survey of the mycobiota of spanish malting barley and evaluation of the mycotoxin producing...

biology 108 (2006) 196–203www.elsevier.com/locate/ijfoodmicro

International Journal of Food Micro

Survey of the mycobiota of Spanish malting barley and evaluationof the mycotoxin producing potential of species

of Alternaria, Aspergillus and Fusarium

Ángel Medina a, Francisco M. Valle-Algarra b, Rufino Mateo b,José V. Gimeno-Adelantado b, Fernando Mateo a, Misericordia Jiménez a,⁎

a Dpto. de Microbiología y Ecologia, Universidad de Valencia, Dr. Moliner 50, E-46100, Burjassot, Valencia, Spainb Dpto. de Química Analítica, Universidad de Valencia, Dr. Moliner 50, E-46100, Burjassot, Valencia, Spain

Received 10 June 2005; received in revised form 16 November 2005; accepted 2 December 2005

Abstract

The present work deals with the toxigenic mycobiota occurring in Spanish malting barley and the capability for producing mycotoxins byseveral important toxigenic fungi. One hundred and eighty seven samples of malting barley were gathered from Spanish breweries beforeprocessing. One hundred and fifty kernels per sample were surface-sanitized with a 2% sodium hypochlorite solution and incubated on threeculture media. The most abundant fungi were species of Alternaria, Aspergillus, Penicillium and Fusarium, which were present in 93%, 82.3%,57.8% and 27.8% of the samples, respectively. To evaluate their mycotoxin producing potential a number of isolates belonging to each genus,except Penicillium, were randomly selected and incubated on culture media known to be appropriate for production of mycotoxins. Alternariol andalternariol monomethyl ether were produced by 26.7% of Alternaria spp. isolates (all belonged to Alternaria alternata). All tested isolates of F.verticillioides produced fumonisin B1 (FB1) and 61.3% of them produced fumonisin B2 (FB2), whereas FB1 was synthesized by 83.3% and FB2 by77.8% of F. proliferatum isolates. Twenty percent of the isolates of the Aspergillus flavus/A. parasiticus group had the capability to produceaflatoxin B1 and aflatoxin B2. Thirty out of 34 isolates of F. graminearum produced deoxynivalenol and zearalenone whereas the other 4 isolatesproduced nivalenol. Ochratoxin Awas detected in 75% and 15% of isolates of Aspergillus section Nigri and A. ochraceus, respectively. This is thefirst survey carried out in Spain on the toxigenic mycobiota contaminating malting barley in breweries and the mycotoxin producing capacity ofseveral species. The information obtained is useful for assessing the risk of mycotoxins in beer.© 2005 Elsevier B.V. All rights reserved.

Keywords: Mycobiota; Mycotoxins; Aspergillus; Alternaria; Fusarium; Toxigenic fungi; Malt; Barley

1. Introduction

Spain is a Mediterranean country with a warm climate suit-able for fungal infection of barley seed both before and afterharvest. The mycobiota of malting barley has been reported inseveral countries (Andersen et al., 1996; Noots et al., 1998;Ackermann, 1998; Gareis, 1999), but no similar study has beencarried out in Spain. The species of fungi occurring in barleyand their enumeration are affected by conditions such as cli-mate, location, time of harvest, and characteristics of the barleyvariety (Haikara et al., 1977).

⁎ Corresponding author. Tel.: +34 963543144; fax: +34 963543202.E-mail address: [email protected] (M. Jiménez).

0168-1605/$ - see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.ijfoodmicro.2005.12.003

Certain fungal species in barley and malt barley can producemycotoxins under suitable conditions. These mycotoxins maypose a health hazard as they can contaminate manufacturedproducts such as beer, thereby entering the human food chain.The most important toxigenic fungi in stored or processed plantproducts are species of Alternaria, Fusarium, Penicillium andAspergillus (Mateo et al., 2004).

Toxigenic Alternaria strains can produce alternariol (AOH)and alternariol monomethyl ether (AME). These mycotoxinshave been found in barley (Gruber-Schley and Thalmann, 1988),although they have not been studied in malting barley or beer.However, some mycotoxins produced by Fusarium spp. suchas fumonisin B1 (FB1) and fumonisin B2 (FB2), and the type Btrichothecenes deoxynivalenol (DON), nivalenol (NIV), and 3-

197Á. Medina et al. / International Journal of Food Microbiology 108 (2006) 196–203

and 15-acetyldeoxynivalenol (3- and 15-ADON) have beendetected in beer (Scott et al., 1993; Shim et al., 1997; Torres etal., 1998; Molto et al., 2000; Taschan et al., 2000).

Aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1

(AFG1) and aflatoxin G2 (AFG2) are produced by toxigenicstrains of Aspergillus flavus and A. parasiticus. They have beenfound in a wide variety of plant products (nuts and grains) and inbeer from several countries (Scott, 1996; Scott and Lawrence,1997; Nakajima et al., 1999).

Ochratoxin A (OTA) can be produced by toxigenic strains ofAspergillus spp. (Varga et al., 1996; Abarca et al., 2001; Baymanet al., 2002; Hesseltine et al., 1972),Penicillium verrucosum (Pitt,1987) and P. nordicum (Larsen et al., 2001). This toxin has alsobeen detected in barley, malting barley, (Trucksess et al., 1999;Gareis, 1999) and beer (Scott and Kanhere, 1995; Nakajima et al.,1999), including Spanish beer (Legarda and Burdaspal, 1998;Medina et al., 2005). Ochratoxin B (OTB), another secondarymetabolite of the ochratoxin group, is also an undesirable com-pound produced by these fungi (Moss, 1996). Although variousmycotoxins have been found in beer, there are few studies of thefungi involved in the production of these mycotoxins (Andersenet al., 1996; Noots et al., 1998; Ackermann, 1998; Gareis, 1999)and till now no study on this topic has been carried out in Spain.

Performing an appropriate sampling on malting barley afterharvest is difficult, as samples must be supplied by maltingcompanies. They cannot be obtained from common commercialsources since the whole production of malting barley varietiesselected for beer manufacturing are brought directly to maltingcompanies. This may explain the very limited number of sci-entific reports on the natural mycobiota of malting barley. Onthe contrary, there is extensive information regarding the naturalmycobiota occurring in other cereals that may be used as adjunctingredients in the brewing process in Spain (Mateo et al., 2004).

In addition to the development of fungi on malting barleyduring pre- and post-harvest stages, the steeping and germinationsteps in the malting process are favourable to further fungalgrowth (Hardwick, 1983). During three stages (pre-harvest, post-harvest and malting), the fungi that infect barley can grow andproduce the mycotoxins that contaminate the fermented beverage(Schwarz et al., 1995).

The aims of the present work were to study the native my-cobiota of malting barley used in Spanish breweries and toevaluate the ability of the fungi to produce mycotoxins in vitro.The results of this survey would likely serve to identify the fungi

Table 1Percentage of malting barley samples contaminated with fungi from different region

Fungi Geographic origin of the samples

Castilla-La Mancha

Albacete (n=46) a Ciudad Real (n=38) Other provin

Alternaria spp. 73.9 100 100Aspergillus spp. 84.8 100 100Penicillium spp. 76.1 55.3 52.0Fusarium spp. 6.5 36.7 24.0Rhizopus spp./Mucor spp. 21.7 10.5 64.0a n denotes the number of samples.

responsible for mycotoxins reported in Spanish beer such asOTA (Legarda and Burdaspal, 1998; Medina et al., 2005) orfumonisins (Torres et al., 1998).

2. Materials and methods

2.1. Sample collection

One hundred and eighty seven samples of malting barleywere collected in the autumn of 2002 just after the harvest from12 malting companies that process this cereal for malt in fourregions of Spain. The regions and the number of samples takenare Castilla-La Mancha (109 samples), Castilla-León (17 sam-ples), Rioja (22 samples), and the Basque Country (36 samples).The first two regions are located in the central plateau of Spainwhereas the two other regions are in northern Spain. Threesamples were of unknown origin (Table 1). Each of the 187samples (about 5 kg) was prepared by probing at two differentplaces three or more different sacks at each company. Sampleswere pooled and mixed thoroughly. Then, 1 kg of each pooledsample was placed in a hermetic sterile plastic bag (laboratorysample) and sent rapidly to our laboratory, where it was stored ina cool place (4–5 °C) and processed within 24 h on arrival.

