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Response to letter to the editor on ‘Fumonisin contamination and fumonisin producing black Aspergilli in dried vine fruits of different origin published in International Journal of Food Microbiology, 143:143–149’
International Journal of Food Microbiology 152 (2012) 46–48
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Letter to the Editor Response to letter to the editor on ‘Fumonisin contamination and fumonisin producing black Aspergilli in dried vine fruits of different origin published in International Journal of Food Microbiology, 143:143–149’
Keywords: Fumonisins Aspergillus Raisin
Since the discovery of a fumonisin gene cluster in the genome of Aspergillus niger (Pel et al., 2007), and proving fumonisin producing abilities of this species (Frisvad et al., 2007), several laboratories have been involved in examining the role of black Aspergilli in potential fumonisin contamination of various food products (Noonim et al., 2009; Mogensen et al., 2010; Logrieco et al., 2009; Knudsen et al., 2011). Among other groups, we clarified that black Aspergilli including A. niger and Aspergillus awamori are responsible for fumonisin contamination of dried vine fruits (Varga et al., 2010). In a recent letter to the editor, Nielssen and Logrieco (in press) criticized several aspects of the work we carried out on identifying black Aspergilli potentially responsible for fumonisin contamination of dried vine fruits. They criticize (i) the amount of fumonisins detected in either the dried vine fruit samples or in the fungal cultures, and (ii) the detection of fumonisins B1 (FB1) and B3 (FB3) in some of the samples. We would like to address these critics in this response. Regarding the criticism about fumonisin content of raisin samples, we would like to point out that our aim was to clarify if black Aspergilli could be responsible for fumonisin contamination of raisins (if there is any contamination). Indeed, we did find high amounts of fumonisins in some of the samples. However, the range cited by Nielssen and Logrieco (in press) is false; it was between 0.4 and 35 mg/kg (omitting the sample showing the highest contamination, the range was 0.4–4.7 mg/kg). Besides, we examined only 7 of the 13 raisin samples because potential fumonisin producing black Aspergilli (A. niger and/or A. awamori) could be isolated only from these ones. As stated in our paper (Varga et al., 2010), we examined the fumonisin content of 1 g of each sample (about 3–5 raisins), since our aim was to clarify if they are contaminated by the same range of isomers produced by the A. niger/A. awamori strains isolated from the samples. We did not intend to do a comprehensive analysis of fumonisin content of the raisin samples examined, so we did not examine larger amounts (200 g) as done by Mogensen et al. (2010). Actually, we think that the average fumonisin content of – say – 200 g of raisins does not say much to the costumer (when I prepare muesli for breakfast for my kids, I use only 10–15 raisins, not 200 g of it). Besides, Knudsen et al. (2011) claim that “analysis of multiple packages from one manufacturer showed a 3-fold package-topackage variation, suggesting that a few raisins per package are contaminated.” Although we did not select for moldy raisins during our work, 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.09.020
there is a chance that we accidentally picked some of these highly contaminated raisins for analysis. Besides, we only examined those samples which were proved to be contaminated by fumonisin producing black Aspergilli. As mentioned above, we did not aim to thoroughly examine the fumonisin content of the raisin samples, but to clarify if black Aspergilli could be responsible for fumonisin contamination. Nielssen and Logrieco (in press) claim that “the levels of fumonisins detected in a recently published surveys (Mogensen et al., 2010) are in the low μg/kg (up to 14 μg/kg) which is more than 1000-fold lower than the levels claimed by Varga et al.” We are aware of the cited paper, but could not find any such data in it. However, we did find similar values in another paper (Knudsen et al., 2011). Similarly to our data, they observed a higher contamination rate in Californian raisins. However, regarding the amounts of fumonisins detected by Knudsen et al. (2011), we are a bit puzzled by the small amounts detected. Apart from raisins, we examined several other commodities, and found much higher contamination levels than that found by Knudsen et al. (2011), for example, ca. 0.3 mg/kg in onion samples (Varga et al., in press), ca. 0.25 mg/kg in a fig sample from Iran, and between 0.5 and 5 mg/kg in date samples (in preparation). Although fumonisins were not detected in onions or dates previously, so we cannot compare our data with those of other laboratories, the fumonisin content detected in figs was in agreement with the average fumonisin contamination found in figs previously (Karbancioglu-Güler and Heperkan, 2009). In view of these levels, we believe that either the extraction procedure used by Knudsen et al. (2011) was not appropriate, or they were “lucky enough” to