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obiology 122 (2008) 93–99www.elsevier.com/locate/ijfoodmicro

International Journal of Food Micr

Influence of nitrogen and carbon sources on the production of ochratoxin Aby ochratoxigenic strains of Aspergillus spp. isolated from grapes

Ángel Medina a, Eva M. Mateo a, Francisco M. Valle-Algarra b, Fernando Mateo c,Rufino Mateo b, Misericordia Jiménez a,⁎

a Dpto. de Microbiologia i Ecologia, Universitat de Valencia, Dr. Moliner 50, E-46100, Burjassot, Valencia, Spainb Dpto. de Química Analítica, Universitat de Valencia, Dr. Moliner 50, E-46100, Burjassot, Valencia, Spain

c Dpto. de Ingeniería Electrónica, Universidad Politécnica de Valencia, Camino de Vera, 14. E-46022, Valencia, Spain

Received 17 April 2007; received in revised form 12 November 2007; accepted 19 November 2007

Abstract

This work studies the influence of nitrogen and carbon source on ochratoxin A production by three Aspergillus isolates A. ochraceus (Aso2),A. carbonarius (Ac25) and A. tubingensis (Bo66), all isolated from grapes. A basal medium (0.01 g/l FeSO4·7H2O, 0.5 g/l MgSO4·7H2O, 0.5 g/lNa2HPO4·2H2O, 1.0 g/l KCl) was prepared. This medium was supplemented with different nitrogen sources, both inorganic [(NH4)3PO4, 0.3 g/lplus NH4NO3, 0.2 g/l] and organic (histidine, proline, arginine, phenylalanine, tryptophan or tyrosine) at two concentrations (0.05 g/l or 0.3 g/l),and different carbon sources (sucrose, glucose, maltose, arabinose or fructose) at three concentrations (10 g/l, 50 g/l or 150 g/l). A medium withsucrose (18 g/l) and glucose (1 g/l) was also tested. After a 10-day incubation period at 25 °C the highest levels of OTA (44.0 ng/ml, 13.5 ng/mland 0.49 ng/ml for A. ochraceus, A. carbonarius and A. tubingensis, respectively) were obtained in the cultures containing 150 g/l of arabinoseand 0.05 g/l of phenylalanine. Analysis of variance of the data showed that there were significant differences (p-value 0.05) among the OTA levelsin the cultures with regard to carbon source and isolate. No significant differences were detected in OTA production regarding nitrogen source,although 0.05 g/l of phenylalanine generally favoured OTA production in the cultures of the three isolates. The dynamics of toxin production inthe cultures of each isolate using the optimized basal medium supplemented with 0.05 g/l of phenylalanine and 150 g/l of arabinose for a period of42 days at 25 °C was also studied. The maximum level of OTA was detected on the 3rd day of incubation in A. tubingensis cultures and on the35th and 43rd days of incubation in A. ochraceus and A. carbonarius, respectively. This is the first study in which defined media have been usedto assess the influence of carbon and nitrogen sources on OTA production by isolates of OTA-producing species isolated from grapes and toanalyse the dynamics of toxin production in these species in a defined culture medium. This optimized medium for OTA production is being usedin current studies aimed at elucidating its biosynthetic pathway.© 2007 Published by Elsevier B.V.

Keywords: Ochratoxin A; Grapes; A. carbonarius; A. Tubingensis; A. ochraceus; Nitrogen source; Carbon source; OTA production

1. Introduction

Ochratoxin A (OTA) is a widespread mycotoxin producedby several Penicillium and Aspergillus species, mainly by P.verrucosum and P. nordicum (Pitt, 1987; Larsen et al., 2001),A. ochraceus and A. carbonarius (Varga et al., 1996; Abarcaet al., 2001; Bayman et al., 2002; Hesseltine et al., 1972)together with a low percentage of closely related species ofAspergillus such as A. niger (Abarca et al., 1994; Heenan

⁎ Corresponding author. Tel.: +34 963543144; fax: +34 963544570.E-mail address: Misericordia.Jimenez@uv.es (M. Jiménez).

