SMALL-SCALE PRODUCTION OF OCHRATOXIN A FOR RAPID DETOXIFICATION ASSAYS

Ochratoxin A (OTA) is one of the most frequently found mycotoxins in food, being produced by several filamentous fungi, majorly Aspergillus species. It has been associated to nephrotoxic, immunotoxic, carcinogenic, genotoxic and teratogenic effects in mammals, which bases the growing interest in developing safe and low-cost decontamination methods for OTA. The objective of this study was to establish a small-scale OTA production method, aiming to facilitate further in vitro assays of biodegradation minimizing the costs and time dedicated to initial steps of such assays. In this way, three different Aspergillus strains – A. ochraceus CMT 00435, A. lacticoffeatus CECT 20581 and A. sclerotioniger CECT 20583 –, known to be able to produce OTA, were chosen to be separately inoculated (inocula concentration of approximately 106 spores/mL) in liquid and solid matrices for mycotoxin production. Thereon, 3 flasks containing 200 mL of tryptic soy broth (TSB) and 3 jars with 100 g of wheat grain respectively received 5 and 25 mL of inoculum each. The inoculated broths were incubated in shaker (25 °C, 110 rpm) for 15 days, with HPLC quantification of OTA on days 7, 12 and 15 – 0.22 µm filtration followed by direct injection in the equipment. The contaminated grains were maintained for 35 days at 25 °C, and sterile water was added (15 mL) on days 7 and 15 to improve fungal growth, and then OTA concentration was determined by HPLC after methanol extraction. For both matrices, A. sclerotioniger produced higher OTA concentrations (maximum levels of 0.85 ppm in broth after 7 d and 5.27 ppm in grains after 35 d), whereas A. lacticoffeatus produced 0.01 and 0.32 ppm and A. ochraceus 0 and 0.05 ppm in broth and grain, respectively. In addition, A. sclerotioniger showed the highest OTA amount in broth at day 7 (0.85 ppm), decreasing through days 12 and 15 (0.72 and 0.68 ppm). This decreasing profile was also observed for A. lacticoffeatus production. This denotes that OTA reaches a maximum level in broth and might be metabolized by the fungi itself. Further tests with solid matrices should be carried out to maximize their application, since higher concentrations of OTA could be achieved – yet after longer incubation periods. Such results may be explored for large scale production of OTA that could be applied for the screening of detoxifying organisms or for animal studies, reducing the costs of purchasing the purified mycotoxin.