CONTAMINATION OF RAISIN BY FILAMENTOUS FUNGI – POTENTIAL PRODUCERS OF OCHRATOXIN A

The forty-one samples of Armenian made and eleven samples of imported raisins collected in several markets in Yerevan were studied. The sample collections were carried out during of years 2004 to 2008. Thirty two species of filamentous fungi from Aspergillus, Penicillium, Alternaria Trichoderma and Syncephalastrum genera were isolated and identified. Among species isolated from studied samples species belonging to Aspergillus genera have a very high frequency of occurrence, 65.2% of all investigated filamentous fungi. Species from Nigri section show the highest occurrence: 66.7% of all isolated fungi belonging to Aspergillus genera. Species A. carbonarius and A. niger were dominated among isolated fungi from section Nigri. Both Armenian and imported samples of raisin had a high contamination level by these fungi which are potential producers of ochratoxin A. In Armenian samples were detected two more ochratoxigenic species belonging to Aspergillus section Nigri: A. sclerotioniger and A. lacticoffeatus. But their frequency of occurred was low. Thirty seven strains of A. flavus were isolated, 92% of them were isolated from Armenian samples. Influence of рН and aw on contamination level of raisin by fungi was studied.  It was revealed that highest contamination level by filamentous fungi occurred in raisins with relatively high aw value. Contamination level of raisin doesn't depend on pH. doi:10.5219/95

ročník 4 29 4/2010 isolated strains of A. niger.A. carbonarius have less frequency but 96% of 48 strains were ochratoxigenic.Naresh and David (2007) reported that in northern Europe the major species of fungi producing ochratoxin A are P. verrucosum, A. ochraceus and species from Aspergillus section Nigri, especially A. carbonarius.O TA was detected in grape, grape juice and dried vine fruit in many countries: Morocco In some cases the level of OTA in products was exceeded maximum allowed quantity.

Sampling
11 samples of imported and 41 samples of Armenian black and white raisins realizing in markets of Yerevan were studied.The sampling has been carried out between 2004 and 2008 by dot method (SANCO/1208/2005-rev. 1).

Mycological analyses of raisin
Samples were sterilized with 3% solution of sodium hypochlorite for 15 minutes.After surface disinfection samples were washed with fresh distillate water.The analyses were carried out with direct plating and dilution plating methods.For plating method direction food particles were placed directly on solidified agar media.For dilution 1:10, 10g of sample was dissolved in 90 ml sterilized water and mixed for 15 minutes (Pitt and Hocking, 1997).For isolation of filamentous fungi CYA (Chapek-Yeast Agar medium, HiMedia Ltd.), GYA (Glucose-Yeast Agar medium, HiMedia Ltd.), and MEA (Malt-Extract Agar medium, HiMedia Ltd.) were used.The plates were incubated at 28° C for 7 days (NF ISO 7954 -88).After incubation the colony forming unit (CFU) was accounted according to NF ISO 7698-91, and frequency of occurrence was detected (El-Kady, Abdel-Hafez and El-Maraghy, 1982).

Determination of aw, pH and sulfur dioxide
Determination of aw was spent with AquaLab (Decagon Devices, Pullman, WA, USA).Definition of pH was spent with pH-meter (Oakton, USA).Sulfur dioxide in samples of raisin was determined by aspiration method (Wood, Foster, Damant et al., 2004).

