Chemical, physical and biological approaches to prevent ochratoxin induced toxicoses in humans and animals.
ABSTRACT Ochratoxins are polyketide derived fungal secondary metabolites with nephrotoxic, immunosuppressive, teratogenic, and carcinogenic properties. Ochratoxin-producing fungi may contaminate agricultural products in the field (preharvest spoilage), during storage (postharvest spoilage), or during processing. Ochratoxin contamination of foods and feeds poses a serious health hazard to animals and humans. Several strategies have been investigated for lowering the ochratoxin content in agricultural products. These strategies can be classified into three main categories: prevention of ochratoxin contamination, decontamination or detoxification of foods contaminated with ochratoxins, and inhibition of the absorption of consumed ochratoxins in the gastrointestinal tract. This paper gives an overview of the strategies that are promising with regard to lowering the ochratoxin burden of animals and humans.
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ABSTRACT: Aflatoxin and ochratoxin levels were determined in maize samples collected from store houses of 15 districts belonging to three agro-ecological zones of Punjab, Pakistan. Toxins were extracted by Aflaochra immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC). Mean moisture content of maize kernels was recorded above the safe storage level of 15%. Results indicated that aflatoxin B1 and B2 contamination was found in 97.3% and 78.9% of the collected samples, respectively. Aflatoxin G1, aflatoxin G2 and ochratoxin A were not detected in any sample. Among positive samples, 77.3% contained aflatoxin B1 and 28% aflatoxin B2, exceeding the legal limits as set by the European Union (EU) and the United States Food and Drug Administration (USFDA). It was concluded that a significant number of samples contained aflatoxin B1 and B2 above the legal limits.Food Additives and Contaminants: Part B Surveillance 03/2014; 7(1):57-62.
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ABSTRACT: Results of a 2-year (2009-2010) survey on the occurrence of ochratoxin A (OTA) in swine feed and in feed for laying hens in Portugal are reported. A total of 664 samples (478 swine feed, 186 feed for laying hens) were analyzed by a HPLC method using fluorescence detection with 2 μg kg(-1) as detection limit. In swine feed, 31 samples (6.49%) were positive for OTA. In feed for laying hens, 12 samples (6.45%) were OTA-positive. The average levels of contamination were low, with median values of positive samples at 3-4 μg kg(-1) in both years and both commodities, although a few samples contained exceptionally high levels (maximum 130 μg kg(-1)). Only the maximum level sample (swine feed) contained OTA at a concentration exceeding the European Commission guidance value. The remaining OTA concentrations found in feed samples were much lower than the guidance values.Mycotoxin Research 05/2012; 28(2):107-10.
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ABSTRACT: Lactic acid bacteria (LAB) are a promising solution to reduce exposure to dietary mycotoxins because of the unique mycotoxin decontaminating characteristic of some LAB. Ochratoxin A (OTA) is one of the most prominent mycotoxins found in agricultural commodities. The present work reports on the ability of Pediococcus parvulus strains that were isolated from Douro wines that spontaneously underwent malolactic fermentation to detoxify OTA. These strains were identified and characterised using a polyphasic approach that employed both phenotypic and genotypic methods. When cultivated on OTA-supplemented MRS media, OTA was biodegraded into OTα by certain P. parvulus strains. The presence of OTα was confirmed using LC-MS/MS. The conversion of OTA into OTα indicates that the OTA amide bond was hydrolysed by a putative peptidase. The rate of OTA biodegradation was found to be dependent on the inoculum size and on the incubation temperature. Adsorption assays with dead P. parvulus cells showed that approximately 1.3%±1.0 of the OTA was adsorbed onto cells wall, which excludes this mechanism in the elimination of OTA by strains that degrades OTA. Under optimum conditions, 50% and 90% of OTA were degraded in 6 and 19h, respectively. Other LAB strains that belonged to different species were tested but did not degrade OTA. OTA biodegradation by P. parvulus UTAD 473 was observed in grape must. Because some P. parvulus strains have relevant probiotic properties, the strains that were identified could be particularly relevant to food and feed applications to counteract the toxic effects of OTA.International journal of food microbiology. 07/2014; 188C:45-52.
