In ternationa l Scholars Journa ls African Journal of Food Science Research ISSN 2375-0723 Vol. 6 (7), pp. 338-346, July, 2018. Available online at www.internationalscholarsjournals.org © International Scholars Journals Author(s) retain the copyright of this article. Full Length Research Paper A study of the fungal populations and their toxins associated with coffee bean seeds Fardos Bokhari* and Magda Mohammad Aly Faculty of Science, Biology Department, King Abd El-Aziz University, P. O. Box 12161, Jeddah 21473, Saudi Arabia. Accepted 03 April, 2018 Thirty samples of coffee beans were collected from different places of Jeddah, Saudi Arabia to determine and identify fungal population. Twenty six species belonging to 7 genera were isolated using potato dextrose agar (PDA) and malt extract agar (MEA) media at 28°C. The most prevalent genera were Aspergillus and Penicillium. Aspergillus was present in 73 and 100% of the samples but Penicillium was present in 86.6 and 100% on the two mentioned media, respectively. Also, Fusarium, Mucor, Rhizopus and Alterneria were recovered in moderate incidences on the two media. Out of the thirty samples of coffee beans collected, thirteen were contaminated with mycotoxin (43.3%). Mycotoxin profiles were also determined in these samples. It was found that aflatoxin G1 (Afl G1) showed the highest incidence rates of occurrence. It occurred in about 23.3% of all samples analyzed and in 54% of the mycotoxin contaminated samples. The other toxins detected were aflatoxins B1 (16.6%), B2 (10%), G2 (6.6%), ochratoxin (10%), patulin (16.6%) and sterigmatocystin (6.6%). The factors affecting the Afl G1 production by Aspergillus flavus were studied. The results clarified that addition of caffeine up to 1 g/l reduced the toxin synthesis (Afl G1) . The effect of some medicinal plants and spices added singly or in combination to the malt extract, on fungal growth and AflG1 production by A. flavus was also studied. Cinnamon and cloves affected fungal growth and mycotoxin production. No growth or toxins were detected in the presence of the two plants. Saffron and ginger did not affect fungal growth or prevented toxin production by A. flavus. In conclusion, coffee beans in Saudi Arabia are highly contaminated with toxigenic fungi, specially, A. flavus, which was found in this study to be the main producer of Afl G1. Medium, temperature, vitamin C, caffeine and some medicinal plants or spices which are used as a traditional additive in Saudi Arabia may affect fungal growth or/and toxin production. Key words: Aflatoxins, mycotoxin, Aspergillus, coffee beans, secondary metabolites, medicinal plant, spices, additives. INTRODUCTION Aspergillus is a filamentous fungus that produces myco- toxins in many food and feed crops. Aflatoxins are poly- ketide-derived, highly toxic, mutagenic, tetratogenic and carcinogenic secondary metabolites for higher vertebra-tes and other animals (Bokhari and Shaker, 2008). They are produced primarily by Aspergillus flavus, Aspergillus parasiticus, Aspergillus nomius, Aspergillus toxicarius and Aspergillus parvisclerotigens (Holmes et al., 2008) on crops such as corn, peanuts, cotton seeds, and coffee beans. Furthermore, the natural occurrence of the toxige-nic Aspergillus species and aflatoxin production in coffee beans was suggested by many authors (Levi, 1980; Daivasikamani and Kannan, 1986; Abdel- Hafez and El- *Corresponding author. E-mail: fmbokh@kau.edu.sa. maghraby, 1992; Nakajima et al., 1997; Batista et al., 2003). Regulatory guidelines issued by the U.S. Food and Drug Administration (FDA) prevent sale of commo- dities if contamination by these fungi or mycotoxins exc- eeds certain levels. Aflatoxins B1 and B2 are produced by A. flavus and A. parasiticus, but the latter species also have been confirmed to form G1 and G2 toxins. On the other hand, ochratoxin was produced by Aspergillus och- receus, Aspergillus niger and Aspergillus carbonarius (Bhatnagar et al., 2004). The fungal isolates were analy- zed for toxins production in an attempt to