0168-1605/$ - see front matter © 2007 Published by Elsevier B.V.doi:10.1016/j.ijfoodmicro.2007.11.055

et al., 1998) and A. tubingensis (Medina et al., 2005; Perroneet al., 2005).

Contamination of food and feed with mycotoxins poses ahigh risk for human and animal health. OTA exhibits intes-tinal fragility, nephrotoxicity, immunosuppresion, teratogeni-city, carcinogenicity (Bondy and Amstrong, 1998; Dirheimer,1998; Huff et al., 1980; Joint Food and AgricultureOrganization of the United Nations/World Health Organiza-tion Expert Committee on Food Additives, 1996; Lea et al.,1989), and cytotoxicity in hepatic cell lines (Bondy andAmstrong, 1998) and induces iron deficiency anaemia (Huffet al., 1979).

94 Á. Medina et al. / International Journal of Food Microbiology 122 (2008) 93–99

Furthermore, it might be involved in the human diseaseknown as Balkan Endemic Nephropathy and in the develop-ment of urinary tract tumours in humans (Abouzied et al.,2002). In 1993, the International Agency for Research onCancer (IARC) classified OTA into group 2B as a possiblehuman carcinogen (IARC, 1993).

Early detection of ochratoxigenic fungi on raw materials,feed and foodstuffs is important for the elimination of OTAfrom the food chain. Application of molecular methods isrequired to achieve this detection, but these methods can best beestablished when based on special genetic features of OTAproducers (Mühlencoert et al., 2004).

To elucidate the genes and enzymes involved in OTAbiosynthesis, it is necessary first to determine the environmentalfactors that regulate production of this mycotoxin.

In Aspergillus species, OTA synthesis depends on severalenvironmental factors and has been reported to be sporadic oreven lost when cultured in the laboratory (Varga et al., 1996).Some authors have reported the effects of culture conditionchanges on OTA production by Aspergillus strains. The resultsobtained were as diverse as the conditions applied. Changes inpH, composition of culture media, trace metals, and carbon (C)sources can have a remarkable influence (Medina et al., 2004,Mühlencoert et al., 2004).

The enzymatic pathways by which aspergilli and penicilliaproduce OTA have been studied. Chlorine is incorporateddirectly (Wei et al., 1971). Phenylalanine is incorporated toOTA (Ferreira and Pitout, 1969) and the polyketide residue issynthesized via the acetate–malonate pathway (Stayn andHolzapfel, 1970). A possible scheme leading to OTA biosynth-esis was published by Huff and Hamilton (1979), but recentlyHarris and Mantle (2001) proposed a different pathway. Theysuggest an unspecified pathway leading from ochratoxin β (de-chlorinated form of ochratoxin α) via ochratoxin α to OTA.

Lack of knowledge about the genetic background of OTAproduction as well as missing data on the phylogeneticrelationship between the relevant species makes it difficult todevelop assays with high specificity either to species or totoxigenic properties.

This current study was aimed at i) to examine the influenceof C and nitrogen (N) sources on OTA production by isolates ofthree producing species (A. ochraceus, A. carbonarius and A.tubingensis) isolated from grapes using defined media, and ii) tostudy the dynamics of toxin production in cultures of the 3isolates under the most favourable, previously selected,conditions. This study may be useful in further research intothe physiology and genetics of OTA-producing species and toknow the culture conditions determining OTA biosynthesis.

