Soil in corn plots was inoculated with nonaflatoxigenic strains of Aspergillus flavus and A. parasiticus during crop years 1994 to 1997 to determine the effect of application of the nontoxigenic strains on preharvest aflatoxin contamination of corn. Corn plots in a separate part of the field were not inoculated and served as controls. Inoculation resulted in significant increases in the total A. flavus/parasiticus soil population in treated plots, and that population was dominated by the applied strain of A. parasiticus (NRRL 21369). In the years when weather conditions favored aflatoxin contamination (1996 and 1997), corn was predominately colonized by A. flavus as opposed to A. parasiticus . In 1996, colonization by wild-type A. flavus was significantly reduced in treated plots compared with control plots, but total A. flavus/parasiticus colonization was not different between the two groups. A change to a more aggressive strain of A. flavus (NRRL 21882) as part of the biocontrol inoculum in 1997 resulted in a significantly ( P < 0.001) higher colonization of corn by the applied strain. Weather conditions did not favor aflatoxin contamination in 1994 and 1995. In 1996, the aflatoxin concentration in corn from treated plots averaged 24.0 ppb, a reduction of 87% compared with the aflatoxin in control plots that averaged 188.4 ppb. In 1997, aflatoxin was reduced by 66% in treated corn (29.8 ppb) compared with control corn (87.5 ppb). Together, the data indicated that although the applied strain of A. parasiticus dominated in the soil, the nonaflatoxigenic strains of A. flavus were more responsible for the observed reductions in aflatoxin contamination. Inclusion of a nonaflatoxigenic strain of A. parasiticus in a biological control formulation for aflatoxin contamination may not be as important for airborne crops, such as corn, as for soilborne crops, such as peanuts.
Addition of sequestering agents to feeds and foods has been proposed as a protective strategy against mycotoxins. To investigate the efficacy of Volclay, a bentonite clay, to protect against aflatoxicosis, rats were fed peanut butter (50% wt/wt)-based diets containing 1,500 ppb aflatoxin (AF), 1,500 ppb aflatoxin with 0.1% Volclay supplementation (AF-LD), or 1,500 ppb aflatoxin with 1% Volclay supplementation (AF-HD) for 8 weeks. The control group was fed a peanut butter-based diet without aflatoxin or Volclay supplementation and a fifth group was fed the control diet with 1.0% Volclay supplementation (VC). No differences in appearance, behavior, or selected hematological and serum chemical variables were observed. Decreased weight gain, decreased food consumption, and liver lesions consistent with hepatic aflatoxicosis were found in AF-fed rats. Weight gain and food consumption of the AF-HD group were comparable to the control and VC groups and were significantly increased compared to AF-fed rats, even though weekly aflatoxin ingestion of AF-HD rats equaled or exceeded that of the AF group. Body weight and food consumption of the AF-LD group were slightly increased compared to AF group and decreased compared to the control, VC, and AF-HD groups, but the differences were not statistically significant. Liver lesions were found in all AF and AF-LD rats. Lesions were also detected in eight of 10 AF-HD-fed rats, but were subtle and significantly less extensive than in AF and AF-LD rats. The data suggest that Volclay is nontoxic and may be an efficacious sequestering agent for residual aflatoxin in peanut butter.
A three-year study was conducted to evaluate the use of a nonaflatoxin-producing strain of Aspergillus parasiticus (NRRL 13539) as a biocompetitive agent for the control of preharvest aflatoxin contamination of peanuts. The agent was added to the soil of the environmental control plot facility at the National Peanut Research Laboratory and tested by subjecting peanuts to optimal conditions for the development of aflatoxin contamination. Edible peanuts from the treated soil contained aflatoxin concentrations of 11, 1, and 40 ppb for crop years 1987, 1988, and 1989, respectively, compared to untreated peanuts with 531, 96, and 241 ppb, respectively. In addition, treatment in 1989 with low and high inoculum levels of a UV-induced mutant from the NRRL 13539 strain resulted in aflatoxin concentrations of 29 and 17 ppb, respectively, in edible peanuts. Soil populations of the biocompetitive agents were not higher than populations of wild strains of A. flavus/parasiticus in untreated soil subjected to late-season drought stress. This is an important ecological consideration relative to the utilization of this biocontrol system.
A simple procedure was devised for the routine screening of aflatoxin B 1 (AFB 1 ) in peanut butter using enzyme-linked immunosorbent assay (ELISA). Peanut butter samples (5 g) were artificially contaminated with AFB 1 and extracted by blending with 25 ml of 55% methanol and 10 ml of hexane. The extract was filtered and aqueous filtrate analyzed by a direct competitive ELISA. Recovery of AFB 1 added to peanut butter samples ranged from 85 to 112%, with an average inter-well coefficient of variation of 18.4%. The inter-assay coefficient of variation was 22.7%. Using this procedure, only 3 of 63 commercial samples of peanut butter had detectable levels (>5.0 μg/kg) of AFB 1 .