Mycotoxins are toxic secondary metabolites produced after growth of fungal species of different genera. This mycotoxin production can be found in different substrata, including food and feed. Nowadays there is an increasing interest in the toxigenic properties of the strains isolated from food, feed and raw materials, and in the factors that can avoid this production. Species of the genus Penicillium, together with Aspergillus and Fusarium spp., are the majority in these substrata and also the main producers of mycotoxins. Ochratoxin A is one of the more studied mycotoxins currently, having a recent legislation in the European Union, mainly due to its high toxicity and wide distribution. Its production is associated with different Aspergillus spp., and uniquely to one species of the genus Penicillium: Penicillium verrucosum. This species is principally isolated from cereals and have a wider distribution in cold climate countries.
In the present thesis, it has been evaluated the presence of Penicillium spp. in 178 samples of feed and cereal destined to animal consumption. A total of 152 strains belonging to 34 different species of the genus were isolated, dominating the species of the subgenus Penicillium. The species Penicillium aurantiogriseum has been the only one isolated from every kind of substrata tested. Penicillium verrucosum, the ochratoxin A producer species, has been isolated only from cereals, and mainly from barley.
A series of physiologic (in the culture media CREA, CSN, YES, NSA, Raulin-Thom and in a liquid culture medium with urea) and biochemical (indole) characteristics, that have been proposed during the last years as taxonomic tools for Penicillium spp., have been evaluated in a total of 298 strains. The media CREA and CSN and the biochemical test for detecting indole metabolites have obtained good results for species distinction in the genus Penicillium. In the culture medium YES, the species P. verrucosum have a violet brown colour in the reverse of the colony, but just by around a 50% of the strains. This colour seems to appear more commonly in fresh cultures than in strains subjected to numerous subcultures.
The capacity of the 298 strains of Penicillium spp. to produce different mycotoxins (aflatoxins, citrinin, sterigmatocystin, ochratoxin A, zearalenone, penicillic acid and penitrem A) was also evaluated. A total of 119 strains had the ability of synthesise one or more of these mycotoxins (except for aflatoxins, sterigmatocystin and zearalenone). A percentage of the 58% of strains of P. aurantiogriseum produced penicillic acid, and the 100% of strains of P. citrinum and P. crustosum synthesised citrinin and penitrem A, respectively. An 85% of strains of P. verrucosum were producers of ochratoxin A, in different concentracions, and around the 50% of them produced the mycotoxin citrinin.
The study by the technique RAPD of the genomic DNA of P. verrucosum allowed the distinction in to groups, A and B, between the strains of the species analysed, corresponding with the two recently proposed species, P. verrucosum and P. nordicum. The study of the sequence of the fragment ITS 1-5.8S-ITS 2 of the DNA codifying for the ribosomal DNA was very homogeneous between the strains of P. verrucosum assayed.
The production of the toxins ochratoxin A and citrinin by P. verrucosum was evaluated under different environmental conditions, obtaining the highest concentrations at values of water activity of 0,94 and 0,96, at temperatures between 15 and 25ºC, at a range of pH between 6 and 10 and with saccharose as carbon source (with higher concentrations of ochratoxin A than in media with fructose or glucose, and all those three media with higher concentrations of the toxins than media with wheat, corn, rice and potato starches).