2.2. Reagents and standards

Standards of all mycotoxins, 2-mercaptoethanol, pentafluor-opropionic anhydride (PFPA) and 4-dimethylaminopyridine(DMAP) were purchased from Sigma (Sigma-Aldrich, Alcoben-das, Spain). Acetonitrile, chloroform, acetic acid, methanol (allLC grade) and phosphoric acid (85%, A.R.) were from J.T. Baker(Deventer, the Netherlands). Yeast extract, malt extract, myco-peptone and toluene were from Panreac (Montcada i Reixac,Barcelona, Spain). Pure water was obtained from a Milli-QPlus apparatus (Millipore, Billerica, MA, USA). Dichloran(2,4-dichloro-4-nitroaniline) was purchased from Tecnidex(Paterna, Valencia, Spain). Tween 80, corn steep liquor and stan-dardized 70–230 mesh aluminium oxide 90 were from Merck(Darmstadt, Germany). Glass microfibre filters (GF/C) and filterpapers (Whatman No. 4) were from Whatman (Maidstone, UK).Celite 545, activated charcoal (Norit) and o-phthaldialdehyde(OPA)were purchased fromFluka (Alcobendas, Spain). Sep PackPlus C18 cartridges were supplied by Waters Assoc. (Milford,MA, USA).

s of Spain

Rioja(n=22)

Basque Country Castilla-León Unknown(n=3)

Overallaverage(n=187)

ces (n=25) Álava (n=36) Burgos (n=17)

100 100 100 66.7 93.045.5 100 23.5 66.7 82.350.0 25.0 100 66.7 57.89.1 69.4 5.8 33.3 27.8

27.3 13.8 88.2 33.3 30.5

198 Á. Medina et al. / International Journal of Food Microbiology 108 (2006) 196–203

2.3. Fungal isolation

Three culture media were used to isolate and enumerate thefungi. PotatoDextroseAgar (PDA)was prepared by boiling 300 gof potato inwater for 1 h. After filtration, the liquidwas brought to1 l. Then, 2% w/v of agar and 1% w/v of glucose were added andthe mixture was autoclaved at 115 °C for 30 min. PDAwas usedfor enumeration of field fungi Alternaria spp. and Fusarium spp.(Ackermann, 1998).

Malt Salt Agar (MSA)was prepared using 20 g ofmalt extractbroth, 75 g of sodium chloride, 15 g of agar and 1 l of pure water.The mixture was autoclaved at 115 °C for 30 min. Owing to itslow water activity, MSA was appropriate for enumeration ofstorage fungi such as Aspergillus spp. and Penicillium spp.(Ackermann, 1998).

Potato Dichloran Agar was prepared by addition of 1.0 ml of a0.2%w/v solution of dichloran in ethanol to PDA cooled to 60 °Cafter sterilization. Dichloran is a fungal growth inhibitor per-mitting the detection and enumeration of fungi such as Fusariumspp., Alternaria spp. or Aspergillus spp., in the presence of fast-growing molds, such as species of Mucor or Rhizopus.

To reduce surface contamination 100 g of each sample weresurface-sanitized with 0.5 l of a 2% solution of sodium hypo-chlorite for 1 min. Afterwards, the grains were washed twice with0.25 l of sterilized pure water for 1 min. Fifty seeds per sample(5 seeds/9 cm Petri dish) were placed onto each of the 3 solidmedia described above. Therefore, a total of 150 seeds per samplewere used. After incubation at 25 °C for 2–15 days depending onthe fungi to be isolated, the mycelia developing from the seeds inall media were transferred to PDA plates under sterile conditions.The highest count for a fungal genus on one of the three mediawas recorded (Ackermann, 1998). Single spore cultures wereprepared for culture identification and for further testing ofselected isolates for their toxigenic ability (Barnett and Hunter,1972; Nelson et al., 1983; Klick and Pitt, 1988; Pitt, 1991). Fivehundred microliters of a spore suspension of each isolate inskimmed milk was lyophilized for 8 h in a VirTis Sentry equip-ment (SP Industries, NY, USA) provided with an Edwards high-vacuum pump (Edwards High Vacuum International, WestSussex, England). Lyophilized fungi were stored in the fungalcollection of the Department of Microbiology and Ecology of theUniversity of Valencia (Spain).

