Characterization of non-Saccharomyces yeast and its interaction with Saccharomyces cerevisiae with investigation of fermentation kinetics and aromatic composition

Abstract

There is growing evidence that non-Saccharomyces yeasts can be utilized to enhance wine aroma although little research has been done on most non-Saccharomyces species. This study was designed to genetically and phenotypically characterize two local South African non-Saccharomyces species, Kazachstania aerobia and Wickerhamomyces anomalus, in addition to determining their fermentation potential and volatile profiles in synthetic grape must. Genetic differences between isolates were investigated using the RAPD method and phenotypic heterogeneity was determined using plate spotting. Isolates were assessed for heat, alcohol, saline, osmotic and oxidative stress tolerance for phenotypic strain characterization. Eight K. aerobia and thirteen W. anomalus isolates were used to ferment synthetic grape must. After characterization three K. aerobia strains and two W. anomalus strains were then selected for the co-culture fermentations with S. cerevisiae VIN13 and EC1118. Fermentations were done by inoculating yeast simultaneously as well as sequentially, 48 hours apart. Single culture fermentations were used as controls. Aroma compounds in the synthetic wine were quantified using GC-FID. RAPD analysis classified W. anomalus isolates into four distinct strains in accordance to place of origin. Phenotypic variations were also evident in the proposed strains’ resistance to oxidative, saline and osmotic stresses compared to VIN13. Interestingly, there were phenotypic differences observed within the same strain groupings. The K. aerobia isolates showed no marked genetic differences, but with slight variations in stress response. Overall, the CBS strain had a higher growth performance than the other strains with K. aerobia Y965 showing the least growth. In co-inoculation experiments of VIN13 and K. aerobia, the latter persisted until day 9 when VIN13 was introduced on day 2 and until day 7 when VIN13 was introduced on day 0 regardless of isolate’s phenotype. When fermenting with EC1118, W. anomalus had higher cell densities compared to when fermented with VIN13. In sequential fermentations W. anomalus survived until day 9 (when fermenting with strain Y934-C) and day 7 (when fermenting with strain LO632). When inoculating simultaneously with both strains of S. cerevisiae, W. anomalus was detected in the must until day five. Kazachstania aerobia and W. anomalus gives a unique aroma profile to wines. Although as single cultures these yeast do not ferment wines to dryness, they are capable of conferring favourable wine aroma when in association S. cerevisiae strains with no risk of sluggish fermentation. Inoculating S. cerevisiae sequentially to the non-Saccharomyces yeast allows sufficient time for the non-Saccharomyces to impart valuable aroma compounds. This study provides a basis for further work on wine quality improvement through exploitation of non-Saccharomyces yeasts.