differentiate aflatoxigenic from nonaflatoxigenic isolates. BoKhari (2007a) reported that coffee seeds were highly contami- nated with toxigenic fungal isolates and toxins especially ochratoxin A. The occurrence of fungal contamination and mycotoxin production started at the beginning of har- Bokhari and Aly 339 vest due to high moisture content of the seeds and incre- ased during transport, storage, or marketing. Within the last decade, significant advances have been made in mycotoxin detection methods and control strate- gies as well as in studying the effect of environ-mental factors on toxin synthesis (Patterson, 1984; Nakajima et al., 1997) . The most significant environmental factors that influence toxin synthesis are media used especially car-bon and nitrogen sources, pH, temperature, water acti-vity, and plant metabolites (Bhatnagar,et al., 2003; Calvo, et al., 2002). Natural plant metabolites affect toxin prod-uction and fungal development. Earlier research efforts in this, as well as other labs, have shown that plant com-ponents or volatile extracts can alter either Aspergillus growth or aflatoxin production, i.e. volatile aldehydes, vitamin C (Clevstrom et al., 2004), caffeine (Buchanan et al., 1983b) and other compounds from neem leaf (Zerin-gue and Bhatnagar, 1999), cotton leaf (Zeringue and McCormick, 1990) and corn-leaf (Wilson et al., 1981). Anthocyanins and related flavonoids also affect aflatoxin biosynthesis (Norton, 1999). In some cases, growth was not significantly aff-ected by various metabolites, while aflatoxin biosynthesis and fungal development were sig-nificantly decreased (Juglal et al., 2002; Basilico and Basilico, 1999). The plant linoleic acid, its derivatives and their precursor affect sexual and asexual sporulation in Aspergillus nidulans, sclerotial development, and toxin synthesis (Champe and Zayat, 1989). The conversion of oleic acid (18:1) to linoleic acid (18:2) is a critical biosyn-thetic step in the generation of sporogenic psi factors. Similarly, Hitokoto et al. (1980) reported that addition of cloves or cinnamon inhibited Aspergillus growth but Aill- ium cepa or green tea leaves inhibited toxin production. The objectives of the present study were to identify the fungal populations and their toxins associated with coffee bean seeds, collected from Jeddah, Saudi Arabia and to evaluate the potential of the toxigenic isolate (A. flavus) to produce aflatoxins G1 which was more dominant my- cotoxin compared with the other toxins detected. No stu- dies were found dealing with the occurrence of Afl. G1 in coffee bean seeds or the factors affecting its production. Different factors witch affect aflatoxin G1 production and the effect of different traditional additive (saffron, ginger, cinnamon, cloves and cardamom) of coffee used in Saudi Arabia were also studied. MATERIALS AND METHODS Collection of samples Thirty coffee bean samples with different varieties of Coffee Arabica L. (about 1-1.5 kg each) were collected from different markets of Jeddah governorate, where coffee beans were sold in open bags. Each sample consisted of three replicates. The samples were col- lected in a sterile polyethylene bags to minimize the loss of water content and provide sufficient aeration, sealed, transferred imme- diately to the laboratory, kept at 4ºC until mycological and mycoto- xins analysis. Moisture content of the green coffee bean samples The moisture content of the samples was directly determined by dry weight method (Aziz, 1987). About 10 g of each sample was trans- ferred to an oven at ° under vacuum for 12- 24 h and until a con- stant weight. Percentage of water content was calculated. Fungal isolation and identification Isolation was carried out by the method described by Batista et al. (2003) . To isolate fungi associated with the coffee bean seeds, the seeds were placed directly on filter paper, moistened with sterile dist. water. Then, thirty beans were collected randomly from each coffee bean sample. A total of 15 seeds were plated directly on agar plates (3 seeds/plate) and