C RESEARCH NOTE J. Food Sci. Technol., 2001, Vol. 38. No. 4, 407-411 Natural Contamination of Some Egyptian Fruit Juices and Beverages by Mycoflora and Mycotoxins M.A. ABDEL-SATER®, A.A. ZOHRI AND M.A. ISMAIL Botany Department, Faculty of Science, Assiut University, Assiut, Egypt. ‘The mycotiora analysis of some canned fruit juices and beverage species. Forty-four apecies and 2 varieties belonging to 18 genera wi juices and grape, peach and apple beverage showed a wide range of colony forming units (cfu) and collected during the current study from mango, guava ‘on Dicloran Rose-Bengai agar and 20 % sucrose-Czapek’s agar at 26°C. The total Sounts of fungi widely fluctuated between 187-518 and 83-535 colonies/50 mi juice or beverages on the two isolation media ‘The highest contaminated product was guava juice and the lowest ona was the natural grape beverages. The most prevalent fungi in all types of juices and beverages were members of Aspergilis and Penicilium, Tho thin layer chromatographic. analysis cf the chloroform extracts of the five different fruit julca and beverages (five each) revealed that the five apple beverages tested were contaminated with aflatoxin B, and G, with concentrations ranging from 20-20 g/L, while two out of the five guava juice samples tested were naturally contaminated ‘wit aflatoxin B, at 12 g/L. All the samples of mango juice, grape and peach Deverages were free from nalually occurring mycotoxins Keywords : Fruit juices, Fruit beverages, Mycotlora, Mycotoxins, Aflatoxin. The soft fruits such as mango, guava, grape, apple and peach are highly susceptible to mould invasion and mycotoxin production in the field. Improper handling and storage conditions after harvest can also facilitate mould contamination, in processing the juices or beverages from fruits, some isolates of fungi have the ability to tolerate this processing. The high resistance of these organisms allowed them to produce their ‘mycotoxins and spoil the beverages. Mycotoxins may enter the fruit juices or beverages by direct contamination, resulting {rom mould growth on the fruits, or by indirect contamination through the use of contaminated ingredients in processed fruits. Now, there are several investigations focussed on the natural contamination of some fruits, vegetables and their products by mycoflora and mycotoxins (Rale and Vakil 1984; Wojtas-Koziel and Borecka 1990 ; Michailides 1991; Ugwuanyi and Jason 1991; Leistner 1992 ; Pokomn 1994; Beuchat 1995 ; Pao et al. 1996 ; Ejechi ot al. 1998; Pao and Brown 1998). {In Egypt, the information about the presence of mycoflora and mycotoxins in fruits and their products are very scarce (Moubasher et al. 1971; El-Kady et al. 1979). In this background, the present work was undertaken to determine the contamination level of mycoflora and mycotoxins in some Egyptian canned fruit juices and beverages. ‘Mycotoxin standards : All mycotoxin standards used throughout this study were kindly provided by Prof. Dr. LA. El Kady, Botany Department, Faculty of Sciences, Assiut University, Assiut, Egypt Collection of samples : Twenty-five samples of different fruit juices and beverages (5 each) were collected from shops. and markets of different sanitation levels at Assiut Governorate, Egypt. These samples included canned juices of mango (Mangitera indica) and guava (Psidium guajava) and canned beverages of grape (Vitis vinifera), apple (Pysus malus) and peach (Prunus persica). The producing companies and ‘Corresponding Author 407 ingredients of these samples are shown in Table 1. The ‘samples were brought tothe laboratory and kept in a refrigerator (8-5°C) til further analysis. Isolation and identification of