MYCOFLORA AND MYCOTOXIN CONTAMINATION OF SOME DRIED FRUITS IN YEMEN REPUBLIC الفلورا الفطرية والسموم الفطرية الملوثة لبعض الثمار الجافة فى اليمن

MYCOFLORA AND MYCOTOXIN CONTAMINATION OF SOME DRIED FRUITS IN YEMEN REPUBLIC

Saeed M. S. Alghalibi and Abdul-Rahman M. Shater

Biology Department, Faculty of Science, Sana’a University, Sana’a, Yemen.

INTRODUCTION:

In most developing countries, agriculture is the back bone of the economy and export crops are greatly depended upon as a source of foreign exchange to finance productive activities and other essential services. Most of these crops are cereals, fruits, vegetables and oil seeds that are highly susceptible to fungal growth and mycotoxin production (Garbutt, 1997). Mycotoxins are toxic metabolites of fungi which, if ingested, can cause acute or chronic toxic effects such as carcinogenic, mutagenic, teratogenic, atherogenic and oestrogenic effects in human and animals (Van Egmond, 1995). The illnesses caused by mycotoxins are called mycotoxicosis. The mycotoxins are not only hazardous to consumer health but also affect food quality resulting in huge economic losses for these countries (Moss, 1998).

The numbers of microorganisms on most dried fruits vary from a few hundreds per gram of fruits to thousands and they are mostly on the outer surfaces. Spores of bacteria and molds are likely to be the most numerous. When part of the fruit has supported growth and sporulation of mold before or after drying, mold spores may be present in large numbers. If drying trays are not clean and improperly loaded, a marked increase in the numbers of bacteria and fungi may take place during the drying process. Spoilage of most dry fruits usually occurs during storage, handling and transport (Frazier and Westhoff, 1988).

Preservation of fruits by solar drying has been practiced for centuries. It is limited to climates with a hot sun and a dry atmosphere and to certain fruits, such as raisins, prunes, figs, apricots, pears and peaches. The fruits are spread out on trays and be turned during drying.

Although natural occurrence of mycotoxins and fungal contamination of many dried fruits were investigated in many parts of the world (Boyacioglu and Gonul, 1988; Steiner et al., 1988; Herry and Lemetayer, 1992; Zohri and Abdel-Gawad, 1993; Ozay et al., 1995; Elhalouat and Debevere, 1997; Elhalouat et al., 1998; Abdel-Sater and Saber, 1999 and Bayman, et al. 2002), none of these studies were reported in Yemen Republic. Therefore, this investigation was mainly planned to determine the mycoflora and mycotoxin contamination of some Yemeni dry fruits.

MATERIALS AND METHODS:

Collection of samples: Twenty dried samples (500 g each) of each of raisins, figs and dates fruits were collected from shops and markets of different sanitation levels at Sana'a, Taiz, Adan and Ibb governorates, Yemen Republic during the period from June 2002 to May 2003. Each sample was put in a sterile polyethylene bag, sealed and transferred to the laboratory. All samples were kept in a refrigerator (4ºC) till mycoflora and mycotoxin analysis.

Isolation of molds and yeasts: Fungi were isolated using the dilution plate method as described by Johnson and Curl (1972). Twenty five grams of each dried fruit sample was comminuted for 2 min in 250 ml of 0.12% sterile plain agar. Further dilutions were made and one ml of an appropriate final dilution was placed in each petridish. 1% glucose- and 40% sucrose-Czapek's agar media were used for isolation of glucophilic and xerophilic fungi, respectively. Chloramphenicol (20 µg/ml) and rose bengal (30 ppm) were used as bacteriostatic agents. Six plates were used for each sample (3 plates for each type of medium). The plates were incubated at 28ºC for 10 days. The developing fungi were counted and identified morphologically based on macro- and microscopic characteristics and by using the following references: Ainsworth & Bisby (1961), Booth (1971), Ellis (1971), Raper and Fennell (1977), Pitt (1979), Moubasher (1993), Samson et al. (1995) and Barnett & Hunter (1998)

Mycotoxins analysis: Twenty-five grams of each dried fruit sample was defatted by extraction with normal hexane for 10 h using Soxhlet-type extractor. The defatted residue was reextracted for another 10 h with chloroform. The chloroform extract was dried over anhydrous sodium sulphate, filtered and evaporated under vacuum to near dryness. The residue was diluted with chloroform to one ml.

