Mycobiota of spices and aromatic herbs

A total of 67 samples of spices and herbs were tested for mould contamination. From 50.7% of samples, moulds were not isolated. The most dominant genera were Aspergillus and Penicillium . Potential producers of mycotoxins Aspergillus spp. and Penicillium spp. were tested for the ability to produce some mycotoxins. Isolates of potentially toxinogenic species were found to produce various mycotoxins, namely alfatoxin B 1 ( Aspergillus flavus ), cyclopiazonic acid ( Aspergillus flavus ), sterigmatocystin ( Emericella nidulans ), roquefortine C ( Penicillium allii, P. chrysogenum, P. crustosum, P. expansum ), penitrem A ( P. crustosum ) and patulin ( P. expansum ). Some of the tested isolates produce two mycotoxins: A. flavus (aflatoxin B 1 and cyclopiazonic acid), P. crustosum (roquefortine C and patulin) and P. expansum (roquefortine C and patulin). None of the tested isolates of Aspergillus section Nigri screened, appeared to produce ochratoxin A. Totally 11 samples were analysed for the presence of aflatoxins and ochratoxin A. Aflatoxin B 1 was found in 5 (45.5%) out of 11 samples analysed with levels ranging from 0.14 to 2.9 µg.kg -1 . In one sample we detected aflatoxin G 1 . Ochratoxin A was found in 3 samples (27.3%), with levels ranging from 2.2 to 5.19 µg.kg -1 . No sample was contaminated by aflatoxins or ochratoxin A above the maximum admitted threshold established by the European legislation.


INTRODUCTION
Spices have been used for flavour, colours, aroma and preservation of food or beverages for thousands years (Ozbey and Kabak, 2012). Because of their processing and environmental conditions, spices can be heavily contaminated with toxigenic fungi and mycotoxins. Mycotoxins are secondary metabolites produced naturally by filamentous fungi, which are considered toxic substances when present in food for human and food for animals (da Rocha et al., 2014). For spices there are two groups of mycotoxins of concern, aflatoxins and ochratoxin A (Ozbey and Kabak, 2012). Aflatoxins are produced by fungi that belong to Aspergillus genus and especially by Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius (Cary and Ehrlich, 2006, Marín et al., 2009). Ochratoxin A is a secondary metabolite produced by filamentous fungi of the genera Aspergillus and Penicillium present in a wide range of foodstuffs. The most relevant ochratoxin A producing species are Penicillium verrucosum, Aspergillus ochraceus, Aspergillus niger and Aspergillus carbonarius due to their prevalence in foodstuffs (cereals, grapes, coffee, etc.)  The aim of the study was the determination of potentially toxigenic filamentous fungi from genera Aspergillus and Penicillium from spices and herbs. A special emphasis was laid on the ability of isolated Aspergillus and Penicillium species to produce some significant toxic extrolites -mycotoxins.

Samples
Totally 67 samples of spices and herbs from different countries (Table 1) were analysed. The samples (approximately 100 g) were collected from the storage rooms of food factory.

Mycological analysis
Dilute plate technique was used for isolation of fungi from the samples according to Samson et al. (2002). Sample in weigh of 20 g was mixed with 180 ml of saline solution (0.85% sodium chloride) with 0.05% Tween 80 in homogenizer. Then 0.1 ml of appropriate dilution made up to 10 -2 was applied on DRBC (Dichloran Rose Bengal Chloramphenicol agar). After 5 to 7 days of incubation at 25 ± 1°C, in dark resulting colonies were transferred onto appropriate identification media.
The identification of Aspergillus species. Conidial suspensions were inoculated at three equidistant points both on Czapek-yeast Extract agar (CYA), Czapek-yeast with 20% Sucrose (CY20S) and malt extract agar (MEA) and incubated in the dark at 25 ± 1 °C, 7 days. Species identification was done according to Klich ( The cultivation for screening of extracellular metabolites (aflatoxin B 1 , aflatoxin G 1 , citrinin, patulin, ochratoxin A) were carried out on YES (Yeast Sucrose agar) and for intracellular (cyclopiazonic acid, penitrem A, roquefortin C, sterigmatocystin) on CYA (Czapek-yeast Extract agar); conditions of cultivation in dark at 25 °C, 14 days. In each tested isolate, 3 pieces of mycelium together with cultivation medium of approximately 5 x 5 mm area were cut from colonies and extracted in 1000 ml of chloroform:methanol (2:1, v/v) on vortex for 2 minutes. Then 20 μl of liquid phase of extracts along with standards (Sigma, Germany) were applied on TLC plate (Marchey-Nagel, Germany) and consequently developed in solvent system toluene:ethylacetate:formic acid (5:4:1, v/v/v/). The visualisation of extrolites was carried out as follows: cyclopiazonic acid directly in daylight after spraying with the Ehrlich reagent (violet-tailed spot); patulin by spraying with 0.5% methylbenzothiazolone hydrochloride in methanol, heated at 130 °C for 8 min and then detectable as a yellow-orange spot; penitrem A after spraying with 20% AlCl 3 in 60% ethanol, heated at 130 °C for 8 min and then detectable as a dark green to black spot on daylight; roquefortin C after spraying with Ce(SO 4 ) 2 x 4 H 2 O visible as an orange spot. Directly under UV light (365 nm) were visualised following mycotoxins: aflatoxin B 1 (blue spot), aflatoxin G 1 (green), citrinin (yellow-green), ochratoxin A (bluish-green), sterigmatocystin (reddish).

