Food Control 43 (2014) 121e128

Contents lists available at ScienceDirect

Food Control

journal homepage: www.elsevier.com/locate/foodcont

Aflatoxin B1 production by parasiticus and strains of Aspergillus section Nigri in currants of Greek origin

Paraskevi Kostarelou a, Alexandros Kanapitsas a, Ioanna Pyrri b, Evangelia Kapsanaki-Gotsi b, Panagiota Markaki a,* a Department of Food Chemistry, Faculty of Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, GR-15784 Athens, Greece b Department of Ecology and Systematics, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece article info abstract

Article history: Aflatoxin B1 (AFB1) mostly produced by Aspergillus flavus and Aspergillus parasiticus, is an extremely toxic Received 4 November 2013 and carcinogenic metabolite. Currants are used in the Mediterranean diet as a food with antioxidant Received in revised form properties. Four strains of Aspergillus section Nigri have been isolated from currants originated from Crete 28 January 2014 and Corinth. In this study AFB production by A. parasiticus and the four strains of Aspergillus section Nigri Accepted 8 February 2014 1 in Cretan and Corinthian currants (Vitis vinifera L.) is investigated. AFB determination was performed by Available online 17 March 2014 1 HPLCeFID. Results revealed that the four strains Aspergillus section Nigri, as well as the aflatoxigenic 1 strain A. parasiticus produced AFB1 (0.0052e1.31 mg AFB1 15 g , corresponding to 0.0003e0.087 mg Keywords: 1 AFB1 g ) in both type of currants (Cretan and Corinthian) on the 12th day of observation. Moreover, Aflatoxin B1 Aspergillus parasiticus AFB1 production, by A. parasiticus in the synthetic Yeast Extract Sucrose (YES) medium was also studied. Aspergillus section Nigri The ability of AFB1 production has been affected by the special characteristics of each isolate and the Currants currants substrate. Yeast extract sucrose Ó 2014 Published by Elsevier Ltd. HPLC

