Fungal isolate and growth medium
This study was carried out on two ochratoxigenic isolates of A. carbonarius from wine grapes in Greece. The isolates were deposited in the mycological collection of the Faculty of Biology of the University of Athens as A. carbonarius ATHUM 5659 and A. carbonarius ATHUM 5660. Both strains were initially tested for OTA production capability on CYA medium, using the method developed by Bragulat et al. (2001), showing mycotoxin potential >8 μg g−1 substrate. A synthetic nutrient medium (SNM), similar to grape composition between véraison and ripeness was used in this study, with the following composition: d(+) glucose, 70 g; d(−) fructose, 30 g; l(−) tartaric acid, 7 g; l(−) malic acid, 10 g; (NH4)2HPO4, 0·67 g; KH2PO4, 0·67 g; MgSO4·7H2O, 1·5 g; NaCl, 0·15 g; CuCl2, 0·0015 g; FeSO4·7H2O, 0·021 g; ZnSO4·7H2O, 0·0075 g; (+)Catechin hydrate, 0·05 g; agar, 25 g; distilled water, c. 1000 ml. The pH of the medium was adjusted to 3·5 with KOH (2 mol l−1). The aw of the unmodified medium was 0·980, measured by a Novasina Thermoconstander RTD 33 (Novasina AG, Zürich, Switzerland) water activity meter at 20°C, and it was used as the control treatment. The water activity of the SNM growth medium was modified to 0·850, 0·900, 0·930 and 0·960 aw by adding different amounts of glycerol (Mitchell et al. 2004).
Inoculation and incubation conditions
Fungi were grown on SNM medium for 10 days at 25°C to obtain sporulating cultures. Spore suspensions were obtained by flooding the plates with 15 ml sterile phosphate buffer solution (pH 7·0) containing 0·1% of a wetting agent (Tween 80; Merck, Darmstadt, Germany) and gently scraping the surface of the medium with a sterile spatula. After filtering through sterile medical tissue (Aseptica, Athens, Greece), the final concentration of spores was assessed by a Neubauer counting chamber (Brand, Wertheim, Germany) and adjusted to 106 spores per ml. A 5 μl volume of spore suspension was inoculated in the centre of 90 mm Petri dishes containing 20 ml solidified SNM growth medium. The inoculated plates were sealed with parafilm, wrapped in polyethylene bags to avoid desiccation and incubated at seven different temperatures from 10 to 40°C (at 5°C intervals). The effect of temperature and water activity on fungal growth was examined by means of a full factorial design. For each strain, aw/temperature combinations were carried out in triplicate and the whole experiment was repeated twice (n = 6).
Fungal growth measurement and model development
Fungal growth was established by diametric measurements (expressed in mm) at right angles to each other on a daily basis. The mean value of the two diameters was used in the modelling. Measurements were carried out for a maximum of 50 days or until Petri dishes were completely colonized. A standard two-step approach was followed to develop a model for the influence of temperature and aw on fungal growth. First, estimates of the maximum specific colony growth rates (μmax) were obtained by applying Baranyi’s primary model (Baranyi et al. 1993; Baranyi and Roberts 1994). An explicit version of the model is the following:
where t is time, y0 is the initial colony diameter, ymax is the maximal colony diameter, μmax is the maximum specific colony growth rate (mm day−1), m is a curvature parameter and A(t) is a delayed time variable (lag phase).
The average estimates of μmax were then fitted to secondary models to describe the single and combined effects of temperature and water activity on fungal growth.
A quadratic response surface model was the first model used. The following transformation of water activity was applied, as introduced by Gibson et al. (1994):
Therefore, the quadratic expression of the natural logarithm of maximum colony growth rate had the following form:
where a0…a5 are design parameters estimated by nonlinear regression. The natural logarithm transformation was introduced to stabilize the variance of the fitted values for growth rate (Gibson and Hocking 1997).
The extended combined model proposed by Parra and Magan (2004), based on the Gibson-type aw dependence [eqn (2)] and the Ratkowsky-type temperature dependence on growth rate, was the second modelling approach to study the effect of temperature and aw on the growth of A. carbonarius. The model has the general form:
where μmax is the maximum specific growth rate (mm day−1) and a0…a4 are regression coefficients.
The model of Miles et al. (1997) was the third approach followed to study the effect of the entire biokinetic range of temperatures and aw levels on the growth of the fungus. The model is based on the following equation:
where b, c and d are coefficients to be fitted and Tmin, Tmax, aw min, aw max are the minimum and maximum values of temperature and water activity, respectively, beyond which growth is not possible.
The linear Arrhenius–Davey equation (Davey 1989) was the forth model tested, based on the following equation:
where T is absolute temperature (°K), aw is water activity and a0…a4 are coefficients to be determined.
Finally, the Rosso equation (Rosso et al. 1995; Rosso and Robinson 2001) for the effect of temperature and water activity on fungal growth was selected:
The terms Tmin, Tmax, aw min, aw max correspond to the values of temperature and water activity, respectively, below and above which no growth occurs. Additionally, Topt and aw opt are the values of temperature and water activity at which μmax is equal to its optimal value (μopt).
The in-house programme DMFit (Institute of Food Research, Norwich, UK) was used to fit the growth curves and estimate the maximum specific colony growth rate (μmax). Non-linear regression was carried out using the Quasi-Newton algorithm of the NLIN procedure of Statistica release 6.0 (Statsoft Inc., Tulsa, OK, USA) to fit the secondary growth models. The indices used for statistical comparison of models were the regression coefficient (R2) and the root mean squared error (RMSE).