Mycotoxin Research. Vol. 12 (1996)

Cyclopiazonic acid and aflatoxins production by Aspergillus (favus isolated from Argentinian corn S.L. Resnik(1,2)", H.H.L. Gonzalez(3,4), A.M. Pacin(2,5), M. Viora(6), G.M. Caballero(1) and E.G. Gros(1) 1 Facultad de Ciencias Exactas y Naturales, UBA 2 Comisi6n de Investigaciones Cientfficas de la Provincia de Buenos Aires 3 Facultad de Ingenieria, UBA 4 Consejo Nacional de Investigaciones Cientificas y Tecnicas 5 Universidad Nacional de Lujan-Centro de Investigaciones en Micotoxinas 6 Facultad de Veterinaria, UBA * Address for correspondence:

S.L. Resnik, Departamento de Industrias, FCEyN-UBA Ciudad Universitaria (1428) Buenos Aires - Argentina, Tel/Fax: (541) 784-0208

Abstract Thirty-four isolates of Aspergillus (favus obtained from the main Argentinian corn production area were tested for their ability to produce both cyclopiazonic acid (CPA) on corn and on liquid media and aflatoxins on corn. Aflatoxins and CPA were quantified by comparison with standards. The last one was confirmed by mass spectrometry. All but one of the isolates produced CPA on liquid medium in a range between 3120 to 62500 Jjg/kg, 27/34 isolates produced CPA on corn at levels ranging from 833 to 10000 Jjg/kg and 5/34 isolates produced aflatoxin 81 in a range between 29 to 115 Jjg/kg. According to these findings, the percentage of Aspergillus (favus isolates with CPA production ability and their levels of CPA production were higher than the observed elsewhere. It was observed significant differences (p<0,01) between CPA production on corn (median: 1761 Jjg/Kg)and in liquid medium (median: 27950 Jjg/Kg). These data represent the first report of the co-production of CPA and aflatoxin 81 by isolates of Aspergillus (favus obtained from corn in Argentina. Introduction Cyclopiazonic acid (CPA), is a toxic indole tetramic acid produced by a number of species of Penicillium and Aspergillus (1,2). In 1977, Luk et al. (1) reported for the first time that CPA could be produced by cultures of Aspergillus jlavus Link. This was an important finding becauseA.flavus is a widely distributed fungus affecting many major

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crops such as corn and peanuts, and is a well known producer of highly toxic mycotoxins as aflatoxins. Toxicity of aflatoxins has been broadly studied in animal species (3) and CPA toxicological effects on different animal species like pigs, dogs, monkey, guinea pigs, poultry and rabbits has also been studied (4-12). Moreover, CPA could be accumulated in animal tissue or transmited to milk and white eggs (13-15). CPA has been found as a natural contaminant of corn, peanuts and cheese (2,16), and aflatoxins have been found in these same agricultural products. The presence of both toxins in food or feed may pose a greater hazard than either mycotoxin alone. The natural occurrence of aflatoxins in Argentinian corn has been detected (17), but there are not surveys with respect to CPA occurrence. Thus we would like to know if A. flavus which contaminate the Argentinian corn could produce CPA. The purposes of this study were I) to evaluate CPA and aflatoxins production by A. flavus isolated from Argentinian corn and 2) to compare CPA production on corn and in liquid medium.

Materials and methods Microorganisms: Thirty-four isolates of A. flavus Link were chosen at random from a 175 strain collection of A. flavus isolated from corn harvested in the main production area in Argentina (18). Inoculum preparation: Pure isolates of A. flavus were grown on potato dextrose agar slants and incubated at 28°e. After 7 days conidial suspensions were prepared by adding sterile distilled water (5.0 ml) to each slant and gently scraping the agar surface with a wire loop to give a turbid suspension. Aflatoxins and cyclopiazonic acid (CPA) production on corn: The fermentation for aflatoxins and CPA production by the A. flavus isolates, was carried out in 500 ml erlenmeyer flasks containing cracked corn (100 g). Moisture was adjusted to 40% by adding distilled water, before autoclaving at 121°C for 60 minutes. Autoclaved flasks were shaken by hand to loosen corn, and each flask was inoculated with 5 ml of the conidial suspension ( 107 conidia/ml). Flasks were incubated at 28°C during 7 days. At the end of the incubation time cultures were autoclaving at 121°C for 15 minutes and freezed until analyzed. Cyclopiazonic production in liquid medium: The conidial suspensions (5 ml) were transferred to freshly prepared synthetic liquid medium (19) and were incubated shaked mechanically in the dark at 28°C for 7 days. Portions of the medium, 100 ml, were poured into a 500 ml erlenmeyer sealed with cotton plugs, and autoclaved for 20 minutes at 120°e. Cyclopiazonic acid analysis: After incubation a long stem funnel was placed in the flask through the cotton plug and 100 ml chloroform was added and occasionally shaking for 24 hours. The mixture was heated in a steam bath until it boiled. After cooling, 10 ml of the chloroform layer was withdrawn and filtered through 5 g anhydrous sodium sulfate and then was washed with 2 ml chloroform and was collected in the same vial. The extract was evaporated to dryness on a steam bath under a stream of nitrogen. CPA were analyzed by thin layer chromatography (TLC) on silica gel 60 precoated glass plates (Merck 5763). The TLC plates were dipped in 2% alcoholic oxalic acid solution and dried at 80°C for I h. The residues were dissolved in 200 fJI chloroform and were applied on the plate

