Isolation and Characterisation of Yeasts with Biotechnological Properties

Abstract

The primary aim of this research was to identify and characterise novel non-Saccharomyces species from a vineyard and assess their potential use as starter cultures, either as an alternative or complement to Saccharomyces cerevisiae. The first step towards this aim was to access the vast potential of non-Saccharomyces yeasts to produce desired oenological characteristics which may be absent in S. cerevisiae with a view towards their application in winemaking. In particular, their ability to produce high levels of aroma compounds (esters, higher alcohols) and secrete enzymes (β-glucosidases, esterases, lipases, proteases) releases aroma compounds from odourless precursors to positively enhance the sensorial profile of wines. The 77 yeast isolates previously isolated from a South Australian vineyard were identified by sequencing the internal transcribed space regions of the 5.8S rRNA gene (ITS1-5.8S-ITS2) region. The isolates consisted of 7 species belonging to 5 genera (Aureobasidium, Kazachstania, Meyerozyma, Wickerhamomyces and Torulaspora). The indigenous isolates were evaluated for oenological properties, specifically ethanol tolerance, enzymatic activities, and hydrogen sulfide production. To improve the overall wine aroma complexity, research has devised various techniques from cultivation and harvesting (of grapes) to vinification. One such method is the use of mixed starter cultures (non-Saccharomyces and S. cerevisiae) to impart distinctive aroma/flavour profiles. Whilst commercial non-Saccharomyces belonging to Metschnikowia pulcherrima, Lachancea thermotolerans and Torulaspora delbrueckii are available as commercial wine starter cultures there is a diverse range of indigenous non-Saccharomyces unexplored. The next step was to conduct fermentation trials with 17 representative strains from each species in chemically defined grape juice media (CDGJM). None of the isolates could complete fermentation (as monocultures) in CDGJM (200 g/L sugar), with T. delbrueckii isolates utilising sugar the fastest, following by Kazachstania aerobia, Kazachstania servazzii and Wickerhamomyces anomalus. Based on their fermentation profile in CDGJM, 7 isolates were selected for use in sequential fermentation with a commercial S. cerevisiae strain in sterile Viognier juice to evaluate their contribution to fermentation kinetics and production of key metabolites, including volatile compounds. These laboratory-scale fermentations showed that species belonging to Kazachstania (K. aerobia and K. servazzii) produced wines with elevated levels of phenylethyl alcohol and isoamyl alcohol, as well as their corresponding acetate esters. The Kazachstania spp. sequential wines also reduced alcohol (ethanol), by ~1% (v/v) compared to the S. cerevisiae control. Because of this interesting result the fermentative efficiency of Kazachstania spp. in non-sterile red musts (Merlot and Shiraz) was evaluated. Three isolates of Kazachstania spp. (2x K. aerobia and 1x K. servazzii) were inoculated in sequential fermentations with S. cerevisiae. In contrast to S. cerevisiae, Kazachstania spp. wines had significantly increased phenylethyl acetate and isoamyl acetate in both Merlot and Shiraz, as well as increased glycerol concentrations and decreased ethanol concentration (Merlot only, not in Shiraz due to the high initial °Brix of Shiraz must). With respect to Shiraz wines, Kazachstania spp. treatments enhanced the wine sensory appeal; the wines were perceived as ‘jammy’, ‘red fruit’ and higher aroma intensity compared to S. cerevisiae control (‘cooked vegetable’, ‘earthy’, ‘forest floor’ and ‘savoury’). In addition to the collection of 77 isolates, 5 isolates belonging to Hanseniaspora uvarum isolated from a Victorian vineyard were included for characterisation. The effects of H. uvarum on the terpene content of white wines were initially evaluated in Viognier, which was overall lower in the sequential wines compared to the S. cerevisiae control. To further validate this phenomenon, tests were conducted in chemically defined grape juice medium spiked with linalool, and two more aromatic varietals (Muscat and Riesling), which included uninoculated treatments as negative controls. The results show that H. uvarum neither increased or reduced the linalool concentrations, except in Riesling which had higher linalool concentration, but still lower compared to the S. cerevisiae control. These findings suggest that the terpene content in white wines could be matrix- and/or temperature-dependent. As Kazachstania spp. consistently produced higher concentrations of acetate esters compared to S. cerevisiae in both white and red wines, their genomic and metabolic features (particularly acetate ester biosynthesis) were investigated. Complete genome sequences of K. aerobia and K. servazzii isolates were obtained at contig level (de novo assembly) based on PacBio sequencing reads. The genome size and GC content was 12.5 Mb and 35.8% for K. aerobia and 12.3 Mb and 34.4% for K. servazzii. Furthermore, comparative analyses were performed with putative orthologous genes involved in acetate ester and higher alcohol formation. Unlike S. cerevisiae, where both alcohol acetyltransferase (AATase) encoding genes ATF1 and ATF2 are present, both K. aerobia and K. servazzii have only one orthologue in their genome. This study will expand the knowledge on the application of non-Saccharomyces yeasts in winemaking, particularly Kazachstania spp. which have demonstrated their potential to be employed as pure or mixed-starter cultures.