Influence of Environmental Factors on the Production of Penitrems A-F by Penicillium crustosum

Abstract

Filamentous fungi produce a multitude of secondary metabolites, some of them known as mycotoxins, which are toxic to vertebrates and other animal groups in low concentrations. Among them, penitrems, which belong to the group of indole-diterpene mycotoxins, are synthesized by Penicillium and Aspergillus genera and exhibit potent tremorgenic effects. This is the first complex study of the penitrems A-F production under the influence of different abiotic factors, e.g., media, incubation time, temperature, pH, light, water activity, and carbon and nitrogen source as well as oxidative and salt stress. For this purpose, penitrems A-F were isolated from Penicillium crustosum cultures and used as analytical standards. Among the carbon sources, glucose supplemented to the media at the concentration of 50 g/L, showed the strongest inducing effect on the biosynthesis of penitrems. Among nitrogen sources, glutamate was found to be the most favorable supplement, significantly increasing production of these secondary metabolites. CuSO4-promoted oxidative stress was also shown to remarkably stimulate biosynthesis of all penitrems. In contrast, the salt stress, caused by the elevated concentrations of NaCl, showed an inhibitory effect on the penitrem biosynthesis. Finally, cheese model medium elicited exceptionally high production of all members of the penitrems family. Obtained results give insides into the biosynthesis of toxicologically relevant penitrems A-F under different environmental factors and can be utilized to prevent food contamination.

Keywords: Penicillium crustosum; cheese model medium; flash chromatography; liquid chromatography; micro-scale extraction; penitrem; stress factor.

Figure 1

Structures of penitrems and their analogs.

Figure 2

Effect of: media (a); temperature (b); pH (c); and light (d) on Pen A (Penitrem A) production by P. crustosum after 7, 14 and 21 days of incubation in mg/g dry mass, * no penitrems were detected. Results are mean of five replicates ± standard deviation and given in mg mycotoxin related to g dry mass (mg/g).

Figure 3

HPLC-UV-DAD (high performance liquid chromatography with ultraviolet diode array detection) chromatograms at 236 nm of the isolated penitrems A–F (10 μg/mL) (a); and of P. crustosum crude extract cultured on modified CDA (Czapek–Dox agar) supplemented with 15 g/L of glycerol (b).

Figure 3

HPLC-UV-DAD (high performance liquid chromatography with ultraviolet diode array detection) chromatograms at 236 nm of the isolated penitrems A–F (10 μg/mL) (a); and of P. crustosum crude extract cultured on modified CDA (Czapek–Dox agar) supplemented with 15 g/L of glycerol (b).

Figure 4

Production of penitrems A–F (mg/g dry mass) after 7, 14 and 21 days of incubation by P. crustosum cultured on CDA in the dark at 22 °C (standard conditions). Results are mean of five replicates ± standard deviation and given in mg mycotoxin related to g dry mass (mg/g).

Figure 5

Production of penitrems A–F (mg/g dry mass) after 7, 14 and 21 days by P. crustosum cultured on modified CDA supplemented with different concentrations of glucose: 5 g/L (a); 50 g/L (b); 100 g/L (c); and 250 g/L (d). Results are mean of five replicates ± standard deviation and given in mg mycotoxin related to g dry mass (mg/g).

Figure 6

Production of penitrems A–F (mg/g dry mass) after 7, 14 and 21 days by P. crustosum cultured on modified CDA supplemented with different concentration of tryptophan: 1.02 g/L (5 mM) (a); and 4.09 g/L (20 mM) (b). Results are mean of five replicates ± standard deviation and given in mg mycotoxin related to g dry mass (mg/g).

Figure 7

Production of penitrems A–F (mg/g dry mass) after 7, 14 and 21 days by P. crustosum cultured on modified CDA supplemented with different concentration of: glycerol 15 g/L (a_w 0.992) (a); 70 g/L (a_w 0.986) (b); 200 g/L (a_w 0.971) (c); and 250 g/L (a_w 0.958) (d). Results are mean of five replicates ± standard deviation and given in mg mycotoxin related to g dry mass (mg/g).

Figure 8

Production of penitrems A–F (mg/g dry mass) after 7, 14 and 21 days by P. crustosum cultured on modified CDA supplemented with different concentration of: NaCl 5 g/L (a); 10 g/L (b); 30 g/L (c); and 50 g/L (d). Results are mean of five replicates ± standard deviation and given in mg mycotoxin related to g dry mass (mg/g).

Figure 9

Production of penitrems A–F (mg/g dry mass) after seven days by P. crustosum cultured on modified CDA supplemented with different concentration of H₂O₂ (0.034 g/L–1 mM, 0.1 g/L–3 mM, and 0.2 g/L–6 mM). Results are mean of five replicates ± standard deviation and given in mg mycotoxin related to g dry mass (mg/g).

Figure 10

Production of penitrems A–F (mg/g dry mass) after seven days by P. crustosum cultured on modified CDA supplemented with different concentration of CuSO₄: (a) 10 mg/L; and (b) 30 mg/L. Results are mean of five replicates ± standard deviation and given in mg mycotoxin related to g dry mass (mg/g).

Figure 11

Production of penitrem A (mg/g dry mass) after 14 days by P. crustosum cultured on different food substrates at 22 °C. Results are mean of five replicates ± standard deviation and given in mg mycotoxin related to g dry mass (mg/g).

Figure 12

Production of penitrems A–F (mg/g dry mass) after: 7 (a); and 14 (b) days by P. crustosum cultured on cheese model medium. Results are mean of five replicates ± standard deviation and given in mg mycotoxin related to g dry mass (mg/g).