New Trends in Sparkling Wine Production: Yeast Rational Selection

NEW TRENDS IN SPARKLING WINE PRODUCTION: YEAST RATIONAL SELECTION

11

Paola Di Gianvito, Giuseppe Arfelli, Giovanna Suzzi, Rosanna Tofalo Faculty of BioScience and Technology for Food, Agriculture and Environment, University of Teramo, Teramo, Italy

11.1 Introduction Sparkling wine is an enjoyable food product that provides pleasure to the consumer, for this reason it is the beverage chosen to celebrate “certain social occasions” (Fountain and Fish, 2010). As happened with the origin of wine, serendipity presumably played a part in the genesis of sparkling wine, but its origin nevertheless still remains unclear (Liger-Belair, 2017). It was already produced from Roman times with the name of “vinum titillum,” but the steps of the process were studied only from the 17th century. In particular, the monk Dom Perignon wrote a book entitled “The art of tending vineyards and the wines of Champagne” was explained the rules to produce this special wine. Sparkling wine diffusion grew during the time and its market has expanded in the recent years, boosted by a high global consumer demand (Garofalo et al., 2016). In fact, because of its high added value, the economic impact of sparkling wine is very important, even if its production is lower compared to that of still wines (MartínezLapuente et al., 2016), only about the 6.6% of world wine production (World Organization of Vine and Wine OIV, 2014). The main appreciated characteristic of this luxury wine is its effervescence (from the Latin fervere: to boil). In fact, after the opening of the bottle, several ascending bubbles are released from the wine due to the difference of pressure between bottle and room (Liger-Belair, 2017) giving rise to the perlage. These bubbles chains are due to the release of carbon dioxide (CO2). The supersaturation of the wine with this gas is also responsible for the very characteristic tingling sensation in mouth. According to European Union terminology (Council Regulation EC 479/2008, 2008), the term “sparkling wine” can be used Alcoholic Beverages. https://doi.org/10.1016/B978-0-12-815269-0.00011-8 © 2019 Elsevier Inc. All rights reserved.

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only for wines whose yeasts produce CO2 during a second alcoholic fermentation or “prise de mousse.” As reported in this regulation, sparkling wines are divided in three different categories: − sparkling wine (CO2 overpressure >3 bar at 20°C and alcoholic strength >9.5%); − quality sparkling wine (CO2 overpressure >3.5 bar at 20°C and alcoholic strength >10%); and − aromatic quality sparkling wine (CO2 overpressure >3 bar at 20°C and alcoholic strength >10%; the base wine is obtained by grapes derived from specific varieties). White, red, or rosè, generally sparkling wines can contain different amount of sugars (Commission Regulation EC 607/2009, 2009) and for this reason they can be served as an aperitif or used to drink with meals or desserts (Fig. 11.1). The main sparkling wines producers are France, Italy, Spain, Germany, and Russia (OIV, 2014). These wines have different

Fig. 11.1  Sparkling wines classification on the basis of the sugar content (Commission Regulation EC 607/2009—Appendix XIV).

Chapter 11  New Trends in Sparkling Wine Production: Yeast Rational Selection   349

c­haracteristics; in fact, several factors determine the final quality. Among them in the winemaking technology, grape variety (PozoBayón et al., 2010), the clone used (Jones et al., 2014), environmental factors (soil morphology, agro-pedological characteristics, climate and viticultural practices, usually described with the French term “terroir”) (Welke et  al., 2014; Caliari et  al., 2014; Jones et  al., 2014; Alessandrini et al., 2017), bentonite addition (García et al., 2009), CO2 amount (Cilindre et al., 2014), harvest and base wine production, the quality of the base wine (Vigentini et al., 2017), the extent to which it is fined and filtered, the yeast strain used (Bozdoğan and Canbaş, 2012; Benucci et al., 2016; Vigentini et al., 2017) ethanol, acidity (MartinezRodriguez et al., 2002; Bozdoğan and Canbaş, 2012), and the duration of aging on lees (Kemp et al., 2015) were studied. The production of a sparkling wine can take from a few weeks to several years, which often justifies a difference in added value for the consumer. In fact, sparkling wine maturation is a slow process regulated by national legislation, thus it takes from a minimum of nine months for the “Cava” (Spain) to 12 months for “Talento” (Italy) or “Champagne” (France) wines (EC Regulation No. 606/2009, 2009). Table 11.1 shows the main sparkling wines produced all over the world

11.2  Sparkling Wine Production The production method is one of the most important factors that determine the style and quality of white and rose´ sparkling wines (Culbert et al., 2017). It is possible to distinguish seven methods, but the main used technologies are three: (1) traditional, (2) MartinottiCharmat, and (3) transfer (Fig. 11.2). When the second alcoholic fermentation occurs in the bottle that is ultimately sold to consumers, the process is variously termed. In particular, the name Champenoise can be used only for sparkling wines produced in the Champagne region (Commission Regulation EC 3309/85, 1985). In other parts of the world, it is called classic method (Talento wines and Spumanti Metodo Classico, Italy), traditional method (Cava, Spain), bottle-fermented, méthode Cap Classique in South Africa, and so on. This technology is the most appreciated method because obtained sparkling wines show the highest volatile compounds concentration (Caliari et al., 2015) and complexity. Traditional method was the only used process until the 1916 when Eugene Charmat realized the idea of Federico Martinotti of a system for large quantities of sparkling wine production. In this method known as Martinotti-Charmat or simply Charmat, secondary fermentation is performed in pressurized tanks equipped with stirring mechanisms that mix the yeast cells into the base wine. Simpler and cheaper than the traditional method, it offers the advantage of preserving varietal

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Table 11.1  Summary of Main Sparkling Wines Produced, Categorized by Their Country of Origin and Varieties Used Country

Name

Grape Cultivar

Method

France

Champagne

Chardonnay blanc, Pinot noir and Pinot Meunier, Arbanne, Petit Meslier, Pinot Blanc Chenin Blan, Chardonnay, Cabernet Franc, Pineau D'Aunis, Grolleau, Gamay, Aligote, Melon, Sacy Chenin Blan, Chardonne, Sauvignon Blanc, Cabernet Franc, Cabernet Sauvignon, Gamay, Grollo, Malbec, Pineau D'Aunis, Pinot Noir Chardonnay blanc, Pinot blanc, Pinot noir, Pinot Gris, Pinot Meunier Chardonnay and Pinot blanc, Pinot noir Glera, Verdiso, Chardonnay blanc, Pinot blanc, Pinot noir, Pinot Gris, Bianchetta trevigiana, Perera Muscat petit grain, Muscat white, Brachetto, Malvasia di Casorzo, Malvasia di Schierano

Champenoise

Muscat white Chardonnay, Pinot Noir, Pinot Blanc, Pinot Meunier Macabeo, Xarel lo, Parellada, Subirat, Chardonnay (blanc), Garnacha tinta, Monastrell (red), Pinot Noir, Malvasıa, Trepat Bical, Cercial, Maria Gomes and Baga

Asti Classic Traditional

Silvaner, Pinot blanc, Pinot noir, and Pinot gris, Riesling

Traditional/ Charmat Traditional

Crémants Vin mousseux Italy

Spain Portugal Germany England Austria Hungary

Talento Franciacorta Prosecco Brachetto d'Acqui DOCG Asti Trento DOC Cava Sparkling wine Sekt Sparkling wine Sekt Pezsgő

Russia

Chardonnay and other classic Champagne grapes, Auxerrois, Seyval Blanc, Müller-Thurgau, Reichensteiner, and Bacchus Welschriesling and Grüner Veltliner, Blaufränkisch Chardonnay, Pinot Noir, Riesling, Muscat Ottonel, Muscat Lunel, Olaszrizling, Kékfrankos, Furmint, Királyleányka, Hárslevelű, Kéknyelű, and Junfark Pinot Noir, Pinot Gris, Chardonnay, Sauvignon, Aligote, Riesling

Ancestrale Charmat

Classic Classic Bulk/Classic Marinotti

Traditional

Traditional Traditional/ Charmat Transfer

Other countries Africa America

Cup Classique Sparkling wine

Sauvignon Blanc, Chenin Blanc, Chardonnay, Pinot Noir

Cup Classique

Chardonnay, Pinot Noir, Pinot Meunier, Pinot Blanc

Traditional/ Charmat

Chapter 11  New Trends in Sparkling Wine Production: Yeast Rational Selection   351

Table 11.1  Summary of Main Sparkling Wines Produced, Categorized by Their Country of Origin and Varieties Used—cont’d Country

Name

Grape Cultivar

Method

Ontario

Sparkling wine Sparkling icewine, Cuvée Close Sparkling wine Sparkling Shiraz

Chardonnay, Pinot Noir, Cabernet franc

Traditional

Chardonnay, Riesling, Vidal, Pinot Noir, Gamay noir

Charmat

Chardonnay, Pinot Noir and Pinot Meunier

Traditional/ Charmat Traditional

Australia

Shiraz

Fig. 11.2  Similitudes and differences among the three main methods for sparkling wine production.

