Research on the production of some analytical mycotoxin standards used in food and feed analysis

In this study, it was aimed to produce at least one of aflatoxin, ochratoxin A (OTA), deoxynivalenol (DON) and zearalenone (ZEA) mycotoxins at high purity (≥%90) using mold isolates. Firstly, a total of 126 mold isolates were obtained from food samples supplied from the Turkish market. The isolates that may belong to Aspergillus, Penicillium or Fusarium sp. were determined by making macroscopic and microscopic observations. Species identification of selected isolates was made by comparing the sequence results of the Internal Transcribed Spacer (ITS1-4) regions with the NCBI database. Potentially mycotoxigenic isolates were grown in broth medium and mycotoxin amounts were determined using High Performance Liquid Chromatography. According to the analysis results, it was determined that IK5-D2 A. flavus (AFB1 and AFB2 producer) and B1-D1 F. culmorum (ZEA producer) isolates were able to produce high amounts of mycotoxins. A Box-Behnken experimental design was created with three independent variables (pH, temperature and incubation time) to increase the production amount of the specified mycotoxins. The results were evaluated according to Response Surface Methodology and the process was mathematically modeled. The amounts of AFB1, AFB2 and ZEA produced under optimized conditions were found as 27244.5 ng/mL, 681.8 ng/mL and 6645.9 ng/mL, respectively. It has been determined that the experimental results were compatible with the predictions obtained from mathematical models. Mycotoxins extracted from the medium were purified using semi-preparative chromatographic method. The purity ratios of the mycotoxin fractions which prepared in acetonitrile solution were 90.3%, 99.6% and 95.4% for AFB1, AFB2 and ZEA, respectively. Recovery rates from the medium were calculated as 73.2%, 82.8% and 91.0%, respectively. It is thought that mycotoxin solutions produced at high purity have the potential to be evaluated in toxicological studies or as analytical standard material in food and feed analysis.