COLONIZATION OF GRAPES BERRIES AND CIDER BY POTENTIAL PRODUCERS OF PATULIN

The aim of this study was to detect potential producers of mycotoxin patulin from grapes (berries, surface sterilized berries endogenous mycobiota and grape juice) of Slovak origin. We analyzed 47 samples of grapes, harvested in 2011, 2012 and 2013 from various wine-growing regions. For the isolation of species we used the method of direct plating berries and surface-sterilized berries (using 1% freshly pre-pared chlorine) berries on DRBC (Dichloran Rose Bengal Chloramphenicol agar). For the determination of fungal contamination of grape juice we used plate-dilution method and DRBC and DG18 (Dichloran 18% Glycerol agar) as media. The cultivation in all modes of inoculation was carried at 25 ±1 °C, for 5 to 7 days. After incubation Aspergillus and Pencillium isolates were inoculated on the identification media. The potential producers of patulin were isolated from 23 samples berries, 19 samples of surface-sterilized berries and 6 samples of grape juice. Overall, the representatives of producers of patulin were detected in 32 (68.1%) samples (75 isolates). In this work we focused on the detection of potential producers of patulin, Penicillium expansum (the most important producer of patulin in fruits), Penicillium griseofulvum and Aspergillus clavatus were isolated. Chosen isolates of potential patulin producers were tested for the ability to produce relevant mycotoxins in in vitro conditions using thin layer chromatography method. The ability to produce patulin in in vitro condition was detected in 82% of isolates of Penicillium expansum, 65% of Penicillium griseofuvum and 100% of Aspergillus clavatus. Some isolates of Penicillium expansum were able to produce citrinin and roquefortine C, Penicillium griseofulvum cyclopiazonic acid, griseofulvin and roquefortin C,


Mycological analysis
For analysis intact berries have been used. For the isolation of species we used the method of direct plating berries, surface-sterilized berries (using 1% freshly pre-pared chlorine) and non-sterilized berries on DRBC (Dichloran Rose Bengal Chloramphenicol agar). For each analysis were used 50 berries. The cultivation was carried at 25 ±1 °C, for 5 to 7 days in dark. Only undamaged berries have been used for analysis. After incubation, the colonies of Aspergillus and Penicillium were transferred onto appropriate identification media.
Dilute plate technique was used for isolation of fungi from the samples of cider according to Samson et al. (2002). Sample 20 ml of cider was mixed with 180 ml of saline solution (0.85% sodium chloride) with 0.05% Tween 80 on homogenizer. Then 0.1 ml of appropriate dilutions made up to 10 -3 was applied on DRBC and DG18 (Dichloran 18% Glycerol agar). After 5 to 7 days of incubation at 25 ±1 °C in dark, resulting colonies were transferred onto appropriate identification media.
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 dark at 25 ±1 °C, 7 days. Species identification was done according to Klich ( Cultivation for screening extracellular metabolites (citrinin, griseofulvin patulin) was carried out on YES (Yeast Sucrose agar) and for intracellular (cyclopiazonic acid and 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; roquefortin C after spraying with Ce(SO4) 2 x 4 H2O visible as orange spot. Directly under UV light (365 nm) were visualised following mycotoxins: citrinin (yellow-green) and griseofulvin (blue spot).

RESULTS AND DISCUSSION
Mycotoxins are abiotic hazards produced by certain fungi that can grow on a variety of crops (Marin et al., 20013). In the current study from 68.1% of samples were isolated potential producers of patulin. We isolated species genera Aspergillus (Aspergillus clavatus) and Penicillium (Penicillium expansum and Penicillium griseofulvum), as potential producers of this mycotoxin. According Dombrink-Kurtzman and Engberg (2006) the Byssochamys nivea strains produced patulin in amounts comparable to Penicillium expansum strains. Interest in the genus Byssochlamys is related to the ability of its ascospores to survive pasteurization and cause spoilage of heat-processed fruit products worldwide. Genus Byssochlamys was not detected in our samples. The number of isolates and isolation frequency of species recovered from the sample are listed in Table 1. According Zouaoui et al. (2015) patulin is a secondary metabolite, which is mainly produced by certain species of Aspergillus and Penicillium fungi.