fungi : Requisite quantities of appropriate dilutions (in triplicates) of the samples were surface plated with Dicloran Rose Bengal medium-DRBC (King et al. 1979) and 20% Sucrose-Czapek Dox agar (Moubasher 1993). The plates were incubated at 28°C for 1- 2 weeks during which period the colonies of fungi were counted, identified and calculated per 10 mi for each sample, Fungal colonies showing characteristic morphology were isolated and subjected to identification (Raper and Funnell 1965; Moubasher 1999). ‘Mycotoxins analysis : Sample extracts for mycotoxin analysis were prepared from 50 mi quantities of each sample according to the method of El-Kady ot al (1979) Chromatographic analysis. of the chloroform extracts was ‘achieved on percolated silica gel plate type 60 F 254 (Merck, Darmstadt) for the presence of aflatoxins B,, B,, G, and G,, cittinin, diacetoxy-scipenol, ochratoxins, patulin, rubratoxins, sterigmatocystin, T-2 toxin and zeralenone (Scott et al. 1970; Roberts and Patterson 1975 and Gimeno 1979), Allatoxins present in the extracts were quantitatively determined according to the method of Jones (1972). The aflatoxin concentration was estimated visually in the chloroform extracts by quantitative thin layer chromatogrophy by comparison with aflatoxins B,, 8, G, and G, standards. The detectable limit was about 5 js/kg for aflatoxin B, and total aflatoxins (Przyhyeski 1975). For confirmation, the plates were sprayed with 50% sulphuric acid in methanol (AOAG 1980). Fungi recovered on DCRB : Thirty-five species and 2 varieties covering 13 genera were isolated from the five fruit products, when plated on REC. The total viable fungal ropagules were found to range from 187-518 colonies/50 mi juice or beverages. The highest contaminated ssmple was quava juice and the lowest one was the natural grape408 TABLE 1 icefboverages Ingredients ‘ype Mango juice Fresh fnits, natural mango, pulp, water Total soluble sols = 18% Guava juice Guava pulp, sugar, otic acid Total soluble solids = 15 % Natural overages grape Total Soluble solids = 12% Natural beverages apple Total ‘soluble solids = 13% Poach beverages benzoate, synthetic colour flavours [Natural grape juice 20%, sugar syrup, natural colour, ciic acid, natural aroma, vitamin C Natural apple juice 20%, natural colour, sugar, small syrup, cite acid, natura’ aroma, vitamin C. Poach core, sugars, water, cite acid, sodium INGREDIENTS AND PRODUCING COMPANIES OF THE DIFFERENT-FRUITS AND BEVERAGES INVESTIGATED Producing company ‘Alexancria Confectionary and Chocolate Co, Hawamdioh Factories, Egypt Eéfina Co,, Egypt tsmaitia National Co. for food industries (Foodico), Egypt, under licence trom Wiid- Heldelberg, Germany, National Co, for food Industries (Foedico), Egypt, under licence from Wild-Heidelberg, Germany Eufina Co., Egypt beverages (Table 2). Rale and Vakil (1984) reported variable ‘counts of fungal colonies in guava fruits, when plated on modified Czapek agar, malt extract-yeast extract glucose Peptone agar and modified molybdate agar. The distribution pattem of fungal species in frut juices and beverages, when plated on DRBC is presented in Table 2. Aspergilus spp. was found to be the most predominant ‘organism showing a 100 % frequency in almost all the samples analysed. Of the 11 Aspergillus spp. and 2 varieties isolated, A flavus, A, fumigatus, A fumigatus var. albus, A. nigerand ‘A terreus were the common species. A frequency of 79.5 % of Aspergilus was reported in a study, relating to parasitic and fungal contamination of fruits and vegetables (Peter et al. 1990). Ugwuanyi and Jason (1991) reported that about 17 % ‘of mango fruit samples contained heat resistant fungi including