642 Journal of Food Protection, Vol. 59, No.6, 1996, Pages 642-644 Copyright ©, International Association of Milk, Food and Environmental Sanitarians A Convenient Method for Assessing Mycotoxin Production in Cultures of Aspergilli and Penicilliat D. ABRAMSON1' and R. M. CLEAR2 lAgriculture and Agri-Food Canada, Winnipeg Research Center, Winnipeg, Manitoba R3T 2M9; Downloaded from http://meridian.allenpress.com/jfp/article-pdf/59/6/642/1660166/0362-028x-59_6_642.pdf by guest on 28 September 2021 and 2Grain Research Laboratory, Canadian Grain Commission, Winnipeg, Manitoba, Canada (MS#95-171:Received17July 1995/Accepted27October1995) ABSTRACT time-consuming step for concentration of the extract. The agar-sampling methods, while more rapid, involve a subjec- Production of aflatoxins, sterigmatocystin, citrinin, and ochra- tive decision as to which sector(s) of the culture to sample, toxin A by Aspergillus and Penicillium species on agar in 50-ml and skillful manipulation of agar plugs onto the TLC flasks after 21 days was detected and confirmed by thin-layer surface. Care is needed while dripping solvent onto the plug chromatography. Toxins were extracted with 2.5 ml of acidified in attempting to produce a compact spot at the TLC origin. methylene chloride for 20 to 60 min. Mycotoxin concentrations Since all the extract is used, confirmatory analysis of the were estimated by liquid chromatography.Addition of formic acid to the methylene chloride extraction solvent effected an increase in same agar sample is not possible. Furthermore, there is the recovery of ochratoxin A from Czapek-yeast (CY) agar from 20% hazard of exposure to toxic vapors during the solvent to 91%. Recoveries of added mycotoxins were generally higher extraction of the plug. In both methods the recoveries of from CY agar than from yeast extract-sucrose (YES) agar and toxins from the media are unknown, and weak toxin ranged from 11% (citrinin from YES agar) to 91% (ochratoxin A producers might be construed as nonproducers. from CY agar). The method described is convenient, rapid, and The proposed method was developed using four com- reproducible, since it samples the entire culture surface. Human mon Aspergillus and Penicillium mycotoxins. It offers a exposure to volatile solvents is also minimized. time-saving alternative to the broth method and an extrac- tion and TLC application which are more precise, more Key words: Mycotoxin, Aspergillus, Penicillium, production reproducible, and more convenient than the agar-sampling method. This procedure also reduces the possibility of Although many analytical methods have been devel- exposure of laboratory workers to toxic vapors. oped to detect mycotoxins in agricultural products (4, 13), it is not feasible to assay all commodities for all mycotoxins MATERIALS AND METHODS (9). To reduce this problem to manageable dimensions, it is expedient to determine the fungi characteristically present in Recovery studies agricultural products from specific locales, and the mycotox- For recovery studies of mycotoxins from Czapek-yeast (CY) and yeast extract-sucrose (YES) agars, 5.0 ml of sterile melted agar ins that can be expected (6). (Difco Laboratories, Detroit, MI) was added to 50-ml Erlenmeyer To do this effectively, many fungal isolates need to be flasks to give a nutrient thickness of approx 4 mm. Before the agar screened for mycotoxins and other secondary metabolites. solidified, I00 ~I of ethanol, containing 50 to 60 ~g of mycotoxin Various methods have been proposed. Fungi have been (aflatoxin B[ and ochratoxin A from Aldrich Chemical Co., cultured using semimicro broth techniques (11, 12) and by Milwaukee, WI; sterigmatocystin and citrinin from Sigma Chemi- sampling cultures from agar plates (5). The metabolites have cal Co., St. Louis, MO), was aseptically added to the flasks to give been analyzed by thin-layer chromatography (TLC) and 10 to 12 ~g of mycotoxin per ml of nutrient; agar in control flasks high-performance liquid chromatography (HPLC) (7). Many contained no mycotoxins. Flasks were plugged with cotton and left Aspergillus and Penicillium species and chemotypes have at 22°C for 21 days. been screened in this manner. After addition of 1.0 ml of methylene chloride or methylene In the examination of Aspergillus and Penicillium chloride/formic acid 25:I (using formic acid 97.7%, Sigma Chemi- cal Co., St. Louis, MO), the flasks were capped with aluminum foil species for their mycotoxin production, difficulties often to minimize evaporation. After 20 min, 40 min, or 60 min at 22°C, a arise using the above techniques. The semimicro broth 0.2 ml aliquot of extraction solvent was mixed with 1.0 ml of method involves a lengthy extraction with hot solvent and a methanol in a quartz cuvette, and the absorbance of trial samples against control samples was measured. The following wavelengths * Authorforcorrespondence.Tel:204-984-5536;Fax:204-983-4604. were