A neutral red desiccated coconut agar for rapid detection of aflatoxigenic fungi and visual determination of aflatoxins

Abstract

Desiccated coconut agar is the conventional medium used for the detection of aflatoxigenic fungi and direct visual determination of aflatoxins. In this study, an improved medium was developed by the incorporation of 0.2% (v/v) neutral red dye into desiccated coconut agar. The medium was formulated by a 2×3 factorial design of neutral red and phenol red stains at three concentration levels. The formulated medium was evaluated for performance by screening for the minimal time required by each Aspergillus species to produce pigments and fluorescence of agar. The medium was also employed for detection of aflatoxigenic fungi and direct visual determination of aflatoxins in foods and fish-meal. The neutral red desiccated coconut agar (NRDCA) as compared to the conventional desiccated coconut agar (DCA) had a light pink background as opposed to the white background of the DCA which often interferes with the visibility of fluorescence. The time of pigmentation and fluorescence production on NRDCA was 28 and 38 h respectively as compared with 33 and 44 h of DCA and 41 and 48 h of palm kernel agar (PKA: an alternative culture medium for cultivation of aflatoxigenic fungi with a reddish pink background). Furthermore, aflatoxigenic moulds were detected in all food commodities and fish-meal after 60 hours of incubation. The highest percentage of aflatoxigenic moulds (62.5%) was detected in yam flour with NRDCA while the lowest percentage (4.46%) was detected with PKA on rice. In addition, aflatoxins were produced in high amounts in food commodities in which aflatoxigenic moulds were detected and there was a significant positive correlation (r=0.4, P<0.05) between the isolates and aflatoxin concentration of the food samples. Rice (a major staple food for Nigerians) had the highest total aflatoxin concentration of 140, 220 and 205 µg/kg on DCA, NRDCA and PKA respectively, while ‘gari’ had the least concentration of 45, 50 and 40 µg/kg. These values were far above the NAFDAC recommended level of 10 µg/kg for unprocessed foods in Nigeria and therefore a source of concern. In addition the study also reveals that Aspergillus nomius can produce aflatoxins B1 in copious amounts on NRDCA, contrary to previous reports of its production in minute quantities on laboratory media. The benefit of this study lies in the rapid analysis and simplified technique for the detection of aflatoxigenic fungi and visual determination of aflatoxins.