International Journal of Food Microbiology 145 (2011) 229–237

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International Journal of Food Microbiology

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Influence of light on food relevant fungi with emphasis on ochratoxinproducing species

Markus Schmidt-Heydt a, Corinna Rüfer a, Frank Raupp a, Anja Bruchmann a,Giancarlo Perrone b, Rolf Geisen a,⁎a Max Rubner Institut, Department for Safety and Quality of Fruit and Vegetables, Haid-und-Neu-Str. 9, 76131 Karlsruhe, Germanyb Institute of Sciences of Food Production, National Research Council (ISPA-CNR), Via Amendola 122/O, 70126 Bari, Italy

⁎ Corresponding author. Tel.: +49 721 6625 450; faxE-mail address: rolf.geisen@mri.bund.de (R. Geisen)

0168-1605/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.ijfoodmicro.2010.12.022

a b s t r a c t

a r t i c l e i n f o

Article history:Received 17 August 2010Received in revised form 7 December 2010Accepted 22 December 2010

Keywords:Blue lightOchratoxinCitrininPenicilliumAspergillus

The influence of light of varying wavelength on growth and ochratoxin A biosynthesis of Aspergilluscarbonarius, A. niger, A. steynii and on Penicillium nordicum and P. verrucosum was analysed. For comparisonthe influence of light on various other food relevant fungi, including citrinin producers, was also tested.Generally the Aspergilli seem to be more resistant to light treatment than the Penicillia. Interestinglywavelengths from both sides of the spectrum, e. g. red (long wavelength, 627 nm) and blue (short wavelength470–455 nm) had the strongest inhibitory effects on growth and ochratoxin A biosynthesis. Blue lightgenerally had a stronger effect. Light of moderate wavelength, 590 to 530 nm, (yellow to green) had more apositive than a negative influence on growth or ochratoxin A biosynthesis compared to the control (darkincubation). The light effect on growth and ochratoxin A biosynthesis was dependent on the growth medium.In contrast to malt extract medium (MEA), YES medium, as an especially nutrient rich medium, had anattenuating effect on the reactivity against light. However the tendency of the response in both media was thesame. Moreover, the light intensity strongly influences how the fungus reacts. Depending on the intensity andthe resistance of the species a complete cessation of growth and/or inhibition of ochratoxin A biosynthesiscould be achieved. Light irradiation has the opposite effect on ochratoxin A than citrinin, two mycotoxinswhich can be produced simultaneously in P. verrucosum. Citrinin was produced essentially under lightconditions which inhibited ochratoxin A biosynthesis. The same was true for a derivative of ochratoxin, inparticular a derivative of ochratoxin β in A. carbonarius. A. carbonarius produced high amounts of theochratoxin β derivative under blue light when the production of ochratoxin A was ceased at the mostinhibiting conditions used (MEA, royal blue light, 455 nm, 1700 lx). Light has a growth stalling but notinactivating effect on aerial mycelia. If a non-growing colony under light is shifted to the dark it immediatelygrows normally. However on spores blue light has a deactivating effect. After incubation of spores ofP. verrucosum for 24 h under blue light up to 97% of the spores were no longer able to germinate. Again thespores of the Aspergilli were much more resistant.

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1. Introduction

Ochratoxin A is a nephrotoxic mycotoxin produced by severalspecies of the genera Aspergillus and Penicillium. Important producingspecies of the Aspergilli are A. ochraceus, from which the toxin wasfirst isolated (van der Merwe et al., 1965), A. carbonarius and to alesser extent A. niger. Recently A. steynii and A. westerdijkiaewere splitas separate species from A. ochraceus. Both are strong and consistentochratoxin A producing species with respect to the toxigenicity ofA. ochraceus. The Aspergilli are mainly responsible for the occurrenceof ochratoxin A on food commodities such as grapes or grape products(A. carbonarius, A. niger), spices, coffee and cocoa (A. ochraceus,

A. westerdijkiae, A. steynii, A. carbonarius). On the other hand twospecies of the genus Penicillium, namely P. verrucosum and P. nordicumare able to produce the toxin as well. P. verrucosum is solelyresponsible for the occurrence of ochratoxin A in cereals and productsthereof, whereas P. nordicum occurs as a contaminant in NaCl richfermented foods such as ham, cheeses or olives (Heperkan et al., 2009;Lund and Frisvad, 2003). P. verrucosum is able to produce citrininbeside ochratoxin A. Another important citrinin producer isP. expansum. The production of ochratoxin and citrinin is somewhatinterdependent (Larsen et al., 2001) because both pathways use thesame precursors.

