Fungal Population and Distribution of Aflatoxigenic Fungi in Commercial Almond Powder Products

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1 90 Vol. 48, No , Fungal Population and Distribution of Aflatoxigenic Fungi in Commercial Almond Powder Products Tomoko K:C?D 1,Yuka IH=>9: 1,Koji ADN6B6 2 and Masakatsu I8=>CD: 1, 1 Tokyo Kasei University: Kaga, Itabashi-ku, Tokyo , Japan; 2 IAA; Food and Agricultural Materials Inspection Center, Fertilizer and Feed Inspection Department: Chuo-ku, Saitama-shi, Saitama , Japan; Corresponding author The fungal population and distribution of aflatoxin-producing fungi in 30 samples of imported almond powder products purchased from retail markets were examined in this study. Total counts of fungi ranged from under colony-forming units (CFU)/g to CFU/g as determined with the dilution plating technique. The predominant fungi in the mouldcontaminated almond samples were Aspergillus niger, A. flavus and the related species, Penicillium, Cladosporium and Rhizopus. Aflatoxin-producing ability in the isolates of A. flavus and related fungi were tested by thin layer chromatography using 2 yeast extract and 15 sucrose broth culture. Four di#erent aflatoxigenic fungi were detected in the isolates; aflatoxins B 1 and B 2 were produced by some strains of A. flavus and A. parvisclerotigenus, and aflatoxins B 1,B 2,G 1,G 2 were produced by all tested strains of A. parasiticus and A. nomius. Identification of the strains was based on morphological and metabolic characters. (Received December 7, 2006) Key words: almond; fungal populations; aflatoxin; aflatoxin-producing fungi; Aspergillus flavus; Aspergillus nomius; Aspergillus parasiticus; Aspergillus parvisclerotigenus (Prunus amygdalus) (P. amygdalus var. dulcis) (P. amygdalus var. amara) 1) ) 3) 4) 30 Aspergillus flavus B (B 1,B 2 ) G (G 1,G 2 )

2 August A-7, A-8; A-17, A-23, A-24, A-25; A- 34, A-35, A g ml 1 1mL 9mL ml 2 Dichloran Rose Bengal Chloramphenicol (DRBC, Oxoid) Aspergillus Flavus Parasiticus Agar (AFPA, Oxoid) 25, 7 Aspergillus flavus Potato-dextrose-agar A. flavus 2 (Difco), 15 YES (YES broth) 7mL 30, ml TLC TLC 60 (Art , Merck) 10 ml (Sigma A9441) n- (85 : 15 : 20) 360 nm 10 ml 60 (Sigma C1530) (TEF; 5 : 4 : 1) (BEM; 90 : 5 : 7) (4-dimetyl-aminobenzaldehyde in 96 EtOH) 50 AFPA ) 0.5 ml 0.1 ml 15 (9 : 1) 0.9 ml 5,000 g 5 HPLC HPLC Agilent 1,100 Series (LC-04) Mightysil RP-18 GP 4.6 mm i.d. 250 mm, 5 mm 40 (1 :3:6) 1.0 ml/min 20 ml 365 nm, 450 nm Table CFU/g CFU/g A CFU/g Table 1. Detection of fungi in commercial almond powder sample Origin No. of sample 20 g (9 : 1) 50 ml 30 (Multi- Sep 226 AflaZon, Romer Labs.) 1mL Deoxidizer application Detected fungal counts (CFU/g) U.S.A. 21 Yes No 4 Italy 2 Yes 1 1 Spain 1 No 1 Unknown 6 Yes 3 No 3 Total

3 92 Vol. 48, No. 4 Table 2. Distribution of fungi in selected almond powder samples (CFU/g) Sample No. Total A. flavus A. niger Cladosporium Penicillium Rhizopus A A A A A A A A A A A including Aspergillus flavus and related species; : not detected Table 3. Incidence of aflatoxigenic fungi and aflatoxins in almomd samples Sample No. A. flavus and related fungi No. of tested Aflatoxin-producing fungi No. of aflatoxin positive strains Aflatoxins (ng/g) B 1 B 2 G 1 G 2 A /g 9 9 (B 1,B 2) ND ND A /g (B 1) ND ND A /g 3 2 (B 1,B 2,G 1,G 2 1) 0.1 ND ND ND (B 1,B 2 1) A /g 15 7 (B 1,B 2,G 1,G 2 6) ND ND ND ND (B 1,B 2 1) A /g 10 7 (B 1,B 2,G 1,G 2 3) ND ND ND ND (B 1,B 2 4) A /g 10 6 (B 1,B 2,G 1,G 2 4) ND ND ND ND (B 1,B 2 2) A /g 10 5 (B 1,B 2 5) 0.5 ND ND ND A /g 8 3 (B 1,B 2,G 1,G 2 2) 0.6 ND ND ND (B 1 1) A /g 8 4 (B 1,B 2 4) ND ND ND: not detected Table 4. Morphological and metabolic characters of aflatoxigenic isolates Strain No. Identification Colony color on ME agar Sclerotia formation Aflatoxins B 1 B 2 G 1 G 2 CPA A-23-3 A. parasiticus dark green none A-23-6 A. flavus yellow green large scl. A A. nomius yellow green barrel scl. A A. nomius yellow green barrel scl. A-24-1 A. parasiticus dark green none A-24-3 A. flavus yellow green large scl. A-25-4 A. parasiticus dark green none A-25-7 A. flavus yellow green none A-25-9 A. nomius yellow green barrel scl. A-34-1 A. parvisclerotigenus purplish grey small scl. A-34-2 A. parvisclerotigenus purplish grey small scl. A-35-1 A. parvisclerotigenus purplish grey small scl. A-35-8 A. parasiticus dark green none A-36-2 A. flavus yellow green large scl. poor sporulation; CPA: cyclopiazonic acid 6 A-34, A-35, A-36 3

August 2007 93 Plate Fig. 1. Fungal colonies developed from almond sample on Aspergillus Flavus and Parasiticus agar (AFPA) incubated for 48 hr at 30.

