Yeasts for low (and high) alcohol

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1 Yeasts for low (and high) alcohol Ana Hranilovic ASVO Adelaide Seminar ARC Training Centre for Innovative Wine Production adelaide.edu.au/tc-iwp/

2 Earlier, shorter, hotter vintages are stressful for winemakers

3 and for the yeast

4 Stress Osmotic stress Ethanol toxicity Oxygen depletion Nutrient limitation Low ph Temperature shock Toxins and inhibitors

5 Requirements Stress Sugar depletion Timely fermentation Aromatic enhancement Ethanol management Osmotic stress Ethanol toxicity Oxygen depletion Nutrient limitation Low ph Temperature shock Toxins and inhibitors

6 High alcohol in wine - mitigation strategies Lower sugar accumulation Fermentation management Low ethanol yielding yeast green harvest vineyard management partial fermentation Sugar stripping Post-fermentative dealcoholisation

7 Microbiological tools GM S. cerevisiae strains various modifications to divert sugars away from ethanol production glycerol as the most carbon promising sink (Kutyna et al. 2010, Tilloy et al. 2015) not permitted in current legislative settings Non-GM S. cerevisiae strains limited variability Comparison of 56 commercial wine yeasts Min % ABV Max % ABV Palacios et al. (2007) Adapted from Varela et al. (2008)

8 You can t teach an old dog new tricks. Luckily, dogs don t carry out the fermentation!

9 Evolving a non-gm low alcohol S. cerevisiae Directed evolution long-term cultivation in a selective environment: - to induce mutations - and generate superior phenotypes Tilloy et al (2014)

10 Lower ethanol non-gm wine yeast evolution of EC1118 in highsalt medium (KCl) followed by crossing of isolates Analytical parameter Ethanol production (% ABV) Parental strain Evolved strain increase in glycerol unrelated to acetic acid production Grams glucose for 1% ethanol Glycerol (g/l) Adapted from Tilloy et al (2014)

11 How about training puppies? aka non-saccharomyces yeasts

12 Non-Saccharomyces yeast > 40 yeast species indigenous to wine-related environment grapes and wine winery surfaces harvesting and other equipment originally regarded as spoilage yeasts re-evaluated as legitimate contributors to wine quality source of natural variation for oenologically-relevant traits

13 Dichotomy... spontaneous fermentation non-saccharomyces at the initial stages overtaken by Saccharomyces population composition dynamics variable and unpredictable limited industrial applicability inoculated fermentation suppression of indigenous yeasts with Saccharomyces inoculum reliable and predictable outcome potentially inferior quality

14 ...becomes a trichotomy controlled multistarter fermentation: sequential or simultaneous inoculation 1. non-saccharomyces - contribute to the chemical and sensory profile 2. S. cerevisiae - completes the fermentation = 4 non-saccharomyces genera; 7 products >200 Saccharomyces strains

15 McLaren Vale 2015 Shiraz vintage trial Assessment of commercially available non-saccharomyces inocula: earlier harvest - overproduction of aroma and flavour-active compounds to boost wine quality commercial ripeness - performance of yeasts

16 McLaren Vale 2015 Shiraz vintage trial 12 kg Shiraz fermentation (TWE) standardised cap management and skin contact stabilisation and aging chemical and sensory analysis

17 Sensory analysis results - earlier harvested fruit and both PDM controls PCA Biplot Dimension 2 (25.7%) PDM Wild Fruit Confectionary Acidic Flavia sweetness Alpha Green Red fruit Prelude Floral Red fruit Bitter 0 Biodiva Flavour Intensity Concerto Melody Spice Surface smoothness Palate fullness Astringency Aroma Intensity -1.6 Hotness PDM Dimension 1 (43.9%)

18 Fermentation performance Fermentation duration: 8 12 days Ethanol concentrations: 13.48% Melody 14.06% PDM Ethanol (% ABV) PDM inoculation 12 Melody Alpha BIodiva Concerto Prelude Flavia Wild ferment PDM 11 Glycerol (g/l) Biodiva Alpha Prelude PDM Melody Concerto Wild ferment Flavia

19 Performance of a commercial non-saccharomyces ADWY - sum up Potential to increase the appeal of wines made from earlier harvested fruit complimentary, rather than alternative, approach in wine alcohol management the pursuit of low-ethanol yielding non-saccharomyces

20 Low ethanol non-saccharomyces highlights! Sequential inoculation of M. pulcherrima AWRI 1149 and S. cerevisiae 1.6% and 0.9% less ethanol (Contreras et al 2014) Important factors: species interactions: M. pulcherrima and S. uvarum synergistic effect inoculation regime Ethanol (% ABV) S. cerevisiae M. pulcherrima AWRI 1149 Ethanol reduction (% ABV) Inoculation rate (x10 6 ) Adapted from Contreras et al (2014) Adapted from Contreras et al (2015)

21 Still a lot of diversity to uncover characterisation and selection of isolates in 100 ml synthetic grape juice fermentations sequential inoculations in grape juice with promising strains

22 and to generate! Directed evolution of a wine non-saccharomyces: T. delbrueckii of positive oenological characteristics and lower ethanol yield Robust strain capable of completing the fermentation whilst retaining low ethanol yield Continuous fermentation in grape juice(s) with increasing ethanol concentration

23 Concluding remarks Robust Saccharomyces for high sugar fermentations available GM strains on-hold in laboratories Non-GM improved strains arising Large diversity of non-saccharomyces yet to be fully explored

24 Acknowledgements Supervisors Vladimir Jiranek and Paul Grbin (University of Adelaide) Tertius van der Westhuizen (Laffort Australia) Independent advisor Vanessa Stockdale (Treasury Wine Estates) Colleagues Sijing Li Simon Dillon and Yalumba winery Nick van Holst Wine Microbiology and Biotechnology Lab ARC Training Centre for Innovative Wine Production Funding bodies ARC Training Centre for Innovative Wine Production Wine Australia

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