It was recently shown that, at least in Penicillium, the production ofochratoxin A is circadian regulated (Schmidt-Heydt et al., 2010). Theproduction of ochratoxin A is high during the dark phases and lowunder light conditions. Under these conditions even a partial

230 M. Schmidt-Heydt et al. / International Journal of Food Microbiology 145 (2011) 229–237

degradation of ochratoxin A could be observed. This was attributed toa toxic effect of ochratoxin A on the production organism itself underincubation in light. The intensity of the light was important to exert aneffect. Also other researches observed that light can influence theproduction of secondary metabolites. Häggblom and Unestam (1979)for example showed that the production of Alternaria toxins can bereduced under blue light. They found a reduction between 69 and 77%of alternariol and alternariol methyl ether. Red light gave no reductionin both toxins in their analysis. Häggblom and Niehaus (1986)showed that light pulses are sufficient to reduce polyketidebiosynthesis in Alternaria. In this analysis also a reduction ofalternariol methyl ether by blue light was reported.

The influence of light on mycotoxin biosynthesis has been analysedon the molecular level also. The product of the veA gene, which is aregulatory protein, is transported into the nucleus during incubation inthe dark (Calvo et al., 2002; Stinnet et al., 2007). In concert with lightreceptors (lreA, lreB; wc1, and wc2) it activates secondary metaboliteproduction during the dark (Bayram et al., 2008; Purschwitz et al.,2008). These results clearly demonstrate the influence of light onsecondary metabolite production. However despite this knowledgeabout the influence of light/dark incubation conditions on mycotoxinbiosynthesis no comprehensive systematic analyses of the light effectson various food-borne fungi have been performed.

During this work the influence of wavelength and light intensity ongermination, growth and toxin biosynthesis has been carried out indetail. Emphasis was laid on ochratoxin A producing Aspergilli andochratoxin A and citrinin producing Penicillia and differences that couldbe found in the reaction behaviour betweenboth species. In addition theinfluence of the light conditions used here were also tested on otherimportant food relevant mycotoxin producing species like A. parasiticus(aflatoxin) F. graminearum (DON) and Alternaria alternata (alternariolmethyl ether), however these data are only briefly described.

2. Materials and methods

2.1. Strains and growth conditions

A. carbonarius ITEM5008 (ITEM: Agri-Food Toxigenic Fungi CultureCollection of the Institute of Sciences of Food Production, CNR, Bari,Italy), A. steynii STE3.53 (STE: strain collection of the Department ofGenetics, Complutense University of Madrid, Spain), A. parasiticusBFE96p, P. nordicumBFE487, P. verrucosumBFE808, P. expansumBFE189,F. graminearum BFE1006, A. alternata BFE376 (BFE: strain collectionof the Max Rubner Institut, Germany), were grown on YES agar(20 g/l yeast extract, 150 g/l sucrose, 15 g/l agar) and on malt extractmedium (MEA, Merck, Darmstadt, Germany) for the determinationof the light influence on growth and toxin biosynthesis. The cultureswere incubated at 20 °C for at least 5 days under controlled lightconditions.

2.2. The light incubation equipment

Three light boxes were constructed to enable incubation of thecultures under differentwavelengths of light, and different intensities ofroyal blue light respectively. Light box 1 was subdivided into 6chambers. Each chamber was equipped with 5 Luxeon high power5 W Light Emitting Diodes (Philips Lumileds Lighting Company, SanJose, California, USA)with the followingdifferent emittingwavelengths:chamber 1, royal blue (455 nm, 3350 lx); chamber 2, blue (470 nm,2357 lx); chamber 3, green (530 nm, 7250 lx); chamber 4, yellow(590 nm, 6400 lx); chamber 5, red (627 nm, 7700 lx); and chamber 6,white light (17,750 lx). The distance between the LEDs and the agarplateswas 18 cm. No heating effect by the LEDs could be detected at thisdistance. An irradiance of 3.8 mW/cm2 was reached under theseconditions. A dark chamber was used as a control. For the royal bluewavelength two different light intensities were used. This was achieved

by using 5 LEDs to produce low light intensity (light box 2) and 25 LEDsto produce high intensity (light box 3) irradiating an area of 660 cm2.The light intensities of these boxes were measured with a Testo 435luminometer (TestoAG, Lenzkirch, Germany) at a distance of 18 cmandwere 200 lx in light box 2 and 1700 lx in light box 3. According to acalculation formula provided by the manufacturer of the LED, the Luxintensities measured by the luminometer fitted very well with thecalculated datawhen the distance of 18 cmand the area of irradiation of660 cm2 were included. The boxes had sufficient capacity for airexchange and were placed in a temperature and humidity controlledroom. Agar plates were single point inoculated with a spore suspension(106spores/ml) from a 5 day old culture, grown on the same mediumand placed immediately into the light box without further preincuba-tion. YES and MEA were used as inoculation media. Inoculated agarplates were placed into the boxes and incubated at 20 °C and a relativehumidity of 85% for different time periods.