2.4. Mycotoxin producing potential of the isolates

2.4.1. Culture media and incubation conditionsSpecies of Alternaria and Aspergillus were grown in liquid

media whereas the Fusaria were grown on autoclaved grain.Ninety Alternaria spp. isolates were tested for AOH and AMEproduction using Malt Broth (MB) (Fábrega et al., 2002). FiftyA. flavus/A. parasiticus isolates were tested for aflatoxin prod-uction using Aflatoxin Production Ability (APA) broth (Hara etal., 1974) and 40 isolates of Aspergillus section Nigri and 20isolates of A. ochraceus were tested for OTA and OTB prod-uction using Yeast Extract Sucrose (YES) broth supplementedwith 5% (w/v) of bee pollen (Medina et al., 2004). All theisolates were taken randomly. For each of the 3 liquid media 50

ml were poured into 100-ml Erlenmeyer flasks. The flasks wereplugged with cotton, covered with aluminium foil and auto-claved at 115 °C for 30 min. One milliliter of a spore suspensionof each isolate made in aqueous Tween 80 (0.05%) having1×105 spores/ml was used to inoculate sterilized liquid media,which were then incubated at 25 °C for 4 weeks. Three flasksper isolate were assayed for mycotoxin accumulation. Controlsconsisted of non-inoculated sterilized liquid media incubatedunder the same conditions.

Thirty-four F. graminearum Schwabe isolates were tested fortype B trichothecene and ZEA production. The assay was per-formed using maize kernels as a culture medium (Llorens et al.,2004a,b). Thirty-one isolates of F. verticillioides (Sacc.)Nirenberg and 18 isolates of F. proliferatum (Matsushima)Nirenberg were tested for FB1 and FB2 production. The assaywas performed using rice seeds as a culture medium (Hinojo,2004). All the isolates were selected randomly.

One hundred grams of maize or rice were placed into 250-mlErlenmeyer flasks and water activity (aw) was adjusted to 0.98by adding the appropriate volume of pure water. The amount ofadded water was calculated from a moisture adsorption curve.An aw sprint RTD 502 unit (Novasina GmbH, Pfäffikon,Switzerland) was used to measure the aw of equilibrated grains.The instrument was calibrated using salt solutions provided bythe manufacturer. The aw values were confirmed aftersterilization using controls consisting in 100 g of grain placedin 250-ml Erlenmeyer flasks in each set of experiments. In somecases, it was necessary to re-hydrate the grains by addition ofsterile pure water to reach the desired aw levels. The flasks wereplugged with cotton, covered with aluminium foil andautoclaved at 115 °C for 30 min. Sterilized kernels wereinoculated with 100 μl of a spore suspension made in aqueousTween 80 (0.05%) having 1×106 spores/ml. Incubation was for4 weeks at the temperatures considered best for production ofthe target mycotoxins. For ZEA and fumonisins, the temper-ature was 20 °C (Hinojo, 2004; Llorens et al., 2004a), whereasfor type B trichothecenes it was 25 °C (Llorens et al., 2004b).All cultures were assayed in triplicate for mycotoxin accumu-lation. Controls consisted of non-inoculated sterilized grainsthat were incubated under the same conditions.

2.4.2. Mycotoxin extraction, clean up and analysisExtraction of alternariols from MB was performed using

liquid–liquid extraction. Five milliliters of liquid culture com-bined with 6 ml of chloroform in a separatory funnel wereintensively shaken by hand. The organic phase was separated,and the aqueous phase was re-extracted twice with 4 ml ofchloroform. The organic extracts were mixed and evaporated ina rotary evaporator at 40 °C. The residue was dissolved in 250μl methanol and transferred to a vial. LC analysis of alternariolswas made according to Motta and Valente-Soares (2000), withsome modifications to increase peak resolution. A gradient oftwo phases: A (methanol–0.3% w/v ZnSO4·H2O aqueoussolution, 20 :80 v/v) and B (methanol) was used. The gradientstarted with 100% of A, after 7 min, the mixture changed to40% A and 60% B, after 2 min the composition changed to 30%A and 70% B, which was maintained for 2 min and changed to


20% A and 80%B, which was maintained for 6 min. Finally, thecomposition was returned to the initial conditions gradually.The mobile phase flow-rate was 0.8 ml/min. Alternariols weredetected by a photodiode array detector (DAD).

Extraction of aflatoxins from APA was made using liquid–liquid extraction. Ten milliliters of liquid culture was intensivelymixed with 7 ml of chloroform in a separatory funnel. Thechloroform phase was separated and the aqueous phase was re-extracted twice with 7 ml of chloroform. The organic extractswere combined and evaporated in rotary evaporator at 40 °C.The residue was dissolved in 250 μl of methanol–water–acetonitrile (18:64:18, v/v/v) and transferred to a vial. Themethodology described byAbdulkadar et al. (2004) was followedfor LC analysis of aflatoxins. The optimum mobile phase flow-rate was 0.8 ml/min.

