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UNIVERSITAT ROVIRA I VIRGILI YEAST STRESS RESPONSES DURING ACCLIMATION FOR SPARKLING WINE PRODUCTION Anna Borrull Riera Anna Borrull Riera Yeast stress responses to acclimation for sparkling wine production Doctoral Thesis Directed by Dr. Nicolas Rozès and Dra. Montse Poblet Universitat Rovira i Virgili Departament of Biochemistry and Biotechnology Tarragona, 2016 UNIVERSITAT ROVIRA I VIRGILI YEAST STRESS RESPONSES DURING ACCLIMATION FOR SPARKLING WINE PRODUCTION Anna Borrull Riera This doctoral thesis was carried out between 2009-2016 within the Food Biotechnology Microbiology research group (Department of Biochemistry and Biotechnology, Faculty of Oenology) at the University Rovira i Virgili. The doctoral thesis was supervised by Professors Nicolas Rozès and Montse Poblet. The research was financially supported by a Spanish Government project (AGL2010- 22001-C02-02). The doctoral research was support by the Universitat Rovira i Virgili with a doctoral fellowship (2009BRDI/12/13) and then supported by the Fundació Catalana per la Recerca (FCR). The researcher‟s three months stay at Institut für Biochemie a Technische Universität of Graz (Graz, Austria) was funded by an AEE grant from Universitat Rovira i Virgili. Editing of the thesis book was supported by Postgraduate and Doctoral School of the Universitat Rovira i Virgili. UNIVERSITAT ROVIRA I VIRGILI YEAST STRESS RESPONSES DURING ACCLIMATION FOR SPARKLING WINE PRODUCTION Anna Borrull Riera UNIVERSITAT ROVIRA I VIRGILI YEAST STRESS RESPONSES DURING ACCLIMATION FOR SPARKLING WINE PRODUCTION Anna Borrull Riera Al meu pare. UNIVERSITAT ROVIRA I VIRGILI YEAST STRESS RESPONSES DURING ACCLIMATION FOR SPARKLING WINE PRODUCTION Anna Borrull Riera CONTENTS OBJECTIVES 1 CHAPTER I: Research and literature review 3 CHAPTER II: Material and methods 56 Article 1: Lipidome methodology Article 2: Metabolome methodology CHAPTER III: Yeast ethanol stress during fermentation 104 Article 3: Ethanol stress (Screening study) CHAPTER IV: Yeast acclimation for cava production 120 Article 4: Physiological state of acclimated cells Article 5: Transcriptome Article 6: Lipidome Article 7: Metabolome CHAPTER V: General discussion 190 REFERENCES 197 ANNEX: Publications 217 ACKNOWLEDGEMENTS 219 UNIVERSITAT ROVIRA I VIRGILI YEAST STRESS RESPONSES DURING ACCLIMATION FOR SPARKLING WINE PRODUCTION Anna Borrull Riera OBJECTIVES The second fermentation for sparkling wines production, due to an accumulation of stressful factors, requires an extra effort for yeasts to survive and carry on with fermentation. Some stress factors are the same as those found in first fermentation, although with a different intensity: ethanol level, low pH, low nitrogen content, accumulation of toxic sub-products such as medium-chain fatty acids (MCFA) and organic acids. Others are found in the second fermentation: low temperature, low oxygen avaibaility, CO2 overpressure, presence of SO2 and total acidity (Juroszek et al. 1987). Some of these stress factors have a synergic effect between themselves. Progressive acclimation of Saccharomyces cerevisiae is required before being inoculated into the base wine. Acclimation process defined by Tai et al. (2007) permits yeast to activate stress metabolism response to ensure the successful of prise de mousse. The main features of this method were first described by Françot and Geoffroy (1957) and the effects of ethanol (major stress factor to acclimate to) on yeast during its acclimation were analysed by Juroszek et al. (1987). Some variations of the yeast acclimation process and its subsequent use, known as the Champenoise method, have mainly been described by Valade and Moulin (1983), and Laurent and Valade (1994, 1998, 2007). Although winemakers apply procedures in order to adapt yeast, there is always a lag phase after inoculation followed by a short cell proliferation, indicating that the cells are still not completely adapted. However, without this acclimation process, cells are highly susceptible to death. The effect of ethanol on yeast has been widely studied (Aguilera et al. 2006; Bauer and Pretorius 2000). It has been reported that the alteration of membrane fluidity is one of the key factors in terms of their tolerance (D'Amore and Stewart 1987; Alexandre et al. 1994; You et al. 2003). However, yeast response to stress during fermentation, secondary fermentation or even acclimation process, remains not deeply studied due to the multifactorial effect of many stress factors. Within this framework, the working hypothesis of this thesis is: 1 UNIVERSITAT ROVIRA I VIRGILI YEAST STRESS RESPONSES DURING ACCLIMATION FOR SPARKLING WINE PRODUCTION Anna Borrull Riera The characterisation of yeasts during acclimation to ethanol and other stress, can improve second fementation success. In order to validate this hypothesis the following objectives were attained: 1. Testing ethanol effect on certain commercial wine yeast strains in a defined growth media at low pH (3.5) in the presence of weak acids and classify them in function of their ethanol tolerance. Then, evaluate the yeast growth parameters in the presence of certain factors can affect yeast viability (pH, presence of glycerol, cysteine and MCFA). The yeast‟s behavioural response to these factors should enable an improvement in the acclimation of commercial wine yeasts to ethanol in order to achieve the secondary fermentation properly for sparkling winemaking. Results are reported and discussed in Chapter III. 2. Physiological study of yeast cells during acclimation to relate it with to their performance during the secondary fermentation. To this end, the effect of aeration and external sugar content during the cell acclimation process on certain physiological parameters was analysed. These parameters were in relation to the carbohydrate reserve (glycogen and trehalose), the activity of vacuoles, Reactive Oxygen Species (ROS) intracellular accumulation and the lipid metabolism, trying to correlate the achievement of secondary fermentation and some physiological parameters analysed during the cell acclimation process. Results are reported and discussed in Chapter IV. 3. The characterisation of cells during acclimation process by the way of omics studies such as transcriptomic, lipidomic and metabolomic were executed to permit us to specify the yeast behavior in these conditions. Results are reported and discussed in Chapter IV. 4. Improve and design new methods for the omics analysis by optimising (i) a GC-MS method to cover major yeast lipids in a single run by analysing a lipid extract and (ii) an extraction protocol to get the major intracellular metabolites from cell yeasts in a single run by using a GC-MS method. Results are reported and discussed in Chapter II. 2 UNIVERSITAT ROVIRA I VIRGILI YEAST STRESS RESPONSES DURING ACCLIMATION FOR SPARKLING WINE PRODUCTION Anna Borrull Riera Chapter I CHAPTER I Research and literature review 1. A brief history of sparkling wine production 1.1. The history of sparkling wine 1.2. Sparkling wine production 1.2.1. Description and classification 1.2.2. The traditional method 1.3. Sparkling wine market 1.3.1. The producers 1.3.2. The consumers 1.3.3. The international market 2. Yeast stress responses 2.1. Secondary fermentation 2.1.1. Base wine composition 2.1.1.1. Ethanol 2.1.1.2. Low nutrient content 2.1.1.3.