2.3. Thin layer chromatographic (TLC) and HPLC/MS/MS analysis

For determination of mycotoxin production, the strains weregrown at 20 °C for 5 days at the respective experimental conditions.Agar plugs (250 mg) were taken from the region between centre andedge of the colonywith the aid of a sterile corer. These agar plugs withthe adhering mycelium were transferred into 2 ml micro reactiontubes and 1 ml of chloroform was added. Triplicates of the fungalmycelia were extracted for 30 min at room temperature on a rotaryshaker; the mycelia were discarded and the chloroform extract wasevaporated to dryness in a vacuum concentrator (Speed Vac, SavantInstruments, Farmingdale, USA). This extraction was carried out atleast three times for each sample. The residues were redissolved in20 μl methanol and spotted onto TLC plates (Silica gel 60, Merck,Darmstadt, Germany). The limit of detection for ochratoxin A was2 ng/250 mg. As mobile phase for the determination of therespective mycotoxin the following compositions were used. Forochratoxin: toluol:methanol:acetic acid (90:5:5, [v:v:v]); for citrinin:toluol: ethylacetate:formic acid (60:30:10, [v:v:v]); for aflatoxin:toluol:ethylacetate:acetic acid (50:30:4, [v:v:v]); for deoxynivalenol:toluol:methanol:acetone (40:20:10, [v:v:v]) and for alternariolmonomethyl ether: toluol:ethylacetate:formic acid (60:35:5, [v:v:v]). The respective puremycotoxin (Sigma, St. Louis, USA)was used asstandard. The spots were visualized under UV light (366 nm). Fordetermination of DON the TLC plate was first sprayed with 20% AlCl3(methanol:water 50:50 [v:v]) and backed for 10 min at 100 °C forderivatisation. For quantitative determination of ochratoxin A byHPLC/LCMS-MS, the residues were redissolved in methanol andsubjected to HPLC according to the following method: HPLC analysiswas performed on an HP 1200 system (Agilent TechnologiesWaldbronn, Germany) equipped with an auto-injector, column ovenand fluorescence detector. The auto-injector was set to 10 °C and thecolumn oven to 25 °C. Separation was carried out on a Prontosil(150 mm x4 mm i.d., particle size 3 μm) reversed-phase column(Bischoff, Leonberg, Germany). Solvent A consisted of 0.1% formicacid in water (pH 3) and solvent B of acetonitrile. A linear gradientwas used: from 10 to 100% B in 30 min. The flow rate was set to1.0 mL/min, and the injection volume was 60 to 100 μL. The HPLCsystem was directly coupled to a hybrid triple-quadrupole/linear iontrapmass spectrometer (QTrap® 3200; Applied Biosystems, Darmstadt,Germany) equipped with a TurboIonSpray source. Analytes weredetected in the positive ion mode at a vaporizer temperature of600 °C and ion spray voltage of 4.5 kV. Spectral datawere recordedwithN2 (CAD=4) as collision gas and a declustering potential of 50 V. Dataacquisition was performed in theMRMmodemonitoring the transitionof [M+H]+m/z 404 in Q1 tom/z 239 in Q3 as quantifier and tom/z 102in Q3 as qualifier. The collision energies were 31 and 97 V, respectively.Quantification was performed by external calibration using thecommercially available reference compound. A calibration curve was

231M. Schmidt-Heydt et al. / International Journal of Food Microbiology 145 (2011) 229–237

constructed in the range of 325 pg/ml to 13 mg/ml inwhich the linearityof the response was given (R2=0.9969). The limit of detection was 7 pgon column. Data collection and handling was done with Analyst 1.5.

2.4. HPLC/MS/MS determination of the ochratoxin β derivative

For HPLC, the same conditions as described above were used. Thefluorescence detection settings were an excitation wavelength of336 nm and an emission wavelength of 530 nm. For MS/MS analysisthe analyte was detected in the positive ion mode at a vaporizertemperature of 600 °C and ion spray voltage of 5.5 kV. Spectral datawere recorded with N2 (CAD=4) as collision gas, a declusteringpotential of 30 V and a collision energy of 20 V. Data acquisition wasperformed in the EMS as well as EPI mode. Spectra were scanned overa mass range of m/z 100–600.

2.5. Growth assessment

For analysing growth and morphology the strains were singlepoint inoculated on MEA, grown for 5 days under the respective light

Fig. 1. Morphology of various food borne fungi in response to different light colours as indicdark, w = white, r = red, y = yellow, g = green, rb = royal blue.

conditions and photographed. The diameters of the colonies weremeasured in two directions at right angles to each other. Threereplicates were measured.