Ochratoxin extraction and LC analysis was made according toMedina et al. (2004). Two milliliters of culture medium wasmixed with 200 μl of a 0.1 M solution of phosphoric acid,extracted with 3×3 ml chloroform and, after evaporation of thesolvent, the residue was dissolved in 0.5 ml of acetonitrile–water–acetic acid, 49.5:49.5:1 (v/v/v).

For type B trichothecene analysis, 50 g of culture dried in aforced air stove at 45 °Cwas finely ground in a laboratorymill and5 g of the ground culture was placed into a 100-ml Erlenmeyerflask. After adding 40 ml of acetonitrile–water (84:16, v/v), themixture was blended in a high-speed blender for 10 min. Afterfiltering through Whatman No. 4 filter paper, the filtrate wasstored in a tightly closed glass bottle at −20 °C until use. Cleanup of the filtrate was made by solid-phase extraction usingcartridges containing 1.16 g of alumina–charcoal–C18 silica(75:1 :40, w/w/w) as described by Valle-Algarra et al. (2005).After clean up, extracts were derivatized with DMAP andPFPA and the trichothecene derivatives were analyzed bysplitless GC-ECD using a HP-5 [5% methyl phenyl siloxane,30 m×0.32 mm I.D., 0.25 μm film thickness] fused-silicacapillary column (Agilent Technologies, Waldbronn, Germany)according to Valle-Algarra et al. (2005).

Zearalenone analysis was made following the methodologydescribed byMateo et al. (2002). Five grams of culture dried in aforced air stove at 45 °C and finely ground in a laboratory millwas extracted with methanol–1% aqueous NaCl (80 :20, v/v)(2×25 ml). Sep-Pak Florisil cartridge was used for clean up andthe purified extract was separated in a liquid chromatographequipped with fluorescence and photodiode array detectors(arranged in tandem).

Table 2Percentage of kernels infected with fungi in malting barley samples harvested in the

Fungi Geographic origin of the samples

Castilla-La Mancha

Albacete Ciudad Real Other provinces

Alternaria spp. 83.23 96.48 87.98Aspergillus spp. 24.82 6.33 12.23Penicillium spp. 13.11 8.58 3.53Fusarium spp. 3.18 3.76 1.21Rhizopus spp./Mucor spp. 25.34 0.78 19.23

Fumonisin analysis was performed using acetonitrile–water(50 :50, v/v) for extraction and C18 cartridges for clean up, asdescribed by Mateo et al. (2002). Derivatization of fumonisinswas carried out with OPA. The fumonisin derivatives were an-alyzed using a LC-fluorescence detection system. A gradientprogramwas used. Themobile phaseswere (A)methanol–0.05Maqueous NaH2PO4 adjusted to pH 5.0 with 2 M NaOH (50 :50,v/v) and (B) acetonitrile–water (80 :20, v/v). The gradient pro-gram was 100% A during 5 min followed by a change to 50% Awith a final hold of 15 min. The column was kept at 30 °C with aflow-rate of 1.0 ml/min. The excitation and emission wave-lengths were set at 335 and 440 nm, respectively.

2.4.3. Chromatographic equipmentThe LC system consisted of a Waters 600 pump equipped

with a Waters 717 automatic injector and a Waters 474 scanningfluorescence detector (Waters). A Waters 996 photodiode arraydetector was set after the fluorescence detector to provideconfirmation of OTA identity by comparison of UV spectra withthat of the standard. Millennium 32® software version 3.01.05(Waters) was used to control the chromatograph and to processthe signals. Separation was performed on a stainless steelLiChrospher 100 C18 reversed-phase column (250×4 mm, 5 μmparticle size) connected to a guard column (4×4 mm, 5 μmparticle size) (Agilent Technologies) filled with the same phase.The column was kept at 40 °C.

The GC system consisted of a HP-6890 Plus gas chromato-graph, equipped with a 63Ni ECD (Hewlett-Packard, Avondale,PA, USA) and an Agilent 7683 Series injector (Agilent Tech-nologies). Signals were processed by HP GC ChemStation soft-ware version A.07:01(682) (Hewlett-Packard). A fused-silicacapillary column HP-5 (Agilent Technologies) was used.

2.5. Statistics

One-way analysis of variance (ANOVA) of data was per-formed using the Statgraphics Plus 5.1 statistical package(StatPoint, Inc., VA, USA).