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aromas when aromatic grape varieties are used due to the less time in contact with the yeast lees (Torchio et al., 2012). In this case, the wine is filtered before the bottling. The transfer method is a combination of both traditional and Charmat technologies. In particular, it follows the same first steps of the traditional method (secondary fermentation inside the bottle) but, after about 2 months from the bottling, wine is transferred to a tank maintained under isobaric conditions to prevent loss of the gas, filtered under pressure at −3°C (26°F) and then rebottled into a fresh bottle. In this way, all advantages of fermentation in bottle are saved avoiding the disgorging step and reducing the loss of wine and production costs.

11.2.1  The Base Wine Preparation Whatever is the chosen technology, the first steps follow the same vinification practices typical for still wines. It is possible to employ white (blanc de blancs) or red grapes (blanc de noirs) vinified as a white wine (elimination of must maceration and intensification of clarification). In general, authorized grape varieties for sparkling wine production are legislated by designations of origin and, therefore these are the same from years (Table 11.1). For example, for Champagne production only Chardonnay, Pinot noir, Pinot Meunier, and rarely Petit Meslier and Arbanne are permitted. Each of these three main varieties contribute individual attributes to the final product, described as “finesse and elegance” for Chardonnay, “body” for Pinot noir, and “fruitiness and roundness” for Pinot Meunier (Jackson, 2008). Even if, recently a new strategy of product diversification was used as the alternative grape varieties for sparkling wine production (Kemp et al., 2015), winemakers know that some grape varieties have better foaming properties than others or that certain treatments performed in the winery can also affect wine foam quality (Vanrell et al., 2007; Vincenzi et al., 2014). Given that the alcohol content increases during secondary fermentation, its best amount in a base wine should be around 9% (v/v) of ethanol and not higher than 10%–10.5% (v/v). Another important component to consider is the acidity that should be adequate to ensure long aging potential (around 5.5 g/L of tartaric acid). In general, grapes grown in cool climate regions may not reach full maturity in each growing season resulting in higher acidity and not fully ripened fruit. These grapes are desired in sparkling wine production as they typically have higher titratable acidity (TA), lower pH, and lower soluble solids measured in Brix. Grapes for sparkling wine are typically harvested at lower sugar levels than grapes for table wines (MartínezLapuente et al., 2013, 2015; Jones et al., 2014). The dates of harvesting have a key role in the sparkling wine quality, in fact, Esteruelas et al.

Chapter 11  New Trends in Sparkling Wine Production: Yeast Rational Selection   353

(2015) studying grapes with different ripening grade demonstrated that advancing the dates of the harvest the foaming properties of wines were improved, while grapes picked earlier led to sparkling wines with more intense herbaceous notes (Coelho et al., 2009). After the harvesting, grapes are gently pressed (≤1.5 bar), transferred in a tank and prepared for the first fermentation. Two pressings can be used. The first one give rise to the highest quality must called the “cuvée,” while the second pressing, known as “tailles” (or tails) has a lesser quality. Selected yeasts (mainly Saccharomyces cerevisiae) generally conduct primary fermentation because the wide clarification processes, the low pH, the low temperature (around 16°C), the presence of sulfur dioxide (SO2), and the absence of grape skins make difficult to complete this process. While there is limited research on non-Saccharomyces used for the secondary fermentation and its exploitation for base wine production is becoming widely diffused. Recently, it was demonstrated that a sequential culture with Torulaspora delbrueckii or Metschnikowia pulcherrima together with S. cerevisiae permitted the release of a high amount of polysaccharides improving foam ability, foam persistence, and changing the aromatic profile of the obtained base wines by increasing smoky and flowery notes (González-Royo et al., 2015). In particular, the use of a sequential inoculation of T. delbrueckii and S. cerevisiae during the primary fermentation caused an increase of the protein concentration into the base wine probably due to the autolysis of the non-Saccharomyces cells and then of the maximal height of the foam (HM) of the base wine (Medina-Trujillo et al., 2016). Sometimes, an additional malolactic fermentation can occur (Blasco et al., 2011) after or simultaneously to the first alcoholic fermentation due to a cofermentation of yeasts with lactic acid bacteria (LAB). This process positively or negatively influences sparkling wine production process and the final quality (for a review see Kemp et al., 2015). After the primary fermentation, often several different still wines are mixed (blending or coupage) because it is rare that a base wine coming from a single vintage, a single vineyard, and a grape variety has the right balance of sugar concentration, flavor, and acidity suitable for sparkling winemaking. Blending is a key step and sometimes up to 80 different wines from different grape varieties, vineyards and vintages can be mixed to produce one champagne (Liger-Belair et al., 2012, 2017).

11.2.2  Pied-de-cuve Preparation Generally, yeast strains chosen to conduct the prise de mousse belong to the S. cerevisiae (la Gatta et al., 2016). During secondary fermentation, several stress factors are present, either common to all

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winemaking styles although with a different intensity, or specific to second fermentation. This condition requires an extra effort for cells to withstand the very unfavorable conditions (Borrull et al., 2015, 2016). To overcome these stresses and have a quick and efficient achievement of the secondary fermentation, the yeast population must be appropriately adapted prior to be inoculated into the base wine (Martí-Raga et  al., 2016a,b; Tai et  al., 2007). The “acclimation process” can be defined as the adaptation of yeast cells to one or different stresses over a period of several hours or days. This procedure, known as pied-de-cuve preparation, consists of two phases: adaptation and proliferation (Borrull et al., 2016). There is not a standard composition for the adaptation medium, but the starter culture must be inoculated in increasing concentrations of ethanol and sugar (Martí-Raga et al., 2015, 2016a). Recently, Martí-Raga et al. (2015, 2016a) demonstrated that different nitrogen sources used in the pied-de-cuve modulate the fermentation kinetics in a strain-dependent manner. In particular, the use of an adaptation media with poor nitrogen sources confers to the yeasts the capacity to maintain their viability for a long period reaching its highest pressure. Furthermore, it was found that nitrogen liberation into the sparkling wine can be improved by adding inorganic sources in the base wine. Divergently, the addition of inactive dry yeast determines the increase of the polysaccharide concentration and foaming properties of the sparkling wine (Martí-Raga et al., 2016b). During the yeast acclimation process, several factors have to be considered to ensure the completion of secondary fermentation such as nitrogen intake, yeast strain, fermentation temperature, and the type of yeast metabolism. In fact, Borrull et  al. (2016) demonstrated that only “NOXI” cells that had a fermentative metabolism (grown in semi-­ anaerobic conditions and in the presence of sugar) were able to complete secondary fermentation because these cells were in the best physiological state (they displayed the best microvacuolation, the highest vacuolar activity, and the lowest reactive oxygen species accumulation). These authors also found differences in the membrane composition, in fact, NOXI cells presented the lower ratio between unsaturated and saturated fatty acids, connected with a minor membrane fluidity and consequently a major ethanol stress resistance (Ding et al., 2009). In general, in order to have an adequate onset of secondary fermentation, the 2%–5% of the final volume should be constituted by the inoculum, approximately 106 CFU/mL (Carrascosa et  al., 2011): a higher number of cells accelerate the process but may increase the production of hydrogen sulfide (H2S) and additional off-odors (Jackson, 2000; Ribéreau-Gayon et al., 2006). Typically, the cuvée is combined with the liqueur de tirage because the base wine has lost much of its nutrients content and cannot support further fermentations.

