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Ochratoxin A (OTA) is one of the most widespread mycotoxins, and is produced by several Aspergillus or Penicillium species. Human exposure to OTA is mainly by intake of contaminated food, with cereal products, followed by coffee and red wine as the main sources of OTA. In this study, the OTA production of four ochratoxigenic fungi (two Aspergillus and two Penicillium species) was investigated in four different media, i.e. wheat and coffee model media as food-based media and two standard labor...Ochratoxin A (OTA) is one of the most widespread mycotoxins, and is produced by several Aspergillus or Penicillium species. Human exposure to OTA is mainly by intake of contaminated food, with cereal products, followed by coffee and red wine as the main sources of OTA. In this study, the OTA production of four ochratoxigenic fungi (two Aspergillus and two Penicillium species) was investigated in four different media, i.e. wheat and coffee model media as food-based media and two standard laboratory media (malt extract glucose agar, MEA and yeast extract sucrose agar, YES). Colony growth was documented and OTA concentrations in cultures were determined at day 2, 4 and 8 of incubation at 25°C by high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC). OTA production clearly depended upon time of incubation, fungal species, and medium composition. On coffee based medium, moderate OTA levels were produced by A. ochraceus BFE635 (9.8 μg/g) and by A. niger BFE632 (10.6 μg/g) on day 8 of incubation. In wheat-based medium, these strains produced much more OTA than in coffee. The highest OTA concentration (83.8 μg/g on day 8) was formed by A. ochraceus BFE635 followed by the other Aspergillus niger BFE632 (49 μg/g). Lower OTA levels were produced by P. verrucosum BFE550 and P. nordicum BFE487, in both wheat and in YES medium, whilst OTA was hardly detectable in coffee and in MEA in case of P. nordicum. Colony growth of the tested strains on different media was not indicative of OTA production. Guttation droplets developed on wheat-based medium with the Aspergillus strains within a week, and this phenomenon coincided with the high OTA amounts formed by these species. Results from this study add to our knowledge on the behaviour of ochratoxigenic fungal species when cultured on food based media. Keywords Ochratoxin A Aspergillus Penicillium Guttation droplets HPTLC Electronic supplementary material The online version of this article (doi:10.1007/s12550-011-0100-0 [Titel anhand dieser DOI in Citavi-Projekt übernehmen] ) contains supplementary material, which is available to authorized users. Download fulltext PDF Introduction Mycotoxins are toxic secondary metabolites of fungi belonging mainly to the genera Aspergillus, Penicillium and Fusarium. Their occurrence in plant-derived foods and feeds can have a serious negative impact on human health and animal productivity. Since it is estimated that 25% of the world’s food crops, including many basic foods, are affected by mycotoxin-producing fungi, this is also associated with significant economic losses (FAO 2001). Ochratoxin A (OTA) is a mycotoxin produced by several species of the genus Aspergillus or Penicillium. OTA is a potent nephrotoxin in pigs and rats, and it is suggested to be an etiological agent for Balkan endemic nephropathy (BEN) in humans (EFSA 2006; Pfohl-Leszkowicz et al. 2002). This mycotoxin was classified as a possible human carcinogen (group 2B) by the International Agency for Research on Cancer (IARC 1993) based on its carcinogenic activity in rodent studies. Moreover, OTA has teratogenic and immunosuppressive properties in some animal species, which was reviewed by Petzinger and Weidenbach (2002). Levels of OTA detected in human biological fluids are indicative of a frequent exposure of the human population in different countries as reviewed by Scott (2005). This exposure occurs mainly by ingestion of contaminated plant-based food such as cereals (Czerwiecki 2001; EFSA 2006; Vega et al. 2009), wine and beer (Mateo et al. 2007), coffee (Studer-Rohr et al. 1995), and/or consumption of animal-derived food with OTA residues (Dall’Asta et al. 2007). Also, other non-conventional sources of exposure have been described, e.g. inhalation of OTA with airborne dusts (Richard et al. 1999; Gareis and Meussdoerffer 2000; Degen et al. 2007; Duarte et al. 2009). In Europe (EC 2002), cereals contribute around 50% to the total OTA dietary intake, followed by