the other were disinfected with 1% sodium hypochloride for 2 min in order to permit isolation of fungi present in the interior of the beans. The media used are either pota- to dextrose agar or Malt extract agar (Merck, Germany) for isolation of different types of fungi. The compositions of malt agar were as the following: malt extract, 20 (g/l); glucose, 20 (g/l); peptone, 15 (g/l); agar, 20 (g/l). Ten plates for each sample (five plates for each medium) were used. The plates were incubated at 28°C for 7-10 days and the developing fungi were counted and identified accord- ing to macro and microscopic characteristics as described in Raper and Fennell (1965), Moubasher (1993) and Samson et al. (1995). The total counts (T.C.) of each species of fungi were calculated and were divided by the weight of the coffee bean seeds used in milli- gram (TC/mg). Extraction of mycotoxins from coffee bean seed samples A one hundred grams of each sample was extracted with 100 ml chloroform twice times and following the procedures described by Sorenson et al. (1967). The chloroform extract was dried over anhy- drous sodium sulfate, filtered, then concentrated under vacuum and the dry material was transferred to a dark vial with a small amount of chloroform, which was evaporated to near dryness. Detection of mycotoxins Thin layer chromatographic technique of the clean extract was done on percolated silica gel plates (Merck, Silica Gel 60, 25 mm, 20 x 20). Detection of the different mycotoxins was carried out according to standard procedures described by Roberts and Patterson (1984) and El-Shanawany et al. (2005). A mixture of toluene-ethyl acetate- 90% formic acid (50:40:10) was used as a mobile phase. Mycoto- xins were visualized under ultraviolet light at 366 nm in a chromato- visor. Aflatoxin, ochratoxin A, patulin and sterigmatocystin, isolated from fungi contaminated the coffee beans, had a retention time and fluorescent spot similar to the standard mycotoxins being tested (Sigma). Chemical confirmation of mycotoxins Chemical confirmation of mycotoxins was performed directly on the developed TLC plate. Two spraying reagents were used to visuali- zation and increase the fluorescence intensity of the mycotoxins (Grabarkiewicz-Saczesna et al., 1985) . The plates were then spra- yed with either 20% AlCl3 solution or 20% sulfuric acid, heated to 110ºC and examined under UV light (365 nm) as described by Bok- hari (1993). 340 Afr. J. Food Sci. Res. Cultivation of A. flavus isolated from coffee bean seeds in liquid media A. flavus was cultured in liquid media to study different factors aff- ecting growth and toxin production. Each treatment had three repli- cates. For inoculum preparation, A. flavus was grown on Czapex - Dox agar containing rose bengal and chloramphenicol (25 mg/ml) in Petri dishes at 28°C until extensive formation of conidia was obser- ved. The use of a combination of rose bengal at a concentration of about 1- 30,000 and chloramphenicol in fungus plating media to prevent growth of bacteria and restrict size of colonies was found to be far superior to the older standard procedure. Thus, a disc of 10 days old culture was used as a source of conidia for inoculating of the different flasks. Extraction of Aspergillus toxins After incubation, the content of each flask (medium + mycelium) was homogenized for 5 min in a high-speed blender with 100 ml chloroform. The extracts procedure was repeated three times. The chloroform extracts were combined, washed, dried, filtered, and concentrated near to dryness, cleaned and mycotoxins detected as previously described (Dos Santos et al., 2003). Effect of different temperature on growth and Aflatoxin G1 production Erlenmeyer flasks (250 ml) containing 50 ml of sterile Malt extract medium were inoculated with about 3 ml of heavy spore suspension containing 2 x 10 4 spore/ml (optical density, 0.65). The flasks were incubated at different temperatures (4, 10, 16, 22, 28, 30, 34°C). After 7 days of incubation, the contents of each flask were mixed with 120 ml of