Process development for the elucidation of mycotoxin formation in Alternaria alternata

Abstract

The black mould Alternaria alternata produces a wide diversity of mycotoxins which are of particular health concern. Since no maximum allowable limits are set for Alternaria toxins in food and feed, prevention of Alternaria infestations and mycotoxin spoilage is the only way to avoid health risks. Thus, the understanding of mycotoxin biosynthesis is essential. For that purpose, a reliable batch process in a 2 L bioreactor was established which enables the study of several parameters influencing the production of the mycotoxins alternariol (AOH), alternariol monomethylether (AME) and tenuazonic acid (TA) by A. alternata DSM 12633. Modified Czapek-Dox medium was used with glucose as carbon source and ammonium and nitrate as nitrogen sources. Consumption of carbon and nitrogen sources as well as formation of the three mycotoxins were monitored; the average data of five independent fermentations was plotted and fitted using a logistic equation with four parameters. Maximum mycotoxin concentrations of 3.49 ± 0.12 mg/L AOH, 1.62 ± 0.14 mg/L AME and 38.28 ± 0.1 mg/L TA were obtained.In this system the effect of different aeration rates (0.53 vvm-0.013 vvm) was tested which exerted a great influence on mycotoxin production. The use of the semi-synthetic Czapek-Dox medium allowed the exchange of carbon and nitrogen sources for acetate and aspartic acid. The use of acetate instead of glucose resulted in the sole production of alternariol whereas the exchange of ammonium and nitrate for aspartate enhanced the production of both AOH and AME while TA production was not affected.

Figure 1

Production of the mycotoxins tenuazonic acid (TA) (a), alternariol (AOH) and alternariol monomethylether (AME) (b) and consumption of the nutrients glucose, nitrate and ammonium (a, b) with A. alternata DSM 12633 in a 2 L bioreactor. Measured glucose, nitrate, ammonium, TA, AOH and AME concentrations are given as averages of five independent fermentations. All lines represent logistic fittings of the concentrations based on Eq. 1.

Figure 2

Calculated averaged relative glucose consumption rate and mycotoxin production rates of five independent fermentations of A. alternata DSM 12633 in a 2 L bioreactor. TA: tenuazonic acid; AOH: alternariol; AME: alternariol monomethylether.

Figure 3

Mycotoxin production with A. alternata in a 2 L bioreactor system using different aeration rates. Results are mean of two replicates. TA: tenuazonic acid; AOH: alternariol; AME: alternariol monomethylether.