red wine (13%) and coffee (10%). Taking into account the nephrotoxic and carcinogenic potential of OTA, tolerable daily intake values for adults have been proposed (Nordic Working Group 1991; JECFA 1996; Kuiper-Goodman et al. 2010), and maximum limits for food and feed (EC European commission 2006) are set by several countries to reduce human exposure. The main species known to produce OTA are Penicillium verrucosum, P. nordicum, Aspergillus carbonarius, A. niger and A. ochraceus (Frisvad et al. 2006; Cabañes et al. 2010). Recently, A. westerdijkiae and A. steynii, two very potent OTA producing new species, have been separated from A. ochraceus, which is only a moderate producing organism (Frisvad et al. 2004). These species are commonly found to contaminate raw materials and food commodities during drying and subsequent storage or transport, i.e. usually, in processes following the harvest stage (Magan and Aldred 2007). Environmental conditions, such as temperature and moisture expressed as water activity (aw), pH and nutrient content of the matrix, incubation time, and light, are decisive factors for fungal OTA production (Abellana et al. 2001; Astoreca et al. 2007; Esteban et al. 2004; Magan and Aldred 2005; Richter et al. 2001; Schmidt-Heydt et al. 2010, 2011). Penicillium species are known as OTA-producers in regions with temperate and cold climates. OTA presence in feedstuffs has been frequently associated with porcine and avian nephropathy in countries such as Denmark, Norway or Sweden (Krogh 1992). P. verrucosum is the main OTA-producing species in cereals such as wheat and barley, and cereal products (Frisvad et al. 2006; Cabañes et al. 2010). In salt-rich proteinaceous food products, P. nordicum is the most predominant ochratoxigenic species (Larsen et al. 2001). The genus Aspergillus is commonly associated with warmer and tropical climates. Coffee berries and grapes have been frequently reported to be infested with Aspergillus species. Contamination of legumes, mixed feed, and less frequently cereals, with Aspergillus species have been also described (Accensi et al. 2004). In warmer areas of coffee production, the main isolated species are A. ochraceus (Suárez-Quiroz et al. 2004a), A. westerdijkiae (Morello et al. 2007), or A. steynii (Noonim et al. 2008), respectively. In a study done in Brazil, A. niger was the species found most commonly, but only 3% of them were able to produce OTA. A. ochraceus also occurred frequently (31% of isolates), and interestingly 75% of those studied species were capable to produce OTA (Taniwaki et al. 2003); however, the recent taxonomic reclassification has to be kept in mind. A. carbonarius has been often linked to wine production, being often found in grape samples (Lucchetta et al. 2010), and it may also develop on grape bunches during veraison (Battilani et al. 2003; Hocking et al. 2007). Knowledge of mycotoxin production by ochratoxigenic species in food-based media could help to estimate OTA levels in foods under unfavourable environmental conditions. In light of the frequent detection of OTA and considering that commodities with a major contribution to overall OTA exposure are cereals and coffee, the aim of this work was to study the toxin production and morphological features of four OTA-producing strains on coffee- and wheat-based media and in two standard laboratory culture media. Materials and methods Standards and reagents Toluene, ethyl acetate, methanol, acetonitrile, o-phosphoric acid and formic acid used as mobile phase were HPLC (high-performance liquid chromatography) grade and obtained from Merck (Darmstadt, Germany). Reference compounds (OTA and OTB, purity 98%) were purchased from Sigma (Taufkirchen, Germany); the toxins were dissolved in methanol and used as standards in a calibration curve. Fungal strains and growth conditions Aspergillus and Penicillium strains from the culture collection of the Max Rubner Institut (BFE numbers) in Karlsruhe (Germany) were used. A. ochraceus BFE635 (CBS589.68; Centraalbureau voor Schimmelcultures, Utrecht, Netherland) A. niger var. niger BFE632, P. nordicum BFE487 and P. verrucosum BFE550 were cultured on four different agar media. Malt extract glucose (MEA) and yeast extract sucrose (YES) media (dry powder) were purchased from Merck (Darmstadt, Germany) and prepared according to the suggestions of the manufacturer. Coffee and wheat medium were prepared as follows: coffee beans and wheat grains, purchased in an “eco-shop” at the local market, were