Toxins 2010, 2, 1718-1750; doi:10.3390/toxins2071718
Chemical, Physical and Biological Approaches to Prevent
Ochratoxin Induced Toxicoses in Humans and Animals
János Varga 1,*, Sándor Kocsubé 1, Zsanett Péteri 1,2, Csaba Vágvölgyi 1 and Beáta Tóth 3
1 Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Közép
fasor 52, H-6726 Szeged, Hungary; E-Mails: firstname.lastname@example.org (S.K.);
email@example.com (Z.P.); firstname.lastname@example.org (C.V.)
2 PannonPharma Company, Mária dűlő 36, H-7634 Pécs, Hungary
3 Cereal Research Non-Profit Limited Company, Alsókikötő sor 9, H-6726 Szeged, Hungary;
E-Mail: email@example.com (B.T.)
* Author to whom correspondence should be addressed; E-Mail: firstname.lastname@example.org;
Tel.: +36-62544-515; Fax: +36-62-544-823.
Received: 6 May 2010; in revised form: 25 June 2010 / Accepted: 29 June 2010 /
Published: 1 July 2010
Abstract: Ochratoxins are polyketide derived fungal secondary metabolites with
nephrotoxic, immunosuppressive, teratogenic, and carcinogenic properties. Ochratoxin-
producing fungi may contaminate agricultural products in the field (preharvest spoilage),
during storage (postharvest spoilage), or during processing. Ochratoxin contamination of
foods and feeds poses a serious health hazard to animals and humans. Several strategies
have been investigated for lowering the ochratoxin content in agricultural products. These
strategies can be classified into three main categories: prevention of ochratoxin
contamination, decontamination or detoxification of foods contaminated with ochratoxins,
and inhibition of the absorption of consumed ochratoxins in the gastrointestinal tract. This
paper gives an overview of the strategies that are promising with regard to lowering the
ochratoxin burden of animals and humans.
Keywords: ochratoxin; detoxification; adsorption; spoilage
Toxins 2010, 2
Ochratoxin A (OTA) is a mycotoxin that contaminates different plant products, including cereals,
coffee beans, nuts, cocoa, pulses, beer, wine, spices, and dried vine fruits . Ochratoxins are cyclic
pentaketides: dihydroisocoumarin derivatives linked to an L-phenylalanine moiety. OTA was first
discovered in 1965 in an Aspergillus ochraceus isolate . Since then, several Aspergillus and
Penicillium species have been described as producers of this mycotoxin . OTA proved to exhibit
nephrotoxic, immunosuppressive, teratogenic, and carcinogenic properties . Several nephropathies
affecting animals as well as humans have been attributed to OTA; e.g., this mycotoxin is the
etiological agent of Danish porcine nephropathy and renal disorders observed in other animals . In
humans, OTA is frequently cited as the possible causative agent of Balkan endemic nephropathy, a
syndrome characterized by contracted kidneys with tubular degeneration, interstitial fibrosis, and
hyalinization of the glomeruli [6,7]. In 1993, the International Agency for Research on Cancer (IARC)
classified OTA as a possible human carcinogen (Group 2B) and concluded that there was sufficient
evidence in experimental animals, but inadequate evidence in humans for the carcinogenicity of OTA
. OTA has also been suggested to play a role in chronic karyomegalic interstitial nephropathy and
chronic interstitial nephropathy in Tunisia , urothelial tumors (end-stage renal disease) in Egypt
, and testicular cancer .
Maximum levels for OTA in commodities have been set by Commission Regulation (EC) for
several agricultural products destined to be used as food or feed ingredients . There are also national
laws and regulations in the European Union. Several strategies have been proposed to prevent the toxic
effects of mycotoxins in general, and of ochratoxins in particular, in foods and feeds :
(i) prevention of mycotoxin contamination;
(ii) decontamination or detoxification of foods contaminated with mycotoxins;
(iii) inhibition of the absorption of consumed mycotoxin in the gastrointestinal tract.