Qualitative analysis of mycotoxins: The chloroform extracts were analyzed on precoated silica gel plate type 60F 254 (Merck) for the presence of different mycotoxins: Aflatoxins (B1, B2, G1 and G2), citrinin, sterigmatocystin, patulin, ochratoxin A, diacetoxyscirpenol, T-2 toxin and zearalenone toxin using thin layer chromatographic technique according to standard procedures (Scott et al. 1970, Thrane, 1986 and Van Egmond, 1995).

Quantitative determination of aflatoxins: The spots of aflatoxin B1 including the standards were removed from the plates, eluded with methanol and estimated spectrophoto-metrically using Spectrophotometer (UNICAM Helios Gamma, Helios Delta) according to the methods described by Scott (1995).

Quantitative determination and confirmation of ochratoxin A: According to the method of Nesheim et al. (1976).

Bioassay of toxins: Brine shrimps (Artemia salina L.) were used for mycotoxins bioassay test according to the method described by Brown (1969) and Reiss (1993).

RESULTS AND DISCUSSION:

1-Glucophilic fungi (isolated on 1% glucose-Czapek's agar):

Data in Table (1) show that twenty one species and one variety belonging to 13 genera were collected from dried fruits of raisins (10 genera, 16 species +1 var.), figs (7 genera, 11 species) and dates (8 genera, 15 species + 1 var.) on 1% glucose-Czapek's agar at 28ºC. The gross total counts of glucophilic fungi in dried raisins, figs and dates were 2240, 640 and 1540 colonies per g dry weight in all samples tested respectively (Table 1). Most of the recovered fungi were isolated from dry fruits in many part of the world as reported by several investigators (Herry and Lemetayer, 1992; Zohri and Abdel-Gawad, 1993; Ozay et al., 1995; Elhalouat and Debevere, 1997; Abdel-Sater and Saber, 1999), but almost all of them were firstly isolated from dried raisins, figs and dates in Yemen.

Aspergillus was the most frequently isolated genus. It occurred in 70%, 30% and 75% of the samples comprising 63.4%, 59.4% and 71.4% of total fungi in dried raisins, figs and dates, respectively. Peter et al. (1990) studied the fungal contamination of fruits and vegetables and indicated that Aspergillus was isolated from 79.5% of the samples. In Egypt, Abdel-Sater and Saber (1999) found that Aspergillus was the predominant genus isolated from dried raisins and dates (90% and 100% of samples respectively), while Zohri and Abdel-Gawad (1993) found that Penicillium was the most predominant genus isolated from dried apricots, figs, prunes and raisins. Of the Aspergillus, 6 species and one variety were isolated of which A. niger and A. flavus were the most prevalent species followed by A. fumigatus and A. ochraceus. The remaining species were less frequently isolated and A. versicolor was only isolated from dried raisins. Most of the Aspergillus species recovered during the current investigation were isolated previously from different dried fruits in different parts of the world, but with variable frequencies and populations (Peter et al., 1990; Zohri and Abdel-Gawad, 1993; Elhalouat and Debevere, 1997; Abdel-Sater and Saber, 1999).

Rhizopus stolonifer was the second most common fungus isolated from dried fruits in Yemen. It occurred in 30%, 15% and 25% of the samples comprising 10.7%, 12.5% and 11.7% of total fungi in dried raisins, figs and dates, respectively (Table 1). It was not recorded by Abdel-Sater and Saber (1999) in their investigation and isolated in low frequency from two samples of dry raisins and one sample of each of dry figs and plums by Zohri and Abdel-Gawad (1993) in Egypt. Also, it was not isolated in Morocco by Elhalouat and Debevere (1997) from dried raisins and prunes.