select less contaminated packs (or we accidentally examined some more contaminated raisin samples). Regarding the extraction procedure, ultrasonication was found to be a useful tool for getting higher recoveries for fumonisin isomers (Varga et al., submitted). Another possible reason for the relatively high fumonisin contents observed in some of our samples could be fumonisin accumulation during storage. We collected the raisin samples examined in our survey for nearly 2 years (from 2008 till the end of 2009), and – although we kept them at 4 °C until examination – there is a good chance that fumonisin content of the samples increased during storage. Besides, we do not know how long were they kept on the shelves in the stores. Nielssen and Logrieco (in press) claim that “when we artificially infected raisins and allowed the fungus overgrown them to an extreme degree, the total maximum amount of fumonisins B2 and B4 was still only 7 mg/kg” (Mogensen et al., 2010). We should mention that these levels were reached after 7 days of incubation according to the authors. Why is it so surprising to get higher levels after several months of storage? Knudsen et al. (2011) also claim that “fumonisins are produced mainly during the drying process concomitant with the decreasing water activity.” Longer storage could lead to the accumulation of fumonisins as observed in our case too, as mentioned in Varga et al. (2010) as well (“…the drying process could have led to the accumulation of these toxins in dried vine fruits.”). Nielssen and Logrieco (in press) also state that “we have worked with hundreds of cultures of A. niger and related species we have
Letter to the Editor
only observed fumonisin levels up to 7–26 mg/kg (……) in pure cultures on the best possible media.” We observed a similar range (see Table 3 of Varga et al., 2010), in line with the results of Palumbo et al. (2011), so it is unclear that what the problem with these values was. Regarding the observation of FB1 and FB3 in some of the fungal cultures, we would like to stress that apart from a single isolate (6.7, Table 3, Varga et al., 2010), we observed only minute amounts of FB1 in our cultures, and FB3 was observed only in isolate 6.7. Actually, the amount of FB1 observed was close to the limit of detection (signal to noise ratio 3:1). Here we would like to mention that recently Mansson et al. (2010) described a “new” type of fumonisins, fumonisin B6 from an A. niger culture, which turned out to be the same as an FB1 isomer (No. 28) described previously by Bartók et al. (2010a) from Fusarium verticillioides. As far as we know, it is presently unclear how this FB1 isomer is synthesized in Aspergilli (or in Fusaria). F. verticillioides is able to produce up to 28 different isomers of FB1, and some (6) very new isomers which carry 3 acyl groups instead of the usual 2 (Bartók et al., 2006, 2010a, 2010b). Although it is unclear how these isomers are produced, it can be speculated that either mutations or transcriptional or translational errors could lead to the production of small amounts of these metabolites. Taken F. verticillioides as an example, it is unclear why it is so surprising that using a technique which has an excellent sensitivity, some FB1 isomers (including FB6 and FB1 itself) are also produced by black Aspergilli, although in trace amounts. They could be either precursors of FB6, or synthesized from it by a simple modification step. Regarding the isolate which produced high amounts of both FB1 and FB3 (6.7 in Varga et al., 2010), further studies are in progress, including analytical work and molecular analysis of the fumonisin gene cluster. Some preliminary results of this molecular work are under publication (Varga et al., 2011). Fumonisins B1 and B3 have also been detected in the raisin samples. Besides, some other isomers not produced by black Aspergilli (FB5, isoFB5) were also present (Varga et al., 2010). As mentioned in the paper, “either we did not isolate the A. niger isolate producing it, the A. niger strain did not produce it on CYA20S medium, or this sample was contaminated by a Fusarium species.” Indeed, we believe that apart from black Aspergilli, other (presumably Fusarium) species could have also contributed to fumonisin contamination of the samples, although we could not detect Fusaria in any of the samples (Varga et al., 2010). We would also like to emphasize the need of clarification of the nomenclature of fumonisins. The identification of FB6 as a structural isomer of FB1 (Bartók et al., 2010b; Mansson et al., 2010), and the fact that FB3 is actually a structural isomer of FB2 emphasize the importance of clarification of the naming to avoid unnecessary introduction of new names, consequently to avoid further confusion. We are aware of the fact that “none of the DNA sequences from the three completely sequenced A. niger strains contains a homolog of the FUM2 gene” (Nielssen and Logrieco, in press), which is thought to be required for the production of fumonisins B1 and B3. However, this is a cytochrome P450 monooxgenase enzyme (3 such genes can be found in the fumonisin gene cluster itself), which group of enzymes is