RESULTS AND DISCUSSION
Fungal flora of raisin A comparative analysis of 41 samples of Armenian raisin and 11 samples of imported raisin from Iran were carried out.32 species of filamentous fungi belonging to two classes: Zygomycetes and Hyphomycetes, and 6 genera: Mucor, Aspergillus, Penicillium, Alternaria, Trichoderma and Syncephalastrum, were isolated and identified (Table 1).  2 and 3).Studied samples of raisin have a high contamination level by fungi from Mucor genera.The presence of these fungi is a result of secondary contamination of raisin.These fungi were detected only on surface of samples.Surface disinfection of samples with 3% solution of sodium hypochlorite inhibited the growth of species of M. mucedo and M. racemosus.The comparative analyses of local and imported raisin have shown that contamination level of Armenian samples was much higher than Iranian samples.No filamentous fungi were identified in some imported samples.The quantity of species isolated from Armenian samples and belonging to Aspergillus genera exceeds the quantity of species isolated from imported raisin samples.Only 6 species of Aspergillus genera were isolated from imported raisin and 14 from local samples (Table 3).and A. tubingensis occurred nearly in all samples.But these fungi were detected at a higher frequency in locally produced raisin rather than in imported (Table 4).2007) not only specie A. carbonarius is potential producer of ochratoxin A but also species of A. niger, A. lacticoffeatus and A. sclerotioniger among filamentous fungi from Aspergillus Nigri section.These species were found in studied samples.The contamination of raisin by fungi was quite high especially by pathogenic fungi A. carbonarius and A. niger.The frequency of occurrence of A. carbonarius was higher in Armenian raisins -59%, rather than in imported raisins -35%.39 strains of A. carbonarius were isolated, 26 of which from Armenian and 10 from imported samples.
A. foetidus, A. sclerotioniger and A. sclerotiicarbonarius species were identified only in locally produced samples.Among ochratoxigenic species only A. niger and A. carbonarius were detected in imported samples.Toxigenic fungi occurred more frequently in locally produced raisin (Figure 1).
The results showed that the contamination level of raisin by filamentous fungi sharply increases every year and new species from Aspergillus genera are appeared.In our samples of 2007-2008 years frequency of occurrence of A. carbonarius, A. foetidus and A. tubingensis species is comparatively higher than in samples collected between 2004 and 2006.The occurrence of ochratoxigenic fungi A. carbonarius has sharply increased (from 70 to 80 %) recently in both locally produced and imported samples (Figure 2).
Species belonging to Aspergillus flavi section: A. flavus and A. nomius have average frequency of occurrence.These species are potential producers of mycotoxins from aflotoxins group.Specie A. flavus has been found in both Armenian and imported raisin (23.5% frequency). A. nomius was detected only in local samples at a low frequency (17%).Contamination level of raisin by A. flavus has also been raised during of recent years.In 2007 to 2008, for example, high contamination level of raisin by A. flavus was observed especially in local samples (Figure 3).37 strains of A. flavus were isolated, 82% of which from Armenian raisin.

Influence of рН, aw and sulfur dioxide content on contamination level
For growth of filamentous fungi and mycotoxin production some growth factors such as pH, water activity (aw) and others are considered.Therefore, influence of рН and aw on contamination level of raisin by fungi was studied (Table 5).The correlation between pH and contamination level is not simple.As such contamination level of raisin doesn't depend on pH.Values of pH of analyzed samples were 4.42-4.8which is favorable for growth and sporulation of fungi (including toxigenic).A positive correlation between water activity and contamination level of Armenian raisin by filamentous fungi is revealed.These results are presented in table 5.The highest contamination levels are detected in samples of Armenian raisin with relatively high value of aw.Colony forming units (cfu.g -1 ) of Iranian samples was very low -0 to 40, despite of high water activity.This is possible because Iranian raisins have a high content of sulfur dioxide."Free" SO 2 in Iranian gold raisin is about 45to 80 mg/kg, which can inhibit fungal growth.Total SO 2 was about 900-1200 mg/kg.It is noteworthy, however the quantity of sulfur dioxide in samples of Iranian gold raisin did not exceed the maximum allowed level such as 2000 mg.kg -1 (Christensen, 2000).Sulfur dioxide was not detected in Armenian black raisins.Armenian black raisins were not treated with sulfur dioxide as commonly used to prevent natural color development.Subsequently, the frequency of occurrence of filamentous fungi was high in these samples.
It can be implied that the contamination of raisins by filamentous fungi can be prevented by use of control environmental conditions.

CONCLUSION
As a result of this and many other reports it is obvious that raisin is a favorable substrate for fungi, particularly for potential producers of mycotoxins such as ochratoxin A (A. carbonarius, A. niger).Six species of potential producers of mycotoxins, A. carbonarius, A. niger, A. sclerotioniger, A. lacticoffeatus, A. flavus and A. nomius, were isolated. A. sclerotioniger, A. lacticoffeatus and A. nomius occurred only in Armenian samples of raisin (Figure 1).In connection with climate change in recent years in Armenia the contamination level of raisin by potential ochratoxigenic fungi such as A. carbonarius has been increased.The frequency of occurrence of A. niger is turned to be very high.In 2004 it was about 66.7%, compare to 75% for recent five years (Figure 2).However, the occurrence of A. carbonarius has risen to 54.2%.Potential producer of aflatoxin A species such as A. flavus and A. nomius have occurred more often in raisin.And contamination of raisin by these species of toxigenic fungus has also risen within recent years, which is an actual and serious problem in Armenia.
Figure 3 frequency of occurrence of A. flavus from years 2004 to 2008