chloroform: water (100:10, v/v) and were shaken vigorously by a rotary shaker (200 rpm) overnight. The extract was sequentially filtered through anhydrous sodium sulfate. The chlo- roform extract was collected, dried and Afl. G1 was detected as a fluorescent green color under the UV light. Effect of different media on growth and aflatoxin G1 prod- uction by A. flavus A. flavus was cultured in different media. The media used were coffee broth, coffee dextrose, potato dextrose or malt extract. After incubation at 28°C for 7 days, the fungal growth and the quantity of Afl. G1 were determined as described above. Effect of different concentrations of caffeine or vitamin C on growth and aflatoxin G1 production by A. flavus Different concentrations of either vitamin C or caffeine were added to the growth media of A. flavus in 250 ml Erlenmeyer flasks con- taining 50 ml of sterile malt extract medium. The vitamin C concen- trations were 0, 1.1, 2.2, 3.3 and 4.4 g/l as well as the caffeine concentrations ranged from 0 to 1 g/l. The growth and the quantity of Afl G1 were determined and compared with the results of control (containing no caffeine or vitamin C). The effect of different medicinal plants on A. flavus growth and aflatoxin G1 production The ability of A. flavus to grow and produce Afl G1 in a medium containing different types of medicinal plants or spices was investi- gated. The medium used was malt extract; and pH of the medium was adjusted to 5.5. The plants used were cardamom, cinnamon, cloves, saffron, and ginger. They were collected, washed, cut into pieces, dried at 60ºC, milled and sieved with 1 mm mesh and add- ed to the fungal medium (1 g/l). After 10 days of incubation at the appropriate temperature, the growth and Afl G1 were quantified.. Statistical analysis Each experiment has three replicates and three determinations were conducted. The numerical data were presented as mean + standard deviation. The Student t-test was used to compare bet- ween numerical data of control and treated. P-value < 0.05 was considered statistically significant. RESULTS AND DISCUSSION The results in Table 1 clearly show that coffee beans samples, collected from Jeddah, were highly contamina- ted with fungi which were represented by seven genera and twenty six species. More fungal species were coun- ted by Abdel-Hafez and Maghraby (1992) in Egypt and Bucheli et al. (1998) in Thailand. Abdel-Hafez and Magh- raby (1992) could isolate 26 fungal species belonging to 16 genera from coffee beans samples. The total viable counts of mold in all coffee beans samples were 419 and 346 colonies/mg dry coffee seeds on each malt extract agar and potato dextrose agar, respectively. The levels or occurrences of the different fungal population encoun- tered in the current study ranged from high, moderate to rare and were almost in agreement to those observed in coffee beans studied by Batista et al. (2003). Examina- tion of coffee bean samples for the presence of toxigenic and non toxigenic fungi was carried out. It has been found that Aspergillus and Penicillium followed by Fus- arium and Mucor were the most prevalent genera on the two used isolation media. These results were similar to a great extent to results obtained by Panneerselvam et al. (2001). They studied microflora of coffee beans and rep- orted that the genera Aspergillus, Penicillium, Cladospo- rium, Trichoderma and Mucor were the most dominant genera. The same findings were obtained by many other (Nunnes et al., 2001; Bokhari, 2007a). Aspergillus was the commonest genus in all samples examined. It was represented by 9 species and appeared in 73 and 100% of the samples tested on the two used media. It was represented by 63-65 % of the total fungi examined. A. flavus was the commonest species. It was represented by 24 and 14% of the total Aspergillus species recovered on Malt extract and Potato dextrose agar, respectively. A. candidus, A. fumigatus, A. niger, A. ochraeceous, A. sydowii, A. terreus, and A. versicolor were less dominant compared to A. Flavus. Abdel-Hafez (1984) reported that coffee seeds were highly contaminated by the genus As- pergillus, followed by Penicillium and Rhizopus. The sec- ond highest incidence rate was represented by genus Penicillium. It was recovered from 86 to 100% of coffee beans seeds samples. Most of Penicillium species were prevalent on Malt extract agar medium and with mode- Bokhari and Aly 341 Table 1. Total counts (TC per mg), number of cases of isolation out of 30 samples and occurrence remarks of fungal genera and species recovered from coffee bean samples at 28ºC for 10 days at two types of media (Malt dextrose agar and Potato dextrose agar). Isolated Fungi Potato dextrose agar Malt extract agar Occurrence No. of T.C. /mg Occurance No. of T.C. remarks appearance Dry remarks appearance /mg /30 sample seed /30 sample Dry seed Aspergillus H 30 234 H 22 275 A. candidus M 9 21 M 6 11 A. flavus H 22 33 H 19 67 A. fumigatus H 19 22 H 14 33 A. melles R 3 11 R 1 5 A. niger H 25 7 R 2 22 A. ochraeceous H 18 33 H 17 45 A. sydowii M 10 36 R 3 31 A. terreus H 19 40 H 19 32 A. versicolor M 11 31 M 9 29 Penicillium H 30 50 H 26 70 P. variable H 15 13 H 19 34 P. canescens R 0 0 R 5 7 P. chrysogenum H 30 7 R 3 7 P. citrinum R 4 11 M 6 11 P. glabrum R 0 0 R 1 4 P. rubrum M 12 16 R 1 2 P. oxalicum R 0 0 R 2 5 P. italicum R 5 3 R 0 0 Fusarium H 18 8 H 19 23 F. oxisporium M 7 5 R 5 3 F. moniliforme M 11 3 H 14 20 Mucor M 21 14 M 15 21 M. racemosus M 9 10 M 7 3 M. circinelloides 0 0 0 H 7 11 M. hiemalis M 12 4 R 1 7 Rhizopus M 8 5 R 4 9 R. stolonifer M 8 5 R 4 9 Alterneria M 9 25 M 11 11 A. chlamydospora M 6 9 M 11 11 A. .solani M 8 5 R 4 9 A. alternata M 9 25 M 9 9 H, High occurrence (more than 15); M, Moderate (less than 15 and more than 5); R, Rare (less than 5). rate incidence or completely absent on PDA medium. From the genus Penicillium, 8 species were identified of which Penicillium chrysogenum; Penicillium duclauxii and Penicillium janczweskii were the most prevalent. Fusa- rium and Mucor were also common genera, recovered at the average of 63 and 50% of all samples examined, constituting, 5.5 and 5% of total fungi on Malt extract agar medium and at the average of 37 and 50% of all sam- ples, constituting 3.4 and 6% of the total fungi examined on PDA. The remaining genera and species were remar- ked in low or rare frequencies of occurrence on one or the two isolation media (Table 1). As shown in Table 2, thirty samples of coffee beans were collected from differ- ent places and % of water content was determined. The contamination of seeds with different mycotoxins was de- termined qualitatively. It was found that mycotoxin prod- uction capability by fungi can be limited or impeded by water activity. Increasing water content of the sample increased the number of toxins detected. Four samples (No. 1, 5, 10, and 26) had moisture contents ranged from 13.6–14% and contaminated by at least two mycotoxins. The % of moisture content of samples 18 342 Afr. J. Food Sci. Res. Table 2. Moisture content, number of toxigenic isolates and natural occurrence of mycotoxins recorded in the different coffee bean samples, collected from various sources. Sam % No. of No. of % of Quality of the toxin detected NO. Moisture fungal toxigenic toxigenic B1 B2 G1 G2 Och. A Patulin Streg. content isolates isolates isolates 1 13.6 22 3 13.6 + + 2 12.9 9 1 11.1 + + 3 13.4 6 0 0.0 4 12.8 12 2 15 + 5 13.8 21 2 9.5 + 6 12.4 12 0 0.0 7 14.1 19 1 5.2 + 8 13.0 20 0 0.0 9 13.9 24 4 16.0 10 13.6 14 4 29 + + 11 12.8 10 0 0.0 12 12.9 7 0 0.0 13 12.4 11 0 0.0 14 13.8 3 1 33.5 15 12.8 5 0 0.0 16 12.8 14 0 0.0 17 12.8 9 0 0.0 18 12.0 27 3 11.1 + + + 19 11.8 13 0 0.0 20 12.9 19 0 0.0 21 11.7 9 0 0.0 22 12.8 11 0 0.0 23 12.3 12 0 0.0 24 12.0 12 2 11.1 + 25 12.1 5 1 20.6 26 14.0 4 2 50 + + 27 12.8 8 0 0.0 28 11.2 4 0 0.0 29 12.0 14 0 0.0 30 12.6 6 2 33.5 + Sam: Sample number, B1, B2, G1 and G2: Aflatoxin B1, B2, G1 and G 2, Och.A :Ochratoxin A, Strg. : Stergmatocystin, +: Detected. from 12-12.6% and found to be contaminated with four different toxins. All the 30 coffee bean