A. faves Penicilium spp. also exhibited a similar degree of frequency to that of Aspergilus spp. Among the 10 species ‘isolated, P. duciauxi/and P. funiculosum were more common in occurrence. Combrink et al (1985) also reported that P. funiculosum was predominantly associated with core rot of starking apples. Emericella (represented by E. nidulans, E. quadiiineata) and Paecilomyces (P. variti) were isolated from four kinds of fruit juices or beverages. Paecilomyces varioti, a heat resistant fungus was present in 17% of mango fruits analysed in Southern Nigeria (Ugwuanyi and Jason 1991). ‘Acremonium (2 species), Cladosporium (2) and Syncephalastrum (1) were recorded from three products of which “A. strictu, C. cladesporioides and S. racemosumwere the most common. Most of these species were reported Previously from different fruit products but with variable incidences (Beuchat 1995 ; Pao et al. 1998). Other moulds such as Alternaria altemata, Cochliobolus Iunatus and Cunninghamella echimulata were isolated only from ‘wo products. Moubasher et al (1971) isolated A. alternata from cittus plants in rare frequency, while Combrink et al (1985) isolated it from 59.1 % of discoloured tissue of starking apples. Also, Wojtas-Koziel and Borecka (1990) isolated 206 isolates of Altemaria spp. {rom apple fruits of which A. alterata was the most common and the highest number of isolates was pathogenic to apples. Xerophillic fungi recovered on 20% sucrose-Czapek ‘agar : Twenty seven species and one variety of each of A. flavus and A. fumigatus appertaining to 8 genera were collected from the fruit juices and beverages on 20 % Sucrose-Czapek’s agar at 28°C. The total counts of viable fungal propagules Widely fluctuated between 83-535 colonies/50 ml and the ighest count was found in guava juice and the lowest in grape beverages similar to those observed with DRBC medium (Table 3) Aspergillus (11 species and 2 varieties) and Penicilium (8 species) were the most common genera as found to occur in 80-100 % of the samples. Tanaka and Miller (1963) isolated 124 mould isolates from 48 spoiled samples of dried California prunes and noted that A. nigerwas found in 15% and other _aspergil in 4%, while Pencillum comprised 33°% of the total Eurotiumwas the second most common genus and isolated from all types of samples, except for peach beverages. Species of Eurotiumare known to survive the commercial processing of fruit juices. This ability is due to the production of ascospores in thelr teleomorphic state (Pitt and Hocking 1985) Alternaria alternata, A. tenuissima, Cladosporium cladosporioides, Gibberella intricans, Paecilomyces variotiiand Pleospora herbarum were isolated only from cone or two kinds of juices or beverages. All the above species have been isolated from ditferent fruit juices and beverages and other fruit parts as reported by several researchers all over the world (Moubasher et al. 1971; El-Kady et al. 1979; Combrink et al. 1985; Wojtas-Koziel and Borecka 1990). Natural occurrence of mycotoxins : Thin layer ‘chromatograph analysis of the chioroform extracts of the different frut juices and beverages clearly showed that ail the {ive apple beverages tested were naturally contaminated with aflatoxins, B, and G, with concentrations ranging from 20-30 ug/L. Only two of five guava juice samples were also found to be contaminated, but only with aflatoxin B, at a concentration of 12 g/L. All samples of mango juices, grape and peach beverages (5 each) were free from mycotoxins. These findings had certain similarities with earlier studies. Sutic et al (1979)409 TABLE 2 TOTAL FUNGAL COUNTS (TFC) AND PERCENTAGE FREQUENCY (%F) OF FUNGAL GENERA AND SPECIES RECOVERED FROM DIFFERENT KINDS OF FRUIT JUICES AND BEVERAGES ON DICLORAN ROSE-BENGAL MEDIUM (OREC) AT 28°C Genera and Species Guava juice Natural grape Natural apple Peach beverages beverages beverages beverages TFC %F TFC %F TFC %F TFC %F ‘Acremonium : 1 2% 1 2 - ‘A. rutlum W. Gams a 5 6 fee coe eee ee A. strctum W. Gams CeO ee pe ‘Atemaria altomata (Fries) Keiss! B 8 3 fee Oe a0 Aspergilus 271100. 