liquid medium after less than 10 days. In further studies, we concentrated on the OTA degradation or adsorbing activities of the isolate, CBS 5905 of Ph. rhodozyma. This Phaffia isolate could degrade more than 90% of the OTA in about 7 days at 20 °C. Interestingly, a significant amount of OTA was found to be bound by the cells after two days, indicating that the OTA is also adsorbed by the cells. The ferment broth of either induced or uninduced cells was unable to degrade OTA. These observations indicate that the enzyme responsible for OTA degradation is not excreted into the ferment broth; thus, the enzyme responsible for OTA degradation is cell-bound. Our data indicate that Ph. rhodozyma is able to convert OTA to OTα, and that this conversion is possibly mediated by an enzyme related to carboxypeptidases. Chelating agents like EDTA and 1,10-phenanthroline inhibit the OTA degradation caused by Ph. Rhodozyma, indicating that the OTA degrading enzyme, like carboxypeptidase A, is a metalloprotease. The temperature optimum of this enzyme was found to be above 30 °C, which is much higher than the temperature optimum for the growth of Ph. rhodozyma cells, which is around 20 °C. Both viable and heat-treated (dead) Ph. rhodozyma cells were also able to adsorb significant amounts of OTA. Further studies are in progress to identify the enzyme responsible for OTA degradation in Ph. rhodozyma [176].

Cell cultures of plants like maize, tomato, and wheat have been found to be able to transform OTA into a number of compounds. The transformation reactions included hydrolysis of ester and peptide bonds, methylation and hydroxylation, some of which led to a loss in toxicity [181,182,183,184]. OTA was found to be toxic to several invertebrates, such as the corn ear worm (Helicoverpa zea) and Carpophilus hemipterus, while others, e.g., the Mediterranean flour moth (Anagasta kuehniella) were not affected [185]. Since these insensitive animals possibly produce some factors which are able to inactivate OTA, they are regarded as promising sources of OTA detoxifying enzymes [184].

Based on an EFSA survey, Trichosporon mycotoxinivorans was found to be the only microorganism that shows the potential to degrade OTA and meets the prerequisites for use as an animal feed additive [186].