ground under liquid nitrogen, to avoid the fat migration from the grains, and sterilized by γ-irradiation (15 kGy); 175 g of coffee powder or wheat flour and 15 g agar were dissolved in 1 l distilled water, and poured into Petri dishes. The plates were single point inoculated by applying centrally 100 μl of a dense (107-108/ml) spore suspension and incubated at 25°C for up to 8 days, unless otherwise indicated. The experiments were done in duplicate. Growth assessment For analysing the growth rate the strains were single-point inoculated on the four media, grown for 8 days at 25°C. At days 2, 4 and 8, the colony diameters were measured in two directions at right angles to each other. The colonies were photographed at the same time. All experiments were done in duplicate. pH measurement Acidity was measured in all plates at different times in culture (days 2–8) by placing a piece of pH-indicator paper (Neutralit and Acilit from Merck) in the middle of the colony for some seconds, and comparison of the coloration obtained with the pH scale of the respective indicator paper. Sample preparation OTA was extracted from cultures with acidified chloroform. Agar plugs (diameter 1 cm) were taken from the edge, the centre, and midway between the edge and the centre of each colony. Two grams of the homogenized sample were extracted with 2 ml chloroform/formic acid (99:1, v/v), by using a vortex for 30 min. To ensure that OTA was extracted from mycelia and spores into the liquid phase, the mixture was treated for 5 min in a sonic bath. The liquid phase was then separated by centrifugation and 500 μl of the chloroform fraction was evaporated to dryness under a gentle stream of nitrogen at 40°C. The extract was reconstituted in 500 μl methanol and filtered using a 0.22-μm Teflon membrane before the analysis. Exudate droplets from some cultures were collected with a micropipette, and analyzed directly after dilution with methanol. Mycotoxin analysis Extracts were analyzed by high-performance thin-layer chromatography (HPTLC) and HPLC. OTA was quantified by external standard method, and presence of OTB was qualitatively analyzed. HPTLC analysis Sample extracts and standard solutions were applied to HPTLC plates (20 × 10 cm) precoated with silica gel F254 (Merck) by means of an automatic TLC Sampler IV (ATS 4) (CAMAG, Muttenz, Switzerland). Chromatography was done in a 20 × 10-cm twin-trough developing chamber (CAMAG) using toluene, ethyl acetate and formic acid (60:30:10, v/v/v) as mobile phase (Scott et al. 1970). Then the plate was dried for 15 min at 120°C to evaporate the mobile phase prior to detection in a TLC Scanner 3 (CAMAG) under UV light at 366 nm. All HPTLC instruments were managed via the software winCATS 1.4.3 Planar Chromatography Manager (CAMAG). OTA and OTB in samples were identified as bluish-green fluorescent spots with the same mobility as that of the standards (Betina 1989). Confirmation of OTA was done by post chromatographic treatment using ammonia vapour, which caused a shift in the fluorescence from bluish-green to deep-blue (Varga et al. 1996). HPLC analysis Extracts were analyzed using a Merck-Hitachi system composed of a quaternary pump Lachrom L-7000, Rheodyne injector with a 20-μl loop, fluorescence detector F-1000 and data acquisition system Varian Star 4.0. Separation was done on a Waters Symmetry C18 column (3.9 × 150 mm, 5 μm), with acetonitrile, methanol and o-phosphoric acid 0.15 M (1:1:1, v/v/v) as mobile phase. The flow rate was 0.8 ml/min; the fluorescence detector was set at 333 nm excitation and 470 nm emission wavelengths (Vega et al. 2009). Confirmation of OTA in samples was done following the method of Zimmerli and Dick (1995) for formation of the methyl ester derivative. The retention times were 3.5 min for OTB, 5.6 min for OTA and 8.5 min for OTA methyl ester using the same chromatographic conditions. Results Growth and morphological observations All strains showed visible growth after 2 days of incubation and the colony diameters expanded progressively during the incubation period (Supplementary Figs. 5–8). P. nordicum BFE487 and P. verrucosum BFE550 showed a similar pattern of growth, characterized by a relatively slow colony expansion on all media (Fig. 1). Only on YES medium was the growth rate slightly higher compared with the other media. In comparison, the Aspergillus colonies grew relatively fast. After growth on YES medium, all three strains covered the dishes completely after 8 days of incubation (Fig. 