414 © 100148100249 100 a9 100, ‘A. flaws Link 55 100 83 10028100 41006100 ‘A. flavus var. columnaris Raper and Fennell - 4 4 2 2 : : A. fumigatus Fresenius 186 100267 100 108-100-180 100225100 ‘A. fumigatus var. albus Ral, Tewari and Agarwal GOO © 0 0 to & A. japonicus Saito 1 40 5 4 AA iger Van Tiegher DOO of OO OO 0 mm & i A oryzae (Aho) Cohn 1 2 2 wo - - : ‘A. sydowil (Bain. and Sart) Thom and Church 2 »o - : - A. tamari Kita ee 1 20 A terreus Thom 2 4 7 wm - - 2 @ 2 « A. ustus (Bain) Thom and Church oo Se 28 co 20) ‘A. versicolor (Vull) Tiraboschi = et mw tlm ‘A, zonalus Kwon and Fennell sa : - 2 2 (Cladosporium 2 4 - + 8 6 2 4 - C. cladosporioides (Fres.) de Viies 2 4 - = 7 6 2 4 - C. herbarum (Pers.) Link ex Gray ee) hoes Cochiiobolus lunatus Nelson and Haasis ie 20) : aco Cunninghamella echinuiata (Thaxt) Thaxt. ox Blakes! : eee cn) Emericela 2 40 2 4 1 wm - ea) nidulans (Eidam) Vullerin a 8 1 mM =. - , quadhlineata (Thom and Raper) Benjamin 2 4 1 @ 1 2 1 2 Gibberaiafyjkuroi (Sawada) Ito 2 40 : ao ‘Mucor hiemalis Wehmer Beco) : 58 ‘Noctria haematococca Berkeley and Brown 2 40 : : : Paeciomyces varioti Bainer 2 @ +1 20 fee a0 ee) Penicitlum 54 100-99 10025100 8100S 82100 . aurantiogriseum Dierckx 5 6 - 5 2 3 8 2 w . brevicompacturn Dierckx - 4 wm 4 ‘i . chnysogenum Thom 9 6 8 6 2 2 - 1 P. cttrioum Thom 3 60 a P. coryophitum Dierckx : - 8 4 oF 100 P, auclauxi Delacroix 6 6 7 100 10 6 2 4 12 40° . funieulosum Thom 1% 100 10 «440 «62 ho . islandicum Sopp 3 2 3 2 15 10 5 60 . piceum Raper and Fennel fies 20) : - 3 6 1 2 . verruculosum Peyronel a = aa - ‘Syncephalastum racemesum (Cohn) Schvoetet 1 20 - 1m 2 Total count 340 518 187 302 3088 Number of genera = 13 8 6 7 a 6 Number of specios = 35 + 2 varitios 1941 var. M842 var A742 var 2041 var, 1841 var TFC = Total fungal count calculated per 50 ml, product, feported that presence of aflatoxins in fruit juices and vegetables at Serbia (Yugoslavia). Neelakantan et al (1983) examined several fruit samples. in Tamil Nadu region (India) and reported that one out of six samples of apple analysed was contaminated with aflatoxin B, ‘Although several samples wore contaminated with various toxic fungi, only aflatoxins were detected in the crude extracts of some these samples. This clearly shows that the presence of mycotoxic fungi in a product does not automatically indicate the presence of mycotoxins, especially if growih has not occurred. On the other hand, the toxins may persist long alter vegetative growth has occurred and the moulds have died. With regard to patuln, it was not completely detected in any ofthe 25 samples tested, whereas numerous investigations410 TABLE 3. TOTAL FUNGAL COUNTS (TFC) AND PERCENTAGE FREQUENCY (%F) OF FUNGAL GENERA AND SPECIES RECOVERED FROM DIFFERENT KINDS OF FRUIT JUICES AND BEVERAGES ON 20% SUCROSE-CZAPERK'S MEDIUM AT 28°C Genera and Species ‘Mango juice Guava juice Natural grape Natural apple Peach beverages beverages beverages. beverages beverages TFC O%F TFC %F TFC %F TFC %F TFC %F ‘Alteran : rr ‘A. alterata (Fries) Kelesier Be tm : A. tenuissima (kunzo: Pers.) Wiltshire Be eee Aspergilus m2 100 «407100 tw 100 ‘A favs Link 61 100 «119 «00 ae www] ‘A flavus var. columnaris Raper and Fennell Ee ee ee A. tumigatus Fresenius 9 100 199 «10014 dw ‘A. fumigatus