taking part in various processes in fungal cells, including the biosynthesis of various mycotoxins and phytotoxins. Additionally, Proctor et al. (2006) proved that “altered fumonisin production phenotypes of naturally occurring F. verticillioides variants can result from single point mutations in the FUM cluster.” Such events could have happened in A. niger/A. awamori too. In connection with their criticisms, Nielssen and Logrieco (in press) claim that some error must have happened during analysis. One of their claims is that perhaps the samples were misidentified. Regarding the purity of the fungal cultures, all isolates were subjected to calmodulin sequence-based identification before fumonisin analysis as mentioned in the paper (Varga et al., 2010). All of them could unambiguously be assigned to A. niger or A. awamori based on the
47
sequence data, and all cultures were pure. Of course there is always the possibility that cultures or extracts are mislabeled or mixed up in the laboratory. However, at the time when these samples were analyzed (end of 2009) we were not involved in any work related to fumonisin producing Fusarium species. Another possibility is carry-over in the HPLC system. Of course every injector has some kind of a “memory effect,” and it is higher in the case of the injector of HP 1090 than in the case of the injectors of e.g. Agilent 1100 or 1200. However, we checked the order of the samples as they were applied to the apparatus, and it clearly showed that memory effect cannot be responsible for carry-over. More exactly, several samples in which we could not detect fumonisins were analyzed before or after samples in which fumonisins were present (e.g. fumonisins were detected in fungal samples 1–3, not in samples 4–6 and 8, but they were present in samples 7 and 9). Besides, before applying the samples, a blank gradient run and a blank injection including only the solvent have also been applied. The base line was clear in both cases, indicating that the HPLC apparatus including the injector, the column, the capillary system, the ESI ion source and parts of the vacuum area were free from fumonisins. Furthermore, during analysis of the samples, an injector program was applied to wash the outer surface of the injector needle in a washing vial containing a mixture of acetonitrile/ water 1/1 (v/v). We developed a technique for the detection of fumonisin isomers using a special HPLC column developed for the identification of structural isomers, a technique which has a very good resolution, but at the same time, it has a relatively long analysis time (120 min/sample, Bartók et al., 2010a, b; Varga et al., 2010). The main drawback of the method is naturally the long analysis time, at the same time we could separate such compounds (e.g. 3-epi-FB3, FB3; 3-epi-FB4, FB4) which could not be separated from each other in former works. Besides high resolution, there is a further advantage of the long analysis time: after injection, the inner surface of the capillary system is continuously rinsed for 120 min, giving less chance for carry-over of fumonisins. Yet another possibility is carry-over in glassware, which is a possible source of error in analytical laboratories. The primary amino group of fumonisins (where it is present) can easily adsorb to the silanol groups of glassware if they were not deactivated e.g. by silylation. That is the reason why extraction, centrifugation and analysis steps were performed in low cost, disposable polypropylene vessels including centrifuge tubes and HPLC microvials; consequently vessels applied could not cause cross-contamination between samples. In conclusion, we hope that we addressed the criticisms posted by Nielssen and Logrieco (in press) appropriately, regarding both the relatively high amounts of fumonisins observed in some of the raisin samples, and the detection of FB1 and FB3 in raisins and some of the fungal cultures. Further work is in progress in our lab to clarify the genetic background of fumonisin production in black Aspergilli, and examination of other agricultural products is also in progress. Results of these analyses have already been submitted or will be published soon in peerreviewed international journals. As we believe that the data published in Varga et al. (2010) are to our best knowledge correct, and the main conclusions – ie. that (i) A. niger and A. awamori are mainly responsible for fumonisin contamination of dried vine fruits worldwide, (ii) fumonisins do occur in dried vine fruits, and (iii) black Aspergilli produce a range of fumonisin isomers not detected before – are justified, we do not intend to retract or modify our paper. Of course, everybody has got the right to be wrong, so as soon as it will turn out that we made a mistake somewhere during our analyses, we will either publish it as an erratum in IJFM, or as a separate paper in appropriate media. References Bartók, T., Szécsi, Á., Szekeres, A., Mesterházy, Á., Bartók, M., 2006. Detection of new fumonisin mycotoxins and fumonisin-like compounds by reversed-phase high performance liquid chromatography/electrospray ionization ion trap mass spectrometry. Rapid Communications in Mass Spectrometry 20, 2447–2462.