samples were tes- ted qualitatively for naturally occurring mycotoxin conta- minations and showed that 57% of the samples were mycotoxin-free (Table 2). It was found that five samples were contaminated by aflatoxins B1 and/or B2 while, Afl. G1 was recovered in the extract of seven coffee bean samples, thus, it was selected for more studies. Only samples (No. 4, 18) as in Table 2 were contaminated with Afl. G2. Also, the results revealed that 17% of the tested samples (5 out of 30) were contaminated by aflatoxins B1 and/or B2; 30% contains aflatoxins G1 or G2; 10%, och- rotoxin; 17%, patulin; but only 7%, sterigmatocystin. Buc- heli et al. (1998) recorded that A. flavus, A. niger, A. fumi- gatus were isolated from the coffee seeds contaminated by fungi. On contrast, Fusarium solani was the common- est fungus in coffee seeds (Muthappa, 1984) but A. fla- vus, A. niger, A. funiculosum and Cladosporium were the commonest in coffee bean samples collected from Egypt. Nunes et al. (2001) reported that coffee seeds were con- taminated by the genus Aspergillus, especially, A. ochra- ceus (10.9%) and A. niger (22.9%). Out of 79 fungal iso- lated belonging to the genus Aspergillus and Penicillium, only 8.9% were toxigenic isolates, 7,6% belonging to genus Aspergillus, especially, to A. flavus which was rep- resented by 38% of the total toxigenic isolates of the genus Aspergillus. Similar results were obtained by Sinha and Sinha (1991), who found that out of 48 strains isola- ted in India, 33 belonged to toxigenic isolates of the genus A. flavus. the total toxigenic isolates of the genus Aspergillus. Similar results were obtained by Sinha and Sinha (1991), who found that out of 48 strains isolated in Bokhari and Aly 343 Table 3. The effect of different media on growth and aflatoxin production by Aspergillus flavus, grown for 10 days at 28ºC. Media used Fungal growth (mg/ml) Aflatoxin G1 Quantity of Aflatoxin production produced ng/g Coffee dextrose 7.3 + 1.3 ++ 9.6 + 1.3 Potato dextrose 8.0 + 2.4 11 +0 .9 Malt extract 13.0+ 1.0* +++ 16+ 1.3* Sabouraud 3.4+ 0.6 + 6.4+ 0.3 Czapek's- Glucose 5.0+ 1.3 + 6.0+ 0.9 *:significant results at p<0.05, +++: high production, ++: moderate production, +: low production Table 4. The effect of different temperature on growth and aflatoxin production by Aspergillus flavus grown on Malt extract for 10 days. Temperature Fungal growth Aflatoxin G1 Quantity of aflatoxin (mg/ml) production produced ng/g 4 °C 1.4 + 1.3* ND ND 10 °C 6.8 + 2.4* ND ND 16°C 8.2+ 1.0* + 11.5* 22 °C 11.4+ 0.6* ++ 12.0 25 °C 12.0+ 1.3 13.0 28 °C 13.3 + 1.3 +++ 16.0 30°C 8.0 + 2.4* ++ 12.0* 35 °C 7.0+ 1.0* + 4.8* 38 °C 5.4+ 0.6* ND ND *:significant results at p<0.05, +++: high production, ++: moderate production, +: low production. ND: not detected India, 33 belonged to toxigenic isolates of the genus A. flavus. A. flavus, which was isolated from coffee beans was the best producer for aflatoxins especially G1. The fac- tors affecting the growth and production of Afl. G1 were studied. The effect of five different media on fungal grow- th of A. flavus and Afl. G1 production was detected (Ta- ble 3). It was found that the semi synthetic malt extract broth was the best for both fungal growth and Afl. G1 pro- duction, followed by potato dextrose and coffee dextrose. On the other hand, inoculation of A. flavus in Sabouraud broth medium decreased the growth and Afl. G1 by 30 and 40% respectively compared to inoculation in malt dextrose broth. Natural media, especially PDA increased mycotoxin production compared with the synthetic media (Abramson et al., 1990; Cortes-Esposa et al., 2006; Al- Arjani, 2008). As can be seen in Table 4, temperature affects the gro- wth and toxin production by A. flavus . Increasing the tem-perature enhanced the growth up to 28ºC where the gro-wth was maximum and got to 13.3 g/l. Increasing tem-perature more than 28ºC decreased both fungal growth and Afl. G1 production. No toxin was recorded at 4, 10 and 38ºC. Davis and Diener (1978) recorded that the aflatoxins B1 and G1 were produced