Penicillium was isolated from five samples of raisins (25%), four samples of dates (20%) and two samples of figs (10%). Similar finding was reported by Abdel-Sater and Saber (1999) in Egypt who found that Penicillium species isolated in low frequency from dry raisins (35%) and dates (30%). In contrast to our finding, Zohri and Abdel-Gawad (1993) found that Penicillium was the most prevalent genus and encountered from all samples of dry figs, prunes, apricots and raisins. It was represented by four species of which P. chrysogenum was the common species in the three dry fruits. This species was also the most common species recovered by Zohri and Abdel-Gawad (1993), Abdel-Sater and Saber (1999) in Egypt and by Elhalouat and Debevere (1997) in Morocco.

The remaining genera and species were isolated from one or two substrates with low frequency and total counts as represented in Table (1). Most of these fungi were isolated previously from various dry fruits, seeds and vegetables in many parts of the world as reported by several researchers (Herry and Lemetayer, 1992; Benkhemmar et al., 1993; Zohri and Abdel-Gawad 1993; Ozay et al., 1995; Elhalouat and Debevere 1997 and Abdel-Sater and Saber 1999).

Table (1): Average total counts (ATC, calculated per g dry weight of fruit) and number of cases of isolation (NCI) of various fungal genera and species isolated from dried fruits on 1% sucrose-Czapek’s agar at 28ºC.

2-Xerophilic fungi (isolated on 40% sucrose-Czapek's agar):

Nine species belonging to seven genera were isolated from dried raisins, figs and dates on 40% sucrose-Czapek's agar at 28ºC, of which Eurotium amstelodami, Zygosaccharomyces rouxii, Aspergillus niger and Penicillium chrysogenum were the most prevalent fungi (Table 2). Elhalouat and Debevere (1997) isolated 7 spoilage molds and yeasts from dried raisins and prunes. They found that the predominant spoilage fungi were Zygosaccharomyces rouxii, Eurotium amstelodami, Aspergillus niger, Penicillium chrysogenum and Fusarium spp. In Egypt, Zohri and Abdel-Gawad (1993) found that Euratium amstelodami, E. chevalieri, P. chrysogenum, A. niger, A. versicolor, A. wentii and Cladosporium cladosporioides were isolated in high frequency on 40% sucrose-Czapek's agar medium from dried apricots, prunes, figs and raisins.

Eurotium amstelodami was recovered from 60%, 30% and 20% of dried samples of raisins, dates and figs constituting 35%, 23% and 17% of total fungi, respectively. It was the most prevalent fungus isolated from dried raisins, while A. niger and Z. rouxii were more prevalent in dried figs and dates.

Zygosaccharomyces rouxii is well known xerophilic fungus; emerging in 23%, 21% and 23% of dried samples of the three substrates in Yemen respectively. It was the most predominant fungus isolated from hydrated raisins and prunes in Morocco (Elhalouat and Debevere 1997), but it was not reported in Egypt from dried raisins, figs, dates, apricots and plums (Zohri and Abdel-Gawad, 1993, Abdel-Sater and Saber, 1999).

Aspergillus niger was the most predominant fungus isolated from dried dates on 40% sucrose-Czapek's agar medium. It was recovered from 45%, 40% and 30% of dried samples of dates, raisins and figs respectively. P. chrysogenum was also isolated in high frequency from raisin samples (40%) and in low frequency from date samples (10%). Abdel-Hafez et al. (1990) rated most of these fungi as osmotolerant, but with variable degrees (osmotic potential ranging from -0.32 to -4.64 MPa on sucrose-Czapek's medium). The remaining genera and species were isolated from one or two substrates with rare frequency of occurrence as shown in Table (2).

Table (2): Average total counts (ATC, calculated per g dry weight of fruits) and number of cases of isolations (NCI) of various fungal genera and species isolated from dried fruits on 40% sucrose-Czapek’s agar at 28ºC.