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Letter to the Editor
Bartók, T., Tölgyesi, L., Szekeres, A., Varga, M., Bartha, R., Szécsi, Á., Bartók, M., Mesterházy, Á., 2010a. Detection and characterization of twenty-eight isomers of fumonisin B1 (FB1) mycotoxin in a solid rice culture infected with Fusarium verticillioides by reversedphase high-performance liquid chromatography/electrospray ionization time-offlight and ion trap mass spectrometry. Rapid Communications in Mass Spectrometry 24, 35–42. Bartók, T., Tölgyesi, L., Mesterházy, Á., Bartók, M., Szécsi, Á., 2010b. Identification of the first fumonisin mycotoxins with three acyl groups by ESI-ITMS and ESI-TOFMS following RP-HPLC separation: palmitoyl, linoleoyl and oleoyl EFB1 fumonisin isomers from a solid culture of Fusarium verticillioides. Food Additives and Contaminants 27, 1714–1723. Frisvad, J.C., Smedsgaard, J., Samson, R.A., Larsen, T.O., Thrane, U., 2007. Fumonisin B2 production by Aspergillus niger. Journal of Agricultural and Food Chemistry 55, 9727–9732. Karbancioglu-Güler, F., Heperkan, D., 2009. Natural occurrence of fumonisin B1 in dried figs as an unexpected hazard. Food Chemistry and Toxicology 47, 289–292. Knudsen, P.B., Mogensen, J.M., Larsen, T.O., Nielsen, K.F., 2011. Occurrence of fumonisins B2 and B4 in retail raisins. Journal of Agricultural and Food Chemistry 59, 772–776. Logrieco, A., Ferracane, R., Haidukowski, M., Cozzi, G., Visconti, A., Ritieni, A., 2009. Fumonisin B2 production by Aspergillus niger from grapes and natural occurrence in must. Food Additives & Contaminants: Part A 26, 1495–1500. Mansson, M., Klejnstrup, M.L., Phipps, R.K., Nielsen, K.F., Frisvad, J.C., Godtfredsen, C.H., Larsen, T.O., 2010. Isolation and characterization of fumonisin B6, a new fumonisin from Aspergillus niger. Journal of Agricultural and Food Chemistry 58, 949–953. Mogensen, J.M., Frisvad, J.C., Thrane, U., Nielsen, K.F., 2010. Production of fumonisin B2 and B4 by Aspergillus niger in raisins and grapes. Journal of Agricultural and Food Chemistry 58, 954–958. Nielssen, K.F., Logrieco, A.F., 2011. Letter to the editor on ‘Fumonisin contamination and fumonisin producing black Aspergilli in dried vine fruits of different origin. IJFM. 143:143–149’. International Journal of Food Microbiology (in press). Noonim, P., Mahakarnchanakul, W., Nielsen, K.F., Frisvad, J.C., Samson, R.A., 2009. Fumonisin B2 production by Aspergillus niger from Thai coffee beans. Food Additives and Contaminants 26, 94–100. Palumbo, J.D., O'Keeffe, T.L., McGarvey, J.A., 2011. Incidence of fumonisin B2 production within Aspergillus section Nigri populations isolated from California raisins. Journal of Food Protection 74, 672–675. Pel, H.J., deWinde, J.H., Archer, D.B., Dyer, P.S., Hofmann, G., Schaap, P.J., Turner, G., deVries, R.P., Albang, R., Albermann, K., Andersen, M.R., Bendtsen, J.D., Benen, J.A.E., van den Berg, M., Breestraat, S., Caddick, M.X., Contreras, R., Cornell, M., Coutinho, P.M., Danchin, E.G.J., Debets, A.J.M., Dekker, P., van Dijck, P.W.M., van Dijk, A., Dijkhuizen, L., Driessen, A.J.M., d'Enfert, S., Geysens, C., Goosen, C., Groot, G.S.P., deGroot, P.W.J., Guillemette, T., Henrissat, B., Herweijer, M., van den Hombergh, J.P.T.W., van den Hondel, C.A.M.J., van der Heijden, R.T.J.M., van der Kaaij, R.M., Klis, F.M., Kools, H.J., Kubicek, C.P., van Kuyk, P.A, Lauber, J., Lu, X., van derMaarel, M.J.E.C., Meulenberg, R., Menke, H., Mortimer, M.A., Nielsen, J., Oliver, S.G., Olsthoorn, M., Pal, K., van Peij, N.N.M.E., Ram, A.F.J., Rinas, U., Roubos, J.A., Sagt, C.M.J., Schmoll, M., Sun, J.B., Ussery, D., Varga, J., Vervecken, W., de Vondervoort, P.J.J.V., Wedler, H., Wosten, H.A.B., Zeng, A.P., van Ooyen, A.J.J., Visser, J., Stam, H., 2007. Genome
sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nature Biotechnology 25, 221–231. Proctor, R.H., Plattner, R.D., Desjardins, A.E., Busman, M., Butcko, A.E., 2006. Fumonisin production in the maize pathogen Fusarium verticillioides: genetic basis of naturally occurring chemical variation. Journal of Agricultural and Food Chemistry 54, 2424–2430. Varga, J., Kocsubé, S., Suri, K., Szigeti, G., Szekeres, A., Varga, M., Tóth, B., Bartók, T., 2010. Fumonisin contamination and fumonisin producing black Aspergilli in dried vine fruits of different origin. International Journal of Food Microbiology 143, 143–149. Varga, J., Frisvad, J.C., Kocsubé, S., Brankovics, B., Tóth, B., Szigeti, G., Samson, R.A., 2011. New and revisited species in Aspergillus section Nigri. Studies in Mycology 69, 1–17. Varga, J., Kocsubé, S., Szigeti, G., Man, V., Tóth, B., Vágvölgyi, C., Bartók, T., in press. Black Aspergilli and fumonisin contamination in onions purchased in Hungary. Acta Alimentaria.
János Varga⁎ Sándor Kocsubé Department of Microbiology, Faculty of Science and Informatics, University of Szeged, H-6726 Szeged, Közép fasor 52, Hungary ⁎Corresponding author. Tel.: + 36 62544515. E-mail addresses: [email protected] (J. Varga), [email protected] (S. Kocsubé).
Beáta Tóth Cereal Research Non-Profit Ltd., H-6726 Szeged, Alsókikötő sor 9, Hungary E-mail address: [email protected].