1). A. ochraceus showed the typical yellow-gold spore pigment on all media (Fig. 2). A. niger BFE632 was the strain with the fastest growth rate in all four media. As expected, this species developed a characteristic black coloration. The Penicillium species developed the distinctive grey/green coloration (Fig. 2). Open image in new window Fig. 1 Colony diameters of the ochratoxigenic species A. ochraceus BFE635, A. niger BFE632, P. nordicum BFE487 and P. verrucosum BFE550. Strains were incubated at 25°C on MEA, YES, and wheat- and coffee–based media; colony diameter (in cm) was measured at days 2, 4 and 8 of incubation Open image in new window Fig. 2 Colony morphology of Aspergillus ochraceus BFE635, Aspergillus niger var. niger BFE632 and Penicillium nordicum BFE487 colonies after 8 days of growth on standard media (MEA and YES) and food-based media (coffee and wheat) at 25°C in darkness Changes in pH values In the pure culture media, the pH values were constant over time (days 2–8) in a range between 6.0 and 6.5. No major variations were observed between most species in the pH of the medium directly surrounding the colonies, with pH values ranging between 5.5 and 6.5. An exception was A. niger, where on day 2 the pH ranged between 3.0 and 3.5 in the middle of the colony and 6.0 in the agar. Over the following days, on YES medium the pH values in the colony declined to 1.0. On coffee- and wheat-based medium, the lowest pH values for A. niger colonies were between 2.0 and 2.5, observed on day 8 of incubation. A. niger is known to be able to produce citric or gluconic acid, which would cause this drop in pH. OTA production by fungal strains cultured on different media OTA and OTB were measured using HPTLC and HPLC with fluorescence detection (Fig. 3, and Supplementary Figs. 9, 10). Both techniques allowed a sensitive detection of the mycotoxins. HPLC with fluorescence detection is most widely used for the analysis of OTA, due its low limit of detection (Scott 2002). On the other hand, HPTLC offers the possibility of a screening for fluorescent or non-fluorescent metabolites in fungal culture extracts (Scott et al. 1970). As expected, all species were able to produce OTA, although the amounts produced in culture varied with strain, time of incubation and medium. In addition to OTA, the presence of other bands/spots was observed on several HPTLC plates (Supplementary Fig. 9). In culture extracts of A. ochraceus BFE635 grown on YES, coffee- and wheat-based medium, OTA was clearly detectable, but non-detectable in MEA cultures. This was confirmed by the shift in fluorescence colour/intensity. In the chromatogram of compounds produced in coffee medium, violet-blue bands, with lower RF values than OTA, were also observed. Moreover, bands with a higher RF value than OTA were present in extracts of this and other fungi when cultured on coffee medium. They were not further identified, but probably are constitutive ingredients of that particular medium (Supplementary Fig. 9). Open image in new window Fig. 3 HPTLC plate of culture extracts from the strain A. ochraceus BFE635 as viewed under UV light at 366 nm. Extracts of cultures after growth for 2, 4 and 8 days (d) on different media (MEA, YES, Coffee, Wheat) were analysed as described in “Materials and methods” In contrast to these results, A. niger BFE632 was able to produce OTA on all media very consistently. Furthermore, yellow and orange-brown fluorescent bands were seen in all media, but were not identified. Like A. niger BFE632, P. nordicum BFE487 showed a consistent production of ochratoxin on all media, albeit at lower quantities. Ochratoxin B (OTB), a metabolite which can co-occur with OTA, was present in P. nordicum extracts from yeast, coffee- and wheat-based medium too. The extracts of P. nordicum showed additional bands with a yellow fluorescence and a slower migration than OTA on YES, coffee and in lower amount on wheat medium. Also, P. verrucosum was able to produce both OTA and OTB, in variable proportions in the different media, whilst no other bands with uncharacteristic fluorescence were observed. Interestingly, on YES and on wheat agar OTA production was highest for this strain. The results are summarized in Fig. 4. As expected, the highest OTA levels were usually measured on day 8 and the lowest on day 2. Production of OTA was relatively high in YES medium for all four fungi studied. On the other hand, MEA medium supported fungal growth (Fig. 1), but not an efficient OTA production. P. nordicum