Chapter 11  New Trends in Sparkling Wine Production: Yeast Rational Selection   355

The liqueur de tirage consists of a mix of still wine, sugar (usually sucrose cane or beet sugar at 20–25 g/L, must or concentrated must), the yeast starter culture, adjuvant (mainly bentonite 3 g/100 L) to promote yeast cells separation from wine after aging), nutrients, and other compounds useful to stimulate yeast growth. It is possible to add also yeast lees that confer a more aged taste to the final product in a shorter time (la Gatta et al., 2016). Once the liqueur de tirage is added, secondary fermentation starts. Regarding the traditional and the transfer methods, bottles are filled and closed with a cup-shaped plastic insert known as a bidule and a metal crown cap.

11.2.3 Secondary Fermentation and Aging Secondary fermentation is the key step for sparkling wines production. In this phase, in fact, active yeasts produce the desired foam and aroma, traits typical of these wines and, for this reason, it is known with the French name prise de mousse (literally translated as “catch the foam”). In this period, wine is stacked at a low, stable temperature (<16°C), and light exposure is avoided. Bottles, for traditional and transfer methods are placed horizontally in order to allow the maximum interface area between yeast cells and cuvée. Several factors determine the secondary fermentation duration and rate such as the yeast strain (Martinez-Rodriguez et  al., 2002; Torrens et  al., 2008; Martí-Raga et  al., 2015; Martínez-García et  al., 2017), yeast’s initial cell number, viability and physiological state (Borrull et al., 2016); temperature (Martí-Raga et al., 2015), CO2 pressure (Kunkee and Ough, 1966), and the chemical composition of the cuvée (Martí-Raga et al., 2015, 2017). Generally, under secondary fermentation conditions, yeast’s growth and metabolism are low because there is an increase of ethanol and CO2 amounts and a decrease of carbon and nitrogen levels. Despite the adaptation step, after the inoculum there is a lag phase, followed by a short cell proliferation step, in which the population reaches about 107 CFU/mL (Carrascosa et al., 2011). At a constant temperature, secondary fermentation usually lasts approximately 2–3 months and subsequently the cell viability fall down, until no viable cells are detected at 9 months (Martí-Raga et al., 2016a,b; Lombardi et al., 2015). Subsequently, the sparkling wines obtained by the traditional or transfer methods are left in contact with lees for months to years, while aging of Charmat style sparkling wines lasts for a few days to some weeks. Generally, a slow wine aging increases the positive effects of the yeast autolysis, improving the final quality of the wine, but it considerably increases the production costs (Lombardi et al., 2015). Lees consists of microorganisms (predominately yeasts) and, in minor proportion, of technological coadjuvants, phenolic compounds, inorganic

356  Chapter 11  New Trends in Sparkling Wine Production: Yeast Rational Selection

matter, and tartaric acid (Kemp et al., 2015). This period known as sur lie aging is necessary to improve and define the organoleptic quality of sparkling wines (Lombardi et al., 2015) because autolysis takes place and new sensory characteristics described as sweet, yeasty, toasty, biscuity, or brioche-like are developed. Autolysis (literally means “self-destruction”) is a slow process in sparkling wines with repercussions on the aroma, mouthfeel, and foaming properties of wines (Suárez-Lepe and Morata, 2012). It is an irreversible lytic event due to the action of hydrolytic enzymes, which release cytoplasmic (peptides, amino acids, fatty acids, and nucleotides) and cell wall compounds (glucans, mannoproteins) into the wine (Fig. 11.3). This step provides unique characteristics to each bottle (Webber et al., 2017) (for a review see Alexandre and Guilloux-Benatier, 2006; Pozo-Bayón et al., 2009a; Torresi et al., 2011; Kemp et al., 2015). Compositional changes during aging sur lie were postulated as the result of the activity of enzymes released by the dead cells together with the release of intracellular compounds (Tudela et al., 2012). It is generally recognized that four main phases occur during aging on lees (Pozo-Bayón et al., 2009a): (1) during the secondary fermentation the amount of amino acids and proteins decrease while peptides are released; (2) viable and dead cells coexist and peptides are degraded and a release of amino acids and proteins occurred; (3) when no viable cells are present, the release of both proteins and peptides predominates; and (4) about 9 months after the tirage, there is a decrease of amino acids concentration. In sparkling winemaking, yeast autolysis starts 2–4 months after the end of secondary fermentation (Alexandre and Guilloux-Benatier, 2006) and in bottle it may last at least 9 months or longer (OIV, 2016). In fact, this process occurs far from ideal conditions (low pH, low temperature, and in the presence of ethanol) and in particular the low temperature causes a low death and enzymatic reaction rates (Tudela et al., 2012). Depending on the duration of the biological aging, two different categories of traditional sparkling wines are known: Reserva, with at least 15 months in contact with the lees and Gran Reserva, with at least 30 months’ contact (Serra-Cayuela et  al., 2013; Pérez-Bernal et  al., 2017). Instead, for Charmat technology if aging lasts between 1 and 3 months, we have the short method that gives rise to sparkling wines with young, fresh, and fruity aroma. Divergently, whether sparkling wine is left in contact with lees for 6 months (long Charmat) obtained beverage has aroma characteristics more similar to wines produced by traditional method (Buxaderas and López-Tamames, 2012). Sometimes, autolysis can be induced by pasteurization of the wine at temperatures of between 33°C and 70°C for 2–5 days.

tid

as

es

s

Gl

se

yc o

na

ca

sid

lu

as

G

es

th

(in ve r

P

ta ro t s au chi es, eas m tin β-g e at as lu s in- es c a lik e and nas pr es ot , ein s)

Pe p

Proteins/peptides/aminoacids Volatile compounds binding Tensioactive function Bioactive function (antioxidant, antimicrobial, antihypertensive)

Nucleotides/nucleosides Flavour compounds Flavour enhancer

Wine aroma retention Foamability Foam stability

Fig. 11.3  Autolysis in sparkling wine.

Wine aroma

Lipids Release of fatty acids Foaming properties???

Aroma precursors Foamability and stability

Vitispirane

Polysaccharides

TDN (1,1,6-trimethyl1,2-dihydronaphthalene)

(glucans and mannoproteins)

Wine aroma

Tensioactive function

Improve tartaric stability Improve stability against protein haze Foaming properties

Chapter 11  New Trends in Sparkling Wine Production: Yeast Rational Selection   357

DNases/RNases

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Aging sur lie duration influences both foaming properties and aroma composition of sparkling wines (Pérez-Magariño et al., 2015a). In fact, it is well known that the volatile compounds have different origins: the grape cultivar (varietal aroma), the yeasts metabolism (fermentative aroma), or the aging stage (postfermentative aroma) (Torresi et al., 2011; Torchio et al., 2012; Bertagnolli et al., 2017). During this last period, the sorption mechanism of the yeast cell walls can modify the changes occurred during the secondary fermentation (Torrens et al., 2010; Pérez-Magariño et al., 2013), reducing aroma compounds concentration in sparkling aged wines, mainly the most hydrophobic ones (Gallardo-Chacón et al., 2009, 2010a). In this way, the aging duration determine the type and amount of the volatile compounds present in the sparkling wine (Riu-Aumatell et al., 2006; Gallardo-Chacón et al., 2010a). In fact, some compound such as vitispirane (eucalyptus aroma) and TDN (1,2-dihydro-1,1,6-trimethylnaphthalene) (petrol aroma) or diethyl succinate has been used to differentiate between shorter and longer lees aging in sparkling wines (Riu-Aumatell et al., 2006; Pozo-Bayón et  al., 2009a) while ethyl dihydrocinnamate was purposed as volatile marker of the secondary fermentation in bottle (Muñoz-Redondo et al., 2017).