3-Mycotoxin contamination of dried fruits:

Thin layer chromatographic analysis of 60 samples tested of dried raisins, figs and dates (20 each), showed that six samples of dried raisins and two samples of each of dried figs and dates were naturally contaminated with aflatoxin B1. The concentrations of aflatoxin were ranged between 130-350 µg/kg of raisins, 120-250 µg/kg of figs and 110-180 µg/kg of dates. In France, Herry and Lemetayer (1992) reported that aflatoxin B1 was detected in dried raisins fruit collected from shops and supermarkets during 1989-1990. Ozay et al. (1995) recorded the presence of aflatoxin in dried figs and raisins in Turkey. Akerstrand and Moller (1989) reported that out of 103 samples of fig collected in Sweden, 53 samples were contaminated with aflatoxin and the concentration of aflatoxin ranged from 5-203 µg/kg. In Egypt, Abdel-Sater & Saber (1999) surveyed the presence of aflatoxin in 60 samples of dried raisins, dates and figs. They found that aflatoxin B1 was detected in one sample of raisin (550 µg/kg), 2 samples of dates (300-390 µg/kg) and 5 samples of figs (600-780 µg/kg). In the other hand, Stoloff, (1976) recorded no detectable aflatoxin in 108 samples of Raisins and 62 samples of dates in USA, while only 3-6% of tested figs examined (165 samples) contained aflatoxin in the range of 2-29 µg/kg.

The results of thin layer chromatographic analysis of chloroform extracts of 60 samples of dried fruits revealed that two samples of dried figs were naturally contaminated with ochratoxin A at levels between 70 to 160 µg/kg of fig. Similar to our findings, Ozay et al.(1995) recorded the presence of ochratoxin A in dried figs in Turkey. In USA, Bayman, et al. (2002) examined 50,000 figs for fungal infections and measured ochratoxin content in figs with visible fungal colonies. Pooled figs infected with Aspergillus alliaceus contained ochratoxin A, figs infected with the A. ochraceus group had little or none, and figs infected with Penicillium had none. In Egypt, Zohri and Abdel-Gawad (1993) reported that all the samples tested of dried fruits were contaminated by ochratoxin A and the concentrations ranged between 50-110 µg/kg of apricots, 60-120 µg/kg of figs and 210-280 µg/kg of prunes. They also found that all samples of raisins were naturally free from mycotoxins. Abdel-Sater and Saber (1999) recorded a detectable amount of ochratoxin A in two samples of dates in Assiut, Egypt (360-450 µg/kg). Ochratoxin A is a secondary metabolite produced by several species of Aspergillus and Penicillium that has been found in a wide variety of cereal grains, coffee beans, cocoa, beer, red wine and recently found in raisins produced in several countries (Trucksess et al., 1999). It also has been identified in tissues and blood of animals fed contaminated feed and in human blood in Balkans, Scandinavia, Germany, France and Canada. It is nephrotoxic to all animal species studied so far, teratogenic, immunotoxic, genotoxic, mutagenic and carcinogenic which lead to life-threatening pathologies (Creppy, 1999).

Results of the brine shrimp bioassay revealed that nearly 25% of all samples were toxic to brine shrimp Artemia salina larvae (Table 3). Similar observation was reported by Abdel-Sater and Saber (1999) in Egypt who found that 30% of dried raisins, dates and figs samples were proved to be toxic to the test organism Artemia salina. High ratio of toxicity using brine shrimp bioassay test may be due to that some fungi elaborate naturally occurring fatty acids that are toxic to brine shrimp. Thus toxicity towards brine shrimp should be confirmed with at least additional test organism like chicken embryos (Curtis et al. 1974).

CONCLUSION:

The present work indicated that dried fruits examined were contaminated with several glucophilic and xerophilic fungi especially members of Aspergillus, Penicillium, Eurotium, Zygosaccharomyces and Rhizopus. Many of these fungi are capable of producing mycotoxins such as aflatoxin B1 and ochratoxin A. These findings indicate that there may be a risk of human exposure to mycotoxins through the consumption of dry fruits or juices and jams manufactured with dried fruits. So, strict hygiene microbiological measured must be applied during different steps during harvest, handling, transport, storage and drying to avoid contamination of dried fruits by mycoflora and mycotoxins which are harmful to human health.

Table (3) Number of dried fruits samples contaminated with mycotoxins out of 20 and the toxicity of their extracts against Artemia salina larvae.

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