Biologia 63/4: 542—547, 2008 Section Cellular and Molecular Biology DOI: 10.2478/s11756-008-0083-2 Concentration of ergosterol in small-grained naturally contaminated and inoculated cereals Juliusz Perkowski, Maciej Bu´ sko, Kinga Stuper, Marian Kostecki, Anna Matysiak & Lidia Szwajkowska-Michalek Department of Chemistry, August Cieszkowski Agricultural University of Pozna´ n, ul. Wojska Polskiego 75, 60–625 Pozna´ n, Poland; e-mail: julperk@au.poznan.pl Abstract: Ergosterol (ERG) is a major sterol constituent of most fungi. Its concentration is negligible in higher plants, but can be used as a chemical marker of the presence of fungal contaminations. In this study, ERG concentration was assessed in randomly collected samples of naturally contaminated grain (wheat, barley and oat) and in samples of grain (wheat, barley, triticale and oat) harvested after inoculation of heads with conidia of different Fusarium species. Wheat samples were analysed at three stages of grain development. The lowest ERG concentration was found in non-inoculated samples at the first stage of grain development. This concentration was increasing with grain ripening. In naturally contaminated samples collected after harvest, ERG concentration was lower in wheat than in barley and oat. ERG concentrations in inoculated samples varied significantly, but were always significantly higher than in naturally contaminated samples. In the above cereal samples it was much lower than the levels assayed in laboratory cultures inoculated with fungi from genus Fusarium. The content of ERG was also analyzed in milling products of small-grained cereals and other foodstuffs, where a considerable variation was observed. The lowest ERG amounts were assayed in flours with a high degree of purification, while the highest ones in case of flours and products with a low purification rate. The results indicate the potential application of HPLC combined with microwave-assisted extraction both when assaying samples with low ERG concentrations (naturally contaminated) and those characterized with high contents of fungal biomass (strongly infected, artificially inoculated). It also facilitates analyses of fungal biomass in technological processes, where results may be expected to vary considerably. Key words: ergosterol; natural contamination; inoculation with Fusarium spp.; HPLC analysis. Abbreviations: ACC, average accuracy; CV, coefficient of deviation; DON, deoxynivalenol; ERG, ergosterol; MAE, microwave-assisted extraction; RSD, relative standard deviation; SD, standard deviation. Introduction Fungal contamination of cereals results in considerable economic losses, because of direct damage to crops, dis- coloration, off-odours, reduced yields and loss of nutri- tive value. Ergosterol (ERG; 22E-Ergosta-5,7,22–trien- 3β-ol; CAS No.: 57-87-4) is a sterol occurring in fungi, bacteria, algae and plants (Anonymous 2000). It was found to be a major sterol in many fungal genera, i.e. Fusarium, Alternaria, Cladosporium, Mucor, Rhizopus, Aspergillus, Penicillium and Paecilomyces (M¨ uller & Schwadorf 1990a). Most of the above-mentioned fungi can be isolated from grain and other parts of cere- als. While some fungi constitute the natural microflora of kernels, others can cause mould invasion and toxic contamination of grain. This is associated with my- cotoxin production during growth and crop storage (under European climatic conditions most commonly ochratoxin A and Fusarium toxins), and the harmful effects are observed in both humans and animals (Bot- talico 1999; Perkowski 1999; Che lkowski et al. 2001; Šrobárová & Pavlová 2001). The concentrations of these toxins are usually significantly correlated with ERG content of contaminated grain. For analyzed wheat ker- nels Abramson et al. (1998) reported values of the cor- relation coefficient between ERG and deoxynivalenol (DON) to be as high as 0.83. In turn, Wi´ sniewska & Bu´ sko (2005) reported the correlation coefficient to be 0.91 at significance level P =0.01. Lower values of this coefficient, statistically significant at P =0.01, were given for rye grain in a study by Miedaner & Perkowski (1996). Considerable variation in the correlation be- tween ERG and DON contents in samples of wheat grain and chaff inoculated with F. culmorum was pre- sented by Snijders & Krechting (1992). For that rea- son, the development of fast and reliable methods of mould contamination detection is very important in ce- real production and processing (Snijders & Krechting 1992; Perkowski & Miedaner 1994; Reid et al. 1999). ERG is regarded as the principal sterol of fungi, with the exception of certain Phycomycetes and rust fungi (Gourama & Bullerman 1995) and plays an im- portant role in cell membranes. Basic methods used to determine amounts of fun- c 2008 Institute of Molecular Biology, Slovak Academy of Sciences