1. Introduction Aflatoxins (AFs) are highly toxic secondary metabolites pro- duced by the species of Aspergillus (A.), especially Aspergillus flavus Raisins constitute an important component of the Mediterra- and Aspergillus parasiticus (Wejdan et al., 2010). AFs are of great nean diet. They are considered to be a rich source of ascorbic acid, concern because of their harmful effects on the health of humans citric acid, malic and tartaric acids with plenty minerals, such as and animals, including carcinogenic, mutagenic, teratogenic and potassium, calcium and magnesium (Nour, Trandafir, & Ionica, immunosuppressive effects (Zinedine & Mañes, 2009). Aflatoxin B1 2011). Moreover, currants contain polyphenolic compounds such (AFB1) is the most potent hepatocarcinogen in mammals and it is as anthocyanins (Nour et al., 2011), vanillic acid, caffeic, gallic and classified by the International Agency of Research on Cancer as p-coumaric acids and quercetin. The consumption of currants Group 1 carcinogen (IARC, 1993a). contributes to the intake of antioxidants (Chiou et al., 2007) and These -producing mold species can grow on a wide moreover they have potent anticarcinogenic properties against range of agricultural commodities in the field, but also during post- several cancers like hepatocellular carcinogenesis in rats (Bishayee harvest operations and storage conditions (Zinedine & Mañes, et al., 2011). 2009). Major food commodities affected are nuts, figs and other dried fruits, spices, crude vegetable oils, cocoa beans and maize ac- cording to many authors (Iamanaka, Castle de Menezes, Vicente, Leite, & Taniwaki, 2007; Imperato, Campone, Piccinelli, Veneziano, & Rastrelli, 2011; Luttfullah & Hussain, 2011). The European Commis- Abbreviations: AFB1,Aflatoxin B1; AFB2a,Aflatoxin B1 hemiacetal derivative; A., Aspergillus; AFPA, Aspergillus flavus parasiticus agar; CFU, Colony forming units; CZA, sion has established the maximum level for AFB1 in dried fruits for 1 Czapek Dox agar; HPLC, High performance liquid chromatography; ATHUM, ATHens direct human consumption to 2 ng g (European Commission, 2006). University Mycology; NOAEL, No Observed e Adverse Effect Level; PMTDI, Provi- The Aspergillus section Nigri (also known as black-spored As- sional Maximum Tolerable Daily Intake; RSD, Relative Standard Deviation; TDI, pergilli) are an important group of fungi because of their impact on Tolerable Daily Intake; YES, Yeast Extract Sucrose. * Corresponding author. Tel.: þ30 210 7274489; fax: þ30 210 7274476. food safety, medical mycology and industrial biotechnology E-mail address: [email protected] (P. Markaki). (Palencia, Klich, Glenn, & Bacon, 2009). The black Aspergilli are http://dx.doi.org/10.1016/j.foodcont.2014.02.011 0956-7135/Ó 2014 Published by Elsevier Ltd. 122 P. Kostarelou et al. / Food Control 43 (2014) 121e128 among the most common fungi causing food spoilage and biode- HewlettePackard 1050 (Waldborn, Germany) Liquid Chromato- terioration of other materials (Schuster, Dunn-Coleman, Frisvad, & graph (pump and injection system) equipped with a JASCO FP-920 van Dijck, 2002). They have been isolated mainly from soil, but they (Co, LTD, Japan) fluorescence detector and an HP integrator 3395. also have been found in several other substrates (Magnoli et al., The HPLC column used was a C18 Nova-Pak (60 E, 4 mm 2007). Reports by several authors (Magnoli et al., 2004; Magnoli, 4.6 250 mm) (Waters, Millipore; Milford, MA). The mobile phase Violante, Combina, Palacio, & Dalcero, 2003; Romero et al., 2005; for AFB1 [water:acetonitrile:methanol (20:4:3, v:v:v)] determina- Spadaro, Patharajan, Lorè, Garibaldi, & Gullino, 2012) showed that tion was filtered through Millipore HA-VLP (0.45 mm) filters before black Aspergilli, especially Aspergillus carbonarius, infect vines and use. Detection of the AFB1 hemiacetal derivative (AFB2a) was car- grapes and produce , mainly ochratoxins. ried out at lex 365 nm and lem 425 nm. The flow rate was 1 In the literature, the occurrence of AFB1 in dried vine fruits is not 1 mL min and the retention time was 14.68 (0.28) min for AFB2a. extensively investigated. However, AFB1 was found in dried vine fruits from India (Saxena and Mehrotra 1990) and Egypt (Abdel 2.3. Reagents Sater & Saber, 1999; Youssef, Abo-Dahab, & Abou-Seidah, 2000). The main purpose of our study was to investigate the potential AFB1 standard was purchased from SigmaeAldrich (St. Louis, of four strains of Aspergillus section Nigri, which have been isolated Missouri, USA). The Millipore filters and the C18 Nova-Pak HPLC from black currants originated from Corinth and Crete, to support column were from Waters (Millipore; Milford, MA, USA). The Aflatest AFB1 production, in these substrates. The AFB1 production by the immunoaffinity columns were obtained from Vicam (Watertown, four Aspergillus section Nigri strains was compared to the produc- MA, USA). All reagents used were of analytical grade (Sigma Aldrich) tion by the aflatoxigenic strain A. parasiticus (control). while HPLC solvents were of HPLC grade and were purchased from Fisher Chemical (Leicestershire, UK). Hexane and methanol (pro 2. Materials and methods analysis) were from Merck (Darmstadt, Germany) and trifluoroacetic acid was from Fluka (Steinheim, The Netherlands). 2.1. Strains and culture conditions 2.4. Media Strains of Aspergillus section Nigri were isolated from currants which originated from Peloponnese e Corinth (1 strain) and Crete Aspergillus Flavus Parasiticus Agar (AFPA) was prepared by dis- (3 strains). The strains were incubated for 7 days at 30 C on slopes solving 4 g of yeast extract (Oxoid, Basingstoke, Hampshire, UK), 2 g of Czapek Dox agar (CZA) and were maintained at 5 C on the same of bacteriological peptone (Oxoid), 0.1 g of ferric ammonium citrate medium. The aflatoxigenic strain A. parasiticus Speare (IMI 283883) (Merck, Germany), 0.2 mL of Dichloran 0.2% in ethanol (Fluka used throughout this study as strain control, was obtained from the Steinheim, The Netherlands), 0.02 g of chloramphenicol (Oxoid), and International Mycological Institute (Egham, Surrey, UK). The 3 g of agar (Oxoid) in 200 mL of distilled water, final pH 6.0e6.5. Aspergillus isolates were identified as Aspergillus section Nigri ac- Czapek Dox agar (CZA) was prepared by dissolving 0.4 g of sodium cording to their morphological characteristics and have been nitrate (Merck), 0.1 g of potassium chloride (Merck), 0.1 g of mag- deposited at the ATHUM Culture Collection of Fungi, in the Myce- nesium sulfate (Merck), 0.002 g of ferric sulfate (Merck), 0.2 g of totheca of the University of Athens. The four strains isolated from dipotassium phosphate (Merck), 6 g of sucrose (Merck), 3 g of agar the currants were the following: Aspergillus sp. isolated from cur- (Merck), 0.002 g of zinc sulfate (Merck), and 0.001 g of copper sulfate rants originated from Corinth ATHUM 6997, Aspergillus sp. isolated (Merck) in 200 mL of distilled water, final pH 6.0e6.5 (Vergopoulou, from currants originated from Crete ATHUM 6998, Aspergillus sp. Galanopoulou, & Markaki, 2001). Yeast Extract Sucrose (YES) broth isolated from currants originated from Crete ATHUM 6999, Asper- was prepared by dissolving 2 g of yeast extract and 15 g of sucrose in gillus sp. isolated from currants originated from Crete ATHUM 7000. 100 mL distilled water, final pH 6.0e6.5 (Pitt, 1986). For the phenotypic examination of the Aspergillus strains, cul- The media for the identification of Aspergillus isolates have been tures were inoculated using suspension at three points onto prepared according to Frisvad and Samson (2004). Czapek yeast agar (CYA), malt extract agar (MEA), yeast extract sucrose agar (YES) and creatine agar (CREA) according to Frisvad 2.5. Sampling and treatment and Samson (2004). The inoculated Petri dishes were incubated in the dark at 25 C. In addition, CYA plates were inoculated and Currants originated from Corinth and Crete were purchased incubated at 30 C in the dark. After 7 days of incubation the colony from the market of Athens (Greece), taken from open containers. diameters, the degree of sporulation, the colony colors in the For the total experimental requirements, a quantity of 3 and 4.5 kg obverse and reverse and the production of soluble pigments were of Corinthian and Cretan currants respectively were used. The two recorded in detail. All the isolates were examined for the produc- types of currants were separately washed with water, cut into tion of alkaloids by the Ehrlich test with the filter paper method pieces, so that the whole mass of the currants would be used as (Lund, 1995). Microscopic examination was performed by tearing substrate. They were finally autoclaved at 110 C for 2 min, in order apart a small piece of the colony from MEA in a drop of 60% lactic to eliminate their natural microflora (Leontopoulos, Siafaka, & acid on a glass slide and placing a coverslip on it. Mounts were Markaki, 2003). The above technique was preferred, as the use of observed under a microscope AxioImager.A1 (Zeiss, Germany) with chlorinated water was not suitable, since it was excessively absor- plain light or with Differential Interference Contrast at 1000 bed by the currant substrate. magnification. The microscopic characteristics of conidia, phialides, After treatment, the damaged currants were randomly divided and metulae, vesicles and stipes were studied in detail. samples were obtained which were distributed aseptically into sterile flasks forming a solid mass. Each flask contained 15 g of currants. 2.2. Apparatus Currants were stored at 4 C until using them in the experiments.