showed a moderate OTA production in all media, whereas P. verrucosum predominantly produced OTA on YES and wheat-based medium. The Aspergillus species showed a high OTA production on YES and wheat based medium too, albeit lower production on coffee medium (Fig. 4). OTA production by A. ochraceus BFE635 reached a level of 83.8 μg/g agar at day 8 of culture on wheat medium, and a level of 9.8 μg/g agar on coffee-based medium (Fig. 4). A. niger BFE632 generated OTA in all media by on day 2 of culture. In YES medium, A. niger reached a peak of OTA production at day 4 (when colony growth reached a plateau, Fig. 1); a slight reduction of OTA levels was observed at day 8 (Fig. 4), indicative of some degradation in the colony. Open image in new window Fig. 4 Ochratoxin A production by A. ochraceus BFE635, A. niger BFE632, P. nordicum BFE487 and P. verrucosum BFE550 on standard media (MEA and YES) and food-based media (coffee and wheat) at days 2, 4 and 8 of culture Discussion The present study analyzed the OTA production of four ochratoxigenic fungi (two Aspergillus and two Penicillium species) in four different media (wheat and coffee as food-based media, MEA and YES as standard media) along with additional parameters, including colony growth and morphology. The OTA production was clearly dependent on the strain and influenced by the time of incubation and composition of the medium. All four fungi produced rather high levels of OTA on YES and wheat-based medium (Fig. 4). YES is generally regarded as a very supportive medium for OTA biosynthesis (Skrinjar and Dimic 1992; Bragulat et al. 2001), but many fungi do not develop morphologically distinct colonies on this medium. In contrast, MEA medium supported fungal growth and the formation of a typical colony morphology rather well (Fig. 1), but, with the exception of Aspergillus niger, OTA production was relatively weak (Fig. 4). Wheat-based medium stimulated OTA production by the Aspergilli in a manner similar to YES medium. Interestingly, although P. verrucosum was not able to produce OTA on MEA and coffee-based medium, this strain was able to produce OTA on YES and wheat-based medium. Wheat is the natural habitat of this species, and the results described here show that apparently the natural environment actively supports OTA biosynthesis by this fungus, comparable with the activation of the biosynthesis by the very supportive YES medium. Aspergilli are not commonly associated with production of important amounts of OTA in wheat at lower temperature (described in Magan and Aldred 2005). However, A. ochraceus BFE635 and A. niger BFE632 produced the highest amounts of OTA (>50 μg OTA/g agar) at 25°C in wheat-based medium. This may indicate that the composition of wheat significantly favours OTA production, even at lower temperatures. This finding is also supported by previous studies done in rice-based medium, where the production of OTA by A. ochraceus was higher than the OTA production by P. verrucosum after 10 days of incubation at 25°C (Saxena et al. 2001). Although A. ochraceus is a well-known OTA producer in coffee beans (Taniwaki et al. 2003), the OTA levels quantified in coffee-based medium were even sixfold lower than in wheat-based medium or YES (Fig. 4). This finding may be attributed to the culture temperature (Pardo et al. 2006; Ramos et al. 1998). For instance, alternating and higher temperatures favour OTA production by A. ochraceus in raw coffee (Palacios-Cabrera et al. 2001; Suárez-Quiroz et al. 2004b). Yet, rather than incubation temperature (25°C in this study), nutritional factors may account for lower OTA formation on coffee than wheat medium. Interestingly, also A. niger BFE632 was capable of producing OTA on coffee-based medium, albeit at much lower levels than on MEA, wheat and YES medium (Fig. 4). In a previous study, A. niger isolates from coffee (RCC4 and RCC20) were unable to produce OTA under similar conditions in this medium (Astoreca et al. 2007), and the authors discuss the possibility that OTA production is inhibited by caffeine or other coffee components. The results described here confirm this view and show that coffee-based medium in fact allows OTA biosynthesis, but at a reduced rate compared with other substrates. Whilst OTA production by A. niger increased over time in three media, we observed a slight decrease in OTA levels in YES medium after 8 days of incubation (Fig. 4). This could be due to reduction/biodegradation of OTA by Aspergillus section Nigri in