2. Materials and methods

2.1. Reagents and standards

The OTA standard was purchased from Sigma (Sigma-Aldrich, Alcobendas, Spain). Acetonitrile, ethyl acetate, aceticacid, methanol (all LC grade), phosphoric acid (85%, A.R.),sucrose and potassium chloride were from J.T. Baker (Deventer,

The Netherlands). Yeast extract, ferrous sulphate heptahydrate,ammonium phosphate, ammonium nitrate, magnesium sulphateheptahydrate and sodium phosphate dibasic dihydrate wereobtained from Panreac (Montcada i Reixac, Barcelona, Spain).Glucose, fructose, maltose and arabinose were purchased fromFluka (Sigma-Aldrich, Alcobendas, Spain). All the amino acidsassayed (histidine, proline, arginine, phenylalanine, tryptophanand tyrosine) were purchased from Sigma. Pure water wasobtained from a Milli-Q Plus apparatus (Millipore, Billerica,Ma., USA) and was used when water was required.

2.2. Fungi and basal medium

One isolate of each A. ochraceus (Aso2), A. carbonarius(Ac25) and A. tubingensis (Bo66), all obtained from grapes inour laboratory, were assayed. The A. tubingensis strain wasdeposited in the Spanish Collection for Type Cultures (CECT,University of Valencia, Spain) with reference No. 20544. Thiswas the same strain where OTA production by A. tubingensiswas first described (Medina et al., 2005). Before doing thisstudy, the three isolates had been characterised as OTAproducers in Yeast Extract Sucrose (YES) medium supplemen-ted with 5% bee pollen following the methodology described byMedina et al. (2004).

With a view to studying the influence of N and C source onOTA production by the different isolates, a basal medium (BM)for OTA production was designed. The BM was prepared bydissolving 0.01 g FeSO4·7H2O, 0.5 g MgSO4·7H2O, 0.5 gNa2HPO4·2H2O and 1 g KCl in 1 l of pure water and adjustingthe pH to 6.5. This medium was supplemented with differentorganic and inorganic N sources and different C sources (asdescribed in the following sections). These C and N sourceswere chemically pure compounds and therefore all the resultingmedia were defined media. An aw sprint RTD 502 unit(Novasina GmbH, Pfäffikon, Switzerland) was used to measurethe aw of culture media. The instrument was calibrated usingsalt solutions provided by the manufacturer.

2.3. Nitrogen sources

The BM was supplemented with different N sources:(NH4)3PO4, 0.3 g/l and NH4NO3, 0.2 g/l or amino acids(histidine, proline, arginine, phenylalanine, tryptophan ortyrosine) at two concentrations (0.05 g/l or 0.3 g/l). In addition,18 g/l of sucrose and 1 g/l glucose were added as C sources to allthese media. Before adding to the BM, solutions of the differentN sources were prepared in sterilised pure water and werefiltered through 0.2 µm Teflon filters. The BM containing the Csource was autoclaved at 115 °C for 30 min and cooled to about40 °C before the aseptic addition of the N source. The finalvolume of media in the 100-ml Erlenmeyer flasks was 50 ml.The media were inoculated with about 107 spores from fungalcultures made on Potato Dextrose Agar incubated for 7–10 daysat 25 °C. A spore suspension was prepared using salt solution(0.9% NaCl) and the concentration of spores was calculatedusing a Thoma counting chamber. The Erlenmeyer flasks wereincubated for 10 days at 25 °C in the dark.

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2.4. Carbon sources

The BM was supplemented with different C sources(sucrose, glucose, maltose, arabinose or fructose) at threeconcentrations (10 g/l, 50 g/l or 150 g/l). A medium withfructose (18 g/l) and glucose (1 g/l) was also tested. In addition,0.05 g/l phenylalanine was added to all these media as N source.The sugars were added before autoclaving the BM at 115 °C for30 min. Phenylalanine was added afterwards as stated in theprevious section. The final volume of media in each 100-mlErlenmeyer flask was 50 ml. The flasks were inoculated,incubated as indicated in the previous section, and analysed forOTA.

