The role of Paecilomyces lilacinus (Thom) Samson and other fungal species in biodegradation of ochratoxin A

Abstract

Nine isolates of fungi of genera Aspergillus, Fusarium, Paecilomyces and Penicillium were cultured on the modified Vogel's medium with the addition of crude ochratoxin A (OTA) extract. This crude OTA extract was derived from a natural solid substrate on which Aspergillus ochraceus strain CBS 108.08 was cultivated. OTA was isolated, partially purified, dried by evaporating and dissolved in ethanol (1 mg ml-1), and added to the test medium up to the final concentration of 10 μg ml-1. The presence of OTA residues was determined after 7 and 14 day cultivation of fungi in the test medium at 27±1°C. The Paecilomyces lilacinus isolate (Inf. 2/A), which completely degraded OTA (150 μg) after only seven days, was selected for further studies. Wet sterile rice grains (50 g + 25 ml distilled water) were inoculated with individual isolates of fungi A. ochraceus (strain CBS 108.08) and P. lilacinus (isolate Inf. 2/A), and with their combination. In the case of P. lilacinus monoculture, 0.9 mg of crude OTA was also added into cultivation substrate. Each test was done in three replications. After the four week cultivation of individual and combined fungi at 27±1°C, inoculated rice grains were dried to the constant weight and pulverized. OTA was determined in these samples by the application of standard TLC method for fodder analysis. OTA in the amount of 61.310 μg kg-1 dry matter (DM) was determined only in the samples inoculated with a producer of ochratoxin A (A. ochraceus, strain CBS 108.08). On the other hand, a much smaller amount of OTA (80 μg kg-1 DM) was detected in samples inoculated with combined cultures of A. ochraceus and P. lilacinus isolates. Gained results indicate that P. lilacinus degraded, on average, 99.8% of OTA. After four week cultivation, the same fungal isolate in the samples of wet sterile rice kernels with the addition of 0.9 mg of crude OTA, completely degraded added crude OTA ( lt 8 μg kg-1).

Devet izolata gljiva iz rodova Aspergillus, Fusarium, Paecilomyces i Penicillium gajeno je na modifikovanoj Vogelovoj podlozi sa dodatkom sirovog ekstrakta ohratoksina A (OTA). Sirovi ekstrakt OTA je dobijen iz čvrstog prirodnog supstrata na kojem je gajen soj Aspergillus ochraceus CBS 108.08. Izolovan i delimično prečišćen OTA, uparen do suvog ostatka i rastvoren u etanolu (1 mg ml-1), dodat je u test podlogu do finalne koncentracije 10 μg ml-1. Nakon sedam i 14 dana gajenja kultura gljiva u test podlozi na 27 ± 1°C de terminisano je prisustvo rezidua OTA primenom modifikovane metode Filtenborg-a i sar. (1983). Od devet testiranih izolata za dalja ispitivanja je odabran izolat Paecilomyces lilacinus (Inf. 2/A), koji je već posle sedam dana u potpunosti razgradio inicijalnu količinu OTA (150 μg). U drugom delu eksperimenta vlažno sterilno zrno pirinča (50 g + 25 ml destilovane vode) zasejano je sa pojedinačnim izolatima A. ochraceus (CBS 108.08) i P. lilacinus (Inf. 2-A), kao i kombinacijom oba izolata. U slučaju mono-kulture P. lilacinus u podlogu je dodat i sirovi OTA (0,9 mg). Svaki od testova je urađen u 3 ponavljanja. Nakon četiri nedelje gajenja monokultura i mešanih kul tura gljiva na 27±1°C, inokulisana zrna su osušena do konstantne težine i sa mlevena do finog praha. U ovim uzorcima izvršena je determinacija OTA primenom standardne metode tankoslojne hromatografije za analizu stočne hrane. U uzorcima koji su bili zasejani samo sa producentom OTA (A. ochraceus, soj CBS 108.08) detektovan je OTA u prosečnoj količini od 61.310 μg kg-1 suvog ostatka. U uzorcima koji su bili zasejani kombinovanim kulturama izolata A. ochraceus i P. lilacinus utvrđena je znatno manja prosečna količina OTA (80 μg kg-1). Ovi rezultati ukazuju da je izolat P. lilacinus razgradio prosečno 99,8% OTA prisutnog u podlozi za kultivaciju. U uzorcima vlažnog sterilnog zrna pirinča sa dodatkom 0,9 mg sirovog OTA isti gljivični izolat je posle četiri nedelje kultivacije kompletno biorazgradio dodat sirovi OTA ( lt 8 μg kg-1).