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along with CPA standards (standard» I00 ng/Ill chloroform). The plate was developed with benzene:acetic acid: methanol (90:5:7) and then dried at room temperature. CPA was visualized by spraying with 1% p-dimetilaminobenzaldehyde in 75 ml ethanol and 25 ml HC!. The color was allowed to develop for 10 minutes at room temperature. CPA appeared as a bluish purple spot with Rf 0.60. CPA was confirmed by mass spectrometry. Chemical ionization mass spectra were obtained on a Trio-2 VG MassLab quadrupole mass spectrometer (Fisons, Biotech, Chesire, UK) equipped with an EI/CI ion chamber, operating under the following conditions: source temperature, 150°C; ionization energy, 40 e V; trap current, 600 IlA; reagent gas (methane) pressure, 33,6.10-4 mBar. Samples were introduced using the direct insertion technique at probe temperature of I 50-290°C. The spectra were recorded under the following scan parameters: mass range, 30-700 u; scan time, 0,90 s; interscan time, 0, lOs. Amyl acetate was employed for tunning, and heptacosa for calibration.

Aflatoxin analysis: Fifty grams of corn culture were blended 3 min at high speed with acetonitrile:water (84: 16). Approximately 8 ml of the acetonitrile:water filtrate was pushed through a multifunctional Mycosep 224 column (Romer Labs., Inc.) and 4 ml of clear extract was evaporated to dryness in a water bath at 60°C under vacuum (20). Each residue was disolved in 100 III of benzene:acetonitrile (98:2) and spotted on precoated silica gel plates (Merck N° 5553). The concentrations of the standard solution used were AFB I =2.4llg/ml, AFB2 = I ug/rnl, AFG I =2.4llg/ml and AFG2 = l ug/rnl. The mobile phase was acetone:chloroform (90: 10). Chromatograms were observed under 365 and 254 nm UV light and toxins were identified and quantified visually by comparying the intensities of fluorescence of sample spots with those of standards. Aflatoxins were confirmed by spraying with sulphuric acid solution (30%). Table 1 - Aflatoxin and CPA production on corn and CPAproduction in liquid mediumat 28°C by Aspergillus flavus isolates from Argentinian corn.

CIM NQ AFB1 (I-Ig/kg)

(1 ) (I-Ig/I)

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016

6250 25000 40750 8330 42000 NO 6250 42500 40000 12500 62500 7576 15000 30500 22500 50500 50500

NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO 36

(2) (I-Ig/kg) NO 833 1667 10000 NO NO 833 1000 NO 6667 1667 1111 1111 1944 1667 1389 8889

CIM NQ 2017 2018 2019 2020 2021 2022 2023 2024 2025 2026 2027 2028 2029 2030 2031 2032 2033

AFB1 (I-Ig/kg) NO NO NO NO ND 29 NO NO 115 NO NO NO 115 NO 115 NO NO

(1 ) (I-Ig/I)

(2) (I-Ig/kg)

41250 3120 25000 56250 40000 25000 24999 38636 62500 28409 18250 31250 52273 27500 38636 6250 22725

8056 3889 5000 2778 NO 3703 3511 3333 1667 5744 NO 1855 8889 NO 4078 5922 2589

CIM: Centro de Investigaci6n en Micotoxinas NO: Not detected CPAproduction in liquid media - (2) CPAproduction on corn. (1 )

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Results and discussion Table I lists CPA and aflatoxins results for the 34A.jlavus isolates screened. The presence of CPA in the A. jlavus cultures extracts was confirmed by comparing the standard and culture extracts mass spectra as is showed in Figure I. Spectra obtained from the culture extracts and CPA standard were esentially identical. All theA. jlavus isolates but one produced CPA in liquid medium in a range between 3120 to 62500 Ilg/1 and 27 of 34 strains produced CPA on ground corn at 28°C at levels ranging from 833 to 10000 ug/kg. The strain CIM 2005 did not produce neither CPA in liquid medium nor on corn. The CPA production in both sustrates was statiscally compared. It can be seen that CPA production in liquid medium was higher than it was on corn. Performing the Wilk Shapiro test for normality (21), the hypothesis of normal distribution of data was rejected for CPA production on corn. Running the Wilcoxon signed rank test (22) on the CPA production in the two sustrates, the hypothesis of equality of medians was rejected (p < 0.0 I) so the CPA production of each isolate in both substrates differ significantly. According to these findings, the percentage ofA.jlavus isolates with CPA production ability was higher than the observed elsewhere (19,23-26). The level of CPA production in liquid medium by the ArgentinianA.jlavus isolates was high in comparison with those observed by Trucksess et al. (19) in northamerican isolates. Figure 1 - Chemical ionization mass spectra of cyclopiazonic acid. (A) standard, and (8) CIM 2007 strain extract obtained from liquid media. CPZIQ 112(1.667) 100

'"

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Only five A. jlavus isolates (Table I) produced AFB I in a range between 36 to 115 ug/kg, Both the percentage of positive strains and the level of AFB I production were low in comparison with the data found in the literature (23,27-31). In conclusion the finding of CPA-producing A. jlavus isolates from corn harvested in the main production area in Argentina and the observed level of CPA production, would seem to justify the study of natural occurrence of this metabolite in the corn harvested in Argentina and also the investigation of other types of foods, including corn meal, for the presence and prevalence of CPA-producing molds and for CPA itself.

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Mycotoxin Research, Vol. 12 (1996)

Acknowledgments Weexpress appreciation to M.E. M6dena and G. Cano for competent technical assistance, to E. Martinez for statistical assistance. We also acknowledge the financial support from Cargill Foundation, Consejo Nacional de Investigaciones Cientfficas y Tecnicas, Comisi6n de Investigaciones Cientfficas de la Provincia de Buenos Aires.Universidad de Buenos Aires, and Merck Qufrnica Argentina for donating some chemicals.

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