2.6. Inactivation of spores

To determine the ability of light to irreversibly inactivate spores ofselected ochratoxigenic fungi, a spore suspension of the respectivefungus with a concentration of 107spores/ml was incubated in 1 ml ofliquid YES medium in tissue culture plates with 24 wells (Greiner bio-one, Kremsmünster, Austria). The culture plates were then incubatedin either the light or the dark as a control. After 24 h of incubation thespore suspension was diluted by 5 logs down to a minimalconcentration of 102spores/ml and the dilutions were plated out onsolid YES agar plates and incubated at 25 °C for 5 days to enablesurviving spores to germinate. The resulting colonies were countedand compared with the control whose number of viable colonies wasset as 100%. These experiments were done at least three times and anaverage value is given.

ated. The fungi were either grown on MEA medium for 5 days and photographed. d =

Table 1

Species Toxin amount1 Light color

YES MEA

Ochratoxin producing Aspergilli

A. carbonarius ITEM5008 OTA/OTβD++++ OTA ++++ OTβD ++++ OTA − OTβD Dark+ OTA ++++ OTβD + OTA + OTβD White++ OTA ++++ OTβD ++ OTA ++ OTβD Red++ OTA ++++ OTβD +++ OTA +++ OTβD Yellow++ OTA ++++ OTβD ++ OTA ++ OTβD Green+ OTA ++++ OTβD − OTA ++++ OTβD Blue+ OTA ++++ OTβD − OTA +++ OTβD Royal blue

A. steynii STE3.53 OTA++++ ++ Dark++++ ++ White++++ ++ Red++++ +++ Yellow++++ +++ Green++++ + Blue++++ + Royal blue

A. niger BFE631 OTA+++ ++ Dark− − White− − Red++ +/− Yellow++ − Green− − Blue− − Royal blue

Ochratoxin and citrinin producing Penicillia

P. nordicum BFE847 OTA++++ ++++ Dark+/− − White+++ ++ Red++++ ++++ Yellow++++ ++++ Green+/− − Blue+/− − Royal blue

P. verrucosum BFE808 OTA/CIT++ OTA − CIT +/− OTA ++ CIT Dark− OTA − CIT − OTA − CIT White+ OTA − CIT − OTA − CIT Red++ OTA ++ CIT − OTA ++ CIT Yellow++ OTA − CIT − OTA ++ CIT Green− OTA − CIT − OTA ++ CIT Blue− OTA − CIT − OTA − CIT Royal blue

P. expansum BFE189 CIT+++ − Dark++++ ++++ White++++ ++ Red++ − Yellow+ − Green++++ +++++ Blue++++ +++++ Royal blue

Miscellaneous mycotoxin producing food borne fungi

A. parasiticus BFE96p AFL+++ +++ Dark+++ + White+++ + Red+++ ++ Yellow+++ +++ Green+/− − Blue+/− − Royal blue

A. alternata BFE376 AME++ + Dark+++ ++ White++ + Red+++ + Yellow+++ ++ Green+++ +/− Blue+++ + Royal blue

F. graminearum BFE1006 DON++ − Dark++++ + White

232 M. Schmidt-Heydt et al. / International Journal of Food Microbiology 145 (2011) 229–237

Table 1 (continued)

Species Toxin amount1 Light color

YES MEA

F. graminearum BFE1006 DON+ − Red+ − Yellow++ + Green++++ + Blue++++ + Royal blue

The number of crosses gives the amount of toxin produced, which was estimated visually. OTA = ochratoxin A, OTβD = OTβ derivative, CIT = citrinin, AFL = aflatoxin B1, AME =Alternariol methyl ether, DON = Deoxynivalenol.1: at least 3 replicates of the experiments were carried out.

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3. Results

3.1. Influence of light of different wavelengths on growth of various foodborne fungi

To gain an overview of the influence of various light wavelengths onthe growth of certain food relevant spoilage and toxin producing fungi,various species were inoculated on MEA and incubated at 20 °C for5 days under exposure of different light wavelengths (light box 1). Theresults are shown in Fig. 1. Several clear cut differences and regularitiescould be observed after this experiment. White light has a positiveinfluence on growth of most the Aspergilli tested (A. carbonariusITEM5008, A. niger BFE631) compared to growth under dark conditions(in terms of diameters, quantitative data support this view, but are notshown). Generally the Aspergilli are quite resistant against the activityof various light colours, however slight changes in the morphology ofthe colonies occurred. In particular, A. niger BFE631 did not sporulateunder royal blue light (455 nm) conditions.

The white light responsiveness of the Penicillia was quite contraryto that of the Aspergilli. For these species white light had an inhibitoryeffect on growth in comparison to growth in the dark. Red and bluelight also inhibited growth. In the case of P. verrucosum absolutely nogrowth occurred at 455 nm (royal blue). For the ochratoxigenicPenicillia no aerial mycelium but only substrate mycelium wasproduced under these conditions. Light at a wavelength of 590–530 nm (yellow to green) were much less inhibitory and seems tosupport increased sporulation. Interestingly P. expansum BFE189,which is a strong citrinin producer, was much better adapted to thesedifferent light conditions than the ochratoxin A producing Penicillia.

When the colonies grown under blue light were shifted to darkconditions, they immediately start to grow, sometimes even fastercompared to the dark control. They also started to produce ochratoxinA immediately (data not shown). This indicates that the lightconditions used in the described experiments only stall the growthof a colony without killing it.

