Improvement of non-Saccharomyces yeast dominance during must fermentation by using spontaneous mutants resistant to SO₂, EtOH and high pressure of CO₂

Abstract

AIM: A genetic study of four wine T. delbrueckii strains was done. Spore clones free of possible recessive growth‐retarding alleles with enhanced resistance to winemaking stressing conditions were obtained from these yeasts.

METHODS: The genetic marker of resistance to cycloheximide (cyhR) allows easy monitoring of the new mutants obtained from these yeasts. Identity of the high pressure resistant (HPR) mutants was verified by analysis of cell morphology, killer phenotype, cyhR marker, presence of viral dsRNA, RFLPs of mtDNA, and sequencing of Internal Transcribed Spacer ofribosomal DNA (ITS).

RESULTS: T. Delbrueckii mutants were isolated from some spore clones. Papillae resistant to SO2 were isolated. Subsequently, new spontaneous mutants capable of growing on YEPD plates with 10% ethanol were isolated. Rosé sparkling wine (cava) was made using these mutants. Two mutants, with the best fermentation kinetics and closest to the reference yeast Sc 85R4, were isolated from some bottles with high CO2 pressure and some were selected there after (Td MutHP41 and Td MutHP42). They had better fermentation kinetics and dominance than their parental yeast. Td MutHP41 showed great improvement for industrial base wine fermentation with respect to its parental yeast. Re-isolation and selection procedure to obtain new reinforced HPR mutants from previously selected HPR mutants was not a sound strategy to continue improving the fermentative capability of T. delbrueckii under high CO2 pressure. Continuous shaking during inocula preparation further improved the fermentative capability of T. delbrueckii yeasts.

CONCLUSIONS: Isolation of spontaneous mutants resistant to SO2 and ethanol seems to be a good strategy to slightly improve the fermentative efficiency of T. delbrueckii in must and base wine. The new mutants were genetically stable enough to be considered for industrial production, and their fermentative capability was further improved by continuously supplying oxygen during the conditioning stage before yeast culture inoculation in base wine.