2. Prevention of Mycotoxin Contamination
2.1. Preharvest management
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 . Indeed, Rousseau and
Blateyron  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 . The organophosphate fungicide, dichlorvos, was found to inhibit OTA production of
A. sulphureus, P. verrucosum, and A. ochraceus [15,16]. Another fungicide, iprodione, has
Toxins 2010, 2
successfully been used in agricultural commodities to prevent the growth of various fungal species,
including OTA producers , and was found to be able to decrease OTA production of
A. westerdijkiae . 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 , or captan,  were found to be effective against black aspergilli,
which colonize grape berries. Recently, Lo Curto et al.  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 . 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 [21–23]. The fungicide Switch
contains cyprodinil and fludioxonil, which belong to the pyrimidine and pyrrolnitrin classes of
fungicides, respectively . The observation that fludioxonil can be used against aspergilli is not
surprising, since pyrrolnitrin was found previously to be effective against black aspergilli . 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 . 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 . Belli et al.  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 . For example, carbendazim has been found to reduce fungal
biota, but to stimulate OTA production , while fenhexamid, mancozeb, and copper hydroxide plus
copper also enhanced infection and OTA production in grapes . 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 .
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 . 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 . 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 . Bacillus
thuringiensis was also found to significantly inhibit the growth of OTA-producing fungi on grapes in
another study .
Toxins 2010, 2
In coffee, the coffee berry borer (also called broca), Hypotenemus hampei, has been shown to be a
vector of A. ochraceus . 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 , entomopathogenic fungi (e.g., Beauveria bassiana), or the insect
parasitoids Cephalonomia stephanoderis or Prorops nasuta . 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 .
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 . 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 . This isolate was
also able to degrade OTA in in vitro experiments. Other epiphytic yeasts, including Candida
guilliermondii, Acremonium cephalosporium , Issatchenkia orientalis, Metschnikowia
pulcherrima, Issatchenkia terricola, and Candida incommunis  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 .
Biological control using non-toxigenic Aspergillus niger isolates against toxin-producing black
aspergilli has also been applied successfully in grapes . 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 .
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 . 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 . 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 . 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 , 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 .
Toxins 2010, 2
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 .
2.2. Postharvest management
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 : 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
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 . 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 .
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 .
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 . 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 . Temperature also influences fungal contamination during storage.
Toxins 2010, 2
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
, and essential oils extracted from plants, such as Thymus vulgaris, Aframomum danielli [60,61],
cinnamon, and clove leaf , which affect mold growth and OTA synthesis. Other essential oils
extracted from plants (thyme, cinnamon, marigold, spearmint, basil, quyssum, caraway, and anise)
, 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 . 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 . Indeed,
flavonoids, including rutin, quercetin, and caffeic acid were found to inhibit both growth and OTA
synthesis in A. carbonarius . 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 .
Several reports have dealt with the antifungal properties of various lactic acid bacteria . 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 .
Another possibility of inhibiting the growth of mycotoxigenic fungi is the application of modified
atmospheres or gases, such as CO2 , 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 . Paster et al.  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 . Postharvest control of grape rot caused by black aspergilli (among
other fungi) has also been successfully carried out using acetaldehyde vapors .
Irradiation of fresh fruits, including grapes and figs, can significantly decrease fungal counts .
-irradiation has also been used successfully to decompose OTA in liquid media .
Recently, fungicide treatment of stored products have also been claimed as a promising tool for the
prevention of OTA accumulation in agricultural products . 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.
Toxins 2010, 2
2.3. HACCP approaches
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 . Magan 
and Magan and Aldred  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 [25,62,75–77].