A laminar flow (Telstar Bio II A, Madrid, Spain), an autoclave, 2.6. Preparation of spore inoculum Selecta-Autester-E Dry (PBI Milano, Italy), an incubator WTB Binder (Tuttlinger, Germany) and a centrifuge Sorvall RC-5B (HS-4) (Nor- A. parasiticus Speare (IMI 283883) was used throughout this walk, USA) were used in this study. HPLC was performed on a study as strain control. An inoculum was obtained by growing the P. Kostarelou et al. / Food Control 43 (2014) 121e128 123 mold on a slant of stock cultures of CZA, which was maintained at distilled water. The mixture was transferred into an Aflatest 5 C. Spore inoculum was prepared by growing A. parasiticus on CZA immunoaffinity column (flow rate 3 mL min 1), washed twice with for 7 days at 30 C and were harvested aseptically using 10 mL of distilled water each time. The column was then allowed to 10 mL of sterile 0.01% v/v Tween 80 solution. AFB1 carried over from dry by passing air through it. AFB1 was eluted with 2 mL of the initial growth was minimized by centrifuging the spore sus- acetonitrile (flow rate ¼ 0.3 mL min 1). Before derivatization, the pension (1000 g for 10 min) and resuspending the biomass in 10 mL eluate was evaporated to dryness on a water bath under a gentle of sterile Tween 80 solution twice. Dilutions (10 1,10 2,10 3,10 4) steam of nitrogen (Daradimos, Markaki, & Koupparis, 2000). A from the initial spore suspension in sterile tubes containing 10 mL derivative of AFB1 (AFB2a, hemiacetal of AFB1) was prepared by of Tween 80 0.05% v/v were prepared (Vergopoulou et al., 2001). adding 200 mL of hexane and 200 mL of trifluoroacetic acid to the The spore concentration was determined by the spread plate sur- evaporated solution of AFB1 eluate, heating at 40 C in a water bath face count technique using 0.1 mL of each dilution on four AFPA for 10 min, evaporating to dryness under a gentle stream of nitro- plates after incubation at 30 C for 48 h. For obtaining an inoculum gen, redissolving in 200 mL with water:acetonitrile (9:1) and containing 102 conidia, plates with 10e100 colony-forming units analyzing by HPLC (volume injected ¼ 40 mL). (cfu) were selected and the desired 102 spore quantity used in the present study was estimated. The quantity of 102 spores flask 1 was 3. Results and discussion chosen as it was the minimum concentration found in the literature producing detectable amounts of AFB by Aspergillus (El-Refai, 1 The in-house characterization of the method for determining Awadalla, & Abou Zid, 1995). It must be cited that the same pro- AFB in YES medium has been reported in detail by Vergopoulou cedure was carried out for all the four Aspergillus section Nigri 1 et al. (2001). The recovery factor of the method was 95.3% (RSD strains used in the present study. % ¼ 9.6) and the detection limit of the derivatized AFB1 (AFB2a)was found to be 0.02 ng AFB mL 1 of YES. 2.7. Experimental design 1 AFB2a shows enhanced fluorescence compared to AFB1 (Stubblefield, 1987). In addition to that, AFB is less toxic compared In the present study the following nine groups of cultures were 2a to AFB because of its protein binding properties, since it is not examined: 1 absorbed from the gastrointestinal tract and it is not toxic to a. YES medium (10 mL) inoculated with 100 conidia of A. par- experimental animals. Moreover, AFB does not interact with nu- asiticus IMI 83883; b. Corinthian currants (15 g) treated non inoc- 2a clear DNA (Patterson, 1976). ulated; c. Corinthian currants (15 g) inoculated with 100 conidia of The analytical protocol for AFB determination in currants was A. parasiticus IMI 83883; d. Corinthian currants (15 g) inoculated 1 in-house characterized in detail (Kollia, Kanapitsas, & Markaki, with 100 conidia of Aspergillus section Nigri ATHUM 6997 isolated 2014). The recovery of the method was 97.6% (RSD% ¼ 6.46) and from currants originated from Corinth; e. Cretan currants (15 g) the detection limit of the derivatized AFB1 (AFB2a) was 0.045 ng treated non inoculated; f. Cretan currants (15 g) inoculated with 1 1 AFB1 g of currant corresponding to 0.68 ng AFB1 15 g of cur- 100 conidia of A. parasiticus IMI 283883; g. Cretan currants (15 g) rants or flask 1. inoculated with 100 conidia of Aspergillus section Nigri ATHUM As far as the analytical method applied for the determination 6998 isolated from currants originated from Crete; h. Cretan cur- and detection of AFB is concerned, it must be mentioned that: 1) rants (15 g) inoculated with 100 conidia of Aspergillus section Nigri 1 The cleaning step was performed by using immunoaffinity mini- ATHUM 6999 isolated from currants originated from Crete; i. Cre- columns, which are selective for AFB isolation; 2) Before the tan currants (15 g) inoculated with 100 conidia of Aspergillus sec- 1 determination by HPLC, the AFB eluted from the minicolumns is tion Nigri ATHUM 7000 isolated from currants originated from 1 derivatized to its hemiacetal AFB thus, AFB identification is Crete. 2a 1 confirmed. In Fig. 1, representative chromatograms of AFB1 pro- duction by all strains in all substrates examined are exposed. 2.8. Statistical analysis Concerning the phenotypic characteristics of the four Aspergillus section Nigri isolates, they all have biseriate conidial heads with Data were analyzed by one-way ANOVA and t-test. The mean small-spored conidia which are quite different from the spores of differences which are significantly different were examined by A. carbonarius commonly present in grapes. However, they present using the Turkey test (Fowler & Cohen, 1997). slight morphological differences that differentiate them at the species level. All the strains have been processed for molecular 2.9. Inoculation analysis in order to confirm the identification at the species level. Three flasks for each day of observation and from each group mentioned in the experimental design have been studied in this 3.1. AFB1 production by A. parasiticus in yeast extract sucrose work. medium and currants originated from Crete and Corinth All flasks with YES and currants, inoculated and non-inoculated, were incubated under stationary conditions at 30 C. Immediately A. parasiticus was used as strain control because it is less studied after autoclaving for 30 min at 115 C as suggested for safety rea- and produces AFB1 with larger stability (Yu et al., 1995). YES was sons (Sinha, Williams, & Hake, 1993), the AFB1 content of each flask chosen since it is the most optimum medium for the biosynthesis of was measured. AFB1 was assayed on days 0, 3, 7, 9, 12 and 15 of high levels of AFB1, compared to other microbiological media, as incubation. The experiment was repeated in triplicate. well as, it is easy and cheap to be prepared (Luchese & Harrigan, 1993). 2.10. AFB1 determination, derivatization and HPLC analysis In the present work, A. parasiticus produced AFB1 after inocu- lation in currants originated from Crete and Corinth. The maximum The content of each flask (containing 15 g of currants or 10 mL of amount of AFB1 produced in Cretan and Corinthian currants was 1 YES medium and the mycelium) was mixed with 30 mL of meth- observed on the 7th day (5.51 ng AFB1 15 g of currants) and 12th 1 anol e water (80:20 v:v) and shaken well for 10 min. After filtration, day (1310 ng AFB1 15 g of currants) of incubation, respectively an aliquot of 1 mL from the filtrate was mixed with 10 mL of (Tables 1 and 2, Fig. 1). 124 P. Kostarelou et al. / Food Control 43 (2014) 121e128