2.5. OTA determination

After incubation, 20 ml of culture medium was filteredthrough No. 4 Whatman paper and tested for OTA by a rapiddetection method optimised in our laboratory. The filteredsample was extracted with 15 ml ethyl acetate (10+ 5 ml) afteracidification with 0.5 ml 1 M phosphoric acid. The water phasewas discarded and the organic phases were combined andevaporated to dryness in a rotary evaporator. The residue wasredissolved in 250 µl of water–acetonitrile–acetic acid(55:44:1, v/v/v), which was also the composition of the mobilephase used in the liquid chromatography (LC) system. Onehundred µl was injected into the LC system.

2.6. Kinetics of OTA production

To study the dynamics of OTA production by each isolate themost favourable medium was selected. This medium was theBM supplemented with 150 g/l arabinose and 0.05 g/lphenylalanine prepared as described above. Three hundred mlof this medium was then aseptically poured into a 500-mlErlenmeyer flask. The sterile medium was then inoculated with6x107 spores and incubated at 25 °C for 6 weeks. All cultureswere assayed in triplicate for OTA accumulation at 3, 5, 7, 10,12, 15, 20, 27, 35 and 42 days after inoculation. On each day ofsampling 6 ml of medium was aseptically collected and filtered

Fig. 1. LC-FLD chromatogram showing OTA produced by A. carbonarius in Basal1 g/l KCl) supplemented with 10 g/l arabinose, 0.3 g/l (NH4)3PO4 and 0.2 g/l NH4N

from each flask. Five ml of the filtered sample was thenacidified with 0.2 ml of 1 M phosphoric acid after which it wasextracted twice, each time with 5 ml of ethyl acetate. The waterphase was discarded and the organic phases were reunified andevaporated to dryness in a rotary evaporator. The residue wasredissolved in 250 µl of water–acetonitrile–acetic acid(55:44:1, v/v/v) and 100 µl was injected into the LC system.

Blanks consisting in liquid sterilized media withoutinoculums incubated in the same conditions were used tocontrol each experiment.

2.7. LC system

The LC system consisted of a Waters 600 pump equippedwith a Waters 717 automatic injector and a Waters 474 scanningfluorescence detector (FLD) (Waters Co., Milford, MA, USA).AWaters 996 photodiode array detector was usually set after thefluorescence detector. Millennium 32® software version 4.0(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 μm particle size) connected to a guard column (4×4 mm,5 μm particle size) (Agilent Technologies, Waldbronn,Germany) filled with the same phase. The mobile phasecomposition was acetonitrile–water–acetic acid, 58:40:2 (v/v/v)and the flow rate was 1 ml/min. The FLD excitation and emissionwavelengths were 330 and 460 nm, respectively. The columnwas kept at 35 °C.

The recovery rate of the method was (74–86%) and the limitof detection was 0.01 ng OTA/ml medium, based on a signal-to-noise ratio of 3:1. The limit of quantification was 0.03 ng OTA/ml medium.

2.8. Statistical analysis

The statistical analysis was performed using StatgraphicsCenturion XV version 15.1.02 (StatPoint, Virginia, USA).Analysis of variance (ANOVA) was used and significantdifferences among mean values were reported at p-valueb0.05.Tukey-honestly significant difference (Tukey-HSD) multiple

Medium (0.01 g/l FeSO4·7H2O; 0.5 g/l MgSO4·7H2O; 0.5 g/l Na2HPO4·2H2O;O3.

Table 1OTA production by Aspergillus spp. isolated from grapes cultured in a definedmedium supplemented with different N sources

N source a [g/l] OTA (ng/ml)

