African Journal of Biotechnology
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African Journal of Biotechnology Vol. 2 (10), pp. 365-368, October 2003 ISSN 1684-5315 © 2003 Academic Journals
Full Length Research Paper
Mycoflora and mycotoxins in kolanuts during storage
L.
O. ADEBAJO* and O. J. POPOOLA Department of Biological Sciences, Olabisi Onabanjo University**, P.M.B. 2002, Ago–Iwoye, Ogun State, Nigeria. *Correspondence author; E-mail: lawadebajo@yahoo.com. ** Former name: Ogun State University. Accepted 20 September 2003 |
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The mycoflora, levels of afllatoxins and the presence of ochratoxin A and zearalenone in nuts of Cola acuminata and C. nitida were determined immediately after curing and after 3, 6 and 9 months of storage in leaf-lined baskets. Five field fungi and 11 storage fungi were isolated. Aspergillus, Penicillium and Fusarium were the predominant genera. None of the target toxins was detected immediately after curing. Increasing quantities (5 to 160 ppb) of each of the aflatoxins B1, B2, G1 and G2 were recorded as from the 3rd month while zearalenone and ochratoxin A were detected only after the 6th and 9th month, respectively. Key words: Kolaunts, mycoflora, mycotoxins, storage.
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Kolanuts are the cotyledons of some species of Cola, a genus of trees belonging to the family Sterculiaceae (Purseglove, 1974). They are a major stimulating masticatory in West Africa. They are used for pharmaceuticals and for flavouring soft drinks and in the preparation of choca-cola and wine (Opeke, 1982).
The cultivation, processing and storage of the nuts are undertaken in the warm, humid rain forest zone where there is high mould infection. However, so highly esteemed are the nuts that fairly mouldy samples are commonly ingested. This is in spite of the numerous toxic metabolies (Bacha et al., 1988, Jimenez et al., 1991) frequently associated with mould-contaminated foods and the consequent risk (Campbell and Stoloff, 1974; Frazier and Westhoff, 1978) posed to their consumers. The present report is on the evaluation of moulds and mycotoxins (aflatoxins, ochratoxin A and zearalenone) in the nuts of ‘abata’ kola or Cola acuminata (P. Beauv.) Schott and Endl. and ‘gbanja’ kola or Cola nitida (Vent.) Schott and Endl. immediately after curing and during storage. |
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Collection
and preparation of samples Fresh kolanuts were obtained immediately after a 5-day curing period from kola merchants in Ago-Iwoye and Sagamu, both in Ogun State and from Ikorodu, Lagos State, Nigeria. Altogether, 15 samples (approx. 6 kg each) were collected for each of C. acuminata and C. nitida. On the day of collection, 30 nuts were randomly taken from each sample before the remaining nuts were packaged, following a traditional method, inside a basket lined with leaves of Thaumatococcus daniellii (Benn.) Benth. The packages (15) were kept inside a clean and well-ventilated room. After 3, 6 and 9 months, 10 nuts were randomly picked from each of three different points (surface, middle, bottom), making a total of 30 nuts from each basket or sample.
Evaluation of mycoflora During each investigation, 225 of the 450 nuts obtained randomly from the 15 packages were carefully observed with the naked eye for any evidence of mould infection.
The cotyledons were then separated by hand and the state of the inner surfaces was also noted. Each cotyledon was subsequently cut into four pieces (about 10 by 18 mm each) and surface disinfected with a 2% aqueous solution of sodium hypochlorite for 2 min. This was followed by washing with six changes of sterile distilled water before the four cotyledon-pieces were plated together equispaced on potato dextrose agar (PDA). The latter contained streptomycin sulphate (5 mg/ml) as an antibacterial antibiotic. Plates were incubated at 28oC for 5 to 10 days during which time the fungi that emerged were counted, isolated and identified with the aid of appropriate manuals. The percentage incidence of the fungal isolate per 400 cotyledons was calculated. Extraction and
determination of mycotoxins The remaining 225 nuts were similarly observed with the naked eye for signs of mould infection. Subsequently, the split cotyledons were dry-milled and mixed thoroughly before three 25-g portions were taken and independently analysed for mycotoxins. Extraction was carried out and the components were separated by 2-dimensional thin layer chromatography (Patterson and Roberts, 1979). Aflatoxins (B1, B2, G1, G2), ochratoxin A and zearalenone were detected by their fluorescence under ultraviolet (UV) light. These toxins were positively identified by coincidence of migration in several solvent systems with reference compounds (Robb and Norval, (1983). Confirmation of aflatoxins and ochratoxin A was by reaction with dilute sulphuric acid and dilute sodium hydroxide, respectively. Zearalenone was confirmed by ratio of fluorescence under 254 and 365 nm UV light (Blaney et al., 1984). For aflatoxins only, concentrations were determined by visual comparison of fluorescent intensity of sample spots with standard following suitable dilution.
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At collection time, only about 2% of the kolanuts had visible mould infection. After 3, 6 and 9 months the proportions had increased to approximately 15, 39 and 88% respectively. This significant increase in mould growth and development during storage necessitates a closer appraisal of the traditional storage technique with the aim of preventing mould deterioration of the nuts. A total of 5 field fungi and 11 storage fungi were obtained from the nuts investigated. The frequency of isolation of the former and the latter fungi decreased and increased, respectively with storage time (Table 1). Field fungi usually colonize seeds before harvest. Their water activity (aw) growth optima are above 0.96 (Hudson 1986), thus, they gradually die during storage (Christensen and Sauer, 1982) due to decrease in aw of produce caused by loss of moisture. On the whole, Aspergillus niger, A, flavus, Penicillium funiculosum, Fusarium moniliforme, A, tamarii, Botryodiplodia theobromae, F. oxysporum and A. orchraceus were the most commonly isolated fungi (Table 1). Aspergillus, Penicillum and Fusarium are well known producers of potent mycotoxins (Bamburg et al., 1969; Jimenez et al., 1991) and they are considered to be the most significant toxigenic moulds at the present time (Chelkowski et al., 1983). Consequently, control of moisture and temperature levels to prevent mould growth and mycotoxin elaboration (Smith and Moss, 1985; Adebajo, 1992) has became mandatory for safe storage of kolanuts and also to avert possible mycotoxic problems in consumers.
Table 1. Incidencea of fungi in nuts of Cola acuminata (Ca) and C. nitida (Cn).
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