Fig. 1. Chromatographs of AFB2a production by: a) standard AFB1 (2.5 ng/mL); b) Aspergillus section Nigri 6998 inoculated in currants originated from Crete, on the 15th day of observation (corresponding to 9.87 ng AFB1/15 g); c) Aspergillus section Nigri 6999 inoculated in currants originated from Crete, on the 15th day of observation (corresponding to 10.04 ng AFB1/15 g); d) Aspergillus section Nigri 7000 inoculated in currants originated from Crete, on the 12th day of observation (corresponding to 5.28 ng AFB1/15 g); e) Aspergillus section Nigri 6997 inoculated in currants originated from Corinth, on the 12th day of observation (corresponding to 74.58 ng AFB1/15 g); All samples were derivatized to AFB2a. The retention time was 14.68 (0.28) min and the injection volume was 40 mL.

3 1 Statistical analysis (Table 3) indicated that AFB1 production by high levels (75.53 10 ng 10 mL of YES medium) (Table 1). The A. parasiticus in Corinthian currants was not significantly different AFB1 production in YES medium on the 12th day of observation, compared to AFB1 production in currants from Crete during the was approximately w13,629 and 50-fold higher, compared to the whole period of incubation (15 days). However, the results applied production in currants originated from Crete and Corinth, respec- to these two types of currants mentioned above only for the 12th tively, on the same day (Tables 1 and 2). Therefore, this is an day of observation, showed that AFB1 production by A. parasiticus in approval that AFB1 may be produced in YES medium at high levels. Corinthian currants was significantly different compared to AFB1 Chiou et al. (2007) confirmed the occurrence of antioxidants and production in currants from Crete (Table 3). Hence, it can be noticed antimicrobial constituents in currants. From the literature, it is that the substrate of Corinthian currants is more favorable than that known that the oxidative stress stimulates aflatoxin biosynthesis of Cretan currants. by A. parasiticus (Jayashree & Subramanyam, 2000; Reverberi et al., On the other hand, AFB1 production by A. parasiticus in YES 2005). On the other hand, antioxidants could interfere reducing the medium continued until the last day of incubation (15th day) at pool of nicotinamide adenine dinucleotide phosphate (reduced P. Kostarelou et al. / Food Control 43 (2014) 121e128 125

Table 1

AFB1 production by A. parasiticus and strain of Aspergillus section Nigri ATHUM 6997.

Cultures Days YES þ A. parasiticus (control)a ng 10 mL 1 (SD) Currants þ A. parasiticus (control)b ng 15 g 1 (SD) Currants þ 6997c,d ng 15 g 1 (SD)

0 N.D.e N.D.e 14 (1.53) 3 18.10 103 (1.82 103)13(5.77) 17 (1.53) 7 41.62 103(1.40 103)21(4.04) 18 (3.51) 9 47.83 103(5.67 103) 142 (15.95) 28 (1.53) 12 65.83 103(4.59 103) 1310 (131.20) 87 (12.42) 15 75.53 103(2.93 103) 387 (25.50) 42 (5.51)

a YES medium inoculated with A. parasiticus. b Currants treated originated from Corinth inoculated with A. parasiticus. c Currants treated originated from Corinth inoculated with Aspergillus section Nigri ATHUM 6997. d AFB1 production by treated non inoculated currants originated from Corinth was not detectable. e Non-Detectable.

Table 2 1 AFB1 production (ng 15 g of currants) by: A. parasiticus and strains of Aspergillus section Nigri in currants originated from Crete.

Currants originated from Crete

Days Non inoculateda ng 15 g 1 Currants þ A. parasiticusb ng 15 g 1 Currants þ 6998c ng 15 g 1 Currants þ 6999d ng 15 g 1 Currants þ 7000e ng 15 g 1 (SD) (SD) (SD) (SD) (SD)

0 0.68 (0.01) 3.36 (0.42) 3.43 (0.23) 2.98 (0.43) 1.34 (0.15) 3 0.68 (0.01) 4.27 (0.73) 3.59 (0.51) 3.26 (0.63) 2.26 (0.68) 7 0.69 (0.01) 5.51 (0.30) 3.71 (0.17) 3.51 (0.15) 2.65 (0.17) 9 0.75 (0.05) 5.03 (0.18) 3.73 (0.11) 3.63 (0.16) 2.97 (0.09) 12 0.70 (0.01) 4.83 (0.23) 4.62 (0.08) 4.28 (0.04) 5.15 (0.12) 15 0.68 (0.01) 3.01 (0.17) 10.08 (0.27) 10.29 (0.22) 4.22 (0.10)