used: ochratoxin A, 333 nm; aflatoxinB1'362 nm; sterigmato- t Contributionnumber1636oftheWinnipegResearchCenter,andnumber cystin, 329 nm; citrinin, 332 nm. The recoveries are shown in 740oftheGrainResearchLaboratory. Table 1 and Table 2. ASSESSING MYCOTOXIN PRODUCTION IN CULTURES 643 Qualitative examination for mycotoxins TABLE 1. Recovery of ochratoxin A from Czapek-yeast agar after YES agar with 0.5% magnesium sulfate (8) and CY agar were extraction with methylene chloride-formic acid 25: 1 or methylene steam sterilized, and 5.0 ml was added to 50-ml Erlenmeyer flasks. chloride alone. Ochratoxin A (10 I-Lg/ml)was added to the liquid The flasks were capped and set aside to cool. Spore suspensions agar following steam sterilization; flasks were assayed after 3 from fungi cultured on potato dextrose agar plates were made and weeks at 22°C diluted with sterile 0.85% NaCl containing 0.1 % peptone (Difco) Solvent Time (min) Recovery (mean %) SE n to give 103 spores per ml. Three flasks per fungal isolate were each inoculated with 0.5 ml, plugged with cotton, and loosely capped Methylene chloride 20 16.1 2.4 4 with foil. 40 19.6 3.7 4 After 21 days in the dark at 22OC, the flasks were opened and 60 19.5 2.0 4 2.5 ml of methylene chloride/formic acid 25: I were added. Each Methylene chloride/ 20 81.5 6.8 4 flask was tightly recapped with foil and, after 60 min, a 10-/-11 formic acid 25: 1 40 89.0 3.3 4 disposable glass capillary pipette was inserted through the foil and 60 90.8 2.5 4 filled. The pipette contents were applied to the origin of the TLC plates. Silica plates (Whatman Inc., Clifton, NJ) were developed Downloaded from http://meridian.allenpress.com/jfp/article-pdf/59/6/642/1660166/0362-028x-59_6_642.pdf by guest on 28 September 2021 with the following solvents: for ochratoxin A, ethyl acetate/ chloroform/formic acid 60:40: 1; for aflatoxins, chloroform/acetone Large differences in recovery of mycotoxins from CY 9: 1; for sterigmatocystin, benzene/acetic acid 9: 1; for citrinin, ethyl and YES agars are apparent in Table 2. Generally, recoveries acetate/acetone/water 5:5:2. CIS reverse-phase plates (E. Merck, followed the series ochratoxin A> aflatoxin Bl > sterigmato- Darmstadt, Germany) were simultaneously run for confirmation of cystin > citrinin, and ranged from 90.8% (ochratoxin A, CY identity, using methanol-acidified water mixtures of 7:3, 7:3, 9: I agar, 60 min) to 9.5% (citrinin, YES agar, 20 min). Because and 6:4, respectively (2). The mycotoxins were visualized by their of relatively low recoveries of sterigmatocystin and citrinin, natural fluorescence at 365 nm and 254 nm; sterigmatocystin was the final amount taken for TLC or HPLC analysis when detected by additional spraying with 10% AICI 3 in ethanol, heating these toxins are suspected should be increased four- or at 110°C, and examining under 365 nm light. five-fold to ensure adequate sensitivity. Low recoveries of citrinin from most substrates are common, and may be due Measurement of mycotoxin production to reaction with cysteine (10). Except for sterigmatocystin, One milliliter of the methylene chloride/formic acid 25: 1 recoveries of mycotoxins from CY agar were generally mixture was taken from each flask and evaporated in a 1.5-ml better than from YES agar. This may be related to the polypropylene microcentrifuge tube. Residues were redissolved in 1.0 ml of acetonitrile and clarified by centrifugation at 13,000 X g for 1 min. Aflatoxins BI and G1 were converted to the B2a and G2a TABLE 2. Recovery of aflatoxin B1 (AFBl), sterigmatocystin (ST), derivatives (3) and sterigmatocystin was determined after acetyla- ochratoxin A (OA) and citrinin (CT) from Czapek-yeast (CY) and tion (1). yeast extract-sucrose (YES) agars with methylene chloride-formic Mycotoxins and their derivatives were determined using a acid 25:1. Mycotoxins (10-12 I-Lg/mleach) were added to the liquid Hewlett-Packard 1084 liquid chromatograph and a Schoeffel agar following steam sterilization; flasks were assayed after 3 FS-970 fluorescence detector. Aliquots of 20 IJl were injected onto weeks at 22°C a 250-mm-long by 4.6-mm-inner-diameter column of 10 flIl1 Lichrospher RP-8 (E. Merck) and run at 45°C with mixtures of Mycotoxin Agar Time (min) Recovery (mean %) SE n methanol and acidified water (containing 0.2% phosphoric acid). Sterigmatocystin was assayed after acetylation using 50% metha- AFBI CY 20 60.9 1.6 4 nol for elution and 256 nm fluorescence irradiation. Aflatoxins, 40 70.3 2.3 4 citrinin, and ochratoxin A were assayed using 35%, 50%, and 60% 60 78.4 0.8 4 methanol for elution, and 365, 333 and 333 nm fluorescence YES 20 29.9 0.7 4 irradiation, respectively. 40 41.7 2.2 4 60 42.8 2.8 4 ST CY 20 16.5 1.1 4 RESULTS AND DISCUSSION 40 19.2 0.2 4 60 19.2 0.6 4 Methylene chloride was initially chosen because it YES 20 24.7 2.3 4 23.8 1.3 4 extracted aflatoxin B, efficiently and was volatile.