by A. flavus on a semi-synthetic medium and their quantities were affected by the media used and temperature of incubation. The ideal temperature for aflatoxin production is 29–30ºC (Schroeder, and Hein, 1967; Payne, 1998). Aflatoxin pro- duction is significantly decreased at temperatures below 25ºC, but is completely inhibited at 37ºC or above. As shown in Table 5, addition of caffeine to the growth medium up to 1 g/l decreased the fungal growth and afla- toxin G1 production to about 67 and 23%, respectively. Similar results were obtained by Soliman (2002), who reported that the highest concentrations of the aflatoxins produced by A. flavus were detected in the decaffeinated green coffee bean compared with the normal green cof- fee bean samples. He added that addition of 1-2% caffei- ne reduced the growth of A. flavus in liquid medium by 50% and the level of aflatoxin in the medium was undet- ectable. Complete inhibition of ochratoxin A, produced by A. ochraceus, was carried out by addition of 1 g/l caffeine to YES growth medium (Nehad et al., 2005), but 2 g/l caffeine inhibited aflatoxins production by A. parasiticus in the same medium (Buchanan and Lewis, 1984). Simi- lar reductions in Afl. B1, ochratoxins and zearalenone were observed by a number of authors (Tsubouchi et al., 1987; Buchanan et al., 1982, 1983a; Nunes et al., 2001). Table 6 shows that addition of vitamin C to the growth 344 Afr. J. Food Sci. Res. Table 5.The effect of different concentration of caffeine on growth and aflatoxin production by Aspergillus flavus grown on Malt extract for 10 days at 28ºC. Caffeine concentration Fungal growth Aflatoxin G1 Quantity of aflatoxin g/l (mg/ml production produced ng/g 0.0 (control) 13.2+1.3 +++ 16.5 0.2 11.3 + 1.3* ++ 11.0* 0.4 10.6+ 0.3* 7.0* 0.6 8.3 + 0.9* + 4.8* 0.8 8.3 + 1.1* + 4.0* 1.0 8.2+ 2.3* ND 2.8* *:significant results at p<0.05, +++: high production, ++: moderate production, +: low production. ND: not detected Table 6. The effect of different concentrations of vitamin C on growth and aflatoxin production by Aspergillus flavus grown on Malt extract for 10 days at 28ºC. Vitamine C concentration Fungal growth Aflatoxin G1 Quantity of Aflatoxin g/l (mg/ml) production produced ng/g 0.0 (control) 13.2+ 2.3 +++ 16.0 1.1 13.3 + 1.3 ++ 13.7 2.2 14.6+ 0.3 14.0 3.3 16.3 + 0.9* +++ 15.8 4.4 17.3 + 1.1* +++ 17.0 *:significant results at p<0.05, +++: high production, ++: moderate production, +: low production. ND: not detected Table 7. The effect of different addition of medicinal plants or spices on growth and aflatoxin production by Aspergillus flavus grown on Malt extract for 10 days at 28ºC. Type of Spices Fungal growth Aflatoxin G1 Quantity of flatoxin (mg/ml) production produced ng/g Control 13.2+ 2.3 +++ 16.0 Saffron 13.3 + 1.5 ++ 13.7* Ginger 13.6+ 0.3 14.0* Cinnamon 6.3 + 0.6* ND ND Cloves 7.3 + 1.1* ND ND Cardamom 11.2+ 2.3 + 10.0 Cloves+ Cinnamon 6.3 + 1.3* ND ND *:significant results at p<0.05, +++: high production, ++: moderate production, +: low production. ND: not detected medium up to 4.4 g/l enhanced the fungal growth and mycotoxin production by A. flavus. Similar results were obtained by Clevstrom et al. (2004). They found that afla- toxins produced by A. flavus which was either isolated from cereal, barley, or coffee were enhanced up to 800 times with the presence of vitamin C in the culture medi- um. The increase in growth and patulin production got to 30% by the addition of Vit. C to the Czapex Dox broth medium (Podgorska, 1992). Similarly, the effect of additions of five different plant materials to the growth medium of A. flavus was studied as can be seen in Table 7. It was found that cinnamon, cloves or both inhibited both growth (50%) and Afl. G1 production (100%) of A. flavus . On contrast, the presen- ce of either saffron or ginger did not significantly affect the Aspergillus's growth or toxin production, but carda- mom decreased fungal growth with no significant effect on the quantity of Afl.G1 production. 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