a Currants treated non inoculated. b Currants treated inoculated with A. parasiticus. c Currants treated inoculated with Aspergillus section Nigri ATHUM 6998. d Currants treated inoculated with Aspergillus section Nigri ATHUM 6999. e Currants treated inoculated with Aspergillus section Nigri ATHUM 7000. form) available for the AF biosynthetic reactions (Maggio-Hall, niger, A. carbonarius and the new isolates with ATHUM 6997, 6998, Wilson, & Keller, 2005). Furthermore, a survey of inhibitors of AF 6999, 7000, which were identified as Aspergilli section Nigri (Mar- biosynthesis has illustrated that many inhibitors have antioxidant kaki, unpublished data). activity (Holmes, Boston, & Payne, 2008). Saffron (10 mg mL 1 of Perrone (2007) states that black Aspergilli are one of the most YES), known for its antioxidant properties, inhibited by 99.9% AFB1 difficult groups concerning classification and identification. Some production in YES medium (Tzanidi, Proestos, & Markaki, 2012). On fungal secondary metabolites were reported to be produced also by the contrary, according to Passone, Rossob, and Etcheverry (2012), A. niger (Blumenthal, 2004). Yassin et al. (2010) reported that sub-lethal antioxidant doses can lead to fungal growth, increase mycotoxin investigations using HPLC revealed that A. niger isolates structure resistance, stimulation of aflD gene expression and AFB1 produced aflatoxins in sorghum grains. Previously Schuster et al. accumulation. Therefore, high concentration of antioxidants is (2002), stated that A. niger has not been proven to produce needed for AFB1 reduction. Consequently, one of the parameters aflatoxins. which affect the limited AFB1 production in currants could be the Strains isolated from currants of Greek origin, which were antioxidant capacity of their constituents, regardless of the type of identified as Aspergilli section Nigri, produced AFB1 in Corinthian currants examined. and Cretan currants (Tables 1 and 2). In the literature, there is no information concerning AFB1 production by strains of Aspergillus 3.2. AFB1 production by strains of Aspergillus section Nigri in section Nigri isolated from Greek currants. currants originated from Crete and Corinth AFB1 production by Aspergillus section Nigri ATHUM 6998 and ATHUM 6999 in currants from Crete achieved their maximum state 1 Before treatment the natural microflora of the samples exam- on the 15th day of observation (10.08 and 10.29 ng AFB1 15 g of ined consisted of bacteria <1000/g cfu, fungi such as Aspergillus currants, respectively) (Table 2, Fig. 1). Furthermore, Aspergillus

Table 3

Statistical analysis (t-test) of AFB1 production in currants inoculated by strains A. parasiticus and strains section Nigri.

Paired samples Df Texp Ttheor Statistical significance A. parasiticus þ A. section Nigri ATHUM 6997a 5 1.410 2.571 No A. parasiticus þ A. section Nigri ATHUM 6997a (only for the 12th of incubation) 2 14.809 4.303 Yes A. parasiticus þ A. section Nigri ATHUM 6998b 5 0.392 2.581 No A. parasiticus þ A. section Nigri ATHUM 6999c 5 0.229 2.581 No A. parasiticus þ A. section Nigri ATHUM 7000d 5 1.883 2.581 No

a Strain isolated from Corinthian currants. b Strain isolated from Cretan currants. c Strain isolated from Cretan currants. d Strain isolated from Cretan currants. 126 P. Kostarelou et al. / Food Control 43 (2014) 121e128

Table 4

One way ANOVA statistical analysis of the AFB1 production in currants inoculated by strains section Nigri.

Samples Df Fexp Ftheor Statistical significance A. section Nigri ATHUM 6997a þ A. section Nigri ATHUM 6998b þ A. section Nigri 3, 20 6.938 3.098 Yes ATHUM 6999c þ A. section Nigri ATHUM 7000d A. section Nigri ATHUM 6998b þ A. section Nigri ATHUM 6999c þ A. section Nigri 2, 15 1.020 3.682 No ATHUM 7000d A. section Nigri ATHUM 6998b þ A. section Nigri ATHUM 6999c þ A. section Nigri 2, 6 847.467 5.43 Yes ATHUM 7000d (only for the 12th of incubation)

a Strain isolated from Corinthian currants. b Strain isolated from Cretan currants. c Strain isolated from Cretan currants. d Strain isolated from Cretan currants. section Nigri ATHUM 7000 in Cretan currants and Aspergillus sec- 6997 in currants from Crete and Corinth where they have been tion Nigri ATHUM 6997 in Corinthian currants produced their originally isolated. maximum amount of AFB1 on the 12th day of observation (5.15 and The low AFB1 amounts could be explained, besides the antiox- 1 87 ng AFB1 15 g of currants, respectively) (Tables 1 and 2, Fig. 1). idant activity of the currant substrate, by the fact that Aspergilli One way ANOVA analysis, showed that there was significant section Nigri are mainly producers of ochratoxin A (OTA). Therefore, difference of aflatoxin B1 production between all strains of Asper- the potential of OTA occurrence in currants may inhibit the afla- gillus section Nigri, during the whole period of incubation (15 days) toxigenic mold growth and the AFB1 production. This statement is (Table 4). Comparing the maximum production by all strains of in agreement with Dimitrokallis, Meimaroglou, and Markaki (2008) Aspergillus section Nigri regardless the day appeared, it was found who reported that OTA’s presence inhibited AFB1 production. that the AFB1 production by the strain ATHUM 6997 in Corinthian Studies from the literature have shown that A. niger strains, pro- currants was 8.5, 8.6 and 16.9-fold higher than that by the strains of duce OTA under optimal conditions (Martins, Martins, Bernardo, & Aspergillus section Nigri (ATHUM 6998, 6999 and 7000 respec- Gimeno, 2005; Soares, Calado, & Venâncio, 2013). In recent years, tively) in Cretan currants. black Aspergillus species (section Nigri) have been described as the By applying one-way ANOVA for AFB1 production by strains of main source of OTA contamination in grapes and also in dried Aspergillus section Nigri 6998, 6999 and 7000 in currants from grapes worldwide (Battilani & Pietri, 2002; Serra, Abrunhosa, Crete, no significant difference was observed, during the whole Kozakiewicz, & Venancio, 2003). Recently, the presence of OTA period of incubation (15 days). Nevertheless, applying one-way and black Aspergilli, in raisins from Western Greece, has been re- ANOVA, only for the 15th day of incubation, it is ascertained that ported (Perrone, De Girolamo, Sarigiannis, Haidukowski, & Visconti, the alteration of AFB1 production between Aspergillus section Nigri 2013). Furthermore, it was found that all samples of dried vine ATHUM 6998, 6999 and 7000 in currants from Crete was statisti- fruits, examined in our laboratory, including currants, from which cally different (Table 4). the strains (Aspergillus section Nigri ATHUM 6997, 6998, 6999 and Although the strains of Aspergillus section Nigri ATHUM 6998, 7000) were isolated, were contaminated from 2.8 to 138.3 ng OTA/ 6999 and 7000, have been isolated from the same type of currants gr and that a percentage of 69.23% was over the limit established by (Cretan), they produced different amounts of AFB1, especially on legislation 10 ng/g (Kollia et al., 2014). Hence, a further study of the the 15th day of incubation. Both strains of Aspergillus section Nigri strains of Aspergillus section Nigri (ATHUM 6997, 6998, 6999 and ATHUM 6998 and 6999 produced AFB1 w2.4-fold higher (10.08 and 7000) and their potential to produce OTA is needed. 1 10.29 ng AFB1 15 g of currants, respectively) compared to the It must be reported that the AFB1 production was also examined production by Aspergillus section Nigri ATHUM 7000 (4.22 ng by the four Aspergillus section Nigri strains in YES medium and has 1 AFB1 15 g of currants), on the 15th day of incubation (Table 2). been revealed that it was from 98.3 to 99.9% lower, in comparison Therefore, the aflatoxigenic potential of the strains Aspergillus to the production by A. parasiticus on the same substrate (Markaki, section Nigri ATHUM 6998, 6999, is superior than that of the strain unpublished data). Aspergillus section Nigri ATHUM 7000 in currants from Crete. What is more, a t-test has been applied and results indicated 3.3. Comparison of Cretan and Corinthian currants as a substrate fi that there was no signi cant difference in AFB1 production be- for AFB1 production tween A. parasiticus and the strain of Aspergillus section Nigri