A. ochraceus(Aso2)

A. carbonarius(Ac25)

A. tubingensis(Bo66)

(NH4)3PO4+NH4NO3

0.30+0.20 20.8±0.7 2.13±0.03 0.34±0.02

Histidine 0.05 15.8±0.5 1.07±0.04 0.073±0.025Proline 0.05 17.3±0.4 1.62±0.03 0.34±0.04Arginine 0.05 21.9±0.6 4.02±0.08 0.41±0.03Phenylalanine 0.05 23.2±0.6 4.12±0.06 0.86±0.07Tryptophan 0.05 21.9±0.9 3.45±0.06 0.076±0.021Tyrosine 0.05 22.6±0.5 3.86±0.03 0.044±0.011Histidine 0.30 16.2±0.6 1.34±0.05 0.18±0.03Proline 0.30 16.6±0.7 2.84±0.04 0.14±0.04Arginine 0.30 15.7±0.5 3.99±0.06 0.073±0.015Phenylalanine 0.30 19.7±0.8 1.87±0.08 0.093±0.017Tryptophan 0.30 19.9±0.4 1.79±0.07 0.041±0.012Tyrosine 0.30 18.8±0.8 1.45±0.07 0.043±0.011

Results are mean of three replicates±standard deviation.a N source was added to a basal medium (0.01 g FeSO4·7H2O; 0.5 g

MgSO4·7H2O; 0.5 g Na2HPO4·2H2O; 1 g KCl) supplemented with 1 g glucoseand 18 g sucrose in 1 l of pure water (pH was adjusted to 6.5). Inoculated mediawere cultured at 25 °C for 10 days.

Table 2OTA production by Aspergillus spp. isolated from grapes cultured in a definedmedium supplemented with different C sources

C source a [g/l] OTA (ng/ml)

A. ochraceus(Aso2)

A. carbonarius(Ac25)

A. tubingensis(Bo66)

Sucrose+Glucose 18+1 20.8±0.9 2.13±0.04 0.34±0.03Sucrose 10 1.42±0.04 2.14±0.05 0.15±0.06Glucose 10 4.87±0.06 1.85±0.07 0.13±0.05Maltose 10 9.44±0.04 1.65±0.08 0.11±0.03Arabinose 10 3.29±0.09 2.12±0.06 0.19±0.05Fructose 10 2.95±0.08 0.34±0.03 0.091±0.016Sucrose 50 13.15±0.25 0.82±0.03 0.29±0.05Glucose 50 15.11±0.23 5.36±0.06 0.21±0.06Maltose 50 9.00±0.12 3.62±0.04 0.19±0.04Arabinose 50 19.9±0.3 9.33±0.09 0.35±0.08Fructose 50 7.95±0.18 1.41±0.03 0.11±0.04Sucrose 150 14.06±0.23 1.32±0.04 0.33±0.05Glucose 150 35.3±0.5 6.31±0.06 0.26±0.06Maltose 150 14.07±0.22 4.52±0.05 0.28±0.04Arabinose 150 43.9±0.4 13.5±0.3 0.49±0.09Fructose 150 8.21±0.13 2.30±0.03 0.20±0.03

Results are mean of three replicates±standard deviation.a C source was added to a basal medium (0.01 g FeSO4·7H2O; 0.5 g

MgSO4·7H2O; 0.5 g Na2HPO4·2H2O; 1 g KCl) supplemented with 0.05 gphenylalanine and brought to 1 l with pure water (pH was adjusted to 6.5).Inoculated media were cultured at 25 °C for 10 days.

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range test at 95% confidence level was used to group the casesinto homogeneous groups with regard to the differentparameters used.

3. Results

3.1. Influence of C and N source on OTA production

Many experiments (data not shown) were required to designa minimally defined medium, to permit fungal growth and OTAproduction by all the isolates used after being supplementedwith the different N and C sources assayed. The combination ofnutrients used in this study fulfilled both requirements.However, OTA concentrations in the fungal cultures preparedon the defined media were lower than those observed oncomplex media such as YES supplemented with bee pollen(Medina et al., 2004) designed to optimise OTA production inA. ochraceus cultures. Nevertheless, the simplicity of themedia used here permitted OTA extraction from large matrixvolumes (20 ml) without this being a problem in thechromatographic analysis where high injection volumes(100 µl) were also used. No interference with OTA wasobserved in the chromatograms (Fig. 1).