Tibor Bartók Department of Food Engineering, Faculty of Engineering, University of Szeged, Moszkvai krt. 5/7, H-6724 Szeged, Hungary E-mail address: [email protected]. 29 July 2011
Contents lists available at SciVerse ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Letter to the Editor Response to letter to the editor on ‘Fumonisin contamination and fumonisin producing black Aspergilli in dried vine fruits of different origin published in International Journal of Food Microbiology, 143:143–149’
Keywords: Fumonisins Aspergillus Raisin
Since the discovery of a fumonisin gene cluster in the genome of Aspergillus niger (Pel et al., 2007), and proving fumonisin producing abilities of this species (Frisvad et al., 2007), several laboratories have been involved in examining the role of black Aspergilli in potential fumonisin contamination of various food products (Noonim et al., 2009; Mogensen et al., 2010; Logrieco et al., 2009; Knudsen et al., 2011). Among other groups, we clarified that black Aspergilli including A. niger and Aspergillus awamori are responsible for fumonisin contamination of dried vine fruits (Varga et al., 2010). In a recent letter to the editor, Nielssen and Logrieco (in press) criticized several aspects of the work we carried out on identifying black Aspergilli potentially responsible for fumonisin contamination of dried vine fruits. They criticize (i) the amount of fumonisins detected in either the dried vine fruit samples or in the fungal cultures, and (ii) the detection of fumonisins B1 (FB1) and B3 (FB3) in some of the samples. We would like to address these critics in this response. Regarding the criticism about fumonisin content of raisin samples, we would like to point out that our aim was to clarify if black Aspergilli could be responsible for fumonisin contamination of raisins (if there is any contamination). Indeed, we did find high amounts of fumonisins in some of the samples. However, the range cited by Nielssen and Logrieco (in press) is false; it was between 0.4 and 35 mg/kg (omitting the sample showing the highest contamination, the range was 0.4–4.7 mg/kg). Besides, we examined only 7 of the 13 raisin samples because potential fumonisin producing black Aspergilli (A. niger and/or A. awamori) could be isolated only from these ones. As stated in our paper (Varga et al., 2010), we examined the fumonisin content of 1 g of each sample (about 3–5 raisins), since our aim was to clarify if they are contaminated by the same range of isomers produced by the A. niger/A. awamori strains isolated from the samples. We did not intend to do a comprehensive analysis of fumonisin content of the raisin samples examined, so we did not examine larger amounts (200 g) as done by Mogensen et al. (2010). Actually, we think that the average fumonisin content of – say – 200 g of raisins does not say much to the costumer (when I prepare muesli for breakfast for my kids, I use only 10–15 raisins, not 200 g of it). Besides, Knudsen et al. (2011) claim that “analysis of multiple packages from one manufacturer showed a 3-fold package-topackage variation, suggesting that a few raisins per package are contaminated.” Although we did not select for moldy raisins during our work, 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.09.020
there is a chance that we accidentally picked some of these highly contaminated raisins for analysis. Besides, we only examined those samples which were proved to be contaminated by fumonisin producing black Aspergilli. As mentioned above, we did not aim to thoroughly examine the fumonisin content of the raisin samples, but to clarify if black Aspergilli could be responsible for fumonisin contamination. Nielssen and Logrieco (in press) claim that “the levels of fumonisins detected in a recently published surveys (Mogensen et al., 2010) are in the low μg/kg (up to 14 μg/kg) which is more than 1000-fold lower than the levels claimed by Varga et al.” We are aware of the cited paper, but could not find any such data in it. However, we did find similar values in another paper (Knudsen et al., 2011). Similarly to our data, they observed a higher contamination rate in Californian raisins. However, regarding the amounts of fumonisins detected by Knudsen et al. (2011), we are a bit puzzled by the small amounts detected. Apart from raisins, we examined several other commodities, and found much higher contamination levels than that found by Knudsen et al. (2011), for example, ca. 0.3 mg/kg in onion samples (Varga et al., in press), ca. 0.25 mg/kg in a fig sample from Iran, and between 0.5 and 5 mg/kg in date samples (in preparation). Although fumonisins were not detected in onions or dates previously, so we cannot compare our data with those of other laboratories, the fumonisin content detected in figs was in agreement with the average fumonisin contamination found in figs previously (Karbancioglu-Güler and Heperkan, 2009). In view of these levels, we believe that either the extraction procedure used by Knudsen et al. (2011) was not appropriate, or they were “lucky enough” to select less contaminated packs (or we accidentally examined some more contaminated raisin samples). Regarding the extraction