Fungal strains, materials, and culture conditions
Pleurotus ostreatus var. columbinus (“Blue Oyster”) and P. ostreatus (“Pearl Oyster”) were obtained from Fungi Perfecti, LLC (Olympia, WA); and P. ostreatus (N001) was obtained from the Spanish Type Culture Collection (CECT; Paterna, Spain). All three strains are commercial and dikaryotic. Cultures were maintained in water vouchers at 4 °C until required for experiments. Cultures were grown out on malt yeast peptone agar (MYPA) consisting of 7 g L−1 malt extract, 0.5 g L−1 yeast extract, 1 g L−1 peptone and 15 g L−1 agar at room temperature (~23 °C) for 10 days in 100 mm × 15 mm Petri dishes before use for substrate inoculation. A. flavus AF13 (ATCC 96044) was provided by Dr. Peter Cotty, USDA–ARS, School of Plant Sciences, University of Arizona. AF13 was grown for spore production on 5-2 Agar (Cotty and Misaghi 1984) consisting of 20 g L−1 Bacto™ agar (Becton, Dickinson and Company, Franklin Lakes, NJ) and 50 mL L−1 V8™ Juice (CSC Brands, L.P., Camden, NJ) adjusted to pH 6.0 for 7 days at 31 °C. Conidia were harvested from AF13 cultures after 7 days of growth, dispensed into sterile water, enumerated using an Orbeco Hellige TB300 IR Turbidimeter (Orbeco Hellige Inc., Sarasota, FL), diluted to 1.5 × 10−7 conidia mL−1, and used on the same day to inoculate maize for aflatoxin production. Nature’s Match™ whole corn (maize) (Land O’ Lakes Purina Feed LLC, Shoreview, Minnesota) was purchased from a local feed store. Maize was homogenized and stored at room temperature until use. Three 50 g subsamples of maize were taken to establish baseline levels of aflatoxin contamination. Baseline moisture content (MC) was measured using a Mettler Toledo HB43 Halogen Moisture Analyzer (Mettler-Toledo International Inc., Columbus, OH).
Aflatoxin production
Maize was added to quart size mason jars (150 g jar−1), adjusted to 20% MC, sterilized, and inoculated with 105 AF13 conidia g−1. Inoculated maize was adjusted to 30% MC and mixed to distribute spores evenly. Jars were fitted with pre-sterilized lids containing seven ½” holes and synthetic filter discs, and incubated at 31 °C for 7 days. Colonized jars were autoclaved for 30 min at 121 °C and 15 psi to kill the AF13 culture. Aflatoxin-contaminated maize was dried to completeness in a horizontal airflow oven (VWR International LLC, Radnor, PA) set at 45 °C for 7 days. The dried and contaminated maize was homogenized using a Bunn® G3 Coffee Mill (Bunn, Springfield, Illinois), quantitated, and stored at −20 °C until use for substrate preparation.
Aflatoxin quantitation
Baseline aflatoxin levels and 10× concentrated mushroom samples were quantitated using the Neogen® Reveal® Q+ test kit and AccuScan Pro Reader (Neogen Corp., Lansing, MI) per manufacturer instructions. AFB1 from other samples was quantitated via thin layer chromatography (TLC) by fluorometric measurement with a CAMAG TLC Scanner 3 densitometer (CAMAG Scientific, Inc., Wilmington, NC) (Stoloff and Scott 1984; Pons et al. 1966). Limits of detection for this method were established using a ½ log dilution series of AFB1 (Sigma-Aldrich Corp., St. Louis, MO). All maize samples that fell below the detection limit after decontamination were concentrated 100× and re-quantitated fluorometrically.
Substrate preparation
Maize that was uncontaminated (<5 ng g−1, but assumed to be 0 ng g−1 for calculations and figures herein) or contaminated (25, 250, or 2500 ng g−1) with aflatoxin was used for controls or as substrate for P. ostreatus growth and mushroom production. Maize samples were adjusted to 45% MC, and 100 g each were added to 4″ × 3″ × 18″ high density polypropylene bags containing a 0.5 µm filter patch. The bags containing maize samples were autoclaved at 121 °C and 15 psi for 60 min. Samples were either mock inoculated with ten 6 mm diameter sterile agar plugs or inoculated with ten 6 mm agar plugs of N001, Pearl, or Blue strains, respectively. Thus, there were 16 sample types including treatment groups and positive and negative controls, with 5 biological replicates used for each sample type (N = 80).
Colonization and fructification
Samples were incubated at 25 °C for 21 days, and subsequently placed in a fruiting chamber at 90–99% relative humidity, <600 ppm CO2, and 23 °C until mushrooms were ready for harvest. Radial mycelial growth could not be measured because the 100 g maize samples were in three-dimensional substrate blocks, so growth was checked daily and the number of days required to completely colonize the substrate was recorded. Bags inoculated with P. ostreatus were sliced upon being placed in the fruiting chamber. Bags that were mock inoculated were not sliced to prevent contamination, but were kept in the fruiting chamber until all mushrooms were harvested. The number of days required for mushrooms to mature was recorded to evaluate potential inhibition. Fresh weights of mushrooms were measured immediately after being harvested and data were expressed as biological efficiency (%BE = fresh mushroom weight/dry substrate weight × 100). Processed samples and mushrooms were stored at −20 °C until they were removed to be dried to completeness at 45 °C, and were again stored at −20 °C until analyzed.
Acid reversion assay
A subset of 10 g substrate samples from all control and treatment groups originally contaminated at 2500 ng g−1 AFB1, in addition to uncontaminated controls, were subjected to conditions approximating physiological conditions of the human stomach (37 °C and pH 2) for 2 h by addition of 20 mL 0.2 M HCl. Samples were dried in the dark for 48 h at 45 °C and −10 kPa in a vacuum oven, and re-quantitated fluorometrically to assess the extent of chemical reversion to AFB1. Data were expressed as percent degradation relative to control samples before and after being subjected to acid reversion conditions.
Salmonella typhimurium (Ames) mutagenicity assay
The Salmonella (Ames) mutagenicity assay with metabolic activation was used as a proxy to measure the carcinogenicity of degradation products (Ames et al. 1975). Tester strain TA1535 (Molecular Toxicology Inc., Boone, NC) was used because it has a –G–G–G– DNA target and is susceptible to the same base substitutions that AFB1 induces (Foster et al. 1983). A subset of samples from P. ostreatus-treated and mock inoculated groups originally contaminated at 2500 ng g−1AFB1, in addition to uncontaminated control substrate samples, were used for this assay. A serial dilution of pure AFB1 in 70% MeOH ranging from 0 to 10,000 ng g−1 was used as a reference. Controls containing only 70% MeOH and S-9 metabolic mix without aflatoxin were also used. Samples from both runs of the degradation experiment were assayed concurrently and compared to a single set of control samples. Three biological and three technical replicates were used for each treatment and control group. Colonies were enumerated using OpenCFU software (Geissmann 2013). Data are given as the number of revertant colony forming units (CFUs).
Data analyses
All experiments were duplicated unless otherwise noted. Results from duplicated experiments are only shown if they were inconsistent with the initial results. Normality of data was assessed using the Shapiro–Wilk goodness-of-fit test. One-way analysis of variance (ANOVA) tests were conducted for the number of days until colonization, number of days required for mushroom formation, %BE, degradation percentage, and acid reversion data. If significant differences were detected by ANOVA at the 95% confidence level, Tukey’s HSD was used to compare means and generate connecting letter reports. Statistical analyses were conducted using JMP Statistical Discovery Software v10.0 (SAS, Cary, NC).