11.2.4  Final Steps in Sparkling Wine Production When aging is complete, yeast lees are separated from the wine. For traditional and transfer methods two phases are necessary: riddling (remuage) and disgorging (dégorgement). In order to recruit lees into the bidule, bottles are gradually turned around their central axis with increasing inclination until they are perpendicular to the floor. When sparkling wine is clear, it is refrigerated to avoid CO2 leakage and the neck of the bottle is placed in a calcium chloride or a glycol solution. This process allows the freezing of the lees trapped in the bidule. Bottles are then uncorked and the pressure ejects the bidule with its ice plug. Even if bottles are immediately corked or recapped, a small amount of wine is lost when wines are disgorged and, for this reason, liqueur d’expédition or dosage solution (containing wine, grape must or a blend of wine and grape must, to which can be added cane or beet sugar, dextrose or liquid sugar, rectified concentrated grape must, oxidized wine, SO2, citric acid, tannin, and occasionally brandy or other spirits) is added either in bottles at filling, or in the closed tank after the fermentation and before bottling (OIV, 2016; Crumpton et al., 2017). The liqueur d’expédition permits to balance the wine and can confer a unique flavor to the wine (Kemp et al., 2017). Furthermore, its addition deeply modifies sparkling wine quality because it modifies the viscosity (Liger-Belair, 2017). It was demonstrated, for example, that the more is the amount of sucrose added at dosage, the more

Chapter 11  New Trends in Sparkling Wine Production: Yeast Rational Selection   359

­ btained sparkling wine is characterized by a quickly and higher foam o when compared to a Brut nature wine (Crumpton et al., 2017). Finally, bottles are filled and corked. Sparkling wines obtained by traditional and transfer methods can also be subjected to a further aging period, also known as “marriage” in which the volatile composition of the beverage could undergo to considerable changes.

11.3  Specialized Yeasts for Sparkling Wine Production Despite several methods and research are conducted one of the best and easily methods to improve sparkling wine production and final quality is the choice of a good starter. In fact, the yeast strain used for the first and second fermentation influences the taste, flavor, bouquet, and even the color of the wine (for a review see Suárez-Lepe and Morata, 2012). Starter selection for sparkling wine production is a hard challenge because secondary fermentation conditions represent a hostile environment and often winemakers believe that the yeast has the only role to increase the pressure without affect other characteristics of the final product. Furthermore, strain selection requires long times of testing to verify the effect on sparkling wines characteristics and the interactions among environmental and technological factors are difficult to be elucidated (Vigentini et al., 2017). Generally, for sparkling wines production specialized S. cerevisiae strains are used because they can grow in base wine. It represents a difficult condition due to the presence of high alcohol concentration, sulfites, and glycerol, low pH, high total acidity, nutrient starvation and CO2 pressure, low temperature and accumulation of toxic fermentation subproducts, such as medium-chain fatty acids (MCFA, C6-C12), and organic acids (Borrull et al., 2015). MCFAs are produced during alcoholic fermentation in hypoxic conditions and decanoic (C10) acid is the most toxic. These fatty acids interact with various stress factors and are able to extend the lag phase, decrease the maximum growth rate and the biomass yield. For this reason, yeast strain has to possess additional technological features respect to starters used for the first fermentation (Fig. 11.4). Fig. 11.4 shows that sparkling wine starter have to be able to grow in base wine and secondary fermentation conditions and to be capable to modify wine characteristics through their metabolism and autolysis. While some stress factors can be depleted such as the nutrient starvation (by the addiction of nutrients in the tirage solution) or the impact of ethanol (by acclimatization step), other stresses are

360  Chapter 11  New Trends in Sparkling Wine Production: Yeast Rational Selection

Fig. 11.4  Technological features of strains for primary and secondary fermentation.

Chapter 11  New Trends in Sparkling Wine Production: Yeast Rational Selection   361

strongly determinant for the starter choice. Among them, two technological features are particularly important for sparkling wine production: autolytic ability and flocculation capacity. The importance of autolytic phenomenon was already discussed. It is interesting to know that different strains can have a different amount of released compounds. In fact, it is well known that sparkling wines obtained by different yeasts can differ from the concentration of peptides and single amino acids, of aroma compounds and polysaccharides. Either the qualitative composition of yeast cell walls or the formation of their wall polysaccharides varies depending on the background of the strains (Schiavone et al., 2015). Moreover, different yeasts may have different polysaccharide release kinetics (Tao et al., 2014).

11.3.1  Yeast Flocculation Mechanism and Genetic Basis In S. cerevisiae flocculation is an asexual, calcium dependent, natural evolved trait in which cells adhere to each other to form flocs consisting of thousands of cells that rapidly sediment to the bottom of the wells/bottles. This phenotype offers a more cost-effective and environmental friendly method for biomass recovery than centrifugation and filtration (Wan et al., 2015; Di Gianvito et al., 2017). This cell-cell adhesion is due to the presence of a family of ­lectin-like proteins called “flocculins” or “adhesins” that protrude to the cell wall and selectively bind mannose molecules on the surface of adjacent cells. Among them, Flo1, Flo5, Flo9, and Flo10 are involved in the cell-cell adhesion, while Flo11p (or Muc1p) is the main responsible of cell-surface interactions. For a long time, flocculation mechanism was not clear and several hypotheses were formulated. Actually, it is known that this phenotype is a time-dependent process in which firstly there is a glycan-glycan interaction, followed by a binding between mannose residues and flocculins (Goossens et al., 2015). During time, the number of interactions increases (El-Kirat-Chatel et al., 2015) supported by calcium ions (Ca2+) that are bridging molecule between sugars (Goossens et  al., 2015) and sugar-proteins (Veelders et al., 2010). Flo proteins have a common structure constituted by three domains (C-terminal, central region, and N-terminal). The C domain permits the link between the protein and the cell surface by the presence of a glycosylphosphatidylinositol structure (GPI anchor). The central region is long, flexible, and rich in serine/threonine residues and it is a spacer. This domain can have different length in Flo proteins, compared to determine a different exposition of the N-terminal of the proteins (Verstrepen et al., 2005). The N-terminal is recognized

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as CRD (carbohydrate-recognition domain) because it is responsible of the specific carbohydrate recognition (Kobayashi et al., 1998; Sim et al., 2013). Characterization of flocculation has defined two subcategories: the Flo1 and NewFlo. The first one phenotype is inhibited only by mannose, while the NewFlo-type, commonly found in brewing strains, is inhibited by multiple sugars including mannose, maltose, glucose, and sucrose, as well as by ammonium ions (Stratford and Assinder, 1991). Flocculation is a complex phenomenon strongly influenced by the expression of specific genes, including FLO genes, cell wall protein genes (CWP, TIR, and DAN genes), mitochondrial genes (OLI1 e OXI2), and genes involved in the cell wall biosynthesis (WAL and ABS) (Panteloglou et  al., 2012; Tofalo et  al., 2016). Recently, it was demonstrated that other two genes (AMN1 and RGA1) have a key role in flocculation development (Li et al., 2013). RGA1 is implicated in control of septin organization, pheromone response, and haploid invasive growth, while AMN1 is required for daughter cell separation and it could be more important than FLO1 gene in flocculation development (Li et al., 2013). FLO family is considered the most important for flocculation development and it includes 13 genes, 5 of which have been recognized as dominant structural genes (FLO1, FLO5, FLO9, FLO10, and FLO11), while FLO8 encodes for the transcriptional activator. It was demonstrated that, while FLO11 gene is involved in cell-surface adhesion, FLO1, FLO5, FLO9, and FLO10 genes are responsible of cell-cell adhesion. In Table 11.2 are reported the most important FLO family genes and their characteristics. The structure of FLO genes is essentially the same, constituted by a promoter sequence followed by the N-terminal domain, a central region, and the C-terminal. The central domain significantly affect FLO genes expression in response to environmental changes (Yue et  al., 2013) and stability because it is rich of tandem repeats (Verstrepen et al., 2005). Furthermore, FLO1, FLO5, FLO9, and FLO10 are in subtelomeric position, while FLO11 is near the centromere of the chromosome IX (Verstrepen et  al., 2004). The position and the presence of tandem repetition cause recombination events (Verstrepen et  al., 2005; Christiaens et  al., 2012) and the creation of new adhesins or pseudogenes (Govender et al., 2008; Van Mulders et al., 2009; Tofalo et al., 2014; Alvarez et al., 2014). Often proteins originated from these genes do not work because they have not all the functional domains (Bojsen et al., 2012). The high number of intra- and intergenic recombination events, the existence of different flocculation degrees and phenotypes, the fact that different FLO proteins have dissimilar responses to