ATHUM 6997 during the whole period of incubation (15 days) in It was revealed that AFB1 production by either the aflatoxigenic Corinthian currants (Tables 1 and 3). However, when a t-test was strain A. parasiticus or the strain of Aspergillus section Nigri ATHUM applied, only for the 12th day of observation, it showed that AFB1 6997 in Corinthian currants was higher in comparison to the pro- production by A. parasiticus, was higher compared to AFB1 pro- duction by A. parasiticus and the strains of Aspergillus section Nigri duction by Aspergillus section Nigri ATHUM 6997 in currants from ATHUM 6998, 6999 and 7000 in Cretan currants (Tables 1 and 2, Corinth (Table 3). Fig. 1). Moreover, the t-test of AFB1 production by A. parasiticus and the Furthermore, it is important to be cited, that AFB1 production in three strains of Aspergillus section Nigri (ATHUM 6998, 6999 and treated non-inoculated currants from Corinth was not detectable 7000) separately examined, in currants from Crete, showed that during the whole period of incubation. On the other hand, in there was no difference in AFB1 production between them, during treated non inoculated currants from Crete, AFB1 production was the whole period of incubation (15 days) (Table 3). This is an observed at low levels (maximum production on the 9th day 1 additional approval that Cretan currants substrate, is not favorable 0.75 ng AFB1 15 g of currants). Cretan currants have been fl for AFB1 production either for the a atoxigenic A. parasiticus or the contaminated by aflatoxigenic fungi and AFB1 was present, even strains of Aspergillus section Nigri. after the currant treatment (Table 2). In the present study, the AFB1 biosynthesis is reported, by the These results mentioned above could probably explain the dif- strains of Aspergillus section Nigri ATHUM 6998, 6999, 7000 and ference between AFB1 production in the two types of currants. The P. Kostarelou et al. / Food Control 43 (2014) 121e128 127 existence of AFB1 even at low levels in Cretan currants could affect The production of AFB1 exposes consumers to risk, since it is the ability of the three strains of Aspergillus section Nigri (ATHUM extremely toxic. Aspergillus spp. may persist through the whole life 6998, 6999 and 7000) and the aflatoxigenic A. parasiticus to grow cycle of currant production, even at low levels. Furthermore, it is and produce AFB1. The AFB1 occurrence in non-inoculated Cretan important to ensure that collection, handling, storage, and mar- currants could interact with the strains after inoculation, resulting keting practices are appropriate in order to prevent fungal attack. to lower AFB1 production in comparison to the Corinthian currants In conclusion, AFB1 is produced by the specific strains (of where AFB1 was not detectable in non-inoculated samples. Aspergillus section Nigri) isolated by Greek currants, under the Additionally, maximum AFB1 production by the strains conditions of our experiments. Moreover, AFB1 production depends A. parasiticus and Aspergillus section Nigri ATHUM 6997, in currants on the exact parameters of the system under investigation, since from Corinth, was w44-fold and w3-fold higher respectively, than the biosynthesis of mycotoxins is a very complicated process and that defined by the legislation (2 ng g 1). In that case, the pro- many factors are involved. duction can be considered as significant. In addition maximum AFB1 production by A. parasiticus and the Acknowledgment three strains of Aspergillus section Nigri (ATHUM 6998, 6999 and 7000) in currants from Crete, separately examined, was from 3 to 6- This work was supported in a small part by the University of fi 1 fold lower than that de ned by the legislation (2 ng AFB1 g ) Athens, Special Account for Research Grants 70/4/8786. (European Commission, 2006). This confirms that the substrate of Cretan currants inhibited AFB1 production by the strains mentioned References above. Generally, the production of AFB1 in the present study cannot be Abdel Sater, M. A., & Saber, S. M. (1999). Mycoflora and mycotoxins of some Egyptian dried fruits. Bulletin of the Faculty of Science of Assiut University D, 28, considered as negligible, as AFB1 is potent carcinogen and the 92e107 (Chemical Abstracts 133, 3874 p. 2000). currant consumption contributes to the daily intake of AFB1 from Battilani, P., & Pietri, A. (2002). Ochratoxin A in grapes and wine. European Journal of diet. Plant Pathology, 108, 639e643. Bishayee, A., Mbimba, T., Thoppil, R. J., Haznagy-Radnai, E., Sipos, P., Darvesh, A. S., 3.4. Risk assessment for AFB et al. (2011). Anthocyanin-rich black currant (Ribes nigrum L.) extract affords 1 chemoprevention against diethylnitrosamine-induced hepatocellular carcino- genesis in rats. The Journal of Nutritional Biochemistry, 22(11), 1035e1046. Aflatoxin B1 is the most potent natural carcinogen known and is Blumenthal, C. Z. (2004). Production of toxic metabolites in Aspergillus niger, fi usually the major aflatoxin produced by toxigenic strains. However, Aspergillus oryzae, and Trichoderma reesei: justi cation of mycotoxin testing in e food grade enzyme preparations derived from the three fungi. Regulatory the No Observed Adverse Effect Level (NOAEL) is not appropriate Toxicology and Pharmacology, 39,214e228. for genotoxic carcinogens such as AFB1 because no threshold can be Cano-Sancho, G., Sanchis, V., Marín, S., & Ramos, A. J. (2013). Occurrence and assumed in this case (Cano-Sancho, Sanchis, Marín, & Ramos, 2013). exposure assessment of aflatoxins in Catalonia (Spain). Food and Chemical Toxicology, 51,188e193. Therefore, most agencies have not set a tolerable daily intake (TDI) Chiou, A., Karathanos, V., Mylona, A., Salta, F., Preventi, F., & Andrikopoulos, N. for AFB1. Nevertheless, Kuiper-Goodman (1998) estimated a Pro- (2007). Currants (Vitis vinifera L.) content of simple phenolics and antioxidant visional Maximum Tolerable Daily Intake (PMTDI) 1.0 ng activity. Food Chemistry, 102,516e522. 