World Mycotoxin Journal, February 2010; 3 (1): 49-57 P U B L I S H E R S
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Wageningen Academic World Mycotoxin Journal, February 2010; 3 (1): 49-57 P u b l i s h e r s Aflatoxigenesis induced in Aspergillus flavus by oxidative stress and reduction by phenolic antioxidants from tree nuts N. Mahoney1, R.J. Molyneux1,2, J.H. Kim1, B.C. Campbell1, A.C. Waiss1 and A.E. Hagerman3 1U.S. Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Plant Mycotoxin Research Unit, 800 Buchanan Street, Albany, CA 94710, USA; 2Current adress: College of Pharmacy, University of Hawaii at Hilo, 200 W. Kawili Street, Hilo, HI 96720-4091, USA; 3Miami University, Department of Chemistry and Biochemistry, Oxford, OH 45056, USA; [email protected] Received: 27 October 2009 / Accepted: 23 December 2009 © 2010 Wageningen Academic Publishers Abstract Almonds, pistachios, and walnuts grown in California have an aggregate value of over $3.3 billion, with a large proportion of the crop being exported. Mycotoxin contamination affects this value, because importing nations, and in particular the European Community (EC), apply strict regulatory levels for aflatoxins, formed by infection of the product by Aspergillus species. Conformance with such limits can best be achieved by capitalising on natural resistance factors inherent to the nuts. Differential resistance of tree nut species to aflatoxin formation, with walnuts being least susceptible, led to research which established that the hydrolysable tannins in walnut pellicle were responsible for this effect. This observation has now been extended to show that the addition to Aspergillus flavus growth media of natural antioxidants present in tree nuts, such as hydrolysable tannins, flavonoids and phenolic acids, decrease maximum aflatoxin by as much as 99.8%. There is evidence that aflatoxin formation is exacerbated by oxidative stress on the fungus and therefore compounds capable of relieving such stress should limit aflatoxin accumulation. The addition of tertiary-butyl hydroperoxide to the media induced oxidative stress in A. flavus and stimulated peak aflatoxin production with high levels being maintained, rather than declining as in control samples. Aflatoxin formation was significantly inhibited by incorporation into the media of the antioxidant, tannic acid. A similar effect was seen for drought-stressed cultures, with increased aflatoxigenesis being reduced in the presence of gallic or caffeic acids. Increasing antioxidant natural products in tree nuts may restrict the ability of A. flavus to biosynthesise aflatoxins, thus achieving conformity with regulatory limits. Keywords: aflatoxins, hydrolysable tannins, almonds, pistachios, walnuts 1. Introduction safety concerns arising from potential contamination by mycotoxins, concurrent with a rise in the number of alert Almonds, pistachios and walnuts, collectively classified and information notifications for incoming shipments as tree nuts, are among the most valuable crops (ca. $3.34 issued by the European Commission (EC) Rapid Alert billion in 2008) in the State of California, where virtually all System for Food and Feed (RASSF) (EC, 2009). In 2008 of the US commercial production is located (USDA-NASS, the highest number of notifications (931) concerned 2009). The volume of these crops, especially almonds, mycotoxins, with 902 involving aflatoxins, of which 710 were has increased rapidly over the past decade, with 40-70% in nuts, nut products and seeds; fumonisins, ochratoxin of production entering the export market, in particular A, patulin, and zearalenone were not found in nut crops. to the European Union (USDA-FDA, 2009). However, Aflatoxins are metabolites produced by certain strains of the income from this source is jeopardised by increased food fungi Aspergillus flavus and Aspergillus parasiticus which ISSN 1875-0710 print, ISSN 1875-0796 online, DOI 10.3920/WMJ2009.1185 49 N. Mahoney et al. can infect many agricultural crops, with tree nuts being relief of such stress by natural antioxidants suppresses or particularly susceptible. The European Community (EC) eliminates aflatoxin formation. The objectives of this study and a number of other countries have set tolerance levels were therefore to investigate levels of aflatoxin and the time- as low as 2 ng/g for aflatoxin B1 and 4 ng/g total aflatoxins course of production by A. flavus cultured in vitro under because of a presumed carcinogenic potential (EC, 2006a). oxidative stress induced by tertiary-butyl hydroperoxide (t-BuOOH) or low humidity, and the counteracting effect of In response to the rise in RASSF notifications, import phenolic antioxidants known to occur in tree nuts. Aflatoxin controls for US almonds and tree nuts from other countries control by constituents inherently present in crops offers were imposed in early 2007, requiring that all shipments considerable advantages because no artificial compounds to Europe after September 1, 2007 be subject to 100% or organisms are required; it is independent