Table 1 shows the OTA levels found in the differentexperiments carried out to examine the influence of concentra-tion and type of N source on OTA production by the threeisolates. Although ANOVA (p=95%) of the data showed thatthere were no significant differences in toxin production inrelation to the N source, some results are remarkable. In A.ochraceus the highest OTA production (23.2 ng/ml) by thisspecies was obtained when 0.05 g of phenylalanine/l was added.The levels of OTA produced, 23.2, 22.6, 21.9 and 21.9 ng/ml,when phenylalanine, tyrosine, arginine and tryptophan, respec-

tively, were added at the lowest concentration (0.05 g/l), slightlyexceeded the levels of toxin found (20.8 ng/ml) when inorganicN sources [(NH4)3PO4, 0.3 g/l plus NH4NO3, 0.2 g/l] wereused. The reverse occurred for histidine and proline, as OTAproduction was somewhat lower in the media spiked with thesetwo amino acids. In general, the OTA levels found in A.ochraceus cultures in relation to N source varied between15.7 ng/ml and 23.2 ng/ml.

In A. carbonarius cultures, the lowest concentration(0.05 g/l) of the amino acids phenylalanine, arginine, tyrosineand tryptophan increased OTA production (4.12, 4.02, 3.86and 3.45 ng/ml, respectively) while arginine and prolinefavoured OTA production at the highest concentration (0.3 g/l). The highest level of OTA in the cultures of this species(4.12 ng/ml) was detected when 0.05 g of phenylalanine/l wasadded to the medium. In A. carbonarius cultures, the OTAlevels detected in the tests studying the influence of N sourceon production of the toxin varied between 1.07 ng/ml and4.12 ng/ml.

A. tubingensis (Bo66) produced the lowest amounts of OTAunder all the conditions assayed. The highest concentration oftoxin (0.86 ng/ml) was detected in the cultures with 0.05 g ofphenylalanine/l. OTA concentrations in the cultures of thisspecies in relation to the N source varied between 0.04 ng/mland 0.86 ng/ml.

It was found that when inorganic salts were used as the soleN source, the three isolates produced intermediate OTA levelswith regard to the levels found for the amino acids. The resultswere closely dependent on the isolate. A. ochraceus producedthe highest OTA levels and A. tubingensis the lowest.Phenylalanine added at 0.05 g/l produced the highest OTAlevels in the cultures of the three strains. The fungi can use

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indiscriminately either inorganic or amino acidic N tobiosynthesize OTA.

Table 2 shows the OTA concentrations determined in theexperiments carried out with the different sugars. The influenceof the presence in the culture medium of a disaccharide (sucroseor maltose) or a monosaccharide (glucose, arabinose or fructose)on OTA production was studied. A medium added with sucrose(18 g/l) and glucose (1 g/l) was also included as control. TheANOVA showed significant differences (p-valueV0.05) in OTAproduction depending on concentration and type of sugar used asthe sole C source. Differences were also observed in relation tothe isolate. In general, the highest OTA levels, irrespective of theisolate, were found in the media supplemented with 150 g/l ofarabinose. Toxin concentrations in A. ochraceus, A. carbo-narius and A. tubingensis cultures were 44.0 ng/ml, 13.5 ng/ml and 0.49 ng/ml, respectively, at this concentration ofarabinose. After arabinose, the highest OTA levels were foundin cultures of A. ochraceus and A. carbonarius containingglucose at concentrations of 50 and 150 g/l. In A. tubingensiscultures, sucrose produced the second highest OTA level. Ingeneral, fructose was the least favourable sugar for OTAproduction by the three isolates.

The range for aw-values in the culture media was 0.999–0.986 depending of the sugar added and its concentration.