4 August Plate Fig. 1. Fungal colonies developed from almond sample on Aspergillus Flavus and Parasiticus agar (AFPA) incubated for 48 hr at 30. Aspergillus ﬂavus and related species produced orange colors in the reverse. Fig. 2. Aspergillus ﬂavus (left) and A. parasiticus (right) on MEA, 25, 7-day culture. Fig. 3. Aspergillus nomius (left) and A. parvisclerotigenus (right) on MEA, 25, 7-day culture.

5 94 1g 10 3 CFU 11 Table 2 A. flavus CFU/g A. flavus A. niger, Cladosporium, Penicillium, Rhizopus A-7 A-8 A-17 A-23, A-24, A-25 A-34 A-35, A-36 A-33 Rhizopus, Mucor A. flavus DRBC A. flavus AFPA A. flavus (Plate Fig. 1) A. flavus YES-broth TLC Table 3 A-7 A CFU/g, CFU/g A. flavus B B A-17 A-23, A-24, A-25 3 B A. flavus B G A. parasiticus, A. nomius A ng/g B 1 A-34, A-35, A-36 A. flavus, A. parasiticus, A. parvisclerotigenus B 1 0.5, 0.6, 2.1 ng/g A. flavus Aspergillus flavus (Malt ext. agar, Oxoid) YES-broth Vol. 48, No. 4 6) 14 Table 4 A. flavus (Plate Fig. 2) A. parasiticus (Plate Fig. 2) B G A. nomius (Plate Fig. 3) B G (Plate Fig. 3) A. parvisclerotigenus B 1, B 2 A. flavus 6) (5 : 4 :1) TLC (90 :5:7) A. flavus A. nomius A. parasiticus A. parvisclerotigenus B G 7) A. flavus A. parasiticus 8) A. flavus A. niger, Rhizopus Cladosporium, Penicillium, yeast

6 August A. flavus 9) 10 3 /g ). B ng/g (ppb) B 1 10 ppb B 1,B 2 A. flavus B G 1,G 2 G A. parasiticus, A. nomius B G B 1 A-34, A-35 B 1,B 2 Saito Tsuruta 11) A. flavus var. parvisclerotigenus Frisvad 12) A. parvisclerotigenus A. flavus, A. parasiticus 2 A. parvisclerotigenus A. nomius AFPA A. flavus, A. parvisclerotigenus A. parasiticus 30, 48 6) A. nomius AFPA 30 DRBC AFPA CFU/g Aspergillus niger, A. flavus Penicillium, Cladosporium, Rhizopus A. flavus 90 YES broth-tlc B 1,B 2 A. flavus, A. parvisclerotigenus B G A. parasiticus, A. nomius B G ng/g B 1, ng/g B ) 1999, p (ISBN ) 2) 1998, p (ISBN ) 3) Hasegawa, A., Tanaka, T., Aoki, N., Yamamoto, S., Toyazaki, N., Matsuda, Y., Udagawa, S., Isolation and identification of Aspergillus flavus from imported nuts and their aflatoxin producibility. Maikotokishin (Proc. Jpn. Assoc. Mycotoxicol.), 25, (1987). 4) Tabata, S., Kamimura, H., Ibe, A., Hashimoto, H., Iida, M., Tamura, Y., Nishima, T., Aflatoxin contamination in foods and foodstu#s intokyo: J. AOAC Int., 76, (1993). 5) ) Samson, R. A., Hoekstra, E. S., Frisvad, J. C. eds., Introduction to food- and air-borne fungi. 7th Ed. Utrecht, Centraalbureau voor Schimmelcultures, (ISBN )

7 96 Vol. 48, No. 4 7) Schade, J. E., McGreevy, K., King, Jr., A. D., Mackey, B., Fuller, G., Incidence of aflatoxin in California almonds. Appl. Microbiol., 29, (1975). 8) Phillips, D. J, Mackey, B., Ellis, W. R., Hansen, T. N., Occurrence and interaction of Aspergillus flavus with other fungi on almonds. Phytopathology, 69, (1979). 9) King, Jr., A. D., Schade, J. E., Influence of almond harvest, processing and storage on fungal population and flora. J. Food Sci., 51, (1986). 10) Yamamoto, K., Statistical consideration of sampling plan for mycotoxin test of granular agricultural products. Shokuhin Eiseigaku Zasshi (J. Food Hyg. Soc. Japan), 32, (1991). 11) Saito, M., Tsuruta, O., A new variety of Aspergillus flavus from tropical soil in Thailand and its aflatoxin productivity. Maikotokishin (Proc. Jpn. Assoc. Mycotoxicol.), 37, (1993). 12) Frisvad, J. C., Skouboe, P., Samson, R. A., Taxonomic comparison of three di#erent groups of aflatoxin producers and a new e$cient producer of aflatoxin B 1, sterigmatocystin and 3-O-methylsterigmatocystin, Aspergillus rambellii sp. nov. Systematic Appl. Microbiol., 28, (2005).

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