3. Decontamination/Detoxification Approaches
The ideal solution for reducing the health risk of mycotoxins is to prevent contamination of foods
with them. Unfortunately, contamination cannot be completely avoided. Therefore, there is an
increased focus on effective methods of detoxification for mycotoxins present in foods, and on the
inhibition of mycotoxin absorption in the gastrointestinal tract. Decontamination or detoxification
procedures are useful in order to recondition mycotoxin contaminated commodities. While certain
treatments have been found to reduce levels of specific mycotoxins, no single method has been
developed that is equally effective against the wide variety of mycotoxins, which may co-occur in
different commodities. Mycotoxin decontamination processes should meet the following criteria :
- They must destroy, inactivate, or remove mycotoxins;
- They must not produce or leave toxic, carcinogenic, or mutagenic residues in the final products
or in food products obtained from animals fed by decontaminated feed;
- They should not adversely affect the desirable physical and sensory properties of the product;
- They must be capable of destroying fungal spores and the mycelium in order to avoiding
mycotoxin formation under favorable conditions;
- They have to be technically and economically feasible.
Several strategies are available for the detoxification or decontamination of commodities containing
ochratoxins. These can be classified as physical, physicochemical, chemical, and (micro)biological
3.1. Physical methods
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
Toxins 2010, 2
the OTA content of barley . 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 .
OTA is generally stable at temperatures used during ordinary cooking. Boudra et al.  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% , 80–90% , and 90–100% , although
opposing values of 0–12%  and 2–28%  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. 
have found a loss of 84%, van der Stegen et al.  of more than 69%, Romani et al.  of more
than 90%, and Pérez de Obanos et al.  of 13–93%. Urbano et al.  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. 
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 .
Table 1. OTA reduction during coffee roasting.
Origin of OTA
200 °C, 10–20 min
180 °C, 10 min
200 ± 5 °C, 20 min
200 ± 5 °C, 20 min
200 °C, 3 min
180–240 °C, 5–12 min
5–6 min, dark roasting
5–6 min, dark roasting
250 °C, 150 sec
250 °C, 150 sec
223 °C, 4 min
Light to dark
175–204 °C, 7–9 min
200–220 °C, 10–15 min
Reduction in OTA content (%) References
Toxins 2010, 2
Regarding other coffee processing techniques, Wilkens and Jörissen  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% .
Coffee steaming might also promote OTA removal (reduction of about 25%) . 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 . More recently, Pérez de Obanos et al.  have pointed out that, depending on the
brew preparation method used, the OTA losses vary in the range 15–50%. Heilmann et al.  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. .
A freezing (−20 °C) defrost (26 °C) process and UV and irradiation treatments were found to be
able to destroy the conidia of mycotoxin producing fungi, but only -irradiation was found to destroy
OTA itself [72,98–100].
Wine filtration through a 0.45 μm membrane showed an 80% decrease of OTA . 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
. 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.
3.2. Physicochemical methods
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  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
Toxins 2010, 2
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. , 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 . According to the results of Olivares-Marin et al.
, 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.  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
. Bejaoui et al.  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
-1,3-glucan through a connecting -1,6-glucan moiety, have been suggested to be responsible for
OTA adsorption on the cells wall [117–119]. 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 . Other promising adsorbent materials are modified zeolites, as they have
shown good results in foodstuff decontamination [127–130]. Organophilic bentonites showed the
ability to bind OTA up to 100% independently of the pH of the test medium buffer .
3.3. Chemical approaches
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
Toxins 2010, 2
most attention for detoxification of aflatoxin- or ochratoxin-contaminated feeds and has been used
successfully in several countries . Ammoniation almost completely decomposes OTA in corn,
wheat, and barley . 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 . Consequently, ammoniation was not recommended for the
detoxification of OTA-contaminateed feeds . 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
[135–137]. 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 . Nevertheless, their in vivo application is not possible due to the undesirable changes
caused in the nutritional and sensory quality of the substrate .
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 . 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 .
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 . 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).
3.4. Microbiological methods
Microbes or their enzymes can be applied for mycotoxin detoxification; such biological approaches
are widely studied [12,129,135,137,145–147]. 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 , and was not observed in rat
Toxins 2010, 2
hepatocytes , nor in rabbit and rat liver . Up to 12 mg kg−1 of OTA in feed was estimated to
be converted to ochratoxin (OT) in cows . 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 . Studies in mice suggested that OTA circulates from the liver into the bile and
into the intestine, where it is also hydrolysed to OT . 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 OT in
cows and rodents are carboxypeptidase A and chymotrypsin . The human intestinal microflora
can also partially degrade OTA .