Penicillium spp.
Penicillium species are ubiquitous, opportunic saprophytes. A majority of the described species are soil fungi, and their occurrence in food is more of less accidental and rarely of consequence. However, quite numbers of species are closely associated with human food supplies. Some species are more specialised: several are destructive pathogen on fruit (e.g. Penicillium digitatum, Penicillium expansum, Penicillium italicum) (Pitt and Hocking, 2009). Penicillium expansum is one of the most common fruit pathogens; it causes soft rot known as "a blue mould rot" on a variety of fruits were tested for ability to produce patulin in in vitro condition. From 45 tested isolates 37 (82.2%) produced patulin. Postharvest diseases are the most important factors that limit commercial export of Chilean table grapes. In recent years, blue mould decay caused by Penicillium expansum has frequently appeared on Red Globe grapes after long period (>60 days) of cold storage, causing significant economical losses (Franck et al., 2005). Penicillium expansum causes significant economic losses to the fruit industry and is also one of potential public health concern because it produces toxic secondary metabolites including patulin, citrinin, and chaetoglogosins (Andersen et al., 2004). 48.9% of tested isolates Penicillium expansum produced citrinin and 93.3% roquefortine C in vitro conditions, as well. Bragulat et al. (2008) reported, that 100% isolates Penicillium expansum, detected from grapes, were able to produce citrinin and 60% patulin. Citrinin is a quinone methide with a powerful antibacterial effect, but toxic to humans and animals. This mycotoxin is mainly hepato-nephrotoxic (Zaied et al., 2012). Roquefortine C (another mycotoxin produced by Penicillium expansum) is a very widespread fungal secondary metabolite. The acute toxicity of roquefortine C is not very high (Cole and Cox, 1981). Furthermore, we have identified species Penicillium griseofulvumother producer of patulin; 42 isolates from the berries and 25 from cider (2 positive samples). Penicillium griseofulvum is a very efficient producer of high levels of patulin in pure culture, and it may potentially produce patulin in cereals, pasta and similar products (Frisvad et al., 2006, Frisvad  et al., 2007b). 11(65%) tested isolates (Table 2) were able to produce patulin. Some isolates produced cyclopiazonic acid (94%), roquefortine C (88%) and griseofulvin (88%), also. Incidence of Penicillium griseofulvum on grapes also described Serra et al. (2005), Bragulat et al. (2008) and other authors. As mentioned above, the Penicillium griseofulvum also produced cyclopiazonic acid. Persistently studies with this mycotoxin have shown that targets correspond to muscles, liver and spleen (Burdock and Flamm, 2000). The last detected mycotoxin was griseofulvin. Griseofulvin is active against dermatophytic fungi of different species in the genera Microsporum, Trychophyton and Epidermophyton. Prolonged griseofulvin treatment in experimental animals provoked biochemical changes consisting mainly of disturbances of porphyrin metabolism, variation in the microsomal cytochrome levels and formation of Mallory bodies (De Carli and Larizza, 1988).

Aspergillus sp.
Species of Aspergillus are among the most economically important fungi, on the positive side being very widely used for synthesis of chemicals, for biosynthetic transformations and enzyme production. On the negative side, they are of great importance in food spoilage and they produce important mycotoxins (Pitt and Hocking, 2009). Lopez-Diaz and Flannigan reported that Aspergillus clavatus, Aspergillus longivesica, Aspergillus giganteus and other species are very efficient producers of patulin laboratory, but only Aspergillus clavatus may play role in human health (Frisvad et al., 2007b). 30 isolates from grapes berries and 25 isolates from cider (2 positive samples) have been identified as Aspergillus clavatus. All tested isolates (Table 2) were able to produce patulin.

CONCLUSION
Potential producers of patulin were isolated from 68.1% of the analysed samples. We isolated species genera Aspergillus and Penicillium. The ability to produce patulin in in vitro condition was detected in 82% of isolates of Penicillium expansum, 65% of Penicillium griseofuvum and 100% of Aspergillus clavatus. Some isolates of Penicillium expansum were able to produce citrinin and roquefortine C, Penicillium griseofulvum cyclopiazonic acid, griseofulvin and roquefortin C, also. Table 1 Filamentous fungipotential producers of patulin identified from grape berries and cider from 47 samples.

Berries
Surface-sterilized berries Cider ** -number of tested isolates, * -number of isolates with ability to produce mycotoxin, TLCthin layer chromatography