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Table 11.2 Principal FLO Genes and Their Characteristics Gene

Characteristics

Milestones

FLO1

Chromosome: I Function: lectin-like protein, cell-cell adhesion Systematic name: YAR050W Aliases: FLO2 , FLO4 Paralog: FLO5 ORF length (in S288C):4614 bp Protein length: 1537 amino acids Repeated region: from 819 to 4303 bp 18 (135 bp), 2 (60 bp), 3 (153 bp), 3 (27 bp) Chromosome: VIII Function: lectin-like protein, cell-cell adhesion Systematic name: YHR211W Paralog: FLO1 ORF length (in S288C): 3228 bp Protein length: 1075 amino acids Repeated region: from 819 to 2844 bp = 8 (135 bp), 2 (60 bp), 3 (153 bp) Chromosome: I Function: lectin-like protein, cell-cell adhesion Systematic name: YAL063C ORF length (in S288C): 3969 bp Protein length: 1322 amino acid Repeated region: from 819 to 3558 bp = 13 (135 bp), 3 (45 bp), 3 (153 bp) Chromosome: XI Function: lectin-like protein, cell-cell adhesion Systematic name: YKR102W ORF length (in S288C): 3510 bp Protein length: 1169 amino acids Repeated region: from 303 to 608 bp = 4 (108 bp), 5 (81 bp), 1 (72 bp)

FLO1 is considered the most important gene of FLO family (Kobayashi et al., 1998; Van Mulders et al., 2009). Floc-forming ability conferred by FLO1 is chymotrypsin sensitive and heat resistant (Hodgson et al., 1985). Smukalla et al. (2008) purposed FLO1 as green-beard gene, demonstrating that it leads the direct cooperation toward the cells that carry it. FLO1 can be activated by FLO8 gene and in a Flo8pindependent manner (Bester et al., 2006; Shen et al., 2006; Fichtner et al., 2007) FLO5 has been identified as a dominant flocculation gene (Teunissen and Steensma, 1995). The duplication of FLO1 gene on chromosome VIII give rise to FLO5 gene (Teunissen and Steensma, 1995). Floc-forming ability conferred by FLO5 is chymotrypsin-­ resistant but heat-labile (Hodgson et al., 1985). Di Gianvito et al. (2017) purposed FLO5 as green-beard gene for wine yeasts, demonstrating that it drives flocculation in a flocculent wine S. cerevisiae strain. FLO9 gene is 94% similar to the FLO1 Its N-terminal shares the 89% of similarity with Flo5A domain (Veelders et al., 2010)

FLO5

FLO9

FLO10

FLO10 gene confers flocculation and invasive and pseudohyphal growth when overexpressed (Guo et al., 2000; Van Mulders et al., 2009). Its N-terminal shares the 64% of similarity with Flo5A domain (Veelders et al., 2010) FLO10 expression is regulated by metastable epigenetic silencing (Halme et al., 2004).

Continued

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Table 11.2 Principal FLO Genes and Their Characteristics—cont’d Gene

Characteristics

Milestones

FLO11

Chromosome: IX Function: cell wall glycoprotein, cellsurface adhesion Systematic name: YIR019C Aliases: MUC1, STA4 ORF length (in S288C): 4104 bp Protein length: 1367 amino acids N° Repeats: 4 (10 aa), 2 (13 aa), 3 (32 aa), 22 (15 aa), 15 (12 aa), 1 (16 aa)

FLO8

Chromosome: VIII Function: transcriptional activator Systematic name: YER109C Aliases: PHD5 , YER108C , STA10 ORF length (in S288C): 2400 bp Protein length: 799 amino acids

FLO11 is required for flocculation only in S. cerevisiae var. diastaticus (Bayly et al., 2005). It is responsible for formation of “flor velum,” biofilm on plastic and liquid surfaces, haploid invasive growth and diploid pseudohyphae formation, for the formation of fibrous interconnections between cells of colony-grown S. cerevisiae wild strains (Váchová et al., 2011; Tofalo et al., 2014). N-Flo11p does not bind mannose (Goossens and Willaert, 2012). FLO11 expression is under many conventional regulatory mechanisms, epigenetic and posttranscriptional control (Soares, 2011). Transcription factor of FLO1 (Kobayashi et al., 1999), FLO11 (Rupp et al., 1999), FLO9 (Verstrepen et al., 2003), STA1 (Kim et al., 2004). It is required for flocculation, diploid filamentous growth and haploid invasive growth (Kim et al., 2004). S. cerevisiae genome reference strain S288C contains an internal in-frame stop at codon 142, which in other strains encodes tryptophan (Liu et al., 1996).

­ roteases or heat treatments or still, bind different sugars could p partially explains the existence of a large family of genes with a considerable sequence homology (Di Gianvito et  al., 2017). However, the evolutionary advantage linked to the presence of a large family of genes involved in the same phenotype is still unclear (Rossouw et al., 2015). It was purposed that yeast evolution in response of different environments or to the microbial populations could bring to the expansion of a specific FLO gene (Green-beard gene) that would become essential for the social behavior (Rossouw et  al., 2015; Di Gianvito et al., 2017). It was established that in laboratory strain S288C FLO1 is the greenbeard gene responsible of flocculation (Smukalla et al., 2008). Greenbeards are genes that can recognize copy of themselves in the genome of other individuals, and their expression leads a social behave of cooperation with other bearers individuals. However, the importance of

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FLO5 gene was demonstrated in some industrial strains (Watari et al., 1990; Tofalo et al., 2014, 2016; Di Gianvito et al., 2017). For this reason, it is reasonable to think that the presence of multiple alleles of FLO genes that discriminate among different allotypes, could exist multiple “colors” of beards (Smukalla et al., 2008).

11.3.1.1  The Importance of Flocculation in Sparkling Wine Production As previously explained, flocculation provides an easy and low cost method for cells recovery during sparkling wine production, able to reduce riddling time to 2 days using automated riddling machines with 504 bottles in each cage (Kemp et al., 2015). However, flocculation confers to yeasts also an advantage in term of major stress resistance because the floc is a protective structure that physically isolates cells from the outer environment (Smukalla et al., 2008; Veelders et al., 2010; Goossens et al., 2015). Furthermore, floc is not only physical unit of hundreds of yeast cells, but also a microsociety (Zhao and Bai, 2009) and inside it, individuals can cooperate to overcome rapidly the stress factors. In particular, flocculent yeasts are more resistant to ethanol stress (Smukalla et al., 2008; Tofalo et al., 2014), antifungal agents (Smukalla et al., 2008), and lignocellulosic inhibitors such as acetic acid, furfural, and hydroxymethyl furfural (Brandberg et al., 2004; Purwadi et al., 2007; Westman et al., 2012, 2014; Landaeta et al., 2013). The mechanisms purposed to explain stress resistance of flocculent strains are different: (1) physical protection: the outer cells of the floc (called “cheaters” because are nonflocculent) are exposed to the harsh condition, while cells in the core found a low amount of inhibitors (Zhao and Bai, 2009); (2) altruistic suicide: under stressing conditions some cells embedded in the floc can undergo autolysis and provide nutrients (proteins, carbohydrates and vitamins) which can support the survival of the younger and healthier cells of the floc (Herker et al., 2004; Smukalla et al., 2008); (3) inhibitors degradation: external cells convert inhibitor compounds and leave subinhibitory levels for inner cells. (Purwadi et al., 2007; Westman et al., 2014). These outer cells are equipped with drug-efflux pumps (Váchová et  al., 2011; Štovíček et  al., 2012) and enter in a stationary phase of growth becoming more resistant to environmental stress (Wloch-Salamon, 2014). (4) deficit of ergosterol: the oxygen and nutrient starvation inside the flocs results in a deficit of ergosterol, the principal target of several antifungal drugs (Smukalla et al., 2008);