3. Results

Alternaria spp. were present in 93% of samples (Table 1)and infected between 64% and 100% of seeds (Table 2). Onaverage 82.8% of the seeds were infected by Alternaria spp.(Table 2).

autumn of 2002 from different regions of Spain

Rioja Basque Country Castilla-León Unknown Overallaverage

Álava Burgos

79.77 69.84 98.45 64.21 82.771.76 21.62 0.56 8.65 14.570.44 2.67 3.34 11.26 7.074.63 8.23 0.80 25.10 4.2119.97 33.53 1.42 5.56 18.63


The second most abundant genus in terms of percentage ofcontaminated samples was Aspergillus, which was isolated in82.3% of the samples, although the infection level was not veryhigh (14.57% of grains) (Tables 1 and 2). Aspergillus sectionNigri, A. ochraceus and the A. flavus/A. parasiticus group oc-curred in 15%, 29% and 64% of the samples, respectively, andinfected 2.0%, 3.1%, and 9.4%, of seeds, respectively. The dis-tribution of the different species of Aspergillus varied with thegeographic origin of the samples as those from Castilla-LaMancha and the Basque Country showed the highest level ofinfected grains. The distribution of isolates was as follows:44.71% belonged to the A. flavus/A. parasiticus group, 13.81%belonged to Aspergillus section Nigri, 6.5% belonged to A.ochraceus and the remainder belonged to other Aspergillus spe-cies (about 35%).

Although 57.8% of samples were contaminated with Penicil-lium spp. (Table 1), the average infection level was only 7.07% ofgrains. Samples from Albacete (a province in Castilla-LaMancha) had the highest level of seed infection by Penicilliumspp. at 13.11% (Table 2). P. verrucosum Dierckx was isolatedonly occasionally.

Fusarium spp. were found in 27.8% of samples (Table 1) and4.21% of kernels, although no sample had over 25% seedinfection (Table 2). Toxigenic species such as F. graminearum,F. culmorum, F. verticillioides and F. proliferatum were amongthe most frequently isolated Fusarium spp. Mucor spp. andRhizopus spp. were isolated in 30.5% of samples but were thesecond most often detected group of fungi, infecting 18.63% ofkernels (Tables 1 and 2).

The statistical analysis (ANOVA) of the data showed theexistence of significant differences (pb0.01) with regard to sam-ple infection among the different toxigenic genera of fungicontaminating malting barley from Spain, with a clear dominanceof Alternaria spp. and Aspergillus spp. However, no significant

Table 3Mycotoxin producing potential of fungi isolated from malting barley grown in Spai

Fungi Mycotoxins

Alternaria spp.a AlternariolAlternariol monomethyl ether

Aspergillus flavus/A. parasiticusb Aflatoxin B1

Aflatoxin B2

Aflatoxin G1

Aflatoxin G2

Aspergillus section Nigri c Ochratoxin AOchratoxin B

Aspergillus ochraceusc Ochratoxin AOchratoxin B

F. graminearumd NIVDON3-/15-ADONZEA

F. verticillioidese FB1

FB2

F. proliferatume FB1

FB2

The culture media were: a) Malt Broth, b) Aflatoxin Production Ability broth, c) Yeae) rice kernels. f) Data are in nanogram per gram dried culture medium.

differences appear in sample infection level with regard to thegeographic origin of the samples.

The mycotoxins AOH and AME were detected in 24 out of90 (26.6%) isolates of Alternaria spp. assayed by LC-DAD(Table 3). All the mycotoxin producing isolates were identifiedas Alternaria alternata. The average level of AOH (213 ng/ml)was higher than the average level of AME (152 ng/ml). AOHlevels ranged from 78 to 682 ng/ml, while AME levels rangedfrom 32 to 229 ng/ml (Table 3).

AFB1 and AFB2 were detected in 10 out of 50 tested isolates(20%) of theA. flavus/A. parasiticus group (Table 3), but only oneisolate produced all four compounds. The average levels ofAFB1,AFB2, AFG1 and AFG2, within the producing isolates were 6.2,8.6, 0.44 and 0.62 μg/ml, respectively. The highest level of AFB1

and AFB2 produced was 16.25 and 21.15 μg/ml, respectively.They were found in the same isolate.

Thirty out of 40 isolates in the Aspergillus section Nigri wereOTA producers. Twenty six producing isolates were classified asA. carbonarius and four belonged to the Aspergillus niger ag-gregate. The respective OTA production ranges were 0.063–0.567 ng/ml (average 0.153 ng/ml) and 0.036–0.189 ng/ml(average 0.089 ng/ml). The average level found in cultures of the30 OTA-producing isolates was 0.145 ng/ml. OTB was found inthe culture of only one isolate of A. carbonarius where its levelwas even higher (0.78 ng/ml) than the OTA level (0.26 ng/ml) inthe same culture.