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 , and some commercial proteases  have also been identified as being able
to carry out this reaction (Figure 1).
Figure 1. Degradation of ochratoxin A by carboxypeptidase A.
Acinetobacter calcoaceticus has also been found to degrade OTA to OTα . These authors
hypothesize that an extracellular esterase enzyme is responsible for OTA degradation. The toxicity of
OTA decreased since OT is almost nontoxic [165,166], although it has been found to exhibit
genotoxic effects . Actually, most OTA degrading microbes have been found to be able to
remove the phenylalanine moiety from OTA, which leads to the accumulation of OT. 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 . More recently, Angioni et al.  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 and phenylalanine
suggested other degradation pathways of OTA than what was observed in most other microorganisms.
Silva et al.  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 . Similarities between OTA degradation kinetics by
Aspergillus niger and Bacillus isolates and the detection of the degradation product, OT in the
ferment broth of B. licheniformis suggest that carboxypeptidase A activity may be responsible for
OTA decomposition by these isolates .
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
Toxins 2010, 2
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 . Varga et al.  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 . 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 . 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.
bacteria belonging to
sp., Stenotrophomonas nitritreducens,
Table 2. Microbes and enzymes able to degrade ochratoxin A.
Microbes or enzymes
Lactobacillus, Streptococcus, Bifidobacterium sp.
Bacillus subtilis, B. licheniformis
Toxins 2010, 2
Table 2. Cont.
Nocardia corynebacterioides, Rhodococcus erythropolis, Mycobacterium sp.
Eubacterium callenderi, E. ramulus, Streptococcus pleomorphus, Lactobacillus vitullinus,
Sphingomonas paucimobilis, S. saccharolytica, Stenotrophomonas nitritreducens, Ralstonia
eutropha, R. basilensis, Ochrobactrum sp., Agrobacterium sp.
Pseudomonas cepacia, P. putida, Rhodococcus erythropolis, Agrobacterium tumefaciens,
Aspergillus niger, A. fumigatus
Aspergillus niger, A. versicolor, A. wentii, A. ochraceus
Aspergillus niger, A. japonicus
Saccharomyces cerevisiae, S. bayanus
Rhizopus stolonifer, R. microsporus, R. homothallicus, R. oryzae
Phaffia rhodozyma, Xanthophyllomyces dendrorhous
Saccharomyces cerevisiae, Kloeckera apiculata
Cryptococcus flavus, C. laurentii, C. curvatus, C. humicolus, Trichosporon ovoides, T.
dulcitum, T. guehoae, T. mucoides, T. coremiiforme, T. cutaneum, T. laibachii, T.
monilifotrme, Rhodotorula mucilaginosa, R. fujisanensis
Commercial proteases (Pancreatin from porcine pancreas, Protease A and Prolyve PAC
from A. niger)
Commercial hydrolases (Amano A, crude lipase preparation from A. niger)
A. niger hydrolytic metalloenzyme
Abrunhosa et al.  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 . In the cases of A. ochraceus and
A. wentii, OT was not detected in the medium as a breakdown product of OTA . Similarly,
some strains of A. carbonarius, A. niger aggregate, and A. japonicus isolated from French grapes
degraded more than 80% of the OTA to OT in liquid medium . Some yeasts belonging to the
genera Rhodotorula, Cryptococcus, and Pichia have also been found to be able to degrade OTA .
We recently examined the OTA degrading and adsorbing activities of astaxanthin-producing yeast
isolates (Phaffia rhodozyma and Xanthophyllomyces dendrorhous) . 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
Toxins 2010, 2
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 OT, 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 .
Figure 2. Comparison of the partial amino acid sequences of the carboxypeptidase A (cpa)
gene of Aspergillus niger to those of metalloproteases from other fungi. Black boxes
indicate highly conserved domains coding for Zn ion binding sites.
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 [181–184]. OTA was
found to be toxic to several invertebrates, such as the corn ear worm (Helicoverpa zea) and