the culture. Similar results have been also reported by other authors in food-based media after 7 days of incubation (Astoreca et al. 2007; Bejaoui et al. 2006). It has been suggested that the strains remove and assimilate the phenylalanine moiety from the OTA molecule when other nitrogen sources of the culture media become exhausted (Téren et al. 1996). Furthermore, Aspergillus species, including some of the section Nigri, are apparently able to convert OTA into OTα (Varga et al. 2005). Also, for the Penicillia a balance between degradation and production was described (Schmidt-Heydt et al. 2010), indicating that the degradation of OTA over time is a general phenomenon in conjunction with this metabolite. OTA production of Penicillium species (P. nordicum and P. verrucosum) at 25°C was lower than those of the Aspergillus species studied. This is probably related to their slower growth in culture (Fig. 1), since formation of secondary metabolites occurs primarily at high states of differentiation (Calvo et al. 2002). In accordance with previous data (Larsen et al. 2001), we observed that P. nordicum produced OTA and OTB in all media, although OTB production was significantly lower than that of OTA. Also, P. verrucosum was able to produce both OTA and OTB, in variable proportions in the different media. On the other hand, we found no direct evidence in the HPTLC analysis for production of citrinin, another secondary metabolite which can be formed by P. verrucosum along with OTA (as reviewed by Cabañes et al. 2010). Recently, Schmidt-Heydt et al. (2011) showed that P. verrucosum (strain BFE808) produced citrinin as well as OTA on YES and MEA medium upon incubation at 20°C under blue light, whilst incubation in the dark, as in our case, favoured only OTA production. Preliminary evidence exists (data not shown) that the production of ochratoxin and citrinin by P. verrucosum is mutually regulated, meaning that under certain conditions the production of ochratoxin is high, whereas that of citrinin is low and vice versa. This maybe attributed to the fact that both mycotoxins use the same precursor, e.g. acetyl-CoA (Larsen et al. 2001), but other explanations are also possible. The conditions described here apparently support OTA biosynthesis. However, it has also to be kept in mind that not all strains of P. verrucosum are able to produce citrinin. Finally, an interesting morphological observation is the occurrence of exudate/guttation droplets in several, but not with all of our fungal cultures. The Aspergillus strains (A. ochraceus BFE635 and A. niger var. niger BFE632) which produced the highest OTA levels on wheat (Fig. 4) also produced abundant exudation droplets on the surface of their colonies on wheat medium and less on coffee medium (Fig. 3). Although formation of guttation droplets is a common feature on the surface of fungal mycelia, their content has been rarely analyzed for mycotoxins, at least in ochratoxigenic species. Recently, Gareis and Gareis (2007) examined the occurrence of liquid droplets in several Penicillium strains (P. nordicum and P. verrucosum) cultured on Czapek yeast extract agar for 10–14 days at 25°C, and reported that high concentrations of OTA (and OTB) were excreted into the exudates. Similar to the results of Gareis and Gareis (2007) for Penicillium, we measured also much higher OTA levels in guttation droplets of Aspergillus cultures than in mycelium and agar (data not shown). That guttation droplets were not observed in our Penillium cultures can be attributed to the shorter incubation times, 2–8 days rather than 10–14 days, as in the study by Gareis and Gareis (2007). The clear (and in the case of A. niger apparently pigment-free) exudate droplets contain rather pure and highly concentrated OTA, and may thus serve as a good source for this toxin for laboratory purposes. Conclusion The results from this study with four ochratoxigenic strains from the genera Aspergillus and Penicillium proved production of significant amounts of OTA on YES, but also on wheat- and coffee-based medium. Under the conditions of this study, A. niger BFE632 was the only strain which produced OTA on MEA medium despite good colony growth of all fungi studied. OTA production increased over time and was high in fungal cultures which presented exudation droplets on the surface of the colonies. The presence of toxins in guttation droplets should be further studied. » weiterlesen» einklappen

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