2.2. Must from dried grapes and fermentation conditions
The must used was obtained from sun-dried grapes of the Pedro
Ximenez variety grown in the MontillaeMoriles Protected Designation of Origin (Cordoba, Spain). The sugar content of the must
was 545 g/L. The must was aseptically centrifuged at 5000 g for
5 min to withdraw part of the microorganisms and solid particles.
The initial pH, 4.3, was lowered to 3.8 by addition of tartaric acid,
and was also supplied with a 100 mg/L concentration of SO2.
Fermentation batches were conducted in 500 mL Erlenmeyer flasks
filled with 400 mL of must each. Those using biocapsules were
performed as follows: before biocapsules were used, they were
easily removed from the production medium using a sterilized
strainer and then were washed twice with sterile distilled water at
4 C and then with fresh must at the same temperature. Next, they
were confined and packed in stainless steel cylinders 62 cm3 in
volume having holes 3 mm in diameter, all under aseptic conditions. The cylinders were hanged on Nylon wires and immersed in
the Erlenmeyer flasks (Fig. 1). This system facilitated easy withdrawal of the immobilized yeasts and their reuse. A control
fermentation batch with free yeast cells was also performed; the
free cells were centrifuged at 5000 g and washed twice with
sterile distilled water at 4 C before their use in fermentation. 500-
mL Erlenmeyer flasks filled with 400 mL of raisin juice were inoculated with 5 106 free viable cells of the yeast per millilitre or
with biocapsules reaching the same concentration of yeast cells.
The number of yeast cells in the biocapsule and fermentation broth
was measured according to García-Martínez et al. (2012).
Once the desired alcohol content (13% v/v) was reached, the
metal cylinders containing the biocapsules were withdrawn from
the fermentation medium and placed in another Erlenmeyer flask
containing fresh raisin must for subsequent reuse. Raisin grape
must and fermentation batches were stopped by adding wine
alcohol up to a final ethanol content of 15% v/v and refrigerating at
2 C. The obtained wines for this study were traditional Pedro
Ximenez wine without fermentation, which was obtained by
fortification of the raisin must with 15% (v/v) wine ethanol (T),
sweet wine from free yeast cells (F) and sweet wines from repeated
batch fermentations with immobilized yeast cells (B1eB5). All
fermentation batches were performed at 24 C in triplicate.