Three of 20 isolates of A. ochraceus were OTA producers andthe average level for this toxin in the cultures was 106 ng/ml (2.2–254 ng/ml). Two isolates were able to produce OTB and the levelsfor this toxin were 0.4 and 34.3 ng/ml (Table 3).

Thirty-four isolates of F. graminearum were assayed forproduction of ZEA and type B trichothecenes (Table 3). NIVwas detected in 11.8% of F. graminearum cultures at anaverage level of 40 ng/g, whereas DON was found in 88.2% of

n

Number of isolates Toxin production (ng/ml)

Assayed Positive Average Range

90 24 213 78–68290 24 152 32–22950 10 6180 340–1625050 10 8570 230–2115050 1 440 44050 2 620 380–83040 30 0.145 0.036–0.56740 1 0.778 0.77820 3 106 2.2–25420 2 17.3 0.4–34.334 4 40f 30–60f

34 30 560f 30–1130f

34 26 550f 110–1370f

34 30 2620f 20–8330f

31 31 8880f 99–30280f

31 19 8430f 13–20470f

18 15 586500f 367900–1327000f

18 14 198000f 35900–342900f

st Extract Sucrose supplemented with 5% (w/v) of bee pollen, d) maize kernels,


cultures at an average level of 560 ng/g. The average levelfound for the sum of 3- and 15-ADON was 550 ng/g. ZEAwasalso detected in 88.2% of the isolates at levels ranging from 20to 8330 ng/g.

All 31 isolates of F. verticillioides and 15 out of 18 isolates ofF. proliferatum (83.3%) produced FB1 (average levels 8.88 and586.5 μg/g, respectively), whereas only 61.3% of F. verticil-lioides isolates and 77.8 % of F. proliferatum isolates producedFB2. The average level of FB2 in the cultures was 8.43 μg/g forF. verticillioides and 198 μg/g for F. proliferatum (Table 3).

4. Discussion

This is the first study in Spain identifying, enumerating andtesting the toxigenic fungi infectingmalting barley. Therefore, it isvery difficult to compare the results from this study with thosefrom other authors.

The dominant species in the malting barley samples weremembers of the genus Alternaria, which agrees with resultsreported by other authors (Haikara et al., 1977; Lacey et al., 1980;Gyllang et al., 1981; Andersen et al., 1996; Ackermann, 1998).Although Haikara et al. (1977) report that 61% of grains werecontaminated with Alternaria spp. the other authors found thisgenus infecting almost 100% of the seeds. Alternariol productionwas detected in 26.6% of Alternaria spp. isolates although thelevels of AOH and AMEwere not high. Tests for AOH and AMEin malting barley have not been done but others have detectedthese toxins in barley (Gruber-Schley and Thalmann, 1988). Thefrequency of Alternaria spp. in the samples could pose a risk ofalternariols being present in malting barley and beer. It wouldtherefore be prudent to test Spanish malting barley for thesecompounds.

Contamination levels by Aspergillus spp. exceeded those in-dicated by other authors (Lacey et al., 1980; Gyllang et al., 1981;Ackermann, 1998). According to Table 2 the overall averagecontamination of grains by this genus was 14.57%.

Aflatoxins and ochratoxins are the toxins that attract mostattention. Production of aflatoxins by isolates of the A. flavus/A.parasiticus group from Spanish malting barley warns about thepossible presence of these toxins in this cereal and supports theresults from Nakajima et al. (1999), who found aflatoxins in beerfrom several countries including Spain.

A high percentage of isolates from the Aspergillus sectionNigri was capable of producing OTA though the levels were notvery high (Table 3). If one considers the wide occurrence of thesefungi in grain their contribution to the presence ofOTA in Spanishbeer samples, as reported by Legarda and Burdaspal (1998) andMedina et al. (2005), could be significant. The low incidence ofA.ochraceus, a species reported to produce both OTA and OTB,would seem to indicate that the very frequent presence of OTA inSpanish beer samples is due mainly to other fungi, probably in theAspergillus section Nigri or Penicillium spp. OTB was detectedonly in one isolate and combined with its low toxicity (Moss,1996), is unlikely to be an important toxin in Spanish maltingbarley.

