Maceration during

alcoholic fermentation was achieved by punching down fermentation caps three times per day. The residual glucose–fructose concentration was monitored on a daily basis with a balling meter. When residual glucose–fructose levels were approximately 10 g L−1 (sixth day), the wines were hydraulically pressed (2 bar) from grape skins. The pressed wine (4.4 L) including lees was dispensed into 4.5-L glass jars equipped with fermentation airlocks and fermentation was allowed to proceed to dryness (residual sugar ≤1.95 g L−1). Racking entailed that wines from each fermentation were carefully siphoned-off (avoiding lees sediment carryover), sulphited to 40 g mL−1 (free sulphur) and bottled (5 × 750-mL dark green glass bottles). Putative wild-type and transgenic yeast populations from completed wine fermentations were established by plating out www.selleckchem.com/products/pci-32765.html 100 μL of a dilution series onto YEPD plates containing 25 mg L−1 kanamycin sulphate (Roche, Germany) and 30 mg L−1 chloramphenicol (Sigma-Aldrich, MO). After incubation at 30 °C for 2–3 days, colonies representing VX-809 clinical trial putative transgenic yeast strains were randomly selected from plates (25 colonies per replicate sample) and assessed

for their resistance to SM, flocculation ability (HSP30p-FLO5 transformants) or lack of invasiveness (HSP30p-FLO11 transformants). Genomic DNA isolated from 25 colonies per replicate sample, putative wild-type BM45 and VIN13 isolates were S. cerevisiae strain-typed using PCR with primers that are specific for δ sequences (Ness et al., 1993). Isolated genomic DNA from S. cerevisiae BM45, EC1118, NT50, VIN13 and WE372 industrial wine yeast wild-type strains served as controls. The lees component (5 mL aliquots) from individual MycoClean Mycoplasma Removal Kit batch fermentations was washed three times with an equal volume of sterile 0.9% saline and stored at −20 °C for flocculation and sedimentation analysis. The lees was recovered by centrifugation and resuspended in 50 mL 100 mM EDTA by vigorous vortexing. Thereafter, the faster

settling amorphous solid debris was allowed to sediment for 20–30 min and the fraction containing only suspended yeast cells was recovered from just below the meniscus. Microscopic evaluation of cellular fractions determined whether extractions were to be repeated. Filter-sterilized Merlot must [24.4% sugar (glucose and fructose), 6 g L−1 titratable acidity and pH 5.2] was sulphited to 40 mg L−1 was prepared as described above. Yeast precultures in YEPD were prepared and processed as described previously (Govender et al., 2008). The flocculation potentials of wild-type and transgenic yeast strains were assessed in small-scale aerobic shake-flask experiments at 27 °C using 100 mL aliquots of filter-sterilized Merlot must. Small-scale batch alcoholic fermentation of 100 mL aliquots of filter-sterilized Merlot must were performed by the inoculation of preacclimatized yeast cell populations at a density of 2 × 106 cells mL−1.