procedure, ultrasonication was found to be a useful tool for getting higher recoveries for fumonisin isomers (Varga et al., submitted). Another possible reason for the relatively high fumonisin contents observed in some of our samples could be fumonisin accumulation during storage. We collected the raisin samples examined in our survey for nearly 2 years (from 2008 till the end of 2009), and – although we kept them at 4 °C until examination – there is a good chance that fumonisin content of the samples increased during storage. Besides, we do not know how long were they kept on the shelves in the stores. Nielssen and Logrieco (in press) claim that “when we artificially infected raisins and allowed the fungus overgrown them to an extreme degree, the total maximum amount of fumonisins B2 and B4 was still only 7 mg/kg” (Mogensen et al., 2010). We should mention that these levels were reached after 7 days of incubation according to the authors. Why is it so surprising to get higher levels after several months of storage? Knudsen et al. (2011) also claim that “fumonisins are produced mainly during the drying process concomitant with the decreasing water activity.” Longer storage could lead to the accumulation of fumonisins as observed in our case too, as mentioned in Varga et al. (2010) as well (“…the drying process could have led to the accumulation of these toxins in dried vine fruits.”). Nielssen and Logrieco (in press) also state that “we have worked with hundreds of cultures of A. niger and related species we have
Letter to the Editor
only observed fumonisin levels up to 7–26 mg/kg (……) in pure cultures on the best possible media.” We observed a similar range (see Table 3 of Varga et al., 2010), in line with the results of Palumbo et al. (2011), so it is unclear that what the problem with these values was. Regarding the observation of FB1 and FB3 in some of the fungal cultures, we would like to stress that apart from a single isolate (6.7, Table 3, Varga et al., 2010), we observed only minute amounts of FB1 in our cultures, and FB3 was observed only in isolate 6.7. Actually, the amount of FB1 observed was close to the limit of detection (signal to noise ratio 3:1). Here we would like to mention that recently Mansson et al. (2010) described a “new” type of fumonisins, fumonisin B6 from an A. niger culture, which turned out to be the same as an FB1 isomer (No. 28) described previously by Bartók et al. (2010a) from Fusarium verticillioides. As far as we know, it is presently unclear how this FB1 isomer is synthesized in Aspergilli (or in Fusaria). F. verticillioides is able to produce up to 28 different isomers of FB1, and some (6) very new isomers which carry 3 acyl groups instead of the usual 2 (Bartók et al., 2006, 2010a, 2010b). Although it is unclear how these isomers are produced, it can be speculated that either mutations or transcriptional or translational errors could lead to the production of small amounts of these metabolites. Taken F. verticillioides as an example, it is unclear why it is so surprising that using a technique which has an excellent sensitivity, some FB1 isomers (including FB6 and FB1 itself) are also produced by black Aspergilli, although in trace amounts. They could be either precursors of FB6, or synthesized from it by a simple modification step. Regarding the isolate which produced high amounts of both FB1 and FB3 (6.7 in Varga et al., 2010), further studies are in progress, including analytical work and molecular analysis of the fumonisin gene cluster. Some preliminary results of this molecular work are under publication (Varga et al., 2011). Fumonisins B1 and B3 have also been detected in the raisin samples. Besides, some other isomers not produced by black Aspergilli (FB5, isoFB5) were also present (Varga et al., 2010). As mentioned in the paper, “either we did not isolate the A. niger isolate producing it, the A. niger strain did not produce it on CYA20S medium, or this sample was contaminated by a Fusarium species.” Indeed, we believe that apart from black Aspergilli, other (presumably Fusarium) species could have also contributed to fumonisin contamination of the samples, although we could not detect Fusaria in any of the samples (Varga et al., 2010). We would also like to emphasize the need of clarification of the nomenclature of fumonisins. The identification of FB6 as a structural isomer of FB1 (Bartók et al., 2010b; Mansson et al., 2010), and the fact that FB3 is actually a structural isomer of FB2 emphasize the importance of clarification of the naming to avoid unnecessary introduction of new names, consequently to avoid further confusion. We are aware of the fact that “none of the DNA sequences from the three completely sequenced A. niger strains contains a homolog of the FUM2 gene” (Nielssen and Logrieco, in press), which is thought to be required for the production of fumonisins B1 and B3. However, this is a cytochrome P450 monooxgenase enzyme (3 such genes can be found in the fumonisin gene cluster itself), which group of enzymes is taking part in various processes in fungal cells, including the biosynthesis of various mycotoxins and phytotoxins. Additionally, Proctor et al. (2006) proved that “altered fumonisin production phenotypes of naturally occurring F. verticillioides variants can result from single point mutations in the FUM cluster.” Such events could have happened in A. niger/A. awamori too. In connection with their criticisms, Nielssen and Logrieco (in press) claim that some error must have happened during analysis. One of their claims is that perhaps the samples were misidentified. Regarding the purity of the fungal cultures, all isolates were subjected to calmodulin sequence-based identification before fumonisin analysis as mentioned in the paper (Varga et al., 2010). All of them could unambiguously be assigned to A. niger or A. awamori based on the