Two food-borne species from other genera which were also tested,F. graminearum and A. alternata, were almost unaffected by the lighttreatment with respect to their growth and morphology. They alsocould grow better under white light compared to the dark andshowed a slight increased growth rate under short wavelength, e. g.under blue light (data not shown).

These results suggest that Penicillia, especially ochratoxin Aproducing Penicillia, are very sensitive to visible light of long andshort wavelength with respect to their growth and morphology. Inparticular, blue light leads to a drastic reduction in growth. Bycomparison, growth and morphology of the Aspergilli, Alternaria andFusarium are affected only weakly.

3.2. Influence of light of different wavelength on ochratoxin A biosynthesisby Aspergillus and on ochratoxin A and citrinin biosynthesis by Penicillium.

To test the influence of light of different wavelengths onochratoxin A and citrinin biosynthesis of the Aspergilli and the

Penicillia, samples from the same colonies tested for the growthability on YES and MEA were taken and subjected to TLC. The resultsare shown in Table 1. The amount of ochratoxin produced wasdependent on the growth substrate (either YES or MEA) and on thewavelength of the light. The effect of some light wavelengths onochratoxin biosynthesis was more pronounced on MEA compared toYES. In the case of A. niger BFE640, which is a weak ochratoxin Aproducer a clear effect could be seen. This strain produces moderateamounts of ochratoxin A on YES as well as on MEA medium in thedark. After incubation under white light the amount is drasticallyreduced on both media to non-detectable levels. The same situationcould be found after incubation under red, blue or royal blue light,Yellow and green light had only a slight reducing effect on YES and amoderate reducing effect on MEA. A. carbonarius ITEM5008 producedhigh amounts of ochratoxin A on YES and MEA medium in the dark,but only low amounts under white light on both media. However, inaddition to ochratoxin A, an ochratoxin β derivative (see below) wasproduced on YES and, dependent on the light conditions, on MEAmedium. This metabolite was confirmed and partially identified byHPLC/MS/MS analysis. Blue and royal blue light inhibited ochratoxin Abiosynthesis to low levels on YES and completely on MEA. Theproduction of the ochratoxin β derivative was not affected at all underdark and all light conditions on YES medium, indicating that growthon this medium supported its production. On MEA medium, lightobviously strongly activated ochratoxin β derivative synthesis, whilein the dark it was not detected. Its production increased withdecreasingwavelength on thismedium, being highest under exposureto blue light (470 nm), when there is no biosynthesis of ochratoxin A.This might indicate that the production of the ochratoxin β derivativeis a reaction against unfavorable light conditions (at least duringgrowth on MEA medium). The biosynthesis of ochratoxin A byA. steynii was quite insensitive to light after incubation on YESmedium. However on MEA yellow and green enhanced, and blue androyal blue reduced, ochratoxin biosynthesis.

As with growth the situation of the ochratoxin A reactivity of thePenicillia was similar but more pronounced because of the greatersensitivity to light of these species. P. nordicum produced highamounts of ochratoxin A in the dark but only scarce amounts underwhite light on YES and none onMEA. Again red light partially reducedthe production on both media, and blue, paticularly royal blue,stopped production completely on MEA. Green and yellow gave thesame amounts as incubation in the dark. The same behavior was truefor P. verrucosum after growth on YES, even though P. verrucosumseemed to bemore sensitive to light than P. nordicum because onMEAmedium ochratoxin A biosynthesis was completely inhibited with alllight wavelengths. However P. verrucosum was also able to producecitrinin. Growth on MEA, but not on YES, obviously induced citrininproduction in the dark. No citrinin was produced onMEA after growthunder white, red and royal blue light, but high amounts wereproduced under yellow, green and even blue light. The lattercondition otherwise lead to a complete cessation of ochratoxin Abiosynthesis on YES as well as onMEAmedium. This result shows thatthe citrinin production window in P. verrucosum is different from

Fig. 2. Representative HPLC chromatogram of the purified extract.

234 M. Schmidt-Heydt et al. / International Journal of Food Microbiology 145 (2011) 229–237

ochratoxin production with respect to different wavelengths. Thisdifference in light responsiveness between ochratoxin A and citrininwas even more pronounced in P. expansum which is a typical citrininproducing species and is not known to produce ochratoxin A.Interestingly in this species citrinin biosynthesis was induced underexactly the conditions which lead to cessation of ochratoxin Abiosynthesis, i.e. under white, red, blue and royal blue high amountsof citrinin were produced. This is somewhat different from thereaction of P. verrucosum where citrinin production was high underyellow and green light.

