Estrategias de uso de Saccharomyces cerevisiae en fermentaciones aeróbicas stars

Abstract

Wine is a food product with an undeniable traditional character, especially in the Mediterranean area where its production dates back to the first human populations. Spain is currently the world’s leading vineyard, with 13% of the total vine cultivation surface devoted to wine production (2021 data available at www.fev.es/sector-cifras/), which translates into a contribution of 2.2% of gross value added and the maintenance of 2.4% of employment in Spain between direct and indirect jobs (Analistas Financieros Internacionales, 2020). The wine sector is present in all the Spanish regions, being an important income source for rural areas, acting as an accelerator of the territory and helping to solve the challenge of territorial depopulation, thanks to the direct production of wine, the presence of accessory industries (timber, cork industry, vineyards, etc.) or through wine tourism (International Financial Analysts, 2020). In recent years there has been an unprecedented increase of temperatures (NOAA Global Climate Report for 2020), accompanied by changes in the level and distribution of precipitation. This context of climate change is affecting a large number of crops, especially in drier regions such as the Mediterranean area. In the specific case of vines, a typical crop in the region, a clear change has been observed in the ripening pattern of the berries due to these climatic alterations. In recent decades, an increasing gap has been observed between the accumulation of sugars within the berry (technological maturity) and organoleptic maturity due to the accumulation of phenolic and aromatic compounds (Jones and Webb, 2010; Holland and Smith, 2010; Keller, 2010; Mira de Orduña, 2010). Producing a wine with sufficient organoleptic quality to be accepted by the consumer involves delaying the harvest beyond the optimal point of sugar concentration. This increase in sugars results, through the fermentation process itself, in an increase in the concentration of ethanol, and therefore in an increase in the alcohol content of the wine. It is estimated that since 1980 commercial wines have increased their alcohol content by 1% every decade. This pattern of increase has been observed in all types of wines and productive areas (Jones et al., 2005; van Leeuwen and Darriet, 2015), being even more severe in warmer regions, traditionally with higher wine productions (Alston et al., 2011; Godden et al., 2015). The increase in alcohol content is a serious problem for the wine industry from several points of view: High ethanol concentrations can seriously affect the winemaking process by preventing both yeasts (alcoholic fermentation) and lactic acid bacteria (malolactic fermentation) from optimal development (Bisson, 1999; Bruescher et al., 2001; Coulter et al., 2008; Graça da Silveira et al., 2002), resulting in stuck fermentations and/or unpalatable wines. Even if the winemaking process is completed, an excess of alcohol generates a sensory imbalance by increasing the solubility of certain volatile compounds, affecting their perception (Goldner et al., 2009; Pickering et al., 1998; Hartmann et al., 2002; Le Berre et al., 2007; Robinson et al., 2009). In addition, today’s society is highly interested in healthy lifestyle and moderate alcohol consumption, so high alcoholic beverages have a reduced place in the market (Schmidtke et al., 2012; Saliba et al., 2013). There are even public measures for highly alcoholic beverages that penalise their international marketing. These measures could even affect the export/import of wine (de Barros Lopes et al., 2003; Contreras et al., 2014). There are numerous approaches to try to alleviate this problem, from crop management to avoid ripening delays (like leaf removal, soil management, or crop relocation) (Stoll et al., 2010; Whiting, 2010; Schmidtke et al., 2011; Ozturk and Anli, 2014), to wine de-alcoholization through filtration and evaporation systems, that require subsequent wine restructuring (Akyereko et al, 2021; Catarino and Mendes, 2011; Labanda et al., 2009; Gonçalves et al., 2013; Diban et al., 2008; Fedrizzi et al., 2014), through to the removal of part of the sugar content from the must by dilution, filtration or enzymatic treatments (Pickering et al., 1998; 1999). All of them have advantages and disadvantages, being approaches that are difficult to implement at the industrial level, would entail serious cost overruns, or could negatively impact consumer perception. Nevertheless, some of them are valid solutions that are being investigated and optimised for transfer to the industrial sector. In this doctoral thesis, a microbiological approach is presented as a possible solution to the increasing alcohol content of wines. This consists of the modification of the alcoholic fermentation process to reduce the ethanol yield of the yeasts, with the objective of minimising the changes in the final product. It could be approached along two main lines: the modification of the ethanol yield of Saccharomyces cerevisiae and/or the use of alternative yeasts outside the Saccharomyces genus. S. cerevisiae is an organism highly specialised in alcoholic fermentation. It is the main agent of the fermentation process and there are lines specifically adapted to the fermentation of grape must (wine strains) (Liti et al., 2009; Peter et al., 2018). These strains have a high tolerance to the stress factors inherent to the oenological environment, such as high sugar concentrations (high osmotic pressure), low pH, high pressures, the presence of sulphite (SO32-), a wide range of temperatures, and ethanol, which they produce in large quantities. This combination of characteristics means that S. cerevisiae, although initially found in a very low proportion, is capable of very quickly displacing the rest of the microbiota present in greater abundance in the must at the beginning of fermentation. This high specialization in the fermentation process is related to an ethanol yield which is not only higher than that of other species but is also a very homogeneous characteristic throughout the species, as described in Chapter 1. On the other hand, there are a plethora of yeast species with a fermentation profile more suited to the objective of a lower ethanol yield. However, these species, even when they survive the initial phases of fermentation, end up being displaced by S. cerevisiae, precisely because of their lesser adaptation to must fermentation, making it necessary to improve their resistance to such crucial stress factors as the presence of sulphite (SO32-), temperature, or low pH. This doctoral thesis proposes the use of S. cerevisiae to reduce the alcohol content of wine by diverting sugar consumption from ethanol production to CO2 generation through the respiratory capacity of yeasts. Specifically, the aim is to solve a problem inherent to this approach by means of different strategies. It has been shown that although it is possible to reduce ethanol production by favouring respiration through the maintenance of aerobic conditions during fermentation, this same aerobiosis produces, for reasons as yet unknown, an undesirable increase in acetic acid production, which means that this strategy cannot be applied in wine production. Chapter 1 explores the natural diversity of S. cerevisiae in search of strains with lower acetic acid production, identifying possible candidate strains for industrial application, and the aeration process for a selected strain is fine-tuned. Chapter 2 addresses the study of the relationship between the flexibilization of carbon catabolite repression (ability to consume secondary sugars in the presence of glucose) and lower acetic acid production, which serves as the basis for the design of a directed evolution to obtain strains with low acetic acid production in aerobiosis carried out. Finally, Chapter 3 explores the reduction of acetic acid by regulating aerobic conditions, not just by controlling airflow but through oxygen consumption by the alternative yeast Metschnikowia pulcherrima (Chapter 3).