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sequence data, and all cultures were pure. Of course there is always the possibility that cultures or extracts are mislabeled or mixed up in the laboratory. However, at the time when these samples were analyzed (end of 2009) we were not involved in any work related to fumonisin producing Fusarium species. Another possibility is carry-over in the HPLC system. Of course every injector has some kind of a “memory effect,” and it is higher in the case of the injector of HP 1090 than in the case of the injectors of e.g. Agilent 1100 or 1200. However, we checked the order of the samples as they were applied to the apparatus, and it clearly showed that memory effect cannot be responsible for carry-over. More exactly, several samples in which we could not detect fumonisins were analyzed before or after samples in which fumonisins were present (e.g. fumonisins were detected in fungal samples 1–3, not in samples 4–6 and 8, but they were present in samples 7 and 9). Besides, before applying the samples, a blank gradient run and a blank injection including only the solvent have also been applied. The base line was clear in both cases, indicating that the HPLC apparatus including the injector, the column, the capillary system, the ESI ion source and parts of the vacuum area were free from fumonisins. Furthermore, during analysis of the samples, an injector program was applied to wash the outer surface of the injector needle in a washing vial containing a mixture of acetonitrile/ water 1/1 (v/v). We developed a technique for the detection of fumonisin isomers using a special HPLC column developed for the identification of structural isomers, a technique which has a very good resolution, but at the same time, it has a relatively long analysis time (120 min/sample, Bartók et al., 2010a, b; Varga et al., 2010). The main drawback of the method is naturally the long analysis time, at the same time we could separate such compounds (e.g. 3-epi-FB3, FB3; 3-epi-FB4, FB4) which could not be separated from each other in former works. Besides high resolution, there is a further advantage of the long analysis time: after injection, the inner surface of the capillary system is continuously rinsed for 120 min, giving less chance for carry-over of fumonisins. Yet another possibility is carry-over in glassware, which is a possible source of error in analytical laboratories. The primary amino group of fumonisins (where it is present) can easily adsorb to the silanol groups of glassware if they were not deactivated e.g. by silylation. That is the reason why extraction, centrifugation and analysis steps were performed in low cost, disposable polypropylene vessels including centrifuge tubes and HPLC microvials; consequently vessels applied could not cause cross-contamination between samples. In conclusion, we hope that we addressed the criticisms posted by Nielssen and Logrieco (in press) appropriately, regarding both the relatively high amounts of fumonisins observed in some of the raisin samples, and the detection of FB1 and FB3 in raisins and some of the fungal cultures. Further work is in progress in our lab to clarify the genetic background of fumonisin production in black Aspergilli, and examination of other agricultural products is also in progress. Results of these analyses have already been submitted or will be published soon in peerreviewed international journals. As we believe that the data published in Varga et al. (2010) are to our best knowledge correct, and the main conclusions – ie. that (i) A. niger and A. awamori are mainly responsible for fumonisin contamination of dried vine fruits worldwide, (ii) fumonisins do occur in dried vine fruits, and (iii) black Aspergilli produce a range of fumonisin isomers not detected before – are justified, we do not intend to retract or modify our paper. Of course, everybody has got the right to be wrong, so as soon as it will turn out that we made a mistake somewhere during our analyses, we will either publish it as an erratum in IJFM, or as a separate paper in appropriate media. References Bartók, T., Szécsi, Á., Szekeres, A., Mesterházy, Á., Bartók, M., 2006. Detection of new fumonisin mycotoxins and fumonisin-like compounds by reversed-phase high performance liquid chromatography/electrospray ionization ion trap mass spectrometry. Rapid Communications in Mass Spectrometry 20, 2447–2462.
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Letter to the Editor