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(5) extracellular matrix (ECM) production: cells in the floc secrete an ECM that blocks the permeation of large molecules (e.g., antibodies) (Beauvais et al., 2009; Váchová et al., 2011); (6) increase of mating efficiency: inside the floc the contacts between cells and the zygote formation are promoted. Furthermore, the diploid offspring may contain genetic variations that could affect the survivor and the evolution (Goossens et al., 2015); and (7) sporulation induction: genes associated with “sporulation” and “spore wall assembly” are upregulated in flocculent strains (Smukalla et al., 2008; Goossens et al., 2015). Even if the exploitation of flocculent yeast starter is widespread for traditional sparkling wine production, few studies are focused on the behavior of these strains during secondary fermentation or their influence in the quality of the final product. Some hybrids obtained by crossing a flocculent strain of S. cerevisiae with a nonflocculent yeast of Saccharomyces uvarum were tested for sparkling winemaking and showed performance intermediate respect to the parental strains. In particular, these yeasts were able to complete secondary fermentation in a wide range of temperature (from 6°C to 36°C). Riddling was easy because they were flocculent and obtained sparkling wines had characteristic and excellent aromatic profiles that were not inferior to wines fermented with the parental strains (Coloretti et al., 2006). Some S. cerevisiae strains with different flocculent degree were tested for 6 months-sparkling wine fermentation/aging in laboratory. The authors found that higher was the flocculation degree, the highest was the ability of strains to survive in stress conditions (Tofalo et al., 2016). These yeasts showed also a different amino acids and protein release into the sparkling wine demonstrating that autolytic ability is independent to the flocculation degree (Perpetuini et al., 2016). The study of FLO genes expression highlighted that the most expressed gene during secondary fermentation was FLO5 and this gene was the only overexpressed after 6 months of aging (Tofalo et  al., 2016). Previously, a higher expression of FLO5 was also correlated to the best ethanol stress resistance (Tofalo et al., 2014) and it was purposed that this compound could be a quorum sensing molecule for flocculent strains (Smukalla et al., 2008). Probably high ethanol concentrations could induce FLO genes expression through the numerous stressresponsive heat-shock proteins (HSP), which were found in the promoter region of these genes (Verstrepen et al., 2003; Claro et al., 2007). The genome wide expression of a flocculent strain during first and secondary fermentation, in fact, highlighted the upregulation of several HSP determining an enhanced unfolded protein response (UPR) (Di Gianvito et al., 2018a). This condition was associated with a higher resistance to ethanol stress (Navarro-Tapia et al., 2016).

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Also higher alcohols like 2-phenylethanol (rose aroma) are quorum sensing molecule in flocculent strains (Chen and Fink, 2006), in fact, during secondary fermentation an overexpression of ARO9 and ARO10 genes (enzymes involved in the first reactions of higher alcohols production) was found (Di Gianvito et al., 2018b). Moreover, yeast flocculation seems to be associated with the enhancement of ester production (Soares, 2011). In fact, sparkling wines fermented with flocculent strains presented a good floral and fruity aroma already after 3 months of secondary fermentation/aging (Di Gianvito et al., 2018a).

11.4  New Trends in Sparkling Wines Research Wine plays an increasing role in our daily life and nowadays the consumers control the new market trends (Giovenzana et al., 2016). Due to the high value and the growing sales of this special wine, producers need to understand, accelerate, and improve its production to meet market requests. For this reason, researchers and winemakers are constantly engaged for product improvements. Studies are conducted in three main areas of sparkling wines production: technological, health, and quality-related aspects.

11.4.1  Technological-Related Improvements Nowadays sparkling wine production is far from the empirical rules coded in the 17th century by Dom Perignon. Previously, winemakers were committed in the decrease of both the time and cost of sparkling wine production and new industrial production methods were developed. Instead, at present environmental issues, such as wine production sustainability is studied (Pomarici and Vecchio, 2014). For quality purposes, base wine fermentation is conducted at low temperature; grape must is cooled at the beginning and during all the process with an expense of energy. Giovenzana et al. (2016) tested a S. cerevisiae wine strain, selected for its sensorial performances and low SO2 production, at higher temperatures than the standard ones. These authors demonstrated that the use of this yeast in Franciacorta base wine production had positive effects on energy saving without compromising sensory, chemical, and aromatic profiles. Other studies were conducted to increase the performance of yeasts during secondary fermentation improving the Pied-de-cuve preparation (Martí-Raga et al., 2016a,b; Borrull et al., 2016). It is well known that the most expensive (in terms of energy, space and time) step of bottle-fermented sparkling wines is the separation

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of yeasts from the medium, in fact riddling and disgorging represent the 43%–50% of the total expenses for the production of a bottle of Champagne (Efremenko et al., 2006). Traditionally, to simplify these phases, bentonite is added into the bottles. This riddling agent is allowed to facilitate lees flocculation. However, bentonite is a nonspecific absorber that can interact with wine, proteins, and compounds responsible for aroma and colors (Jaeckels et al., 2017). To avoid these problems, the use of immobilized yeasts was purposed (Wada et al., 1979; Veliky and Williams, 1981). Several studies were conducted introducing inside the bottles yeast cells immobilized in beads or gels made with different supports in order to diminish and simplify or even eliminate the riddling and disgorging procedures (Genisheva et al., 2014). It was demonstrated that obtained sparkling wines were equivalent to those prepared with free cells in the content of ethanol, organic acids, total nitrogen, higher alcohols, and sensory parameters (Fumi et al., 1987, 1988; Yokotsuka et al., 1997; Vanrell et al., 2007; Bozdoğan and Canbaş, 2012). Furthermore, this technology permits to control the fermentation, minimize its duration and protects partially cells from the toxicity of the ethanol (Tataridis et  al., 2005; Berovic et  al., 2014), resulting in less storage time being needed in the winery. Otherwise, in some cases a cells discharge from the support was observed (Martynenko et al., 2004) and other methods of immobilization were studied. Supports used in winemaking should be cheap, of food-grade purity, available in nature, stable, easy to prepare and to use at industrial scale, of good biocompatibility and able to sustain low-temperature fermentation (Duarte et al., 2013). Different immobilization techniques were tested such as carrageenan entrapping, collagen casting, chitosan/glutaraldehyde molding, and most notably calcium-alginate gel or films entrapping (Santos et  al., 2018) that is the most promising and used for sparkling wine production. Recently, Berovic et al. (2014) developed a new separation method based on the magnetization of the yeast cells. These authors obtained magnetically responsive yeasts by the adsorption of superparamagnetic, iron-oxide maghemite (γ-Fe2O3) nanoparticles onto the surfaces of the cells. The application of a magnetic force attracted magnetically responsive cells in the direction of the increasing magnetic-field density allowing the separation. It was demonstrated that the magnetic nanoparticles did not penetrate inside the cells and increased the metabolic activity of the yeast during primary and secondary fermentations probably for the presence of free iron ions in the medium. It was also found that this technology did not influence the final wine composition and the amount of Fe3+ was in the range of permissible iron concentrations (Berovic et al., 2014). However, the effect on sparkling wine quality as well as yeast multiplication, foaming, oxidative potential, and longterm lees aging is currently unclear (Kemp et al., 2015).

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Furthermore, the natural and spontaneous immobilization/coimmobilization of S. cerevisiae was considered. For example, the coimmobilization between S. cerevisiae and the filamentous fungus Penicillium chrysogenum in biocapsules was studied (Peinado et al., 2006). During the fermentation the fungus died remaining only as a mere inert support for yeasts (García-Martínez et al., 2011), while the yeast maintain most of its catalytic activity. These biocapsules were tested in some fermentative processes such as sweet wines, bioethanol, and sparkling wine production (Peinado et  al., 2006; GarcíaMartínez et al., 2008; Puig-Pujol et al., 2013). At the same time, yeast flocculation can be seen as a natural way of yeast immobilization (Verbelen et al., 2006). This capacity is really sought in yeasts for sparkling wine production by traditional method and, for this reason, the presence of this phenotype was purposed as a new additional technological feature for starter selection (PozoBayón et al., 2009a; Kurec and Brányik, 2011; Tofalo et al., 2014, 2016; Perpetuini et al., 2016; Garofalo et al., 2016). Flocculation mechanism and genetic basis are discussed in Section 11.3.1.