Penicillium spp. were more frequently detected in this studythan in some other countries (Haikara et al., 1977; Andersen

et al., 1996; Ackermann, 1998). There are many differentmycotoxins produced by species of Penicillium (Frisvad andSamson, 1991), although they are less toxic than the othermycotoxins reported here. Characterization to species level ofall Penicillium isolates and the study of their mycotoxinproducing ability need a broad and thorough work focused onthis genus. Its occurrence in malting barley was high and thepossible presence of its toxins in beer has not been assessed.This study will be the object of further research.

Both Aspergillus spp. and Penicillium spp. are normallyconsidered as fungi developing in stored commodities. However,both fungi had a high incidence in the recently harvested samplesof malting barley studied in this work. Some authors (Magan andLacey, 1984) have reported that classification into field andstorage species is applicable in temperate climates, but that inwarmer regions some species usually considered as storage fungimay invade pre-harvest grain. In Spain malting barley is grown inareas where the temperature during cereal ripening is high.

Fusarium spp. showed an irregular distribution depending onthe geographic origin of the samples. Likely the humidity of eachregion where the samples were taken had an important effect onthe colonization of kernels byFusarium spp. Species of this genuswere detected in nearly 70% of samples from Alava (northernSpain) which can probably be due to the frequent precipitationand the high relative humidity in this area. The overall incidenceof species of Fusarium in infected grains of malting barley islower (4.21%) than levels reported in countries from northernEurope where average levels were 10–22% (Ackermann, 1998;Haikara et al., 1977). Thus, with the exception of a few locations,levels of Fusarium species in Spanish malting barley in thissurvey were not relevant. However, we must underline that allisolates of F. verticillioides and 83.3% of the F. proliferatumstudied in this work were able to synthesize FB1 and that fu-monisins are not affected by brewery processes (Alberts et al.,1990; Scott et al., 1995). In the first stages of the malting processthe level of Fusarium spp. increases (Haikara et al., 1977), whichmay explain why FB1 has been detected in Spanish beer (Torres etal., 1998; Mateo et al., 2004). However, the origin of this myco-toxin could also be the cereals used as adjuncts in the breweryprocess of some beer brands (Castellá et al., 1999).

Although in Spain there have been no studies examining thelevels of NIV, DON, 3- and 15-ADON or ZEA in malting barleyor beer, MacDonald et al. (2004) reported 0.11 mg/kg of DONin malting barley grown in the UK. Scott et al. (1993) andTaschan et al. (2000) detected DON in beer from severalcountries. Molto et al. (2000) reported the presence of DON inbeer from Argentina. Shim et al. (1997) studied the presence ofNIV and DON in Korean and imported beer, which were foundin 80% and 26% of beer samples, respectively, whereas ZEAwas not found. ZEA is metabolized largely to β-zearalenol, ametabolite with lower acivity than the parent compound, bybrewing strains of Saccharomyces cerevisiae (Scott et al., 1992;Boswald et al., 1995). Therefore, this metabolite should betested in beer, a prediction made earlier by Schoental (1984).However, ZEA was occasionally found to occur naturally inAfrican beers (Lovelace and Nyathi, 1977; Martin and Keen,1978; Okoye, 1986). It was also detected in one European beer


sample (Payen et al., 1983), although several other Europeansurveys were negative. The ZEA levels produced by the isolatesof Fusarium spp. from malting barley studied in this work arelower than the levels produced by isolates of the same speciesfrom other hosts (Llorens et al., 2004a). Although toxin prod-uction in vivo and in vitro are not really comparable, con-sidering these results and what was previously discussed, wemay conclude that ZEA does not pose a serious health risk tobeer consumers.

In the studied year due to their frequency and toxigenic abi-lities the fungi of most concern in malting barley grown in Spainwere Alternaria spp. and Aspergillus spp. Species of other generasuch as Fusarium were less frequent except in malting barleygrown in northern Spain. Presence of fumonisins reported inSpanish beer might be due to regional differences in growingconditions, crop rotations, other cereals used as adjuncts or to theincrease in Fusaria level during the malting process. The irregulardistribution of Fusarium spp. in malting barley as influenced bygrowing conditions, especially the relative humidity would in-fluence the occurrence of Fusarium toxins in beer from differentcountries.

Acknowledgements

The authors wish to acknowledge financial support from theSpanish Government “Ministerio de Educación y Ciencia” (Pro-jects AGL-2001-2974-C05-01 and AGL-2004-07549-C05-02/ALI). The authors also acknowledge the Valencian Government(Conselleria d'Empresa, Universitat i Ciencia) for financing byProject GV04B-111 and for a research grant.

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