The determination of mycotoxins in sample
In the 11 samples were determined following mycotoxins: aflatoxin B 1 , B 2 , G 1 , G 2 and ochratoxin A. Analyses were performed by HPLC method (high-pressure liquid chromatography) in an external accredited laboratory.

RESULTS AND DISCUSSION
In the current study from 50.7% of the samples, moulds were not isolated (basil, crushed black pepper, granulated garlic, curry, cumin powder, salvia, crushed chillies, crushed bay leaves, paprika (spicy), dill, crushed green pepper, savory). These findings are similar to data reported by Witkowska et al. (2011), where in 50% of samples of commercial herbs and spices were detected moulds. The fungal species recovered from the samples are listed in Table 2. Species of 11 genera were isolated and identified. The Aspergillus and Penicillium were the most common genera. Hashem and Alamri (2010) from 15 spices isolated as the most common genera Aspergillus, Penicillium and Rhizopus. Rhizopus (Rhizopus stolonifer) was isolated from granulated onion, only. In the Aspergillus section Nigri, A. niger and A. carbonarius produce ochratoxin A (Almela et al.,  2007). The isolates of this section were the most frequent in our study.
Totally 11 samples were analysed for the presence of aflatoxins and ochratoxin A (Table 4). Aflatoxin B 1 was found in 5 (45.5%) out of 11 samples analysed with levels ranging from 0.14 to 2.9 µg.kg -1 . No sample was contaminated by aflatoxin B 1 above the maximum admitted threshold established by the European legislation (Commission regulation, 2010b). In one sample we detected aflatoxin G 1 . Ochratoxin A was found in 3 of samples (27.3%), with levels ranging from 2.2 to 5.1 µg.kg -1 . No sample was contaminated by ochratoxin A above the maximum admitted threshold established by the European legislation (Commission regulation, 2010a). Prelle et al. (2014) showed that 15.4% and 23.8% of samples were contaminated with aflatoxins and ochratoxin A, respectively. In our study, 2.3% of spice samples contaminated by ochratoxin A get over the threshold admitted by European Regulation. Zhao et al. (2013) presented that about 11% of the 480 Chinese spices samples tested contained detectible levels of aflatoxin B 1 , with the highest concentrations found in chili, prickly ash and pepper. Zinedine et al. (2006) reported the higher level of aflatoxin B 1 contamination in red paprika (9.68 µg.kg -1 ). The analysis of the spice samples contamination (in Morocco) with aflatoxin B 1 revealed that paprika is frequently contaminated, since 95% were contaminated with that mycotoxin and 40% of samples exceeded European regulation for that contaminant (Mahgubi et al., 2013). Co-occurrence of aflatoxin B 1 and ochratoxin A in samples of crushed chillies and paprika was detected in our study. Ozbey a Kabak (2012) reported co-occurrence of these mycotoxins in 62.5% of red chilli flake, 40.9% of red chilli powder and 4.3% pepper of powder samples.

CONCLUSION
From 50.7% of samples, moulds were not isolated. The most dominant genera were Aspergillus and Penicillium. The isolates of potentially toxinogenic species were found to produce various mycotoxins (alfatoxin B 1 , cyclopiazonic acid, sterigmatocystin, roquefortine C, penitrem A and patulin). None of the tested isolates Aspergillus section Nigri screened appeared to produce ochratoxin A. Totally 11 samples were analysed for the presence of aflatoxins and ochratoxin A. Aflatoxin B 1 was found in 45.5% out of 11 samples analysed. Ochratoxin A was found in 27.3% of samples.  <0.10 <0.10 <0.10 <0.10 <0.20 AFB1 -aflatoxin B 1 , AFB2 -aflatoxin B 2 , AFG1 -aflatoxin G 1 , AFG2 -aflatoxin G 2 , OTA -ochratoxin A