11.4.2  Health-Related Improvements In recent years, one of the new market strategies in the oenological industry is based on the discovery that moderate alcohol consumption may prevent cardiovascular heart disease. This theory known as “French Paradox” is independent of the type of alcoholic beverage (liquor, beer, and wine), but some studies found that wine consumption has the major effects on global mortality, cardiovascular mortality, and incidence of cancer (Satué-Gracia et  al., 1999; Gronbaek et  al., 2000; Theobald et al., 2003). The mechanisms by which moderate alcohol consumption is associated to atherosclerosis are not completely known (Vázquez-Agell et al., 2007), but researchers attributed this action to ethanol itself and also to the presence of phenolic compounds. These molecules, in fact, contribute to sensory quality and have antioxidant properties. Some studies were conducted to demonstrate that alcoholic beverages with medium-level polyphenols content like sparkling wines have antioxidant properties (Vázquez-Agell et  al., 2007; Jordão et al., 2010; Stefenon et al., 2014), especially when elaborated with β-glucanases and yeast preparations that could have a potential improvement of sparkling wine antioxidant capacity (RodriguezNogales et  al., 2012b). Furthermore, during aging this characteristic was also linked to the presence of yeast lees (Gallardo-Chacón et al., 2010b) because these have an affinity for oxygen much higher than wine polyphenols (Salmon, 2005; Martín et al., 2013). Give that sparkling wine consumption has good properties, some studies were conducted improve the heath quality of this ­beverage.

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In particular, one topic was the reduction of the sulfite content in base wine (Giovenzana et  al., 2016) as well as in the final product (Fracassetti et al., 2016; Webber et al., 2017). SO2 is traditionally used to prevent the oxidation and also for its antimicrobial action, but it is toxic and may cause similar allergenic reaction in sulfite-sensitive people (Pozo-Bayón et al., 2012; Webber et al., 2017). The strategies of reduction suggested the substitution of this molecule with antioxidant compounds, but results are still not satisfactory. For example, Fracassetti et al. (2016) evaluated the addition in the liqueur de tirage of three different polyphenols-based antioxidant formulas into an Italian sparkling white wine in order to find a potential substitute of SO2. Even if, obtained results demonstrated that the tested mixtures were unsuitable to completely avoid the use of SO2, it was highlighted that further investigations could bring to find the best antioxidant formula to substitute SO2. With the same purpose, after the disgorging, Webber et al. (2017) introduced variables concentrations of l-glutathione (GSH) reduced form in sparkling wines. GSH is a tripeptide with antioxidant properties and its use was recently included among the oenological practices recommended by the OIV (OIV, 2016). Despite the promising results presented in the previous studies in model wines, these authors found that the addition of 20 mg/L (allowed limit) showed little influence on the preservation of the sparkling wine. These studies demonstrate that we are still further from the substitution of SO2, but this is the right way.

11.4.3  Quality-Related Improvements The major number of studies conducted on sparkling wines was focused on the improvement of their quality. As explained before, sparkling wine is the result of several factors and for this reason, different topics were considered. A general tendency was the diversification of the products and, for this purpose, the potentiality of different grape varieties was explored. Among them, several autochthonous cultivars were considered and it was found that they can confer a varietal fingerprint that makes unique and the obtained wine is peculiar (García et  al., 2009; Pozo-Bayón et  al., 2010; Pérez-Magariño et  al., 2013; Caliari et  al., 2014, 2015; la Gatta et al., 2016; Coldea et al., 2016; Ruiz-Moreno et al., 2017). Even if there is a growing interest for these grapes, we are still far from the widespread use and the designation of origin. As for the cultivars, the research of autochthonous yeasts was also conducted (Hidalgo et al., 2004; Vigentini et al., 2017). These microorganisms, in fact, are potentially adapted to a specific grape must and reflect the biodiversity of a particular area, which supports the

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idea that indigenous yeast strains can be associated with a “terroir” (Bokulich et al., 2014; Mas et al., 2016). This idea is so well established that it was also purposed to make a secondary fermentation using the natural population of high ethanol resistant strains collected after the spontaneous fermentation of a selected must (Vigentini et al., 2017). However, sparkling wine fermentation is a hard challenge for yeasts because of the harsh conditions and for this reason yeast strains sealed as starters for secondary fermentation are a small number and mainly belong to the S. cerevisiae (Torresi et  al., 2011; Borrull et  al., 2015; Vigentini et  al., 2015, 2017; Perpetuini et  al., 2016). Moreover, the use of non-Saccharomyces yeasts in sparkling winemaking could increase the glycerol content of the wine and affect viscosity (decrease of foaming properties), mouthfeel, and wine flavor (Kemp et al., 2015). The conditions of secondary fermentation as well as the production technology determine the starter choice and the features that must be investigated, for this reason a rational selection must be done. Divergently from young wines, a key step to improving sparkling wines quality is the aging sur lie. Its duration is a key hurdle in bringing these special wines to the market and influences production costs (Pérez-Bernal et  al., 2017). Therefore, the decrease of aging time is highly desirable to reduce the microbial spoilage (Alexandre and Guilloux-Benatier, 2006; Comuzzo et al., 2015, 2017). Aging time varies but, for quality sparkling wines of European origin (e.g., Champagne in France, Cava in Spain, Sekt in Germany, and Franciacorta, Asti, and Prosecco in Italy), it usually takes anywhere from a few months to several years (Tudela et al., 2012). Some new approaches include the design of procedures able to accelerate the natural process of yeast autolysis in order to obtain high-quality wines in a shorter time. Until now, various ways of accelerating autolytic phenomenon have been examined as reported in the next sections.

11.4.3.1  Increase of the Storage Temperature During sparkling winemaking autolysis needs several months to occur because wine is far from the ideal conditions: 45°C and pH 5 in the absence of ethanol. Because the pH of the wine cannot be changed the first purpose to accelerate autolysis was the increase of aging temperature (Charpentier and Feuillat, 1993). These authors found that whether wine was heated at 45–55°C, there was a fast release of considerable amounts of nitrogen into the wine. Nevertheless, it was also noticed that if the time/temperature was too aggressive, a destruction of intracellular proteases occurred, so slow yeast autolysis cannot continue (Feuillat and Charpentier, 1982). While, if enzyme activity was maintained, the increase of storage temperature generated off-flavors lately described as “overly toasty,” therefore this procedure is not used.

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11.4.3.2  Yeast Components/Enzyme Addition Another strategy for accelerating the autolytic process is the addition of yeast components to the base wine. In general these preparations, mixed at the tirage step, are obtained from S. cerevisiae and can be classified in four types depending on the manufacture process employed for their production: inactivated dry yeasts (IDY), yeast autolysates, yeast hulls or walls, and yeast extracts (Pozo-Bayón et  al., 2009b). Among them, supplementation with IDY as additional source of proteins, mannoproteins, and polysaccharides has been proposed (Martínez-Lapuente et  al., 2013). Their use in the tirage phase premised to obtain sparkling wines with higher glycerol content and in general better appreciated from the sensory point of view than the control (sparkling wine without IDY) (Medina-Trujillo et  al., 2017). In the same work, IDY from S. cerevisiae and T. delbrueckii were exploited and better results were obtained for non-Saccharomyces IDY (Medina-Trujillo et al., 2017). Different concentrations of a yeast mannoproteins extracted by mild heat procedures were also tested and a positive influence on foaming properties (Núñez et al., 2006) and volatile profile (Pérez-Magariño et al., 2015b) were highlighted. Unfortunately, results using IDY are controversy because the thermal treatments used during their manufacturing can lead to the formation of off-flavors that may be released into the wine, negatively affecting its sensory properties (Pozo-Bayón et al., 2009a,b; Comuzzo et al., 2015). Other strategies were developed based on the idea that the release of intracellular compounds during autolysis is due to the action of hydrolytic enzymes (β-glucanases, mannosidases, proteases, etc.). Enzymes were widely used in winemaking either before, during or after the fermentation for several purposes. Among them, β-glucanases were purposed to accelerate autolysis during sparkling wine production. Even if in oenology, β-glucanases are exploited from years, studies during sparkling winemaking are more recent. The use of exogenous β-glucanases sourced from Trichoderma spp. and Aspergillus niger was investigated (Torresi et al., 2014). The monitoring of aging on lees showed that tested enzymes were able to enhance the cell disorganization and to improve the release of amino acids into the wines. Divergently, influence on total proteins content or foam characteristics was not found (Torresi et al., 2014). Furthermore, the addition of yeast derivatives (yeast cell walls and yeast autolysates) or β-glucanases on the chemical composition of sparkling wines was compared. It was found that β-glucanases addition determined an increase of the aging characteristics in the sparkling wines, while yeast derivatives (yeast cell walls and yeast autolysates) improved their fruity and flowery character (RodriguezNogales et al., 2012a).