Bartók, T., Tölgyesi, L., Szekeres, A., Varga, M., Bartha, R., Szécsi, Á., Bartók, M., Mesterházy, Á., 2010a. Detection and characterization of twenty-eight isomers of fumonisin B1 (FB1) mycotoxin in a solid rice culture infected with Fusarium verticillioides by reversedphase high-performance liquid chromatography/electrospray ionization time-offlight and ion trap mass spectrometry. Rapid Communications in Mass Spectrometry 24, 35–42. Bartók, T., Tölgyesi, L., Mesterházy, Á., Bartók, M., Szécsi, Á., 2010b. Identification of the first fumonisin mycotoxins with three acyl groups by ESI-ITMS and ESI-TOFMS following RP-HPLC separation: palmitoyl, linoleoyl and oleoyl EFB1 fumonisin isomers from a solid culture of Fusarium verticillioides. Food Additives and Contaminants 27, 1714–1723. Frisvad, J.C., Smedsgaard, J., Samson, R.A., Larsen, T.O., Thrane, U., 2007. Fumonisin B2 production by Aspergillus niger. Journal of Agricultural and Food Chemistry 55, 9727–9732. Karbancioglu-Güler, F., Heperkan, D., 2009. Natural occurrence of fumonisin B1 in dried figs as an unexpected hazard. Food Chemistry and Toxicology 47, 289–292. Knudsen, P.B., Mogensen, J.M., Larsen, T.O., Nielsen, K.F., 2011. Occurrence of fumonisins B2 and B4 in retail raisins. Journal of Agricultural and Food Chemistry 59, 772–776. Logrieco, A., Ferracane, R., Haidukowski, M., Cozzi, G., Visconti, A., Ritieni, A., 2009. Fumonisin B2 production by Aspergillus niger from grapes and natural occurrence in must. Food Additives & Contaminants: Part A 26, 1495–1500. Mansson, M., Klejnstrup, M.L., Phipps, R.K., Nielsen, K.F., Frisvad, J.C., Godtfredsen, C.H., Larsen, T.O., 2010. Isolation and characterization of fumonisin B6, a new fumonisin from Aspergillus niger. Journal of Agricultural and Food Chemistry 58, 949–953. Mogensen, J.M., Frisvad, J.C., Thrane, U., Nielsen, K.F., 2010. Production of fumonisin B2 and B4 by Aspergillus niger in raisins and grapes. Journal of Agricultural and Food Chemistry 58, 954–958. Nielssen, K.F., Logrieco, A.F., 2011. Letter to the editor on ‘Fumonisin contamination and fumonisin producing black Aspergilli in dried vine fruits of different origin. IJFM. 143:143–149’. International Journal of Food Microbiology (in press). Noonim, P., Mahakarnchanakul, W., Nielsen, K.F., Frisvad, J.C., Samson, R.A., 2009. Fumonisin B2 production by Aspergillus niger from Thai coffee beans. Food Additives and Contaminants 26, 94–100. Palumbo, J.D., O'Keeffe, T.L., McGarvey, J.A., 2011. Incidence of fumonisin B2 production within Aspergillus section Nigri populations isolated from California raisins. Journal of Food Protection 74, 672–675. Pel, H.J., deWinde, J.H., Archer, D.B., Dyer, P.S., Hofmann, G., Schaap, P.J., Turner, G., deVries, R.P., Albang, R., Albermann, K., Andersen, M.R., Bendtsen, J.D., Benen, J.A.E., van den Berg, M., Breestraat, S., Caddick, M.X., Contreras, R., Cornell, M., Coutinho, P.M., Danchin, E.G.J., Debets, A.J.M., Dekker, P., van Dijck, P.W.M., van Dijk, A., Dijkhuizen, L., Driessen, A.J.M., d'Enfert, S., Geysens, C., Goosen, C., Groot, G.S.P., deGroot, P.W.J., Guillemette, T., Henrissat, B., Herweijer, M., van den Hombergh, J.P.T.W., van den Hondel, C.A.M.J., van der Heijden, R.T.J.M., van der Kaaij, R.M., Klis, F.M., Kools, H.J., Kubicek, C.P., van Kuyk, P.A, Lauber, J., Lu, X., van derMaarel, M.J.E.C., Meulenberg, R., Menke, H., Mortimer, M.A., Nielsen, J., Oliver, S.G., Olsthoorn, M., Pal, K., van Peij, N.N.M.E., Ram, A.F.J., Rinas, U., Roubos, J.A., Sagt, C.M.J., Schmoll, M., Sun, J.B., Ussery, D., Varga, J., Vervecken, W., de Vondervoort, P.J.J.V., Wedler, H., Wosten, H.A.B., Zeng, A.P., van Ooyen, A.J.J., Visser, J., Stam, H., 2007. Genome
sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nature Biotechnology 25, 221–231. Proctor, R.H., Plattner, R.D., Desjardins, A.E., Busman, M., Butcko, A.E., 2006. Fumonisin production in the maize pathogen Fusarium verticillioides: genetic basis of naturally occurring chemical variation. Journal of Agricultural and Food Chemistry 54, 2424–2430. Varga, J., Kocsubé, S., Suri, K., Szigeti, G., Szekeres, A., Varga, M., Tóth, B., Bartók, T., 2010. Fumonisin contamination and fumonisin producing black Aspergilli in dried vine fruits of different origin. International Journal of Food Microbiology 143, 143–149. Varga, J., Frisvad, J.C., Kocsubé, S., Brankovics, B., Tóth, B., Szigeti, G., Samson, R.A., 2011. New and revisited species in Aspergillus section Nigri. Studies in Mycology 69, 1–17. Varga, J., Kocsubé, S., Szigeti, G., Man, V., Tóth, B., Vágvölgyi, C., Bartók, T., in press. Black Aspergilli and fumonisin contamination in onions purchased in Hungary. Acta Alimentaria.
János Varga⁎ Sándor Kocsubé Department of Microbiology, Faculty of Science and Informatics, University of Szeged, H-6726 Szeged, Közép fasor 52, Hungary ⁎Corresponding author. Tel.: + 36 62544515. E-mail addresses: [email protected] (J. Varga), [email protected] (S. Kocsubé).
Beáta Tóth Cereal Research Non-Profit Ltd., H-6726 Szeged, Alsókikötő sor 9, Hungary E-mail address: [email protected].
Tibor Bartók Department of Food Engineering, Faculty of Engineering, University of Szeged, Moszkvai krt. 5/7, H-6724 Szeged, Hungary E-mail address: [email protected]. 29 July 2011