Red and blue light also had an inhibiting effect on aflatoxin B1production by the analyzed strain of A. parasiticus BFE96p and onalternariol methyl ether biosynthesis by A. alternata. However incontrast to these species F. graminearum showed a differentbehaviour. DON biosynthesis in F. graminearum seemed to beincreased under the exact conditions that inhibited ochratoxin A,i.e. blue and royal blue light. This indicates that DON biosynthesisis obviously not negatively regulated by light of shorter wave-length, which corresponds to the fact that growth of F. grami-nearum was also little affected. These observations could beconfirmed by results with other Fusarium toxins obtained underthe same light conditions (publication in preparation; FrancescaFanelli, personal communication).

3.3. HPLC/MS/MS characterization of the ochratoxin β derivative

The purified extracted TLC band was analyzed by reversed-phaseHPLC with fluorescence detection. A representative HPLC profile isdepicted in Fig. 2. One metabolite was clearly detectable. Theidentification of this metabolite was elucidated using HPLC/MS/MS.

Fig. 3. Mass spectrum of ochratoxin β derivative.

The positive mass spectrum displays a quasi-molecular ion [M+H]+ at m/z 301 (Fig. 3). Furthermore, the sodiated cation [M+Na]+ atm/z 323 can clearly be distinguished. The intense fragment ion [M+H]+

at m/z 238, derived by the loss of 63 amu, can be assigned as amethylated form of ochratoxin β (m/z 223). However, with the HPLC/MS/MS system used the position of the CH3-group in the isodehydro-coumarin moiety as well as the nature of the leaving group of 63 amuremainsunclear. Forfinal identificationNMRanalysis is necessary. In thisstudy we tentatively assign the peak as ochratoxin β derivative.

3.4. Influence of the light intensity on ochratoxin A biosynthesis inAspergilli and Penicillia

As described above blue light has an inhibiting effect onochratoxin A production by Penicillia and Aspergilli; however thelatter are more resistant. In the experiments described above byusing light box 1 only one light intensity was used for allwavelengths. To test the influence of the blue light intensityexposure devices were constructed (light box 2 and light box 3)which allowed incubation under weak (200 lx) or strong (1700 lx)royal blue light of the same wavelength used in light box 1. Thefungal strains were incubated on MEA agar plates for 5 days underthese light conditions and the ochratoxin produced was analyzed byTLC and quantified by HPLC-MS/MS. The ochratoxin biosynthesisprofiles in response to different light intensities were very similarfor all species (Fig. 4). In particular, P. nordicum, A. steynii andA. carbonarius produced high amounts of ochratoxin A whenincubated in the dark (Fig. 4). Because MEA medium was used assubstrate P. verrucosum produced ochratoxin A and citrinin afterincubation in the dark. Incubation under weak blue light (200 lx)resulted in a decrease in ochratoxin A biosynthesis in P. nordicum,A. carbonarius and A. steynii. P. verrucosum in contrast did notproduce any ochratoxin A under these conditions, but citrinin wasstill produced. When the intensity of blue light was increased from200 lx to 1700 lx, a further drastic reduction in secondarymetabolite biosynthesis occurred. In A. carbonarius only traces ofochratoxin A could be detected after TLC, whereas neitherP. nordicum and P. verrucosum nor A. steynii were able to produceany detectable amount of ochratoxin A. P. verrucosum did notproduce any detectable citrinin. In parallel to the reduction ofochratoxin A, A. carbonarius was however able to produce theochratoxin β derivative under these conditions.

The quantitative analysis of this reduction is shown in Table 2.Exposure of P. verrucosum to weak blue light (200 lx) resulted in areduction of ochratoxin A biosynthesis by a factor of 6, P. nordicumby a factor of 1.5, A. carbonarius by a factor of 3.5 and A. steynii by afactor of 7.6. A further increase in the light intensity to 1700 lx leadsto a complete abolition of detectable ochratoxin biosynthesis. OnlyA. carbonarius and P. nordicum were able to produce very lowamounts of ochratoxin A under these conditions, detectable only byHPLC/MS.

3.5. Influence of blue light on the viability of conidiospores of ochratoxinA producing Penicillia

As described above blue light halts the growth of aerial mycelia ofsensitive fungi. In order to test the influence of blue light on sporegermination 107 conidiospores of the respective fungus wereincubated under blue light (light box 1). After that incubation thesurviving spores were counted by dilution plating and compared withthe control which had been incubated in the dark. This analysis wasperformed with P. verrucosum as a species sensitive to blue light andA. carbonarius as a more resistant species. In the case of incubation ofthe spores in the dark no negative effect on the viability could beobserved. Nearly all of the P. verrucosum spores (97.2%) andA. carbonarius spores (98.9%) germinated and could outgrow to

Fig. 4. TLC chromatogram of the chloroform extracts of P. nordicum (A), P. verrucosum (B), A. steynii (C) and A. carbonarius (D) after growth on MEA for 5 days under royal blue light(455 nm) at different intensities. Three replicates are shown for each incubation conditions. The lane with the OTA standards is indicated. The colonies grown under the various lightconditions and used for extraction of ochratoxin A are shown for comparison. The TLC migration relationships of ochratoxin A, ochratoxin B and the ochratoxin β derivative areshown in D.

