Inhibition of mycelial growth of mycotoxigenic fungi by phenolic antioxidants

During a recent review of the literature, it became evident that information on the fungicidal and fungistatic activity of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butylhydroquinone (TBHQ), and propyl gallate (PG) has largely been focused on controlling growth and aflatoxin by Aspergillusflavus Link and A. parasiticus Speare (Chang and Branen, 1975; Fung et al., 1977; Beuchat and Jones, 1978; Ahmand, 1979; Lin and Fung, 1983; and Thompson, 1991). However, other species of mycotoxic genera include Fusarium and Penicillium, but the fungitoxic activity from antioxidants against these species has received limited attention. The present study was undertaken to determine the minimum inhibitory concentration of six phenolic antioxidants against known mycotoxin-producing species of Aspergillus, Fusarium, and Penicillium. The presence of agar in the test medium has been reported as confounding the effects observed from several inhibitory chemicals, specifically as they relate to spore germination (Ko et al., 1975). Hence a comparison was also made in liquid and agar media to evaluate the effect of agar on mycelial growth. The fungal cultures were obtained from the American Type Culture Collection (Rockville, Maryland). Stock cultures were maintained at 5 C on slants of potato dextrose agar (PDA) (Difco, Detroit, Michigan). The phenolic antioxidants BHA, BHT, PG, propyl paraben (PP), TBHQ, and 2,4,5-trihydroxybutyrophenone (THBP) were purchased from Fluka Chemical Company (Ronkonkoma, New York). A stock solution of each phenolic antioxidant was prepared in 95% ethanol at 1% concentration. The appropriate concentration was added to the medium before autoclaving. Mycelial discs were prepared as described by Moleyar and Narasimham (1986). Twenty ml of PDA medium, in petri plates, were inoculated with 1 ml of a spore suspension (50,000 spores/ ml) of the test fungus. The suspension was uniformly spread over the medium using a bent glass rod. The inoculated medium was then incubated at 27 C for 48 h. Mycelial discs of 0.6 cm diam were removed from the petri plates with a cork borer and used as inocula for PDA and potato dextrose broth (PDB) media to determine antifungal activity. The antifungal activity on agar medium was determined by incorporating antioxidants (0, 100, 250, 500, and 1000 ,ug/ml), which was then dispensed as 20-ml quantities into sterile plastic petri plates (9 cm). The center of each plate was inoculated with a mycelial agar disc (0.6 cm) placed upside down on the medium, and incubated at 27 C for 7 days. At the end of the incubation period, the plates were examined for mycelial growth. The lowest concentration of the test compound in which no growth occurred on the PDA medium was recorded as the minimum inhibitory concentration (MIC). The control was prepared by incorporating an appropriate volume of 95% ethanol in 20 ml of PDA. Three replicate plates per treatment were used and the experiments were repeated two times. To assess the antifungal activity of antioxidants in liquid medium, the antioxidants were incorporated in PDB to yield final concentrations of 0, 100, 250, 500 and 1000 ,tg/ml. Fifty ml of the amended medium were dispensed in 250-ml flasks. A mycelial agar disc (0.6 cm) was used to inoculate the liquid medium. The fungal cultures, incubated stationarily at 27 C for 7 days, were examined for mycelial growth. Appropriate controls included ethanol which was incorporated in the PDB. Three replicate flasks per treatment were prepared and experiments were repeated two times. The effect of six phenolic antioxidants on mycelial growth of 16 species of toxigenic fungi on agar medium is shown in TABLE I. No fungi showed a response at the 100 jtg/ml level. Also 791