Livro das Actas - advid

nitrogen broth (6.7 g/L YNB with 10% ethanol, adjusted to pH 3.50 ± 0.01 and sterilized
by membrane filtration (0.22 µm pore size), incubated at 25 ºC with orbital shaking (120
rpm). Experimental wine was obtained by blending several commercial red wines followed
by filtration through membranes of 0.22 μm pore size. Incubation was carried out
at 25ºC and cellular culturability was measured by plating onto GYP agar.
2.3. Chemical analysis
The 4-ethylphenol production was measured according to a protocol described by
Bertrand (1981). A GC-FID (Varian CP-3800) series with a capillary column from FactorFour
(ID, 0.25 mm; length 15.0 m; film, 0.25 µm) was used. The concentrations of
sugars, acids and alcohols were assessed by HPLC using a flow rate of 0.6 mL/min of
an aqueous solution of 2.5 mM H 2 SO 4 (mobile phase). The separation was performed
in a size-exclusion and ion-exchange column (8.0 x 300 mm, SH1011, Shodex) at 60
ºC. The quantification and integration was done using refractive index detector (Waters
2410) and chromatography data software Empower-2, respectively.
2.4. Experimental design and statistical analysis
The influence of three factors viz., sugar, ethanol and sulfur was studied using a central
composite rotatable design (CCRD). The results of the 17 treatments were analyzed
using the software “Statistica”, version 7, from Statsoft, Tulsa, USA. The linear (L)
and quadratic (Q) effects of these factors, as well as their linear interactions, were calculated
and their significance was evaluated by analysis of variance. A four-dimensional
surface, described by a first- or a second-order polynomial equation was fitted to each
set of experimental data points. First- and second-order coefficients were generated by
regression analysis. The fit of the models was evaluated by the determination coefficients
(R 2 ) and adjusted R 2 (R adj 2 ) (WEISBERG, 1985).
3. RESULTS AND DISCUSSION
Table-1 shows the polynomial equations of the surface responses fitted to experimental
data points. The significant effects (P