235M. Schmidt-Heydt et al. / International Journal of Food Microbiology 145 (2011) 229–237

colonies after plating on solid YES medium. If the P. verrucosum sporesuspension however was incubated under blue light (470 nm) only53.8% were able to outgrow to colonies. This inhibiting effect was evenmore pronounced by using blue light with a shorter wavelength (royalblue, 455 nm). In this case almost all of the spores were inactivatedresulting in a germination rate of 3%. In contrast, the blue lightincubation had amuch lower effect on the viability of the A. carbonariusspores. After treatment with royal blue light (455 nm) 63.8% of thespores were still able to outgrow to colonies on YES medium.

These results clearly show that light has a stalling effect on thegrowth of aerial mycelium, but has an inactivating effect ongerminating spores. This inactivation effect is dependent on thespecies, the wavelength and the intensity of light.

Table 2HPLC/MS/quantification of ochratoxin A from various species grown under differentblue light intensities.

Growthconditions

Ochratoxin A [ng/250 mg]

P. verrucosum P. nordicum A. steynii A. carbonarius

Dark 3.23 5257.77 3349.91 9041.66LED (200 lx) 0.49 3542.22 440.00 2589.33LED (1700 lx) nd 6.03 nd 91.37

nd = not detectable.

4. Discussion

The influence of light on growth, morphology and toxin produc-tion of ochratoxin A producing Aspergilli and Penicillia were analyzedduring this work. It has already been shown that light has an influenceon the production of toxins by A. alternata (Häggblom and Unestam,1979), on sterigmatocystin by A. nidulans (Bayram et al., 2008;Purschwitz et al., 2008) and on aflatoxin by A. parasiticus (Bennetet al., 1981). However no comprehensive systematic analyses aboutthis influence on ochratoxin A producing fungi exist. In the currentanalysis it was shown that light has an influence on growth,morphology and ochratoxin A biosynthesis in Aspergillus but moreso in Penicillium. Depending on light wavelength and intensity acomplete cessation of growth could be achieved for Penicillium.Moreover the biosynthesis of ochratoxin A could be completelyceased in Penicillium and Aspergillus.

As shown by Purschwitz et al. (2008), the Aspergilli carry a redand a blue light receptor. For P. nordicum and P. verrucosumhomologues of the blue light receptor have also already beenidentified (Dominic Stoll, Diploma thesis, University of Karlsruhe).The red light receptor of A. nidulans (FphA) has been characterized byBlumenstein et al. (2005). Sexual development is repressed in redlight. In combination with the blue light receptor (lreA, lreB) and veAand velB it forms a complex and can regulate expression of

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downstream genes in response to light (Bayram et al., 2008;Purschwitz et al., 2008).

In the current analysis it could be confirmed that especially redlight and blue light has an influence on growth and ochratoxinbiosynthesis, however the influence was genus dependent. Generallythe Aspergilli grow better after exposure to white light than in thedark. This was also true for some other species analyzed such asF. graminearum and A. alternata (data not shown). Häggblom andNiehaus (1987) also demonstrated that growth and conidiation ofA. alternata were not affected by light under the conditions used. Justthe opposite was true for the Penicillia analyzed. They grew better inthe dark compared to light incubation. The Penicillia, especially theochratoxin A producing species, were generally more sensitive thanall other species analyzed. Light of longwavelength (red, 627 nm) andof short wavelength (blue, 455–470 nm) lead to a reduction ingrowth. Light of moderate wavelength usually supported growth(590–530 nm, yellow to green).

In the case of ochratoxin biosynthesis again red and blue lightweremost effective. Blue light especially reduced or completely abolishedochratoxin A biosynthesis depending on the species treated. A clearcorrelation between intensity of the blue light and the inhibitoryeffect could be established. Weak royal blue light (455 nm; 200 lx)lead to a moderate reduction of ochratoxin A for all species tested. Afurther increase in the intensity of the royal blue light to 1700 lxhowever clearly further reduced ochratoxin A biosynthesis to eithernon-detectable levels (P. verrucosum, A. steynii) or to very lowamounts (A. carbonarius, P. nordicum). Taken together these resultsshow that light intensity is an important factor in inactivation ofochratoxin A biosynthesis in the different species and it can beassumed that the low production of ochratoxin A by A. carbonarius,and P. nordicum can be completely inhibited by further increasing theintensity of the blue light. Häggblom and Niehaus (1987) showed forA. alternata that the fluency rate (flux density) of the blue light playsan important role.