1 Daradimos, E., Markaki, P., & Koupparis, M. (2000). Evaluation and validation of two AFB1 kg bw for adults and children without hepatitis B. fluorometric HPLC methods for the determination of aflatoxin B1 in olive oil. The AFB1 production (maximum production) risk assessment in Food Additives & Contaminants, 17(1), 65e73. currants examined in the present work, was estimated for a con- Dimitrokallis, V., Meimaroglou, D. M., & Markaki, P. (2008). Study of the ochratoxin A effect on Aspergillus parasiticus growth and aflatoxin B1 production. Food and sumption of 50 g by an adolescent (50 kg) and by an adult (70 kg) as Chemical Toxicology, 46(7), 2435e2439. well. El-Refai, I. M., Awadalla, O. A., & Abou Zid, A. M. (1995). Effects of some insect fl fl An adolescent displayed an AFB1 daily intake by the strains growth regulators on growth of Aspergillus avus and its productivity of a a- e ATHUM 6998, 6999 and 7000, 1.46e2.92 fold lower than the PMTDI toxin B1 and lipids. Food Additives & Contaminants, 12(4), 585 590. European Commission. (2006). Commission Regulation (EC) No. 1881/2006 of and by the strain 6997 a daily intake 5.8 fold higher than the PMTDI. December 2006 setting maximum levels for certain contaminants in foodstuffs. fi On the other hand, the AFB1 daily intake for the non-inoculated Of cial Journal of the European Union, L364,5. fi samples of currants was found to be 20 fold lower than the PMTDI. Fowler, J., & Cohen, L. (1997). Practical statistics for eld biology. Chichester, England: John Wiley & Sons. An adult displayed an AFB1 daily intake by the strains ATHUM Frisvad, J. C., & Samson, R. A. (2004). Polyphasic of Penicillium subgenus 6998, 6999 and 7000 from 2.08 to 4.16 fold lower than the PMTDI Penicillium. A guide to identification of food and air-borne terverticillate and by the strain 6997, a daily intake 4.14 fold higher than the Penicillia and their mycotoxins. Studies in Mycology, 49,1e174. Holmes, R. A., Boston, R. S., & Payne, G. A. (2008). Diverse inhibitors of aflatoxin PMTDI. On the other hand, the AFB1 daily intake for the non- biosynthesis. Applied Microbiology and Biotechnology, 78(4), 559e572. inoculated samples of currants was found to be 27.8 fold lower Iamanaka, B., Castle de Menezes, H., Vicente, E., Leite, R., & Taniwaki, M. (2007). than the PMTDI. Aflatoxigenic fungi and aflatoxins occurrence in sultanas and dried figs commercialized in Brazil. Food Control, 18, 454e457. Thus, the potential risk of an exposure level is depended on the IARC. (1993a). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans.In quantities of the currants consumed daily and the consumer’s Some naturally occurring substances: Food items and constituents, heterocyclic weight. amines and mycotoxins (Vol. 56); (pp. 245e395). Lyon: IARC Press. Imperato, R., Campone, L., Piccinelli, A. L., Veneziano, A., & Rastrelli, L. (2011). Survey of aflatoxins and ochratoxin a contamination in food products imported in Italy. 4. Conclusions Food Control, 22, 1905e1910. Jayashree, T., & Subramanyam, C. (2000). Oxidative stress as a prerequisite for fl The results of the present study indicated that currants origi- a atoxin production by Aspergillus parasiticus. Free Radical Biology and Medi- cine, 29(10), 981e985. nated from Crete and Corinth are not a favorable substrate for AFB1 Kollia, E., Kanapitsas, A., & Markaki, P. (2014). Occurrence of aflatoxin B1 and production by the aflatoxigenic strain of A. parasiticus. Furthermore, ochratoxin A in dried vine fruits from Greek market. Food Additives & Con- e the four strains of Aspergillus section Nigri (ATHUM 6998, 6999, taminants: Part B: Surveillance, 7(1), 11 16. Kuiper-Goodman, T. (1998). Food safety: mycotoxins and phycotoxins in perspec- 7000 and 6997) originating from Cretan and Corinthian currants tive. In M. Miraglia, H. van Edmond, C. Brera, & J. Gilbert (Eds.), Mycotoxins and e were able to produce AFB1 in currants. So they could be charac- phycotoxins-developments in chemistry: Toxicology and food safety (pp. 25 48). Fort Collins, Colo: Alaken Inc. terized as aflatoxigenic. The ability of the AFB1 production depends Leontopoulos, D., Siafaka, A., & Markaki, P. (2003). Black olives as substrate for on the currants substrate and mainly on the special characteristics Aspergillus parasiticus growth and aflatoxin B1 production. Food Microbiology, of each isolate. 20,119e126. 128 P. Kostarelou et al. / Food Control 43 (2014) 121e128