3.2. Kinetics of OTA production

To perform this experiment, a combination of nutrientswas chosen which, in general, encouraged OTA production bythe three isolates of Aspergillus spp. used in this study. The

Fig. 2. Evolution of OTA accumulation in cultures of Aspergillus spp. isolatedfrom grapes in BM supplemented with 0.05 g/l phenylalanine and 150 g/larabinose. Incubation temperature 25 °C.

chosen culture medium was the basal medium supplementedwith 150 g of arabinose/l and 0.05 g of phenylalanine/l. OTAproduction by each of the isolates over a 42-day period isshown in Fig. 2, where it can be seen that the kinetics oftoxin production is different for each isolate. A. tubingensiscultures showed maximum OTA levels on the third day ofincubation after which the level dropped sharply andremained low but with slight variations right to the end ofthe incubation period. The dynamics of OTA production in A.ochraceus and A. carbonarius were quite similar but differentto the dynamics observed in A. tubingensis, with themaximum toxin level for these two isolates found in thecultures assayed on the 35th and 42nd day, respectively. Inboth cases, OTA levels progressively increased over timethroughout the entire period studied.

In general, as Fig. 2 shows, OTA concentrations in samplestaken at different times over the whole incubation period werehigher in A. ochraceus cultures followed by those found in A.carbonarius and A. tubingensis, in this order.

4. Discussion

Different authors have studied the influence of cultureconditions on the production of OTA by producing species(Parra and Magan, 2005; Cairns-Fuller et al., 2005; Ribeiro etal., 2006), and some of these studies have focused specificallyon finding a culture medium that favours toxin production by A.ochraceus (Medina et al., 2004). These studies proposeundefined or complex media and it is not possible to studythe influence of specific nutrients, so that their use in metabolicand biosynthetic pathway studies is problematic. There are veryfew studies which use defined media prepared only withchemically pure nutrients (Ferreira and Pitout, 1969; Lai et al.,1970; Mühlencoert et al., 2004). These media are very useful inmetabolic and biosynthetic studies, although, in general, thelevel of metabolites produced by the microorganism is alwayslower than that produced in complex media where they haveabundant nutrients and growth factors. In this study, definedmedia are used and the influence of C and N sources on OTAproduction by three isolates of Aspergillus spp. is studied.Although A. ochraceus has a low incidence rate in grape(Medina et al., 2005; Chulze et al., 2006), it has a high OTA-producing capacity and is often isolated from other substratessuch as cereals (Mateo et al., 2004). The importance of thisspecies together with others that have often been isolated fromgrape and described more recently as OTA producers, such asA. carbonarius (Codex Alimentarius Commission, 1999) andA. tubingensis (Medina et al., 2005; Perrone et al., 2005), hasgiven rise to the need for studies aimed at finding theirbiosynthetic pathways and genetics.

OTA production by modifying the culture medium withdifferent N sources was done by Ferreira and Pitout (1969).These authors obtained the best toxin yields (100 mg OTA/l)using NH4NO3 or organic nitrogen sources, such as glutamicacid or proline. Our results indicate that other amino acids suchas phenylalanine can improve the amount of OTA producedcompared to those when proline is used (Table 1).

98 Á. Medina et al. / International Journal of Food Microbiology 122 (2008) 93–99

On the other hand, Mühlencoert et al. (2004) demonstratedthat if A. ochraceus (NRRL 3174) was provided with(NH4)2SO4 rather than with NH4NO3 as the sole N source,then the amount of OTA was reduced by 25% withoutmodification of the mycelial dry weight. In our experiments,the simultaneous use of (NH4)3PO4 and NH4NO3 as inorganic Nsources did not provide the best OTAyields, although acceptablelevels were generally obtained with that combination.