The production of citrinin by P. verrucosum is influenced by lightsomewhat differently from ochratoxin A. In contrast to ochratoxin A,blue light leads to an increased production of citrinin in P. verrucosum.This behavior is even more pronounced in P. expansum, a typicalcitrinin producing species. In P. expansum citrinin production is highlyinduced under exactly the conditions which inhibit ochratoxin Abiosynthesis in the analyzed Penicillia, i.e. under white, red, blue androyal blue light. Stormer et al. (1998) hypothesized that citrinin mayact as a sun protectant. Interestingly growth of P. expansum is muchless affected under blue light compared to growth of the citrinin non-producing P. nordicum. Moreover also P. verrucosum could grow betterafter exposure to blue light (470 nm) where it produces citrinin,compared to the non-producing P. nordicum. This was demonstratedby analysing the diameter of the colonies of both species (data notshown). This suggests some kind of protection by the production ofcitrinin. A similar mutual regulation between two secondarymetabolites by the effect of light could be observed in A. carbonariusafter growth on MEA. A. carbonarius ITEM5008 showed a reducedochratoxin A production under certain conditions (blue light, MEA)but produced high amounts of an ochratoxin β derivative instead. Thisderivative is not a degradation product of ochratoxin A, becauseexperimental degradation of ochratoxin A under light conditions leadto fragmentation products with chromatographic characteristicsdifferent from the ochratoxin β derivative (data not shown). Inparallel to the situation in Penicillium it might be possible thatA. carbonarius has developed this shift from ochratoxin A to theochratoxin β derivative for protection against strong light exposure.This hypothesis is also supported by the fact that the growth rate ofA. carbonarius increases with the intensity of the blue light (Fig. 2D).For ochratoxin producing Penicillia it was shown that the productionof ochratoxin A, but not B is unfavorable under intense lightconditions (Schmidt-Heydt et al., 2010). Under these conditions

much more ochratoxin B is produced. Ochratoxin B is much less toxiccompared to ochratoxin A (Stander et al., 2000). Ochratoxin Bcontains the isodehydrocoumarin moiety ochratoxin β. Coumarinsare antioxidants (Fylaktakidou et al., 2004) and light, especially bluelight has an oxidative effect (Miyzaki et al., 2001). A. carbonariususually occurs on grapes in the southern hemisphere (Perrone et al.,2008) and is exposed to high intensities of daylight. Taken togetherthe ochratoxin A producing Penicillia and Aspergilli seem to havedeveloped two mutually regulated secondary metabolite pairs(ochratoxin A/citrinin and ochratoxin A/ochratoxin β derivative)which counter react under light conditions the regulation of whichseem to play a role in light protection.

According to the results reported here light has a growthretarding effect on ochratoxin A producing Penicillia. If a growingcolony of P. nordicum is exposed to light of a certain intensity andwavelength it stops growth. If this colony is then incubated in thedark, it starts to grow and to produce ochratoxin A again. Lighttreatment of a colony is not uniform. Cells in the exposed areas of thecolony are not protected against light and stop growth or may evenbe killed if the intensity is high enough. It can be assumed that thepenetration of light into the deeper parts of the colonies is low due tophysical protection or to absorbing metabolites so that cells in thispart of the colony are shielded against the action of light and are notaffected, or are less affected, which explains the immediate start ofgrowth after shift to the dark. Spores on the other hand are uniformlyexposed to light and are not protected. On germinating spores lighthas an inactivating effect depending on the light wavelength and thespecies. As was the general case for the Aspergilli, also the spores ofA. carbonarius survived the blue light treatment better than theP. verrucosum spores.

Taken together, the results show that under certain circum-stances light can completely inhibit the biosynthesis of ochratoxin Aor even growth of the ochratoxin A producing fungi. Ochratoxin Aproducing Penicillia seem to be especially sensitive but, at higherlight intensities, ochratoxin production in Aspergilli can also benegatively influenced. The analysed species of the genus Alternariaand Fusarium were highly resistant under blue light conditions withrespect to growth. The F. graminearum strain even produced moreDON under these conditions. There was a tendency for growth ofthese resistant species to be favoured under blue light (data notshown). These differences in sensitivity might either be due todifferent sensitive blue and red light receptors or to the production ofprotecting substances like spore pigments or other secondarymetabolites. The production of the ochratoxin β derivative andcitrinin are induced under certain light treatments which hypothet-ically suggest a protective function of these two secondarymetabolites. For citrinin at least, preliminary results point in thatdirection. The fact that in YES medium the production of somesecondary metabolites analysed here (e.g. ochratoxin β derivative)was not directly correlated to the light conditions, can be attributedto the fact the YES is generally a very conducive medium formycotoxin production. Further analyses revealed that light, espe-cially blue light, also has a reducing or inhibiting effect on aflatoxin B1

production by A. parasiticus and alternariol methyl ether productionby A. alternata (Table 1). However, in contrast, the production of DONby F. graminearum increased under these otherwise inhibitoryconditions, which suggests that Fusarium has a different responseto these light conditions.

Acknowledgement

We thank Michaela Ebli for skillful technical assistance andJessica Gil Serna and Maria Gonzales Jaen for the strain of A. steynii.This work was financially supported by EC KBBE-2007-222690-2MYCORED.

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