var. albus Rai, Tewari and Agarwal - = 6 00 : ‘A. riger Van Tieghom (eat e040) ee00 ero eeleuie tot (00 solar 100 A. ochracous Wilhelm BOR 20) see ‘A oryzae (Rib) Cohn oS ie oo) A. parasiticus Spi : 5 2 : : ‘A. sydowil (Bain. and Sart) Thom and Church : ac 1 20 A. tamaci Kita : eo 2 «0 A terreus Thorn 6 0 0 9 3 2 2 4 A. ustus (Bain.) Thom and Church - ge ge A. versicolor (Vull) Trabeschi iia sf ee 1a ee 000 ee (Ee 0) (00) ee Cladosporium cladasporioides (Fres.) de Vries 5 4 3 m2 - 2s : Eurotium 7 @ 2 2» 7 wo 3 wo - E amstelodami Mangin (Oe coe ee eee oe oo E. chevalier’ Mangin re go E repens De Barry oe See oa 120 ete Gbverela inticans Wolenw. : cn 1 om Pasclomyces varioti Bainet - 4 mw ee Ponicitiam 121 1001221002100 P. aurantiogrisoum Dietckx m8 P. brevicompactum Dierckx BG P. chrysogenum Thom et tee 00 ees) 00) 00) . cltrinum Thoin pat oyna) P, auctaun Delacroix 2m 4 - = oe 6 funiculosum Thom ss 60 «14 8) ws PP. isandicum Sopp 2 8 3 2 - 4 . oxaicum Curtie and Thom re 5 a Gg Gg Preaspora herbarum (Ft. Rabenh. Ex Ces, and de Not) 2 OM ee ee Total count 387 535 cy 187 88 Number of genera = 8 5 5 5 5 2 Number of species = 27 + 2 vatietios 5 1542 var, " 9 10+1 var TF ‘ola! fungal count calculated per 50 ml, product have reported its presence as the main myctoxin in fruit juices especialy apple juice (Brackett and Marth 1973; Bata et al 1983). Inthe present work, thin layer chromatographic analysis was done and this method was found suitable only for samples with high patulin content (above 50 yg/Kg) as mentioned by Bata et al (1983). In many countries, the maximum permissible patulin content for apple pulps is 20-90 g/kg. Brackett and Marth (1979) examined 20 samples of apple juices in Wisconsin (USA) and reported that most samples contained less than 50 hig of patulin per litre. Bata et al (1983) found the patulin Concentration in apple juice to range from zero to 13 ughkg in Budapest (Hungary) Also, citinin, diacetoxyscirpenol, ochratoxins, rubratoxins, sterigmatocystin, T-2 toxin and zearalenon were not detected in any sample of frit juices or beverages tested In conclusion, from the mycological analyses of the juice and beverage samples, it was revealed that all the samples were contaminated with a large variety of fungal species, but, low in their numbers. The most contaminated samples were those of guava juices and the lowest were those of grape beverages. Also it was found that A. flavus, A. fumigatus and A nigerwere recovered from all samples of fruit juices and beverages. Penicillum funiculasum was common in all samples TLC analysis of the chloroform extracts of the five samples of each of mango juice, grape and peach beverages showed no detectable levels of mycotoxins. However, the five apple beverage samples contained aflatoxins B, and G, at concentrations of about 20-30 ug/L. Also, two (out of five)‘quava samples contained, aflatoxin B, at 12 ug/L. The detection of such toxins in apple and guava juices is considered to be first record in Egypt, which can threaten human health. In this background, concepts of good manufacturing practices and hazard analysis critical control points are to be implemented in the fruit juices and beverages industry. References AOAC (1980) Association of Official Analytical Chemists. 13th edn. Official Methods of Analysis. Washington, DC Bata A, lasztity R, Sandor GS (1983) Capillary gas liquid ‘chromatographic sereoning for patulin in apple juice and apple ‘concentrates. In : Proceedings of Intemational Symposium on Mycotoxins (Sopt 68, 1982, Cairo, Egypl) pp 567-572 Beuchat LR (1995) Pathogenic microorganisms associated with fresh produce. 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