In relation to the C source, the results obtained in the presentstudy suggest that significant differences (p-valueV0.05) inOTA production by the different isolates are observed with theC sources used. Ferreira and Pitout (1969) also studied theinfluence of various C sources on OTA biosynthesis. Thehighest yields of toxin were obtained with sucrose andconsiderably less with glucose. When fructose or lactose wasused as C source no OTAwas detected in the cultures, and theyconcluded that these sugars do not lead to OTA production. Oneyear later, Lai et al. (1970) observed that in modified Adey-Mateles synthetic medium (AM medium) (Adey and Mateles,1964) at pH 7 highest OTA production by A. sulphureus (NRRL4077) occurred with glucose and sucrose as C sources.Mühlencoert et al. (2004) tested glucose and sucrose in AMmedium using A. ochraceus (NRRL 3174). The results obtainedindicated that the amount of OTA increased when sucrose wasused but OTA production was delayed in time. The resultsobtained in our work with these sugars only partially agree withthose obtained by the above authors.

The combination of glucose (1 g/l) and sucrose (18 g/l)usually gave higher OTA production when the sugars wereadded individually at a concentration of 50 g/l. This may be dueto the fact that glucose is an easily assimilated sugar whichproduces a faster increase in the growth of the fungus and thusgreater accumulation of OTA in the medium after 10 days.

The analytical methodology for determining the toxin hasimproved noticeably over recent years (Maier et al., 2004;Bacaloni et al., 2005; Medina et al., 2006; Aresta et al., 2006)permitting lower detection limits. Although in this workfructose provided lower levels of toxin in the medium,production was not totally inhibited and this is important interms of food safety. In our study, the sugar that provided thehighest toxin levels in the cultures of the three isolates wasarabinose with the lowest levels given by fructose.

As Table 2 shows, the use of higher concentrations (150 g/l)of sugar in general encouraged OTA production, consideringthat the cultures used to study the influence of C source and itsconcentration on OTA production were only 10-day old, thisincreased production can hardly be explained as being due toexhaustion of the C source in the cultures with 50 g/l of thedifferent sugars. Increase in OTA production was explained forAspergillus section Nigri on the basis of the stress undergone byfungi when aw decreases (Bellí et al., 2004). However, in ourcase, addition of C sources at the different concentrationsassayed produced only small differences in aw-values (0.999–0.986) and water availability does not seem to be a problem forthe fungi. Thus, the differences found in OTA level in culturescan be related to the nature of the sugars. Some factorsuncontrolled in our experiments and linked to the isolate may

also influence. Arabinose, at the highest level assayed,positively influences OTA production in the cultures of thethree isolates.

Due to their utility in industrial applications, many of thegenes whose products are involved in the degradation of plantcell wall polysaccharides, like arabinose, have been cloned andcharacterized in detail from Aspergillus. The capacity of theisolates included in this study to use arabinose as the sole Csource is very important not only in terms of OTA productionbut also in terms of their capacity for vegetable colonisation.

The dynamics of OTA production in A. ochraceus, A.carbonarius and A. tubingensis has been studied in general andin particular in a defined culture medium for the first time. Theincubation period during which the maximum levels of toxinwere found in the culture medium, the evolution of productionover time and toxin concentrations recorded in the medium overthe whole period vary significantly in the different isolates ofthe three assayed species of Aspergillus. This particularbehaviour in fungal isolates in terms of mycotoxin productionhas been described previously for other fungal species (Llorenset al., 2004). Significant differences were observed in produc-tion not only between isolates of different species but alsobetween isolates of a same species. However we have made ourstudies over time. With regard to the decrease in OTAconcentration once reached the maximum level, it may be dueto degradation produced by the fungi, which has been reportedin Aspergillus section Nigri species by Bejaoui et al. (2006).

Physiological and genetic studies to explain the OTA-producing capacity of the different isolates are required. Thiswould explain the distribution of these fungi and, in many cases,the specificity of the guest for which the media used in thisstudy could prove useful.

Acknowledgements

The authors are grateful to the Spanish Ministry forEducation and Science for the financial support (ProjectAGL-2004-07549-C05-02/ALI and a research grant). Theauthors would also like to thank the Valencian RegionalGovernment (Conselleria d'Empresa, Universitat i Ciencia) forfinancing a research grant.

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