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However, enzyme preparations are costly and thus less expensive techniques should be assayed (Martín et al., 2013).

11.4.3.3  Use of Killer/Killer Sensitive Strains Another explored method to overcome the delay of the autolysis during sparkling wine production was the providing of a pool of dead yeast cells immediately after the end of the secondary fermentation. In particular, it was purposed to exploit the K2 (Todd et  al., 2000) or Klus (Rodríguez-Cousiño et  al., 2011) toxins of S. cerevisiae against the sensitive yeast of the same species. These authors demonstrated that in a coculture with a killer and a killer sensitive strain, the first one dominated during the fermentation in synthetic medium at low pH and in the presence of ethanol. Furthermore, the cocultures presented a higher amount of released proteins respect to the pure cultures: the use of this strategy either for the propagation of the tirage starter culture and as a mixed culture inoculum during the secondary fermentation was purposed. The effect of this strategy in real conditions was later explored with contrasting results. Lombardi et al. (2015) demonstrated the use of the mix of killer/killer sensitive strains caused the release of higher amount of total proteins, amino acids, and polysaccharides than the wines fermented with pure cultures already after 3 months. Otherwise, other authors found significant differences only in the increase of mannose content (from mannan) in the mixed-inoculated sparkling wines, but not the total polysaccharide and protein contents after 6 months of secondary fermentation/aging (Velázquez et al., 2016). Both the studies presented a general major sensorial acceptability of wines obtained from the killer sensitive yeast coculture, already after 3 months (Lombardi et  al., 2015) and a positive influence on the foam ability, height, and stability, without negatively affecting the fermentation kinetics (Velázquez et al., 2016). Also in this case, the selection of the appropriate couple of killer/ killer sensitive yeasts has a key role because it is well known that different sensitive yeasts can have a dissimilar degree of sensitivity to the toxin. Furthermore, killer strains can present differences in the amount of toxin produced and in the behavior during secondary fermentation (condition far from those optimal for S. cerevisiae killer activity).

11.4.3.4  Other Technologies In recent years, due to the importance of autolysis for the development of peculiar characteristics of wines, other technologies were purposed to accelerate this phenomenon. Among them, high pressure homogenization (HPH), pulsed electric field (PEF), and ultrasounds (US) technologies were suggested.

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HPH is a sustainable nonthermal treatment for large-scale microbial cell disruption, intracellular bioproduct recovery, or enzyme activity modulation. Cell wall breakdown is induced through cavitation, shear, and turbulence phenomena suffered during the passage of the microorganisms through the homogenization valves (Comuzzo et al., 2017). The exploitation of autolysates obtained by HPH was explored for still (Comuzzo et al., 2015) and sparkling wines (Patrignani et al., 2013) production. The advantage of the use of HPH to produce yeast autolysates consists in the possibility to accelerate the yeasts autolysis (modification of ester profile with lower medium- and long-chain fatty acid concentrations) and to tailor the composition and aroma profile of yeast derivatives (Comuzzo et al., 2017). In fact, it leads to an enhancement of enzyme activities and to a change of their specificity (Lanciotti et al., 2007). Despite the positive results obtained on the aroma profile of sparkling wines, further studies are necessary to understand the effects of HPH on sensory attributes of sparkling wine and foaming ability (Kemp et al., 2015). Even if studies in real condition are not yet performed, also PEF (application of intermittent electric fields of high intensity for short periods) (Barba et  al., 2015) or US were able to reduce yeast cell viability and increase the release of intracellular compounds in model systems or in still wines (Martín et al., 2013; Liu et al., 2016). For this reason, they could be potentially used for sparkling winemaking because they are relatively low-cost, nonhazardous and environmentally friendly techniques.

11.4.3.5  Use of Yeasts With High Autolytic Capacity Several studies demonstrated that yeasts are the protagonists in sparkling winemaking and each of them can be considered a specific autolytic profile (Rodriguez-Nogales et  al., 2012b). Sometimes the solutions previously explained to reduce autolysis time gave contradictory results or are expensive and for this reason, the use of yeasts with high autolytic capacity is the easier tool. The use of selected starters or of a mix of flocculent strains differing in autolytic activity (Perpetuini et  al., 2016) or of genetically modify yeasts (Nunez et al., 2005; Cebollero et al., 2005; Tabera et al., 2006) was purposed as new strategies for improving sparkling wine quality. While the exploitation of yeasts that have a naturally high autolytic ability does not have problems, the use of genetically modified yeasts is not premised in all the countries and often have poor consumer acceptance. Despite this issue, some studies were conducted. Through random mutagenesis, Gonzalez et  al. (2003) obtained autolytic mutants from an industrial second fermentation strain (S. ­cerevisiae IFI473) and the mutant IFI473I presented the highest release of nitrogen compounds in a model system. Subsequently, this strain was

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tested for sparkling wine elaboration and it showed a high release of mannose as well as a high-quality foam (Nunez et al., 2005). Further studies started from the confirmed idea that autophagy occurs in oenological conditions and it is involved in the early stages of autolysis (Cebollero and Gonzalez, 2006; Tudela et al., 2012). Autophagy is an intracellular degradation and recycling pathway highly conserved in eukaryotes. During autophagy, cellular material is engulfed in a double membrane vesicle (autophagosome) that fuses with the vacuole. The vesicle and its contents are degraded by the resident hydrolases, and the catabolites are exported to the cytoplasm and recycled (for a review see Galluzzi et al., 2017; Torggler et al., 2017). The modification of the expression of autophagy-related genes can promote the cell death. It can occur with the deletion of these genes or with their overexpression. For example, Tabera et al. (2006) demonstrated that a partial deletion of BCY1 gene (a regulatory subunit of cAMP-dependent protein kinase A, pathway important for the control of metabolism, stress resistance, proliferation, and filamentous growth) produced a lack of autophagy, so an autolytic phenotype under simulated secondary fermentation conditions. On the contrary, also excessive autophagy levels can promote the autophagy-dependent cell death. It was demonstrated that the overexpression of CSC1-1 caused a constitutive autophagy phenotype resulting in accelerated autolysis in a model system (Cebollero et al., 2005). However, this mutant strain was not tested in sparkling wine conditions. Further studies are necessary to better understand autophagy/autolysis machinery and how to modulate these phenomena.

11.5 Conclusions Sparkling wine technology is an ancient and complex art. Due to the high added value, researchers are constantly engaged to study the mechanisms and reactions which make this wine unique and peculiar. Factors that affect its quality are several and different and to find the right balance between these elements is a hard challenge for winemakers. One of the most important aspects to consider is the starter selection. Even if often the number of isolates and characters to consider are numerous the yeast rational selection offers the best way to obtain strains of S. cerevisiae with technological properties that might improve the sensorial profile of wines as well as the production technology. Two oenological features required to a good starter for sparkling wine production are autolytic ability and flocculation capacity. In particular, flocculent yeasts are able to make easy and eco-friendly the riddling step. Furthermore, these yeasts have good performance

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during secondary fermentation and could have a good sensorial impact in the final product; in fact, they can carry out autolysis at high levels, depending on the strains but not on the degree of flocculation (Perpetuini et al., 2016). Further studies are necessary understand the real effect of flocculent yeasts in sparkling wines final quality and to select a tailored flocculent yeast capable to conduct secondary fermentation and to confer peculiar aroma to the wine.

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