of multi- inspection (EC, 2006b). To address this issue, a Voluntary factorial vectors of infection and contamination; it can be Aflatoxin Sampling Plan (VASP) was created by the Almond manipulated through breeding, selection, or agronomic Board of California (2007). The VASP protocol is designed practices; and the bioactivity is generally present throughout to have a level of detection equivalent to that currently used growth, harvest, processing, and distribution. by the European Union so that only 5% of consignments accompanied by a VASP certificate would be subject to 2. Materials and methods control on entry. Since warehousing and replacement could cost $10,000 for each rejected consignment, and even Instrumentation more if the almonds have to be reprocessed or destroyed, it is obviously desirable to ensure that the product is NMR spectra were obtained at 25 °C from samples dissolved essentially aflatoxin-free, whether analysis is done at point in CD3OD with TMS as an internal standard on a Bruker of shipment or point of entry. The nature of the crop and ARX400 spectrometer (Bruker Optics, Billerica, MA, its consumption (i.e. with minimal processing) requires USA) at frequencies of 400.13 MHz (1H) and 100.62 MHz that aflatoxin control be acceptable to consumers. Thus, (13C). A 90° pulse at a 7-8 s repetition rate was used for the use of fungicides, or processing to destroy aflatoxins 1H experiments, and a 30° pulse at a 2.3 s repetition rate once they are formed, are not likely to be approved and was used for 13C experiments. The number of attached natural methods of control must be applied. Procedures protons for 13C signals was determined from DEPT90 and that reduce aflatoxin levels prior to the VASP are therefore DEPT135 assays. economically advantageous in reducing costs associated with sorting to reduce potentially contaminated kernels. Chemicals In previous research we have shown both species and Pentagalloyl-O-glucose was prepared by methanolysis of varietal differences in the susceptibility of tree nuts to tannic acid as previously described (Chen and Hagerman, aflatoxin contamination (Mahoney et al., 2003). In 2004). California, tree nuts in the retail trade have been found to be infected primarily by Aspergillus species, especially A. 3,4-di-O-galloylquinic acid was isolated from hulls of flavus and A. niger, and the microorganism profiles are very ‘Kerman’ pistachios. In brief, fresh hulls were separated similar in almonds, pistachios and walnuts (Bayman et al., by hand from mature pistachios and lyophilised. Dry 2002). However, in vitro experiments with walnuts showed hulls (675 g) were sequentially extracted with 4 litre each that they were much less capable of supporting aflatoxin of hexane, ethyl acetate, acetone, methanol, and water production by A. flavus than almonds. This is confirmed using a Polytron homogeniser (Kinematica, Riverview, by the EC sampling program, in which almonds comprise FL, USA) followed by filtration through #4 filter paper the majority of notifications issued for tree nut shipments (Whatman, Piscataway, NJ, USA). The water extract was from the US. For example, notifications for almonds centrifuged and the supernatant stirred overnight with 2 increased steadily from 28 in 2005 to 66 in 2007; pistachios kg Amberlite XAD-2 (Acros Organics, Morris Plains, NJ, remained fairly constant at 13 and 11 notifications for the USA). The XAD-2 resin was filtered and washed with 4 same two years; and there was only a single notification for litres methanol, followed by solvent removal under reduced walnuts in the three-year period from 2005-2007. This led pressure and lyophilisation (yield 18 g). The dried, XAD- us to hypothesise that specific constituents in these tree retained fraction was dissolved in 0.1% acetic acid and nuts, with variable degrees of bioactivity, were capable applied to a 60×4.8 cm Sephadex G25 fine column (GE of inhibiting aflatoxin formation. In the case of walnuts, Healthcare, Piscataway, NJ, USA), followed by elution hydrolysable tannins present in the pellicle (seed coat) and with 0.1% acetic acid and detection at 254 nm. The eluate their hydrolysis products, gallic and ellagic acid, were found containing 3,4-di-O-galloylquinic acid was collected and to suppress aflatoxin formation in vitro (Mahoney and lyophilised (430 mg yield). Molyneux, 2004). Since all of these compounds are phenolic 1 antioxidants a reasonable hypothesis is that biosynthesis of The H NMR (CD3OD) spectrum obtained was as follows: aflatoxins in the fungus is induced by oxidative stress, and δ 2.15, J=14.0, 6.0 Hz, (1H, dd, H-6ax), 2.31, J=6.3Hz (2H, 50 World Mycotoxin Journal 3 (1) Aflatoxin reduction by antioxidants in tree nuts br d, H-2ax, 2eq), 2.35, J=13.9, 2.9 Hz (1H, dd, H-6eq), 4.44, or G2. Spores were collected on a swab and transferred to J=6.0, 3.0, 3.0 Hz (1H, H-5), 5.22, J=8.4, 3.0 Hz (1H, H-4), 0.05% Tween 80. Spore concentration was calculated using 5.68, J= 14.7, 6.8 Hz (1H, H-3), 6.99 (2H, s, H2’ or 6’), 7.09 a Neubauer counting chamber.