AGRIVULTURAL UNIVERSITY OF ATHENS Dept. of Food Science and Human Nutrition Laboratory of Microbiology and Biotechnology of Foods Probabilistic models for the effect of temperature, water activity and sodium metabisulphite concentration on the growth and OTA production boundaries of Aspergillus carbonarius isolated from Greek wine grapes. Kogkaki, E., Natskoulis, P., Kizis, D., Nychas, G.-J.E., Panagou, E.Z. Lab. of Microbiology and Biotechnology of Foods, Dept. of Food Science and Human Nutrition, School of Food, Biotechnology and Development, Agricultural University of Athens, 75 Iera Odos str., Athens, Greece 11855, E-mail: stathispanagou@aua.gr INTRODUCTION Black Aspergilli and in particular Aspergillus carbonarius have a central role in OTA contamination of grapes and grape products (Counil et al., 2005). Climatic factors could result in fungal contamination and increase the risk of mycotoxins in these products. OTA may present several toxicological manifestations, such as teratogenic, mutagenic, carcinogenic, and immunosuppressive effects to susceptible animals and humans, while it was associated with the Balkan Endemic Nephropathy (Richard 2007). The purpose of the present work was to develop a probabilistic modelling approach to determine the growth and OTA production boundaries of an A. carbonarius isolate on a grape medium as a function of water activity, temperature and sodium metabisulphite (NaMBS) concentration as an antifungal agent. MATERIALS AND METHODS A wild strain (Ac-57) of an A. carbonarius isolate from Greek vineyards was selected in this work. The OTA potential of the fungus had been determined on CYA medium and found to be ca. 745 ng OTA/g. Experiments were undertaken on a Grape Juice Medium, consisted of 50% white grape juice (‘Georgas Family’ organic pasteurised grape juice) and 1.5% agar (Pateraki et al., 2005) in distilled water and modified with glycerol at aw levels of 0.88, 0.90, 0.93, and 0.98 (Belli et al., 2004). Sodium metabisulfite (NaMBS) was added after sterilisation resulting in concentrations of 0, 50, 100, and 150 ppm. The final pH of the substrate was 3.5. The medium was poured into 90 mm Petri dishes and the fungal strain was inoculated centrally with 5 μl from a 10-6 spore/ml suspension. The dishes were Incubated at 15, 20, 25, 30, and 35°C. Growth and OTA production responses were evaluated for a period of 28 days. Fungal growth was assessed visually and OTA production was determined by HPLC (Bragulat et al., 2001). Visible growth/no growth and OTA/no OTA production were scored as values of 1 or 0, respectively. Data were fitted to a logistic regression model: Where P is the probability of fungal growth or OTA production (in the range 0-1), βi are coefficients to be estimated, and xi are the quantitative variables under study (water activity of the grape juice medium, temperature, sodium metabisulphite, and time). The model included single and quadratic terms as well as second order interactions of quantitative variables (Tassou et al., 2009). Figure 1: Growth/no growth (a) and OTA/no OTA (b) interface of A. carbonarius Ac-57 after 7 days of incubation on a grape juice medium. Red symbols indicate growth or OTA production. Open symbols indicate no growth or OTA production. Lines indicate different probabilities at 0.1, 0.5, and 0.9. RESULTS No fungal growth and OTA were detected at 15°C and 0.88 aw irrespective of NaMBH concentration. The highest level of NaMBS assayed (150 ppm) was effective to suppress growth and OTA production at all temperatures and aw in the range of 0.88-0.93. A typical example of fungal growth and OTA production is illustrated in Figure 1 after 7 days of incubation at 25°C. It is evident that the fungus can grow at low aw levels, especially at 0 and 50 ppm of NaMBS, however no OTA could be detected at these conditions and the fungus needed high water activity levels (0.93 upwards) to produce the toxin. Taking into account the predicted probability of growth with respect to time, it can be concluded that no growth could be observed at 150 ppm NaMBS with the exception of high aw levels (0.96 and 0.98) (Figure 2). Taking into account the probability of 0.5 as a threshold value for fungal growth, it can be inferred that at 100 ppm NaMBS the time for visible growth was 25, 16 and 4 days at aw 0.88, 0.90, and 0.93, respectively. Decreasing the concentration of the antifungal agent by 50% (i.e., 50 ppm) the time for visible fungal growth was drastically reduced to 8 and 5 days at aw 0.88 and 0.90, respectively. Plots of probability of growth for aw, temperature and NaMBS at 7 days of incubation are shown in Figure 3. It is characteristic that increasing levels of NaMBS allow fungal growth only at high aw levels. At 150 ppm NaMBS and aw 0.96 the fungus could grow between 22-34°C, whereas at aw 0.98 growth can occur from as low as 14°C. Figure 2: Effect of water activity (aw) and NaMBS concentration of the predicted probability of growth of A. carbonarius Ac-57 at 25°C across incubation time. REFERENCES Counil, E., Verger, P., Volatier, J.-L. 2005. Fitness-for-purpose of dietary survey duration: A case-study with the assessment of exposure to ochratoxin A. Food Chem. Toxicol. 44: 499-509. Bellí, N., Marín, S., Sanchis, V., Ramos, A.J. 2004. Influence of water activity and temperature on growth of isolates of Aspergillus section Nigri obtained from grapes. Int. J. Food Microbiol. 96:19-27. Bragulat, M.R., Abarca, M.L., Cabañes, F.J. 2001. An easy screening method for fungi producing ochratoxin A in pure culture. Int. J. Food Microbiol. 71: 139-144. Pateraki, M., Dekanea, A., Mitchell, D., Lydakis, D., Magan, N. 2007. Influence of sulphur dioxide, controlled atmospheres and water availability on in vitro germination, growth and ochratoxin A production by strains of Aspergillus carbonarius isolated from grapes. Postharvest Biol. Technol. 44: 141-149. Tassou, C.C., Natskoulis, P.J., Magan, N., Panagou, E.Z. 2009. Effect of temperature and water activity on growth and ochratoxin A production boundaries of two Aspergillus carbonarius isolates on a simulated grape juice medium. J. Appl. Microbiol. 107: 257-268. Figure 3: Effect of water activity (aw), temperature and NaMBS concentration of the predicted probability of growth of A. carbonarius Ac-57 on a grape juice medium for 7 days of incubation. This work has been supported by the project ‘Design and development of innovative tools for the detection of ochratoxigenic fungi in wine and table grapes – FungalPrognosis_242’ co‐financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) ‐ Research Funding Program: ARISTEIA-I.