Luchese, R. H., & Harrigan, W. F. (1993). Biosynthesis of aflatoxin-the role of Romero, S. M., Comerio, R. M., Larumbe, G., Ritieni, A., Vaamonde, G., & Pinto, F. nutritional factors. Journal of Applied Bacteriology, 74,5e14. (2005). Toxigenic fungi isolated from dried vine fruits in Argentina. Interna- Lund, F. (1995). Differentiating Penicillium species by detection of indole metabo- tional Journal of Food Microbiology, 104,43e49. lites using a filter paper method. Letters in Applied Microbiology, 20, 228e231. Saxena, J., & Mehrotra, B. S. (1990). The occurrence of mycotoxins in some dry fruits Luttfullah, G., & Hussain, A. (2011). Studies on contamination level of aflatoxins in retail marketed in Nainital district of India. Acta Alimentaria, 19,221e224. some dried fruits and nuts of Pakistan. Food Control, 22, 426e429. Schuster, E., Dunn-Coleman, N., Frisvad, J. C., & van Dijck, P. W. M. (2002). On the Maggio-Hall, L. A., Wilson, R. A., & Keller, N. P. (2005). Fundamental contribution of safety of Aspergillus niger e a review. Applied Microbiology and Biotechnology, b-oxidation to polyketide mycotoxin production in planta. Molecular Plant- 59, 426e435. Microbe Interactions, 18(8), 783e793. Serra, R., Abrunhosa, L., Kozakiewicz, Z., & Venancio, A. (2003). Black Aspergillus Magnoli, C., Astoreca, A., Ponsone, M. L., Combina, M., Palacio, G., Rosa, C., et al. species as ochratoxin A producers in Portuguese wine grapes. International (2004). Survey of mycoflora and ochratoxin A in dried vine fruits from Journal of Food Microbiology, 88,63e68. Argentina markets. Letters in Applied Microbiology, 39, 326e331. Sinha, N. R., Williams, R. E., & Hake, S. (1993). Overexpression of the maize homeo Magnoli, C., Astoreca, A., Ponsone, M. L., Fernández-Juri, M. G., Barberis, C., & box gene, Knotted-1, causes a switch from determinate to indeterminate cell Dalcero, A. M. (2007). Ochratoxin A and Aspergillus section Nigri in fates. Genes & Development, 7, 787e795. seeds at different months of storage in Córdoba, Argentina. International Journal Soares, C., Calado, T., & Venâncio, A. (2013). Mycotoxin production by Aspergillus of Food Microbiology, 119,213e218. niger aggregate strains isolated from harvested maize in three Portuguese re- Magnoli, C., Violante, M., Combina, M., Palacio, G., & Dalcero, A. (2003). Mycoflora gions. Revista Iberoamericana de Micología, 30(1), 9e13. and ochratoxin-producing strains of Aspergillus section Nigri in wine grapes in Spadaro, D., Patharajan, S., Lorè, A., Garibaldi, A., & Gullino, M. L. (2012). Ochra- Argentina. Letters in Applied Microbiology, 37,179e184. toxigenic black species of Aspergilli in grape fruits of Northern Italy identified Martins, H. M., Martins, M. L., Bernardo, F., & Gimeno, A. (2005). Ability of wild by an improved PCR-RFLP procedure. Toxins, 4(2), 42e54. strains of Aspergillus niger to produce ochratoxin A in cracked corn. Revista Stubblefield, R. D. (1987). Optimum conditions for formation of aflatoxin M1-tri- Portuguesa de Ciências Veterinárias, 100,189e192. fluoroacetic acid derivative. Journal of the Association of Official Analytical Nour, V., Trandafir, I., & Ionica, M. E. (2011). Ascorbic acid, anthocyanins, organic Chemists, 7,1047e1049. acids and mineral content of some black and red currant cultivars. Fruits, 66, Tzanidi, C., Proestos, C., & Markaki, P. (2012). Saffron (Crocus sativus L.) inhibits 353e362. aflatoxin B1 production by Aspergillus parasiticus. Advances in Microbiology, 2, Palencia, E., Klich, M., Glenn, A., & Bacon, C. (2009). Use of a rep-PCR system to 310e316. predict species in the Aspergillus section Nigri. Journal of Microbiological Vergopoulou, S., Galanopoulou, D., & Markaki, P. (2001). Methyl jasmonate stimu- Methods, 79,1e7. lates aflatoxin B1 biosynthesis by Aspergillus parasiticus. Journal of Agricultural Passone, M. A., Rossob, L. C., & Etcheverry, M. (2012). Influence of sub-lethal anti- and Food Chemistry, 7, 3494e3498. oxidant doses, water potential and temperature on growth, sclerotia, aflatoxins Wejdan, S. K., Bahruddin, S., Chew, B. Y., Nor, H. H., Abdussalam Salhin, M. A., and aflD(¼nor-1) expression by Aspergillus flavus RCP08108. Microbiological Muhammad Idiris, S., et al. (2010). Determination of aflatoxins in animal Research, 167,470e477. feeds by HPLC with multifunctional column clean-up. Food Chemistry, 118, Patterson, D. S. P. (1976). Structure, metabolism and toxicity of aflatoxin. Cahiers de 882e886. Nutrition et de Diététique, 2,71e78. Yassin, M. A., El-Samawaty, A. R., Bahkali, A., Moslem, M., Abd-Elsalam, K. A., & Perrone, G. (2007). Biodiversity of Aspergillus species in some important agricul- Hyde, K. D. (2010). Mycotoxin-producing fungi occurring in sorghum grains tural products. Studies in Mycology, 59,53e66. from Saudi Arabia. Fungal Diversity, 44(1), 45e52. Perrone, G., De Girolamo, A., Sarigiannis, Y., Haidukowski, M. E., & Visconti, A. Youssef, M. S., Abo-Dahab, N. F., & Abou-Seidah, A. A. (2000). Mycobiota and (2013). Occurrence of ochratoxin A, fumonisin B2 and black aspergilli in raisins mycotoxin contamination of dried raisins in Egypt. African Journal of Mycology from Western Greece regions in relation to environmental and geographical and Biotechnology, 8,69e86. Chemical Abstracts 135, 317617r, 2001. factors. Food Additives & Contaminants e Part A, 30(7), 1339e1347. Yu, J., Chang, P. K., Payne, G. A., Cary, J. W., Bhatnagar, D.d., & Cleveland, T. E. (1995). Pitt, J. J. (1986). Methods for the mycological examination of food. NATO Advanced Comparison of the omtA genes encoding O-methyltransferases involved in Science Institutes Series No.122.F. aflatoxin biosynthesis from Aspergillus parasiticus and A. flavus. Gene, 163(1), Reverberi, M., Fabbri, A. A., Zjalic, S., Ricellil, A., Punelli, F., & Fanelli, C. (2005). 121e125. Antioxidant enzymes stimulation in Aspergillus parasiticus by Lentinula edodes Zinedine, A., & Mañes, J. (2009). Occurrence and legislation of mycotoxins in food inhibits aflatoxin production. Applied Microbiology and Biotechnology, 69(2), and feed from Morocco. Food Control, 20(4), 334e344. 207e215.